CN117979990A - Compositions and methods for treating melanoma - Google Patents

Abstract

The present disclosure provides compositions and methods for treating melanoma.

Description

Compositions and methods for treating melanoma

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Application No. 63/227,323 filed on July 29, 2021 and U.S. Application No. 63/256,377 filed on October 15, 2021, the entire contents of each of which are hereby incorporated by reference.

Background Art

Cancer is the second leading cause of death worldwide. Conventional treatments such as chemotherapy, radiotherapy, surgery, and targeted therapies (e.g., including recent advances in immunotherapy) have improved outcomes in patients with advanced solid tumors. Over the past few years, the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) have approved checkpoint inhibitors (ipilimumab targeting the CTLA-4 pathway, and targeting programmed death receptor/ligand [PD/PD-L1], including atezolizumab, avelumab, durvalumab, nivolumab, cemiplimab, and pembrolizumab) for the treatment of patients with a variety of cancer types (primarily solid tumors, including melanoma). However, these treatments have not shown success in treating advanced patients with refractory tumors. Similarly, clinical efforts to treat cancer using vaccines that stimulate targeted immune responses against tumors have also been unsuccessful in such advanced patients.

Summary of the invention

The poor prognosis of certain cancers (e.g., such as melanoma) highlights the need for additional treatment methods. The present disclosure provides, among other things, the insight that pharmaceutical compositions (e.g., immunogenic compositions, such as, for example, vaccines, in some embodiments) that deliver RNA molecules encoding melanoma tumor-associated antigens (TAAs) (e.g., melanoma TAAs) represent particularly effective treatment options for patients with melanoma. Such RNA molecules can, for example, target dendritic cells in lymphoid tissue. The present disclosure also provides, among other things, the insight that the pharmaceutical compositions described herein are particularly useful and/or effective when administered to patients with advanced melanoma (e.g., stage III or stage IV melanoma). Advanced cancers, such as advanced melanoma, are also referred to as "late stage" cancers. In addition, the present disclosure provides such particular insight that patients with no evidence of disease (e.g., in some embodiments, patients whose melanomas have been completely resected) may still benefit from anti-tumor immunity induced by such pharmaceutical compositions when first administered with the pharmaceutical compositions described herein.

Without wishing to be bound by any particular theory, since TAAs are typically non-mutated self-antigens, central T cell tolerance may lead to the largely weak, clinically ineffective T cell responses observed in certain clinical trials of cancer vaccines. The present disclosure provides, among other things, the insight that a combination of tumor-associated antigens comprising a New Yorkoesophageal squamous cell carcinoma (NY-ESO-1) antigen, a melanoma-associated antigen A3 (MAGE-A3) antigen, a tyrosinase antigen, and a transmembrane phosphatase with tensin homology (TPTE) antigen represent a particularly useful group of tumor-associated antigens for targeted immunotherapy. Without wishing to be bound by any particular theory, the present disclosure notes that the restricted normal tissue expression of such a combination of tumor-associated antigens and their high prevalence in melanoma (e.g., more than 90% of melanoma patients express at least one of the tumor-associated antigens NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, and TPTE antigen) may contribute to their usefulness in the treatment of melanoma.

The present disclosure also provides insight that the compositions disclosed herein can induce de novo antigen-specific anti-tumor immune responses and enhance pre-existing immune responses against vaccine antigens.

Still further, the present disclosure provides particular insight that delivery of tumor-associated antigens (NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, and TPTE antigen) by RNA via lipid particles (e.g., lipoplexes or lipid nanoparticles) targeting dendritic cells (e.g., immature dendritic cells) may be a particularly beneficial strategy for cancer vaccines, wherein the RNA is translated for antigen presentation (e.g., enhanced presentation) on HLA class I and class II molecules. Without wishing to be bound by a particular theory, in some embodiments, the RNA compositions described herein may temporospatially align vaccine antigen delivery with costimulation of type I interferon-driven antiviral immune mechanisms mediated by toll-like receptors (TLRs), and result in significant expansion of antigen-specific T cells. The present disclosure also provides, among other things, the insight that the RNA compositions described herein are not only effective as a monotherapy for treating melanoma, but can also synergize with immune checkpoint inhibitors (e.g., anti-PD1 therapy) in melanoma patients, who may have previously been treated with immune checkpoint inhibitors in some embodiments. To date, no treatments comprising cancer vaccines containing ribonucleic acids and lipid particles (e.g., lipoplexes or lipid nanoparticles) encoding tumor-associated antigens have been approved for the treatment of cancer (e.g., melanoma). Those skilled in the art will be aware of the emerging field of nucleic acid therapeutics and, in addition, RNA (e.g., mRNA) therapeutics (see, e.g., mRNA encoding proteins and/or cytokines). Multiple embodiments of the technology provided herein can utilize specific features of the developed RNA (e.g., mRNA) therapeutic technology and/or delivery system. For example, in some embodiments, the administered RNA (e.g., mRNA) may include non-nucleoside-modified nucleotides. In some embodiments, the administered RNA (e.g., mRNA) may include one or more modified nucleotides (e.g., but not limited to pseudouridine), nucleosides, and/or linkages. As an alternative or in addition, in some embodiments, the RNA (e.g., mRNA) administered may include a modified polyA sequence (e.g., a disrupted polyA sequence) that enhances stability and/or translation efficiency. As an alternative or in addition, in some embodiments, the RNA (e.g., mRNA) administered may include a specific combination of at least two 3'UTR sequences (e.g., a combination of sequence elements of the amino-terminal enhancer of the split RNA and a sequence derived from the mitochondrial encoded 12S RNA). As an alternative or in addition, in some embodiments, the RNA (e.g., mRNA) administered may include a '5 UTR sequence derived from human α-globin mRNA. As an alternative or in addition, in some embodiments, the RNA (e.g., mRNA) administered may include a 5' cap analog, for example, for co-transcriptional capping. As an alternative or in addition, in some embodiments, the RNA (e.g., mRNA) administered may include a secretion signal coding region (e.g., a human secretion signal coding sequence) with reduced immunogenicity. In some embodiments, the RNA (e.g., mRNA) administered may include an MHC trafficking domain. In some embodiments, the administered RNA may be formulated in or with one or more delivery vehicles (eg, lipid particles, eg, lipoplexes or lipid nanoparticles).

In one aspect, the present disclosure provides, inter alia, a method comprising: administering to a patient suffering from cancer at least one dose of a pharmaceutical composition, wherein the pharmaceutical composition comprises: (a) one or more RNA molecules collectively encoding (i) a New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) a melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) a tyrosinase antigen, (iv) a transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) a lipid particle.

In some embodiments, patients suitable for the techniques described herein (including, for example, methods and/or pharmaceutical compositions, etc.) are classified as having evidence of disease at the time of administration.

In some embodiments, patients suitable for the techniques described herein (including, for example, methods and/or pharmaceutical compositions, etc.) are classified as having no evidence of disease at the time of administration.

Thus, certain aspects of the present disclosure provide methods comprising: administering to a patient at least one dose of a pharmaceutical composition comprising: (a) one or more RNA molecules collectively encoding (i) a New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) a melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) a tyrosinase antigen, (iv) a transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) a lipid particle; wherein the patient was diagnosed with cancer prior to the time of administration, but the patient was classified as having no evidence of disease at the time of administration.

In some embodiments, evidence of disease or no evidence of disease is determined by applying the immune-related Response Evaluation Criteria In Solid Tumor (irRECIST) criteria or RECIST 1.1 criteria.

In some embodiments, the technology described herein relates to a pharmaceutical composition comprising one or more RNA molecules, the one or more RNA molecules comprising: (i) a first RNA molecule encoding a NY-ESO-1 antigen, (ii) a second RNA molecule encoding a MAGE-A3 antigen, (iii) a third RNA molecule encoding a tyrosinase antigen, and (iv) a fourth RNA molecule encoding a TPTE antigen. In some embodiments, a single RNA molecule of the one or more RNA molecules encodes at least two of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen.

In some embodiments, the technology described herein relates to pharmaceutical compositions comprising a single RNA molecule encoding a multi-epitopic polypeptide, wherein the multi-epitopic polypeptide comprises at least two of a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and a TPTE antigen.

In some embodiments, one or more RNA molecules present in the pharmaceutical compositions described herein may further comprise at least one sequence encoding a CD4+ epitope. For example, in some embodiments, the CD4+ epitope is delivered by the same RNA molecule encoding at least one of a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and a TPTE antigen.

In some embodiments, one or more RNA molecules present in the pharmaceutical compositions described herein may also include at least one sequence encoding tetanus toxoid P2, a sequence encoding tetanus toxoid P16, or both. In some embodiments, the inclusion of P2 and/or P16 in the RNA molecule improves immune stimulation compared to comparable RNA molecules without P2 or P16. Without wishing to be bound by any particular theory, P2 and/or P16 may provide CD4 ⁺ mediated T cell assistance during sensitization. Demotz et al. 1989, Dredge et al. 2002, Livingston et al. 2013, each of which is incorporated herein by reference in its entirety.

In some embodiments, one or more RNA molecules present in the pharmaceutical compositions described herein may include at least one of the following: a sequence encoding an MHC class I trafficking domain; a 5' cap or a 5' cap analog; a sequence encoding a signal peptide; at least one non-coding regulatory element; at least one polyadenine tail; at least one 5' untranslated region (UTR) and/or at least one 3' UTR; and combinations thereof. In some embodiments, the polyadenine tail to be included in one or more RNA molecules is a modified adenine sequence or comprises a modified adenine sequence.

In some embodiments, one or more RNA molecules present in the pharmaceutical compositions described herein may comprise, in 5' to 3' order: (i) a 5' cap or a 5' cap analog; (ii) at least one 5' UTR; (iii) a signal peptide; (iv) a coding region encoding at least one of a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and a TPTE antigen; (v) at least one sequence encoding tetanus toxoid P2, tetanus toxoid P16, or both; (vi) a sequence encoding an MHC class I trafficking domain; (vii) at least one 3' UTR; and (viii) a polyadenine tail.

In some embodiments, one or more RNA molecules present in the pharmaceutical compositions described herein comprise natural ribonucleotides. In some embodiments, one or more RNA molecules present in the pharmaceutical compositions described herein comprise modified ribonucleotides or synthetic ribonucleotides.

In some embodiments, at least one of the tumor-associated antigens encoded by one or more RNA molecules (e.g., those described herein) is a full-length antigen. In some embodiments, at least one of the tumor-associated antigens encoded by one or more RNA molecules (e.g., those described herein) is a truncated antigen. In some embodiments, at least one of the tumor-associated antigens encoded by one or more RNA molecules (e.g., those described herein) is a non-mutated antigen. For example, in some embodiments, at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen is a full-length, non-mutated antigen. In some embodiments, the NY-ESO-1 antigen is a full-length antigen (e.g., in some embodiments, a full-length, non-mutated antigen). In some embodiments, the MAGE-A3 antigen is a full-length antigen (e.g., in some embodiments, a full-length, non-mutated antigen). In some embodiments, the tyrosinase antigen is a truncated antigen (e.g., in some embodiments, a truncated, non-mutated antigen). In some embodiments, the TPTE antigen is a truncated antigen (e.g., in some embodiments, a truncated, non-mutated antigen).

In some embodiments, at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen is expressed by dendritic cells in lymphoid tissue of the patient. In some embodiments, at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen is present in cancer.

In some embodiments, the lipid particles of the pharmaceutical compositions described herein comprise liposomes. In some embodiments, the lipid particles of the pharmaceutical compositions described herein comprise cationic liposomes. In some embodiments, the lipid particles of the pharmaceutical compositions described herein comprise lipid nanoparticles.

In some embodiments, the lipid particles of the pharmaceutical compositions described herein comprise N,N,N-trimethyl-2-3-dioleyloxy-1-chloropropylamine (DOTMA), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine phospholipids (DOPE), or both.

In some embodiments, the lipid particles of the pharmaceutical compositions described herein include at least one ionizable amino lipid. In some embodiments, the lipid particles of the pharmaceutical compositions described herein include at least one ionizable amino lipid and a helper lipid. In some embodiments, an exemplary helper lipid is a phospholipid or includes a phospholipid. In some embodiments, an exemplary helper lipid is a sterol or includes a sterol. In some embodiments, the lipid particles of the pharmaceutical compositions described herein include at least one polymer-conjugated lipid (e.g., in some embodiments, a PEG-conjugated lipid).

In some embodiments, the technology provided herein can be used for human patients. In some embodiments, the technology provided herein can be used to treat cancer and/or extend the time to recurrence. In some embodiments, the cancer is epithelial cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is late. In some embodiments, the cancer is II phase, III phase, or IV phase. In some embodiments, the cancer is IIIB phase, IIIC phase, or IV phase melanoma. In some embodiments, the cancer is completely removed, there is no evidence of disease, or both.

In some embodiments, the methods described herein include administering a second dose of a provided pharmaceutical composition (e.g., those described herein) to a patient (e.g., in some embodiments, a patient with melanoma or a patient with no evidence of disease). In some embodiments, the methods described herein include administering at least two doses of a pharmaceutical composition to a patient (e.g., in some embodiments, a patient with melanoma or a patient with no evidence of disease). In some embodiments, the methods described herein include administering at least three doses of a pharmaceutical composition to a patient (e.g., in some embodiments, a patient with melanoma or a patient with no evidence of disease).

In some embodiments, the disclosure provides a dosing regimen that is particularly useful for the purposes described herein. For example, in some embodiments, at least one of the at least three doses is administered to the patient (e.g., in some embodiments, a patient with melanoma or a patient without disease evidence) within 8 days after the patient has received another dose of the at least three doses. In some embodiments, at least one of the at least three doses is administered to the patient (e.g., in some embodiments, a patient with melanoma or a patient without disease evidence) within 15 days after the patient has received another dose of the at least three doses. In some embodiments, the dosing regimen according to the disclosure includes administering at least 8 doses of the pharmaceutical composition described herein to the patient (e.g., in some embodiments, a patient with melanoma or a patient without disease evidence) within 10 weeks. In some embodiments, the dosing regimen according to the disclosure includes administering the dosage of the pharmaceutical composition described herein to the patient (e.g., in some embodiments, a patient with melanoma or a patient without disease evidence) weekly for 6 weeks, and then administering the dosage of the pharmaceutical composition described herein every two weeks for 4 weeks. In some embodiments, a dosing regimen according to the present disclosure includes administering a dose of a pharmaceutical composition described herein to a patient (e.g., in some embodiments, a patient with melanoma or a patient with no evidence of disease) monthly after an initial dosing regimen (e.g., an initial dosing regimen comprising at least 8 doses). In some embodiments, the dosing regimen includes administering a dose of a pharmaceutical composition described herein to a patient (e.g., in some embodiments, a patient with melanoma or a patient with no evidence of disease) weekly for a period of 7 weeks. In some embodiments, the dosing regimen includes administering a dose of a pharmaceutical composition described herein to a patient (e.g., in some embodiments, a patient with melanoma or a patient with no evidence of disease) every three weeks.

In some embodiments, the dosage (e.g., the first dose and/or the second dose) is 5 μg to 500 μg of total RNA. In some embodiments, the dosage (e.g., the first dose and/or the second dose) is 7.2 μg to 400 μg of total RNA. In some embodiments, the dosage (e.g., the first dose and/or the second dose) is 10 μg to 20 μg of total RNA. In some embodiments, the dosage (e.g., the first dose and/or the second dose) is about 14.4 μg of total RNA. In some embodiments, the dosage (e.g., the first dose and/or the second dose) is about 25 μg of total RNA. In some embodiments, the dosage (e.g., the first dose and/or the second dose) is about 50 μg of total RNA. In some embodiments, the dosage (e.g., the first dose and/or the second dose) is about 100 μg of total RNA. In some embodiments, administration can be performed systemically. In some embodiments, administration can be performed intravenously. In some embodiments, administration can be performed intramuscularly. In some embodiments, administration can be performed subcutaneously.

In some embodiments, the pharmaceutical compositions described herein can be administered as a single treatment. In some embodiments, the pharmaceutical compositions described herein can be administered as part of a combination therapy. In some embodiments, the combination therapy can include the provided pharmaceutical compositions and immune checkpoint inhibitors. In some embodiments, the technology described herein can be used for patients who have previously received immune checkpoint inhibitors. In some embodiments, the technology described herein can also include administering immune checkpoint inhibitors to patients. Some examples of immune checkpoint inhibitors include, but are not limited to: PD-1 inhibitors, PDL-1 inhibitors, CTLA4 inhibitors, Lag-3 inhibitors, or a combination thereof. In some embodiments, immune checkpoint inhibitors are antibodies or include antibodies. In some embodiments, immune checkpoint inhibitors are the following or include the following: inhibitors listed in Table 4 or Example 8 herein. In some embodiments, immune checkpoint inhibitors are the following or include the following: ipilimumab, nivolumab, pembrolizumab, avelumab, cemiplimab, atezolizumab, durvalumab, or a combination thereof. In some embodiments, immune checkpoint inhibitors that may be particularly useful according to the present disclosure are ipilimumab or include ipilimumab. In some embodiments, immune checkpoint inhibitors that may be particularly useful according to the present disclosure are or comprise the following: ipilimumab and nivolumab. In some embodiments, immune checkpoint inhibitors that may be particularly useful according to the present disclosure are or comprise cimiprilimab.

In some embodiments, the technology described herein can be used to induce an immune response in a patient receiving a pharmaceutical composition described herein. In some embodiments, the pharmaceutical composition described herein can induce an immune response in the patient.

In some embodiments, the method described herein may also include determining the level of an immune response in a patient. In some embodiments, such methods described herein may also include comparing the immune response level in a patient with the immune response level in a second patient to which a pharmaceutical composition has been administered, wherein the second patient was diagnosed with cancer before the administration time and was classified as having disease evidence at the administration time. In some such embodiments, the administered pharmaceutical composition induces an immune response level in a patient that is comparable to the immune response level in a second patient to which the pharmaceutical composition has been administered, the second patient having previously been diagnosed with cancer and being classified as having disease evidence at the administration time. In some embodiments, the level of an immune response is a de novo immune response induced by a pharmaceutical composition described herein.

In some embodiments, the methods described herein also include determining the level of immune response in the patient before and after administering the pharmaceutical composition described herein. In some such embodiments, the method also includes comparing the level of immune response in the patient after administering the pharmaceutical composition with the level of immune response in the patient before administering the pharmaceutical composition. In some embodiments, the level of immune response in the patient after administering the pharmaceutical composition is increased compared to the level of immune response in the patient before administering the pharmaceutical composition. In some embodiments, the level of immune response in the patient after administering the pharmaceutical composition is maintained compared to the level of immune response in the patient before administering the pharmaceutical composition.

In some embodiments, the technology described herein can induce adaptive responses in patients receiving the pharmaceutical compositions described herein. In some embodiments, the technology described herein can induce T cell responses in patients receiving the pharmaceutical compositions described herein. In some embodiments, the T cell response is a CD4+ response or comprises a CD4+ response. In some embodiments, the T cell response is a CD8+ response or comprises a CD8+ response. Methods for determining the level of immune response are known in the art. In some embodiments, the level of immune response in patients can be determined using interferon-γ enzyme-linked immunosorbent spot (ELISpot) assay.

In some embodiments, the methods described herein further comprise measuring the level of one or more of NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, and TPTE antigen in lymphoid tissue of the patient. In some embodiments, the methods described herein further comprise measuring the level of one or more of NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, and TPTE antigen in cancer.

In some embodiments, the methods described herein also include measuring the metabolic activity level in the spleen of the patient. In some embodiments, the methods described herein also include measuring the metabolic activity level in the spleen of the patient before and after administering the pharmaceutical composition described herein. The metabolic activity level in the spleen of the patient can be measured by using a suitable method known in the art, for example, in some embodiments, using positron emission tomography (positron emissiontomography, PET), computerized tomography (computerized tomography, CT) scanning, magnetic resonance imaging (magnetic resonance imaging, MRI), or a combination thereof to measure.

In some embodiments, the method described herein also includes measuring the amount of one or more cytokines in the patient's plasma. In some embodiments, the method described herein also includes measuring the amount of one or more cytokines in the patient's plasma before and after administering the pharmaceutical composition described herein. Some non-limiting examples of one or more cytokines to be measured include: interferon (interferon, IFN) -α, IFN-γ, interleukin (interleukin, IL) -6, IFN-inducible protein (inducible protein, IP) -10, IL-12 p70 subunit or a combination thereof.

In some embodiments, the methods described herein further include measuring the number of cancerous lesions in the patient. In some embodiments, the methods described herein further include measuring the number of cancerous lesions in the patient before and after administering the pharmaceutical composition described herein. In some such embodiments, fewer cancerous lesions are detected in the patient after administering the pharmaceutical composition than before administering the pharmaceutical composition.

In some embodiments, the method described herein also includes measuring the number of T cells induced by the pharmaceutical composition described herein in the patient. In some embodiments, the method described herein also includes measuring the number of T cells induced by the pharmaceutical composition described herein in the patient at multiple time points after the pharmaceutical composition is administered. In some embodiments, the method described herein also includes measuring the number of T cells induced by the pharmaceutical composition in the patient after administering the first dose of the pharmaceutical composition and administering the second dose of the pharmaceutical composition. In some such embodiments, compared with administering the first dose of the pharmaceutical composition, the number of T cells induced by the administered pharmaceutical composition in the patient after administering the second dose of the pharmaceutical composition is greater.

In some embodiments, the methods described herein further comprise determining the phenotype of T cells induced by the pharmaceutical composition in the patient after administering the pharmaceutical composition. In some embodiments, at least a portion of the T cells induced by the administered pharmaceutical composition in the patient have a T helper-1 phenotype. In some embodiments, the T cells induced by the administered pharmaceutical composition in the patient comprise T cells having a PD1+ effector memory phenotype.

In some embodiments, the technology described herein can be used to be applied to patients classified as having evidence of disease. In some such embodiments, the method described herein for patients classified as having evidence of disease also includes measuring the size of one or more cancerous lesions. In some embodiments, the method described herein also includes measuring the size of one or more cancerous lesions in patients before and after applying the pharmaceutical composition described herein. In some embodiments, the method described herein also includes comparing the size of one or more cancerous lesions in patients before and after applying the pharmaceutical composition. In some such embodiments, the size of at least one cancerous lesion in a patient after applying the pharmaceutical composition is equal to or less than the size of at least one cancerous lesion before applying the pharmaceutical composition.

In some embodiments, the method described herein for patients classified as having disease evidence also includes monitoring progression-free survival duration. In some such embodiments, the method described herein includes comparing the progression-free survival duration of the patient with a reference progression-free survival duration. In some embodiments, an exemplary reference progression-free survival duration is the average progression-free survival duration of a plurality of comparable patients who do not receive the pharmaceutical composition described herein. In some embodiments, the progression-free survival duration of the patient applying the pharmaceutical composition described herein is longer in time compared to the reference progression-free survival duration.

In some embodiments, the method described herein for patients classified as having disease evidence also includes measuring the disease stabilization duration. In some embodiments, disease stabilization can be determined by applying irRECIST or RECIST 1.1 standards. In some embodiments, the method described herein also includes comparing the disease stabilization duration of the patient with the reference disease stabilization duration. In some embodiments, such a reference disease stabilization duration is the average disease stabilization duration of a plurality of comparable patients who do not receive the pharmaceutical composition described herein. In some embodiments, compared with the reference disease stabilization duration, the patient applying the pharmaceutical composition described herein shows an improved disease stabilization duration.

In some embodiments, the method described herein for patients classified as having disease evidence also includes measuring the tumor responsiveness duration. In some embodiments, tumor responsiveness is determined by applying irRECIST or RECIST1.1 standards. In some embodiments, the method described herein also includes comparing the tumor responsiveness duration of the patient applying the pharmaceutical composition described herein with the reference tumor responsiveness duration. In some embodiments, such a reference tumor responsiveness duration is the average tumor responsiveness duration of multiple comparable patients who do not receive the pharmaceutical composition described herein. In some embodiments, compared with the reference tumor responsiveness duration, the patient applying the pharmaceutical composition described herein shows an improved tumor responsiveness duration.

In some embodiments, the technology described herein can be used to be applied to patients classified as having no evidence of disease. In some such embodiments, the methods described herein also include monitoring disease-free survival duration. In some embodiments, the methods described herein also include comparing the disease-free survival duration of the patient with a reference disease-free survival duration. In some embodiments, such a reference disease-free survival duration is the average disease-free survival duration of a plurality of comparable patients who have not received the pharmaceutical composition described herein. In some embodiments, compared with the reference disease-free survival duration, the patient applying the pharmaceutical composition described herein shows an improved disease-free survival duration.

In some embodiments, the method described herein for patients classified as having no evidence of disease may also include measuring the duration to disease recurrence. In some embodiments, disease recurrence is determined by applying irRECIST or RECIST1.1 standards. In some embodiments, the method described herein also includes comparing the duration to disease recurrence of the patient applying the pharmaceutical composition described herein with the duration to disease recurrence with reference to the duration to disease recurrence. In some embodiments, such a duration to disease recurrence with reference to the duration to disease recurrence is the average duration to disease recurrence of multiple comparable patients who do not receive the pharmaceutical composition described herein. In some embodiments, compared with the duration to disease recurrence with reference to, the patient applying the pharmaceutical composition described herein shows the duration to disease recurrence of improvement.

In some embodiments, the technology described herein can be used to extend the overall survival of patients. In some embodiments, patients are classified as having disease evidence. In some embodiments, patients are classified as having no disease evidence.

In some aspects, also provided herein are pharmaceutical compositions for inducing an immune response against cancer in a patient. In some embodiments, such patients are classified as having no evidence of disease, but have previously been diagnosed with cancer. In some embodiments, the pharmaceutical composition comprises: (a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase (TPTE) antigen with tensin homology, or (v) a combination thereof; and (b) lipid particles.

In some aspects, pharmaceutical compositions for treating cancer are also provided herein. In some embodiments, such patients are classified as having no evidence of disease, but have previously been diagnosed with cancer. In some embodiments, the pharmaceutical composition comprises: (a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase (TPTE) antigen with tensin homology, or (v) a combination thereof; and (b) lipid particles. In some embodiments, the pharmaceutical compositions described herein are particularly useful for administration to patients with melanoma.

The use of the pharmaceutical compositions described herein is also within the scope of the present disclosure. In some embodiments, the pharmaceutical compositions described herein can be used to induce an immune response against cancer in a patient, the patient, for example, in some embodiments, being classified as a patient with no evidence of disease but previously diagnosed with cancer. In some embodiments, the pharmaceutical compositions described herein can be used to treat cancer in a patient, the patient, for example, in some embodiments, being classified as a patient with no evidence of disease but previously diagnosed with cancer. In some embodiments, the cancer is melanoma. In some embodiments, the pharmaceutical composition comprises: (a) one or more RNA molecules, which together encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase (TPTE) antigen with tensin homology, or (v) a combination thereof; and (b) lipid particles.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1a to 1d depict exemplary TAA constructs, experimental designs, and vaccine-mediated immune activation. Figure 1a, structure of TAA RNA. The 5'-cap analog, 5'- and 3'-untranslated regions (UTRs), and poly (A) tails were optimized for stability and translation efficiency. In addition, the TAA coding sequence was labeled with a signal peptide (SP), tetanus toxoid CD4+ epitopes P2 and P16, and MHC class I trafficking domain (MITD) for enhanced HLA presentation and immunogenicity. Figure 1b, clinical trial design. Figure 1c, metabolic activity in the spleen, measured by transaxial [18F] FDG-PET/CT at baseline (before) and 4 hours after the sixth vaccine injection (after). Figure 1d, cytokine plasma levels (before each vaccine injection and 2 hours, 6 hours and 24 hours (and in some cases, 48 hours) and body temperature in patients (from cohort V) injected with six ascending doses per week. The horizontal dashed line indicates the upper limit of normal.

Figures 2a to 2k depict T cell immunity and clinical activity of FixVac. Figures 2a, 2c, Proportion of patients with vaccine-induced T cell responses (de novo or expanded) before and after vaccination as analyzed by IFN-γ ELISpot, measured ex vivo (a; n=50) or after IVS (c; n=20). PBL, peripheral blood lymphocytes. Figure 2b, ex vivo CD8+ T cell responses of patient A2-09, measured using CD4-depleted PBMCs pulsed with TAA PepMix. Control, PBMCs with culture medium. Figure 2d, post-IVS CD4+ T cell responses of patient 42-06, measured using autologous dendritic cells loaded with TAA PepMix as targets. Control, luciferase-transfected dendritic cells. Figure 2e, ex vivo frequency of NY-ESO-1-specific T cells from patient 12-01 (cohort 1, six vaccine doses) stained with HLA multimers. Dashed lines indicate vaccination. Figures 2f to 2i, de novo induced HLA-B*3503-restricted NY-ESO-1-specific T cells from patient A2-09 (cohort A, continued vaccination). Dashed lines indicate vaccination. Figure 2f, phenotype of PBMCs stained with NY-ESO-1/HLA-B*3501 multimers. Multimer-positive CD8+ T cells are shown in red. BV421 and BV650 are immunofluorescence markers. Figure 2g, left, multimer analysis; and right, ICS of T cells stimulated with single peptides or PepMix. Figure 2h, ICS of CD8+ T cells stimulated with NY-ESO-1 peptides ex vivo. Figure 2i, specific lysis of melanoma cell lines by healthy donor CD8+ T cells transfected with cloned HLA-B*3503-restricted NY-ESO-1-specific TCRs from patient A2-09 (effector: target (E: T) ratio = 20: 1). SK-MEL-37 and SK-MEL-28 are melanoma cell lines. PD-Cy7 is an immunofluorescence marker. Figure 2j, Figure 2k, clinical activity, which is assessed as the effect of FixVac without/with anti-PD1 antibody on target lesions (n=38; 4 patients had no target lesions at baseline). Figure 2j, asterisks indicate the combination with anti-PD1 antibody. PD, progressive disease; PR, partial response.

Figures 3a to 3g depict T cell immunity in patient 53-02 treated with FixVac monotherapy. Figure 3a, top, NY-ESO-196-104-specific Cw*0304-restricted CD8+T cells analyzed by HLA multimer staining. Control, cytomegalovirus (CMV)-pp65 multimer. Bottom, exemplary flow cytometry. Figure 3b, melanoma lesions as assessed by CT scan. Lesions smaller than quantifiable size are drawn as 0.1 mm in diameter. NT, non-target lesions; T, target lesions; according to the solid tumor immune-related response evaluation criteria (irRECIST) version 1.1. Figure 3c, top, ex vivo frequency of NY-ESO-196-104-specific, cytokine-secreting CD8+T cells analyzed by ICS. Bottom, exemplary flow cytometry. Figure 3d, top, killing of melanoma cell lines by CD8+T cells from IVS cultures (E:T=20:1). Bottom, frequencies of NY-ESO-196-104 multimer-specific CD8+T cells from PBMCs after IVS at different treatment time points (-1, baseline; day 22, after 3 vaccinations; day 64, after 7 vaccinations). Figure 3e, cytotoxicity of two HLA-B*4001-restricted NY-ESO-1-specific TCRs cloned and transfected into healthy donor CD8+T cells from samples after vaccination to melanoma cell lines (E:T=20:1). Figure 3f, TCR frequencies from e in peripheral blood, measured by ex vivo TCR repertoire analysis. TRB, T cell receptor-β. Figure 3g, top, kinetics of ex vivo frequencies of MAGE-A3167-176-specific, cytokine-secreting CD8+T cells. Bottom, exemplary flow cytometry.

Figures 4a to 4g depict T cell immunity in partially responding patients treated with a FixVac/anti-PD1 combination. Figures 4a to 4c, patient C2-28. a, size of target lesions; Figure 4b, de novo MAGE-A3-specific CD8+T cells analyzed by HLA multimer staining (top), and exemplary flow cytometry (bottom). Figure 4c, melanoma cell recognition by MAGE-A3168-176-specific TCR. Figure 4d, CT scan of lung lesions in patient C2-31. Figures 4e to 4f, patient C1-40. Figure 4e, MAGE-A3168-176-specific HLA-A*0101-restricted T cells analyzed by HLA-multimer staining. Figure 4f, top, lysis of melanoma cell lines by CD8+T cells from IVS cultures of PBMCs collected before and during treatment (E:T=8.5:1). Bottom, MAGE-A3168-176-specific CD8+ T cells after IVS. Figure 4g, Correlation of FixVac TAA transcript expression with the number of non-synonymous single-nucleotide variants (snSNVs) in melanomas from three independent cohorts (n=50). RPKM, reads per kilobase per million mapped reads.

Fig. 5 depicts patient subgroups.At baseline, patients had advanced melanoma with radiographically measurable disease or unmeasurable disease.Immune monitoring was performed on 49 patients in all subgroups.Clinical antitumor activity was evaluated for 42 patients (1 unresected III C stage, 41 IV stages) in a total of 56 patients with measurable disease at baseline, whose follow-up imaging data were available at data cutoff (25 were treated with FixVac monotherapy, and 17 were treated with FixVac in combination with anti-PD1 therapy).The remaining 14 patients (5 received FixVac monotherapy, and 9 received a combination with anti-PD1 therapy) were not included in the efficacy analysis for the reasons described in the previous sentence.PD, progressive disease;PR, partial response;SD, stable disease (according to the best objective overall response of irRECIST 1.1).According to irRECIST1.1, CR* refers to the metabolic complete response of patients with SD as the best response. Thirty-three patients with radiographically nonmeasurable disease at baseline did not undergo exploratory analysis for objective best overall response and are being followed up for relapse-free survival.

Figures 6a to 6c depict characterization of cytokine secretion. Figures 6a, 6b, for: Figure 6a, all available patients; and Figure 6b, patients treated with 50 μg or 100 μg of RNA-lipid complex (lipoplex, LPX) target dose alone ("Mono") or in combination with anti-PD1 therapy ("aPD1"), peak plasma cytokine levels (6 hours after vaccine injection) and body temperature (4 hours after vaccine injection). The box shows the 25th to 75th quantiles, where the line represents the median; the whisker shows the minimum to maximum value; the gray dots show the individual values of each dose level; the dotted line indicates the upper limit of normal. The sample number (number, n) is indicated in the figure. Figure 6c, correlation of plasma cytokine levels (y axis) with plasma IFN-α concentration 6 hours after RNA-LPX administration (for IFN-γ, IL-12 p70 and IL-6, n = 147; for IP-10, n = 147).

Figures 7a to 7f depict T cell immunity induced by FixVac. Figure 7a, phenotype (left) and quality (middle and right) of TAA-specific T cells measured by IFN-γ ELISpot after IVS (left and middle) or in vitro (right). Only positive responses are shown. Figure 7b, exemplary flow cytometry of PBMCs from patient 12-01 stained with NY-ESO-192-100/Cw*0304 polymers. Figure 7c, flow cytometry gating strategy for characterization of polymer+T cell phenotype. Upper row, from left to right: starting with events obtained with constant flow stream and fluorescence intensity, single events (singlet) are identified. Dump-negative events (viable, CD4-, CD14-, CD16-, CD19-) and lymphocytes are identified and gated. In lymphocytes, CD8+HLA polymer-positive T cells are gated for further analysis. Bottom row, left panel: Different subsets of CD8+ T cells (indicated in black) and NY-ESO-1 multimer-positive CD8+ T cells (red) were gated into four subsets based on CD45RA and CCR7 expression, and CD27 and CD28 expression were analyzed in the right panel - central memory (CCR7+CD45RA-), naive (CCR7+CD45RA+), effector memory (CCR7-CD45RA-), and effector memory re-expressing RA (CCR7-CD45RA+). PD1 and OX40 expression was analyzed for multimer-positive (red) and multimer-negative (black) CD8+ T cells. Figure 7d, Detection of IFN-γ and TNF secreting CD8+ T cells in patient A2-09 after stimulation with MAGE-A3212-220 peptide. FIG. 7e , Comparison of fold induction of spot counts ex vivo after vaccination between patients with measurable (n=27) or non-measurable (n=30) disease (left), patients treated with different vaccine doses (14.4 μg (n=17), 50 μg (n=10), 100 μg (n=24); middle), and patients treated with FixVac alone (Mono (n=44)) or in combination with anti-PD1 therapy (aPD1 (n=12); right). Only positive responses at the visit after vaccination are shown. A fold change of more than 2 compared to baseline was considered a response to the vaccine. If both CD4 and CD8 results were positive after treatment, only the ratio of higher spot counts is shown. FIG. 7f , The proportion of patients with vaccine-induced T cell responses (de novo or expanded) determined by IFN-γ ELISpot before and after vaccination, measured ex vivo from patients treated with FixVac alone (n=14) or in combination with anti-PD1 therapy (n=12). Only data from patients with measurable disease are shown.

Figures 8a to 8d depict disease responses and treatment regimens for patients evaluated for clinical activity. Figures 8a and 8b are lane diagrams of patients that can be used to evaluate efficacy assessments from the start of treatment to disease progression or continued treatment. Figure 8a is a patient treated with melanoma FixVac monotherapy. The numbers on the y-axis represent individual patients. CR = complete response; PR = partial response; SD = stable disease; and PD = progressive disease. The gray line indicates the time when the initial treatment phase ends and when continued treatment begins. Figure 8a contains data obtained from patients with evidence of disease (ED patients) who received BNT111 as a monotherapy. Figure 8b is a patient treated with FixVac and anti-PD1 therapy. The dark green triangle indicates the start and completion of treatment. The dark green arrow shows a patient who is still receiving treatment. The red cross marks disease progression; patients are classified by the best overall response and progression-free survival time (CR, PD, PR, SD). The light green star indicates the first recorded objective response, and the light green arrow indicates ongoing disease control. The black vertical line marks the date planned for the eighth vaccination (study day 64). A single asterisk indicates the patient for the clinical course and treatment regimen shown in d. CR**, according to irRECIST1.1, a metabolic complete response in patients with stable disease as the best response. Patients with radiologically unmeasurable disease at baseline are being followed up for recurrence-free survival and have not undergone clinical efficacy assessment. Figure 8c, tumor burden at baseline associated with clinical response after FixVac treatment. PD, progressive disease; PR, partial response; SD, stable disease. Figure 8d, clinical course and treatment regimen of patients Pt 53-02, A2-09, C2-28, A2-10, C2-31 and C1-40. FD, first diagnosis of melanoma at any stage. FD IV stage, first diagnosis of melanoma in stage IV. * New bone lesions diagnosed and treated with radiotherapy.

Figures 9a to 9j depict T cell immunity in patient 53-02 with a partial response under FixVac monotherapy. Figure 9a, CT scans of the right lower and middle lobes before (before) and after (after) initiation of melanoma FixVac treatment. Figure 9b, Kinetics of NY-ESO-196-104-specific, HLA-Cw*0304-restricted CD8+ T cell responses (see also Figure 3a). Figures 9c to f, Discovery and characterization of NY-ESO-196-104-specific HLA-Cw*0304-restricted TCRs. Figure 9c, Sorting gate for multimer-positive CD8+ T cells for TCR cloning (gating within a single, live CD3+ lymphocyte population). Control, fluorescence minus one (FMO) sample. Fig. 9d, Recognition of peptide-pulsed HLA-Cw*0304-transfected K562 cells by NY-ESO-1-TCR-transfected CD8+ T cells in IFN-γ ELISpot. Control, HIV-gag PepMix; NY-ESO-1, NY-ESO-1 PepMix. Fig. 9e, Cytotoxicity of NY-ESO-1-TCR-transfected healthy donor CD8+ T cells after 24 hours of co-culture with HLA-transfected melanoma cell lines (SK-MEL-37 and SK-MEL-28; E:T=50:1). Fig. 9f, Kinetics of NY-ESO-1-specific TCR clonotype frequencies in TCR repertoire data obtained from PBMCs before and after vaccination. Figs. 9g to 9i, Discovery and characterization of two NY-ESO-1124-133-specific HLA-B*4001-restricted TCRs. Figure 9g, PBMCs were stimulated with NY-ESO-1 PepMix and single IFN-γ positive CD8+T cells were sorted by flow cytometry for TCR cloning (control, HIV-gag PepMix). Figures 9h, 9i, HLA restriction and epitope specificity of NY-ESO-1-TCR analyzed using IFN-γ ELISpot after co-culture of TCR-transfected CD8+T cells with peptide-pulsed HLA-transfected K562 cells. NY-ESO-1, NY-ESO-1 PepMix. Figure 9j, Cytotoxicity of NY-ESO-1-specific TCRs identified in samples after vaccination of patients. TCR-transfected healthy donor CD8+T cells were stimulated for 12 hours with HLA-transfected melanoma cell lines (SK-MEL-37, SK-MEL-28) at an effector to target ratio of 20:1.

Figures 10a to 10i depict T cell immunity in patients A2-10, C2-31 and C1-40. Figures 10a to 10f, Patient A2-10 with CPI-refractory melanoma, partially responded under FixVac monotherapy. Figure 10a, CT scans of inguinal lymph node metastases obtained before and after the start of vaccination. Figure 10b, CD4+ T cell responses after IVS before vaccination and after eight vaccinations, restimulated in IFN-γ ELISpot assays with autologous dendritic cells transfected with RNA (encoding one of the TAAs or luciferase as a control) or with PepMix pulsed with TAAs and unpulsed dendritic cells (no peptide). Figure 10c, CD8+ and CD4+ T cells secreting cytokines after intradermal challenge with NY-ESO-1 RNA. Skin infiltrating lymphocytes were recovered from punch biopsies 15 days after 8 weekly vaccinations and stimulated with PepMix encoding NY-ESO-1 or tyrosinase. Figures 10d to 10f, Discovery and characterization of HLA II restricted TAA-specific TCRs. d, CD4+T cells from IVS cultures were restimulated with dendritic cells pulsed with PepMix and sorted by flow cytometry for TCR cloning (control, HIV-gag PepMix). APC and PE are fluorescent dye markers. Figure 10e, HLA restriction and epitope specificity were determined by IFN-γ ELISpot using TCR-transfected healthy donor CD4+T cells and RNA-transfected or peptide-pulsed HLA-transfected K562 cells. DRA, DRB, DQA and DQB numbers refer to specific HLA alleles. Control, K562 cells without peptide (-). Fig. 10f, Kinetics of TCR clonotype frequencies in peripheral blood by ex vivo TCR library analysis. Fig. 10g, TAA-specific CD8+ and CD4+ T cell responses of patient C2-31 by IFN-γ ELISpot on peptide-loaded autologous dendritic cells after IVS with TAA PepMix. Control, dendritic cells loaded with irrelevant peptide. Fig. 10h, 10i, Clinical and immune responses of patient C1-40 with CPI-refractory melanoma who had a partial response under Melanoma FixVac in combination with nivolumab. Fig. 10h, CT scans of the right middle and left lower lobes before and after starting Melanoma FixVac treatment. Fig. 10i, Ex vivo frequencies of MAGE-A3168-176-specific A*0101-restricted (left panel) and NY-ESO-192-100-specific HLA_Cw*0304-restricted (right panel) CD8+ T cells analyzed by HLA multimer staining.

Figure 11 depicts the gating strategy for the flow cytometry analysis of the data shown in Figure 2e (Pt 12-01 at most the 50th day). The gating strategy for the flow cytometry of T cells for identifying vaccine induction. (Upper row, from left to right) Starting from the events obtained with constant flow stream and fluorescence intensity, single events were identified. Viable cells and lymphocytes were identified and gated. In lymphocytes, Dump-negative events (CD4-, CD14-, CD16-, CD19-negative) were gated to exclude them for further analysis. In Dump-negative events, CD8+HLA multimer positive T cells were gated for further analysis (lower row).

Figure 12 depicts the gating strategy for the flow cytometry analysis of the data shown in Figure 2f, Figure 2g (Pt A2-09), Figure 2e (Pt 12-01 after the 50th day), Figure 3a (Pt53-02) and Figure 7c (Pt A2-09), Figure 9b (Pt 53-02). The gating strategy for the flow cytometry gating of the phenotypic characterization of vaccine-induced T cells. (Top row, from left to right) Starting from the events obtained with constant flow stream and fluorescence intensity, single events were identified. Dump-negative events (viable, CD4-negative, CD14-negative, CD16-negative, CD19-negative) and lymphocytes were identified and gated. In lymphocytes, CD8+HLA multimer-positive T cells were gated for further analysis. (Middle row left figure). Analyzed the PD1 and OX40 expression of multimer-positive (red) and multimer-negative (black) CD8+T cells (middle row middle and right). Based on CD45RA and CCR7, the different subpopulations of CD8+T cells (indicated in black) and multimer-positive CD8+T cells (highlighted in red) were gated into four subpopulations: central memory (CD45RA-CCR7+), naive (CD45RA+CCR7+), effector memory (CD45RA-CCR7-), and effector memory re-expressing RA (CD45RA+CCR7-). The expression of CD27 and CD28 in each subpopulation was analyzed.

Figure 13 depicts the gating strategy for the flow cytometry analysis of the data shown in Fig. 2h, Fig. 2g (Pt A2-09) and Fig. 7d (Pt A2-09).The gating strategy for the flow cytometry gating of cytokine responses in the T cells for identifying vaccine induction.(upper row, from left to right) starting from the event obtained with constant flow stream and fluorescence intensity, a single event was identified.Dump-negative events (viable, CD14-, CD16-, CD19-negative) and lymphocytes were identified and gated.In lymphocytes, CD8+ and CD4+T cells were gated for further analysis (lower row left figure).The generation of effector cytokines TNF and IFNγ in CD8+ (lower row middle figure) and CD4+T cells (lower row right figure) was gated and analyzed.

Figure 14 depicts the gating strategy for the flow cytometry analysis of the data shown in Fig. 3 c and Fig. 4 g (Pt 53-02).The gating strategy for the flow cytometry gating of cytokine responses in the T cells for identifying vaccine induction.(upper row, from left to right) starting from the event obtained with constant flow stream and fluorescence intensity, a single event is identified.In the next step, lymphocytes are identified and gated.In lymphocytes, CD8+ and CD4+T cells are gated for further analysis (lower row left figure).The generation of effector cytokines TNF and IFNγ in CD8+ (lower row middle figure) and CD4+T cells (lower row right figure) is gated and analyzed.

FIG. 15 depicts a gating strategy for flow cytometry based on the detection of multimer-positive T cells for patient 53-02 after the IVS shown in FIG. 3 d. To detect NY-ESO-196-104 multimer-specific T cells, single events and lymphocytes were first identified. Within single lymphocytes, CD3+ viable cells were gated. CD8+/multimer+ was identified within viable CD3+ cells. The gating strategy for sample day 64 is shown as an example of the multimer analysis depicted in FIG. 3 d.

Figure 16 depicts a flow cytometry gating strategy for single cell sorting of TAA-specific T cells for TCR cloning shown in Figures 9c, 9g and 10d. In order to detect TAA-specific T cells based on (a) polymer staining or (b, c) IFNy secretion, single events and lymphocytes were first identified. Within a single lymphocyte, CD3+ viable cells were gated. Within viable CD3+ cells, (a) CD8+/polymer+, (b) CD8+/IFNy+ or (c) CD4+/IFNy+ T cells were gated. The sorting gate is highlighted in red. After (a) polymer staining or (b) IFNγ secretion assay, the gating strategy for NY-ESO-1-specific T cells of patient 53-02 is shown correspondingly to the data depicted in Figures 9c, 9g, and the gating strategy for MAGE-A3-specific T cells of patient A2-10 is shown correspondingly to Figure 10d.

Figure 17 depicts the gating strategy for the flow cytometry analysis of the data shown in Figure 10c (Pt A2-10). The gating strategy for the flow cytometry gating of cytokine responses in T cells for identifying vaccine induction. (Upper row, from left to right) Starting from the events obtained with constant flow stream and fluorescence intensity, single events were identified. Dump-negative events (viable cells) and lymphocytes were identified and gated. In lymphocytes, CD8+ and CD4+T cells were gated for further analysis (lower row left figure). The generation of effector cytokines TNF and IFNγ in CD8+ (lower row middle figure) and CD4+T cells (lower row right figure) was gated and analyzed.

Figure 18 depicts the gating strategy for flow cytometry analysis of the data shown in Figure 4b (Pt C2-028), Figure 4e (Pt C1-040) and Figure 10i (Pt C1-40). Flow cytometry gating strategy for identifying vaccine-induced T cells. (Top row, from left to right) Starting with events obtained with constant flow stream and fluorescence intensity, single events were identified. Dump-negative events (viable, CD4-, CD14-, CD16-, CD19-negative) and lymphocytes were identified and gated. Within lymphocytes, CD8+HLA multimer-positive T cells were gated for further analysis (bottom row).

Figure 19 depicts the flow cytometry gating strategy for detecting multimer-positive T cells of patient C1-40 after IVS shown in Figure 4f. To detect MAGE-A3168-176 multimer-specific T cells, single events and lymphocytes were first identified. Within single lymphocytes, CD3+ viable cells were gated. Within viable CD3+ cells, CD8+/multimer+ was identified. The gating strategy for sample day 129 is shown as an example of the multimer analysis depicted in Figure 4f.

Figures 20a to 20c depict ex vivo ELISPOT CD4+ or CD8+ (Figure 20a), CD8+ (Figure 20b) or CD4+ (Figure 20c) responses. The frequency of patients with vaccine-induced (expanded or de novo) responses. The numbers in the bar segments represent the number of patients evaluated in each segment. Only patients treated with a single treatment were included.

Figure 21 depicts ex vivo ELISPOT responses by cell type. The number and percentage of ELISPOT responses can be evaluated. Only non-abundant measurements with evaluable results for both CD4 and CD8 from patients treated with a single therapy were included.

Figure 22 depicts vaccine-induced ex vivo ELISPOT CD4+ or CD8+ responses to any cell type. Fractions of de novo and expanded responses. Only patients treated with a single treatment were included.

Figures 23a to 23c depict ex vivo ELISPOT CD4+ or CD8+ (Figure 23a), CD8+ (Figure 23b) or CD4+ (Figure 23c) responses. The frequency of patients with vaccine-induced (expanded or de novo) responses. The numbers in the bar segments represent the number of patients evaluated in each segment. Only patients treated with a single treatment were included.

Figure 24 depicts the ex vivo ELISPOT CD4+ or CD8+ responses of the clinical best responses of patients with unevaluable disease. The numbers in the bar segments represent the number of patients with the ex vivo ELISPOT measurements evaluated in each segment. Only patients treated with a single treatment were included. Patients without evaluable ELISPOT results or recorded clinical best were excluded.

Figure 25 depicts ex vivo ELISPOT CD4+ or CD8+ responses by clinical best responses of evaluable disease patients. The numbers in the bar segments represent the number of patients with evaluated ex vivo ELISPOT measurements in each segment. Only patients treated with a single treatment were included. Patients without evaluable ELISPOT results or recorded clinical best were excluded.

Figures 26a-26b depict a summary of disease-free survival data for NED patients, and a Kaplan-Meier summary of disease-free survival data for NED patients.

Figures 27a to 27f depict a summary of the overall survival data for ED patients (Figure 27a), NED patients (Figure 27b), and the combination of NED and ED patients (Figure 27c); and a Kaplan-Meier summary of the overall survival data for ED patients (Figure 27d), NED patients (Figure 27e), and the combination of ED and NED patients (Figure 27f).

Figures 28a to 28c depict a summary of adverse events for ED patients (Figure 28a), NED patients (Figure 28b), and ED and NED patients combined (Figure 28c).

Figure 29 depicts patient treatment data.In the sum of 89 patients, 3 patients recruited twice were counted only once with their first recruitment (2 patients were treated in group CI and subsequently recruited in group CIII, and 1 patient from group CII was subsequently recruited in expansion group Exp.A). The starting dose is blue, and the target dose is orange.In groups CII to CVII and Exp.A, B and C, patients received 8 doses of melanoma FixVac (at the 1st, 8, 15, 22, 29, 36, 50 and 64 days). The patient in group CI only received 6 doses (at the 1st, 8, 15, 22, 29 and 43 days). Patients with measurable disease at baseline were allowed to choose to continue treatment (Q4W) until disease progression or drug-related toxicity.When FixVac was combined with anti-PD1 therapy, this situation occurred from the first dose, except that a patient (asterisk) of anti-PD1 therapy was added during treatment.

FIG30 depicts the characteristics and prior treatments of patients in the clinical analysis group.

Figure 31 depicts data of spleen FDR upstate as measured by PET/CT imaging. FDG uptake in the spleen was assessed by PET/CT imaging and quantified in selected patients at baseline and at different time points after the fourth (71-27), fifth (C1-45) or sixth (C1-44) vaccination cycle. Total and relative FDG uptake in the spleen are shown. SUV, standardized uptake value.

FIG. 32 depicts data for relevant adverse events that occurred following treatment in more than 5% of patients.

FIG. 33 depicts data for antigen-specific α/β TCRs isolated from single T cells from melanoma patients.

FIG. 34 includes schematic diagrams showing exemplary mRNA molecules as described herein and the mode of action of the mRNA complexed in a lipoplex.

35 includes a table providing various characteristics associated with patients participating in the safety and efficacy studies of an exemplary composition described herein (BNT111).

36 includes a table providing various characteristics associated with patients participating in the safety and efficacy studies of an exemplary composition described herein (BNT111).

Figure 37 includes lanes sorted by best clinical response and duration of disease-free survival. The length of the bar indicates the duration of disease control. The dotted line indicates the approximate date of the last administration of BNT111 during the initial trial treatment according to the clinical trial protocol. DFS = disease-free survival; LTFU = long-term follow-up; PD refers to progressive disease. The data in this work (ploy) were obtained from patients treated with BNT111 monotherapy.

Figure 38 includes a bar graph showing ex vivo responses from patients as determined by ELISpot. Ex vivo responses were detected in 14/22 (64%) and 19/28 (68%) ED and NED patients, respectively.

Figure 39 includes a bar graph showing responses after in vitro stimulation from patients as determined by ELISpot. ELISpot after in vitro stimulation (IVS) was performed in 9 ED patients and 6 NED patients (small sample size due to limited sample availability). In all 15 patients, T cell responses against at least one TAA were observed.

FIG. 40 includes a bar graph showing treatment-emergent serious adverse events with a ≥ 10% incidence in any subgroup of patients following treatment with an exemplary composition described herein (BNT111).

41 includes a bar graph showing relevant treatment-emergent serious adverse events with a Common Terminology Criteria for Adverse Events grade greater than or equal to 3 following treatment with an exemplary composition described herein (BNT111).

FIG. 42 includes a table providing a summary of preliminary efficacy in patients with evaluable disease according to irRECIST.

43 includes waterfall plots of the best changes from baseline observed in target lesions according to irRECIST in patients with measurable disease at baseline who were treated with an exemplary monotherapy (BNT111) or in combination with a PD-1 inhibitor or BRAF/MEK inhibition.

Some definitions

About or approximately: As used herein, when applied to one or more target values, the term "about" or "approximately" refers to a value similar to the reference value. Generally, those skilled in the art who are familiar with the context will understand the relevant degree of variation covered by "about" or "approximately" in the context. For example, in some embodiments, the term "about" or "approximately" can be contained in 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value.

Administration: As used herein, the term "administering" and its variations generally refer to administering a composition to a subject to achieve delivery of a medicament as a composition or a medicament contained in a composition to a target site or site to be treated. One of ordinary skill in the art will recognize that a variety of routes can be used for administration to a subject (e.g., a human) where appropriate. For example, in some embodiments, administration can be ocular, oral, parenteral, topical, etc. In some specific embodiments, administration can be bronchial (e.g., by bronchial instillation), buccal, transdermal (which can be or include, for example, one or more of the surface to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, in a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In some embodiments, administration can be parenteral. In some embodiments, administration can be oral. In some specific embodiments, administration can be intravenous. In some specific embodiments, administration can be subcutaneous. In some embodiments, administration can only involve a single dose. In some embodiments, administration can involve applying a fixed number of doses. In some embodiments, administration can involve intermittent administration (e.g., multiple doses separated in time) and/or periodic administration (e.g., separate doses separated by a common time period). In some embodiments, administration can involve continuous administration (e.g., perfusion) in at least a selected time period. In some embodiments, administration can include a primary immunization and a booster regimen. The primary immunization and booster regimen can include administering a first dose of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine), followed by administering a second dose of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) after a time interval. In the case of an immunogenic composition, the primary immunization and booster regimen can result in an improved immune response in the patient.

Antibody: As used herein, the term "antibody agent" refers to an agent that specifically binds to a specific antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex comprising an immunoglobulin structural element sufficient to confer specific binding. In some embodiments, an antibody agent is a polypeptide or comprises a polypeptide whose amino acid sequence comprises structural elements recognized by those skilled in the art as immunoglobulin variable domains. In some embodiments, an antibody agent is a polypeptide protein having a binding domain that is homologous or largely homologous to an immunoglobulin binding domain.

Exemplary antibody agents include, but are not limited to, monoclonal antibodies or polyclonal antibodies. In some embodiments, the antibody agent may comprise one or more constant region sequences that are specific to mouse, rabbit, primate or human antibodies. In some embodiments, as known in the art, the antibody agent may comprise one or more sequence elements that are humanized, primatized, chimeric, etc. In many embodiments, the term "antibody agent" is used to refer to one or more constructs or forms known in the art or developed for utilizing antibody structural and functional characteristics in alternative presentations. For example, in some embodiments, the antibody agent used in accordance with the present disclosure is selected from, but not limited to, the following forms: complete IgA, IgG, IgE or IgM antibodies; bispecific or multispecific antibodies (e.g. , etc.); antibody fragments, such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments and isolated complementarity determining regions (CDRs) or groups thereof; single-chain Fv; polypeptide-Fc fusions; single-domain antibodies (e.g., shark single-domain antibodies, such as IgNAR or fragments thereof); camel antibodies; masked antibodies (e.g., ); Small modular immunopharmaceuticals (“SMIPsTM”); Single-chain or tandem diabodies VHH; microantibodies; Ankyrin repeat protein or DART;TCR-like antibody; MicroProteins; and In some embodiments, the antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, the antibody may comprise a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.] or other side groups [e.g., polyethylene glycol, etc.]).

Associated with: As the term is used herein, two events or entities are "associated with" one another if the presence, level, and/or form of one event or entity is associated with the presence, level, and/or form of another event or entity. For example, a particular biological phenomenon is considered to be associated with a particular disease, disorder, or condition (e.g., cancer) if its presence is associated with the incidence and/or susceptibility to, or likelihood of response to, a treatment for, such a disease, disorder, or condition (e.g., in a relevant population).

Blood-derived sample: As used herein, the term "blood-derived sample" refers to a sample derived from a blood sample (i.e., a whole blood sample) of a subject in need thereof. Examples of blood-derived samples include, but are not limited to, plasma (including, for example, fresh frozen plasma), serum, blood fractions, plasma fractions, serum fractions, blood fractions (including red blood cells (RBC), platelets, white blood cells, etc.), and cell lysates containing fractions thereof (e.g., cells such as red blood cells, white blood cells, etc. can be harvested and lysed to obtain cell lysates). In some embodiments, the blood-derived sample that can be used for characterization described herein is a plasma sample.

Cancer: The term "cancer" as used herein generally refers to a disease or condition in which cells of a tissue of interest exhibit relatively abnormal, uncontrolled and/or autonomous growth such that they exhibit an abnormal growth phenotype characterized by a significant loss of control over cell proliferation. In some embodiments, cancer may comprise precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non-metastatic cells. In some embodiments, cancer may be characterized by solid tumors. In some embodiments, cancer may be characterized by hematological tumors. In general, examples of different types of cancer known in the art include, for example, hematopoietic cancers, including leukemias, lymphomas (Hodgkin's and non-Hodgkin's), myelomas, and myeloproliferative disorders; sarcomas; melanomas; adenomas; solid tissue cancers; squamous cell carcinomas of the mouth, larynx, pharynx, and lung; liver cancer; genitourinary cancers, such as prostate cancer, cervical cancer, bladder cancer, uterine cancer, and endometrial cancer; and renal cell carcinoma; bone cancer; pancreatic cancer; skin cancer; cutaneous or intraocular melanoma; cancers of the endocrine system, thyroid cancer; parathyroid cancer; head and neck cancer; ovarian cancer; breast cancer; glioblastoma; colorectal cancer; gastrointestinal cancer and cancers of the nervous system; benign lesions such as papilloma, etc. In some embodiments, the cancer may be melanoma.

Cap: As used herein, the term "cap" refers to a structure comprising or essentially consisting of a nucleoside-5'-triphosphate that is typically attached to the 5' end of an uncapped RNA (e.g., an uncapped RNA with a 5'-diphosphate). In some embodiments, the cap is or comprises a guanine nucleotide. In some embodiments, the cap is or comprises a naturally occurring RNA 5' cap, including, for example, but not limited to, a 7-methylguanosine cap with a structure named "m7G". In some embodiments, the cap is or comprises a synthetic cap analog that is similar to an RNA cap structure and has the ability to stabilize RNA if attached thereto, including, for example, but not limited to, anti-reverse cap analogs (ARCA) known in the art. It will be understood by those skilled in the art that methods for attaching a cap to the 5' end of an RNA are known in the art. For example, in some embodiments, a capped RNA can be obtained by capping an RNA with a 5' triphosphate group or an RNA with a 5' diphosphate group in vitro with a capping enzyme system (including, for example, but not limited to, a vaccinia capping enzyme system or a cerevisiae capping enzyme system). Alternatively, capped RNA can be obtained by in vitro transcription (IVT) of a single-stranded DNA template using methods known in the art, wherein the IVT system comprises a dinucleotide cap analog (including, for example, an m7GpppG cap analog or an N7-methyl, 2'-O-methyl-GpppG ARCA cap analog or an N7-methyl, 3'-O-methyl-GpppG ARCA cap analog) in addition to GTP.

Co-administration: As used herein, the term "co-administration" refers to the use of a pharmaceutical composition described herein and an additional therapeutic agent (e.g., a chemotherapeutic agent described herein). The combined use of a pharmaceutical composition described herein and an additional therapeutic agent (e.g., a chemotherapeutic agent described herein) can be performed simultaneously or separately (e.g., in any order sequence). In some embodiments of the pharmaceutical composition described herein, the pharmaceutical composition described herein and the additional therapeutic agent (e.g., a chemotherapeutic agent described herein) can be combined in a pharmaceutically acceptable carrier, or it can be placed in a separate carrier and delivered to a target cell or administered to a subject at different times. Each of these situations is considered to fall within the meaning of "co-administration" or "combination", provided that the pharmaceutical composition described herein and the additional therapeutic agent (e.g., a chemotherapeutic agent described herein) are delivered or administered in a sufficiently close time so that the biological effects produced by each on the subject or target cell being treated have at least some temporal overlap.

Combination therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is exposed to two or more treatment regimens (e.g., two or more therapeutic agents) simultaneously. In some embodiments, two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of the first regimen are administered before any doses of the second regimen are administered); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, the "administration" of a combination therapy may involve administering one or more agents or modalities to a subject receiving the other agents or modalities in the combination. For clarity, combination therapy does not require that the separate agents be administered together in a single composition (or even necessarily at the same time), but in some embodiments, two or more agents or their active portions may be administered together in a combined composition.

Comparable: As used herein, the term "comparable" refers to two or more agents, entities, situations, condition groups, etc., which may not be identical to each other but are similar enough to allow comparisons between them, so that those skilled in the art will understand that conclusions can be reasonably drawn based on the observed differences or similarities. In some embodiments, the group of comparable conditions, environments, individuals or groups is characterized by a plurality of substantially identical features and one or a small amount of changes. It will be understood by those of ordinary skill in the art that in the context, in any given case, two or more such agents, entities, situations, condition groups, etc. require what degree of identity to be considered comparable. For example, it will be understood by those of ordinary skill in the art that when the following environments, individuals or groups are comparable to each other: characterized by having substantially the same features of sufficient number and type to ensure a reasonable conclusion, i.e., the differences in the results or observed phenomena obtained under or under different environments, individuals or groups are caused by or indicate the changes in the features of these changes.

Complementary: As used herein, the term "complementary" is used to refer to oligonucleotide hybridization in relation to the base pairing rules. For example, the sequence "C-A-G-T" is complementary to the sequence "G-T-C-A". Complementarity can be partial or complete. Therefore, any degree of partial complementarity is intended to be included within the scope of the term "complementary", provided that the partial complementarity allows oligonucleotide hybridization. Partial complementarity is according to the base pairing rules, in which one or more nucleic acid bases do not match. Full or complete complementarity between nucleic acids is where each and every nucleic acid base matches another base under the base pairing rules.

Contacting: As used interchangeably herein, the terms "delivery" and variations thereof or "contacting" refer to the introduction of ssRNA or a composition comprising it into a target cell (e.g., the cytosol of a target cell). The target cell may be cultured in vitro or ex vivo, or present in a subject (in vivo). The method of introducing ssRNA or a composition comprising it into a target cell may vary with in vitro, ex vivo, or in vivo applications. In some embodiments, ssRNA or a composition comprising it may be introduced into a target cell in a cell culture by in vitro transfection. In some embodiments, ssRNA or a composition comprising it may be introduced into a target cell by a delivery vehicle (e.g., a lipid nanoparticle described herein). In some embodiments, ssRNA or a composition comprising it may be introduced into a target cell in a subject by administering a pharmaceutical composition described herein to the subject.

Detection: The term "detection" is used broadly herein to include appropriate means for determining whether a target entity or any form of measurement of a target entity is present in a sample. Thus, "detection" may include determining, measuring, evaluating or determining the presence or absence, level, quantity and/or position of a target entity. Quantitative and qualitative determinations, measurements or evaluations are included, including semi-quantitative. Such determinations, measurements or evaluations may be relative (e.g., when the target entity is detected relative to a control reference) or may be absolute. Thus, when used in the context of quantifying a target entity, the term "quantification" may refer to absolute or relative quantification. Absolute quantification can be accomplished by associating the level of the detected target entity with a known control standard (e.g., by generating a standard curve). Alternatively, relative quantification can be accomplished by comparing the detection levels or amounts between two or more different target entities to provide a relative quantification of each of the two or more different target entities (i.e., relative to each other).

Disease: As used herein, the term "disease" refers to a disorder or condition that impairs the normal function of a tissue or system, typically in a subject (e.g., a human subject) and typically manifests as characteristic signs and/or symptoms. In some embodiments, an exemplary disease is cancer.

Encoding: As used herein, the term "encoding" or its variations refers to the sequence information of a first molecule that directs the production of a second molecule having a defined nucleotide sequence (e.g., mRNA) or a defined amino acid sequence. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process including a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Therefore, if the transcription and translation of the mRNA corresponding to a gene in a cell or other biological system produces a polypeptide, the gene, cDNA, or ssRNA (e.g., mRNA) encodes a polypeptide. In some embodiments, the coding region of an ssRNA encoding a tumor-associated antigen (TAA) refers to a coding strand whose nucleotide sequence is identical to the mRNA sequence of such a tumor-associated antigen. In some embodiments, the coding region of an ssRNA encoding a TAA refers to a non-coding strand of such a TAA, which can be used as a template for transcription of a gene or cDNA.

Epitope: As used herein, the term "epitope" includes any portion specifically recognized by the patient's immune system. For example, an epitope can be any portion specifically recognized by a T cell, a B cell, an immunoglobulin (e.g., an antibody or receptor), an immunoglobulin (e.g., an antibody or receptor), a binding component, or an aptamer. In some embodiments, an epitope is composed of a plurality of chemical atoms or groups on an antigen. In some embodiments, when the antigen adopts a related three-dimensional conformation, such chemical atoms or groups are surface exposed. In some embodiments, when the antigen adopts such a conformation, such chemical atoms or groups are physically close to each other in space. In some embodiments, when the antigen adopts an alternative conformation (e.g., linearized), at least some of such chemical atoms are groups that are physically separated from each other.

Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of the RNA transcript (e.g., by splicing, editing, 5' cap formation and/or 3' end formation); (3) translation of the RNA into a polypeptide or protein; and/or (4) post-translational modification of the polypeptide or protein.

5' untranslated region (five prime untranslated region): As used herein, the term "5' untranslated region" or "5'UTR" refers to the sequence of an mRNA molecule between the transcription start site and the start codon of the RNA coding region. In some embodiments, "5'UTR" refers to such a sequence of an mRNA molecule that starts at the transcription start site and ends one nucleotide (nt) before the start codon (usually AUG) of the RNA coding region, such as in its natural environment.

Homology: As used herein, the term "homology" or "homologs" refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules are considered to be "homologous" to each other if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules are considered to be "homologous" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known to those of ordinary skill in the art, certain amino acids are typically classified as being similar to each other as "hydrophobic" or "hydrophilic" amino acids, and/or having "polar" or "non-polar" side chains. Replacing one amino acid with another amino acid of the same type can generally be considered a "homologous" replacement.

Identity: As used herein, the term "identity" refers to the overall correlation between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, if the sequence between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical, it is considered to be "substantially identical" to each other. For example, the calculation of the identity percentage of two nucleic acid or polypeptide sequences can be performed by comparing the two sequences for the purpose of optimal comparison (e.g., for the purpose of optimal comparison, a gap can be introduced in one or both of the first and second sequences, and different sequences can be ignored for the purpose of comparison). In certain embodiments, the length of the sequence compared for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or substantially 100% of the length of the reference sequence. The nucleotides at the corresponding positions are then compared. When the position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. Considering the number of spaces and the length of each space (needing to be introduced for the best comparison of the two sequences), the percent identity between the two sequences is a function of the number of identical positions shared by the sequences. The comparison of sequences and the determination of the percent identity between the two sequences can be completed using a mathematical algorithm. For example, the algorithm of Meyers and Miller, 1989 (which has been incorporated into the ALIGN program (version 2.0)) can be used to determine the percent identity between two nucleotide sequences. In some exemplary embodiments, nucleic acid sequence comparisons performed with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package using the NWSgapdna.CMP matrix.

RECIST criteria: As used herein, the term "RECIST" or "RECIST criteria" refers to the Response Evaluation criteria for In Solid Tumors. For example, the RECIST criteria are as described in Eisenhauer et al. (European J. Cancer 45: 228-247 (2009), which is incorporated herein by reference in its entirety). In some embodiments, the RECIST criteria are RECIST 1.1. In some embodiments, the RECIST criteria are iRECIST. For example, the iRECIST criteria are as described in Seymour, L. et al. (Lancet Oncol. 18: 3 e143-e152 (2017), which is incorporated herein by reference in its entirety). In some embodiments, the RECIST criteria are "irRECIST criteria", which are solid tumor immune-related response evaluation criteria. For example, the irRECIST criteria are as described in Nishino et al. (Clin Cancer Res 19: 3936-43 (2013), which is incorporated herein by reference in its entirety). In some embodiments, the irRECIST criteria are irRECIST 1.1. In some embodiments, the RECIST criteria are "imRECIST criteria", which are immune-modified response evaluation criteria for solid tumors (immune-modified Response Evaluation Criteria for In Solid Tumors). For example, the irRECIST criteria are as described in Hodi et al. (J Clin Oncol 36: 850-8 (2018), which is incorporated herein by reference in its entirety).

Locally advanced tumor: As used herein, the term "locally advanced tumor" or "locally advanced cancer" refers to its art-recognized meaning, which can vary with different types of cancer. For example, in some embodiments, a locally advanced tumor refers to a tumor that is larger but has not yet spread to another part of the body. In some embodiments, a locally advanced tumor is used to describe a cancer that has grown outside the tissue or organ in which it began but has not yet spread to distant sites in the subject's body. By way of example only, in some embodiments, locally advanced pancreatic cancer generally refers to stage III disease in which the tumor extends to adjacent organs (e.g., lymph nodes, liver, duodenum, superior mesenteric artery and/or celiac trunk) but has no signs of metastatic disease; however, complete surgical resection with negative pathological margins is not possible.

Nucleic acid/polynucleotide: As used herein, the term "nucleic acid" refers to a polymer of at least 10 or more nucleotides. In some embodiments, the nucleic acid is DNA or comprises DNA. In some embodiments, the nucleic acid is RNA or comprises RNA. In some embodiments, the nucleic acid is a peptide nucleic acid (PNA) or comprises a peptide nucleic acid (PNA). In some embodiments, the nucleic acid is a single-stranded nucleic acid or comprises a single-stranded nucleic acid. In some embodiments, the nucleic acid is a double-stranded nucleic acid or comprises a double-stranded nucleic acid. In some embodiments, the nucleic acid comprises both a single-stranded portion and a double-stranded portion. In some embodiments, the nucleic acid comprises a backbone containing one or more phosphodiester bonds. In some embodiments, the nucleic acid comprises a backbone containing both phosphodiester and non-phosphodiester bonds. For example, in some embodiments, the nucleic acid may comprise a backbone containing one or more phosphorothioates or 5'-N-phosphoramidite bonds and/or one or more peptide bonds, for example, as in "peptide nucleic acids". In some embodiments, the nucleic acid comprises one or more or all natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, the nucleic acid comprises one or more or all non-natural residues. In some embodiments, the non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, inserted bases, and combinations thereof). In some embodiments, the non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) compared to those in the natural residue. In some embodiments, nucleic acid has a nucleotide sequence encoding a functional gene product such as RNA or a polypeptide. In some embodiments, nucleic acid has a nucleotide sequence comprising one or more introns. In some embodiments, nucleic acid can be prepared by separation from natural sources, enzymatic synthesis (e.g., by polymerization based on complementary templates, e.g., in vivo or in vitro, in recombinant cells or systems, propagation or chemical synthesis). In some embodiments, the nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides in length.

Nucleic acid particles: "Nucleic acid particles" can be used to deliver nucleic acids to target sites of interest (e.g., cells, tissues, organs, etc.). Nucleic acid particles can be formed from at least one cationic or cationically ionizable lipid or lipid-like substance, at least one cationic polymer such as protamine, or a mixture thereof, and nucleic acids. Nucleic acid particles include lipid nanoparticle (LNP)-based formulations and lipoplex (LPx)-based formulations.

Nucleotide: As used herein, the term "nucleotide" refers to its art-recognized meaning. When the number of nucleotides is used as an indication of size (e.g., of a polynucleotide), the specific number of nucleotides refers to the number of nucleotides on a single strand (e.g., of a polynucleotide).

Patient: As used herein, the term "patient" refers to any organism suffering from a disease or disorder or condition or at risk of a disease or disorder or condition. Typical patients include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates and/or humans). In some embodiments, the patient is a human. In some embodiments, the patient suffers from or is susceptible to one or more diseases or disorders or conditions. In some embodiments, the patient exhibits one or more symptoms of a disease or disorder or condition. In some embodiments, the patient has been diagnosed with one or more diseases or disorders or conditions. In some embodiments, the disease or disorder or condition to which the provided technology is applicable is cancer or includes cancer, or there is one or more tumors. In some embodiments, the patient is receiving or has received a specific treatment to diagnose and/or treat a disease, disorder or condition. In some embodiments, the patient is a cancer patient.

Polypeptide: As used herein, the term "polypeptide" generally has its art-recognized meaning: a polymer of at least three or more amino acids. It will be understood by those of ordinary skill in the art that the term "polypeptide" is intended to be sufficiently general to encompass not only polypeptides having the complete sequences described herein, but also polypeptides that represent functional, biologically active or characteristic fragments, portions or domains of such complete polypeptides (e.g., fragments, portions or domains that retain at least one activity). In some embodiments, the polypeptide may contain L-amino acids, D-amino acids, or both and/or may contain any of a variety of amino acid modifications or analogs known in the art. Available modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, the polypeptide may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof (e.g., may be peptidomimetics or comprise peptidomimetics).

Reference/reference standard: As used herein, "reference" describes a standard or control relative to which a comparison is made. For example, in some embodiments, a target agent, animal, individual, colony, sample, sequence or value is compared with a reference or control agent, animal, individual, colony, sample, sequence or value. In some embodiments, a reference or control is tested and/or measured substantially simultaneously with the target test or determination. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. In some embodiments, a reference or control is or comprises a set of specifications (e.g., acceptance criteria). Typically, as will be understood by those skilled in the art, a reference or control is measured or characterized under conditions or circumstances comparable to the conditions or circumstances under evaluation. Those skilled in the art will understand when there is enough similarity to justify reliance on a specific possible reference or control and/or to compare with a specific possible reference or control.

Ribonucleotide: As used herein, the term "ribonucleotide" encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications, including but not limited to, for example: (a) terminal modifications, such as 5' terminal modifications (e.g., phosphorylation, dephosphorylation, conjugation, reverse connection, etc.), 3' terminal modifications (e.g., conjugation, reverse connection, etc.), (b) base modifications, such as replacement with modified bases, stable bases, destabilizing bases, or bases that pair with an extended partner library or conjugated bases, (c) sugar modifications (e.g., at the 2' position or the 4' position) or sugar replacement, and (d) internucleoside bond modifications, including modification or replacement of phosphodiester bonds. The term "ribonucleotide" also encompasses ribonucleotide triphosphates, including modified and unmodified ribonucleotide triphosphates.

Ribonucleic acid (RNA): As used herein, the term "RNA" refers to a polymer of ribonucleotides. In some embodiments, RNA is single-stranded. In some embodiments, RNA is double-stranded. In some embodiments, RNA comprises both single-stranded and double-stranded portions. In some embodiments, RNA may comprise a backbone structure as described in the definition of "nucleic acid/polynucleotide" above. RNA may be a regulatory RNA (e.g., siRNA, microRNA, etc.) or a messenger RNA (mRNA). In some embodiments, wherein RNA is mRNA. In some embodiments in which RNA is mRNA, RNA typically comprises a poly (A) region at its 3' end. In some embodiments in which RNA is mRNA, RNA typically comprises a cap structure recognized in the art at its 5' end, for example, for identifying mRNA and connecting it to a ribosome to initiate translation. In some embodiments, RNA is synthetic RNA. Synthetic RNA includes RNA synthesized in vitro (e.g., by enzymatic synthesis methods and/or by chemical synthesis methods).

Selective or specific: It is understood by those skilled in the art that when used in connection with an active agent herein, the term "selective" or "specific" means that the agent distinguishes between potential target entities, states, or cells. For example, in some embodiments, if an agent preferentially binds to its target in the presence of one or more competing alternative targets, it is said to be "specifically" bound to the target. In many embodiments, specific interactions depend on the presence of specific structural features (e.g., epitopes, clefts, binding sites) of the target entity. It should be understood that specificity does not have to be absolute. In some embodiments, specificity can be evaluated relative to the specificity of the target binding portion of one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to the specificity of a reference specific binding portion. In some embodiments, specificity is evaluated relative to the specificity of a reference nonspecific binding portion.

Specific binding: As used herein, the term "specific binding" refers to the ability to distinguish between possible binding partners in the environment in which binding occurs. When there are other potential targets, an antibody agent that interacts with a specific target is said to "specifically bind" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining the degree of association between the CDR of the antibody agent and its partner; in some embodiments, specific binding is assessed by detecting or determining the degree of dissociation of the antibody agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining the ability of the antibody agent to compete for alternative interactions between its partner and another entity. In some embodiments, specific binding is assessed by performing such detection or determination over a range of concentrations.

Subject: As used herein, the term "subject" refers to an organism to which the compositions described herein are to be administered, for example, for experimental, diagnostic, preventive and/or therapeutic purposes. Typical subjects include animals (e.g., mammals, such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, the subject is a human subject. In some embodiments, the subject suffers from a disease, disorder or condition (e.g., cancer). In some embodiments, the subject is susceptible to a disease, disorder or condition (e.g., cancer). In some embodiments, the subject exhibits one or more symptoms or features of a disease, disorder or condition (e.g., cancer). In some embodiments, the subject exhibits one or more non-specific symptoms of a disease, disorder or condition (e.g., cancer). In some embodiments, the subject does not exhibit any symptoms or features of a disease, disorder or condition (e.g., cancer). In some embodiments, the subject is a person with one or more features of susceptibility or risk characteristics to a disease, disorder or condition (e.g., cancer). In some embodiments, the subject is a patient. In some embodiments, the subject is an individual to whom diagnosis and/or treatment is administered and/or has been administered.

Suffering from: An individual who is "suffering from" a disease, disorder, and/or condition has been diagnosed as having and/or exhibits one or more symptoms of the disease, disorder, and/or condition.

Synthetic: As used herein, the term "synthetic" refers to an entity that is artificial, or an entity made by human intervention, or an entity that is produced by synthesis rather than natural occurrence. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule that is chemically synthesized (e.g., by solid phase synthesis in some embodiments). In some embodiments, the term "synthetic" refers to an entity made outside of a biological cell. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule (e.g., RNA) produced by in vitro transcription using a template.

Therapeutic agent: As used interchangeably herein, the phrases "therapeutic agent" or "treatment" refer to an agent or intervention that has a therapeutic effect and/or induces a desired biological and/or pharmacological effect when administered to a subject or patient. In some embodiments, a therapeutic agent or treatment is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and/or reduce the incidence of one or more symptoms or features of a disease, disorder, and/or condition. In some embodiments, a therapeutic agent or treatment is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to alleviate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and/or reduce the incidence of one or more symptoms or features of a disease, disorder, and/or condition.

3' untranslated region: As used herein, the term "3' untranslated region" or "3'UTR" refers to the sequence of an mRNA molecule that begins after the stop codon of the coding region of the open reading frame sequence. In some embodiments, the 3'UTR begins immediately after the stop codon of the coding region of the open reading frame sequence, such as in its natural environment. In other embodiments, the 3'UTR does not begin immediately after the stop codon of the coding region of the open reading frame sequence, such as in its natural environment.

Threshold level (e.g., acceptance criteria): As used herein, the term "threshold level" refers to a level used as a reference to obtain information about a measurement result (e.g., a measurement result obtained in an assay) and/or to classify a measurement result (e.g., a measurement result obtained in an assay). For example, in some embodiments, a threshold level means a value measured in an assay that defines a dividing line between two subpopulations of a population (e.g., a batch that meets a quality control standard and a batch that does not meet a quality control standard). Thus, a value equal to or higher than the threshold level defines a subpopulation of the population, and a value below the threshold level defines another subpopulation of the population. The threshold level may be determined based on one or more control samples or between control sample populations. The threshold level may be determined before, simultaneously with, or after the measurement of the purpose is performed. In some embodiments, the threshold level may be a range of values.

Treatment: As used herein, the term "treatment" and variations thereof refer to any method for partially or completely alleviating, ameliorating, alleviating, inhibiting, preventing, delaying the onset of, reducing the severity of, and/or reducing the incidence of one or more symptoms or features of a disease, disorder, and/or condition. Treatment may be administered to a subject that does not show signs of a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject that only shows early signs of a disease, disorder, and/or condition, for example, for the purpose of reducing the risk of developing a pathological condition associated with the disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject that is in the late stages of a disease, disorder, and/or condition.

Unresectable tumor: As used herein, the term "unresectable tumor" generally refers to a tumor that cannot be removed surgically. In some embodiments, an unresectable tumor refers to a tumor that involves and/or grows into essential organs or tissues (including blood vessels that may not be reconstructed), and/or is located in a position that cannot be easily accessed or otherwise causes unreasonable risk of damage to one or more other critical or essential organs and/or tissues (including blood vessels). In some embodiments, an unresectable tumor refers to a tumor that cannot be surgically removed without causing a risk of damage to the patient, and the risk is determined in reasonable medical judgment to exceed the benefits expected to be obtained by the patient through resection. In some embodiments, the "unresectable" of a tumor refers to the possibility of achieving a negative margin (R0) resection. In the case of pancreatic cancer, the presence of encasement of a tumor to a large blood vessel such as the superior mesenteric artery (SMA) or the celiac axis, portal vein obstruction, and celiac or para-aortic lymphadenopathy is generally considered to exclude R0 surgery. Those skilled in the art will understand the parameters for determining whether a tumor is unresectable.

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, liposome transfection). Enzymatic reactions and purification techniques can be performed according to the manufacturer's instructions or as commonly practiced in the art or as described herein. The aforementioned techniques and operations can generally be performed according to conventional methods known in the art, and as described in various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.

DETAILED DESCRIPTION

For patients with recurrent or refractory advanced solid tumors, the outcome of standard of care (SOC) remains poor. Treatment options also include palliative chemotherapy (which may be poorly tolerated after previous repeated exposure to cytotoxic compounds) or best supportive care, as well as unproven beneficial investigational treatments. Treatment in this population is not curative, and the expected overall survival is several months. Vaccines have become an effective treatment option for some cancers with high unmet medical needs. However, vaccine trials for the treatment of patients with treatment-resistant tumors have mostly been unsuccessful. Therefore, the medical needs for developing vaccines to treat a variety of cancer types, including treatment of refractory cancers, remain high.

The present disclosure provides, inter alia, insights and techniques for treating cancer (e.g., melanoma (e.g., advanced melanoma)) using pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) comprising RNA encoding a tumor-associated antigen (TAA). The present disclosure provides, inter alia, insights that the pharmaceutical compositions described herein may be particularly useful and/or effective when administered to patients with no evidence of disease at the time of the first administration, demonstrating that the pharmaceutical compositions induce T cell immunity even in the absence of a detectable tumor.

In some embodiments, the disclosure provides, in particular, a method of administering to a patient at least one dose of a pharmaceutical composition described herein (e.g., an immunogenic composition, such as a vaccine), the pharmaceutical composition comprising an RNA molecule and a lipid particle (e.g., a lipid complex or a lipid nanoparticle). In some embodiments, one or more RNA molecules encode one or more tumor-associated antigens (TAA), and when administered to a patient, the tumor-associated antigens are combined to induce a strong adaptive immune response (e.g., CD4 ⁺ and/or CD8 ⁺ T cell immune response) to one or more TAAs encoded by the one or more RNA molecules. Without wishing to be bound by any particular theory, the disclosure proposes that such a pharmaceutical composition can achieve antigen-specific T cell immunity and a lasting objective response in cancer patients (e.g., patients with unresectable cancer (e.g., melanoma), patients with or receiving checkpoint inhibitors, or patients with both). In particular, the disclosure also teaches that by administering a pharmaceutical composition as described herein (e.g., an immunogenic composition, such as a vaccine) to a patient, the patient is diagnosed with cancer before the administration time, but wherein the patient is classified as having no disease evidence at the time of administration.

No evidence of disease can be a classification according to RECIST criteria. In some embodiments, no evidence of disease does not mean that the patient does not have any disease, but rather no evidence of the presence of disease, particularly as determined according to RECIST criteria.

In some embodiments, the present disclosure provides, inter alia, the insight of encoding an mRNA comprising an amino acid sequence of a tumor-associated antigen (TAA), an immunogenic variant thereof, or an immunogenic fragment of a TAA or an immunogenic variant thereof. Thus, the mRNA encodes a peptide or protein comprising at least an epitope of a TAA or an immunogenic variant thereof for inducing an immune response against the TAA. In some embodiments, the present disclosure provides, inter alia, RNA technology for delivering one or more RNA molecules to a patient, wherein the one or more RNA molecules collectively encode: (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof. In some embodiments, a single RNA molecule encodes all of the following: (i) New York Esophageal Squamous Cell Carcinoma (NY-ESO-1) antigen, (ii) Melanoma-Associated Antigen A3 (MAGE-A3) antigen, (iii) Tyrosinase antigen, and (iv) Transmembrane Phosphatase with Tensin Homology (TPTE) antigen. In some embodiments, the sequence encoding the following is not present on a single RNA molecule: (i) New York Esophageal Squamous Cell Carcinoma (NY-ESO-1) antigen, (ii) Melanoma-Associated Antigen A3 (MAGE-A3) antigen, (iii) Tyrosinase antigen, and (iv) Transmembrane Phosphatase with Tensin Homology (TPTE) antigen. For example, a first RNA molecule may encode two of the following: (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, and (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, and a second RNA molecule may encode the remaining two. As another example, the sequences encoding the following may each be present on a different RNA molecule such that each RNA molecule encodes only one antigen: (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, and (iv) transmembrane phosphatase with tensin homology (TPTE) antigen.

In some embodiments, the present disclosure provides, among other things, the insight that a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) is formulated with a lipid particle (e.g., a lipoplex or a lipid nanoparticle) for administration to a patient (e.g., intravenous (IV), intramuscular, or subcutaneous administration). In particular, a pharmaceutical composition comprising one or more RNA (e.g., mRNA) molecules encoding at least one TAA (e.g., a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and/or a TPTE antigen) or an immunogenic fragment thereof is formulated with a lipid particle (e.g., a lipoplex or a lipid nanoparticle) for administration to a patient (e.g., IV, intramuscular, or subcutaneous administration). Without wishing to be bound by any particular theory, as described herein, a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) can be taken up by immature dendritic cells and the RNA molecules are translated for enhancing antigen presentation on HLA class I and class II molecules. In some embodiments, the TAA (e.g., NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, and/or TPTE antigen) is expressed from RNA (e.g., mRNA) that is, for example, engineered to be minimally immunogenic and/or formulated in lipid nanoparticles (e.g., LNPs). In some embodiments, the RNA (e.g., mRNA) encoding at least one TAA (e.g., NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, and/or TPTE antigen) may comprise modified nucleotides (e.g., but not limited to, pseudouridine).

In some embodiments, the present disclosure provides, inter alia, a method of administering to a patient at least one dose of a pharmaceutical composition comprising: (a) one or more RNA molecules collectively encoding: (i) a New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) a melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) a tyrosinase antigen, (iv) a transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) a lipid particle (e.g., a lipoplex or a lipid nanoparticle); wherein the patient was diagnosed with cancer prior to the time of administration, but the patient was classified as having no evidence of disease at the time of administration (e.g., no evidence of disease was determined by applying Response Evaluation Criteria in Solid Tumors (RECIST) criteria, such as RECIST1.1 criteria or irRECIST criteria).

In some embodiments, the present disclosure provides, inter alia, a method of administering at least one dose of a pharmaceutical composition to a patient suffering from cancer, wherein the pharmaceutical composition comprises: (a) one or more RNA molecules that collectively encode: (i) a New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) a melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) a tyrosinase antigen, (iv) a transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) a lipid particle (e.g., a lipoplex or a lipid nanoparticle).

In some embodiments, the present disclosure provides, inter alia, a pharmaceutical composition for inducing an immune response against cancer in a patient, wherein the pharmaceutical composition comprises: (a) one or more RNA molecules that collectively encode: (i) a New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) a melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) a tyrosinase antigen, (iv) a transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) a lipid particle (e.g., a lipoplex or a lipid nanoparticle); and wherein the patient is classified as having no evidence of disease but has previously been diagnosed with cancer (e.g., melanoma).

In some embodiments, the present disclosure provides, inter alia, a pharmaceutical composition for treating cancer, wherein the pharmaceutical composition comprises: (a) one or more RNA molecules that collectively encode: (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) lipid particles (e.g., lipid complexes or lipid nanoparticles); and wherein the patient is classified as having no evidence of disease but has previously been diagnosed with cancer (e.g., melanoma). No evidence of disease can be determined according to RECIST criteria, such as RECIST1.1 criteria or irRECIST criteria.

In some embodiments, the present disclosure provides, inter alia, a pharmaceutical composition for inducing an immune response against cancer in a patient, wherein the pharmaceutical composition comprises: (a) one or more RNA molecules that collectively encode: (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) a lipid particle (e.g., a lipoplex or a lipid nanoparticle).

In some embodiments, the present disclosure provides, inter alia, a pharmaceutical composition for treating cancer, wherein the pharmaceutical composition comprises: (a) one or more RNA molecules that collectively encode: (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) a lipid particle (e.g., a lipoplex or a lipid nanoparticle).

I. Existing Methods

The present disclosure provides a technique for treating cancer. An exemplary cancer that can be treated by the techniques described herein is melanoma. The health risks associated with melanoma can be significant, and advanced or metastatic melanoma (e.g., unresectable stage III, stage IV) is still a fatal disease. For example, for systemic treatment of unresectable stage III/IV and recurrent melanoma, there are currently two methods that have shown improvement in progression-free survival (PFS) and overall survival (OS) in randomized trials. These two methods are: (1) checkpoint inhibition (PD-1/PD-L1 inhibition, CTLA-4 inhibition), and (2) targeting mitogen-activated protein kinase (MAPK) pathway. Although these methods have achieved a certain degree of success, both have experienced challenges and can benefit from a combination with the techniques described herein or be replaced by the techniques described herein. An overview of the current method is described below.

A. Systemic treatment

1. Checkpoint inhibitors

Immune checkpoint inhibitors (CPIs) targeting cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4; for example, ipilimumab) and programmed death 1 (PD-1; for example, nivolumab and pembrolizumab) have been approved for the treatment of advanced or metastatic melanoma alone or in combination. USPI; USPI; USPI, each of which is incorporated herein by reference in its entirety). In first-line treatment, nivolumab and ipilimumab combination therapy was associated with an improved overall response rate (ORR; 57% vs. 19% vs. 44%) and median PFS (11.5 months vs. 2.9 months vs. 6.9 months) compared to single-agent ipilimumab or nivolumab, respectively. However, the combination is associated with significant toxicity, and the effect of combination therapy on overall survival has not been fully determined (Wolcok et al. 2017, which is incorporated herein by reference in its entirety). For patients who are not candidates for combination therapy, monotherapy with anti-PD-1 therapy (e.g., pembrolizumab or nivolumab) or CTLA-4 inhibitors (e.g., ipilimumab) is also an option.

2. Signal transduction inhibitors

About half of patients with metastatic cutaneous melanoma harbor activating mutations of the proto-oncogene B-Raf (BRAF), which is an intracellular signaling kinase in the MAPK pathway. BRAF inhibitors (e.g., vemurafenib and dabrafenib) show clinical activity in melanomas with BRAF V600 mutations. BRAF inhibitors have monotherapy efficacy in patients with BRAF mutant melanoma, but half of the patients relapse within about 6 months due to drug resistance. For patients with previously untreated unresectable or metastatic disease, combined treatment with BRAF and MEK inhibitors circumvents resistance and has better efficacy (e.g., improved ORR, duration of response, PFS, and OS) than monotherapy with BRAF inhibitors. However, 50% of patients who responded to the combination therapy still progressed within the first 12 months (Mackiewicz et al. 2018, Gellrich et al. 2020, each of which is incorporated herein by reference in its entirety). Pembrolizumab and nivolumab have also been approved for first-line treatment of patients with BRAF mutations. For patients with BRAF V600 mutant tumors that are not very progressive, the currently recommended treatment sequence is immunotherapy (e.g., anti-PD-1 therapy) followed by targeted therapy with BRAF/MEK inhibitors (Michielin et al. 2019, which is incorporated herein by reference in its entirety).

3. Intralesional treatment

Talimogene laherparepvec (T-vec, trade name ) is a genetically modified oncolytic virus therapy, which indicates local treatment of unresectable skin, subcutaneous and lymph node lesions in patients with recurrent melanoma after primary surgery. T-vec is a modified herpes simplex virus type 1 (herpes simplex virus, type 1, HSV-1), which undergoes genetic modification (insertion of 2 copies of human cytokine granulocyte macrophage colony stimulating factor [granulocyte macrophage-colony stimulating factor, GM-CSF] gene) to promote selective viral replication in tumor cells, while reducing viral pathogenicity and promoting immunogenicity. In a randomized Phase III trial, intratumoral T-vec showed an objective response rate of 26% relative to 5.7% compared with subcutaneous GM-CSF. However, the differences observed in overall survival did not reach statistical significance, and the response rate in visceral lesions was poor (Rehman et al. 2016, which is incorporated herein by reference in its entirety). Therefore, this treatment option may be applicable to selected patients.

4. Other treatments

Treatment options for patients with advanced or metastatic melanoma who have progressed on targeted therapy or immunotherapy may include high-dose interleukin (IL)-2 or other cytotoxic therapies (e.g., dacarbazine, carboplatin/paclitaxel, nab-paclitaxel). These agents have moderate response rates of less than 20% in the first-line and second-line settings, but there are no data in the post-PD-1 setting. In addition, little consensus exists regarding the best standard chemotherapy (Swetter et al. 2021, which is incorporated herein by reference in its entirety). Initial promising results with c-kit-inhibitors were reported with a response rate of 23.3% (Guo et al. 2011, which is incorporated herein by reference in its entirety), whereas sorafenib, a multikinase inhibitor targeting both the MAPK-cascade and the VEGF and PDGF-cascades, did not improve median PFS compared to placebo in a phase III, randomized, double-blind, placebo-controlled trial in combination with carboplatin and paclitaxel (Hauschild et al. 2009, which is incorporated herein by reference in its entirety).

5. Adjuvant therapy

Adjuvant therapy is recommended for the treatment of patients with completely resected cutaneous melanoma (no evidence of disease) in stage III and completely resected stage IV (Swetter et al. 2021, which is incorporated herein by reference in its entirety).

For these patient groups, adjuvant therapy is based on a large number of prospective clinical trials with immune checkpoint inhibitors and BRAF-targeted therapy. Clinical trials in the adjuvant setting have shown that immune checkpoint inhibitors and BRAF-targeted therapy improve relapse-free survival (RFS) or disease-free survival when compared with conventional treatment, and provide higher overall survival (OS) rates at 3 or 5 years. However, there are concerns about the toxicity of adjuvant therapy, for example, after adjuvant immune checkpoint inhibition, 25% to 41% of patients have grade 3 to 4 adverse events (AEs) and a low proportion of patients have lifelong AEs (mostly immune-related) (Gershenwald et al. 2017, which is incorporated herein by reference in its entirety).

6. Overview of Exemplary Features of the Described Technology

Based on the above treatment options, significant progress has been made using approved treatments for the treatment of stage III and stage IV melanoma. However, approximately 40% to 45% of patients have been reported to experience no response to initial treatment, manifesting as primary resistance; and an additional 30% to 40% of patients have been reported to experience an initial response but ultimately progress, with secondary resistance (Mooradian and Sullivan 2019, which is incorporated herein by reference in its entirety). These subpopulations of patients with primary refractory disease or secondary recurrence represent a population with unmet medical needs, justifying the development of new treatments for patients with unresectable stage III and stage IV melanoma to induce higher initial response rates, thereby reducing primary resistance, and for patients with recurrent melanoma (Testori et al. 2020, which is incorporated herein by reference in its entirety). In addition, adding new treatments to anti-PD-1 therapy may improve responses relative to anti-PDI treatment alone.

The tolerability of currently available treatment options precludes the use of adjuvant therapy in patients with stage IIB or stage IIC high-risk disease and in some patients with stage III disease. New systemic therapies with better tolerability profiles may allow treatment of these patient subsets and improve available adjuvant treatment options for patients with completely resected disease.

The exemplary compositions described herein include TAAs: NY-ESO-1, tyrosinase, MAGE-A3, and TPTE. These cancer vaccine targets were selected based on the following criteria, among other reasons:

Low or absent expression in toxicity-related organs.

• Expression in a significant fraction of melanoma cells.

· The ability to induce antigen-specific immune responses.

· Role in tumor biology based on literature.

In addition, these TAAs were selected at least in part due to tissue expression analysis in the Phase I Lipo-MERIT trial. In this trial, approximately 8% of the screened patients did not express any of the four antigens at detectable levels in tumors or metastases. Taking into account the clonal heterogeneity of cancer and the limitations of clinically available samples (only one position), the present disclosure provides such a recognition that more than 92% of the observed rates in patients actually express at least one selected TAA. In addition, in a significant percentage of patients, several of these TAAs were found to be co-expressed. Therefore, the present disclosure provides such an insight: a significant population of melanoma patients is expected to develop a multi-epitope, vaccine-induced immune response and benefit from treatment with the composition described herein. As used herein, the term "BNT111" refers to a pharmaceutical composition comprising NY-ESO-1 antigen, tyrosinase antigen, MAGE-A3 antigen and TPTE antigen, preferably a pharmaceutical composition formulated as shown in Table 3.

In some embodiments, the compositions described herein (e.g., BNT111) can sensitize, activate and/or expand CD4 ⁺ and CD8 ⁺ T cell specificity and thereby generate a T cell specific complement pool against non-mutant TAAs that are frequently expressed in human melanoma regardless of the mutational burden of the tumor.

The liposome formulation of compositions described herein (e.g., BNT111) is designed to deliver antigens to secondary lymphoid tissues, and utilizes the inherent and adaptive immune mechanisms of antiviral to induce efficient antigen-specific T cell responses. Compositions described herein (e.g., BNT111) administered intravenously can be delivered to secondary lymphoid tissues (e.g., spleen, lymph nodes and bone marrow) and are rapidly taken up by antigen-presenting cells (antigen-presenting cell, APC). Proteins translated by RNA components of compositions described herein (e.g., BNT111) can be processed and presented on individual groups of both HLA-I and HLA-II class molecules of patients (Kranz et al. 2016, which are incorporated herein by reference in their entirety). The close proximity of APC to T cells in lymphoid tissues represents an ideal microenvironment for effective sensitization and expansion of CD8 ⁺ and CD4 ⁺ T cell responses (Zinkemagelet al. 1997, which are incorporated herein by reference in their entirety). The components of the compositions described herein activate APCs through toll-like receptor signaling, which leads to the pulsed release of proinflammatory cytokines (e.g., IFN-α, IL-6, IFN-γ, and IP-10). In addition, the secretion of type I interferons accompanied by effective antigen presentation stimulates immune cells and directly inhibits regulatory T cells (Srivastava et al. 2014, which is incorporated herein by reference in its entirety), which, in combination with homologous CD4 ⁺ T cell assistance, is necessary to overcome tolerance to self-antigens. Based on this dual mechanism of action, repeated administration of the compositions described herein (e.g., BNT111) enables effective sensitization and rapid expansion of antigen-specific CD8 ⁺ T cell responses.

Together with the TAA expression data and the observed dual mechanism of action, the present disclosure provides the expectation that most melanoma patients will mount a de novo or boosted multi-epitopic, vaccine-induced, antigen-specific immune response and gain benefit from treatment with the compositions described herein.

The activation, expansion and differentiation of initial T cells are physiologically associated with the induction of the immunomodulatory checkpoint molecule PD-1 (Sharpe and Pauken 2018, which is incorporated herein by reference in its entirety). Therefore, as further discussed herein, anti-PD-1/anti-PD-L1 blockade will enhance the activity of T cell responses induced by the compositions herein (e.g., BNT111), as supported by non-clinical data in mouse tumor models. In patients treated with PD-1/PD-L1 blockade, one reason for treatment failure is the lack of preformed antigen-specific T lymphocytes that recognize relevant tumor antigens. In some embodiments, such T lymphocytes are caused by the compositions described herein (e.g., BNT111), which induce effective antigen-specific CD4 ⁺ and CD8 ⁺ T cell responses. These T cells not only perform direct anti-tumor activity through their cytotoxicity after recognizing their target antigens on tumor cells, but also induce inflammation (e.g., IFN-γ secretion) in the tumor microenvironment, thereby sensitizing tumor cells to the therapeutic effects of checkpoint inhibitors.

In some embodiments, for patients who are refractory to anti-PD-1/anti-PD-L1 therapy or who relapse after anti-PD-1/anti-PD-L1 therapy (meaning that activation of pre-existing memory T cells alone is insufficient to mediate clinical activity), the addition of a PD-1 inhibitor (which will rescue the newly sensitized T cell specificity from exhaustion) will expand the effect of the compositions described herein (e.g., BNT111).

In some embodiments, the objective response rate of the combination of a composition described herein (e.g., BNT111) and a PD-1 inhibitor is 25% and the disease control rate is 22% in patients with a median of 5 prior treatments, which may be higher if the treatment is used in a less pre-treated patient population.

I. Tumor-associated antigens

In some embodiments, the disclosure provides, among other things, one or more RNA molecules encoding antigens. In some embodiments, the antigen is a tumor-associated antigen (TAA). The disclosure provides insights that a significant percentage of melanoma patients cumulatively express at least one of the four TAAs, regardless of the mutational load of the tumor. In some embodiments, one or more RNA molecules collectively encode: (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof. A high incidence of these antigens is observed in melanoma patients. It is reported that these antigens are also selectively expressed in cancer cells. The disclosure provides insights that selective expression of NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, and/or TPTE antigen may provide low risk of on-target/off-tumor toxicity. In some embodiments, one or more RNA molecules encoding antigens (e.g., TAAs, e.g., NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, and/or TPTE antigen) can be expected to induce multi-epitopic CD8 ⁺ and CD4 ⁺ T cell responses that result in killing of tumor cells expressing at least one targeted antigen.

In some embodiments, at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen is a full-length, non-mutated antigen. In some embodiments, all of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen are full-length, non-mutated antigens. In some embodiments, the NY-ESO-1 antigen, the MAGE-A3 antigen, and the TPTE antigen are full-length, non-mutated antigens. In some embodiments, the NY-ESO-1 antigen and the MAGE-A3 antigen are full-length, non-mutated antigens. In some embodiments, at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen is not a full-length antigen. For example, in some embodiments, the tyrosinase antigen is not full-length, but only comprises a portion of the tyrosinase. In some embodiments, the tyrosinase antigen comprises a signal peptide, an EGF-like domain, a CμA domain, a CμB domain, or a combination thereof. In some embodiments, the TPTE antigen is not full-length, but only comprises a portion of the TPTE antigen.

In some embodiments, following administration of one or more RNA molecules (e.g., one or more RNA molecules collectively encoding: (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, (iv) a TPTE antigen, or (v) a combination thereof), at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen is expressed by dendritic cells in lymphoid tissue of the patient.

In some embodiments, at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen is present in a cancer (e.g., a melanoma). In some embodiments, the methods described herein include determining the presence and/or abundance (e.g., level or amount) of at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen in a patient's cancer. For example, in some embodiments, a sample (e.g., a blood or blood component (e.g., serum or plasma) sample or a tumor biopsy) is isolated from a patient, and the sample is assessed for the presence and/or abundance (e.g., level or amount) of one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen.

New York esophageal squamous cell carcinoma (NY-ESO-1) antigen: The NY-ESO-1 antigen is a member of the cancertestis antigen (CTA) gene family. About 50% of all CTA genes form a multigene family on the X chromosome and are referred to as CT-X genes. These CTAs are located in a specific cluster in the chromosome, with the highest density in the Xq24 to q28 region (see Thomas et al., Front. Immunol. 9: 947 (2018), which is incorporated herein by reference in its entirety). Without wishing to be bound by theory, it is generally believed that NY-ESO-1 expression is primarily confined to testicular germ cells and placental trophoblasts and is either absent or lowly expressed at the transcript or protein level in normal healthy adult somatic cells. NY-OSO-1 is expressed in a variety of human cancers, including melanoma (Giavina-Bianchi et al. J. Immunol. Res. 2015, which is incorporated herein by reference in its entirety). According to at least one report. NY-ESO-1 protein is detected in approximately 20% of invasive melanomas (Giavina-Bianchi).

In some embodiments, an RNA molecule of one or more RNA molecules as described herein encodes a New York Esophageal Squamous Cell Carcinoma (NY-ESO-1) antigen or an immunogenic fragment thereof. In some embodiments, a single RNA molecule encoding a NY-ESO-1 antigen is a full-length, non-mutated antigen. In some embodiments, an RNA molecule of one or more RNA molecules as described herein encodes a NY-ESO-1 antigen that does not contain an amino acid substitution associated with melanoma cancer progression (e.g., a wild-type amino acid sequence of a NY-ESO-1 antigen).

In some embodiments, the NY-ESO-1 antigen comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the NY-ESO-1 antigen comprises or consists of the amino acid sequence of SEQ ID NO: 1.

In some embodiments, the NY-ESO-1 antigen is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of SEQ ID NO:2.

Melanoma-associated antigen A3 (MAGE-A3) antigen: The MAGE-A3 antigen is a member of the MAGEA gene family. The MAGEA gene is clustered at chromosome Xq28. It is associated with some genetic disorders such as congenital dyskeratosis. MAGE-A3 is proposed to enhance the ubiquitin ligase activity of RING-type zinc-finger-containing E3 ubiquitin protein ligases, and can enhance the ubiquitin ligase activity of TRIM28 and stimulate p53/TP53 ubiquitination through TRIM28. MAGE-A3 is also proposed to play a role by recruiting and/or stabilizing Ubl conjugating enzymes (E2) at the E3: substrate complex. MAGE-A3 is believed to play a role in embryonic development and is re-expressed in tumor transformation or tumor progression. In some embodiments, in vitro expression promotes cell viability of melanoma cell lines. It is known that the MAGE-A3 antigen is recognized by T cells when expressed on melanoma.

In some embodiments, the RNA molecule in one or more RNA molecules as described herein encodes a melanoma associated antigen A3 (MAGE-A3) antigen or an immunogenic fragment thereof. In some embodiments, a single RNA molecule encodes a full-length, non-mutated MAGE-A3 antigen. In some embodiments, the RNA molecule in one or more RNA molecules as described herein encodes a MAGE-A3 antigen that does not contain amino acid substitutions associated with melanoma cancer progression (e.g., a wild-type amino acid sequence of a MAGE-A3 antigen).

In some embodiments, the MAGE-A3 antigen comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 3. In some embodiments, the MAGE-A3 antigen comprises or consists of the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the MAGE-A3 antigen is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO:4.

Tyrosinase antigen: Tyrosinase antigen is encoded by the TYR gene and is a member of the tyrosinase family or protein, which is widely distributed in animals. This gene encodes a melanosome enzyme belonging to the tyrosinase family and plays an important role in the melanin biosynthesis pathway. Tyrosinase is known to be expressed in many cancers including melanoma (see Osella-Abate et al., Br. J. Cancer 89 (8): 1457-62 (2003), which is incorporated herein by reference in its entirety).

In some embodiments, a single RNA molecule in one or more RNA molecules as described herein encodes a tyrosinase antigen or its immunogenic fragment. In some embodiments, the RNA molecule encodes a full-length, non-mutated tyrosinase antigen. In some embodiments, the RNA molecule in one or more RNA molecules as described herein encodes such a tyrosinase antigen: it does not include the amino acid replacement (for example, the wild-type amino acid sequence of the tyrosinase antigen) associated with melanoma cancer progression. In some embodiments, the tyrosinase antigen is not full-length, but only includes a part of tyrosinase. In some embodiments, the tyrosinase antigen includes a signal peptide, an EGF-like domain, a CμA domain, a CμB domain, or a combination thereof.

In some embodiments, the tyrosinase antigen comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the tyrosinase antigen comprises or consists of the amino acid sequence of SEQ ID NO: 5.

In some embodiments, the tyrosinase antigen is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO:6.

Transmembrane phosphatase with tensin homology (TPTE) antigen: TPTE antigen is a member of the cancer-testis antigen (CTA) family. CTA antigen expression is highly tissue-restricted. TPTE is a transmembrane phosphatase with tensin homology that can play a role in the signal transduction pathways of endocrine or spermatogenic function of the testis. TPTE mRNA expression in healthy adult tissues is restricted to the testis, and in all other normal tissue samples, transcript levels are below the detection limit of highly sensitive RT-PCR. (Simon P, et al. Functional TCR retrieval from single antigen specific human T cells reveals multiple novel epitopes. In Cancer Immunol Res. 2(12): 1230-44 (2014), which is incorporated herein by reference in its entirety).

In some embodiments, the RNA molecule in one or more RNA molecules as described herein encodes a TPTE antigen or an immunogenic fragment thereof. In some embodiments, the RNA molecule encodes a full-length, non-mutated TPTE antigen. In some embodiments, the RNA molecule encodes a truncated TPTE antigen. In some embodiments, the RNA molecule encodes a truncated, non-mutated TPTE antigen. In some embodiments, the RNA molecule in one or more RNA molecules as described herein encodes a TPTE antigen that does not contain an amino acid substitution associated with melanoma cancer progression (e.g., a wild-type amino acid sequence of a TPTE antigen).

In some embodiments, the RNA molecule of one or more RNA molecules as described herein encodes a TPTE antigen or an immunogenic fragment thereof as described in WO2005/026205, the entire contents of which are incorporated herein by reference for the purposes described herein.

In some embodiments, the TPTE antigen comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7. In some embodiments, the TPTE antigen comprises or consists of the amino acid sequence of SEQ ID NO: 7.

In some embodiments, the TPTE antigen is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of SEQ ID NO:8.

In some embodiments, exemplary nucleic acid sequences encoding TAA as described herein and amino acid sequences of TAA as described herein are provided in Table 1 below.

Table 1: Sequences of TAA

T cell epitopes: In some embodiments, the present disclosure provides, inter alia, pharmaceutical compositions comprising: one or more RNA molecules that collectively encode (i) a NY-ESO-) antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, (iv) a TPTE antigen, or (v) a combination thereof; and a T cell epitope.

As used herein, the term "T cell epitope" refers to a portion or fragment of a protein that is recognized by a T cell when presented in the context of an MHC molecule. The term "major histocompatibility complex" and the abbreviation "MHC" include MHC class I and MHC class II molecules, and relate to a gene complex present in all vertebrates. MHC proteins or molecules are important for signal transduction between lymphocytes and antigen presenting cells or diseased cells in an immune response, wherein MHC proteins or molecules bind peptide epitopes and present them to be recognized by T cell receptors on T cells. Proteins encoded by MHC are expressed on the cell surface and display both autoantigens (peptide fragments from the cell itself) and non-autoantigens (e.g., fragments of invading microorganisms) to T cells. In the case of MHC class I/peptide complexes, binding peptides are generally about 8 to about 10 amino acids long, but longer or shorter peptides may also be effective. In the case of MHC class II/peptide complexes, binding peptides are generally about 10 to about 25 amino acids long, and particularly about 13 to about 18 amino acids long, but longer and shorter peptides may also be effective.

In some embodiments, the RNA molecule in the one or more RNA molecules encodes a CD4 epitope or an immunogenic fragment thereof. In some embodiments, the CD4 epitope comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the CD4 epitope described as the "P2P16" domain in SEQ ID NO: 11, 12, 15, 16, 19, 20, 23 or 24.

In some embodiments, the CD4 epitope comprises tetanus toxoid P2, tetanus toxoid P16, or both. In some embodiments, tetanus toxoid P2 comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the CD4 epitope described as the "P2" domain in SEQ ID NO: 11, 12, 15, 16, 19, 20, 23 or 24. In some embodiments, tetanus toxoid P16 comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of the CD4 epitope described as the "P16" domain in SEQ ID NO: 11, 12, 15, 16, 19, 20, 23 or 24.

II. Some exemplary embodiments of RNA encoding provided tumor-associated antigens

In some embodiments, the present disclosure provides, inter alia, pharmaceutical compositions comprising one or more RNA molecules that collectively encode: (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, (iv) a TPTE antigen, or (v) a combination thereof. In some embodiments, a single RNA molecule may encode at least two of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen. In some embodiments, a single RNA molecule may encode at least three of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen. In some embodiments, a single RNA molecule may encode each of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen.

In some embodiments, a single RNA molecule can encode a multi-epitope polypeptide. For example, in some embodiments, a single RNA molecule encodes a multi-epitope polypeptide comprising at least two of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen. In another example, in some embodiments, a single RNA molecule encodes a multi-epitope polypeptide comprising at least three of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen. In another example, in some embodiments, a single RNA molecule encodes a multi-epitope polypeptide comprising each of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen.

CD4+ epitope: In some embodiments, the present disclosure provides, among other things, a pharmaceutical composition comprising one or more RNA molecules, one or more RNA molecules collectively encoding: (i) NY-ESO-1 antigen, (ii) MAGE-A3 antigen, (iii) tyrosinase antigen, (iv) TPTE antigen, or (v) a combination thereof; and a CD4 ⁺ epitope. In some embodiments, the CD4+ epitope is delivered by the same RNA molecule that collectively encodes a tumor-associated antigen described herein. In some embodiments, the CD4+ epitope is delivered by a separate RNA molecule. In some embodiments, the CD4+ epitope is or comprises a non-specific antigen (e.g., an antigen that is not associated with melanoma). In some embodiments, the CD4+ epitope is or comprises a non-specific antigen that provides an auxiliary effect. For example, in some embodiments, the CD4+ epitope may include, but is not limited to, a tetanus toxoid antigen polypeptide, for example, in some embodiments, a tetanus toxoid P2 polypeptide and/or a tetanus toxoid P16 polypeptide.

MHC trafficking domain: In some embodiments, the RNA molecules described herein comprise a sequence encoding an MHC trafficking domain. In some embodiments, the MHC trafficking domain is or comprises the transmembrane region and cytoplasmic region of a chain of an MHC molecule (e.g., an MHC class I molecule), for example, in some embodiments as described in International Patent Publication No. WO 2005/038030, the contents of which are incorporated herein by reference in their entirety for the purposes described herein. In some embodiments, the MHC trafficking domain is or comprises an MHC class I trafficking domain. In some embodiments, the MHC class I trafficking domain comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of the MHC class I trafficking domain described as a "MITD" domain in SEQ ID NO: 11, 12, 15, 16, 19, 20, 23 or 24. In some embodiments, the MHC class I trafficking domain comprises an amino acid sequence identical to the amino acid sequence of the MHC class I trafficking domain described as a “MITD” domain in SEQ ID NO: 11, 12, 15, 16, 19, 20, 23, or 24.

Signal peptide coding region: In some embodiments, the RNA molecules described herein comprise a sequence encoding a signal peptide. In some embodiments, the inclusion of such a signal peptide can be used to improve the processing and presentation of antigens. In some embodiments, the signal peptide is or comprises a secretory signal peptide. In some embodiments, the secretory signal peptide may correspond to a sequence encoding a human MHC class I complex alpha chain or a fragment thereof. In some embodiments, the secretory signal peptide may correspond to a 70 to 80 bp fragment encoding a secretory signal peptide, which in some embodiments may direct the translocation of a nascent polypeptide chain into the endoplasmic reticulum. In some embodiments, the signal peptide comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence of the signal peptide coding region described as "Sec" in SEQ ID NO: 11, 12, 15, 16, 19, 20, 23 or 24. In some embodiments, the signal peptide comprises an amino acid sequence identical to the amino acid sequence of the signal peptide described as "Sec" in SEQ ID NO: 11, 12, 15, 16, 19, 20, 23, or 24. In some embodiments, the signal peptide is linked to the N-terminus of the antigen contained in the RNA molecule.

In some embodiments, the RNA molecules described herein include at least one non-coding sequence element. In some embodiments, such non-coding sequence elements are included in the RNA molecule to enhance RNA stability and/or translation efficiency. Some examples of non-coding sequence elements include, but are not limited to: 3' untranslated region (UTR), 5'UTR, cap structure, polyadenine (polyA) tail, and any combination thereof.

UTR (5'UTR and/or 3'UTR): In some embodiments, the RNA molecules provided include nucleotide sequences encoding a 5'UTR and/or a 3'UTR of interest. It will be appreciated by those skilled in the art that the untranslated regions of the mRNA sequence (e.g., 3'UTR and/or 5'UTR) may contribute to mRNA stability, mRNA localization, and/or translation efficiency.

In some embodiments, the RNA molecules provided may include 5'UTR nucleotide sequences and/or 3'UTR nucleotide sequences. In some embodiments, such 5'UTR sequences may be operably connected to the 3' of a coding sequence (e.g., one or more coding regions). In addition or in lieu thereof, in some embodiments, 3'UTR sequences may be operably connected to the 5' of a coding sequence (e.g., one or more coding regions).

In some embodiments, the 5' and 3' UTR sequences contained in the RNA molecules described herein may consist of or include the following: 5' and 3' UTR sequences that are naturally present or endogenous to the open reading frame of the target gene. Alternatively, in some embodiments, the 5' and/or 3' UTR sequences contained in the RNA molecules are not endogenous to the coding sequence (e.g., it covers one or more coding regions); in some such embodiments, such 5' and/or 3' UTR sequences can be used to modify the stability and/or translation efficiency of the transcribed RNA sequence. For example, the skilled person will understand that AU-rich elements in the 3' UTR sequence can reduce the stability of the mRNA. Therefore, as the skilled person will understand, 3' and/or 5' UTRs can be selected or designed based on the characteristics of UTRs known in the art to improve the stability of the transcribed RNA.

For example, it will be appreciated by those skilled in the art that, in some embodiments, a nucleotide sequence consisting of or comprising a Kozak sequence of an open reading frame sequence of a target gene or nucleotide sequence can be selected and used as a nucleotide sequence encoding a 5'UTR. As will be appreciated by the skilled artisan, it is known that Kozak sequences improve the translation efficiency of some RNA transcripts, but not necessarily all RNAs require Kozak sequences to enable efficient translation. In some embodiments, the RNA molecule provided may include a nucleotide sequence encoding a 5'UTR derived from an RNA virus, the RNA genome of which is stable in the cell. In some embodiments, a variety of modified ribonucleotides (e.g., as described herein) can be used for 3' and/or 5'UTR, for example to prevent exonuclease degradation of the transcribed RNA sequence.

In some embodiments, the 5'UTR contained in the RNA molecules described herein can be derived from human α-globin mRNA combined with a Kozak region.

In some embodiments, the RNA molecule may include one or more 3'UTRs. For example, in some embodiments, the RNA molecule may include two copies of a 3'-UTR derived from a globin mRNA (e.g., such as α2-globin, α1-globin, β-globin (e.g., human β-globin) mRNA). In some embodiments, two copies of a 3'UTR derived from human β-globin mRNA may be used, for example, in some embodiments, it may be placed between the coding sequence and the poly (A) tail of the RNA molecule to improve protein expression levels and/or extend the persistence of the mRNA. In some embodiments, a 3'UTR derived from human β-globin as described in WO2007/036366 may be included in the RNA molecule described herein, the contents of which are incorporated herein by reference in their entirety for the purposes described herein.

In some embodiments, the 3'UTR contained in the RNA molecule can be or contain one or more (e.g., 1, 2, 3 or more) of the 3'UTR sequences disclosed in WO 2017/060314, the entire contents of which are incorporated herein by reference for the purposes described herein. In some embodiments, the 3'-UTR can be a combination of at least two sequence elements derived from an "amino terminal enhancer of split" (AES) mRNA (referred to as F) and a mitochondrially encoded 12S ribosomal RNA (referred to as I) (FI element). These are identified by an in vitro selection process for sequences that confer RNA stability and enhance total protein expression (see WO 2017/060314, which is incorporated herein by reference).

PolyA tail: In some embodiments, the ssRNA provided may include a nucleotide sequence encoding a polyA tail. The polyA tail is a nucleotide sequence comprising a series of adenosine nucleotides, and its length can vary (e.g., at least 5 adenine nucleotides) and can be as many as hundreds of adenosine nucleotides. In some embodiments, the polyA tail is a nucleotide sequence comprising at least 30 adenosine nucleotides or more (including, for example, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, at least 100 or more) adenosine nucleotides. In some embodiments, the polyA tail is a nucleotide sequence comprising at least 120 adenosine nucleotides. In some embodiments, the polyA tail as described in WO 2007/036366 may be included in the RNA molecules described herein, and its contents are incorporated herein by reference as a whole for the purposes described herein.

In some embodiments, the polyA tail is or comprises a polyA homopolymeric tail. In some embodiments, the polyA tail may comprise one or more modified adenosine nucleosides including, but not limited to, cordiocipin and 8-azaadenosine.

In some embodiments, the polyA tail may include one or more non-adenosine nucleotides. In some embodiments, the polyA tail may be or include a destroyed or modified polyA tail as described in WO 2016/005324 (the entire contents of which are incorporated herein by reference for the purposes described herein). For example, in some embodiments, the polyA tail included in the RNA molecule described herein may be or include a modified polyA sequence, which includes: a linker sequence; a first sequence of at least 20 A consecutive nucleotides, which is 5' of the linker sequence; and a second sequence of at least 20 A consecutive nucleotides, which is 3' of the linker sequence. In some embodiments, the modified polyA sequence may include: a linker sequence comprising at least ten non-A nucleotides (e.g., T, G and/or C nucleotides); a first sequence of at least 30 A consecutive nucleotides, which is 5' of the linker sequence; and a second sequence of at least 70 A consecutive nucleotides, which is 3' of the linker sequence.

5' cap: In some embodiments, the RNA molecules described herein may include a 5' cap, which may be incorporated into such RNA molecules during transcription, or attached to such RNA molecules after transcription. In some embodiments, the RNA molecules may include an anti-reverse cap analog (ARCA). In some embodiments, the RNA molecules may include the cap analog β-S-ARCA (D1) (m ₂ ^{7, 2'-O} Gpp _s pG) as shown below:

In some embodiments, the RNA molecule may comprise an S-ARCA cap structure as disclosed in WO2011/015347 or WO2008/157688, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

In some embodiments, the RNA molecule may include a 5' cap structure for co-transcriptional capping of mRNA. Some examples of cap structures for co-transcriptional capping are known in the art, including, for example, as described in WO 2017/053297, the entire contents of which are incorporated herein by reference for the purposes described herein. In some embodiments, the 5' cap included in the RNA molecule described herein is or includes m7G(5')ppp(5')(2'OMeA)pG. In some embodiments, the 5' cap included in the RNA molecule described herein is or includes Cap1 structure [for example, but not limited to _m27,3' ^- OGppp( _m12' ^-O )ApG].

In some embodiments, one or more RNA molecules collectively encoding NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, TPTE antigen, or a combination thereof comprise natural ribonucleotides. In some embodiments, one or more RNA molecules collectively encoding NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, TPTE antigen, or a combination thereof comprise at least one modified or synthetic ribonucleotide. In some embodiments, modified or synthetic ribonucleotides are included in RNA molecules to increase their stability and/or reduce their cytotoxicity. For example, in some embodiments, at least one of the A, U, C, and G ribonucleotides of the RNA molecules described herein can be replaced by a modified ribonucleotide. For example, in some embodiments, some or all cytidine residues present in the RNA molecule can be replaced by a modified cytidine, which in some embodiments can be, for example, 5-methylcytidine. Alternatively or additionally, in some embodiments, some or all uridine residues present in the RNA molecule can be replaced by a modified uridine, which in some embodiments can be, for example, a pseudouridine, such as, for example, 1-methylpseudouridine. In some embodiments, all uridine residues present in the RNA molecule are replaced with pseudouridine, such as 1-methylpseudouridine.

In some embodiments, the present disclosure provides, inter alia, pharmaceutical compositions comprising one or more RNA molecules, wherein the RNA molecules comprise, from 5' to 3': (i) a 5' cap or a 5' cap analog; (ii) at least one 5'UTR; (iii) a signal peptide; (iv) a coding region encoding at least one of a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and a TPTE antigen; (v) at least one sequence encoding a CD4 ⁺ epitope; (vi) a sequence encoding an MHC trafficking domain; (vii) at least one 3'UTR; and (viii) a polyadenine tail. For example, in some embodiments, the cap structure contained in the RNA molecules described herein can be a cap structure that can increase the resistance of the RNA molecule to degradation by extracellular and intracellular RNases and result in higher protein expression. In some embodiments, an exemplary cap structure is or comprises β-S-ARCA (D1) (m ₂ ^{7, 2'-O} Gpp _s pG). In some embodiments, the exemplary 5'UTR sequence element included in the RNA molecule described herein is or includes a characteristic sequence and a Kozak consensus sequence from human α-globin. In some embodiments, the exemplary 3'UTR sequence element included in the RNA molecule described herein may be or include two copies of the 3'UTR derived from human β-globin, or a combination (FI element) of two sequence elements derived from "amino terminal split enhancer" (AES) mRNA (referred to as F) and mitochondrial encoded 12S ribosomal RNA (referred to as I). See, for example, WO2007/036366 and WO2017/060314, each of which is incorporated herein by reference in its entirety for the purposes described herein. In some embodiments, the poly (A) tail included in the RNA molecule described herein can be designed to enhance RNA stability and/or translation efficiency. In some embodiments, the exemplary poly (A) tail is or includes a continuous poly (A) sequence of at least 120 adenosine nucleotides in length. In some embodiments, an exemplary poly(A) tail is or comprises a modified poly(A) sequence of 110 nucleotides in length comprising a stretch of 30 adenosine residues followed by a 10-nucleotide linker sequence and another stretch of 70 adenosine residues (A30L70).

Linker: In some embodiments, at least one sequence encoding a linker may be present in an RNA molecule to separate separate components present in an RNA molecule. For example, in some embodiments, at least one sequence encoding a linker may be present between a coding region encoding one or more tumor-associated antigens described herein and a sequence encoding a CD4+ epitope. In some embodiments, at least one sequence encoding a linker may be present between a sequence encoding a CD4+ epitope and a sequence encoding an MHC trafficking domain. In some embodiments, the sequence encoding a linker may encode a peptide linker. In some embodiments, a peptide linker may be rich in glycine and/or serine. In some embodiments, a peptide linker rich in glycine and/or serine may include at least one amino acid that is not glycine or serine. In some embodiments, the length of a peptide linker may be 3 to 20 amino acids, or 3 to 15 amino acids, or 3 to 10 amino acids. In some embodiments, the length of a peptide linker may be 10 amino acids.

In some embodiments, one or more RNA molecules described herein are or comprise one or more mRNAs.

In some embodiments, the pharmaceutical composition comprises: (i) an RNA molecule encoding a NY-ESO-1 antigen as disclosed in Table 2 below; an RNA molecule encoding a MAGE-A3 antigen as disclosed in Table 2 below; an RNA molecule encoding a tyrosinase antigen as disclosed in Table 2 below; and an RNA molecule encoding a TPTE antigen as disclosed in Table 2 below. In some such embodiments, the pharmaceutical composition can be prepared by mixing RNA molecules each encoding a tumor-associated antigen as described herein at a molar ratio of about 1:1:1:1. In other words, in some embodiments, if the total RNA dose is 100 μg, the pharmaceutical composition can be prepared to include 25 μg of NY-ESO-1 antigen encoding RNA, 25 μg of MAGE-A3 antigen encoding RNA, 25 μg of tyrosinase antigen encoding RNA, and 25 μg of TPTE antigen encoding RNA. In some embodiments, this can be achieved by forming, for example, NY-ESO-1 antigen lipid particles (e.g., NY-ESO-1 antigen lipid complexes or lipid nanoparticles), MAGE-A3 antigen lipid particles (e.g., MAGE-A3 antigen lipid complexes or lipid nanoparticles), tyrosinase antigen lipid particles (e.g., tyrosinase antigen lipid complexes or lipid nanoparticles), and TPTE antigen lipid particles (e.g., TPTE antigen lipid complexes or lipid nanoparticles). In this method, the RNA-lipid particles can then be mixed. In other words, mixing can be performed after the RNA and lipid particles form RNA-lipid particles (e.g., RNA-lipid complexes or RNA-lipid nanoparticles).

Table 2: Exemplary constructs of RNA molecules each encoding a tumor-associated antigen described herein

GS = glycine/serine linker; MITD = MHC class I trafficking domain; sec = secretion signal peptide; UTR = untranslated region; hAg = human α-globin; P2P16 = P2 and P16 helper epitopes derived from tetanus toxoid; 2hBg = 2 copies of human β-globin; A120 = polyA tail of 120 A in length; A30L70 = two consecutive stretches of adenine nucleotides separated by a linker (one stretch is 30 A long and the other stretch is 70 A long); FI = a combination of at least two sequence elements derived from the "amino-terminal split enhancer" (AES) mRNA (designated F) and the mitochondrial-encoded 12S ribosomal RNA (designated I).

In some embodiments, the RNA molecule encoding the NY-ESO-1 antigen is or comprises the nucleotide sequence of RBL001.1 or RBL001.3. In some embodiments, the RNA molecule encoding the NY-ESO-1 antigen comprises a sequence encoding a polypeptide having the amino acid sequence of RBL001.1 or RBL001.3. In the following, a sequence alignment of RBL001.1 and RBL003.1 is given for both the nucleotide sequence of the full-length RNA and for the translated protein (where the amino acid is located below the third nucleotide of the corresponding codon triplet). Sequence elements as shown in Figure 1a are shown above the nucleotide sequence. Differences in nucleotide and amino acid sequences are indicated by "*". SEQ ID NO: 9 represents RBL001.1 RNA; SEQ ID NO: 10 represents RBL001.3 RNA; SEQ ID NO: 11 represents RBL001.1 protein; SEQ ID NO: 12 represents RBL001.3 protein.

In some embodiments, the RNA molecule encoding the tyrosinase antigen is or comprises the nucleotide sequence of RBL002.2 or RBL002.4. In some embodiments, the RNA molecule encoding the tyrosinase antigen comprises a sequence encoding a polypeptide having the amino acid sequence of RBL002.2 or RBL002.4. In the following, a sequence alignment of RBL002.2 and RBL002.4 is given for both the nucleotide sequence of the full-length RNA and for the translated protein (wherein the amino acid is located below the third nucleotide of the corresponding codon triplet). Sequence elements such as those shown in Figure 1a are shown above the nucleotide sequence. Differences in nucleotide and amino acid sequences are indicated by "*". SEQ ID NO: 13 represents RBL002.2 RNA; SEQ ID NO: 14 represents RBL002.4 RNA; SEQ ID NO: 15 represents RBL002.2 protein; SEQ ID NO: 16 represents RBL002.4 protein.

In some embodiments, the RNA molecule encoding the MAGE-A3 antigen is or comprises the nucleotide sequence of RBL003.1 or RBL003.3. In some embodiments, the RNA molecule encoding the MAGE-A3 antigen comprises a sequence encoding a polypeptide having the amino acid sequence of RBL003.1 or RBL003.3. In the following, a sequence alignment of RBL003.1 and RBL003.3 is given for both the nucleotide sequence of the full-length RNA and for the translated protein (wherein the amino acid is located below the third nucleotide of the corresponding codon triplet). Sequence elements as shown in Figure 1a are shown above the nucleotide sequence. Differences in nucleotide and amino acid sequences are indicated by "*". SEQ ID NO: 17 represents RBL003.1 RNA; SEQ ID NO: 18 represents RBL003.3 RNA; SEQ ID NO: 19 represents RBL003.1 protein; SEQ ID NO: 20 represents RBL003.3 protein.

In some embodiments, the RNA molecule encoding the TPTE antigen is or comprises the nucleotide sequence of RBL004.1 or RBL004.3. In some embodiments, the RNA molecule encoding the TPTE antigen comprises a sequence encoding a polypeptide having the amino acid sequence of RBL004.1 or RBL004.3. In the following, a sequence alignment of RBL004.1 and RBL004.3 is given for both the nucleotide sequence of the full-length RNA and for the translated protein (wherein the amino acid is located below the third nucleotide of the corresponding codon triplet). Sequence elements as shown in Figure 1a are shown above the nucleotide sequence. Differences in nucleotide and amino acid sequences are indicated by "*". SEQ ID NO: 21 represents RBL004.1 RNA; SEQ ID NO: 22 represents RBL004.3 RNA; SEQ ID NO: 23 represents RBL004.1 protein; SEQ ID NO: 24 represents RBL004.3 protein.

B. Exemplary Manufacturing Methods

Individual RNA molecules can be produced by methods known in the art. For example, in some embodiments, single-stranded RNA can be produced, for example, by in vitro transcription using a DNA template. Plasmid DNA used as an in vitro transcription template to generate RNA molecules described herein is also within the scope of the present disclosure.

In the presence of an appropriate RNA polymerase (e.g., a recombinant RNA polymerase, such as T7 RNA polymerase) and ribonucleotide triphosphates (e.g., ATP, CTP, GTP, UTP), a DNA template is used for in vitro RNA synthesis. In some embodiments, RNA molecules (e.g., RNA molecules described herein) can be synthesized in the presence of modified ribonucleotide triphosphates. By way of example only, in some embodiments, N1-methylpseudouridine triphosphate ( ^m1 ΨTP) can be used in place of uridine triphosphate (UTP). As will be clear to those skilled in the art, during in vitro transcription, RNA polymerase (e.g., as described and/or used herein) typically passes through at least a portion of a single-stranded DNA template in the 3'→5' direction to produce a single-stranded complementary RNA in the 5'→3' direction.

In some embodiments in which the RNA molecule comprises a polyA tail, those skilled in the art will appreciate that such a polyA tail can be encoded in the DNA template, for example by using appropriately tailed PCR primers, or it can be added to the RNA molecule after in vitro transcription, for example by enzymatic treatment (e.g., using a poly(A) polymerase, such as E. coli Poly(A) polymerase).

In some embodiments, those skilled in the art will appreciate that adding a 5' cap to an RNA (e.g., mRNA) can aid in RNA recognition and in connecting RNA to a ribosome to initiate translation and enhance translation efficiency. Those skilled in the art will also appreciate that the 5' cap can also protect the RNA product from 5' exonuclease-mediated degradation, thereby increasing half-life. Capping methods are known in the art; those of ordinary skill in the art will appreciate that, in some embodiments, capping can be performed after in vitro transcription in the presence of a capping system (e.g., an enzyme-based capping system, such as a capping enzyme of a vaccinia virus). In some embodiments, a cap can be introduced during in vitro transcription, as well as a plurality of ribonucleotide triphosphates, so that the cap is incorporated into the RNA molecule ssRNA during transcription (also referred to as co-transcriptional capping).

After RNA transcription, the DNA template is digested. In some embodiments, digestion can be achieved using DNase I under appropriate conditions.

In some embodiments, RNA molecules can be purified after the in vitro transcription reaction, for example, to remove components used or formed during the production process, such as, for example, proteins, DNA fragments and/or nucleotides. A variety of nucleic acid purifications known in the art can be used according to the present disclosure. In some embodiments, RNA molecules can be purified using magnetic bead-based purification, which in some embodiments can be or include magnetic bead-based chromatography. In some embodiments, RNA molecules can be purified using hydrophobic interaction chromatography (HIC) followed by diafiltration.

In some embodiments, dsRNA can be obtained as a by-product during in vitro transcription. In some such embodiments, a second purification step can be performed to remove dsRNA contamination. For example, in some embodiments, cellulose materials (for example, microcrystalline cellulose) can be used to remove dsRNA contamination, for example, in some embodiments in the form of chromatography. In some embodiments, cellulose materials (for example, microcrystalline cellulose) can be pre-treated to deactivate potential RNA enzyme contamination, for example, in some embodiments, by autoclaving and then incubating with alkaline aqueous solution (for example NaOH). In some embodiments, cellulose materials can be used according to the method described in WO 2017/182524 (the entire contents of which are incorporated herein by reference) to purify RNA molecules.

In some embodiments, the ssRNA batch can be further processed by one or more filtration and/or concentration steps. For example, in some embodiments, the RNA molecules (e.g., after removing dsRNA contamination) can be further diafiltered, e.g., to adjust the concentration of the ssRNA to a desired RNA concentration and/or to exchange the buffer for a drug substance buffer.

In some embodiments, RNA molecules can be processed by 0.2 μm filtration before they are filled into a suitable container.

In some embodiments, RNA quality control can be performed and/or monitored at any time during the production process of RNA molecules and/or compositions comprising the same. For example, in some embodiments, RNA quality control parameters can be evaluated and/or monitored after each or certain steps of the RNA molecule manufacturing process, such as after in vitro transcription and/or after each purification step.

In some embodiments, one or more assessments can be used during manufacture or other preparation or use of the RNA molecule (e.g., as a release test).

In some embodiments, one or more quality control parameters can be evaluated to determine whether the RNA molecules described herein meet or exceed predetermined acceptance criteria (e.g., for subsequent formulation and/or release for distribution). In some embodiments, such quality control parameters may include, but are not limited to, RNA integrity, RNA concentration, residual DNA template, and/or residual dsRNA. Methods for evaluating RNA quality are known in the art.

In some embodiments, one or more characteristics of a batch of RNA molecules can be evaluated to determine the next course of action. For example, if the RNA quality assessment indicates that a batch of single-stranded RNA meets or exceeds the acceptance criteria, such a batch of single-stranded RNA can be designated for one or more additional steps of manufacturing and/or formulation and/or distribution. Otherwise, if such a batch of single-stranded RNA does not meet or exceed the acceptance criteria, alternative measures can be taken (e.g., discarding the batch).

In some embodiments, batches of RNA molecules having exemplary evaluation results can be used in one or more additional steps of manufacturing and/or formulation and/or distribution.

III. RNA delivery technology

The provided pharmaceutical compositions (e.g., one or more RNA molecules encoding one or more TAAs) can be delivered for therapeutic applications described herein using any suitable method known in the art, including, for example, delivery as naked RNA, or delivery mediated by viral and/or non-viral vectors, polymer-based vectors, lipid-based vectors, nanoparticles (e.g., lipid nanoparticles, polymer nanoparticles, lipid-polymer hybrid nanoparticles, etc.), and/or peptide-based vectors. See, for example, Wadhwa et al. "Opportunities and Challenges in the Delivery of mRNA-Based Vaccines" Pharmaceutics (2020) 102 (page 27), the contents of which are incorporated herein by reference for information about various methods that can be used to deliver RNA molecules described herein.

In some embodiments, one or more RNA molecules can be formulated with lipid particles for delivery (eg, in some embodiments, by intravenous injection).

In some embodiments, lipid particles can be designed to protect RNA molecules (e.g., mRNA) from the influence of extracellular RNases and/or are modified for delivery of RNA systemically to target cells (e.g., dendritic cells). In some embodiments, when RNA molecules are administered intravenously to objects in need thereof, such lipid particles can be particularly useful for delivery of RNA molecules (e.g., mRNA).

In some embodiments, the lipid particles comprise liposomes. In some embodiments, the lipid particles comprise cationic liposomes.

In some embodiments, the lipid particles comprise lipid nanoparticles.

In some embodiments, the lipid particle comprises a lipid complex.

In some embodiments, lipid particles include N, N, N trimethyl-2-3-dioleyloxy-1-chlorinated propylamine (DOTMA), 1,2-dioleoyl-sn-glyceryl-3-phosphoethanolamine phospholipids (DOPE), or both. In some embodiments, lipid particles include at least one ionizable amino lipid. In some embodiments, lipid particles include at least one ionizable amino lipid and auxiliary lipid. In some embodiments, auxiliary lipid is or includes phospholipid. In some embodiments, auxiliary lipid is or includes sterol. In some embodiments, lipid particles include at least one polymer-conjugated lipid.

RNA lipid complex particles: In some embodiments, the RNA molecules described herein can be delivered by liposome formulations. In some embodiments, the negatively charged RNA molecules described herein are complexed with cationic liposomes to form RNA lipid complex particles. In some embodiments, the RNA molecules described herein are embedded in the (phospho) lipid bilayer structure within the RNA lipid complex particles. In some embodiments, the cationic liposomes may include cationic lipids or ionizable amino lipids (e.g., those described herein) and optionally additional or auxiliary lipids (e.g., at least one neutral lipid as described herein) to form an injectable particle formulation.

In some embodiments, RNA lipid complex particles can be prepared by mixing liposomes with RNA molecules described herein. In some embodiments, liposomes can be obtained by injecting a solution of lipids in ethanol into water or a suitable aqueous phase. In some embodiments, cationic liposomes are stabilized in aqueous formulations, for example, as described in WO 2016/046060, the entire contents of which are incorporated herein by reference for the purposes described herein. In some embodiments, cationic liposomes can be produced by the following methods: For example, as described in WO 2019/077053, the entire contents of which are incorporated herein by reference for the purposes described herein.

In some embodiments, spleen-targeted RNA lipoplex particles that can be used to deliver RNA molecules described herein are described in WO 2013/143683, the entire contents of which are incorporated herein by reference for the purposes described herein. In some embodiments, RNA molecules and positively charged liposomes are mixed so that cationic lipids and RNA are present in a charge ratio of 1.3:2. It has been determined that such a charge ratio effectively targets RNA to the spleen.

In some embodiments, the RNA lipid complex particles comprise a cationic lipid or an ionizable amino lipid (e.g., those described herein) and an RNA molecule described herein. In some embodiments, such RNA lipid complex particles may also comprise additional or auxiliary lipids (e.g., those described herein). Without wishing to be bound by theory, electrostatic interactions between the positively charged liposomes and the negatively charged RNA lead to the complexation and spontaneous formation of the RNA lipid complex particles.

In some embodiments, where a cationic lipid or an ionizable amino lipid (e.g., those described herein) and a helper lipid are used, such a cationic lipid or an ionizable amino lipid and such a helper lipid may be present in a molar ratio of 2: 1. In some embodiments, the cationic lipid or the ionizable amino lipid may be or include DOTMA. In some embodiments, the helper lipid may be or include a neutral lipid. In some embodiments, the neutral lipid may be or include DOPE.

In some embodiments, the RNA lipid complex particles are nanoparticles. In some embodiments, the particle size (e.g., Z-average) of the RNA lipid complex nanoparticles can be about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm.

RNA lipid nanoparticles: In some embodiments, the RNA molecules described herein can be delivered by lipid nanoparticle formulations. In some embodiments, RNA lipid nanoparticles can be prepared by mixing lipids with RNA molecules described herein. In some embodiments, at least a portion of the RNA molecules are encapsulated by lipid nanoparticles. In some embodiments, at least 90% or more (including, for example, at least 95%, 96%, 97%, 98%, 99% or more) of the RNA molecules are encapsulated by lipid nanoparticles.

In a plurality of embodiments, the average size (e.g., Z-means) of lipid nanoparticles can be from about 100nm to 1000nm, or from about 200nm to 900nm, or from about 200nm to 800nm, or from about 250nm to about 700nm. In some embodiments, the particle size (e.g., Z-means) of lipid nanoparticles can be from about 30nm to about 200nm, or from about 30nm to about 150nm, from about 40nm to about 150nm, from about 50nm to about 150nm, from about 60nm to about 130nm, from about 70nm to about 110nm, from about 70nm to about 100nm, from about 80nm to about 100nm, from about 90nm to about 100nm, from about 70 to about 90nm, from about 80nm to about 90nm, or from about 70nm to about 80nm. In some embodiments, the average size of lipid nanoparticles is determined by measuring particle diameter.

In certain embodiments, when RNA molecules (eg, mRNA) are present in provided lipid nanoparticles, they are resistant to degradation by nucleases in aqueous solution.

In some embodiments, the lipid nanoparticle is a cationic lipid nanoparticle comprising one or more cationic lipids (e.g., those described herein). In some embodiments, the cationic lipid nanoparticle may include at least one cationic lipid, at least one polymer-conjugated lipid and at least one auxiliary lipid (e.g., at least one neutral lipid).

1. Helper lipids

In some embodiments, the lipid particles for delivering the RNA molecules described herein include at least one helper lipid, which can be a neutral lipid, a positively charged lipid, or a negatively charged lipid. In some embodiments, the helper lipid is a lipid that can be used to improve the effectiveness of lipid-based particles (e.g., particles based on cationic lipids) delivered to target cells. In some embodiments, the helper lipid can be or include a structural lipid, the concentration of which is selected to optimize particle size, stability, and/or encapsulation.

In some embodiments, lipid particles used to deliver RNA molecules described herein comprise a neutral helper lipid. Some examples of such neutral helper lipids include, but are not limited to, phosphatidylcholines, such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), phosphatidylethanolamine (e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelin (SM), ceramide, cholesterol, steroids (e.g., sterols), and derivatives thereof. Neutral lipids may be of synthetic or natural origin. Other neutral helper lipids known in the art, for example, as disclosed in WO 2017/075531 and WO 2018/081480 (each of which is incorporated herein by reference in its entirety for the purposes described herein), can also be used for the lipid particles described herein. In some embodiments, the lipid particles for delivering the RNA molecules described herein include DSPC and/or cholesterol.

In some embodiments, the lipid particles used to deliver the RNA molecules described herein comprise at least one helper lipid (e.g., those described herein). In some such embodiments, the lipid particles may comprise DOPE.

2. Cationic lipids

In some embodiments, the lipid particles for delivering RNA molecules described herein include cationic lipids. Cationic lipids are generally lipids with a net positive charge, such as in some embodiments at a specific pH. In some embodiments, cationic lipids may include one or more positively charged amine groups. In some embodiments, cationic lipids may include cationic head groups, meaning positively charged head groups. In some embodiments, cationic lipids may have a hydrophobic domain (e.g., one or more domains of neutral lipids or anionic lipids), provided that the cationic lipid has a net positive charge. In some embodiments, cationic lipids include polar head groups, and in some embodiments, they may include one or more amine derivatives, such as primary amines, secondary amines and/or tertiary amines, quaternary ammoniums, various combinations of amines, ammonium salts, or guanidines and/or imidazoles and pyridine, piperazine and amino acid head groups (e.g., lysine, arginine, ornithine and/or tryptophan). In some embodiments, the polar head groups of cationic lipids include one or more amine derivatives. In some embodiments, the polar head groups of cationic lipids include quaternary ammoniums. In some embodiments, the head groups of cationic lipids may include multiple cationic charges. In some embodiments, the head group of the cationic lipid comprises a cationic charge. Some examples of monocationic lipids include, but are not limited to, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and/or 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 2,3-di-tetradecyloxy-propyl-(2-hydroxyethyl)-dimethyl nitrogen bromide (DMRIE), didodecyl (dimethyl) nitrogen bromide (DDAB), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), 3P-[N-(N\N'-dimethylamino-ethane)carbamoyl] cholesterol (DC-Choi) and/or dioleyl ether phosphatidylcholine (DOEPC).

In some embodiments, the positively charged lipid structure described herein may also include one or more other components that are generally useful for forming vesicles (e.g., for stabilization). Some examples of such other components include, but are not limited to, fatty alcohols, fatty acids, and/or cholesterol esters or any other pharmaceutically acceptable excipients that may affect surface charge, membrane fluidity, and contribute to the incorporation of lipids into the lipid assembly. Some examples of sterols include cholesterol, cholesterol hemisuccinate, cholesterol sulfate, or any other derivative of cholesterol. In some embodiments, a cationic lipid includes DMEPC and/or DOTMA. In some embodiments, the cationic lipid includes DOTMA.

In some embodiments, cationic lipids are ionizable, so that they can exist in positively charged form or neutral form according to pH. For example, in some embodiments, cationic lipids are ionizable amino lipids. Such ionization of cationic lipids can affect the surface charge of lipid particles under different pH conditions, which can affect the ability of plasma protein absorption, blood clearance and/or tissue distribution and the formation of endosome non-bilayer structure in some embodiments. Therefore, in some embodiments, cationic lipids can be or include pH responsive lipids. In some embodiments, pH responsive lipids are fatty acid derivatives or other amphipathic compounds that can form a readily soluble lipid phase, and their pKa values are pH 5 to pH 7.5. This means that lipids are uncharged at pH higher than pKa values, and positively charged at pH lower than pKa values. In some embodiments, pH responsive lipids can be used to supplement or replace cationic lipids, such as by combining one or more RNA molecules with lipids or lipid mixtures at low pH. pH responsive lipids include but are not limited to 1,2-dienyloxy-3-dimethylamino-propane (DODMA).

In some embodiments, the lipid particle may comprise one or more cationic lipids as described in WO 2017/075531 (e.g., as shown in Tables 1 and 3 therein) and WO 2018/081480 (e.g., as shown in Tables 1 to 4 therein), the entire contents of each of which are incorporated herein by reference for the purposes described herein.

In some embodiments, cationic lipids that can be used according to the present disclosure are amino lipids, which include titratable tertiary amino head groups connected to at least two saturated alkyl chains by ester bonds, and the ester bonds can be easily hydrolyzed to promote rapid degradation and/or excretion by renal pathways. In some embodiments, the apparent pK _a of such amino lipids is about 6.0 to 6.5 (for example, the apparent pK _a in one embodiment is about 6.25), resulting in substantially completely positively charged molecules at acidic pH (for example, pH 5). In some embodiments, such amino lipids, when incorporated into lipid particles, can impart different physicochemical properties of particle formation, cellular uptake, fusogenicity and/or endosome release of regulating RNA molecules. In some embodiments, the RNA aqueous solution is introduced into the lipid mixture comprising such amino lipids at pH 4.0 and can cause electrostatic interactions between negatively charged RNA backbones and positively charged cationic lipids. It is not desirable to be bound by any particular theory, such electrostatic interactions cause particle formation consistent with the effective encapsulation of RNA drug substances. After RNA encapsulation, the pH of the medium surrounding the resulting lipid nanoparticles is adjusted to a more neutral pH (e.g., pH 7.4), resulting in neutralization of the surface charge of the lipid nanoparticles. When all other variables remain constant, such charge-neutral particles show longer in vivo circulation life and better delivery to hepatocytes than charged particles that are rapidly cleared by the reticuloendothelial system. After endosomal uptake, the low pH of the endosomal body causes the lipid nanoparticles containing such amino lipids to fuse and allows RNA to be released into the cytosol of the target cell.

Cationic lipids can be used alone or in combination with neutral lipids (eg, cholesterol and/or neutral phospholipids), or in combination with other known lipid assembly components.

3. Polymer-conjugated lipids

In some embodiments, lipid nanoparticles for delivering RNA molecules described herein may comprise at least one polymer-conjugated lipid. A polymer-conjugated lipid is typically a molecule comprising a lipid portion and a polymer portion conjugated thereto.

In some embodiments, the polymer-conjugated lipid is a PEG-conjugated lipid. In some embodiments, the PEG-conjugated lipid is designed to sterically stabilize the lipid particles by forming a protective hydrophilic layer of a protective hydrophobic lipid layer. In some embodiments, when such lipid particles are administered in vivo, the PEG-conjugated lipid can reduce its association with serum proteins and/or the resulting uptake by the reticuloendothelial system.

A variety of PEG-conjugated lipids are known in the art and include, but are not limited to, pegylated diacylglycerol (PEG-DAG) (e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimamyristoylglycerol (PEG-DMG)), pegylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEG succinate diacylglycerol, PEG-S-DAG) (e.g., 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinic acid diethyl ester (PEG-S-DMG)), pegylated ceramide (PEG-DAG) (e.g., 1-(monomethoxy-polyethylene glycol)-2,3-dimamyristoylglycerol (PEG-DMG)), pegylated phosphatidylethanolamine (PEG-PE), PEG diacylglycerol succinate (PEG-S-DAG) (e.g., 4-O-(2',3'-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)succinic acid diethyl ester (PEG-S-DMG)), pegylated ceramide, PEG-cer) or PEG dialkoxypropyl carbamate (for example, ω-methoxy(polyethoxy)ethyl N-(2,3-di(tetradecyloxy)propyl)carbamate or 2,3-di(tetradecyloxy)propyl N-(ωmethoxy(polyethoxy)ethyl)carbamate) and the like.

Some PEG-conjugated lipids (also referred to as PEGylated lipids) have obtained clinical approval, and they have shown safety in clinical trials. It is known that PEG-conjugated lipids affect cellular uptake, which is a prerequisite for endosomal localization and payload delivery. The pharmacology of encapsulated nucleic acids can be controlled in a predictable manner by adjusting the alkyl chain length of the PEG-lipid anchor. In some embodiments, the PEG-conjugated lipid can be designed and/or selected based on reasonable solubility characteristics and/or its molecular weight to effectively perform the function of the space barrier. For example, in some embodiments, the PEGylated lipid does not show obvious surfactant or permeability enhancement or interference to the biomembrane. In some embodiments, the PEG in such PEG-conjugated lipids can be connected to the diacyl lipid anchor with a biodegradable amide bond, thereby promoting rapid degradation and/or excretion. In some embodiments, the LNP comprising the PEG-conjugated lipid retains a complete and sufficient number of PEGylated lipids. In the blood compartment, such PEGylated lipids dissociate from the particles over time, showing more fused particles that are more easily absorbed by cells, ultimately leading to the release of the RNA payload.

In some embodiments, lipid particles (e.g., lipid nanoparticles) may include one or more PEG-conjugated lipids or PEGylated lipids, as described in WO 2017/075531 and WO 2018/081480, each of which is incorporated herein by reference in its entirety for the purposes described herein. For example, in some embodiments, PEG-conjugated lipids that can be used according to the present disclosure may have the following structure as described in WO 2017/075531 or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof:

Wherein: R ₈ and R ₉ are each independently a straight or branched, saturated or unsaturated alkyl chain containing 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and the average value of w is 30 to 60. In some embodiments, R ₈ and R ₉ are each independently a straight, saturated alkyl chain containing 12 to 16 carbon atoms. In some embodiments, the average value of w is 43 to 53. In other embodiments, the average w is about 45.

In some embodiments, the lipids forming the lipid nanoparticles described herein include: polymer-conjugated lipids; cationic lipids; and auxiliary neutral lipids. In some such embodiments, the total polymer-conjugated lipids may be present at about 0.5 to 5 mol%, about 0.7 to 3.5 mol%, about 1 to 2.5 mol%, about 1.5 to 2 mol%, or about 1.5 to 1.8 mol% of the total lipids. In some embodiments, the total polymer-conjugated lipids may be present at about 1 to 2.5 mol% of the total lipids. In some embodiments, the molar ratio of total cationic lipids to total polymer-conjugated lipids (e.g., PEG-conjugated lipids) may be about 100: 1 to about 20: 1, or about 50: 1 to about 20: 1, or about 40: 1 to about 20: 1, or about 35: 1 to about 25: 1.

In some embodiments involving polymer-conjugated lipids, cationic lipids, and auxiliary neutral lipids in lipid nanoparticles described herein, the total cationic lipids are present at about 35 to 65 mol%, about 40 to 60 mol%, about 41 to 49 mol%, about 41 to 48 mol%, about 42 to 48 mol%, about 43 to 48 mol%, about 44 to 48 mol%, about 45 to 48 mol%, about 46 to 48 mol%, or about 47.2 to 47.8 mol% of the total lipids.

In some embodiments of polymer-conjugated lipids, cationic lipids and auxiliary neutral lipids in lipid nanoparticles described herein, total neutral lipids are present at about 35 to 65 mol%, about 40 to 60 mol%, about 45 to 55 mol%, or about 47 to 52 mol% of total lipids. In some embodiments, total neutral lipids are present at 35 to 65 mol% of total lipids. In some embodiments, total non-steroidal neutral lipids (e.g., DPSC) are present at about 5 to 15 mol%, about 7 to 13 mol%, or 9 to 11 mol% of total lipids. In some embodiments, total non-steroidal neutral lipids are present at about 9.5, 10 or 10.5 mol% of total lipids. In some embodiments, the molar ratio of total cationic lipids to non-steroidal neutral lipids is about 4.1: 1.0 to about 4.9: 1.0, about 4.5: 1.0 to about 4.8: 1.0, or about 4.7: 1.0 to 4.8: 1.0. In some embodiments, total steroid neutral lipid (e.g., cholesterol) is present at about 35 to 50mol%, about 39 to 49mol%, about 40 to 46mol%, about 40 to 44mol%, or about 40 to 42mol% of total lipid. In certain embodiments, total steroid neutral lipid (e.g., cholesterol) is present at about 39, 40, 41, 42, 43, 44, 45, or 46mol% of total lipid. In certain embodiments, the molar ratio of total cationic lipid to total steroid neutral lipid is about 1.5: 1 to 1: 1.2 or about 1.2: 1 to 1: 1.2.

In some embodiments, the lipid composition comprising cationic lipids, polymer-conjugated lipids, and neutral lipids can have the individual lipids present at a specific molar percentage of the total lipids or at a specific molar ratio (relative to each other) as described in WO 2018/081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

IV. Provided Pharmaceutical Compositions

The present disclosure provides, among other things, pharmaceutical compositions for delivering antigens (e.g., TAAs) to patients. In some embodiments, the pharmaceutical composition comprises one or more RNA molecules encoding NY-ESO-1 antigens, MAGE-A3 antigens, tyrosinase antigens, TPTE antigens, or a combination thereof; and lipid particles (e.g., lipoplexes or lipid nanoparticles). In some embodiments, the pharmaceutical composition comprises one or more RNA molecules encoding NY-ESO-1 antigens, MAGE-A3 antigens, tyrosinase antigens, and TPTE antigens together; and lipid particles (e.g., lipoplexes or lipid nanoparticles). In some embodiments, the pharmaceutical composition comprises at least four populations of RNA-lipid particles (e.g., lipoplexes or lipid nanoparticles), wherein each RNA-lipid particle comprises an RNA molecule and a lipid particle, and wherein the RNA molecule of each of the four RNA lipid particles is different, e.g., each RNA encodes a different TAA as described herein.

In some embodiments, one or more RNA molecules can be formulated with lipid nanoparticles (e.g., those described herein) for administration to a patient. Thus, in some embodiments, the pharmaceutical composition comprises one or more RNA molecules encoding NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, TPTE antigen, or a combination thereof; and lipid particles (e.g., lipid complexes or lipid nanoparticles), wherein the one or more RNA molecules are encapsulated with the lipid particles (e.g., forming RNA-lipid particles). In some embodiments, the RNA-lipid particles are RNA-lipid complex particles. In some embodiments, the RNA-lipid particles are RNA-lipid nanoparticles.

In some embodiments, the pharmaceutical composition is administered as a monotherapy. In some embodiments, the pharmaceutical composition is administered as part of a combination therapy.

In some embodiments, the pharmaceutical composition comprises a first RNA molecule encoding a NY-ESO-1 antigen, a second RNA molecule encoding a MAGE-A3, a third RNA molecule encoding a tyrosinase antigen, and a fourth RNA molecule encoding a TPTE antigen, and the first RNA molecule, the second RNA molecule, the third RNA molecule, and the fourth RNA molecule may be present in the pharmaceutical composition in approximately equimolar amounts (e.g., a molar ratio of about 1:1:1:1).

In some embodiments, the concentration of total RNA in a pharmaceutical composition described herein (e.g., the total concentration of all of one or more RNA molecules) is about 0.01 mg/mL to about 0.5 mg/mL, or about 0.05 mg/mL to about 0.1 mg/mL.

The pharmaceutical preparation may additionally comprise a pharmaceutically acceptable excipient, as used herein, which includes any and all solvents, dispersion media, diluents or other liquid carriers, dispersion or suspension aids, surfactants, isotonic agents, thickeners or emulsifiers, preservatives, solid binders, lubricants, and the like, such as those suitable for the desired particular dosage form. Remington's The Science and Practice of Pharmacy, 21st edition, A.R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; which is incorporated herein by reference in its entirety) discloses a variety of excipients for formulating pharmaceutical compositions and known techniques for their preparation. Unless any conventional excipient medium is incompatible with the substance or its derivatives, such as by producing any undesirable biological effect or in other cases interacting in a harmful manner with any other component of the pharmaceutical composition, its use is contemplated within the scope of the present disclosure.

In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the United States Food and Drug Administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopoeia (United States Pharmacopoeia, USP), the European Pharmacopoeia (European Pharmacopoeia, EP), the British Pharmacopoeia (British Pharmacopoeia) and/or the International Pharmacopoeia (International Pharmacopoeia).

Pharmaceutically acceptable excipients for making pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and/or granulating agents, surfactants and/or emulsifiers, disintegrants, adhesives, preservatives, buffers, lubricants and/or oils. Such excipients may optionally be included in the pharmaceutical preparation. According to the formulator's judgment, excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweeteners, flavoring agents and/or aromatics may be present in the composition.

General considerations in the formulation and/or manufacture of medicaments can be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (which is incorporated herein by reference in its entirety).

In some embodiments, the pharmaceutical compositions provided herein can be formulated according to conventional techniques with one or more pharmaceutically acceptable carriers or diluents and any other known adjuvants and excipients, such as those disclosed in Remington: The Science and Practice of Pharmacy 21st Edition, Lippincott Williams & Wilkins, 2005, which is incorporated herein by reference in its entirety.

The pharmaceutical compositions described herein can be administered by suitable methods known in the art. As will be appreciated by those skilled in the art, the route and/or mode of administration may depend on a variety of factors, including, for example but not limited to, the stability and/or pharmacokinetics and/or pharmacodynamics of the pharmaceutical compositions described herein.

In some embodiments, the pharmaceutical compositions described herein are formulated for parenteral administration, which includes modes of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

In some embodiments, the pharmaceutical compositions described herein are formulated for intravenous administration. In some embodiments, pharmaceutically acceptable carriers useful for intravenous administration include sterile aqueous solutions or dispersions and sterile powders for the preparation of sterile injectable solutions or dispersions.

In some embodiments, the pharmaceutical compositions described herein are formulated for subcutaneous administration. In some embodiments, the pharmaceutical compositions described herein are formulated for intramuscular administration.

The therapeutic composition must be sterile and stable under manufacturing and storage conditions generally. The composition can be formulated into solutions, dispersions, powders (e.g., lyophilized powders), microemulsions, lipid nanoparticles or other ordered structures suitable for high drug concentrations. The carrier can be a solvent or a dispersion medium, which comprises, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol and liquid polyethylene glycol, etc.), and a suitable mixture thereof. Suitable fluidity can be maintained, for example, by using a coating (e.g., lecithin), by maintaining the required particle size in the case of a dispersion and by using a surfactant. In many cases, it will be preferred to include an isotonic agent in the composition, such as sugar, polyols (e.g., mannitol, sorbitol) or sodium chloride. In some embodiments, the extended absorption of the injectable composition can be achieved by including an agent (e.g., monostearate and gelatin) that delays absorption in the composition.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.

In some embodiments, dispersions are prepared by incorporating the active compound into a sterile carrier that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying (lyophilization), which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Some examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils (e.g., olive oil), and injectable organic esters (e.g., ethyl oleate). Proper fluidity can be maintained, for example, by the use of coating materials (e.g., lecithin), by maintaining the desired particle size in the case of dispersions, and by the use of surfactants.

These compositions may also include adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. The presence of microorganisms can be prevented by sterilization procedures and by including multiple antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, etc.). It may also be desirable to include isotonic agents, such as sugar, sodium chloride, etc., in the pharmaceutical compositions described herein. In addition, the extended absorption of injectable drug forms may be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin).

The formulations of the pharmaceutical compositions described herein can be prepared by any method known in the art of pharmacology or developed thereafter. In general, such preparation methods include the steps of associating the active ingredient with a diluent or another excipient and/or one or more other auxiliary ingredients, and then, if necessary and/or desired, shaping, and/or packaging the product into desired single or multiple dose units.

Pharmaceutical compositions according to the present disclosure can be prepared, packaged and/or sold in bulk as a single unit dose and/or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of at least one RNA product produced using the systems and/or methods described herein.

The relative amounts of the one or more RNA molecules encapsulated in the LNP, the pharmaceutically acceptable excipients, and/or any additional ingredients in the pharmaceutical composition may vary depending on the subject, target cell, disease or condition to be treated, and may further depend on the route by which the composition is administered.

In some embodiments, the pharmaceutical compositions described herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art. The actual dosage level of the active ingredient (e.g., one or more RNA molecules encapsulated in lipid nanoparticles) in the pharmaceutical compositions described herein can be varied to obtain an amount of the active ingredient that is nontoxic to the patient in order to effectively achieve the desired therapeutic response of a specific patient, composition, and mode of administration. The selected dosage level will depend on a variety of pharmacokinetic factors, including the activity of the specific composition of the present disclosure employed, the route of administration, the time of administration, the excretion rate of the specific compound employed, the duration of treatment, other drugs, compounds and/or substances used in combination with the specific composition employed, the age, sex, weight, condition, general health and previous medical history of the patient treated, and similar factors known in the medical field.

A physician or veterinarian with ordinary skills in the art can easily determine and prescribe the effective amount of the desired pharmaceutical composition. For example, a physician or veterinarian can start with a dose of the active ingredient (e.g., one or more RNA molecules encapsulated in lipid nanoparticles) used in the pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dose until the desired effect is achieved. For example, the exemplary doses described in Example 7 can be used to prepare a pharmaceutically acceptable dosage form.

In some embodiments, the pharmaceutical composition is formulated (eg, for intravenous administration) to deliver a dose of about 7.2 μg to about 400 μg (or any subrange subsumed therein) of total RNA, eg, as described in Example 7.

In some embodiments, the pharmaceutical composition described herein may also include one or more additives, for example, in some embodiments, it can enhance the stability of such compositions under specific conditions. Some examples of additives may include, but are not limited to, salts, buffer substances, preservatives, and carriers. For example, in some embodiments, the pharmaceutical composition may also include a cryoprotectant (e.g., sucrose) and/or an aqueous buffer solution, in some embodiments, it may include one or more salts, including, for example, alkali metal salts or alkaline earth metal salts, for example, such as sodium salts, potassium salts, and/or calcium salts.

Exemplary formulations include, but are not limited to, those listed in Table 3.

Table 3: Exemplary pharmaceutical composition formulations

[1]: The RNA comprises a first RNA molecule encoding a NY-ESO-1 antigen, a second RNA molecule encoding a MAGE-A3 antigen, a third RNA molecule encoding a tyrosinase antigen, and a fourth RNA molecule encoding a TPTE antigen.

In some embodiments, the pharmaceutical compositions described herein may also include one or more active agents in addition to RNA (e.g., one or more RNA molecules, e.g., one or more mRNA molecules). For example, in some embodiments, the pharmaceutical composition includes an immune checkpoint inhibitor (also referred to as a "checkpoint inhibitor"). In some embodiments, an exemplary immune checkpoint inhibitor may be or include an immune checkpoint inhibitor indicated for the treatment of cancer (e.g., melanoma), including, for example, but not limited to, PD-1 inhibitors, PDL-1 inhibitors, CTLA4 inhibitors, LAG-3, or a combination thereof. In some embodiments, the immune checkpoint inhibitor is an antibody. Checkpoint inhibitors may include, for example, but not limited to, those listed in Table 4.

Table 4: Exemplary immune checkpoint molecules and inhibitors of these checkpoint molecules

In some embodiments, the active agent that may be included in the pharmaceutical composition described herein is or includes the following: a therapeutic agent administered in a combination therapy described herein. The pharmaceutical composition described herein may be administered in combination therapy, i.e., in combination with other agents. In some embodiments, such therapeutic agents may include agents that cause exhaustion or functional inactivation of regulatory T cells. For example, in some embodiments, the combination therapy may include a provided pharmaceutical composition and at least one immune checkpoint inhibitor.

In some embodiments, the pharmaceutical compositions described herein may be administered in conjunction with radiation therapy and/or autologous peripheral stem cell or bone marrow transplantation.

In some embodiments, the pharmaceutical compositions described herein may be combined with a checkpoint inhibitor (e.g., an inhibitor of PD-1, PD-L1, CTLA4, and/or its associated pathways). In some embodiments, the checkpoint inhibitor may include ipilimumab, nivolumab, pembrolizumab, or a combination thereof.

In some embodiments, the pharmaceutical compositions described herein may be combined with a signal transduction inhibitor. In some embodiments, the signal transduction inhibitor may include a BRAF inhibitor (e.g., vemurafenib or dabrafenib). In some embodiments, the signal transduction inhibitor may include a MEK inhibitor.

In some embodiments, the pharmaceutical compositions described herein can be combined with intralesional therapy (eg, talimogenelaherparepvec).

In some embodiments, the pharmaceutical compositions described herein can be combined with a cytotoxic therapy (eg, IL-2, dacarbazine, carboplatin/paclitaxel, nab-paclitaxel).

In some embodiments, the pharmaceutical compositions described herein can be frozen to allow for long-term storage.

Although the descriptions of pharmaceutical compositions provided herein are primarily directed to pharmaceutical compositions suitable for administration to humans, it will be appreciated by those skilled in the art that such compositions are generally suitable for administration to animals of all kinds. Modifications of pharmaceutical compositions suitable for administration to humans to render the compositions suitable for administration to a variety of animals are well understood, and a veterinary pharmacologist of ordinary skill can design and/or perform such modifications with only ordinary (if any) experimentation.

To ensure suitable quality of the components useful in the pharmaceutical compositions described herein (e.g., one or more RNA molecules collectively encoding (i) New York Esophageal Squamous Cell Carcinoma (NY-ESO-1) antigen, (ii) Melanoma-Associated Antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof), one or more quality assessments and/or standards (e.g., RNA quality assessments) may be performed and/or monitored.

The present disclosure provides, inter alia, methods of characterizing one or more characteristics of one or more RNA molecules, or compositions thereof, wherein the one or more RNA molecules encode part or all of an antibody agent.

In some embodiments, RNA integrity assessment of one or more RNA molecules (e.g., in some embodiments, a pharmaceutical composition comprising one or more RNA molecules collectively encoding a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof) can be performed by adaptation of a capillary gel electrophoresis assay.

Additionally or alternatively, in some embodiments, the RNA ratio of a pharmaceutical composition comprising one or more RNA molecules each encoding a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof can be measured by droplet digital PCR.

Additionally or alternatively, in some embodiments, residual DNA template and residual dsRNA are measured as in-process controls according to acceptance criteria for drug substance intermediate levels to ensure the quality of the individual RNAs prior to mixing into the drug substance (e.g., prior to mixing two or more RNA molecules each encoding a different TAA or TAA combination (e.g., NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, TPTE antigen, or a combination thereof)).

Additionally or alternatively, in some embodiments, residual host cell DNA and/or host cell proteins can be measured in a composition comprising RNA molecules.

V. Patient Groups

The technology provided herein can be used to treat diseases or conditions associated with cancer.In some embodiments, the technology provided herein can be used to treat diseases and conditions associated with epithelial cancers.

One type of cancer that the technology described herein can be used to treat is melanoma. Melanoma is a malignant tumor of melanocytes. Melanoma can occur in the skin, but it can also originate from mucosal surfaces or be located at other sites to which neural crest cells migrate, including the uveal tract. (Kuk et al. 2016, which is incorporated herein by reference in its entirety). Mucosal and uveal melanomas differ significantly from skin melanomas in incidence, prognostic factors, molecular characteristics, and treatment (van der Kooii et al. 2019, which is incorporated herein by reference in its entirety).

In the United States, it is estimated that approximately 106,110 patients will be diagnosed with skin melanoma in 2021, and there will be approximately 7,180 deaths (Siegel et al. 2021, which is incorporated herein by reference in its entirety). Although the age-standardized incidence of melanoma is lower when compared to non-melanoma skin cancer (3.4/100,000 vs. 11.0/100,000 in 2020, respectively), it has a high mortality rate (Globocan 2020, Coricovac et al. 2018, each of which is incorporated herein by reference in its entirety). Invasive melanoma accounts for approximately 1% of skin cancers, but causes the most deaths caused by skin cancer (ACS 2021, which is incorporated herein by reference in its entirety).

The outcome of melanoma depends on the stage of presentation. For patients with early-stage disease (e.g., localized disease), 5-year survival is about 99% of patients, and for patients in the regional stage (e.g., spread to the lymph nodes), 5-year survival is 66% of patients. However, for patients with distant disease, 5-year survival is only about 27% (SEER CRS 2021; Swetter et al. 2021, each of which is incorporated herein by reference in its entirety).

In some embodiments, the technology provided herein can be used to treat melanoma. In some embodiments, the technology provided herein can be used to treat skin melanoma. In some embodiments, the technology provided herein can be used to treat advanced cancer (e.g., melanoma). Some examples of advanced cancer include but are not limited to phase II, phase III, or phase IV. In some embodiments, the technology provided herein can be used to treat diseases or conditions associated with phase IIIB, phase IIIC, or phase IV melanoma. In some embodiments, cancer is completely excised. In some embodiments, there is no evidence of disease (e.g., cancer). In some embodiments, cancer is completely excised and there is no evidence of disease.

In some embodiments, the technology provided herein can be used to treat patients (e.g., adult patients) with metastatic melanoma. In some embodiments, the technology provided herein can be used to treat patients (e.g., adult patients) with unresectable melanoma, for example, in some embodiments where surgical resection may lead to severe morbidity. In some embodiments, the technology provided herein can be used to treat patients (e.g., adult patients) with locally advanced melanoma. In addition or alternatively, in some embodiments, the cancer in such patients may progress after treatment, or such cancer patients may not have satisfactory alternative treatments. In some embodiments, patients receiving treatment described herein may have received other cancer treatments, such as, but not limited to, chemotherapy.

In some embodiments, the technology provided herein can be used to treat advanced melanoma.In some embodiments, the technology provided herein can be used to treat patients with unresectable melanoma who have experienced checkpoint inhibitors (CPIs).

In some embodiments, the technology provided herein can be used to treat patients diagnosed with cancer before the administration time of the pharmaceutical composition, but wherein the patient is classified as no evidence of disease (NED) at the time of administration. In some embodiments, patients classified as NED at the time of administration are patients whose melanoma has been completely removed (e.g., by surgery). In some embodiments, patients classified as NED at the time of administration are patients who have previously been diagnosed with clinical phase 3 or 4 melanoma (or pathological phase 3 or 4 melanoma) and whose melanoma has been completely removed (e.g., by surgery). In some embodiments, patients classified as NED at the time of administration are patients whose melanoma has been completely removed and will continue to receive adjuvant therapy. In some embodiments, patients classified as NED at the time of administration are patients who have previously been diagnosed with clinical phase 3 or 4 melanoma (or pathological phase 3 or 4 melanoma) and whose melanoma has been completely removed and will continue to receive adjuvant therapy. Without wishing to be bound by a particular theory, in some embodiments, "no evidence of disease" is determined by applying RECIST criteria, such as RECIST 1.1 criteria or Immune-related Response Evaluation Criteria in Solid Tumors (irRECIST) criteria.

For the sake of clarity, patients classified as NED at the time of administration are different from patients classified as suffering from "unmeasurable disease". Patients suffering from "unmeasurable disease" mean that there is disease evidence, but cannot be reliably measured according to RECIST criteria, such as Eisenhauer et al. "New response evaluation criteria in solid tumours: Revised RECIST guideline (version 1.1)" European Journal of Cancer (2009) 45: 228-247 (the entire content of which is incorporated herein by reference for the purposes described herein) RECIST1.1 criteria described in the lesions. Some examples of lesions that are considered to be unmeasurable lesions include, but are not limited to, bone lesions, pleural effusions, ascites, "complex irregular" lesions in tissues or organs. In other words, patients suffering from "unmeasurable disease" mean that according to RECIST criteria, such as RECIST1.1 criteria discussed above, patients with tumor lesions that are not considered to be "measurable" lesions. Thus, the difference between non-measurable disease and NED is that the former means that the disease is present but cannot be measured, whereas the latter (NED) means that the disease is not present and is therefore not assessable and clearly not measurable.

Thus, administering a pharmaceutical composition as described herein to a NED patient may seem counterintuitive. However, the present disclosure recognizes that a patient may be determined to be cancer-free or in remission, but the cancer may reappear. Thus, the present disclosure provides insight that such a patient may benefit from receiving a pharmaceutical composition as described herein because it may, for example, enhance the patient's immune response to cancer. Enhancing the patient's immune response to cancer may allow the patient's body to attack cancer cells, e.g., cancer cells that are not detected or that are developing.

In some embodiments, the technology provided herein can be used to treat melanoma patients with measurable disease.

In some embodiments, the technology provided herein can be used to treat melanoma patients with non-measurable disease.

In some embodiments, the technology provided herein can be used to treat patients in remission.

In some embodiments, the object of administering the pharmaceutical composition described herein may have received a previous anticancer treatment. Some examples of previous anticancer treatments include, but are not limited to, chemotherapy, interferon and interleukin, monoclonal antibodies, protein kinase inhibitors, radiotherapy, immune checkpoint inhibitors, or combinations thereof. For example, in some embodiments, the object of administering the pharmaceutical composition described herein may have received immune checkpoint inhibitors, but has not experienced tumor regression. In another example, in some embodiments, the object of administering the pharmaceutical composition described herein may have received immune checkpoint inhibitors and experienced tumor regression. Some examples of such immune checkpoint inhibitors include, but are not limited to, PD-1 inhibitors, PDL-1 inhibitors, CTLA-4 inhibitors, or combinations thereof. In some embodiments, immune checkpoint inhibitors are antibodies (e.g., but not limited to, ipilimumab and nivolumab). Some other examples of checkpoint inhibitors are included in Table 4 above or in Example 8.

In some embodiments, patients who meet one or more of the disease-specific inclusion criteria as described in Example 12 are suitable for treatment described herein (e.g., receive a provided pharmaceutical composition as a monotherapy or as part of a combination therapy). In some embodiments, such patients who are administered a treatment described herein may also meet one or more of the other inclusion criteria as described in Example 12.

In some embodiments, a cancer patient having melanoma but meeting one or more of the exclusion criteria as described in Example 13 is not administered a treatment described herein.

VI. Readout of Patients Administered with Pharmaceutical Compositions

In some embodiments, administration of a pharmaceutical composition comprising one or more RNA molecules that collectively encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof induces an immune response. In some embodiments, the methods described herein further comprise determining the level of immune response in a patient (e.g., a patient classified as having no evidence of disease at the time of administration) administered the pharmaceutical composition. For example, in some embodiments, determining the level of immune response in a patient occurs before and after administration of the pharmaceutical composition.

Some non-limiting examples of methods for determining the level of immune response in a patient are described in Examples 1 to 3. For example, in some embodiments, enhanced glucose consumption after administration of a pharmaceutical composition can be utilized by performing a [18F]-fluoro-2-deoxy-2-d-glucose (FDG)-positron emission tomography (PET)/computed tomography (CT) scan of the spleen after administration of the pharmaceutical composition. Without wishing to be bound by theory, (FDG)-(PET)/(CT) scans are used to indicate targeted lymphoid tissue-resident immune cells and at least transient activation of lymphoid tissue-resident immune cells. In some embodiments, as described in Example 1, an interferon-γ enzyme-linked immunosorbent spot (ELISpot) assay is used to determine the level of immune response in a patient. In some embodiments, positron emission tomography (PET), computed tomography (CT) scans, magnetic resonance imaging (MRI), or a combination thereof are used to measure the level of metabolic activity in a patient's spleen. In some embodiments, positron emission tomography (PET) and computed tomography (CT) scans are used to measure the level of metabolic activity in a patient's spleen. In some embodiments, positron emission tomography (PET) and magnetic resonance imaging (MRI) are used to measure the level of metabolic activity in the patient's spleen.

In some embodiments, determining the level of immune response in a patient (e.g., a patient classified as having no evidence of disease at the time of administration) after receiving the pharmaceutical composition comprises comparing the level of immune response in the patient with the level of immune response in a second patient to whom the pharmaceutical composition has been administered. In some embodiments, the second patient was diagnosed with cancer prior to the time of administration and was classified as having evidence of disease at the time of administration.

In some embodiments, the pharmaceutical composition induces an immune response level in a patient (e.g., a patient classified as having no evidence of disease at the time of administration) that is comparable to the immune response level in a second patient to whom the pharmaceutical composition has been administered. In some embodiments, the second patient has previously been diagnosed with cancer and is classified as having evidence of disease at the time of administration. In some embodiments, if the immune response level in a patient is different from the immune response level in the second patient, the immune response level in the patient is comparable if it differs by less than 20%, less than 15%, less than 10%, or less than 5%.

In some embodiments, the level of immune response in a patient (e.g., a patient classified as having no evidence of disease at the time of administration) after administration of the pharmaceutical composition is compared to the level of immune response in a patient before administration of the pharmaceutical composition. For example, in some embodiments, the level of immune response in a patient (e.g., a patient classified as having no evidence of disease at the time of administration) after administration of the pharmaceutical composition is increased compared to the level of immune response in a patient before administration of the pharmaceutical composition. In some embodiments, the level of immune response in a patient (e.g., a patient classified as having no evidence of disease at the time of administration) after administration of the pharmaceutical composition is maintained compared to the level of immune response in a patient before administration of the pharmaceutical composition.

In some embodiments, the level of immune response is a de novo immune response induced by a pharmaceutical composition. In some embodiments, a de novo immune response is an immune response that occurs in response to a pharmaceutical composition. In some embodiments, a de novo immune response does not include background or pre-existing levels of an immune response.

In some embodiments, administration of a pharmaceutical composition comprising one or more RNA molecules that co-encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof to a patient (e.g., a patient classified as having no evidence of disease at the time of administration) induces an adaptive immune response. For example, in some embodiments, the immune response in the patient is a T cell response, wherein the T cell response includes a CD4 ⁺ and/or CD8 ⁺ T cell response. In some embodiments, administration of a pharmaceutical composition comprising one or more RNA molecules that co-encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof to a patient (e.g., a patient classified as having no evidence of disease at the time of administration) induces CD4 ⁺ and/or CD8 ⁺ T cell immunity.

In some embodiments, the methods described herein include determining the level of immune response in a patient by measuring the amount of one or more cytokines in the patient's plasma. For example, as described in Example 2, the presence and/or amount of one or more cytokines (e.g., IFN-α, IFN-γ, interleukin (IL) -6, IFN--induced protein (IP) -10, IL-12p70 subunit, or a combination thereof) associated with an immune response can be used to determine the level of immune response in a patient. In some embodiments, measuring the amount of one or more cytokines in the patient's plasma occurs before and after administering the pharmaceutical composition.

In some embodiments, the methods described herein include measuring the number of cancer lesions in a patient. For example, in some embodiments, the methods described herein include measuring the number of cancer lesions in a patient before and after administering the pharmaceutical composition. In some embodiments, administering the pharmaceutical composition to a patient (e.g., a patient classified as having no evidence of disease at the time of administration) reduces the number of cancer lesions compared to the number of cancer lesions in the patient before administration of the pharmaceutical composition comprising one or more RNA molecules encoding a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof.

In some embodiments, the methods described herein include measuring the number of T cells induced by the pharmaceutical composition in the patient. For example, in some embodiments, the methods described herein include measuring the number of T cells induced by the pharmaceutical composition in the patient at multiple time points after administering the pharmaceutical composition. In another example, the methods described herein include measuring the number of T cells induced by the pharmaceutical composition in the patient after administering the first dose of the pharmaceutical composition and after administering the second dose of the pharmaceutical composition. In some embodiments, the number of T cells induced by the pharmaceutical composition in the patient after administering the second dose of the pharmaceutical composition is greater than after administering the first dose of the pharmaceutical composition.

In some embodiments, the methods described herein include determining the phenotype of T cells induced by the pharmaceutical composition in a patient after administering the pharmaceutical composition. For example, in some embodiments, after administering the pharmaceutical composition, at least a portion of the T cells induced by the pharmaceutical composition in the patient have a T helper-1 phenotype. In some embodiments, the phenotype of T cells induced by the pharmaceutical composition in the patient has a PD1 ⁺ effector memory phenotype. In some embodiments, the phenotype of T cells induced by the pharmaceutical composition in the patient has a T-helper-1 and PD1 ⁺ effector memory phenotype.

In some embodiments, the methods described herein include, for patients classified as having evidence of disease, measuring the size of one or more cancer lesions in the patient. For example, in some embodiments, the methods described herein include measuring the size of one or more cancer lesions in the patient before and after administering the pharmaceutical composition. In some embodiments, administering the pharmaceutical composition to a patient (e.g., a patient classified as having no evidence of disease at the time of administration) maintains or reduces the size of one or more cancer lesions compared to the size of one or more cancer lesions in the patient before administering the pharmaceutical composition comprising one or more RNA molecules encoding NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, TPTE antigen, or a combination thereof. In other words, after administering the pharmaceutical composition described herein, the size of one or more cancer lesions does not increase.

In some embodiments, the methods described herein include monitoring progression-free survival duration for patients classified as having disease evidence. In some embodiments, the methods described herein include comparing the progression-free survival duration of the patient with a reference progression-free survival duration. In some embodiments, the reference progression-free survival duration is the average progression-free survival duration of a plurality of comparable patients who have not received the pharmaceutical composition described herein. In some embodiments, the progression-free survival duration of the patient is longer in time compared to the reference progression-free survival duration.

In some embodiments, the methods described herein include, for patients classified as having disease evidence, measuring the duration of disease stabilization. In some embodiments, disease stabilization is determined by applying irRECIST or RECIST 1.1 standards. In some embodiments, the methods described herein include comparing the duration of disease stabilization of the patient with the duration of disease stabilization of the reference disease. In some embodiments, the duration of disease stabilization of the reference disease is the average duration of disease stabilization of a plurality of comparable patients who have not received the pharmaceutical composition. In some embodiments, compared with the duration of disease stabilization of the reference disease, the patient applying the pharmaceutical composition described herein shows an increased duration of disease stabilization.

In some embodiments, the methods described herein include measuring the tumor responsiveness duration for patients classified as having disease evidence. In some embodiments, tumor responsiveness is determined by applying irRECIST or RECIST 1.1 standards. In some embodiments, the methods described herein include comparing the patient's tumor responsiveness duration with a reference tumor responsiveness duration. In some embodiments, the reference tumor responsiveness duration is the average tumor responsiveness duration of a plurality of comparable patients who have not received the pharmaceutical composition. In some embodiments, compared with the reference tumor responsiveness duration, the patient applying the pharmaceutical composition described herein shows an improved tumor responsiveness duration.

In some embodiments, the methods described herein include monitoring disease-free survival duration for patients classified as having no evidence of disease. In some embodiments, the methods described herein include comparing the disease-free survival duration of the patient with a reference disease-free survival duration. In some embodiments, the disease-free survival duration in the patient applying the pharmaceutical composition described herein is shown to be longer in time compared to the reference disease-free survival duration. In some embodiments, the reference disease-free survival duration is the average disease-free survival duration of a plurality of comparable patients who have not received the pharmaceutical composition. In some embodiments, the patient applying the pharmaceutical composition described herein shows an improved disease-free survival duration compared to the reference disease-free survival duration.

In some embodiments, the method described herein includes, for patients classified as having no evidence of disease, measuring the duration to disease recurrence. In some embodiments, disease recurrence is determined by applying irRECIST or RECIST 1.1 standards. In some embodiments, the method described herein includes comparing the duration to disease recurrence of the patient with the duration to disease recurrence with reference to disease recurrence. In some embodiments, the duration to disease recurrence with reference to the average duration to disease recurrence of multiple comparable patients who do not receive the pharmaceutical composition. In some embodiments, compared with the duration to disease recurrence with reference to, the patient applying the pharmaceutical composition described herein shows an increased duration to disease recurrence.

VII. Treatment

In some embodiments, the pharmaceutical compositions described herein can be taken up by target cells (eg, dendritic cells) for translation of antigen-encoding RNA, thereby inducing CD4 ⁺ and CD8 ⁺ T cell immunity against the antigen.

Therefore, another aspect of the present disclosure relates to methods of using the pharmaceutical compositions described herein. For example, one aspect provided herein is such a method, which includes administering the provided pharmaceutical composition to an object suffering from cancer. In some embodiments, the provided pharmaceutical composition is administered by intravenous injection or infusion. Some examples of cancer include, but are not limited to, epithelial cancers, including, but not limited to, melanoma (e.g., skin melanoma, stage IIIB, stage IIIC, or stage IV melanoma).

Dosing Regimens: Those skilled in the art will appreciate that cancer therapeutics are typically administered using a varying range of pharmaceutical compositions that may be administered over a dosing cycle.

In some embodiments, a pharmaceutical composition described herein is administered in eight doses within 64 days from the first administration, eg, using a prime and boost regimen.

In some embodiments, a pharmaceutical composition described herein is administered in six doses within 43 days from the first administration, eg, using a prime and boost regimen.

In some embodiments, a pharmaceutical composition described herein is administered monthly after an initial dosing cycle (eg, a prime and boost regimen) is completed.

In some embodiments, the pharmaceutical compositions described herein are administered in one or more dosing cycles.

In some embodiments, a dosing cycle is at least 7 days or more (including, for example, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, at least 29 days, at least 30 days, at least 40 days, at least 50 days or at least 60 days). In some embodiments, a dosing cycle is at least 28 days. In some embodiments, a dosing cycle is at least 35 days. In some embodiments, a dosing cycle is at least 42 days. In some embodiments, a dosing cycle is at least 49 days. In some embodiments, a dosing cycle is at least 56 days. In some embodiments, a dosing cycle is at least 63 days.

In some embodiments, a dosing cycle may involve multiple doses, for example, according to such a pattern: for example, doses may be administered regularly within the cycle, or doses may be administered every 6 days, every 7 days, every 8 days, every 9 days, every 10 days, every 12 days, or every 14 days within the cycle. In some embodiments, a dosing cycle may involve at least 2 doses, including, for example, at least 3 doses, at least 4 doses, at least 5 doses, at least 6 doses, at least 7 doses, at least 8 doses or more. In some embodiments, a dosing cycle may involve up to 8 doses, which may be administered weekly, every two weeks, or a combination thereof.

In some embodiments, multiple cycles can be applied. For example, in some embodiments, at least 2 cycles (including, for example, at least 3 cycles, at least 4 cycles, at least 5 cycles, at least 6 cycles, at least 7 cycles, at least 8 cycles, at least 9 cycles, at least 10 cycles or more) can be applied. In some embodiments, the number of dosing cycles to be applied can vary with the type of treatment (for example, single treatment vs. combination therapy). In some embodiments, at least 2 dosing cycles can be applied. In some embodiments, the first dosing cycle can be different from the second dosing cycle. In some embodiments, the first dosing cycle can include 6 to 8 weekly and/or biweekly dosages, and the second dosing cycle after the first dosing cycle can include at least one monthly dosage.

In some embodiments, there may be a "resting period" between cycles; in some embodiments, there may be no resting period between cycles. In some embodiments, there may be a resting period between cycles sometimes and not sometimes.

In some embodiments, the length of the resting period can range from a few days to a few months. For example, in some embodiments, the length of the resting period can be at least 3 days or longer, including, for example, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days or longer. In some embodiments, the length of the resting period can be at least 1 week or longer, including, for example, at least 2 weeks, at least 3 weeks, at least 4 weeks or longer.

Dosage: The dosage of the pharmaceutical compositions described herein can vary with many factors, including, for example, but not limited to, the weight of the subject to be treated, the type of cancer and/or the stage of cancer, and/or whether it is a monotherapy or a combination therapy. In some embodiments, a dosing cycle involves administering a set number and/or pattern of doses. For example, in some embodiments, the pharmaceutical compositions described herein are administered in at least one dose/dosing cycle, including, for example, at least two doses/dosing cycles, at least three doses/dosing cycles, at least four doses/dosing cycles, or more.

In some embodiments, the dosing cycle involves, for example, a cumulative dose set within a specific time period and optionally administered by multiple doses, which can be administered, for example, at set intervals and/or according to a set pattern. In some embodiments, the cumulative dose set can be administered by multiple doses at set intervals, so that there is at least some time overlap in the biological and/or pharmacokinetic effects produced by such multiple doses on target cells or on the treated object. In some embodiments, the cumulative dose set can be administered by multiple doses at set intervals, so that the biological and/or pharmacokinetic effects produced by such multiple doses on target cells or on the treated object can be additive. By way of example only, in some embodiments, the set cumulative dose of X mg can be administered by two doses and each dose is X/2 mg, wherein the time of administration of such two doses is close enough so that the biological and/or pharmacokinetic effects produced by each X/2 mg dose on the target cell or on the treated object can be additive.

In some embodiments, each dose or cumulative dose (e.g., for intravenous administration) is administered at a level such that one or more RNA molecules collectively encoding a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof are expected to reach a level (e.g., plasma level and/or tissue level) high enough for translation and antigen presentation in antigen presenting cells (e.g., dendritic cells or immature dendritic cells) that induce CD4 ⁺ and CD8 ⁺ T cell immunity to the one or more antigens throughout the dosing cycle.

In some embodiments, each dose is about 7.2 μg to about 400 μg (eg, any subrange herein) of total RNA.

In some embodiments, the methods provided herein include dose escalation. Exemplary methods including dose escalation are described, for example, in WO2018/0077942.

In some embodiments, the methods provided herein include 7 dose escalation cohorts (3+3 design) and 3 expansion cohorts. For example, Table 5 provides an exemplary dosing regimen.

Table 5: Exemplary Dosing Regimens

In some embodiments, administration may be adjusted based on the response of the subject being treated. For example, in some embodiments, if one or more parameters used for safety pharmacology assessment indicate that the previous dose may not meet the medical safety requirements according to the physician, administration may involve the administration of a higher dose followed by a lower dose. In some embodiments, dose escalation may be performed at one or more of the levels shown in Table 5 of Example 7; in some embodiments, dose escalation may involve the administration of at least one lower dose from Table 5 followed by at least one higher dose from Table 5. Without wishing to be bound by any particular theory, the present disclosure provides, in particular, such insights: a pharmaceutically guided dose escalation (PGDE) method may be applied to determine the appropriate dose of the pharmaceutical composition described herein. An exemplary dose escalation study is provided in Example 7.

Also provided herein are methods for determining a dosing regimen for a pharmaceutical composition comprising one or more RNA molecules that collectively encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof. For example, in some embodiments, such methods include the following steps: (A) administering a pharmaceutical composition (e.g., a pharmaceutical composition described herein) to a subject having melanoma or a subject that has been classified as having no evidence of disease under a predetermined dosing regimen; (B) regularly monitoring or measuring the subject for evidence of disease (e.g., tumor lesion size and/or metastasis) over a period of time; (C) evaluating the dosing regimen based on the monitoring or measurement results and/or outcomes. For example, if a decrease in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is not treatment-related, the dose and/or dosing frequency may be increased; or if a decrease in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is treatment-related but shows adverse effects (e.g., toxic effects) in the subject, the dose and/or dosing frequency may be reduced. If the reduction in tumor size following administration of a pharmaceutical composition (eg, a pharmaceutical composition described herein) is therapeutically relevant and does not indicate adverse effects (eg, toxic effects) in the subject, no changes are made to the dosing regimen.

In some embodiments, such a method of determining a dosing regimen for a pharmaceutical composition can be performed in a group of animal subjects (e.g., mammalian non-human subjects) each bearing a human melanoma xenograft tumor, the pharmaceutical composition comprising one or more RNA molecules collectively encoding a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof. In some such embodiments, if less than 30% of the animal subjects exhibit a reduction in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) and/or the degree of reduction in tumor size exhibited by the animal subjects is not therapeutically relevant, the dose and/or dosing frequency can be increased; or if the reduction in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is therapeutically relevant, but significant adverse effects (e.g., toxic effects) are exhibited in at least 30% of the animal subjects, the dose and/or dosing frequency can be reduced. If the reduction in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is therapeutically relevant and no significant adverse effects (e.g., toxic effects) are exhibited in the animal subjects, no changes are made to the dosing regimen.

Although the dosing regimens (e.g., dosing schedules and/or dosages) provided herein are primarily suitable for administration to humans, it will be appreciated by those skilled in the art that equivalent dosage regimens can be determined for administration to all species of animals. A veterinary pharmacologist of ordinary skill can design and/or perform such determinations using only ordinary experimentation, if any.

Monotherapy: In some embodiments, the pharmaceutical compositions described herein may be administered to a patient as a monotherapy.

Combination Therapies: The present disclosure provides, among other things, the insight that the ability of the pharmaceutical compositions described herein to induce CD4 ⁺ and CD8 ⁺ T cell immunity against antigens encoded by one or more RNA molecules can enhance the cytotoxic effects of chemotherapy and/or other anti-cancer therapies (e.g., immune checkpoint inhibitors), the pharmaceutical compositions comprising one or more RNA molecules that collectively encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof. In some embodiments, such combination therapies can prolong progression-free survival and/or overall survival, for example, relative to individual therapies administered alone and/or relative to another suitable reference. Thus, in some embodiments, the pharmaceutical compositions described herein can be administered to patients with cancer (e.g., melanoma) in combination with other anti-cancer agents.

Without wishing to be bound by a particular theory, the present disclosure observes that certain immune checkpoint inhibitors, such as, for example, PD-1 inhibition, PDL-1 inhibition, and CTLA4 inhibition, act synergistically with the pharmaceutical compositions described herein when administered as a combination therapy to patients with tumors experiencing CPI.

The present disclosure provides, inter alia, the insight that the pharmaceutical compositions described herein may be particularly useful and/or effective when administered to patients with no evidence of disease at the time of the first administration, thereby demonstrating that the pharmaceutical compositions induce T cell immunity even in the absence of detectable tumors.

In some embodiments, the pharmaceutical composition provided can be applied as part of a combined treatment comprising such a pharmaceutical composition and an immune checkpoint inhibitor. Therefore, in some embodiments, the pharmaceutical composition provided can be applied to an object with cancer (e.g., melanoma) that has received an immune checkpoint inhibitor or chemotherapeutic agent, or an object that has received an immune checkpoint inhibitor or chemotherapeutic agent and is classified as having no evidence of disease. In some embodiments, the pharmaceutical composition provided can be co-administered with an immune checkpoint inhibitor to an object with cancer (e.g., melanoma) or has been classified as having no evidence of disease. In some embodiments, the pharmaceutical composition provided and the immune checkpoint inhibitor can be applied simultaneously or sequentially. For example, in some embodiments, the first dose of an immune checkpoint inhibitor can be applied after the pharmaceutical composition provided (e.g., at least 30 minutes thereafter). In some embodiments, immune checkpoint inhibitors and the pharmaceutical composition provided are administered concomitantly.

For example, in some embodiments, the immune checkpoint inhibitor comprises one or more inhibitors selected from Table 4 above (see, e.g., Mafin-Acevdeo et al., J. Hematology & Oncology, 14:45 (2021), which is incorporated herein by reference in its entirety) or as described in Example 8.

Combination therapy with anti-cancer therapy comprising ipilimumab: In some embodiments, the administered therapy comprising the provided pharmaceutical composition can be co-administered or overlapped with an immune checkpoint inhibitor comprising ipilimumab. Ipilimumab blocks cytotoxic T-lymphocyte antigen-4 (CTLA-4), a key negative regulator of anti-tumor T cell responses. Blocking CTLA-4 inhibits T cell activation, thereby allowing pre-existing antigen-specific T cells to expand.

Combination therapy with anti-cancer therapy comprising nivolumab: In some embodiments, the administered therapy comprising a provided pharmaceutical composition can be co-administered or overlapped with an immune checkpoint inhibitor comprising nivolumab. Nivolumab is a monoclonal antibody that binds to the PD-1 receptor and blocks interaction with PD-L1 and PD-L2. Blocking this interaction releases PD-1-mediated suppression of immune responses (including anti-tumor T cell responses) pathways, allowing pre-existing antigen-specific T cells to expand.

Combination therapy with anti-cancer therapy comprising pembrolizumab: In some embodiments, the administered therapy comprising a provided pharmaceutical composition can be co-administered or overlapped with an immune checkpoint inhibitor comprising pembrolizumab. Pembrolizumab is a monoclonal antibody that binds to the PD-1 receptor and blocks interaction with PD-L1 and PD-L2. Blocking this interaction releases PD-1-mediated suppression of immune responses (including anti-tumor T cell responses) pathways, allowing pre-existing antigen-specific T cells to expand.

Combination therapy with anti-cancer therapy comprising cemiplimab: In some embodiments, the administered therapy comprising the provided pharmaceutical composition can be co-administered or overlapped with an immune checkpoint inhibitor comprising cemiplimab. Cemiplimab is a monoclonal antibody that binds to the PD-1 receptor and blocks interaction with PD-L1 and PD-L2. Blocking this interaction releases PD-1-mediated suppression of immune responses (including anti-tumor T cell responses) pathways, allowing pre-existing antigen-specific T cells to expand.

Efficacy Monitoring: In some embodiments, patients receiving the provided treatment may be monitored periodically during the dosing regimen to assess the efficacy of the administered treatment. For example, in some embodiments, the efficacy of the administered treatment may be assessed periodically (e.g., every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, or longer) by intra-treatment imaging.

Exemplary embodiments

Some exemplary embodiments provided below are also within the scope of the present disclosure:

Embodiment 1. A method, comprising:

Administering to a patient at least one dose of a pharmaceutical composition comprising:

(a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and

(b) lipid particles;

wherein the patient was diagnosed with cancer prior to the time of administration, but the patient was classified as having no evidence of disease at the time of administration.

Embodiment 2. The method of embodiment 1, wherein the absence of evidence of disease is determined by applying the immune-related response evaluation criteria in solid tumors (irRECIST) criteria or RECIST 1.1 criteria.

Embodiment 3. A method, comprising:

Administering at least one dose of a pharmaceutical composition to a patient suffering from cancer, wherein the pharmaceutical composition comprises:

(a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and

(b) Lipid particles.

Embodiment 4. The method of Embodiment 3, wherein the patient is classified as having no evidence of disease at the time of administration.

Embodiment 5. The method of Embodiment 3, wherein the patient is classified as having evidence of disease at the time of administration.

Embodiment 6. The method of embodiment 4 or 5, wherein the presence or absence of evidence of disease is determined by applying the immune-related response evaluation criteria in solid tumors (irRECIST) criteria or RECIST 1.1 criteria.

Embodiment 7. The method of any one of Embodiments 1 to 6, wherein the one or more RNA molecules comprise:

(i) a first RNA molecule encoding the NY-ESO-1 antigen,

(ii) a second RNA molecule encoding a MAGE-A3 antigen,

(iii) a third RNA molecule encoding a tyrosinase antigen, and

(iv) a fourth RNA molecule encoding the TPTE antigen.

Embodiment 8. The method of any one of Embodiments 1 to 7, wherein a single RNA molecule of the one or more RNA molecules encodes at least two of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen.

Embodiment 9. The method of any one of embodiments 1 to 8, wherein a single RNA molecule of the one or more RNA molecules encodes a multi-epitope polypeptide, wherein the multi-epitope polypeptide comprises at least two of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen.

Embodiment 10. The method of any one of Embodiments 1 to 9, wherein the one or more RNA molecules further comprise at least one sequence encoding a CD4+ epitope.

Embodiment 11. The method of any one of Embodiments 1 to 9, wherein the one or more RNA molecules further comprise at least one sequence encoding tetanus toxoid P2, a sequence encoding tetanus toxoid P16, or both.

Embodiment 12. The method of any one of Embodiments 1 to 11, wherein the one or more RNA molecules comprise a sequence encoding an MHC class I trafficking domain.

Embodiment 13. The method of any one of Embodiments 1 to 12, wherein the one or more RNA molecules comprise a 5' cap or a 5' cap analog.

Embodiment 14. The method of any one of Embodiments 1 to 13, wherein the one or more RNA molecules comprise a sequence encoding a signal peptide.

Embodiment 15. The method of any one of Embodiments 1 to 14, wherein the one or more RNA molecules comprise at least one non-coding regulatory element.

Embodiment 16. The method of any one of Embodiments 1 to 15, wherein the one or more RNA molecules comprise a polyadenine tail.

Embodiment 17. The method of Embodiment 16, wherein the polyadenine tail is or comprises a modified adenine sequence.

Embodiment 18. The method of any one of Embodiments 1 to 17, wherein the one or more RNA molecules comprise at least one 5' untranslated region (UTR) and/or at least one 3'UTR.

Embodiment 19. The method of embodiment 18, wherein the one or more RNA molecules comprise, in 5' to 3' order:

(i) a 5' cap or a 5' cap analog;

(ii) at least one 5′UTR;

(iii) signal peptide;

(iv) a coding region encoding at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen and the TPTE antigen;

(v) at least one sequence encoding tetanus toxoid P2, tetanus toxoid P16, or both;

(vi) a sequence encoding an MHC class I trafficking domain;

(vii) at least one 3'UTR; and

(viii) Polyadenine tail.

Embodiment 20. The method of any one of Embodiments 1 to 19, wherein the one or more RNA molecules comprise natural ribonucleotides.

Embodiment 21. The method of any one of Embodiments 1 to 20, wherein the one or more RNA molecules comprise modified or synthetic ribonucleotides.

Embodiment 22. The method of any one of Embodiments 1 to 21, wherein at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen is a full-length, non-mutated antigen.

Embodiment 23. The method of any one of Embodiments 1 to 22, wherein all of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen are full-length, non-mutated antigens.

Embodiment 24. The method of any one of Embodiments 1 to 23, wherein at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen is expressed by dendritic cells in the lymphoid tissue of the patient.

Embodiment 25. The method of any one of Embodiments 1 to 24, wherein at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen is present in the cancer.

Embodiment 26. The method of any one of Embodiments 1 to 25, wherein the lipid particles comprise liposomes.

Embodiment 27. The method of any one of Embodiments 1 to 26, wherein the lipid particles comprise cationic liposomes.

Embodiment 28. The method of any one of Embodiments 1 to 25, wherein the lipid particles comprise lipid nanoparticles.

Embodiment 29. The method of any one of embodiments 1 to 28, wherein the lipid particles comprise N,N,N-trimethyl-2-3-dioleyloxy-1-chloropropylamine (DOTMA), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine phospholipids (DOPE), or both.

Embodiment 30. The method of any one of Embodiments 1 to 29, wherein the lipid particle comprises at least one ionizable amino lipid.

Embodiment 31. The method of any one of Embodiments 1 to 30, wherein the lipid particle comprises at least one ionizable amino lipid and a helper lipid.

Embodiment 32. The method of any one of Embodiments 31, wherein the helper lipid is or comprises a phospholipid.

Embodiment 33. The method of any one of Embodiments 31 or 32, wherein the helper lipid is or comprises a sterol.

Embodiment 34. The method of any one of Embodiments 1 to 33, wherein the lipid particle comprises at least one polymer-conjugated lipid.

Embodiment 35. The method of any one of Embodiments 1 to 34, wherein the patient is a human.

Embodiment 36. The method of any one of Embodiments 1 to 35, wherein the cancer is an epithelial cancer.

Embodiment 37. The method of any one of Embodiments 1 to 36, wherein the cancer is melanoma.

Embodiment 38. The method of Embodiment 37, wherein the melanoma is cutaneous melanoma.

Embodiment 39. The method of any one of Embodiments 1 to 38, wherein the cancer is advanced.

Embodiment 40. The method of any one of Embodiments 1 to 39, wherein the cancer is stage II, stage III, or stage IV.

Embodiment 41. The method of any one of Embodiments 1 to 40, wherein the cancer is stage IIIB, stage IIIC, or stage IV melanoma.

Embodiment 42. The method of any one of Embodiments 1 to 41, wherein the cancer is completely resected, there is no evidence of disease, or both.

Embodiment 43. The method of any one of Embodiments 1 to 42, further comprising administering to the patient a second dose of the pharmaceutical composition.

Embodiment 44. The method of any one of Embodiments 1 to 43, further comprising administering at least two doses of the pharmaceutical composition to the patient.

Embodiment 45. The method of any one of Embodiments 1 to 44, further comprising administering at least three doses of the pharmaceutical composition to the patient.

Embodiment 46. The method of embodiment 45, wherein at least one of the at least three doses is administered to the patient within 8 days of the patient having received another of the at least three doses.

Embodiment 47. The method of embodiment 45 or 46, wherein at least one of the at least three doses is administered to the patient within 15 days of the patient having received another of the at least three doses.

Embodiment 48. The method of any one of Embodiments 1 to 47, comprising administering to the patient at least 8 doses of the pharmaceutical composition within 10 weeks.

Embodiment 49. The method of embodiment 48, comprising administering to the patient a dose of the pharmaceutical composition weekly for a period of 6 weeks, and then administering a dose of the pharmaceutical composition every two weeks for a period of 4 weeks.

Embodiment 50. The method of Embodiment 48 or 49, further comprising administering to said patient monthly doses of said pharmaceutical composition after said at least 8 doses.

Embodiment 51. The method of any one of Embodiments 1 to 47, comprising administering to said patient a dose of said pharmaceutical composition weekly for a period of 7 weeks.

Embodiment 52. The method of embodiment 51, further comprising administering a dose of the pharmaceutical composition to the patient every three weeks.

Embodiment 53. The method of any one of Embodiments 1 to 52, wherein the first dose and/or the second dose is 5 μg to 500 μg of total RNA.

Embodiment 54. The method of any one of Embodiments 1 to 53, wherein the first dose and/or the second dose is 7.2 μg to 400 μg total RNA.

Embodiment 55. The method of any one of Embodiments 1 to 54, wherein the first dose and/or the second dose is 10 μg to 20 μg of total RNA.

Embodiment 56. The method of any one of Embodiments 1 to 55, wherein the first dose and/or the second dose is about 14.4 μg total RNA.

Embodiment 57. The method of any one of Embodiments 1 to 56, wherein the first dose and/or the second dose is about 25 μg of total RNA.

Embodiment 58. The method of any one of Embodiments 1 to 54, wherein the first dose and/or the second dose is about 50 μg of total RNA.

Embodiment 59. The method of any one of Embodiments 1 to 54, wherein the first dose and/or the second dose is about 100 μg of total RNA.

Embodiment 60. The method of any one of Embodiments 1 to 59, wherein the first dose and/or the second dose is administered systemically.

Embodiment 61. The method of any one of Embodiments 1 to 60, wherein the first dose and/or the second dose is administered intravenously.

Embodiment 62. The method of any one of Embodiments 1 to 60, wherein the first dose and/or the second dose is administered intramuscularly.

Embodiment 63. The method of any one of Embodiments 1 to 60, wherein the first dose and/or the second dose is administered subcutaneously.

Embodiment 64. The method of any one of Embodiments 1 to 63, wherein the pharmaceutical composition is administered as a monotherapy.

Embodiment 65. The method of any one of Embodiments 1 to 63, wherein the pharmaceutical composition is administered as part of a combination therapy.

Embodiment 66. The method of Embodiment 65, wherein the combination therapy comprises the pharmaceutical composition and an immune checkpoint inhibitor.

Embodiment 67. The method of any one of Embodiments 1 to 66, wherein the patient has previously received an immune checkpoint inhibitor.

Embodiment 68. The method of any one of Embodiments 1 to 63 and 65 to 67, further comprising administering an immune checkpoint inhibitor to the patient.

Embodiment 69. The method of any one of Embodiments 66 to 68, wherein the check point inhibitor is or comprises: a PD-1 inhibitor, a PDL-1 inhibitor, a CTLA4 inhibitor, a Lag-3 inhibitor, or a combination thereof.

Embodiment 70. The method of any one of Embodiments 66 to 69, wherein the check point inhibitor is or comprises an antibody.

Embodiment 71. The method of any one of Embodiments 66 to 70, wherein the checkpoint inhibitor is or comprises an inhibitor listed in Table 4 herein.

Embodiment 72. The method of any one of Embodiments 66 to 71, wherein the checkpoint inhibitor is or comprises ipilimumab, nivolumab, pembrolizumab, avelumab, cemiplimab, atezolizumab, durvalumab, or a combination thereof.

Embodiment 73. The method of any one of Embodiments 66 to 72, wherein the checkpoint inhibitor is or comprises ipilimumab.

Embodiment 74. The method of any one of Embodiments 66 to 72, wherein the checkpoint inhibitors are or comprise ipilimumab and nivolumab.

Embodiment 75. The method of any one of Embodiments 1 to 74, wherein the pharmaceutical composition induces an immune response in the patient.

Embodiment 76. The method of any one of Embodiments 1 to 76, further comprising determining the level of immune response in the patient.

Embodiment 77. The method of Embodiment 76, which compares the level of immune response in the patient with the level of immune response in a second patient who has been administered the pharmaceutical composition, wherein the second patient was diagnosed with cancer prior to the time of administration and was classified as having evidence of disease at the time of administration.

Embodiment 78. The method of Embodiment 77, wherein the pharmaceutical composition induces a level of immune response in the patient that is comparable to a level of immune response in a second patient to whom the pharmaceutical composition has been administered, the second patient having been previously diagnosed with cancer and classified as having evidence of disease at the time of administration.

Embodiment 79. The method of any one of Embodiments 75 to 78, wherein the level of the immune response is a de novo immune response induced by the pharmaceutical composition.

Embodiment 80. The method of any one of Embodiments 1 to 79, further comprising determining the level of immune response in the patient before and after administration of the pharmaceutical composition.

Embodiment 81. The method of Embodiment 80, which compares the level of immune response in the patient after administration of the pharmaceutical composition with the level of immune response in the patient before administration of the pharmaceutical composition.

Embodiment 82. The method of Embodiment 81, wherein the level of immune response in the patient is increased after administration of the pharmaceutical composition compared to the level of immune response in the patient before administration of the pharmaceutical composition.

Embodiment 83. The method of Embodiment 81, wherein the level of immune response in the patient is maintained after administration of the pharmaceutical composition compared to the level of immune response in the patient before administration of the pharmaceutical composition.

Embodiment 84. The method of any one of Embodiments 75 to 83, wherein the immune response in the patient is an adaptive immune response.

Embodiment 85. The method of any one of Embodiments 75 to 84, wherein the immune response in the patient is a T cell response.

Embodiment 86. The method of Embodiment 85, wherein the T cell response is or comprises a CD4+ response.

Embodiment 87. The method of Embodiment 85 or 86, wherein the T cell response is or comprises a CD8+ response.

Embodiment 88. The method of any one of Embodiments 75 to 87, wherein the level of immune response in the patient is determined using an interferon-gamma enzyme-linked immunosorbent spot (ELISpot) assay.

Embodiment 89. The method of any one of Embodiments 1 to 88, further comprising measuring the level of one or more of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen in the patient's lymphoid tissue.

Embodiment 90. The method of any one of Embodiments 1 to 89, further comprising measuring the level of one or more of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen in the cancer.

Embodiment 91. The method of any one of Embodiments 1 to 90, further comprising measuring the level of metabolic activity in the spleen of the patient.

Embodiment 92. The method of any one of Embodiments 1 to 91, further comprising measuring the level of metabolic activity in the patient's spleen before and after administration of the pharmaceutical composition.

Embodiment 93. The method of embodiment 91 or 92, wherein the level of metabolic activity in the patient's spleen is measured using positron emission tomography (PET), computed tomography (CT) scanning, magnetic resonance imaging (MRI), or a combination thereof.

Embodiment 94. The method of any one of Embodiments 1 to 93, further comprising measuring the amount of one or more cytokines in the patient's plasma.

Embodiment 95. The method of any one of Embodiments 1 to 94, further comprising measuring the amount of one or more cytokines in the patient's plasma before and after administration of the pharmaceutical composition.

Embodiment 96. The method of embodiment 94 or 95, wherein the one or more cytokines comprise interferon (IFN)-α, IFN-γ, interleukin (IL)-6, IFN-induced protein (IP)-10, IL-12 p70 subunit, or a combination thereof.

Embodiment 97. The method of any one of Embodiments 1 to 96, further comprising measuring the number of cancer lesions in the patient.

Embodiment 98. The method of any one of Embodiments 1 to 97, further comprising measuring the number of cancer lesions in the patient before and after administration of the pharmaceutical composition.

Embodiment 99. The method of Embodiment 98, wherein there are fewer cancer lesions in the patient after administration of the pharmaceutical composition than before administration of the pharmaceutical composition.

Embodiment 100. The method of any one of Embodiments 1 to 99, further comprising measuring the number of T cells induced by the pharmaceutical composition in the patient.

Embodiment 101. The method of any one of Embodiments 1 to 100, further comprising measuring the number of T cells induced by the pharmaceutical composition in the patient at multiple time points after administration of the pharmaceutical composition.

Embodiment 102. The method of any one of Embodiments 1 to 101, further comprising measuring the number of T cells induced by the pharmaceutical composition in the patient after administration of the first dose of the pharmaceutical composition and after administration of the second dose of the pharmaceutical composition.

Embodiment 103. The method of Embodiment 102, wherein the number of T cells induced by the pharmaceutical composition in the patient is greater after administration of the second dose of the pharmaceutical composition than after administration of the first dose of the pharmaceutical composition.

Embodiment 104. The method of any one of Embodiments 1 to 103, further comprising determining the phenotype of T cells induced by the pharmaceutical composition in the patient after administration of the pharmaceutical composition.

Embodiment 105. The method described in Embodiment 104, wherein at least a portion of the T cells induced by the pharmaceutical composition in the patient have a T helper-1 phenotype.

Embodiment 106. The method described in Embodiment 104 or 105, wherein the T cells induced in the patient by the pharmaceutical composition comprise T cells having a PD1+ effector memory phenotype.

Embodiment 107. The method of any one of Embodiments 3 to 106, further comprising, for patients classified as having evidence of disease, measuring the size of one or more cancer lesions.

Embodiment 108. The method of any one of Embodiments 3 to 107, further comprising, for patients classified as having evidence of disease, measuring the size of one or more cancer lesions in the patient before and after administration of the pharmaceutical composition.

Embodiment 109. The method of Embodiment 108, further comprising comparing the size of one or more cancer lesions in the patient before and after administration of the pharmaceutical composition.

Embodiment 110. The method of Embodiment 109, wherein the size of at least one cancer lesion in the patient after administration of the pharmaceutical composition is equal to or smaller than the size of the at least one cancer lesion before administration of the pharmaceutical composition.

Embodiment 111. The method of any one of Embodiments 3 to 110, further comprising monitoring progression-free survival duration for patients classified as having evidence of disease.

Embodiment 112. The method of Embodiment 111, which compares the patient's progression-free survival duration with a reference progression-free survival duration.

Embodiment 113. The method described in Embodiment 112, wherein the reference progression-free survival duration is the average progression-free survival duration of a plurality of comparable patients who have not received the pharmaceutical composition.

Embodiment 114. The method of Embodiment 112 or 113, wherein the patient's progression-free survival duration is longer in time compared to a reference progression-free survival duration.

Embodiment 115. The method of any one of Embodiments 3 to 114, further comprising measuring the duration of disease stabilization for patients classified as having evidence of disease.

Embodiment 116. The method of 115, wherein stable disease is determined by applying irRECIST or RECIST 1.1 criteria.

Embodiment 117. The method of Embodiment 115 or 116, further comprising comparing the patient's duration of disease stabilization with a reference duration of disease stabilization.

Embodiment 118. The method described in Embodiment 117, wherein the reference duration of disease stabilization is the average duration of disease stabilization of a plurality of comparable patients who have not received the pharmaceutical composition.

Embodiment 119. The method of Embodiment 118, wherein the patient exhibits an improved duration of disease stabilization compared to the reference duration of disease stabilization.

Embodiment 120. The method of any one of Embodiments 3 to 119, further comprising measuring the duration of tumor responsiveness for patients classified as having evidence of disease.

Embodiment 121. The method of 120, wherein tumor responsiveness is determined by applying irRECIST or RECIST 1.1 criteria.

Embodiment 122. The method of Embodiment 120 or 121, further comprising comparing the patient's tumor responsiveness duration with a reference tumor responsiveness duration.

Embodiment 123. The method described in Embodiment 122, wherein the reference tumor responsiveness duration is the average tumor responsiveness duration of multiple comparable patients who have not received the pharmaceutical composition.

Embodiment 124. The method of Embodiment 123, wherein the patient exhibits an improved duration of tumor responsiveness compared to the reference duration of tumor responsiveness.

Embodiment 125. The method of any one of Embodiments 1 to 106, further comprising monitoring the duration of disease-free survival for patients classified as having no evidence of disease.

Embodiment 126. The method of Embodiment 125, further comprising comparing the patient's disease-free survival duration to a reference disease-free survival duration.

Embodiment 127. The method described in Embodiment 126, wherein the reference disease-free survival duration is the average disease-free survival duration of a plurality of comparable patients who have not received the pharmaceutical composition.

Embodiment 128. The method of Embodiment 127, wherein the patient exhibits an improved disease-free survival duration compared to the reference disease-free survival duration.

Embodiment 129. The method of any one of Embodiments 1 to 106 and 125 to 128, further comprising, for patients classified as having no evidence of disease, measuring the duration to disease recurrence.

The method of embodiment 130.129, wherein disease recurrence is determined by applying irRECIST or RECIST 1.1 criteria.

Embodiment 131. The method of Embodiment 129 or 130, further comprising comparing the patient's duration to disease recurrence with a reference duration to disease recurrence.

Embodiment 132. The method described in Embodiment 131, wherein the reference duration to disease recurrence is the average duration to disease recurrence of a plurality of comparable patients who have not received the pharmaceutical composition.

Embodiment 133. The method of Embodiment 132, wherein the patient exhibits an increased duration to disease recurrence compared to the reference duration to disease recurrence.

Embodiment 134. A pharmaceutical composition for inducing an immune response against cancer in a patient, wherein the pharmaceutical composition comprises:

(a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and

(b) lipid particles;

and wherein the patient was classified as having no evidence of disease but had been previously diagnosed with cancer.

Embodiment 135. A pharmaceutical composition for treating cancer, wherein the pharmaceutical composition comprises:

(a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and

(b) lipid particles;

and wherein the patient was classified as having no evidence of disease but had been previously diagnosed with cancer.

Embodiment 136. A pharmaceutical composition for inducing an immune response against cancer in a patient, wherein the pharmaceutical composition comprises:

(a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and

(b) Lipid particles.

Embodiment 137. A pharmaceutical composition for treating cancer, wherein the pharmaceutical composition comprises:

(a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and

(b) Lipid particles.

Embodiment 138. The pharmaceutical composition of Embodiment 136 or 137, wherein the patient is classified as having no evidence of disease at the time of administration.

Embodiment 139. The pharmaceutical composition of Embodiment 136 or 137, wherein the patient is classified as having evidence of disease at the time of administration.

Embodiment 140. The pharmaceutical composition of any one of Embodiments 134 to 139, wherein the presence or absence of evidence of disease is determined by applying the immune-related response evaluation criteria in solid tumors (irRECIST) criteria or RECIST 1.1 criteria.

Embodiment 141. The pharmaceutical composition of any one of Embodiments 134 to 140, wherein the cancer is melanoma.

Embodiment 142. The pharmaceutical composition of any one of Embodiments 134 to 141, wherein the one or more RNA molecules comprise:

(i) a first RNA molecule encoding the NY-ESO-1 antigen,

(ii) a second RNA molecule encoding a MAGE-3 antigen,

(iii) a third RNA molecule encoding a tyrosinase antigen, and

(iv) a fourth RNA molecule encoding the TPTE antigen.

Embodiment 143. The pharmaceutical composition of any one of Embodiments 134 to 142, wherein a single RNA molecule of the one or more RNA molecules encodes at least two of the NY-ESO-1 antigen, the MAGE-3 antigen, the tyrosinase antigen, and the TPTE antigen.

Embodiment 144. The pharmaceutical composition of any one of Embodiments 134 to 143, wherein a single RNA molecule of the one or more RNA molecules encodes a multi-epitope polypeptide, wherein the multi-epitope polypeptide comprises at least two of the NY-ESO-1 antigen, the MAGE-3 antigen, the tyrosinase antigen, and the TPTE antigen.

Embodiment 145. The pharmaceutical composition of any one of Embodiments 134 to 144, wherein the one or more RNA molecules further comprise at least one sequence encoding a CD4+ epitope.

Embodiment 146. The pharmaceutical composition of any one of Embodiments 134 to 145, wherein the one or more RNA molecules comprise at least one sequence encoding tetanus toxoid P2, a sequence encoding tetanus toxoid P16, or both.

Embodiment 147. The pharmaceutical composition of any one of Embodiments 134 to 146, wherein the one or more RNA molecules comprise a sequence encoding an MHC class I trafficking domain.

Embodiment 148. The pharmaceutical composition of any one of Embodiments 134 to 147, wherein the one or more RNA molecules comprise a 5' cap or a 5' cap analog.

Embodiment 149. The pharmaceutical composition of any one of Embodiments 134 to 148, wherein the one or more RNA molecules comprise a sequence encoding a signal peptide.

Embodiment 150. The pharmaceutical composition of any one of Embodiments 134 to 149, wherein the one or more RNA molecules comprise at least one non-coding regulatory element.

Embodiment 151. The pharmaceutical composition of any one of Embodiments 134 to 150, wherein the one or more RNA molecules comprise a polyadenine tail.

Embodiment 152. The pharmaceutical composition of Embodiment 151, wherein the polyadenine tail is or comprises a modified adenine sequence.

Embodiment 153. The pharmaceutical composition of any one of Embodiments 134 to 152, wherein the one or more RNA molecules comprise at least one 5' untranslated region (UTR) and/or at least one 3'UTR.

Embodiment 154. The pharmaceutical composition of Embodiment 153, wherein the one or more RNA molecules comprise, in 5' to 3' order:

(i) a 5' cap or a 5' cap analog;

(ii) at least one 5′UTR;

(iii) signal peptide;

(iv) a coding region encoding at least one of the NY-ESO-1 antigen, the MAGE-3 antigen, the tyrosinase antigen and the TPTE antigen;

(v) at least one sequence encoding tetanus toxoid P2, tetanus toxoid P16, or both;

(vi) a sequence encoding an MHC class I trafficking domain;

(vii) at least one 3'UTR; and

(viii) Polyadenine tail.

Embodiment 155. The pharmaceutical composition of any one of Embodiments 134 to 154, wherein the one or more RNA molecules comprise natural ribonucleotides.

Embodiment 156. The pharmaceutical composition of any one of Embodiments 134 to 155, wherein the one or more RNA molecules comprise modified or synthetic ribonucleotides.

Embodiment 157. The pharmaceutical composition of any one of Embodiments 134 to 156, wherein at least one of the NY-ESO-1 antigen, the MAGE-3 antigen, the tyrosinase antigen, and the TPTE antigen is a full-length, non-mutated antigen.

Embodiment 158. The pharmaceutical composition of any one of Embodiments 134 to 157, wherein all of the NY-ESO-1 antigen, the MAGE-3 antigen, the tyrosinase antigen, and the TPTE antigen are full-length, non-mutated antigens.

Embodiment 159. The pharmaceutical composition of any one of Embodiments 134 to 158, wherein at least one of the NY-ESO-1 antigen, the MAGE-3 antigen, the tyrosinase antigen, and the TPTE antigen is expressed by dendritic cells in the lymphoid tissue of the patient.

Embodiment 160. The pharmaceutical composition of any one of Embodiments 134 to 159, wherein at least one of the NY-ESO-1 antigen, the MAGE-3 antigen, the tyrosinase antigen, and the TPTE antigen is present in the cancer.

Embodiment 161. The pharmaceutical composition of any one of Embodiments 134 to 160, wherein the lipid particles comprise liposomes.

Embodiment 162. The pharmaceutical composition of any one of Embodiments 134 to 160, wherein the lipid particles comprise cationic liposomes.

Embodiment 163. The pharmaceutical composition of any one of Embodiments 134 to 162, wherein the lipid particles comprise lipid nanoparticles.

Embodiment 164. The pharmaceutical composition of any one of Embodiments 134 to 163, wherein the lipid particles comprise N,N,N-trimethyl-2-3-dioleyloxy-1-propylammonium chloride (DOTMA), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine phospholipids (DOPE), or both.

Embodiment 165. The pharmaceutical composition of any one of Embodiments 134 to 164, wherein the lipid particle comprises at least one ionizable amino lipid.

Embodiment 166. The pharmaceutical composition of any one of Embodiments 134 to 165, wherein the lipid particle comprises at least one ionizable amino lipid and a helper lipid.

Embodiment 167. The pharmaceutical composition of any one of Embodiments 166, wherein the helper lipid is or comprises a phospholipid.

Embodiment 168. The pharmaceutical composition of any one of Embodiments 166 or 167, wherein the helper lipid is or comprises a sterol.

Embodiment 169. The pharmaceutical composition of any one of Embodiments 134 to 168, wherein the lipid particle comprises at least one polymer-conjugated lipid.

Embodiment 170. The pharmaceutical composition of any one of Embodiments 134 to 169, wherein the patient is a human.

Embodiment 171. The pharmaceutical composition of any one of Embodiments 134 to 170, wherein the cancer is an epithelial cancer.

Embodiment 172. The pharmaceutical composition of any one of Embodiments 134 to 171, wherein the cancer is melanoma.

Embodiment 173. The pharmaceutical composition of Embodiment 172, wherein the melanoma is cutaneous melanoma.

Embodiment 174. The pharmaceutical composition of any one of Embodiments 134 to 173, wherein the cancer is in an advanced stage.

Embodiment 175. The pharmaceutical composition of any one of Embodiments 134 to 174, wherein the cancer is stage II, stage III, or stage IV.

Embodiment 176. The pharmaceutical composition of any one of Embodiments 134 to 175, wherein the cancer is stage IIIB, IIIC, or IV melanoma.

Embodiment 177. The pharmaceutical composition of any one of Embodiments 134 to 176, wherein the cancer is completely resected, there is no evidence of disease, or both.

Embodiment 178. Use of a pharmaceutical composition for inducing an immune response against cancer in a patient, wherein the pharmaceutical composition comprises:

(a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and

(b) lipid particles;

and wherein the patient was classified as having no evidence of disease but had been previously diagnosed with cancer.

Embodiment 179. Use of a pharmaceutical composition for treating cancer, wherein the pharmaceutical composition comprises:

(a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and

(b) lipid particles;

and wherein the patient was classified as having no evidence of disease but had been previously diagnosed with cancer.

Embodiment 180. The use of Embodiment 178 or 179, wherein the cancer is melanoma.

Embodiment 181. Use of a pharmaceutical composition for inducing an immune response against cancer in a patient, wherein the pharmaceutical composition comprises:

(a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and

(b) Lipid particles.

Embodiment 182. Use of a pharmaceutical composition for treating cancer, wherein the pharmaceutical composition comprises:

(a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and

(b) Lipid particles.

Embodiment 183. The use of Embodiment 181 or 182, wherein the patient is classified as having no evidence of disease at the time of administration.

Embodiment 184. The use of Embodiment 181 or 182, wherein the patient is classified as having evidence of disease at the time of administration.

Embodiment 185. The use of any one of Embodiments 178 to 184, wherein the presence or absence of evidence of disease is determined by applying the Immune-related Response Evaluation Criteria in Solid Tumors (irRECIST) criteria or RECIST 1.1 criteria.

Embodiment 186. The use of any one of Embodiments 178 to 185, wherein the cancer is melanoma.

Embodiment 187. The use of any one of Embodiments 178 to 186, wherein the one or more RNA molecules comprise:

(i) a first RNA molecule encoding the NY-ESO-1 antigen,

(ii) a second RNA molecule encoding a MAGE-3 antigen,

(iii) a third RNA molecule encoding a tyrosinase antigen, and

(iv) a fourth RNA molecule encoding the TPTE antigen.

Embodiment 188. The use of any one of Embodiments 178 to 187, wherein a single RNA molecule of said one or more RNA molecules encodes at least two of said NY-ESO-1 antigen, said MAGE-3 antigen, said tyrosinase antigen, and said TPTE antigen.

Embodiment 189. The use of any one of Embodiments 178 to 188, wherein a single RNA molecule of the one or more RNA molecules encodes a multi-epitope polypeptide, wherein the multi-epitope polypeptide comprises at least two of the NY-ESO-1 antigen, the MAGE-3 antigen, the tyrosinase antigen, and the TPTE antigen.

Embodiment 190. The use of any one of Embodiments 178 to 189, wherein the one or more RNA molecules further comprise at least one sequence encoding a CD4+ epitope.

Embodiment 191. The use of Embodiment 190, wherein the one or more RNA molecules comprise at least one sequence encoding tetanus toxoid P2, a sequence encoding tetanus toxoid P16, or both.

Embodiment 192. The use of any one of Embodiments 178 to 191, wherein the one or more RNA molecules comprises a sequence encoding an MHC class I trafficking domain.

Embodiment 193. The use of any one of Embodiments 178 to 192, wherein the one or more RNA molecules comprises a 5' cap or a 5' cap analog.

Embodiment 194. The use of any one of Embodiments 178 to 193, wherein the one or more RNA molecules comprises a sequence encoding a signal peptide.

Embodiment 195. The use of any one of Embodiments 178 to 194, wherein the one or more RNA molecules comprise at least one non-coding regulatory element.

Embodiment 196. The use of any one of Embodiments 178 to 195, wherein the one or more RNA molecules comprises a polyadenine tail.

Embodiment 197. The use described in Embodiment 196, wherein the polyadenine tail is or comprises a modified adenine sequence.

Embodiment 198. The use of any one of Embodiments 178 to 197, wherein the one or more RNA molecules comprise at least one 5' untranslated region (UTR) and/or at least one 3'UTR.

Embodiment 199. The use of Embodiment 198, wherein the one or more RNA molecules comprise, in 5' to 3' order:

(i) a 5' cap or a 5' cap analog;

(ii) at least one 5′UTR;

(iii) signal peptide;

(iv) a coding region encoding at least one of the NY-ESO-1 antigen, the MAGE-3 antigen, the tyrosinase antigen and the TPTE antigen;

(v) at least one sequence encoding tetanus toxoid P2, tetanus toxoid P16, or both;

(vi) a sequence encoding an MHC class I trafficking domain;

(vii) at least one 3'UTR; and

(viii) Polyadenine tail.

Embodiment 200. The use of any one of Embodiments 178 to 199, wherein the one or more RNA molecules comprise natural ribonucleotides.

Embodiment 201. The use of any one of Embodiments 178 to 200, wherein the one or more RNA molecules comprise modified or synthetic ribonucleotides.

Embodiment 202. The use of any one of Embodiments 178 to 201, wherein at least one of the NY-ESO-1 antigen, the MAGE-3 antigen, the tyrosinase antigen, and the TPTE antigen is a full-length, non-mutated antigen.

Embodiment 203. The use of any one of Embodiments 178 to 202, wherein all of the NY-ESO-1 antigen, the MAGE-3 antigen, the tyrosinase antigen, and the TPTE antigen are full-length, non-mutated antigens.

Embodiment 204. The use of any one of Embodiments 178 to 203, wherein at least one of the NY-ESO-1 antigen, the MAGE-3 antigen, the tyrosinase antigen, and the TPTE antigen is expressed by dendritic cells in the lymphoid tissue of the patient.

Embodiment 205. The use of any one of Embodiments 178 to 204, wherein at least one of the NY-ESO-1 antigen, the MAGE-3 antigen, the tyrosinase antigen, and the TPTE antigen is present in the cancer.

Embodiment 206. The use of any one of Embodiments 178 to 205, wherein the lipid particle comprises a liposome.

Embodiment 207. The use of any one of Embodiments 178 to 205, wherein the lipid particles comprise cationic liposomes.

Embodiment 208. The use of any one of Embodiments 178 to 207, wherein the lipid particles comprise lipid nanoparticles.

Embodiment 209. The use of any one of Embodiments 178 to 208, wherein the lipid particles comprise N,N,N-trimethyl-2-3-dioleyloxy-1-propylammonium chloride (DOTMA), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine phospholipids (DOPE), or both.

Embodiment 210. The use of any one of Embodiments 178 to 209, wherein the lipid particle comprises at least one ionizable amino lipid.

Embodiment 211. The use of any one of Embodiments 178 to 210, wherein the lipid particle comprises at least one ionizable amino lipid and a helper lipid.

Embodiment 212. The use of any one of Embodiments 211, wherein the helper lipid is or comprises a phospholipid.

Embodiment 213. The use of any one of Embodiments 211 or 212, wherein the helper lipid is or comprises a sterol.

Embodiment 214. The use of any one of Embodiments 178 to 213, wherein the lipid particle comprises at least one polymer-conjugated lipid.

Embodiment 215. The use of any one of Embodiments 178 to 214, wherein the patient is a human.

Embodiment 216. The use of any one of Embodiments 178 to 215, wherein the cancer is an epithelial cancer.

Embodiment 217. The use of any one of Embodiments 178 to 216, wherein the cancer is melanoma.

Embodiment 218. The use of Embodiment 217, wherein the melanoma is cutaneous melanoma.

Embodiment 219. The use of any one of Embodiments 178 to 218, wherein the cancer is in an advanced stage.

Embodiment 220. The use of any one of Embodiments 178 to 219, wherein the cancer is stage II, stage III, or stage IV.

Embodiment 221. The use of any one of Embodiments 178 to 220, wherein the cancer is stage IIIB, stage IIIC, or stage IV melanoma.

Embodiment 222. The use of any one of Embodiments 178 to 221, wherein the cancer is completely resected, there is no evidence of disease, or both.

Example

Example 1: Experimental Design and Materials and Methods

Lipo-MERIT clinical trial design. The main objectives of this trial (NCT02410733) are: to evaluate the safety and tolerability of melanoma FixVac, its preliminary efficacy and progression-free survival; to study vaccine-induced antigen-specific immune responses; and to determine the Phase II dose. As used herein, the term "FixVac" refers to a pharmaceutical composition comprising one or more RNA molecules and lipid particles (e.g., lipoplexes or lipid nanoparticles) as shown in Figure 1. BNT111 is an embodiment of FixVac. Since this is the first human Phase I trial and consistent with the purpose, statistical methods were not used to predetermine the sample size. The researchers were not blinded to the allocation during the experiment and outcome assessment.

This trial was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines and was approved by an independent ethics committee. All patients provided written informed consent.

Eligible patients had stage III B to C or IV malignant melanoma (American Joint Committee on Cancer, AJCC 2009 melanoma classification), both resected and unresected, and therefore had measurable and non-measurable disease at baseline, and expressed at least one of the four vaccine TAAs. Patients were also at least 18 years of age and had adequate hematologic and end-organ function. Inclusion criteria required that subjects were ineligible for, or refused, any other available approved treatment after all available treatment options had been transparently disclosed. Key exclusion criteria were the presence of clinically relevant autoimmune disease, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), or active brain metastases. Patients received eight RNA-LPX injections (prime/repeated boost regimen) over 64 days, except for patients from cohort 1, who received only six injections over 43 days. For patients with measurable disease who did not show disease progression or drug-related toxicity, optional continuation of monthly vaccine doses was provided. Patients were treated in seven dose escalation cohorts where target doses ranged from 14.4 μg to 400 μg total RNA and in three expansion cohorts that further explored dose levels of 14.4 μg, 50 μg, and 100 μg. RNA-LPX administration was performed by four consecutive intravenous slow push injections using an intravenous catheter.

Additional information about the patients who participated in this study is contained in Figures 35 and 36.

Key study assessments. Safety and tolerability were evaluated based on changes in physical examination or vital signs, clinical laboratory analysis, and reports of any adverse events (including clinically significant laboratory abnormalities). Adverse events were graded according to the National Cancer Institute Common Terminology Criteria (NCI CTC 4.03 version). Safety was characterized from grade 1 to grade 5 according to these criteria.

Imaging of the chest, abdomen, and brain was performed by CT scan and magnetic resonance imaging (MRI) at baseline and every 90 days thereafter according to local imaging guidelines and irRECIST version 1.1 ( Ref. 25 ).

Prior to and 4 hours after the administration of FixVac, vital signs (temperature, heart rate, and blood pressure) were measured as clinically indicated.

In order to assess the immune response induced by the vaccine, blood was sampled at baseline, before the fourth, sixth and eighth vaccine administrations, and 7 to 14 days and 19 to 33 days after the eighth administration. In group 1, blood was sampled at baseline, before the third, fourth, fifth and sixth vaccine administrations, and 7 to 14 days after the sixth administration. During the continuation of treatment, blood samples were obtained before each administration. PBMCs were separated from peripheral blood or leukocyte apheresis samples by Ficoll-Hypaque (Amersham Biosciences) density gradient centrifugation.

For cytokine analysis, serum was sampled before and 2, 6, 24, or 48 hours after treatment and shipped at -80°C. Samples were analyzed in duplicate (MLM Medical Labs) using a human pan-IFN-α ELISA (PBL Assay Science) and a multisport assay system (Meso Scale Discovery). The sample amounts for each analysis were: IFN-α vs. IP-10, n=166; IFN-α vs. IFN-γ, n=167; IFN-α vs. IL-6, n=167; IFN-α vs. IL-12 p70, n=167; from 72 patients, with up to 6 data points per patient.

Delayed-type hypersensitivity (DTH) reactions were evaluated in detail in some patients. Skin-infiltrating lymphocytes (SIL) were recovered from punch biopsies after intradermal injection of RNA diluted in concentrated (×2.67) Ringer's solution (manufactured by BAG Health Care under good manufacturing practice (GMP) guidelines) and cultured in medium (RPMI 1640, 7% human AB serum, 1× antifungal) containing high-dose IL-2 (50,000 U·ml-1) for 2 to 3 weeks.

Data reporting. This is an ongoing exploratory, open-label, non-randomized first-in-human Phase I clinical trial. The data presented are based on an exploratory interim analysis, with a data extraction date of July 29, 2019. This exploratory analysis was conducted to inform and initiate the design of a randomized Phase 2 trial of FixVac/anti-PD1 combination therapy for patients experiencing CPI. The analysis was triggered by the availability of baseline to three-month comparative immunogenicity data for approximately half of the study population (n=51) in the dose cohort, and the availability of at least three months of follow-up data for two subgroups of patients treated with FixVac monotherapy and FixVac/anti-PD1 combination. The exploratory interim analysis focuses specifically on vaccine-induced immune responses (secondary endpoints). In addition, preliminary high-level data on study drug tolerance (primary endpoint) and responses (secondary endpoints) in patients with measurable disease according to irRECIST 1.1 were reported. The clinical data shown are preliminary and not fully verified source data. When this article was accepted for publication, 109 (95%) of 115 patients were recruited.

Exemplary Materials and Methods. The following materials and methods were used in the following examples.

FDG-PET/CT imaging. After a 4 to 6 hour fasting period (resulting in blood glucose levels below 130 mg dl-1) and after a 60 to 70 minute allotment time, about 2 MBq kg-1 FDG was applied to assess [18F] FDG uptake in the spleen by PET/CT imaging. According to clinical routine, an EARL-certified Philips Gemini time-of-flight (TOF) PET/CT scanner was used to collect data for 2 to 2.5 minutes per bed position. The average standardized uptake value (SUV) was measured in a 2 cm sphere centered in the spleen.

TAA expression profiling. Total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) samples of patients (RNeasy FFPE kit, Qiagen). Complementary DNA was synthesized (Peqstar, VWR International) according to good clinical laboratory practice (GCLP) guidelines, and the expression of NY-ESO-1, tyrosinase, MAGE-A3, and TPTE RNA, as well as a reference gene encoding hypoxanthine guaninephosphoribosyltransferase (HPRT1) were analyzed by quantitative polymerase chain reaction (PCR; Applied Biosystems 7300 Real-Time PCR System, Thermo Fisher Scientific). The median quantitative cycle (Cq) value of each TAA was normalized to the median Cq of the reference gene to obtain the relative expression ACq value, which was classified as positive or negative based on the TAA-specific cutoff point.

Manufacturing of RNA-LPX. RNA, liposomes, and RNA-LPX were manufactured under GMP conditions. RNA was manufactured by in vitro transcription of a DNA plasmid template encoding the full-length sequence of NY-ESO-1, MAGE-A3, TPTE, or tyrosinase amino acids 1 to 477. Manufacturing, analysis, and release of the four TAA-encoding RNA drug products were performed as previously described (Ref. 26).

Liposomes with a net cationic charge were used to complex RNA to form RNA-LPX. Cationic liposomes were made from cationic synthetic lipids (R)-N,N,N trimethyl-2-3-dioleyloxy-1-propylammonium chloride (R-DOTMA) (Merck and Cie) and phospholipids 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine phospholipids (DOPE) (Corden Pharma) based on an ethanol injection technique (reference 28). Release analysis of the liposomes included determination of appearance, lipid concentration, RNase presence, particle size, osmotic concentration, pH, subvisible particles, pyrogen testing, and sterility.

The injectable RNA-LPX drug product is prepared in a dedicated pharmacy according to a proprietary (Ref. 27) protocol by incubating a single concentrated RNA drug product with an isotonic NaCl solution (0.9%) (Fresenius Kabi) and cationic liposomes. The RNA-LPX preparation protocol is derived from the protocol for nucleotide lipid complex formation as described in (Refs. 8, 29). Prior to injection, the RNA-LPX is further diluted to the desired concentration using an isotonic NaCl solution (0.9%) (Fresenius Kabi). Regular quality control of the RNA-LPX drug product includes determination of RNA content, RNA integrity, particle size, and polydispersity index.

In vitro stimulation of PBMCs. CD4+ and CD8+ T cells were isolated from cryopreserved PBMCs using microbeads (Miltenyi Biotec). For IVS, RNA or peptides encoding TAAs were used. For IVS using RNA, CD4- or CD8-depleted PBMCs were electroporated with RNA encoding vaccine antigens, enhanced green fluorescent protein (eGFP), influenza matrix protein 1 (M1), or tetanus p2/p16 sequences (influenza M1 and tetanus p2/p16 were positive controls for CD4+ and CD8+ T cells, respectively) after overnight resting. The cells were then placed at 37°C for 3 hours and irradiated at 15 Gy. Overnight resting CD4+/CD8+ T cells were combined with electroporated and irradiated antigen presenting cells at an effector to target (E:T) ratio of 2:1. For peptide IVS, CD4+T cells were expanded in the presence of fast dendritic cells (E:T=10:1) pulsed with PepMix encoding MAGE-A3, tyrosinase, TPTE or NY-ESO-1. To expand CD8+T cells, CD4-depleted PBMCs were co-cultured with purified CD8+T cells (E:T=1:10) in the presence of IL-4 and granulocyte macrophage colony-stimulating factor (GM-CSF) (1,000U·ml-1 each) and the corresponding peptide. One day after starting IVS, fresh culture medium containing 10U ml-1 IL-2 (Proleukin S, Novartis) and 5ng ml-1 IL-15 (Peprotech) was added. CD8IVS cultures stimulated with peptides additionally received IL-4 and GM-CSF (1,000U ml-1 each). For tumor cell lysis experiments, peptide-pulsed bulk PBMCs were used for IVS and harvested after 6 to 8 days of culture. For longer cultures, IL-2 was supplemented 7 days after setting up the IVS cultures. After 11 days of stimulation, cells were analyzed by flow cytometry and used for ELISpot assays.

IFN-γ ELISpot. ELISpot analysis was performed on 51 patients (50 patients ex vivo and 20 patients after IVS). In addition to the 49 patients shown in Figure 5, two patients who received BRAF/MEK inhibitors were tested in the IFN-γ ELISPOT. Multiscreen filter plates (Merck Millipore) pre-coated with antibodies specific for IFN-γ (Mabtech) were washed with phosphate-buffered saline (PBS) and blocked with X-VIVO 15 (Lonza) containing 2% human serum albumin (CSL-Behring) for 1 to 5 hours. Next, 0.5×10 ⁵ to 3×10 ⁵ effector cells per well were stimulated for 16 to 20 hours with peptides (ex vivo), autologous dendritic cells electroporated with RNA or loaded with peptides (after IVS), or K562 cells transfected with HLA class I or class II loaded with peptides (for TCR validation). In order to analyze T cell responses in vitro, cryopreserved PBMCs were left to stand for 2 to 5 hours at 37°C before ELISpot. Alternatively, CD4 ^- or CD8 ^- depleted PBMCs were used as CD8 or CD4 effectors. All tests were performed in duplicate or triplicate, and positive controls (Staphylococcus enterotoxin B (Sigma Aldrich), anti-CD3 (Mabtech)) and cells from reference donors with known reactivity were included. Spots were visualized with biotin-conjugated anti-FNγ antibodies (Mabtech) followed by incubation with ExtrAvidin-alkaline phosphatase (Sigma-Aldrich) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) (Sigma-Aldrich). Alternatively, secondary antibodies directly conjugated to alkaline phosphatase were used (ELISpot-Pro kit, Mabtech). The plates were scanned using an ImmunoSpot Series S5 Versa ELISpot analyzer (CTL, S5Versa-02-9038) or a classic robotic ELISPOT reader (AID) and analyzed by ImmunoCapture version 6.3 or AID ELISPOT 7.0 software. Spot counts were summarized as the median of each triplicate or duplicate. T cell responses stimulated by RNA or peptides encoding vaccine antigens were compared with responses elicited by target cells electroporated with control RNA (luciferase) or by unloaded cells. Responses were defined as positive with a minimum of 5 spots/1×10 ⁵ cells in an ex vivo setting, or 25 spots/5×10 ⁴ cells in a post-IVS setting, and spot counts more than twice as high as the corresponding controls.

Flow cytometry. Antigen-specific CD8 ⁺ T cells were identified using fluorophore-conjugated HLA multimers (Immudex). Cells were first stained for multimers and then for the following (antibody clones in parentheses) cell surface markers: CD28 (CD28.8), CD197 (150503), CD45RA (HI100), CD3 (UCHT1 or SK7), CD16 (3G8), CD14 CD19 (SJ25C1), CD27 (L128), CD279 (EH12), CD134 (ACT35), and CD8 (RPA-T8 or SK1), all purchased from BD Biosciences; CD19 (HIB19) and CD4 (OKT4), from Biolegend. Live-dead staining was also performed using 4', 6-diamidino-2-phenylindole (DAPI; BD) or the fixable viability dyes eFluor 780 or eFluor 506 (eBioscience). Single peak, live, multimeric positive events were identified among CD3 ⁺ (or CD8 ⁺ ), CD4 ^- CD14 ^- CD16 ^- CD19 ^-, or CD3 ⁺ (or CD8 ⁺ )CD4 - events. To detect antigen-specific T cells after IVS, singlet, live CD3 ⁺ , CD8 ⁺ multimer+ lymphocytes were gated.

For staining of intracellular cytokines, autologous dendritic cells electroporated with RNA encoding individual neo-epitopes were added at an E:T ratio of 10:1 and cultured for approximately 16 hours at 37°C in the presence of brefeldin A and monensin. Cells were stained for viability (using the fixable viability dyes eFluor 506 or eFluor 780, eBioscience) and for surface markers including CD8 (RPA-T8 or SK1), CD16 (3G8), CD14 (all from BD Biosciences), CD19 (HIB19) or CD4 (OKT4) (from Biolegend). After permeabilization, intracellular cytokine staining was performed using antibodies against IFN-γ (B27, BD Biosciences) and against TNF (Mab11, BD or Biolegend). IFN-γ+ and TNF+ events were identified in CD8 ^{+ and CD4+ cells} pre-gated on singlets, live and CD14 ^- CD16 ^- CD19- (not used in all experiments) populations.

The cell surface expression of the transfected TCR genes was analyzed using anti-TCR antibodies (Beckman Coulter) and CD8- or CD4-specific antibodies (SK-1, BD; REA623, Miltenyi Biotec) for the appropriate variable region family or constant region of the TCR-β chain. The HLA antigens of antigen-presenting cells used to evaluate the function of TCR-transfected T cells were detected by staining with HLA class II specific antibodies (9-49, Beckman Coulter) and HLA class I specific antibodies (DX17, BD Biosciences). Collection was performed on LSR Fortessa SORP, FACSCelesta or FACSCanto II cell analyzers (BD Biosciences) and analyzed by FlowJo software (Tree Star).

Cloning of HLA antigens. HLA antigens were synthesized by Eurofins Genomics Germany GmbH based on the corresponding high-resolution HLA typing results. HLA DQA sequences were amplified from donor-specific cDNA using DQA1_s (Pho GCC ACC ATG ATC CTA AAC AAA GCT CTG MTG C) and DQA1_as (TAT GCG ATC GCT CAC AAK GGC CCY TGG TGT CTG) primers with 2.5 U Pfu polymerase. HLA antigens were cloned into appropriately digested IVT vectors (reference 10).

RNA is transferred into cells. RNA is added to cells suspended in X-VIVO 15 culture medium (Lonza) in precooled 4-mm gap sterile electroporation cuvette (Bio-Rad). Electroporation is performed using BTX ECM 830 square wave electroporation system under conditions previously established for each cell type (T cells, 500V, 3 milliseconds/pulse, one pulse; Immature dendritic cells, 300V, 12 milliseconds/pulse, one pulse; SK-MEL-29, 250V, 3 milliseconds/pulse, three pulses; Jurkat cells, 275V, 10 milliseconds/pulse, one pulse; K562 cells, 200V/8 milliseconds/three pulses).

Peptides. Pools of overlapping peptides encoding full-length NY-ESO-1, tyrosinase, MAGE-A3, and TPTE or short (8- to 11-mer) epitopes derived from these antigens (PepMix), as well as HIV gag encoding PepMix were used as controls. All synthetic peptides were purchased from JPT Peptide Technologies GmbH and dissolved in water with 10% dimethyl sulfoxide (DMSO) to a final concentration of 3 mM (short peptides) or dissolved in 100% DMSO (PepMix).

Cell lines. K562 and SK-MEL-28 cell lines were obtained from ATCC. SK-MEL-29 cell line was obtained from Memorial Sloan Kettering Cancer Center, New York. SK-MEL-37 cell line is described in reference 30. Jurkat T cell line was prepared by Promega, and the Jurkat T cell line expresses a luciferase reporter driven by a nuclear factor of activated T cell (NFAT)-response element of activated T cells. Re-certification of cell lines was performed by short tandem repeat (STR) spectrum analysis at the American Type Culture Collection (ATCC) and Eurofins. All cell lines used were tested negative for mycoplasma contamination. Common misidentified cell lines were not used.

Single cell sorting. Based on stimulation-induced IFN-γ secretion or multimer binding, single antigen-specific T cells were sorted using ex vivo PBMC or IVS cultures. For stimulation, PBMCs were pulsed with overlapping peptides encoding related antigens or control antigens, and T cells amplified after IVS were cultured with dendritic cells pulsed with autologous peptides. After 4 hours, cells were harvested and stained with viability dye eFluor 780 (eBioscience) and fluorescent dye-conjugated antibodies for CD3, CD8 and CD4 (all BD Biosciences) and IFNγ secretion assay kit (Miltenyi Biotec) with fluorescent dye-conjugated antibodies for IFNγ. Alternatively, PBMCs were stained with corresponding multimers. Sorting of single neoantigen-specific T cells was performed using BD FACSDiva or BD FACSChorus software on FACSAria or FACSMelody flow cytometers (both from BD Biosciences). Antigen-specific T cells were identified relative to control samples stimulated with control antigens or stained in the absence of polymers. One T cell per well (gate set on single live CD3+ and CD8+IFN-γ+, CD4+IFN-γ+ or CD8+ polymers+lymphocytes) was harvested in a 96-well V-bottom plate (Greiner Bio-One), which contained 6 μl of mild hypotonic cell lysis buffer/well (composed of 0.2% Triton X-100, 0.2 μl RiboLock RNA enzyme inhibitor (Thermo Scientific), 5ng poly (A) carrier RNA (Qiagen) and 1 μl dNTP mixture (10mM, Biozym) in RNase-free water). After sorting, the plate was sealed, centrifuged and directly stored at -65°C to -85°C.

Cloning of antigen-specific TCR. As described in 10, TCR genes were cloned from single T cells and modified as follows. Plates with sorted cells were thawed and template-switched cDNA synthesis was performed using RevertAid H reverse transcriptase (Thermo Fisher) with primers specific for TCR-α and TCR-β constant genes (TRAC, 5′-catcaeaggaactttctgggctg-3′; TRBC1, 5′-gctggtaggacaccgaggtaaagc-3′; TRBC2 5′-gctggtaagactcggaggtga agc-3′), followed by pre-amplification using PfuUltra Hotstart DNA polymerase (Agilent). After both cDNA synthesis and PCR, the remaining primers were removed by treatment with 5U exonuclease I (NEB). Aliquots of cDNA were used for Vα/Vβ gene-specific multiplex PCR. Products were analyzed on a capillary electrophoresis system (Qiagen). Band is carried out size classification on agarose gel at the sample of 430bp to 470bp place, and band is cut off and purified using gel extraction kit (Qiagen). Purified fragment is sequenced and uses IMGT/V-Quest instrument to analyze corresponding V (D) J to connect (reference 31). Use NotI to digest the DNA of the new and effectively rearranged corresponding TCR chain, and clone it in the pST1 vector comprising suitable constant region, for complete TCR-α/β chain 10 of in vitro transcription.

Single-cell TCR sequencing. For selected patients, TCRs from sorted single cells were obtained by a next-generation sequencing (NGS)-based single-cell TCR sequencing (scTCR-seq) workflow. Here, template-switched cDNA synthesis was performed using primers specific for TCR-α and TCR-β constant genes (TRAC, 5′-catcacaggaactttctgggctg-3′; TRBC, 5′-cacgtggtcggggwagaagc-3′), followed by treatment with 5U of exonuclease I. The PCR products were PCR-PCR prepared using 2.5 U PfuUltra Hotstart DNA polymerase (Agilent), 1× PCR buffer, 0.2 mM dNTPs, 0.2 μM of one of the eight labeled forward primers (Tag130-RBCx-TS 5′-cgatccagactagacgctcaggaagxxxxxaagcagtggtatcaacgcagagt-3′), and 0.1 μM of each of the labeled nested TCR-α and TCR-β constant gene-specific primers (Tag146-TRAC, 5′-caatatgtgaccgccgagtcccaggttagagtctc tcagctggtacacggcag-3′; Tag146-TRBC, 5′-caatatgtgaccgccgagtccc aggggctcaaacacagcgacctcgggtg-3′), each cDNA was PCR amplified and row-by-row barcoded (95°C for 2 min; 5 cycles of 94°C for 30 sec, 61°C for 30 sec, 72°C for 1 min; 5 cycles of 94°C for 30 sec, 64°C for 30 sec, 72°C for 1 min; 8 cycles of 94°C for 30 sec, 72°C for 2 min; 72°C for 6 min) (RBC, row barcode; TS, template switching primer). Samples from each column were pooled and purified twice using AMPure XP beads (Agencourt) with exonuclease I treatment in between. For each pool, one third of the purified TCR cDNA was further amplified by PCR (95°C for 1 min; 24 cycles of 94°C for 20 sec, 64°C for 20 sec, 72°C for 30 sec; 72°C for 3 min) using 1 μl PfuUltra II Fusion Hotstart DNA polymerase (Agilent), 1× reaction buffer, 0.2 mM dNTPs, forward primer (Tag-130 5′-(n)nnnncgatccagactagacgctcaggaag-3′) and one of 12 Tag-146 reverse oligonucleotides containing different barcodes for each column (5′-xxxxxcaatatgtgaccgccgagtcccagg-3′). PCR products were pooled and purified with AMPure XP beads and exonuclease I, followed by generation of TCR sequencing libraries using the TruSeq DNA Nano kit (Illumina). Paired end 300-bp sequencing was used on Illumina MiSeq to sequence the scTCR library in the case of 10,000 reads/wells at sequencing depth. Sequencing data was demultiplexed to single cell level using bcl2fastq software (Illumina) subsequently by internal Python script. TCR sequences were then obtained using MiXCR-2.1.5 (reference 32). Selected pairing α and β V (D) J fragments (Eurofins Genomics) were synthesized and cloned as above for subsequent in vitro transcription.

Large-scale TCR sequencing. Total RNA was isolated from 1×106 flash-frozen PBMCs collected at multiple time points during vaccination using the RNeasy Mini kit (Qiagen). Libraries were generated using the SMARTer Human TCR-α/β Repertoire Analysis Kit (Clontech) and sequenced using the Illumina MiSeq system. The number of total TCR reads per sample ranged from 1×106 to 4×106. Data were analyzed using VDJtools (reference 33) and MiXCR.

Functional TCR characterization. TCR-transfected CD4+ or CD8+ T cells from healthy donors were co-cultured with peptide-pulsed HLA class I or class II transfected K562 cells and tested by IFN-γ ELISpot assay. Alternatively, Jurkat cells of T cell activation bioassay (NFAT, Promega) were transfected with RNA encoding CD8-α and TCR-α/β and tested for target cells (Fig. 4c). After adding Bio-Glo reagent (Promega), T cell activation was analyzed by luminescence measurement (InfiniteF200 PRO, Tecan).

Cytotoxicity assay. T cell-mediated cytotoxicity was assessed by cell index impedance measurement using the xCELLigence MP system (OMNI Life Science) according to the supplier's instructions. CD8+T cells transfected with OKT3-activated TCRs from healthy donors or CD8+T cells derived from patients in IVS cultures were used as effector cells. Melanoma cell lines transfected with the corresponding HLA alleles and seeded in 96-well PET E plates (ACEABiosciences) at a concentration of 2×104 cells/well were used as target cells. After 24 hours, effector T cells were added at different E:T ratios, and the cell index values were monitored every 30 minutes using the xCELLigence system for up to 48 hours. Based on negative controls (for TCR, mock transfected T cells; for IVS cells, pre-treated IVS cultures), specific lysis was calculated after the specified time of co-culture (Figures 2i, 3e, 12 hours; Figure 3d, 63 hours; Figure 4f, 8 hours).

Mutation discovery and gene expression. Detect mutations as described in (reference 26). In essence, the genomic sequence reads from each patient are aligned with the human reference genome hg19 using Burrows-Wheeler Aligner (BWA) software (reference 34). The exons from tumors and matched normal samples are compared to retrieve single nucleotide variants (SNVs). In order to retain high-confidence SNVs, loci with presumed homozygous genotypes are filtered, and suspected sites from presumed heterozygous mutation events are filtered to remove false positives. For the final list of high-confidence mutations, genomic coordinates and genes known to the University of California at Santa Cruz (UCSC) genome browser are incorporated to associate variants with genes. Non-synonymous mutations are selected for further processing.

Tumor RNA sequencing data were used to calculate gene expression values using Sailfish (ref. 35) and UCSC known gene transcripts as reference. Transcript counts were normalized to transcripts per million (TPM).

To compare mutational load with gene expression, in cases where genes were represented by several transcript isoforms in the UCSC database, the average of transcript expression values was used. Mutational load and expression levels were correlated using patient data from three melanoma cohorts: 13 patients from the NCT02035956 trial (reference 26), 25 patients from a published melanoma cohort (reference 22), and metastasis data from 12 patients in the MET500 cohort (reference 36).

Statistics and reproducibility. The sample size (n) represents the number of patients analyzed, except for the sum of multiple measurements from 72 patients (up to 6 times for each patient) designated as n in Figure 6c. If not otherwise specified, the central value represents the mean value, with the symbol repeated. For cytotoxicity experiments in which a single replicate value cannot be displayed, the dispersion of all technical triplicates calculated for lysis is expressed as standard deviation. By Spearman'correlation (Fig. 6c, rs: Spearman's rank correlation coefficient), Pearson correlation, Kruskal-Wallis test, followed by Dunn post-hoc test (Fig. 6b) or Brown-Forsythe and Welch analysis of variance (ANOVA), followed by Dunnett T3 multiple comparison test (Fig. 9d) to determine statistical significance (P). All analyses were two-tailed and performed using GraphPad Prism 8.4. All experiments were performed once. The experiment was not randomized.

The following example summarizes the results of an exploratory interim analysis of 89 patients (ended on July 29, 2019) (Figure 5), which focused on the immune response induced by melanoma FixVac. The best objective response to FixVac alone or in combination with anti-PD1 antibodies in patients with measurable disease was also evaluated (Figure 29).

Example 2: In vivo characterization of immune activation mediated by an exemplary RNA composition described herein

This example demonstrates in vivo characterization of immune activation after administration of an exemplary pharmaceutical composition comprising: one or more RNA molecules that collectively encode NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, TPTE antigen, or a combination thereof; and a lipid particle (e.g., a lipoplex or lipid nanoparticle). Figure 1a shows an exemplary schematic diagram of one or more RNA molecules that collectively encode NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, and TPTE antigen.

FixVac targeting in spleen. This example shows that FixVac is targeted to spleen by taking advantage of the enhanced glucose consumption of cells after TLR ligand stimulation (reference 12). In this example, [18F]-fluoro-2-deoxy-2-d-glucose (FDG)-positron emission tomography (PET)/computed tomography (CT) scans were performed shortly after injection of FixVac. Shortly after injection, a significant increase in metabolic activity was observed, particularly in the spleen, indicating rapid targeting and transient activation of lymphoid tissue-resident immune cells (Fig. 1c).

Adjuvantity. In order to determine the adjuvantity of FixVac after administration to patients, the amount of plasma cytokines was measured (reference 8). The increase in the levels of interferon (IFN)-α, IFN-γ, interleukin (IL)-6, IFN-induced protein (IP)-10 and IL-12 p70 subunits was consistent with the FixVac dose, accompanied by a transient increase in body temperature (Fig. 1d; Fig. 6a). Cytokine secretion is pulsatile, transient and self-limiting, reaching a peak value 2 to 6 hours after treatment and normalizing within 24 hours (Fig. 1d). Combining FixVac with anti-PD1 antibodies does not affect cytokines (Fig. 6b). The plasma concentration of IFN-α is well correlated with all other measured cytokines (see Spearman correlation ( _rs ) to IFNα as shown in Fig. 6c).

Adverse event spectrum. Consistent with the cytokine pattern, the clinical adverse event spectrum is dominated by mild to moderate flu-like symptoms (e.g., fever and chills). Most of the adverse events are early-onset, transient, and controlled with antipyretics, and resolve within 24 hours. In vivo observations summarize the findings in mice, where the mode of action of FixVac is driven by the translation of antigen-encoding RNA in dendritic cells residing in the lymphoid compartment and by the accompanying inflammatory response induced by TLRs on antigen-presenting cells (references 8, 13, and 20). However, the concentration of FixVac that triggers cytokine release in humans is more than 1,000 times lower than in mice (Kranz et al. 2014, which is incorporated herein by reference in its entirety).

Additional details about adverse events detected during administration are included in Figures 40 and 41. As shown, the most frequently occurring related TEAEs were fever, followed by chills, headache, fatigue, nausea, arthralgia, vomiting, and tachycardia. The frequency of these related TEAEs was similar between the ED and NED subgroups. Most of these symptoms were CTCAE grade 1 or 2 and were expected to be reactogenic due to the inherent adjuvant properties of RNA-LPX. When compared to the NED subgroup, a higher proportion of patients experienced ≥ grade 3 related TEAEs in the ED subgroup (10 patients [26.3%] vs. 3 patients [9.1%], respectively). In the ED and NED subgroups, 4/38 patients (10.5%) and 1/33 patients (3.0%) experienced TESAEs that were considered related to the trial treatment, respectively (data not shown).

Example 3 : Immunogenicity of the pharmaceutical composition

This example shows the immunogenicity of samples collected from melanoma patients (e.g., patients with stage III B to C or stage IV malignant melanoma (American Joint Committee on Cancer (AJCC) 2009 melanoma classification), both resected and unresected, and therefore with measurable and non-measurable disease at baseline, and expressing at least one of the four TAAs included in FixVac) after administration of FixVac, after in vitro stimulation (IVS). In this example, the immunogenicity of FixVac was measured by IFN-γ ELISpot after IVS.

For 50 patients, before or after vaccination (after eight injections of FixVac), a large number of or ^CD4- or CD8 ^- depleted peripheral blood mononuclear cells (PBMCs) incubated with overlapping peptides (so-called PepMix) representing the full-length sequence of TAAs described herein were subjected to ex vivo IFN-γ ELISpot (Figures 2a and 2b). Samples from 20 patients were also analyzed using IFN-γ ELISpot after IVS (Figure 2c), where autologous dendritic cells loaded with TAA PepMix were used as targets. Samples from all 20 of these patients showed T cell responses to at least one TAA (Figure 2c), mainly CD4 ⁺ responses alone or CD8 ⁺ and CD4 ⁺ responses (Figure 7a). Vaccine-induced de novo responses (responses that could not be detected before vaccination) were more frequent than enhancements of pre-vaccine responses (Figure 7a). Of the samples from 50 patients analyzed using ex vivo IFN-γ ELISpot, more than 75% showed an immune response against at least one TAA (Figure 2a). The majority of these high-magnitude T cell responses were CD8 ⁺ (Figure 2a).

De novo CD8 ⁺ T cells were measured in vitro by HLA multimer analysis and intracellular cytokine staining (ICS). The antigen-specific T cells that rose to single-digit or low double-digit percentages of circulating CD8 ⁺ T cells within 4 to 8 weeks (Fig. 2e to g; Fig. 3a, Fig. 7b, Fig. 11) had PD1 ⁺ CCR7 ^- CD27 ^{+/- CD45RA -} effect memory phenotype (Fig. 2f, Fig. 7c and Fig. 12), and secreted IFN-γ and tumor necrosis factor (TNF) (Fig. 2h, Fig. 7d and Fig. 13) after antigen-specific restimulation. Most patients had multi-epitope CD8 ⁺ immune responses (Fig. 2b, Fig. 2g). In patients who experienced monthly maintenance vaccinations after the first 8 vaccinations, the frequency of TAA-specific T cells continued to increase or remained stable for more than one year (Fig. 2g). In patients without continuous vaccinations, memory T cells still existed for several months and had a slow downward trend (Fig. 2e and Fig. 7b).

Example 4: TAA-specific T-cell receptors from vaccine-expanded T cells isolated from patients TCR) characterization

This example characterizes T cell receptors from expanded T cells following administration of FixVac.

Transfection of TAA-specific T cell receptors (TCRs) from vaccine-expanded T cells (Figure 33) into healthy donor T cells effectively killed TAA-positive melanoma cells (Figure 2i). T cell responses were not affected by the presence or absence of radiologically measurable disease at baseline, the dose of FixVac treatment, or whether FixVac was administered alone or in combination with anti-PD1 antibodies (Figures 7e and 7f).

Example 5: Best Objective Response in 42 Patients with Measurable Metastatic Disease

This example shows responses in melanoma patients with measurable metastatic disease for whom one scan at baseline and at least one scan after treatment was available. 41 patients were in stage IV who had previously undergone a line of systemic therapy and were checkpoint inhibitor (CPI) experienced; 35 of these had been exposed to antibodies against both PD1 and cytotoxic T-lymphocyte-associated protein 4 (CTLA4) (Figure 30).

In the FixVac monotherapy group (n=25), 3 patients experienced partial responses and 7 patients had stable disease (Figure 2j, Figure 5). Another patient showed complete metabolic remission of metastatic lesions in [18F]-FDG-PET/CT imaging. In the FixVac/anti-PD1 combination group, 6 of 17 patients had partial responses. Target lesion regression occurred in all doses, but the rate of partial response was highest in patients treated with 100 μg melanoma FixVac plus anti-PD1 (5 of 10 patients; objective response rate 50%) (Figure 2j). Most patients with partial response or stable disease showed durable disease control (over a maximum observation period of two years) (Figure 2k; Figure 8a and Figure 8b). Objective response was associated with tumor burden at baseline (Figure 8c).

Example 6: Immunostaining from melanoma patients receiving FixVac monotherapy and FixVac/anti-PD1 combination Signs of immune response

This example shows the response of a specific patient following treatment with a combination therapy of FixVac and PD-1 inhibition.

Several patients with partial responses (patients 53-02 and A2-10 receiving FixVac monotherapy, and patients C2-28, C2-31, and C1-40 receiving the FixVac/anti-PD1 combination; FIG8 d ) had adequate blood samples for detailed characterization of the immune response.

Patient 53-02 entered the trial after progression on pembrolizumab. On FixVac monotherapy, the patient experienced a partial response that lasted for 8 months and regression of multiple metastases (Figure 3b and Figure 9a). A few weeks after stopping vaccination at the patient's request, regrowth of metastatic lesions was diagnosed. The patient was rechallenged with pembrolizumab and remained stable for another seven months (Figure 8d).

For this patient, strong de novo immune responses to NY-ESO-1 and MAGE-A3 were detected by ex vivo ELISpot. Vaccine-induced HLA-Cw*0304-restricted CD8 ⁺ T cell responses to NY-ESO-196-104 epitope 15, identified by HLA multimer staining, increased dramatically to over 10% of peripheral blood CD8+ T cells and remained high with continued vaccination (Figures 3a and 9b). ICS determined that NY-ESO-1 reactive IFN-γ+ T cells expanded to a maximum of 15% of the entire peripheral blood CD8+ T cell population (Figures 3c and 14). Short-term IVS cultures of PBMCs after vaccination that expanded against the NY-ESO-196-104 epitope effectively killed endogenous NY-ESO-1+ melanoma cells (Figures 3d and 15).

HLA-Cw*0304 restricted (Figures 9c to 9f) and HLA-B*4001 restricted (Figures 9g to 9j) NY-ESO-1 specific TCRs were identified by single cell cloning from T cells using HLA multimer binding and antigen-specific cytokine secretion, respectively (Figure 16). All TCRs mediated killing of NY-ESO-1 ⁺ melanoma cells (Figures 3e, 9e, and 9j). TCR-β clonotype analysis determined that these T cells were de novo (Figures 3f and 9f). The patient also produced MAGE-A3 _167-176 specific T cells for a long time, which accounted for about 2% of the total CD8 ⁺ T cells (Figure 3g).

Patient A2-10 showed rapid progression of multi-metastatic disease under treatment with ipilimumab and nivolumab (Figure 8d). On FixVac monotherapy, the patient experienced a partial response over a 6-month duration and regression of multiple lymph node and lung metastases (Figure 10a). Due to progressive disease in the inguinal lymph nodes, FixVac was stopped after eight months. The patient received pembrolizumab monotherapy again and experienced a partial response.

IFN-γ ⁺ CD4 ⁺ T cell responses to MAGE-A3 and NY-ESO-1 were detected in the PBMC after treatment from this patient (Figure 10b). In the skin infiltrating lymphocytes from delayed-type hypersensitivity (DTH) reactions obtained after eight vaccinations, NY-ESO-1 specific CD4+T cells were detected (Figure 10c and Figure 17). Several NY-ESO-1-directed, tyrosinase-directed and MAGE-A3-directed TCR clonotypes from CD4+T cells in PBMC after treatment were cloned (Figure 10d, Figure 10e and Figure 33). These clonotypes include TCRs that recognize the MAGE-A3281-295 epitope, which is reported as immunodominant and promiscuously present on multiple HLA-DRB1 alleles 16 (Figure 10e). Most of the TCR frequencies cannot be detected by TCR clonotype spectrum analysis, and are increased to easily detected frequencies (Figure 10f) in the case of vaccination.

Patient C2-28 had numerous liver and subcutaneous metastases that initially progressed under treatment with the ipilimumab/nivolumab combination and subsequently stabilized under continued nivolumab monotherapy. The patient was switched to the FixVac/nivolumab combination treatment and experienced a partial response (Figures 4a and 8d) with reduction in liver and subcutaneous target lesions (tumor burden decreased from 91 mm to 15 mm). After 11 months of treatment, the patient developed a single bone metastasis, which was irradiated and maintained under continued vaccination.

For this patient, NY-ESO-1 and MAGE-A3 T cells were detected by ELISpot after IVS (data not shown). De novo HLA-A*0101 restricted T cell responses against MAGE-A3168-176 epitope 17 were increased to up to 2% of peripheral blood CD8+ T cells (Figure 4b and Figure 18). Two TCRs cloned from MAGE-A3168-176 multimer binding T cells specifically recognized endogenous MAGE-A3+ melanoma cells (Figure 4c and Figure 33).

Patient C2-31 had locally recurrent melanoma with recent systemic metastatic spread. The patient had progressed under pembrolizumab treatment within 7 months and had multiple metastases in the lungs, liver, and lymph nodes. FixVac was added to the ongoing pembrolizumab treatment, and the patient rapidly experienced a partial response (Fig. 4d and Fig. 8d). CD4+T cell responses to MAGE-A3, TPTE, and NY-ESO-1, as well as CD8+T cell responses to NY-ESO-1 and MAGE-A3 were detected, most of which were de novo (Fig. 10g).

Patient C1-40 has a history of pembrolizumab-responsive metastatic melanoma and experienced progressive disease with multiple rapidly progressing lung lesions seven months after stopping pembrolizumab. Initially treated with nivolumab, melanoma FixVac was added to it eight weeks later. The patient experienced a partial response with shrinkage of lung metastases (Fig. 8d and Fig. 10h). HLA multimer staining showed strong vaccine-induced T cell responses for MAGE-A3 _168-176 and NY-ESO-1 _92-100 epitopes (Fig. 4e and Fig. 10i). Short-term cultures of lymphocytes after vaccination effectively killed MAGE-A3+ melanoma cells, indicating the functionality of vaccine-induced T cells (Fig. 4f and Fig. 19).

Summary of findings. The data provided in Examples 1 to 6 collectively provide certain key findings. First, the transient cytokine response of FixVac-induced T cells and the high magnitude and T-helper-1 phenotype show that the RNA-LPX vaccine class has the same effective mode of action in humans, which has been characterized as being key to anti-tumor effects in mouse models (references 8, 18). Several full-length TAAs were delivered together, and patients generated polyclonal CD4 ⁺ and CD8 ⁺ T cell responses. As indicated by the kinetics of HLA multimer-positive T cells, the prime/repeated boost regimen expanded the pool of circulating antigen-specific T cells (particularly those targeting NY-ESO-1 and MAGE-A3) by several orders of magnitude over time.

Patients who experienced a partial response were those with the most dramatic and diverse T-cell responses. However, the possibility of bias due to the fact that these responders remained in the trial for a longer period of time cannot be ruled out, which allowed the collection of sufficient blood for epitope identification and multimer analysis—the most informative assay for analyzing T-cell frequency.

T cells induced by FixVac were fully functional, recognized their target epitopes on melanoma cells, and exhibited strong cytotoxic activity. Long-term immune monitoring data obtained for some patients showed that vaccine-induced T cells were maintained by continued vaccination for more than one year.

Second, the observations described in Examples 1 to 6 suggest that, although Melanoma FixVac is active as a single agent, it also synergizes with anti-PD1 therapy in patients with tumors that experience CPI. Patients 53-02 and A2-10 began Melanoma FixVac therapy after anti-PD1 failure, experienced tumor regression under Melanoma FixVac monotherapy, eventually re-progressed, and subsequently responded to re-exposure to anti-PD1 therapy. T cells induced by Melanoma FixVac have a PD1+ effector memory phenotype and are therefore stimulated by anti-PD1 antibodies. Consistent with this view, PD1 blockade enhanced the anti-tumor effect of RNA-LPX vaccines in a mouse model with advanced tumors that are insensitive to anti-PD1 monotherapy (Reference 18). Notably, the tumor regression rates observed with the melanoma FixVac/anti-PD1 combination in pre-treated, CPI-experienced patients (over 35%) were within the range of the objective response rates achieved with PD1 blockade alone in patients with CPI-naive metastatic melanoma (Ref. 19).

Third, the findings presented in Examples 1 to 6 support the usefulness of non-mutated shared TAAs as cancer vaccine targets. Over the past two decades, clinical effects in TAA-based cancer vaccine trials have been largely disappointing in patients with advanced cancer and are often associated with relatively weak vaccine-induced immunity (reference 20). T cells against cancer mutations have been identified as drivers of CPI-mediated clinical efficacy—along with advances in technology that enable personalized cancer vaccination—promote the view that cancer mutations that are not affected by central tolerance mechanisms are more attractive vaccine targets. However, the data shown in Examples 1 to 6 show that T cell tolerance to non-mutant TAAs can be overcome by effective vaccine categories. PD1 blockade works by amplifying pre-existing antigen-specific T cells, many of which target new antigens of mutation origin (reference 21). More than half of patients with metastatic melanoma: have moderate to low mutation loads, which are associated with a lower probability of pre-formed new antigen-specific T cells; and are at a higher risk of anti-PD1 treatment failure and thus disease progression (reference 22). Given that the four TAAs targeted here are highly prevalent in human melanoma (Refs. 10, 23, 24), and their expression is not associated with tumor mutational burden (Fig. 4g), melanoma FixVac sensitized, activated, and expanded a complementary pool of CD4 ⁺ and CD8 ⁺ T cells. Therefore, vaccines based on non-mutant TAAs in combination with anti-PD1 therapy may have particular clinical utility for tumor control in patients with lower mutational burden, including those who have undergone CPI therapy.

Example 7: Exemplary Dosing (eg, Dose Escalation)

In some embodiments, the pharmaceutical compositions provided herein can be administered to patients with melanoma as a monotherapy and/or in combination with other anti-cancer treatments (e.g., such as immune checkpoint inhibitors). In some embodiments, the melanoma patient to be treated is a patient with anti-PD1 refractory/recurrent, unresectable stage III or IV melanoma.

In some embodiments, administration involves at least 8 doses within 10 weeks. In some embodiments, administration may also involve monthly doses following a 10-week dosing regimen.

In some embodiments, administration involves 6 weekly doses of a pharmaceutical composition described herein (e.g., FixVac), followed by 2 biweekly doses of a pharmaceutical composition described herein (e.g., FixVac). In some embodiments, administration may also involve monthly doses after 2 biweekly doses.

In some embodiments, administration involves 5 weekly doses of a pharmaceutical composition described herein (e.g., FixVac), followed by 2 biweekly doses of a pharmaceutical composition described herein (e.g., FixVac). In some embodiments, administration may also involve monthly doses after 2 biweekly doses.

In some embodiments where a combination therapy is administered, a pharmaceutical composition described herein (e.g., FixVac) may be administered on the same day as an immune checkpoint inhibitor. In some such embodiments, a pharmaceutical composition described herein (e.g., FixVac) and an immune checkpoint inhibitor may be administered separately.

In some embodiments, a pharmaceutical composition described herein (e.g., FixVac) is administered on the same day as immune checkpoint inhibitor therapy.

In some embodiments, dose escalation can be performed. In some such embodiments, administration can be performed at one or more levels shown in Table 6; in some embodiments, dose escalation can involve administering at least one lower dose from Table 6, followed by administering at least one higher dose from Table 6.

Table 6: Exemplary Dosing

Dosage Levels Dosage (μg total RNA) 1 7.2 2 14.4 3 29 4 50 5 75 6 100 7 200 8 400

In some embodiments, additional or alternative dosage levels may be evaluated, for example, including, for example, 7.5, 8, 9, 10, 11, 12, 13, 1415, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400 μg of total RNA. The efficacy of the treatment can be assessed by immune monitoring and/or clinical anti-tumor activity.

Example 8: Some exemplary immune checkpoint inhibitors that can be used in combination with the pharmaceutical compositions described herein

Approved immune checkpoint inhibitors can be used to treat certain cancers, including melanoma. Some non-limiting examples of FDA-approved immune checkpoint inhibitors include ipilimumab, cemiplimab, nivolumab, pembrolizumab, atezolizumab, avelumab and durvalumab. Some other examples of immune checkpoint inhibitors currently under study may include dostarlimab, INCMGA00012, toripalimab, SHR-1210, INCB086550 (oral PD-1 inhibitor), PDR001, HX008 and CX-072.

In some embodiments, immune checkpoint inhibitors can be administered according to a regimen indicated as monotherapy for the treatment of certain cancers, for example, every 3 weeks in some embodiments.

Example 9: Exemplary Adverse Events

In some embodiments, one or more indicators of potential adverse events of the object of monotherapy as described herein can be monitored within a period of time of the treatment regimen. The clinical adverse event spectrum is dominated by mild to moderate flu-like symptoms (e.g., fever and chills). Most of the adverse events are early-onset, transient and controlled with antipyretics, and disappear within 24 hours (Figure 32). In some embodiments, particularly for the object of receiving monotherapy as described herein, one or more fever, chills, headache, fatigue, nausea, tachycardia, feeling cold, joint pain, limb pain, vomiting, lymphocyte count reduction, interferon gamma level improvement, hypertension, dizziness, diarrhea, alpha tumor necrosis factor improvement, flu-like illness and white blood cell count reduction of the object can be monitored.

Example 10: Exemplary Stopping Criteria

In some embodiments, treatment as described herein may be discontinued if, for example, (i) a patient experiences an adverse event (AE) that meets drug limiting toxicity (DLT) criteria; (ii) a patient experiences an AE that meets DLT criteria after a dosing cycle that fails to resolve to ≤ Grade 1 within a predetermined time period; (iii) dosing is delayed beyond the dosing cycle due to toxicity that may be related to the administered treatment; (iv) a drug-related or life-threatening Grade 4 AE (excluding asymptomatic Grade 4 elevations in non-hematologic laboratory values that resolve to ≤ Grade 2 [with or without medical intervention1] within 14 days) that does not meet DLT criteria, unless otherwise approved by a medical monitor; (v) a second occurrence of a Grade ≥ 3 infusion related reaction (IRR) despite premedication prior to a second administration; and/or (vi) a first occurrence of an allergic reaction or Grade 4 IRR.

Example 11: Exemplary Evaluations and/or Criteria for RNA Molecules Described herein

In some embodiments, one or more assessments as described herein can be used during manufacture or other preparation or use of an RNA molecule (e.g., as a release test).

In some embodiments, one or more quality control parameters may be evaluated to determine whether the RNA molecules described herein meet or exceed acceptance criteria (e.g., for subsequent formulation and/or release for distribution). In some embodiments, such quality control parameters may include, but are not limited to, RNA integrity, RNA concentration, residual DNA template, and/or residual dsRNA. Methods for evaluating RNA quality are known in the art; for example, one skilled in the art will recognize that in some embodiments, one or more analytical tests may be used for RNA quality assessment, such as, for example, capillary gel electrophoresis for RNA integrity, UV absorption spectrophotometry for RNA content and/or concentration, quantitative PCR for residual DNA template, immuno-based assays for residual dsRNA, and detection of translated antigens.

In some embodiments, the RNA batch can be evaluated, for example, for RNA integrity, RNA content and/or concentration, residual DNA template, residual dsRNA, antigen expression, or a combination thereof to determine the next course of action. For example, if the RNA quality assessment indicates that such a batch of RNA molecules meets or exceeds a predetermined acceptance criterion, the batch of RNA molecules can be designated for one or more additional steps of manufacturing and/or formulation and/or distribution. Otherwise, if such a batch of RNA molecules does not meet or exceed the acceptance criterion, an alternative action can be taken (e.g., discarding the batch).

Example 12: Exemplary inclusion criteria

In some embodiments, cancer patients who meet one or more of the following disease-specific inclusion criteria are selected for treatment with the compositions and/or methods described herein:

Group I: Stage IV malignant melanoma (AJCC 2009 melanoma classification)

Cohorts II to VII and end of expansion cohort: Stage IIIB to IIIC or IV malignant melanoma (AJCC 2009 Melanoma Classification), expansion cohort C only for patients with stage IV melanoma (AJCC 2009 Melanoma Classification) with measurable disease (at least one target lesion according to irRECIST 1.1) [applicable to all patients after approval of protocol version 10.0]

Treatment only for subjects who are ineligible for or refuse any other available approved treatment after all available treatment options have been transparently disclosed (to be documented).

Expression of any of the four TAAs determined by RT-qPCR analysis from FFPE

Age ≥ 18 years

Written informed consent

ECOG performance status (PS) 0 to 1

Life expectancy >/= 6 months

WBC ≥ 3 × 10E9/L

Hemoglobin ≥ 9 g/dL

Platelet count ≥100,000/ ^mm3

ALT/AST < 3 × ULN (except patients with liver metastases)

Negative pregnancy test (measured by beta-HCG) for women of childbearing age

Example 13: Exemplary Exclusion Criteria

In some embodiments, the cancer patient has melanoma that is not amenable to the compositions and/or methods described and/or used herein.

In some embodiments, cancer patients who (i) have recently received cancer treatment; (ii) are concurrently receiving systemic steroid therapy; (iii) have recently undergone major surgery; (iv) have an active infection and are being treated with anti-infective therapy; and/or (v) have been diagnosed with growing brain or leptomeningeal metastases are not suitable for the compositions and/or methods described and/or used herein.

In some embodiments, the following cancer patients may not be recommended for treatment with the pharmaceutical compositions described herein. Exclusion criteria include:

Pregnancy or breastfeeding

Primary ocular melanoma

A second malignancy other than squamous cell or basal cell carcinoma, inactive prostate cancer, or cervical cancer in situ or inactive treated urothelial carcinoma

Brain metastases

○ Patients with a history of treated or inactive brain metastases are eligible for treatment in expansion cohort C if they meet all of the following criteria:

○ Measurable disease outside the brain (except inactive brain metastases);

○ No ongoing need for corticosteroids as treatment for brain metastases,

○ Corticosteroids discontinued ≥1 week prior to Visit 2 (Day 1) and no ongoing symptoms attributable to brain metastases;

○ Screening brain radiographic imaging ≥ 4 weeks from completion of radiation therapy

Patients after splenectomy

Known hypersensitivity to the active substance or to any of the excipients

Severe local infection (e.g., cellulitis, abscess) or systemic infection (e.g., pneumonia, sepsis) requiring systemic antibiotic therapy within 2 weeks prior to the first dose of study drug

A positive test for acute or chronic active hepatitis B or C infection

Clinically relevant active autoimmune disease

Systemic immunosuppression:

HIV disease

○Use of chronic oral or systemic steroid medications (topical or inhaled steroids are permitted)

○Other clinically relevant systemic immunosuppression

Symptomatic congestive heart failure (NYHA 3 or 4)

Unstable angina

Radiation therapy and minor surgery within 14 days before the first dose of study treatment

Myelosuppressive chemotherapy within 14 days before the first study treatment administration and after blood value reconstitution

Received ipilimumab within 28 days prior to the first dose of study treatment

Treatment with a BRAF inhibitor, MEK inhibitor, or combination of both, and an anti-PD-1 antibody within 14 days prior to the first dose of study treatment (at the discretion of the investigator, not applicable to patients receiving parallel treatment in expansion cohorts A, B, or C)

Receipt of interferon, major surgery, vaccinations, or other investigational agents within 28 days or 5 half-lives (whichever is longer) prior to first treatment

· Use of approved BRAF inhibitors vemurafenib or dabrafenib, approved anti-PD-1 inhibitors nivolumab or pembrolizumab, and approved MEK inhibitor trametinib, or a combination of approved BRAF-MEK inhibitors in patients in the dose escalation cohort. Following analysis of safety data collected for the dose escalation cohort and DSMB approval, concomitant treatment with approved BRAF inhibitors, approved anti-PD-1 antibodies or MEK inhibitors, and a combination of approved BRAF-MEK inhibitors will be allowed for patients included in the expansion cohort. Local irradiation will also be allowed as a concurrent treatment for patients in the expansion cohort.

- Following approval of protocol version 10.0, only anti-PD-1 antibodies were allowed to treat patients in expansion cohort C.

Fertile males and females who are unwilling to use a highly effective birth control method (less than 1% per year, such as spermicide-containing condoms, spermicide-containing diaphragms, birth control pills, injections, patches, or intrauterine devices) during study treatment and for at least 28 days (male patients) and 90 days (female patients of childbearing potential) after the last dose of study treatment

Serious concurrent illness or other conditions (e.g., psychological, family, social, or geographic circumstances) that would preclude adequate follow-up and adherence to the protocol

Example 14: Exemplary Efficacy Assessment and/or Detection

In some embodiments, cancer patients administered a pharmaceutical composition described herein as a monotherapy or in combination with an additional anti-cancer treatment may be periodically monitored for treatment efficacy and/or adjustment of treatment dosage/regimen.

In some embodiments, the efficacy of treatment can be assessed by computed tomography and/or magnetic resonance imaging scans. In some embodiments, MRI scans can be performed using a 3 Tesla whole body instrument. In some embodiments, when evaluating lesions for efficacy assessment, one or more of the following criteria can be used:

o Complete response: All target lesions disappear. Any pathological lymph node (whether target or non-target) must be reduced to <10 mm in short axis.

○ Partial response: A decrease of at least 30% in the sum of diameters of target lesions, with the baseline sum diameter as reference.

○ Progressive disease: The sum of the diameters of the target lesions increases by at least 20%, taking as reference the smallest sum on study (including the baseline sum if it is the smallest sum on study). In addition to the 20% relative increase, the sum must also show an absolute increase of at least 5 mm. The appearance of one or more new lesions is also considered progression.

○ Stable disease: neither sufficient shrinkage to qualify for a PR nor sufficient improvement to qualify for progressive disease, using the smallest sum diameter on study as reference.

Example 15: Immune Responses in Patients with Evidence of Disease versus Patients without Evidence of Disease

This example shows ex vivo characterization of immune responses following administration of an exemplary pharmaceutical composition comprising one or more RNA molecules collectively encoding a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof, and lipid particles to patients with evidence of disease (ED) and patients without evidence of disease (NED).

Background: Lipo-MERIT is an ongoing, first-in-human, open-label, dose-escalating Phase I trial that studies the safety, tolerability, and immunogenicity of BNT111 in patients with advanced melanoma. BNT111 is a ribonucleic acid lipoplex (RNA-LPX) vaccine that targets melanoma tumor-associated antigens (TAA) New York esophageal squamous cell carcinoma 1 (NY-ESO-1), tyrosinase, melanoma-associated antigen 3 (MAGE-A3), and transmembrane phosphatases (TPTEs) with tensin homology. As shown in Examples 1 to 6, BNT111 alone or in combination with immune checkpoint inhibitors (CPIs) has a favorable adverse event (AE) spectrum, generating antigen-specific T cell responses and inducing durable objective responses in CPI-experienced patients with unresectable melanoma. This example shows the immunogenicity, efficacy, and safety data of patients with no evidence of disease (NED) in a trial that included a subgroup of BNT111 monotherapy.

Methods: BNT111 was administered intravenously to patients with stage IIIB, IIIC, and IV cutaneous melanoma according to a prime and boost regimen. Patients were treated in seven dose-escalation cohorts (dose range: 7.2 μg to 400 μg total RNA) and three expansion cohorts that further explored dose levels of 14.4 μg, 50 μg, and 100 μg. In this analysis, patients receiving BNT111 monotherapy were grouped as having evidence of disease (ED) or NED, and immunogenicity, efficacy (by solid tumor immune-related response evaluation criteria), and safety were evaluated. Vaccine-induced immune responses were directly analyzed in vitro using interferon-γ enzyme-linked immunosorbent spot (ELISpot) assays.

Results: As of May 24, 2021, 115 patients received BNT111 in the Lipo-MERIT trial. Of the 71 patients treated with BNT111 monotherapy, 38 had ED after prior treatment and 33 had NED. Baseline characteristics were similar between the two groups. ELISpot data showed comparable BNT111-induced T cell responses against at least one TAA in ED vs. NED patients (14/22 [64%] and 19/28 [68%] patients with available ELISpot-evaluable samples, respectively), suggesting that BNT111 has the ability to induce T cell immunity even in the absence of detectable tumors. In NED patients, clinical efficacy was promising, and the median disease-free survival was 34.8 months (95% confidence interval: 7.0 to not reached). The safety profile in ED and NED patients was similar, with 38/38 (100%) and 32/33 (97%) patients, respectively, experiencing relevant treatment-emergent AEs (TEAEs), most of which were mild to moderate influenza-like symptoms.

In particular, samples from patients with ED and NED were analyzed using ex vivo ELISpot (Figures 20a to c), where autologous dendritic cells loaded with TAA PepMix were used as targets. Figures 20a to c show the frequency of patients with vaccine-induced (expanded or de novo) responses: CD4 ⁺ or CD8 ⁺ (Figure 20a); CD4 ⁺ (Figure 20b); or CD8 ⁺ (Figure 20c) responses. The numbers in the bar segments represent the number of patients evaluated in each segment. Only patients treated with a single treatment were included. Unexpectedly, samples from NED patients showed stronger vaccine-induced responses (e.g., CD4 ⁺ or CD8 ⁺ (Figure 20a); CD4 ⁺ (Figure 20b); or CD8 ⁺ (Figure 20c)) than samples from ED patients.

The results of the ex vivo ELISPOT were also compared by cell type. As shown in Figures 21 to 22, TAAs induced more pronounced and diverse immune responses in NED patients compared to ED patients. The de novo response was compared to the expanded response in the ex vivo ELISPOT assay (assessing CD4 ⁺ or CD8 ⁺ responses to any cell type), showing that the de novo response in each (4/4 antigen) NED patient population was 100% compared to half of the ED (2/4 antigen) patient population.

Samples from patients with ED and NED were analyzed using ELISpot after IVS (Figures 23a to c), where autologous dendritic cells loaded with TAA PepMix were used as targets. Figures 23a to c show the frequency of patients with vaccine-induced (expanded or de novo) responses: CD4 ⁺ or CD8 ⁺ (Figure 23a); CD4 ⁺ (Figure 23b); or CD8 ⁺ (Figure 23c) responses. The numbers in the bar segments represent the number of patients evaluated in each segment. Only patients treated with a single treatment were included. Unexpectedly, samples from NED patients showed stronger vaccine-induced responses than samples from ED patients (e.g., CD4 ⁺ or CD8 ⁺ (Figure 23a); CD4 ⁺ (Figure 23b); or CD8 ⁺ (Figure 23c)).

As shown in Figures 24 and 25, the upper figure shows patients with non-evaluable disease, and the lower figure shows patients with evaluable disease. The numbers in the bar segments represent the number of patients with evaluated ex vivo ELISPOT measurements in each segment. Only patients treated with a single treatment are included.

Figure 26a shows the disease-free survival data of NED patients based on the number of events (eg, death, relapse, and new treatment started) and the number of censors. Figure 26b shows the Kaplan-Meier summary of the disease-free survival data of NED patients.

Figures 27a to 27c show the overall survival data for ED patients (Figure 27a), NED patients (Figure 27b), and ED and NED patients combined (Figure 27c) based on the number of events (e.g., death, relapse, and new treatment started) and the number of censoring. Figures 27d to 27f show the Kaplan-Meier summary of the overall survival data for ED patients (Figure 27d), NED patients (Figure 27e), and ED and NED patients combined (Figure 27f).

Figures 28a to 28c show a summary of adverse events for ED patients (Figure 28a), NED patients (Figure 28b), and ED and NED patients combined (Figure 28c).

Conclusions: The immunogenicity and safety profiles of BNT111 as monotherapy were comparable in ED and NED patients, and promising signs of clinical activity were observed in NED patients.

Example 16: Pharmacology and immune responses after administration of BNT111

This example shows the immune response detected after administration of BNT111 to a patient.

Cytokines, such as IFN-γ, IFN-α, TNF-α, IP-10, IL-2, IL-6, IL-10 and IL-12 (p70), are analyzed at different time points up to 36 days after the vaccination from baseline (i.e. before the vaccination), and are frequently sampled during the first 48 hours after the vaccination. The patient shows a dose-dependent, short-term increase and elevated body temperature of the plasma levels of different cytokine profiles. Cytokine release is pulsed and reaches a peak value in approximately 2 to 6 hours after administration and the value returns to baseline in 24 hours or earlier. Observe that the cytokine based on IFN-α includes the activation of IFN-γ and continuous IP-10 patterns and the activation of IL-12, IL-6 and TNF-α.

In blood samples from 20 patients analyzed with IFN-γ-ELISpot after in vitro expansion, T cell responses against at least one TAA were observed in each patient. These included T cell specificities that were not detected at baseline and induced de novo by the vaccine, as well as T cell specificities that were present at low levels at baseline and expanded and amplified by the vaccine antigens.

In 80 patients, IFN-γ-ELISpot was performed ex vivo without prior in vitro stimulation. In 72.5% of these patients, a robust immune response against at least one TAA was induced to levels detectable ex vivo.

All four TAAs were immunogenic. Most patients showed either a single CD4 ⁺ response or a simultaneous CD4 ⁺ and CD8 ⁺ T cell response to a single TAA.

T cell responses, including de novo sensitized T cell responses, were found to be rapidly induced to high magnitude within 4 to 8 weeks and to persist for several months. In some patients, antigen-specific CD8 ⁺ T cell responses accounting for more than 10% of all peripheral blood CD8 ⁺ T cells were observed.

In selected cases, expansion of T cell specificity was observed in parallel with a reduction in tumor burden.

Example 17: Efficacy data after administration of BNT111

This example provides a summary of the preliminary efficacy data observed following administration of BNT111.

Preliminary efficacy is presented for BNT111 monotherapy, BNT111 with nivolumab or pembrolizumab, and for BNT111 in combination with BRAF/MEK inhibitors. Figure 42 provides details of the best overall response for each of these treatment groups according to the highest dose administered.

Of the 115 patients, 75 (68%) patients with unresectable stage III or IV melanoma showed evaluable disease at baseline, including 4 patients with only non-target lesions. The subgroup efficacy analysis group included patients with evaluable disease at baseline who received at least one dose of BNT111 and had baseline and at least one treatment/after treatment tumor response assessment (N=75).

Efficacy is given for 36 patients receiving BNT111 monotherapy, 36 patients receiving BNT111 with nivolumab or pembrolizumab, and three patients receiving BNT111 with BRAF/MEK inhibitors. All 36 BNT111 monotherapy patients had received prior treatment with checkpoint inhibitors, and among patients treated with BNT111 in combination with PD-1 inhibitors, 35/36 patients had received prior treatment with checkpoint inhibitors. Most patients had progressive disease at the start of treatment.

Among the 36 patients with evaluable disease at baseline who were treated with BNT111 monotherapy, the best overall response (the best response recorded from the start of trial treatment until disease progression/relapse) included 1 patient with CR (3%), 3 patients with PR (8%), and 9 patients with SD (25%). The overall response rate was 11%, and the disease control rate was 36%. The median duration of response was 8.4 months (95% confidence interval [CI]: 6.2 to 33.3 months).

Among the 36 patients evaluable for efficacy analysis who were treated with BNT111 cancer vaccine in combination with nivolumab or pembrolizumab, the best overall response included 9 patients (25%) who achieved PR and 8 patients (22%) who had SD, resulting in an overall response rate of 25% and a disease control rate of 47%. The median duration of response was 22.9 months (95% CI: 3.0 to 22.9 months).

Of the three patients evaluable for efficacy and treated with the BNT111 cancer vaccine in combination with a BRAF/MEK inhibitor, 1 (33.3%) patient achieved SD.

[0046] Figure 43 depicts the best change from baseline in target lesions according to irRECIST in patients with measurable disease treated with monotherapy or in combination with nivolumab or pembrolizumab or BRAF/MEK inhibition.

Example 18: Safety Analysis

This example provides an assessment of the safety of exemplary compositions described herein.

BNT111 was administered to 115 patients with melanoma. BNT111 demonstrated a favorable safety and tolerability profile as a monotherapy (n=38). Thirty-eight patients received BNT111 in combination with pembrolizumab or nivolumab, both of which were administered according to their respective product labels. The favorable safety and tolerability of the combination was also demonstrated.

Almost all patients in the subgroup had TEAEs related to the study drug. The overall safety profile between the treatment subgroups (e.g., BNT111 monotherapy relative to BNT111 combined with PD-1 inhibitors or BRAF/MEK) was comparable, with only small differences noted. However, the number of patients treated in combination with BRAF/MEK inhibitors was too small to draw any conclusions.

The overall safety profile of the combination therapy with PD-1 inhibitors relative to BNT111 monotherapy was comparable in terms of flu-like symptoms (reactogenicity) (e.g., fever, chills, tachycardia, and headache). The most important TEAEs that were more common in the PD-1 combination therapy subgroup compared to BNT111 monotherapy were syncope (13% vs. 0%) and melanocytic naevus (13% vs. 3%).

Differences in gastrointestinal AEs such as nausea (55% vs. 17%), vomiting (29% vs. 17%), diarrhea (11% vs. 3%), and decreased appetite (13% vs. 3%) were noted in the PD-1 inhibitor combination subgroup versus the BNT111 monotherapy subgroup.

In addition, differences in hypotension were noted between the PD-1 inhibitor combination subgroup relative to the BNT111 monotherapy subgroup (24% vs. 9%). A higher number of joint pains was reported in the BNT111 monotherapy subgroup (31% vs. 11%). In the Lipo-MERIT Phase I trial, no dose-limiting toxicity (DLT) was reported during dose escalation (from 7.2 μg to the highest administered dose of 400 μg total RNA).

No drug-related deaths were reported. 11/115 (8%) patients died within the main course of the trial, i.e. within 90 days after the last trial treatment. No deaths were considered related to BNT111. Most patients died due to disease progression and general physical health deterioration.

TEAEs considered to be related to study drug were transient, mostly flu-like symptoms, and Common Terminology Criteria for Adverse Events (CTCAE) grades 1 and 2.

Thirteen of 115 patients (11%) experienced treatment-related TESAEs; 30/115 (26%) patients experienced treatment-related TEAEs of CTCAE grade ≥3; 19/115 (17%) patients had treatment-related TEAEs that led to permanent trial treatment discontinuation, and 19/115 (17%) patients had treatment-related TEAEs that led to dose reduction. Table 7 provides an overview of TEAEs by category.

Table 7: Lipo-MERTT - Summary of the number and percentage of patients with at least one TEAE by subgroups ^{1 to 4}

1Data extraction date/eCRF data extraction is as of May 24, 2021.

2 AEs lacking actions taken with BNT111 were not conservatively considered dose reductions. For one event, this entry was missing in the eCRF: In one patient in the expansion cohort C (BNT111 + PD-1 inhibitor treatment group), there was an event of increased CRP (not yet MedDRA coded, CTCAE grade not reported, considered unrelated).

3 AEs lacking causality were conservatively considered not to be related to BNT111. This applies to the following events: one event of right flank pain (not yet MedDRA coded in eCRF); in one patient in expansion cohort C (BNT111 + PD-1 inhibitor treatment group), the CTCAE grade was not provided in the eCRF.

4 One patient who received BNT111 as monotherapy at the first recruitment and BNT111 + BRAF/MEK at the second recruitment is represented in this table. Therefore, there is a difference between the totals shown and the sum of the individual treatments.

AE = adverse event; CRP = C-reactive protein; CTCAE = Common Terminology Criteria for Adverse Events; DLT = dose-limiting toxicity; eCRF = electronic case report form; MedDRA = Medical Dictionary for Regulatory Activities; MEK = mitogen-activated protein kinase kinase; PD-1 = programmed death 1; PT = preferred term; SAE = serious adverse event; TE = treatment-emergent; TEAE = treatment-emergent adverse event; TESAE = treatment-emergent serious adverse event.

Table 8 provides a summary of the frequency of relevant treatment-emergent serious adverse events (TESAEs) by worst CTCAE grade, and Table 9 provides a summary of the same data by treatment subpopulation. Table 8: Lipo-MERIT - Number of Patients with Worst CTCAE Grade Relevant TESAE ¹ by PT (N=115) ²

1 TESAEs were defined as occurring after initiation of study drug until 90 days after the last intake of study drug. The table includes TESAEs from both treatment groups for four dual-enrolled patients.

2Data extraction date/eCRF data extraction is as of May 24, 2021.

AE = adverse event; PT = preferred term; TESAE = treatment-emergent serious adverse event.

Table 9: Lipo-MERIT - Number of patients with relevant TESAEs treated with BNT111 monotherapy or PD-1 inhibitor combination (N=115) ^{1 to 4}

1Data extraction date/eCRF data extraction is as of May 24, 2021.

2 TESAEs were defined as occurring after the start of study drug administration until 90 days after the last intake of study drug.

3A patient may have a TESAE coded with more than one preferred term.

4 One patient who received BNT111 as monotherapy at the first recruitment and BNT111 + BRAF/MEK at the second recruitment is represented in this table. Therefore, there is a difference between the totals shown and the sum of the individual treatments.

PD-1 = programmed death 1; TESAE = treatment-emergent serious adverse event.

It is noteworthy that in the Lipo-MERIT trial, eight patients are still treated with BNT111 monotherapy (n=2) or a combination of BNT111 and PD-1 inhibitors (n=6) for trial treatment. All eight patients who used multiple previous series of treatments are in so-called "continuation treatment", and their treatment duration is 15 to 52 months. Initially, "continuation treatment" was provided only when all IMP components (based on four precursor RNARBL001.1, RBL002.2, RBL003.1 and RBL004.1) were in stock. However, since these eight patients with a large number of pre-treatments have achieved at least stable disease or response (according to irRECIST, partial remission or complete remission), and therefore continue to obtain clinical benefits from trial treatment, the trial is not stopped, but instead continues to provide further trial treatment (so-called "extended treatment") with current BNT111 substances. For the benefit of these patients, the trial is allowed to continue.

Example 19: Pharmacological data obtained in mice

Mice can be a relevant species for evaluating the primary and secondary pharmacological effects as well as potential toxicological effects of RNA-LPX complexes, and thus capture the potential substance-specific (i.e., RNA molecule-specific) toxicity of RNA-LPX. Mice exhibit all primary and secondary pharmacological effects, from inducing CD4+ and/or CD8+ T cell responses to enhancing immune responses and leading to subsequent immunomodulatory effects of TLR triggering, cell activation, and cytokine secretion. However, given the species specificity of the BNT111 TAA and the unique set of MHC molecules in each patient that can present a large set of antigenic peptides, there are no relevant and conclusive mouse tumor models for human melanoma TAAs encoded by BNT111, and it is not feasible to conduct pharmacodynamic studies in mice for BNT111 as a single agent or in combination with checkpoint blockade. Therefore, most of the primary pharmacodynamic, mechanism of action, and antitumor activity studies were conducted in mice with RNA-LPX vaccines encoding model antigens.

Nonclinical studies performed have shown that in mice, vaccination with RNA LPX induced DC maturation and activation of major lymphocyte subsets in the spleen, and released systemic cytokines within the first 3 to 6 hours, including IFNα, TNFα, IP-10, and IL 6 in response to TLR7 triggered by single-stranded RNA (Kranz et al. 2016, which is incorporated herein by reference in its entirety). Transient leukopenia coincided with peak IFNα levels and can be attributed to IFNα downstream effects.

Vaccination with RNA LPX in mice effectively primed and expanded de novo cytotoxic CD4+ and CD8+ T cells targeting BNT111-encoded antigens NY ESO 1, tyrosinase, MAGE A3, TPTE, and other melanoma-associated or model antigens. Following in vitro co-incubation, human DCs loaded with BNT111 RNA were able to stimulate IFN-γ production by antigen-specific CD8+ T cells expressing the corresponding TCR in an RNA dose-dependent manner.

The induced antigen-specific CD8+ T cells were shown to be able to infiltrate mouse tumors, and RNA LPX vaccination was associated with polarization of the tumor microenvironment toward a pro-inflammatory, cytotoxic, and less immunosuppressive context. RNA LPX vaccination was shown to trigger antigen release from tumors, which enabled further expansion of vaccine-induced tumor-specific T cells, even after treatment cessation.

Tumor-infiltrating CD8+ T cells upregulated the expression of PD 1 in response to RNA LPX vaccination, and PD L1 was significantly expressed by tumors. T cells with high PD 1 expression are considered to have high antigen affinity. As hypothesized, the combination of RNA LPX vaccination with PD 1/PD L1 checkpoint blockade acted synergistically in inhibiting tumor growth and improving survival by sensitizing PD 1/PD L1 blockade-resistant mouse tumors to the therapeutic combination. The enhancement of vaccine-induced breaking of tolerance to self-antigens expressed by B16 melanoma by PD 1/PD L1 blockade further suggests the strong anti-tumor activity of the combination of RNA LPX vaccination with PD 1/PD L1 blockade.

Table 10 summarizes the nonclinical primary pharmacodynamic studies conducted with BNT111.

Table 10: Summary of major non-clinical pharmacodynamic studies of BNT111

DC = dendritic cell; dsRNA = double-stranded RNA; HA = influenza hemagglutinin; hiDC = human immature DC; HPV = human papillomavirus; IFN = interferon; IFNAR1 = interferon alpha and beta receptor subunit 1; IL = interleukin; IP10 = interferon-gamma-induced protein 10; IV = intravenous; MHC = major histocompatibility complex; NK = natural killer cell; OVA = ovalbumin; PBMC = peripheral blood mononuclear cell; pDC = plasmacytoid DC; PD-1 = programmed death ligand 1; PD-L1 = programmed death protein 1; SD = single dose; RD = repeated dose; TAM = tumor-associated macrophage; TCR = T cell receptor; tg = transgenic; TIL = tumor infiltrating leukocyte; TLR = toll-like receptor; TME = tumor microenvironment; TNF = tumor necrosis factor; Treg = CD4 ⁺ CD25 ⁺ FoxP3 + T regulatory cells; TRP = tyrosinase-related protein; WB = whole blood. *The initial development (applied to the Lipo-MERIT trial) was based on four prodrug products, RBL001.1, RBL002.2, RBL003.1 and RBL004.1, encoding the same target but with slightly improved properties, such as translatability and stability, against RNA.

To further elucidate the main pharmacodynamics, mechanism of action, and antitumor activity of BNT111 and to provide a rationale for the combination of BNT111 with PD-1/PD-L1 checkpoint blockade, RNA-LPX vaccines encoding model antigens (e.g., human papillomavirus 16 oncoprotein E7) or other melanoma-associated antigens (tyrosinase-related proteins 1 and 2) were applied.

Table 11: Summary of major nonclinical pharmacodynamic studies supporting BNT111

CCL=CC chemokine ligand, CCR=CC chemokine receptor, CTLA=cytotoxic T lymphocyte-associated protein, CXCL=CXC chemokine ligand, HA=influenza hemagglutinin, HPV=human papillomavirus, ICOS=inducible T-cell co-stimulation, IFN=interferon, IL=interleukin, gzm=granzyme, PD-1=programmed death-1, PD-L1=programmed death ligand 1, SC=subcutaneous, TAM=tumor-associated macrophage, TBX=T-box transcription factor, TCR=T cell receptor, tg=transgenic, TIL=tumor infiltrating leukocytes, TLR=toll-like receptor, TME=tumor microenvironment, TNF=tumor necrosis factor, Treg=CD4 ⁺ CD25 ⁺ FoxP3 ⁺ T regulatory cells, TRP=tyrosinase-related protein, WB=whole blood.

References

Throughout this disclosure, reference may be made to the following references. Each is incorporated herein by reference in its entirety.

1. Melero, I. et al. Therapeutic vaccines for cancer: an overview of clinical trials. Nat. Rev. Clin. Oncol. 11, 509-524 (2014).

2. Romero, P. et al. The Human Vaccines Project: a roadmap for cancervaccine development. Sci. Transl. Med. 8, 334ps9 (2016).

3. Coulie, P.G., Van den Eynde, B.J., van der Bruggen, P. & Boon, T. Turnoutantigens recognized by T lymphocytes: at the core of cancerimmunotherapy. Nat. Rev. Cancer 14, 135-146 (2014).

4. Kyewski, B. & Derbinski, J. Self-representation in the thymus: an extended view. Nat. Rev. Immunol. 4, 688-698 (2004).

5. Holtkamp, S. et al. Modification of antigen-encoding RNA increases stability, translational efficacy, and T-cell stimulatory capacity of dendritic cells. Blood 108, 4009-4017 (2006).

6. Orlandini von Niessen, A.G. et al. Improving mRNA-based therapeuticgene delivery by expression-augmenting 3′UTRs identified by cellular libraryscreening. Mol. Ther. 27, 824-836 (2019).

7. Kreiter, S. et al. Increased antigen presentation efficiency by coupling antigens to MHC class I trafficking signals. J. Immunol. 180, 309-318 (2008).

8. Kranz, L.M. et al. Systemic RNA delivery to dendritic cells exploits antiviral defense for cancer immunotherapy. Nature 534, 396-401 (2016).

9. De Vries, J. & Figdor, C. Immunotherapy: cancer vaccine triggers antiviral-type defences. Nature 534, 329-331 (2016).

10. Simon, P. et al. Functional TCR retrieval from single antigen-specific human T cells reveals multiple novel epitopes. Cancer Immunol. Res. 2, 1230-1244 (2014).

11. Cheerer, M.A. et al. The prioritization of cancer antigens: a national cancer institute pilot project for the acceleration of translational research. Clin. Cancer Res. 15, 5323-5337 (2009).

12. Pektor, S. et al. Toll like receptor mediated immune stimulation can be visualized in vivo by [18F]FDG-PET. Nucl. Med. Biol. 43, 651-660 (2016).

13. Reinhard, K. et al. An RNA vaccine drives expansion and efficacy of claudin-CAR-T cells against solid tumors. Science 367, 446-453 (2020).

14. Pektor, S. et al. In vivo imaging of the immune response upon systemic RNA cancer vaccination by FDG-PET. EJNMMI Res. 8, 80 (2018).

15. Jackson, H. et al. Striking immunodominance hierarchy of naturally occurring CD8+ and CD4+ T cell responses to tumor antigen NY-ESO-I.J. Immunol. 176, 5908-5917 (2006).

16. Hu, Y. et al. Immunologic hierarchy, class II MHC promiscuity, and epitope spreading of a melanoma helper peptide vaccine. Cancer Immunol. Immunother. 63, 779-786 (2014).

17.Hanagiri, T., van Baren, N., Neyns, B., Boon, T. & Coulie, P.G.Analysis of arare melanoma patient with a spontaneous CTL response to a MAGE-A3 peptide presented by HLA-A1.Cancer Immunol.Immunother .55, 178-184(2006).

18. Grunwitz, C. et al. HPV16 RNA-LPX vaccine mediates complete regression of aggressively growing HPV-positive mouse tumors and establishesprotective T cell memory. OncoImmunology 8, e1629259 (2019).

19. Robert, C. et al. Pembrolizumab versus ipilimumab in advanced melanoma. N. Engl. J. Med. 372, 2521-2532 (2015).

20. Rosenberg, S.A., Yang, J.C. & Restifo, N.P. Cancer immunotberapy: moving beyond current vaccines. Nat. Med. 10, 909-915 (2004).

21. Ribas, A. & Wolchok, J.D. Cancer immunotherapy using checkpointblockade. Science 359, 1350-1355 (2018).

22. Hugo, W. et al. Genomic and transcriptomic features of response to anti-PD-1 therapy in metastatic melanoma. Cell 165, 35-44 (2016).

23. Simpson, A.J.G., Caballero, O.L., Jungbluth, A., Chen, Y.-T. & Old, L.J. Cancer/testis antigens, gametogenesis and cancer. Nat. Rev. Cancer 5, 615-625 (2005).

24.Hofbauer, G.F., Kamarashev, J., Geertsen, R., R. & Dummer, R. Tyrosinaseimmunoreactivity in formalin-fixed, paraffin-embedded primary and metastatic melanoma: frequency and distribution. J. Cutan. Pathol. 25, 204-209 (1998).

25. Nishino, M., Gargano, M., Suda, M., Ramaiya, N.H. & Hodi, F.S. Optimizing immune related tumor response assessment: does it reduce the number of lesions impact response assessment in melanoma patients treated with ipilimumab? J. Immunother. Cancer 2, 17 (2014).

26. Sahin, U. et al. Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer. Nature 547, 222-226 (2017).

27. Grabbe, S. et al. Translating nanoparticulate-personalized cancervaccines into clinical applications: case study with RNA-lipoplexes for the treatment of melanoma. Nanomedicine 11, 2723-2734 (2016).

28.Batzri, S. & Kom, E.D. Single bilayer liposomes prepared without sonication. Biochim. Biophys. Acta 298, 1015-1019 (1973).

29.Barichello, J.M., Ishida, T. & Kiwada, H. Complexation of siRNA and pDNA with cationic liposomes: the important aspects in lipoplex preparation. Methods Mol. Biol. 605, 461-472 (2010).

30.Carey, T.E., Takahashi, T., Resnick, L.A., Oettgen, H.F. & Old, L.J.Cellsurface antigens of human malignant melanoma: mixed hemadsorption assays forhumoral immunity to cultured autologous melanoma cells.Proc.Natl Acad.Sci.USA73, 3278- 3282(1976).

31.Brochet, (2008).

32. Bolotin, D.A. et al. MiXCR: software for comprehensive adaptiveimmunity profiling. Nat. Methods 12, 380-381 (2015).

33. Shugay, M. et al. VDJtools: unifying post-analysis of T cell receptorrepertoires. PLOS Comput. Biol. 11, e1004503 (2015).

34. Li, H. & Durbin, R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 25, 1754-1760 (2009).

35. Patro, R., Mount, S.M. & Kingsford, C. Sailfish enables alignment-freeisoform quantification from RNA-seq reads using lightweight algorithms. Nat. Biotechnol. 32, 462-464 (2014).

36. Robinson, D.R. et al. Integrative clinical genomics of metastatic cancer. Nature 548, 297-303 (2017).

37. American Cancer Society: Cancer Facts & Figures 2021. Atlanta, GA: American Cancer Society; 2021. Available from: https://www.cancer.org/content/dam/eancer-org/research/cancer-facts-and-statistics /annual-cancer-facts-and-figures/2021/cancer-facts-and-figures-2021.pdf (accessed on August 26, 2021).

38. Banchereau J, Ueno H, Dhodapkar M, et al.Immune and clinical outcomes in patients with stage IV melanoma vaccinated with peptide-pulsed dendriticcells derived from CD34+progenitors and activated with type I interferon.JImmunother.2005;28(5): 505-16.

39.Brichard VG, Lejeune D.GSK's antigen-specific cancer immunotherapy program: pilot results leading to Phase III clinical development.Vaccine.2007;27;25 Suppl 2:B61-71.

40. Carrasco J, Van Pel A, Neyns B, et al. Vaccination of a melanomapatient with maturedendritic cells pulsed with MAGE-3 peptides triggers the activity of nonvaccine anti-tumorcells. J Immunol 2008; 180(5): 3585-93.

41. Chen Q, Jackson H, Shackleton M, et al. Characterization of antigen-specific CD8+T lymphocyte responses in skin and peripheral blood following intradermal peptide vaccination. Cancer Immun. 2005; 5:5.

42. Coricovac D, Dehelean C, Moaca EA, et al. Cutaneous Melanoma-A LongRoad from Experimental Models to Clinical Outcome: A Review. Int J MolSci. 2018; 19(6): 1566.

43. Demotz S, Lanzaveechia A, Eisel U, et al. Delineation of several DR-restricted tetanus toxin T cell epitopes. J Immunol. 1989; 142(2): 394-402.

44. Dredge K, Marriott JB, Todryk SM, Dalgleish AG. Adjuvants and the promotion of Thl-type cytokines in tumor immunotherapy. Cancer Immunol Immunother. 2002; 51(10): 521-31.

45. Gellrich FF, Schmitz M, Beissert S, Meier F. Anti-PD-1 and novel combinations in the treatment of melanoma-an update. J Clin Med. 2020; 14; 9(1): 223.

46. Gershenwald JE, Scolyer RA, Hess, DR, et al. Melanoma staging: evidence-based changes in the American Joint Committee on Cancer Eighth Edition CancerStaging Manual. CA Cancer J Clin 2017;67:472-492.

47.Guo J, Si L, Kong Y, et al.Phase II, open-label, single-arm trial ofimatinib mesylate in patients with metastatic melanoma harboring c-Kitmutation or amplification.J Clin Oncol 2011;29(21):2904 -9.

48. Hauschild A, Kahler KC, Schafer M, Fluck M. Interdisciplinary management recommendations for toxicity associated with interferon-alfatherapy. J Dtsch Dermatol Ges. 2008; 6(10): 829-38.

49. Holtkamp S, Kreiter S, Selmi A, et al. Modification of antigen-encoding RNA increases stability, translational efficacy, and T cellstimulatory capacity of dendritic cells. Blood. 2006; 108(13): 4009-17.

50.International Agency for Research on Cancer.GLOBOCAN 2020: Population factsheets.Available at: hrtps://gco.iarc.fr/today/data/factsheets/populations/908-europe-fact-sheets.pdf.Accessed: 16 JUN 2021.

51. United States Prescribing Information. Available at: https://www.merck.com/product/usa/pi_circulars/k/keytruda/keytruda_pi.pdf (accessed on August 26, 2021).

52. Kranz LM, Diken M, Haas H, et al. Systemic RNA delivery to dendritic cells exploits antiviral defense for cancer immunotherapy. Nature. 2016; 534(7607): 396-401.

53.Kreiter S, Selmi A, Diken M, et al.Increased antigen presentationefficiency by coupling antigens to MHC class I trafficking signals.JImmunol.2008;180(1):309-18.

54. Kuk D, Shoushtari AN, Barker CA, et al. Prognosis of mucosal, uveal, acral, nonacral cutaneous, and unknown primary melanoma from the time of first metastasis. The Oncologist. 2016;21:848-854.

55. United States Prescribing Information. Available at: https://www.accessdata.fda.gov/drugsatfda_docs/label/2021/761097s007lbl.pdf (accessed on August 26, 2021).

56. Livingston KA, Jiang X, Stephensen CB. CD4 T-helper cell cytokinephenotypes and antibody response following tetanus toxoid boosterimmunization. J lmmunol Methods. 2013; 390(1-2): 18-29.

57. Mackiewicz J, Mackiewicz A. BRAF and MEK inhibitors in the era of immunotherapy in melanoma patients. Contemp Oncol 2018; 22(1A): 68-72.

58. Marchand M, Punt CJ, Aamdal S, et al. Immunisation of metastatic cancer patients with MAGE-3 protein combined with adjuvant SBAS-2: a clinical report. Eur J Cancer 2003;39(1):70-7.

59. Michielin O, van Akkooi ACJ, Ascierto PA, et al. Cutaneous melanoma: ESMO Clinical Practice. Guidelines for diagnosis, treatment and follow-up. Annals of Oncology. 2019;30:1884-1901.

60. Mooradian MJ, Sullivan RI. What to do when anti-PD-1 therapy fails in patients with melanoma. Oneology (Williston Park). 2019; 33(4): 141-48.

61. United States Prescribing Information. Available at: https://packageinserts.bms.com/pi/pi_opdivo.pdf (accessed on August 26, 2021).

62. Orlandini von Niessen AG; Poleganov MA, Rechner C, et al. ImprovingmRNA-Based Therapeutic Gene Delivery by Expression-Augmenting 3′UTRsIdentified by Cellular Library Screening. Mol Ther. 2019; 27(4): 824-36.

63.Oshita C, Takikawa M, Kume A, et al.Dendritic cell-based vaccination in metastatic melanoma patients: phase II clinical trial.Oncol Rep 2012;28(4):1131-8.

64. Rehman H, Silk AW, Kane MP, Kaufman HL. Into the clinic: Talomogeneaherparepvec (TVEC), a first-in-class intratumoral oncolytic viral therapy. JImmunother Cancer 2016;4:53.

65.Sanderson K, Scotland R, Lee P, et al. (2005): Autoimmunity in a phaseI trial ora fully human anti-cytotoxic T-lymphocyte antigen-4 monoclonalantibody with multiple melanoma peptides and Montanide ISA 51 for patients with resected stages III and IV melanoma.J Clin Oncol.2005;23(4):741-50.

66.SEER Cancer Statistics Review(CSR)1975-2017.National CancerInstitute.16.Melanoma of the Skin.https://seer.cancer.gov/csr/1975_2017/results_merged/sect_16_melanoma_skin.pdf.(Table 16.8)(accessed on August 26, 2021).

67. Shackleton M, Davis ID, Hopkins W, et al. The impact of imiquimod, a Toll-like receptor-7 ligand (TLR7L), on the immunogenicity of melanoma peptidevaccination with adjuvant Flt3 ligand. Cancer Immun. 2004; 4:9.

68. Sharpe AH, Pauken KE. The diverse functions of the PD1 inhibitorypathway. In Nature reviews. Immunology. 2018; 18(3): 153-67.

69. Siegal RL, Miller KD, Fuchs HE, et al. Cancer statistics 2021. CACancer J Clin. 2021;71:7-33.

70. Slingluff CL Jr, Petroni GR, Yamshchikov GV, et al. Clinical and immunologic results of a randomized phase II trial of vaccination using fourmelanoma peptides either administered in granulocyte-macrophage colony-stimulating factor in adjuvant or pulsed on dendritic cells. J ClinOncol. 2003;21(21):4016-26.

71. Srivastava S, Koch MA, Pepper M, Campbell DJ. Type I interferons directly inhibit regulatory T cells to allow optimal antiviral T cell responses during acute LCMV infection. J Exp Med. 2014; 211(5): 961-74.

72. Swetter SM, Thompson JA, Albertini MR, et al. (2021). Melanoma: Cutaneous-NCCN Clinical Practice Guidelines in Oncology. Version 2.2021-February 19, 2021.

73. Testori AAE, Chellino S, and van Akkooi ACJ. Adjuvant therapy formelanoma: past, current, and future developments. Cancers 2020;12:1-15.

74. Toungouz M, Libin M, Bulté F, et al. Transient expansion of peptide-specific lymphocytes producing IFN-gamma after vaccination with dendritic cells pulsed with MAGE peptides in patients with mage-A1/A3-posifive tumors. JLeukoe Biol 2001; 69 (6):937-43.

75. Tyagi P, Mirakhur B. MAGRIT: the largest-ever phase III lung cancer trial aims to establish a novel tumor-specific approach to therapy. Clin LungCancer. 2009; 10(5): 371-74.

76. Van der Kooij MK, Speetjens FM, van der Burg SH, et al. Uveal versus cutaneous melanoma; same origin, very distinct tumor types. Cancers. 2019; 11: 3-16.

77. Weide B, Pascolo S, Scheel B, et al. Direct injection of protamine-proteeted mRNA: results of a phase 1/2 vaccination trial in metastaticmelanoma patients. J Immunother. 2009; 32(5): 498-07.

78. Wilgenhof S, Van Nuffel AM, Corthals J, et al. Therapeutic vaccination with an autologousmRNA electroporated dendritic cell vaccine in patients with advanced melanoma. JImmunother 2011;34(5):448-56.

79. Wolchok JD, Chiarion-Sileni V, Gonzalez R, et al. Overall survival with combined nivolumab and ipilimumab in advanced melanoma. N Engl JMed. 2017; 377(14): 1345-1356.

80. United States Prescribing Information. Available at: https://packageinserts.bms.com/pi/pi_yervoy.pdf (accessed on August 26, 2021).

81. Zinkernagel RM, Ehl S, Aichele P, et al. Antigen localization regulates immune responses in a dose-and time-dependent fashion: ageographic view of immune reactivity. Immunol Rev. 1997; 156: 199-209.

Equivalent solution

Those skilled in the art will recognize or be able to determine many equivalents to the specific embodiments of the invention described herein using only routine experimentation. It should be understood that the present invention encompasses all variations, combinations and arrangements in which one or more of the listed claims, limitations, elements, clauses, descriptive terms, etc., are introduced into another claim (or any other claim as related) that is subordinate to the same base claim, unless otherwise stated, or unless it is obvious to one of ordinary skill in the art that a contradiction or inconsistency will occur. In addition, it should also be understood that any embodiment or aspect of the present invention may be expressly excluded from the claims, regardless of whether a specific exclusion is recorded in the specification. The scope of the present invention is not intended to be limited to the above description, but as set forth in the appended claims.

Claims (145)

1. A method, comprising: Administering to a patient at least one dose of a pharmaceutical composition comprising: (a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) lipid particles; wherein the patient was diagnosed with cancer prior to the time of administration, but the patient was classified as having no evidence of disease at the time of administration. 2. The method of claim 1, wherein absence of evidence of disease is determined by applying the immune-related response evaluation criteria in solid tumors (irRECIST) criteria or RECIST 1.1 criteria. 3. A method, comprising: Administering at least one dose of a pharmaceutical composition to a patient suffering from cancer, wherein the pharmaceutical composition comprises: (a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) Lipid particles. 4. The method of claim 3, wherein the patient is classified as having no evidence of disease at the time of administration. 5. The method of claim 3, wherein the patient is classified as having evidence of disease at the time of administration. 6. The method of claim 4 or 5, wherein the presence or absence of evidence of disease is determined by applying the immune-related response evaluation criteria in solid tumors (irRECIST) criteria or RECIST 1.1 criteria. 7. The method of any one of claims 1 to 6, wherein the one or more RNA molecules comprise: (i) a first RNA molecule encoding the NY-ESO-1 antigen, (ii) a second RNA molecule encoding a MAGE-A3 antigen, (iii) a third RNA molecule encoding a tyrosinase antigen, and (iv) a fourth RNA molecule encoding the TPTE antigen. 8. The method of any one of claims 1 to 7, wherein a single RNA molecule of the one or more RNA molecules encodes at least two of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen. 9. The method of any one of claims 1 to 8, wherein a single RNA molecule of the one or more RNA molecules encodes a multi-epitope polypeptide, wherein the multi-epitope polypeptide comprises at least two of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen. 10. The method of any one of claims 1 to 9, wherein the one or more RNA molecules further comprise at least one sequence encoding a CD4+ epitope. 11. The method of any one of claims 1 to 9, wherein the one or more RNA molecules further comprise at least one sequence encoding tetanus toxoid P2, a sequence encoding tetanus toxoid P16, or both. 12. The method of any one of claims 1 to 11, wherein the one or more RNA molecules comprise a sequence encoding an MHC class I trafficking domain. 13. The method of any one of claims 1 to 12, wherein the one or more RNA molecules comprise a 5' cap or a 5' cap analog. 14. The method of any one of claims 1 to 13, wherein the one or more RNA molecules comprise a sequence encoding a signal peptide. 15. The method of any one of claims 1 to 14, wherein the one or more RNA molecules comprise at least one non-coding regulatory element. 16. The method of any one of claims 1 to 15, wherein the one or more RNA molecules comprise a polyadenine tail. 17. The method of claim 16, wherein the polyadenine tail is or comprises a modified adenine sequence. 18. The method of any one of claims 1 to 17, wherein the one or more RNA molecules comprise at least one 5' untranslated region (UTR) and/or at least one 3' UTR. 19. The method of claim 18, wherein the one or more RNA molecules comprise, in 5' to 3' order: (i) a 5' cap or a 5' cap analog; (ii) at least one 5′UTR; (iii) signal peptide; (iv) a coding region encoding at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen and the TPTE antigen; (v) at least one sequence encoding tetanus toxoid P2, tetanus toxoid P16, or both; (vi) a sequence encoding an MHC class I trafficking domain; (vii) at least one 3'UTR; and (viii) Polyadenine tail. 20. The method of any one of claims 1 to 19, wherein the one or more RNA molecules comprise natural ribonucleotides. 21. The method of any one of claims 1 to 20, wherein the one or more RNA molecules comprise modified or synthetic ribonucleotides. 22. The method of any one of claims 1 to 21, wherein at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen is a full-length, non-mutated antigen. 23. The method of any one of claims 1 to 22, wherein all of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen are full-length, non-mutated antigens. 24. The method of any one of claims 1 to 23, wherein at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen is expressed by dendritic cells in lymphoid tissue of the patient. 25. The method of any one of claims 1 to 24, wherein at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen is present in the cancer. 26. The method of any one of claims 1 to 25, wherein the lipid particle comprises a liposome. 27. The method of any one of claims 1 to 26, wherein the lipid particles comprise cationic liposomes. 28. The method of any one of claims 1 to 25, wherein the lipid particles comprise lipid nanoparticles. 29. The method of any one of claims 1 to 28, wherein the lipid granules comprise N,N,N trimethyl-2-3-dioleyloxy-1-propylamine chloride (DOTMA), 1,2-dioleoyl-sn-glyceryl-3-phosphoethanolamine phospholipids (DOPE), or the two. 30. The method of any one of claims 1 to 29, wherein the lipid particle comprises at least one ionizable amino lipid. 31. The method of any one of claims 1 to 30, wherein the lipid particle comprises at least one ionizable amino lipid and a helper lipid. 32. The method of any one of claims 31, wherein the helper lipid is or comprises a phospholipid. 33. The method of any one of claims 31 or 32, wherein the helper lipid is or comprises a sterol. 34. The method of any one of claims 1 to 33, wherein the lipid particle comprises at least one polymer-conjugated lipid. 35. The method of any one of claims 1 to 34, wherein the patient is a human. 36. The method of any one of claims 1 to 35, wherein the cancer is an epithelial cancer. 37. The method of any one of claims 1 to 36, wherein the cancer is melanoma. 38. The method of claim 37, wherein the melanoma is cutaneous melanoma. 39. The method of any one of claims 1 to 38, wherein the cancer is advanced. 40. The method of any one of claims 1 to 39, wherein the cancer is stage II, stage III, or stage IV. 41. The method of any one of claims 1 to 40, wherein the cancer is stage IIIB, stage IIIC, or stage IV melanoma. 42. The method of any one of claims 1 to 41, wherein the cancer is completely resected, there is no evidence of disease, or both. 43. The method of any one of claims 1 to 42, further comprising administering to the patient a second dose of the pharmaceutical composition. 44. The method of any one of claims 1 to 43, further comprising administering to the patient at least two doses of the pharmaceutical composition. 45. The method of any one of claims 1 to 44, further comprising administering to the patient at least three doses of the pharmaceutical composition. 46. The method of claim 45, wherein at least one of the at least three doses is administered to the patient within 8 days of the patient having received another of the at least three doses. 47. The method of claim 45 or 46, wherein at least one of the at least three doses is administered to the patient within 15 days of the patient having received another of the at least three doses. 48. The method of any one of claims 1 to 47, comprising administering to the patient at least 8 doses of the pharmaceutical composition within 10 weeks. 49. The method of claim 48, comprising administering to the patient a dose of the pharmaceutical composition weekly for a period of 6 weeks, and then administering a dose of the pharmaceutical composition every two weeks for a period of 4 weeks. 50. The method of claim 48 or 49, further comprising administering to the patient monthly doses of the pharmaceutical composition after the at least 8 doses. 51. The method of any one of claims 1 to 47, comprising administering to the patient a dose of the pharmaceutical composition weekly for a period of 7 weeks. 52. The method of claim 51, further comprising administering a dose of the pharmaceutical composition to the patient every three weeks. 53. The method of any one of claims 1 to 52, wherein the first dose and/or the second dose is 5 μg to 500 μg of total RNA. 54. The method of any one of claims 1 to 53, wherein the first dose and/or the second dose is 7.2 μg to 400 μg total RNA. 55. The method of any one of claims 1 to 54, wherein the first dose and/or the second dose is 10 μg to 20 μg of total RNA. 56. The method of any one of claims 1 to 55, wherein the first dose and/or the second dose is about 14.4 μg total RNA. 57. The method of any one of claims 1 to 56, wherein the first dose and/or the second dose is about 25 μg of total RNA. 58. The method of any one of claims 1 to 54, wherein the first dose and/or the second dose is about 50 μg of total RNA. 59. The method of any one of claims 1 to 54, wherein the first dose and/or the second dose is about 100 μg of total RNA. 60. The method of any one of claims 1 to 59, wherein the first dose and/or the second dose is administered systemically. 61. The method of any one of claims 1 to 60, wherein the first dose and/or the second dose is administered intravenously. 62. The method of any one of claims 1 to 60, wherein the first dose and/or the second dose is administered intramuscularly. 63. The method of any one of claims 1 to 60, wherein the first dose and/or the second dose is administered subcutaneously. 64. The method of any one of claims 1 to 63, wherein the pharmaceutical composition is administered as a monotherapy. 65. The method of any one of claims 1 to 63, wherein the pharmaceutical composition is administered as part of a combination therapy. 66. The method of claim 65, wherein the combination therapy comprises the pharmaceutical composition and an immune checkpoint inhibitor. 67. The method of any one of claims 1 to 66, wherein the patient has previously received an immune checkpoint inhibitor. 68. The method of any one of claims 1 to 63 and 65 to 67, further comprising administering an immune checkpoint inhibitor to the patient. 69. The method of any one of claims 66 to 68, wherein the checkpoint inhibitor is or comprises a PD-1 inhibitor, a PDL-1 inhibitor, a CTLA4 inhibitor, a Lag-3 inhibitor, or a combination thereof. 70. The method of any one of claims 66 to 69, wherein the check point inhibitor is or comprises an antibody. 71. The method of any one of claims 66 to 70, wherein the checkpoint inhibitor is or comprises an inhibitor listed in Table 4 herein. 72. The method of any one of claims 66 to 71, wherein the checkpoint inhibitor is or comprises ipilimumab, nivolumab, pembrolizumab, avelumab, cemiplimab, atezolizumab, durvalumab, or a combination thereof. 73. The method of any one of claims 66 to 72, wherein the checkpoint inhibitor is or comprises ipilimumab. 74. The method of any one of claims 66 to 72, wherein the checkpoint inhibitors are or comprise ipilimumab and nivolumab. 75. The method of any one of claims 1 to 74, wherein the pharmaceutical composition induces an immune response in the patient. 76. The method of any one of claims 1 to 76, further comprising determining the level of an immune response in the patient. 77. The method of claim 76, which compares the level of immune response in the patient with the level of immune response in a second patient who has been administered the pharmaceutical composition, wherein the second patient was diagnosed with cancer prior to the time of administration and was classified as having evidence of disease at the time of administration. 78. The method of claim 77, wherein the pharmaceutical composition induces a level of immune response in the patient that is comparable to a level of immune response in a second patient to whom the pharmaceutical composition has been administered, the second patient having been previously diagnosed with cancer and classified as having evidence of disease at the time of administration. 79. The method of any one of claims 75 to 78, wherein the level of immune response is a de novo immune response induced by the pharmaceutical composition. 80. The method of any one of claims 1 to 79, further comprising determining the level of immune response in the patient before and after administration of the pharmaceutical composition. 81. The method of claim 80, which compares the level of immune response in the patient after administration of the pharmaceutical composition with the level of immune response in the patient before administration of the pharmaceutical composition. 82. The method of claim 81, wherein the level of immune response in the patient is increased after administration of the pharmaceutical composition compared to the level of immune response in the patient before administration of the pharmaceutical composition. 83. The method of claim 81, wherein the level of immune response in the patient is maintained after administration of the pharmaceutical composition compared to the level of immune response in the patient before administration of the pharmaceutical composition. 84. The method of any one of claims 75 to 83, wherein the immune response in the patient is an adaptive immune response. 85. The method of any one of claims 75 to 84, wherein the immune response in the patient is a T cell response. 86. The method of claim 85, wherein the T cell response is or comprises a CD4+ response. 87. The method of claim 85 or 86, wherein the T cell response is or comprises a CD8+ response. 88. The method of any one of claims 75 to 87, wherein the level of immune response in the patient is determined using an interferon-gamma enzyme-linked immunosorbent spot (ELISpot) assay. 89. The method of any one of claims 1 to 88, further comprising measuring the level of one or more of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen in lymphoid tissue of the patient. 90. The method of any one of claims 1 to 89, further comprising measuring the level of one or more of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen in the cancer. 91. The method of any one of claims 1 to 90, further comprising measuring the level of metabolic activity in the patient's spleen. 92. The method of any one of claims 1 to 91, further comprising measuring the level of metabolic activity in the patient's spleen before and after administration of the pharmaceutical composition. 93. The method of claim 91 or 92, wherein the level of metabolic activity in the patient's spleen is measured using positron emission tomography (PET), computed tomography (CT) scan, magnetic resonance imaging (MRI), or a combination thereof. 94. The method of any one of claims 1 to 93, further comprising measuring the amount of one or more cytokines in the patient's plasma. 95. The method of any one of claims 1 to 94, further comprising measuring the amount of one or more cytokines in the patient's plasma before and after administration of the pharmaceutical composition. 96. The method of claim 94 or 95, wherein the one or more cytokines comprise interferon (IFN)-α, IFN-γ, interleukin (IL)-6, IFN-induced protein (IP)-10, IL-12p70 subunit, or a combination thereof. 97. The method of any one of claims 1 to 96, further comprising measuring the number of cancerous lesions in the patient. 98. The method of any one of claims 1 to 97, further comprising measuring the number of cancer lesions in the patient before and after administering the pharmaceutical composition. 99. The method of claim 98, wherein there are fewer cancer lesions in the patient after administration of the pharmaceutical composition than before administration of the pharmaceutical composition. 100. The method of any one of claims 1 to 99, further comprising measuring the number of T cells induced by the pharmaceutical composition in the patient. 101. The method of any one of claims 1 to 100, further comprising measuring the number of T cells induced by the pharmaceutical composition in the patient at multiple time points after administration of the pharmaceutical composition. 102. The method of any one of claims 1 to 101, further comprising measuring the number of T cells induced by the pharmaceutical composition in the patient after administering a first dose of the pharmaceutical composition and after administering a second dose of the pharmaceutical composition. 103. The method of claim 102, wherein the number of T cells induced by the pharmaceutical composition in the patient is greater after administration of the second dose of the pharmaceutical composition than after administration of the first dose of the pharmaceutical composition. 104. The method of any one of claims 1 to 103, further comprising determining the phenotype of T cells induced by the pharmaceutical composition in the patient after administration of the pharmaceutical composition. 105. The method of claim 104, wherein at least a portion of the T cells induced by the pharmaceutical composition in the patient have a T helper-1 phenotype. 106. The method of claim 104 or 105, wherein the T cells induced in the patient by the pharmaceutical composition comprise T cells having a PD1+ effector memory phenotype. 107. The method of any one of claims 3 to 106, further comprising, for patients classified as having evidence of disease, measuring a size of one or more cancerous lesions. 108. The method of any one of claims 3 to 107, further comprising, for patients classified as having evidence of disease, measuring the size of one or more cancer lesions in the patient before and after administration of the pharmaceutical composition. 109. The method of claim 108, further comprising comparing the size of one or more cancer lesions in the patient before and after administration of the pharmaceutical composition. 110. The method of claim 109, wherein the size of at least one cancer lesion in the patient after administration of the pharmaceutical composition is equal to or smaller than the size of the at least one cancer lesion before administration of the pharmaceutical composition. 111. The method of any one of claims 3 to 110, further comprising monitoring progression-free survival duration for patients classified as having evidence of disease. 112. The method of claim 111, comparing the patient's progression-free survival duration to a reference progression-free survival duration. 113. The method of claim 112, wherein the reference progression-free survival duration is the average progression-free survival duration of a plurality of comparable patients who did not receive the pharmaceutical composition. 114. The method of claim 112 or 113, wherein the patient's progression-free survival duration is longer in time compared to a reference progression-free survival duration. 115. The method of any one of claims 3 to 114, further comprising, for patients classified as having evidence of disease, measuring duration of disease stabilization. 116. The method of 115, wherein stable disease is determined by applying irRECIST or RECIST 1.1 criteria. 117. The method of claim 115 or 116, further comprising comparing the patient's duration of disease stabilization to a reference duration of disease stabilization. 118. The method of claim 117, wherein the reference duration of disease stabilization is an average duration of disease stabilization of a plurality of comparable patients who have not received the pharmaceutical composition. 119. The method of claim 118, wherein the patient exhibits an increased duration of disease stabilization compared to the reference duration of disease stabilization. 120. The method of any one of claims 3 to 119, further comprising measuring duration of tumor responsiveness for patients classified as having evidence of disease. 121. The method of 120, wherein tumor responsiveness is determined by applying irRECIST or RECIST 1.1 criteria. 122. The method of claim 120 or 121, further comprising comparing the patient's tumor responsiveness duration to a reference tumor responsiveness duration. 123. The method of claim 122, wherein the reference tumor responsiveness duration is the average tumor responsiveness duration of a plurality of comparable patients who did not receive the pharmaceutical composition. 124. The method of claim 123, wherein the patient exhibits improved duration of tumor responsiveness compared to the reference duration of tumor responsiveness. 125. The method of any one of claims 1 to 106, further comprising monitoring duration of disease-free survival for patients classified as having no evidence of disease. 126. The method of claim 125, further comprising comparing the patient's disease-free survival duration to a reference disease-free survival duration. 127. The method of claim 126, wherein the reference disease-free survival duration is the average disease-free survival duration of a plurality of comparable patients who did not receive the pharmaceutical composition. 128. The method of claim 127, wherein the patient exhibits an improved disease-free survival duration compared to the reference disease-free survival duration. 129. The method of any one of claims 1-106 and 125-128, further comprising, for patients classified as having no evidence of disease, measuring the duration to disease recurrence. 130. The method of 129, wherein disease recurrence is determined by applying irRECIST or RECIST 1.1 criteria. 131. The method of claim 129 or 130, further comprising comparing the patient's duration to disease recurrence to a reference duration to disease recurrence. 132. The method of claim 131, wherein the reference duration to disease recurrence is the average duration to disease recurrence of a plurality of comparable patients who did not receive the pharmaceutical composition. 133. The method of claim 132, wherein the patient exhibits an increased duration to disease recurrence compared to the reference duration to disease recurrence. 134. A pharmaceutical composition for inducing an immune response against cancer in a patient, wherein the pharmaceutical composition comprises: (a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) lipid particles; and wherein the patient was classified as having no evidence of disease but had been previously diagnosed with cancer. 135. A pharmaceutical composition for treating cancer, wherein the pharmaceutical composition comprises: (a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) lipid particles; and wherein the patient was classified as having no evidence of disease but had been previously diagnosed with cancer. 136. The pharmaceutical composition of claim 134 or 135, wherein the cancer is melanoma. 137. Use of a pharmaceutical composition for inducing an immune response against cancer in a patient, wherein the pharmaceutical composition comprises: (a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) lipid particles; and wherein the patient was classified as having no evidence of disease but had been previously diagnosed with cancer. 138. Use of a pharmaceutical composition for treating cancer, wherein the pharmaceutical composition comprises: (a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) lipid particles; and wherein the patient was classified as having no evidence of disease but had been previously diagnosed with cancer. 139. The use of claim 137 or 138, wherein the cancer is melanoma. 140. A pharmaceutical composition for inducing an immune response against cancer in a patient, wherein the pharmaceutical composition comprises: (a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) Lipid particles. 141. A pharmaceutical composition for treating cancer, wherein the pharmaceutical composition comprises: (a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) Lipid particles. 142. The pharmaceutical composition of claim 140 or 141, wherein the cancer is melanoma. 143. Use of a pharmaceutical composition for inducing an immune response against cancer in a patient, wherein the pharmaceutical composition comprises: (a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) Lipid particles. 144. Use of a pharmaceutical composition for treating cancer, wherein the pharmaceutical composition comprises: (a) one or more RNA molecules that collectively encode (i) New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) melanoma associated antigen A3 (MAGE-A3) antigen, (iii) tyrosinase antigen, (iv) transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof; and (b) Lipid particles. 145. The use of claim 143 or 144, wherein the cancer is melanoma.