HK1255016A1 - Methods of treating solid or lymphatic tumors by combination therapy - Google Patents

Description

Methods of treating solid or lymphoid tumors by combination therapy

Technical Field

The present invention relates to the topical administration of an infectious agent in combination with one or more immunomodulators for cancer immunotherapy.

Background

The human immune system of innate and adaptive immunity is a very complex system that has not been successfully used to combat cancer. One explanation is that since cancer usually develops in the later part of life, the development of an immune response against cancer is not important to the theory of survival of the fittest in the course of evolution. In all possibilities, the different aspects of the human immune system are not specifically designed for the purpose of intending to kill cells considered as "self". Even after extensive removal of the primary tumor, it is still problematic to prevent the formation of metastases due to the growth of micro-metastases already present at the time of surgery or the formation of new metastases from tumor cells or tumor stem cells that have not been completely removed or reattached after surgery. Basically, surgery and/or radiation therapy can only take into account macroscopic lesions for later stages of cancer, while most patients have recurrent cancer and are not suitable for further therapy.

Recently, the FDA has approved two immunotherapeutic agents against prostate cancer and melanoma. The first agent, Propofol (Provenge), utilizes GM-CSF fusion molecules with prostate antigens to activate monocytes or antigen-presenting cells in late stage cancer patients in vitro and to prolong the overall survival of such patients. The second agent is an anti-CTLA-4 monoclonal antibody, which has been shown to produce a significantly enhanced effect in T-effector cell production. Oncolytic virus CG0070 has also been shown to trigger long-term complete responses in bladder cancer patients after a series of six weekly intravesical treatments (see Burke JM et al, journal of Urology Dec,188(6)2391-7, 2012).

Current cancer immunotherapy approaches face a variety of fundamental challenges. For example, tumor-specific immune T lymphocytes in cancer patients, even when present, often only occur systemically at low frequencies. Possible reasons are that the antigenicity and specific immunogenicity of the tumor antigens of common cancers are often poor, and that there is a tremendous amount of repressor activity via the interleukins and regulatory cells (e.g., tregs, tumor-associated macrophages, etc.). In addition, the old concept of using non-specific components to boost an immune response to a specific component has been found to be less successful because the human body's ability to generate an extremely specific immune response against its own cells is naturally limited. After all, most cancer cells are not sufficiently immunogenic to be different from normal cells. Such immune responses, if generated, derived from non-specific immune components will be short lived.

For at least the reasons discussed above, attempts have been made to use in vitro and pre-formulated therapeutic cancer vaccines with available tumor antigens and adjuvants for decades without much success. There is a clear need for cancer immunotherapy methods with improved performance.

The disclosures of all publications, patents, patent applications, and published patent applications cited herein are hereby incorporated by reference in their entirety.

Disclosure of Invention

The present application provides methods, compositions (including pharmaceutical compositions) and kits for treating a solid or lymphatic tumor in an individual comprising topically administering to the site of the tumor an infectious agent and an immunomodulator (including a combination of immunomodulators). Such methods, compositions, and kits may also include tumor cells that are not activated or uses thereof. Local administration of therapeutic components (e.g., infectious agents, immunomodulators and non-activated tumor cells) to the tumor site is a key requirement of the present invention.

Without being bound by any theory or hypothesis, the present invention is based in part on an "at" tumor site concept for delivering therapeutic components and eliciting an immune response, which can be used to overcome potentially insurmountable obstacles in treating solid or resistant lymphoid tumors. According to the concept of "at" the tumor site, the therapeutic component is delivered "at" the tumor site, in the correct effective amount, at the correct time, and in the correct order. The effective amount, time, and sequence of the therapeutic component can each independently be adjusted based on the particular condition of the tumor. For example, administration using a combination of an infectious agent, an immunomodulator and optionally non-activated tumor cells "at the tumor site concept can produce an appropriate amount of an immune-related molecule (e.g., GM-CSF) selected from the infectious agent, the individual's own body response, and/or optionally administered live tumor cells, resulting in the release of a tolerance-destroying antigen (TBA), which facilitates the confirmation of an immune signal (e.g., 1, 2, 3 signals of CD4 and CD8T cells) and the generation of effector cells also" at the tumor site. Thus, it is believed that the "complete" specific cancer immunotherapy response with a strong and durable effect occurs "at the tumor site.

Without being bound by any theory or hypothesis, it is believed that the release of a previously unknown tumor specific or selective resistance-destroying antigen (TBA) at the tumor site by the real-time infection process may play a critical role in this process, since such antigens can only be released at the exact time and site of cell death. TBA may consist of antigens derived from tumors or even from structures vital to tumors (e.g., stromal cells), and TBA may not be previously transcribed by AIRE (an autoimmune regulator gene in the thymus). In addition, it is believed that the release of TBA is a transient phenomenon that must be captured at the tumor site.

Within the framework of the concept of "at" the tumor site, it is further believed that the use of immune modulators (e.g., immune checkpoint inhibitors) and immune stimulants can greatly help provide a synergistic effect with the use of an infectious agent and non-activated tumor cells just "at" the tumor site. Depending on the dose, route of administration, and other pharmacokinetic and pharmacodynamic factors, immunomodulators can exert different effects on the body and in particular on the immune system. The concept of "at" the tumor site in the present invention requires that the immunomodulator be administered at a dose and schedule that is adjustable, rather than being expressed, for example, at a fixed dose from a transgene. For example, increasing doses of IV administration of anti-CTLA-4 antagonist antibodies are associated with systemic increases in immunosuppressive agent cells (e.g., tregs). At sufficiently high systemic levels of anti-CTLA-4 antibodies, patients can only gain benefit in local tumor sites and draining lymph nodes, such as increased CD8/CD4 ratios and upregulation of IL12 and IFN γ, among others, which is associated with significant immune-related adverse events and exacerbation of autoimmune conditions (including irreversible and fatal events). In contrast, in the present invention, an immunomodulator (e.g., an anti-CTLA-4 antibody) is administered "at" the tumor site, such that the "complete" specific cancer immunotherapy response happens to occur "at" the tumor site, including the "live" mixture of specific cancer cell death and release of TBA (less normal cell death to confound the system), "real-time" maturation and migration of antigen presenting cells and immune cells, confirmation of immune signals via immunomodulators (e.g., co-stimulatory factors in immune cell activation, function, survival, expansion and memory, antagonists and agonists of inhibitory checkpoint molecules). All of the above immune events occur exactly at the tumor site, contrary to conventional wisdom in the art that relies solely on a central or systemic immune response via secondary lymphoid organs to eradicate tumor cells.

without being bound by any theory or hypothesis, it is believed that IL6 in combination with TGF diverts Treg and other CD4 cells to the Th17 immune pathway if this transition occurs just immediately upon cancer cell death and antigen presentation and immune cell activation, which is necessary for effector T cells to destroy so-called "autoimmune" cancer cells, otherwise, when only the Th1 pathway is temporarily conferred without releasing IL6 and targeting to the Th17 pathway, cancer cells are "tolerated" by effector T cells.

Accordingly, one aspect of the present patent application provides a method of treating a solid or lymphatic tumor in a subject (e.g., a human subject), the method comprising: a) locally administering to the tumor site an effective amount of an infectious agent; and b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, there is provided a method of inhibiting metastasis of a solid or lymphatic tumor in a subject (e.g., a human subject), the method comprising: a) locally administering to the tumor site an effective amount of an infectious agent; and b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators).

In some embodiments according to any of the methods provided above, the infectious agent is a virus, including a non-oncolytic virus or an oncolytic virus, such as a virus selected from the group consisting of: adenovirus, herpes simplex virus, vaccinia virus, mumps virus, newcastle disease virus, poliovirus, measles virus, Seneca valleyvirus, coxsackie virus, rieo virus, vesicular stomatitis virus, maraba (maraba) and rhabdovirus, and parvovirus. In some embodiments, the infectious agent is a bacterium, such as Bacillus Calmette-Guerin (BCG), mycobacterial cell wall-DNA complex ("MCNA"), or listeria monocytogenes (listeria monocytogenes).

In some embodiments according to any one of the methods provided above, the infectious agent is an oncolytic virus. In some embodiments, the oncolytic virus is an oncolytic adenovirus. In some embodiments, the oncolytic virus preferentially replicates in cancer cells. In some embodiments, an oncolytic virus comprises a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication. In some embodiments, the tumor specific promoter is the E2F-1 promoter. In some embodiments, the tumor specific promoter is a human E2F-1 promoter. In some embodiments, the E2F-1 promoter comprises the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4.

In some embodiments according to any one of the methods provided above, the infectious agent and/or immunomodulator is administered directly into the tumor. In some embodiments, the infectious agent is administered directly into the tumor. In some embodiments, the immunomodulator is administered directly into the tumor.

In some embodiments according to any one of the methods provided above, the infectious agent and/or immunomodulator is administered to a tissue having a tumor. In some embodiments, the infectious agent is administered to a tissue having a tumor. In some embodiments, the immunomodulatory agent is administered to a tissue having a tumor.

In some embodiments according to any one of the methods provided above, the infectious agent and the immunomodulator are administered sequentially. In some embodiments, the infectious agent is administered prior to administration of the immunomodulator. In some embodiments, the infectious agent is administered after administration of the immunomodulator.

In some embodiments according to any one of the methods provided above, the infectious agent and the immunomodulator are administered simultaneously. In some embodiments, the infectious agent and the immunomodulator are administered in the same composition.

In some embodiments according to any one of the methods provided above, the immune modulator is a modulator of an immune checkpoint molecule selected from the group consisting of: CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the immunomodulatory agent is an inhibitor of CTLA-4, such as an anti-CTLA-4 antibody (e.g., Ipilimumab). In some embodiments, the anti-CTLA-4 antibody is selected from the group consisting of: ipilimumab, tremelimumab (Tremilimumab) and single chain anti-CTLA-4 antibody. In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin that specifically recognizes CTLA-4, e.g., an anti-transportin molecule that specifically binds to CTLA-4.

In some embodiments according to any of the methods provided above, the immunomodulatory agent is an immunostimulatory agent (e.g., an agonist of an immunostimulatory molecule). In some embodiments, the immunostimulant is an activator of OX40, 4-1BB, or CD 40. In some embodiments, the immunostimulatory agent is a stimulator of CD40, e.g., an agonist antibody to CD 40.

in some embodiments according to any of the methods provided above, the method further comprises locally administering to the tumor site an immune-related molecule (e.g., a cytokine, a chemokine, or PRRago (i.e., a pathogen recognition receptor agonist)). in some embodiments, the immune-related molecule is selected from the group consisting of GM-CSF, IL-2, IL-12, an interferon (e.g., a type 1, type 2, or type 3 interferon, such as interferon gamma), CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I, MDA5, LGP2, and LT α β. in some embodiments, the immune-related molecule is administered separately from the infectious agent.

In some embodiments according to any of the methods provided above, the infectious agent is a virus comprising a viral vector, and wherein the viral vector comprises a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine). In some embodiments, the nucleic acid encoding the immune-related molecule is operably linked to a viral promoter. In some embodiments, the virus is an adenovirus and the viral promoter is the E3 promoter. In some embodiments, the infectious agent is adenovirus serotype 5, wherein the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced with the human E2F-1 promoter and nucleic acid encoding human GM-CSF. In some embodiments, the infectious agent is CG 0070.

in some embodiments according to any of the methods provided above, the method further comprises locally administering a pretreatment composition to the tumor site prior to administration of the infectious agent.

in some embodiments according to any one of the methods provided above, the individual (e.g., at the tumor site completely or only) undergoes prior therapy prior to administration of the infectious agent and the immunomodulator.

In some embodiments according to any of the methods provided above, the method further comprises locally administering to the tumor site an effective amount of inactivated tumor cells. In some embodiments, the tumor cells that are not activated are autologous. In some embodiments, the tumor cells that are not activated are allogeneic. In some embodiments, the tumor cells that are not activated are from a tumor cell line. In some embodiments, the non-activated tumor cells are inactivated by irradiation.

In some embodiments according to any one of the methods provided above, the infectious agent and the tumor cells that are not activated are administered simultaneously. In some embodiments, the infectious agent and the non-activated tumor cells are administered in a single composition. In some embodiments, the infectious agent and the non-activated tumor cells are mixed immediately prior to administration.

In some embodiments according to any one of the methods provided above, the solid or lymphoid tumor is bladder cancer (e.g., muscle-invasive bladder cancer or non-muscle-invasive bladder cancer). In some embodiments, the infectious agent is administered intravesically. In some embodiments, the immunomodulatory agent is administered intravesically.

In some embodiments according to any of the methods provided above, the infectious agent and/or immunomodulator is administered weekly.

In some embodiments according to any of the methods provided above, the individual has high expression of one or more biomarkers in the tumor (e.g., tumor cells or tumor-derived immune cells) selected from the group consisting of: PD-1, PD-L1 and PD-L2. In some embodiments, the individual has high expression of one or more biomarkers in the mature dendritic cells derived from the tumor selected from the group consisting of: CD80, CD83, CD86 and HLA-class II antigens. In some embodiments, the individual has high expression of one or more biomarkers selected from the group consisting of: CXCL9, CXCL10, CXCL11, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1, HES4, MTIB, MTIE, MTIG, MTIH, GADD45A, LAMP3 and miR-155.

Another aspect of the present patent application provides a kit for treating a solid or lymphatic tumor in an individual, comprising: a) an infectious agent, b) an immunomodulator (including combinations of immunomodulators), and c) a device for local administration of the infectious agent or immunomodulator to a tumor site. In some embodiments, the infectious agent is a virus, e.g., a non-oncolytic virus or an oncolytic virus. In some embodiments, the infectious agent is an oncolytic adenovirus that preferentially replicates in cancer cells.

In some embodiments according to any one of the kits provided above, the immune modulator is a modulator of an immune checkpoint molecule selected from the group consisting of: CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the immunomodulatory agent is an inhibitor of CTLA-4, such as an anti-CTLA-4 antibody (e.g., ipilimumab). In some embodiments, the anti-CTLA-4 antibody is selected from the group consisting of: ipilimumab, tremelimumab and single chain anti-CTLA-4 antibody. In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin that specifically recognizes CTLA-4, e.g., an anti-transportin molecule that specifically binds to CTLA-4.

In some embodiments according to any one of the kits provided above, the immunomodulatory agent is an immunostimulatory agent (e.g., an agonist of an immunostimulatory molecule). In some embodiments, the immunostimulant is an activator of OX40, 4-1BB, or CD 40. In some embodiments, the immunostimulatory agent is a stimulator of CD40, e.g., an agonist antibody to CD 40.

in some embodiments, the infectious agent comprises a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine). in some embodiments, the immune-related molecule is selected from the group consisting of GM-CSF, IL-2, IL12, an interferon (e.g., a type 1, type 2, or type 3 interferon, such as interferon gamma), CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I, MDA5, LGP2, and LT α β. in some embodiments, the infectious agent is a virus comprising a viral vector, and wherein the viral vector comprises a nucleic acid encoding an immune-related molecule.

In some embodiments according to any one of the kits provided above, the kit further comprises an immune-related molecule selected from the group consisting of: STING activators (e.g., CDN), PRRago (e.g., CpG, imiquimod or poly I: C), TLR stimulators (e.g., GS-9620, AED-1419, CYT-003-QbG10, AVE-0675 or PF-7909), and RLR stimulators (e.g., RIG-I, Mda5 or LGP2 stimulators).

in some embodiments according to any one of the kits provided above, the kit further comprises a pretreatment composition comprising a transduction enhancer.

In some embodiments according to any one of the kits provided above, the kit further comprises a plurality of non-activated tumor cells. In some embodiments, the kit further comprises instructions to mix the infectious agent with the non-activated tumor cells prior to administration. In some embodiments, the device for local administration is used to administer a plurality of non-activated tumor cells and an infectious agent simultaneously.

In some embodiments according to any one of the kits provided above, the device for topical administration is for administering an infectious agent and/or an immunomodulator directly into a tumor. In some embodiments, the device for topical administration is used to administer an infectious agent and/or an immunomodulator to a tissue having a tumor.

In one aspect of the present patent application there is additionally provided a pharmaceutical composition comprising: a) an infectious agent, b) an immunomodulator (including combinations of immunomodulators), and c) a pharmaceutically acceptable excipient suitable for topical administration of the composition to the site of a tumor. In some embodiments, the pharmaceutically acceptable excipient is a polymer, such as a hydrogel.

In some embodiments according to any one of the pharmaceutical compositions provided above, the infectious agent is a virus, e.g., a non-oncolytic virus or an oncolytic virus. In some embodiments, the infectious agent is an oncolytic adenovirus that preferentially replicates in cancer cells.

In some embodiments according to any one of the pharmaceutical compositions provided above, the immune modulator is a modulator of an immune checkpoint molecule selected from the group consisting of: CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the immunomodulatory agent is an inhibitor of CTLA-4, such as an anti-CTLA-4 antibody (e.g., ipilimumab). In some embodiments, the anti-CTLA-4 antibody is selected from the group consisting of: ipilimumab, tremelimumab and single chain anti-CTLA-4 antibody. In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin that specifically recognizes CTLA-4, e.g., an anti-transportin molecule that specifically binds to CTLA-4.

In some embodiments according to any one of the pharmaceutical compositions provided above, the immunomodulatory agent is an immunostimulatory agent (e.g., an agonist of an immunostimulatory molecule). In some embodiments, the immunostimulant is an activator of OX40, 4-1BB, or CD 40. In some embodiments, the immunostimulatory agent is a stimulator of CD40, e.g., an agonist antibody to CD 40.

in some embodiments of any of the pharmaceutical compositions provided above, the infectious agent comprises a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine). in some embodiments, the immune-related molecule is selected from the group consisting of GM-CSF, IL-2, IL12, an interferon (e.g., a type 1, type 2, or type 3 interferon, such as interferon gamma), CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I, MDA5, LGP2, and LT α β. in some embodiments, the infectious agent is a virus comprising a viral vector, and wherein the viral vector comprises a nucleic acid encoding an immune-related molecule.

In some embodiments of the pharmaceutical composition provided above, the pharmaceutical composition further comprises an immune-related molecule selected from the group consisting of: STING activators (e.g., CDN), PRRago (e.g., CpG, imiquimod or poly I: C), TLR stimulators (e.g., GS-9620, AED-1419, CYT-003-QbG10, AVE-0675 or PF-7909), and RLR stimulators (e.g., RIG-I, Mda5 or LGP2 stimulators).

in some embodiments according to any one of the pharmaceutical compositions provided above, the pharmaceutical composition further comprises a pretreatment composition comprising a transduction enhancer.

In some embodiments according to any one of the pharmaceutical compositions provided above, the pharmaceutical composition further comprises a plurality of non-activated tumor cells. In some embodiments, the plurality of non-activated tumor cells are autologous. In some embodiments, the plurality of non-activated tumor cells are allogeneic. In some embodiments, the plurality of non-activated tumor cells are from a tumor cell line. In some embodiments, the plurality of non-activated tumor cells are inactivated by irradiation.

Also provided is the use of any of the infectious agents described herein and any of the immunomodulators (including combinations of immunomodulators) for treating a solid or lymphatic cancer (e.g., for inhibiting tumor metastasis), and the use of any of the infectious agents described herein and any of the immunomodulators (including combinations of immunomodulators) for the manufacture of a medicament for treating a solid or lymphatic cancer (e.g., for inhibiting tumor metastasis).

Such and other aspects and advantages of the present invention will become apparent from the following detailed description and appended claims. It will be understood that one, some or all of the features of the various embodiments described herein may be combined to form further embodiments of the invention.

Drawings

FIG. 1 is a schematic representation of CG0070 and wild-type (wt) adenovirus type 5. CG0070 is based on adenovirus serotype 5, but the endogenous E1a promoter and E319 kD coding regions have been replaced by the cDNA coding regions of the human E2F-1 promoter and human GM-CSF, respectively.

Figure 2 shows the groups of animals and dosing schedule in the in vivo study of example 9.

Figure 3 is a scatter plot showing the distribution of the listed metastatic lesions for each animal group on day 23 of the in vivo study of example 9. The horizontal line corresponds to the average value. The two-tailed statistical analysis was performed at P-0.05. Test results were considered insignificant (ns) at P >0.05, significant (indicated by symbol) at 0.01< P <0.05, significant (×) at 0.001< P <0.01, and significant (×) at P < 0.001.

Figure 4 is a box and whisker plot showing tumor volume for each animal group at day 19 in the in vivo study of example 9. Boxes represent the 25 th and 75 th percentiles of observations, lines represent median observations, and whiskers represent extreme observations.

Fig. 5 is a graph showing the dosing schedule of the in vivo study in example 10.

Detailed Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims the benefit of U.S. provisional patent application 62/243,512 filed on 10/19/2015, the contents of which are incorporated herein by reference in their entirety.

Submission of sequence Listing in ASCII text documents

The following submissions of ASCII text documents are incorporated by reference herein in their entirety: sequence Listing in Computer Readable Form (CRF) (filename: 744442000140SEQLIST. txt, recording date: 2016, 10, 14 days, size: 3 KB).

The present invention provides methods and compositions for treating a solid or lymphatic tumor in an individual, including inhibiting tumor metastasis, by locally administering to the tumor site an effective amount of an infectious agent (e.g., an oncolytic virus, optionally expressing or combined with an interleukin such as GM-CSF) and an effective amount of an immunomodulatory agent (including combinations of immunomodulatory agents, e.g., an immune stimulant and/or an immune checkpoint inhibitor). Such methods and compositions may also include the local administration of non-activated tumor cells. The infectious agent and/or immunomodulator and/or non-activated tumor cells may be administered directly into the tumor. Alternatively, the infectious agent and/or immunomodulator and/or inactive tumor cells are administered to a tissue having tumor cells. For example, one exemplary tumor suitable for the methods described herein is bladder cancer, and the infectious agent and/or immunomodulator may be administered intravesically.

The present invention provides live, real-time, "in vivo" cancer vaccine systems generated in humans by the local (e.g., intratumoral) delivery of therapeutic components, including an infectious agent, one or more immunomodulatory agents, and live cancer cells. Without being bound by any theory or hypothesis, it is believed that the in vivo site and real-time infection system results in the release of previously unknown tolerance-breaking antigens ("TBAs"), which may be essentially transient phenomena. Thus, effective adaptive immunotherapy against solid and lymphoid tumors can be achieved in the presence of all three components described herein (infectious agent, immunomodulator and live cancer cells present at or administered to the tumor site).

One requirement of the methods described herein is the local administration of an infectious agent, an immunomodulator (including combinations of immunomodulators), and optionally non-activated tumor cells to the tumor site. The direct effect of topical administration is important because if the components are not provided directly to the tumor cells (e.g., upon systemic administration), the components will produce pharmacokinetic and pharmacodynamic changes by or on the human body. Such changes would suggest a fine balance between tumor inhibition and activation in the wrong direction of the complex and subtle immune response required for success.

Thus, it is believed that the combinations described herein will allow full exploitation of both oncolytic and immunogenic responses in an individual and increase the therapeutic potential of cancer immunotherapy. It will be understood by those skilled in the art that the combination therapy methods described herein require that one agent or composition be administered in combination with another agent. The dosage, dosing schedule, route of administration, and order of administration of each agent in the combination therapies provided herein (e.g., infectious agent, each immunomodulator, and non-activated tumor cells) can be independently optimized to provide optimal therapeutic results. Such methods may also be further combined with pretreatment, such as local irradiation, or local administration of an interleukin, chemokine or other beneficial therapeutic agent, to increase the chance of success of the therapy.

In one aspect, there is provided a method of treating a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an infectious agent; and b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an infectious agent; b) locally administering to the tumor site an effective amount of an immunomodulator (including a combination of immunomodulators); and c) locally administering to the tumor site an effective amount of non-activated tumor cells. In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of an infectious agent; and b) intravesically administering an effective amount of an immunomodulator (including combinations of immunomodulators).

Also provided are compositions (e.g., pharmaceutical compositions), kits, and articles of manufacture useful for the methods described herein. In one aspect, a kit for treating a solid or lymphatic tumor in an individual is provided, comprising: a) an infectious agent, b) an immunomodulator (including combinations of immunomodulators), and c) a device for local administration of the infectious agent or immunomodulator to a tumor site. In one aspect, a kit for treating a solid or lymphatic tumor in an individual is provided, comprising: a) an infectious agent, b) an immunomodulator (including combinations of immunomodulators), c) a plurality of non-activated tumor cells; and d) a device for locally administering an infectious agent, an immunomodulator or a plurality of non-activated tumour cells to the tumour site. In another aspect, a pharmaceutical composition is provided comprising: a) an infectious agent, b) an immunomodulator (including combinations of immunomodulators), and c) a pharmaceutically acceptable excipient suitable for topical administration of the composition to the site of a tumor. In another aspect, a pharmaceutical composition is provided comprising: a) an infectious agent, b) an immunomodulator (including combinations of immunomodulators), c) a plurality of non-activated tumor cells; and d) a pharmaceutically acceptable excipient suitable for topical administration of the composition to the tumor site.

Definition of

As used herein, "treatment" or "treating" is a method for obtaining beneficial or desired results, including clinical results. For purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by a disease, reducing the extent of a disease, stabilizing a disease ((e.g., preventing or delaying disease progression), preventing or delaying disease spread (e.g., metastasis), preventing or delaying disease recurrence, reducing the rate of recurrence of a disease, delaying or delaying disease progression, improving disease status, providing remission (partial or total) of a disease, reducing the dose of one or more other agents needed to treat a disease, delaying disease progression, improving quality of life, and/or prolonging survival.

"adjuvant setting" refers to a clinical setting in which an individual has a history of cancer and is typically (but not necessarily) responsive to therapy, including but not limited to surgery (e.g., surgical resection), radiation therapy, and chemotherapy. Treatment or administration in the "adjuvant setting" refers to the subsequent mode of treatment.

By "lead setting" is meant a clinical setting in which the method is performed prior to the initial/established therapy. Lead setting herein also refers to any "tumor site preparation" treatment modality that is used in a sequential manner in conjunction with a therapeutic component as described herein (e.g., an infectious agent and an immunomodulator; or an infectious agent, an immunomodulator and non-activated tumor cells).

As used herein, "infectious agent" refers to a virus, including non-oncolytic or oncolytic viruses, including but not limited to adenovirus, herpes simplex virus, vaccinia virus, mumps virus, newcastle disease virus, polio virus, measles virus, seneca valley virus, coxsackie virus, rio virus, vesicular stomatitis virus, malaba and rhabdo virus, and parvovirus. In addition, the infectious agent may also be a bacterium, such as bacillus calmette-guerin (BCG), mycobacterial cell wall-DNA complex ("MCNA"), or listeria monocytogenes.

The term "effective amount" as used herein refers to an amount of a compound or composition sufficient to treat a given disorder, condition, or disease (e.g., ameliorate, alleviate, reduce, and/or delay one or more of its symptoms). In reference to cancer, an effective amount includes an amount sufficient to cause shrinkage of the tumor and/or reduce the growth rate of the tumor (e.g., inhibit tumor growth) or prevent or delay proliferation of other undesired cells in the cancer. In some embodiments, an effective amount is an amount sufficient to delay the development of cancer. In some embodiments, an effective amount is an amount sufficient to prevent or delay relapse. In some embodiments, an effective amount is an amount sufficient to reduce the recurrence rate in an individual. In some embodiments, an effective amount is an amount sufficient to inhibit tumor metastasis in an individual. An effective dose may be administered in one or more administrations. An effective amount of the drug or composition may: (i) reducing the number of cancer cells; (ii) reducing tumor size; (iii) inhibit, delay, slow to some extent, and preferably stop cancer cell infiltration into peripheral organs; (iv) inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; (v) inhibiting tumor growth; (vi) preventing tumorigenesis and/or recurrence; (vii) delay of tumorigenesis and/or recurrence; (viii) (iii) reducing the rate of recurrence of the tumor, and/or (ix) alleviating to some extent one or more symptoms associated with the cancer. As understood in the art, an "effective amount" may be one or more doses, i.e., a single dose or multiple doses may be required to achieve a desired therapeutic endpoint.

"in combination with …" or "in combination with …" refers to the administration of one treatment modality in addition to another, e.g., the administration of an infectious agent as described herein in addition to another agent (e.g., an immunomodulator and/or non-activated tumor cell) to the same individual under the same treatment plan. Thus, "in conjunction with …" or "in combination with …" refers to administration of one treatment modality before, during, or after delivery of another treatment modality to the individual.

The term "concurrently administering" as used herein means that the first and second therapies in the combination therapy are administered concurrently. Where the first and second therapies are administered simultaneously, the first and second therapies may be included in the same composition (e.g., the composition includes both the first and second therapies) or in separate compositions (e.g., the first therapy is included in one composition and the second therapy is included in another composition).

The term "sequentially administering" or "in order" as used herein means that the first and second therapies in a combination therapy are administered at intervals, for example, of greater than about 1 minute, such as greater than about any one of: 5. 10, 15, 20, 30, 40, 50, 60 or more minutes. In some instances, the term "sequentially administering" means that the first and second therapies in the combination therapy are administered at intervals of more than about 1 day, e.g., more than about any one of: from 1 day to 1 week, 2 weeks, 3 weeks, 4 weeks, 8 weeks, 12 weeks, or more. The first therapy or the second therapy may be administered first. The first and second therapies are included in separate compositions, which may be included in the same or different packages or kits.

The term "administered immediately prior to …" means that the first therapy is administered no more than about any of 15 minutes, such as no more than 10, 5, or 1 minutes, prior to the administration of the second therapy. The term "administered immediately after …" means that the first therapy is administered no more than about any one of 15 minutes, such as no more than 15, 10, or 1 minutes after the second therapy is administered.

As used herein, "specific" or "specificity" or "selectivity" when used in describing a compound as an inhibitor means that the compound interacts (e.g., binds, modulates and inhibits) preferentially with a particular target (e.g., proteins and enzymes) over a non-target.

The terms "transduction" and "transduction" as used herein include all methods known in the art for the use of infectious agents (e.g., viruses) or otherwise to introduce DNA into cells for expression of a protein or molecule of interest. In addition to viruses or virus-like agents, there are also chemical-based transfection methods, for example using calcium phosphate, dendrimers, liposomes or cationic polymers (e.g. DEAE-polydextrose or polyethyleneimine); non-chemical methods such as electroporation, cell extrusion, sonic perforation, optical transfection, puncture infection, protoplast fusion, plastid or transposon delivery; particle-based methods, e.g., using gene guns, magnetic or magnet-assisted transfection, particle bombardment; and hybridization methods, such as nuclear transfection.

The term "tumor-site preparation" as used herein describes a single treatment modality or a combination of more than one treatment modality to be used in combination with a therapeutic component (e.g., an infectious agent and an immunomodulator; or an infectious agent, an immunomodulator and non-activated tumor cells) in a sequential manner, and wherein a treatment modality or modality is administered directly or indirectly (e.g., via IV therapy) to a tumor site (e.g., cancer cells or tissue containing cancer cells). Exemplary therapeutic modalities for tumor site preparation include, but are not limited to, administration of immune-related molecules, irradiation, and administration of therapeutic agents. All tumor site preparation described herein may include administration of a single molecule or agent, or a combination of more than one molecule and/or agent.

It is to be understood that the embodiments of the invention described herein include "consisting of an embodiment" and/or "consisting essentially of an embodiment".

Reference herein to "about" a value or parameter includes (and describes) variations that are directed to that value or parameter itself. For example, descriptions that refer to "about X" include descriptions of "X".

As used herein, reference to "not" a value or parameter generally means and states "different from" the value or parameter. For example, a method not used to treat type X cancer means that the method is used to treat a cancer other than type X.

The term "about X-Y" as used herein has the same meaning as "about X to about Y".

As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

Methods of treating solid or lymphoid tumors

The present invention provides a method of treating a solid or lymphoid tumour (e.g. bladder cancer) in a subject (e.g. a human), the method comprising: a) locally administering to the tumor site an effective amount of an infectious agent; and b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, there is provided a method of inhibiting metastasis of a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an infectious agent; and b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, the infectious agent is a virus, for example a virus selected from the group consisting of: adenovirus, herpes simplex virus, vaccinia virus, mumps virus, newcastle disease virus, poliovirus, measles virus, seneca valley virus, coxsackie virus, rio virus, vesicular stomatitis virus, malaba and rhabdovirus, and parvovirus. In some embodiments, the infectious agent is a bacterium, such as a mycobacterium and derivatives thereof (e.g., bacillus calmette-guerin ("BCG"), or mycobacterium cell wall-DNA complex ("MCNA" or "MCC"), such as UROCIDIN^TM) Or listeria monocytogenes. In some embodiments, the infectious agent is a wild-type infectious agent. In some embodiments, the infectious agent is genetically modified. In some embodiments, the infectious agent is attenuated (e.g., via multiple passages, withoutActivation or genetic modification). In some embodiments, the infectious agent is only one or more portions of a wild-type infectious agent that can cause infection, inflammation, or infection-like effects. In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the methods comprise locally administering a combination of immune modulators comprising one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the infectious agent and/or immunomodulator (including combinations of immunomodulators) is administered directly into the tumor. In some embodiments, the infectious agent and/or immunomodulator (including combinations of immunomodulators) is administered to a tissue having a tumor. In some embodiments, both the infectious agent and the immunomodulator (including the combination of immunomodulators) are administered directly into the tumor. In some embodiments, both the infectious agent and the immunomodulator (including combinations of immunomodulators) are administered to a tissue having a tumor. In some embodiments, the infectious agent is administered weekly. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered weekly. In some embodiments, the method further comprises administering the infectious agent and/or immunomodulator (including combination of immunomodulators) by a route of administration other than topical administration.

Exemplary viruses suitable for use as infectious agents in the present invention include, but are not limited to, adenoviruses, such as H101CG-TG-102(Ad5/3-D24-GM-CSF) and CG 0070; herpes simplex viruses, e.g. Talimogene laherparapavec (T-VEC) and HSV-1716Rio viruses, e.g.Vaccinia viruses, such as JX-594; seneca valley viruses, such as NTX-010 and SVV-001; newcastle disease viruses such as NDV-NS1 and GL-ONC 1; polioviruses, such as PVS-RIPO; measles viruses, such as MV-NIS; coxsackie viruses, e.g. Cavatak^TM(ii) a Vesicular stomatitis virus; malaba and rhabdoviruses; parvovirus and mumps virus. In some embodiments, the virus is a non-oncolytic virus. In some embodiments, the virus is an oncolytic virus. In some embodiments, the virus is competent for replication. In some embodiments, the virus preferentially replicates in tumor cells.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus); and b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, there is provided a method of inhibiting metastasis of a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus); and b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, the oncolytic virus is a wild-type oncolytic virus. In some embodiments, the oncolytic virus is genetically modified. In some embodiments, the oncolytic virus is attenuated (e.g., via multiple passages, inactivation, or genetic modification). In some embodiments, the oncolytic virus is competent for replication. In some embodiments, the oncolytic virus preferentially replicates in cancer cells. In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the methods comprise locally administering a combination of immune modulators comprising one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the oncolytic virus and/or immunomodulator (including combinations of immunomodulators) is administered directly into the tumor. In some embodiments, an oncolytic virus and/or an immunomodulator (including a combination of immunomodulators) is administered to a tissue having a tumor. In some embodiments, both the oncolytic virus and the immunomodulator (including a combination of immunomodulators) are administered directly into the tumor. In some embodiments, both the oncolytic virus and an immunomodulator (including a combination of immunomodulators) are administered to a tissue having a tumor. In some embodiments, the oncolytic virus is administered weekly. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered weekly. In some embodiments, the method further comprises administering the oncolytic virus and/or an immunomodulator (including a combination of immunomodulators) by a route of administration other than topical administration.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication; and b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, there is provided a method of inhibiting metastasis of a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication; and b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the methods comprise locally administering a combination of immune modulators comprising one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4. In some embodiments, the oncolytic virus and/or immunomodulator (including combinations of immunomodulators) is administered directly into the tumor. In some embodiments, an oncolytic virus and/or an immunomodulator (including a combination of immunomodulators) is administered to a tissue having a tumor. In some embodiments, both the oncolytic virus and the immunomodulator (including a combination of immunomodulators) are administered directly into the tumor. In some embodiments, both the oncolytic virus and an immunomodulator (including a combination of immunomodulators) are administered to a tissue having a tumor. In some embodiments, the oncolytic virus is administered weekly. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered weekly. In some embodiments, the method further comprises administering the oncolytic virus and/or an immunomodulator (including a combination of immunomodulators) by a route of administration other than topical administration.

in some embodiments, the methods described herein further comprise locally administering to the tumor site an immune-related molecule (e.g., an interleukin, a chemokine, or PRRago (i.e., a pathogen recognition receptor agonist)). in some embodiments, the immune-related molecule is selected from the group consisting of GM-CSF, IL-2, IL-12, an interferon (e.g., a type 1, type 2, or type 3 interferon, such as interferon gamma), CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I, MDA5, LGP2, and LT α β. in some embodiments, the immune-related molecule is selected from the group consisting of a stimulator of a STING (i.e., stimulator of interferon genes) activator (e.g., a cyclic dinucleotide), a PRRago (e., a stimulator of an interferon gene), CpG (e.g., a stimulator of interferon genes) CpG (e., a stimulator of a CpG, e., a cyclic dinucleotide), a PRRago, a polynucleotide), a polynucleotide encoding a polypeptide, a dna, a protein.

The present invention is based, in part, on the undisclosed results of clinical trials we conducted between 2005 and 2008. Without being bound by any theory or hypothesis, it is believed that the viral infectious agent CG0070, which is specifically designed to replicate only in cancer cells, provides "the appropriate amount" of GM-CSF "in" real time "at the tumor site during cancer cell death. This "at" tumor site delivery of GM-CSF by the infectious agent during cancer cell death is believed to be critical for antigen presenting cell maturation and cross-presentation of the currently established antigens, neoantigens and tolerance-breaking antigens (TBA) from this cell death mixture to activated T cells. In this therapeutic context, a suitable amount of GM-CSF is required at the tumor site, since a high dose of GM-CSF would render the immune system unfocused and trigger a transient increase in local and systemic suppressors; whereas low doses of GM-CSF will not be sufficient to activate the inflammatory process and the associated immune cells. It is believed that a fine balance of tumor sites comprising appropriate amounts of GM-CSF and a mixture of local "live" cancer cell death elicits an adaptive immune response specific for cancer cells. Thus, it is believed that a cancer-specific and oncolytic infectious agent delivered "at the tumor site, in combination with a suitable amount of GM-CSF or other suitable immune-related molecule expressed by the infectious agent or secreted by the body's defense in response to any infectious agent during cell death, infection or inflammation, is a desirable choice for effective cancer immunotherapy.

in some embodiments, immune-related molecules enhance an immune response in an individual, the immune-related molecules may include, but are not limited to, interleukins, chemokines, stem cell growth factors, lymphotoxins, hematopoietic factors, Colony Stimulating Factors (CSF), erythropoietin, thrombopoietin, tumor necrosis factor- α (TNF), TNF- β, granulocyte-colony stimulating factor (G-CSF), granulocyte macrophage-colony stimulating factor (GM-CSF), interferon- α, interferon- β, interferon-gamma, interferon-lambda, stem cell growth factor designated "S1 factor", human growth hormone, N-methanesulphonyl human growth hormone, bovine growth hormone, parathyroid hormone, thyroxine, insulin, proinsulin, relaxin, Follicle Stimulating Hormone (FSH), Thyroid Stimulating Hormone (TSH), stimulating hormone (LH), liver growth factor, prostaglandins, fibroblast growth factor, prolactin, placental lactogen, OB protein, Mullerian hormone (CSF), rat hormone, sinomin, angiostatin (IL-17), angiostatin (IL-17), TNF-IL-17, TNF-IL, TNF-17, TNF-IL-7, TNF-IL, TNF-7, TNF-IL-5, TNF-IL, TNF-5, TNF-IL, TNF-IL, TNF-7, TNF-IL, TNF-IL-7, TNF-IL, TNF-5, TNF-7, TNF-IL, TNF-5, TNF-IL, TNF-5, TNF.

The immune-related molecule can be any of the molecular modalities known in the art, including but not limited to aptamers, mRNA, siRNA, microrna, shRNA, peptides, antibodies, anti-transporters, spherical nucleic acids, TALENs, zinc finger nucleases, CRISPR/Cas9, and small molecules.

Immune-related molecules can be used alone or in combination. For example, any number (e.g., any of 1, 2, 3, 4, 5, 6, or more) of immune-related molecules can be administered simultaneously or sequentially.

Thus, for example, in some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine) operably linked to the viral promoter; and b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, there is provided a method of inhibiting metastasis of a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine) operably linked to the viral promoter; and b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the methods comprise locally administering a combination of immune modulators comprising one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4. In some embodiments, the viral promoter operably linked to the nucleic acid encoding the immune-related molecule is the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF. In some embodiments, the oncolytic virus and/or immunomodulator (including combinations of immunomodulators) is administered directly into the tumor. In some embodiments, an oncolytic virus and/or an immunomodulator (including a combination of immunomodulators) is administered to a tissue having a tumor. In some embodiments, both the oncolytic virus and the immunomodulator (including a combination of immunomodulators) are administered directly into the tumor. In some embodiments, both the oncolytic virus and an immunomodulator (including a combination of immunomodulators) are administered to a tissue having a tumor. In some embodiments, the oncolytic virus is administered weekly. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered weekly. In some embodiments, the method further comprises administering the oncolytic virus and/or an immunomodulator (including a combination of immunomodulators) by a route of administration other than topical administration.

In some embodiments, the infectious agent is adenovirus serotype 5. In some embodiments, the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced with the human E2F-1 promoter and nucleic acid encoding human GM-CSF. In some embodiments, a polyadenylation signal (PA) is inserted 5' to the E2F-1 promoter. In some embodiments, the nucleic acid encoding human GM-CSF is operably linked to the E3 promoter. In some embodiments, the vector backbone of adenovirus serotype 5 further comprises the same E2, E4, late protein regions, or Inverted Terminal Repeats (ITRs) as the wild-type adenovirus serotype 5 genome. In some embodiments, the infectious agent has a genomic structure as shown in figure 1. In some embodiments, the infectious agent undergoes conditional replication. In some embodiments, the infectious agent replicates preferentially in cancer cells. In some embodiments, the cancer cell is an Rb pathway deficient cancer cell. In some embodiments, the infectious agent is CG 0070.

Thus, for example, in some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of adenovirus serotype 5, wherein the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced by the human E2F-1 promoter and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine, such as GM-CSF); and b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, there is provided a method of inhibiting metastasis of a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of adenovirus serotype 5, wherein the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced by the human E2F-1 promoter and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine, such as GM-CSF); and b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the methods comprise locally administering a combination of immune modulators comprising one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the tumor specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the adenovirus and/or the immunomodulator (including the combination of immunomodulators) is administered directly into the tumor. In some embodiments, an adenovirus and/or an immunomodulator (including a combination of immunomodulators) is administered to a tissue having a tumor. In some embodiments, both the adenovirus and the immunomodulator (including the combination of immunomodulators) are administered directly into the tumor. In some embodiments, both the adenovirus and the immunomodulator (including the combination of immunomodulators) are administered to a tissue having a tumor. In some embodiments, the adenovirus is administered weekly. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered weekly. In some embodiments, the method further comprises administering the adenovirus and/or the immunomodulator (including the combination of immunomodulators) by a route of administration other than topical administration.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual comprising: a) locally administering to the tumor site an effective amount of CG 0070; and b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, there is provided a method of inhibiting metastasis of a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of CG 0070; and b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the methods comprise locally administering a combination of immune modulators comprising one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, CG0070 and/or an immunomodulatory agent (including combinations of immunomodulatory agents) are administered directly into the tumor. In some embodiments, CG0070 and/or an immunomodulatory agent (including combinations of immunomodulatory agents) are administered to a tissue having a tumor. In some embodiments, both CG0070 and an immunomodulatory agent (including a combination of immunomodulatory agents) are administered directly into the tumor. In some embodiments, both CG0070 and an immunomodulatory agent (including a combination of immunomodulatory agents) are administered to a tissue having a tumor. In some embodiments, CG0070 is administered weekly. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered weekly. In some embodiments, the method further comprises administering CG0070 and/or an immunomodulator (including a combination of immunomodulators) by a route of administration other than topical administration.

In some embodiments, the infectious agent and the immunomodulator (including the combination of immunomodulators) discussed above are administered sequentially, i.e., the infectious agent is administered before or after the immunomodulator (including the combination of immunomodulators) is administered. In some embodiments, the infectious agent is administered prior to administration of the immunomodulator (including the combination of immunomodulators). In some embodiments, no more than about any of the following is administered with the infectious agent prior to administration of the immunomodulator (including the combination of immunomodulators): 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours. In some embodiments, the infectious agent is administered about day or week (e.g., about any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more) prior to administration of the immunomodulator (including the combination of immunomodulators). In some embodiments, the infectious agent is administered after administration of the immunomodulator (including the combination of immunomodulators). In some embodiments, no more than about any of the infectious agents are administered after administration of the immunomodulator (including the combination of immunomodulators): 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours. In some embodiments, the infectious agent is administered about day or week (e.g., about any of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more) after administration of the immunomodulator (including the combination of immunomodulators). In some embodiments, the infectious agent and immunomodulator (including combination of immunomodulators) are administered immediately after one another (e.g., within 5 minutes or less between administrations). For example, in some embodiments, the infectious agent is administered immediately prior to administration of the immunomodulator (including the combination of immunomodulators). In some embodiments, the infectious agent is administered immediately after administration of the immunomodulator (including the combination of immunomodulators).

In some embodiments, the infectious agent and the immunomodulator (including a combination of immunomodulators) are administered simultaneously. In some embodiments, the infectious agent and the immunomodulator (including the combination of immunomodulators) are administered simultaneously via separate compositions. In some embodiments, the infectious agent and the immunomodulator (including the combination of immunomodulators) are administered as a single composition. In some embodiments, the infectious agent and the immunomodulator (including the combination of immunomodulators) are mixed prior to administration of the composition (e.g., immediately prior to administration of the composition, e.g., within less than about 10, 5, or 1 minute prior to administration of the composition). In some embodiments, a composition comprising an infectious agent and an immunomodulator (including a combination of immunomodulators) is pre-prepared and stored for at least about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or more prior to administration.

Immunomodulatory agents discussed herein include both immune stimulants and immune checkpoint inhibitors. The immunomodulator may be any of the molecular modalities known in the art, including but not limited to aptamers, mRNA, siRNA, microrna, shRNA, peptides, antibodies, anti-transporters, spherical nucleic acids, TALENs, zinc finger nucleases, CRISPR/Cas9, and small molecules.

In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the immunostimulatory agent is a natural or engineered ligand for an immunostimulatory molecule, including, for example, a ligand for OX40 (e.g., OX40L), a ligand for CD28 (e.g., CD80, CD86), a ligand for ICOS (e.g., B7RP1), a ligand for 4-1BB (e.g., 4-1BBL, Ultra4-1BBL), a ligand for CD27 (e.g., CD70), a ligand for CD40 (e.g., CD40L), and a ligand for a TCR (e.g., an MHC class I or class II molecule, IMCgp 100). In some embodiments, the immunostimulatory agent is an antibody selected from the group consisting of: anti-CD 28 (e.g., TGN-1412), anti-OX 40 (e.g., MEDI6469, MEDI-0562), anti-ICOS (e.g., MEDI-570), anti-GITR (e.g., TRX518, INBRX-110, NOV-120301), anti-41-BB (e.g., BMS-663513, PF-05082566), anti-CD 27 (e.g., BION-1402, Vallulumab, and hCD27.15), anti-CD 40 (e.g., CP870,893, BI-655064, BMS-986090, APX005M), anti-CD 3 (e.g., (MEDI 6469, MEDI-0562)For example, brimonimab, muromab (muromonab)), and anti-HVEM. In some embodiments, the antibody is an agonistic antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an antigen binding fragment selected from the group consisting of: fab, Fab ', F (ab') of full-length antibody₂Fv, scFv, and other antigen binding subsequences. In some embodiments, the antibody is a human, humanized or chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other functional variant or derivative thereof.

in some embodiments, the immune checkpoint inhibitor is a natural or engineered ligand of an inhibitory immune checkpoint molecule, including, for example, ligands of CTLA-4 (e.g., B7.1, B7.2), ligands of TIM3 (e.g., galectin-9), ligands of the A2a receptor (e.g., adenosine, Regadenson), ligands of LAG3 (e.g., MHC class I or MHC class II molecules), ligands of BTLA (e.g., HVEM, B7-H4), ligands of KIR (e.g., MHC class I or MHC class II molecules), ligands of PD-1 (e.g., PD-L1, PD-L2), ligands of IDO (e.g., NKTR-218, indomethamod (indomethaod), NLG919) and ligands of CD2 (e.g., P- α receptor), in some embodiments, the immune inhibitor is an anti-checkpoint antibody selected from anti-murine mAb, anti-103, anti-mAbI-A1010, PCT/US2001/020964, MPDL3280A, AMP-224, daplizumab (Dapirolizumab pegol) (CDP-7657), MEDI-4920), anti-CD 73 (e.g., AR-42(OSU-HDAC42, HDAC-42, AR42, AR42, OSU-HDAC-42, NSC D736012, HDAC42, HDAC42, NSCD736012, NSC-D736012), MEDI-9447), anti-B7-H3 (e.g., MGA271, DS-5573a, 8H9), anti-CD 47 (e.g., CC-90002, TTI-621, VLST-007), anti-BTLA, anti-VISTA, anti-A2 aR, anti-B7-1, anti-B7-H4, anti-CD 52 (e.g., azulen-55), anti-TGF-11, anti-TGF-Fab, anti-TGF-antibody (e.g., in some embodiments, anti-MAIL-35, anti-MAIL-MAb-35, in some embodiments, anti-MAb-MAIL-MAb, such as a fragment, in some embodiments, an anti-MAIL-MAB-MAb-binding antibody, an anti-antibody in some embodiments, such as a, an anti-antibody, such as a fragment, an anti-antibody, an anti-monoclonal antibody₂Fv, scFv, and other antigen binding subsequences. In some embodiments, the antibody is a human, humanized or chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other functional variant or derivative thereof.

In some embodiments, the method comprises topically administering a single immunomodulator. In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immunostimulatory agent.

In some embodiments, the method comprises topically administering at least two (e.g., any of 2, 3, 4, 5, 6, or more) immunomodulators. In some embodiments, all or a portion of at least two immunomodulators are administered simultaneously, e.g., in a single composition. In some embodiments, all or a portion of at least two immunomodulators are administered sequentially. In some embodiments, the method comprises topically administering a combination of immune modulators comprising an immune checkpoint inhibitor and an immune stimulant. In some embodiments, the method comprises topically administering a combination of immunomodulators comprising two or more (e.g., any of 2, 3, 4, 5, 6 or more) checkpoint inhibitors. In some embodiments, the method comprises topically administering a combination of immunomodulatory agents comprising two or more (e.g., any of 2, 3, 4, 5, 6, or more) immunostimulatory agents. In some embodiments, the method comprises topically administering a combination of an immune checkpoint inhibitor comprising any number (e.g., any of 1, 2, 3, 4, 5, 6, or more) of immune checkpoint inhibitors and any number (e.g., any of 2, 3, 4, 5, 6, or more) of immune modulators of immune stimulants. For example, in some embodiments, the method comprises: a) locally administering to the tumor site an effective amount of an infectious agent (e.g., a virus, such as an oncolytic virus); and b) topically administering to the individual an effective amount of a first immune modulator (e.g., an immune checkpoint inhibitor); and c) locally administering to the tumor site an effective amount of a second immunomodulator (e.g., an immunostimulant). In some embodiments, the methods comprise administering a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab), or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)) and a CD40 agonist (e.g., an agonistic anti-CD 40 antibody, e.g., APX 005M). In some embodiments, the methods comprise administering a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab), or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)) and a4-1BB agonist (e.g., an agonistic anti-4-1 BB antibody, e.g., PF-05082566). In some embodiments, the method comprises administering a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody) and a PD-L1 inhibitor (e.g., an anti-PD-L1 antibody).

In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody. Any of the anti-CTLA-4 antibodies known in the art can be used in the present invention, including but not limited to ipilimumab, tremelimumab, and KAHR-102. In some embodiments, the anti-CTLA-4 antibody is(ipilimumab). In some embodiments, the anti-CTLA-4 antibodyIs a monoclonal antibody or a polyclonal antibody. In some embodiments, the anti-CTLA-4 antibody is an antigen-binding fragment selected from the group consisting of: fab, Fab ', F (ab') of full-length anti-CTLA-4 antibody₂Fv, scFv, and other antigen binding subsequences. In some embodiments, the anti-CTLA-4 antibody is a human, humanized or chimeric antibody. In some embodiments, the anti-CTLA-4 antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other functional variant or derivative thereof. In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin that specifically recognizes CTLA-4 (e.g., an anti-transportin molecule that specifically binds to CTLA-4). In some embodiments, the inhibitor of CTLA-4 is a natural or engineered ligand for CTLA-4, e.g., B7.1 or B7.2.

Thus, for example, in some embodiments, there is provided a method of treating a solid or lymphatic tumor in a subject (e.g., a human), the method comprising: a) locally administering to the tumor site an effective amount of an infectious agent; and b) locally administering to the tumor site an effective amount of an inhibitor of CTLA-4 (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab), or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)). In some embodiments, there is provided a method of inhibiting metastasis of a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an infectious agent; and b) locally administering to the tumor site an effective amount of an inhibitor of CTLA-4 (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab), or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)). In some embodiments, the infectious agent is a non-oncolytic virus. In some embodiments, the infectious agent is an oncolytic virus. In some embodiments, the infectious agent is a wild-type infectious agent. In some embodiments, the infectious agent is genetically modified. In some embodiments, the infectious agent is attenuated (e.g., via multiple passages, inactivation, or genetic modification). In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody (e.g., ipilimumab). In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin, e.g., an anti-transporter that specifically recognizes CTLA-4. In some embodiments, the method further comprises topically administering a second immunomodulator, such as an immunostimulant (e.g., a CD40 activator or a4-1BB activator). In some embodiments, the agent and/or inhibitor of CTLA-4 is administered directly into the tumor. In some embodiments, the agent is administered to a tissue having a tumor. In some embodiments, both the infectious agent and the inhibitor of CTLA-4 are administered directly into the tumor. In some embodiments, both the infectious agent and the inhibitor of CTLA-4 are administered to a tissue having a tumor. In some embodiments, the infectious agent is administered weekly. In some embodiments, the inhibitor of CTLA-4 is administered weekly. In some embodiments, the infectious agent and the inhibitor of CTLA-4 are administered sequentially. In some embodiments, the infectious agent is administered prior to (e.g., immediately prior to) administration of the inhibitor of CTLA-4. In some embodiments, the infectious agent is administered after (e.g., immediately after) administration of the inhibitor of CTLA-4. In some embodiments, the infectious agent and the inhibitor of CTLA-4 are administered simultaneously (e.g., in a single composition). In some embodiments, the method further comprises administering the infectious agent and/or the inhibitor of CTLA-4 by a route of administration other than local administration.

For example, in some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus); and b) locally administering to the tumor site an effective amount of an inhibitor of CTLA-4. In some embodiments, there is provided a method of inhibiting metastasis of a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus); and b) locally administering to the tumor site an effective amount of an inhibitor of CTLA-4. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4).

In some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication; and b) locally administering to the tumor site an effective amount of an inhibitor of CTLA-4. In some embodiments, there is provided a method of inhibiting metastasis of a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication; and b) locally administering to the tumor site an effective amount of an inhibitor of CTLA-4. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine) operably linked to the viral promoter; and b) locally administering to the tumor site an effective amount of an inhibitor of CTLA-4. In some embodiments, there is provided a method of inhibiting metastasis of a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine) operably linked to the viral promoter; and b) locally administering to the tumor site an effective amount of an inhibitor of CTLA-4. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4. In some embodiments, the viral promoter operably linked to the nucleic acid encoding the immune-related molecule is the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual comprising: a) locally administering to the tumor site an effective amount of adenovirus serotype 5, wherein the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced by the human E2F-1 promoter and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine, such as GM-CSF); and b) locally administering to the tumor site an effective amount of an inhibitor of CTLA-4. In some embodiments, there is provided a method of inhibiting metastasis of a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of adenovirus serotype 5, wherein the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced by the human E2F-1 promoter and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine, such as GM-CSF); and b) locally administering to the tumor site an effective amount of an inhibitor of CTLA-4. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4). In some embodiments, the tumor specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual comprising: a) locally administering to the tumor site an effective amount of CG 0070; and b) locally administering to the tumor site an effective amount of an inhibitor of CTLA-4. In some embodiments, there is provided a method of inhibiting metastasis of a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of CG 0070; and b) locally administering to the tumor site an effective amount of an inhibitor of CTLA-4. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4). In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody (e.g., ipilimumab). In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin, e.g., an anti-transporter that specifically recognizes CTLA-4. In some embodiments, the inhibitor of CG0070 and/or CTLA-4 is administered directly into the tumor. In some embodiments, the inhibitor of oncolytic virus and/or CTLA-4 is administered to a tissue having a tumor. In some embodiments, both the CG0070 and the inhibitor of CTLA-4 are administered directly into the tumor. In some embodiments, the inhibitors of CG0070 and CTLA-4 are administered to a tissue having a tumor. In some embodiments, CG007 is administered weekly. In some embodiments, the inhibitor of CTLA-4 is administered weekly. In some embodiments, the inhibitor of CG0070 and CTLA-4 is administered sequentially. In some embodiments, the CG0070 is administered prior to (e.g., immediately prior to) administration of the inhibitor of CTLA-4. In some embodiments, the CG0070 is administered after (e.g., immediately after) administration of the inhibitor of CTLA-4. In some embodiments, the inhibitor of CG0070 and CTLA-4 are administered concurrently (e.g., in a single composition). In some embodiments, the method further comprises administering an inhibitor of CG0070 and/or CTLA-4 by a route of administration other than topical administration.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in an individual, the method comprising: a) administering an effective amount of CG0070 intratumorally; and b) inhibition of intratumoral administration of an effective amount of CTLA-4An agent (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)), wherein the effective amount of CG0070 is about 1X 10 per week⁸To about 1X 10¹⁴Viral particles (vp) (e.g., about 1X 10 per week)⁸To about 1X 10¹⁰About 1X 10¹⁰To about 1X 10¹²Or about 1X 10¹²To about 1X 10¹⁴Any one of vp), wherein the effective amount of the inhibitor of CTLA-4 is about 0.1mg/Kg to about 10mg/Kg per week (e.g., any one of about 0.1mg/Kg to about 1mg/Kg, about 1mg/Kg to about 5mg/Kg, or about 5mg/Kg to about 10mg/Kg per week), and wherein the inhibitor of CTLA-4 is administered immediately (e.g., no more than 5 minutes after administration) after administration of CG 0070. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody (e.g., ipilimumab) (e.g.,). In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin, e.g., an anti-transporter that specifically recognizes CTLA-4. In some embodiments, an effective amount of DDM is further administered intratumorally as a transduction enhancer to the individual in combination with the administration of CG 0070. In some embodiments, the inhibitor of CG0070 and CTLA-4 is administered by injection into a tissue having a tumor. In some embodiments, the inhibitors of CG0070 and CTLA-4 are administered by direct injection into the tumor. In some embodiments, CG0070 is administered for about 1 to about 6 weeks as a course of treatment. In some embodiments, the course of treatment is repeated every about 2 months to about 3 months. In some embodiments, the solid or lymphoid tumor is selected from the group consisting of: head and neck cancer, breast cancer, colorectal cancer, liver cancer, pancreatic adenocarcinoma, gallbladder and bile duct cancer, ovarian cancer, cervical cancer, small cell lung cancer, non-small cell lung cancer, renal cell cancer, bladder cancer, prostate cancer, bone cancer, mesothelioma, brain cancer, soft tissue sarcoma, uterine cancer, thyroid cancer, nasopharyngeal cancer, and melanoma. In some embodiments, the solid or lymphoid tumor is refractory to prior therapy. In some embodiments, the method further comprises the topical administration of a second immunomodulator, e.g., immunizationAn irritant. In some embodiments, the second immunomodulator is a CD40 activator, e.g., an agonist anti-CD 40 antibody (e.g., APX 005M). In some embodiments, the second immunomodulator is a4-1BB activator, e.g., an agonist anti-4-1 BB antibody (e.g., PF-05082566). In some embodiments, the second immunomodulator is a PD-L1 inhibitor. In some embodiments, the method further comprises pretreatment, such as irradiation or administration of a therapeutic agent (e.g., an interleukin, such as CCL 21).

In some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in an individual, the method comprising: a) administering an effective amount of CG0070 intratumorally; and b) intratumorally administering an effective amount of an inhibitor of CTLA-4 (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)); and c) intratumorally administering an effective amount of an activator of CD40 (e.g., an agonistic anti-CD 40 antibody), wherein the effective amount of CG0070 is about 1X 10 per week⁸To about 1X 10¹⁴Viral particles (vp) (e.g., 5X 10 per week)¹⁰vp、1×10¹¹vp、5×10¹¹vp or 1X 10¹²About any of vp), wherein the effective amount of the inhibitor of CTLA-4 is about 0.1mg to about 100mg (e.g., no more than about any of 1mg, 3mg, 6mg, 12mg, or 24mg per week), and wherein the effective amount of the CD40 activator is about 0.1mg to about 100mg (e.g., no more than about any of 1mg, 3mg, 6mg, 12mg, or 24mg per week). In some embodiments, the inhibitor of CTLA-4 and the activator of CD40 are administered immediately (e.g., no more than 5 minutes after administration) after administration of CG 0070. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody (e.g., ipilimumab) (e.g.,). In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin, e.g., an anti-transporter that specifically recognizes CTLA-4. In some embodiments, the CD40 activator is an agonistic anti-CD 40 antibody, e.g., APX 005M. In some embodiments, the combination with administration of CG0070 further comprises administering to the individual a therapeutically effective amount of a compound of formula iAn effective amount of DDM is administered intratumorally as a transduction enhancer. In some embodiments, the CG0070, the inhibitor of CTLA-4, and the activator of CD40 are administered by injection into a tissue having a tumor. In some embodiments, the CG0070, the inhibitor of CTLA-4, and the activator of CD40 are administered by direct injection into the tumor. In some embodiments, CG0070 is administered for about 1 to about 6 weeks as a course of treatment. In some embodiments, the course of treatment is repeated every about 2 months to about 3 months. In some embodiments, the solid or lymphoid tumor is selected from the group consisting of: head and neck cancer, breast cancer, colorectal cancer, liver cancer, pancreatic adenocarcinoma, gallbladder and bile duct cancer, ovarian cancer, cervical cancer, small cell lung cancer, non-small cell lung cancer, renal cell cancer, bladder cancer, prostate cancer, bone cancer, mesothelioma, brain cancer, soft tissue sarcoma, uterine cancer, thyroid cancer, nasopharyngeal cancer, and melanoma. In some embodiments, the solid or lymphoid tumor is refractory to prior therapy. In some embodiments, the method further comprises pretreatment, such as irradiation or administration of a therapeutic agent (e.g., an interleukin, such as CCL 21).

In some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in an individual, the method comprising: a) administering an effective amount of CG0070 intratumorally; and b) intratumorally administering an effective amount of an inhibitor of CTLA-4 (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)); and c) intratumorally administering an effective amount of a4-1BB activator (e.g., an agonistic anti-4-1 BB antibody), wherein the effective amount of CG0070 is about 1X 10 per week⁸To about 1X 10¹⁴Viral particles (vp) (e.g., 5X 10 per week)¹⁰vp、1×10¹¹vp、5×10¹¹vp or 1X 10¹²About any of vp), wherein the effective amount of the inhibitor of CTLA-4 is about 0.1mg to about 100mg (e.g., no more than about any of 1mg, 3mg, 6mg, 12mg, or 24mg per week), and wherein the effective amount of the 4-1BB activator is about 0.1mg to about 100mg (e.g., no more than about any of 1mg, 3mg, 6mg, 12mg, or 24mg per week). In some embodiments, the administration of CG0070 is immediately followed by (e.g., not followed by) administration of CG0070Over 5 minutes) administering an inhibitor of CTLA-4 and a4-1BB activator. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody (e.g., ipilimumab) (e.g.,). In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin, e.g., an anti-transporter that specifically recognizes CTLA-4. In some embodiments, the 4-1BB activator is an agonistic anti-4-1 BB antibody, such as PF-05082566. In some embodiments, an effective amount of DDM is further administered intratumorally as a transduction enhancer to the individual in combination with the administration of CG 0070. In some embodiments, the inhibitor of CG0070, CTLA-4, and the 41-BB activator are administered by injection into a tissue having a tumor. In some embodiments, the CG0070, the inhibitor of CTLA-4, and the 41-BB activator are administered by direct injection into the tumor. In some embodiments, CG0070 is administered for about 1 to about 6 weeks as a course of treatment. In some embodiments, the course of treatment is repeated every about 2 months to about 3 months. In some embodiments, the solid or lymphoid tumor is selected from the group consisting of: head and neck cancer, breast cancer, colorectal cancer, liver cancer, pancreatic adenocarcinoma, gallbladder and bile duct cancer, ovarian cancer, cervical cancer, small cell lung cancer, non-small cell lung cancer, renal cell cancer, bladder cancer, prostate cancer, bone cancer, mesothelioma, brain cancer, soft tissue sarcoma, uterine cancer, thyroid cancer, nasopharyngeal cancer, and melanoma. In some embodiments, the solid or lymphoid tumor is refractory to prior therapy. In some embodiments, the method further comprises pretreatment, such as local irradiation or administration of a therapeutic agent (e.g., an interleukin, such as CCL 21).

In some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual comprising: a) administering an effective amount of CG0070 intratumorally; and b) administering intratumorally an effective amount of an inhibitor of CTLA-4; and c) administering an effective amount of a PD-L1 inhibitor intratumorally. In some embodiments, there is provided a method of inhibiting metastasis of a solid or lymphatic tumor in an individual, the method comprising: a) administering an effective amount of CG0070 intratumorally; and b) intratumoral administrationAn effective amount of an inhibitor of CTLA-4; and c) administering an effective amount of a PD-L1 inhibitor intratumorally. In some embodiments, an effective amount of CG0070 is about 1 × 10 per week⁸To about 1X 10¹⁴Viral particles (vp) (e.g., 5X 10 per week)¹⁰vp、1×10¹¹vp、5×10¹¹vp or 1X 10¹²About any of vp). In some embodiments, an effective amount of an inhibitor of CTLA-4 is from about 0.1mg to about 100mg (e.g., no more than about any of 1mg, 3mg, 6mg, 12mg, or 24mg per week). In some embodiments, an effective amount of a PD-L1 inhibitor is about 0.1mg to about 100mg (e.g., no more than about any one of 1mg, 3mg, 6mg, 12mg, or 24mg per week). In some embodiments, the inhibitor of CTLA-4 and the inhibitor of PD-L1 are administered immediately (e.g., no more than 5 minutes after administration) after administration of CG 0070. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody (e.g., ipilimumab) (e.g.,). In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin, e.g., an anti-transporter that specifically recognizes CTLA-4. In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody, such as KY-1003, MCLA-145, RG7446, BMS935559, MPDL3280A, MEDI4736, avilumab (Avelumab), or STI-a 1010. In some embodiments, the CG0070, the inhibitor of CTLA-4, and the PD-L1 inhibitor are administered by injection into a tissue having a tumor. In some embodiments, the CG0070, the inhibitor of CTLA-4, and the PD-L1 inhibitor are administered by direct injection into the tumor. In some embodiments, CG0070 is administered for about 1 to about 6 weeks as a course of treatment. In some embodiments, the course of treatment is repeated every about 2 months to about 3 months. In some embodiments, the solid or lymphoid tumor is selected from the group consisting of: head and neck cancer, breast cancer, colorectal cancer, liver cancer, pancreatic adenocarcinoma, gallbladder and bile duct cancer, ovarian cancer, cervical cancer, small cell lung cancer, non-small cell lung cancer, renal cell cancer, bladder cancer, prostate cancer, bone cancer, mesothelioma, brain cancer, soft tissue sarcoma, uterine cancer, thyroid cancer, nasopharyngeal cancer, and melanoma. In thatIn some embodiments, the solid or lymphoid tumor is refractory to prior therapy. In some embodiments, the method further comprises pretreatment, such as irradiation or administration of a therapeutic agent (e.g., an interleukin, such as CCL 21).

In some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual comprising: a) subjecting a solid or lymphatic tumor to local irradiation; followed by b) intratumoral administration of an effective amount of CG 0070; and c) administering an effective amount of an inhibitor of CTLA-4 intratumorally. In some embodiments, there is provided a method of inhibiting metastasis of a solid or lymphatic tumor in an individual, the method comprising: a) subjecting a solid or lymphatic tumor to local irradiation; followed by b) intratumoral administration of an effective amount of CG 0070; and c) administering an effective amount of an inhibitor of CTLA-4 intratumorally. In some embodiments, an effective amount of CG0070 is about 1 × 10 per week⁸To about 1X 10¹⁴Viral particles (vp) (e.g., 5X 10 per week)¹⁰vp、1×10¹¹vp、5×10¹¹vp or 1X 10¹²About any of vp). In some embodiments, an effective amount of an inhibitor of CTLA-4 is from about 0.1mg to about 100mg (e.g., no more than about any of 1mg, 3mg, 6mg, 12mg, or 24mg per week). In some embodiments, the inhibitor of CTLA-4 and CG0070 are administered simultaneously. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody (e.g., ipilimumab) (e.g.,). In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin, e.g., an anti-transporter that specifically recognizes CTLA-4. In some embodiments, the inhibitor of CG0070 and CTLA-4 is administered by injection into a tissue having a tumor. In some embodiments, the inhibitors of CG0070 and CTLA-4 are administered by direct injection into the tumor. In some embodiments, CG0070 is administered for about 1 to about 6 weeks as a course of treatment. In some embodiments, the course of treatment is repeated every about 2 months to about 3 months. In some embodiments, the solid or lymphoid tumor is selected from the group consisting of: head and neck cancer, breast cancer, colorectal cancer, liver cancer, pancreasAdenocarcinoma, gallbladder and bile duct cancer, ovarian cancer, cervical cancer, small cell lung cancer, non-small cell lung cancer, renal cell carcinoma, bladder cancer, prostate cancer, bone cancer, mesothelioma, brain cancer, soft tissue sarcoma, uterine cancer, thyroid cancer, nasopharyngeal carcinoma, and melanoma. In some embodiments, the solid or lymphoid tumor is refractory to prior therapy.

In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. Any of the anti-PD-1 antibodies known in the art may be used in the present invention, including but not limited to nivolumab, pembrolizumab, pidilizumab, BMS-936559, and atilizumab, lanbruzumab, MK-3475, AMP-224, AMP-514, STI-A1110, and TSR-042. In some embodiments, the anti-PD-1 antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the anti-PD-1 antibody is an antigen-binding fragment selected from the group consisting of: fab, Fab ', F (ab') of full-length anti-PD-1 antibody₂Fv, scFv, and other antigen binding subsequences. In some embodiments, the anti-PD-1 antibody is a human, humanized, or chimeric antibody. In some embodiments, the anti-PD-1 antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other variant or derivative thereof. In some embodiments, the PD-1 inhibitor is a natural or engineered ligand for PD-1, such as PD-L1 or PD-L2. In some embodiments, the PD-1 inhibitor is an inhibitor of the interaction between PD-1 and its ligand, for example, an inhibitor of the PD-1/PD-L1 interaction or an inhibitor of the PD-1/PD-L2 interaction. In some embodiments, the PD-1 inhibitor is an inhibitor of a PD-1 ligand, such as an inhibitor of PD-L1 (e.g., an anti-PD-L1 antibody) or an inhibitor of PD-L2 (e.g., an anti-PD-L2 antibody). Any inhibitor of the interaction between PD-1 and its ligand can be used in the present invention, see for example US patent nos. US7709214, US7432059, US7722868, US8217149, US 83796 and US 9102725. In some embodiments, the PD-1 inhibitor is an Fc fusion protein comprising a PD-1 ligand, such as an Fc-fusion of PD-L2 (e.g., AMP-224).

Thus, for example, in some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in a subject (e.g., a human), the method comprising: a) locally administering to the tumor site an effective amount of an infectious agent; and b) topically administering an effective amount of a PD-1 inhibitor (e.g., an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, or pidilizumab) or an Fc fusion protein of a PD-1 ligand (e.g., AMP-224)) to the tumor site. In some embodiments, the infectious agent is a non-oncolytic virus. In some embodiments, the infectious agent is an oncolytic virus. In some embodiments, the infectious agent is a wild-type infectious agent. In some embodiments, the infectious agent is genetically modified. In some embodiments, the infectious agent is attenuated (e.g., via multiple passages, inactivation, or genetic modification). In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody, e.g., nivolumab, pembrolizumab, or pidilizumab. In some embodiments, the PD-1 inhibitor is an inhibitor of the interaction between PD-1 and its ligand, for example, an inhibitor of the PD-1/PD-L1 interaction or an inhibitor of the PD-1/PD-L2 interaction. In some embodiments, the PD-1 inhibitor is an Fc fusion protein comprising a PD-1 ligand, such as an Fc-fusion of PD-L2 (e.g., AMP-224). In some embodiments, the method further comprises topically administering a second immunomodulator, such as an immunostimulant (e.g., a CD40 activator or a4-1BB activator). In some embodiments, the infectious agent and/or PD-1 inhibitor is administered directly into the tumor. In some embodiments, the infectious agent and/or PD-1 inhibitor is administered to a tissue having a tumor. In some embodiments, both the infectious agent and the PD-1 inhibitor are administered directly into the tumor. In some embodiments, both the infectious agent and the PD-1 inhibitor are administered to a tissue having a tumor. In some embodiments, the infectious agent is administered weekly. In some embodiments, the PD-1 inhibitor is administered weekly. In some embodiments, the infectious agent and the PD-1 inhibitor are administered sequentially. In some embodiments, the infectious agent is administered prior to (e.g., immediately prior to) administration of the PD-1 inhibitor. In some embodiments, the infectious agent is administered after (e.g., immediately after) the administration of the PD-1 inhibitor. In some embodiments, the infectious agent and the PD-1 inhibitor are administered simultaneously (e.g., in a single composition). In some embodiments, the method further comprises administering the infectious agent and/or the PD-1 inhibitor by a route of administration other than topical administration.

For example, in some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus); and b) topically administering an effective amount of a PD-1 inhibitor (e.g., an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, or pidilizumab) or an Fc fusion protein of a PD-1 ligand (e.g., AMP-224)) to the tumor site.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication; and b) topically administering an effective amount of a PD-1 inhibitor (e.g., an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, or pidilizumab) or an Fc fusion protein of a PD-1 ligand (e.g., AMP-224)) to the tumor site. In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine) operably linked to the viral promoter; and b) topically administering an effective amount of a PD-1 inhibitor (e.g., an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, or pidilizumab) or an Fc fusion protein of a PD-1 ligand (e.g., AMP-224)) to the tumor site. In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4. In some embodiments, the viral promoter operably linked to the nucleic acid encoding the immune-related molecule is the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in an individual, the method comprising: a) locally administering to the tumor site an effective amount of adenovirus serotype 5, wherein the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced by the human E2F-1 promoter and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine, such as GM-CSF); and b) topically administering an effective amount of a PD-1 inhibitor (e.g., an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, or pidilizumab) or an Fc fusion protein of a PD-1 ligand (e.g., AMP-224)) to the tumor site. In some embodiments, the tumor specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in an individual, the method comprising: a) locally administering to the tumor site an effective amount of CG 0070; and b) topically administering an effective amount of a PD-1 inhibitor (e.g., an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, or pidilizumab) or an Fc fusion protein of a PD-1 ligand (e.g., AMP-224)) to the tumor site. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody, e.g., nivolumab, pembrolizumab, or pidilizumab. In some embodiments, the PD-1 inhibitor is an inhibitor of the interaction between PD-1 and its ligand, for example, an inhibitor of the PD-1/PD-L1 interaction or an inhibitor of the PD-1/PD-L2 interaction. In some embodiments, the PD-1 inhibitor is an Fc fusion protein comprising a PD-1 ligand, such as an Fc-fusion of PD-L2 (e.g., AMP-224). In some embodiments, both the CG0070 and the PD-1 inhibitor are administered directly into the tumor. In some embodiments, the oncolytic virus and/or PD-1 inhibitor is administered to a tissue having a tumor. In some embodiments, both the CG0070 and the PD-1 inhibitor are administered directly into the tumor. In some embodiments, both the CG0070 and the PD-1 inhibitor are administered to a tissue having a tumor. In some embodiments, CG007 is administered weekly. In some embodiments, the PD-1 inhibitor is administered weekly. In some embodiments, the CG0070 and the PD-1 inhibitor are administered sequentially. In some embodiments, CG0070 is administered prior to (e.g., immediately prior to) administration of the PD-1 inhibitor. In some embodiments, CG0070 is administered after (e.g., immediately after) administration of the PD-1 inhibitor. In some embodiments, the CG0070 and the PD-1 inhibitor are administered simultaneously (e.g., in a single composition). In some embodiments, the method further comprises administering the CG0070 and/or PD-1 inhibitor by a route of administration other than topical administration.

In some embodiments, the immune checkpoint inhibitor is an inhibitor of a PD-1 ligand (e.g., PD-L1 and/or PD-L2). In some embodiments, the inhibitor of a PD-1 ligand is an anti-PD-L1 antibody. In some embodiments, the inhibitor of a PD-1 ligand is an anti-PD-L2 antibody. Exemplary anti-PD-L1 antibodies include, but are not limited to, KY-1003, MCLA-145, RG7446 (also known as acilizumab), BMS935559 (also known as MDX-1105), MPDL3280A, MEDI4736, avizumab (also known as MSB0010718C), and STI-a 1010. In some embodiments, the anti-PD-L1 or anti-PD-L2 is a monoclonal or polyclonal antibody. In some embodiments, the anti-PD-1 or anti-PD-L2 antibody is an antigen-binding fragment selected from the group consisting of: fab, Fab ', F (ab') of full-length anti-PD-1 or anti-PD-L2 antibodies₂Fv, scFv, and other antigen binding subsequences. In some embodiments, the anti-PD-L1 or anti-PD-L2 antibody is a human, humanized, or chimeric antibody. In some embodimentsThe anti-PD-L1 or anti-PD-L2 antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other variant or derivative thereof. In some embodiments, the inhibitor of a PD-1 ligand is an inhibitor (e.g., a peptide, protein, or small molecule) of both PD-L1 and PD-L2. Exemplary inhibitors of both PD-L1 and PD-L2 include, but are not limited to, AUR-012 and AMP-224. In some embodiments, an inhibitor of PD-L1 and an inhibitor of PD-L2 are used interchangeably in any of the methods of treatment described herein.

Thus, for example, in some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in a subject (e.g., a human), the method comprising: a) locally administering to the tumor site an effective amount of an infectious agent; and b) locally administering to the tumor site an effective amount of an inhibitor of a PD-1 ligand (e.g., an anti-PD-L1 or anti-PD-L2 antibody, or an inhibitor of both PD-L1 and PD-L2). In some embodiments, the infectious agent is a non-oncolytic virus. In some embodiments, the infectious agent is an oncolytic virus. In some embodiments, the infectious agent is a wild-type infectious agent. In some embodiments, the infectious agent is genetically modified. In some embodiments, the infectious agent is attenuated (e.g., via multiple passages, inactivation, or genetic modification). In some embodiments, the inhibitor of a PD-1 ligand is an anti-PD-L1 antibody, such as KY-1003, MCLA-145, RG7446, BMS935559, MPDL3280A, MEDI4736, avizumab, or STI-A1010. In some embodiments, the inhibitor of a PD-1 ligand is an anti-PD-L2 antibody. In some embodiments, the inhibitor of a PD-1 ligand is an inhibitor (e.g., a peptide, protein, or small molecule) of both PD-L1 and PD-L2, such as AUR-012 and AMP-224. In some embodiments, the method further comprises topically administering a second immunomodulator, such as an immunostimulant (e.g., a CD40 activator or a4-1BB activator). In some embodiments, the agent and/or the inhibitor of a PD-1 ligand is administered directly into the tumor. In some embodiments, the agent is administered to a tissue having a tumor. In some embodiments, both the infectious agent and the inhibitor of the PD-1 ligand are administered directly into the tumor. In some embodiments, both the infectious agent and the inhibitor of PD-1 ligand are administered to a tissue having a tumor. In some embodiments, the infectious agent is administered weekly. In some embodiments, the inhibitor of PD-1 ligand is administered weekly. In some embodiments, the infectious agent and the inhibitor of the PD-1 ligand are administered sequentially. In some embodiments, the infectious agent is administered prior to (e.g., immediately prior to) administration of the inhibitor of PD-1 ligand. In some embodiments, the infectious agent is administered after (e.g., immediately after) administration of the inhibitor of the PD-1 ligand. In some embodiments, the infectious agent and the inhibitor of the PD-1 ligand are administered simultaneously (e.g., in a single composition). In some embodiments, the method further comprises administering an inhibitor of the infectious agent and/or the PD-1 ligand by a route of administration other than topical administration.

For example, in some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus); and b) locally administering to the tumor site an effective amount of an inhibitor of a PD-1 ligand (e.g., an anti-PD-L1 or anti-PD-L2 antibody, or an inhibitor of both PD-L1 and PD-L2).

In some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication; and b) locally administering to the tumor site an effective amount of an inhibitor of a PD-1 ligand (e.g., an anti-PD-L1 or anti-PD-L2 antibody, or an inhibitor of both PD-L1 and PD-L2). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine) operably linked to the viral promoter; and b) locally administering to the tumor site an effective amount of an inhibitor of a PD-1 ligand (e.g., an anti-PD-L1 or anti-PD-L2 antibody, or an inhibitor of both PD-L1 and PD-L2). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4. In some embodiments, the viral promoter operably linked to the nucleic acid encoding the immune-related molecule is the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in an individual, the method comprising: a) locally administering to the tumor site an effective amount of adenovirus serotype 5, wherein the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced by the human E2F-1 promoter and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine, such as GM-CSF); and b) locally administering to the tumor site an effective amount of an inhibitor of a PD-1 ligand (e.g., an anti-PD-L1 or anti-PD-L2 antibody, or an inhibitor of both PD-L1 and PD-L2). In some embodiments, the tumor specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in an individual, the method comprising: a) locally administering to the tumor site an effective amount of CG 0070; and b) locally administering to the tumor site an effective amount of an inhibitor of a PD-1 ligand (e.g., an anti-PD-L1 or anti-PD-L2 antibody, or an inhibitor of both PD-L1 and PD-L2). In some embodiments, the inhibitor of a PD-1 ligand is an anti-PD-L1 antibody, such as KY-1003, MCLA-145, RG7446, BMS935559, MPDL3280A, MEDI4736, avizumab, or STI-A1010. In some embodiments, the inhibitor of a PD-1 ligand is an anti-PD-L2 antibody. In some embodiments, the inhibitor of a PD-1 ligand is an inhibitor (e.g., a peptide, protein, or small molecule) of both PD-L1 and PD-L2, such as AUR-012 and AMP-224. In some embodiments, the inhibitor of CG0070 and/or PD-1 ligand is administered directly into the tumor. In some embodiments, the inhibitor of an oncolytic virus and/or PD-1 ligand is administered to a tissue having a tumor. In some embodiments, both the CG0070 and the inhibitor of the PD-1 ligand are administered directly into the tumor. In some embodiments, both the CG0070 and the inhibitor of the PD-1 ligand are administered to the tissue having the tumor. In some embodiments, CG007 is administered weekly. In some embodiments, the inhibitor of PD-1 ligand is administered weekly. In some embodiments, the inhibitor of CG0070 and PD-1 ligand is administered sequentially. In some embodiments, CG0070 is administered prior to (e.g., immediately prior to) administration of the inhibitor of PD-1 ligand. In some embodiments, CG0070 is administered after (e.g., immediately after) administration of the inhibitor of the PD-1 ligand. In some embodiments, the inhibitor of the CG0070 and PD-1 ligand is administered simultaneously (e.g., in a single composition). In some embodiments, the method further comprises administering an inhibitor of CG0070 and/or PD-1 ligand by a route of administration other than topical administration.

In some embodiments, the immunostimulant is an activator of CD 40. In some embodiments, the activator of CD40 is an agonistic anti-CD 40 antibody. Any of the known anti-CD 40 antibodies can be used in the present invention, including but not limited to CP-870,893, daclizumab (also known as SGN-40), ChiLob 7/4, APX005, and APX005M, BI-655064, and BMS-986090. In some embodiments, the agonistic anti-CD 40 antibody is a monoclonal antibody or a polyclonal antibody. In some embodiments, the agonistic anti-CD 40 antibody is an antigen selected from the group consisting ofBinding fragment: fab, Fab ', F (ab') of full-length anti-CD 40 antibody₂Fv, scFv, and other antigen binding subsequences. In some embodiments, the agonistic anti-CD 40 antibody is a human, humanized, or chimeric antibody. In some embodiments, the agonistic anti-CD 40 antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other variant or derivative thereof. In some embodiments, the activator of CD40 is a natural or engineered CD40 ligand, such as CD 40L. In some embodiments, the activator of CD40 is an inhibitor of the interaction between CD40 and CD 40L. In some embodiments, an activator of CD40 increases signaling of CD 40.

Thus, for example, in some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in a subject (e.g., a human), the method comprising: a) locally administering to the tumor site an effective amount of an infectious agent; and b) locally administering to the tumor site an effective amount of an activator of CD40 (e.g., an agonistic anti-CD 40 antibody, such as CP-870,893, daclizumab, ChiLob 7/4, or APX 005M). In some embodiments, the infectious agent is a non-oncolytic virus. In some embodiments, the infectious agent is an oncolytic virus. In some embodiments, the infectious agent is a wild-type infectious agent. In some embodiments, the infectious agent is genetically modified. In some embodiments, the infectious agent is attenuated (e.g., via multiple passages, inactivation, or genetic modification). In some embodiments, the activator of CD40 is an agonistic anti-CD 40 antibody, such as CP-870,893, daclizumab, ChiLob 7/4, or APX 005M. In some embodiments, the method further comprises locally administering a second immune modulator, e.g., an immune checkpoint inhibitor. In some embodiments, the second immunomodulator is an inhibitor of CTLA-4, e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4). In some embodiments, the infectious agent and/or the activator of CD40 is administered directly into the tumor. In some embodiments, the infectious agent and/or the activator of CD40 is administered to a tissue having a tumor. In some embodiments, both the infectious agent and the activator of CD40 are administered directly into the tumor. In some embodiments, both the infectious agent and the activator of CD40 are administered to a tissue having a tumor. In some embodiments, the infectious agent is administered weekly. In some embodiments, the activator of CD40 is administered weekly. In some embodiments, the infectious agent and the activator of CD40 are administered sequentially. In some embodiments, the infectious agent is administered prior to (e.g., immediately prior to) administration of the activator of CD 40. In some embodiments, the infectious agent is administered after (e.g., immediately after) administration of the activator of CD 40. In some embodiments, the infectious agent and the activator of CD40 are administered simultaneously (e.g., in a single composition). In some embodiments, the method further comprises administering the infectious agent and/or the activator of CD40 by a route of administration other than topical administration.

For example, in some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus); and b) locally administering to the tumor site an effective amount of an activator of CD40 (e.g., an agonistic anti-CD 40 antibody, such as CP-870,893, daclizumab, ChiLob 7/4, or APX 005M).

In some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication; and b) locally administering to the tumor site an effective amount of an activator of CD40 (e.g., an agonistic anti-CD 40 antibody, such as CP-870,893, daclizumab, ChiLob 7/4, or APX 005M). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine) operably linked to the viral promoter; and b) locally administering to the tumor site an effective amount of an activator of CD40 (e.g., an agonistic anti-CD 40 antibody, such as CP-870,893, daclizumab, ChiLob 7/4, or APX 005M). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4. In some embodiments, the viral promoter operably linked to the nucleic acid encoding the immune-related molecule is the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in an individual, the method comprising: a) locally administering to the tumor site an effective amount of adenovirus serotype 5, wherein the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced by the human E2F-1 promoter and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine, such as GM-CSF); and b) locally administering to the tumor site an effective amount of an activator of CD40 (e.g., an agonistic anti-CD 40 antibody, such as CP-870,893, daclizumab, ChiLob 7/4, or APX 005M). In some embodiments, the tumor specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor (e.g., inhibiting tumor metastasis) in an individual, the method comprising: a) locally administering to the tumor site an effective amount of CG 0070; and b) locally administering to the tumor site an effective amount of an activator of CD40 (e.g., an agonistic anti-CD 40 antibody, such as CP-870,893, daclizumab, ChiLob 7/4, or APX 005M). In some embodiments, the activator of CD40 is an agonistic anti-CD 40 antibody, such as CP-870,893, daclizumab, ChiLob 7/4, or APX 005M. In some embodiments, the activator of CG0070 and/or CD40 is administered directly into the tumor. In some embodiments, an activator of oncolytic virus and/or CD40 is administered to a tissue having a tumor. In some embodiments, both activators of CG0070 and CD40 are administered directly into the tumor. In some embodiments, both activators of CG0070 and CD40 are administered to the tissue having the tumor. In some embodiments, CG007 is administered weekly. In some embodiments, the activator of CD40 is administered weekly. In some embodiments, the activators of CG0070 and CD40 are administered sequentially. In some embodiments, CG0070 is administered prior to (e.g., immediately prior to) administration of the activator of CD 40. In some embodiments, CG0070 is administered after (e.g., immediately after) administration of the activator of CD 40. In some embodiments, the activators of CG0070 and CD40 are administered simultaneously (e.g., in a single composition). In some embodiments, the method further comprises administering an activator of CG0070 and/or CD40 by a route of administration other than topical administration.

In some embodiments, the method comprises administering two or more (e.g., any of 2, 3, 4, 5, 6, or more) infectious agents. For example, in some embodiments, the method comprises: a) locally administering to the tumor site an effective amount of a first infectious agent (e.g., a virus, such as an oncolytic virus); and b) topically administering to the individual an effective amount of a second infectious agent (e.g., a bacterium, such as BCG, MCNA, or listeria monocytogenes); and c) locally administering an effective amount of an immunomodulator (including a combination of immunomodulators) to the site of the tumor.

in some embodiments, the pretreatment composition includes a transduction enhancer, such as N-dodecyl- β -D-maltoside (DDM), DDM is a non-ionic surfactant including maltose derivatized with a single 12 carbon chain, and serves as a mild detergent and solubilizer.

The pretreatment composition can be administered directly into the tumor or to the tissue having the tumor. In some embodiments, the pretreatment composition comprises a solution of a transduction enhancing agent (e.g., DDM). Suitable concentrations of the pretreatment composition (e.g., DDM solution) include, but are not limited to, about any of 0.01%, 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1%, 2%, 3%, 4%, or 5% of a transduction enhancer (e.g., DDM). In some embodiments, the pretreatment composition comprises a transduction enhancer (e.g., DDM) of any one of about 0.01% to about 0.05%, about 0.05% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 1% to about 2%, about 2% to about 3%, about 3% to about 4%, about 4% to about 5%, about 0.01% to about 1%, about 0.05% to about 2%, about 1% to about 5%, or about 0.1% to about 5%.

In some embodiments, the pretreatment (e.g., DDM) is administered immediately prior to (e.g., no more than 5 minutes) administration of the infectious agent. In some embodiments, the pretreatment (e.g., DDM) is administered no more than about any of 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 90 minutes, 2 hours, 3 hours, or 4 hours prior to administration of the infectious agent. In some embodiments, the pretreatment (e.g., DDM) is administered no more than about 2 hours prior to administration of the infectious agent.

Suitable dosages of the pretreatment composition (e.g., DDM) include, but are not limited to, about any of the following: 0.1mg/kg, 0.5mg/kg, 1mg/kg, 1.5mg/kg, 2mg/kg, 2.5mg/kg, 5mg/kg, 10mg/kg, 25mg/kg, 50mg/kg, 100mg/kg, 150mg/kg, 200mg/kg, 250mg/kg, 300mg/kg, 400mg/kg, 500mg/kg, 0.1mg/kg to 0.5mg/kg, 0.5mg/kg to 1mg/kg, 1mg/kg to 2mg/kg, 2mg/kg to 5mg/kg, 5mg/kg to 10mg/kg, 10mg/kg to 25mg/kg, 25mg/kg to 50mg/kg, 50mg/kg to 100mg/kg, 100mg/kg to 150mg/kg, 150mg/kg to 200mg/kg, 200mg/kg to 250mg/kg, 250mg/kg to 500mg/kg or 0.5mg/kg to about 5 mg/kg. In some embodiments, a suitable dose of the pretreatment composition is about any of 0.1g, 0.2g, 0.5g, 0.75g, 1g, 1.5g, 2g, 2.5g, 5g, or 10g of a transduction enhancer (e.g., DDM).

Prior to administration of the infectious agent and immunomodulator (including combinations of immunomodulators), the individual (e.g., either completely or only at the tumor site) undergoes prior therapy. In some embodiments, the prior therapy is prepared at the tumor site using one or more (e.g., 1, 2, 3, 4, 5, or more) treatment modalities, including but not limited to radiation therapy, administration of one or more immune-related molecules, administration of other therapeutic agents, and combinations thereof. It is believed that the addition of other pretreatment preparations increases the chances of success of the above-described process. Without being bound by any theory or hypothesis, for example, local radiation or chemotherapy with or without lymphoablative effects may increase the chances of an infectious process and may deplete the more sensitive tregs at the tumor site, thereby restoring depleted or tolerant T memory cells. Similarly, the preparation of a tumor site prior to or concurrent with the administration of a combination of the invention "at the tumor site" may involve interleukins, chemokines, small molecules and other well-known beneficial immunomodulators, such as IL2, IL12, OX40, CD40 and 4-1BB agonists. Such tumor site preparation modalities may be combined or administered sequentially, as desired.

In some embodiments, the prior therapy is radiation therapy (e.g., with or without chemotherapy). In some embodiments, radiation therapy is combined with chemotherapy. In some embodiments, the prior therapy is radiation therapy directed to the whole body. In some embodiments, the prior therapy is radiation therapy directed only to the tumor site. In some embodiments, the prior therapy is radiation therapy directed to tissue having a tumor. In some embodiments, the prior therapy is radiation therapy directed only to the tumor site selected for local administration of the infectious agent and the immunomodulator. In some embodiments, the prior therapy is radiation therapy directed only to tissue with a tumor selected for local administration of an infectious agent and an immunomodulator. In some embodiments, the dose of radiation therapy is insufficient to eradicate the tumor cells. For example, a suitable dose of radiation therapy is about any one of: 1Gy, 5Gy, 10Gy, 15Gy, 20Gy, 25Gy, 30Gy, 35Gy, 40Gy, 45Gy, 50Gy, 55Gy, 60Gy, 65Gy, 70Gy, 75Gy, 80Gy, 90Gy, or 100 Gy. In some embodiments, the dose of radiation therapy is no more than about any of the following: 1Gy, 5Gy, 10Gy, 15Gy, 20Gy, 25Gy, 30Gy, 35Gy, 40Gy, 45Gy, 50Gy, 55Gy, 60Gy, 65Gy, 70Gy, 75Gy, 80Gy, 90Gy, or 100 Gy. In some embodiments, the dose of radiation therapy is any one of: about 1Gy to about 5Gy, about 5Gy to about 10Gy, about 10Gy to about 15Gy, about 15Gy to about 20Gy, about 20Gy to about 25Gy, about 25Gy to about 30Gy, about 30Gy to about 35Gy, about 5Gy to about 15Gy, about 10Gy to about 20Gy, about 20Gy to about 30Gy, about 30Gy to about 40Gy, about 40 to about 50Gy, about 50Gy to about 60Gy, about 60Gy to about 70Gy, about 70Gy to about 80Gy, about 80Gy to about 100Gy, about 10 to about 30Gy, about 20Gy to about 40Gy, about 1 to about 25Gy, about 25 to about 50Gy, about 30 to about 60Gy, about 60Gy to about 80Gy, or about 10Gy to about 60 Gy. The appropriate dose of radiation therapy may also depend on the type, stage and location of the tumor.

In some embodiments, radiation therapy is administered in more than one way (e.g., about any of 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 16, 18, 20, or more ways). In some embodiments, the radiation therapy portion is administered over the course of about any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or longer. In some embodiments, the radiation therapy portion is administered during the course of any one of: about 1 day to about 5 days, about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 5 weeks to about 6 weeks, about 6 weeks to about 7 weeks, about 2 weeks to about 4 weeks, about 4 weeks to about 6 weeks, or about 1 week to about 6 weeks. In some embodiments, the radiation therapy is administered in about two fractions per day. In some embodiments, each fraction of radiation therapy is about 1.8Gy to about 2Gy per day for an adult (five days a week), or about 1.5Gy to about 1.8Gy per day for a child (five days a week). In some embodiments, each portion of radiation therapy is about any one of: 1Gy, 1.5Gy, 2Gy, 2.5Gy, 5Gy, 10Gy, 15Gy, 20Gy, 30Gy, 40Gy, 50Gy, or more. In some embodiments, each portion of radiation therapy is any one of the following: about 1Gy to about 1.5Gy, about 1.5Gy to about 2Gy, about 1Gy to about 2.5Gy, about 2.5Gy to about 5Gy, about 5Gy to about 10Gy, about 10Gy to about 15Gy, about 15Gy to about 20Gy, about 20Gy to about 30Gy, about 25Gy to about 50Gy, about 1Gy to about 10Gy, or about 2Gy to about 20 Gy. In some embodiments, the radiation therapy is administered in a single fraction.

In some embodiments, the radiation therapy is intended for lymphatic clearance, in single dose fractions per day or in multiple fractions over several days to weeks. In some embodiments, the lymphoablative radiation therapy is administered as systemic irradiation. In some embodiments, lymphatic clearance is only to the local tumor site or tissue with the tumor. In some embodiments, the lymphoablative radiation therapy is administered in two fractions per day. In some embodiments, each fraction of lymphoablative radiation therapy is about 1Gy to about 2Gy per day for adults (five days a week), or about 0.5Gy to about 1.8Gy per day for children (five days a week). In some embodiments, each portion of radiation therapy is about any one of: 1Gy, 1.5Gy, 2Gy, 2.5Gy, 5Gy, 10Gy, 15Gy, 20Gy, 30Gy, 40Gy, 50Gy, or more. In some embodiments, each portion of radiation therapy is any one of the following: about 1Gy to about 1.5Gy, about 1.5Gy to about 2Gy, about 1Gy to about 2.5Gy, about 2.5Gy to about 5Gy, about 5Gy to about 10Gy, about 10Gy to about 15Gy, about 15Gy to about 20Gy, about 20Gy to about 30Gy, about 25Gy to about 50Gy, about 1Gy to about 10Gy, or about 2Gy to about 20 Gy. In some embodiments, the lymphoablative radiation therapy is administered with or without chemotherapeutic agents, such as, but not limited to, cyclophosphamide (cycloposphamide) and fludarabine (fludarabine).

Any of the known methods of radiation therapy may be used in the present invention, including, but not limited to, external beam radiation therapy (EBRT or XRT), teletherapy, brachytherapy, sealed source radiation therapy, whole body radioisotope therapy (RIT), unsealed source radiation therapy, intraoperative radiation therapy (IORT), targeted intraoperative radiation therapy (targitt), magnitude-modulated radiation therapy (IMRT), volume-modulated arc therapy (VMAT), particle therapy, and drill therapies.

In some embodiments, there is provided a method of treating an individual having a solid or lymphoid tumor, the method comprising: (a) topically applying radiation therapy; b) locally administering to the tumor site an effective amount of an infectious agent (e.g., an oncolytic virus, e.g., CG 0070); and c) topically administering to the individual an effective amount of an immunomodulator (including a combination of immunomodulators (e.g., immune checkpoint inhibitors and/or immunostimulants). In some embodiments, there is provided a method of inhibiting metastasis of a solid or lymphatic tumor in an individual, the method comprising: (a) topically applying radiation therapy; b) locally administering to the tumor site an effective amount of an infectious agent (e.g., an oncolytic virus, e.g., CG 0070); and c) topically administering to the individual an effective amount of an immunomodulator (including a combination of immunomodulators (e.g., immune checkpoint inhibitors and/or immunostimulants). In some embodiments, radiation therapy is administered prior to administration of the infectious agent and/or immunomodulator (including combinations of immunomodulators). In some embodiments, radiation therapy is administered from about 1 day to about 1 week (e.g., about 2 days) prior to administration of the infectious agent and the immunomodulator (including the combination of immunomodulators). In some embodiments, radiation therapy, and/or an infectious agent and/or an immunomodulator (including a combination of immunomodulators) is administered directly to a solid or lymphoid tumor. In some embodiments, radiation therapy, and/or an infectious agent and/or an immunomodulator (including a combination of immunomodulators) is administered to a tissue having a solid or lymphoid tumor. In some embodiments, the immunomodulator is a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)). In some embodiments, the immunomodulatory agent is a CD40 agonist (e.g., an agonistic anti-CD 40 antibody, e.g., CP-870,893, daclizumab, ChiLob 7/4, or APX 005M). In some embodiments, the methods comprise local administration of a combination of immunomodulators, comprising a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)) and a CD40 agonist (e.g., an agonistic anti-CD 40 antibody, e.g., CP-870,893, daclizumab, ChiLob 7/4, or APX 005M). In some embodiments, the radiation therapy is administered at a dose of about 1Gy to about 10Gy (e.g., about 4Gy) per tumor site. In some embodiments, radiation therapy is administered weekly at about 2 divided doses (e.g., daily for about 2 days). In some embodiments, the radiation therapy is administered for no more than about 4 weeks. In some embodiments, the infectious agent and immunomodulator (including combination of immunomodulators) are administered weekly for about 2 weeks to about 8 weeks (e.g., about 6 weeks). In some embodiments, the infectious agent and immunomodulator (including combination of immunomodulators) are administered every other two weeks for about 1 month to about 4 months (e.g., about 2 months). In some embodiments, the infectious agent and immunomodulator (including combinations of immunomodulators) are additionally administered monthly as maintenance therapy.

in some embodiments, the therapeutic agent is any one or combination of chemotherapeutic agents known in the art (e.g., cyclophosphamide). in some embodiments, the therapeutic agent is any one or combination of agents known in the art to target or block the cell signaling pathway, e.g., BRAF inhibitors. in some embodiments, the therapeutic agent is any one or combination of cell therapies known in the art, e.g., TIL cells, T/T cells, and/or TCR/T cells. in some embodiments, the therapeutic agent is an agent that increases the level of interleukins involved in the immunogenic pathway. any of the immune-related molecules described herein may be used as therapeutic agents, including, but not limited to, interleukins (e.g., IL6, IL8 and IL18 (such interleukins may have an inflammatory and/or anti-inflammatory effect, or some may promote the development of neoplasms and interleukin such as interleukin 06793, e.g-798), interleukin proliferation, e.g-T, VEGF-leukocyte proliferation, e.g-T, VEGF-stimulating factor, e.g-T, VEGF-T cell proliferation, or tumor proliferation stimulating factor, e.g-stimulating factor, e.g. tnf-T-5, e.g. VEGF-tpt-3, and VEGF-stimulating hormone receptor, e.g. tnf-T-9, tnf-5, tnf-tpt cell stimulating hormone-9, and tnf-5, e.g. stimulating hormone-9, interleukin-3, interleukin-stimulating factor, interleukin-3, interleukin-stimulating.

Any of the one or more therapeutic agents described herein (e.g., chemotherapeutic agents, agents that target or block cell signaling pathways, interleukins, chemokines, cell therapies, etc.) can be administered, either alone or in combination, directly or indirectly (e.g., via intravenous administration) to the tumor site.

In some embodiments, a method of treating an individual having a solid or lymphoid tumor (e.g., inhibiting tumor metastasis) comprises: (a) topically administering a therapeutic agent (e.g., CCL 21); b) locally administering to the tumor site an effective amount of an infectious agent (e.g., an oncolytic virus, e.g., CG 0070); and c) topically administering to the individual an effective amount of an immunomodulator (including a combination of immunomodulators (e.g., immune checkpoint inhibitors and/or immunostimulants). In some embodiments, the therapeutic agent comprises a chemokine. In some embodiments, the chemokine is CCL 21. In some embodiments, CCL21 is a nanocapsule. In some embodiments, the therapeutic agent is administered prior to administration of the infectious agent and/or immunomodulator (including combinations of immunomodulators). In some embodiments, the therapeutic agent (e.g., CCL21) is about 1 day to about 1 week (e.g., about 2 days) prior to administration of the infectious agent and the immunomodulatory agent (including the combination of immunomodulatory agents). In some embodiments, the therapeutic agent, and/or infectious agent and/or immunomodulator (including combinations of immunomodulators) is administered directly to a solid or lymphoid tumor. In some embodiments, a therapeutic agent, and/or an infectious agent and/or an immunomodulator (including a combination of immunomodulators) is administered to a tissue having a solid or lymphoid tumor. In some embodiments, the immunomodulator is a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)). In some embodiments, the immunomodulatory agent is a CD40 agonist (e.g., an agonistic anti-CD 40 antibody, e.g., CP-870,893, daclizumab, ChiLob 7/4, or APX 005M). In some embodiments, the methods comprise local administration of a combination of immunomodulators, comprising a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)) and a CD40 agonist (e.g., an agonistic anti-CD 40 antibody, e.g., CP-870,893, daclizumab, ChiLob 7/4, or APX 005M). In some embodiments, a therapeutic agent (e.g., CCL21) is administered at a dose of about 10 μ g to about 100mg per tumor site. In some embodiments, the dose of therapeutic agent (e.g., CCL21) per tumor site depends on the size of the tumor, e.g., from about 100 μ g to about 10mg (e.g., about 400 μ g) for tumors having a longest dimension of about 5cm or more, from about 50 μ g to about 5mg (e.g., about 200 μ g) for tumors having a longest dimension of about 2cm to about 5cm, or from about 25 μ g to about 2.5mg (e.g., about 100 μ g) for tumors having a longest dimension of about 0.5cm to about 2 cm. In some embodiments, the therapeutic agent (e.g., CCL21), infectious agent, and immunomodulator (including combinations of immunomodulators) are administered weekly in about 2 weeks to about 8 weeks (e.g., about 6 weeks). In some embodiments, the therapeutic agent (e.g., CCL21), the infectious agent, and the immunomodulator (including the combination of immunomodulators) are administered every other two weeks for about 1 month to about 4 months (e.g., about 2 months). In some embodiments, a therapeutic agent (e.g., CCL21), an infectious agent, and an immunomodulator (including a combination of immunomodulators) are additionally administered monthly as maintenance therapy.

In some embodiments, a method of treating an individual having a solid or lymphoid tumor (e.g., inhibiting tumor metastasis) comprises: (a) local administration of therapeutic agents (e.g., CpG ODN); b) locally administering to the tumor site an effective amount of an infectious agent (e.g., an oncolytic virus, e.g., CG 0070); and c) topically administering to the individual an effective amount of an immunomodulator (including a combination of immunomodulators (e.g., immune checkpoint inhibitors and/or immunostimulants). In some embodiments, the therapeutic agent comprises PRRago. In some embodiments, the chemokine is a CpG ODN, such as CpG 7909. In some embodiments, the therapeutic agent is administered prior to administration of the infectious agent and/or immunomodulator (including combinations of immunomodulators). In some embodiments, the therapeutic agent (e.g., CpG ODN) is about 1 day to about 1 week (e.g., about 2 days) prior to administration of the infectious agent and the immunomodulator (including the combination of immunomodulators). In some embodiments, the therapeutic agent, and/or infectious agent and/or immunomodulator (including combinations of immunomodulators) is administered directly to a solid or lymphoid tumor. In some embodiments, a therapeutic agent, and/or an infectious agent and/or an immunomodulator (including a combination of immunomodulators) is administered to a tissue having a solid or lymphoid tumor. In some embodiments, the immunomodulator is a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)). In some embodiments, the immunomodulator is an OX40 agonist (e.g., an agonist anti-OX 40 antibody, e.g., MEDI-6469). In some embodiments, the methods comprise local administration of a combination of immunomodulators, comprising a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)) and an OX40 agonist (e.g., an agonist anti-OX 40 antibody, e.g., MEDI-6469). In some embodiments, the therapeutic agent (e.g., CpG ODN) is administered at a dose of about 10 μ g to about 100mg per tumor site. In some embodiments, the dose of therapeutic agent (e.g., CpG ODN) per tumor site depends on the size of the tumor, e.g., from about 200 μ g to about 20mg (e.g., about 2mg) for tumors having a longest dimension of about 5cm or greater, from about 100 μ g to about 10mg (e.g., about 1mg) for tumors having a longest dimension of about 2cm to about 5cm, or from about 50 μ g to about 5mg (e.g., about 500 μ g) for tumors having a longest dimension of about 0.5cm to about 2 cm. In some embodiments, the therapeutic agent (e.g., CpG ODN), infectious agent, and immunomodulator (including combinations of immunomodulators) are administered weekly in about 2 weeks to about 8 weeks (e.g., about 6 weeks). In some embodiments, the therapeutic agent (e.g., CpG ODN), infectious agent, and immunomodulator (including combinations of immunomodulators) are administered every other two weeks for about 1 month to about 4 months (e.g., about 2 months). In some embodiments, a therapeutic agent (e.g., CpG ODN), an infectious agent, and an immunomodulator (including a combination of immunomodulators) are additionally administered monthly as maintenance therapy.

The appropriate dosage of the infectious agent depends on factors such as: the nature of the infectious agent, the type of solid or lymphoid tumor being treated and the route of administration. As used herein, "particle" when in relation to an infectious agent means the collective number of physical singular units of the infectious agent (e.g., virus or bacteria). This number translates into or is equivalent to another number that means infectious titer units, such as plaque forming units (pfu) or international units, as determined by infectivity assays as known in the art. In some embodiments, the infectious agent is administered at a dose of about any one of: 1X 10⁵1X 10 particles of⁶1X 10 particles of⁷1X 10 particles of⁸1X 10 particles of⁹1X 10 particles of¹⁰Granule, 2X 10¹⁰Granule, 5X 10¹⁰1X 10 particles of¹¹Granule, 2X 10¹¹Granule, 5X 10¹¹1X 10 particles of¹²Granule, 2X 10¹²Granule, 5X 10¹²1X 10 particles of¹³Granule, 2X 10¹³Granule, 5X 10¹³1X 10 particles of¹⁴Single particle or 1X 10¹⁵And (4) granules. In some embodiments, the infectious agent is administered at a dose of any one of: about 1X 10⁵To about 1X 10⁶Particles, about 1X 10⁶To about 1X 10⁷Particles, about 1X 10⁷To about 1X 10⁸Particles, about 1X 10⁸To about 1X 10⁹Particles, about 1X 10⁹To about 1X 10¹⁰Particles, about 1X 10¹⁰Each granuleGranulating to about 1 × 10¹¹Particles, about 1X 10¹¹To about 5X 10¹¹Particles, about 5X 10¹¹To about 1X 10¹²Particles, about 1X 10¹²To about 2X 10¹²Particles, about 2X 10¹²To about 5X 10¹²Particles, about 5X 10¹²To about 1X 10¹³Particles, about 1X 10¹³To about 1X 10¹⁴Per particle or about 1X 10¹⁴To about 1X 10¹⁵And (4) granules.

In some embodiments, the infectious agent is administered daily. In some embodiments, the infectious agent is about any one of at least 1, 2, 3, 4, 5, 6, or 7 (i.e., daily) administrations per week. In some embodiments, the infectious agent is administered weekly. In some embodiments, the infectious agent is administered continuously weekly; two out of three weeks are administered weekly; three weeks out of four weeks weekly; once every 2 weeks; once every 3 weeks; once every 4 weeks; once every 6 weeks; administered once every 8 weeks, monthly or every 2 to 12 months. In some embodiments, the interval between each administration is less than about any of: 6 months, 3 months, 1 month, 20 days, 15 days, 12 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the interval between each administration exceeds about any of the following: 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, or 12 months. In some embodiments, there is no break in the dosing schedule. In some embodiments, each administration is separated by no more than about one week.

The administration of the infectious agent may be over an extended period of time (e.g., about 1 month up to about 7 years). In some embodiments, the infectious agent is administered for a period of time of at least about any one of: 1. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 48, 60, 72, or 84 months. In some embodiments, the infectious agent is administered over a period of at least 4 weeks or 6 weeks. In some embodiments, the infectious agent is administered weekly in 4 weeks every 3 months. In some embodiments, the infectious agent is administered weekly every 3 months for 6 weeks.

Suitable dosages of immunomodulators (including combinations of immunomodulators) depend on factors such as: the nature of the immunomodulator or combination of immunomodulators, the type of solid or lymphoid tumor to be treated and the route of administration. Exemplary doses of immunomodulators (including combinations of immunomodulators) include, but are not limited to, about any of the following: 1mg/m²、5mg/m²、10mg/m²、20mg/m²、50mg/m²、100mg/m²、200mg/m²、300mg/m²、400mg/m²、500mg/m²、750mg/m²、1000mg/m²Or more. In some embodiments, the dose of the immunomodulator (including the combination of immunomodulators) is included in any one of the following ranges: about 1 to about 5mg/m²About 5 to about 10mg/m²About 10 to about 20mg/m²About 20 to about 50mg/m²About 50 to about 100mg/m²About 100mg/m²To about 200mg/m²About 200 to about 300mg/m²About 300 to about 400mg/m²About 400 to about 500mg/m²About 500 to about 750mg/m²Or from about 750 to about 1000mg/m². In some embodiments, the dose of the immunomodulatory agent is about any one of: 1. mu.g/kg, 2. mu.g/kg, 5. mu.g/kg, 10. mu.g/kg, 20. mu.g/kg, 50. mu.g/kg, 0.1mg/kg, 0.2mg/kg, 0.3mg/kg, 0.4mg/kg, 0.5mg/kg, 1mg/kg, 2mg/kg, 5mg/kg, 10mg/kg, 20mg/kg, 50mg/kg, 100mg/kg or more. In some embodiments, the dose of the immunomodulator (including the combination of immunomodulators) is any one of: about 1 μ g/kg to about 5 μ g/kg, about 5 μ g/kg to about 10 μ g/kg, about 10 μ g/kg to about 50 μ g/kg, about 50 μ g/kg to about 0.1mg/kg, about 0.1mg/kg to about 0.2mg/kg, about 0.2mg/kg to about 0.3mg/kg, about 0.3mg/kg to about 0.4mg/kg, about 0.4mg/kg to about 0.5mg/kg, about 0.5mg/kg to about 1mg/kg, about 1mg/kg to about 5mg/kg, about 5mg/kg to about 10mg/kg, about 10mg/kg to about 20mg/kg, about 20mg/kg to about 50mg/kg, about 50mg/kg to about 100mg/kg, or about 1mg/kg to about 100 mg/kg. In some embodiments, an immunomodulator (including a combination of immunomodulators)) Is about any one of the following: 1 μ g, 10 μ g, 50 μ g, 100 μ g, 500 μ g, 1mg, 2mg, 4mg, 6mg, 12mg, 18mg, 24mg, 50mg, 100mg, 500mg or 1000 mg. In some embodiments, the dose of the immunomodulator (including the combination of immunomodulators) is any one of: about 1 μ g to about 10 μ g, about 10 μ g to about 5010 μ g, about 50 μ g to about 100 μ g, about 100 μ g to about 500 μ g, about 500 μ g to about 1mg, about 1mg to about 5mg, about 5mg to about 10mg, about 10mg to about 25mg, about 25mg to about 50mg, about 50mg to about 100mg, about 100mg to about 500mg, about 500mg to about 1000mg, about 1 μ g to about 1mg, about 1mg to about 1000mg, or about 1 μ g to about 1000 mg. In some embodiments, the dose of immunomodulator (including combination of immunomodulators) administered at each tumor site is no more than about any one of: 10 μ g, 50 μ g, 100 μ g, 500 μ g, 1mg, 2mg, 4mg, 6mg, 12mg, 18mg, 24mg, 50mg or 100 mg. In some embodiments, the dose of immunomodulatory agent (including combinations of immunomodulatory agents) administered per tumor site is any one of: about 10 μ g to about 50 μ g, about 50 μ g to about 100 μ g, about 100 μ g to about 500 μ g, about 100 μ g to about 1mg, about 1mg to about 2mg, about 2mg to about 5mg, about 5mg to about 10mg, about 10mg to about 15mg, about 10mg to about 25mg, about 25mg to about 50mg, about 50mg to about 100mg, about 1mg to about 50mg, or about 100 μ g to about 10 mg. In some embodiments, the dose of immunomodulator (including combination of immunomodulators) administered at each tumor site is based on the size of the tumor.

In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered daily. In some embodiments, an immunomodulator (including a combination of immunomodulators) is administered at least about any one of 1, 2, 3, 4, 5, 6, or 7 times a week (i.e., daily). In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered weekly. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered weekly on an episodic basis; two out of three weeks are administered weekly; three weeks out of four weeks weekly; once every 2 weeks; once every 3 weeks; once every 4 weeks; once every 6 weeks; administered once every 8 weeks, monthly or every 2 to 12 months. In some embodiments, the interval between each administration is less than about any of: 6 months, 3 months, 1 month, 20 days, 15 days, 12 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the interval between each administration exceeds about any of the following: 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, or 12 months. In some embodiments, there is no break in the dosing schedule. In some embodiments, each administration is separated by no more than about one week. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered on the same dosing schedule as the infectious agent. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered on a different dosing schedule than the infectious agent. In some embodiments, the infectious agent is administered weekly for 4 weeks, and the immunomodulator (including the combination of immunomodulators) is administered weekly for three of four weeks.

Administration of the immunomodulator (including the combination of immunomodulators) can be over an extended period of time (e.g., about 1 month up to about 7 years). In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered over a period of at least about any one of: 1. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 48, 60, 72, or 84 months. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered over a period of at least 3 weeks or 6 weeks. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered weekly in three of four weeks every 3 months. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered weekly over 6 weeks every 3 months.

Exemplary routes of administration of the infectious agent, immunomodulator (including combinations of immunomodulators), interleukin and/or pretreatment composition include, but are not limited to, intratumoral, intravesical, intramuscular, intraperitoneal, intravenous, intraarterial, intracranial, intrapleural, subcutaneous and epidermal routes, or delivery into lymph glands, body spaces, organs or tissues known to contain such living cancer cells (e.g., intrahepatic or intrapancreatic injection). In some embodiments, administration is by direct injection of the agent into the tumor. In some embodiments, the administering is performed by injecting the agent directly into a site proximate to the tumor cell. The specific route of administration depends on the nature of the solid or lymphoid tumor and is discussed further below above and below different types of solid or lymphoid tumors.

In some embodiments, in which the infectious agent and/or immune modulator (including combinations of immune modulators) are administered intratumorally (e.g., intratumoral injection), the total volume administered does not exceed about any of the following: 0.5mL, 1mL, 1.5mL, 2mL, 2.5mL, 5mL, or 10 mL. In some embodiments, the volume of the infectious agent and/or immunomodulator (including combination of immunomodulators) administered intratumorally (e.g., intratumorally injected) at each tumor site is dependent on the size of the tumor site. Tumor size can be measured as the tumor volume or longest dimension of the tumor. For example, for tumors having a longest dimension greater than about 5cm, the intratumoral administration volume is no more than about 2 mL; an intratumoral administration volume of about 1mL for a tumor having a longest dimension of about 2cm to about 5 cm; an intratumoral administration volume of about 0.5mL for a tumor having a longest dimension of about 0.75cm to about 2 cm; and an intratumoral administration volume of about 0.1mL for tumors having a longest dimension of less than about 0.75 cm. In some embodiments, an infectious agent and/or an immunomodulator (including a combination of immunomodulators) is administered to all tumor sites. In some embodiments, an infectious agent and/or an immunomodulatory agent (including combinations of immunomodulatory agents) is administered to about any of 1, 2, 3, 4, 5, 6, or more tumor sites. In some embodiments, the infectious agent and/or immunomodulator (including combinations of immunomodulators) is administered to a tumor site with the largest size.

In some embodiments, the amount of the infectious agent in combination with the immunomodulatory agent is effective to inhibit tumor metastasis in the individual. In some embodiments, metastasis is inhibited by at least about 10% (including, for example, about any of at least 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, methods of inhibiting metastasis to a lymph node are provided. In some embodiments, methods of inhibiting metastasis to the lung are provided. Metastasis can be assessed by any method known in the art, for example, by blood testing, bone scanning, x-ray scanning, CT scanning, PET scanning, and biopsy.

In some embodiments, the amount of infectious agent combined with the immunomodulator is effective to prolong the survival (e.g., disease-free survival) of the individual. In some embodiments, survival is extended by at least about 2, 3, 4, 5, 6, 12, or 24 months. In some embodiments, there is provided a method of prolonging survival of an individual having a solid or lymphoid tumor, the method comprising: (a) locally administering to the tumor site an effective amount of an infectious agent (e.g., CG 0070); and (b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators).

In some embodiments, the amount of infectious agent combined with the immunomodulator is effective to cause remission (partial or complete) of the disease in the individual. In some embodiments, there is provided a method of causing remission (partial or complete) of a disease in an individual having a solid or lymphoid tumor, the method comprising: (a) locally administering to the tumor site an effective amount of an infectious agent (e.g., CG 0070); and (b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators).

In some embodiments, the amount of infectious agent combined with the immunomodulator is effective to improve the quality of life of the individual. In some embodiments, there is provided a method of improving the quality of life of an individual having a solid or lymphatic tumor, the method comprising: (a) locally administering to the tumor site an effective amount of an infectious agent (e.g., CG 0070); and (b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators).

In some embodiments, the amount of infectious agent combined with the immunomodulatory agent is effective to inhibit the growth or reduce the size of a solid or lymphoid tumor. In some embodiments, the size of the solid or lymphatic tumor is reduced by at least about 10% (including, e.g., at least about any of 20%, 30%, 40%, 60%, 70%, 80%, 90%, or 100%). In some embodiments, there is provided a method of inhibiting growth or reducing size of a solid or lymphoid tumor in a subject, the method comprising: (a) locally administering to the tumor site an effective amount of an infectious agent (e.g., CG 0070); and (b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators).

Solid or lymphoid tumors discussed herein include, but are not limited to, Hodgkin's lymphoma (Hodgkin lymphoma), non-Hodgkin's lymphoma, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, Kaposi's sarcoma, soft tissue sarcoma, uterosynoviosarcoma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor (Wilm's tumor), Cervical cancer, testicular tumor, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

In some embodiments, the solid or lymphoid tumor is selected from the group consisting of: squamous cell carcinoma of the head and neck, breast cancer, colorectal cancer, pancreatic adenocarcinoma, ovarian cancer, non-small cell lung cancer, prostate cancer, and melanoma. The methods are applicable to all stages (according to the American Joint Committee on Cancer, AJCC) of staged groups including stages I, II, III and IV) of solid or lymphoid tumors. In some embodiments, the solid or lymphoid tumor is: early stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, cancer in remission, cancer in an adjuvant setting, or cancer in a lead setting. In some embodiments, the solid or lymphoid tumor is locally resectable, locally unresectable, or unresectable. In some embodiments, the solid or lymphatic tumor is locally resectable or marginally resectable. In some embodiments, the cancer is refractory to a prior therapy.

In some embodiments, the solid or lymphoid tumor is a head and neck cancer. In some embodiments, the cancer of the head and neck is squamous cell carcinoma in the head and neck. In some embodiments, the cancer of the head and neck is hypopharynx cancer, cancer of the larynx, cancer of the lip and oral cavity, metastatic squamous neck cancer with occult primary, nasopharyngeal cancer, oropharyngeal cancer, paranasal sinus and nasal cavity cancer, or salivary gland cancer. In some embodiments, the squamous cell carcinoma of the head and neck is an early stage head and neck cancer, a non-metastatic head and neck cancer, a late stage head and neck cancer, a locally advanced head and neck cancer, a metastatic head and neck cancer, a head and neck cancer in remission, a head and neck cancer in a secondary setting, or a head and neck cancer in a lead setting. In some embodiments, the head and neck cancer is in a pre-conductant setting. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by injection into head and neck tissue having a head and neck tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct injection into a head and neck tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct injection into a metastatic site of a head and neck tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by injection into head and neck tissue proximal to a head and neck tumor.

In some embodiments, the solid or lymphoid tumor is breast cancer. In some embodiments, the breast cancer is early stage breast cancer, non-metastatic breast cancer, advanced breast cancer, stage IV breast cancer, locally advanced breast cancer, metastatic breast cancer, breast cancer in remission, breast cancer in a helper setting, or breast cancer in a lead setting. In some embodiments, the breast cancer is in a pre-conductive setting. In some embodiments, the breast cancer is at a late stage. In some embodiments, breast cancer (which may be HER2 positive or HER2 negative) includes, for example, advanced breast cancer, stage IV breast cancer, locally advanced breast cancer, and metastatic breast cancer. In some embodiments, the breast cancer is a triple negative breast cancer. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by intramammary injection into breast tissue having a breast tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct intramammary injection into a breast tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct injection into a metastatic site of a breast tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by intramammary injection into breast tissue proximal to a breast tumor.

In some embodiments, the cancer is renal cell carcinoma. In some embodiments, the renal cell carcinoma is adenocarcinoma. In some embodiments, the renal cell carcinoma is clear cell renal cell carcinoma, papillary renal cell carcinoma (also referred to as invasive renal cell carcinoma), refractory renal cell carcinoma, collecting duct renal cell carcinoma, granular renal cell carcinoma, mixed granular renal cell carcinoma, renal angiomyolipoma, or spindle renal cell carcinoma. In some embodiments, the renal cell carcinoma is in any of stages I, II, III, or IV according to the American Joint Committee for Cancer (AJCC) staging group. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by intrarenal injection into renal tissue having a renal tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct intrarenal injection into a renal tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct injection into a metastatic site of a renal tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by intrarenal injection into renal tissue proximal to the renal tumor.

In some embodiments, the solid or lymphoid tumor is prostate cancer. In some embodiments, the prostate cancer is adenocarcinoma. In some embodiments, the prostate cancer is a sarcoma, neuroendocrine tumor, small cell carcinoma, ductal carcinoma, or lymphoma. In some embodiments, the prostate cancer is at any one of four stages A, B, C or D according to the Jewett staging system. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by intraprostatic injection into prostate tissue having a prostate tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct intraprostatic injection into a prostate tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct injection into the metastatic sites of prostate tumors. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by intraprostatic injection into prostate tissue proximal to the prostate tumor.

In some embodiments, the solid or lymphoid tumor is lung cancer. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). Examples of NSCLC include, but are not limited to, large cell carcinoma, adenocarcinoma, neuroendocrine lung tumor, and squamous cell carcinoma. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by intrapulmonary injection into lung tissue having a lung tumor. In some embodiments, the lung cancer is Small Cell Lung Cancer (SCLC). In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct intrapulmonary injection into a lung tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct injection into a metastatic site of a lung tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by intrapulmonary injection into lung tissue proximal to a lung tumor.

In some embodiments, the solid or lymphoid tumor is melanoma. In some embodiments, the melanoma is superficial spreading melanoma, malignant lentigo melanoma, nodular melanoma, mucosal melanoma, polypoid melanoma, desmoplastic melanoma, melanomas without melanin, soft tissue melanoma, or acral lentigo melanoma. In some embodiments, the melanoma is in any of stage I, II, III, or IV according to the joint committee for cancer (AJCC) staging group. In some embodiments, the melanoma is relapsed. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by injection into skin tissue having a melanoma tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct injection into a melanoma tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct injection into a metastatic site of a melanoma tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by injection into lung tissue proximal to a melanoma tumor.

In some embodiments, the solid or lymphoid tumor is ovarian cancer. In some embodiments, the ovarian cancer is epithelial ovarian cancer. In some embodiments, the ovarian cancer is stage I (e.g., IA, IB, or IC), stage II (e.g., HA, HB, or IIC), stage III (e.g., IIIA, HIB, or HIC), or stage IV. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by intra-ovarian injection into ovarian tissue having an ovarian tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct intra-ovarian injection into an ovarian tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct injection into the metastatic site of the ovarian tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by intra-ovarian injection into ovarian tissue proximal to the ovarian tumor.

In some embodiments, according to any one of the methods above, the solid or lymphatic tumor is pancreatic cancer. In some embodiments, the pancreatic cancer is a serous cystic neoplasm, a mucinous cystic neoplasm, an intraductal papillary mucinous tumor, a pancreatic adenocarcinoma, an adenosquamous carcinoma, a squamous cell carcinoma, a ring cell carcinoma, an undifferentiated carcinoma with giant cells, a solid pseudopapillary neoplasm, a ampulla carcinoma, or a pancreatic neuroendocrine tumor. In some embodiments, the pancreatic cancer is pancreatic adenocarcinoma. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by intraparenchymal injection into pancreatic tissue having a pancreatic tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct intrapancreatic injection into a pancreatic tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct injection into a metastatic site of a pancreatic tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by intraparenchymal injection into pancreatic tissue proximal to a pancreatic tumor.

In some embodiments, the solid or lymphoid tumor is endometrial cancer. In some embodiments, the endometrial cancer is an adenocarcinoma, a carcinosarcoma, a squamous cell carcinoma, an undifferentiated carcinoma, a small cell carcinoma, or a transitional cell carcinoma. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by intra-endometrial injection into endometrial tissue having an endometrial tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct intra-endometrial injection into an endometrial tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct injection into a metastatic site of an endometrial tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by intra-endometrial injection into endometrial tissue adjacent to an endometrial tumor.

In some embodiments, according to any one of the above methods, the solid or lymphoid tumor is colorectal cancer. In some embodiments, the colorectal cancer is adenocarcinoma, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, leiomyosarcoma, melanoma, or squamous cell carcinoma. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by injection into colorectal tissue having a colorectal tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct injection into a colorectal tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct injection into the metastatic site of a colorectal tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by injection into colorectal tissue proximal to a colorectal tumor.

In some embodiments, according to any one of the methods above, the solid or lymphoid tumor is hepatocellular carcinoma (HCC). In some embodiments, the HCC is early stage HCC, non-metastatic HCC, primary HCC, advanced HCC, locally advanced HCC, metastatic HCC, HCC in remission, or HCC in relapse. In some embodiments, the HCC is locally resectable (i.e., a tumor limited to a portion of the liver that allows for complete surgical removal), locally unresectable (i.e., a local tumor may be unresectable because it involves critical vascular structures or because it is damaged), or unresectable (i.e., a tumor involves all lobes of the liver and/or has spread to involve other organs (e.g., lungs, lymph nodes, bones) N1 tumor (regional lymph node metastasis) or M1 tumor (distant metastasis). In some embodiments, the HCC is stage T1, T2, T3, or T4 HCC according to AJCC (american joint committee for cancer) staging guidelines. In some embodiments, the HCC is any one of hepatocellular carcinoma, a fibrolamellar variant of HCC, and mixed hepatocellular cholangiocarcinoma. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by intrahepatic injection into liver tissue with HCC. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct intrahepatic injection into HCC. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct injection into the metastatic sites of HCC. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by intrahepatic injection into tissue proximate HCC.

In some embodiments, the solid or lymphoid tumor is a lymphoma according to any of the above methods. In some embodiments, the lymphoma is a B cell neoplasm, a T cell neoplasm, and/or a putative NK-cell neoplasm. Examples of B cell neoplasms include, but are not limited to, precursor B cell neoplasms (e.g., precursor B-lymphoblastic leukemia/lymphoma) and peripheral B cell neoplasms (e.g., B cell chronic lymphocytic leukemia/prolymphocytic leukemia/small lymphocytic lymphoma (small lymphocytic (SL) NHL), lymphoplasmacytoid lymphoma/immunocytoma, mantle cell lymphoma, follicular central lymphoma, follicular lymphoma (e.g., cytological grade: I (small cell), II (mixed small and large cell), III (large cell), and/or subtypes: diffuse and major small cell type), low grade/follicular non-hodgkin's lymphoma (NHL), intermediate grade/follicular NHL, marginal zone B cell lymphoma (e.g., extranodal (e.g., MALT-type mononuclear-like B cells), and/or nodules (e.g., +/-monocyte-like B cells)), splenic marginal zone lymphomas (e.g., +/-villous lymphocytes), hairy cell leukemia, plasmacytoma/plasma cell myeloma (e.g., myeloma and multiple myeloma), diffuse large B cell lymphoma (e.g., primary mediastinal (thymus) B cell lymphoma), moderate diffuse NHL, berkitt's lymphoma, high B cell lymphoma, berkitt's, high immunoblastic NHL, high lymphoblastic NHL, high small non-dividing cell NHL, giant disease NHL, AIDS-related lymphoma, and fahrenheit macroglobulinemia (Waldenstrom's macroglobulinemia)). Examples of T-cell and/or putative NK-cell neoplasms include, but are not limited to, precursor T-cell neoplasms (precursor T-lymphoblastic lymphoma/leukemia) and peripheral T-cell and NK-cell neoplasms (e.g., T-cell chronic lymphocytic leukemia/prolymphocytic leukemia and large granular lymphocytic leukemia (LGL) (e.g., T-cell type and/or NK-cell type), cutaneous T-cell lymphoma (e.g., mycosis fungoides/Sezary syndrome), unspecified primary T-cell lymphoma (e.g., cytological classes (e.g., medium-cell, mixed medium-cell, and large-cell), large-cell, lymphoepithelial cell, subtype hepatosplenic γ δ T-cell lymphoma, and subcutaneous lipid membrane inflammatory T-cell lymphoma), angioimmunoblastic T-cell lymphoma (AILD), Angiocentric lymphoma, intestinal T-cell lymphoma (e.g., +/-bowel disease-associated), adult T-cell lymphoma/leukemia (ATL), degenerative large cell lymphoma (ALCL) (e.g., CD30+, T-and naked cell types), degenerative large cell lymphoma, and hodgkin's lymphoma). In some embodiments, the lymphoma is hodgkin's disease or non-hodgkin's lymphoma (NHL). For example, hodgkin's disease may be predominantly lymphobulbar, tuberous sclerosing, mixed cell, missing and/or enriched with lymphocytes. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by intralymphatic injection into a lymph node having a lymphoid tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct intralymphatic injection into a lymphoid tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct injection into a metastatic site of a lymphoid tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by intralymphatic injection into tissue proximal to a lymphoid tumor.

In some embodiments, according to any one of the methods above, the solid or lymphoid tumor is mesothelioma. In some embodiments, the mesothelioma is pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma, or mesothelioma between dermal tissues affecting other organs covered. In some embodiments, the mesothelioma is benign mesothelioma or malignant mesothelioma. In some embodiments, the mesothelioma is epithelial mesothelioma, sarcoma-like mesothelioma, biphasic mesothelioma, or papillary mesothelioma. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by injection into mesothelioma bearing mesothelioma. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct injection into a mesothelioma. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct injection into a metastatic site of a mesothelioma. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by injection into the dermal tissue between adjacent mesotheliomas.

In some embodiments, according to any one of the methods above, the solid or lymphoid tumor is a brain tumor. In some embodiments, the brain tumor is a primary brain tumor or a secondary (or metastatic) brain tumor. In some embodiments, the brain tumor is a glioma (e.g., an astrocytoma, an oligodendroglioma, or an ependymoma), a meningioma, a schwannoma, a craniopharyngioma, a germ cell tumor, or a pineal area tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by injection into brain tissue having a brain tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct injection into a brain tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct injection into a metastatic site of a brain tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by injection into brain tissue proximate a brain tumor.

In some embodiments, the solid or lymphatic tumor is a gallbladder and bile duct tumor according to any of the above methods. In some embodiments, the gallbladder and bile duct tumors are carcinomas, adenocarcinomas, biliary tract carcinomas, papillary tumors, small cell (neuroendocrine) carcinomas, adenosquamous carcinomas, or rhabdomyosarcomas. In some embodiments, the gallbladder and bile duct tumor is gallbladder cancer, extrahepatic bile duct cancer, or intrahepatic bile duct cancer. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by injection into the gallbladder or bile duct tissue having a gallbladder and bile duct tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct injection into the gallbladder and bile duct tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct injection into the metastatic sites of gallbladder and bile duct tumors. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by injection into the gallbladder or bile duct tissue proximal to the gallbladder and bile duct tumor.

In some embodiments, according to any of the above methods, the solid or lymphatic tumor is a soft tissue sarcoma. In some embodiments, the soft tissue sarcoma is adult fibrosarcoma, alveolar sarcoid, angiosarcoma, clear cell sarcoma, desmoplastic small round cell tumor, epithelioid sarcoma, fibrosarcoma, liposarcoma, malignant metaphylloma, malignant peripheral nerve sheath tumor (e.g., neurofibrosarcoma, malignant nerve sheath tumor, or neurogenic sarcoma), mucinous fibrosarcoma, synovial sarcoma, undifferentiated polymorphic sarcoma, cutaneous fibrosarcoma bulge, fibromatosis, angioendothelioma, infantile fibrosarcoma, solitary fibroma, elastofibroma, fibroma, fibrosarcoma, hemangioblastoma, granular cell tumor, hemangioma, hibernating lipoma, leiomyoma, lipoblastoma, lymphangioma, myxoma, neurofibroma, pea, rhabdomyoma, granuloma, hemangioblastoma, neurofibroma, and neurofibromyalgia, Schwannoma, tenosynovium giant cell tumor, spindle cell tumor, or a neoplastic-like condition of the soft tissue. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by injection into tissue with soft tissue sarcoma. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct injection into soft tissue sarcoma.

In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct injection into a metastatic site of soft tissue sarcoma. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by injection into tissue proximal to soft tissue sarcoma. In some embodiments, according to any one of the above methods, the solid or lymphatic tumor is a uterine tumor. In some embodiments, the uterine tumor is uterine cancer, a uterine sarcoma (e.g., endometrial stromal sarcoma, undifferentiated sarcoma, or uterine leiomyosarcoma), or a uterine carcinosarcoma (e.g., a malignant mixed mesodermal tumor or a malignant mixed muller's tumor). In some embodiments, the uterine tumor is a fibroid tumor, such as a leiomyoma, adenofibroma, or adenomyoma. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by intrauterine injection into uterine tissue having a uterine tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct intrauterine injection into a uterine tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct injection into a metastatic site of a uterine tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by intrauterine injection into uterine tissue proximal to a uterine tumor.

In some embodiments, according to any one of the methods above, the solid or lymphoid tumor is a cervical tumor. In some embodiments, the cervical tumor is a squamous cell carcinoma, adenocarcinoma, or adenosquamous carcinoma. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by endocervical injection into cervical tissue having a cervical tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct endocervical injection into a cervical tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct injection into a metastatic site of a cervical tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by endocervical injection into cervical tissue proximate to a cervical tumor.

In some embodiments, according to any one of the methods above, the solid or lymphatic tumor is a thyroid tumor. In some embodiments, the thyroid tumor is an undifferentiated thyroid tumor (e.g., papillary carcinoma, follicular carcinoma or hurthley cell carcinoma), medullary thyroid carcinoma, degenerative carcinoma, thyroid lymphoma, thyroid sarcoma, or parathyroid tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by injection into thyroid tissue with thyroid tumors. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct injection into a thyroid tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct injection into a metastatic site of a thyroid tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by injection into thyroid tissue proximal to a thyroid tumor.

In some embodiments, according to any one of the above methods, the solid or lymphoid tumor is a nasopharyngeal carcinoma. In some embodiments, the nasopharyngeal carcinoma is a keratinized squamous cell carcinoma, a non-keratinized differentiated or undifferentiated carcinoma (e.g., a lymphoepithelial carcinoma), an oral and oropharyngeal tumor, a nasal and paranasal sinus tumor, or a salivary gland tumor. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by injection into nasopharyngeal tissue having nasopharyngeal carcinoma. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by direct injection into nasopharyngeal carcinoma. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is performed by direct injection into a metastatic site of nasopharyngeal carcinoma. In some embodiments, administration of the infectious agent, immunomodulator (including combinations of immunomodulators), and/or pretreatment composition is by injection into the nasopharyngeal tissue adjacent to the nasopharyngeal carcinoma.

In some embodiments, the subject is a human subject. In some embodiments, an individual being treated for a solid or lymphoid tumor is identified as having one or more of the conditions described herein. The identification of a condition as described herein by a skilled physician is common in the art (e.g., via blood examination, X-ray, acoustic, CT scan, PET/CT scan, MRI scan, PET/MRI scan, nuclear medicine radioisotope scan, endoscopic procedures, biopsy, angiography, CT-angiography, etc.) and may also be suspected by the individual or others by, for example, tumor growth, bleeding, ulceration, pain, enlarged lymph nodes, cough, jaundice, swelling, weight loss, cachexia, sweating, anemia, secondary tumor phenomena, thrombosis, and the like. In some embodiments, an individual is selected for any of the treatment methods described herein based on any one or more of a variety of risk factors and/or diagnostic methods known to those of skill in the art, including but not limited to genetic profiling, family history, medical history (e.g., appearance of related conditions and history of viral infection), lifestyle, or habits.

In some embodiments, the degree of expression of one or more biomarkers (including, but not limited to, immune checkpoint molecules, co-stimulatory molecules, interleukins, chemokines, other immune-related molecules, and HLA-class II antigens) is selected from the group consisting of the individual for any of the methods of treatment described herein. In some embodiments, an individual is selected for treatment based on the degree of expression (e.g., high degree of expression) of one or more inhibitory immune checkpoint molecules (including, but not limited to, CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, 2B4, and their ligands). In some embodiments, an individual is selected for a method of treatment based on the degree of expression (e.g., low degree of expression) of one or more stimulatory immune checkpoint molecules or co-stimulatory molecules, including but not limited to OX40, 4-1BB, CD40, and their ligands. In some embodiments, an individual is selected for treatment based on the degree of expression (e.g., high degree of expression) of one or more biomarkers in a tumor (e.g., tumor cells and/or immune cells within a tumor) selected from the group consisting of: PD-1, PD-L1 and PD-L2. In some embodiments, the individual is selected for treatment based on the degree of expression (e.g., high degree of expression) of one or more biomarkers selected from the group consisting of: CD80, CD83, CD86 and HLA-class II antigens in mature dendritic cells of tumor origin. In some embodiments, the individual is selected for treatment based on the degree of expression (e.g., high degree of expression) of one or more biomarkers selected from the group consisting of: CXCL9, CXCL10, CXCL11, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1, HES4, MTIB, MTIE, MTIG, MTIH, GADD45A, LAMP3 and miR-155.

in some embodiments, the individual has a high expression of one or more stimulatory immune checkpoint molecules, in some embodiments, the individual has a high expression of one or more biomarkers selected from the group consisting of PD-1, PD-L1, and PD-L2 in a tumor (e.g., tumor cells and/or immune cells within a tumor), in some embodiments, PD-L1, and PD-L2 are used as biomarkers for selecting a patient or as ligands for inhibiting PD-1, in some embodiments, the individual has a high expression of one or more biomarkers selected from the group consisting of CD80, CD83, CD86, and HLA-II class antigens in tumor-derived mature cells, including but not limited to tumor-specific antigens and tumor-associated antigens expressed in solid or lymphoid tumors, and tumor-associated antigens, e.g., CD 73738, CD 38, CD 9626, CD 36, CD 48, CD-II class antigens expressed in a tumor-derived mature cell, and miR, CD-L-b 2, CD-b-.

The degree of expression of a biomarker can be measured at the nucleic acid level (e.g., gene copy number, DNA methylation or chromatin remodeling levels, mRNA levels), or protein level (including the level of post-translational modification of a protein, such as the level of phosphorylation of a protein corresponding to the biomarker). The degree of expression can be determined using any of the methods known in the art. For example, suitable methods for determining the extent of mRNA expression of a biomarker include, but are not limited to, reverse transcription polymerase chain reaction (RT-PCR), quantitative PCR, microarrays, and RNA sequencing. For example, suitable methods for determining the extent of protein expression of a biomarker include, but are not limited to, immunohistochemistry, western blotting, and mass spectrometry.

The extent of expression of a biomarker can be determined using a fresh or archived sample of the individual (including, but not limited to, solid or lymphoid tumor tissue, normal tissue adjacent to solid or lymphoid tumor tissue, normal tissue remote from solid or lymphoid tumor tissue, or peripheral hemoglobulins). In some embodiments, the sample is solid or lymphoid tumor tissue. In some embodiments, the sample is a biopsy containing tumor cells, such as fine needle aspiration of tumor cells. In some embodiments, prior to analysis, the biopsy cells are centrifuged into a pellet, fixed and embedded in paraffin. In some embodiments, the cells under examination are snap frozen prior to analysis. In some embodiments, the sample is a bodily fluid, such as a blood sample or a plasma sample. In some embodiments, the sample comprises circulating metastatic cancer cells. In some embodiments, the sample is obtained by sorting Circulating Tumor Cells (CTCs) from blood.

In some embodiments, a sample of an individual is used to determine the extent of expression of one or more biomarkers in a specific cell population of the individual. In some embodiments, the sample comprises immune cells isolated from or derived from a solid or lymphoid tumor. Exemplary immune cells relevant to biomarker expression assays include, but are not limited to, dendritic cells (e.g., immature or mature dendritic cells), B cells, T cells (e.g., Th1 cells, Th2 cells, Th17 cells, NK T cells, Treg cells, etc.), Natural Killer (NK) cells, monocytes, macrophages, neutrophils, and combinations thereof. In some embodiments, the sample comprises tumor-infiltrating lymphocytes. In some embodiments, the sample comprises mature dendritic cells derived from a tumor. Specific cell populations can be isolated from a test sample, such as a tumor sample (e.g., a tumor biopsy or resection) or a bodily fluid (e.g., a blood sample), using methods known in the art, such as flow cytometry methods, based on the expression of specific cell surface molecules in the cell population.

High or low expression levels of the biomarker are determined as compared to a standard level of expression of the biomarker known in the art (e.g., a clinically accepted normal level in a standardized test) or as compared to the level of expression of the biomarker in a control sample. In some embodiments, the degree of expression of the biomarker in the individual is compared to the degree of expression of the biomarker in a plurality of control samples. In some embodiments, a plurality of control samples are used to generate statistics for classifying the level of a biomarker in an individual having a solid or lymphoid tumor. The control sample can be obtained from the same source (e.g., individuals and tissues) and method as the non-control sample. In some embodiments, control samples are obtained from different individuals (e.g., individuals without solid or lymphoid tumors; individuals with benign or non-and late forms of solid or lymphoid tumors; and/or individuals sharing similar ethnic groups, ages, and sexes). In some embodiments, the control sample is cultured tissue or cells that are determined to be an appropriate control. In some embodiments where the sample is a solid or lymphoid tumor tissue sample, the control sample may be a non-cancerous sample from the same individual. In some embodiments, multiple control samples (e.g., from different individuals) are used to determine the range of levels of a biomarker in a particular tissue, organ, or cell population. In some embodiments, the degree of expression of a biomarker in a sample of an individual is classified as high, medium, or low according to a scoring system (e.g., an immunohistochemistry-based scoring system). In some embodiments, the high expression of the biomarker in the test sample of the individual as compared to the control test sample is at least about any of the following: 1.5 times, 2 times, 3 times, 5 times, 10 times, 20 times, 50 times, 100 times, 200 times, 500 times, 1000 times or more. In some embodiments, low expression of a biomarker as compared to a control sample is no more than about any of the following for the degree of expression of the biomarker in the individual's sample: 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01%, 0.001% or less. In some embodiments, the degree of expression of two or more biomarkers is combined, e.g., using a statistical model, to determine an expression score for selecting or recommending an individual for treatment.

Treatment of bladder by intravesical administrationMethod of cancer

One aspect of the present patent application relates to the treatment of bladder cancer. In this context, topical administration of the infectious agent and the immunomodulator (including the combination of immunomodulators) may encompass intravesical administration of one or both components. Any of the methods described herein can be used to inhibit bladder tumor growth, inhibit bladder tumor metastasis, prolong survival (e.g., disease-free survival) of a subject with bladder cancer, cause remission of a disease in a subject with bladder cancer, and/or improve quality of life of a subject with bladder cancer.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of an infectious agent; and b) intravesically administering an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, the infectious agent is a virus, for example a virus selected from the group consisting of: adenovirus, herpes simplex virus, vaccinia virus, mumps virus, newcastle disease virus, poliovirus, measles virus, seneca valley virus, coxsackie virus, rio virus, vesicular stomatitis virus, malaba and rhabdovirus, and parvovirus. In some embodiments, the infectious agent is a bacterium, such as a mycobacterium and derivatives thereof (e.g., bacillus calmette-guerin ("BCG"), or mycobacterium cell wall-DNA complex ("MCNA" or "MCC"), such as UROCIDIN^TM) Or listeria monocytogenes. In some embodiments, the infectious agent is a wild-type infectious agent. In some embodiments, the infectious agent is genetically modified. In some embodiments, the infectious agent is attenuated (e.g., via multiple passages, inactivation, or genetic modification). In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the methods comprise the local administration of a combination of immune modulators comprising one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant)In combination). In some embodiments, the infectious agent is administered weekly. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered weekly. In some embodiments, the method further comprises administering the infectious agent and/or immunomodulator (including combination of immunomodulators) by a route of administration other than intravesical administration.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus); and b) intravesically administering an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, the oncolytic virus is a wild-type oncolytic virus. In some embodiments, the oncolytic virus is genetically modified. In some embodiments, the oncolytic virus is attenuated (e.g., via multiple passages, inactivation, or genetic modification). In some embodiments, the oncolytic virus is competent for replication. In some embodiments, the oncolytic virus preferentially replicates in cancer cells. In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the methods comprise locally administering a combination of immune modulators comprising one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the oncolytic virus is administered weekly. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered weekly. In some embodiments, the method further comprises administering the oncolytic virus and/or an immunomodulator (including a combination of immunomodulators) by a route of administration other than intravesical administration.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication; and b) intravesically administering an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the methods comprise locally administering a combination of immune modulators comprising one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4. In some embodiments, the oncolytic virus is administered weekly. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered weekly. In some embodiments, the method further comprises administering the oncolytic virus and/or an immunomodulator (including a combination of immunomodulators) by a route of administration other than intravesical administration.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication and a nucleic acid encoding an interleukin operably linked to the viral promoter; and b) intravesically administering an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the methods comprise locally administering a combination of immune modulators comprising one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4. In some embodiments, the viral promoter operably linked to a nucleic acid encoding an interleukin is the E3 promoter. In some embodiments, the interleukin is GM-CSF. In some embodiments, the oncolytic virus is administered weekly. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered weekly. In some embodiments, the method further comprises administering the oncolytic virus and/or an immunomodulator (including a combination of immunomodulators) by a route of administration other than intravesical administration.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of adenovirus serotype 5, wherein the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced with the human E2F-1 promoter and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine); and b) intravesically administering an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the methods comprise locally administering a combination of immune modulators comprising one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the tumor specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the adenovirus is administered weekly. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered weekly. In some embodiments, the method further comprises administering the adenovirus and/or the immunomodulator (including the combination of immunomodulators) by a route of administration other than intravesical administration.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of CG 0070; and b) intravesically administering an effective amount of an immunomodulator (including combinations of immunomodulators). In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the methods comprise locally administering a combination of immune modulators comprising one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, CG0070 is administered weekly. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered weekly. In some embodiments, the method further comprises administering CG0070 and/or an immunomodulatory agent (including a combination of immunomodulatory agents) by a route of administration other than intravesical administration.

The methods described herein can be used to treat a variety of bladder cancer conditions. In some embodiments, the bladder cancer is low grade bladder cancer. In some embodiments, the bladder cancer is a high grade bladder cancer. In some embodiments, the bladder cancer is muscle invasive (e.g., T2, T3, or T4). In some embodiments, the bladder cancer is non-invasive (e.g., Ta, T1Cis, Cis and Ta, and/or T1).

In some embodiments, the bladder cancer is transitional cell carcinoma or urothelial cancer (e.g., metastatic urothelial cancer), including but not limited to papillary tumors and squamous carcinomas. In some embodiments, the bladder cancer is metastatic urothelial cancer. In some embodiments, the bladder cancer is urothelial cancer of the bladder. In some embodiments, the bladder cancer is urothelial cancer of the ureter. In some embodiments, the bladder cancer is urothelial cancer of the urethra. In some embodiments, the bladder cancer is urothelial cancer of the renal pelvis.

In some embodiments, the bladder cancer is squamous cell carcinoma. In some embodiments, the bladder cancer is a non-squamous cell carcinoma. In some embodiments, the bladder cancer is adenocarcinoma. In some embodiments, the bladder cancer is small cell carcinoma.

In some embodiments, the bladder cancer is early stage bladder cancer, non-metastatic bladder cancer, non-invasive bladder cancer, non-muscle invasive bladder cancer, primary bladder cancer, advanced bladder cancer, locally advanced bladder cancer (e.g., unresectable locally advanced bladder cancer), metastatic bladder cancer, or remitting bladder cancer. In some embodiments, the bladder cancer is locally resectable, locally unresectable, or unresectable. In some embodiments, the bladder cancer is a high-grade non-muscle invasive cancer refractory to standard intravesical infusion (intravesical) therapy.

The methods provided herein can be used to treat an individual (e.g., a human) diagnosed with or suspected of having bladder cancer. In some embodiments, the individual undergoes a tumor resection. In some embodiments, the subject refuses surgery. In some embodiments, the individual is medically inoperable. In some embodiments, the subject is in a clinical stage of Ta, Tis, T1, T2, T3a, T3b, or T4 bladder cancer. In some embodiments, the subject is in a clinical stage of Tis, CIS, Ta, or T1.

in some embodiments, the individual was previously treated for bladder cancer (also referred to as "prior therapy"). in some embodiments, the individual was previously treated with standard therapy for bladder cancer.

In some embodiments, the individual has recurrent bladder cancer (e.g., bladder cancer at the clinical stage of Ta, Tis, T1, T2, T3a, T3b, or T4) following a prior therapy (e.g., prior standard therapy, e.g., treatment with BCG). For example, the subject may initially respond to treatment with a prior therapy, but develop bladder cancer after about any of about 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 24, 36, 48, or 60 months after cessation of the prior therapy.

Any of the immunomodulatory agents described herein (including immunostimulants and immune checkpoint inhibitors) may be used in combination therapy for intravesical administration. The immunomodulator may be any of the molecular modalities known in the art, including but not limited to aptamers, mRNA, siRNA, microrna, shRNA, peptides, antibodies, anti-transporters, spherical nucleic acids, TALENs, zinc finger nucleases, CRISPR/Cas9, and small molecules.

In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the immunostimulatory agent is a natural or engineered ligand for an immunostimulatory molecule, including, for example, a ligand for OX40 (e.g., OX40L), a ligand for CD28 (e.g., CD80, CD86), a ligand for ICOS (e.g., B7RP1), a ligand for 4-1BB (e.g., 4-1BBL, Ultra4-1BBL), a ligand for CD27 (e.g., CD70), a ligand for CD40 (e.g., CD40L), and a ligand for a TCR (e.g., an MHC class I or class II molecule, IMCgp 100). In some embodiments, the immunostimulatory agent is an antibody selected from the group consisting of: anti-CD 28 (e.g., TGN-1412), anti-OX 40 (e.g., MEDI6469, MEDI-0562), anti-ICOS (e.g., MEDI-570), anti-GITR (e.g., TRX518, INBRX-110, NOV-120301), anti-41-BB (e.g., BMS-663513, PF-05082566), anti-CD 27 (e.g., BION-1402, Wallimumab, and hCD27.15), anti-CD 40 (e.g., CP870,893, BI-655064, BMS-986090, APX005M), anti-CD 3 (e.g., brilimumab, Moluomab), and anti-HVEM. In some embodiments, the antibody is agonisticA motile antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an antigen binding fragment selected from the group consisting of: fab, Fab ', F (ab') of full-length antibody₂Fv, scFv, and other antigen binding subsequences. In some embodiments, the antibody is a human, humanized or chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other functional variant or derivative thereof.

in some embodiments, the immune checkpoint inhibitor is a natural or engineered ligand of an inhibitory immune checkpoint molecule, including, for example, ligands of CTLA-4 (e.g., B7.1, B7.2), ligands of TIM3 (e.g., galectin-9), ligands of the A2a receptor (e.g., adenosine, Regadenoson), ligands of LAG3 (e.g., MHC class I or MHC class II molecules), ligands of BTLA (e.g., HVEM, B7-H4), ligands of KIR (e.g., MHC class I or MHC class II molecules), ligands of PD-1 (e.g., PD-L1, PD-L2), ligands of IDO (e.g., NKTR-218, indomethamine, NLG919) and ligands of CD 39p-alpha receptor in some embodiments, the immune inhibitor is an anti-checkpoint inhibitor of HDAC receptor binding domain III receptor, such as anti-MAPPA-III ligand of HDAC receptor, HDAC-NO-III, (E) receptor of HDAC receptor, HDAC receptor-NO-1, NO-7, anti-NO-III, (E) and anti-III) ligand of anti-5, such as anti-IgG-VEGF-5, anti-103, anti-VEGF-5, anti-VEGF-IgG-5, anti-VEGF-103, anti-VEGF-103, anti-VEGF-IgG-VEGF-103, anti-VEGF-103, anti-VEGF-IgG-103, anti-VEGF-103, anti-VEGF-103, anti-VEGF-103, anti-VEGF-III-VEGF-103, anti-VEGF-III, anti-103, anti-VEGF-103, anti-VEGF-III, anti-VEGF-HDAC-42, HDAC42, NSCD736012, NSC-D736012), MEDI-9447), anti-B7-H3 (e.g., MGA271, DS-5573a, 8H9), anti-CD 47 (e.g., CC-90002, TTI-621, VLST-007), anti-BTLA, anti-VISTA, anti-A2 aR, anti-B7-1, anti-B7-H4, anti-CD 52 (e.g., alemtuzumab), anti-IL-10, anti-IL-35, and anti-TGF- β (e.g., fresolimumab)₂Fv, scFv, and other antigen binding subsequences. In some embodiments, the antibody is a human, humanized or chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other functional variant or derivative thereof.

In some embodiments, the method comprises intravesically administering a single immunomodulator. In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immunomodulatory agent is an immunostimulatory agent.

In some embodiments, the method comprises intravesically administering at least two (e.g., any one of 2, 3, 4, 5, 6, or more) immunomodulators. In some embodiments, all or a portion of at least two immunomodulators are administered simultaneously, e.g., in a single composition. In some embodiments, all or a portion of at least two immunomodulators are administered sequentially. In some embodiments, the method comprises intravesically administering a combination of immunomodulators comprising an immune checkpoint inhibitor and an immune stimulant. In some embodiments, the method comprises intravesically administering a combination of immunomodulators comprising two or more (e.g., any of 2, 3, 4, 5, 6 or more) checkpoint inhibitors. In some embodiments, the method comprises intravesically administering a combination of immunomodulatory agents comprising two or more (e.g., any of 2, 3, 4, 5, 6, or more) immunostimulatory agents. In some embodiments, the method comprises intravesically administering a combination of an immune checkpoint inhibitor comprising any number (e.g., any of 1,2, 3, 4, 5, 6, or more) of immune checkpoint inhibitors and any number (e.g., any of 2, 3, 4, 5, 6, or more) of immune modulators. For example, in some embodiments, the method comprises: a) intravesically administering to the tumor site an effective amount of an infectious agent (e.g., a virus, such as an oncolytic virus); and b) intravesically administering to the individual an effective amount of a first immune modulator (e.g., an immune checkpoint inhibitor); and c) intravesically administering to the tumor site an effective amount of a second immunomodulator (e.g., an immunostimulant). In some embodiments, the methods comprise intravesically administering a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab), or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)) and a CD40 agonist (e.g., an agonistic anti-CD 40 antibody, e.g., APX 005M). In some embodiments, the methods comprise intravesically administering a CTLA-4 inhibitor (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab), or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)) and a4-1BB agonist (e.g., an agonistic anti-4-1 BB antibody, e.g., PF-05082566).

Thus, for example, in some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of an infectious agent; and b) intravesically administering an effective amount of an inhibitor of CTLA-4 (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)). In some embodiments, the infectious agent is a non-oncolytic virus. In some embodiments, the infectious agent is an oncolytic virus. In some embodiments, the infectious agent is a wild-type infectious agent. In some embodiments, the infectious agent is genetically modified. In some embodiments, the infectious agent is attenuated (e.g., via multiple passages, inactivation, or genetic modification). In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody (e.g., ipilimumab). In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin, e.g., an anti-transporter that specifically recognizes CTLA-4. In some embodiments, the method further comprises intravesically administering a second immunomodulator, such as an immunostimulant (e.g., a CD40 activator or a4-1BB activator). In some embodiments, the infectious agent is administered weekly. In some embodiments, the inhibitor of CTLA-4 is administered weekly. In some embodiments, the infectious agent and the inhibitor of CTLA-4 are administered sequentially. In some embodiments, the infectious agent is administered prior to (e.g., immediately prior to) administration of the inhibitor of CTLA-4. In some embodiments, the infectious agent is administered after (e.g., immediately after) administration of the inhibitor of CTLA-4. In some embodiments, the infectious agent and the inhibitor of CTLA-4 are administered simultaneously (e.g., in a single composition). In some embodiments, the method further comprises administering the agent and/or the inhibitor of CTLA-4 by a route of administration other than intravesical administration.

For example, in some embodiments, there is provided a method of treating bladder cancer in an individual comprising: a) intravesically administering an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus); and b) intravesically administering an effective amount of an inhibitor of CTLA-4 (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)).

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication; and b) intravesically administering an effective amount of an inhibitor of CTLA-4 (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine) operably linked to the viral promoter; and b) intravesically administering an effective amount of an inhibitor of CTLA-4 (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4. In some embodiments, the viral promoter operably linked to the nucleic acid encoding the immune-related molecule is the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of adenovirus serotype 5, wherein the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced with the human E2F-1 promoter and a nucleic acid encoding an immune-related molecule (e.g., an interleukin, a chemokine, such as GM-CSF); and b) intravesically administering an effective amount of an inhibitor of CTLA-4 (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)). In some embodiments, the tumor specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of CG 0070; and b) intravesically administering an effective amount of an inhibitor of CTLA-4 (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)). In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody (e.g., ipilimumab). In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin, e.g., an anti-transporter that specifically recognizes CTLA-4. In some embodiments, CG007 is administered weekly. In some embodiments, the inhibitor of CTLA-4 is administered weekly. In some embodiments, the inhibitor of CG0070 and CTLA-4 is administered sequentially. In some embodiments, the CG0070 is administered prior to (e.g., immediately prior to) administration of the inhibitor of CTLA-4. In some embodiments, the CG0070 is administered after (e.g., immediately after) administration of the inhibitor of CTLA-4. In some embodiments, the inhibitor of CG0070 and CTLA-4 are administered concurrently (e.g., in a single composition). In some embodiments, the method further comprises administering an inhibitor of CG0070 and/or CTLA-4 by a route of administration other than intravesical administration.

In some embodiments, there is provided a method of treating bladder cancer (e.g., muscle invasive bladder cancer) in an individual, the method comprising: a) intravesically administering an effective amount of CG 0070; and b) intravesically administering an effective amount of an inhibitor of CTLA-4 (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)), wherein the effective amount of CG0070 is about 1 x 10 per week¹²Individual virus particles (vp) wherein the effective amount of the inhibitor of CTLA-4 is about 0.1mg/Kg, but does not exceed a total of 20mg per dose per week. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody (e.g., ipilimumab) (e.g.,). In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin, e.g., an anti-transporter that specifically recognizes CTLA-4. In some embodiments, the method further comprises a pretreatment comprising intravesically administering an effective amount of a transduction enhancing agent prior to (e.g., immediately prior to or no earlier than 2 hours prior to) administration of CG 0070. In some embodiments, the transduction enhancer is DDM. In some embodimentsIn a case, CG0070 is administered within about four weeks. In some embodiments, the inhibitor of CTLA-4 is administered within about three weeks, e.g., in 2, 3, and 4 weeks of the four week course of CG0070 administration. In some embodiments, the inhibitor of CTLA-4 is administered immediately after (e.g., no more than 5 minutes after) administration of CG 0070. In some embodiments, the subject further receives cystectomy or pelvic lymphadenectomy. In some embodiments, the muscle invasive bladder cancer is transitional cell (i.e., urinary tract) bladder cancer. In some embodiments, MIBC is stage T2-4a, Nx-1, M0 according to the United states Joint Committee for cancer (AJCC) criteria.

In some embodiments, there is provided a method of treating bladder cancer (e.g., muscle invasive bladder cancer) in an individual, the method comprising: a) intravesically administering an effective amount of CG 0070; and b) intravesically administering an effective amount of an inhibitor of CTLA-4 (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)), wherein the effective amount of CG0070 is about 1 x 10 per week¹²Individual virus particles (vp) wherein the effective amount of the inhibitor of CTLA-4 is about 0.2mg/Kg, but does not exceed a total of 20mg per dose per week. In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin, e.g., an anti-transporter that specifically recognizes CTLA-4. In some embodiments, the method further comprises a pretreatment comprising intravesically administering an effective amount of a transduction enhancing agent prior to (e.g., immediately prior to or no earlier than 2 hours prior to) administration of CG 0070. In some embodiments, the transduction enhancer is DDM. In some embodiments, the method further comprises intravesically administering a second immunomodulator, such as an immunostimulant (e.g., a CD40 activator or a4-1BB activator). In some embodiments, CG0070 is administered within about four weeks. In some embodiments, the inhibitor of CTLA-4 is administered within about three weeks, e.g., in 2, 3, and 4 weeks of the four week course of CG0070 administration. In some embodiments, the inhibitor of CTLA-4 is administered immediately after (e.g., no more than 5 minutes after) administration of CG 0070. In some embodiments, the subject further receives cystectomy or pelvic lymph nodeAnd (4) excision. In some embodiments, the muscle invasive bladder cancer is transitional cell (i.e., urinary tract) bladder cancer. In some embodiments, MIBC is stage T2-4a, Nx-1, M0 according to the United states Joint Committee for cancer (AJCC) criteria.

In some embodiments, there is provided a method of treating bladder cancer (e.g., muscle invasive bladder cancer) in an individual, the method comprising: a) intravesically administering an effective amount of CG 0070; and b) intravesically administering an effective amount of an inhibitor of CTLA-4 (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)), wherein the effective amount of CG0070 is about 1 x 10 per week¹²Individual virus particles (vp) wherein the effective amount of the inhibitor of CTLA-4 is about 0.3mg/Kg, but does not exceed a total of 20mg per dose per week. In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin, e.g., an anti-transporter that specifically recognizes CTLA-4. In some embodiments, the method further comprises a pretreatment comprising intravesically administering an effective amount of a transduction enhancing agent prior to (e.g., immediately prior to or no earlier than 2 hours prior to) administration of CG 0070. In some embodiments, the transduction enhancer is DDM. In some embodiments, the method further comprises intravesically administering a second immunomodulator, such as an immunostimulant (e.g., a CD40 activator or a4-1BB activator). In some embodiments, CG0070 is administered within about four weeks. In some embodiments, the inhibitor of CTLA-4 is administered within about three weeks, e.g., in 2, 3, and 4 weeks of the four week course of CG0070 administration. In some embodiments, the inhibitor of CTLA-4 is administered immediately after (e.g., no more than 5 minutes after) administration of CG 0070. In some embodiments, the subject further receives cystectomy or pelvic lymphadenectomy. In some embodiments, the muscle invasive bladder cancer is transitional cell (i.e., urinary tract) bladder cancer. In some embodiments, MIBC is stage T2-4a, Nx-1, M0 according to the United states Joint Committee for cancer (AJCC) criteria.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of an infectious agent; and b) intravesically administering an effective amount of a PD-1 inhibitor (e.g., an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, or pidilizumab) or an Fc fusion protein of a PD-1 ligand (e.g., AMP-224)). In some embodiments, the infectious agent is a wild-type infectious agent. In some embodiments, the infectious agent is genetically modified. In some embodiments, the infectious agent is attenuated (e.g., via multiple passages, inactivation, or genetic modification). In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody, e.g., nivolumab, pembrolizumab, or pidilizumab. In some embodiments, the PD-1 inhibitor is an inhibitor of the interaction between PD-1 and its ligand, for example, an inhibitor of the PD-1/PD-L1 interaction or an inhibitor of the PD-1/PD-L2 interaction. In some embodiments, the PD-1 inhibitor is an Fc fusion protein comprising a PD-1 ligand, such as an Fc-fusion of PD-L2 (e.g., AMP-224). In some embodiments, the method further comprises intravesically administering a second immunomodulator, such as an immunostimulant (e.g., a CD40 activator or a4-1BB activator). In some embodiments, the infectious agent is administered weekly. In some embodiments, the PD-1 inhibitor is administered weekly. In some embodiments, the infectious agent and the PD-1 inhibitor are administered sequentially. In some embodiments, the infectious agent is administered prior to (e.g., immediately prior to) administration of the PD-1 inhibitor. In some embodiments, the infectious agent is administered after (e.g., immediately after) the administration of the PD-1 inhibitor. In some embodiments, the infectious agent and the PD-1 inhibitor are administered simultaneously (e.g., in a single composition). In some embodiments, the method further comprises administering the infectious agent and/or the PD-1 inhibitor by a route of administration other than intravesical administration.

For example, in some embodiments, there is provided a method of treating bladder cancer in an individual comprising: a) intravesically administering an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus); and b) intravesically administering an effective amount of a PD-1 inhibitor (e.g., an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, or pidilizumab) or an Fc fusion protein of a PD-1 ligand (e.g., AMP-224)).

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication; and b) intravesically administering an effective amount of a PD-1 inhibitor (e.g., an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, or pidilizumab) or an Fc fusion protein of a PD-1 ligand (e.g., AMP-224)). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine) operably linked to the viral promoter; and b) intravesically administering an effective amount of a PD-1 inhibitor (e.g., an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, or pidilizumab) or an Fc fusion protein of a PD-1 ligand (e.g., AMP-224)). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4. In some embodiments, the viral promoter operably linked to the nucleic acid encoding the immune-related molecule is the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of adenovirus serotype 5, wherein the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced with the human E2F-1 promoter and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or chemokine, such as GM-CSF); and b) intravesically administering an effective amount of a PD-1 inhibitor (e.g., an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, or pidilizumab), or an Fc fusion protein of a PD-1 ligand (e.g., AMP-224)). In some embodiments, the tumor specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of CG 0070; and b) intravesically administering an effective amount of a PD-1 inhibitor (e.g., an anti-PD-1 antibody (e.g., nivolumab, pembrolizumab, or pidilizumab) or an Fc fusion protein of a PD-1 ligand (e.g., AMP-224)). In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody, e.g., nivolumab, pembrolizumab, or pidilizumab. In some embodiments, the PD-1 inhibitor is an inhibitor of the interaction between PD-1 and its ligand, for example, an inhibitor of the PD-1/PD-L1 interaction or an inhibitor of the PD-1/PD-L2 interaction. In some embodiments, the PD-1 inhibitor is an Fc fusion protein comprising a PD-1 ligand, such as an Fc-fusion of PD-L2 (e.g., AMP-224). In some embodiments, CG007 is administered weekly. In some embodiments, the PD-1 inhibitor is administered weekly. In some embodiments, the CG0070 and the PD-1 inhibitor are administered sequentially. In some embodiments, CG0070 is administered prior to (e.g., immediately prior to) administration of the PD-1 inhibitor. In some embodiments, CG0070 is administered after (e.g., immediately after) administration of the PD-1 inhibitor. In some embodiments, the CG0070 and the PD-1 inhibitor are administered simultaneously (e.g., in a single composition). In some embodiments, the method further comprises administering the CG0070 and/or PD-1 inhibitor via a route of administration other than intravesical administration.

In some embodiments, there is provided a method of treating bladder cancer in a subject (e.g., a human), comprising: a) intravesically administering an effective amount of an infectious agent; and b) intravesically administering an effective amount of an inhibitor of a PD-1 ligand (e.g., an inhibitor of anti-PD-L1 or anti-PD-L2 antibody, or both PD-L1 and PD-L2). In some embodiments, the infectious agent is a wild-type infectious agent. In some embodiments, the infectious agent is genetically modified. In some embodiments, the infectious agent is attenuated (e.g., via multiple passages, inactivation, or genetic modification). In some embodiments, the inhibitor of a PD-1 ligand is an anti-PD-L1 antibody, such as KY-1003, MCLA-145, RG7446, BMS935559, MPDL3280A, MEDI4736, avizumab, or STI-A1010. In some embodiments, the inhibitor of a PD-1 ligand is an anti-PD-L2 antibody. In some embodiments, the inhibitor of a PD-1 ligand is an inhibitor (e.g., a peptide, protein, or small molecule) of both PD-L1 and PD-L2, such as AUR-012 and AMP-224. In some embodiments, the method further comprises intravesically administering a second immunomodulator, such as an immunostimulant (e.g., a CD40 activator or a4-1BB activator). In some embodiments, the infectious agent is administered weekly. In some embodiments, the inhibitor of PD-1 ligand is administered weekly. In some embodiments, the infectious agent and the inhibitor of the PD-1 ligand are administered sequentially. In some embodiments, the infectious agent is administered prior to (e.g., immediately prior to) administration of the inhibitor of PD-1 ligand. In some embodiments, the infectious agent is administered after (e.g., immediately after) administration of the inhibitor of the PD-1 ligand. In some embodiments, the infectious agent and the inhibitor of the PD-1 ligand are administered simultaneously (e.g., in a single composition). In some embodiments, the method further comprises administering an inhibitor of the infectious agent and/or the PD-1 ligand by a route of administration other than intravesical administration. In some embodiments, an inhibitor of PD-L1 and an inhibitor of PD-L2 are used interchangeably in any of the methods of treatment described herein.

For example, in some embodiments, there is provided a method of treating bladder cancer in an individual comprising: a) intravesically administering an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus); and b) intravesically administering an effective amount of an inhibitor of a PD-1 ligand (e.g., an inhibitor of anti-PD-L1 or anti-PD-L2 antibody, or both PD-L1 and PD-L2).

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication; and b) intravesically administering an effective amount of an inhibitor of a PD-1 ligand (e.g., an inhibitor of anti-PD-L1 or anti-PD-L2 antibody, or both PD-L1 and PD-L2). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine) operably linked to the viral promoter; and b) intravesically administering an effective amount of an inhibitor of a PD-1 ligand (e.g., an inhibitor of anti-PD-L1 or anti-PD-L2 antibody, or both PD-L1 and PD-L2). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4. In some embodiments, the viral promoter operably linked to the nucleic acid encoding the immune-related molecule is the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of adenovirus serotype 5, wherein the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced with the human E2F-1 promoter and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or chemokine, such as GM-CSF); and b) intravesically administering an effective amount of an inhibitor of a PD-1 ligand (e.g., an inhibitor of anti-PD-L1 or anti-PD-L2 antibody, or both PD-L1 and PD-L2). In some embodiments, the tumor specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of CG 0070; and b) intravesically administering an effective amount of an inhibitor of a PD-1 ligand (e.g., an inhibitor of anti-PD-L1 or anti-PD-L2 antibody, or both PD-L1 and PD-L2). In some embodiments, the inhibitor of a PD-1 ligand is an anti-PD-L1 antibody, such as KY-1003, MCLA-145, RG7446, BMS935559, MPDL3280A, MEDI4736, avizumab, or STI-A1010. In some embodiments, the inhibitor of a PD-1 ligand is an anti-PD-L2 antibody. In some embodiments, the inhibitor of a PD-1 ligand is an inhibitor (e.g., a peptide, protein, or small molecule) of both PD-L1 and PD-L2, such as AUR-012 and AMP-224. In some embodiments, CG007 is administered weekly. In some embodiments, the inhibitor of PD-1 ligand is administered weekly. In some embodiments, the inhibitor of CG0070 and PD-1 ligand is administered sequentially. In some embodiments, CG0070 is administered prior to (e.g., immediately prior to) administration of the inhibitor of PD-1 ligand. In some embodiments, CG0070 is administered after (e.g., immediately after) administration of the inhibitor of the PD-1 ligand. In some embodiments, the inhibitor of the CG0070 and PD-1 ligand is administered simultaneously (e.g., in a single composition). In some embodiments, the method further comprises administering an inhibitor of CG0070 and/or PD-1 ligand by a route of administration other than intravesical administration.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of an infectious agent; and b) intravesically administering an effective amount of an activator of CD40 (e.g., an agonistic anti-CD 40 antibody, e.g., CP-870,893, daclizumab, ChiLob 7/4, or APX 005M). In some embodiments, the infectious agent is a wild-type infectious agent. In some embodiments, the infectious agent is genetically modified. In some embodiments, the infectious agent is attenuated (e.g., via multiple passages, inactivation, or genetic modification). In some embodiments, the activator of CD40 is an agonistic anti-CD 40 antibody, such as CP-870,893, daclizumab, ChiLob 7/4, or APX 005M. In some embodiments, the method further comprises intravesically administering a second immune modulator, e.g., an immune checkpoint inhibitor (e.g., an inhibitor of CTLA-4, e.g., an anti-CTLA-4 antibody or an anti-transporter that specifically binds to CTLA-4). In some embodiments, the infectious agent is administered weekly. In some embodiments, the activator of CD40 is administered weekly. In some embodiments, the infectious agent and the activator of CD40 are administered sequentially. In some embodiments, the infectious agent is administered prior to (e.g., immediately prior to) administration of the activator of CD 40. In some embodiments, the infectious agent is administered after (e.g., immediately after) administration of the activator of CD 40. In some embodiments, the infectious agent and the activator of CD40 are administered simultaneously (e.g., in a single composition). In some embodiments, the method further comprises administering the infectious agent and/or the activator of CD40 by a route of administration other than intravesical administration.

For example, in some embodiments, there is provided a method of treating bladder cancer in an individual comprising: a) intravesically administering an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus); and b) intravesically administering an effective amount of an activator of CD40 (e.g., an agonistic anti-CD 40 antibody, e.g., CP-870,893, daclizumab, ChiLob 7/4, or APX 005M).

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication; and b) intravesically administering an effective amount of an activator of CD40 (e.g., an agonistic anti-CD 40 antibody, e.g., CP-870,893, daclizumab, ChiLob 7/4, or APX 005M). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine) operably linked to the viral promoter; and b) intravesically administering an effective amount of an activator of CD40 (e.g., an agonistic anti-CD 40 antibody, e.g., CP-870,893, daclizumab, ChiLob 7/4, or APX 005M). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4. In some embodiments, the viral promoter operably linked to the nucleic acid encoding the immune-related molecule is the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of adenovirus serotype 5, wherein the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced with the human E2F-1 promoter and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or chemokine, such as GM-CSF); and b) intravesically administering an effective amount of an activator of CD40 (e.g., an agonistic anti-CD 40 antibody, e.g., CP-870,893, daclizumab, ChiLob 7/4, or APX 005M). In some embodiments, the tumor specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

In some embodiments, there is provided a method of treating bladder cancer in an individual, the method comprising: a) intravesically administering an effective amount of CG 0070; and b) intravesically administering an effective amount of an activator of CD40 (e.g., an agonistic anti-CD 40 antibody, e.g., CP-870,893, daclizumab, ChiLob 7/4, or APX 005M). In some embodiments, the activator of CD40 is an agonistic anti-CD 40 antibody, such as CP-870,893, daclizumab, ChiLob 7/4, or APX 005M. In some embodiments, CG007 is administered weekly. In some embodiments, the activator of CD40 is administered weekly. In some embodiments, the activators of CG0070 and CD40 are administered sequentially. In some embodiments, CG0070 is administered prior to (e.g., immediately prior to) administration of the activator of CD 40. In some embodiments, CG0070 is administered after (e.g., immediately after) administration of the activator of CD 40. In some embodiments, the activators of CG0070 and CD40 are administered simultaneously (e.g., in a single composition). In some embodiments, the method further comprises administering an activator of CG0070 and/or CD40 by a route of administration other than intravesical administration.

Intravesical administration of an infectious agent and/or immunomodulator (including combinations of immunomodulators) provides a unique opportunity for relatively convenient but effective exposure of an intravesical tumor to the infectious agent and/or immunomodulator (including combinations of immunomodulators), as well as potential reduced toxicity to other tissues. Suitable dosages and dosing frequency of the infectious agent and the immunomodulator (including the combination of immunomodulators) are within the same ranges as described for topical administration of the infectious agent and immunomodulator (including the combination of immunomodulators), respectively, in the preceding section.

In some embodiments, the infectious agent and/or immunomodulator (including combination of immunomodulators) is administered via a catheter in solution by instillation. In some embodiments, the total volume of the solution for intravesical instillation is about any of: 1mL, 10mL, 50mL, 75mL, 100mL, 125mL, 150mL, 200mL, 250mL, 300mL, 400mL, or 500 mL. In some embodiments, the total volume of the solution for intravesical instillation is any one of: about 1mL to about 10mL, about 10mL to about 50mL, about 50mL to about 75mL, about 75mL to about 100mL, about 100mL to about 125mL, about 75mL to about 125mL, about 100mL to about 150mL, about 150mL to about 200mL, about 200mL to about 300mL, about 300mL to about 400mL, about 400mL to about 500mL, about 50mL to about 250mL, or about 100mL to about 250 mL.

In some embodiments, the infectious agent is at about 1 × 10⁸To about 1X 10¹⁵A particle (e.g. about 1X 10)¹¹To about 1X 10¹⁴Particles, e.g. about 1X 10¹²Individual particles). In some embodiments, the infectious agent is administered by instillation in a volume of about 50 to about 500mL (e.g., about 100 mL).

In some embodiments, the immunomodulator (including combinations of immunomodulators) is administered at a dose of from about 0.1mg/Kg to about 100mg/Kg (e.g., from about 0.1mg/Kg to about 0.3mg/Kg, from about 0.1mg/Kg to about 0.5mg/Kg, from about 0.5mg/Kg to about 1mg/Kg, from about 1mg/Kg to about 10mg/Kg, from about 10mg/Kg to about 50mg/Kg, from about 50mg/Kg to about 100mg/Kg, or from about 1mg/Kg to about 100 mg/Kg). In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered at a dose not exceeding about any one of the following per administration: 500mg, 400mg, 300mg, 200mg, 100mg, 80mg, 60mg, 40mg, 20mg or 10 mg. In some embodiments, the immunomodulator (including the combination of immunomodulators) is administered by instillation in a volume of from about 1mL to about 500mL (e.g., about 100 mL).

A solution of the infectious agent and/or immunomodulator (including the combination of immunomodulators) may be retained in the bladder for a specific amount of time prior to voiding to achieve uniform distribution in the bladder tumor cells or sufficient exposure to the infectious agent and/or immunomodulator (including the combination of immunomodulators). In some embodiments, the solution retains at least about any one of the following in the bladder of the subject: 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, or more. In some embodiments, the solution retains any one of the following in the bladder of the subject: from about 5 minutes to about 10 minutes, from about 10 minutes to about 15 minutes, from about 10 minutes to about 20 minutes, from about 20 minutes to about 30 minutes, from about 30 minutes to about 45 minutes, from about 45 minutes to about 50 minutes, from about 50 minutes to about 1 hour, from about 5 minutes to about 15 minutes, from about 10 minutes to about 30 minutes, from about 30 minutes to about 1 hour, or from about 1 hour to about 2 hours. In some embodiments, the infectious agent (e.g., an oncolytic virus, e.g., CG0070) is retained in the bladder of the individual for about 45 minutes to about 50 minutes. In some embodiments, the immunomodulator (including the combination of immunomodulators) is retained in the bladder for about 45 minutes to 1 hour. In some embodiments, the efficiency of intravesical administration of the infectious agent is further enhanced by a pretreatment comprising intravesical administration of an effective amount of a transduction enhancing agent (e.g., DDM).

In some embodiments, the pretreatment step is carried out by contacting the luminal surface of the bladder of the individual with a pretreatment composition followed by administration of an infectious agent and an immunomodulator (including a combination of immunomodulators). For example, the pretreatment composition can include about 0.01% to about 0.5% (e.g., 0.05 to about 0.2%, e.g., about 0.1%) of a transduction enhancer (e.g., DDM). In some embodiments, the total volume of the pretreatment composition (e.g., DDM) is about 10mL to about 1000mL (e.g., about 10mL to about 100mL, about 100mL to about 500mL, or about 500mL to about 1000 mL). In some embodiments, a suitable dose of the pretreatment composition is about any one of 0.1g, 0.2g, 0.5g, 0.75g, 1g, 1.5g, 2g, 2.5g, 5g, or 10g of a transduction enhancer (e.g., DDM). In some embodiments, the effective amount of the pretreatment composition is about 1g of DDM (e.g., 100mL of a 0.1% DDM solution).

In some embodiments, the pretreatment composition (e.g., DDM) is administered immediately prior to (e.g., no more than 5 minutes) administration of the infectious agent. In some embodiments, the pretreatment composition (e.g., DDM) is not more than about any of the following administered prior to administration of the infectious agent: 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 90 minutes, 2 hours, 3 hours, or 4 hours. In some embodiments, the pretreatment composition (e.g., DDM) is administered no more than about 2 hours prior to administration of the infectious agent. In some embodiments, the pretreatment composition (e.g., DDM solution) remains in the bladder for about any of at least 5 minutes, 10 minutes, 15 minutes, or 20 minutes. In some embodiments, the pretreatment composition (e.g., DDM solution) remains in the bladder for any of about 5 minutes to about 10 minutes, about 10 minutes to about 15 minutes, about 12 minutes to about 15 minutes, about 15 minutes to about 20 minutes, or about 10 minutes to about 20 minutes. In some embodiments, the pretreatment composition (e.g., DDM solution) remains in the bladder for about 12 minutes to about 15 minutes.

In some embodiments, the pretreatment step is carried out by contacting the luminal surface of the bladder of the individual with a pretreatment composition followed by administration of an infectious agent and an immunomodulator (including a combination of immunomodulators).

In some embodiments, the method further comprises washing the luminal surface of the bladder contacted with the pretreatment composition. In some embodiments, the method further comprises washing the luminal surface of the bladder after contacting the bladder with the pretreatment composition prior to administering the infectious agent.

In some embodiments, the pretreatment step comprises one or more tumor site preparation steps as described in the methods of treating solid or lymphoid tumors section.

in some embodiments, the pretreatment comprises intravesically administering an effective amount of an immune-related molecule (e.g., an interleukin, a chemokine, or PRRago) in some embodiments, the immune-related molecule is selected from the group consisting of GM-CSF, IL-2, IL12, an interferon (e.g., a type 1, type 2, or type 3 interferon, such as interferon gamma), CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I, MDA5, LGP2, LT α β, a stng activator (e.g., CDN), PRRago (e.g., CpG, imiquimod or poly I: C), a TLR stimulator (e.g-9620, AED-1419, CYT-003-QbG10, AVE-0675, or PRRago-795), and the administration of an immune-related molecule, as a separate or separate, in a transfection-related nucleic acid molecule, or a transfection-associated transfection molecule, or a cell-associated with an immune-associated antigen, or a cell-associated transfection molecule, and/or a cell-transfection molecule, wherein the administration of the immune-associated molecule comprises administering a cell, a cell-transfection molecule, a cell-associated transfection molecule, a cell-associated transfection-expressing, a cell-associated transfection molecule, a cell-associated transfection-expressing, a cell-associated transfection-associated molecule, a cell-associated cell, a cell-.

In some embodiments, the pre-treatment step comprises administering an effective amount of radiation therapy to the bladder of the individual prior to administering the infectious agent and the immunomodulator (including the combination of immunomodulators). In some embodiments, radiation therapy is combined with chemotherapy. In some embodiments, radiation therapy is administered without chemotherapy. In some embodiments, the radiation therapy comprises irradiating the whole body. In some embodiments, the radiation therapy is irradiation of only the tumor site. In some embodiments, the radiation therapy is irradiation of tissue having a tumor. In some embodiments, the radiation therapy is irradiation of only the tumor site selected for local administration of the infectious agent and the immunomodulator. In some embodiments, the radiation therapy is irradiation of only the tissue with the tumor selected for local administration of the infectious agent and the immunomodulator. In some embodiments, the dose of radiation therapy is insufficient to eradicate the tumor cells. For example, a suitable dose of radiation therapy is about any one of: 1Gy, 5Gy, 10Gy, 15Gy, 20Gy, 25Gy, 30Gy, 35Gy, 40Gy, 45Gy, 50Gy, 55Gy, 60Gy, 65Gy, 70Gy, 75Gy, 80Gy, 90Gy, or 100 Gy. In some embodiments, the dose of radiation therapy is no more than about any of the following: 1Gy, 5Gy, 10Gy, 15Gy, 20Gy, 25Gy, 30Gy, 35Gy, 40Gy, 45Gy, 50Gy, 55Gy, 60Gy, 65Gy, 70Gy, 75Gy, 80Gy, 90Gy, or 100 Gy. In some embodiments, the dose of radiation therapy is any one of: about 1Gy to about 5Gy, about 5Gy to about 10Gy, about 10Gy to about 15Gy, about 15Gy to about 20Gy, about 20Gy to about 25Gy, about 25Gy to about 30Gy, about 30Gy to about 35Gy, about 5Gy to about 15Gy, about 10Gy to about 20Gy, about 20Gy to about 30Gy, about 30Gy to about 40Gy, about 40 to about 50Gy, about 50Gy to about 60Gy, about 60Gy to about 70Gy, about 70Gy to about 80Gy, about 80Gy to about 100Gy, about 10 to about 30Gy, about 20Gy to about 40Gy, about 1 to about 25Gy, about 25 to about 50Gy, about 30 to about 60Gy, about 60Gy to about 80Gy, or about 10Gy to about 60 Gy. In some embodiments, radiation therapy is administered in more than one way (e.g., about any of 2, 3, 4, 5, 6, 7,8, 9, 10, 12, 15, 16, 18, 20, or more ways). In some embodiments, the radiation therapy portion is administered over the course of about any one of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or longer. In some embodiments, the radiation therapy portion is administered during the course of any one of: about 1 day to about 5 days, about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 3 weeks to about 4 weeks, about 4 weeks to about 5 weeks, about 5 weeks to about 6 weeks, about 6 weeks to about 7 weeks, about 2 weeks to about 4 weeks, about 4 weeks to about 6 weeks, or about 1 week to about 6 weeks. In some embodiments, the radiation therapy is administered in about two fractions per day. In some embodiments, each fraction of radiation therapy is about 1.8Gy to about 2Gy per day for an adult (five days a week), or about 1.5Gy to about 1.8Gy per day for a child (five days a week). In some embodiments, each portion of radiation therapy is about any one of: 1Gy, 1.5Gy, 2Gy, 2.5Gy, 5Gy, 10Gy, 15Gy, 20Gy, 30Gy, 40Gy, 50Gy, or more. In some embodiments, each portion of radiation therapy is any one of the following: about 1Gy to about 1.5Gy, about 1.5Gy to about 2Gy, about 1Gy to about 2.5Gy, about 2.5Gy to about 5Gy, about 5Gy to about 10Gy, about 10Gy to about 15Gy, about 15Gy to about 20Gy, about 20Gy to about 30Gy, about 25Gy to about 50Gy, about 1Gy to about 10Gy, or about 2Gy to about 20 Gy.

In some embodiments, the radiation therapy is administered in a single fraction. In some embodiments, the radiation therapy is intended for lymphatic clearance, in single dose fractions per day or in multiple fractions over several days to weeks. In some embodiments, the lymphoablative radiation therapy is administered as a systemic irradiation. In some embodiments, lymphatic clearance is only to the local tumor site or tissue with the tumor. In some embodiments, the lymphoablative radiation therapy is administered in two fractions per day. In some embodiments, each fraction of lymphoablative radiation therapy is about 1Gy to about 2Gy per day for adults (five days a week), or about 0.5Gy to about 1.8Gy per day for children (five days a week). In some embodiments, each portion of radiation therapy is about any one of: 1Gy, 1.5Gy, 2Gy, 2.5Gy, 5Gy, 10Gy, 15Gy, 20Gy, 30Gy, 40Gy, 50Gy, or more. In some embodiments, each portion of radiation therapy is any one of the following: about 1Gy to about 1.5Gy, about 1.5Gy to about 2Gy, about 1Gy to about 2.5Gy, about 2.5Gy to about 5Gy, about 5Gy to about 10Gy, about 10Gy to about 15Gy, about 15Gy to about 20Gy, about 20Gy to about 30Gy, about 25Gy to about 50Gy, about 1Gy to about 10Gy, or about 2Gy to about 20 Gy. In some embodiments, lymphoablative radiation therapy is administered with or without chemotherapeutic agents (such as, but not limited to, cyclophosphamide and fludarabine).

Any of the known methods of radiation therapy may be used in the present invention, including, but not limited to, external beam radiation therapy (EBRT or XRT), teletherapy, brachytherapy, sealed source radiation therapy, whole body radioisotope therapy (RIT), unsealed source radiation therapy, intraoperative radiation therapy (IORT), targeted intraoperative radiation therapy (targitt), magnitude-modulated radiation therapy (IMRT), volume-modulated arc therapy (VMAT), particle therapy, and drill therapies.

in some embodiments, the pretreatment step comprises administering to the luminal surface of the bladder of the individual an effective amount of a therapeutic agent directly or indirectly (e.g., via an intravenous route) prior to administration of the infectious agent and an immunomodulatory agent (including a combination of immunomodulatory agents). in some embodiments, the therapeutic agent is any one or combination of chemotherapeutic agents known in the art (e.g., cyclophosphamide). in some embodiments, the therapeutic agent is any one or combination of agents known in the art that target or block a cellular signaling pathway, such as a BRAF inhibitor.

Combination therapy with tumor cells

One aspect of the present application relates to a method of treating a solid or lymphatic tumor in an individual (e.g., a human), the method comprising: a) locally administering to the tumor site an effective amount of an infectious agent; b) locally administering to the tumor site an effective amount of an immunomodulator (including a combination of immunomodulators); and c) locally administering to the tumor site an effective amount of non-activated tumor cells. Such at least three component combination therapy methods may include any of the embodiments of the methods described above for combination therapies comprising an infectious agent and an immunomodulatory agent (including combinations of immunomodulatory agents). The combination therapy methods of the invention comprising non-activated tumor cells are advantageous over other cancer immunotherapy methods involving similar components, since the administration parameters (e.g., dose, frequency of administration, and/or route of administration) for each of the three components (i.e., the infectious agent (e.g., an oncolytic virus, such as an oncolytic adenovirus), an immunomodulator (including combinations of immunomodulators), and non-activated tumor cells) can be independently adjusted to optimize performance of therapy on an individual and minimize toxicity. Any of the methods described herein can be used to inhibit growth of a solid or lymphoid tumor, inhibit metastasis of a solid or lymphoid tumor, prolong survival (e.g., disease-free survival) of a subject having a solid or lymphoid tumor, cause remission of a disease in a subject having a solid or lymphoid tumor, and/or improve the quality of life of a subject having a solid or lymphoid tumor.

Without being bound by any theory or hypothesis, it is believed that in this three component combination therapy, an external source of non-activated but viable tumor cells (also referred to herein as "viable cancer cells" or "viable tumor cells"), whether autologous or allogeneic in origin, may provide an additional, but important source of neoantigens upon administration at the tumor site. In this context, by exogenous source is meant that such tumor cells have been previously removed from the same individual or from another individual. The cells may further undergo in vitro culture for expansion, cryopreservation, thawing, and characterization. It is believed that this external source of non-activated tumor cells may sometimes not only stimulate T cell responses, but may also respond to B cells, and sometimes trigger a number of antibody responses in concert with infectious agents (e.g., viruses) and immunomodulators (including combinations of immunomodulators) as previously described.

Thus, in some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an infectious agent; b) to the sameLocal administration of an effective amount of an immunomodulator (including combinations of immunomodulators) at a tumor site; and c) locally administering to the tumor site an effective amount of non-activated tumor cells. In some embodiments, the infectious agent is a virus, for example a virus selected from the group consisting of: adenovirus, herpes simplex virus, vaccinia virus, mumps virus, newcastle disease virus, poliovirus, measles virus, seneca valley virus, coxsackie virus, rio virus, vesicular stomatitis virus, malaba and rhabdovirus, and parvovirus. In some embodiments, the infectious agent is a non-oncolytic virus. In some embodiments, the infectious agent is an oncolytic virus. In some embodiments, the infectious agent is a bacterium, such as bacillus calmette-guerin ("BCG"), listeria monocytogenes, or a mycobacterial cell wall-DNA complex ("MCNA" or "MCC", e.g., UROCIDIN)^TM). In some embodiments, the infectious agent is a wild-type infectious agent. In some embodiments, the infectious agent is genetically modified. In some embodiments, the infectious agent is attenuated (e.g., via multiple passages, inactivation, or genetic modification). In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immune modulator is a modulator of an immune checkpoint molecule selected from the group consisting of: CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the immunostimulant is an activator of OX40, 4-1BB, or CD 40. In some embodiments, the methods comprise locally administering a combination of immune modulators comprising one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the tumor cells that are not activated are autologous. In some embodiments, the tumor cells that are not activated are allogeneic. In some embodiments, the tumor cells that are not activated are from a tumor cell line. In some embodiments, the inactivation is by irradiationThe tumor cells that are activated are not activated. In some embodiments, the infectious agent and the non-activated tumor cells are administered simultaneously (e.g., in a single composition). In some embodiments, the infectious agent and the non-activated tumor cells are mixed immediately prior to administration. In some embodiments, the infectious agent and the non-activated tumor cells are administered sequentially. In some embodiments, the infectious agent and the non-activated tumor cells are mixed at the site of administration immediately after administration. In some embodiments, the infectious agent, immunomodulator (including combinations of immunomodulators), and/or non-activated tumor cells are administered to a tissue having a tumor. In some embodiments, the infectious agent, immunomodulator (including combinations of immunomodulators), and/or non-activated tumor cells are administered directly into a tumor. In some embodiments, the method further comprises administering the infectious agent and/or immunomodulator (including combination of immunomodulators) and/or inactive tumor cells by a route of administration other than topical administration.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus); b) locally administering to the tumor site an effective amount of an immunomodulator (including a combination of immunomodulators); and c) locally administering to the tumor site an effective amount of non-activated tumor cells. In some embodiments, the oncolytic virus is a wild-type oncolytic virus. In some embodiments, the oncolytic virus is genetically modified. In some embodiments, the oncolytic virus is attenuated (e.g., via multiple passages, inactivation, or genetic modification). In some embodiments, the oncolytic virus is competent for replication. In some embodiments, the oncolytic virus preferentially replicates in cancer cells. In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immune modulator is a modulator of an immune checkpoint molecule selected from the group consisting of: CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the immunostimulant is an activator of OX40, 4-1BB, or CD 40. In some embodiments, the methods comprise locally administering a combination of immune modulators comprising one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the tumor cells that are not activated are autologous. In some embodiments, the tumor cells that are not activated are allogeneic. In some embodiments, the tumor cells that are not activated are from a tumor cell line. In some embodiments, the non-activated tumor cells are inactivated by irradiation. In some embodiments, the oncolytic virus and the non-activated tumor cells are administered simultaneously (e.g., in a single composition). In some embodiments, the oncolytic virus and the non-activated tumor cells are mixed immediately prior to administration. In some embodiments, the oncolytic virus and the non-activated tumor cell are administered sequentially. In some embodiments, the oncolytic virus and the non-activated tumor cells are mixed at the site of administration immediately after administration. In some embodiments, an oncolytic virus, an immunomodulator (including a combination of immunomodulators), and/or a non-activated tumor cell is administered to a tissue having a tumor. In some embodiments, the oncolytic virus, an immunomodulator (including a combination of immunomodulators), and/or non-activated tumor cells are administered directly into a tumor. In some embodiments, the method further comprises administering the oncolytic virus and/or an immunomodulator (including a combination of immunomodulators) and/or the inactivated tumor cell by a route of administration other than topical administration.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication; b) locally administering to the tumor site an effective amount of an immunomodulator (including a combination of immunomodulators); and c) locally administering to the tumor site an effective amount of non-activated tumor cells. In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immune modulator is a modulator of an immune checkpoint molecule selected from the group consisting of: CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the immunostimulant is an activator of OX40, 4-1BB, or CD 40. In some embodiments, the methods comprise locally administering a combination of immune modulators comprising one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4. In some embodiments, the tumor cells that are not activated are autologous. In some embodiments, the tumor cells that are not activated are allogeneic. In some embodiments, the tumor cells that are not activated are from a tumor cell line. In some embodiments, the non-activated tumor cells are inactivated by irradiation. In some embodiments, the oncolytic virus and the non-activated tumor cells are administered simultaneously (e.g., in a single composition). In some embodiments, the oncolytic virus and the non-activated tumor cells are mixed immediately prior to administration. In some embodiments, the oncolytic virus and the non-activated tumor cell are administered sequentially. In some embodiments, the oncolytic virus and the non-activated tumor cells are mixed at the site of administration immediately after administration. In some embodiments, an oncolytic virus, an immunomodulator (including a combination of immunomodulators), and/or a non-activated tumor cell is administered to a tissue having a tumor. In some embodiments, the method further comprises administering the oncolytic virus and/or an immunomodulator (including a combination of immunomodulators) and/or the inactivated tumor cell by a route of administration other than topical administration.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual comprising: a) locally administering to the tumor site an effective amount of an oncolytic virus (e.g., an oncolytic adenovirus) comprising a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for viral replication and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine) operably linked to the viral promoter; b) locally administering to the tumor site an effective amount of an immunomodulator (including a combination of immunomodulators); and c) locally administering an effective amount of non-activated tumor cells to the tumor site, wherein the non-activated tumor cells are inactivated. In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immune modulator is a modulator of an immune checkpoint molecule selected from the group consisting of: CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the immunostimulant is an activator of OX40, 4-1BB, or CD 40. In some embodiments, the methods comprise locally administering a combination of immune modulators comprising one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4. In some embodiments, the viral promoter operably linked to the nucleic acid encoding the immune-related molecule is the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF. In some embodiments, the tumor cells that are not activated are autologous. In some embodiments, the tumor cells that are not activated are allogeneic. In some embodiments, the tumor cells that are not activated are from a tumor cell line. In some embodiments, the non-activated tumor cells are inactivated by irradiation. In some embodiments, the oncolytic virus and the non-activated tumor cells are administered simultaneously (e.g., in a single composition). In some embodiments, the oncolytic virus and the non-activated tumor cells are mixed immediately prior to administration. In some embodiments, the oncolytic virus and the non-activated tumor cell are administered sequentially. In some embodiments, the oncolytic virus and the non-activated tumor cells are mixed at the site of administration immediately after administration. In some embodiments, an oncolytic virus, an immunomodulator (including a combination of immunomodulators), and/or a non-activated tumor cell is administered to a tissue having a tumor. In some embodiments, the oncolytic virus, an immunomodulator (including a combination of immunomodulators), and/or non-activated tumor cells are administered directly into a tumor. In some embodiments, the method further comprises administering the oncolytic virus and/or an immunomodulator (including a combination of immunomodulators) and/or the inactivated tumor cell by a route of administration other than topical administration.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual comprising: a) locally administering to the tumor site an effective amount of adenovirus serotype 5, wherein the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced by the human E2F-1 promoter and a nucleic acid encoding an immune-related molecule (e.g., an interleukin, a chemokine, such as GM-CSF); b) locally administering to the tumor site an effective amount of an immunomodulator (including a combination of immunomodulators); and c) locally administering to the tumor site an effective amount of non-activated tumor cells. In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immune modulator is a modulator of an immune checkpoint molecule selected from the group consisting of: CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the immunostimulant is an activator of OX40, 4-1BB, or CD 40. In some embodiments, the methods comprise locally administering a combination of immune modulators that includes one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the tumor specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4. In some embodiments, the viral promoter operably linked to the nucleic acid encoding the immune-related molecule is the E3 promoter. In some embodiments, the tumor cells that are not activated are autologous. In some embodiments, the tumor cells that are not activated are allogeneic. In some embodiments, the tumor cells that are not activated are from a tumor cell line. In some embodiments, the non-activated tumor cells are inactivated by irradiation. In some embodiments, the adenovirus and the non-activated tumor cell are administered simultaneously (e.g., in a single composition). In some embodiments, the adenovirus and the inactivated tumor cell are mixed immediately prior to administration. In some embodiments, the adenovirus and the non-activated tumor cell are administered sequentially. In some embodiments, the adenovirus and the non-activated tumor cell are mixed at the site of administration immediately after administration. In some embodiments, an adenovirus, an immunomodulator (including a combination of immunomodulators), and/or a non-activated tumor cell is administered to a tissue having a tumor. In some embodiments, the adenovirus, immunomodulator (including combinations of immunomodulators), and/or non-activated tumor cell is administered directly into a tumor. In some embodiments, the method further comprises administering the adenovirus and/or the immunomodulator (including the combination of immunomodulators) and/or the inactivated tumor cell by a route of administration other than topical administration.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual comprising: a) locally administering to the tumor site an effective amount of CG 0070; and b) locally administering to the tumor site an effective amount of an immunomodulator (including combinations of immunomodulators); and c) locally administering to the tumor site an effective amount of non-activated tumor cells. In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immune modulator is a modulator of an immune checkpoint molecule selected from the group consisting of: CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the immunostimulant is an activator of OX40, 4-1BB, or CD 40. In some embodiments, the methods comprise locally administering a combination of immune modulators comprising one or more immune checkpoint inhibitors and/or one or more immune stimulants (e.g., a combination of at least two immune checkpoint inhibitors, at least two immune stimulants, or at least one immune checkpoint inhibitor and at least one immune stimulant). In some embodiments, the tumor cells that are not activated are autologous. In some embodiments, the tumor cells that are not activated are allogeneic. In some embodiments, the tumor cells that are not activated are from a tumor cell line. In some embodiments, the non-activated tumor cells are inactivated by irradiation. In some embodiments, CG0070 and the non-activated tumor cells are administered simultaneously (e.g., in a single composition). In some embodiments, CG0070 and the non-activated tumor cells are mixed immediately prior to administration. In some embodiments, CG0070 and the non-activated tumor cells are administered sequentially. In some embodiments, CG0070 and the non-activated tumor cells are mixed at the site of administration immediately after administration. In some embodiments, CG0070, an immunomodulator (including a combination of immunomodulators), and/or non-activated tumor cells are administered to a tissue having a solid or lymphoid tumor. In some embodiments, CG0070, an immunomodulator (including a combination of immunomodulators), and/or non-activated tumor cells are administered directly into the tumor. In some embodiments, the method further comprises administering CG0070 and/or an immunomodulator (including a combination of immunomodulators) and/or inactivated tumor cells by a route of administration other than topical administration.

In some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual comprising: a) administering an effective amount of CG0070 intratumorally; and b) intratumorally administering an effective amount of an inhibitor of CTLA-4 (e.g., an anti-CTLA-4 antibody (e.g., ipilimumab) or an engineered lipocalin (e.g., an anti-transportan that specifically recognizes CTLA-4)); c) intratumorally administering an effective amount of a4-1BB activator (e.g., an agonistic anti-4-1 BB antibody, e.g., PF-05082566); and d) intratumorally administering an effective amount of non-activated tumor cells (e.g., allogeneic non-activated tumor cells) to the tumor site, wherein the effective amount of CG0070 is about 1X 10 cells per week⁸To about 1X 10¹⁴Individual viral particles (vp) (e.g., about any of the following: 5X 10 per week¹⁰vp、1×10¹¹vp、5×10¹¹vp or 1X 10¹²vp), wherein the effective amount of the inhibitor of CTLA-4 is from about 0.1mg to about 100mg (e.g., no more than about any of 1mg, 3mg, 6mg, 12mg, or 24mg per week), wherein the effective amount of the 4-1BB activator is from about 0.1mg to about 100mg (e.g., no more than about any of 1mg, 3mg, 6mg, 12mg, or 24mg per week), and wherein the effective amount of non-activated tumor cells is at least about 10 of the effective amount of CG0070⁴. In some embodiments, the inhibitor of CTLA-4 and the 4-1BB activator are administered immediately (e.g., no more than 5 minutes after administration) after administration of CG0070 and the non-activated tumor cells. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody (e.g., ipilimumab) (e.g.,). In some embodiments, the inhibitor of CTLA-4 is an engineered lipocalin, e.g., an anti-transporter that specifically recognizes CTLA-4. In some embodiments, the 4-1BB activator is an agonistic anti-4-1 BB antibody, such as PF-05082566. In some embodiments, an effective amount of DDM is further administered intratumorally as a transduction enhancer to the individual in combination with the administration of CG 0070. In some embodiments, the non-activated tumor cells are inactivated by irradiation. In some embodiments, CG0070 and the non-activated tumor cells are administered simultaneously (e.g., in a single composition). In some embodiments, CG0070 and the non-activated tumor cells are mixed immediately prior to administration. In some embodiments, the CG0070, the inhibitor of CTLA-4, the 41-BB activator, and the non-activated tumor cells are administered by injection into a tissue having a tumor. In some embodiments, the CG0070, the inhibitor of CTLA-4, the 41-BB activator, and the non-activated tumor cells are administered by direct injection into the tumor. In some embodiments, CG0070, the inhibitor of CTLA-4, the 41-BB activator, and the non-activated tumor cells are administered weekly as a course of treatment in about 1 week to about 8 weeks (e.g., about 4 weeks or about 6 weeks). In some embodiments, the course of treatment is repeated every about 2 months to about 3 months. In some embodiments, the solid or lymphoid tumor is selected from the group consisting of: head and neck cancer, breast cancer, colorectal cancer, liver cancer, pancreatic adenocarcinoma, gallbladder and bile duct cancer, ovarian cancer, cervical cancer, small cell lung cancer, non-small cell lung cancer, renal cell cancer, bladder cancer, prostate cancer, bone cancer, mesothelioma, brain cancer, soft tissue sarcoma, uterine cancer, thyroid cancer, nasopharyngeal cancer, and melanoma. In some embodiments, the solid or lymphoid tumor is refractory to prior therapy.

The non-activated tumor cells can be obtained from a variety of sources, including but not limited to autologous sources, allogeneic sources, tumor cell lines, and combinations thereof. Typically, the tumor cells that are not activated are of the same type, or express one or more of the same tumor antigens and the solid or lymphoid tumor being treated. In some embodiments, the tumor cells that are not activated consist of a single population of tumor cells. In some embodiments, the tumor cells that are not activated comprise multiple (e.g., 2, 3, 4, 5, 6, or more) populations of tumor cells.

In some embodiments, the non-activated tumor cells are derived from an allogeneic source. In some embodiments, the non-activated tumor cells are derived from different individuals having tumors (e.g., solid or lymphoid tumors of the same type). In some embodiments, the non-activated tumor cells and the solid or lymphoid tumor of the treated individual express at least one shared tumor antigen (e.g., a tumor-associated antigen and/or a tumor-specific antigen).

In some embodiments, the tumor cells that are not activated are derived from a tumor cell line that shares the same or similar origin or genetic profile (e.g., tumor antigen expression profile) as a solid or lymphoid tumor of an individual. In some embodiments, an individual that does not have activated tumor cells and has a tumor expresses at least one shared tumor antigen (e.g., a tumor-associated antigen and/or a tumor-specific antigen). For example, where the solid or lymphoid tumor being treated is prostate cancer, the prostate tumor cells are selected from the group consisting of: DU145, PC-3 and LnCaP.

In some embodiments, the non-activated tumor cells are derived from the same individual with a solid or lymphoid tumor. In some embodiments, the non-activated tumor cells are derived from a tissue having a solid or lymphoid tumor. In some embodiments, the tumor cells that are not activated are derived from a solid or lymphoid tumor (e.g., a tumor biopsy or resection tumor). In some embodiments, the non-activated tumor cells are derived from a metastatic site of a solid or lymphoid tumor of the individual. In some embodiments, the non-activated tumor cells provide one or more cells, interleukins, chemokines and/or antigenic components during death of the non-activated tumor cells in vivo, wherein the one or more components are sampled and cross-presented by antigen presenting cells (e.g., dendritic cells) of the individual to stimulate an immune response against the solid or lymphoid tumor.

in some embodiments, the non-activated tumor cells are modified, e.g., genetically modified, e.g., transduced or transfected in vitro or in vivo, e.g., by an infectious agent, in some embodiments, the non-activated tumor cells are modified to express or secrete an immune-related molecule, in some embodiments, the immune-related molecule is an interleukin, a chemokine, or another immune-related molecule, in some embodiments, the immune-related molecule is selected from the group consisting of an IL-2, IL-12, an interferon (e.g., a type 1, type 2, or type 3 interferon, e.g., interferon gamma), CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, LG9, CpG 10, RIG-I, MDA5, RLP 2, and α β in some embodiments, the immune-related molecule is selected from the group consisting of a CYG-96003, a TLR-9623, a TLR-9, a TLR-968, a TLR-638, a TLR-IL-9, a TLR-9, such as a TLR-9, a TNF-9, or a polypeptide such as a TNF-IL-9, or a polypeptide such as a TLR-9, or a polypeptide.

In some embodiments, the non-activated tumor cells are modified to express or secrete one or more immunomodulators. In some embodiments, the one or more immunomodulatory agents comprise an immunostimulatory agent. In some embodiments, the immunostimulatory agent is a natural or engineered ligand for an immunostimulatory molecule, including, for example, a ligand for OX40 (e.g., OX40L), a ligand for CD28 (e.g., CD80, CD86), a ligand for ICOS (e.g., B7RP1), a ligand for 4-1BB (e.g., 4-1BBL, Ultra4-1BBL), a ligand for CD27 (e.g., CD70), a ligand for CD40 (e.g., CD40L), and a ligand for a TCR (e.g., a MHC class I or class II molecule, IMCgp 100). In some embodiments, the immunostimulatory agent is an antibody selected from the group consisting of: anti-CD 28 (e.g., TGN-1412), anti-OX 40 (e.g., MEDI6469, MEDI-0562), anti-ICOS (e.g., MEDI-570), anti-GITR (e.g., TRX518, INBRX-110, NOV-120301), anti-41-BB (e.g., BMS-663513, PF-05082566), anti-CD 27 (e.g., BION-1402, Wallimumab, and hCD27.15), anti-CD 40 (e.g., CP870,893, BI-655064, BMS-986090, APX005M), anti-CD 3 (e.g., brilimumab, Moluomab), and anti-HVEM. In some embodiments, the antibody is an agonistic antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an antigen binding fragment selected from the group consisting of: fab, Fab ', F (ab') of full-length antibody₂And Fv, scFv or other antigen binding subsequences. In some embodiments, the antibody is a human, humanized or chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific anti-antibodyA body, a single domain antibody, a fusion protein comprising an antibody portion, or any other functional variant or derivative thereof.

in some embodiments, the immune checkpoint inhibitor is a natural or engineered ligand of an inhibitory immune checkpoint molecule, including, for example, a ligand of CTLA-4 (e.g., B7.1, B7.2), a ligand of TIM3 (e.g., galectin-9), a ligand of the A2a receptor (e.g., adenosine, Regadenoson), a ligand of LAG3 (e.g., MHC class I or MHC class II molecules), a ligand of BTLA (e.g., HVEM, B7-H4), a ligand of KIR (e.g., MHC class I or MHC class II molecules), a ligand of PD-1 (e.g., PD-L1, PD-L2), a ligand of IDO (e.g., NKTR-218, indomethacin, NLG919) and a ligand of CD47 (e.g., anti-alpha receptor), in some embodiments, the immune inhibitor is an anti-CD 6019, anti-CD 20068, anti-IgG-NO-1, such as anti-7, anti-NOIn the case, the antibody is a monoclonal antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an antigen binding fragment selected from the group consisting of: fab, Fab ', F (ab') of full-length antibody₂And Fv, scFv or other antigen binding subsequences. In some embodiments, the antibody is a human, humanized or chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other functional variant or derivative thereof.

In some embodiments, the non-activated tumor cells are transduced and genetically modified by the infectious agent used in the combination therapy.

Tumor cells can be isolated from the tissue, excised tumor, or tumor biopsy by any of the methods known in the art, including but not limited to mechanical, enzymatic isolation methods, and combinations thereof. For example, a mixture of collagenase, DNA hydrolase and hyaluronidase may be used to incubate a tumor sample to obtain non-activated tumor cells. In some embodiments, the plurality of batches of isolated autologous tumor cells are obtained from a solid or lymphatic tumor or metastatic site of the individual during the course of treatment. In some embodiments, the inactivated tumor cells are cryopreserved prior to inactivation.

Since cancer cells, especially in metastatic sites, are heterogeneous mixtures of different cell lineages that undergo rapid replication and frequent mutation, it is sometimes preferable to have specific components that can accommodate such changes as they occur. Autologous tumor cells can be prepared from initial surgical samples, biopsy, or later removal of metastatic lesions. One advantage of this approach is that autologous tumor cells can be altered depending on the availability of the patient's response tumor sample. For example, tumor-infectious agent (e.g., virus) live and in vivo vaccine systems generated in the primary tumor stage may be different from those later using tumor cells from metastatic sites. In some embodiments, the ultimate goal is to adapt the immunotherapeutic response according to the predominant tumor type, an advantage not found in recent developments of pathway-targeted therapies or monoclonal antibody-directed therapies.

The non-activated tumor cells are inactivated prior to administration. Generally, non-activated tumor cells are competent for proliferation. Tumor cells can be inactivated using any of the methods known in the art. In some embodiments, the non-activated tumor cells are inactivated by irradiation. In some embodiments, the non-activated tumor cells are irradiated at a dose of about 50 to about 200rad/min or about 120 to about 140rad/min prior to administration to a patient. In some embodiments, the non-activated tumor cells are irradiated at a total dose of about any of 2,500rad, 5,000rad, 10,000rad, 15,000rad, or 20,000 rad. In some embodiments, the non-activated tumor cells are irradiated at a total dose of about 10,000 to about 20,000 rad. In some embodiments, the non-activated tumor cells are irradiated at a total dose sufficient to inhibit substantially 100% of the cells from further proliferation. In some embodiments in which the non-activated tumor cells are genetically modified, the total dose of irradiation is insufficient to inhibit expression or secretion of an immune-related molecule (e.g., GM-CSF). In some embodiments, the total dose of radiation is insufficient to inhibit transduction or genetic modification of non-activated tumor cells by the infectious agent when administered. In some embodiments, the non-activated tumor cells are cryopreserved prior to administration.

Non-activated tumor cells are administered intratumorally, e.g., by intratumoral injection. The appropriate dose of non-activated tumor cells for administration depends on the state (e.g., microenvironment, type, stage, etc.) of the solid or lymphoid tumor and other diagnostic and risk factors of the individual. In some embodiments, a suitable dose of non-activated tumor cells is 1 × 10³、1×10⁴、1×10⁵、2×10⁵、5×10⁵、1×10⁶、2×10⁶、5×10⁶、1×10⁷、5×10⁷Or 1X 10⁸About any one of the individual cells. In some embodiments, a suitable dose of non-activated tumor cells is about 1 × 10³To about 1X 10⁴About 1X 10⁴To about 1X 10⁵About 1X 10⁵To about 2×10⁵About 2X 10⁵To about 5X 10⁵About 5X 10⁵To about 10⁶About 10⁶To about 2X 10⁶About 2X 10⁶To about 5X 10⁶About 5X 10⁶To about 1X 10⁷About 1X 10⁷To about 5X 10⁷Or about 5X 10⁷To about 1X 10⁸Any one of the individual tumor cells. In some embodiments, the dose of non-activated tumor cells is calculated as cells/Kg body weight.

In some embodiments, the relative ratio of infectious agent (e.g., virus) to non-activated tumor cells is based on a fold infection (MOI) index calculated using the number of particles of infectious agent versus the number of tumor cells that are not activated alone or versus the total number of viable tumor cells (including the number of tumor cells that are not activated and the estimated number of viable tumor cells at the site of administration). In some embodiments, the MOI is at least 1,2, 5, 10, 50, 100, 200, 500, 1000, 5000, 10⁴、10⁵、10⁶Or about any of the greater. In some embodiments, the infectious agent is provided in an amount proportional to the estimated volume of the tumor site. In some embodiments, the tumor cells that are not activated are provided in an amount limited by: preparations from tumor biopsy, tumor resection, tumor cell culture, and other methods known in the art for isolating tumor cells. In some embodiments, the infectious agent is at about 1 × 10⁵To about 1X 10¹⁴A particle (e.g., about 1X 10)¹²Individual particles) are provided in the composition. In some embodiments, the tumor cells that are not activated are at about 1 × 10³Cell to about 1X 10⁸Individual cell (e.g., about 1X 10⁵Non-activated tumor cells) are provided in the composition.

In some embodiments, the non-activated tumor cells are administered daily. In some embodiments, the tumor cells that are not activated are administered at least about any one of 1,2, 3, 4, 5, 6, or 7 times a week (i.e., daily). In some embodiments, the non-activated tumor cells are administered weekly. In some embodiments, the tumor cells that are not activated are administered biweekly; no interruption is carried out every week; two out of three weeks are administered weekly; three weeks out of four weeks weekly; once every 2 weeks; once every 3 weeks; once every 4 weeks; once every 6 weeks; administered once every 8 weeks, monthly or every 2 to 12 months. In some embodiments, the interval between each administration is less than about any of: 6 months, 3 months, 1 month, 20 days, 15 days, 12 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the interval between each administration exceeds about any of the following: 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, or 12 months. In some embodiments, there is no break in the dosing schedule. In some embodiments, the interval between each administration is no more than about one week. In some embodiments, the tumor cells that are not activated are administered on the same dosing schedule as the infectious agent. In some embodiments, the non-activated tumor cells are administered on a different dosing schedule than the infectious agent.

Administration of non-activated tumor cells can be over an extended period of time (e.g., about 1 month up to about 7 years). In some embodiments, the non-activated tumor cells are administered for a period of at least about any one of: 1. 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 18, 24, 30, 36, 48, 60, 72, or 84 months. In some embodiments, the non-activated tumor cells are administered over a period of at least 3 weeks or 6 weeks. In some embodiments, the non-activated tumor cells are administered weekly in three of four weeks every 3 months. In some embodiments, the non-activated tumor cells are administered weekly in 6 weeks every 3 months.

In some embodiments, the infectious agent is administered weekly. In some embodiments, the immunomodulator is administered weekly. In some embodiments, the non-activated tumor cells are administered weekly.

In some embodiments, the infectious agent is administered daily. In some embodiments, the immunomodulator is administered daily. In some embodiments, the non-activated tumor cells are administered daily.

In some embodiments, the infectious agent is first administered multiple times daily or weekly (e.g., any of 1,2, 3, 4, 5, 6, 7,10, or more times) over a first course of treatment, then administered multiple times daily or weekly (e.g., any of 1,2, 3, 4, 5, 6, 7,10, or more times) over a second course of treatment, and then the course of treatment is maintained every month or every few (e.g., any of 2, 3, 4, 5, 6, or more) months. In some embodiments, the immunomodulator is first administered multiple times daily or weekly (e.g., any of 1,2, 3, 4, 5, 6, 7,10 or more times) over a first treatment course, then administered multiple times daily or weekly (e.g., any of 1,2, 3, 4, 5, 6, 7,10 or more times) over a second treatment course, and then the treatment course is maintained every month or every few (e.g., any of 2, 3, 4, 5, 6 or more) months. In some embodiments, the non-activated tumor cells are first administered multiple times daily or weekly (e.g., any of 1,2, 3, 4, 5, 6, 7,10, or more times) over a first course of treatment, then administered multiple times daily or weekly (e.g., any of 1,2, 3, 4, 5, 6, 7,10, or more times) over a second course of treatment, and then the course of treatment is maintained every month or every few months (e.g., any of 2, 3, 4, 5, 6, or more).

In some embodiments, the infectious agent, immunomodulator, and inactive cells are administered using any combination of the above dosing schedules. Each course of treatment may include administration over the course of days, weeks, or months. The course of treatment may be repeated as long as necessary.

In some embodiments, the infectious agent and the non-activated tumor cells discussed above are administered sequentially, i.e., administration of the infectious agent is administered before or after administration of the non-activated tumor cells. In some embodiments, the infectious agent is administered prior to administration of the tumor cells that are not activated. In some embodiments, the infectious agent is administered no more than about any one of the following prior to administration of the non-activated tumor cells: 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours. In some embodiments, the infectious agent is administered about day or week (e.g., about any of: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more) prior to administration of the non-activated tumor cells. In some embodiments, the infectious agent is administered after administration of the tumor cells that are not activated. In some embodiments, the infectious agent is administered no more than about any one of the following after administration of the non-activated tumor cells: 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, or 24 hours. In some embodiments, the infectious agent is administered about day or week (e.g., about any of: 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, or more) after the administration of the non-activated tumor cells. In some embodiments, the infectious agent and the non-activated tumor cells are administered to one immediately after the other (e.g., within 5 minutes or less between administrations). For example, in some embodiments, the infectious agent is administered immediately prior to administration of the non-activated tumor cells. In some embodiments, the infectious agent is administered immediately after administration of the non-activated tumor cells.

In some embodiments, the infectious agent and the non-activated tumor cells are administered simultaneously. In some embodiments, the infectious agent and the non-activated tumor cells are administered simultaneously via separate compositions. In some embodiments, the infectious agent and the non-activated tumor cells are administered in a single composition. In some embodiments, the infectious agent and the non-activated tumor cells are mixed prior to administration of the composition (e.g., immediately prior to administration of the composition, e.g., within less than about 10, 5, or 1 minute prior to administration of the composition). In some embodiments, a composition comprising an infectious agent and non-activated tumor cells is pre-prepared and stored for at least about any of 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or longer prior to administration. In some embodiments, the inactivated tumor cells and the infectious agent are completely separated until the time of administration to the individual. In some embodiments, the infectious agent and the non-activated tumor cells do not require pre-incubation prior to administration.

Kit and pharmaceutical composition

In another aspect, kits, unit doses, and articles of manufacture are provided that are useful in any of the methods described herein.

for example, in some embodiments, there is provided a kit for treating a solid or lymphoid tumor in an individual (e.g., for inhibiting tumor metastasis) comprising a) an infectious agent, B) an immune modulator (including a combination of immune modulators), and C) a device for locally administering an infectious agent and/or an immune modulator (including a combination of immune modulators) to the tumor site in some embodiments, the infectious agent is a virus, e.g., an oncolytic adenovirus in some embodiments, the infectious agent comprises a nucleic acid encoding an immune-related molecule (e.g., an interleukin or chemokine), in some embodiments, the immune-related molecule is selected from the group consisting of GM-CSF, IL-2, IL, an interferon (e.g., type 1, type 2 or type 3 interferon, e.g., interferon gamma), CCL, CXCL, TLR, lgg, TLR, CpG, TLR, a tumor-B, a tumor-cell-activating agent in some embodiments, a tumor-cell-stimulating, a tumor-cell stimulating, a tumor-cell-stimulating, a tumor-cell, a tumor.

in some embodiments, there is provided a kit for treating a solid or lymphoid tumor in an individual (e.g., for inhibiting tumor metastasis) comprising a) a replication competent oncolytic virus (e.g., oncolytic adenovirus), B) an immunomodulatory agent (including a combination of immunomodulatory agents), and c) means for locally administering to the tumor site an oncolytic virus and/or an immunomodulatory agent (including a combination of immunomodulatory agents) in some embodiments, the oncolytic virus comprises a tumor specific promoter (e.g., E2F-1 promoter) operably linked to a gene necessary for viral replication (e.g., E1A, E1B, or E4 gene) in some embodiments, the oncolytic virus comprises a nucleic acid encoding an immune related molecule (e.g., an interleukin or a chemotactic agent) in some embodiments, the nucleic acid encoding an immune related molecule is operably linked to a viral promoter, e.g., E3 promoter, in some embodiments, the immune stimulator is OX 23, 4-1BB or CD 24 (e.g., an agonist or CD 9-B-t-B-c-B-c-B-c-B-c-.

in some embodiments, a kit is provided for treating a solid or lymphoid tumor in an individual (e.g., for inhibiting tumor metastasis) comprising a) an adenovirus serotype 5, wherein the endogenous E1a promoter and E319 kD coding region of native adenovirus are replaced by a human E2-1 promoter and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine, such as GM-CSF), B) an immunomodulator (including a combination of immunomodulators), and c) means for locally administering to the tumor site an oncolytic virus and/or immunomodulator (including a combination of immunomodulators) in some embodiments, the immunostimulant is an activator of OX40, 4-1BB or CD40 (e.g., an agonist antibody), in some embodiments, the immunomodulator is a modulator of an immune checkpoint molecule selected from the group consisting of (e.g., antibodies) PD-4, PD-1, PD-L5, PD-2, B58 3, B7, a tumor-activating ligand, a combination of multiple tumor cells in some embodiments, including a tumor cell activation kit, a tumor activation kit, a kit comprising a tumor cell activation kit, a direct or multiple tumor cell activation kit, a tumor cell activation kit comprising multiple tumor activator, a multiple activator, a tumor cell activating agent, a tumor activating kit comprising multiple, a tumor cell activating agent, a tumor cell activating kit, a multiple, a tumor cell activating kit, a tumor activating kit, a multiple activating kit, a multiple activating kit, a kit.

in some embodiments, there is provided a kit for treating a solid or lymphoid tumor in an individual (e.g., for inhibiting tumor metastasis) comprising a) CG0070, B) an immune modulator (including a combination of immune modulators), and c) means for locally administering to the tumor site an oncolytic virus and/or an immune modulator (including a combination of immune modulators) in some embodiments, the immune modulator is an activator (e.g., agonist antibody) of OX40, 4-1BB or CD40 in some embodiments, the immune modulator is a modulator of an immune checkpoint molecule selected from the group consisting of CTLA-4, PD-1, PD-L1, PD-L2, 3, B7-H3, B7-H4, LAG-3, KIR, and ligands thereof, in some embodiments, the kit comprising a combination of immune modulators including one or more than one immune inhibitor in a kit for use in a combined administration of tumor activation regimen, or a combined administration of multiple tumor cell activation regimen, including a tumor activation regimen, a combined administration of an autologous tumor-activating agent for use in a tumor activation regimen, a tumor-activating regimen, and/tumor-activating composition comprising a combined administration of one or multiple tumor cell therapies for use in a combined administration of an autologous tumor-activating composition, a tumor-activating composition comprising a tumor-activating composition, and/or a tumor cell, and a tumor-activating composition, in some embodiments, a kit comprising one or a tumor-activating composition, in a kit comprising one or a tumor cell, a combined administration of an immune modulator for use in a tumor cell, a tumor-activating composition for use in some embodiments, a combined administration of an immune modulator for use in a tumor-activating composition, a combined administration of a tumor-activating composition, and/or a tumor-activating composition, a kit comprising a tumor-activating composition, and/or a kit comprising a tumor-activating composition, and/.

The kit may also include instructions for selection of individuals suitable for treatment. For example, a package may include instructions for selection of individuals based on expression of one or more biomarkers (e.g., PD-1, PD-L1, or PD-L2). In some embodiments, the kit further comprises reagents for assessing the extent of expression of a biomarker (e.g., PD-1, PD-L1, or PD-L2). The instructions supplied in the kit of the invention are typically written instructions on a label or package insert (e.g., a sheet of paper included in the kit), but may also be machine-readable instructions (e.g., instructions on a magnetic or optical storage disc).

Also provided is a tumor cell preparation kit comprising materials and instructions for performing tumor dissociation and preparation, enzyme and/or viral vector transduction reagents, cryopreservation vials, and the like, and a package insert containing instructions for use. The tumor cell preparation kit can be used to provide tumor cells that are not activated, and the kit can be used in combination with any of the kits for treating solid or lymphoid tumors described above for performing a combination therapy comprising an infectious agent, an immunomodulator (including a combination of immunomodulators), and isolated and inactivated tumor cells.

Instructions relating to the use of infectious agents (e.g., oncolytic adenoviruses, such as CG0070) and immunomodulators (including combinations of immunomodulators) generally include information on the dosage, schedule of administration and route of administration for the intended treatment. The container may be a unit dose, a bulk package (e.g., a multi-dose package), or a sub-unit dose. For example, a kit can be provided containing sufficient doses of an infectious agent as disclosed herein and an immunomodulator (including a combination of immunomodulators) to provide effective treatment of an individual for an extended period of time (e.g., any of one week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or longer). The kit may also include a plurality of unit doses of the infectious agent and immunomodulator (including combinations of immunomodulators) and instructions for use, packaged in amounts sufficient for storage and use in pharmacies, such as animal pharmacies and compound pharmacies.

The kit of the invention is in a suitable package. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. The kit may optionally provide additional components, such as buffers and explanatory information. Thus, the present application also provides articles of manufacture including vials (e.g., sealed vials), bottles, jars, flexible packaging, and the like.

The article of manufacture may include a container and a label or package insert located on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. Such containers may be formed from a variety of materials, such as glass or plastic. Typically, the container contains a composition effective to treat the diseases or conditions described herein, and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a) an infectious agent; or b) an immunomodulator (including combinations of immunomodulators). The indicia or package insert indicates that the composition is used to treat a particular condition in an individual. The label or package insert will also include instructions for administering the composition to the individual. Also encompassed are articles of manufacture and kits comprising the combination therapies described herein.

The package insert refers to instructions typically included in commercial packaging for therapeutic products that contain information about the indications, usage, dosage, administration, contraindications, and/or warnings regarding the use of such therapeutic products. In some embodiments, the package insert indicates that the composition is for use in treating a solid or lymphoid tumor (e.g., bladder cancer, renal cell carcinoma, or melanoma).

Additionally, the article of manufacture may additionally comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution, and dextrose solution. It may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles and syringes.

Medical devices for local administration (e.g., intravesical or intratumoral injection) of infectious agents, immunomodulators (including combinations of immunomodulators), and/or inactive tumor cells are known in the art. For example, a medical device for intravesical delivery may include a catheter, such as a Rusch 173430 Foley catheter and a BARD LUBRI-SIL Foley catheter number 70516 SI. Medical devices for intratumoral injection may include a syringe, a needle or needle array, and a plurality of outlets. The intratumoral injection device may be designed, inter alia, to ensure a uniform distribution of infectious agents, immunomodulators (including combinations of immunomodulators) and/or inactive tumor cells in the tumor site. In some embodiments, the intratumoral injection device comprises a forced air nozzle.

in another aspect, a pharmaceutical composition (e.g., cocktail) comprising an infectious agent and an immune modulator (including a combination of immune modulators) is provided, e.g., in some embodiments, a pharmaceutical composition comprising a) an infectious agent, B) an immune modulator (including a combination of immune modulators), and C) a pharmaceutically acceptable excipient suitable for local administration of the composition to a tumor site, in some embodiments, the infectious agent is a virus, e.g., an oncolytic adenovirus, in some embodiments, the infectious agent comprises a nucleic acid encoding an immune-related molecule (e.g., an interleukin or chemokine), in some embodiments, the immune-related molecule is selected from the group consisting of GM-CSF, IL-2, IL, an interferon (e.g., type 1, type 2 or type 3 interferon, e.g., interferon gamma), CCL, CXCL, TLR, lgg, CpG, TLR, rld, TLR, and optionally, in some embodiments, in a pharmaceutical composition comprising a combination of a plurality of immune-cytokine-stimulating agent, a tumor-stimulating agent, e.g-stimulating agent, a tumor-stimulating agent, e.g., a tumor-stimulating agent, a tumor-cell stimulating agent, e, a tumor-stimulating agent, e, a tumor-cell stimulating agent, e, a tumor-stimulating agent, e, a tumor-cell stimulating agent, a tumor-cell stimulating agent, e, a tumor-stimulating agent, e.g-stimulating agent, a tumor-cell stimulating agent, a tumor-stimulating agent, e.g-cell stimulating agent, a tumor-stimulating agent, e.g-stimulating agent, a tumor-cell stimulating agent, a tumor-cell stimulating agent, a tumor-stimulating agent, e, a tumor-stimulating agent, a tumor-cell stimulating agent, a tumor-stimulating agent, e, a tumor-cell stimulating agent, e, a tumor-cell stimulating agent, a tumor-stimulating agent, e.g., a tumor-stimulating agent, a tumor-cell stimulating agent, a tumor-cell stimulating agent.

In some embodiments, there is provided a pharmaceutical composition comprising: a) a replication-competent oncolytic virus (e.g., an oncolytic adenovirus), b) an immunomodulator (including combinations of immunomodulators), and c) a pharmaceutically acceptable excipient suitable for topical administration of the composition to a tumor site. In some embodiments, the oncolytic virus comprises a tumor specific promoter (e.g., the E2F-1 promoter) operably linked to a gene essential for viral replication (e.g., the E1A, E1B, or E4 gene). In some embodiments, an oncolytic virus comprises a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine). In some embodiments, the nucleic acid encoding the immune-related molecule is operably linked to a viral promoter, such as the E3 promoter. In some embodiments, the pharmaceutical composition further comprises a plurality of non-activated tumor cells. In some embodiments, the plurality of non-activated tumor cells are autologous, allogeneic, derived from a tumor cell line, or a combination thereof. In some embodiments, the plurality of non-activated tumor cells are inactivated by irradiation. In some embodiments, the immunostimulant is an activator of OX40, 4-1BB, or CD40 (e.g., an agonist antibody). In some embodiments, the immune modulator is a modulator of an immune checkpoint molecule selected from the group consisting of (e.g., an antibody): CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the pharmaceutical composition comprises a combination of immune modulators including one or more immune checkpoint inhibitors and/or one or more immune stimulators (e.g., a combination of a CTLA-4 inhibitor and a CD40 activator, or a combination of a CTLA-4 inhibitor and a4-1BB activator). In some embodiments, the excipient is suitable for direct administration of the oncolytic virus and/or immunomodulators (including combinations of immunomodulators) and/or non-activated tumor cells into a tumor. In some embodiments, the excipient is suitable for administering an oncolytic virus and/or an immunomodulator (including a combination of immunomodulators) and/or non-activated tumor cells to a tissue having a tumor. In some embodiments, the excipient is a polymer, such as a hydrogel. In some embodiments, the polymer (e.g., hydrogel) is adapted to delay the release of oncolytic viruses, and/or immunomodulators (including combinations of immunomodulators) and/or inactive tumor cells.

In some embodiments, there is provided a pharmaceutical composition comprising: a) adenovirus serotype 5, in which the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced by the human E2F-1 promoter and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine, such as GM-CSF); b) an immunomodulator (including combinations of immunomodulators), and c) a pharmaceutically acceptable excipient suitable for topical administration of the composition to a tumor site. In some embodiments, the pharmaceutical composition further comprises a plurality of non-activated tumor cells. In some embodiments, the plurality of non-activated tumor cells are autologous, allogeneic, derived from a tumor cell line, or a combination thereof. In some embodiments, the plurality of non-activated tumor cells are inactivated by irradiation. In some embodiments, the immunostimulant is an activator of OX40, 4-1BB, or CD40 (e.g., an agonist antibody). In some embodiments, the immune modulator is a modulator of an immune checkpoint molecule selected from the group consisting of (e.g., an antibody): CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the pharmaceutical composition comprises a combination of immune modulators including one or more immune checkpoint inhibitors and/or one or more immune stimulators (e.g., a combination of a CTLA-4 inhibitor and a CD40 activator, or a combination of a CTLA-4 inhibitor and a4-1BB activator). In some embodiments, the excipient is suitable for direct administration of the adenovirus and/or the immunomodulator (including combinations of immunomodulators) and/or the non-activated tumor cell into a tumor. In some embodiments, the excipient is suitable for administering the adenovirus and/or the immunomodulator (including combinations of immunomodulators) and/or the non-activated tumor cell to a tissue having a tumor. In some embodiments, the excipient is a polymer, such as a hydrogel. In some embodiments, the polymer (e.g., hydrogel) is adapted to delay the release of adenovirus, and/or immunomodulator (including combinations of immunomodulators), and/or non-activated tumor cells.

In some embodiments, there is provided a pharmaceutical composition comprising: a) CG 0070; b) an immunomodulator (including combinations of immunomodulators), and c) a pharmaceutically acceptable excipient suitable for topical administration of the composition to a tumor site. In some embodiments, the pharmaceutical composition further comprises a plurality of non-activated tumor cells. In some embodiments, the plurality of non-activated tumor cells are autologous, allogeneic, derived from a tumor cell line, or a combination thereof. In some embodiments, the plurality of non-activated tumor cells are inactivated by irradiation. In some embodiments, the immunostimulant is an activator of OX40, 4-1BB, or CD40 (e.g., an agonist antibody). In some embodiments, the immune modulator is a modulator of an immune checkpoint molecule selected from the group consisting of (e.g., an antibody): CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands. In some embodiments, the pharmaceutical composition comprises a combination of immune modulators including one or more immune checkpoint inhibitors and/or one or more immune stimulators (e.g., a combination of a CTLA-4 inhibitor and a CD40 activator, or a combination of a CTLA-4 inhibitor and a4-1BB activator). In some embodiments, the excipient is suitable for administering CG0070 and/or an immunomodulator (including a combination of immunomodulators) and/or non-activated tumor cells directly into a tumor. In some embodiments, the excipient is suitable for administering CG0070 and/or an immunomodulatory agent (including combinations of immunomodulatory agents) and/or non-activated tumor cells to a tissue having a tumor. In some embodiments, the excipient is a polymer, such as a hydrogel. In some embodiments, the polymer (e.g., hydrogel) is adapted to delay the release of CG0070, and/or an immunomodulatory agent (including combinations of immunomodulatory agents) and/or non-activated tumor cells.

The pharmaceutical composition may include any suitable excipient, including active or passive excipients for drug delivery,such as polymeric and non-polymeric systems. In some embodiments, the excipient is a natural polysaccharide, such as an exopolysaccharide hydrogel. Exemplary polymers suitable for use as excipients for pharmaceutical compositions include, but are not limited to, non-biodegradable polymers such as silicones, crosslinked PVA, and EVA; biodegradable natural polymers such as gelatin, collagen, telogen, scleroglucan, gellan gum and guar gum; biodegradable synthetic polymers such as PLA, PGA, PLGA, polycaprolactone, poly-p-dioxanes, polyphosphoesters, polyanhydrides, and polyphosphazenes. Other systems that may be used as excipients include microspheres and nanospheres with or without polymers, including "smart" polymer systems, including pH-reactive dendrimers, such as poly-amidoamide (PAMAM) dendrimers, poly (propyleneimine) dendrimers, poly (L-iononate) esters, poly (hydroxyproline), poly (propylacrylic acid), poly (methacrylic acid), poly (methyl methacrylate), poly (ethyl methacrylate),poly-silicon amine,S-100、L-100, chitosan, poly (methacrylic acid) (PMMA), PMAA-PEG copolymer, Maleic Anhydride (MA), N-dimethylaminoethyl methacrylate (DMAEMA); temperature-reactive polymers, e.g. poloxamersPlastin (Prolastin), poly (N-substituted acrylamide), poly (organophosphazene), cyclotriphosphazene with poly (ethylene glycol) and amino acid ester, block copolymers of poly (ethylene glycol)/poly (lactic acid-co-glycolic acid), poly (ethylene glycol) (PEG), poly (propylene glycol) (PPG), PMAA, poly (vinyl alcohol) (PVA), various silk-elastin like polymers, poly (silicon amine), poly (vinyl methyl ether) (PVME), poly (vinyl methyl oxazolinone) (PVMO), poly (vinyl pyrrolidone) (PVP), poly (N-vinyl pyrrolidone) (PVP)Caprolactam), poly (N-vinyl isobutyl amide), poly (vinyl methyl ether), poly (N-vinyl caprolactam) (PVCL), poly (siloxyethylene glycol), poly (dimethylaminoethyl methacrylate), triblock copolymers poly (DL-lactide-co-glycolide-b-glycol-b-DL-lactide-co-glycolide) (PLGA-PEG-PLGA), cellulose derivatives, alginates, gellan gum, xyloglucan; magnetic field sensitive polymers such as poly (N-isopropylacrylamide) (PNIPAAm); a hydrogel comprising a ferromagnetic material PNIPAAm-co-acrylamide; electrical signal sensitive polymers such as chitosan, sulfonated polystyrene, poly (thiophene), poly (ethyl oxazoline); ionic polymers, e.g. sodium alginate (Ca)²⁺) Chitosan (Mg)²⁺) (ii) a And photosensitive polymers such as modified poly (acrylamide).

In some embodiments, the infectious agent, immunomodulator (including combinations of immunomodulators), and non-activated tumor cell may be formulated separately or together in a polymer (e.g., a hydrogel) in a pharmaceutical composition. The polymer (e.g., hydrogel) may enable delayed release of one or more components of the pharmaceutical composition, i.e., any one or combination of the infectious agent, the immunomodulator (including combinations of immunomodulators), and the non-activated tumor cell. One or more components of the polymer (e.g., hydrogel) formulation may delay release of the component at the site of administration for at least any one of 1 minute, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, or longer. The polymer (e.g., hydrogel) may comprise any of the suitable materials, such as natural or synthetic polymers known in the art. In some embodiments, the polymer is biodegradable and biocompatible.

The components of the compositions (e.g., pharmaceutical compositions) described herein, including the infectious agent, the immunomodulator (including a combination of immunomodulators) and the plurality of non-activated tumor cells, can be present at a specific relative ratio to each other. In some embodiments, the relative ratio of infectious agent to non-activated tumor cells is based on the fold infection (MOI) index, which is the number of particles of the infectious agent used versus the number of particles of the infectious agent aloneThe number of tumor cells that are not activated or the total number of viable tumor cells (including the estimated number of tumor cells that are not activated and viable tumor cells at the site of administration). In some embodiments, the MOI is at least about 1,2, 5, 10, 50, 100, 200, 500, 1000, 5000, 10⁴、10⁵、10⁶Or any of the larger. In some embodiments, the infectious agent is provided in an amount proportional to the estimated volume of the tumor site. In some embodiments, the tumor cells that are not activated are provided in an amount limited by: preparation from tumor biopsy, tumor resection, tumor cell culture, and other methods known in the art for isolating tumor cells. In some embodiments, the infectious agent is at about 1 × 10⁵To about 1X 10¹⁴A particle (e.g. about 1X 10)¹²Individual particles) are provided in the composition. In some embodiments, the tumor cells that are not activated are at about 1 × 10³Cell to about 1X 10⁸One cell (e.g., about 1X 10)⁵Non-activated tumor cells) are provided in the composition. In some embodiments, the immunomodulator (including the combination of immunomodulators) is provided in the composition at from about 0.1mg/Kg to about 100mg/Kg body weight (e.g., about 1mg/Kg body weight).

In some embodiments, the total amount of the composition is sufficient for a single, locally administered (e.g., intratumoral injection or intravesical administration) full dose. In some embodiments, the total amount of the composition is sufficient for a single locally administered (e.g., intratumorally injected) divided dose to one of the plurality of tumor sites. In some embodiments, the total amount of the composition is sufficient for multiple local administrations (including a combination of a single local administration (e.g., intratumoral injection) in one tumor site and multiple divided dose administrations at multiple tumor sites).

Infectious agent

The methods and compositions described herein pertain to infectious agents, including, but not limited to, bacteria (e.g., BCG) and viruses (including viral vectors, such as oncolytic viruses, e.g., oncolytic adenoviruses). The infectious agent may be a natural infectious agent or a genetically modified infectious agent, such as an attenuated infectious agent, and/or an infectious agent with additional advantageous characteristics (e.g., preferentially replicates or encodes an immune-related molecule in cancer cells).

In some embodiments, the infectious agent is a virus. Exemplary viruses suitable for use in the present invention include, but are not limited to, adenoviruses, such as H101CG-TG-102(Ad5/3-D24-GM-CSF) and CG 0070; herpes simplex viruses, e.g. Talimogene laherparapavec (T-VEC) and HSV-1716Rio viruses, e.g.Vaccinia viruses, such as JX-594; seneca valley viruses, such as NTX-010 and SVV-001; newcastle disease viruses such as NDV-NS1 and GL-ONC 1; polioviruses, such as PVS-RIPO; measles viruses, such as MV-NIS; coxsackie viruses, e.g. Cavatak^TM(ii) a Vesicular stomatitis virus; malaba and rhabdoviruses; parvovirus and mumps virus.

In some embodiments, the infectious agent is a bacterium, such as a mycobacterium and derivatives thereof, or listeria monocytogenes. Exemplary mycobacteria and derivatives thereof include, but are not limited to, bacillus calmette-guerin ("BCG") and mycobacterial cell wall-DNA complexes ("MCNA" or "MCC", e.g., UROCIDIN)^TM)。

In some embodiments, the infectious agent is a wild-type infectious agent. In some embodiments, the infectious agent is genetically modified. In some embodiments, the infectious agent is attenuated (e.g., via multiple passages, inactivation, or genetic modification). In some embodiments, the infectious agent is only one or more portions of a wild-type infectious agent that can cause infection, inflammation, or infection-like effects.

In some embodiments, the infectious agent is a non-oncolytic virus. In some embodiments, the non-oncolytic virus is a wild-type non-oncolytic virus. In some embodiments, the non-oncolytic virus is genetically modified. In some embodiments, the non-oncolytic virus is attenuated (e.g., via multiple passages, inactivation, or genetic modification). In some embodiments, the non-oncolytic virus does not replicate. In some embodiments, the non-oncolytic virus is replication competent. In some embodiments, the non-oncolytic virus preferentially replicates in cancer cells. In some embodiments, the non-oncolytic virus comprises a tumor cell specific promoter operably linked to a viral gene essential for viral replication and a viral vector comprising a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine) operably linked to the viral promoter. In some embodiments, the immune-related molecule is GM-CSF.

In some embodiments, the infectious agent is an oncolytic virus, such as an oncolytic adenovirus. In some embodiments, the oncolytic virus is a wild-type oncolytic virus. In some embodiments, the oncolytic virus is genetically modified. In some embodiments, the oncolytic virus is attenuated (e.g., via multiple passages, inactivation, or genetic modification). In some embodiments, the oncolytic virus is competent for replication. In some embodiments, the oncolytic virus preferentially replicates in cancer cells.

In some embodiments, the infectious agent is an oncolytic virus (e.g., an oncolytic adenovirus) that includes a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication. In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as a human E2F-1 promoter or an E2F-1 promoter comprising a nucleotide sequence set forth in SEQ ID NO:1, shown below. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4.

SEQ ID NO:1

In some embodiments, the infectious agent is an oncolytic virus (e.g., an oncolytic adenovirus) that includes a viral vector comprising a tumor cell-specific promoter operably linked to a viral gene essential for viral replication and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine) operably linked to the viral promoter. In some embodiments, the tumor specific promoter is an E2F-1 promoter, such as the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO. 1. In some embodiments, the viral genes necessary for viral replication are selected from the group consisting of: E1A, E1B and E4. In some embodiments, the viral promoter operably linked to the nucleic acid encoding the immune-related molecule is the E3 promoter. In some embodiments, the immune-related molecule is GM-CSF.

In some embodiments, the infectious agent is adenovirus serotype 5, wherein the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced with the human E2F-1 promoter and a nucleic acid encoding an immune-related molecule (e.g., an interleukin or a chemokine, such as GM-CSF). In some embodiments, the tumor specific promoter is the human E2F-1 promoter or the E2F-1 promoter comprising the nucleotide sequence set forth in SEQ ID NO: 1.

In some embodiments, the infectious agent is CG0070, an adenovirus serotype 5 with the E2F promoter of the E1a gene and GM-CSF expression of the E3 gene.

CG0070 is a conditionally replicating oncolytic adenovirus (serotype 5) designed to preferentially replicate in and kill Rb pathway deficient cancer cells. This vector is transcriptionally regulated by a promoter that is upregulated in Rb pathway deficient tumor cells (e.g., the E2F-1 promoter). In about 85% of all cancers, one or more genes of the Rb pathway (e.g., the tumor suppressor Rb gene) are mutated. CG0070 encodes, in addition to its proliferation-limiting, the human interleukin GM-CSF, which is selectively expressed in infected tumor cells to stimulate an immune response against uninfected distant (e.g., metastasis) and local tumor foci.

The genomic structure of the oncolytic adenoviral vector CG0070 is schematically shown in FIG. 1. The product of the adenovirus early E1A gene is required for adequate expression of other regions of the adenovirus genome. CG0070 is engineered to express the E1A gene under the control of the human E2F-l promoter, which human E2F-l promoter provides tumor specificity for the E1A gene product. To prevent transcriptional readthrough activation of E1A expression, a polyadenylation signal (PA) was inserted 5' to the E2F-1 promoter. CG0070 includes the entire wild type E3 region except the 19 kD-coding region. Direct comparison of the E3-containing oncolytic adenoviral vector with the E3-deleted oncolytic adenoviral vector shows the advantage of the E3-containing vector in tumor spread and efficacy. In place of the 19kD gene, CG0070 carries the cDNA of human GM-CSF under the control of the endogenous E3 promoter (E3P). Since the E3 promoter is in turn activated by E1A, both viral replication and GM-CSF expression are ultimately under the control of the E2F-1 promoter. The remainder of the viral vector backbone, including E2, E4, late protein regions, and Inverted Terminal Repeats (ITRs), is identical to the wild-type Ad5 genome.

CG0070 was prepared in HeLa-S3 cells and released from infected HeLa-S3 cells by detergent lysis. CG0070 was purified from the lysate by chromatography, followed by formulation in 5% sucrose, 10mM Tris, 0.05% polysorbate-80, 1% glycine, 1mM magnesium chloride (pH 7.8).

CG0070 was supplied as a sterile, slightly opalescent, frozen liquid in stoppered glass vials. The particle concentration per mL (vp/mL) is listed on the certificate of analysis for each batch of CG 0070.

An additional potential anti-tumor activity of CG0070 is that it carries the cDNA of human GM-CSF, a key cytokine for the generation of long-lasting anti-tumor immunity. Thus, CG0070 is a selectively replicating oncolytic vector with the potential to attack tumors by two mechanisms: direct cytotoxicity as a replicating vector and induction of host immune response. In vitro and in vivo studies performed to characterize tumor selectivity and anti-tumor activity and safety of CG0070 are summarized in the following section.

Immunomodulator

In some embodiments, the methods of the invention comprise administering an infectious agent with an immunomodulator.

an "immunomodulator" refers to an agent that alters, inhibits or stimulates the immune system of the body in the presence, an immunomodulator may target a specific molecule (e.g., a checkpoint molecule), or non-specifically modulate an immune response, an immunomodulator may include a composition or formulation that activates the immune system (e.g., an adjuvant or activator) or a composition or formulation that down-regulates the immune system, an adjuvant may include an aluminum-based composition, and a composition that includes a bacterial or mycobacterial cell wall component, an activator may include a molecule that activates antigen presenting cells to stimulate a cellular immune response, for example, an activator may be an immunostimulatory peptide, an activator may include, but is not limited to, agonists of toll-like receptors TLR-2, 3, 4,6, 7,8 or 9, granulocyte macrophage colony stimulating factor (GM-CSF), TNF, CD L, CD28, FLT-3 ligands, or interleukins, such as IL-1, IL-2, IL-4, IL-7, IL-12, IL-15 or IL-21, may include agonists of activated receptors on T cells (including co-receptor agonists, such as agonists of activation receptors), or agonists such as IL-1, IL-2, IL-638, or IL-7, IL-11, IL-7, IL-12, IL-21, or IL-2 antagonist, such as a antagonist, or IL-11 antagonist, such as a antagonist, or a antagonist, such as a immunosuppressive antagonist, or antagonist for immune receptor antagonist, such as a antagonist, or antagonist, such as a antagonist for immune receptor antagonist, or a antagonist, such as a antagonist, or a for immune receptor antagonist, for example, or a antagonist, such as an immune receptor antagonist for the like immune receptor antagonist, such as a antagonist of immune receptor antagonist, or for the like immune receptor antagonist of immune receptor antagonist, such as a antagonist, or a for the like immune receptor antagonist, or for the general antagonist of immune receptor antagonist, such as a antagonist of immune receptor antagonist, such as IL-6, or for example, such as a antagonist of immune receptor antagonist of immune system, such as IL-6, or for the immune receptor antagonist.

Immune modulators of particular interest in the present invention include immune stimulants and immune checkpoint inhibitors. As used herein, the terms "immune checkpoint inhibitor", "checkpoint inhibitor" and the like refer to a compound that inhibits the activity of the control mechanisms of the immune system. Immune system checkpoints or immune checkpoints are inhibitory pathways in the immune system that are commonly used to maintain self-tolerance or to modulate the duration and magnitude of physiological immune responses to minimize collateral tissue damage. Checkpoint inhibitors may inhibit immune system checkpoints in a pathway by stimulating the activity of a stimulatory checkpoint molecule or inhibiting the activity of an inhibitory checkpoint molecule. Stimulatory checkpoint molecules are molecules (e.g., proteins) that stimulate or positively modulate the immune system. Inhibitory checkpoint molecules are molecules (e.g., proteins) that inhibit or negatively regulate the immune system. Immune system checkpoint molecules include, but are not limited to, cytotoxic T-lymphocyte antigen 4(CTLA-4), programmed cell death 1 protein (PD-1), programmed cell death 1 ligand 1(PD-L1), programmed cell death 1 ligand 2(PD-L2), lymphocyte activation gene 3(LAG3), B7-1, B7-H3, B7-H4, T cell membrane protein 3(TIM3), B-and T-lymphocyte detoxifying agents (BTLA), T cell activation suppressors containing V-domain immunoglobulin (Ig) (VISTA), killer immunoglobulin-like receptors (KIR), and A2A adenosine receptor (A2 aR). Thus, checkpoint inhibitors include antagonists of CTLA-4, PD-1, PD-L1, PD-L2, LAG3, B7-1, B7-H3, B7-H4, BTLA, VISTA, KIR, A2aR, or TIM 3. For example, antibodies that bind to CTLA-4, PD-1, PD-L1, PD-L2, LAG3, B7-1, B7-H3, B7-H4, BTLA, VISTA, KIR, A2aR, or TIM3 and antagonize their function are checkpoint inhibitors. In addition, any molecule (e.g., peptide, nucleic acid, small molecule, etc.) that inhibits the inhibitory function of an immune system checkpoint is a checkpoint inhibitor.

The immunomodulator may be any of the molecular modalities known in the art, including but not limited to aptamers, mRNA, siRNA, microrna, shRNA, peptides, antibodies, anti-transporters, spherical nucleic acids, TALENs, zinc finger nucleases, CRISPR/Cas9, and small molecules.

In some embodiments, the immunomodulatory agent is an immunostimulatory agent. In some embodiments, the immunostimulatory agent is a natural or engineered ligand for an immunostimulatory molecule, including, for example, a ligand for OX40 (e.g., OX40L), a ligand for CD28 (e.g., CD80, CD86), a ligand for ICOS (e.g., B7RP1), a ligand for 4-1BB (e.g., 4-1BBL, Ultra4-1BBL), a ligand for CD27 (e.g., CD70), a ligand for CD40 (e.g., CD40L), and a ligand for a TCR (e.g., an MHC class I or class II molecule, IMCgp 100). In some embodiments, the immunostimulatory agent is an antibody selected from the group consisting of: anti-CD 28 (e.g., TGN-1412), anti-OX 40 (e.g., MEDI6469, MEDI-0562), anti-ICOS (e.g., MEDI-570), anti-GITR (e.g., TRX518, INBRX-110, NOV-120301), anti-41-BB (e.g., BMS-663513, PF-05082566), anti-CD 27 (e.g., BION-1402, Wallimumab, and hCD27.15), anti-CD 40 (e.g., CP870,893, BI-655064, BMS-986090, APX005M), anti-CD 3 (e.g., brilimumab, Moluomab), and anti-HVEM. In some embodiments, the antibody is an agonistic antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an antigen binding fragment selected from the group consisting of: fab, Fab ', F (ab') of full-length antibody₂Fv, scFv, and other antigen binding subsequences. In some embodiments, the antibody is a human, humanized or chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other functional variant or derivative thereof.

In some embodiments, the immune modulator is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a natural or engineered ligand of an inhibitory immune checkpoint molecule, including, for example, a ligand of CTLA-4 (e.g., B7.1, B7.2), a ligand of TIM3 (e.g., galectin-9), a ligand of the A2a receptor (e.g., adenosine, Regadenoson), a ligand of LAG3 (e.g., MHC class I or MHC class II molecules), a ligand of BTLA (e.g., HVEM, B7-H4), a ligand of KIR (e.g., MHC class I or MHC class II molecules),(iii) a ligand of PD-1 (e.g., PD-L1, PD-L2), a ligand of IDO (e.g., NKTR-218, indoximol, NLG919) and a ligand of CD47 (e.g., SIRP- α receptor). in some embodiments, the immune checkpoint inhibitor is an antibody targeting an inhibitory immune checkpoint protein. in some embodiments, the immunomodulator is an antibody selected from the group consisting of anti-CTLA-4 (e.g., ipilimumab, tremelimumab, KAHR-102), anti-TIM 3 (e.g., F38-2E2, ENUM005), anti-LAG 3 (e.g., anti-LAG-986016, IMP701, IMP321, C9B7W), anti-KIR (e.g., rillizumab and IPH2101), anti-PD-1 (e.g., nivolumumab, pidilizumab, pembrolizumab, BMS-6559, azulizumab, Ak-34601514, and anti-IPH 2101), anti-PD-CD-NO, anti-CD-NO-7, anti-CD-NO-7, anti-NO-7, such as anti-NO-7, anti-NO-7, anti-NO-7, anti-NO-7, NO-7, NO-7, NO-7, NO-7, NO-NO₂Fv, scFv, and other antigen binding subsequences. In some embodiments, the antibody is a human, humanized or chimeric antibody. In some embodiments, the antibody is a bispecific antibody, a multispecific antibody, a single domain antibody, a fusion protein comprising an antibody portion, or any other functional variant or derivative thereof.

The immunomodulators can be used alone or in combination. For example, any number (e.g., any of 1,2, 3, 4, 5, 6, or more) of immune checkpoint inhibitors may be used simultaneously or sequentially, or any number (e.g., any of 2, 3, 4, 5, 6, or more) of immune stimulants may be used simultaneously or sequentially. Alternatively, any number (e.g., any of 1,2, 3, 4, 5, 6, or more) of immune checkpoint inhibitors in combination with any number (e.g., any of 2, 3, 4, 5, 6, or more) of immunostimulatory agents may be used simultaneously or sequentially. Sequential administration of the immunomodulator may be separated by hours, days or weeks. The route of administration of the two or more immunomodulators may be the same or different. For example, one immunomodulator may be administered intratumorally and a second immunomodulator may be administered intravenously; or both immunomodulators may be administered intratumorally.

Exemplary immune checkpoint molecules and immune modulators thereof are discussed below. It is understood that other suitable immune checkpoint molecules and immune modulators known in the art are also within the scope of the present patent application.

CTLA-4

CTLA-4 is an immune checkpoint molecule that is upregulated on activated T cells. anti-CTLA-4 mabs can block CTLA-4 interactions with CD80/86 and switch off the mechanism of immunosuppression and enable continuous T cell stimulation by DCs. Examples of anti-CTLA-4 antibodies are ipilimumab (see US patents 6,984,720, 7,452,535, 7,605,238, 8,017,114 and 8,142,778), tremelimumab (see US patents 6,68,736, 7,109,003, 7,132,281, 7,411,057, 7,807,797, 7,824,679 and 8,143,379) and other anti-CTLA-4 antibodies (including single chain antibodies) (see, e.g., US patents 5,811,097, 6051,227 and 7,229,628 and US patent publication US 20110044953).

Two IgG mabs, ipilimumab and tremelimumab, against CTLA-4 have been tested in clinical trials for a variety of indications. Ipilimumab is approved by the FDA for the treatment of melanoma, e.g., for advanced stage melanoma patients. The complete prescription information is fully described in(Bristol Meyers).(ipilimumab) appears as a 50mg single use vial.

Anti-transporters are engineered proteins capable of recognizing and binding with high affinity to specific targets. It is an antibody mimetic, but it is structurally unrelated to antibodies. Rather, it is a human lipocalin derived from a family belonging to the natural binding protein. Anti-carrier protein was used instead of monoclonal antibody, but about 1/8 of the monoclonal antibody was about 180 amino acids in size and about 20kDa in mass. Anti-transporter proteins are described in us patent 7,250,297. Anti-transporters have been developed that bind CTLA-4 with high affinity and specificity, as described, for example, in international patent application publication WO 2012072806. Any of the CTLA-4-binding transporters can be used in the present application. In some embodiments, the CTLA-4 binding anti-transporter is PRS-010(Piers AG).

PD-1

PD-1 is part of the B7/CD28 family of co-stimulatory molecules that regulate T cell activation and tolerance, and therefore, antagonist anti-PD-1 antibodies can be used to overcome tolerance. PD-1 is defined as the receptor for B7-4. B7-4 inhibits immune cell activation when bound to inhibitory receptors on immune cells. Engagement of the PD-1/PD-L1 pathway results in the inhibition of T cell effector functions, interleukin secretion and proliferation. (Turnis et al, OncoImmunology 1(7):1172-1174, 2012). High levels of PD-1 are associated with depleted or chronically stimulated T cells. Furthermore, increased PD-1 expression is associated with decreased survival of cancer patients.

Agents used to down-regulate the interaction between PD-1, B7-4 and B7-4 in immune cells and the PD-1 inhibitory signal cause an enhancement of the immune response. Any of the anti-PD-1 antibodies known in the art may be used in the present invention, see for example US7101550, US5698520, US6808710, US7029674, US7794710, US7892540, US8008449, US8088905, US8163503, US8168757, US8354509, US8460927, US8609089, US8747833, US 0058779105, US 8987, US8952136, US8981063, US8993731, US9062112, US9067999, US9073994, US9084776, US9102728 and US 7488802; and US patent publications US20020055139, US 20140044738. For example, nivolumab is a human mAb against PD-1 approved by the FDA for the treatment of unresectable or metastatic melanoma as well as squamous non-small cell lung cancer.

PD-L1/PD-L2

PD-L1 (programmed cell death-ligand 1) is also known as Cluster of differentiation 274(CD274) or B7 homolog 1 (B7-H1). PD-L1 acts as a ligand for PD-1 to play a major role in suppressing the immune system during specific events (e.g., pregnancy, tissue allograft, autoimmune diseases and other disease states such as hepatitis and cancer). The formation of the PD-1 receptor/PD-L1 ligand complex transmits inhibitory signals that reduce the proliferation of CD8+ T cells of lymph nodes.

Any of the anti-PD-L1 antibodies are known to be useful in the present invention, see, e.g., U.S. patents US7943743, US7722868, US8217149, US 83796, US8552154, and US 9102725; and U.S. patent application publications US20140341917 and US 20150203580; and international patent application PCT/US 2001/020964. For example, anti-PD-L1 antibodies in clinical development include BMS935559 (also known as MDX-1105), MPDL3280A, MEDI4736, avizumab (also known as MSB0010718C), KY-1003, MCLA-145, RG7446 (also known as acilizumab), and STI-a 1010.

PD-L2 (programmed cell death 1 ligand 2) is also known as B7-DC. PD-L2 acts as a ligand for PD-1. In certain instances, PD-L2 and its inhibitors are useful as a replacement for PD-L1 and its inhibitors, respectively.

CD40

CD40 (cluster of differentiation 40) is a costimulatory protein found on antigen presenting cells and is required for activation of such cells. T is_HBinding of CD40L (CD154) to CD40 on the cell activates antigen presenting cells and induces a variety of downstream effects to stimulate an immune response.

Agents that stimulate the activity of CD40 may be used as immunostimulants. Any of the agonistic anti-CD 40 antibodies are known to be useful in the present invention, see, for example, US patents US5786456, US5674492, US5182368, US5801227, US7824683, US6843989, US7618633, US7537763, US5677165, US5874082, US6051228, US6312693, US6315998, US6413514, US6838261, US6843989, US6946129, US7063845, US7172759, US7193064, US7288251, US7338660, US7547438, US7563442, US 7612, US 8778345; and U.S. patent publications US 2003059427, US 20020142358, and US 20050136055; international patent publications WO 02/088186, WO 01/56603, WO 88/06891, WO 94/04570 and WO 05/63289; schlossman et al, Leucocyte Typing,1995,1: 547-556; and Paulie et al, 1984, cancer Immunol.Immunother.17: 165-179. For example, agonistic anti-CD 40 antibodies in clinical development include CP-870,893, daclizumab (also known as SGN-40), and ChiLob 7/4 or APX 005M.

OX40

OX40 (also known as CD134 and TNFRSF4) is a member of the TNFR-superfamily of receptors. OX40 is a costimulatory immune checkpoint molecule expressed after 24 to 72 hours after T cell activation. The interaction of OX40L with OX40 will sustain T cell proliferation and immune responses and memory beyond the first two days. Methods of enhancing immune responses to tumor antigens during or shortly after T cell priming by antigen by engaging OX40 receptors on the T cell surface by OX40 receptor binding agents OX40L or OX40 agonists are useful as immune checkpoint inhibitors in CLIVS.

LAG-3

The use of LAG-3 (lymphocyte activation gene-3) and in a more common way MHC class II ligands or MHC class II-like ligands as adjuvants for vaccines to boost antigen-specific immune responses has been successful in preclinical models. Antibodies or agents directed against or modulating the LAG-3 gene product may be helpful in the present invention. For details regarding LAG-3 related patents and claims, see U.S. patent 5773578, cited and referenced patents.

Examples

The following examples are intended to be illustrative of the invention only and therefore should not be construed as limiting the invention in any way. The following examples and detailed description are provided by way of illustration and not by way of limitation.

Example 1: intravesical administration of CG0070 and CTLA-4 inhibitors in patients with muscle-invasive bladder cancer Combined phase I/II clinical study of

This example illustrates a clinical study of intravesical administration of CG0070 in combination with anti-CTLA-4 antibodies in patients with Muscle Invasive Bladder Cancer (MIBC). Muscle invasive bladder cancer is chosen as an example herein, since CG0070 has been shown to be active in bladder cancer. In addition, all muscle invasive bladder cancer patients require cystectomy, thereby providing a good tumor sample to prepare the tumor cells required for this vaccine system. In addition, despite the use of lead chemotherapy, the prognosis for patients with muscle invasive bladder cancer (T3-4) is poor. Most of these patients are over 60 years old and rarely can experience the severe side effects of chemotherapy. There is an unmet need for effective agents that can minimize the risk of disease recurrence in this patient population.

This clinical study is a phase I/II, one-armed, open label, interventional dose escalation safety and performance study of combinations of intravesical CG0070 and CTLA-4 inhibitors as a lead therapy in patients with transitional cell muscle invasive bladder cancer disease who were selected for radical cystectomy and pelvic lymphadenectomy. The primary safety objective of the study was to investigate whether CG0070 and CTLA 4 blockade prior to cystectomy was safe and tolerated for leading treatment of MIBC patients. The primary performance goal of the study was to measure changes in tumor PD-L1 or PD-1 content following treatment leading to CG0070 and CTLA-4 inhibitors. Secondary study objectives included assessing 2-year disease-free survival (DFS), 2-year progression-free survival (PFS), Overall Survival (OS), the proportion of pathological complete responses at cystectomy (proportion of p 0), the proportion of pathological staged reductions at cystectomy, and the proportion of organ-restricted disease at cystectomy.

In phase I portion of the study, a cohort of (e.g., 3 to 6) patients received intravesical CG0070 and CTLA 4 blockade at one of 4 dose values. The first dose value consisted of CG0070 only. Each patient received 4 instillations of an intravesical CTLA-4 inhibitor (e.g., ipilimumab) 4 times per week (e.g., on day 1 of each week) and 3 times per week from two weeks (e.g., on days 8, 15, and 22) at one of 4 dose values, and administration of the CTLA-4 inhibitor was after CG 0070.

Dose escalation follows a modified Fibonacci sequence, where dose increments become smaller as the dose increases. For example, if none of the first three patients in the cohort experienced dose-limiting toxicity, the other three patients would be treated with the next higher dose value. However, if one of the first three patients experiences dose-limiting toxicity, the other three patients will be treated with the same dose value. Dose escalation continues until at least 2 patients in the cohort of 3 to 6 patients experience dose limiting toxicity (i.e., > 33% of the patients have dose limiting toxicity at this dose value). The recommended dose for the next stage or phase of the assay is generally defined as the value of the dose just below the toxic dose value. Dose-limiting toxicity (DLT) is defined using the common adverse event evaluation criteria (CTCAE) version 4. DLT is defined as ≧ 3 drug-related Adverse Events (AEs) from day 1 of week 1 to day 1 of week 4 of treatment, including any grade 3 or higher toxicity requiring discontinuation of study treatment for more than 3 consecutive weeks and/or discontinuation permanently due to immune-related toxicity, but excluding tumor-exacerbated grade 3 AEs (defined as localized pain, irritation, or rash located at a known or suspected tumor site) and grade 3 immune-mediated events of the skin (rash, itch) or endocrine system (hypothyroidism, hyperthyroidism, hypophysis, adrenal insufficiency, hypogonadism, and Cushingoid syndrome), which return to grade 1 or baseline within 3 weeks with or without administration of a steroid. Liver immunotoxicity is defined as a grade 3 or higher increase in aspartate aminotransferase, alanine aminotransferase or total bilirubin. A significant D-dimer increase (20% increase from baseline at least 1 μ g/mL) combined with >2 grade changes in INR, PT, PTT, platelets, or fibrinogen for >7 days was considered DLT. In addition, clinically significant thrombosis or hemorrhage associated with CG0070 treatment was considered DLT. Patients who have treatment delayed more than 21 days due to toxicity associated with study treatment are considered to have treatment-equivalent DLTs. Patients who were delayed in treatment in the replacement cohort by more than 7 days or who exited the study before 3 administrations for reasons other than treatment-related toxicity. The Maximum Tolerated Dose (MTD) is the dose immediately before 2DLT is produced. The highest dose administered without 2DLT will be MFD if the maximum feasible dose (MTD) is undefined. The patient dose in this study was not allowed to decrease. However, if at least 2 of the 6 patients in dose value 1 experience a DLT, then 3 patients will be eligible for dose value 1. Furthermore, if at least 2 of the 6 patients in dose value 1 experience a DLT, then 3 patients will be eligible for a dose value of 2.

For example, dosage values I include those at 1X 10¹²Doses of individual virus particles (vp) were intravesically administered CG0070 alone once a week for 4 weeks. Dosage values II include: (1) at 1 × 10¹²A dose of individual viral particles (vp) administered intravesically, once a week for 4 weeks, CG 0070; and (2) intravesical administration of a CTLA-4 inhibitor (e.g., ipilimumab) at a dose of 0.1mg/Kg, but not more than 20mg total per dose, immediately after CG0070 instillation and drainage over 3 weeks, starting at week 2 and ending at week 4. Dosage values III include: (1) at 1 × 10¹²A dose of individual viral particles (vp) administered intravesically, once a week for 4 weeks, CG 0070; (2) CTLA-4 inhibitor (e.g., ipilimumab) was administered intravesically at a dose of 0.2mg/Kg, but not more than 20mg total per dose, immediately after CG0070 instillation and drainage, weekly over 3 weeks, starting at week 2 and ending at week 4. Dosage values IV include: (1) at 1 × 10¹²A dose of individual viral particles (vp) administered intravesically, once a week for 4 weeks, CG 0070; and (2) 0.3mg/Kg immediately after CG0070 instillation and drainage, but not exceeding total per doseThe CTLA-4 inhibitor (e.g., ipilimumab) is administered intravesically weekly over a dose of 20mg for 3 weeks, starting at week 2 and ending at week 4.

In phase II portion of the study, each patient was administered intravesically a combination of CG0070 and CTLA-4 inhibitor over the course of 4 weeks of treatment at the dose values determined in phase I portion of the study. During both phase I and phase II portions of the study, prior to administration of intravesical therapy, each patient was evaluated for adverse events and samples (e.g., blood and urine samples) were collected for laboratory evaluation. For example, prior to the first intravesical administration of CG0070, blood and urine samples are collected from each patient to assess GM-CSF levels, as well as CG0070 and wild-type adenovirus levels. Prior to each of the week 2, 3, and 4 administrations, samples of the patients were collected for laboratory evaluation of hematology (e.g., CBC with differences, chemistry, and coagulation), serum chemistry (e.g., sodium, potassium, chloride, BUN, creatinine, glucose, total protein, albumin, calcium, total bilirubin, direct bilirubin, alkaline phosphate, LDH, AST, ALT, and thyroid function), and urinalysis. Vital signs (including blood pressure, pulse, respiration, and temperature) were recorded before each CG0070 treatment and every hour for a total of 2 hours during the treatment to ensure that the patient was clinically stable.

CG0070 and CTLA-4 inhibitors may be administered as follows. Patients are advised not to drink fluid for 4 hours prior to treatment and should void their bladder prior to treatment administration. On the day of the study, each patient received pre-treatment with intravesically administered transduction enhancing agent (DDM) via catheter (Rusch 173430 Foley catheter and BARD LUBRI-SIL Foley catheter No. 70516 SI). Pretreatment included an intravesical wash with 100mL of saline followed by 75mL of 0.1% DDM. The patient then received an intravesical instillation of 100mL of 0.1% DDM, which was retained in the bladder for 12-15 minutes, and then rinsed with 100mL saline. If the patient is unable to tolerate DDM pretreatment for at least 5 minutes, further treatment with CG0070 and CTLA-4 inhibitors should be discontinued for the treatment. If intravesical infusion of CG0070 is delayed by more than 2 hours after DDM pretreatment, the patient will not receive CG0070 and must spend 2 daysDDM and CG0070 treatments are rescheduled shortly thereafter. If treatment is delayed by more than 2 weeks, the patient must continue to meet eligibility criteria before being retreated. After pretreatment with DDM, each patient received 100mL via a catheter (e.g., Rusch 173430 Foley catheter and BARD LUBRI-SIL Foley catheter number 70516SI) at a concentration of 1.0X 10¹⁰Single intravesical instillation of vp/mL CG0070 with residence time of 45 to 50 minutes. Treatment must occur at least 14 days after any prior bladder biopsy. Patients who experience bleeding during catheterization (traumatic catheterization) should not be treated with CG 0070. While CG0070 remains in the bladder, the patient should be repositioned from left to right, and should also lie on the back and abdomen to maximize exposure of the bladder surface to CG 0070. The patient position was changed every 10-12 minutes for a total of 45 to 50 minutes. CG0070 is then discharged into the treatment bag via the catheter. Once the CG0070 solution is excreted from the bladder, an appropriate dose of CTLA-4 inhibitor (e.g., ipilimumab, e.g., ipilimumab)) (e.g., dose values for phase I study, excluding any CTLA-4) were diluted into 100ml of saline and instilled into the bladder. Following instillation, the urethral catheter is then withdrawn and the patient is asked to remain for an additional 45min to 1 hour (or as long as possible) before being emptied by urination.

After a 6-week course of treatment in the phase II portion of the study, each patient received cystectomy. Cystectomy is performed 10 to 14 days after the last intravesical treatment (e.g., about day 40) or once any treatment-related toxicity has subsided and the medical condition is suitable for surgery. Following cystectomy, tumor samples are obtained from the patient and evaluated in a pathology laboratory, and laboratory evaluations are performed to determine whether the patient is responsive to treatment. This evaluation included pathological and immunological evaluation of resected tumors: (1) tumor stage and grade (if present); (2) tumor immune parameters such as Treg, CD4, CD8, and other T cell subsets; (3) detecting the expression status of tumor PD-L1 by immunohistochemical method; (4) lymph node invasion; (5) macroscopic photographs before and after treatment were compared. Each patient was evaluated at months 3, 6, 12, 18 and 24 (plus or minus 2 weeks) from the date of cystectomy to monitor long-term response and toxicity of CG0070, disease recurrence or progression and subsequent therapy and response. After 2 years, patients were exposed once a year to evaluate long-term toxicity associated with gene therapy (e.g., new malignancies, autoimmune diseases, neurological and hematological disorders, etc.) and five years of survival following the first intravesical CG0070 therapy. Patients were followed up to a total of 5 years after treatment with CG0070 or according to current FDA guidelines and current standards of care.

The primary outcome measure of the study was determined as follows. Patients were followed throughout the study and after completion of the study to evaluate AE, SAE and SUSAR to determine the safety and tolerability of treatment. In addition, at the time of cystectomy, the performance of the treatment was assessed by determining the rate of change of PD-L1 and PD-1 status, defined as the difference in the proportion of patients who were PDL1 or PD1 positive before and after at least three or more completed intravesical instillations interventions.

Secondary outcome measures for the study were determined as follows. At the time of cystectomy, the proportion of pathologically complete response (p0 proportion) of each T-stage cystectomy was assessed by determining the proportion of patients with pathologically complete tumor response at the primary tumor site after the cystectomy intervention (by T-staging and further stratification for the entire patient group). The rate of pathological staging reduction at cystectomy, defined as the proportion of patients with staged or graded reduction of the tumor at the primary tumor site following cystectomy intervention, was also determined at cystectomy; and organ-restricted disease proportion at cystectomy, defined as the proportion of patients who did not find positive lymph nodes at cystectomy. Up to 2 years after cystectomy, patients are followed to determine disease-free survival for 2 years, defined as the number of months from the date of cystectomy to early relapse or death (for whatever reason); and 2 years progression free survival of patients with residual disease after cystectomy, defined as the number of months from the date of cystectomy to early disease progression or death (whatever the cause). Up to 5 years after cystectomy, patients were followed to determine overall survival, defined as the number of months from the date of cystectomy to the date of death (for whatever reason).

In addition, exploratory outcome measures to be evaluated during the course of the study include, but are not limited to, changes in immune function within the primary tumor site in the patient, including the evaluation of tregs (CD4+ CD25+ Foxp3+), CD4, CD8, CD4RO45, and CD4ICOShigh, among others, before and after intervention; macroscopic changes in primary tumor sites by photographs taken before and after intervention; systemic absolute lymphocyte count; and systemic interleukin patterns.

Patients must meet all of the following conditions to be eligible for the study.

1.18 years old or older;

2. patients with pathologically diagnosed transitional cell (urinary tract) bladder cancer; wherein the radical cystectomy with curative intent is suitable for muscle invasive disease (i.e., United states Joint Committee for cancer (AJCC) stage T2-4a, N_X-1M0). Patients must be able to enter the study within five weeks of their most recent diagnostic procedures, typically a diagnostic biopsy, a bladder tumor transurethral resection (turbo) procedure, or other diagnostic scans (e.g., CT, MRI, and PET procedures);

3. histopathologically confirmed transitional cell (urinary tract) cancer. Urinary tract tumors with mixed histology (but with < 50% variants) were eligible;

4. due to medical conditions that can be confirmed by researchers, it is not appropriate to receive leading chemotherapy. (e.g., renal impairment may be based on a calculated creatinine clearance of about <60ml/min or a hearing loss ≧ 25dB, as measured by audiometry, averaged over 3 contiguous test frequencies in at least 1 ear; or other significant cardiac dysfunction, vascular disease or chronic obstructive pulmonary disease, etc.), or refusal to accept lead chemotherapy following specific informed consent for relapse and increased risk of morbidity with the introduction of lead-free chemotherapy;

5. the energy status of the clinical research cooperative organization for eastern Bank cancer (ECOG) is less than or equal to 2;

6. non-pregnant or lactating;

7. consent to study informed consent and HIPAA authorization to publish personal health information;

8. baseline CBC and adequate liver function are defined as follows:

a.WBC>3000 cells/mm³，ANC>1,000 cells/mm³Heme (hemoglobin)>9g/dL and platelet count>80,000/mm³；

b. Bilirubin, AST, and ALT are less than the upper limit of the normal value of 2.5 x;

c. adequate clotting with acceptable PT/INR, PTT and fibrinogen (less than the upper normal limit of 1.5 or according to institutional specifications);

d. the absolute lymphocyte count is more than or equal to 800/mu L.

Patients meeting any of the following exclusion criteria were excluded from the study:

1. history of anaphylaxis following exposure to human or human therapeutic humanity or humans, history of anaphylaxis to GM-CSF, clinically meaningful anaphylaxis, or any known allergy history or previous reaction history to any formulation excipient in the study drug;

2. known HIV, HBV or HCV infections;

3. the intended use of chemotherapy or radiation therapy not specified in the study protocol at the time of study;

4. any potential medical condition that would, in the investigator's opinion, render the administration of the study drug harmful to the patient would obscure the interpretation of the adverse event or surgical resection;

5. systemic treatment for any investigational clinical trial within 28 days prior to enrollment;

6. concurrent therapy with other immunosuppressive or immunomodulatory agents, including any systemic steroid (with the exception of steroids that allow for inhalation or topical administration, and acute and chronic standard dose NSAIDs. short courses of therapy (i.e.. ltoreq.1 day) with glucocorticoids are acceptable to prevent response to IV imaging for CT scans;

7. the study entered immunosuppressive therapy within 3 months, including: cyclosporine, antithymocyte globulin, or tacrolimus (tacrolimus);

a history of stage III or greater cancer, excluding urothelial cancer. Basal or squamous cell skin cancers must be adequately treated and the subject must be disease-free at the time of enrollment. Patients with a history of stage I or II cancer must be adequately treated and have been disease free for more than 2 years at the time of registration;

9. concomitant active autoimmune diseases (e.g., rheumatoid arthritis, multiple sclerosis, autoimmune thyroid disease, uveitis);

10. progressive or current viral or bacterial infection. Before being placed in the study, all infections had to resolve and the patients had to remain free of fever for 7 days without antibiotics.

Example 2: intratumoral administration of CG0070 and CTLA-4 inhibition in patients with refractory injectable physical tumors Phase I/II clinical study of combinations of agents

This example illustrates a phase I/II clinical study of CG0070 in combination with a CTLA-4 inhibitor (e.g., anti-CTLA-4 monoclonal antibody or blocking agent) in patients with refractory injectable physical tumors. This study is a multicenter, single-arm, open-label, interventional study aimed at assessing the safety and performance of combination therapy involving intratumoral administration of CG0070 and CTLA-4 inhibitors in patients with physical tumors (including cutaneous or visceral lesions, such as head and neck squamous cell carcinoma, breast cancer, colorectal cancer, pancreatic adenocarcinoma, ovarian cancer, non-small cell lung cancer, prostate cancer, and melanoma). CG0070 administration may include pretreatment with a transduction agent (e.g., DDM).

The clinical study in phase I is divided into three phases. In phase 1, CG0070 is administered to each subject via intratumoral injection every week for 6 weeks (e.g., on day 1 of each week). A cohort of (e.g., 3 to 6) patients received intratumoral CG0070 (e.g., pretreated with DDM) for one of the four dose values. The dose escalation procedure is as described in example 1, and the MTD/MFD determined in phase 1 is used for the start of phase 2.

In phase 2 of phase I, CTLA-4 inhibitor (e.g., anti-CTLA-4 mAb or blocking agent, e.g., ipilimumab) is administered to each subject via intratumoral injection every week (e.g., on day 1 of every week) for 6 weeks. A cohort of (e.g., 3 to 6) patients received the intratumoral CTLA-4 inhibitor at one of three dose values. The dose escalation procedure is as described in example 1, and the MTD/MFD determined in phase 1 is used for the start of phase 3.

In phase 3 of phase I, each subject was administered a combination of CG0070 (e.g., pre-treatment with DDM) at the dose determined in phase 1 of the study and CTLA-4 inhibitor at the dose determined in phase 2 of the study via intratumoral injection every week (e.g., on day 1 of each week) for 6 weeks. A cohort of (e.g., 3 to 6) patients received intratumoral CG0070 (e.g., pretreated with DDM) and CTLA-4 inhibitor sequentially for 6 weeks at one of the three dose values. The dose escalation procedure was as described in example 1. Once the MTD or MFD is reached, the patient receives a repeated 6-week course of treatment 3 months after the first injection (once per week for six weeks constituting a course) and then progresses every 3 months until complete response, disappearance of all injectable tumors, confirmed disease progression, or intolerance to the study treatment, whichever occurs first. Patients in a dose escalation phase of phase 1 or phase 2 portion of the study may be enrolled in a repeat MTD or MFD course study after a period of three months from the last intervention of a fully successful enrollment assessment.

There are two main outcome measures for this study: (1) safety and tolerability; and (2) performance. Safety and tolerability were assessed from the start of each phase until 3 months after enrollment of the last subject in each phase. Stage 1 determines the safety and tolerability of CG0070 (e.g., pretreated with DDM) as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory solid tumors. Stage 2 the safety and tolerability of CTLA-4 inhibitors (e.g., anti-CTLA-4 mabs or blockers) as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory solid tumors. Stage 3 determines the safety and tolerability of the combination of CG0070 (e.g., pretreated with DDM) with CTLA-4 inhibitors (e.g., anti-CTLA-4 mabs or blockers) as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory physical tumors. Performance was assessed from the start of each session until 24 months after enrollment of the last subject of each session. Performance was assessed by confirmed Objective Response Rate (ORR) of stage 1 treatment with CG0070 alone (e.g., using DDM pretreatment), stage 2 treatment with CTLA-4 inhibitor alone (e.g., anti-CTLA-4 mAb or blocker), and stage 3 treatment with a combination of CG0070 (e.g., using DDM pretreatment) and CTLA-4 inhibitor (e.g., anti-CTLA-4 mAb or blocker) in patients with injectable refractory physical tumors.

Secondary outcome measures for this study are as follows. Safety secondary outcomes were evaluated from the beginning of each session until 24 months after enrollment of the last subject of each session. For all three phases, the secondary safety outcome measure includes the incidence of the following events: all Adverse Events (AEs), AE grade 3 or higher, events requiring discontinuation of study drug, local effects on tumors, clinically significant laboratory changes, and clinically significant changes in vital signs. Performance secondary outcomes were evaluated 24 months from the start of each session until the last subject enrolled in each session. Secondary outcome measures of performance include optimal overall response rate (BOR), Disease Control Rate (DCR), Durable Response Rate (DRR), duration of response (DOR), Time To Response (TTR), Progression Free Survival (PFS), Overall Survival (OS), 1 year and 2 year survival for all three stages.

Eligibility of patients of both sexes for the study was determined based on the following inclusion criteria:

1. patients must have histologically confirmed physical tumors that failed standard therapy (surgery, chemotherapy, radiation therapy, or endocrine therapy) and for which there is no curative choice, including but not limited to: squamous cell carcinoma of the head and neck, squamous cell carcinoma of the skin, breast cancer, malignant melanoma, colorectal cancer, pancreatic adenocarcinoma, ovarian cancer, non-small cell lung cancer, and prostate cancer;

2. the patient may already have any kind and number of prior cancer therapies;

3. the patient must have measurable lesions that can be assessed by RECIST method;

4. the tumor mass to be treated must be sufficient for injection (i.e., more than 2cm away from the major vascular structures) and measurement by RECIST;

5. the patient must be more than or equal to 18 years old;

6. the life expectancy of the patient must be more than or equal to 12 weeks;

7. patients must have a us eastern bank cancer clinical research cooperative organization (ECOG) physical status of 0, 1 or 2;

8. the patient must have adequate liver function, defined as follows:

a. the total bilirubin content is less than or equal to 1.5 multiplied by the upper limit of normal value (ULN); and

b. if liver metastasis is present, the AST/ALT content is less than or equal to 2.5 × ULN, or less than or equal to 5 × ULN;

9. the patient must have adequate renal function, as defined below: serum creatinine < 1.5 × ULN, or for creatinine>Patient creatinine clearance (calculated) of 1.5 × ULN ≥ 60mL/min/1.73m²；

10. The patient must have adequate bone marrow function, as defined below:

a. the absolute neutrophilic count is more than or equal to 1,200/mu L; and

b. the platelet count is more than or equal to 80,000/mu L;

11. the patient must be free of known hemorrhagic diathesis or clotting lesions, which would render intratumoral injection or biopsy unsafe;

12. men and women with fertility potential must agree to use appropriate contraceptive measures and for up to six months before study entry;

13. women with fertility potential must be tested for negative urine or serum pregnancy within one week before starting treatment; and

14. the patient must be able to understand and be willing to sign a written informed consent document.

The following patients were excluded from the study:

1. patients receiving chemotherapy, immunotherapy or radiation therapy, or adverse events >1 grade (with the exception of hair loss) resulting from agents administered more than 4 weeks prior to screening within 4 weeks prior to screening;

2. patients with a history of significant tumor bleeding or clotting or bleeding disorders;

3. patients with target tumors that can potentially invade major vascular structures (e.g., innominate arteries, carotid arteries) based on explicit imaging findings as determined by a radiologist;

4. patients with grade 1 or greater pre-existing neurological abnormalities (CTCAE version 4.0);

5. patients hospitalized during the 30 days prior to study entry for an urgent condition requiring hospitalization assessment, treatment or procedure. In addition, an urgent condition requiring hospitalization assessment, treatment or procedure must have resolved or be medically stable and not severe 30 days prior to entering the study;

6. patients with clinically significant Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV) or Epstein-Barr virus (EBV) infection. Testing patients for HIV during pre-treatment screening;

patients receiving steroids or immunosuppressants, e.g., against rheumatoid arthritis;

8. patients concurrently with any other study medication;

9. patients with central nervous system metastases or with a history of such diseases;

10. a female who is pregnant or lactating or who wishes to become pregnant during the study period;

11. patients with uncontrolled intercropping (including but not limited to ongoing or active infection, symptomatic congestive heart failure, unstable angina, cardiac arrhythmia, or psychiatric disease/social situation that would limit compliance to research requirements).

Example 3: intratumoral administration of CG0070 to patients with advanced stage physical tumors (e.g., melanoma) Phase I/II clinical study of combinations of CTLA-4 inhibitors and CD40 agonists

This example illustrates a phase I/II clinical study of patients with solid or lymphoid tumors in combination with a CTLA-4 inhibitor (e.g., anti-CTLA-4 monoclonal antibody or blocker) and a CD40 agonist (e.g., agonistic anti-CD 40 antibody). The phase I study was a dose escalation study of patients with refractory physical tumors. Phase II studies are single-armed, open label, interventional studies aimed at assessing performance, safety and tolerance of repeated intratumoral injections of CG0070, CTLA-4 inhibitors and CD40 agonists in patients with physical tumors (e.g., refractory unresectable, or metastatic stage III/IV malignant melanoma). CG0070 administration may include transduction enhancing agents, such as DDM.

The clinical study in phase I is divided into three phases. Phase 1 is a dose escalation study with intratumoral injection of CG0070 only. A cohort of (e.g., 3 to 6) patients received weekly intratumoral injections of CG0070 (e.g., with DDM) for 4 weeks at one of the following four dose values: 5X 10¹⁰vp、1×10¹¹vp、5×10¹¹vp or 1X 10¹²vp. For example, virus CG0070 was reconstituted in 0.1% DDM in saline. The total volume of each dose was 2 mL. The concentration of CG0070 solution for the lowest dose is about 2.5X 10¹⁰vp/ml and about 5X 10 for the highest dose ¹¹vp/ml. If the patient has a single lesion (which must be greater than 2cm), then the total volume of CG0070 solution is injected into the lesion. If there are two or more lesions, the maximum injection volume based on lesion size as shown in table 1 is followed. If the largest lesion is at least 2cm, any remaining volume is injected into the largest lesion. If the largest lesion is less than 2cm, the remaining volume is divided between the two larger lesions. The maximum number of lesions injected was 3. The total dose is administered regardless of the total number and size of lesions. The dose escalation procedure is as described in example 1, and the MTD/MFD is named dose value stage 1, which is used at the beginning of stage 2.

TABLE 1 injection volume per lesion based on tumor size

Tumor size (longest dimension)	Maximum injection volume
		≥5.0cm	2.0mL
Not less than 2.0cm to 5.0cm	1.0mL
		>0.5cm to 2.0cm	0.5mL

Stage 2 of phase I is a combination of a dose escalating intratumoral injection dose of a CTLA-4 inhibitor (e.g., anti-CTLA-4 mAb or a blocking agent, e.g., ipilimumab) and CG0070 of stage 1. A cohort of (e.g., 3 to 6) patients received weekly intratumoral injections of a fixed dose of CG0070 (e.g., with DDM) in combination with a CTLA-4 inhibitor (e.g., ipilimumab) at one of the following three dose values for 6 weeks: 6mg, 12mg or 18 mg. For each administration, CG0070 was first injected intratumorally according to the injection volume per lesion as defined in stage 1. CTLA-4 inhibitors were administered immediately after each CG0070 injection. The total volume at each dose value, and the maximum injection volume based on the lesion size of two or more injection lesions are listed in table 2 below. The maximum number of lesions injected is 3, and the total dose of CTLA-4 inhibitor is administered regardless of the total number and size of lesions. Any remaining volume of CTLA-4 inhibitor is administered subcutaneously around the injected lesion. Provided that if the lesion is completely resolved before the last planned treatment, both CG0070 and a CTLA-4 inhibitor (e.g., ipilimumab) may be administered to the previously non-injected lesion. If all lesions resolve before the end of the course of treatment, only a CTLA-4 inhibitor (e.g., ipilimumab) may be injected in the subcutaneous region at or around the previous lesion. The dose escalation procedure is as described in example 1, and the MTD/MFD is named dose value stage 2, which is used at the beginning of stage 3.

TABLE 2 injection volume of immunomodulator per lesion based on tumor size

Stage 3 of phase I is a combination of a CD40 agonist (e.g., CD40 agonistic antibody, e.g., APX005M) with a CTLA-4 inhibitor (e.g., anti-CTLA-4 mAb or a blocking agent, e.g., ipilimumab) and CG0070 at increasing intratumoral injection dose values stage 2. A cohort of (e.g., 3 to 6) patients received weekly intratumoral injections of fixed doses of CG0070 (e.g., with DDM) and a CTLA-4 inhibitor (e.g., ipilimumab) in combination with a CD40 agonist (e.g., APX005M) at one of the following three dose values for 6 weeks: 6mg, 12mg or 18 mg. For each administration, the dose value stage 2 CG0070 and CTLA-4 inhibitor (e.g., ipilimumab) were adjusted to 2mL and intratumoral injections were performed according to the injection volume per lesion as defined in table 1. A CD40 agonist (e.g., APX005M) is administered immediately after each CG0070/CTLA-4 inhibitor injection. The total volume at each dose value, and the maximum injection volume based on the lesion size of two or more injection lesions are listed in table 2 below. The maximum number of lesions injected was 3, and the total dose of CD40 agonist (e.g., APX005M) was administered regardless of the total number and size of lesions. Any remaining volume of CD40 agonist (e.g., APX005M) was administered subcutaneously around the injected lesion. Provided that if the lesion is completely resolved before the last planned treatment, CG0070, a CTLA-4 inhibitor (e.g., ipilimumab), and a CD40 agonist (e.g., APX005M) may be administered to the previously non-injected lesion. If all lesions resolve before the end of the course of treatment, only CD40 agonist (e.g., APX005M) may be injected in the subcutaneous region at or around the previous lesion. The dose escalation procedure was as described in example 1, and the MTD/MFD was named the study dose, which was used in phase II.

For phase II of the study, the cohort of patients first received a study dose of a three-component combination of CG0070 (e.g., with DDM), a CTLA-4 inhibitor (e.g., ipilimumab), and a CD-40 agonist (e.g., APX005M) measured in phase 3 of phase I for 4 weeks per week, followed by four intratumoral injections of the three-component combination every 2 weeks. Thereafter, monthly intratumoral injections of the three-component combination were administered for maintenance therapy until complete response, disappearance of all injectable tumors, confirmed disease progression, or intolerance to the study therapy, whichever occurred first. Patients in a phase I dose escalation phase (e.g., phase 1,2, or 3) may be enrolled in the phase II study as long as there is a rest period of at least four weeks from the last dose. For each administration, the lesion was first injected with GC0070, followed by a CTLA-4 inhibitor (e.g., ipilimumab) and a CD40 agonist (e.g., APX 005M). The largest injectable tumor (as determined by PI) was the first tumor to be injected, and the injection volumes and doses were according to tables 3 and 4. Any remaining volume of drug was injected into the next largest injectable tumor (as determined by PI) and the injection volumes and doses were according to tables 3 and 4. This procedure was repeated for the other remaining volumes until the entire total volume and dose as determined in phase I was injected. CG0070 injections were omitted at the site of the specific injection when the focus no longer survived at that site. However, even when the lesions disappear, CTLA-4 inhibitor and CD40 agonist injections are also administered into the same site until the end of the course of treatment. Each patient received a minimum of 8 injections of a CTLA-4 inhibitor and a CD40 agonist.

TABLE 3 injection volume per lesion based on tumor size

Tumor size (longest dimension)	Maximum injection volume
		≥5.0cm	2.0mL
Not less than 2.0cm to 5.0cm	1.0mL
		>0.75cm to 2.0cm	0.5mL
<0.75cm	0.1mL

TABLE 4 dosage of agent (immunomodulator and/or immune-related molecule) per lesion based on tumor size

There are two main outcome measures for this study: (1) safety and tolerability; and (2) performance. Safety and tolerability were assessed from the start of each phase until 3 months after enrollment of the last subject in each phase or phase II. Stage 1 determines the safety and tolerability of CG0070 (e.g., with DDM) as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory physical tumors. Stage 2 the safety and tolerability of the combination of CTLA-4 inhibitors (e.g., anti-CTLA-4 mabs or blockers, e.g., ipilimumab) with CG0070 as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory solid tumors. Stage 3 and II determine the safety and tolerability of CD40 agonists (agonistic anti-CD 40 antibodies, e.g., APX005M) in combination with CG0070 and CTLA-4 inhibitors as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory physical tumors. Performance was assessed from the start of each phase or phase II until 24 months after enrollment of the last subject of each phase or phase II. Performance was assessed by confirmed Objective Response Rate (ORR) of combined treatment of CG0070 alone (e.g., with DDM) in stage 1, CG0070 and a CTLA-4 inhibitor (e.g., anti-CTLA-4 mAb or a blocking agent, e.g., ipilimumab) in stage 2, CG0070, CTLA-4 inhibitor and CD40 agonist (e.g., agonistic anti-CD 40 antibody, e.g., APX005M) in stages 3 and II in patients with injectable refractory solid tumors.

Secondary outcome measures for this study are as follows. Safety secondary outcomes were evaluated 24 months after enrollment from the start of each phase until the last subject of each phase or phase II. For all three phases and phase II, the secondary safety outcome measure included the incidence of the following events: all Adverse Events (AEs), AE grade 3 or higher, events requiring discontinuation of study drug, local effects on tumors, clinically significant laboratory changes, and clinically significant changes in vital signs. Performance secondary outcomes were evaluated 24 months after enrollment from the beginning of each phase or phase II until the last subject of each phase or phase II. Secondary outcome measures of performance include best overall response rate (BOR), Disease Control Rate (DCR), Durable Response Rate (DRR), duration of response (DOR), Time To Response (TTR), Progression Free Survival (PFS), Overall Survival (OS), 1 year and 2 year survival for all three stages and phase II.

Eligibility of patients of both sexes for the study was determined based on the following inclusion criteria:

1. patients must have histologically confirmed physical tumors that failed standard therapy (surgery, chemotherapy, radiation therapy, or endocrine therapy) and for which there is no curative choice, including but not limited to: squamous cell carcinoma of the head and neck, squamous cell carcinoma of the skin, breast cancer, malignant melanoma, colorectal cancer, pancreatic adenocarcinoma, ovarian cancer, non-small cell lung cancer, and prostate cancer;

2. the patient may already have any kind and number of prior cancer therapies;

3. the patient must have measurable lesions that can be assessed by RECIST method;

4. the tumor mass to be treated must be evaluable via the cutaneous route and sufficient for injection (i.e. more than 2cm away from the major vascular structures) and measurement by RECIST;

5. the patient must be more than or equal to 18 years old;

6. the life expectancy of the patient must be more than or equal to 12 weeks;

7. patients must have a us eastern bank cancer clinical research cooperative organization (ECOG) physical status of 0, 1 or 2;

8. the patient must have adequate liver function, defined as follows:

a. the total bilirubin content is less than or equal to 1.5 multiplied by the upper limit of normal value (ULN); and

b. if liver metastasis is present, the AST/ALT content is less than or equal to 2.5 × ULN, or less than or equal to 5 × ULN;

9. the patient must have adequate renal function, as defined below: serum creatinine < 1.5 × ULN, or for creatinine>Patient creatinine clearance (calculated) of 1.5 × ULN ≥ 60mL/min/1.73m²；

10. The patient must have adequate bone marrow function, as defined below:

a. the absolute neutrophilic count is more than or equal to 1,200/mu L; and

b. the platelet count is more than or equal to 80,000/mu L;

11. the patient must be free of known hemorrhagic diathesis or clotting lesions, which would render intratumoral injection or biopsy unsafe;

12. men and women with fertility potential must agree to use appropriate contraceptive measures and for up to six months before study entry;

13. women with fertility potential must be tested for negative urine or serum pregnancy within one week before starting treatment; and

14. the patient must be able to understand and be willing to sign a written informed consent document.

The following patients were excluded from the study:

1. patients receiving chemotherapy, immunotherapy or radiation therapy, or adverse events >1 grade (with the exception of hair loss) resulting from agents administered more than 4 weeks prior to screening within 4 weeks prior to screening;

2. patients with a history of significant tumor bleeding or clotting or bleeding disorders;

3. patients with target tumors that can potentially invade major vascular structures (e.g., innominate arteries, carotid arteries) based on explicit imaging findings as determined by a radiologist;

4. patients with grade 1 or greater pre-existing neurological abnormalities (CTCAE version 4.0);

5. patients hospitalized during the 30 days prior to study entry for an urgent condition requiring hospitalization assessment, treatment or procedure. In addition, an urgent condition requiring hospitalization assessment, treatment or procedure must have resolved or be medically stable and not severe 30 days prior to entering the study;

6. patients with clinically significant Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV) or estain-bal virus (EBV) infection. Testing patients for HIV during pre-treatment screening;

patients receiving steroids or immunosuppressants, e.g., against rheumatoid arthritis;

8. patients concurrently with any other study medication;

9. patients with central nervous system metastases or with a history of such diseases;

10. a female who is pregnant or lactating or who wishes to become pregnant during the study period;

11. patients with uncontrolled intercropping (including but not limited to ongoing or active infection, symptomatic congestive heart failure, unstable angina, cardiac arrhythmia, or psychiatric disease/social situation that would limit compliance to research requirements).

Example 4: preparation of non-activated tumor cells.

This example illustrates an exemplary method of preparing non-activated tumor cells that can be used for local administration to a tumor site (e.g., by intratumoral injection) in combination with an infectious agent and an immunomodulator.

The tumor cells may be from a biopsy or resection of the tumor from an autologous or allogeneic source. Alternatively, they may be harvested from established tumor cell lines or separately developed tumor cell lines, from autologous or allogeneic sources. Tumor cells are usually isolated by gradient density centrifugation, plastic adhesion and trypsinization. Isolated tumor cells are expanded over multiple passages to provide sufficient cells for treatment. In the case of tumor cells harvested from cell lines, the cells are further washed, filtered and analyzed for characterization (e.g., expression of tumor antigens), sterility, and survival. Tumor cells are cryopreserved in cell banks or stored as aliquots ready for administration.

Preparation of tumor cells from surgical samples

For the usual surgical samples, one piece of tumor was removed for pathological classification and then the main tumor cell mass was placed in tubes with HBSS with natamycin (gentamycin) and stored at 8 ℃. Within about 8-12 hours, fresh tumor samples were carried to the laboratory where they were further dissociated. Tumor samples were cut into smaller pieces, typically 1cm, with a scalpel³. It was subsequently incubated in an enzyme solution at 37 ℃. The most effective commonly used enzyme solution is a mixture of collagenase, DNA hydrolase and hyaluronidase. After incubation, the resulting suspension was filtered through a nylon mesh with a pore size of 40 μm. Such steps are repeated until all major portions of the tumor sample are dissolved. The resulting cell suspension was then washed three times in HBSS and then prepared for cryopreservation.

Cryopreservation and thawing of tumor cells

Tumor cells isolated in this way were subsequently frozen in 10% human serum albumin and 10% DMSO and frozen in liquid nitrogen at 10%⁷Aliquots of individual cells were stored. Cell freezing can be performed in a freezing computer Kryo 10 series II (Messer-Griesheim). On the day of planned administration, cells were carefully thawed in warm medium supplemented with 10% human serum albumin and subsequently washed three times in this medium.

Inactivation of tumor cells

The proliferative capacity of tumor cells was inactivated with 200Gy using a remote cobalt source prior to administration.

Preparation of non-activated tumor cells for injection into tumor sites

Will 10⁷Aliquots of the non-activated tumor cells were adjusted to a volume suitable for intratumoral injection. See, for example, example 5. Typically, the inactivated cells can be centrifuged and reconstituted to a volume of about 2mL in 37 ℃ media with 10% albumin.

Example 5: intratumoral administration of CG0070 with CTLA-4 inhibitor, 4-1BB kinase in patients with hepatocellular carcinoma Phase I/II clinical study of combinations of mobilizing agents and non-activated tumor cells

This example illustrates a phase I/II, multicenter, open label clinical study to evaluate the performance, safety and tolerability of a combination therapy comprising CG0070, a CTLA-4 inhibitor, a 4-1BB agonist and non-activated tumor cells for treating patients with refractory injectable liver tumors. A combination of CG0070, a CTLA-4 inhibitor, a 4-1BB agonist, and non-activated tumor cells is administered intrahepatically into a hepatic tumor with known progression in patients with hepatocellular carcinoma or in patients with liver metastases from breast adenocarcinoma, colorectal adenocarcinoma, gastroesophageal carcinoma (adenocarcinoma or squamous cell carcinoma), melanoma, non-small cell lung cancer, or clear cell renal cell carcinoma.

In this study, non-activated tumor cell lines were from allogeneic sources. The combination of CG0070 and non-activated tumor cells (hereinafter referred to as "VC" or "VC combination") has a fixed composition, wherein the number of non-activated tumor cells is about at least 4 log lower than the number of CG0070 viral particles. For example, each patient is injected about 1X 10 per administration⁸Or lower number of non-activated tumor cells and 1X 10¹²Combinations of CG0070 at doses of vp. The CTLA-4 inhibitor may be an anticalin that specifically recognizes CTLA-4. The antiporter protein may be formulated in LPGA. Lifting deviceFor example, a75:25 weight ratio of CTLA-4 specific anti-transporter protein in an LPGA formulation (hereinafter referred to as anti-transporter protein/LPGA 75:25) may be used. The 4-1BB agonist may be an agonistic anti-4-1 BB antibody, such as PF-05082566.

Phase I of the clinical study was divided into three phases. In phase 1, VC combinations comprising CG0070 and non-activated tumor cells (e.g., non-activated allogeneic tumor cells) are administered to each patient via intratumoral injection weekly (e.g., on day 1 of each week) over 6 weeks. A cohort of (e.g., 3 to 6) patients receives an intratumoral VC of one of the four dose values. A cohort of (e.g., 3 to 6) patients received weekly intratumoral injections of CG0070 (e.g., with DDM) for four weeks at one of the following four dose values: 5X 10¹⁰CG0070 and 5X 10 of vp⁶A non-activated tumor cell, 1X 10¹¹CG0070 and 1X 10 of vp⁷Non-activated tumor cell, 5X 10¹¹CG0070 and 5X 10 of vp⁷Non-activated tumor cells or 1X 10¹²CG0070 and 1X 10 of vp⁸And (3) non-activated tumor cells. For example, CG0070 and inactivated tumor cells were mixed in a total volume of 2m in saline (e.g., with 0.1% DDM) immediately prior to administration. If the patient has a single lesion (which must be greater than 2cm), then the total volume of VC solution is injected into the lesion. If there are two or more lesions, the maximum injection volume based on lesion size as shown in table 1 is followed. If the largest lesion is at least 2cm, any remaining volume is injected into the largest lesion. If the largest lesion is less than 2cm, the remaining volume is divided between the two larger lesions. The maximum number of lesions injected was 3. The total dose is administered regardless of the total number and size of lesions. The dose escalation procedure is as described in example 1, and the MTD/MFD is named dose value stage 1, which is used at the beginning of stage 2.

Phase 2 of phase I is a combination of a dose escalating intratumoral injection dose value of the CTLA-4 inhibitor of phase 1 (e.g., anti-transporter/LPGA 75:25) in combination with VC. A cohort of (e.g., 3 to 6) patients received weekly intratumoral injections of fixed doses of CG0070 and a combination of non-activated tumor cells with a CTLA-4 inhibitor (e.g., anti-transporter/LPGA 75:25) for 6 weeks at one of the following three dose values: 1.2mg, 2.4mg or 3.6 mg. For each administration, VC combinations were first injected intratumorally according to the injection volume of each lesion as defined in stage 1. CTLA-4 inhibitors (e.g., antiportein/LPGA 75:25) were administered to the same injection site at volumes according to table 5 immediately following each VC injection. The maximum number of lesions injected is 3, and the total dose of CTLA-4 inhibitor is administered regardless of the total number and size of lesions. Any remaining volume of CTLA-4 inhibitor is administered subcutaneously around the injected lesion. Provided that if the lesion is completely resolved before the last planned treatment, both the VC combination and the CTLA-4 inhibitor (e.g., anti-transporter/LPGA 75:25) can be administered to the previously non-injected lesion. If all lesions resolve before the end of the course of treatment, only the CTLA-4 inhibitor (e.g., anti-transporter/LPGA 75:25) may be injected in the subcutaneous region at or around the previous lesion. The dose escalation procedure is as described in example 1, and the MTD/MFD is named dose value stage 2, which is used at the beginning of stage 3.

TABLE 5 injection volume of immunomodulator per lesion based on tumor size

Phase 3 of phase I is a combination of a 4-1BB agonist (e.g., a 4-1BB agonistic antibody, e.g., PF-05082566) with VC and a CTLA-4 inhibitor (e.g., anti-carrier protein/LPGA 75:25) at increasing intratumoral injection dose values of phase 2. A cohort of (e.g., 3 to 6) patients received weekly intratumoral injections of a fixed dose of a VC combination and a combination of a CTLA-4 inhibitor (e.g., antiportein/LPGA 75:25) and a 4-1BB agonist (e.g., PF-05082566) at one of three dose values for 6 weeks: 6mg, 12mg or 18 mg. For each administration, the dose value of the VC combination of stage 2 and CTLA-4 inhibitor (e.g., anti-transporter/LPGA 75:25) was adjusted to 2mL and intratumoral injection was performed according to the injection volume per lesion as defined in table 1. The 4-1BB agonist (e.g., PF-05082566) is administered immediately after each VC/CTLA-4 inhibitor injection. The total volume at each dose value, and the maximum injection volume based on the lesion size of two or more injection lesions are listed in table 2 below. The maximum number of lesions injected is 3, and a total dose of 4-1BB agonist (e.g., PF-05082566) is administered regardless of the total number and size of lesions. Any remaining volume of 4-1BB agonist (e.g., PF-05082566) is administered subcutaneously around the injected lesion. Provided that if the lesion is completely resolved before the last planned treatment, a VC combination, a CTLA-4 inhibitor (e.g., anti-transporter/LPGA 75:25), and a 4-1BB agonist (e.g., PF-05082566) can be administered to the previously non-injected lesion. If all lesions resolve before the end of the treatment course, only 4-1BB agonist (e.g., PF-05082566) may be injected in the subcutaneous region at or around the previous lesion. The dose escalation procedure was as described in example 1, and the MTD/MFD was named the study dose, which was used in phase II.

For phase II of the study, the cohort of patients first received a study dose of a four-component combination of CG0070 (e.g., with DDM), non-activated tumor cells (e.g., non-activated allogeneic tumor cells), CTLA-4 inhibitor (e.g., anti-transporter/LPGA 75:25), and 4-1BB agonist (e.g., PF-05082566) measured in phase 3 of phase I for 4 weeks per week followed by four intratumoral injections of the four-component combination every 2 weeks. Thereafter, monthly intratumoral injections of the four-component combination were administered for maintenance therapy until complete response, disappearance of all injectable tumors, confirmed disease progression, or intolerance to the study therapy, whichever occurred first. Patients in a phase I dose escalation phase (e.g., phase 1,2, or 3) may be enrolled in the phase II study as long as there is a rest period of at least four weeks from the last dose. For each administration, GC0070 and non-activated tumor cells (e.g., allogeneic non-activated tumor cells) were first mixed just prior to administration, injected at the site of the lesion, followed by CTLA-4 inhibitor (e.g., antiporter/LPGA 75:25) and 4-1BB agonist (e.g., PF-05082566). The largest injectable tumor (as determined by PI) was the first tumor to be injected, and the injection volumes and doses were according to tables 3 and 4. Any remaining volume of drug was injected into the next largest injectable tumor (as determined by PI) and the injection volumes and doses were according to tables 3 and 4. This procedure was repeated for the other remaining volumes until the entire total volume and dose as determined in phase I was injected. VC combination injections were omitted at a specific injection site when the lesions no longer survived at that site. However, even when the lesions disappear, CTLA-4 inhibitor and 4-1BB agonist injections are also administered into the same site until the end of the course of treatment. Each patient received a minimum of 8 injections of CTLA-4 inhibitor and 4-1BB agonist.

There are two main outcome measures for this study: (1) safety and tolerability; and (2) performance. Safety and tolerability were assessed from the beginning of each phase or stage II until 3 months after enrollment of the last subject in each phase or stage II. Stage 1 determines the safety and tolerability of CG0070 (e.g., with DDM) in combination with non-activated tumor cells (e.g., allogeneic non-activated tumor cells) as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory liver tumors. Phase 2 determines the safety and tolerability of a combination of CG0070 (e.g., with DDM), non-activated tumor cells (e.g., allogeneic non-activated tumor cells), and CTLA-4 inhibitors (e.g., anti-transporter/LPGA 75:25) as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory liver tumors. Phase 3 determines the safety and tolerability of a combination of CG0070 (e.g., with DDM), non-activated tumor cells (e.g., allogeneic non-activated tumor cells), CTLA-4 inhibitors (e.g., anti-transporter/LPGA 75:25), and 4-1BB agonists (e.g., PF-05082566) as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory liver tumors. Phase II the safety and tolerability of a combination of CG0070 (e.g., with DDM), non-activated tumor cells (e.g., allogeneic non-activated tumor cells), CTLA-4 inhibitors (e.g., anti-transporter/LPGA 75:25), and 4-1BB agonists (e.g., PF-05082566) was determined by the incidence of dose-limiting toxicity (DLT) in patients with refractory liver tumors. Performance was assessed from the beginning of each phase of phase II until 24 months after enrollment of the last subject of each phase. Performance was assessed by the confirmed Objective Response Rate (ORR) of the combination treatment of CG0070 (e.g., with DDM) and non-activated tumor cells (e.g., with non-activated allogeneic tumor cells) in stage 1, CG0070 (e.g., with DDM), non-activated tumor cells (e.g., with non-activated allogeneic tumor cells) and CTLA-4 inhibitors (e.g., anti-transporter/LPGA 75:25) in stage 2, and the combination treatment of CG0070 (e.g., with DDM), non-activated tumor cells (e.g., with non-activated allogeneic tumor cells), CTLA-4 inhibitors (e.g., anti-transporter/LPGA 75:25), and 4-1BB agonists (e.g., PF-05082566) in stages 3 and II.

Secondary outcome measures for this study are as follows. Secondary safety outcomes were evaluated from the beginning of each phase or phase II until 24 months after enrollment of the last subject of each phase or phase II. For all three phases and phase II, the secondary safety outcome measure included the incidence of the following events: all Adverse Events (AEs), AE grade 3 or higher, events requiring discontinuation of study drug, local effects on tumors, clinically significant laboratory changes, and clinically significant changes in vital signs. Performance secondary outcomes were evaluated 24 months after enrollment from the start of each phase until the last subject of each phase or phase II. Secondary outcome measures of performance include best overall response rate (BOR), Disease Control Rate (DCR), Durable Response Rate (DRR), duration of response (DOR), Time To Response (TTR), Progression Free Survival (PFS), Overall Survival (OS), 1 year and 2 year survival for all three stages and phase II.

Eligibility of patients of both sexes for the study was determined based on the following inclusion criteria: (1) the subject must have histologically confirmed breast adenocarcinoma, colorectal adenocarcinoma, gastroesophageal carcinoma (adenocarcinoma or squamous cell carcinoma), melanoma, non-small cell lung cancer, or clear cell renal cell carcinoma with liver metastases, or hepatocellular carcinoma with known disease progression; (2) a non-hepatocellular carcinoma subject must have received at least 1 prior standard of care systemic anti-cancer therapy for its metastatic disease; (3) the subject must have a measurable liver tumor suitable for injection; (4) the competence status of the us east bank cancer clinical research partner organisation must be 0 or 1 and the life expectancy should be greater than 5 months or longer. Sufficient blood, kidney, liver and coagulation function is required; (5) the Child-Pugh score must be A to B7.

The following patients were excluded from the study: (1) the subject must not be a candidate for liver surgery or local therapy of liver tumors with curative intent or planned systemic anti-cancer therapy; (2) liver tumors are estimated to not invade more than about one-third of the liver; (7) before enrollment, liver tumor-targeted therapy, liver surgery, antibody-based therapy or immunotherapy must be performed for <28 days, chemotherapy <21 days and targeted small molecule therapy or hormone therapy <14 days; (8) the subject must not have central nervous system metastases or irradiated, stable brain metastases from breast adenocarcinoma, non-small cell lung cancer, clear cell renal cell carcinoma, or melanoma; (9) the subject must not have a history or evidence of symptomatic autoimmune pneumonia, glomerulonephritis, vasculitis, or other symptomatic autoimmune disease; (10) the subject must not have a symptomatic autoimmune disease or be immunosuppressed; (11) the subject must not have a history of physical organ transplantation; (12) for non-hepatocellular carcinoma, acute or chronic hepatitis B virus or hepatitis C virus infection must not be present; (13) for hepatocellular carcinoma, the hepatitis B and C viral loads must be undetectable and not recently treated with certain antiviral agents; (14) tumors should not invade major portal, hepatic or great veins macroscopically; (15) the subject must not have an active herpetic skin lesion or previous complications of herpetic infection (e.g., herpetic keratitis or encephalitis) and need not be treated with an anti-herpetic drug; (16) the subject must not require concurrent warfarin treatment; (17) female subjects with fertility potential are reluctant to use an acceptable effective contraceptive method during the regimen and 3 months after the last dose of intervention.

Example 6: radiation pretreatment of patients with refractory non-hodgkin's lymphoma, nasopharyngeal carcinoma, and melanoma Phase I/II clinical study of intratumoral administration of CG0070 in combination with CTLA-4 inhibitors and CD40 agonists

This study is a multicenter, single-arm, open-label, interventional study aimed at assessing the safety and performance of combination therapy including radiation pretreatment followed by intratumoral administration of CG0070, a CTLA-4 inhibitor and a CD40 agonist in patients with solid or lymphoid tumors (e.g., non-hodgkin's lymphoma, nasopharyngeal carcinoma or melanoma).

The radiation pretreatment is carried out as follows. 2 days prior to each administration of therapy (e.g., CG0070, a combination of CG0070 and a CTLA-4 inhibitor, or a combination of CG0070, a CTLA-4 inhibitor and a CD40 agonist), 2Gy of a single dose of external radiation was administered daily to each tumor site treated in the patient for 2 days. The maximum dose of radiation is limited to one radiation session per month (2Gy for 2 days) for up to 4 months. After this maximum dose, all radiation is stopped. Total radiation received during a 4 month treatment should not exceed 16 Gy.

The clinical study in phase I is divided into three phases. Phase 1 is a dose escalation study of intratumoral injections of CG0070 in combination with radiation pretreatment. A cohort of (e.g., 3 to 6) patients received weekly radiation pre-treatment followed by intratumoral injection of CG0070 (e.g., with DDM) at one of the following four dose values for 4 weeks: 5X 10¹⁰vp、1×10¹¹vp、5×10¹¹vp or 1X 10¹²vp. For example, virus CG0070 was reconstituted in 0.1% DDM in saline. The total volume of each dose was 2 mL. The concentration of CG0070 solution for the lowest dose is about 2.5X 10¹⁰vp/ml and about 5X 10 for the highest dose¹¹vp/ml. If the patient has a single lesion (which must be greater than 2cm), then the total volume of CG0070 solution is injected into the lesion. If there are two or more lesions, the maximum injection volume based on lesion size as shown in table 1 is followed. If the biggest diseaseFoci of at least 2cm, any remaining volume is injected into the largest lesion. If the largest lesion is less than 2cm, the remaining volume is divided between the two larger lesions. The maximum number of lesions injected was 3. The total dose is administered regardless of the total number and size of lesions. The dose escalation procedure is as described in example 1, and the MTD/MFD is named dose value stage 1, which is used at the beginning of stage 2.

Stage 2 of phase I is a combination of dose escalating intratumoral injection dose of a CTLA-4 inhibitor (e.g., anti-CTLA-4 mAb or a blocking agent, e.g., ipilimumab) with CG0070 and radiation pretreatment of stage 1. A cohort of (e.g., 3 to 6) patients received weekly radiation pre-treatment, followed by intratumoral injection of a fixed dose of CG0070 (e.g., with DDM) in combination with a CTLA-4 inhibitor (e.g., ipilimumab) at one of the following three dose values for 6 weeks: 6mg, 12mg or 18 mg. For each administration, CG0070 was first injected intratumorally according to the injection volume per lesion as defined in stage 1. CTLA-4 inhibitors were administered immediately after each CG0070 injection. The total volume at each dose value, and the maximum injection volume based on the lesion size of two or more injection lesions are listed in table 2 below. The maximum number of lesions injected is 3, and the total dose of CTLA-4 inhibitor is administered regardless of the total number and size of lesions. Any remaining volume of CTLA-4 inhibitor is administered subcutaneously around the injected lesion. Provided that if the lesion is completely resolved before the last planned treatment, both CG0070 and a CTLA-4 inhibitor (e.g., ipilimumab) may be administered to the previously non-injected lesion. If all lesions resolve before the end of the course of treatment, only a CTLA-4 inhibitor (e.g., ipilimumab) may be injected in the subcutaneous region at or around the previous lesion. The dose escalation procedure is as described in example 1, and the MTD/MFD is named dose value stage 2, which is used at the beginning of stage 3.

Stage 3 of phase I is a combination of a CD40 agonist (e.g., CD40 agonistic antibody, e.g., APX005M) with a CTLA-4 inhibitor (e.g., anti-CTLA-4 mAb or a blocking agent, e.g., ipilimumab), CG0070 and radiation pretreatment at increasing intratumoral injection dose values stage 2. A cohort of (e.g., 3 to 6) patients received weekly radiation pre-treatment, followed by intratumoral injection of a fixed dose of CG0070 (e.g., with DDM) and a CTLA-4 inhibitor (e.g., ipilimumab) in combination with a CD40 agonist (e.g., APX005M) at one of the following three dose values for 6 weeks: 6mg, 12mg or 18 mg. For each administration, the dose value stage 2 CG0070 and CTLA-4 inhibitor (e.g., ipilimumab) were adjusted to 2mL and intratumoral injections were performed according to the injection volume per lesion as defined in table 1. A CD40 agonist (e.g., APX005M) is administered immediately after each CG0070/CTLA-4 inhibitor injection. The total volume at each dose value, and the maximum injection volume based on the lesion size of two or more injection lesions are listed in table 2 below. The maximum number of lesions injected was 3, and the total dose of CD40 agonist (e.g., APX005M) was administered regardless of the total number and size of lesions. Any remaining volume of CD40 agonist (e.g., APX005M) was administered subcutaneously around the injected lesion. Provided that if the lesion is completely resolved before the last planned treatment, CG0070, a CTLA-4 inhibitor (e.g., ipilimumab), and a CD40 agonist (e.g., APX005M) may be administered to the previously non-injected lesion. If all lesions resolve before the end of the course of treatment, only CD40 agonist (e.g., APX005M) may be injected in the subcutaneous region at or around the previous lesion. The dose escalation procedure was as described in example 1, and the MTD/MFD was named the study dose, which was used in phase II.

For phase II of the study, the cohort of patients received a weekly radiation pretreatment first followed by intratumoral injection of a study dose of three-component combination of CG0070 (e.g., with DDM), a CTLA-4 inhibitor (e.g., ipilimumab), and a CD-40 agonist (e.g., APX005M) for 4 weeks as determined in phase 3 of phase I, followed by a four-time intratumoral injection of the three-component combination every 2 weeks. Thereafter, monthly intratumoral injections of the three-component combination were administered for maintenance therapy until complete response, disappearance of all injectable tumors, confirmed disease progression, or intolerance to the study therapy, whichever occurred first. Patients in a phase I dose escalation phase (e.g., phase 1,2, or 3) may be enrolled in the phase II study as long as there is a rest period of at least four weeks from the last dose. For each administration, the lesion was first injected with GC0070, followed by a CTLA-4 inhibitor (e.g., ipilimumab) and a CD40 agonist (e.g., APX 005M). The largest injectable tumor (as determined by PI) was the first tumor to be injected, and the injection volumes and doses were according to tables 3 and 4. Any remaining volume of drug was injected into the next largest injectable tumor (as determined by PI) and the injection volumes and doses were according to tables 3 and 4. This procedure was repeated for the other remaining volumes until the entire total volume and dose as determined in phase I was injected. CG0070 injections were omitted at the site of the specific injection when the focus no longer survived at that site. However, even when the lesions disappear, CTLA-4 inhibitor and CD40 agonist injections are also administered into the same site until the end of the course of treatment. Each patient received a minimum of 8 injections of a CTLA-4 inhibitor and a CD40 agonist.

There are two main outcome measures for this study: (1) safety and tolerability; and (2) performance. Safety and tolerability were assessed from the start of each phase until 3 months after enrollment of the last subject in each phase or phase II. Phase 1 the safety and tolerability of CG0070 (e.g., with DDM) with radiation pretreatment as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory physical or lymphoid tumors was determined. Stage 2 determines the safety and tolerability of CTLA-4 inhibitors (e.g., anti-CTLA-4 mabs or blockers, e.g., ipilimumab) in combination with CG0070 and radiation pretreatment as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory physical or lymphoid tumors. The phase 3 and II studies determine the safety and tolerability of CD40 agonists (agonistic anti-CD 40 antibodies, e.g., APX005M) in combination with CG0070 and CTLA-4 inhibitors in combination with radiation pretreatment as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory physical or lymphoid tumors. Performance was assessed from the start of each phase or phase II until 24 months after enrollment of the last subject of each phase or phase II. Performance was assessed by confirmed Objective Response Rate (ORR) of CG0070 (e.g., with DDM) with radiation pretreatment treatment in stage 1, CG0070 and CTLA-4 inhibitors (e.g., anti-CTLA-4 mAb or blockers, e.g., ipilimumab) with radiation pretreatment in stage 2, CG0070, CTLA-4 inhibitors and CD40 agonists (e.g., agonistic anti-CD 40 antibodies, e.g., APX005M)) with radiation pretreatment in stage 3 and II in patients with injectable refractory physical or lymphoid tumors.

Secondary outcome measures for this study are as follows. Safety secondary outcomes were evaluated 24 months after enrollment from the start of each phase until the last subject of each phase or phase II. For all three phases and phase II, the secondary safety outcome measure included the incidence of the following events: all Adverse Events (AEs), AE grade 3 or higher, events requiring discontinuation of study drug, local effects on tumors, clinically significant laboratory changes, and clinically significant changes in vital signs. Performance secondary outcomes were evaluated 24 months after enrollment from the beginning of each phase or phase II until the last subject of each phase or phase II. Secondary outcome measures of performance include best overall response rate (BOR), Disease Control Rate (DCR), Durable Response Rate (DRR), duration of response (DOR), Time To Response (TTR), Progression Free Survival (PFS), Overall Survival (OS), 1 year and 2 year survival for all three stages and phase II.

Eligibility of patients of both sexes for the study was determined based on the following inclusion criteria: (1) the patient must be more than or equal to 18 years old; (2) the patient must be able to understand and be willing to sign a written informed consent document; (3) the patient must have histologically confirmed malignant disease (i.e., stage I: histologically confirmed melanoma, or metastatic nasopharyngeal carcinoma; stage II: histologically confirmed melanoma, non-Hodgkin's lymphoma, or metastatic nasopharyngeal carcinoma); (4) the patient must have failed at least one systemic therapy or be intolerant of at least one prior systemic therapy; (5) patients must have at least two lesions of a size that can be assessed by modified world health organization (mWHO)/Cheson guidelines; one of the two lesions must be suitable for biopsy (core or fine needle aspirate) and intratumoral injection up to 5ml (diameter > -10 mm); (6) patients with asymptomatic brain metastases were eligible; (systemic steroids should be avoided if possible, or the subject should be stable for the lowest clinically effective dose); (7) patients must have a us eastern bank cancer clinical research cooperative organization (ECOG) physical status of 0, 1 or 2; (8) the life expectancy of the patient should not be less than 16 weeks; (9) patients must have baseline (screening/baseline) radiographic images within 6 weeks of the initial study, (e.g., brain, chest, abdomen, pelvis, and bone scans with specific imaging tests to be determined by the attending physician); (10) the patient must have the following laboratory results: white Blood Cells (WBC) > < 2000/uL (about 2 × 10^ 9/L); absolute neutrophilic count > -1000/uL (about 0.5 x 10^ 9/L); platelet count > -75 × 10^3/uL (about 75 × 10^ 9/L); heme > -9 g/dL (transfusible); creatinine <2.0 × upper limit of normal value (ULN); for subjects without liver metastasis, aspartate Aminotransferase (AST)/alanine Aminotransferase (ALT) <2.5 × ULN, < 5 times that of individuals with liver metastasis; and bilirubin <2.0 × ULN (except subjects with Gilbert's syndrome, who must have less than 3.0mg/dL total bilirubin); (11) patients must not have active or chronic infection with Human Immunodeficiency Virus (HIV), hepatitis B or hepatitis C; (12) women with childbearing potential (WOCBP) must use appropriate contraceptive methods throughout the study and up to 26 weeks after the last dose of study product to minimize the risk of pregnancy in order to avoid pregnancy; and (13) men with fertility potential must use appropriate contraceptive methods to avoid conception in a manner that minimizes the risk of pregnancy throughout the study (and for up to 26 weeks after the last dose of study product).

The following patients were excluded from the study: (1) a patient with any other malignant disease, who is free of disease for less than 5 years, with the exception of well-treated and cured basal or squamous cell skin cancer, superficial bladder cancer or carcinoma in situ of the cervix; (2) patients with a history of significant tumor bleeding, or clotting or bleeding disorders; (3) patients with a history of inflammatory bowel disease, including ulcerative colitis and Crohn's disease, were excluded from this study, as well as symptomatic disease (e.g., rheumatoid arthritis, systemic progressive sclerosis [ scleroderma ], systemic lupus erythematosus, autoimmune vasculitis [ e.g., Wegener's Granulomatosis) ]); patients with a history of action neuropathies of autoimmune origin (e.g., Guillain-Barre Syndrome (Guillain-Barre Syndrome) and myasthenia gravis); (4) patients with any potential medical or psychiatric condition, which, from the investigator's perspective, would make administration of an interventional drug harmful or obscure interpretation of Adverse Events (AEs), such as conditions associated with frequent diarrhea; (5) patients with underlying cardiac conditions that are considered unsuitable for surgery by cardiology consultation; (6) a patient for concurrent treatment with any one of: interleukin-2 (IL 2), interferon, or other non-research immunotherapy regimens; cytotoxic chemotherapy; an immunosuppressant; other study therapies; or chronic use of systemic corticosteroids (previous history of AEs with IL-2 or interferon does not prevent the subject from entering the current study); (7) patients receiving any study agent; (8) patients receiving immunosuppressive agents (unless treatment for potential AEs is required); and (9) women with fertility potential (WOCBP) who are unwilling or unable to use acceptable methods of contraception to avoid pregnancy for the duration of their study period and for at least 8 weeks after discontinuation of study medication; at baseline, have a positive pregnancy test, or are pregnant or lactating.

Example 7: intratumoral CCL21 pretreatment followed by intratumoral administration in patients with refractory physical tumors Phase I/II clinical study of CG0070 in combination with CTLA-4 inhibitors and CD40 agonists

This study is a multicenter, single-arm, open-label, interventional study aimed at assessing safety and performance of combination therapy including intratumoral CCL21 pretreatment followed by intratumoral administration of CG0070, a CTLA-4 inhibitor, and a CD40 agonist in patients with refractory physical tumors.

Intratumoral CCL21 pretreatment was performed as follows. Intratumoral CCL21 nanocapsules were administered into each targeted tumor site at a dose of about 200 μ g/mL 2 days prior to each administration of therapy (e.g., CG0070, a combination of CG0070 and a CTLA-4 inhibitor, or a combination of CG0070, a CTLA-4 inhibitor, and a CD40 agonist). For tumors with longest dimension exceeding 5cm, the dose of CCL21 nanocapsules within the tumor is about 2 mL; about 1mL for tumors with a longest dimension of 2cm to 5 cm; and about 0.5mL for tumors with longest dimensions of 0.5cm to 2 cm. Intratumoral CCL21 nanocapsules were administered weekly in 6 weeks in study phase I, or weekly in 4 weeks in study phase II, followed by 4 more cycles every 2 weeks. Thereafter, CCL21 nanocapsules were administered intratumorally once a month until disease progression or a toxic event occurred.

The clinical study in phase I is divided into three phases. Stage 1 is a dose escalation study of intratumoral injections of CG0070 combined with intratumoral CCL21 nanocapsule pretreatment. A cohort of (e.g., 3 to 6) patients received a weekly intratumoral injection of CG0070 (e.g., with DDM) at one of the following four dose values for 4 weeks following a weekly intratumoral CCL21 nanocapsule pretreatment: 5X 10¹⁰vp、1×10¹¹vp、5×10¹¹vp or 1X 10¹²vp. For example, virus CG0070 was reconstituted in 0.1% DDM in saline. The total volume of each dose was 2 mL. The concentration of CG0070 solution for the lowest dose is about 2.5X 10¹⁰vp/ml and about 5X 10 for the highest dose¹¹vp/ml. If the patient has a single lesion (which must be greater than 2cm), then the total volume of CG0070 solution is injected into the lesion. If there are two or more lesions, the maximum injection volume based on lesion size as shown in table 1 is followed. If the largest lesion is at least 2cm, any remaining volume is injected into the largest lesion. If the largest lesion is less than 2cm, the remaining volume is divided between the two larger lesions. The maximum number of lesions injected was 3. The total dose is administered regardless of the total number and size of lesions. The dose escalation procedure is as described in example 1, and the MTD/MFD is named dose value stage 1, which is used at the beginning of stage 2.

Stage 2 of phase I is a combination of dose escalating intratumoral injection dose values of a CTLA-4 inhibitor (e.g., anti-CTLA-4 mAb or a blocking agent, e.g., ipilimumab) of stage 1 in combination with CG0070 and intratumoral CCL21 nanocapsule pretreatment. A cohort of (e.g., 3 to 6) patients received weekly intra-tumor CCL21 nanocapsule pretreatment followed by intra-tumor injection of a fixed dose of CG0070 (e.g., with DDM) in combination with a CTLA-4 inhibitor (e.g., ipilimumab) at one of the following three dose values for 6 weeks: 6mg, 12mg or 18 mg. For each administration, CG0070 was first injected intratumorally according to the injection volume per lesion as defined in stage 1. CTLA-4 inhibitors were administered immediately after each CG0070 injection. The total volume at each dose value, and the maximum injection volume based on the lesion size of two or more injection lesions are listed in table 2 below. The maximum number of lesions injected is 3, and the total dose of CTLA-4 inhibitor is administered regardless of the total number and size of lesions. Any remaining volume of CTLA-4 inhibitor is administered subcutaneously around the injected lesion. Provided that if the lesion is completely resolved before the last planned treatment, both CG0070 and a CTLA-4 inhibitor (e.g., ipilimumab) may be administered to the previously non-injected lesion. If all lesions resolve before the end of the course of treatment, only a CTLA-4 inhibitor (e.g., ipilimumab) may be injected in the subcutaneous region at or around the previous lesion. The dose escalation procedure is as described in example 1, and the MTD/MFD is named dose value stage 2, which is used at the beginning of stage 3.

Stage 3 of phase I is a combination of a CD40 agonist (e.g., CD40 agonistic antibody, e.g., APX005M) with a CTLA-4 inhibitor (e.g., anti-CTLA-4 mAb or a blocking agent, e.g., ipilimumab), CG0070 and intratumoral CCL21 nanocapsule pretreatment at increasing intratumoral injection dose values stage 2. A cohort of (e.g., 3 to 6) patients received weekly intra-tumor CCL21 nanocapsule pretreatment followed by intra-tumor injection of a fixed dose of CG0070 (e.g., with DDM) and a CTLA-4 inhibitor (e.g., ipilimumab) in combination with a CD40 agonist (e.g., APX005M) at one of the following three dose values for 6 weeks: 6mg, 12mg or 18 mg. For each administration, the dose value stage 2 CG0070 and CTLA-4 inhibitor (e.g., ipilimumab) were adjusted to 2mL and intratumoral injections were performed according to the injection volume per lesion as defined in table 1. A CD40 agonist (e.g., APX005M) is administered immediately after each CG0070/CTLA-4 inhibitor injection. The total volume at each dose value, and the maximum injection volume based on the lesion size of two or more injection lesions are listed in table 2 below. The maximum number of lesions injected was 3, and the total dose of CD40 agonist (e.g., APX005M) was administered regardless of the total number and size of lesions. Any remaining volume of CD40 agonist (e.g., APX005M) was administered subcutaneously around the injected lesion. Provided that if the lesion is completely resolved before the last planned treatment, CG0070, a CTLA-4 inhibitor (e.g., ipilimumab), and a CD40 agonist (e.g., APX005M) may be administered to the previously non-injected lesion. If all lesions resolve before the end of the course of treatment, only CD40 agonist (e.g., APX005M) may be injected in the subcutaneous region at or around the previous lesion. The dose escalation procedure was as described in example 1, and the MTD/MFD was named the study dose, which was used in phase II.

For phase II of the study, the cohort of patients first received once weekly intratumoral CCL21 nanocapsule pretreatment followed by intratumoral injection of a study dose of CG0070 (e.g., with DDM), a CTLA-4 inhibitor (e.g., ipilimumab), and a CD-40 agonist (e.g., APX005M) for 4 weeks in a three-component combination determined in phase 3 of phase I for a total of four intratumoral injections every 2 weeks. Thereafter, monthly intratumoral injections of the three-component combination were administered for maintenance therapy until complete response, disappearance of all injectable tumors, confirmed disease progression, or intolerance to the study therapy, whichever occurred first. Patients in a phase I dose escalation phase (e.g., phase 1,2, or 3) may be enrolled in the phase II study as long as there is a rest period of at least four weeks from the last dose. For each administration, the lesion was first injected with GC0070, followed by a CTLA-4 inhibitor (e.g., ipilimumab) and a CD40 agonist (e.g., APX 005M). The largest injectable tumor (as determined by PI) was the first tumor to be injected, and the injection volumes and doses were according to tables 3 and 4. Any remaining volume of drug was injected into the next largest injectable tumor (as determined by PI) and the injection volumes and doses were according to tables 3 and 4. This procedure was repeated for the other remaining volumes until the entire total volume and dose as determined in phase I was injected. CG0070 injections were omitted at the site of the specific injection when the focus no longer survived at that site. However, even when the lesions disappear, CTLA-4 inhibitor and CD40 agonist injections are also administered into the same site until the end of the course of treatment. Each patient received a minimum of 8 injections of a CTLA-4 inhibitor and a CD40 agonist.

There are two main outcome measures for this study: (1) safety and tolerability; and (2) performance. Safety and tolerability were assessed from the start of each phase until 3 months after enrollment of the last subject in each phase or phase II. Stage 1 determines the safety and tolerability of CG0070 (e.g., with DDM) and intratumoral CCL21 pretreatment as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory physical or lymphoid tumors. Stage 2 determines the safety and tolerability of CTLA-4 inhibitors (e.g., anti-CTLA-4 mabs or blockers, e.g., ipilimumab) in combination with CG0070 and intratumoral CCL21 pretreatment as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory physical or lymphoid tumors. The phase 3 and II studies determine the safety and tolerability of CD40 agonists (agonistic anti-CD 40 antibodies, e.g., APX005M) in combination with CG0070 and CTLA-4 inhibitors in combination with intratumoral CCL21 pretreatment as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory physical or lymphoid tumors. Performance was assessed from the start of each phase or phase II until 24 months after enrollment of the last subject of each phase or phase II. Performance was assessed by confirmed Objective Response Rate (ORR) of combined treatment with CG0070 (e.g., with DDM) and intratumoral CCL21 pretreatment in stage 1, CG0070 and CTLA-4 inhibitors (e.g., anti-CTLA-4 mAb or blockers, e.g., ipilimumab) and intratumoral CCL21 pretreatment in stage 2, CG0070, CTLA-4 inhibitors and CD40 agonists (e.g., agonistic anti-CD 40 antibodies, e.g., APX005M)) and intratumoral CCL21 pretreatment in stage 3 and II in patients with injectable refractory solid or lymphoid tumors.

Secondary outcome measures for this study are as follows. Safety secondary outcomes were evaluated 24 months after enrollment from the start of each phase until the last subject of each phase or phase II. For all three phases and phase II, the secondary safety outcome measure included the incidence of the following events: all Adverse Events (AEs), AE grade 3 or higher, events requiring discontinuation of study drug, local effects on tumors, clinically significant laboratory changes, and clinically significant changes in vital signs. Performance secondary outcomes were evaluated 24 months after enrollment from the beginning of each phase or phase II until the last subject of each phase or phase II. Secondary outcome measures of performance include best overall response rate (BOR), Disease Control Rate (DCR), Durable Response Rate (DRR), duration of response (DOR), Time To Response (TTR), Progression Free Survival (PFS), Overall Survival (OS), 1 year and 2 year survival for all three stages and phase II.

Eligibility of patients of both sexes for the study was determined based on the following inclusion criteria: (1) patients must have histologically confirmed physical tumors that failed standard therapy (surgery, chemotherapy, radiation therapy, or endocrine therapy) and for which there is no curative choice, including but not limited to: squamous cell carcinoma of the head and neck, squamous cell carcinoma of the skin, breast cancer, malignant melanoma, colorectal cancer, pancreatic adenocarcinoma, ovarian cancer, non-small cell lung cancer, and prostate cancer; (2) the patient may already have any kind and number of prior cancer therapies; (3) the patient must have measurable lesions that can be assessed by RECIST method; (4) the tumor mass to be treated must be sufficient for injection (i.e., more than 2cm away from the major vascular structures) and measurement by RECIST; (5) the patient must be more than or equal to 18 years old; (6) the life expectancy of the patient must be more than or equal to 12 weeks; (7) patients must have a us eastern bank cancer clinical research cooperative organization (ECOG) physical status of 0, 1 or 2; (8) the patient must have adequate liver function, defined as follows: the total bilirubin content is less than or equal to 1.5 multiplied by the upper limit of normal value (ULN); and if liver metastasis is present, the AST/ALT content is less than or equal to 2.5 × ULN, or less than or equal to 5 × ULN; (9) the patient must have adequate renal function, as defined below: serum creatinine < 1.5 × ULN, or for creatinine>Patient creatinine clearance (calculated) of 1.5 × ULN ≥ 60mL/min/1.73m²(ii) a (10) The patient must have adequate bone marrow function, as defined below: the absolute neutrophilic count is more than or equal to 1,200/mu L; and platelet countNot less than 80,000/mu L; (11) the patient must be free of known hemorrhagic diathesis or clotting lesions, which would render intratumoral injection or biopsy unsafe; (12) men and women with fertility potential must agree to use appropriate contraceptive measures and for up to six months before study entry; (13) women with fertility potential must be tested for negative urine or serum pregnancy within one week before starting treatment; and (14) the patient must be able to understand and be willing to sign a written informed consent document.

The following patients were excluded from the study: (1) patients receiving chemotherapy, immunotherapy or radiation therapy, or adverse events >1 grade (with the exception of hair loss) resulting from agents administered more than 4 weeks prior to screening within 4 weeks prior to screening; (2) patients with a history of significant tumor bleeding or clotting or bleeding disorders; patients with target tumors that can potentially invade major vascular structures (e.g., innominate arteries, carotid arteries) based on explicit imaging findings as determined by a radiologist; (3) patients with grade 1 or greater pre-existing neurological abnormalities (CTCAE version 4.0); (4) patients hospitalized during the 30 days prior to study entry for an urgent condition requiring hospitalization assessment, treatment or procedure. In addition, an urgent condition requiring hospitalization assessment, treatment or procedure must have resolved or be medically stable and not severe 30 days prior to entering the study; (5) patients with clinically significant Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV) or estain-bal virus (EBV) infection. Testing patients for HIV during pre-treatment screening; (6) patients receiving steroids or immunosuppressive agents, for example, against rheumatoid arthritis; (7) patients concurrently with any other study medication; (8) patients with central nervous system metastases or with a history of such diseases; (9) a female who is pregnant or lactating or who wishes to become pregnant during the study period; (10) patients with uncontrolled intercropping (including but not limited to ongoing or active infection, symptomatic congestive heart failure, unstable angina, cardiac arrhythmia, or psychiatric disease/social situation that would limit compliance to research requirements).

Example 8: intratumoral CpG pretreatment followed by intratumoral administration of patients with refractory physical tumors Phase I/II clinical study of CG0070 in combination with CTLA-4 inhibitors and OX40 agonists

This study is a multicenter, one-armed, open label, interventional study aimed at assessing the safety and performance of combination therapy comprising intratumoral CpG pretreatment followed by intratumoral administration of CG0070 in combination with a CTLA-4 inhibitor and an OX40 agonist in patients with refractory physical tumors.

Intratumoral CpG pretreatment was performed as follows. Intratumoral CpG (e.g., CpG 7909) was administered at a dose of about 1mg/mL into each targeted tumor site 2 days prior to each administration of therapy (e.g., CG0070, a combination of CG0070 and a CTLA-4 inhibitor, or a combination of CG0070, a CTLA-4 inhibitor and an OX40 agonist). For tumors with a longest dimension of more than 5cm, the injection volume of intratumoral CpG is about 2 mL; about 1mL for tumors with a longest dimension of 2cm to 5 cm; and about 0.5mL for tumors with longest dimensions of 0.5cm to 2 cm. Intratumoral CpG was administered weekly for 6 weeks in study phase I or weekly for 4 weeks in study phase II, followed by 4 more cycles every 2 weeks. Thereafter, CpG was administered intratumorally once a month until disease progression or toxic events occurred.

The clinical study in phase I is divided into three phases. Stage 1 is a dose escalation study of intratumoral injections of CG0070 combined with intratumoral CpG (e.g., CpG 7909) pretreatment. A cohort of (e.g., 3 to 6) patients received weekly intra-tumor CpG (e.g., CpG 7909) pretreatment followed by intra-tumor injection of CG0070 (e.g., with DDM) at one of the following four dose values for 4 weeks: 5X 10¹⁰vp、1×10¹¹vp、5×10¹¹vp or 1X 10¹²vp. For example, virus CG0070 was reconstituted in 0.1% DDM in saline. The total volume of each dose was 2 mL. The concentration of CG0070 solution for the lowest dose is about 2.5X 10¹⁰vp/ml and about 5X 10 for the highest dose¹¹vp/ml. If the patient has a single lesion (which must be greater than 2cm), then the total volume of CG0070 solution is injected into the lesion. If it is storedAt two or more lesions, the maximum injection volume based on lesion size as shown in table 1 was followed. If the largest lesion is at least 2cm, any remaining volume is injected into the largest lesion. If the largest lesion is less than 2cm, the remaining volume is divided between the two larger lesions. The maximum number of lesions injected was 3. The total dose is administered regardless of the total number and size of lesions. The dose escalation procedure is as described in example 1, and the MTD/MFD is named dose value stage 1, which is used at the beginning of stage 2.

Stage 2 of phase I is a combination of dose escalating intratumoral injection dose values of a CTLA-4 inhibitor (e.g., anti-CTLA-4 mAb or a blocking agent, e.g., ipilimumab) of stage 1 with CG0070 and intratumoral CpG pretreatment. A cohort of (e.g., 3 to 6) patients received weekly intra-tumor CpG (e.g., CpG 7909) pretreatment followed by intra-tumor injection of a fixed dose of CG0070 (e.g., with DDM) in combination with a CTLA-4 inhibitor (e.g., ipilimumab) at one of the following three dose values for 6 weeks: 6mg, 12mg or 18 mg. For each administration, CG0070 was first injected intratumorally according to the injection volume per lesion as defined in stage 1. CTLA-4 inhibitors were administered immediately after each CG0070 injection. The total volume at each dose value, and the maximum injection volume based on the lesion size of two or more injection lesions are listed in table 2 below. The maximum number of lesions injected is 3, and the total dose of CTLA-4 inhibitor is administered regardless of the total number and size of lesions. Any remaining volume of CTLA-4 inhibitor is administered subcutaneously around the injected lesion. Provided that if the lesion is completely resolved before the last planned treatment, both CG0070 and a CTLA-4 inhibitor (e.g., ipilimumab) may be administered to the previously non-injected lesion. If all lesions resolve before the end of the course of treatment, only a CTLA-4 inhibitor (e.g., ipilimumab) may be injected in the subcutaneous region at or around the previous lesion. The dose escalation procedure is as described in example 1, and the MTD/MFD is named dose value stage 2, which is used at the beginning of stage 3.

Stage 3 of phase I is a combination of dose escalating intratumoral injection dose values stage 2 OX40 agonist (e.g., OX40 agonist antibody, e.g., MEDI-6469) in combination with CTLA-4 inhibitor (e.g., anti-CTLA-4 mAb or blocker, e.g., ipilimumab), CG0070 and intratumoral CpG (e.g., CpG 7909) pretreatment. A cohort of (e.g., 3 to 6) patients received weekly intra-tumor CpG pretreatment followed by intratumor injection of a fixed dose of CG0070 (e.g., with DDM) and a CTLA-4 inhibitor (e.g., ipilimumab) in combination with an OX40 agonist (e.g., MEDI-6469) at one of the following three dose values for 6 weeks: 6mg, 12mg or 18 mg. For each administration, the dose value stage 2 CG0070 and CTLA-4 inhibitor (e.g., ipilimumab) were adjusted to 2mL and intratumoral injections were performed according to the injection volume per lesion as defined in table 1. An OX40 agonist (e.g., MEDI-6469) is administered immediately after each CG0070/CTLA-4 inhibitor injection. The total volume at each dose value, and the maximum injection volume based on the lesion size of two or more injection lesions are listed in table 2 below. The maximum number of lesions injected is 3, and a total dose of OX40 agonist (e.g., MEDI-6469) is administered regardless of the total number and size of lesions. Any remaining volume of OX40 agonist (e.g., MEDI-6469) is administered subcutaneously around the injected lesion. Provided that if the lesion is completely resolved before the last planned treatment, CG0070, a CTLA-4 inhibitor (e.g., ipilimumab), and an OX40 agonist (e.g., MEDI-6469) may be administered to the previously non-injected lesion. If all lesions resolve before the end of the treatment process, only OX40 agonist (e.g., MEDI-6469) may be injected in the subcutaneous region at or around the previous lesion. The dose escalation procedure was as described in example 1, and the MTD/MFD was named the study dose, which was used in phase II.

For phase II of the study, the cohort of patients received a weekly intratumoral CpG pretreatment followed by intratumoral injection of a study dose of a three-component combination of CG0070 (e.g., with DDM), a CTLA-4 inhibitor (e.g., ipilimumab), and an OX-40 agonist (e.g., MEDI-6469) for 4 weeks, measured in phase 3 of phase I, four times every 2 weeks. Thereafter, monthly intratumoral injections of the three-component combination were administered for maintenance therapy until complete response, disappearance of all injectable tumors, confirmed disease progression, or intolerance to the study therapy, whichever occurred first. Patients in a phase I dose escalation phase (e.g., phase 1,2, or 3) may be enrolled in the phase II study as long as there is a rest period of at least four weeks from the last dose. For each administration, the lesion is first injected with GC0070, followed by a CTLA-4 inhibitor (e.g., ipilimumab) and an OX-40 agonist (e.g., MEDI-6469). The largest injectable tumor (as determined by PI) was the first tumor to be injected, and the injection volumes and doses were according to tables 3 and 4. Any remaining volume of drug was injected into the next largest injectable tumor (as determined by PI) and the injection volumes and doses were according to tables 3 and 4. This procedure was repeated for the other remaining volumes until the entire total volume and dose as determined in phase I was injected. CG0070 injections were omitted at the site of the specific injection when the focus no longer survived at that site. However, even when the lesion disappears, CTLA-4 inhibitor and OX-40 agonist (e.g., MEDI-6469) injections are also administered into the same site until the end of the course of treatment. Each patient received a minimum of 8 injections of a CTLA-4 inhibitor and an OX-40 agonist (e.g., MEDI-6469).

There are two main outcome measures for this study: (1) safety and tolerability; and (2) performance. Safety and tolerability were assessed from the start of each phase until 3 months after enrollment of the last subject in each phase or phase II. Stage 1 the safety and tolerability of CG0070 (e.g., with DDM) with intratumoral CpG pretreatment as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory physical or lymphoid tumors was determined. Stage 2 determines the safety and tolerability of CTLA-4 inhibitors (e.g., anti-CTLA-4 mabs or blockers, e.g., ipilimumab) in combination with CG0070 and intratumoral CpG pretreatment as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory physical or lymphoid tumors. The phase 3 and phase II studies determine the safety and tolerability of OX40 agonists (agonistic anti-OX 40 antibodies, e.g., MEDI-6469) in combination with CG0070 and CTLA-4 inhibitors and intratumoral CpG pretreatment as assessed by the incidence of dose-limiting toxicity (DLT) in patients with refractory solid or lymphoid tumors. Performance was assessed from the start of each phase or phase II until 24 months after enrollment of the last subject of each phase or phase II. Performance was assessed by confirmed Objective Response Rate (ORR) of combined treatment with CG0070 (e.g., with DDM) and intratumoral CpG pretreatment in stage 1, CG0070 and CTLA-4 inhibitors (e.g., anti-CTLA-4 mAb or blockers, e.g., ipilimumab) and intratumoral CpG pretreatment in stage 2, CG0070, CTLA-4 inhibitors and OX40 agonists (e.g., agonistic anti-OX 40 antibodies, e.g., MEDI-6469)) and intratumoral CpG pretreatment in stage 3 and II in patients with injectable refractory physical or lymphoid tumors.

Secondary outcome measures for this study are as follows. Safety secondary outcomes were evaluated 24 months after enrollment from the start of each phase until the last subject of each phase or phase II. For all three phases and phase II, the secondary safety outcome measure included the incidence of the following events: all Adverse Events (AEs), AE grade 3 or higher, events requiring discontinuation of study drug, local effects on tumors, clinically significant laboratory changes, and clinically significant changes in vital signs. Performance secondary outcomes were evaluated 24 months after enrollment from the beginning of each phase or phase II until the last subject of each phase or phase II. Secondary outcome measures of performance include best overall response rate (BOR), Disease Control Rate (DCR), Durable Response Rate (DRR), duration of response (DOR), Time To Response (TTR), Progression Free Survival (PFS), Overall Survival (OS), 1 year and 2 year survival for all three stages and phase II.

Eligibility of patients of both sexes for the study was determined based on the following inclusion criteria: (1) patients must have histologically confirmed physical tumors that failed standard therapy (surgery, chemotherapy, radiation therapy, or endocrine therapy) and for which there is no curative choice, including but not limited to: squamous cell carcinoma of the head and neck, squamous cell carcinoma of the skin, breast cancer, malignant melanoma, colorectal cancer, pancreatic adenocarcinoma, ovarian cancer, non-small cell lung cancer, and prostate cancer; (2) the patient may already have any kind and number of prior cancer therapies; (3) the patient must have measurable lesions that can be assessed by RECIST method; (4) The tumor mass to be treated must be sufficient for injection (i.e., more than 2cm away from the major vascular structures) and measurement by RECIST; (5) the patient must be more than or equal to 18 years old; (6) the life expectancy of the patient must be more than or equal to 12 weeks; (7) patients must have a us eastern bank cancer clinical research cooperative organization (ECOG) physical status of 0, 1 or 2; (8) the patient must have adequate liver function, defined as follows: the total bilirubin content is less than or equal to 1.5 multiplied by the upper limit of normal value (ULN); and if liver metastasis is present, the AST/ALT content is less than or equal to 2.5 × ULN, or less than or equal to 5 × ULN; (9) the patient must have adequate renal function, as defined below: serum creatinine < 1.5 × ULN, or for creatinine>Patient creatinine clearance (calculated) of 1.5 × ULN ≥ 60mL/min/1.73m²(ii) a (ii) a (10) The patient must have adequate bone marrow function, as defined below: the absolute neutrophilic count is more than or equal to 1,200/mu L; and a platelet count of greater than or equal to 80,000/μ L; (11) the patient must be free of known hemorrhagic diathesis or clotting lesions, which would render intratumoral injection or biopsy unsafe; (12) men and women with fertility potential must agree to use appropriate contraceptive measures and for up to six months before study entry; (13) women with fertility potential must be tested for negative urine or serum pregnancy within one week before starting treatment; and (14) the patient must be able to understand and be willing to sign a written informed consent document.

The following patients were excluded from the study: (1) patients receiving chemotherapy, immunotherapy or radiation therapy, or adverse events >1 grade (with the exception of hair loss) resulting from agents administered more than 4 weeks prior to screening within 4 weeks prior to screening; (2) patients with a history of significant tumor bleeding or clotting or bleeding disorders; patients with target tumors that can potentially invade major vascular structures (e.g., innominate arteries, carotid arteries) based on explicit imaging findings as determined by a radiologist; (3) patients with grade 1 or greater pre-existing neurological abnormalities (CTCAE version 4.0); (4) patients hospitalized during the 30 days prior to study entry for an urgent condition requiring hospitalization assessment, treatment or procedure. In addition, an urgent condition requiring hospitalization assessment, treatment or procedure must have resolved or be medically stable and not severe 30 days prior to entering the study; (5) patients with clinically significant Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV), Hepatitis C Virus (HCV) or estain-bal virus (EBV) infection. Testing patients for HIV during pre-treatment screening; (6) patients receiving steroids or immunosuppressive agents, for example, against rheumatoid arthritis; (7) patients concurrently with any other study medication; (8) patients with central nervous system metastases or with a history of such diseases; (9) a female who is pregnant or lactating or who wishes to become pregnant during the study period; (10) patients with uncontrolled intercropping (including but not limited to ongoing or active infection, symptomatic congestive heart failure, unstable angina, cardiac arrhythmia, or psychiatric disease/social situation that would limit compliance to research requirements).

Example 9: ar20-1004 and anti-CTLA-4 antibodies and/or/and/or combinations thereof in a squamous cell lung carcinoma mouse allograft model Or a combination of anti-PD-L1 antibodies.

This example illustrates an in vivo study of the performance of oncolytic adenovirus Ar20-1004 administered alone or in combination with anti-CTLA-4 antibody 9H10 and/or anti-PD-L1 antibody WBP315 in KLN 205 murine squamous cell lung cancer mouse allograft model. Efficacy was assessed by monitoring tumor growth and metastasis. Ar20-1004 is a conditionally replicating oncolytic adenovirus having the same construct as CG0070 except that it expresses mouse GM-CSF (CG0070 expresses human GM-CSF). Due to the presence of the tumor-selective E2F-1 promoter, Ar20-1004 selectively replicates and selectively kills tumor cells that are Rb-pathway deficient. Cell death events and expressed GM-CSF can stimulate an immune response against distant uninfected metastases. Ar20-1004 has been described in US2008/0118470, incorporated herein by reference.

Materials and methods

Ar20-1004 (1.2X 10^12vp/mL) and anti-PD-L1 (WBP315) (5.6mg/mL) were prepared in Cold Genesys Inc. and stored at-80 ℃ prior to use. anti-CTLA-49H 10 and hamster polyclonal IgG were purchased from BioX cells (West Lebanon, NH) as stocks of 6.15mg/mL and 9.55mg/mL, respectively. All dosing solutions were freshly prepared daily and the solutions were combined for the entire group of animals prior to dosing. anti-PD-L1 (WBP315), anti-CTLA-49H 10, and hamster IgG isotype were each diluted in PBS to create a 1mg/mL dosing solution.

KLN 205 tumor cells were seeded in the right and left flanks of female DBA/2 mice. Tumors were implanted on the left side 4 days after implantation on the right side. When the tumor in the right flank reaches 99-102mm³Group mean volume of (a), treatment was started on day 1 (D) in 8 groups of mice with established subcutaneous KLN 205 tumors (n-10). All agents were administered to the right flank tumors within the tumors on days 1, 4, 7 and 10. At 1 × 10¹⁰pfu/animal Ar20-1004 was administered. anti-CTLA-4, hamster polyclonal IgG and anti-PD-L1 were administered at 20 μ g/animal each. Control animals were untreated. The animal groups and dosing schedule are summarized in figure 2.

Tumors were measured twice weekly on both abdomens. Study endpoint was defined as mean tumor volume in the right flank of control group of 1000mm³Or 35 days, the first arrival is the standard. When the control group reached the end of tumor volume, the study was terminated on day 23. Treatment results were based on percent tumor growth inhibition (% TGI), defined as the percent difference in Median Tumor Volume (MTV) (sum of bilateral tumor volumes) between treated and control mice on day 19. The results were analyzed using Mann-Whitney U test, and tested at P<Statistical significance was considered at 0.05.

Results were also analyzed by counting lung metastasis on day 23 (i.e., the last day of the study). Animals were sacrificed using isoflurane anesthesia at the endpoint and necropsy was performed to identify metastases. The total count was obtained by adding the number of lesions counted in the right superior, middle, inferior and posterior vena cava valves to the number of lesions counted in the left lung. The percent inhibition was defined as the difference between the number of metastatic lesions of the indicated control group and the number of metastatic lesions of the drug-treated group, expressed as a percentage of the number of metastatic lesions of the indicated control.

Inhibition [% 1- (number of drug-treated lesions/number of control lesions) ] × 100.

The results were analyzed using the Kruskal-Wallis test and considered statistically significant when P < 0.05. Treatment tolerance was assessed by Body Weight (BW) measurements and frequent observation of clinical signs of treatment-related (TR) side effects.

Results

This study characterized the anti-tumor response induced by Ar20-1004 in the KLN 205 murine squamous cell lung cancer allograft model. The reaction was also evaluated when Ar20-1004 was administered in combination with anti-CTLA-4 and/or anti-PD-L1. Tumors were measured twice weekly until day 23 and TGI analysis was performed at D19. Lung metastasis was counted on day 23. All treatments were well tolerated.

Ar20-1004, anti-CTLA-4, and anti-PD-L1 alone or in combination did not show significant tumor growth inhibition when intratumorally administered in KLN 205 murine squamous cell lung cancer model in female DBA/2 mice (figure 4). Ar20-1004, anti-CTLA-4, and anti-PD-L1 monotherapies inhibited metastasis by 71%, 60%, and 66%, respectively, but such results were not statistically significant compared to the untreated group. Similarly, the combination of Ar20-1004 with anti-CTLA-4, or dual therapy of anti-CTLA-4 and anti-PD-L1 (without Ar20-1004) inhibited metastasis by 69% and 74%, respectively, which were not statistically significant.

Notably, the combination therapy including Ar20-1004 and anti-PD-L1 caused a significant 84% inhibition of metastatic counts, and the triple combination therapy including Ar20-1004, anti-PD-L1, and anti-CTLA-4 caused a significant 94% inhibition of metastatic lesions (fig. 3).

Example 10: intratumoral administration of Ar20-1004 in a 4T1 syngeneic mouse model with intratumoral anti-CTLA-49H 10 and- Or a combination of localized irradiation.

This example illustrates an in vivo study evaluating the anti-tumor immune response induced by Ar20-1004 alone or in combination with CTLA-4-blocking and/or irradiation at the primary tumor site in a 4T1 syngeneic mouse model and therapeutic impact on metastasis.

4T1 is an Rb-pathway deficient mouse breast cancer cell line. As a preliminary step, the antitumor effect of Ar20-1004, a human adenovirus (Ad5) derivative having a mouse-specific GM-CSF sequence, was evaluated by 4T1 cell viability and toxicity (i.e., GM-CSF production) analysis. Use of LNCap pure line FGC in the same in vitro assay (CRL-1740^TM) Human cell lines served as positive controls. Triplicate wells of cancer cells were infected with Ar20-1004 at MOIs of 10, 100, and 1000, respectively, for 24 hours. Cell viability was assessed via MTT assay at 24 hours, 72 hours and 120 hours post infection. Cell supernatants were collected at 24, 72 and 120 hours post-infection and tested for total GM-CSF protein by ELISA.

In vivo studies, female BALB/c mice 8-12 weeks old were injected orthotopically with 10 injections into the 4 th inguinal mammary fat pad⁴4T1 tumor cells. When the tumor reaches 50-100mm³Pair-wise matching was performed and mice were randomized into treatment groups as shown in table 1 to begin treatment. The dosing regimen is shown in figure 5. 5Gy of radiation was given 1 day before the 1 st dose of Ar20-1004 (treatment protocol 1) and optionally anti-CTLA-4 antibody 9H10(BioXell) or Syrian hamster IgG2 isotype control (BioXell, treatment protocol 2). Mice were treated 4 times at 3 day intervals using treatment regimens 1 and 2 as listed in table 6. All agents were delivered into one syringe at a time for a single dose of intratumoral administration. The total dose volume did not exceed 50 μ l/dose/mouse.

TABLE 6.

The study had two primary endpoints: (1) tumor Growth Inhibition (TGI); and (2) transition count. The animals were also examined for any effect of treatment on normal behavior, such as mobility, visual estimates of food and water consumption, weight gain/loss (body weight was measured on the first week of weekdays and then twice weekly after randomization), eye/hair pads and any other abnormal effects. Any adverse reactions or deaths were reported.

To monitor tumor growth, the volume of the primary tumor was measured using a caliper. Individual animals with > 30% weight loss observed in a single time or with > 25% weight loss measured three consecutive times were euthanized. Any group with mean weight loss > 20% or > 10% mortality stopped dosing, but this group was not euthanized and allowed to recover. In the group with > 20% weight loss, subjects who reached the end of their weight loss were euthanized. If the group treatment-related weight loss returns to within 10% of the original weight, dosing can be resumed at a lower dose or less frequent dosing schedule.

To determine the metastasis counts, 2-3 animals from the observation group were euthanized every two days starting on day 12. The lungs of the animals were removed from each mouse with minimal bronchi and tumor lesions on the lung surface were stained with indian ink and counted. When 50-100 metastatic lesions per lung group were observed in the observation group, a metastatic count endpoint was reached and all mice were euthanized and their lung metastatic lesions were counted. When evaluating all animals for metastasis,% TGI was also calculated based on tumor volume measurements taken on the last day of the study. All animals were also necropsied systemically at termination to identify any metastases by indian ink staining at the injection site, regional lymph nodes, lung, liver, kidney, spleen and brain.

Exemplary embodiments

Embodiment 1: in some embodiments, there is provided a method of treating a solid or lymphatic tumor in an individual, the method comprising: a) locally administering to the tumor site an effective amount of an infectious agent; and b) locally administering to the tumor site an effective amount of an immunomodulatory agent.

Embodiment 2: in some other embodiments of embodiment 1, the infectious agent is a virus.

Embodiment 3: in some other embodiments of embodiment 2, the virus is selected from the group consisting of: adenovirus, herpes simplex virus, vaccinia virus, mumps virus, newcastle disease virus, poliovirus, measles virus, seneca valley virus, coxsackie virus, rio virus, vesicular stomatitis virus, malaba and rhabdovirus, and parvovirus.

Embodiment 4: in some other embodiments of embodiment 2 or embodiment 3, the virus is a non-oncolytic virus.

Embodiment 5: in some other embodiments of embodiment 2 or embodiment 3, the virus is an oncolytic virus.

Embodiment 6: in some other embodiments of embodiment 5, the oncolytic virus is an oncolytic adenovirus.

Embodiment 7: in some other embodiments of embodiment 5 or embodiment 6, the oncolytic virus preferentially replicates in cancer cells.

Embodiment 8: in some other embodiments of embodiment 7, the oncolytic virus comprises a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for replication of the virus.

Embodiment 9: in some other embodiments of embodiment 8, the tumor specific promoter is the E2F-1 promoter.

Embodiment 10: in some other embodiments of embodiment 9, the tumor specific promoter is the human E2F-1 promoter.

Embodiment 11: in embodiment 9 or some other embodiments of embodiment 10, the E2F-1 promoter includes the nucleotide sequence set forth in SEQ ID NO. 1.

Embodiment 12: in some other embodiments of any one of embodiments 8 to 11, the viral gene necessary for replication of the virus is selected from the group consisting of: E1A, E1B and E4.

Embodiment 13: in some other embodiments of embodiment 1, the infectious agent is a bacterium.

Embodiment 14: in some other embodiments of embodiment 13, the bacterium is bacillus calmette-guerin (BCG), mycobacterial cell wall-DNA complex ("MCNA"), or listeria monocytogenes.

Embodiment 15: in some other embodiments of any one of embodiments 1 to 14, the infectious agent is administered directly into the tumor.

Embodiment 16: in some other embodiments of any one of embodiments 1 to 14, the infectious agent is administered into a tissue having the tumor.

Embodiment 17: in some other embodiments of any one of embodiments 1 to 16, the immunomodulator is administered directly into the tumor.

Embodiment 18: in some other embodiments of any one of embodiments 1 to 16, the immunomodulator is administered to a tissue having the tumor.

Embodiment 19: in some other embodiments of any one of embodiments 1 to 18, the infectious agent and the immunomodulatory agent are administered sequentially.

Embodiment 20: in some other embodiments of embodiment 19, the infectious agent is administered prior to administration of the immunomodulatory agent.

Embodiment 21: in some other embodiments of embodiment 19, the infectious agent is administered after administration of the immunomodulatory agent.

Embodiment 22: in some other embodiments of any one of embodiments 1 to 18, the infectious agent and the immunomodulator are administered simultaneously.

Embodiment 23: in some other embodiments of embodiment 22, the infectious agent and the immunomodulator are administered in the same composition.

Embodiment 24: in some other embodiments of any one of embodiments 1 to 23, the immune modulator is a modulator of an immune checkpoint molecule selected from the group consisting of: CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands.

Embodiment 25: in some other embodiments of embodiment 24, the immunomodulatory agent is an inhibitor of CTLA-4.

Embodiment 26: in some other embodiments of embodiment 25, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody.

Embodiment 27: in some other embodiments of embodiment 26, the anti-CTLA-4 antibody is selected from the group consisting of: ipilimumab, tremelimumab and single chain anti-CTLA-4 antibody.

Embodiment 28: in some other embodiments of embodiment 27, the anti-CTLA-4 antibody is ipilimumab.

Embodiment 29: in some other embodiments of embodiment 25, the inhibitor of CTLA-4 is an engineered lipocalin that specifically recognizes CTLA-4.

Embodiment 30: in some other embodiments of embodiment 29, the engineered lipocalin is an anti-transporter molecule that specifically binds to CTLA-4.

Embodiment 31: in some other embodiments of any one of embodiments 1 to 23, the immunomodulator is an immunostimulant.

Embodiment 32: in some other embodiments of embodiment 31, the immunostimulant is an activator of OX40, 4-1BB, or CD 40.

Embodiment 33: in some other embodiments of embodiment 32, the immunostimulant is a stimulator of CD 40.

Embodiment 34: in some other embodiments of embodiment 33, the immunomodulator is an agonist antibody to CD 40.

Embodiment 35: in some other embodiments of any one of embodiments 1-34, the method further comprises locally administering an immune-related molecule to the tumor site.

embodiment 36 in some other embodiments of embodiment 35 the immune-related molecule is selected from the group consisting of GM-CSF, IL-2, IL-12, interferon, CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I, MDA5, LGP2, and LT α β.

Embodiment 37: in some other embodiments of embodiment 35, the immune-related molecule is selected from the group consisting of: STING activators, PRRago, TLR stimulators and RLR stimulators.

Embodiment 38: in some other embodiments of any one of embodiments 35 to 37, the immune-related molecule is administered separately from the infectious agent.

Embodiment 39: in some other embodiments of embodiment 35 or embodiment 36, the immune-related molecule is expressed by the infectious agent, wherein the infectious agent comprises a nucleic acid encoding the immune-related molecule.

Embodiment 40: in some other embodiments of embodiment 39, the infectious agent is a virus comprising a viral vector, and wherein the viral vector comprises the nucleic acid encoding the immune-related molecule.

Embodiment 41: in some other embodiments of embodiment 40, the nucleic acid encoding the immune-related molecule is operably linked to a viral promoter.

Embodiment 42: in some other embodiments of embodiment 41, the virus is an adenovirus and the viral promoter is the E3 promoter.

Embodiment 43: in some other embodiments of any one of embodiments 1 to 42, the infectious agent is adenovirus serotype 5, wherein the endogenous E1a promoter and the E319kD coding region of the native adenovirus are replaced with the human E2F-1 promoter and a nucleic acid encoding human GM-CSF.

Embodiment 44: in some other embodiments of embodiment 43, the infectious agent is CG 0070.

Embodiment 45: in some other embodiments of any one of embodiments 1-44, the method further comprises locally administering a pretreatment composition to the tumor site prior to administering the infectious agent.

Embodiment 46: in some other embodiments of embodiment 45, the pretreatment composition comprises a transduction enhancing agent.

embodiment 47 in some other embodiments of embodiment 46, the transduction enhancing agent is N-dodecyl- β -D-maltoside (DDM).

Embodiment 48: in some other embodiments of any one of embodiments 1 to 47, the individual undergoes prior therapy prior to administration of the infectious agent and the immunomodulator.

Embodiment 49: in some other embodiments of embodiment 48, the prior therapy is radiation therapy.

Embodiment 50: in some other embodiments of embodiment 49, the prior therapy comprises administration of a therapeutic agent.

Embodiment 51: in some other embodiments of embodiment 50, the therapeutic agent is an agent that increases the level of an interleukin involved in an immunogenic pathway.

Embodiment 52: in some other embodiments of embodiment 50, the therapeutic agent is an agent that causes dysfunction of or damage to a structural component of a tumor.

embodiment 53 in some other embodiments of embodiment 52 the therapeutic agent is selected from the group consisting of an anti-VEGF antibody, hyaluronidase, CCL21, and N-dodecyl- β -maltoside.

Embodiment 54: in some other embodiments of any one of embodiments 48-53, the prior therapy is provided at a dose insufficient to eradicate such tumor cells.

Embodiment 55: in some other embodiments of any one of embodiments 1 to 54, the method further comprises locally administering to the tumor site an effective amount of non-activated tumor cells.

Embodiment 56: in some other embodiments of embodiment 55, such non-activated tumor cells are autologous.

Embodiment 57: in some other embodiments of embodiment 55, such non-activated tumor cells are allogeneic.

Embodiment 58: in some other embodiments of embodiment 55, such non-activated tumor cells are from a tumor cell line.

Embodiment 59: in some other embodiments of any one of embodiments 55 to 58, such non-activated tumor cells are inactivated by irradiation.

Embodiment 60: in some other embodiments of any one of embodiments 55 to 59, the infectious agent and the non-activated tumor cells are administered simultaneously.

Embodiment 61: in some other embodiments of embodiment 60, the infectious agent and such non-activated tumor cells are administered in a single composition.

Embodiment 62: in some other embodiments of embodiment 60 or embodiment 61, the infectious agent and such non-activated tumor cells are mixed immediately prior to the administration.

Embodiment 63: in some other embodiments of any one of embodiments 1 to 62, the solid or lymphoid tumor is bladder cancer.

Embodiment 64: in some other embodiments of embodiment 63, the infectious agent is administered intravesically.

Embodiment 65: in some other embodiments of embodiment 63 or embodiment 64, the immunomodulatory agent is administered intravesically.

Embodiment 66: in some other embodiments of any one of embodiments 63 to 65, the bladder cancer is muscle invasive bladder cancer.

Embodiment 67: in some other embodiments of any one of embodiments 63 to 65, the bladder cancer is a non-muscle invasive bladder cancer.

Embodiment 68: in some other embodiments of any one of embodiments 1 to 67, the infectious agent is administered weekly.

Embodiment 69: in some other embodiments of any one of embodiments 1 to 68, the immunomodulator is administered weekly.

Embodiment 70: in some other embodiments of any one of embodiments 1 to 69, the individual has high expression of one or more biomarkers in the tumor selected from: PD-1, PD-L1 and PD-L2.

Embodiment 71: in some other embodiments of any one of embodiments 1 to 70, the individual has high expression of one or more biomarkers selected from the group consisting of: CD80, CD83, CD86 and HLA-class II antigens.

Embodiment 72: in some other embodiments of any one of embodiments 1 to 71, the individual has high expression of one or more biomarkers selected from the group consisting of: CXCL9, CXCL10, CXCL11, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1, HES4, MTIB, MTIE, MTIG, MTIH, GADD45A, LAMP3 and miR-155.

Embodiment 73: in some other embodiments of any one of embodiments 1 to 72, the subject is a human subject.

Embodiment 74: in some embodiments, there is provided a kit for treating a solid or lymphatic tumor in an individual comprising: a) an infectious agent, b) an immunomodulator, and c) a device for local administration of the infectious agent or immunomodulator to a tumor site.

Embodiment 75: in some other embodiments of embodiment 74, the infectious agent is a virus.

Embodiment 76: in some other embodiments of embodiment 75, the virus is an oncolytic adenovirus that preferentially replicates in cancer cells.

Embodiment 77: in some other embodiments of embodiment 75, the virus is a non-oncolytic virus.

Embodiment 78: in some other embodiments of any one of embodiments 74 to 77, the immune modulator is a modulator of an immune checkpoint molecule selected from the group consisting of: CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands.

Embodiment 79: in some other embodiments of embodiment 78, the immunomodulatory agent is an inhibitor of CTLA-4.

Embodiment 80: in some other embodiments of embodiment 79, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody.

Embodiment 81: in some other embodiments of embodiment 80, the anti-CTLA-4 antibody is ipilimumab.

Embodiment 82: in some other embodiments of embodiment 79, the inhibitor of CTLA-4 is an engineered lipocalin that specifically recognizes CTLA-4.

Embodiment 83: in some other embodiments of embodiment 82, the engineered lipocalin is an anti-transporter molecule that specifically binds to CTLA-4.

Embodiment 84: in some other embodiments of any one of embodiments 74 to 83, the immunomodulator is an immunostimulant.

Embodiment 85: in some other embodiments of embodiment 84, the immunostimulant is an activator of OX40, 4-1BB, or CD 40.

Embodiment 86: in some other embodiments of embodiment 85, the immunostimulatory agent is an agonist of CD 40.

Embodiment 87: in some other embodiments of embodiment 86, the immunomodulator is an agonist antibody to CD 40.

Embodiment 88: in some other embodiments of any one of embodiments 74 to 87, the infectious agent comprises a nucleic acid encoding an immune-related molecule.

embodiment 89 in some other embodiments of embodiment 88 the immune-related molecule is selected from the group consisting of GM-CSF, IL-2, IL12, interferon, CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I, MDA5, LGP2, and LT α β.

Embodiment 90: in some other embodiments of embodiment 88 or embodiment 89, the infectious agent is a virus comprising a viral vector, and wherein the viral vector comprises the nucleic acid encoding the immune-related molecule.

Embodiment 91: in some other embodiments of embodiment 90, the nucleic acid encoding the immune-related molecule is operably linked to a viral promoter.

Embodiment 92: in some other embodiments of embodiment 91, the virus is an adenovirus and the viral promoter is the E3 promoter.

Embodiment 93: in some other embodiments of any one of embodiments 74 to 92, the infectious agent is adenovirus serotype 5, wherein the endogenous E1a promoter and the E319kD coding region of the native adenovirus are replaced with the human E2F-1 promoter and a nucleic acid encoding human GM-CSF.

Embodiment 94: in some other embodiments of embodiment 93, the infectious agent is CG 0070.

Embodiment 95: in some other embodiments of any one of embodiments 74 to 94, the kit further comprises a pretreatment composition comprising a transduction enhancer.

embodiment 96 in some other embodiments of embodiment 95, the transduction enhancing agent is N-dodecyl- β -D-maltoside (DDM).

embodiment 97 in some other embodiments of any one of embodiments 74 to 96, the kit further comprises an immune-related molecule selected from the group consisting of GM-CSF, IL-2, IL12, interferon, CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I, MDA5, LGP2, LT α β, a STING activator, PRRago, a TLR stimulant, and an RLR stimulant.

Embodiment 98: in some other embodiments of any one of embodiments 74 to 97, the kit further comprises a plurality of non-activated tumor cells.

Embodiment 99: in some other embodiments of embodiment 98, the kit further comprises instructions to mix the infectious agent and such non-activated tumor cells prior to administration.

Embodiment 100: in some other embodiments of embodiment 98 or embodiment 99, the device for local administration is for administering the plurality of non-activated tumor cells and the infectious agent simultaneously.

Embodiment 101: in some other embodiments of any one of embodiments 74-100, the device for local administration is for administering the infectious agent or the immunomodulatory agent directly into the tumor.

Embodiment 102: in some other embodiments of any one of embodiments 74-101, the device for local administration is for administering the infectious agent or the immunomodulatory agent to a tissue having the tumor.

Embodiment 103: in some embodiments, there is provided a pharmaceutical composition comprising: a) an infectious agent, b) an immunomodulator, and c) a pharmaceutically acceptable excipient suitable for topical administration of the composition to the site of a tumor.

Embodiment 104: in some other embodiments of embodiment 103, the pharmaceutically acceptable excipient is a polymer.

Embodiment 105: in some other embodiments of embodiment 104, the polymer is a hydrogel.

Embodiment 106: in some other embodiments of any one of embodiments 103 to 105, the infectious agent is a virus.

Embodiment 107: in some other embodiments of embodiment 106, the virus is an oncolytic adenovirus that preferentially replicates in cancer cells.

Embodiment 108: in some other embodiments of embodiment 107, the virus is a non-oncolytic virus.

Embodiment 109: in some other embodiments of any one of embodiments 103 to 108, the immune modulator is a modulator of an immune checkpoint molecule selected from the group consisting of: CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands.

Embodiment 110: in some other embodiments of embodiment 109, the immunomodulatory agent is an inhibitor of CTLA-4.

Embodiment 111: in some other embodiments of embodiment 110, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody.

Embodiment 112: in some other embodiments of embodiment 111, the anti-CTLA-4 antibody is ipilimumab.

Embodiment 113: in some other embodiments of embodiment 110, the inhibitor of CTLA-4 is an engineered lipocalin that specifically recognizes CTLA-4.

Embodiment 114: in some other embodiments of embodiment 113, the engineered lipocalin is an anti-transporter molecule that specifically binds to CTLA-4.

Embodiment 115: in some other embodiments of any one of embodiments 103 to 114, the immunomodulator is an immunostimulatory agent.

Embodiment 116: in some other embodiments of embodiment 115, the immunostimulant is an activator of OX40, 4-1BB, or CD 40.

Embodiment 117: in some other embodiments of embodiment 116, the immunostimulant is a stimulator of CD 40.

Embodiment 118: in some other embodiments of embodiment 117, the immunomodulator is an agonist antibody to CD 40.

Embodiment 119: in some other embodiments of any one of embodiments 103 to 118, the infectious agent comprises a nucleic acid encoding an immune-related molecule.

embodiment 120 in some other embodiments of embodiment 119, the immune-related molecule is selected from the group consisting of GM-CSF, IL-2, IL12, interferon, CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I, MDA5, LGP2, and LT α β.

Embodiment 121: in some other embodiments of embodiment 119 or embodiment 120, the infectious agent is a virus comprising a viral vector, and the viral vector comprises a nucleic acid encoding the immune-related molecule.

Embodiment 122: in some other embodiments of embodiment 121, the nucleic acid encoding the immune-related molecule is operably linked to a viral promoter.

Embodiment 123: in some other embodiments of embodiment 122, the virus is an adenovirus and the viral promoter is the E3 promoter.

Embodiment 124: in some other embodiments of any one of embodiments 103 to 123, the infectious agent is adenovirus serotype 5, wherein the endogenous E1a promoter and the E319kD coding region of the native adenovirus are replaced with the human E2F-1 promoter and a nucleic acid encoding human GM-CSF.

Embodiment 125: in some other embodiments of embodiment 124, the infectious agent is CG 0070.

Embodiment 126: in some other embodiments of any one of embodiments 103 to 125, the pharmaceutical composition further comprises a pretreatment composition comprising a transduction enhancer.

embodiment 127 in some other embodiments of embodiment 126, the transduction enhancing agent is N-dodecyl- β -D-maltoside (DDM).

embodiment 128 in some other embodiments of any one of embodiments 103 to 127, the pharmaceutical composition further comprises an immune-related molecule selected from the group consisting of GM-CSF, IL-2, IL12, interferon, CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I, MDA5, LGP2, LT α β, a STING activator, PRRago, a TLR stimulant, and a RLR stimulant.

Embodiment 129: in some other embodiments of any one of embodiments 103 to 128, the pharmaceutical composition further comprises a plurality of non-activated tumor cells.

Embodiment 130: in some other embodiments of embodiment 129, the plurality of non-activated tumor cells are autologous.

Embodiment 131: in some other embodiments of embodiment 129, the plurality of non-activated tumor cells are allogeneic.

Embodiment 132: in some other embodiments of embodiment 129, the plurality of inactivated tumor cells is from a tumor cell line.

Embodiment 133: in some other embodiments of any one of embodiments 129 to 132, the plurality of non-activated tumor cells are inactivated by irradiation.

Sequence listing

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Claims (42)

1. A method of treating a solid or lymphatic tumor comprising: a) topically administering an effective amount of an infectious agent to the site of the tumor; and b) locally administering an effective amount of an immunomodulator to the site of the tumor.

2. The method of claim 1, wherein the infectious agent is a virus.

3. The method of claim 2, wherein the virus is an oncolytic virus.

4. The method of claim 3, wherein the oncolytic virus is an oncolytic adenovirus.

5. The method of claim 3 or claim 4, wherein the oncolytic virus preferentially replicates in cancer cells.

6. The method of claim 5, wherein the oncolytic virus comprises a viral vector comprising a tumor cell specific promoter operably linked to a viral gene essential for replication of the virus.

7. The method of claim 6, wherein the tumor specific promoter is the E2F-1 promoter.

8. The method of claim 6 or claim 7, wherein the viral genes essential for replication of the virus are selected from the group consisting of: E1A, E1B and E4.

9. The method of claim 1, wherein the infectious agent is a bacterium.

10. The method of claim 9, wherein the bacterium is bacillus calmette-guerin (BCG), mycobacterial cell wall-DNA complex ("MCNA"), or listeria monocytogenes.

11. The method of any one of claims 1-10, wherein the infectious agent is administered directly into the tumor.

12. The method of any one of claims 1-10, wherein the infectious agent is administered to a tissue having the tumor.

13. The method of any one of claims 1-12, wherein the infectious agent and the immunomodulator are administered sequentially.

14. The method of any one of claims 1-12, wherein the infectious agent and the immunomodulator are administered simultaneously.

15. The method of any one of claims 1-14, wherein the immune modulator is a modulator of an immune checkpoint molecule selected from the group consisting of: CTLA-4, PD-1, PD-L1, PD-L2, TIM3, B7-H3, B7-H4, LAG-3, KIR, and their ligands.

16. The method of claim 15, wherein the immunomodulator is an inhibitor of CTLA-4.

17. The method of any one of claims 1-14, wherein the immunomodulator is an immunostimulant.

18. The method of claim 17, wherein the immunostimulant is an activator of OX40, 4-1BB, or CD 40.

19. The method of any one of claims 1-18, further comprising locally administering an immune-related molecule to the site of the tumor.

20. the method of claim 19, wherein the immune-related molecule is selected from the group consisting of GM-CSF, IL-2, IL-12, interferon, CCL4, CCL19, CCL21, CXCL13, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, RIG-I, MDA5, LGP2, LT α β, a STING activator, PRRago, a TLR stimulator, and a RLR stimulator.

21. The method of claim 19 or claim 20, wherein the immune-related molecule is administered separately from the infectious agent.

22. The method of claim 19 or claim 20, wherein the immune-related molecule is expressed by the infectious agent, wherein the infectious agent comprises a nucleic acid encoding the immune-related molecule.

23. The method of claim 22, wherein said infectious agent is a virus comprising a viral vector, and wherein said viral vector comprises a nucleic acid encoding said immune-related molecule.

24. The method of claim 23, wherein the nucleic acid encoding the immune-related molecule is operably linked to a viral promoter.

25. The method of claim 24, wherein the virus is an adenovirus and the viral promoter is the E3 promoter.

26. The method of any one of claims 1-25, wherein the infectious agent is adenovirus serotype 5, wherein the endogenous E1a promoter and the E319 kD coding region of the native adenovirus are replaced with the human E2F-1 promoter and a nucleic acid encoding human GM-CSF.

27. The method of claim 26, wherein the infectious agent is CG 0070.

28. The method of any one of claims 1-27, further comprising locally administering a pretreatment composition to the site of the tumor prior to administering the infectious agent.

29. The method of any one of claims 1-28, wherein the individual has undergone prior therapy prior to administration of the infectious agent and the immunomodulator.

30. The method of claim 29, wherein the prior therapy is radiation therapy.

31. The method of claim 29, wherein the prior therapy comprises administration of a therapeutic agent.

32. the method of claim 31, wherein the therapeutic agent is selected from the group consisting of an anti-VEGF antibody, hyaluronidase, CCL21, and N-dodecyl- β -maltoside.

33. The method of any one of claims 29-32, wherein the prior therapy is provided at a dose insufficient to eradicate the tumor cells.

34. The method of any one of claims 1-33, further comprising locally administering to the site of the tumor an effective amount of non-activated tumor cells.

35. The method of any one of claims 1-34, wherein the solid or lymphatic tumor is bladder cancer.

36. The method of claim 35, wherein the infectious agent is administered intravesically.

37. The method of claim 35 or 36, wherein the immunomodulatory agent is administered intravesically.

38. The method of any one of claims 1-37, wherein the individual has high expression of one or more biomarkers in the tumor selected from the group consisting of: PD-1, PD-L1 and PD-L2.

39. The method of any one of claims 1-38, wherein the individual has high expression of one or more biomarkers in the mature dendritic cells of tumor origin selected from the group consisting of: CD80, CD83, CD86 and HLA-class II antigens.

40. The method of any one of claims 1-39, wherein the individual has high expression of one or more biomarkers selected from the group consisting of: CXCL9, CXCL10, CXCL11, CCR7, CCL5, CCL8, SOD2, MT2A, OASL, GBP1, HES4, MTIB, MTIE, MTIG, MTIH, GADD45A, LAMP3 and miR-155.

41. A kit for treating a solid or lymphatic tumor in an individual comprising: a) an infectious agent, b) an immunomodulator, and c) a device for local administration of said infectious agent or immunomodulator to a tumour site.

42. A pharmaceutical composition comprising: a) an infectious agent, b) an immunomodulator, and c) a pharmaceutically acceptable excipient suitable for topical administration of the composition to a tumor site.