CN101822840A - LIGHT-antitumor antigen antibodies for prevention and treatment of primary and metastatic cancer - Google Patents

Abstract

The present invention relates to a tumor-specific antibody linked to LIGHT protein or LIGHT protein fragment, a composition containing the antibody, a method and application for preventing and treating cancer. The LIGHT-antibodies and compositions of the present invention can be used to prevent or treat primary tumors and/or metastatic tumors, reduce, inhibit, and reduce primary tumor growth and/or cancer metastasis.

Description

LIGHT-anti-tumor antigen antibody for the prevention and treatment of primary and metastatic cancer

technical field

The present invention belongs to the field of cancer prevention and treatment. The present invention relates to a tumor-specific antibody linked to LIGHT protein or its fragment, a composition containing the antibody, a method and application for preventing and treating cancer.

Background technique

LIGHT (homologous to lymphotoxin , exhibits inducible expression, and competes with HSV g lycoprotein D for h erpes virus entry mediator, areceptor expressed by T lymphocytes (homologous to lymphotoxin, exhibits inducible expression, and competes with HSV glycoprotein D Competing herpesvirus entry mediator-a receptor expressed by T lymphocytes)) is a recently identified TNF ligand superfamily type II transmembrane glycoprotein. LIGHT (TNFSF 14) is a member of the tumor necrosis factor (TNF) family, which interacts with lymphotoxin β receptor (LTβR, Lymphotoxin β receptor) and herpes virus entry mediator (HVEM, herpes virus) mainly expressed on stromal cells and T cells, respectively. entry mediator) interaction. LTβR signaling is required for the formation of organized lymphoid structures, which may be at least in part due to LTβR's ability to induce the expression of chemokines and adhesion molecules, which can attract naive T cells and dendritic cells (DCs) in lymphoid organs. ). In vivo LIGHT stimulation of LTβR on stromal cells leads to expression of CCL21, which in the absence of LTαβ (another ligand of LTβR) attracts naive T cells in the T cell domain of the spleen. These results confirm that LIGHT is able to interact with LTβR to regulate the expression of CCL21 chemokine. In addition, LIGHT also exhibits potent, CD28-independent, T cell sensitization and expansion co-stimulatory activity, resulting in enhanced antitumor T cell immunity and/or enhanced autoimmunity. Signaling through LTβR is required for organized lymphoid tissue formation. The lymphotoxin beta receptor (LTβR) plays an important role in the formation of lymphoid structures. LTβR is activated by two members of the TNF family, namely, lymphotoxin αβ and LIGHT. LTβR plays a key role in the different organization of T and B regions and the formation of LN in secondary lymphoid organs. Signaling through LTβR regulates the expression of chemokines and adhesion molecules in secondary lymphoid organs. Chemokines and adhesion molecules control the migration and localization of DCs and lymphocytes in the spleen. Overexpression of soluble LT or TNF in nonlymphoid tissues is sufficient to promote functional lymphoid neogenesis.

LIGHT has a unique role in T cell activation and lymphoid tissue formation. LIGHT is a ligand for LTβR and herpesvirus entry mediator (HVEM). LIGHT is mainly expressed on lymphoid tissues. Interaction of LIGHT with LTβR can reestablish lymphoid structure in spleen of LTα ^-/- mice. In addition, upregulation of LIGHT can lead to T cell activation and migration into non-lymphoid tissues and formation of lymphoid structures. In contrast, LIGHT ^-/- mice exhibited impaired T cell activation and delayed cardiac rejection. Thus, LIGHT is a potent co-stimulatory molecule that can also promote lymphoid tissue formation to enhance local immune responses. The lack of efficient naive T cell priming in draining lymphoid tissues and the inability to expand tumor-specific T cells in tumors hampers cancer eradication.

Micrometastases (small aggregates of cancer cells visible under a microscope) can establish at a very early stage in the development of a heterogeneous primary tumor and seed distant tissues before they are clinically detected. For example, detectable metastases can be observed in breast cancer when the primary tumor volume is small. Thus, at the time of diagnosis, many cancer patients already have microscopic metastases, an observation that has led to the development of postoperative adjuvant therapy for patients with solid tumors. Despite these advances, success has been limited, and optimal treatment of metastatic disease remains a significant challenge in cancer treatment.

Metastatic disease is a major cause of cancer morbidity and mortality. Although surgery, chemotherapy, or radiotherapy can often control primary tumor growth, it is rarely successful in eradicating metastases that have spread. An unanswered question is whether this response allows incoming CTLs to be taught to leave the tumor site. Another unanswered question is whether these CTLs are then able to patrol the periphery and efficiently remove spontaneously metastatic tumor cells. Local treatment of tumors with LIGHT can generate a large number of tumor-specific CTLs that leave the primary tumor and infiltrate distant tumors to completely eradicate pre-existing spontaneous metastases.

Naturally occurring T cell responses against malignancy in humans are often insufficient to cause tumor (primary or metastatic) regression. Immunotherapy potentially elicits tumor-reactive T cells that can seek out and destroy tumor-antigen-positive cancer cells that have disseminated, but automatic immunization of tumor-bearing hosts has shown only limited benefit. The lack of well-established antigens in most tumors limits autoimmunization or adoptive transfer therapy. Immunotherapy that is effective even in the absence of specific tumor antigens will be more applicable and therapeutically feasible. Immunotherapy is often used after conventional surgery, radiation and chemotherapy. Surgery can reduce the tumor burden but also removes a major source of tumor antigens, which may signal the withdrawal of the immune response, while radiotherapy and chemotherapy will further impair the existing immune response. It remains unclear when and how to boost the antitumor autoimmune response.

Anti-Her2/neu antibodies inhibit and delay the growth of Her2/neu+ tumors. Appropriate combination therapy may induce synergy between conventional cancer therapy and LIGHT to eradicate established tumors. A key option is to use agents that can selectively kill tumors but not immune cells. HER-2/neu (also known as HER2 or c-erb-B2) is a 185-kDa protein receptor with tyrosine kinase activity and broad similarity to the epidermal growth factor (EGF) receptor. HER-2/neu is expressed in many epithelial tumors and is known to be overexpressed in approximately 20-25% of all ovarian and breast cancers, 35-45% of all pancreatic adenocarcinomas and up to 90% of colorectal cancers. HER-2/neu overexpression is a hallmark of poor prognosis and cancer metastasis. Anti-Her2/neu antibodies can inhibit tumor growth in an FcR-dependent manner (Clynes et al., 2000). HER-2/neu-positive tumor cells are potentially good targets for tumor-reactive cytotoxic T lymphocytes that have been used in immunotherapy trials. Importantly, anti-Her2/neu (Herceptin) antibodies have been tested in several clinical trials and have been shown to be an effective adjuvant therapy for HER-2/neu-positive breast cancer (Piccart-Gebhart et al., 2005; Romond et al ., 2005). However, long-term (52 weeks) use concomitant with paclitaxel immunization often resulted in cardiac side effects. Innate immunity at the T-cell and B-cell level was observed in patients with HER-2/neu-positive tumors, confirming the immunogenicity of HER-2/neu (Ercolini et al., 2005; Kiessling et al., 2002) . The frequency of patients responding to Herceptin is limited, and most patients initially responding to Herceptin develop resistance within a year of treatment (Kiessling et al., 2002). Therefore, there is an urgent need to develop new strategies to eradicate neu+ and neu- tumors. In the present invention, we combine LIGHT and anti-neu therapy to achieve long-term immunity not only to neu but also to other tumor antigens, thereby potentially effectively eradicating primary and/or distant tumors. Human neu+ tumor cell lines treated with Herceptin showed a higher rate of cleavage by HER-2-specific CTLs (Kono et al., 2004). Therefore, it is important to determine whether there is a synergy between LIGHT and anti-Her2 antibody therapy to eradicate local or distant cancers.

The present invention provides a novel method for preventing and treating cancer (including primary tumor and metastases) using antibody-LIGHT. Antibodies against tumor surface antigens will bring LIGHT to the tumor site to attract more immune cells and kill the tumor.

Contents of the invention

In a first aspect, the present invention relates to tumor-specific antibodies linked to LIGHT protein or fragments thereof. The tumor-specific antibody is connected with LIGHT protein or its fragments to form a complex. The complex includes at least two components, one component is tumor-specific antibody, and the other component is LIGHT protein or its fragment.

In the complex of the present invention, the antibody and the LIGHT protein or its fragments may be connected by one or more methods of fusion protein formation, chemical conjugation, and immunoliposome formation. In a preferred embodiment, said antibody and said LIGHT protein or fragment thereof are linked by forming a fusion protein.

In a preferred embodiment, the present invention relates to a fusion protein comprising a tumor-specific antibody and a LIGHT protein or a fragment thereof.

Complexes and fusion proteins of the invention may be isolated, eg, may be recombinantly produced, or may be at least partially purified.

When forming a fusion protein, a linker may or may not be included between the antibody and the LIGHT protein or its fragment. A linker is an amino acid sequence consisting of one or more (eg, 1-50, 1-20, 1-15 or 1-10) amino acid residues. For example, the connecting joint may be the joint shown in Figure 1A.

Fusion proteins may contain signal peptides (targeting peptides) or tags to facilitate purification.

When forming a fusion protein, the antibody is preferably a single chain antibody, preferably a scFv.

In a preferred embodiment, the fusion protein of the present invention is composed of a signal peptide (targeting peptide) and/or a tag that facilitates purification, a tumor-specific antibody (single-chain antibody), a linker, and LIGHT protein or a fragment thereof. In a specific embodiment, the fusion protein of the present invention is composed of a tumor-specific antibody (single-chain antibody) and LIGHT protein or a fragment thereof. In a preferred embodiment, the fusion protein of the present invention consists of a tumor-specific antibody (single-chain antibody), a linker and LIGHT protein or a fragment thereof.

In the fusion protein of the present invention, the LIGHT protein or a fragment thereof may be upstream (N-terminal side) or downstream (C-terminal side) of the tumor-specific antibody (single-chain antibody).

The antibody may be a humanized monoclonal antibody, chimeric antibody, heterominibody, or single chain antibody. The antibody of the present invention is preferably an antibody specific for a tumor antigen. In one embodiment, the tumor antigen is a tumor surface antigen. In one embodiment, the antibody specifically recognizes and binds to a tumor surface antigen.

Antibodies of the invention may be antibody fragments. Antibody fragments capable of specifically binding antigens are known in the art. Preferably, the antibody is an antibody fragment sufficient to recognize a tumor antigen.

The antibodies in the complexes of the invention include, but are not limited to, antibodies specific for the following tumor antigens: HER2, HER4, HER8 and/or EGFR, such as anti-neu antibody and/or anti-Her2 antibody, or 237 and anti-pUA. Antibodies used in the complexes of the invention also include antibodies specific for proteins overexpressed on tumors or mutated transmembrane proteins. Antibodies used in the complexes of the present invention also include antibodies specific to STEAP (six transmembrane epithelial antigen of the prostate), CD55.

An example anti-human Her2/neu scFv is 7.16.4. Another example anti-human Her2/neuscFv is SEQ ID NO:5.

The LIGHT protein and fragments thereof are preferably human. See SEQ ID NO: 1 for the wild-type human LIGHT DNA sequence. See SEQ ID NO: 2 for the amino acid sequence of natural human LIGHT.

Preferably, said LIGHT protein fragment is sufficient to stimulate cytotoxic T lymphocytes.

Preferably, the LIGHT protein fragment comprises or is a fragment of the extracellular domain of the LIGHT protein.

The fragment of the LIGHT protein is preferably a fragment comprising the extracellular domain of the LIGHT protein. In a specific embodiment, the fragment of the LIGHT protein may be the extracellular domain of the LIGHT protein. In one embodiment, the extracellular domain of the LIGHT protein has the following amino acid sequence:

QLHWRLGEMVTRLPDGPAGSWEQLIQERRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV4(SEQ ID NO:

(See also gi|13124597|sp|043557|TNF14_HUMAN[13124597].

Fragments of the LIGHT protein preferably comprise about 100-150 amino acids of LIGHT. In a preferred embodiment, the LIGHT protein fragment comprises: the amino acid sequence of about 85th-239th position of the LIGHT protein.

In a preferred embodiment, the fragment of the LIGHT protein comprises: the amino acid sequence of about 85-239 positions of the LIGHT protein, or the amino acid sequence of about 90-239 positions of the LIGHT protein, or the amino acid sequence of about 90-235 positions of the LIGHT protein . The amino acid number refers to the position of the amino acid sequence shown in SEQ ID NO: 2, or the corresponding amino acid sequence position after the best alignment with the amino acid sequence.

In another embodiment, the LIGHT protein fragment comprises a conserved domain of the LIGHT protein.

In another preferred embodiment, said LIGHT protein or LIGHT protein fragment is a protease resistant LIGHT protein or LIGHT protein fragment.

In a preferred embodiment, the LIGHT protein or LIGHT protein fragment is human LIGHT protein or LIGHT protein fragment, which contains a mutation in the protease recognition sequence EQLI, resulting in protease resistance.

As an illustration, a mutant human LIGHT amino acid sequence lacking EQLI is shown in SEQ ID NO:3.

In a specific embodiment, the complex comprises or has or consists of the following sequence: antibody (eg, anti-Her2 antibody or anti-237 antibody) + LIGHT.

In another aspect, the present invention also relates to an isolated nucleic acid molecule encoding a complex of the present invention, wherein said complex is a fusion protein comprising said antibody and said LIGHT protein or LIGHT protein fragment.

The present invention also relates to an isolated nucleic acid molecule encoding a fusion protein of the present invention comprising a tumor-specific antibody and a LIGHT protein or a fragment thereof.

The present invention also relates to vectors comprising the nucleic acid molecules described above. The vector is preferably an expression vector capable of expressing said nucleic acid molecule in a host cell.

The present invention also relates to host cells comprising the vectors described above. Host cells include animal cells, plant cells, microbial cells such as E. coli cells, animal cell lines, non-reproductive animal cell lines, and the like.

The complex of the present invention can be used to prevent or treat primary tumors and/or metastatic tumors, or reduce, inhibit, or reduce primary tumor growth and/or cancer metastasis, or stimulate the production of naive T cells. At least one of chemokines, adhesion molecules and co-stimulatory molecules, or stimulate tumor-specific T cells against said tumor.

In another aspect, the invention relates to compositions comprising a complex of the invention or a nucleic acid molecule or vector of the invention.

In another aspect, the present invention relates to a pharmaceutical composition for preventing or treating primary tumors and/or metastatic tumors, or reducing, inhibiting, or reducing primary tumor growth and/or cancer metastasis, comprising the complex of the present invention Or nucleic acid molecule or carrier and pharmaceutically acceptable carrier of the present invention.

In one embodiment, the pharmaceutical composition of the present invention is preferably in a form suitable for intravenous administration, such as injection.

In a preferred embodiment, the pharmaceutical composition of the present invention reduces cancer metastasis by stimulating the production of at least one of chemokines, adhesion molecules and co-stimulatory molecules that sensitize naive T cells.

In a preferred embodiment, the pharmaceutical composition of the invention reduces primary tumor growth and/or cancer metastasis by stimulating tumor-specific T cells against said tumor.

In another embodiment, the pharmaceutical composition is for administration in combination with chemotherapeutic agents and/or radiation therapy.

In another aspect, the present invention relates to a combined preparation comprising the tumor-specific antibody linked to the LIGHT protein or a fragment thereof of the present invention and a nucleic acid molecule encoding the LIGHT protein or a fragment thereof.

In another aspect, the present invention relates to the use of the complex of the present invention or the nucleic acid molecule or carrier of the present invention in the preparation of a medicament for preventing or treating primary tumors and/or metastatic tumors, or reducing, inhibiting, or reducing primary tumor growth and/or cancer metastasis, or stimulating the production of at least one of chemokines, adhesion molecules, and co-stimulatory molecules that sensitize naive T cells, or stimulating tumor-specific T cells against said tumors cell.

The cancers/tumors to be prevented and treated by the present invention include but are not limited to breast cancer, lung cancer, prostate cancer, colon cancer, or skin cancer.

In another aspect, the present invention relates to a method for preventing or treating primary tumors and/or metastatic tumors, or reducing, inhibiting, or reducing primary tumor growth and/or cancer metastasis, said method comprising:

administering a complex of the invention or a pharmaceutical composition of a nucleic acid molecule or carrier of the invention; and

The primary tumor growth and/or cancer metastasis is reduced by activating tumor-specific anti-tumor T cells.

In one embodiment of the above method, the pharmaceutical composition is administered intravenously.

In one embodiment of the above methods, the pharmaceutical composition reduces cancer metastasis by stimulating the production of at least one of chemokines, adhesion molecules, and co-stimulatory molecules that sensitize naive T cells

In another aspect, the present invention relates to a method for preventing or treating primary tumors and/or metastatic tumors, or reducing, inhibiting, or reducing primary tumor growth and/or cancer metastasis, said method comprising:

(A) administering to an individual a pharmaceutical composition comprising a complex of the invention or a nucleic acid molecule or carrier of the invention;

(B) Reducing primary tumor growth and/or cancer metastasis by stimulating tumor-specific T cells against the tumor.

In a preferred embodiment, the nucleic acid molecule is delivered to a pre-existing tumor site.

In a preferred embodiment, the nucleic acid molecule is delivered to a site distal to a pre-existing tumor site.

In a preferred embodiment, the method further comprises administering a chemotherapeutic agent.

In a preferred embodiment, the method further comprises the use of radiation therapy.

In one embodiment, said tumor-specific antibody linked to LIGHT protein or a fragment thereof is as defined above.

Description of drawings

Figure 1 shows the construction of antibody-LIGHT fusion protein.

Figure 1A shows the molecular design of the antibody-LIGHT fusion protein. The figure above is the overall strategy for fusion proteins. The figure below is a specific example of the fusion of LIGHT and anti-her2 antibody at the gene level. scFv(neu) is an anti-Her2 single-chain Fv antibody. The example of this antibody given in the figure is 7.16.4. Other anti-Her2 antibodies or antibodies against other antigens can also be used in the present invention. The C-terminus of scFv(neu) was connected to the N-terminus of the LIGHT fragment through the linker L2. There are two non-limiting examples of L2, where L2 short is the long linker (CS), L2 long is the short linker (CL), ECD is the extracellular domain of LIGHT, and p3 is part of the vector.

Figure 1B shows LIGHT-anti-Her2 and their linkers: the sequence of anti-Her2 antibody and short linker (CS) or long linker (CL), and the ECD of mouse LIGHT.

Figure 1C shows an anti-human Her2/neu scfv, which can be used in the present invention, to be fused to LIGHT or a fragment thereof via a linker (or not) to form a fusion protein.

Figure 2 illustrates that delivery of LIGHT to neu+ tumors can enhance anti-neu immunity. Adv-mmlit (adenovirus expressing murine mutant LIGHT) inhibits neu+N202 tumor growth. See embodiment 2 for details.

Figure 3 shows that 237-LIGHT binds Ag104Ld tumor cells as well as LTβR and HVEM. These data showed that 237-LIGHT could bind both Ag104 tumor cells, as well as LTβR and HVEM, suggesting that 237-LIGHT can bind two target sites. Approximately 4 x ¹⁰⁵ Ag104Ld tumor cells were stained using the reagents indicated above each panel. Mean fluorescence of FL2 is marked by peak. The last three panels are overlays of the two stainings shown above the panel.

Figure 4 illustrates that 237-LIGHT can produce co-stimulatory effect on the proliferation of T cells stimulated by anti-CD3. Approximately 3 x ¹⁰ mixed lymph node cells and splenocytes were plated into each well of a 96-well plate that had been coated with anti-CD3 and the indicated reagents. ³ H was added 48 hours after stimulation and plates were harvested 18 hours after ³ H addition. Clearly, much better T cell responses were obtained with 1 ug/ml 237-LIGHT, comparable to anti-CD-28, the most potent anti-co-stimulatory molecule to date. Therefore, these data indicate that the fusion protein of the present invention still maintains the function of LIGHT.

Figure 5 shows that systemic application of low doses of 237-LIGHT fusion protein can limit the growth of established primary tumors. C3B6F1 mice were inoculated subcutaneously on day 0 with approximately 10 ⁵ Ag104Ld tumor cells. Mice were adoptively transferred 3×10 ⁶ activated 2C T cells (activated with SIY peptide in vitro) and injected intravenously with 10 μg of mouse immunoglobulin (mIg, as control) or 237-LIGHT antibody. mIg or 237-LIGHT dosing was repeated on day 15. The results are shown in the figure.

Figure 6 is a graph showing the results of eradication of secondary tumors after removal of primary tumors. B6C3F1 mice were inoculated with Ag104Ald tumor cells as the primary tumor at the first site. A second tumor was inoculated at the distal site 15 days later. Mice were then treated with the fusion protein 237-LIGHT on days 15, 29 and 36 as indicated in the figure.

Figure 7 shows the growth of transplanted Her2+ Tubo tumors when administered with anti-Her2 antibody or isotype IgG (Isotype IgG), where it was found that some Tubo tumor growth disappeared after initial anti-Her2 antibody treatment, but in Re-growth after cessation of treatment. BABL/c mice were inoculated with 10 ⁶ Tubo tumor cells via sc route. 100 ug of anti-Her 2 antibody (7.16.4) or isotype IgG was injected via the ip route on days 10 and 17 after tumor inoculation. Tumor growth was detected at the time points indicated in the graph. The results showed that tumor regrowth occurred in 3 out of 5 mice treated with the anti-Her 2 antibody.

Figure 8 shows tumor growth when Ad-LIGHT alone, anti-Her2 alone, and Ad-LIGHT and anti-Her2 were administered in combination. BABL/c mice were inoculated sc with 10 ⁶ Tubo tumor cells. 1e10Ad-LIGHT or Ad-LacZ viral particles (VP) were injected intratumorally on day 18 after tumor inoculation. 50 ug of anti-Her 2 antibody or isotype IgG was injected ip on days 18 and 25 after tumor inoculation. Tumor growth was detected at the time points indicated in the graph. After day 21, all treatment groups were significantly different from the isotype IgG group. After day 25, the combined Ad-LIGHT and anti-Her2 treatment group was significantly different from the Ad-LIGHT alone or monoclonal antibody-Her2 antibody groups. Statistical analysis was performed using a two-tailed student's t test. Data are expressed as mean + SEM. p<0.05 was considered to be significantly different. The results showed that anti-Her2 antibodies were able to slow tumor growth but not eradicate tumors, leading to tumor eradication only when both anti-Her2 antibodies and ad-LIGHT were given.

Figure 9 shows that spontaneous tumor growth can be controlled in Her2/neu Tg mice by combination therapy. Mice were treated as indicated shortly after tumors were first detected. See Example 6. Mice bearing spontaneous tumors were treated three times at 0, 1, 2 weeks with antibody (100 ug) or adenovirus (VP: 10 ¹⁰ ). In the combination therapy group, anti-her2 monoclonal antibody (mab) and adenovirus expressing murine mutant LIGHT (adv-mmlight) were administered.

Figure 10 shows the tumor volumes of different treatment groups in Example 7. showed that primary neu+ TUBO tumors could be eliminated by short-term treatment with the antibody and the fusion protein anti-HER antibody-LIGHT. "Ctrl" indicates a control group. "Her2" indicates the group of anti-HER antibodies. "Her2+Fab-LIGHT" indicates an anti-HER antibody-LIGHT fusion protein set. See embodiment 7 for details.

Figure 11 shows the number of lung metastases in different treatment groups in Example 8. It shows that the fusion protein anti-HER2 antibody-LIGHT can reduce lung metastases. The untreated group is the control group treated with PBS. The 7.16.4 group is the group treated with the anti-neu antibody 7.16.4 monoclonal antibody. The Fab-LIGHT group is the group treated with anti-HER2 antibody-LIGHT fusion protein. See embodiment 8 for details.

Detailed description of the invention

Targeting tumors with LIGHT-antibody fusion products in the present invention can generate strong immunity against primary tumors and metastases.

The tumor environment often forms an immune barrier that prevents sufficient levels of antigen or antigen-presenting cells from entering the draining lymph nodes thereby resulting in ineffective T cell sensitization. In order to develop a new practical method to generate strong anti-tumor immunity, the inventors directly used TNF superfamily member 14 (TNFSF14), LIGHT, to target tumor tissue, and LIGHT can directly recruit more immune cells to the tumor site and enhance anti-tumor Immunization (Yu et al., 2004, Wang et al., 2006, Fan et al., 2006). To rule out large established tumors, a combination of standard therapy and LIGHT was developed. Targeting tumors with anti-Her2 can effectively reduce tumor burden but cannot completely eradicate tumors, especially metastases. The present inventors have now demonstrated that LIGHT-anti-tumor antigen antibodies can slow down the growth of massive tumors. The inventors also demonstrated that the synergy between LIGHT and anti-Her/neu leads to better control of the primary tumor than either therapy alone. Importantly, metastatic neu+ tumors were eradicated with LIGHT-anti-her2/neu. This study can be extended to LIGHT linked to antibodies against other tumor antigens, especially in the treatment of micrometastases. Equally important, other cytokines can also be linked to anti-tumor antigen antibodies and used against micrometastases.

Early metastases may be inactive or slow growing, which may not be effective in stimulating an immune response. How to eradicate these micrometastases has become a difficult problem. The inventors' data confirmed that tumor-specific antibodies can more effectively bring LIGHT to the tumor site to inhibit tumor growth. For example, postoperative treatment of patients with neu+ tumors is feasible using an anti-neu SvFc fusion with LIGHT (Sv-Fc-neu-LIGHT) that can bring LIGHT to distant tumor sites. In this way, the inventors can facilitate the targeting of immune cells to micrometastases.

Induction of an immune response in tumor tissue by LIGHT-antibody fusions or conjugates can generate sufficient antigen-specific primed effector T cells to leave the tumor and eradicate metastases even after removal of the tumor. Combining surgical resection with targeting of primary tumors using TNFSF14 (LIGHT) is a novel strategy to elicit a better immune response to eradicate spontaneous metastases. Antibody-LIGHT treatment slows the growth of aggressive tumors.

A new approach to treating tumors, especially micrometastasis, where the tumor is not visible with imaging modalities, is to create a lymphoid microenvironment that expresses the Chemokines, adhesion molecules and co-stimulatory molecules required for naive T cell sensitization and resulting expansion of activated T cells. Can generate a wider range of anti-tumor T cells. Direct delivery of fusion proteins linking LIGHT (or other immunostimulatory agents) with anti-tumor antigen antibodies or conjugates of the two can be effective against tumors and tumor metastasis. The inventors' data clearly show that in vivo tumor volume is reduced when tumors are targeted with LIGHT-antibody conjugates or fusion products compared to control treated tumors. Distant tumors or metastases can be reduced after systemic treatment of the primary tumor.

The following specifically disclosed nucleic acid molecules of the present invention encode recombinant human LIGHT including the extracellular domain:

QLHWRLGEMVTRLPDGPAGSWEQLIQERRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV4(SEQ IDNO:

Metastatic disease is the leading cause of death in cancer patients. The initial occultation of metastases or small primary tumors can be attributed to insufficient levels of antigen available to sensitize CD8+ T cells. A method of treatment utilizing an antibody (antibody-LIGHT) conjugated to LIGHT that recognizes an antigen expressed by tumor cells can specifically and effectively target migrating cells after systemically introducing the antibody-LIGHT via intravenous (i.v.) injection. tumor cells.

As an example, in a mouse model, "237" (see literature A Mutant Chaperone Converts a Wild-Type Protein into a Tumor-Specific Antigen. Andrea Schietinger et al., SCIENCE, 13 OCTOBER 2006, VOL 314, Pages 304-308; P.L. Ward, H. Koeppen, T. Hurteau, H. Schreiber, J ExpMed 170, 217 (1989)) is a high-affinity monoclonal antibody against Ag104 tumor cells that accumulates in vivo at high concentrations after intravenous injection inside the tumor. Heterominibody237-LIGHT (linked by conjugation or genetic linking) allows the specific delivery of LIGHT into tumor tissues at various distal locations after its systemic introduction. LIGHT-antibody conjugates (eg, LIGHT-antibody fusion proteins) selectively accumulate in tumor tissue and specifically bind Ag104 tumors in vitro.

Therapeutic approaches utilizing antibodies that recognize antigens expressed by tumor cells coupled to LIGHT (antibody-LIGHT) are designed to specifically and effectively target migrating tumor cells following systemic introduction of the antibody-LIGHT by intravenous injection ( Strategy see Figure 1). Any tumor-specific antibody recognizing any tumor antigen is suitable for conjugation to LIGHT or a functional fragment thereof.

Expression of LIGHT on tumor cells can promote tumor rejection. Tumor Ag104A and its derivatives were used as one of our tumor models. Ag104A was originally derived from spontaneous osteosarcoma in C3H (H-2 ^k ) mice, and even very low doses of Ag104A (10 ⁴ ) were able to grow invasively in C3H or B6C3F1 mice (Jackson laboratory, Maine USA) with Very little infiltration. When the strong antigen L ^d (allogenic antigen) was introduced into the tumor, the tumor still remained resistant to immune recognition, suggesting that there may be a strong tumor barrier. Locally expressed mutant LIGHT can become a strong co-stimulatory molecule that can enhance the direct presentation of tumor antigens to antigen-specific T cells and prevent the anergy of infiltrating T cells in the tumor microenvironment. It appears that LIGHT has multiple functions in mediating tumor immunity. LIGHT can also enhance tumor apoptosis in vivo.

Metastases are rarely successfully eradicated by currently available cancer treatments. Inducing an immune response in primary tumor tissue prior to surgical resection can generate tumor-specific effector T cells sufficient to eradicate distant metastases. Sensitization of tumor-specific CD8+ T cells by, for example, delivery of LIGHT-antibodies in primary tumors can facilitate the subsequent exit of cytotoxic T lymphocytes (CTLs) that can home to distant tumors. Targeting the primary tumor prior to surgical resection can eradicate spontaneous metastases through an immune-mediated approach.

Metastatic tumor cells that have disseminated can remain occult and clinically undetectable for months or even years after surgical resection of the primary tumor, leading to subsequent recurrence of clinical disease. Immunotherapy strategies are suitable for eradicating this micrometastatic disease. For example, delivery of LIGHT-antibodies into primary tumors prevents metastasis formation and produces rejection of established metastases in peripheral tissues. For example, direct delivery of LIGHT as an antibody-LIGHT fusion protein to a tumor (e.g., a primary tumor) can generate sufficient numbers of effector/memory T cells from tumor tissue that can move to distant locations, resulting in a reduction in the strength of the immune response. Overall increase, increased production of inflammatory cytokines, and eradication of spontaneous metastases.

In the presence of LIGHT on the tumor surface, CTLs can be efficiently sensitized and subsequently infiltrate LIGHT-negative distant tumors through the circulation. Without the benefit conferred by the presence of LIGHT in the primary tumor, it would be expected that there would be few activated T cells at the secondary tumor site. It is possible that these effector/memory T cells generated at local tumor sites in the presence of LIGHT are able to leave the tumor and patrol and identify metastatic tumor cells at the periphery. Chemokine receptor (CCR7) has recently been shown to be a key molecule for T cells to exit peripheral tissues, including sites of inflammation, and travel to draining LNs.

As used herein, monoclonal antibodies include "chimeric" antibodies in which a portion of the heavy and/or light chains of the antibody are identical or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, The remainder of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody type or subclass; the term also includes fragments of "chimeric" antibodies, provided that the fragment It is sufficient to exhibit the desired biological activity (see US Patent 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable region antigen-binding sequences derived from a non-human primate and human constant region sequences.

"Antibody fragment" includes a portion of an intact antibody, such as the antigen-binding or variable region of an intact antibody. Examples of antibody fragments include Fab, Fab', F(ab') ₂ , single chain Fv, and Fv fragments; diabodies; linear antibodies (see U.S. Patent 5,641,870; Zapata et al., Protein Eng. 8(10):1057 -1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments have been obtained by proteolytic digestion of intact antibodies. However, these fragments can also be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments are all capable of expression and secretion in E. coli, thus allowing the facile production of large quantities of these fragments. Antibody fragments can be isolated from antibody phage libraries. Antibody fragments can also be "linear" antibodies, eg, as described in US Patent 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

Conjugates of antibodies and co-stimulatory molecules such as LIGHT can be prepared using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio ) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, iminothiolane (IT), imidate difunctional Derivatives (such as dimethyl adipimate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), diazide-based compounds (such as, Bis(p-azidobenzoyl)hexamethylenediamine), dinitrogen derivatives (such as bis-(p-diazoylbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-di isocyanate), and bisactive fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). The extracellular domain of LIGHT or a fragment thereof is conjugated to an antibody or antibody fragment specific for a tumor antigen, preferably a surface tumor antigen.

Alternatively, fusion proteins comprising anti-tumor antigen antibodies and LIGHT can be prepared, eg, by recombinant techniques or peptide synthesis. The length of this DNA may include corresponding regions encoding the two parts of the conjugate, wherein said two parts may be adjacent to each other or a region encoding a linker peptide that does not destroy the desired properties of the conjugate separate.

Figure 1 illustrates possible structures of fusion proteins or conjugates. We also give an example of a fusion protein sequence as a general strategy to guide the construction of antibody-LIGHT.

The LIGHT-antibody complexes disclosed herein can also be formulated in the form of immunoliposomes. "Liposomes" are vesicles composed of various lipids, phospholipids, and/or surfactants that are useful for drug delivery to mammals. The components of liposomes are usually arranged in a bilayer, similar to the lipid arrangement of biological membranes. Antibody-containing liposomes can be prepared by methods known in the art, such as those described in US Pat. Liposomes with increased circulation time are disclosed in US Patent 5,013,556.

For the prevention or treatment of diseases, the dose and mode of administration can be selected by a clinician according to known criteria. The appropriate dosage of the LIGHT-antibody conjugate or fusion product may depend on the type of cancer being treated, the severity and course of the disease, the size of the tumor, the extent of metastasis, whether the antibody is being administered for prophylactic or therapeutic purposes, previous therapy , the patient's clinical history and response to antibodies, and the judgment of the attending physician. The LIGHT-antibody compositions are suitable for administration to a patient at one time or over a series of treatments. Preferably, the composition is administered by intravenous infusion or subcutaneous injection. Depending on the type and severity of the disease, about 1 μg/kg to about 50 μg/kg of body weight (e.g., about 0.1-15 mg/kg/dose) of antibody can be used as an initial candidate dose, for example, by one administration or multiple divided administrations or continuous administration. Administered to the patient by infusion. An advantage of the fusion proteins of the invention is that much lower doses are used than the doses administered with the antibody alone. The dosing regimen may include administering an initial loading dose of about 0.01 mg/kg, followed by a weekly maintenance dose of about 0.22 mg/kg of anti-Her2-LIHGT fusion. However, other dosage regimens may also be useful. A typical daily dosage may range from about 1 μg/kg to 0.1 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, as appropriate, the treatment may be maintained until a desired suppression of disease symptoms is achieved, eg, reduction in tumor size/volume and reduction in metastasis. The course of this treatment can be monitored by conventional methods and assays, based on criteria known to the physician or others skilled in the art.

Suitable tumor surface antigens that can be targeted using LIGHT-antibody fusions or conjugates include the epidermal growth factor receptor family (EGFR), including HER1, HER2, HER4, and HER8 (Nam, N.H., & Parang, K. (2003) , Current targets for anti-cancer drug discovery. Current Drug Targets, 4(2), 159-179), STEAP (six transmembrane epithelial antigens of the prostate; Hubert et al., STEAP: a prostate-specific cell-surface antigen highly expressed in human Prostate tumors., Proc.Natl.Acad.Sci.USA.1999; 96(25):14523-8.), CD55 (Hsu et al., Generation and characterization of monoclonal antibodies directed against the surface antigens of cervical cancer cells., Hybrid Hybridomics. 2004:23(2):121-5).

Antibodies of the present invention can be prepared using methods known in the art. For example, the preparation of anti-neu/Her2 antibody can be found in the following literature: A B7.1-antibody fusion protein retains antibody specificity and ability to activate via the T cell costimulatory pathway. Challita-Eid PM et al., J Immunol.1998 Apr 1, 160 (7 ): 3419-26; and HER-2/neu-specific monoclonal antibodies collaborate with HER-2/neu-targeted granulocyte macrophage colony-stimulating factor secreting whole cell vaccination to augment CD8+T cell effector functionand tumor/tumor-vi 2 neu-transgenic mice. Wolpoe ME et al., J Immunol. 2003 Aug 15, 171(4): 2161-9.

Example

The details of the present invention and related effects thereof will be further described below in conjunction with the examples, but it should be understood that these examples are only for illustrating the present invention, and are not intended to limit the scope of the present invention in any way.

Materials and methods

Preparation of antibody-LIGHT fusion protein

Using standard protocols, a fusion protein 237-LIGHT construct was constructed that allows antibody 237 to specifically target Ag104A while carrying LIGHT to the tumor area. LIGHT employs an extracellular domain (see ECD in Figure 1B for its sequence).

Similarly, the fusion protein 7.16.4-LIGHT construct was constructed. 7.16.4 is an anti-her2 monoclonal antibody (Anti-her2). LIGHT employs an extracellular domain (ECD). The construction and sequence of the fusion protein 7.16.4-LIGHT are shown in Figure 1.

Figure 1 provides a detailed strategy and associated sequences.

Preparation and testing of scFv-LIGHT fusion protein (see Figure 1)

●Expression system used

Eukaryotic expression vector: pFLAG-CMV-1 (sigma) or pSecTag (invitrogen)

Cell line: CHO or 293 cells

Purification method: anti-FLAG system or Ni-NTA system

Among them, the Diabody system is constructed in the IRES system.

●Complete strategy

1. Antigen binding assay of N-terminal or C-terminal LIGHT constructs

(1) Overlap PCR of scFv-LIGHT gene construct

(2) Cloning LIGHT-scFv in pComb3X vector

(3) Expressed in E.coli Top10F'

(4) Purification with Ni-NTA system

(5) Anti-uPA activity test: FACS with HT1080 cells

2. T cell binding assay of N-terminal or C-terminal LIGHT constructs

(1) Transfer of the scFv-LIGHT fusion construct into a eukaryotic vector

(2) Transient transfection into CHO or 293 cells

(3) Purify with anti-FLAG or Ni-NTA system

(4) LIGHT functional test and anti-uPA: FACS

Mice, Cell Lines, and Reagents

Female C3HXC57BL/6F1 (C3B6F1 ) mice, 4-8 weeks old, were purchased from National Cancer Institute, Frederick Cancer Research Facility, (Frederick, MD). C57BL/6-RAG-1 deficient (RAG-1 ^−/− ) mice were purchased from Jackson Laboratories (Bar Harbor, ME). HY TCR transgenic mice (HY mice) with RAG-2 deficiency/B6 background were purchased from Taconic Farms (Germantown, NY). 2C TCR transgenic mice (2C mice) with a RAG-1 deficient background bred into B6 through 10 passages were provided by J. Chen (Massachusetts Institute of Technology, Boston, MA). OT-1 TCR transgenic mice (OT-1 mice) were provided by A. Ma (The University of Chicago). RAG-1 ^-/- , HY, 2C, OT-1 mice were bred and maintained in a special pathogen-free facility at the University of Chicago. Animals were cared for and used according to institutional policies.

AG104A fibrosarcomas arose spontaneously in aged C3H mice and were cultured adaptively as described (Ward 1989 JEM). AG104A overexpressing murine H-2L ^d (AG104-Ld), a transfectant of AG104A cells, has been described previously. These tumor cell lines were maintained in DMEM (Mediatech) supplemented with 10% FCS (Sigma-Aldrich), 100 U/ml penicillin and 100 μg/ml streptomycin (BioWhittaker). Hybridoma cell lines producing anti- ^Ld (clone 30-5-7) and anti-2CTCR (1B2) antibodies were obtained from D. Sachs (National Institute of Health, Bethesda, MD) and T. Gajweski (The University of Chicago), respectively.

Monoclonal antibodies produced by hybridomas were purified from culture supernatants by standard protocols using protein G columns. The 1B2 antibody was conjugated to FITC or biotin by the Monoclonal Antibody Facility at the University of Chicago. PE-conjugated anti-CD8 antibody, Cy-Chrome (CyC)-conjugated streptavidin, CyC-conjugated anti-CD44 antibody, PE-conjugated anti-CD62L antibody and PE-conjugated Th1.2 antibody were purchased from BD Biosciences. FITC-conjugated goat anti-mouse IgG was purchased from Caltag. PE-conjugated streptavidin was purchased from Immunotech. Donkey anti-human IgG conjugated to PE was purchased from Jackson Immunological Research Lab (West grove, PA). Biotinylated goat anti-SLC antibody was purchased from R&Dsystem Inc. (Minneapolis, MN). AP-conjugated rabbit anti-goat Ig antibody was purchased from Vector Laboratories Inc. (Burlingame, CA). Purified goat anti-SLC antibody was purchased from Pepro Tech (Rock hill, NJ). Collagenase (type 4) was purchased from Sigma-Aldrich. CFSE was purchased from Molecular Probes. The HVEM-Ig and LTβR-Ig fusion proteins used in this study have been described previously.

tumor growth in vivo

Tumor cells were injected subcutaneously into the lower back of the mice, ie, 0.5-1 cm above the base of the tail. Tumor growth was measured every 3 to 4 days using calipers. The size (in cubic centimeters) is calculated by the formula V=πabc/6 (where a, b and c are three orthogonal diameters).

Histology

Tumor tissues were collected for histological examination at the indicated times, fixed in 10% neutral buffered formalin, processed for paraffin embedding, and stained with hematoxylin and eosin. For immunohistochemical staining of SLC, tumor tissue was harvested, embedded in OCT compound (Miles-Yeda, Rehovot, Israel), and frozen at -70°C. Frozen sections (5-10 [mu]m thick) were fixed in cold 2% formalin in PBS and permeabilized with 0.1% saponin/PBS. Sections were pre-blocked with 5% goat serum in 0.1% saponin/PBS for half an hour at room temperature in a wet chamber. To stain SLC, it was first incubated with a 1/25 dilution of biotinylated goat anti-SLC antibody (R&D systems Inc. Minneapolis, MN) in blocking buffer. Alkaline phosphatase-conjugated rabbit anti-goat Ig antibody (Vector Laboratories Inc. Burlingame, CA) was added after 2 hours. For immunofluorescence staining, sections were blocked with 2% normal mouse serum, rabbit serum and goat serum in PBS for half an hour at room temperature in a humid chamber. Replace the blocking solution with 50 μl of the primary antibody diluted 1/100 in the blocking solution, PE-conjugated anti-Th1.2 (BD PharMingen) or PE-conjugated anti-CD8 (BD PharMingen), and incubate the slices at room temperature for 1 hour in a wet chamber . Samples were mounted in Mowiol 4-88 (BD Biosciences, La Jolla, CA) containing 10% 1,4-diazabicyclo[2.2.2]octane. Samples were analyzed within 48 hours with a Zeiss Axioplan microscope (Zeiss, Oberkochen, Germany) and a Photometrics PXL CCD camera (Photometrics, Tucson, AZ). No-Neighbor deconvolution was performed using Openlab v2.0.6 (Improvision, Lexington, MA).

ELISA for CCL21

Tumor homogenates were prepared and analyzed for CCL21. Similar amounts of tumor tissue were collected from tumor-bearing mice, weighed, homogenized in PBS containing protease inhibitors, and the supernatant collected by centrifugation. Polystyrene 96-well microtiter plates (Immulon 4, Dynatech Laboratories, Chantilly, VA) were coated with 2 μg/ml goat anti-mouse CCL21 in PBS, and then blocked with 0.1% bovine serum albumin (BSA) in PBS for 30 days at room temperature. minute. After washing, serial dilutions of a known concentration standard (recombinant CCL21, 50 ng/ml, R&D) and samples were added and incubated for 2 hours at room temperature. After 3 washes, biotinylated rabbit anti-SLC Ab was added to the wells. After 2 hours of incubation and washing, 50 μl of 1/1000 diluted alkaline phosphatase-conjugated avidin (Dako) was added and incubated for 1 hour, followed by color development. The developed color was measured at 405 nm on an automated plate reader (Spectra-Max 340, Molecular Devices, Sunnyvale, CA), and the amount of CCL21 was determined from a standard curve by ELISA and normalized to tissue weight. Data are mean ± s.d.

T cell co-stimulation assay

T cells were purified by negative selection in a magnetic field according to the manufacturer's instructions (Miltenyi Biotec, Auburn, California). The purity of the isolated T cells was assessed to be greater than 95% by flow cytometry using an anti-CD3 monoclonal antibody. Plates coated with 0.2 g/ml anti-CD3 monoclonal antibody were further coated with mutated LIGHT-flag at 37°C for 4 hours. After washing, purified T cells (1×10 ⁶ cells/ml) were cultured in the wells. A soluble form of anti-CD28 monoclonal antibody (1 μg/ml) was used. In all experiments, T cell proliferation was assessed by adding 1 Ci/well3H ^- thymidine during the last 15 hours of the 3 day culture period. Incorporation ^of3H -thymidine was measured in a TopCount microplate scintillation counter (Packardinstrument, Meriden, CT).

Cell Isolation from Tumor Tissue

The mice were first bled to reduce blood contamination of the tumor tissue. Tumor tissues were collected, washed in PBS, cut into pieces, resuspended in DMEM supplemented with 2% FCS and 1.25 mg/ml collagenase D (collagenase D solution) and incubated at 37°C in a shaking incubator for 40 minutes. After 40 minutes the single cell suspension was collected, and the cell clumps were digested in collagenase D solution for another 40 minutes until all tumor tissue was dissociated into a single cell suspension.

pharmaceutical composition

Therapeutic compositions used herein can be formulated as pharmaceutical compositions with carriers suitable for the desired method of delivery. Suitable carriers include substances which, when combined with the therapeutic composition, retain the antitumor function of the therapeutic composition. Examples include, but are not limited to, various standard pharmaceutical carriers such as sterile phosphate-buffered saline, bacteriostatic water, and the like. Therapeutic formulations can be dissolved and administered by any route suitable for delivering the therapeutic composition to the tumor site. Potentially effective routes of administration include, but are not limited to, intravenous, parenteral, intraperitoneal, intramuscular, intratumoral, intradermal, intraorgan, orthotopic, and the like. Formulations for intravenous injection contain therapeutics in preserved bacteriostatic aqueous solution, sterile unpreserved water, and/or diluted in polyethylene chloride or polyethylene bags containing sterile sodium chloride for injection combination. For therapeutic protein preparations, lyophilization can be performed and stored in sterile powder form, preferably under vacuum, for reconstitution in bacteriostatic water (containing a preservative such as benzyl alcohol) or sterile water prior to injection. Dosages and dosing regimens for cancer treatment using the methods disclosed herein may vary with the method and target cancer, and generally depend on a variety of factors known and understood in the art.

Determination of cytokines in spleen and tumor

Tumor and spleen homogenates were prepared as described (Yu et al., 2003). Briefly, similar amounts of tumor or spleen tissue were collected, weighed and homogenized in PBS containing protease inhibitors, and the supernatant collected by centrifugation. The amount of cytokines in the supernatant was quantified using the Cytometric bead array kit (CBA) (BD Biosciences) on a FACS Caliber cytometer equipped with CellQuestPro and CBA software (Becton Dickinson) according to the manufacturer's instructions.

Statistical analysis of tumor growth differences

Since tumor growth was observed repeatedly over time in the same mice, the data were analyzed using a random effects model for longitudinal data. For each experiment, it was assumed that tumor growth was treatment dependent and followed a linear growth rate over time. The model gives overall estimates for the intercept and slope of the linear growth for each group. Intercept and slope were allowed to vary between individual mice. Compare the slopes, ie, the growth rates, which are different in the different treatment groups. Actual tumor growth may not follow a linear growth trend throughout the follow-up period. In some experiments, tumor growth increased slowly in the early stages and accelerated in the later stages. A quadratic term was added to the follow-up time in the above random-effects model.

Wild-type human LIGHT DNA sequence (sequence encoding protease site EQLI is underlined):

5'-ATGGAGGAGAGTGTCGTACGGCCCTCAGTGTTTGTGGTGGATGGACAGACCGACATCCCATTCACGAGGCTGGGACGAAGCCACCGGAGACAGTCGTGCAGTGTGGCCCGGGTGGGTCTGGGTCTCTTGCTGTTGCTGATGGGGGCTGGGCTGGCCGTCCAAGGCTGGTTCCTCCTGCAGCTGCACTGGCGTCTAGGAGAGATGGTCACCCGCCTGCCTGACGGACCTGCAGGCTCCTGG GAGCAGCTGATA CAAGAGCGAAGGTCTCACGAGGTCAACCCAGCAGCGCATCTCACAGGGGCCAACTCCAGCTTGACCGGCAGCGGGGGGCCGCTGTTATGGGAGACTCAGCTGGGCCTGGCCTTCCTGAGGGGCCTCAGCTACCACGATGGGGCCCTTGTGGTCACCAAAGCTGGCTACTACTACATCTACTCCAAGGTGCAGCTGGGCGGTGTGGGCTGCCCGCTGGGCCTGGCCAGCACCATCACCCACGGCCTCTACAAGCGCACACCCCGCTACCCCGAGGAGCTGGAGCTGTTGGTCAGCCAGCAGTCACCCTGCGGACGGGCCACCAGCAGCTCCCGGGTCTGGTGGGACAGCAGCTTCCTGGGTGGTGTGGTACACCTGGAGGCTGGGGAGAAGGTGGTCGTCCGTGTGCTGGATGAACGCCTGGTTCGACTGCGTGATGGTACCCGGTCTTACTTCGGGGCTTTCATGGTGTGA-3'(SEQ ID NO：1)

天然人LIGHT氨基酸序列(加下划线显示蛋白酶消化位点)：MEESVVRPSVFVVDGQTDIPFTRLGRSHRRQSCSVARVGLGLLLLLMGAGLAVQGWFLLQLHWRLGEMVTRLPDGPAGSW EQLI QERRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPC GRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV(SEQ ID NO：2)

一种突变的人LIGHT氨基酸序列(缺乏EQLI，以点表示)：MEESVVRPSVFVVDGQTDIPFTRLGRSHRRQSCSVARVGLGLLLLLMGAGLAVQGWFLLQLHWRLGEMVTRLPDGPAGSW....QERRSHEVNPAAHLTGANSSLTGSGGPLLWETQLGLAFLRGLSYHDGALVVTKAGYYYIYSKVQLGGVGCPLGLASTITHGLYKRTPRYPEELELLVSQQSPCGRATSSSRVWWDSSFLGGVVHLEAGEKVVVRVLDERLVRLRDGTRSYFGAFMV(SEQ ID NO：3)

一种抗-人Her2/neu scFv的序列CATATGCAGGTGCAGCTGTTGCAGTCTGGGGCAGAGTTGAAAAAACCCGGGGAGTCTCTGAAGATCTCCTGTAAGGGTTCTGGATACAGCTTTACCAGCTACTGGATCGCCTGGGTGCGCCAGATGCCCGGGAAAGGCCTGGAGTACATGGGGCTCATCTATCCTGGTGACTCTGACACCAAATACAGCCCGTCCTTCCAAGGCCAGGTCACCATCTCAGTCGACAAGTCCGTCAGCACTGCCTACTTGCAATGGAGCAGTCTGAAGCCCTCGGACAGCGCCGTGTATTTTTGTGCGAGACATGACGTGGGATATTGCAGTAGTTCCAACTGCGCAAAGTGGCCTGAATACTTCCAGCATTGGGGCCAGGGCACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGCAGTCTGTGTTGACGCAGCCGCCCTCAGTGTCTGCGGCCCCAGGACAGAAGGTCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTACCAGCAGCTCCCAGGAACAGCCCCCAAACTCCTCATCTATGGTCACACCAATCGGCCCGCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCCATCAGTGGGTTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGGACCAAGCTGACCGTCCTAGGTAGCGGCCGC(SEQ ID NO：5)

Example 1: Coupling or conjugation of LIGHT expression to a tumor targeting agent

In one aspect, to enable delivery of a mutant LIGHT expressing delivery system or an equivalent delivery system, the mutant LIGHT can be coupled or conjugated to a tumor targeting agent such as a tumor specific antibody. For example, tumor-specific antibodies can be conjugated to LIGHT, thereby selectively delivering the fusion protein to tumor sites. In addition, tumor-specific antibodies can be engineered to be expressed on the surface of viral delivery systems, or liposomal vesicles can be coated with tumor-specific antibodies. A delivery vehicle expressing mutated LIGHT and containing a tumor-targeting agent will first target specific tumor cells, and then transform the tumor cells to express mutated LIGHT on the surface of the tumor cells. This targeted expression of mutant LIGHT on the tumor surface will induce chemokines on stromal cells surrounding the tumor to attract T cells and lead to initial sensitization of T cells. This treatment is suitable for all tumors, especially solid tumors. 4T1, MC38, B16, and mastocytomas have been treated with ad-LIGHT and have shown reductions in primary and/or secondary tumors. Therefore, various tumors, especially their metastases, can be targeted using LIGHT-antibodies. For example, by systemic injection, anti-her2/neu antibody-LIGHT can bring LIGHT to the metastatic tumor site expressing HER2/neu, which can then generate a local immune response to clear the tumor. Therefore, the fusion protein can be delivered by any systemic and local route, and due to the specificity of the antibody or other agent for the tumor antigen, the fusion protein will be more localized to the tumor site.

Example 2: Adv-LIGHT can promote the rejection of Her2+ tumors

LIGHT-expressing adenovirus was locally delivered to Her2+ tumors to test whether local administration of LIGHT could promote the rejection of Her2+ tumors.

Figure 2 illustrates that delivery of LIGHT to neu+ tumors can enhance anti-neu immunity. Adv-mmlit (adenovirus expressing murine mutant LIGHT) inhibits neu+N202 tumor growth, even in Her2 transgenic mice.

Transgenic FBV mice expressing Her2 develop mammary tumors in adulthood that resemble human breast cancer and are extremely difficult to treat because of the preexisting Her2 expression in the transgenic mice and resistance to Her2 may have developed. On days 18 and 20, approximately 2×10 ¹⁰ viral particles of adv-lacz or adv-mmlit (adenovirus expressing murine mutant LIGHT, which is resistant to protease degradation; adv-lacz and adv-mmlit) were injected into the tumor. For the construction process of mmlit, see the construction of ad-lacz and ad-mmLIGHT in The Journal of Immunology, 2007, 179: 1960-1968, respectively). Tumor growth was monitored weekly and tumor size was measured. Tumor growth was much slower in the group treated with adv-mmlit compared to the control adv-lacz.

Example 3: Functional activity of LIGHT-antibody fusion proteins

By flow cytometry, LTβR-Ig and HVEM-Ig were used to measure the binding ability of 237-LIGHT (a fusion protein of 237 antibody and LIGHT, see Materials and Methods for its construction) to the receptor of LIGHT (LTβR and HVEM) ( image 3). Figure 3 shows that this fusion protein can still maintain the ability to bind to tumors and LIGHT receptors. The fusion protein can bind LTβR and HVEM after binding to tumor (237 specifically binds to Ag104). To test whether this fusion protein still retains its ability to activate T cells, we first tested 237-LIGHT in vitro for its ability to co-stimulate T cells in the presence of a suboptimal dose of plate-bound anti-CD3, thereby determining its functional activity . The results showed that the functionality of 237-LIGHT was comparable to that of anti-CD28 (see Figure 4). Therefore, it was shown that our strategy for generating fusion proteins is feasible.

To test whether 237-LIGHT fusion protein can inhibit tumor growth in vivo, B6C3HF1 mice were subcutaneously (sc) injected with 5×10 ⁴ Ag104-tumor cells for 10 days, and then treated with 10 μg fusion protein. Small doses of fusion protein, ie, 10 μg, showed inhibition of tumor growth (see Figure 5). Fusion proteins can lead to strong anti-tumor immunity.

This example demonstrates that 237-LIGHT can bind to LIGHT's receptors, LTβR and HVEM, by flow cytometry using LTβR-Ig and HVEM-Ig, respectively, and demonstrates that tumor-specific antibodies conjugated to LIGHT can stimulate immunity to reduce tumor growth .

Example 4 Treatment of residual tumor after surgical resection with LIGHT-antibody fusion protein

In order to test whether the targeting agent antibody-LIGHT can powerfully eliminate small numbers of metastatic tumor cells or residual cancer cells that cannot effectively stimulate the immune system, we designed a two-tumor model to simulate clinical scenarios. Two sites were inoculated with Ag104L ^d tumor cells, one site was inoculated with 10 ⁶ cells, and the other site was inoculated with 1×10 ⁴ cells. Two weeks later, larger tumors ( ¹⁰⁶ ) were treated with Ad-LIGHT (see adv-mmlight above) and removed surgically 10 days after treatment. Mice were treated systemically on days 15, 29 and 36 with 237-LIGHT (see above) at the doses described herein. The results are shown in Figure 6, which shows that the LIGHT-antibody can be used to eradicate secondary tumors after surgical removal of the primary tumor (the experiment was set up in a situation similar to that of most cancer patients). This suggests that systemic antibody-LIGHT therapy can generate a strong immune response to powerfully eliminate small numbers of metastatic tumor cells or residual cancer cells that cannot effectively stimulate the immune system.

Example 5: Synergy of Anti-Her2 Antibodies and LIGHT

Tubo is a tumor line derived from Balb/c Tg mice overexpressing the mutant neu gene. We observed that this tumor line was sensitive to anti-Her2 antibody (7.16.4) treatment both in vivo and in vitro. However, when the tumors were fully established, the effects of both the antibody and LIGHT diminished. And once the anti-neu antibody treatment was discontinued, the tubo would re-grow within 3-4 weeks (see Figure 7).

In another experiment, 10e6 tubo tumor cells were inoculated s.c. into BABL/c mice. On day 18 after tumor inoculation, 10e10 VPs (viral particles) of Ad-LIGHT or Ad-LacZ were injected into the tumor. On days 18 and 25 after tumor inoculation, 50 ug of anti-Her 2 antibody or isotype IgG was injected i.p. Tumor growth was detected at indicated time points. After day 21, all treatment groups were significantly different compared to isotype IgG. After day 25, the Ad-LIGHT combined with anti-Her2 group was significantly different from either the Ad-LIGHT (see above adv-mmlight) alone group or the anti-Her alone group. Statistical analysis was performed by two-tailed Student's t test. Data shown are mean+SEM. p<0.05 was considered to be significantly different. The result is shown in Figure 8. Clearly, no tumors were detected in the case of the combination, in contrast, tumors continued to grow when the monotherapy was used (see Figure 8). All five mice in each of the other groups had tumors except for the combination drug, and all the mice died within 2-3 weeks.

Example 6: Control of spontaneous tumor growth in Her2/neu Tg mice by combination therapy ) (FBV background) routinely develops mammary carcinoma at 4-5 months after birth. These tumors in Tg mice are extremely difficult to treat because of the immune evasion of such tumors in Tg mice. The inventors treated these mice in three groups of mice: Anti-her2+ad-Laz, Anti-Her2+ad-LIGHT and no treatment group. Each group was given 100ug anti-her2 monoclonal antibody (Anti-her2, 7.16.4) and 10 ¹⁰ VP (virus particles) of adenovirus expressing mouse mutant LIGHT (ad-LIGHT, For its construction, see The Journal of Immunology, 2007, 179: 19601968) or an adenovirus expressing Laz (ad-Laz, for its construction, see The Journal of Immunology, 2007, 179: 1960 1968), or without treatment. In the combined treatment group, an anti-her2 monoclonal antibody (Anti-her2, 7.16.4) and an adenovirus expressing a murine mutant LIGHT (ad-LIGHT, see above for its construction) were administered. Without treatment, mice die within 5-6 weeks of first detection of tumor masses. Importantly, tumors in mice treated with the combination therapy did not grow during treatment and remained unchanged for the subsequent 6-7 weeks. As shown in Figure 9.

These data demonstrate that systemic targeting of tumors with antibody-LIGHT can also eradicate distant tumors. Moreover, ad-LIGHT and antibodies can have a synergistic effect, inhibiting spontaneous tumor growth. Therefore, Antibody-LIGHT can be used as a drug to treat patients with metastatic cancer.

Example 7: Therapeutic effect of fusion protein anti-HER antibody-LIGHT on primary neu+ reactive tumors

This example demonstrates that fusion proteins can be used to control HER2/neu+ responsive tumors.

Similar to human HER-2/neu+ tumors, TUBO was able to respond to anti-neu antibodies in vitro. TUBO is a spontaneous tumor from neu Tg (transgenic) mice, and TUBO cells are an in vitro clonal cell line established from spontaneously generated mammary lobular carcinoma in BALB-neuTg mice (Journal of Immunology, 165:5133-42, 2000) . On day 0, 4×10 ⁵ TUBO was inoculated on the back of Balb/c mice. In the anti-HER antibody-LIGHT fusion protein group (i.e. Her2+Fab-LIGHT group; n=5/group), a small dose (50ug each time) of anti-neu antibody 7.16.4 was given on the 15th, 18th and 21st days to reduce Tumor burden, then, on the 21st, 24th and 27th days, a small dose (20ug each time) of the fusion protein anti-HER antibody-LIGHT (ie Fab-LIGHT) was given. Tumor growth is detected.

In the anti-HER antibody group (ie, Her2 group; N=5), a small dose (50 ug) of anti-neu antibody 7.16.4 was administered on the 15th, 18th, and 21st days.

Control group (ie Ctrl group; N=5): no treatment.

Fab-LIGHT is a fusion protein obtained by fusing the anti-neu scFv antibody and the 85th-239th amino acid fragment of mouse LIGHT through a linker. For the construction of the fusion protein, please refer to the construction of the fusion protein in Figure 1.

The results are shown in Figure 10. The results showed that anti-neu antibody could slightly delay tumor growth (see Her2 group in Figure 10), but administration of the fusion protein anti-HER antibody-LIGHT 3 weeks after tumor inoculation eliminated the tumor (see Her2+Fab in Figure 10 -LIGHT group). Therefore, fusion proteins can be used to shorten anti-HER2/neu antibody therapy and eliminate remaining tumors.

Example 8: The therapeutic effect of anti-HER2 antibody-LIGHT fusion protein on metastatic tumors

This example demonstrates that fusion proteins can be used to reduce metastatic tumors.

4T1-neu is the mouse mammary tumor 4T1 transfected with neu (Miller, FR, BEMiller, and GH Heppner. 1983. Invasion Metastasis 3:22-31, 1983), which metastasized spontaneously 10-12 days after subcutaneous inoculation. On day 0, Balb/c mice (n=5 for each group) were inoculated with 2×10 ⁵ 4T1-neu. Three small doses of anti-neu antibody 7.16.4 monoclonal antibody (100ug per injection; 7.16.4 group) or fusion protein anti-HER2 antibody-LIGHT "Fab-LIGHT" ( The doses were 20, 50, 50ug; Fab-LIGHT group), or PBS (untreated group). On day 23, the primary tumor was removed prior to injection of the fusion protein. Lungs were collected on day 33 and lung metastases were counted.

The results are shown in Figure 11. The data showed that the ability of the fusion protein anti-HER2 antibody-LIGHT to reduce lung tumor metastasis was significantly stronger than that of the anti-neu antibody (see Figure 11). Therefore, the fusion protein can be used to reduce or eliminate distant metastatic tumors.

cited publications

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sequence listing

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<120> LIGHT-anti-tumor antigen antibody for prevention and treatment of primary and metastatic cancer

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Claims (27)

CLAIMS 1. A complex comprising a tumor-specific antibody and a LIGHT protein or LIGHT protein fragment linked to the antibody. 2. The complex of claim 1, wherein the antibody and the LIGHT protein or LIGHT protein fragment are linked by one or more methods of fusion protein formation, chemical conjugation, and immunoliposome formation. 3. The complex of claim 1 or 2, wherein said antibody and said LIGHT protein or LIGHT protein fragment are connected by forming a fusion protein, with or without a linker between said antibody and said LIGHT protein or LIGHT protein fragment ; the antibody is preferably a single chain antibody, preferably a scFv. 4. The complex of any preceding claim, wherein the antibody is a humanized monoclonal antibody, chimeric antibody, heterominibody, or single chain antibody. 5. The complex of any one of the preceding claims, wherein the antibody recognizes a tumor surface antigen. 6. The complex of any one of the preceding claims, wherein the antibody is an antibody fragment sufficient to recognize a tumor antigen. 7. The complex of any one of the preceding claims, wherein the LIGHT protein or LIGHT protein fragment is human LIGHT protein or LIGHT protein fragment. 8. The complex of any one of the preceding claims, wherein the LIGHT protein fragment is sufficient to stimulate cytotoxic T lymphocytes. 9. The complex of any one of the preceding claims, wherein the LIGHT protein fragment comprises or is a fragment of the LIGHT protein ectodomain (SEQ ID NO: 4). 10. The complex of claim 9, wherein said fragment comprises about 100-150 amino acids of LIGHT. 11. The complex of claim 9, wherein said fragment comprises: the amino acid sequence of about 85-239 positions of LIGHT protein, or the amino acid sequence of about 90-239 positions of LIGHT protein, or the amino acid sequence of about 90-235 positions of LIGHT protein amino acid sequence. 12. The complex of any preceding claim, wherein the LIGHT protein or LIGHT protein fragment is a protease resistant LIGHT protein or LIGHT protein fragment. 13. The complex of any one of the preceding claims, wherein the LIGHT protein or LIGHT protein fragment is human LIGHT protein or LIGHT protein fragment comprising a mutation in the protease recognition sequence EQLI, such as SEQ ID NO:3. 14. The complex of any one of the preceding claims, wherein the LIGHT protein fragment comprises an extracellular domain with the amino acid sequence shown in SEQ ID NO:4. 15. The complex of any one of the preceding claims, wherein the LIGHT protein fragment comprises a conserved domain of the LIGHT protein. 16. The complex of any one of the preceding claims, wherein the antibody is an antibody specific to the following tumor antigens: epidermal growth factor receptor family (EGFR), including HER1, HER2, HER4 and HER8, STEAP (six times of the prostate transmembrane epithelial antigen), CD55, such as anti-neu antibody and/or anti-Her2 antibody, such as the antibody shown in 7.16.4 or SEQ ID NO:5. 17. An isolated nucleic acid molecule encoding the complex of any one of claims 3-16, wherein said complex is a fusion protein comprising said antibody and said LIGHT protein or LIGHT protein fragment. 18. A vector comprising the nucleic acid molecule of claim 17. 19. A host cell comprising the vector of claim 18. 20. A composition comprising a complex according to any one of claims 1-16 or a nucleic acid molecule according to claim 17 or a vector according to claim 18. 21. A pharmaceutical composition for preventing or treating primary tumors and/or metastatic tumors, or reducing primary tumor growth and/or cancer metastasis, comprising a compound according to any one of claims 1-16 or a claim The nucleic acid molecule of claim 17 or the carrier and pharmaceutically acceptable carrier of claim 18. 22. The pharmaceutical composition of claim 21 in a form suitable for intravenous administration. 23. The pharmaceutical composition of claim 21, wherein the medicament reduces cancer metastasis by stimulating the production of at least one of chemokines, adhesion molecules, and costimulatory molecules that sensitize naive T cells. 24. The pharmaceutical composition of claim 21, wherein said pharmaceutical composition reduces primary tumor growth and/or cancer metastasis by stimulating tumor-specific T cells against said tumor. 25. The pharmaceutical composition of claim 21, wherein said cancer is breast cancer, lung cancer, prostate cancer, colon cancer, or skin cancer. 26. The pharmaceutical composition according to claim 21, wherein said pharmaceutical composition is for administration in combination with chemotherapeutic agents and/or radiotherapy. 27. Use of the complex of any one of claims 1-16 or the nucleic acid molecule of claim 17 or the carrier of claim 18 in the preparation of a medicament for the prevention or treatment of primary tumors and/or metastasis tumors, or reduce primary tumor growth and/or cancer metastasis, or stimulate the production of at least one of chemokines, adhesion molecules, and co-stimulatory molecules that sensitize naive T cells, or stimulate anti-tumor Tumor-specific T cells.

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