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

The present application claims priority from U.S. application Ser. No.63/227,323 filed on 7.29 of 2021 and U.S. application Ser. No.63/256,377 filed on 10.15 of 2021, each of which is incorporated herein by reference in its entirety.

Background

Cancer is the second leading cause of death worldwide. Conventional therapies such as chemotherapy, radiation therapy, surgery, and targeted therapies (e.g., including recent advances in immunotherapy) have improved outcomes in patients with advanced solid tumors. In the last few years, the food and drug administration (Food and Drug Administration, FDA) and the European drug administration (European MEDICINES AGENCY, EMA) have approved checkpoint inhibitors (ipilimumab targeted to CTLA-4 pathway and to programmed death receptor/ligand [ PD/PD-L1], including atuzumab, avermectin (avelumab), dewasuzumab (durvalumab), nivolumab (nivolumab), cimapr Li Shan antibody (cemiplimab) and pembrolizumab (pembrolizumab)) for the treatment of patients with multiple cancer types, mainly 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.

Disclosure of Invention

The poor prognosis of certain cancers (e.g., such as melanoma) highlights the need for additional treatment methods. The present disclosure provides, inter alia, such insight: pharmaceutical compositions (e.g., immunogenic compositions, such as, for example, vaccines in some embodiments) that deliver RNA molecules encoding melanoma tumor associated antigens (tumor-associated antigen, TAA) (e.g., melanoma TAA) represent a particularly effective treatment option for patients with melanoma. Such RNA molecules may, for example, target dendritic cells in lymphoid tissue. The present disclosure also provides, among other things, such insight: the pharmaceutical compositions described herein are particularly useful and/or effective when administered to patients with advanced melanoma (e.g., stage III or IV melanoma). Advanced cancers, such as advanced melanoma, are also known as "advanced (LATE STAGE)" cancers. Furthermore, the present disclosure provides such a particular insight: patients without evidence of disease (e.g., in some embodiments, patients whose melanoma has been completely resected) may still benefit from anti-tumor immunity induced by such pharmaceutical compositions when first administered the pharmaceutical compositions described herein.

Without wishing to be bound by any particular theory, since TAAs are generally non-mutated autoantigens, central T cell tolerance can lead to a largely weak, clinically ineffective T cell response observed in certain clinical trials of cancer vaccines. The present disclosure provides, inter alia, such insight: the combination of tumor-associated antigens comprising New York esophageal squamous cell carcinoma (New York oesophageal squamous cell carcinoma, NY-ESO-1) antigen, melanoma-associated antigen A3 (melanom-associated antigen A3, MAGE-A3) antigen, tyrosinase antigen, and transmembrane phosphatase (transmembrane phosphatase WITH TENSIN homolog, TPTE) antigen with tensin homology represents 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 limited normal tissue expression of such tumor-associated antigen combinations and their high prevalence in melanoma (prevalence) (e.g., more than 90% expression of at least one of tumor-associated antigen NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, and TPTE antigen in melanoma patients) can contribute to their usefulness in melanoma treatment.

The present disclosure also provides the 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 such a particular insight: delivery of tumor-associated antigens (NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, and TPTE antigen) via lipid particles (e.g., lipid complexes or lipid nanoparticles) that target dendritic cells (e.g., immature dendritic cells) through RNA that is translated for antigen presentation (e.g., enhanced presentation) on HLA class I and class II molecules can be a particularly beneficial strategy for cancer vaccines. Without wishing to be bound by a particular theory, in some embodiments, the RNA compositions described herein may align vaccine antigen delivery with co-stimulation of type I interferon-driven antiviral immune mechanisms mediated through toll-like receptors (TLRs) and result in significant expansion of antigen-specific T cells. The present disclosure also provides, among other things, such insight: the RNA compositions described herein are not only effective as monotherapy for treating melanoma, but may also act synergistically with immune checkpoint inhibitors (e.g., anti-PD 1 treatment) in melanoma patients, who in some embodiments may have been previously treated with immune checkpoint inhibitors. To date, no treatment comprising cancer vaccines containing ribonucleic acids encoding tumor-associated antigens and lipid particles (e.g., lipid complexes or lipid nanoparticles) has been approved for the treatment of cancer (e.g., melanoma). Those skilled in the art will recognize the emerging field of nucleic acid therapeutics and, in addition, RNA (e.g., mRNA) therapeutics (see, e.g., mRNA encoding proteins and/or cytokines). Various embodiments of the technology provided herein can take advantage of specific features of the RNA (e.g., mRNA) treatment technology and/or delivery system developed. For example, in some embodiments, the administered RNA (e.g., mRNA) can comprise non-nucleoside modified nucleotides. In some embodiments, the administered RNA (e.g., mRNA) may comprise one or more modified nucleotides (e.g., without limitation, pseudouridine), nucleosides, and/or linkages. Alternatively or additionally, in some embodiments, the administered RNA (e.g., mRNA) may comprise a modified polyA sequence (e.g., a disrupted polyA sequence) that enhances stability and/or translational efficiency. Alternatively or additionally, in some embodiments, the administered RNA (e.g., mRNA) can comprise a specific combination of at least two 3' utr sequences (e.g., a combination of sequence elements of an amino terminal enhancer of a split RNA with sequences derived from a mitochondrially encoded 12S RNA). Alternatively or additionally, in some embodiments, the administered RNA (e.g., mRNA) may comprise a' 5 UTR sequence derived from human α -globin mRNA. Alternatively or additionally, in some embodiments, the administered RNA (e.g., mRNA) may comprise a 5' cap analog, e.g., for co-transcription capping. Alternatively or additionally, in some embodiments, the administered RNA (e.g., mRNA) can comprise a secretion signal encoding region (e.g., a human secretion signal encoding sequence) having reduced immunogenicity. In some embodiments, the administered RNA (e.g., mRNA) may comprise an MHC transport domain. In some embodiments, the administered RNA can be formulated in or with one or more delivery vehicles (e.g., lipid particles, e.g., lipid complexes or lipid nanoparticles).

In one aspect, the present disclosure provides, inter alia, 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) 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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and (b) lipid particles.

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

Accordingly, certain aspects of the present disclosure provide a method comprising: administering at least one dose of a pharmaceutical composition to a patient, the pharmaceutical composition comprising: (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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and (b) lipid particles; wherein the patient is diagnosed with cancer prior to the time of administration, but the patient is 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 an immune-related response assessment criteria (immune-related Response Evaluation CRITERIA IN Solid turner, irRECIST) standard or a RECIST 1.1 standard.

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

In some embodiments, the technology described herein relates to a pharmaceutical composition comprising a single RNA molecule encoding a multi-epitope polypeptide, wherein the multi-epitope 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 the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the 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 tetanus toxoid P2, a sequence encoding tetanus toxoid P16, or both. In some embodiments, inclusion of P2 and/or P16 in the RNA molecule may improve immune stimulation compared to an equivalent RNA molecule without P2 or P16. Without wishing to be bound by any particular theory, P2 and/or P16 may provide CD4 ⁺ -mediated T cell help during priming (prime). 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, the one or more RNA molecules present in the pharmaceutical compositions described herein may comprise at least one of the following: a sequence encoding an MHC class I transport domain; a5 'cap or 5' cap analogue; a sequence encoding a signal peptide; at least one non-coding adjustment element; at least one poly adenine tail; at least one 5 'untranslated region (untranslated region, UTR) and/or at least one 3' UTR; and combinations thereof. In some embodiments, the polyadenylation tail to be included in the one or more RNA molecules is 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 5' cap analogue; (ii) at least one 5' utr; (iii) a signal peptide; (iv) A coding region encoding at least one of an 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 transport domain; (vii) at least one 3' utr; and (viii) a poly adenine tail.

In some embodiments, one or more RNA molecules present in the pharmaceutical compositions described herein comprise a natural ribonucleotide. 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 (e.g., those described herein) encoded by the one or more RNA molecules is a full-length antigen. In some embodiments, at least one of the tumor-associated antigens (e.g., those described herein) encoded by one or more RNA molecules is a truncated antigen. In some embodiments, at least one of the tumor-associated antigens (e.g., those described herein) encoded by one or more RNA molecules 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 a 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 trimethyl-2-3-dioleyloxy-1-propanamine chloride (DOTMA), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine phospholipid (DOPE), or both.

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

In some embodiments, the techniques provided herein may be used in human patients. In some embodiments, the techniques provided herein can be used to treat cancer and/or extend to the time of recurrence. In some embodiments, the cancer is an epithelial cancer. In some embodiments, the cancer is melanoma. In some embodiments, the cancer is advanced. In some embodiments, the cancer is stage II, stage III, or stage IV. In some embodiments, the cancer is stage IIIB, stage IIIC or stage IV melanoma. In some embodiments, the cancer is completely resected, no evidence of disease, or both.

In some embodiments, the methods described herein comprise administering a second dose of the provided pharmaceutical composition (e.g., those described herein) to a patient (e.g., in some embodiments, a patient with melanoma or a patient without evidence of disease). In some embodiments, the methods described herein comprise administering at least two doses of the pharmaceutical composition to a patient (e.g., in some embodiments, a patient with melanoma or a patient without evidence of disease). In some embodiments, the methods described herein comprise administering at least three doses of the pharmaceutical composition to a patient (e.g., in some embodiments, a patient with melanoma or a patient without evidence of disease).

In some embodiments, the present disclosure provides dosing regimens that are 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 within 8 days of the patient having received another of the at least three doses (e.g., in some embodiments, a patient with melanoma or a patient without evidence of disease). In some embodiments, 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 (e.g., in some embodiments, a patient with melanoma or a patient without evidence of disease). In some embodiments, a dosing regimen according to the present disclosure includes administering at least 8 doses of a pharmaceutical composition described herein to a patient (e.g., in some embodiments, a patient with melanoma or a patient without evidence of disease) over 10 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 without evidence of disease) every week for a period of 6 weeks, and then administering a dose of a pharmaceutical composition described herein every two weeks for a period of 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., a patient with melanoma or a patient without evidence of disease in some embodiments) monthly following an initial dosing regimen (e.g., an initial dosing regimen comprising at least 8 doses). In some embodiments, the dosing regimen comprises administering a dose of a pharmaceutical composition described herein to a patient (e.g., in some embodiments, a patient with melanoma or a patient without evidence of disease) for a period of 7 weeks. In some embodiments, the dosing regimen comprises administering a dose of a pharmaceutical composition described herein every three circumferential patients (e.g., in some embodiments, patients with melanoma or patients without evidence of disease).

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

In some embodiments, the pharmaceutical compositions described herein may be administered as a monotherapy. In some embodiments, the pharmaceutical compositions described herein may be administered as part of a combination therapy. In some embodiments, the combination therapy may comprise the provided pharmaceutical compositions and an immune checkpoint inhibitor. In some embodiments, the techniques described herein can be used on patients who have previously received immune checkpoint inhibitors. In some embodiments, the techniques described herein may further comprise administering an immune checkpoint inhibitor to the patient. Some examples of immune checkpoint inhibitors include, but are not limited to: PD-1 inhibitors, PDL-1 inhibitors, CTLA4 inhibitors, lag-3 inhibitors, or combinations thereof. In some embodiments, the immune checkpoint inhibitor is or comprises an antibody. In some embodiments, the immune checkpoint inhibitor is or comprises the following: the inhibitors listed in table 4 or example 8 herein. In some embodiments, the immune checkpoint inhibitor is or comprises the following: ipilimumab, nivolumab, pembrolizumab, avilamab, cimetidine Li Shan, atrazumab, devaluzumab, or combinations thereof. In some embodiments, an immune checkpoint inhibitor that may be particularly useful according to the present disclosure is or comprises ipilimumab. In some embodiments, an immune checkpoint inhibitor that may be particularly useful according to the present disclosure is or comprises the following: ipilimumab and nivolumab. In some embodiments, an immune checkpoint inhibitor that may be particularly useful according to the present disclosure is or comprises a cisapride Li Shan antibody or a cisapride Li Shan antibody.

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

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

In some embodiments, the methods described herein further comprise determining the level of immune response in the patient before and after administration of the pharmaceutical compositions described herein. In some such embodiments, the method further comprises comparing the level of immune response in the patient after administration of the pharmaceutical composition to the level of immune response in the patient prior to administration of the pharmaceutical composition. In some embodiments, the level of immune response in the patient after administration of the pharmaceutical composition is increased compared to the level of immune response in the patient prior to administration of the pharmaceutical composition. In some embodiments, the level of immune response in the patient is maintained after administration of the pharmaceutical composition as compared to the level of immune response in the patient prior to administration of the pharmaceutical composition.

In some embodiments, the techniques described herein can induce an adaptive response in a patient receiving the pharmaceutical compositions described herein. In some embodiments, the techniques described herein can induce a T cell response in a patient receiving a pharmaceutical composition described herein. In some embodiments, the T cell response is or comprises a cd4+ response. In some embodiments, the T cell response is 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 a patient can be determined using an interferon-gamma enzyme-linked immunosorbent spot (enzyme-linked immune absorbent 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 the patient's lymphoid tissue. In some embodiments, the methods described herein further comprise measuring the level of one or more of a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and a TPTE antigen in the cancer.

In some embodiments, the methods described herein further comprise measuring the level of metabolic activity in the spleen of the patient. In some embodiments, the methods described herein further comprise measuring the level of metabolic activity in the spleen of the patient before and after administration of the pharmaceutical composition described herein. The level of metabolic activity in the spleen of a patient may be measured by using suitable methods known in the art, for example, in some embodiments, positron emission tomography (positron emission tomography, PET), computed tomography (computerized tomography, CT) scanning, magnetic resonance imaging (magnetic resonance imaging, MRI), or a combination thereof.

In some embodiments, the methods described herein further comprise measuring the amount of one or more cytokines in the plasma of the patient. In some embodiments, the methods described herein further comprise measuring the amount of one or more cytokines in the patient's plasma before and after administration of the pharmaceutical compositions described herein. Some non-limiting examples of one or more cytokines to be measured include: 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 comprise measuring the number of cancer lesions in the patient. In some embodiments, the methods described herein further comprise measuring the number of cancer lesions in the patient before and after administration of the pharmaceutical compositions described herein. In some such embodiments, fewer cancer lesions are detected in the patient after administration of the pharmaceutical composition than before administration of the pharmaceutical composition.

In some embodiments, the methods described herein further comprise measuring the number of T cells in the patient induced by the pharmaceutical compositions described herein. In some embodiments, the methods described herein further comprise measuring the number of T cells induced by the pharmaceutical compositions described herein in the patient at a plurality of time points after administration of the pharmaceutical composition. In some embodiments, the methods described herein further comprise 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. In some such embodiments, the number of T cells induced by the administered pharmaceutical composition is greater in the patient after administration of the second dose of the pharmaceutical composition than after administration of the first dose of the pharmaceutical composition.

In some embodiments, the methods described herein further comprise determining the phenotype of T cells induced by the pharmaceutical composition in the patient after administration of the pharmaceutical composition. In some embodiments, at least a portion of T cells in a patient induced by an administered pharmaceutical composition have a T helper-1 phenotype. In some embodiments, the T cells induced in the patient by the administered pharmaceutical composition comprise T cells having a pd1+ effector memory phenotype.

In some embodiments, the techniques described herein may be used for administration to patients classified as evidence of disease. In some such embodiments, the methods described herein for patients classified as evidence of disease further comprise measuring the size of one or more cancer lesions. In some embodiments, the methods described herein further comprise measuring the size of one or more cancer lesions in the patient before and after administration of the pharmaceutical composition described herein. In some embodiments, the methods described herein further comprise comparing the size of one or more cancer lesions in the patient before and after administration of the pharmaceutical composition. In some such embodiments, the size of the at least one cancer lesion in the patient after administration of the pharmaceutical composition is equal to or less than the size of the at least one cancer lesion prior to administration of the pharmaceutical composition.

In some embodiments, the methods described herein for patients classified as evidence of disease further comprise monitoring progression-free survival duration. In some such embodiments, the methods described herein comprise comparing the progression-free survival duration of the patient to a reference progression-free survival duration. In some embodiments, one exemplary reference progression-free survival duration is the average progression-free survival duration of a plurality of comparable patients not receiving the pharmaceutical composition described herein. In some embodiments, the progression-free survival duration of a patient administered the pharmaceutical composition described herein is longer in time than a reference progression-free survival duration.

In some embodiments, the methods described herein for patients classified as evidence of disease further comprise measuring the duration of disease stabilization. In some embodiments, disease stability may be determined by application of irRECIST or RECIST 1.1 criteria. In some embodiments, the methods described herein further comprise comparing the patient's disease stability duration to a reference disease stability duration. In some embodiments, such a reference disease-stable duration is the average disease-stable duration of a plurality of comparable patients not receiving the pharmaceutical composition described herein. In some embodiments, the patient administered the pharmaceutical composition described herein exhibits an increased duration of disease stability compared to a reference duration of disease stability.

In some embodiments, the methods described herein for patients classified as evidence of disease further comprise measuring tumor responsiveness duration. In some embodiments, tumor responsiveness is determined by applying irRECIST or RECIST 1.1 criteria. In some embodiments, the methods described herein further comprise comparing the tumor responsiveness duration of the patient administered the pharmaceutical composition described herein to a reference tumor responsiveness duration. In some embodiments, such a reference tumor response duration is the average tumor response duration of a plurality of comparable patients who did not receive the pharmaceutical composition described herein. In some embodiments, the patient administered the pharmaceutical composition described herein exhibits an increased tumor responsiveness duration compared to a reference tumor responsiveness duration.

In some embodiments, the techniques described herein may be used for administration to patients classified as no evidence of disease. In some such embodiments, the methods described herein further comprise monitoring disease-free survival duration. In some embodiments, the methods described herein further comprise comparing the patient's disease-free survival duration to 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 not receiving the pharmaceutical composition described herein. In some embodiments, the patient administered the pharmaceutical composition described herein exhibits an increased disease-free survival duration as compared to a reference disease-free survival duration.

In some embodiments, the methods described herein for patients classified as no evidence of disease may further comprise measuring the duration of time to disease recurrence. In some embodiments, disease recurrence is determined by application of irRECIST or RECIST 1.1 criteria. In some embodiments, the methods described herein further comprise comparing the duration of time to disease recurrence to a patient administered the pharmaceutical composition described herein to the duration of time to disease recurrence referenced. In some embodiments, such a reference is to the duration of disease recurrence is the average of a plurality of comparable patients not receiving the pharmaceutical composition described herein to the duration of disease recurrence. In some embodiments, the patient administered the pharmaceutical composition described herein exhibits an increased duration of relapse to disease compared to the duration of relapse to disease referenced.

In some embodiments, the techniques described herein can be used to prolong the overall survival of a patient. In some embodiments, the patient is classified as having evidence of disease. In some embodiments, the patient is classified as having no evidence of disease.

In some aspects, provided herein are also 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 been previously diagnosed as having cancer. In some embodiments, 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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and (b) lipid particles.

In some aspects, provided herein are also pharmaceutical compositions for treating cancer. In some embodiments, such patients are classified as having no evidence of disease, but have been previously diagnosed as having cancer. In some embodiments, 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 (TPTE) antigen having 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, e.g., in some embodiments, a patient classified as having no evidence of disease but previously diagnosed as having cancer. In some embodiments, the pharmaceutical compositions described herein can be used to treat cancer in patients, for example, in some embodiments, patients classified as having no evidence of disease but having been previously diagnosed as having cancer. In some embodiments, the cancer is melanoma. In some embodiments, 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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and (b) lipid particles.

Drawings

FIGS. 1a to 1d depict exemplary TAA constructs, test designs and vaccine-mediated immune activation. FIG. 1a, TAARNA structure. The 5' -cap analogues, 5' -and 3' -untranslated regions (UTRs) and poly (a) tails were optimized for stability and translation efficiency. In addition, the TAA coding sequence was tagged with signal peptide (SIGNAL PEPTIDE, SP), tetanus toxoid cd4+ epitopes P2 and P16, and MHC class I transport domain (MHC CLASS I TRAFFICKING domain, MITD) for enhanced HLA presentation and immunogenicity. FIG. 1b, clinical trial design. FIG. 1c, metabolic activity in spleen, measured by beam [18F ] FDG-PET/CT at baseline (before) and 4 hours after the sixth vaccine injection. Figure 1d, cytokine plasma levels (2 hours, 6 hours, and 24 hours (and in some cases 48 hours) before and after each vaccine injection) and body temperature for six incremental dose injections per week of patients (from cohort V).

Figures 2a to 2k depict the T cell immune and clinical activity of FixVac. Figures 2a, 2c, ratio of patients with vaccine-induced T cell responses (de novo or expanded) as analyzed by IFN- γ ELISpot before and after vaccination measured ex vivo (a; n=50) or after IVS (c; n=20). PBL, peripheral blood lymphocytes. FIG. 2b, ex vivo CD8+ T cell responses of patients A2-09, measured using TAA PepMix pulsed CD4 depleted PBMC. Control, PBMCs with medium. FIG. 2d, CD4+ T cell response following IVS of patient 42-06, measured using autologous dendritic cells loaded with TAA PepMix as the target. Control, luciferase transfected dendritic cells. FIG. 2e, frequency of HLA multimer staining ex vivo of NY-ESO-1 specific T cells from patient 12-01 (cohort 1, six vaccine doses). The dashed line indicates vaccination. Figures 2f to 2i, head-induced HLA-B3503 restricted NY-ESO-1 specific T cells from patients A2-09 (cohort a, continued vaccination). The dashed line indicates vaccination. FIG. 2f, phenotype of NY-ESO-1/HLA-B.times.3501 multimer-stained PBMC. Polymer positive CD8+ T cells are shown in red. BV421 and BV650 are immunofluorescent markers. FIG. 2g, left, multimeric analysis; and, right, ICS of T cells stimulated with single peptide or PepMix. FIG. 2h ICS of ex vivo NY-ESO-1 peptide stimulated CD8+ T cells. Figure 2i, specific lysis of melanoma cell lines was performed by healthy donor cd8+ T cells transfected with cloned HLA-B-3503 restricted NY-ESO-1 specific TCRs from patients A2-09 (effector: target (E: T) ratio = 20:1). SK-MEL-37 and SK-MEL-28 are melanoma cell lines. PD-Cy7 is an immunofluorescent label. Fig. 2j, fig. 2k, clinical activity, assessed as effect of FixVac without/with anti-PD 1 antibody on target lesions (n=38; 4 patients had no target lesions at baseline). Fig. 2j, asterisks indicate combination with anti-PD 1 antibodies. PD, progressive disease; PR, partial response.

Figures 3a to 3g depict T cell immunity in patient 53-02 treated with FixVac monotherapy. FIG. 3a, top, NY-ESO-196-104 specific Cw.0304 restricted CD8+ T cells analyzed by HLA multimeric staining. Control, cytomegalovirus (CMV) -pp65 multimer. Bottom, exemplary flow cytometry. Fig. 3b, melanoma lesions as assessed by CT scan. Lesions smaller than quantifiable size were plotted as 0.1mm in diameter. NT, non-target lesions; t, target lesions; version 1.1 was evaluated according to the solid tumor immune-related response criteria (irRECIST). FIG. 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, frequency of NY-ESO-196-104 multimer-specific cd8+ T cells following IVS from PBMCs 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 on melanoma cell lines (E: t=20:1) from samples cloned and transfected into healthy donor cd8+ T cells after vaccination. Fig. 3f, TCR frequency from e in peripheral blood, measured by analysis of the ex vivo TCR library (repertoire). TRB, T cell receptor-beta. FIG. 3g, top, kinetics of ex vivo frequency of CD8+ T cells specific for MAGE-A3167-176, secreting cytokines. Bottom, exemplary flow cytometry.

Figures 4a to 4g depict T cell immunity in a partially responsive patient treated with FixVac/anti-PD 1 combination. Fig. 4a to 4C, patients C2-28.a, the size of the target lesion; FIG. 4b, de novo MAGE-A3 specific CD8+ T cells analyzed by HLA multimeric staining (top), and exemplary flow cytometry (bottom). FIG. 4c, melanoma cell recognition by MAGE-A3168-176 specific TCR. Figure 4d, CT scan of lung lesions in patients C2-31. Fig. 4e to 4f, patients C1-40. FIG. 4e MAGE-A3168-176 specific HLA-A 0101 restricted T cells analyzed by HLA-multimeric staining. Figure 4f, top, lysis of melanoma cell lines by cd8+ T cells from IVS cultures of PBMCs collected prior to and at the time of treatment (E: t=8.5:1). Bottom, MAGE-A3168-176 specific CD8+ T cells after IVS. Fig. 4g, correlation of FixVac TAA transcriptional expression with the number of non-synonymous single nucleotide variants (non-synonymous single-nucleotide variant, snSNV) in melanoma from three independent groups (n=50). RPKM, read per kilobase per million map reads.

Fig. 5 depicts a subgroup of patients. At baseline, patients had advanced melanoma with radiographically measurable or non-measurable disease. The immune monitoring was performed on 49 patients in all subgroups. Clinical antitumor activity was assessed in 42 out of a total of 56 patients with measurable disease at baseline (1 unresectable stage III C, 41 stage IV), whose follow-up imaging data was available at data cutoff (25 treated with FixVac monotherapy, 17 treated with FixVac in combination with anti-PD 1 therapy). The remaining 14 patients (5 received FixVac monotherapy and 9 received combination with anti-PD 1 therapy) were not included in the efficacy analysis for the reasons described in the preceding sentence. PD, progressive disease; PR, partial response; SD, stable disease (best objective overall response according to irRECIST 1.1.1). According to irRECIST.1, cr refers to the metabolic complete response of the patient with SD as the best response. Thirty-three patients with radiographically unmeasurable disease at baseline did not undergo exploratory analysis for objective best overall response and were undergoing follow-up for relapse free survival.

Fig. 6a to 6c depict characterization of cytokine secretion. Fig. 6a, 6b, for: FIG. 6a, all available patients; and FIG. 6b, peak plasma cytokine levels (6 hours after vaccine injection) and body temperature (4 hours after vaccine injection) of patients treated with 50 μg or 100 μg of RNA-lipid complex (LPX) target dose alone ("Mono") or in combination with anti-PD 1 treatment ("aPD 1"). Boxes show the 25 th to 75 th quantiles, with the line representing the median; whisker (whisker) shows minimum to maximum; gray dots show individual values for each dose level; the dashed line indicates the normal upper limit. Sample numbers (number, n) are indicated in the figure. FIG. 6c, correlation of plasma cytokine levels (y-axis) with plasma IFN- α concentration 6 hours after RNA-LPX administration (n=147 for IFN- γ, IL-12 p70 and IL-6; n=147 for IP-10).

Figures 7a to 7f depict T cell immunity induced by FixVac. FIG. 7a, phenotype (left) and quality (middle and right) of TAA-specific T cells measured by IFN-. Gamma.ELISPot after IVS (left and middle) or ex vivo (right). Only positive responses are shown. FIG. 7b, exemplary flow cytometry of PBMC from patient 12-01 stained with NY-ESO-192-100/Cw 0304 multimers. FIG. 7c, flow cytometry gating strategy for the phenotypic characterization of multimeric+T cells. Upper row, left to right: starting from events obtained with constant flow and fluorescence intensity, a single event was identified (unimodal (singlet)). Dump-negative events (viable, CD4-, CD14-, CD16-, CD 19-) and lymphocytes were identified and gated. Within lymphocytes, cd8+ HLA multimer positive T cells were gated for further analysis. Lower row, left panel: based on CD45RA and CCR7 expression, different subsets of cd8+ T cells (indicated in black) and NY-ESO-1 multimer positive cd8+ T cells (red) were gated into four subsets, and CD27 and CD28 expression-central memory (ccr7+cd45ra-), initial (ccr7+cd45ra+), effector memory (CCR 7-CD45 RA-) and effector memory re-expressing RA (CCR 7-cd445ra+), were analyzed in the right panel. PD1 and OX40 expression was analyzed for both multimer-positive (red) and multimer-negative (black) cd8+ T cells. FIG. 7d, CD8+ T cells of patient A2-09 secreting IFN-. Gamma.and TNF were examined following stimulation with MAGE-A3212-220 peptide. Figure 7e, comparison of fold induction of ex vivo spot counts after vaccination in 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-PD 1 treatment (agd 1 (n=12); right). Only positive responses at visit after vaccination are shown. Fold changes exceeding 2 from baseline are considered to be responsive to the vaccine. If both CD4 and CD8 results were positive after treatment, only the higher spot count ratio was shown. Fig. 7f, proportion of patients with vaccine-induced T cell responses (de novo or expanded) determined by IFN- γelispot before and after vaccination, measured ex vivo from patients treated with FixVac alone (n=14) or in combination with anti-PD 1 treatment (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, 8b, lane diagrams of patients that can be used to evaluate efficacy assessment from the start of treatment to disease progression or continued treatment. Figure 8a, 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 ended and when the treatment continued started. Figure 8a contains data obtained from patients with evidence of disease (ED patients) who received BNT111 as monotherapy. Figure 8b, patient treated with FixVac and anti-PD 1 treatments. The dark green triangles indicate treatment initiation and completion. Dark green arrows show the patient still receiving treatment. Red crosses mark disease progression; patients were classified by best overall response and progression free survival time (CR, PD, PR, SD). The light green star indicates the objective response of the first record and the light green arrow indicates ongoing disease control. The black vertical line marks the date on which the eighth vaccination was planned (study day 64). The single asterisk indicates the patient for the clinical course and treatment regimen shown in d. CR, according to irec ist1.1, the metabolism of the patient suffering from stable disease as the best response is fully responsive. Patients with a radiology non-measurable disease at baseline were undergoing follow-up for relapse free survival and did not undergo clinical efficacy assessment. Figure 8c, tumor burden at baseline correlated with clinical response following FixVac treatments. PD, progressive disease; PR, partial response; SD, stable disease. FIG. 8d, clinical course and treatment regimen for patients Pt 53-02, A2-09, C2-28, A2-10, C2-31 and C1-40. FD, first diagnosis of melanoma at any stage. FD stage IV, first diagnosis of melanoma at stage IV. * New bone lesions diagnosed and treated with radiation therapy.

Figures 9a to 9j depict T cell immunity in patient 53-02 with partial response under FixVac monotherapy. Figure 9a, CT scan of the lower and middle lobes of the right lung before (anterior) and after (posterior) onset of melanoma FixVac treatment. FIG. 9b, kinetics of HLA-Cw 0304 restricted CD8+ T cell responses specific for NY-ESO-196-104 (see also FIG. 3 a). FIGS. 9c to f, discovery and characterization of NY-ESO-196-104 specific HLA-Cw 0304 restricted TCRs. FIG. 9c, sorting gates for Polymer-positive CD8+ T cells for TCR clones (gates within a single, viable CD3+ lymphocyte population). Control, fluorescence minus one (fluorescence minus one, FMO) sample. FIG. 9d, recognition of K562 cells transfected with peptide pulsed HLA-Cw 0304 by NY-ESO-1-TCR transfected CD8+ T cells in IFN-gamma 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). Figure 9f kinetics of NY-ESO-1 specific TCR clonotype frequency in TCR library data obtained from PBMCs prior to and after vaccination. FIGS. 9g to 9i, discovery and characterization of two NY-ESO-1124-133 specific HLA-B4001 restricted TCRs. FIG. 9g, PBMC were stimulated with NY-ESO-1 PepMix and individual IFN-gamma positive CD8+ T cells were sorted by flow cytometry for TCR cloning (control, HIV-gag PepMix). FIGS. 9h, 9i HLA restriction and epitope specificity of NY-ESO-1-TCR using IFN-. Gamma.ELISPot.assay after co-culture of TCR transfected CD8+ T cells with peptide pulsed HLA transfected K562 cells. NY-ESO-1, NY-ESO-1 PepMix. FIG. 9j cytotoxicity of NY-ESO-1 specific TCR identified in samples after vaccination of patients. TCR transfected healthy donor cd8+ T cells were stimulated with HLA-transfected melanoma cell line (SK-MEL-37, SK-MEL-28) at an effector to target ratio of 20:1 for 12 hours.

FIGS. 10a to 10i depict T cell immunity in patients A2-10, C2-31 and C1-40. FIGS. 10a to 10f, patient A2-10 with CPI refractory melanoma, developed a partial response under FixVac monotherapy. Figure 10a, CT scan of inguinal lymph node metastasis obtained before and after the start of vaccination. FIG. 10b, CD4+ T cell responses before vaccination and after IVS after eight vaccinations, restimulation with autologous dendritic cells transfected with RNA (encoding one of TAA or luciferase as control) or with PepMix pulsed and unpulsed dendritic cells encoding TAA (no peptide) in IFN-. Gamma.ELISPot.assay. FIG. 10c, CD8+ and CD4+ T cells secreting cytokines after intradermal challenge with NY-ESO-1 RNA. Skin infiltrating lymphocytes were recovered from the punch biopsies 15 days after 8 weekly vaccinations and stimulated with PepMix encoding NY-ESO-1 or tyrosinase. Fig. 10d to 10f, discovery and characterization of hla II restricted TAA specific TCRs. d, cd4+ T cells from IVS cultures were re-stimulated with PepMix pulsed dendritic cells and sorted by flow cytometry for TCR cloning (control, HIV-gag PepMix). APC and PE are fluorescent dye labels. FIG. 10e HLA restriction and epitope specificity was determined by IFN-. Gamma.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 (-). Figure 10f kinetics of TCR clonotype frequency in peripheral blood analyzed by ex vivo TCR libraries. FIG. 10g TAA specific CD8+ and CD4+ T cell responses of patient C2-31 on autologous peptide-loaded dendritic cells by IFN-. Gamma.ELISPot after IVS with TAA PepMix. Control, dendritic cells loaded with irrelevant peptide. FIGS. 10h, 10i, clinical and immune responses of patients C1-40 with CPI refractory melanoma, who developed a partial response under melanoma FixVac in combination with nivolumab. Figure 10h, CT scans of right middle and left lower lobes before and after initiation of melanoma FixVac treatment. FIG. 10i, in vitro frequency of MAGE-A3168-176 specific A.times.0101 restricted (left panel) and NY-ESO-192-100 specific HLA_Cw.times.0304 restricted (right panel) CD8+ T cells analyzed by HLA multimeric staining.

FIG. 11 depicts gating strategies for flow cytometry analysis of the data shown in FIG. 2e (Pt 12-01 up to day 50). Flow cytometry gating strategies for identifying vaccine-induced T cells. Starting from events obtained with constant flow stream and fluorescence intensity, a single event was identified (top row, left to right). Viable cells and lymphocytes were identified and gated. In lymphocytes, the Dump-negative events (CD 4-, CD14-, CD16-, CD 19-negative) were gated to exclude them for further analysis. In the Dump-negative event, cd8+ HLA multimer positive T cells were gated for further analysis (bottom row).

FIG. 12 depicts gating strategies for flow cytometry analysis of the data shown in FIG. 2f, FIG. 2g (Pt A2-09), FIG. 2e (Pt 12-01 after day 50), FIG. 3a (Pt 53-02) and FIG. 7c (Pt A2-09), FIG. 9b (Pt 53-02). Flow cytometry gating strategies for phenotypic characterization of vaccine-induced T cells. Starting from events obtained with constant flow stream and fluorescence intensity, a single event was identified (top row, left to right). Dump-negative events (viable, CD 4-negative, CD 14-negative, CD 16-negative, CD 19-negative) and lymphocytes were identified and gated. Among lymphocytes, cd8+ HLA multimer positive T cells were gated for further analysis. (middle left panel). PD1 and OX40 expression was analyzed for both multimer-positive (red) and multimer-negative (black) cd8+ T cells (middle and right panels). Different subsets of cd8+ T cells (indicated in black) and multimer-positive cd8+ T cells (highlighted in red) were gated into four subsets based on CD45RA and CCR 7: central memory (CD 45 RA-ccr7+), initial (cd45ra+ccr7+), effector memory (CD 45RA-CCR 7-) and effector memory re-expressing RA (cd45ra+ccr7-). The expression of CD27 and CD28 in each subpopulation was analyzed.

FIG. 13 depicts gating strategies for flow cytometry analysis of the data shown in FIGS. 2h, 2g (Pt A2-09) and 7d (Pt A2-09). Flow cytometry gating strategies for identifying cytokine responses in vaccine-induced T cells. Starting from events obtained with constant flow stream and fluorescence intensity, a single event was identified (top row, left to right). Dump-negative events (viable, CD14-, CD16-, CD 19-negative) and lymphocytes were identified and gated. Within lymphocytes, cd8+ and cd4+ T cells were gated for further analysis (bottom left panel). Production of the effector cytokines TNF and ifnγ in cd8+ (bottom row of panels) and cd4+ T cells (bottom row of right panels) was gated and analyzed.

FIG. 14 depicts gating strategies for flow cytometry analysis of the data shown in FIGS. 3c and 4g (Pt 53-02). Flow cytometry gating strategies for identifying cytokine responses in vaccine-induced T cells. Starting from events obtained with constant flow stream and fluorescence intensity, a single event was identified (top row, left to right). In the next step lymphocytes are identified and gated. Within lymphocytes, cd8+ and cd4+ T cells were gated for further analysis (bottom left panel). Production of the effector cytokines TNF and ifnγ in cd8+ (bottom row of panels) and cd4+ T cells (bottom row of right panels) was gated and analyzed.

FIG. 15 depicts gating strategies for flow cytometry based upon detection of multimeric positive T cells of patient 53-02 following the IVS shown in FIG. 3 d. To detect NY-ESO-196-104 multimer-specific T cells, a single event and lymphocytes were first identified. Within a single lymphocyte, cd3+ viable cells are gated. Cd8+/multimer+ are recognized within viable cd3+ cells. The gating strategy for sample day 64 is shown as one example of the multimeric analysis depicted in fig. 3 d.

Fig. 16 depicts a flow cytometry gating strategy for single cell sorting of TAA specific T cells for TCR cloning shown in fig. 9c, 9g and 10 d. To detect TAA-specific T cells based on (a) multimeric staining or (b, c) IFNy secretion, a single event and lymphocytes were first identified. Within a single lymphocyte, cd3+ viable cells are gated. Gating was performed on (a) CD8+/multimer+, (b) CD8+/IFNy + or (c) CD4+/IFNy + T cells in viable CD3+ cells. The sorting gate is highlighted in red. Following either (a) multimeric staining or (b) ifnγ secretion assay, gating strategy for NY-ESO-1 specific T cells of patient 53-02 is shown corresponding to the data depicted in fig. 9c, 9g, and gating strategy for MAGE-A3 specific T cells of patient A2-10 is shown corresponding to that in fig. 10 d.

FIG. 17 depicts gating strategies for flow cytometry analysis of the data shown in FIG. 10c (Pt A2-10). Flow cytometry gating strategies for identifying cytokine responses in vaccine-induced T cells. Starting from events obtained with constant flow stream and fluorescence intensity, a single event was identified (top row, left to right). Dump-negative events (viable cells) and lymphocytes were identified and gated. Within lymphocytes, cd8+ and cd4+ T cells were gated for further analysis (bottom left panel). Production of the effector cytokines TNF and ifnγ in cd8+ (bottom row of panels) and cd4+ T cells (bottom row of right panels) was gated and analyzed.

FIG. 18 depicts gating strategies for flow cytometry analysis of the data shown in FIG. 4b (Pt C2-028), FIG. 4e (Pt C1-040) and FIG. 10i (Pt C1-40). Flow cytometry gating strategies for identifying vaccine-induced T cells. Starting from events obtained with constant flow stream and fluorescence intensity, a single event was identified (top row, left to right). Dump-negative events (viable, CD4-, CD14-, CD16-, CD 19-negative) and lymphocytes were identified and gated. Within lymphocytes, cd8+ HLA multimer positive T cells were gated for further analysis (bottom row).

FIG. 19 depicts a flow cytometry gating strategy for detecting multimeric positive T cells of patients C1-40 following the IVS shown in FIG. 4 f. To detect MAGE-A3168-176 multimer-specific T cells, single events and lymphocytes were first identified. Within a single lymphocyte, cd3+ viable cells are gated. Within viable cd3+ cells, cd8+/multimer+ were identified. The gating strategy for sample day 129 is shown as one example of the multimeric analysis depicted in fig. 4 f.

Figures 20a to 20c depict ex vivo ELISPOT cd4+ or cd8+ (figure 20 a), cd8+ (figure 20 b) or cd4+ (figure 20 c) responses. Frequency of patients with vaccine-induced (amplified or de novo) responses. The numbers in the bar segments represent the number of patients evaluated in each segment. Only patients treated with monotherapy are included.

Figure 21 depicts an ex vivo ELISPOT response by cell type. The number and percentage of ELISPOT responses can be evaluated. Only non-large amounts of measurements with evaluable results of both CD4 and CD8 from patients treated with monotherapy were included.

Figure 22 depicts vaccine-induced ex vivo ELISPOT cd4+ or cd8+ responses to any cell type. De novo responses and expanded scores of responses. Only patients treated with monotherapy are included.

Fig. 23a to 23c depict ex vivo ELISPOT cd4+ or cd8+ (fig. 23 a), cd8+ (fig. 23 b) or cd4+ (fig. 23 c) responses. Frequency of patients with vaccine-induced (amplified or de novo) responses. The numbers in the bar segments represent the number of patients evaluated in each segment. Only patients treated with monotherapy are included.

Figure 24 depicts an ex vivo ELISPOT cd4+ or cd8+ response by which the clinically optimal response of a disease patient could not be assessed. The numbers in the bar segments represent the number of patients with the evaluated ex vivo ELISPOT measurements in each segment. Only patients treated with monotherapy are included. Patients with no evaluable ELISPOT results or recorded clinical optima were excluded.

Figure 25 depicts an ex vivo ELISPOT cd4+ or cd8+ response by which the clinical optimal response of a disease patient can be assessed. The numbers in the bar segments represent the number of patients with the evaluated ex vivo ELISPOT measurements in each segment. Only patients treated with monotherapy are included. Patients with no evaluable ELISPOT results or recorded clinical optima were excluded.

Figures 26a to 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.

FIGS. 27a to 27f depict a summary of total survival data for ED patients (FIG. 27 a), NED patients (FIG. 27 b), and NED and ED patient combinations (FIG. 27 c); and Kaplan-Meier summary of total survival data for ED patients (fig. 27 d), NED patients (fig. 27 e), and ED and NED patient combinations (fig. 27 f).

Fig. 28a to 28c depict a summary of adverse events for ED patients (fig. 28 a), NED patients (fig. 28 b), and ED and NED patient combinations (fig. 28 c).

Fig. 29 depicts patient treatment data. Of the total number of 89 patients, 3 patients enrolled twice were counted only once for their first recruitment (2 patients were treated in cohort CI and subsequently enrolled in cohort CIII, and 1 patient from cohort CII subsequently enrolled in the expanded cohort exp.a). The initial dose was blue and the target dose was orange. In groups CII to CVII and exp.a, B and C, patients received 8 doses of melanoma FixVac (on days 1,8, 15, 22, 29, 36, 50 and 64). Patients in cohort CI received only 6 doses (on days 1,8, 15, 22, 29 and 43). Patients with measurable disease at baseline are allowed to choose to continue treatment (Q4W) until disease progression or drug-related toxicity. When FixVac is combined with anti-PD 1 treatment, this occurs from the first dose, except for the case of one patient (asterisk) to whom anti-PD 1 treatment was added during treatment.

Figure 30 depicts features and previous treatments of patients in a clinical analysis group.

Fig. 31 depicts data for 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 cycles. Total FDG uptake and relative FDG uptake in the spleen are shown. SUV, normalized uptake values.

Figure 32 depicts data for related adverse events occurring after treatment in more than 5% of patients.

Figure 33 depicts data for antigen specific alpha/beta TCRs isolated from single T cells of melanoma patients.

Fig. 34 includes a schematic diagram illustrating exemplary mRNA molecules as described herein and modes of action of the mRNA complexing in a lipid complex.

Figure 35 includes a table providing a variety of features related to patients participating in the safety and efficacy studies of the exemplary composition described herein (BNT 111).

Figure 36 includes a table providing a variety of features related to patients participating in the safety and efficacy studies of the exemplary composition described herein (BNT 111).

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

Fig. 38 includes bar graphs showing the in vitro response from patients as determined by ELISpot. Ex vivo responses were detected in 14/22 (64%) and 19/28 (68%) ED and NED patients, respectively.

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

Fig. 40 includes a bar graph showing the following: serious adverse events (treatment-emergent serious ADVERSE EVENT) occurred in treatments with > 10% incidence in any subset of patients after treatment with the exemplary compositions described herein (BNT 111).

Fig. 41 includes a bar chart showing the following: serious adverse events occurring in the treatment associated therewith after treatment with the exemplary compositions described herein (BNT 111) have a general term standard grade of greater than or equal to 3.

Fig. 42 includes a table providing an overview of preliminary efficacy in patients with evaluable disease according to irRECIST.

Fig. 43 includes a waterfall plot of the optimal change from baseline observed in a target lesion according to irRECIST in a patient with measurable disease at baseline treated with exemplary monotherapy (BNT 111) or in combination with PD-1 inhibitor or BRAF/MEK inhibition.

Certain definitions

About or about: as used herein, the term "about" or "approximately" when applied to one or more destination values refers to values similar to the reference value. In general, those skilled in the art who are familiar with the context will understand the relative degree of variation that is covered by the context "about" or "approximately. For example, in some embodiments, the term "about" or "approximately" may be encompassed within 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.

And (3) application: as used herein, the term "administering" and variations thereof generally refers to administering a composition to a subject to effect delivery of an agent as or contained in the composition to a target site or site to be treated. Those of ordinary skill in the art will recognize a variety of routes that may be used to administer to a subject (e.g., a human) where appropriate. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, and the like. In some embodiments, administration may be transbronchial (e.g., by bronchial instillation), buccal (buccal), transdermal (which may be or include, for example, one or more of surface to dermis, intradermal (interdermal), transdermal, etc.), enteral, intraarterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular (intraventricular), within a particular organ (e.g., intrahepatic), transmucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, transtracheal (e.g., by intratracheal instillation), vaginal, vitreous, etc. In some embodiments, the administration may be parenteral. In some embodiments, administration may be oral. In some embodiments, the administration may be intravenous. In some embodiments, the administration may be subcutaneous. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve the administration of a fixed number of doses. In some embodiments, administration may 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 may involve continuous administration (e.g., infusion) for at least a selected period of time. In some embodiments, administration may include priming and boosting regimens. Priming and boosting regimens may include administration of a first dose of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) followed by administration of a second dose of the pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) after a time interval. In the case of immunogenic compositions, priming and boosting regimens can lead to an increase in immune response in patients.

Antibody: as used herein, the term "antibody agent" refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes an immunoglobulin structural element sufficient to confer specific binding. In some embodiments, the antibody agent is or comprises a polypeptide whose amino acid sequence comprises a structural element recognized by one of skill in the art as an immunoglobulin variable domain. In some embodiments, the 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, an antibody agent may comprise one or more constant region sequences that are characteristic of a mouse, rabbit, primate, or human antibody. In some embodiments, an antibody agent may comprise one or more sequence elements, which are humanized, primatized, chimeric, etc., as known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more constructs or forms known or developed in the art for utilizing the structural and functional characteristics of antibodies in alternative presentations. For example, in some embodiments, the antibody agent used according to the present disclosure is in a form selected from, but not limited to, the following: intact IgA, igG, igE or IgM antibodies; bispecific or multispecific antibodies (e.gEtc.); antibody fragments, such as Fab fragments, fab ' fragments, F (ab ') 2 fragments, fd ' fragments, fd fragments, and isolated complementarity determining regions (complementarity determining region, CDRs) or groups thereof; a single chain Fv; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies, such as IgNAR or fragments thereof); camel antibodies; masking antibodies (e.g./>) ; Small modular immunopharmaceuticals ("SMIPsTM"); single-chain or tandem diabodies/>VHH；/>A minibody; /(I)Ankyrin repeat protein or/>DART; TCR-like antibodies; /(I) MicroProteins；/> And/>In some embodiments, the antibody may lack covalent modifications (e.g., linkages of glycans) that it would have if naturally produced. In some embodiments, the antibody may comprise a covalent modification (e.g., attachment of a glycan, payload [ e.g., a detectable moiety, therapeutic moiety, catalytic moiety, etc. ] or other pendent group [ e.g., polyethylene glycol, etc. ]).

Related to: as the term is used herein, an event or entity is "related to" one another if the presence, level, and/or form of the event or entity is related to 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 correlates with the incidence and/or susceptibility or likelihood of response to treatment (e.g., in a related population).

Blood-derived samples: 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 (which includes, for example, fresh frozen plasma), serum, blood fractions, plasma fractions, serum fractions, blood fractions (including Red Blood Cells (RBCs), platelets, leukocytes, etc.), and cell lysates comprising fractions thereof (e.g., cells such as red blood cells, leukocytes, etc., can be harvested and lysed to obtain cell lysates). In some embodiments, the blood-derived sample useful for characterization described herein is a plasma sample.

Cancer: the term "cancer" as used herein generally refers to a disease or disorder 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 of cell proliferation. In some embodiments, the cancer may comprise pre-cancerous (e.g., benign), malignant, pre-metastatic, and/or non-metastatic cells. In some embodiments, the cancer may be characterized by a solid tumor. In some embodiments, the cancer may be characterized by a hematological tumor. Generally, examples of different types of cancers known in the art include, for example, hematopoietic cancers, including leukemia, lymphomas (Hodgkin's and non-Hodgkin), myelomas, and myeloproliferative diseases; sarcoma; melanoma; adenoma; solid tissue cancer; oral, laryngeal, pharyngeal and pulmonary squamous cell carcinoma; liver cancer; genitourinary system cancers, such as prostate cancer, cervical cancer, bladder cancer, uterine cancer, and endometrial cancer; renal cell carcinoma; bone cancer; pancreatic cancer; skin cancer; cutaneous or intraocular melanoma; endocrine system cancer, thyroid cancer; parathyroid cancer; cancer of the head and neck; 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 consisting essentially of nucleoside-5 ' -triphosphates that are typically attached to the 5' end of uncapped RNAs (e.g., uncapped RNAs with 5' -biphosphates). 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 having a structure designated "m 7G". In some embodiments, the cap is or comprises a synthetic cap analogue that is similar to an RNA cap structure and has the ability to stabilize RNA (if linked thereto), including for example, but not limited to, anti-reverse cap analogues (anti-REVERSE CAP analogue, ARCA) known in the art. Those skilled in the art will appreciate that methods for ligating caps to the 5' end of RNA are known in the art. For example, in some embodiments, the capped RNA can be obtained by capping RNA having a 5 'triphosphate group or RNA having a 5' diphosphate group with a capping enzyme system (which includes, for example, but is not limited to, a vaccinia capping enzyme system or a saccharomyces cerevisiae capping enzyme system) in vitro. Alternatively, the capped RNA can be obtained by in vitro transcription of a single stranded DNA template (in vitro transcription, IVT) using methods known in the art, wherein the IVT system comprises a dinucleotide cap analogue (including, for example, an m7GpppG cap analogue or an N7-methyl, 2 '-O-methyl-GPPPG ARCA cap analogue or an N7-methyl, 3' -O-methyl-GPPPG ARCA cap analogue) 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 the pharmaceutical compositions described herein and additional therapeutic agents (e.g., chemotherapeutic agents described herein) may be performed simultaneously or separately (e.g., in any order). In some embodiments of the pharmaceutical compositions described herein, the pharmaceutical composition described herein and the additional therapeutic agent (e.g., chemotherapeutic agent described herein) may be combined in one pharmaceutically acceptable carrier, or they may be placed in separate carriers and delivered to the target cells or administered to the subject at different times. Each of these conditions 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., chemotherapeutic agent described herein) are delivered or administered in sufficiently close time that there is at least some temporal overlap of biological effects produced by each on the subject or target cell being treated.

Combination therapy: as used herein, the term "combination therapy" refers to those situations in which a subject is exposed to two or more therapeutic 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 prior to any dose administration of the second regimen); in some embodiments, such agents are administered in an overlapping dosing regimen. In some embodiments, "administering" of a combination therapy may involve administering one or more agents or modes to a subject receiving other agents or modes in the combination. For clarity, combination therapy does not require that the individual agents be administered together in a single composition (or even must be administered simultaneously), but in some embodiments, two or more agents or active portions thereof may be administered together in a combination composition.

The method is equivalent to that of: as used herein, the term "comparable" refers to two or more agents, entities, conditions, sets of conditions, etc., that may not be identical to each other but that are sufficiently similar to allow comparison therebetween such that one of ordinary skill in the art will understand that a conclusion can be reasonably drawn based on the observed differences or similarities. In some embodiments, a group of comparable conditions, environments, individuals, or populations is characterized by a plurality of substantially identical features and one or a few varying features. Those of ordinary skill in the art will understand what degree of identity is required for two or more such agents, entities, situations, condition sets, etc. in any given context to be considered equivalent. For example, one of ordinary skill in the art will appreciate that groups of environments, individuals, or groups are equivalent to one another when: characterized by having a sufficient number and type of substantially identical features to ensure a reasonable conclusion that the difference in the results or observed phenomena obtained under different circumstances, groups or circumstances of individuals or populations is caused by or indicative of the change in the characteristics of these changes.

Complementary: as used herein, the term "complementary" is used to refer to hybridization of oligonucleotides associated with the base pairing rules. For example, the sequence "C-A-G-T" is complementary to the sequence "G-T-C-A". Complementarity may be partial or complete. Thus, any degree of partial complementarity is intended to be included within the term "complementary," provided that the partial complementarity allows hybridization of the oligonucleotides. Partial complementarity is according to the base pairing rules in which one or more nucleobases are mismatched. Full or complete complementarity between nucleic acids is where each and every nucleic acid base matches another base under the base pairing rules.

Contact: as used interchangeably herein, the term "deliver" and variants thereof or "contact" refers to the introduction of ssRNA or a composition comprising the same into a target cell (e.g., the cytosol of a target cell). The target cells may be cultured in vitro or ex vivo, or present in a subject (in vivo). The method of introducing ssRNA or a composition comprising the same into a target cell may vary with in vitro, ex vivo, or in vivo applications. In some embodiments, ssRNA or a composition comprising the same may be introduced into target cells in a cell culture by in vitro transfection. In some embodiments, ssrnas or compositions comprising the same can be introduced into target cells by delivery vehicles (e.g., lipid nanoparticles described herein). In some embodiments, ssRNA or a composition comprising the same can be introduced into target cells in a subject by administering to the subject a pharmaceutical composition described herein.

And (3) detection: the term "detecting" is used broadly herein to include any suitable means of determining the presence or absence of an entity of interest in a sample or any form of measurement of an entity of interest. Thus, "detecting" may include determining, measuring, assessing or determining the presence or absence, level, quantity and/or location of an entity of interest. Including quantitative and qualitative assays, measurements or evaluations, which include semi-quantitative. Such determination, measurement or evaluation may be relative (e.g., when the entity of interest is detected relative to a control reference) or may be absolute. Thus, the term "quantization" when used in the context of quantizing a destination entity may refer to absolute or relative quantization. Absolute quantification may be accomplished by correlating the level of the detected entity of interest with a known control standard (e.g., by generating a standard curve). Or relative quantification may be accomplished by comparing the detection levels or amounts between two or more different destination entities to provide a relative quantification (i.e., relative to each other) of each of the two or more different destination entities.

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 that is typically manifested as a characteristic sign and/or symptom. In some embodiments, the exemplary disease is cancer.

Encoding: as used herein, the term "encoding" or variants thereof refers to 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 may encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). RNA molecules can encode polypeptides (e.g., by a translation process). Thus, a gene, cDNA or ssRNA (e.g., mRNA) encodes a polypeptide if transcription and translation of mRNA corresponding to the gene in a cell or other biological system produces the polypeptide. In some embodiments, the coding region of ssRNA encoding a tumor-associated antigen (TAA) refers to the coding strand, which has the same nucleotide sequence as the mRNA sequence of such tumor-associated antigen. In some embodiments, the coding region of ssRNA encoding a TAA refers to the non-coding strand of such 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 immune system of a patient. For example, an epitope may be any portion specifically recognized by a T cell, B cell, immunoglobulin (e.g., antibody or receptor), binding component, or aptamer. In some embodiments, an epitope is made up of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface exposed when the antigen adopts the relevant three-dimensional conformation. 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., is 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) Generating an RNA template from the 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) translating 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 initiation site and the RNA coding region initiation codon. In some embodiments, "5' utr" refers to a sequence of an mRNA molecule that begins at the transcription start site and ends one nucleotide (nt) before the RNA coding region start codon (typically AUG), for example in its natural environment.

Homology: as used herein, the term "homology" or "homolog" 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 "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 "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., contain residues with related chemical properties at the corresponding positions). For example, certain amino acids are generally classified as similar to each other as "hydrophobic" or "hydrophilic" amino acids, and/or as having "polar" or "nonpolar" side chains, as known to those of ordinary skill in the art. Substitution of one amino acid for another amino acid of the same type may generally be considered a "homologous" substitution.

Identity: as used herein, the term "identity" 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 "substantially identical" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical. For example, the calculation of the percent identity of two nucleic acid or polypeptide sequences may be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second sequences for optimal alignment, and non-identical sequences may be ignored for comparison purposes). In certain embodiments, the length of the sequences aligned 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 a position in a first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in a second sequence, then the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap (which needs to be introduced for optimal alignment of the two sequences). Comparison of sequences and determination of percent identity between two sequences may be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of MEYERS AND MILLER,1989, which has been incorporated into the ALIGN program (version 2.0). In some exemplary embodiments, the nucleic acid sequence comparison performed with the ALIGN program uses 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 may be determined using the GAP program in the GCG software package using the nwsgapdna.

RECIST standard: as used herein, the term "RECIST" or "RECIST criteria" refers to a solid tumor response evaluation criteria (Response Evaluation criteria for In Solid Tumors). For example, RECSIT standards such as 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 is RECIST 1.1. In some embodiments, the RECIST criteria is iRECIST. For example, iRECIST standards such as 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 is the "irRECIST criteria," which is a solid tumor immune-related response assessment criteria. For example, irRECIST standards such as those described in Nishino et al (CLIN CANCER RES 19:3936-43 (2013), which is incorporated herein by reference in its entirety). In some embodiments, irRECIST standard is irRECIST 1.1.1. In some embodiments, the RECIST criteria is the "imRECIST criteria" which is a solid tumor immune modification response assessment criteria (immune-modified Response Evaluation Criteria for In Solid Tumors). For example, irRECIST standards such as those 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 may 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 body part. In some embodiments, locally advanced tumors are used to describe cancers that have grown outside of their starting tissue or organ but have not spread to distant sites within the subject. 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 trunk abdominal) but has no signs of metastatic disease; however, complete surgical excision with negative pathological margin (margin) 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 or comprises DNA. In some embodiments, the nucleic acid is or comprises RNA. In some embodiments, the nucleic acid is or comprises a peptide nucleic acid (peptide nucleic acid, PNA). In some embodiments, the nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, the nucleic acid is 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 comprising one or more phosphodiester linkages. In some embodiments, the nucleic acid comprises a backbone comprising both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, the nucleic acid may comprise a backbone comprising one or more phosphorothioate or 5' -N-phosphoramidite linkages and/or one or more peptide linkages, e.g., as in "peptide nucleic acids". In some embodiments, the nucleic acid comprises one or more or all of the 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 unnatural 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-deadenosine, 7-deazaguanosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, inserted bases, and combinations thereof). In some embodiments, the non-natural residues comprise one or more modified sugars (e.g., 2 '-fluororibose, ribose, 2' -deoxyribose, arabinose, and hexose) as compared to those in the natural residues. In some embodiments, the nucleic acid has a nucleotide sequence encoding a functional gene product, such as an RNA or polypeptide. In some embodiments, the nucleic acid has a nucleotide sequence comprising one or more introns. In some embodiments, the nucleic acid can be prepared by isolation from a natural source, enzymatic synthesis (e.g., by complementary template-based polymerization, e.g., proliferation in vivo or in vitro, in a recombinant cell or system, or chemical synthesis). In some embodiments, the nucleic acid is at least 3,4,5,6,7,8,9,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,110,120,130,140,150,160,170,180,190,20,225,250,275,300,325,350,375,400,425,450,475,500,600,700,800,900,1000,1500,2000,2500,3000,3500,4000,4500,5000,5500,6000,6500,7000,7500,8000,8500,9000,9500,10,000,10,500,11,000,11,500,12,000,12,500,13,000,13,500,14,000,14,500,15,000,15,500,16,000,16,500,17,000,17,500,18,000,18,500,19,000,19,500, or 20,000 residues or nucleotides in length.

Nucleic acid particles: "nucleic acid particles" can be used to deliver a nucleic acid to a target site of interest (e.g., cell, tissue, organ, etc.). The nucleic acid particles may be formed from at least one cationic or cationically ionizable lipid or lipid-like substance, at least one cationic polymer, such as protamine, or mixtures thereof, and a nucleic acid. Nucleic acid particles include lipid nanoparticle (lipid nanoparticle, LNP) based formulations and lipid complex (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 the size (e.g., of a polynucleotide), the particular 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 having 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 is suffering from or susceptible to one or more diseases or disorders or conditions. In some embodiments, the patient exhibits one or more symptoms of the disease or disorder or condition. In some embodiments, the patient has been diagnosed as having one or more diseases or disorders or conditions. In some embodiments, the disease or disorder or condition for which the provided techniques are applicable is or includes cancer, or the presence of one or more tumors. In some embodiments, the patient is receiving or has received a particular 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 broad 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 (e.g., fragments, portions, or domains that retain at least one activity) of such complete polypeptides. 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. Useful modifications include, for example, terminal acetylation, amidation, methylation, and the like. In some embodiments, the polypeptide may comprise natural amino acids, unnatural amino acids, synthetic amino acids, and combinations thereof (e.g., may be or comprise peptidomimetics).

Reference/reference standard: as used herein, "reference" describes a standard or control against which a comparison is made. For example, in some embodiments, an agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested and/or assayed substantially simultaneously with the test or assay of interest. In some embodiments, the reference or control is a historical reference or control, optionally embodied in a tangible medium. In some embodiments, the reference or control is or comprises a set of specifications (e.g., acceptance criteria). Typically, as will be appreciated by those skilled in the art, the reference or control is assayed or characterized at conditions or conditions comparable to those under evaluation. Those skilled in the art will understand when sufficient similarity exists to prove reliance on and/or compare to a particular possible reference or control.

Ribonucleotides: as used herein, the term "ribonucleotide" encompasses both 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). The modified ribonucleotides may include one or more modifications including, but not limited to, for example: (a) a terminal modification, such as a 5 'terminal modification (e.g., phosphorylation, dephosphorylation, conjugation, reverse ligation, etc.), a 3' terminal modification (e.g., conjugation, reverse ligation, etc.), a base modification, such as a substitution with a modified base, a stabilized base, a destabilized base, or a base that base pairs with an extended partner pool or a conjugated base, (c) a sugar modification (e.g., at the 2 'position or the 4' position) or a sugar substitution, and (d) an internucleoside linkage modification, including a modification or substitution of a phosphodiester linkage. The term "ribonucleotide" also encompasses ribonucleotides that include both modified and unmodified ribonucleotides that are triphosphate.

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

Selective or specific: those of skill in the art understand that the term "selective" or "specific" as used herein in connection with an agent having activity means that the agent distinguishes between potential target entities, states, or cells. For example, in some embodiments, an agent is said to "specifically" bind to one or more competing surrogate targets if it preferentially binds to its target in the presence of the target. In many embodiments, the specific interaction depends on the presence of specific structural features (e.g., epitope, groove (cleft), binding site) of the target entity. It should be understood that the specificity need not be absolute. In some embodiments, specificity may be assessed relative to the specificity of a target binding moiety of one or more other potential target entities (e.g., competitors). In some embodiments, the specificity is assessed relative to the specificity of a reference specific binding member. In some embodiments, specificity is assessed relative to the specificity of a reference non-specific binding member.

Specific binding: as used herein, the term "specific binding" refers to the ability to distinguish between potential binding partners in the environment in which the binding occurs. When other potential targets are present, an antibody agent that interacts with one particular 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 a CDR of an antibody agent and its partner; in some embodiments, specific binding is assessed by detecting or determining the extent of dissociation of the antibody agent-partner complex; in some embodiments, specific binding is assessed by detecting or determining the ability of an 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 assay over a range of concentrations.

The object is: as used herein, the term "subject" refers to an organism to which the compositions described herein are to be administered, e.g., for experimental, diagnostic, prophylactic 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 has 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 human having one or more characteristics of susceptibility or risk characteristics for 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 and/or to whom diagnosis and/or therapy has been administered.

Is provided with: an individual "suffering from" a disease, disorder, and/or condition has been diagnosed with and/or exhibiting one or more symptoms of the disease, disorder, and/or condition.

And (3) synthesis: as used herein, the term "synthetic" refers to an entity that is artificial, or made by human intervention, or that is produced synthetically, rather than naturally. For example, in some embodiments, a synthesized 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 phrase "therapeutic agent" or "treatment" refers to an agent or intervention that, when administered to a subject or patient, has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect. In some embodiments, a therapeutic agent or treatment is any substance that can be used to alleviate, ameliorate, alleviate, 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, the therapeutic agent or treatment is a medical intervention (e.g., surgery, radiation, phototherapy) that may be performed to alleviate, 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 a 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, e.g., 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, e.g., in its natural environment.

Threshold level (e.g., acceptance criteria): as used herein, the term "threshold level" refers to a level that is used as a reference to obtain information about and/or classify a measurement (e.g., a measurement obtained in an assay). For example, in some embodiments, the threshold level means a value measured in an assay that defines a demarcation line between two sub-populations of a population (e.g., a lot that meets quality control criteria and a lot that does not meet quality control criteria). Thus, values at or above the threshold level define one sub-population of the population, and values below the threshold level define another sub-population of the population. The threshold level may be determined based on one or more control samples or between groups of control samples. The threshold level may be determined before, simultaneously with, or after the measurement of interest is made. In some embodiments, the threshold level may be a range of values.

Treatment: as used herein, the term "treatment" and variations thereof refers 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. The treatment may be administered to a subject that does not exhibit signs of the disease, disorder, and/or condition. In some embodiments, the treatment may be administered to a subject that exhibits only early signs of a disease, disorder, and/or condition, e.g., for the purpose of reducing the risk of developing a pathological condition associated with the disease, disorder, and/or condition. In some embodiments, the treatment may be administered to a subject at a later stage of the disease, disorder, and/or condition.

Unresectable tumor: as used herein, the term "unresectable tumor" generally refers to a tumor that cannot be removed by surgery. In some embodiments, a non-resectable tumor refers to a tumor that involves and/or grows into an essential organ or tissue (including a blood vessel that may not be reconstructable) and/or is located in a position that does not readily access a risk of unreasonable damage to one or more other critical or essential organs and/or tissues (including a blood vessel). In some embodiments, a non-resectable tumor refers to a tumor that cannot be resected by surgery without risking damage to the patient, which risk is determined in a sound medical judgment to be beyond the patient's expected benefit from resection. In some embodiments, "unresectability" of a tumor refers to the likelihood of achieving a negative-cut (R0) excision. In the case of pancreatic cancer, the presence of tumor wrapping of large blood vessels such as Superior Mesenteric Artery (SMA) MESENTERIC ARTERY or celiac axis (encasement), portal vein occlusion, and celiac or periaortic lymphadenopathy is generally considered to be a finding that precludes R0 surgery. Those skilled in the art will appreciate parameters that determine whether a tumor is unresectable.

Standard techniques can be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). The enzymatic reaction and purification techniques may be performed according to manufacturer's instructions or as commonly practiced in the art or as described herein. The foregoing techniques and operations may generally be performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the present specification. See, e.g., ,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, standard of Care (SOC) outcomes remain poor. Treatment options also include palliative chemotherapy (which may be less tolerant after previous repeated exposure to cytotoxic compounds) or optimal supportive care, as well as research treatments that are not demonstrated to be beneficial. Treatment in this population is not curable, with overall survival expected to be months. Vaccines have become an effective therapeutic choice for some cancers with highly unmet medical needs. However, vaccine trials for treating patients with treatment-refractory tumors have been largely unsuccessful. Accordingly, there remains a high medical need to develop vaccines to treat a variety of cancer types, including treatment of refractory cancers.

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 RNAs encoding tumor-associated antigens (TAAs). The present disclosure provides, inter alia, such insight: the pharmaceutical compositions described herein may be particularly useful and/or effective when administered to a patient without evidence of disease at the time of first administration, thereby demonstrating that the pharmaceutical composition induces T cell immunity even in the absence of detectable tumor.

In some embodiments, the present disclosure provides, inter alia, methods of administering to a patient at least one dose of a pharmaceutical composition described herein (e.g., an immunogenic composition, such as a vaccine) 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 (TAAs) that combine when administered to a patient to induce a strong adaptive immune response (e.g., CD4 ⁺ and/or CD8 ⁺ T cell immune response) against the one or more TAAs encoded by the one or more RNA molecules. Without wishing to be bound by any particular theory, the present disclosure suggests that such pharmaceutical compositions may achieve antigen-specific T cell immunity and a sustained objective response in cancer patients (e.g., patients with unresectable cancer (e.g., melanoma), patients with or undergoing checkpoint inhibitors, or patients with both). In particular, the present disclosure also teaches that by administering a pharmaceutical composition (e.g., an immunogenic composition, such as a vaccine) as described herein to a patient who was diagnosed with cancer prior to the time of administration, but wherein the patient was classified as having no evidence of disease at the time of administration.

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

In some embodiments, the present disclosure provides, inter alia, such insight: an mRNA encoding an amino acid sequence comprising 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 TAA or an immunogenic variant thereof for inducing an immune response against TAA. In some embodiments, the present disclosure provides, inter alia, RNA techniques for delivering one or more RNA molecules to a patient, the 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 (TPTE) antigen having tensin homology, or (v) a combination thereof. In some embodiments, a single RNA molecule encodes all of the following: (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, and (iv) a Transmembrane Phosphatase (TPTE) antigen with tensin homology. In some embodiments, the sequence encoding the following is not present on a single RNA molecule: (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, and (iv) a Transmembrane Phosphatase (TPTE) antigen with tensin homology. For example, the first RNA molecule may encode two of the following: (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, and (iv) a Transmembrane Phosphatase (TPTE) antigen with tensin homology, and the second RNA molecule can 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) a new york esophageal squamous cell carcinoma (NY-ESO-1) antigen, (ii) a melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) a tyrosinase antigen, and (iv) a Transmembrane Phosphatase (TPTE) antigen with tensin homology.

In some embodiments, the present disclosure provides, inter alia, such insight: pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) are formulated with lipid particles (e.g., lipid complexes or lipid nanoparticles) for administration (e.g., intravenous (IV), intramuscular, or subcutaneous administration) to a patient. In particular, a pharmaceutical composition comprising one or more RNA (e.g., mRNA) molecules encoding at least one TAA (e.g., NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, and/or TPTE antigen) or immunogenic fragment thereof is formulated with lipid particles (e.g., lipid complexes or lipid nanoparticles) for administration (e.g., IV, intramuscular, or subcutaneous administration) to a patient. Without wishing to be bound by any particular theory, a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) as described herein may be taken up by immature dendritic cells and the RNA molecules translated for enhancing antigen presentation on HLA class I and class II molecules. In some embodiments, TAAs (e.g., NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, and/or TPTE antigen) are expressed from RNAs (e.g., mRNA) engineered to be minimally immunogenic and/or formulated in lipid nanoparticles (e.g., LNP), for example. 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) can comprise modified nucleotides (e.g., without limitation, pseudouridine).

In some embodiments, the present disclosure provides, inter alia, methods of administering at least one dose of a pharmaceutical composition to a patient, the pharmaceutical composition comprising: (a) one or more RNA molecules that together 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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and (b) lipid particles (e.g., lipid complexes or lipid nanoparticles); wherein the patient is diagnosed with cancer prior to the time of administration, but the patient is classified as free of evidence of disease at the time of administration (e.g., free of evidence of disease is determined by application of a solid tumor response assessment criterion (RECIST) criteria, such as RECIST1.1 criteria or irRECIST criteria).

In some embodiments, the present disclosure provides, inter alia, methods of administering at least one dose of a pharmaceutical composition to a patient having cancer, wherein the pharmaceutical composition comprises: (a) one or more RNA molecules that together 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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and (b) lipid particles (e.g., lipid complexes or lipid nanoparticles).

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 together 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 (TPTE) antigen having tensin homology, 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 been previously diagnosed as having 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 together 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 (TPTE) antigen having tensin homology, 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 been previously diagnosed as having cancer (e.g., melanoma). No evidence of disease may 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 together 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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and (b) lipid particles (e.g., lipid complexes or lipid nanoparticles).

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 together 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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and (b) lipid particles (e.g., lipid complexes or lipid nanoparticles).

I. Existing methods

The present disclosure provides techniques for treating cancer. One 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) remains a fatal disease. For example, for systemic treatment of unresectable stage III/IV and recurrent melanoma, there are currently two approaches to demonstrate an improvement in Progression Free Survival (PFS) and Overall Survival (OS) in randomized trials. The two methods are as follows: (1) Checkpoint inhibition (PD-1/PD-L1 inhibition, CTLA-4 inhibition), and (2) targeting mitogen-activated protein kinase (mitogen-ACTIVATED PROTEIN KINASE, MAPK) pathways. While these approaches have met with some degree of success, both experience challenges and may benefit from being combined with or replaced by the techniques described herein. An overview of the current process is described below.

A. Systemic treatment

1. Checkpoint inhibitors

Immune checkpoint inhibitors (checkpoint inhibitor, CPI) targeting cytotoxic T lymphocyte-associated antigen 4 (cytoxic T-lymphocyte-associated antigen, CTLA-4, e.g., ipilimumab) and programmed death 1 (programmed death 1, PD-1; e.g., nivolumab and pembrolizumab) have been approved for treatment of advanced or metastatic melanoma @ alone or in combinationUSPI；/>USPI；/>USPI, each of which is incorporated herein by reference in its entirety). In first-line treatment, the combined treatment of nivolumab and nivolumab correlated with increased overall response rate (overall response rate, ORR;57% versus 19% versus 44%) and median PFS (11.5 months versus 2.9 months versus 6.9 months) compared to single agent of ipilimab or nivolumab, respectively. However, the combination is associated with significant toxicity, and the effect of the combination treatment on overall survival has not been fully determined (Wolchok 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 inhibitor (e.g., ipilimab) is also an option.

2. Signal transduction inhibitors

About half of patients with metastatic skin melanoma contain an activating mutation of the (harbor) proto-oncogene B-Raf (BRAF), an intracellular signaling kinase in the MAPK pathway. BRAF inhibitors such as vemurafenib (vemurafenib) and dabrafenib (dabrafenib) show clinical activity in melanoma 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 previously suffering from untreated unresectable or metastatic disease, combination treatment with BRAF and MEK inhibitors circumvents resistance and has better efficacy (e.g., improved ORR, response duration, PFS and OS) than BRAF inhibitor monotherapy. However, 50% of patients who responded to combination therapy still progressed during 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 in patients with BRAF mutations. For patients with BRAF V600 mutant tumors that do not progress very rapidly, the currently recommended therapeutic sequence is immunotherapy (e.g., anti-PD-1 therapy), followed by targeted therapy with a BRAF/MEK inhibitor (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 viral therapy that demonstrates the local treatment of unresectable skin, subcutaneous and lymph node lesions in patients with recurrent melanoma following primary surgery. T-vec is a modified herpes simplex virus type 1 (herpes simplex virus, type 1, HSV-1) that has undergone genetic modification (insertion of 2 copies of the 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% versus 5.7% compared to subcutaneous GM-CSF. However, the observed differences 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). Thus, the treatment options may be applicable to the selected patient.

4. Other treatments

Treatment options for patients with advanced or metastatic melanoma who progress under targeted therapy or immunotherapy may include high doses of Interleukin (IL) -2 or other cytotoxic therapies (e.g., dacarbazine, carboplatin/paclitaxel, albumin-bound paclitaxel). These agents have moderate response rates of less than 20% in both the first and second line environments, but no data in the environment after PD-1 is present. Furthermore, little consensus exists regarding optimal standard chemotherapy (SWETTER ET al 2021, incorporated herein by reference in its entirety). Initial promising results for c-kit-inhibitors were reported with a response rate of 23.3% (Guo et al 2011, incorporated herein by reference in its entirety), whereas in phase III, randomized, double-blind, placebo-controlled trials with carboplatin and paclitaxel combinations, the multi-kinase inhibitor sorafenib (sorafenib), targeting both the MAPK-cascade and both VEGF and PDGF-cascades, did not improve median PFS compared to placebo (Hauschild et al 2009, incorporated herein by reference in its entirety).

5. Adjuvant therapy

For the treatment of patients with fully resected cutaneous melanoma (no evidence of disease) in stage III as well as in stage IV of complete resection, adjuvant therapy is suggested (SWETTER ET al 2021, incorporated herein by reference in its entirety).

For these patient groups, adjuvant therapy was based on a number of prospective clinical trials with immune checkpoint inhibitors and BRAF targeted therapies. Clinical trials in the adjuvant setting showed that immune checkpoint inhibitors and BRAF targeted therapies improved Relapse Free Survival (RFS) or disease free survival rates when compared to conventional therapies and provided higher Overall Survival (OS) rates at 3 or 5 years. However, toxicity concerns regarding adjuvant therapy are significant, e.g., after adjuvant immune checkpoint inhibition, there are grade 3to 4 adverse events (ADVERSE EVENT, AE) in 25% to 41% of patients 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 with approved treatments for the treatment of stage III and IV melanoma. However, it is reported that about 40% to 45% of patients experience no response to initial treatment, exhibiting primary resistance; and an additional 30% to 40% of patients were reported to experience initial responses, but eventually progressed with secondary resistance (Mooradian and Sullivan 2019, incorporated herein by reference in its entirety). These patient sub-populations with primary refractory disease or secondary recurrence represent an unmet medical need population, and it has been proven reasonable to develop new treatments for patients with unresectable stage III and IV melanoma to induce higher initial response rates to reduce primary resistance and new treatments for patients with recurrent melanoma (Testori et al 2020, incorporated herein by reference in its entirety). Furthermore, the addition of new therapies to anti-PD-1 therapy may increase response relative to anti-PDI therapy alone.

The tolerance of currently available treatment options prevents the use of adjunctive therapy in patients with stage IIB or stage IIC high risk diseases and in part in patients with stage III diseases. New systemic treatments with better tolerability profiles may allow for the treatment of these patient sub-populations and improve the available adjunctive treatment options for patients with complete resections of disease.

Exemplary compositions described herein comprise TAA: NY-ESO-1, tyrosinase, MAGE-A3 and TPTE. Among other reasons, these cancer vaccine targets were selected based on the following criteria:

low or absent expression in toxicity related organs.

Expression in a significant fraction of melanoma cells.

The ability to induce an antigen-specific immune response.

Tumor biology according to literature.

Furthermore, these TAAs were selected at least in part due to tissue expression analysis in phase I Lipo-MERIT assays. In this trial, about 8% of the screened patients did not express detectable levels of any of these four antigens in the tumor or metastasis. Given the clonal heterogeneity of cancers and the limitations of clinically available samples (only one location), the present disclosure provides such insight: the ratio of 92% may be in excess of the observed patient to actually express at least one selected TAA. In addition, several of these TAAs were found to be co-expressed in a significant percentage of patients. Accordingly, the present disclosure provides such insight: it is expected that a significant population of melanoma patients will develop a multi-epitope, vaccine-induced immune response and benefit from treatment with the compositions described herein. As used herein, the term "BNT111" refers to a pharmaceutical composition comprising an NY-ESO-1 antigen, a tyrosinase antigen, a MAGE-A3 antigen, and a TPTE antigen, preferably formulated as shown in table 3.

In some embodiments, the compositions described herein (e.g., BNT 111) can sensitize, activate, and/or expand CD4 ⁺ and CD8 ⁺ T cell specificities, and thus produce T cell specific complementation pools for non-mutant TAAs that are frequently expressed in human melanoma independent of the mutant burden of the tumor.

The liposomal formulations of the compositions described herein (e.g., BNT 111) are designed to deliver antigen into secondary lymphoid tissues and utilize the innate and adaptive immune mechanisms of antiviral for inducing efficient antigen-specific T cell responses. Compositions described herein (e.g., BNT 111) administered intravenously can be delivered to secondary lymphoid tissues (e.g., spleen, lymph nodes, and bone marrow) and rapidly taken up by antigen-presenting cells (APC). Proteins translated from the RNA component of the compositions described herein (e.g., BNT 111) can be processed and presented on an individual group of both HLA-I and HLA-II molecules of a patient (Kranz et al 2016, which is incorporated herein by reference in its 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 (Zinkemagel et al 1997, incorporated herein by reference in its entirety). The components of the compositions described herein activate APCs via toll-like receptor signaling, which results in the pulsed release of pro-inflammatory cytokines (e.g., IFN- α, IL-6, IFN- γ, and IP-10). In addition, secretion of type I interferon 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 the cognate CD4 ⁺ T cell helper are necessary to overcome tolerance to autoantigens. Based on this dual mechanism of action, repeated administration of the compositions described herein (e.g., BNT 111) effectively sensitizes and rapidly expands antigen-specific CD8 ⁺ T cell responses.

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

Activation, expansion and differentiation of the naive T cells are physiologically related to induction of the immune modulatory checkpoint molecule PD-1 (SHARPE AND Pauken 2018, incorporated herein by reference in its entirety). Thus, as discussed further herein, anti-PD-1/anti-PD-L1 blockade will enhance the activity of T cell responses induced by the compositions herein (e.g., BNT 111), as supported by non-clinical data in a mouse tumor model. One reason for treatment failure in patients treated with PD-1/PD-L1 blockade is the lack of preformed antigen-specific T lymphocytes that recognize the relevant tumor antigen. In some embodiments, such T lymphocytes are caused by the compositions described herein (e.g., BNT 111), which induce potent antigen-specific CD4 ⁺ and CD8 ⁺ T cell responses. These T cells not only perform direct anti-tumor activity by 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 refractory to anti-PD-1/anti-PD-L1 treatment or relapsed after anti-PD-1/anti-PD-L1 treatment (meaning that activation of pre-existing memory T cells alone is insufficient to mediate clinical activity), the addition of a PD-1 inhibitor (which would rescue the newly sensitized T cells from specific depletion) would augment the effects of the compositions described herein (e.g., BNT 111).

In some embodiments, the objective response rate of the combination of the composition described herein (e.g., BNT 111) and the PD-1 inhibitor is 25% and the disease control rate is 22% in patients with a prior treatment with a median of 5, which may be higher if the treatment is used in a less pre-treated patient population.

I. Tumor associated antigens

In some embodiments, the present disclosure provides, inter alia, one or more RNA molecules encoding an antigen. In some embodiments, the antigen is a Tumor Associated Antigen (TAA). The present disclosure provides such insight: a significant percentage of melanoma patients cumulatively express at least one of the four TAAs, irrespective of the mutational burden of the tumor. In some embodiments, the one or more RNA molecules 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 (TPTE) antigen having tensin homology, or (v) a combination thereof. High incidence of these antigens was observed in melanoma patients. These antigens are also reported to be selectively expressed in cancer cells. The present disclosure provides such insight: selective expression of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen and/or the TPTE antigen can provide low risk of on-target/off-tumor toxicity. In some embodiments, one or more RNA molecules encoding an antigen (e.g., TAA, e.g., NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, and/or TPTE antigen) can be expected to induce multi-epitope 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 comprises only a portion of 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 comprises only a portion of the TPTE antigen.

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

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., melanoma). In some embodiments, the methods described herein comprise determining the presence and/or abundance (e.g., level or amount) of at least one of a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and a 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 tumor biopsy) is isolated from a patient and the presence and/or abundance (e.g., level or amount) of one of the NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, and TPTE antigen of the sample is assessed.

New York esophageal squamous cell carcinoma (NY-ESO-1) antigen: the NY-ESO-1 antigen is a member of the cancer testis antigen (CANCER TESTIS ANTIGEN, CTA) gene family. About 50% of all CTA genes form a polygene family on the X chromosome and are called CT-X genes. These CTAs are located in specific clusters in the chromosome, which are most dense 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 limited to testicular germ cells and placental trophoblasts and is either absent or underexpressed at the transcript or protein level in normal healthy adult somatic cells. NY-0SO-1 is expressed in a variety of human cancers including melanoma (Giavina-Biankhi 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 (Giavina-Biankhi) was detected in about 20% of invasive melanoma.

In some embodiments, an RNA molecule of the 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, the single RNA molecule encoding the NY-ESO-1 antigen is a full-length, non-mutated antigen. In some embodiments, an RNA molecule of one or more RNA molecules described herein encodes an NY-ESO-1 antigen of: it does not contain amino acid substitutions (e.g., the wild-type amino acid sequence of the NY-ESO-1 antigen) associated with progression of melanoma cancer.

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

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

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 Xq 28. It is associated with some genetic disorders such as congenital keratinization disorder. MAGE-A3 was proposed to enhance the ubiquitin ligase activity of RING type zinc finger containing E3 ubiquitin protein ligases and to enhance the ubiquitin ligase activity of TRIM28 and stimulate p53/TP53 ubiquitination by TRIM 28. MAGE-A3 has also been proposed for use in the production of a polypeptide by the reaction of E3: the Ubl conjugated enzyme (E2) is recruited and/or stabilized at the substrate complex to function. MAGE-A3 is thought to play a role in embryonic development and to be re-expressed in neoplastic or tumor progression. In some embodiments, in vitro expression promotes cell viability of the melanoma cell line. MAGE-A3 antigen is known to be recognized by T cells when expressed on melanoma.

In some embodiments, an RNA molecule of 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, an RNA molecule of one or more RNA molecules as described herein encodes such a MAGE-A3 antigen: it does not contain amino acid substitutions (e.g., wild-type amino acid sequence of MAGE-A3 antigen) associated with melanoma cancer progression.

In some embodiments, the MAGE-A3 antigen comprises amino acid sequences as set forth in SEQ ID NO:3, has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical. In some embodiments, the MAGE-A3 antigen comprises SEQ ID NO:3 or a sequence consisting of SEQ ID NO:3, and a polypeptide sequence of 3.

In some embodiments, the MAGE-A3 antigen consists of antibodies directed to SEQ ID NO:4, and a nucleic acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity.

Tyrosinase antigen: tyrosinase antigens are encoded by TYR genes and are members of the tyrosinase family or proteins, which are widely distributed in animals. The gene codes for melanosome enzymes 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 of one or more RNA molecules as described herein encodes a tyrosinase antigen or an immunogenic fragment thereof. In some embodiments, the RNA molecule encodes a full-length, non-mutated tyrosinase antigen. In some embodiments, an RNA molecule of one or more RNA molecules as described herein encodes such a tyrosinase antigen: it does not contain amino acid substitutions (e.g., the wild-type amino acid sequence of tyrosinase antigen) associated with melanoma cancer progression. In some embodiments, the tyrosinase antigen is not full length, but comprises only a portion of 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 tyrosinase antigen comprises a nucleotide sequence that hybridizes to SEQ ID NO:5 has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical. In some embodiments, the tyrosinase antigen comprises SEQ ID NO:5 or a sequence consisting of SEQ ID NO:5, and a polypeptide sequence of 5.

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

Transmembrane Phosphatase (TPTE) antigen with tensin homology: the TPTE antigen is a member of the Cancer Testis Antigen (CTA) family. CTA antigen expression is highly tissue limiting. TPTE is a transmembrane phosphatase with tensin homology that can play a role in the signal transduction pathway of endocrine or spermatogenic functions of testis. TPTE mRNA expression in healthy adult tissue is limited to testes, and transcript levels in all other normal tissue samples are below the detection limit .(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), of highly sensitive RT-PCR, which is incorporated herein by reference in its entirety).

In some embodiments, an RNA molecule of one or more RNA molecules as described herein encodes a TPTE antigen or 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, an RNA molecule of one or more RNA molecules as described herein encodes such a TPTE antigen: it does not contain amino acid substitutions (e.g., the wild-type amino acid sequence of TPTE antigen) associated with melanoma cancer progression.

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

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

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

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

Table 1: sequence of TAA

/>

/>

/>

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

As used herein, the term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" include MHC class I and MHC class II molecules and relate to the gene complexes present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in an immune response, where they bind peptide epitopes and present them for recognition 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, the binding peptide is typically about 8 to about 10 amino acids in length, although longer or shorter peptides may also be effective. In the case of MHC class II/peptide complexes, the binding peptide is typically about 10 to about 25 amino acids in length, and in particular about 13 to about 18 amino acids in length, although longer and shorter peptides may also be effective.

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

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

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

Cd4+ epitope: in some embodiments, the present disclosure provides, inter alia, pharmaceutical compositions comprising one or more RNA molecules that collectively encode: (i) an NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, (iv) a TPTE antigen, or (v) a combination thereof; the 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 a helper effect. For example, in some embodiments, the cd4+ epitope may comprise, but is not limited to, a tetanus toxoid antigen polypeptide, e.g., in some embodiments, a tetanus toxoid P2 polypeptide and/or a tetanus toxoid P16 polypeptide.

MHC transport domain: in some embodiments, the RNA molecules described herein comprise sequences encoding MHC transport domains. In some embodiments, the MHC transport domain is or comprises the transmembrane and cytoplasmic regions of a chain of MHC molecules (e.g., MHC class I molecules), e.g., 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 class I transport domain is or comprises an MHC class I transport domain. In some embodiments, the MHC class I transport domain comprises a sequence identical to the sequence set forth in SEQ ID NO: 11. 12, 15, 16, 19, 20, 23 or 24, the amino acid sequence of the MHC class I transport domain described as the "MITD" domain has an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical. In some embodiments, the MHC class I transport domain comprises a sequence identical to the sequence set forth in SEQ ID NO: 11. 12, 15, 16, 19, 20, 23 or 24, an amino acid sequence identical to the amino acid sequence of the MHC class I transport domain described as the "MITD" domain.

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

In some embodiments, an RNA molecule described herein comprises at least one non-coding sequence element. In some embodiments, such non-coding sequence elements are included in RNA molecules 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, poly adenine (polyA) tail, and any combination thereof.

UTR (5 'UTR and/or 3' UTR): in some embodiments, provided RNA molecules comprise a nucleotide sequence encoding a 5'utr of interest and/or a 3' utr of interest. Those of skill in the art will appreciate that untranslated regions of an mRNA sequence (e.g., the 3'utr and/or the 5' utr) may contribute to mRNA stability, mRNA localization, and/or translation efficiency.

In some embodiments, provided RNA molecules can comprise a 5'utr nucleotide sequence and/or a 3' utr nucleotide sequence. In some embodiments, such 5'utr sequences may be operably linked to a 3' coding sequence (e.g., that encompasses one or more coding regions). Additionally or alternatively, in some embodiments, the 3'utr sequence may be operably linked 5' to a coding sequence (e.g., that encompasses one or more coding regions).

In some embodiments, the 5 'and 3' utr sequences comprised in the RNA molecules described herein may consist of or comprise: the 5 'and 3' UTR sequences are naturally occurring or endogenous to the open reading frame of the gene of interest. Or in some embodiments, the 5 'and/or 3' utr sequences contained in the RNA molecule are not endogenous to the coding sequence (e.g., they encompass one or more coding regions); in some such embodiments, such 5 'and/or 3' utr sequences may be used to modify the stability and/or translation efficiency of the transcribed RNA sequences. For example, the skilled artisan will appreciate that AU-rich elements in the 3' UTR sequence may reduce the stability of mRNA. Thus, as will be appreciated by the skilled artisan, the 3 'and/or 5' UTRs may be selected or designed based on the characteristics of UTRs known in the art to enhance the stability of transcribed RNA.

For example, one of skill in the art will understand that in some embodiments, a nucleotide sequence consisting of or comprising a Kozak sequence of the open reading frame sequence of a gene or nucleotide sequence of interest may be selected and used as the nucleotide sequence encoding the 5' utr. As the skilled artisan will appreciate, kozak sequences are known to increase the translation efficiency of some RNA transcripts, but not necessarily all RNAs require Kozak sequences to enable efficient translation. In some embodiments, provided RNA molecules can comprise a nucleotide sequence encoding a 5' utr derived from an RNA virus whose RNA genome is stable in a cell. In some embodiments, a variety of modified ribonucleotides (e.g., as described herein) can be used in the 3 'and/or 5' utr, e.g., to prevent exonuclease degradation of transcribed RNA sequences.

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

In some embodiments, the RNA molecule may comprise one or more 3' utrs. For example, in some embodiments, an RNA molecule can comprise two copies of a 3' -UTR derived from globin mRNA (e.g., such as α2-globin, α1-globin, β -globin (e.g., human β -globin) mRNA). In some embodiments, two copies of the 3' UTR derived from human β -globin mRNA may be used, e.g., in some embodiments, it may be placed between the coding sequence of an RNA molecule and the poly (A) tail to improve protein expression levels and/or to extend mRNA persistence. In some embodiments, a 3' utr derived from human β -globin as described in WO 2007/036366 may be included in an 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 may be or comprise 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 may be a combination of at least two sequence elements (FI elements) derived from an "amino terminal cleavage enhancer" (amino TERMINAL ENHANCER of split, AES) mRNA (referred to as F) and a mitochondrially encoded 12S ribosomal RNA (referred to as I). These were identified by performing an ex vivo selection procedure on 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 provided ssrnas may comprise a nucleotide sequence encoding a polyA tail. A polyA tail is a nucleotide sequence comprising a series of adenosine nucleotides, which may vary in length (e.g., at least 5 adenine nucleotides) and may be up to hundreds of adenosine nucleotides. In some embodiments, the polyA tail is a nucleotide sequence comprising at least 30 adenosine nucleotides or more (including, e.g., 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 contained in an RNA molecule as described herein, the contents of which are incorporated herein by reference in their entirety for the purposes described herein.

In some embodiments, the polyA tail is or comprises a polyA homopolymer 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 comprise one or more non-adenosine nucleotides. In some embodiments, the polyA tail may be or comprise a disrupted 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 comprised in the RNA molecules described herein may be or comprise a modified polyA sequence comprising: a linker sequence; a first sequence of at least 20 a contiguous nucleotides, which is 5' of the linker sequence; and a second sequence of at least 20 a contiguous nucleotides, which is 3' of the linker sequence. In some embodiments, the modified polyA sequence may comprise: 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 contiguous nucleotides, which is 5' of the linker sequence; and a second sequence of at least 70 a contiguous nucleotides that is 3' of the linker sequence.

5' Cap: in some embodiments, the RNA molecules described herein can comprise a 5' cap that can be incorporated into such RNA molecules during transcription, or linked to such RNA molecules after transcription. In some embodiments, the RNA molecule may comprise an anti-reverse cap analogue (ARCA). In some embodiments, the RNA molecule may comprise the cap analogue β -S-ARCA (D1) (m ₂ ^7,2'-OGpp_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, each of which is incorporated herein by reference in its entirety for the purposes described herein.

In some embodiments, the RNA molecule may comprise a 5' cap structure for mRNA co-transcription capping. Some examples of cap structures for co-transcribing 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 comprised in the RNA molecules described herein is or comprises m7G (5') ppp (5 ') (2' ome) pG. In some embodiments, the 5' Cap included in the RNA molecules described herein is or includes a Cap1 structure [ e.g., without limitation, m ₂ ^7,3'-OGppp(m₁ ^2'-O) ApG ].

In some embodiments, the one or more RNA molecules that collectively encode the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, the TPTE antigen, or a combination thereof, comprise natural ribonucleotides. In some embodiments, the one or more RNA molecules that collectively encode an NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a 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 A, U, C and G ribonucleotides of an RNA molecule described herein can be replaced by a modified ribonucleotide. For example, in some embodiments, some or all of the cytidine residues present in the RNA molecule can be replaced with a modified cytidine, which in some embodiments can be, for example, 5-methylcytidine. Alternatively or additionally, in some embodiments, some or all of the uridine residues present in the RNA molecule may be replaced by a modified uridine, which in some embodiments may be, for example, a pseudouridine, such as, for example, 1-methyl pseudouridine. In some embodiments, all uridine residues present in the RNA molecule are replaced with pseudouridine, e.g. 1-methyl pseudouridine.

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 5' cap analogue; (ii) at least one 5' utr; (iii) a signal peptide; (iv) A coding region encoding at least one of an NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and a TPTE antigen; (v) At least one sequence encoding an epitope of CD4 ⁺; (vi) a sequence encoding an MHC transport domain; (vii) at least one 3' utr; and (viii) a poly adenine tail. For example, in some embodiments, the cap structures included in the RNA molecules described herein may be cap structures that can increase the resistance of the RNA molecules to extracellular and intracellular rnase degradation and result in higher protein expression. In some embodiments, an exemplary cap structure is or comprises β -S-ARCA (D1) (m ₂ ^7,2'-OGpp_s pG). In some embodiments, exemplary 5' utr sequence elements included in RNA molecules described herein are or include a signature sequence from human α -globin and Kozak consensus sequences. In some embodiments, exemplary 3'utr sequence elements included in the RNA molecules described herein may be or include two copies of a 3' utr derived from human β -globin, or a combination of two sequence elements (FI elements) derived from an "amino-terminal cleavage enhancer" (AES) mRNA (referred to as F) and a mitochondrially encoded 12S ribosomal RNA (referred to as I). See, e.g., WO2007/036366 and WO2017/060314, the entire contents of each of which are incorporated herein by reference for the purposes described herein. In some embodiments, the poly (a) tail included in the RNA molecules described herein can be designed to enhance RNA stability and/or translation efficiency. In some embodiments, an exemplary poly (a) tail is or comprises a contiguous 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 (a 30L 70).

And (3) joint: in some embodiments, at least one sequence encoding a linker may be present in the RNA molecule to separate the individual components present in the RNA molecule. For example, in some embodiments, at least one sequence encoding a linker may be present between the coding region encoding one or more tumor-associated antigens described herein and the sequence encoding a cd4+ epitope. In some embodiments, at least one sequence encoding a linker may be present between the sequence encoding the cd4+ epitope and the sequence encoding the MHC transport domain. In some embodiments, the sequence encoding the linker may encode a peptide linker. In some embodiments, the peptide linker may be glycine and/or serine rich. In some embodiments, the glycine and/or serine rich peptide linker may comprise at least one amino acid that is not glycine or serine. In some embodiments, the peptide linker may be 3 to 20 amino acids or 3 to 15 amino acids or 3 to 10 amino acids in length. In some embodiments, the peptide linker may be 10 amino acids in length.

In some embodiments, one or more RNA molecules described herein is or comprises one or more mRNA.

In some embodiments, the pharmaceutical composition comprises: (i) RNA molecules encoding the NY-ESO-1 antigens as disclosed in Table 2 below; RNA molecules encoding MAGE-A3 antigens as disclosed in table 2 below; RNA molecules encoding tyrosinase antigens 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 may be prepared by combining RNA molecules each encoding a tumor-associated antigen as described herein at about 1:1:1:1 in a molar ratio. In other words, in some embodiments, if the total RNA dose is 100 μg, the pharmaceutical composition may be prepared to comprise 25 μg gNY-ESO-1 antigen-encoding RNA, 25 μg MAGE-A3 antigen-encoding RNA, 25 μg tyrosinase antigen-encoding RNA, 25 μg TPTE antigen-encoding RNA. In some embodiments, this may be achieved by forming, for example, the following: 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 may then be mixed. In other words, mixing may 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 transport domain; sec = secretion signal peptide; UTR = untranslated region; hAg = human α -globin; p2p16=tetanus toxoid-derived P2 and P16 helper epitopes; 2hBg = 2 copies of human β -globin; a120 Poly a tail of 120 a in length; a30l70=two consecutive segments of adenine nucleotides separated by a linker (one segment is 30 a long in length and the other segment is 70 a long in length); FI = a combination of at least two sequence elements derived from an "amino terminal cleavage enhancer" (AES) mRNA (referred to as F) and a mitochondrially encoded 12S ribosomal RNA (referred to as I).

In some embodiments, the RNA molecule encoding the NY-ESO-1 antigen is or comprises the nucleotide sequence of RBL001.1 or RBL 001.3. In some embodiments, the RNA molecule encoding the NY-ESO-1 antigen comprises a sequence encoding a polypeptide having the amino acid sequence RBL001.1 or RBL 001.3. In the following, the nucleotide sequences for full-length RNA are given, as well as the sequence alignment of RBL001.1 and RBL003.1 for both the translated protein (with amino acid under the third nucleotide of the corresponding codon triplet). The sequence elements as shown in figure 1a are shown above the nucleotide sequence. Nucleotide and amino acid sequence differences 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 RBL 002.4. In some embodiments, the RNA molecule encoding the tyrosinase antigen comprises a sequence encoding a polypeptide having the amino acid sequence RBL002.2 or RBL 002.4. In the following, the nucleotide sequences for full-length RNA are given, as well as the sequence alignment of RBL002.2 and RBL002.4 for both the translated protein (with amino acids below the third nucleotide of the corresponding codon triplet). The sequence elements as shown in figure 1a are shown above the nucleotide sequence. Nucleotide and amino acid sequence differences 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 RBL 003.3. 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 RBL 003.3. In the following, sequence alignments of RBL003.1 and RBL003.3 are given for both the nucleotide sequence of the full-length RNA and for the translated protein in which the amino acid is below the third nucleotide of the corresponding codon triplet. The sequence elements as shown in figure 1a are shown above the nucleotide sequence. Nucleotide and amino acid sequence differences 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 RBL 004.3. 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 RBL 004.3. In the following, the nucleotide sequences for full-length RNA are given, as well as the sequence alignment of RBL004.1 and RBL004.3 for both the translated protein (with amino acids below the third nucleotide of the corresponding codon triplet). The sequence elements as shown in figure 1a are shown above the nucleotide sequence. Nucleotide and amino acid sequence differences 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

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

DNA templates are used for in vitro RNA synthesis in the presence of suitable RNA polymerases (e.g., recombinant RNA polymerase, e.g., T7 RNA polymerase) and ribonucleoside triphosphates (e.g., ATP, CTP, GTP, UTP). In some embodiments, RNA molecules (e.g., the RNA molecules described herein) can be synthesized in the presence of modified ribonucleotides triphosphates. For example only, in some embodiments, N1-methyl pseudouridine triphosphate (^m1 ψTP) may be used in place of Uridine Triphosphate (UTP). As will be apparent to those of skill 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 the single stranded DNA template in the 3'→5' direction to produce 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 polyA tail may be encoded in the DNA template, for example by using a suitable tailed PCR primer, or it may be added to the RNA molecule after in vitro transcription, for example by enzymatic treatment, for example using a Poly (a) polymerase, for example e.coli (e.coli) Poly (a) polymerase.

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

After RNA transcription, the DNA template is digested. In some embodiments, digestion may be accomplished using dnase I under appropriate conditions.

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

In some embodiments, the dsRNA may be obtained as a byproduct during in vitro transcription. In some such embodiments, a second purification step may be performed to remove dsRNA contamination. For example, in some embodiments, a cellulosic material (e.g., microcrystalline cellulose) may be used to remove dsRNA contamination, e.g., in some embodiments in chromatographic form. In some embodiments, the cellulosic material (e.g., microcrystalline cellulose) may be pretreated to inactivate potential rnase contamination, for example in some embodiments by autoclaving followed by incubation with an aqueous alkaline solution (e.g., naOH). In some embodiments, the RNA molecules can be purified using cellulosic materials according to the methods described in WO 2017/182524 (the entire contents of which are incorporated herein by reference).

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

In some embodiments, the RNA molecule may be treated by 0.2 μm filtration before it is filled into a suitable container.

In some embodiments, RNA quality control may be performed and/or monitored at any time during the production process of the RNA molecule and/or composition comprising the same. For example, in some embodiments, RNA quality control parameters may be assessed and/or monitored after each or some steps of the RNA molecule manufacturing process, e.g., after in vitro transcription and/or after each purification step.

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

In some embodiments, one or more quality control parameters may 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 dispensing). 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 assessing RNA quality are known in the art.

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

In some embodiments, the RNA molecule lot with exemplary evaluation results can be used for one or more additional steps of manufacturing and/or formulation and/or dispensing.

RNA delivery techniques

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 mediated delivery 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, e.g., ,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 to obtain information about a variety of methods that can be used to deliver the RNA molecules described herein.

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

In some embodiments, the lipid particle may be designed to protect RNA molecules (e.g., mRNA) from extracellular rnases and/or engineered for systemic delivery of RNA to target cells (e.g., dendritic cells). In some embodiments, such lipid particles may be particularly useful for delivering RNA molecules (e.g., mRNA) when the RNA molecules are administered intravenously to a subject in need thereof.

In some embodiments, the lipid particle comprises a liposome. In some embodiments, the lipid particle comprises a cationic liposome.

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

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

In some embodiments, the lipid particle comprises N, N trimethyl-2-3-dioleyloxy-1-propanamine chloride (DOTMA), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine phospholipid (DOPE), or both. In some embodiments, the lipid particle comprises at least one ionizable amino lipid. In some embodiments, the lipid particle comprises at least one ionizable amino lipid and a helper lipid. In some embodiments, the helper lipid is or comprises a phospholipid. In some embodiments, the helper lipid is or comprises a sterol. In some embodiments, the lipid particle comprises at least one polymer conjugated lipid.

RNA lipid complex particles: in some embodiments, the RNA molecules described herein can be delivered by a liposome formulation. 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 a (phospho) lipid bilayer structure within the RNA lipid complex particle. In some embodiments, the cationic liposome can comprise a cationic lipid or an ionizable amino lipid (e.g., such as those described herein) and optionally an additional or auxiliary lipid (e.g., at least one neutral lipid as described herein) to form an injectable particulate formulation.

In some embodiments, RNA lipid complex particles can be prepared by mixing liposomes with RNA molecules described herein. In some embodiments, the liposomes can be obtained by injecting a solution of the lipid in ethanol into water or a suitable aqueous phase. In some embodiments, the cationic liposome is stabilized in an aqueous formulation, e.g., as described in WO 2016/046060, the entire contents of which are incorporated herein by reference for the purposes described herein. In some embodiments, the cationic liposome can be produced by the following method: 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 lipid complex particles useful for delivery of the 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, the RNA molecule and positively charged liposome are mixed such that the cationic lipid and RNA are present in a charge ratio of 1.3:2. Such charge ratios were determined to be effective in targeting 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 further comprise additional or helper lipids (e.g., those described herein). Without wishing to be bound by theory, the electrostatic interaction between positively charged liposomes and negatively charged RNAs results in the complexation and spontaneous formation of RNA lipid complex particles.

In some embodiments, wherein a cationic lipid or ionizable amino lipid (e.g., those described herein) and a helper lipid are used, such cationic lipid or ionizable amino lipid and such helper lipid may be present in a molar ratio of 2:1. In some embodiments, the cationic lipid or ionizable amino lipid may be or comprise DOTMA. In some embodiments, the helper lipid may be or comprise a neutral lipid. In some embodiments, the neutral lipid may be or comprise 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 nanoparticle may be about 100nm to 1000nm or about 200nm to 900nm or about 200nm to 800nm or about 250nm to about 700nm.

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

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

In certain embodiments, when an RNA molecule (e.g., mRNA) is present in the provided lipid nanoparticle, it is resistant to degradation with a nuclease 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 comprise at least one cationic lipid, at least one polymer-conjugated lipid, and at least one helper lipid (e.g., at least one neutral lipid).

1. Helper lipids

In some embodiments, the lipid particles for delivering RNA molecules described herein comprise at least one helper lipid, which may 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 increase the effectiveness of delivering the lipid-based particles (e.g., cationic lipid-based particles) to the target cell. In some embodiments, the helper lipid may be or comprise a structural lipid at a concentration selected to optimize particle size, stability, and/or encapsulation.

In some embodiments, the lipid particles for delivering RNA molecules described herein comprise neutral helper lipids. Some examples of such neutral helper lipids include, but are not limited to, phosphatidylcholine, such as 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), phosphatidylethanolamine (e.g., 1, 2-dioleoyl-sn-glycero-3-phosphorylethanolamine (DOPE), sphingomyelin (sphingomyelin, SM), ceramides, cholesterol, steroids (e.g., sterols), and derivatives thereof.

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

2. Cationic lipids

In some embodiments, the lipid particles for delivering RNA molecules described herein comprise a cationic lipid. Cationic lipids are typically lipids having a net positive charge, for example, in some embodiments at a particular pH. In some embodiments, the cationic lipid may comprise one or more positively charged amine groups. In some embodiments, the cationic lipid may comprise a cationic headgroup, meaning a positively charged headgroup. In some embodiments, the cationic lipid can have a hydrophobic domain (e.g., one or more domains of a neutral lipid or an anionic lipid), provided that the cationic lipid has a net positive charge. In some embodiments, the cationic lipid comprises a polar head group, in some embodiments it may comprise one or more amine derivatives, such as primary, secondary and/or tertiary amines, quaternary amines, various combinations of amines, ammonium salts, or guanidine and/or imidazole groups, as well as pyridine, piperazine, and amino acid head groups (e.g., lysine, arginine, ornithine, and/or tryptophan). In some embodiments, the polar head group of the cationic lipid comprises one or more amine derivatives. In some embodiments, the polar head group of the cationic lipid comprises a quaternary amine. In some embodiments, the headgroup of the cationic lipid may comprise a plurality of cationic charges. In some embodiments, the headgroup of the cationic lipid comprises one cationic charge. Some examples of monocationic lipids include, but are not limited to, 1, 2-dimyristoyl-sn-glycero-3-ethyl phosphorylcholine (DMEPC), 1, 2-di-O-octadecenyl-3-trimethylammoniopropane (DOTMA) and/or 1, 2-dioleoyl-3-trimethylammoniopropane (DOTAP), 1, 2-dimyristoyl-3-trimethylammoniopropane (dmtpap), 2, 3-ditetradecyloxy) propyl- (2-hydroxyethyl) -dimethylazaniumbromide (dmriie), didodecyl (dimethyl) nitrogen bromide (DDAB), 1, 2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 3P- [ N- (n\n' -dimethylamino-ethane) carbamoyl ] cholesterol (DC-Choi), and/or dioleylether phosphatidylcholine (DOEPC).

In some embodiments, positively charged lipid structures described herein may also comprise 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 excipient that can affect surface charge, membrane fluidity, and facilitate incorporation of the lipid 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 comprises DMEPC and/or DOTMA. In some embodiments, the cationic lipid comprises DOTMA.

In some embodiments, the cationic lipid is ionizable such that it can exist in a positively charged form or a neutral form depending on pH. For example, in some embodiments, the cationic lipid is an ionizable amino lipid. Such ionization of cationic lipids can affect the surface charge of the lipid particles at different pH conditions, which in some embodiments can affect plasma protein absorption, blood clearance, and/or tissue distribution, as well as the ability to form endosomal non-bilayer structures. Thus, in some embodiments, the cationic lipid may be or comprise a pH-responsive lipid. In some embodiments, the pH-responsive lipid is a fatty acid derivative or other amphiphilic compound capable of forming a readily soluble lipid phase and having a pKa value of from pH 5 to pH 7.5. This means that the lipid is uncharged at pH above the pKa value and positively charged at pH below the pKa value. In some embodiments, the pH-responsive lipids can be used to supplement or replace cationic lipids, for example, by combining one or more RNA molecules with a lipid or a mixture of lipids at a low pH. The pH-responsive lipids include, but are not limited to, 1, 2-dienyloxy-3-dimethylamino-propane (DODMA).

In some embodiments, the lipid particles 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 1-4 therein), each of which is incorporated herein by reference in its entirety for the purposes described herein.

In some embodiments, cationic lipids that can be used in accordance with the present disclosure are amino lipids comprising a titratable tertiary amino headgroup linked to at least two saturated alkyl chains via an ester linkage that can be readily hydrolyzed to facilitate rapid degradation and/or excretion via the renal pathway. In some embodiments, such amino lipids have an apparent pK _a of about 6.0 to 6.5 (e.g., an apparent pK _a of about 6.25 in one embodiment), resulting in a molecule that is substantially fully positively charged at an acidic pH (e.g., pH 5). In some embodiments, such amino lipids, when incorporated into lipid particles, can impart different physicochemical properties that regulate particle formation, cellular uptake, fusibility (fusogenicity), and/or endosomal release of RNA molecules. In some embodiments, the introduction of an aqueous RNA solution into a lipid mixture comprising such amino lipids at pH 4.0 can result in electrostatic interactions between the negatively charged RNA backbone and the positively charged cationic lipids. Without wishing to be bound by any particular theory, such electrostatic interactions result in particle formation consistent with efficient encapsulation of the RNA drug substance. After RNA encapsulation, the pH of the medium surrounding the resulting lipid nanoparticle is adjusted to a more neutral pH (e.g., pH 7.4), resulting in neutralization of the surface charge of the lipid nanoparticle. When all other variables are kept constant, such charge neutral particles exhibit a longer in vivo circulation life and better delivery to hepatocytes than charged particles that are rapidly cleared by the reticuloendothelial system. Following endosomal uptake, the low pH of the endosome fuses the lipid nanoparticles comprising such amino lipids and allows release of RNA into the cytosol of the target cell.

The cationic lipids can be used alone or in combination with neutral lipids (e.g., cholesterol and/or neutral phospholipids), or in combination with other known lipid assembly components.

3. Polymer conjugated lipids

In some embodiments, lipid nanoparticles for delivery of RNA molecules described herein may comprise at least one polymer conjugated lipid. The polymer conjugated lipid is typically a molecule comprising a lipid moiety and a polymer moiety 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 particle by forming a protective hydrophilic layer of a protective hydrophobic lipid layer. In some embodiments, when such lipid particles are administered in vivo, PEG conjugated lipids may reduce their association with serum proteins and/or uptake of the resulting reticuloendothelial system.

A variety of PEG conjugated lipids are known in the art and include, but are not limited to, pegylated diacylglycerol (PEGYLATED DIACYLGLYCEROL, PEG-DAG) (e.g., l- (monomethoxy-polyethylene glycol) -2, 3-dimyristoylglycerol (PEG-DMG)), pegylated phosphatidylethanolamine (PEGYLATED PHOSPHATIDYLETHANOLOAMINE, PEG-PE), PEG diacylglycerol succinate (PEG succinate diacylglycerol, PEG-S-DAG) (e.g., diethyl 4-O- (2 ',3' -di (tetradecyloxy) propyl-1-O- (omega-methoxy (polyethoxy) ethyl) succinate (PEG-S-DMG)), pegylated ceramide (PEGYLATED CERAMIDE, PEG-cer), or PEG dialkoxypropyl carbamate (e.g., omega-methoxy (polyethoxy) ethyl N- (2, 3-di (tetradecyloxy) propyl) carbamate or 2, 3-di (tetradecyloxy) propyl N- (omega methoxy (polyethoxy) ethyl) carbamate), and the like.

Certain PEG conjugated lipids (also known as pegylated lipids) are clinically approved and exhibit safety in clinical trials. PEG conjugated lipids are known to affect cellular uptake, which is a prerequisite for endosomal localization and payload delivery. The pharmacology of the encapsulated nucleic acid can be controlled in a predictable manner by adjusting the alkyl chain length of the PEG-lipid anchor. In some embodiments, PEG conjugated lipids can be designed and/or selected based on reasonable solubility characteristics and/or molecular weights thereof to effectively perform the function of a spatial barrier. For example, in some embodiments, the pegylated lipids do not exhibit significant surfactant or permeability enhancement or interference effects on the biofilm. In some embodiments, PEG in such PEG-conjugated lipids may be linked to the diacyl lipid anchors with biodegradable amide linkages, thereby facilitating rapid degradation and/or excretion. In some embodiments, LNP comprising PEG conjugated lipids retains a full complement 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 the cells, ultimately resulting in release of the RNA payload.

In some embodiments, the lipid particles (e.g., lipid nanoparticles) may comprise one or more PEG-conjugated 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 may 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 from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester linkages; and w has an average value of 30 to 60. In some embodiments, R ₈ and R ₉ are each independently a straight, saturated alkyl chain containing from 12 to 16 carbon atoms. In some embodiments, w has an average value of 43 to 53. In other embodiments, the average w is about 45.

In some embodiments, the lipid forming the lipid nanoparticles described herein comprises: a polymer conjugated lipid; cationic lipids; and helper neutral lipids. In some such embodiments, the total polymer conjugated lipids may be present at about 0.5 to 5mol%, about 0.7 to 3.5mol%, about 1 to 2.5mol%, about 1.5 to 2mol%, or about 1.5 to 1.8mol% of the total lipids. In some embodiments, the total polymer conjugated lipid may be present at about 1 to 2.5mol% of the total lipid. In some embodiments, the molar ratio of total cationic lipid to total polymer conjugated lipid (e.g., PEG conjugated lipid) 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 helper neutral lipids in the lipid nanoparticles described herein, the total cationic lipids are present at about 35 to 65mol%, about 40 to 60mol%, about 41 to 49mol%, about 41 to 48mol%, about 42 to 48mol%, about 43 to 48mol%, about 44 to 48mol%, about 45 to 48mol%, about 46 to 48mol%, or about 47.2 to 47.8mol% of the total lipids.

In some embodiments involving polymer conjugated lipids, cationic lipids, and helper neutral lipids in the lipid nanoparticles described herein, the total neutral lipids are present at about 35 to 65mol%, about 40 to 60mol%, about 45 to 55mol%, or about 47 to 52mol% of the total lipids. In some embodiments, the total neutral lipids are present at 35 to 65mol% of the total lipids. In some embodiments, total non-steroid neutral lipids (e.g., DPSC) are present at about 5 to 15mol%, about 7 to 13mol%, or 9 to 11mol% of the total lipids. In some embodiments, the total non-steroid neutral lipids are present at about 9.5, 10, or 10.5 mole% of the total lipids. In some embodiments, the molar ratio of total cationic lipid to non-steroid neutral lipid 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 lipids (e.g., cholesterol) are present at about 35 to 50mol%, about 39 to 49mol%, about 40 to 46mol%, about 40 to 44mol%, or about 40 to 42mol% of the total lipids. In certain embodiments, the total steroid neutral lipid (e.g., cholesterol) is present at about 39, 40, 41, 42, 43, 44, 45, or 46m01% of the 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, a lipid composition comprising cationic lipids, polymer conjugated lipids, and neutral lipids may have individual lipids present in a particular molar percentage of total lipids or in a particular molar ratio (relative to each other) as described in WO 2018/081480, each of which is incorporated herein by reference in its entirety for the purposes described herein.

IV. pharmaceutical compositions provided

The present disclosure provides, inter alia, pharmaceutical compositions for delivering an antigen (e.g., TAA) to a patient. In some embodiments, the pharmaceutical composition comprises one or more RNA molecules encoding an NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof; and lipid particles (e.g., lipid complexes or lipid nanoparticles). In some embodiments, the pharmaceutical composition comprises one or more RNA molecules that collectively encode an NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and a TPTE antigen; and lipid particles (e.g., lipid complexes or lipid nanoparticles). In some embodiments, the pharmaceutical composition comprises at least four populations of RNA-lipid particles (e.g., lipid complexes 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 an NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof; and lipid particles (e.g., lipid complexes or lipid nanoparticles), wherein 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 particle is an RNA-lipid nanoparticle.

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 an NY-ESO-1 antigen, a second RNA molecule encoding 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 can be present in the pharmaceutical composition in about equimolar amounts (e.g., in a molar ratio of about 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 the one or more RNA molecules) is about 0.01mg/mL to about 0.5mg/mL, or about 0.05mg/mL to about 0.1mg/mL.

The pharmaceutical formulation may additionally comprise pharmaceutically acceptable excipients, as used herein, which include any and all solvents, dispersion media, diluents or other liquid vehicles, dispersion or suspension aids, surfactants, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, such as those suitable for the particular dosage form desired. Remington' S THE SCIENCE AND PRACTICE of Pharmacy, 21 st 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 derivative thereof, such as by producing any undesirable biological effect or in other cases interacting in a deleterious 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 for veterinary use. In some embodiments, the excipient is approved by the U.S. food and drug administration (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 pharmacopeia (United States Pharmacopoeia, USP), the european pharmacopeia (European Pharmacopoeia, EP), the british pharmacopeia (British Pharmacopoeia), and/or the international pharmacopeia (International Pharmacopoeia).

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and/or granulating agents, surfactants and/or emulsifying agents, disintegrants, binders, preservatives, buffers, lubricants and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweeteners, flavoring agents and/or fragrances may be present in the composition at the discretion of the formulator.

General considerations in the formulation and/or manufacture of medicaments can be found, for example, in Remington: THE SCIENCE AND PRACTICE of Pharmacy 21 st edition, lippincott Williams & Wilkins,2005 (which is incorporated herein by reference in its entirety).

In some embodiments, the pharmaceutical compositions provided herein may be formulated according to conventional techniques, such as those described in Remington: THE SCIENCE AND PRACTICE of Pharmacy 21 st edition, lippincott Williams & Wilkins,2005 (which is incorporated herein by reference in its entirety).

The pharmaceutical compositions described herein may be administered by any suitable method known in the art. As will be appreciated by those of skill in the art, the route and/or manner 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, including modes of administration other than enteral and topical administration, typically by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intra-articular, 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.

Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The composition may be formulated as a solution, dispersion, powder (e.g., lyophilized powder), microemulsion, lipid nanoparticle, or other ordered structure suitable for high drug concentrations. The carrier may be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols (e.g., mannitol, sorbitol) or sodium chloride in the composition. In some embodiments, prolonged absorption of the injectable compositions can be brought about by including in the composition agents which delay absorption (e.g., monostearates and gelatins).

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

In some embodiments, the dispersion is prepared by incorporating the active compound into a sterile vehicle 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, and the like) 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 a coating material, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

These compositions may also contain adjuvants, such as preserving, wetting, emulsifying and dispersing agents. Prevention of the presence of microorganisms can be ensured by sterilization procedures and by inclusion of both various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, and the like). It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like in the pharmaceutical compositions described herein. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the inclusion of agents which delay absorption (e.g., aluminum monostearate and gelatin).

The formulations of the pharmaceutical compositions described herein may be prepared by any method known in the pharmacological arts or hereafter developed. Generally, such a preparation method comprises the steps of: associating the active ingredient with a diluent or another excipient and/or one or more other auxiliary ingredients, and then shaping if necessary and/or desired, and/or packaging the product into the desired single or multi-dose unit.

Pharmaceutical compositions according to the present disclosure may be prepared, packaged and/or sold in bulk as single unit doses 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, pharmaceutically acceptable excipients, and/or any additional ingredients in the pharmaceutical composition can vary depending on the subject, target cell, disease or disorder to be treated, and can further depend on the route of administration of the composition.

In some embodiments, the pharmaceutical compositions described herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill 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 in order to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without toxicity to the patient. The selected dosage level will depend on a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or substances used in combination with the particular composition being employed, the age, sex, weight, condition, general health and past history of the patient being treated, and like factors well known in the medical arts.

A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian may begin with a dose of the active ingredient (e.g., the one or more RNA molecules encapsulated in the lipid nanoparticle) below the level required in the pharmaceutical composition to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. For example, exemplary dosages as described in example 7 may be used to prepare pharmaceutically acceptable dosage forms.

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

In some embodiments, the pharmaceutical compositions described herein may further comprise one or more additives, e.g., which may enhance the stability of such compositions under certain conditions in some embodiments. 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 further comprise a cryoprotectant (e.g., sucrose) and/or an aqueous buffer solution, which in some embodiments may comprise one or more salts, including, for example, alkali metal salts or alkaline earth metal salts, e.g., such as sodium, potassium, 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 an 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 further comprise 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 comprises an immune checkpoint inhibitor (also referred to as a "checkpoint inhibitor"). In some embodiments, exemplary immune checkpoint inhibitors may be or include immune checkpoint inhibitors 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 combinations thereof. In some embodiments, the immune checkpoint inhibitor is an antibody. Checkpoint inhibitors may include, for example, but are 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 agents that may be included in the pharmaceutical compositions described herein are or include the following: therapeutic agents administered in the combination therapies described herein. The pharmaceutical compositions 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 result in the depletion or functional inactivation of regulatory T cells. For example, in some embodiments, a combination therapy may comprise a provided pharmaceutical composition and at least one immune checkpoint inhibitor.

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

In some embodiments, the pharmaceutical compositions described herein can be combined with a checkpoint inhibitor (e.g., an inhibitor of PD-1, PD-L1, CTLA4, and/or related pathways thereof). 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 comprise a MEK inhibitor.

In some embodiments, the pharmaceutical compositions described herein may be combined with intralesional treatment (e.g., talimogene laherparepvec).

In some embodiments, the pharmaceutical compositions described herein can be combined with cytotoxic therapies (e.g., IL-2, dacarbazine, carboplatin/paclitaxel, albumin-bound paclitaxel).

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

Although the description of the pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for administration to humans, those skilled in the art will appreciate that such compositions are generally suitable for administration to all kinds of animals. It is well understood that modifications are made to pharmaceutical compositions suitable for administration to humans to a variety of animals, and that a veterinarian of ordinary skill can design and/or make such modifications by merely ordinary (if any) experimentation.

To ensure proper quality of the components useful in the pharmaceutical compositions described herein (e.g., 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 (TPTE) antigen having tensin homology, or (v) a combination thereof), one or more quality assessments and/or criteria (e.g., RNA quality assessments) can 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 that collectively encode an NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof) can be performed by a modified (adaptation) 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 an NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof can be measured by microdroplet digital PCR.

Additionally or alternatively, in some embodiments, the residual DNA template and residual dsRNA are measured as in-process controls according to accepted criteria for drug substance intermediate levels to ensure quality of the individual RNAs prior to mixing to 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 protein may be measured in a composition comprising RNA molecules.

V. patient population

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

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

In the united states, it was estimated that about 106,110 patients would be diagnosed with cutaneous melanoma in 2021, and about 7,180 deaths would occur (Siegel et al 2021, incorporated herein by reference in its entirety). Although melanoma has a lower age-standardized incidence when compared to non-melanoma skin cancers (3.4/100,000 compared to 11.0/100,000, respectively, in 2020), 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 about 1% of skin cancers, but leads to the greatest death caused by skin cancers (ACS 2021, which is incorporated herein by reference in its entirety).

The outcome of melanoma depends on the stage of the onset (presentation). For patients with early stage disease (e.g., localized disease), 5-year survival is about 99% of patients, and for patients at regional stage (regional stage) (e.g., spread to lymph nodes), 5-year survival is 66% of patients. However, for patients with distant disease, the 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 techniques provided herein can be used to treat melanoma. In some embodiments, the techniques provided herein can be used to treat cutaneous melanoma. In some embodiments, the techniques provided herein can be used to treat advanced cancers (e.g., melanoma). Some examples of advanced cancers include, but are not limited to, stage II, stage III, or stage IV. In some embodiments, the techniques provided herein can be used to treat a disease or disorder associated with stage IIIB, stage IIIC, or stage IV melanoma. In some embodiments, the cancer is completely resected. In some embodiments, there is no evidence of disease (e.g., cancer). In some embodiments, the cancer is completely resected and no evidence of disease.

In some embodiments, the techniques provided herein can be used to treat patients (e.g., adult patients) with metastatic melanoma. In some embodiments, the techniques provided herein can be used to treat patients (e.g., adult patients) with unresectable melanoma, e.g., in some embodiments in which surgical excision may result in serious morbidity. In some embodiments, the techniques provided herein can be used to treat patients (e.g., adult patients) with locally advanced melanoma. Additionally or alternatively, in some embodiments, cancer in such patients may progress after treatment, or such cancer patients may not have satisfactory replacement therapy. In some embodiments, a patient undergoing treatment as described herein may have received other cancer treatments, such as, but not limited to, chemotherapy.

In some embodiments, the techniques provided herein can be used to treat advanced melanoma. In some embodiments, the techniques provided herein may be used to treat patients suffering from unresectable melanoma who have undergone checkpoint inhibitors (CPI).

In some embodiments, the techniques provided herein can be used to treat a patient diagnosed with cancer prior to the time of administration of a pharmaceutical composition, but wherein the patient is classified as having no evidence of disease at the time of administration (no evidence of disease, NED). In some embodiments, the patient classified as NED at the time of administration is a patient whose melanoma has been completely resected (e.g., by surgery). In some embodiments, the patient classified as NED at the time of administration is a patient who has been previously diagnosed with clinical stage 3 or stage 4 melanoma (or pathological stage 3 or stage 4 melanoma) and whose melanoma has been completely resected (e.g., by surgery). In some embodiments, the patient classified as NED at the time of administration is a patient whose melanoma has been completely resected and will continue to receive adjuvant therapy. In some embodiments, the patient classified as NED at the time of administration is a patient who was previously diagnosed with clinical stage 3 or stage 4 melanoma (or pathological stage 3 or stage 4 melanoma) and whose melanoma has been completely resected and will continue to receive adjuvant therapy. Without wishing to be bound by a particular theory, in some embodiments, the "no evidence of disease" is determined by applying RECIST criteria, such as RECIST1.1 criteria or solid tumor immune-related response assessment criteria (irRECIST) criteria.

For clarity, patients classified as NED at the time of administration are different from patients classified as having "unmeasurable disease". Patients suffering from "non-measurable disease" are intended to have evidence of disease, but cannot be treated according to RECIST criteria such as, for example, 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 contents of which are incorporated herein by reference for the purposes of the disclosure herein) are incorporated by reference for all purposes. Some examples of lesions that are considered non-measurable lesions include, but are not limited to, bone lesions, hydrothorax ascites, and "complex irregular" lesions in tissues or organs. In other words, a patient suffering from "non-measurable disease" means a patient suffering from a neoplastic lesion that is not considered to be a "measurable" lesion, according to RECIST criteria, such as RECIST1.1 criteria as discussed above. Thus, the difference between an unmeasurable disease and NED is that the former means that the disease is present but cannot be measured, while the latter (NED) means that the disease is absent and therefore is not evaluable and not significantly measurable.

Thus, administration of a pharmaceutical composition as described herein to NED patients may seem counterintuitive. However, the present disclosure recognizes that it may be determined that the patient is not cancer or in remission, but that the cancer may reappear. Accordingly, the present disclosure provides such insight: such patients may benefit from receiving a pharmaceutical composition as described herein, as it may, for example, enhance the patient's immune response to cancer. Boosting a patient's immune response to cancer may cause the patient's body to attack cancer cells, e.g., undetected or ongoing cancer cells.

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

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

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

In some embodiments, a subject administered a pharmaceutical composition described herein may have received a prior anti-cancer treatment. Some examples of previous anti-cancer therapies include, but are not limited to, chemotherapy, interferons and interleukins, monoclonal antibodies, protein kinase inhibitors, radiation therapy, immune checkpoint inhibitors, or combinations thereof. For example, in some embodiments, a subject administered a pharmaceutical composition described herein may have received an immune checkpoint inhibitor, but not experienced tumor regression. In another example, in some embodiments, a subject administered a pharmaceutical composition described herein may have received an immune checkpoint inhibitor 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, the immune checkpoint inhibitor is an antibody (e.g., without limitation, ipilimumab and nivolumab). Further examples of checkpoint inhibitors are included in table 4 above or in example 8.

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

Readout of patients to whom pharmaceutical compositions have been administered

In some embodiments, administration of a pharmaceutical composition comprising one or more RNA molecules that collectively encode an 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 to whom the pharmaceutical composition is administered (e.g., a patient classified as no evidence of disease at the time of administration). For example, in some embodiments, determining the level of immune response in the patient occurs before and after administration of the pharmaceutical composition.

Some non-limiting examples of methods for determining the level of an immune response in a patient are described in examples 1-3. For example, in some embodiments, enhanced glucose consumption after administration of the pharmaceutical composition may be exploited by [18F ] -fluoro-2-deoxy-2-d-glucose (FDG) -Positron Emission Tomography (PET)/Computed Tomography (CT) scanning of the spleen after administration of the pharmaceutical composition. Without wishing to be bound by theory, (FDG) - (PET)/(CT) scans are used to indicate at least transient activation of targeted lymphoid tissue resident immune cells. In some embodiments, the level of immune response in the patient is determined using an interferon-gamma enzyme-linked immunosorbent spot (ELISpot) assay, as described in example 1. In some embodiments, the level of metabolic activity in the spleen of the patient is measured using Positron Emission Tomography (PET), computed Tomography (CT) scanning, magnetic Resonance Imaging (MRI), or a combination thereof. In some embodiments, the level of metabolic activity in the spleen of a patient is measured using Positron Emission Tomography (PET) and Computed Tomography (CT) scans. In some embodiments, the level of metabolic activity in the spleen of a patient is measured using Positron Emission Tomography (PET) and Magnetic Resonance Imaging (MRI).

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

In some embodiments, the pharmaceutical composition induces an immune response level in the patient (e.g., a patient classified as 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 been previously diagnosed with cancer and is classified as having evidence of disease at the time of administration. In some embodiments, the level of immune response in a patient is comparable if the level of immune response in the patient is different from the level of immune response in a second patient, if they differ by less than 20%, less than 15%, less than 10%, or less than 5%.

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

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

In some embodiments, administration of a pharmaceutical composition comprising one or more RNA molecules that collectively encode an 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 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 comprises a CD4 ⁺ and/or CD8 ⁺ T cell response. In some embodiments, administration of a pharmaceutical composition comprising one or more RNA molecules that collectively encode an 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 no evidence of disease at the time of administration) induces CD4 ⁺ and/or CD8 ⁺ T cell immunity.

In some embodiments, the methods described herein comprise determining the level of an 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 associated with an immune response (e.g., IFN- α, IFN- γ, interleukin (IL) -6, IFN-Inducible Protein (IP) -10, IL-12 p70 subunit, or a combination thereof) can be used to determine the level of an 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 administration of the pharmaceutical composition.

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

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

In some embodiments, the methods described herein comprise determining the phenotype of T cells induced by the pharmaceutical composition in the patient after administration of the pharmaceutical composition. For example, in some embodiments, at least a portion of T cells in a patient induced by a pharmaceutical composition have a T helper-1 phenotype after administration of the pharmaceutical composition. 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 measuring the size of one or more cancer lesions in a patient for a patient classified as evidence of disease. For example, in some embodiments, the methods described herein comprise measuring the size of one or more cancer lesions in a patient before and after administration of the pharmaceutical composition. In some embodiments, administration of the pharmaceutical composition to a patient (e.g., a patient classified as no evidence of disease at the time of administration) maintains or reduces the size of one or more cancer lesions as compared to the size of one or more cancer lesions in the patient prior to administration of the pharmaceutical composition comprising one or more RNA molecules that collectively encode the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, the TPTE antigen, or a combination thereof. In other words, the size of the one or more cancer lesions does not increase after administration of the pharmaceutical composition described herein.

In some embodiments, the methods described herein comprise monitoring progression free survival duration for patients classified as evidence of disease. In some embodiments, the methods described herein comprise comparing the progression-free survival duration of the patient to 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 not receiving the pharmaceutical composition described herein. In some embodiments, the patient's progression-free survival duration is longer in time than the reference progression-free survival duration.

In some embodiments, the methods described herein comprise measuring the duration of disease stability for patients classified as evidence of disease. In some embodiments, disease stability is determined by application of irRECIST or RECIST 1.1 criteria. In some embodiments, the methods described herein comprise comparing the patient's disease stability duration to a reference disease stability duration. In some embodiments, the reference disease-stability duration is the average disease-stability duration of a plurality of comparable patients who have not received the pharmaceutical composition. In some embodiments, the patient administered the pharmaceutical composition described herein exhibits an increased duration of disease stability compared to a reference duration of disease stability.

In some embodiments, the methods described herein comprise measuring tumor responsiveness duration for patients classified as evidence of disease. In some embodiments, tumor responsiveness is determined by applying irRECIST or RECIST 1.1 criteria. In some embodiments, the methods described herein comprise comparing the tumor responsiveness duration of the patient to a reference tumor responsiveness duration. In some embodiments, the reference tumor response duration is the average tumor response duration of a plurality of comparable patients who did not receive the pharmaceutical composition. In some embodiments, the patient administered the pharmaceutical composition described herein exhibits an increased tumor responsiveness duration compared to a reference tumor responsiveness duration.

In some embodiments, the methods described herein comprise monitoring disease-free survival duration for patients classified as no evidence of disease. In some embodiments, the methods described herein comprise comparing the patient's disease-free survival duration to a reference disease-free survival duration. In some embodiments, the disease-free survival duration in a patient administered the pharmaceutical composition described herein exhibits a longer time than a 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 administered the pharmaceutical composition described herein exhibits an increased disease-free survival duration as compared to a reference disease-free survival duration.

In some embodiments, the methods described herein comprise measuring the duration of disease recurrence for patients classified as no evidence of disease. In some embodiments, disease recurrence is determined by application of irRECIST or RECIST 1.1 criteria. In some embodiments, the methods described herein comprise comparing the duration of a patient's to disease recurrence to a reference to the duration of disease recurrence. In some embodiments, the reference to the duration of disease recurrence is the average of a plurality of comparable patients not receiving the pharmaceutical composition to the duration of disease recurrence. In some embodiments, the patient administered the pharmaceutical composition described herein exhibits an increased duration of relapse to disease compared to the duration of relapse to disease referenced.

VII treatment of

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

Accordingly, another aspect of the present disclosure relates to methods of using the pharmaceutical compositions described herein. For example, one aspect provided herein is a method comprising administering the provided pharmaceutical composition to a subject having cancer. In some embodiments, the provided pharmaceutical compositions are administered by intravenous injection or infusion. Some examples of cancers include, but are not limited to, epithelial cancers, including, but not limited to, melanoma (e.g., cutaneous melanoma, stage IIIB, stage IIIC, or stage IV melanoma).

Dosing regimen: those skilled in the art recognize that cancer therapeutics are typically administered using a diverse range of pharmaceutical compositions that can be administered during the administration cycle.

In some embodiments, the pharmaceutical compositions described herein are administered in eight doses, e.g., within 64 days from the first administration using a priming and boosting regimen.

In some embodiments, the pharmaceutical compositions described herein are administered in six doses, e.g., within 43 days from the first administration using a priming and boosting regimen.

In some embodiments, the pharmaceutical compositions described herein are administered monthly after the initial dosing cycle (e.g., priming and boosting regimen) is completed.

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

In some embodiments, one 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, one dosing cycle is at least 28 days. In some embodiments, one dosing cycle is at least 35 days. In some embodiments, one dosing cycle is at least 42 days. In some embodiments, one dosing cycle is at least 49 days. In some embodiments, one dosing cycle is at least 56 days. In some embodiments, one dosing cycle is at least 63 days.

In some embodiments, one dosing cycle may involve multiple doses, e.g., according to such patterns: for example, the dose may be administered periodically, as in a cycle, or the dose 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 in a cycle. In some embodiments, one 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 higher. In some embodiments, one dosing cycle may involve up to 8 doses, which may be administered weekly, biweekly, or a combination thereof.

In some embodiments, multiple cycles may 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) may be administered. In some embodiments, the number of dosing cycles to be administered can vary with the type of treatment (e.g., monotherapy vs. combination therapy). In some embodiments, at least 2 dosing cycles may be administered. In some embodiments, the first dosing period may be different from the second dosing period. In some embodiments, the first dosing period may comprise 6 to 8 weekly and/or bi-weekly doses, and the second dosing period after the first dosing period may comprise at least one monthly dose.

In some embodiments, there may be a "rest period" between periods; in some embodiments, there may be no rest periods between periods. In some embodiments, there may be periods of inactivity between periods and there may be no periods of inactivity.

In some embodiments, the length of the rest period may be in the range of days to months. For example, in some embodiments, the length of the resting period may be at least 3 days or more, 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 more. In some embodiments, the length of the resting period may be at least 1 week or more, including, for example, at least 2 weeks, at least 3 weeks, at least 4 weeks, or more.

Dosage is as follows: the dosage of the pharmaceutical compositions described herein may vary depending on a number of 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 the cancer, and/or monotherapy or combination therapy. In some embodiments, the 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 a cumulative dose being set, e.g., over a specific period of time and optionally by multi-dose administration, which may be administered, e.g., at set intervals and/or according to a set pattern. In some embodiments, the set cumulative dose may be administered at set intervals with multiple doses such that there is at least some temporal overlap in the biological and/or pharmacokinetic effects produced by such multiple doses on the target cells or on the subject being treated. In some embodiments, the set cumulative dose may be administered at set intervals by multiple doses, such that the biological and/or pharmacokinetic effects produced by such multiple doses on the target cells or on the subject being treated may be additive. By way of example only, in some embodiments, a set cumulative dose of X mg may be administered by two doses and each dose is X/2 mg, where such two doses are administered in close enough time that the biological and/or pharmacokinetic effects produced by each X/2 mg dose on the target cells or on the subject being treated may be additive.

In some embodiments, each dose or cumulative dose is administered at such a level (e.g., for intravenous administration): such that the one or more RNA molecules collectively encoding the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, the TPTE antigen, or a combination thereof are expected to reach a level (e.g., plasma level and/or tissue level) that is sufficiently high 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 against the one or more antigens throughout the administration cycle.

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

In some embodiments, the methods provided herein comprise dose escalation. An exemplary method comprising dose escalation is described for example in WO 2018/0077442.

In some embodiments, the methods provided herein comprise 7 dose escalation cohorts (3+3 design) and 3 expansion cohorts. For example, table 5 provides exemplary dosing regimens.

Table 5: exemplary dosing regimen

In some embodiments, the administration may be adjusted based on the response of the subject receiving the treatment. For example, in some embodiments, administration may involve administration of a higher dose followed by administration of a lower dose if one or more parameters for safety pharmacology assessment indicate that the previous dose may not meet medical safety requirements according to a physician. 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 administration of at least one lower dose from table 5 followed by administration of at least one higher dose from table 5. Without wishing to be bound by any particular theory, the present disclosure provides, inter alia, such insight: a pharmaceutically-directed dose escalation (pharmaceutically guided dose escalation, PGDE) method may be applied to determine the appropriate dose of the pharmaceutical compositions described herein. An exemplary dose escalation study is provided in example 7.

Also provided herein are methods of determining a dosing regimen of a pharmaceutical composition comprising one or more RNA molecules that collectively encode an NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof. For example, in some embodiments, such a method comprises the steps of: (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) Periodically monitoring or measuring evidence of disease (e.g., tumor lesion size and/or metastasis) in a subject over a period of time; (C) Dosing regimens are evaluated based on the monitoring or measurement results and/or outcomes. For example, if the decrease in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is not therapeutically relevant, the dose and/or frequency of administration may be increased; or if the decrease in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is therapeutically relevant, but exhibits an adverse effect (e.g., a toxic effect) in a subject, the dose and/or frequency of administration can be reduced. If the decrease in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is therapeutically relevant and does not show adverse effects (e.g., toxic effects) in the subject, no change is made to the dosing regimen.

In some embodiments, such methods of determining the dosing regimen of a pharmaceutical composition comprising one or more RNA molecules that collectively encode an NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof, can be performed in a group of animal subjects (e.g., mammalian non-human subjects) each bearing a human melanoma xenograft tumor. In some such embodiments, the dose and/or frequency of administration may be increased if less than 30% of the animal subjects exhibit a decrease in tumor size after administration of the pharmaceutical composition (e.g., the pharmaceutical composition described herein) and/or the extent of decrease in tumor size exhibited by the animal subjects is not therapeutically relevant; or if the decrease in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is therapeutically relevant, but exhibits significant adverse effects (e.g., toxic effects) in at least 30% of the animal subjects, the dose and/or frequency of administration can be reduced. If the decrease in tumor size after administration of a pharmaceutical composition (e.g., a pharmaceutical composition described herein) is therapeutically relevant and does not show significant adverse effects (e.g., toxic effects) in an animal subject, no change is 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, those skilled in the art will appreciate that dosage equivalents for administration to all species of animals can be determined. A veterinarian of ordinary skill can design and/or make such a determination using only ordinary experimentation if present.

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

Combination therapy: the present disclosure provides, inter alia, such insight: 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, comprising one or more RNA molecules that together encode NY-ESO-1 antigen, MAGE-A3 antigen, tyrosinase antigen, TPTE antigen, or a combination thereof, may enhance the cytotoxic effects of chemotherapy and/or other anti-cancer therapies (e.g., immune checkpoint inhibitors). In some embodiments, such combination treatment may prolong progression free survival and/or overall survival, e.g., relative to treatment of an individual administered alone and/or relative to another suitable reference. Thus, in some embodiments, the pharmaceutical compositions described herein may be administered to a patient suffering from 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 a patient suffering from a tumor experiencing CPI.

The present disclosure provides, inter alia, such insight: the pharmaceutical compositions described herein may be particularly useful and/or effective when administered to a patient without evidence of disease at a first administration time, thereby demonstrating that the pharmaceutical composition induces T cell immunity even in the absence of a detectable tumor.

In some embodiments, the provided pharmaceutical compositions may be administered as part of a combination therapy comprising such pharmaceutical compositions and an immune checkpoint inhibitor. Thus, in some embodiments, provided pharmaceutical compositions can be administered to a subject having cancer (e.g., melanoma) that has received an immune checkpoint inhibitor or chemotherapeutic agent, or a subject that has received an immune checkpoint inhibitor or chemotherapeutic agent and is classified as having no evidence of disease. In some embodiments, the provided pharmaceutical compositions can be co-administered with an immune checkpoint inhibitor to a subject having cancer (e.g., melanoma) or a subject that has been classified as having no evidence of disease. In some embodiments, the provided pharmaceutical compositions and immune checkpoint inhibitors may be administered simultaneously or sequentially. For example, in some embodiments, the first dose of the immune checkpoint inhibitor may be administered after (e.g., at least 30 minutes after) administration of the provided pharmaceutical composition. In some embodiments, the immune checkpoint inhibitor and the provided pharmaceutical composition 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. Therapeutics & Oncology,14:45 (2021), which is incorporated herein by reference in its entirety) or as described in example 8.

Combination therapy with an anti-cancer therapy comprising ipilimumab: in some embodiments, the administered treatment comprising the provided pharmaceutical composition may be co-administered or overlapped with an immune checkpoint inhibitor comprising ipilimumab. Irinotecan blocks cytotoxic T lymphocyte antigen-4 (cytoxic T-lymphocyte antigen-4, ctla-4), a key negative regulator of anti-tumor T cell responses. Blocking CTLA-4 inhibits T cell activation, thereby expanding pre-existing antigen-specific T cells.

Combination therapy with an anti-cancer therapy comprising nivolumab: in some embodiments, the administered treatment comprising the provided pharmaceutical composition may 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 interactions with PD-L1 and PD-L2. Blocking this interaction releases inhibition of the PD-1 mediated immune response (including anti-tumor T cell responses) pathway, allowing the expansion of pre-existing antigen-specific T cells.

Combination therapy with anti-cancer therapy comprising pembrolizumab: in some embodiments, the administered treatment comprising the provided pharmaceutical composition may 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 interactions with PD-L1 and PD-L2. Blocking this interaction releases inhibition of the PD-1 mediated immune response (including anti-tumor T cell responses) pathway, allowing the expansion of pre-existing antigen-specific T cells.

Combination therapy with an anti-cancer therapy comprising a sempervirens Li Shan antibody: in some embodiments, the administered treatment comprising the provided pharmaceutical composition may be co-administered or overlapped with an immune checkpoint inhibitor comprising a cimipn Li Shan antibody. Zemipril Li Shan antibody is a monoclonal antibody that binds to the PD-1 receptor and blocks interactions with PD-L1 and PD-L2. Blocking this interaction releases inhibition of the PD-1 mediated immune response (including anti-tumor T cell responses) pathway, allowing the expansion of pre-existing antigen-specific T cells.

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

Exemplary embodiments

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

embodiment 1. A method comprising:

administering at least one dose of a pharmaceutical composition to a patient, the pharmaceutical composition comprising:

(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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and

(B) Lipid particles;

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

Embodiment 2. The method of embodiment 1, wherein no evidence of disease is determined by applying the standard of evaluation of immune-related responses of solid tumors (irRECIST) or the standard of RECIST 1.1.

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) 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 (TPTE) antigen having tensin homology, 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 evidence of disease or no evidence of disease is determined by applying the standard of evaluation of immune-related responses to solid tumors (irRECIST) or the standard of RECIST 1.1.

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 said 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 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 transport 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 5' cap analogue.

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 poly adenine tail.

Embodiment 17. The method of embodiment 16, wherein the poly adenine 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) A5 'cap or 5' cap analogue;

(ii) At least one 5' UTR;

(iii) A signal peptide;

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

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

(vi) A sequence encoding an MHC class I transport domain;

(vii) At least one 3' UTR; and

(Viii) Poly adenine tails.

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 lymphoid tissue of the patient.

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 particle comprises a liposome.

Embodiment 27. The method of any one of embodiments 1 to 26, wherein the lipid particle comprises a cationic liposome.

Embodiment 28. The method of any one of embodiments 1 to 25, wherein the lipid particle comprises a lipid nanoparticle.

Embodiment 29. The method of any of embodiments 1 to 28, wherein the lipid particle comprises N, N trimethyl-2-3-dioleyloxy-1-propanamine chloride (DOTMA), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine phospholipid (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.

The method of any one of embodiments 32, 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 skin 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, no evidence of disease, or both.

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

Embodiment 44 the method of any one of embodiments 1 to 43, further comprising administering at least two doses of said pharmaceutical composition to said 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 said patient at least 8 doses of said pharmaceutical composition over 10 weeks.

Embodiment 49 the method of embodiment 48, comprising administering a dose of the pharmaceutical composition to the patient weekly for a period of 6 weeks, and subsequently 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 a dose of said pharmaceutical composition to said patient monthly following said at least 8 doses.

Embodiment 51 the method of any one of embodiments 1 to 47, comprising administering a dose of the pharmaceutical composition to the patient 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 from 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 of total RNA.

Embodiment 55 the method of any one of embodiments 1 to 54, wherein the first dose and/or the second dose is from 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 of 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 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 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 said combination therapy comprises said 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 to the patient an immune checkpoint inhibitor.

Embodiment 69 the method of any one of embodiments 66 to 68, wherein the checkpoint inhibitor is or comprises: PD-1 inhibitors, PDL-1 inhibitors, CTLA4 inhibitors, lag-3 inhibitors, or combinations thereof.

Embodiment 70 the method of any one of embodiments 66 to 69, wherein the checkpoint 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: the inhibitors listed in table 4 herein.

Embodiment 72 the method of any one of embodiments 66 to 71, wherein the checkpoint inhibitor is or comprises the following: ipilimumab, nivolumab, pembrolizumab, avilamab, cimetidine Li Shan, atrazumab, devaluzumab, or combinations 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 said checkpoint inhibitor is or comprises ipilimumab and nivolumab.

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

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

Embodiment 77 the method of embodiment 76, comparing the level of an immune response in the patient to the level of an immune response in a second patient to whom the pharmaceutical composition has been administered, wherein the second patient is diagnosed with cancer prior to the time of administration and is classified as 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.

The method of any one of embodiments 79, wherein the level of the immune response is an 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, comparing the level of an immune response in the patient after administration of the pharmaceutical composition to the level of an immune response in the patient prior to administration of the pharmaceutical composition.

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

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

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

Embodiment 85 the method of any one of embodiments 75 to 84, wherein the immune response in said 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 said 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 lymphoid tissue of the patient.

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 spleen of the patient 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 spleen of the patient is measured using Positron Emission Tomography (PET), computed Tomography (CT) scan, 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.

The method of embodiment 94 or 95, wherein the one or more cytokines comprise Interferon (IFN) - α, IFN- γ, interleukin (IL) -6, IFN-Inducible 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 is less cancer lesion 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 a plurality of 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 a first dose of the pharmaceutical composition and after administration of a 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 after administration of the second dose of the pharmaceutical composition is greater 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 of embodiment 104, wherein at least a portion of the T cells in the patient induced by the pharmaceutical composition have a T helper-1 phenotype.

Embodiment 106. The method of embodiment 104 or 105, wherein the T cells in the patient induced 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 measuring the size of one or more cancer lesions for a patient classified as evidence of disease.

Embodiment 108 the method of any one of embodiments 3 to 107, further comprising, for a patient classified as 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 the at least one cancer lesion in the patient after administration of the pharmaceutical composition is equal to or less than the size of the at least one cancer lesion prior to 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 evidence of disease.

Embodiment 112 the method of embodiment 111, which compares the progression free survival duration of the patient to a reference progression free survival duration.

Embodiment 113 the method of embodiment 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.

Embodiment 114 the method of embodiment 112 or 113, wherein the patient's progression-free survival duration is longer in time than 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 stability for patients classified as evidence of disease.

The method of embodiment 116.115, wherein the disease stability is determined by application of irRECIST or RECIST 1.1 criteria.

Embodiment 117 the method of embodiment 115 or 116, further comprising comparing the patient's disease stability duration to a reference disease stability duration.

Embodiment 118 the method of embodiment 117, wherein said reference disease stability duration is the average disease stability duration of a plurality of comparable patients who did not receive said pharmaceutical composition.

Embodiment 119 the method of embodiment 118, wherein said patient exhibits an increased disease stability duration as compared to said reference disease stability duration.

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

The method of embodiment 121.120, wherein tumor responsiveness is determined by applying the irRECIST or RECIST 1.1 criteria.

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

Embodiment 123 the method of embodiment 122, wherein said reference tumor response duration is the average tumor response duration of a plurality of comparable patients who did not receive said pharmaceutical composition.

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

Embodiment 125 the method of any one of embodiments 1 to 106, further comprising monitoring disease-free survival duration for patients classified as 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 of embodiment 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.

Embodiment 128 the method of embodiment 127, wherein said patient exhibits an increased disease-free survival duration as compared to said reference disease-free survival duration.

Embodiment 129 the method of any of embodiments 1-106 and 125-128, further comprising measuring the duration of disease recurrence for a patient classified as no evidence of disease.

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

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

Embodiment 132 the method of embodiment 131, wherein the duration of reference to disease recurrence is the average of a plurality of comparable patients not receiving the pharmaceutical composition to the duration of disease recurrence.

Embodiment 133 the method of embodiment 132, wherein the patient exhibits an increased duration of relapse to disease compared to the duration of relapse to disease referenced.

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) 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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and

(B) Lipid particles;

and wherein the patient is classified as having no evidence of disease, but has been previously diagnosed as having cancer.

Embodiment 135 a pharmaceutical composition for treating 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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and

(B) Lipid particles;

and wherein the patient is classified as having no evidence of disease, but has been previously diagnosed as having 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) 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 (TPTE) antigen having tensin homology, 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) 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 (TPTE) antigen having tensin homology, 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 of embodiments 134 to 139, wherein evidence of disease or no evidence of disease is determined by application of a solid tumor immune-related response assessment criteria (irRECIST) criteria or RECIST 1.1 criteria.

The pharmaceutical composition of any one of embodiments 134-140, wherein the cancer is melanoma.

Embodiment 142 the pharmaceutical composition of any one of embodiments 134 to 141, wherein said one or more RNA molecules comprise:

(i) A first RNA molecule encoding said NY-ESO-1 antigen,

(Ii) A second RNA molecule encoding MAGE-3 antigen,

(Iii) A third RNA molecule encoding a tyrosinase antigen, and

(Iv) A fourth RNA molecule encoding TPTE antigen.

Embodiment 143 the pharmaceutical composition of any one of embodiments 134-142, 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 144 the pharmaceutical composition of any one of embodiments 134 to 143, wherein a single RNA molecule of said one or more RNA molecules encodes a multi-epitope polypeptide, wherein said multi-epitope polypeptide comprises at least two of said NY-ESO-1 antigen, said MAGE-3 antigen, a tyrosinase antigen, and said TPTE antigen.

Embodiment 145 the pharmaceutical composition of any of embodiments 134-144, wherein said 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 said one or more RNA molecules comprise a sequence encoding an MHC class I transport domain.

Embodiment 148 the pharmaceutical composition of any of embodiments 134-147, wherein said one or more RNA molecules comprises a5 'cap or 5' cap analogue.

The pharmaceutical composition of any one of embodiments 134-148, 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 said one or more RNA molecules comprise at least one non-coding regulatory element.

Embodiment 151 the pharmaceutical composition of any of embodiments 134 to 150, wherein said one or more RNA molecules comprise a poly adenine tail.

Embodiment 152 the pharmaceutical composition of embodiment 151, wherein the poly-adenine tail is or comprises a modified adenine sequence.

Embodiment 153 the pharmaceutical composition of any one of embodiments 134-152, wherein said 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) A5 'cap or 5' cap analogue;

(ii) At least one 5' UTR;

(iii) A signal peptide;

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

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

(vi) A sequence encoding an MHC class I transport domain;

(vii) At least one 3' UTR; and

(Viii) Poly adenine tails.

Embodiment 155 the pharmaceutical composition of any of embodiments 134-154, wherein said one or more RNA molecules comprise natural ribonucleotides.

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

Embodiment 157 the pharmaceutical composition of any of embodiments 134-156, 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.

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

The pharmaceutical composition of any one of embodiments 134-158, 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 lymphoid tissue of the patient.

The pharmaceutical composition of any one of embodiments 134-159, wherein at least one of said NY-ESO-1 antigen, said MAGE-3 antigen, said tyrosinase antigen, and said TPTE antigen is present in said cancer.

Embodiment 161 the pharmaceutical composition of any of embodiments 134-160, wherein said lipid particle comprises a liposome.

The pharmaceutical composition of any one of embodiments 134-160, wherein the lipid particle comprises a cationic liposome.

The pharmaceutical composition of any one of embodiments 134-162, wherein the lipid particle comprises a lipid nanoparticle.

The pharmaceutical composition of any of embodiments 134-163, wherein the lipid particle comprises N, N trimethyl-2-3-dioleyloxy-1-propanammonium chloride (DOTMA), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine phospholipid (DOPE), or both.

Embodiment 165 the pharmaceutical composition of any of embodiments 134-164, wherein said lipid particle comprises at least one ionizable amino lipid.

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

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

Embodiment 168 the pharmaceutical composition of any of embodiments 166 or 167 wherein the helper lipid is or comprises a sterol.

The pharmaceutical composition of any one of embodiments 134-168, 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 said patient is a human.

The pharmaceutical composition of any one of embodiments 134-170, 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 said melanoma is skin melanoma.

Embodiment 174 the pharmaceutical composition of any of embodiments 134-173, wherein the cancer is advanced.

Embodiment 175 the pharmaceutical composition of any of embodiments 134-174, 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 said cancer is stage IIIB, IIIC or IV melanoma.

Embodiment 177 the pharmaceutical composition of any one of embodiments 134-176, wherein said cancer is completely resected, no evidence of disease, or both.

Embodiment 178 the 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) 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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and

(B) Lipid particles;

and wherein the patient is classified as having no evidence of disease, but has been previously diagnosed as having cancer.

Use of a pharmaceutical composition for treating 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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and

(B) Lipid particles;

and wherein the patient is classified as having no evidence of disease, but has been previously diagnosed as having cancer.

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

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) 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 (TPTE) antigen having tensin homology, 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) 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 (TPTE) antigen having tensin homology, 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 evidence of disease or no evidence of disease is determined by applying the solid tumor immune-related response assessment criteria (irRECIST) criteria or RECIST 1.1 criteria.

The use of any of embodiments 186, 178-185, wherein the cancer is melanoma.

Embodiment 187 the use of any one of embodiments 178 to 186, wherein the one or more RNA molecules comprises:

(i) A first RNA molecule encoding said NY-ESO-1 antigen,

(Ii) A second RNA molecule encoding MAGE-3 antigen,

(Iii) A third RNA molecule encoding a tyrosinase antigen, and

(Iv) A fourth RNA molecule encoding TPTE antigen.

The use of any one of embodiments 178-187, 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.

The use of any one of embodiments 178-188, 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.

The use of any one of embodiments 178-189, 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.

The use of any one of embodiments 178-191, wherein the one or more RNA molecules comprise a sequence encoding an MHC class I transport domain.

The use of any of embodiments 178-192, wherein the one or more RNA molecules comprise a 5 'cap or 5' cap analog.

Embodiment 194 the use of any of embodiments 178-193, wherein said one or more RNA molecules comprises a sequence encoding a signal peptide.

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

The use of any one of embodiments 178-195, wherein the one or more RNA molecules comprise a poly adenine tail.

The use of embodiment 197, embodiment 196, wherein the poly-adenine tail is or comprises a modified adenine sequence.

The use of any one of embodiments 178-197, 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) A5 'cap or 5' cap analogue;

(ii) At least one 5' UTR;

(iii) A signal peptide;

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

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

(vi) A sequence encoding an MHC class I transport domain;

(vii) At least one 3' UTR; and

(Viii) Poly adenine tails.

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

Embodiment 201 the use of any one of embodiments 178 to 200, wherein said 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 said NY-ESO-1 antigen, said MAGE-3 antigen, said tyrosinase antigen, and said TPTE antigen is a full-length, non-mutated antigen.

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

The use of any one of embodiments 178-203, 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 lymphoid tissue of the patient.

The use of any one of embodiments 178-204, wherein at least one of said NY-ESO-1 antigen, said MAGE-3 antigen, said tyrosinase antigen, and said TPTE antigen is present in said cancer.

Embodiment 206 the use of any of embodiments 178-205, wherein said lipid particle comprises a liposome.

Embodiment 207 the use of any of embodiments 178-205, wherein said lipid particle comprises a cationic liposome.

Embodiment 208 the use of any of embodiments 178-207, wherein said lipid particle comprises a lipid nanoparticle.

Embodiment 209, any of embodiments 178 to 208, wherein said lipid particle comprises N, N trimethyl-2-3-dioleyloxy-1-propanammonium chloride (DOTMA), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine phospholipid (DOPE), or both.

The use of any one of embodiments 178-209, wherein the lipid particle comprises at least one ionizable amino lipid.

The use of any one of embodiments 178-210, wherein the lipid particle comprises at least one ionizable amino lipid and a helper lipid.

The use of any one of embodiments 212, 211, wherein said helper lipid is or comprises a phospholipid.

The use of any one of embodiments 211 or 212, wherein said helper lipid is a sterol or comprises a sterol.

The use of any one of embodiments 178-213, wherein the lipid particle comprises at least one polymer conjugated lipid.

The use of any of embodiments 178-214, wherein the patient is a human.

The use of any one of embodiments 178-215, wherein the cancer is an epithelial cancer.

The use of any one of embodiments 178-216, wherein the cancer is melanoma.

Embodiment 218 the use of embodiment 217, wherein the melanoma is skin melanoma.

The use of any one of embodiments 178-218, wherein the cancer is advanced.

The use of any one of embodiments 178-219, wherein the cancer is stage II, stage III or stage IV.

The use of any one of embodiments 178-220, 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 said cancer is completely resected, no evidence of disease, or both.

Example

Example 1: test design, materials and methods

Lipo-MERIT clinical trial design. The main purpose of this test (NCT 02410733) is: assessing the safety and tolerability of melanoma FixVac, its primary efficacy and progression free survival; study of vaccine-induced antigen-specific immune responses; and determining the phase II dose. As used herein, the term "FixVac" refers to a pharmaceutical composition that comprises one or more RNA molecules as shown in fig. 1 and a lipid particle (e.g., a lipid complex or lipid nanoparticle). BNT111 is one embodiment of FixVac. Since this is the first phase I trial of the human body and is consistent with the purpose, no statistical method is used to predetermine the sample amount. The investigator did not take blindness to the assignment during the experimental and outcome assessment.

The test was based on Helsinki declaration (Declaration of Helsinki) and Good clinical practice Guidelines (Good CLINICAL PRACTICE Guidelines), and was developed in the independent ethical committee (INDEPENDENT ETHICS committee) (Ethik-Kommission of the)RHEINLAND PFALZ, mainz, germany) and the regulatory authorities (competent regulatory authority) (Paul-Ehrlich Institute, langen, germany). All patients provided written informed consent.

Eligible patients have stage III B to C or IV malignant melanoma (united states joint committee for cancer (American Joint Committee on Cancer, AJCC) 2009 melanoma classification), both resected and non-resected, and therefore at baseline, had measurable and non-measurable disease, and expressed at least one of the four vaccine TAAs. The patient is also at least 18 years old and has appropriate hematologic and end-organ (end-organ) function. Inclusion criteria require that after all available treatment options have been transparently disclosed, the subject is disqualified for, or refuses, any other available approved treatment. The key exclusion criteria are the presence of clinically relevant autoimmune diseases, human immunodeficiency virus (human immunodeficiency virus, HIV), hepatitis b virus (HEPATITIS B VIRUS, HBV), hepatitis c virus (HEPATITIS C virus, HCV) or active brain metastasis. Patients received eight RNA-LPX injections (prime/repeat boost regimen) within 64 days, which received only six injections within 43 days, except for patients from cohort 1. For patients with measurable disease that do not exhibit disease progression or drug-related toxicity, an optional continued treatment of once-a-month vaccine doses is provided. Patients were treated in seven dose escalation cohorts where the target dose was 14.4 μg to 400 μg of total RNA and in three expanded cohorts further exploring dose levels of 14.4 μg, 50 μg and 100 μg. RNA-LPX administration was performed by four consecutive intravenous slow bolus injections using an intravenous catheter.

Additional information about patients who participated in the study was contained in figures 35 and 36.

Key study evaluation. Safety and tolerability are assessed based on physical examination or changes in vital signs, clinical laboratory analysis, and reporting of any adverse event, including clinically significant laboratory abnormalities. Adverse events were graded according to the national cancer institute common terminology standard (National Cancer Institute Common Terminology Criteria) (NCI CTC version 4.03). Security is characterized from level 1 to level 5 according to these criteria.

Chest, abdomen and brain were imaged by CT scanning and Magnetic Resonance Imaging (MRI) at baseline and every 90 days thereafter according to local imaging guidelines and version irRECIST 1.1.1 (ref 25).

Vital signs (body temperature, heart rate and blood pressure) were measured and used as clinical indicators before FixVac and 4 hours after FixVac.

To evaluate vaccine-induced immune responses, blood was sampled at baseline, before the fourth, sixth and eighth vaccine administration, 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 continued treatment, blood samples were taken prior to each administration. PBMCs were isolated from peripheral blood or leukopenia samples by Ficoll-Hypaque (Amersham Biosciences) density gradient centrifugation.

For cytokine analysis, serum was sampled 2 hours, 6 hours, 24 hours or 48 hours before and after treatment and transported at-80 ℃. Samples were analyzed in duplicate (MLM MEDICAL Labs) using a human pan-IFN- α ELISA (PBL ASSAY SCIENCE) and a multiport (multisport) assay system (Meso Scale Discovery). The sample amounts for each analysis were: IFN- α relative to IP-10, n=166; IFN- α versus IFN- γ, n=167; IFN- α relative to IL-6, n=167; IFN- α relative to IL-12 p70, n=167; from 72 patients, and wherein each patient is up to 6 data points.

Delayed-type hypersensitivity (DTH) responses were evaluated in detail in some patients. Skin infiltrating lymphocytes (skin-INFILTRATING LYMPHOCYTE, SIL) were recovered from a punch biopsy after intradermal injection of RNA diluted in concentrated (. Times.2.67) Ringer's solution (manufactured by BAG HEALTH CARE under good manufacturing practice (good manufacturing practice, GMP) guidelines) in medium (RPMI 1640,7% human AB serum, 1X antifungal) containing high doses of IL-2 (50,000 U.ml-1) for 2 to 3 weeks.

Data reporting. This is an ongoing exploratory, open-label, non-randomized, first-time phase I clinical trial in humans. Presented data was based on a exploratory metaphase (interim) analysis, with data extraction date of 2019, 7, 29. This exploratory analysis was performed to inform and initiate the design of a randomized phase 2 trial for FixVac/anti-PD 1 combination therapy for patients experiencing CPI. The analysis was initiated by baseline to three months comparison of the availability of immunogenicity data for about half of the study population (n=51) in the dose cohort, as well as the availability of at least three months follow-up data for two patient sub-populations treated with FixVac monotherapy and FixVac/anti-PD 1 combination. Exploratory metaphase analysis is particularly focused on vaccine-induced immune responses (secondary endpoints). Furthermore, preliminary high level data are reported regarding study drug tolerance (primary endpoint) and response (secondary endpoint) of patients with measurable disease according to irRECIST 1.1.1. The displayed clinical data is preliminary and incompletely validated source data. 109 of 115 patients (95%) were recruited when this paper was published.

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

FDG-PET/CT imaging. About 2 MBq kg-1 FDG was applied after a 4 to 6 hour fasting period (resulting in blood glucose levels below 130mg dl-1) and after a 60 to 70 minute dispensing time, and [18f ] fdg uptake in the spleen was assessed by PET/CT imaging. According to clinical routine, PHILIPS GEMINI time-of-flight (TOF) PET/CT scanners certified by EARL acquire at 2 to 2.5 minutes per bed position. Average normalized uptake values (standardized uptake value, SUV) were measured in 2cm spheres centered within the spleen.

TAA expression profiling. Total RNA was extracted from formalin-fixed paraffin embedded (FFPE) samples of patients (RNEASY FFPE kit, qiagen). Complementary DNA (Peqstar, VWR Intemational) was synthesized according to the guidelines of the good clinical laboratory specifications (good clinical laboratory practice, GCLP) and analyzed by quantitative polymerase chain reaction (polymerase chain reaction, PCR; applied Biosystems, 7300 real-time PCR system, thermo FISHER SCIENTIFIC) for expression of NY-ESO-1, tyrosinase, MAGE-A3 and TPTE RNA and reference genes encoding hypoxanthine guanine phosphoribosyl transferase (hypoxanthine guanine phosphoribosyltransferase, HPRT 1). The median quantitative cycle (Cq) value for each TAA was normalized to the median Cq of the reference gene to obtain a relative expression ACq value, which was classified as positive or negative based on TAA-specific cut-off.

RNA-LPX production. RNA, liposomes and RNA-LPX are manufactured under GMP conditions. RNA was produced by in vitro transcription of DNA plasmid templates encoding the full length sequences of amino acids 1 to 477 of NY-ESO-1, MAGE-A3, TPTE or tyrosinase. The manufacture, analysis and release of the four TAA-encoding RNA drug products were performed as previously described (reference 26).

Liposomes with a net cationic charge are used to complex RNA to form RNA-LPX. Based on ethanol injection technique (reference 28), cationic liposomes were made from cationic synthetic lipids (R) -N, N trimethyl-2-3-dioleyloxy-1-propanammonium chloride (R-DOTMA) (MERCK AND CIE) and phospholipid 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine phospholipid (DOPE) (Corden Pharma) using the proprietary protocol employed (reference 27). The release analysis of liposomes included determining appearance, lipid concentration, rnase presence, particle size, osmolarity, pH, sub-visible particles (subvisible particle), pyrogen testing, and sterility.

Injectable RNA-LPX drug products were prepared in a dedicated pharmacy according to a proprietary (reference 27) protocol by incubating the concentrated RNA drug product alone with isotonic NaCl solution (0.9%) (Fresenius Kabi) and cationic liposomes. The RNA-LPX preparation protocol was derived from the protocol for nucleotide lipid complex formation as described (references 8, 29). RNA-LPX was further diluted to the desired concentration using isotonic NaCl solution (0.9%) (Fresenius Kabi) prior to injection. Periodic quality control of RNA-LPX pharmaceutical products includes determining 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 peptide encoding TAA is used. For IVS using RNA, CD 4-or CD 8-depleted PBMC were electroporated with RNA encoding vaccine antigen, enhanced green fluorescent protein (ENHANCED GREEN fluorescent protein, eGFP), influenza matrix protein 1 (M1) or tetanus p2/p16 sequences (influenza M1 and tetanus p2/p16 are positive controls for CD4+ and CD8+ T cells, respectively) after overnight rest. The cells were then allowed to stand at 37℃for 3 hours and irradiated to 15 Gy. The overnight resting CD4+/CD8+ T cells were combined with electroporated and irradiated antigen presenting cells at a ratio of effector to target (E: T) of 2:1. For peptide IVS, CD4+ T cells were expanded in the presence of fast dendritic cells pulsed with PepMix encoding MAGE-A3, tyrosinase, TPTE or NY-ESO-1 (E: T=10: 1). To expand cd8+ T cells, CD 4-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 (granulocyte macrophage colony-stimulating factor, GM-CSF) (1,000 u·ml-1 each) and the corresponding peptide. One day after the initiation of IVS, fresh medium containing 10U ml-1 IL-2 (Proleukin S, novartis) and 5ng ml-1 IL-15 (Peprotech) was added. CD8 IVS cultures stimulated with peptides received additional IL-4 and GM-CSF (1,000U ml-1 each). For tumor cell lysis experiments, peptide pulsed large amounts of PBMCs were used for IVS and harvested after 6 to 8 days of culture. For longer cultures, IL-2 was supplemented 7 days after IVS culture was set up. After 11 days of stimulation, cells were analyzed by flow cytometry and used in ELISpot assays.

IFN-. Gamma.ELISPot. ELISpot analysis was performed on 51 patients (50 patients ex vivo, 20 patients after IVS). In addition to the 49 patients shown in fig. 5, two patients who received BRAF/MEK inhibitors were tested in IFN- γ ELISPOT. Multiscreen filter plates (Merck Millipore) pre-coated with antibodies specific for IFN-gamma (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 with peptide (ex vivo), with autologous dendritic cells electroporated with RNA or loaded with peptide (after IVS), or with peptide-loaded HLA class I or II transfected K562 cells (for TCR validation) for 16 to 20 hours. To analyze isolated T cell responses, cryopreserved PBMCs were allowed to stand at 37 ℃ for a period of 2 to 5 hours followed by ELISpot. Or using CD4 ^- or CD8 ^- depleted PBMCs as CD8 or CD4 effectors. All assays were performed in duplicate or triplicate and included positive controls (staphylococcal enterotoxin B (Staphyloccocus enterotoxin B) (Sigma Aldrich), anti-CD 3 (Mabtech)) and cells from reference donors with known reactivity. Spots were visualized with biotin conjugated anti-Fnγ antibody (Mabtech) followed by incubation with ExtrAvidin-alkaline phosphatase (Sigma-Aldrich) and 5-bromo-4-chloro-3-indolyl phosphate (5-bromo-4-chloro-3-indolyl phosphate, BCIP)/nitroblue tetrazolium (nitro blue tetrazolium, NBT) (Sigma-Aldrich). Or using a secondary antibody (ELISPot-Pro kit, mabtech) conjugated directly to alkaline phosphatase. The plates were scanned using an ImmunoSpot series S5Versa ELISpot analyzer (CTL, S5 Versa-02-9038) or a classical robotic ELISpot reader (AID) and analyzed by ImmunoCapture version 6.3 or AID ELISpot 7.0 software. The spot counts were summed up to median of each triplicate or duplicate. The T cell response stimulated by the RNA or peptide encoding the vaccine antigen is compared to the response 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 the ex vivo environment, or 25 spots/5×10 ⁴ cells in the post IVS environment, and a spot count greater than twice as high as the corresponding control.

Flow cytometry. Antigen-specific CD8 ⁺ T cells were identified using fluorophore-conjugated HLA multimers (Immudex). Cells were first stained for multimers and then for cell surface markers as follows (antibody clones in brackets): CD28 (CD 28.8), CD197 (150503), CD45RA (HI 100), CD3 (UCHT 1 or SK 7), CD16 (3G 8), CD14CD19 (SJ 25C 1), CD27 (L128), CD279 (EH 12), CD134 (ACT 35) and CD8 (RPA-T8 or SK 1), all purchased from BD Biosciences; CD19 (HIB 19) and CD4 (OKT 4), from Biolegend. Live-dead staining was also performed using 4',6-diamidino-2-phenylindole (4', 6-diamidino-2-phenylindole, DAPI; BD) or a fixable viability dye eFluor 780 or eFluor 506 (eBioscience). A unimodal, live, multimeric positive event was identified in CD3 ⁺ (or CD8 ⁺)、CD4^-CD14^-CD16^-CD19^- or CD3 ⁺ (or CD8 ⁺) CD 4-event to gate for the detection of antigen-specific T cells after IVS, unimodal, live CD3 ⁺、CD8⁺ multimeric + lymphocytes were gated.

For staining intracellular cytokines, autologous dendritic cells electroporated with RNA encoding a single neoepitope were added at an E:T ratio of 10:1 and incubated in the presence of brefeldin A (brefeldin A) and monensin (monensin) for about 16 hours at 37 ℃. Cells were stained for viability (using the fixable viability dye eFluor 506 or eFluor 780, ebioscience) and for surface markers, CD8 (RPA-T8 or SK 1), CD16 (3G 8), CD14(All from BD Biosciences), CD19 (HIB 19) or CD4 (OKT 4) (from Biolegend). After permeabilization, intracellular cytokine staining was performed using antibodies against IFN- γ (B27, BD Biosciences) and antibodies against TNF (Mab 11, BD or bioleged). IFN-gamma and TNF+ events were identified in CD8 ⁺ and CD4 ⁺ cells of preset gates on unimodal, live and CD14 ^-CD16^-CD19^- (not used in all experiments) populations.

Cell surface expression of transfected TCR genes was analyzed using anti-TCR antibodies (Beckman Coulter) and CD 8-or CD 4-specific antibodies (SK-1,BD;REA623,Miltenyi Biotec) directed against the appropriate variable region family or constant region of the TCR-beta chain. 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 (DX 17, BD Biosciences). The collection was performed on LSR Fortessa SORP, FACSCELESTA or FACSCanto II cell analyzer (BD Biosciences) and analyzed by FlowJo software (Tree Star).

Cloning of HLA antigen. HLA antigens are synthesized by Eurofins Genomics Germany GmbH according to the corresponding high resolution HLA typing results. HLA DQA sequences were amplified from donor-specific cDNA with 2.5U Pfu polymerase 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. HLA antigens were cloned into an appropriately digested IVT vector (ref 10).

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

A peptide. Overlapping peptide pools (PepMix) encoding full length NY-ESO-1, tyrosinase, MAGE-A3 and TPTE or short (8 to 11 mer) epitopes derived from these antigens were used, as well as HIV gag encoding PepMix 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 3mM (short peptide) or in 100% DMSO (PepMix).

A cell line. Both the K562 and SK-MEL-28 cell lines were obtained from ATCC. The SK-MEL-29 cell line was obtained from the New York commemorative Stonekilin cancer center (memory slot KETTERING CANCER CENTER, new York). The SK-MEL-37 cell line is described in reference 30. A Jurkat T cell line was prepared by Promega that expressed a luciferase reporter driven by the nuclear factor (nuclear factor ofactivated T cell, NFAT) -responsive element of activated T cells. Re-certification of cell lines was performed by short tandem repeat (short TANDEM REPEAT, STR) spectroscopic analysis at the American type culture Collection (AMERICAN TYPE Culture Collection, ATCC) and Eurofins. All cell lines used were tested negative for mycoplasma contamination. Common Misidentified (MISIDENTIFIED) cell lines were not used.

Single cell sorting. Individual antigen-specific T cells were sorted using ex vivo PBMC or IVS cultures based on stimulation-induced IFN- γ secretion or multimeric binding. For stimulation, PBMCs were pulsed with overlapping peptides encoding the relevant antigen or control antigen, while T cells expanded after IVS were cultured with autologous peptide pulsed dendritic cells. After 4 hours, cells were harvested and stained with the viability dye eFluor 780 (eBioscience) and the fluorochrome conjugated antibodies to CD3, CD8 and CD4 (all BD Biosciences) and ifny secretion assay kit (Miltenyi Biotec) with the fluorochrome conjugated antibodies to ifny. Or the PBMC were stained with the corresponding multimers. Sorting of individual neoantigen-specific T cells was performed on a FACSAria or FACSMelody flow cytometer (all from BD Biosciences) using BD FACSDiva or BD FACSChorus software, respectively. Antigen-specific T cells were identified relative to control samples stimulated with control antigen or stained without multimers. One T cell per well (gating on single live cd3+ and cd8+ IFN- γ+, cd4+ IFN- γ+ or cd8+ multimer+ lymphocytes) was harvested into a 96 well V-plate (Greiner Bio-One) containing 6 μl of mild hypotonic lysis buffer/well (consisting of 0.2% Triton X-100, 0.2 μ 1 RiboLock RNA enzyme inhibitor (Thermo Scientific), 5ng poly (a) vector RNA (Qiagen) and 1 μl dNTP mix (10 mm, biozym) in rnase-free water). After sorting, the plates were sealed, centrifuged and stored directly at-65 ℃ to-85 ℃.

Cloning of antigen-specific TCRs. The TCR gene was cloned from a single T cell as described in 10, and the following modifications were made. Plates with selected cells were thawed and template-switched cDNA synthesis was performed with REVERTAID H reverse transcriptase (Thermo Fisher) using primers (TRAC,5′-catcaeaggaactttctgggctg-3′;TRBC1,5′-gctggtaggacaccgaggtaaagc-3′;TRBC2 5′-gctggtaagactcggaggtga agc-3′) specific for TCR-a and TCR- β constant genes, 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 V.alpha.V.beta.gene-specific multiplex PCR. The products were analyzed on a capillary electrophoresis system (Qiagen). Samples of the bands at 430bp to 470bp were size fractionated on agarose gels and the bands were excised and purified using a gel extraction kit (Qiagen). The purified fragments were sequenced and the corresponding V (D) J junctions were analyzed using the IMGT/V-Quest tool (ref.31). The DNA of the new and effectively rearranged corresponding TCR chain was digested with NotI and cloned into pST1 vector containing the appropriate constant region for in vitro transcription of the complete TCR-a/β chain 10.

Single cell TCR sequencing. For selected patients, TCRs from the sorted single cells were obtained by single-cell TCR sequencing (single-cell TCR sequencing, scTCR-seq) workflow based on next generation sequencing (next generation sequencing, NGS). Here, template-switched cDNA synthesis was performed using primers specific for TCR-alpha and TCR-beta constant genes (TRAC, 5'-catcacaggaactttctgggctg-3'; TRBC,5 '-CACGTGGTCGGGGWAGAAGC-3'), followed by treatment with 5U exonuclease I. Each cDNA was PCR amplified and line-by-line barcoded (95 ℃ for 2 min; 5 cycles of 94 ℃ for 30 seconds, 61 ℃ for 30 seconds, 72 ℃ for 1 min; 5 cycles of 94 ℃ for 30 seconds, 64 ℃ for 30 seconds, 72 ℃ for 1 min; 8 cycles of 94 ℃ for 30 seconds, 72 ℃ for 2 min; 72 ℃ for 6 min) (RBC, line code; TS, template transfer primer) using 2.5U PfuUltra Hotstart DNA polymerase (Agilent), 1 xpcr buffer, 0.2mM dntps, 0.2 μm of one of the eight labeled forward primers (Tag 130-RBCx-TS 5 '-cgatccagactagacgctcaggaagxxxxxaagcagtggtatcaacgcagagt-3') and 0.1 μm of each labeled nested (linked) TCR-a and TCR- β constant gene-specific primer (Tag146-TRAC,5′-caatatgtgaccgccgagtcccaggttagagtctc tcagctggtacacggcag-3′;Tag146-TRBC,5′-caatatgtgaccgccgagtccc aggggctcaaacacagcgacctcgggtg-3′),. 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℃for 1 min; 24 cycles of 94℃for 20 seconds, 64℃for 20 seconds, 72℃for 30 seconds; 72℃for 3 min) using 1. Mu. l PfuUltra II Fusion Hotstart DNA polymerase (Agilent), 1 Xreaction buffer, 0.2mM dNTPs, forward primer (Tag-130 5'- (n) NNNNCGATCCAGACTAGACGCTCAGGAAG-3') and one of 12 Tag-146 reverse oligonucleotides containing a different barcode for each column (5 '-xxxxxcaatatgtgaccgccgagtcccagg-3'). PCR products were pooled and purified with AMPure XP beads and exonuclease I, followed by TCR sequencing library generation using TruSeq DNA Nano kit (Illumina). The scTCR library was sequenced on an Illumina Miseq using paired-end 300-bp sequencing with a sequencing depth of 10,000 reads/well. Sequencing data was then demultiplexed (demultiplex) to single cell level by internal Python script using bcl2fastq software (Illumina). TCR sequences were then obtained using MiXCR-2.1.5 (ref 32). The selected paired alpha and beta V (D) J fragments (Eurofins Genomics) were synthesized and cloned as above for subsequent in vitro transcription.

A large number of TCRs were sequenced. Total RNA was isolated from 1X 106 quick frozen PBMC, which were collected at various time points during vaccination, using RNEASY MINI kit (Qiagen). Libraries were generated using a smart human TCR- α/β profiling kit (Clontech) and sequenced using the Illumina MiSeq system. The number of total TCR readouts per sample is 1 x 106 to 4 x 106. Data were analyzed using VDJtools (ref 33) and MiXCR.

Characterization of functional TCR. 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 the T cell activation bioassay (NFAT, promega) were transfected with RNA encoding CD8- α and TCR- α/β and tested against target cells (FIG. 4 c). T cell activation was analyzed by luminescence measurement (INFINITE F PRO, tecan) after addition of Bio-Glo reagent (Promega).

Cytotoxicity assay. T cell mediated cytotoxicity was assessed by cell index impedance measurement using the xcelligent MP system (OMNI LIFE SCIENCE) according to the instructions of the supplier. As effector cells, OKT 3-activated TCR-transfected cd8+ T cells from healthy donors or patient-derived cd8+ T cells from IVS cultures were used. Melanoma cell lines transfected with the corresponding HLA allele and seeded in 96-well PET E plates (ACEA Biosciences) at a concentration of 2×104 cells/well were used as target cells. After 24 hours, at different E: t ratio effector T cells were added and cell index values were monitored every 30 minutes using the xcelligent system for a period of up to 48 hours. Specific lysis was calculated after the indicated time of co-culture (FIGS. 2i, 3e,12 hours; FIG. 3d,63 hours; FIG. 4f,8 hours) based on negative control (T cells mock transfected for TCR; pretreatment of IVS cultures for IVS cells).

Mutation discovery and gene expression. Mutations were detected as described (reference 26). Essentially, genomic sequence reads from each patient were aligned with the ginseng genome hg19 using Burrows-WHEELER ALIGNER (BWA) software (reference 34). Exons from tumor and matched normal samples were compared to retrieve single nucleotide variants (single nucleotide variant, SNV). To preserve high confidence SNV, loci with putative homozygous genotypes are filtered, and suspected sites from putative heterozygous mutant events are filtered to remove false positives. For the final list of high confidence mutations, genomic coordinates (genomic coordinate) and genes known to the san cruz division, california university (University of California at Santa Cruz, UCSC) genomic browser were incorporated to correlate variants with genes. Non-synonymous mutations were selected for further processing.

Gene expression values were calculated using tumor RNA sequencing data using Sailfish (reference 35) and known gene transcripts of UCSC as references. Transcript counts were normalized to per million transcripts (TRANSCRIPTS PER million, TPM).

To compare the mutation load with the gene expression, in the case where the genes in the UCSC database are represented by several transcript isoforms, an average of transcript expression values was used. The following patient data from three melanoma groups were used to correlate mutation burden and expression levels: 13 patients from NCT02035956 trial (reference 26), 25 patients from the published melanoma cohort (reference 22), and metastasis data from 12 of MET500 cohort patients (reference 36).

Statistics and reproducibility. The sample amount (n) represents the number of patients analyzed, except in fig. 6c where the sum of the multiple measurements from 72 patients (up to 6 per patient) is designated as n. If not otherwise stated, the center value represents an average value, where the repetition is described as a symbol. For cytotoxicity experiments in which single duplicate values cannot be displayed, the dispersion (dispersion) of all technical triplicate for lysis calculation is expressed as standard deviation. Statistical significance (P) was determined by Spearman' correlation (FIG. 6c, rs: spearman rank correlation coefficient), pearson correlation (Pearson correlation), kruskal-Wallis test, followed by Dunn post-hoc test (FIG. 6 b) or Brown-Forsythe and Welch analysis of variance (ANOVA), followed by Dunnett T3 multiple comparison test (FIG. 9 d). All analyses were double tailed and were performed using GRAPHPAD PRISM 8.4.4. All experiments were performed 1 time. The experiments were not random.

The following examples summarize the results of exploratory metaphase analysis (ending at 2019, 7, 29) for 89 patients (fig. 5), focusing on the immune response induced by melanoma FixVac. The best objective response to FixVac alone or in combination with anti-PD 1 antibodies in patients with measurable disease was also assessed (figure 29).

Example 2: in vivo characterization of immune activation mediated by one 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 collectively encoding an NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof; and lipid particles (e.g., lipid complexes or lipid nanoparticles). FIG. 1a shows an exemplary schematic of one or more RNA molecules that collectively encode an NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and a TPTE antigen.

FixVac targeting in the spleen. This example shows that FixVac is targeted to the spleen by exploiting the enhanced glucose consumption of cells following TLR ligand stimulation (reference 12). In this example, a [18F ] -fluoro-2-deoxy-2-d-glucose (FDG) -Positron Emission Tomography (PET)/Computed Tomography (CT) scan was performed shortly after injection FixVac. Shortly after injection, a significant increase in metabolic activity was observed, especially in the spleen, indicating rapid targeting and transient activation of lymphoid tissue resident immune cells (fig. 1 c).

Adjuvanticity (adjuvanticity). To determine the adjuvanticity of FixVac after administration to a patient, the amount of plasma cytokines was measured (reference 8). The increase in the levels of Interferon (IFN) -alpha, IFN-gamma, interleukin (IL) -6, IFN-Inducible Protein (IP) -10 and IL-12 p70 subunits was consistent with FixVac doses, accompanied by a brief increase in body temperature (FIG. 1d; FIG. 6 a). Cytokine secretion was pulsed, transient and self-limiting, peaking at 2 to 6 hours after treatment and normalizing within 24 hours (fig. 1 d). The combination of FixVac with anti-PD 1 antibody did not affect cytokines (fig. 6 b). The plasma concentration of IFN- α correlated well with all other cytokines measured (see the Szelman correlation (r _s) for IFN- α as shown in FIG. 6 c).

Adverse event profile. Consistent with the cytokine pattern, the spectrum of clinical adverse events is dominated by mild to moderate influenza-like symptoms (e.g., fever and coldness). Adverse events were mostly premature, transient and controlled with antipyretics, and resolved within 24 hours. In vivo observations summarize findings in mice, where FixVac's mode of action is driven by translation of antigen-encoding RNAs in dendritic cells residing in lymphoid compartments, as well as by concomitant inflammatory responses induced by TLRs on antigen presenting cells (references 8, 13 and 20). However, the concentration of FixVac triggering cytokine release in humans is more than 1,000-fold lower in mice (Kranz et al 2014, incorporated herein by reference in its entirety).

Additional details regarding adverse events detected during administration are contained in fig. 40 and 41. As shown, the most frequently occurring related TEAEs are fever followed by chills, headache, fatigue, nausea, joint pain, vomiting and tachycardia. The frequencies of these related TEAEs are similar between ED and NED subgroups. Most of these symptoms are CTCAE grade 1 or grade 2 and are expected to be reactogenic due to the inherent adjuvanticity of RNA-LPX. The proportion of patients in the ED subgroup who experienced ≡3 grade related TEAE was higher (10 patients [26.3% ] vs.3 patients [9.1% ], respectively) when compared to the NED subgroup. In the ED and NED subgroups, 4/38 patients (10.5%) and 1/33 patients (3.0%) experienced TESAE (data not shown) that were considered relevant to the trial treatment.

Example 3: immunogenicity of pharmaceutical compositions

This example shows immunogenicity of samples collected from melanoma patients (e.g., patients with stage III B to C or stage IV malignant melanoma (united states joint cancer committee (AJCC) 2009 melanoma classification) after FixVac administration, both resected and non-resected, and thus had measurable and non-measurable disease at baseline, and expressed at least one of the four TAAs contained in FixVac) after in vitro stimulation (in vitro stimulation, IVs). In this example, the immunogenicity of FixVac was measured by IFN-. Gamma.ELISPot after IVS.

For 50 patients, large numbers of peripheral blood mononuclear cells (PERIPHERAL BLOOD MONONUCLEAR CELL, PBMCs) depleted of either CD4 ^- or CD8 ^- incubated with overlapping peptides representing the full length sequence of TAAs described herein (so-called PepMix) were subjected to ex vivo IFN- γ ELISpot (fig. 2a and 2 b) either before or after vaccination (after eight injections FixVac). Samples from 20 patients were also analyzed using post IVS IFN- γelispot (fig. 2 c), in which 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 (fig. 2 c), predominantly CD4 ⁺ responses alone or both CD8 ⁺ and CD4 ⁺ responses (fig. 7 a). The vaccine-induced de novo response (a response undetectable prior to vaccination) was more frequent than the enhancement of pre-vaccine response (fig. 7 a). 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 (fig. 2 a). Most of these high magnitude T cell responses were CD8 ⁺ (fig. 2 a).

Ex vivo de novo CD8 ⁺ T cells were measured by HLA multimeric analysis and intracellular cytokine staining (intracellular cytokine staining, ICS). Antigen-specific T cells that rose to the single-digit or low two-digit percentage of circulating CD8 ⁺ T cells within 4 to 8 weeks (fig. 2e to g; fig. 3a, 7b, 11) had the PD1 ⁺CCR7^-CD27⁺/^-CD45RA^- effector memory phenotype (fig. 2f, 7c, and 12) and secreted IFN- γ and tumor necrosis factor (tumor necrosis factor, TNF) after antigen-specific restimulation (fig. 2h, 7d, and 13). Most patients had a multi-epitope CD8 ⁺ immune response (fig. 2b, fig. 2 g). In patients undergoing monthly maintenance vaccination after the first 8 vaccinations, the frequency of TAA-specific T cells continued to increase or remained stable for more than one year (fig. 2 g). In patients without continuous vaccination, memory T cells remained present and had a slow downward trend for several months (fig. 2e and fig. 7 b).

Example 4: characterization of TAA-specific T Cell Receptor (TCR) from vaccine expanded T cells isolated from patients

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

Transfection of TAA-specific T Cell Receptors (TCRs) from vaccine expanded T cells (fig. 33) into healthy donor T cells effectively killed TAA-positive melanoma cells (fig. 2 i). T cell responses were unaffected by the presence or absence of a radiology measurable disease at baseline, fixVac therapeutic doses, or whether FixVac was administered alone or in combination with anti-PD 1 antibodies (fig. 7e and 7 f).

Example 5: optimal objective response in 42 patients with measurable metastatic disease

This example shows the response of melanoma patients with measurable metastatic disease for which one scan at baseline and at least one scan after treatment are available. 41 patients were in stage IV who had previously undergone a series of systemic treatments and experienced checkpoint inhibitors (CPI); 35 of these have been exposed to antibodies against both PD1 and cytotoxic T lymphocyte-associated protein 4 (cytoxic T-lymphocyte-associated protein, ctla 4) (fig. 30).

In FixVac monotherapy (n=25), 3 patients experienced partial responses and 7 patients had stable disease (fig. 2j, fig. 5). Another patient showed complete metabolic remission of metastatic lesions in [18F ] -FDG-PET/CT imaging. In the FixVac/anti-PD 1 combination group, 6 out of 17 patients developed partial responses. Target lesion regression occurred in all doses, but the ratio of partial response was highest among patients treated with 100 μg melanoma FixVac plus anti-PD 1 (5 out of 10 patients; objective response rate 50%) (fig. 2 j). Most patients with partial response or stable disease showed persistent disease control (during the up to two years of observation period) (fig. 2k; fig. 8a and 8 b). Objective response correlated with tumor burden at baseline (fig. 8 c).

Example 6: characterization of immune response from melanoma patients receiving FixVac monotherapy and FixVac/anti-PD 1 combination

This example shows the response of a particular patient after treatment with a combination 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 FixVac/anti-PD 1 combination; FIG. 8 d) had sufficient blood samples for detailed characterization of the immune response.

Patient 53-02 entered the trial after progression through pembrolizumab therapy. At FixVac monotherapy, the patient experienced a partial response lasting 8 months and regression of multiple metastases (fig. 3b and 9 a). Regeneration of metastatic lesions was diagnosed several weeks after stopping vaccination as required by the patient. The patient received again (rechallenge) pembrolizumab therapy and remained stable for an additional seven months (fig. 8 d).

For this patient, a strong de novo immune response was detected against NY-ESO-1 and MAGE-A3 by ex vivo ELISPot. The vaccine-induced HLA-Cw 0304 restricted CD8 ⁺ T cell response against NY-ESO-196-104 epitope 15 identified by HLA multimeric staining increased dramatically to over 10% of peripheral blood cd8+ T cells and remained high with continued vaccination (fig. 3a and 9 b). ICS established that NY-ESO-1 reactive IFN-gamma+ T cells expanded to a maximum of 15% of the total peripheral blood CD8+ T cell population (FIGS. 3c and 14). Short term IVS cultures of post-vaccinated PBMC against NY-ESO-196-104 epitope amplification effectively killed endogenous NY-ESO-1+ melanoma cells (FIGS. 3d and 15).

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

Patients A2-10 showed rapid progression of the multiple transfer disease under treatment with ipilimumab and nivolumab (fig. 8 d). At FixVac monotherapy, the patient experienced a partial response over a 6 month duration and multiple lymph nodes and lung metastases resolved (fig. 10 a). FixVac stopped after eight months due to progressive disease of the inguinal lymph nodes. The patient received pembrolizumab monotherapy again and experienced a partial response.

IFN-gamma ⁺CD4⁺ T cell responses to MAGE-A3 and NY-ESO-1 were detected in post-treatment PBMC from this patient (FIG. 10 b). Of the skin infiltrating lymphocytes from Delayed Type Hypersensitivity (DTH) reaction obtained after eight vaccinations, NY-ESO-1 specific cd4+ T cells were detected (fig. 10c and 17). Several NY-ESO-1-directed, tyrosinase-directed and MAGE-A3-directed TCR clonotypes from CD4+ T cells in post-treatment PBMC were cloned (FIG. 10d, FIG. 10e and FIG. 33). These clonotypes include TCRs that recognize the MAGE-a3281-295 epitope, which is reported to be immunodominant and promiscuously present on multiple HLA-DRB1 alleles 16 (fig. 10 e). The TCR frequency was mostly undetectable by TCR clonotype profile analysis and increased to a frequency that was easily detectable in the case of vaccination (figure 10 f).

Patient C2-28 had many liver and subcutaneous metastases that initially progressed on treatment with the ipilimumab/nivolumab combination and then stabilized on continued nivolumab monotherapy. The patient shifted to FixVac/nivolumab combination therapy and experienced a partial response (fig. 4a and 8 d) and reduced liver and subcutaneous target lesions (tumor burden reduced from 91mm 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-A3T cells were detected by post IVS ELISPot (data not shown). The slave head HLA-A 0101 restricted T cell response to MAGE-a3168-176 epitope 17 was increased to up to 2% of peripheral blood cd8+ T cells (fig. 4b and fig. 18). Two TCRs cloned from MAGE-a3168-176 multimer-binding T cells specifically recognized endogenous MAGE-a3+ melanoma cells (fig. 4c and 33).

Patients C2-31 had locally recurrent melanoma and had recently had systemic metastatic spread. The patient progressed on pembrolizumab therapy for 7 months and had multiple metastases in lung, liver and lymph nodes. FixVac was added to the ongoing pembrolizumab treatment and the patient rapidly experienced a partial response (fig. 4d and 8 d). CD4+ T cell responses were detected against MAGE-A3, TPTE and NY-ESO-1, and CD8+ T cell responses against NY-ESO-1 and MAGE-A3, most of which were de novo (FIG. 10 g).

Patients C1-40 had a history of pembrolizumab-responsive metastatic melanoma and experienced progressive disease seven months after cessation of pembrolizumab, with multiple rapidly progressing pulmonary lesions. Treatment with nivolumab was initially performed, to which melanoma FixVac was added after eight weeks. The patient experienced a partial response with a contraction of the lung metastasis (fig. 8d and 10 h). HLA multimeric staining showed strong vaccine-induced T cell responses against MAGE-A3 _168-176 and NY-ESO-1 _92-100 epitopes (FIG. 4e and FIG. 10 i). Short term cultures of lymphocytes after vaccination effectively killed MAGE-a3+ melanoma cells, indicating the functionality of vaccine-induced T cells (fig. 4f and 19).

Summary of findings. The data provided in examples 1 to 6 together provide some key findings. First, the FixVac-induced transient cytokine responses of T cells, as well as the high magnitude and T-helper-1 phenotypes, showed that the RNA-LPX vaccine class has the same effective mode of action in humans, which was characterized in the mouse model as critical for anti-tumor effects (references 8, 18). Several full-length TAAs are delivered together, and the patient generates polyclonal CD4 ⁺ and CD8 ⁺ T cell responses. As indicated by the kinetics of HLA-multimer-positive T cells, prime/repeat boost protocols expanded pools of circulating antigen-specific T cells (particularly those targeted to NY-ESO-1 and MAGE-A3) over time by orders of magnitude.

Patients who experienced partial responses are those with the most pronounced and diverse T cell responses. However, the possibility of bias due to the fact that these responders stay in the assay for a longer time cannot be excluded, which allows to collect enough blood for epitope identification and multimeric analysis-the most informative assay for analyzing T cell frequencies.

The T cells induced by FixVac are fully functional, recognize their target epitopes on melanoma cells, and exhibit 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, observations described in examples 1-6 demonstrate that, although melanoma FixVac is active as a single agent, it also works synergistically with anti-PD 1 treatment in patients with tumors that experience CPI. Patients 53-02 and A2-10 began melanoma FixVac treatment after failure to anti-PD 1, underwent tumor regression under melanoma FixVac monotherapy, eventually progressed again, and subsequently responded to receiving anti-PD 1 treatment again. T cells induced by melanoma FixVac have a pd1+ effector memory phenotype and are therefore stimulated by anti-PD 1 antibodies. Consistent with this view, PD1 blockade enhanced the anti-tumor effect of RNA-LPX vaccines in a mouse model with advanced tumors that is insensitive to anti-PD 1 monotherapy (reference 18). Notably, the tumor regression rate observed with the melanoma FixVac/anti-PD 1 combination in the pre-treated, CPI-experienced patients (over 35%) was within the range of objective response rates exhibited by PD1 blockade alone in patients with metastatic melanoma that did not receive CPI (CPI-naive) (reference 19).

Third, the findings presented in examples 1 to 6 support the usefulness of non-mutated consensus TAAs a target for cancer vaccines. The clinical role in TAA-based cancer vaccine trials has been largely disappointing in patients with advanced cancer and is often associated with relatively weak vaccine-induced immunity over the past two decades (reference 20). T cells directed against cancer mutations are identified as drivers of CPI block-mediated clinical efficacy-along with advances in technology enabling personalized cancer vaccination-have prompted the notion 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 against non-mutant TAAs can be overcome by an effective vaccine class. PD1 blockade works by expanding pre-existing antigen-specific T cells, many of which are directed against mutation-derived neoantigens (reference 21). More than half of patients with metastatic melanoma: with moderate to low mutation loading, which correlates with lower probability of pre-formed neoantigen-specific T cells; and is at higher risk of failing anti-PD 1 therapy and thus disease progression (reference 22). Whereas the four TAAs targeted here are highly prevalent in human melanoma (references 10, 23, 24), and their expression is independent of tumor mutation burden (fig. 4 g), melanoma FixVac sensitized, activated and expanded a complementary pool of CD4 ⁺ and CD8 ⁺ T cells. Thus, non-mutant TAA-based vaccines in combination with anti-PD 1 therapies may have particular clinical utility for tumor control in patients with lower mutational burden, including those already undergoing CPI treatment.

Example 7: exemplary administration (e.g., dose escalation)

In some embodiments, the pharmaceutical compositions provided herein can be administered to a patient suffering from melanoma as monotherapy and/or in combination with other anti-cancer therapies (e.g., such as immune checkpoint inhibitors). In some embodiments, the melanoma patient to be treated is a patient with refractory/recurrent, unresectable stage III or IV melanoma against PD 1.

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

In some embodiments, 6 weekly doses relating to the pharmaceutical composition described herein (e.g., fixVac) are administered followed by 2 weekly doses of the pharmaceutical composition described herein (e.g., fixVac). In some embodiments, administration may also involve a monthly dose following administration of 2 weekly doses.

In some embodiments, 5 weekly doses involving a pharmaceutical composition described herein (e.g., fixVac) are administered followed by 2 weekly doses of a pharmaceutical composition described herein (e.g., fixVac). In some embodiments, administration may also involve a monthly dose following administration of 2 weekly doses.

In some embodiments wherein a combination therapy is administered, the pharmaceutical compositions described herein (e.g., fixVac) may be administered on the same day as the immune checkpoint inhibitor therapy. In some such embodiments, the pharmaceutical compositions (e.g., fixVac) and immune checkpoint inhibitor treatments described herein can be administered separately.

In some embodiments, the pharmaceutical compositions described herein (e.g., fixVac) are administered on the same day as the immune checkpoint inhibitor treatment.

In some embodiments, dose escalation may be performed. In some such embodiments, administration may be at one or more of the levels shown in table 6; in some embodiments, dose escalation may involve administration of at least one lower dose from table 6 followed by administration of at least one higher dose from table 6.

Table 6: exemplary administration of drug

Dosage level	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, the dosage level may be assessed in addition to or as an alternative, e.g., including, e.g., at a dosage level of 7.5,8,9,10,11,12,13,1415,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,125,150,175,200,225,250,275,300,325,350,375,400μg total RNAs. Efficacy of treatment can be assessed by immunological monitoring and/or clinical anti-tumor activity.

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

Approved immune checkpoint inhibitors are useful in the treatment of certain cancers, including melanoma. Some non-limiting examples of FDA-approved immune checkpoint inhibitors include ipilimumab, cimapril Li Shan antibody, nivolumab, pembrolizumab, avilamab, and Dewaruzumab. Some additional examples of immune checkpoint inhibitors currently under investigation may include rituximab (Dostarlimab), INCMGA00012, terlipressin Li Shan (Toripalimab), SHR-1210, INCB086550 (oral PD-1 inhibitor), PDR001, HX008, and CX-072.

In some embodiments, the immune checkpoint inhibitor may be administered according to a regimen indicated as monotherapy for treating certain cancers, e.g., every 3 weeks in some embodiments.

Example 9: exemplary adverse events

In some embodiments, one or more indicators of potential adverse events of a subject administered a monotherapy as described herein may be monitored over a period of time of a treatment regimen. The spectrum of clinical adverse events is dominated by mild to moderate flu-like symptoms (e.g., fever and coldness). Adverse events were mostly premature, transient and controlled with antipyretics, and resolved within 24 hours (fig. 32). In some embodiments, particularly for subjects receiving monotherapy as described herein, one or more of fever, chill, headache, fatigue, nausea, tachycardia, cold sensation, joint pain, limb pain, emesis, reduced lymphocyte count, increased levels of interferon gamma, hypertension, dizziness, diarrhea, increased alpha tumor necrosis factor, influenza-like disease, and reduced white blood cell count of the subject may be monitored.

Example 10: exemplary stop criteria

In some embodiments, if, for example, (i) the patient experiences an Adverse Event (AE) that meets the criteria for drug limiting toxicity (drug limiting toxicity, DLT); (ii) Patients experienced AEs meeting DLT criteria after the dosing cycle that failed to regress to ∈1 within a predetermined period of time; (iii) Dose delays exceeding the dosing period due to toxicity that may be associated with the administered treatment; (iv) Drug-related or life threatening class 4 AEs that do not meet DLT criteria (excluding asymptomatic class 4 elevation in non-hematologic laboratory values, which regresses to class 2 or less within 14 days [ with or without medical intervention 1) unless otherwise approved by a medical monitor; (v) Despite the prior administration of the precursor prior to the second administration, a second infusion-related reaction of ≡3 (infusion related reaction, IRR) occurred; and/or (vi) first occurrence of an allergic reaction or grade 4 IRR, the treatment as described herein may be discontinued.

Example 11: exemplary assays and/or criteria for RNA molecules described herein

In some embodiments, one or more of the evaluations as described herein may 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 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 dispensing). 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 assessing RNA quality are known in the art; for example, one of skill 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, immune-based assays for residual dsRNA, detection of translated antigens.

In some embodiments, the RNA batch may 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 criteria, the batch of RNA molecules may be designated for one or more additional steps of manufacture and/or formulation and/or dispensing. Otherwise, if such a batch of RNA molecules does not meet or exceed the acceptance criteria, then an alternative action may be taken (e.g., discard the batch).

Example 12: exemplary inclusion criteria

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

group I: malignant melanoma stage IV (AJCC 2009 melanoma classification)

Groups II to VII end expanded group: group C expanded to patients with stage iii melanoma (AJCC 2009 melanoma classification) and measurable disease (according to irRECIST 1.1.1 at least one target lesion) only [ applicable to all patients after version 10.0 approval of protocol ]

Treatment only for subjects who do not qualify for any other available approved treatment or who reject any other available approved treatment after all available treatment options have been transparently disclosed (to be recorded).

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

Age equal to or greater than 18 years old

Written informed consent

ECOG performance states (performance status, PS) 0 to 1

Life expectancy >/=6 months

·WBC≥3×10E9/L

Hemoglobin of 9g/dL or more

Platelet count ≡100,000/mm ≡ ³

ALT/AST < 3 XULN (except for patients with liver metastasis)

Negative pregnancy test for women of child-bearing age (measured by β -HCG)

Example 13: exemplary exclusion criteria

In some embodiments, the cancer patient has melanoma that is unsuitable for the compositions and/or methods described and/or used herein.

In some embodiments, (i) has recently received a cancer treatment; (ii) is concurrently receiving systemic steroid therapy; (iii) there has been a major surgery recently; (iv) Has active infections and is being treated with anti-infective therapy; and/or (v) a cancer patient diagnosed with developing brain or pia mater metastasis is 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. The exclusion criteria included:

Pregnancy or lactation

Primary ocular melanoma

Concurrent second malignancy in addition to squamous cell carcinoma or basal cell carcinoma, inactive prostate cancer, or in situ cervical cancer or inactive treated urothelial cancer

Brain metastasis

Patients with a history of treated or inactive brain metastasis were eligible for treatment in the expanded cohort C, provided that they met all of the following criteria:

Measurable disease outside the brain (except for inactive brain metastasis);

there is no continuous need for corticosteroids as a treatment for brain metastases,

Disabling corticosteroid at ≡1 week prior to visit 2 (day 1) and no sustained symptoms attributable to brain metastasis;

screening brain radiographic imaging at 4 weeks or more after completion of the self-radiotherapy

Patients after splenectomy

Known hypersensitivity to the active substance or to any excipient

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

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 drugs (allowing topical or inhalation of steroids)

Other clinically relevant systemic immunosuppression

Symptomatic congestive heart failure (NYHA 3 or 4)

Unstable angina pectoris

Radiation therapy and minor surgery within 14 days prior to administration of the first study treatment

Myelosuppression chemotherapy within 14 days prior to administration of the first study treatment and after reconstitution of blood values

Receiving ipilimumab 28 days prior to administration of the first study treatment

Treatment with BRAF inhibitor, MEK inhibitor or a combination of both anti-PD-1 antibodies within 14 days prior to the first administration of study treatment (determined by the investigator not applicable to patients receiving parallel treatment in the extended cohort A, B or C)

Receiving interferon, major surgery, vaccination and other investigational agents within 28 days or 5 half-lives (depending on the longer range given) prior to first treatment

The combination of the approved BRAF inhibitor vemurafenib or dabrafenib, the approved anti-PD-1 inhibitor nivolumab or pembrolizumab, and the approved MEK inhibitor trametinib (trametinib), or the approved BRAF-MEK inhibitor in patients in the dose escalation cohort. Following analysis and DSMB approval of safety data collected for the dose escalation cohort, concomitant treatment with a combination of an approved BRAF inhibitor, an approved anti-PD-1 antibody or MEK inhibitor, and an approved BRAF-MEK inhibitor is allowed for patients included in the expanded cohort. Local irradiation will also be allowed as simultaneous treatment of patients in an extended cohort.

After version 10.0 approval, only anti-PD-1 antibodies were allowed for treatment of patients in expanded cohort C.

Fertility male and female who are reluctant to use efficient fertility control methods (less than 1% per year, such as spermicidal condoms, spermicidal caps, fertility control pills (birth control pill), injections, patches or intrauterine devices) during the study treatment and at least 28 days (male patients) and 90 days (female patients with fertility potential) after the last dose of study treatment

There are serious complications or other conditions (such as psychological, family, social or geographical environments) that do not allow adequate follow-up and follow-up protocols

Example 14: exemplary efficacy assessment and/or detection

In some embodiments, cancer patients administered with the pharmaceutical compositions described herein as monotherapy or in combination with additional anti-cancer therapies may be monitored periodically for therapeutic efficacy and/or adjustment of therapeutic dose/regimen.

In some embodiments, the efficacy of the treatment may be assessed by computed tomography and/or magnetic resonance imaging scans. In some embodiments, MRI scans may be performed using a3 Tesla (Tesla) whole body instrument. In some embodiments, when evaluating lesions for efficacy assessment, one or more of the following criteria may be used:

complete response: all target lesions disappeared. The minor axis of any pathological lymph node (whether targeted or non-targeted) must be reduced to < 10mm.

Partial response: the sum of diameters of target lesions is reduced by at least 30% with reference to the baseline sum diameter.

Progressive disease: the sum of the diameters of the target lesions is increased by at least 20% with reference to the minimum sum in the study (including the baseline sum if it is the minimum sum in the study). In addition to a relative increase of 20%, the sum must also show an absolute increase of at least 5 mm. The appearance of one or more new lesions is also considered progress.

Stability disease: neither contraction nor improvement in progressive disease qualification was sufficient to qualify for PR, taking as reference the minimum sum diameter in the study.

Example 15: immune response of patients with evidence of disease relative to patients without evidence of disease

This example shows the ex vivo characterization of an immune response 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 lipid particles to patients with Evidence of Disease (ED) and patients without evidence of disease (NED).

Background: lipo-MERIT is an ongoing, first 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 lipid complex (ribonucleic acid lipoplex, RNA-LPX) vaccine that targets melanoma Tumor Associated Antigen (TAA) New York esophageal squamous cell carcinoma 1 (NY-ESO-1), tyrosinase, melanoma associated antigen 3 (MAGE-A3) and Transmembrane Phosphatase (TPTE) with tensin homology. As shown in examples 1 to 6, BNT111 alone or in combination with immune checkpoint inhibitors (CPI) has an advantageous Adverse Event (AE) profile, generating antigen-specific T cell responses and inducing a durable objective response in patients with non-resectable melanoma who have CPI experiences. This example shows immunogenicity, efficacy and safety data for patients without evidence of disease (NED) in a trial incorporating BNT111 monotherapy sub-group.

The method comprises the following steps: BNT111 is administered intravenously to patients with stage IIIB, IIIC and IV cutaneous melanoma according to prime and boost regimens. Patients were treated in seven dose escalation cohorts (dose range: 7.2 μg to 400 μg total RNA) and three expanded cohorts further exploring dose levels of 14.4 μg, 50 μg and 100 μg. In this analysis, patients receiving BNT111 monotherapy were grouped as Evidence of Disease (ED) or NED and were evaluated for immunogenicity, efficacy (by solid tumor immune-related response assessment criteria) and safety. The immune response induced by the vaccine was analyzed directly ex vivo using interferon-gamma enzyme-linked immunosorbent spot (ELISpot) assay.

Results: by 24 days 5 of 2021, 115 patients received BNT111 in the Lipo-MERIT test. Of 71 patients treated with BNT111 monotherapy, 38 had ED after the previous treatment and 33 had NED. Baseline characteristics were similar between the two groups. ELISpot data shows comparable BNT111 induced T cell responses against at least one TAA in ED and NED patients (14/22 [64% ] and 19/28[68% ] patients with available ELISpot evaluable samples, respectively), indicating that BNT111 has the ability to induce T cell immunity even in the absence of detectable tumor. In NED patients, clinical efficacy was promising, and median disease-free survival was 34.8 months (95% confidence interval: 7.0 to unrealized). ED patients were similar to the safety profile in NED patients, with 38/38 (100%) and 32/33 (97%) patients experiencing related treatment-emergent AE (treatment-EMERGENT AE, TEAE), 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 (fig. 20a to c), in which autologous dendritic cells loaded with TAA PepMix were used as targets. Figures 20a to c show the presence of vaccine induced (amplified or de novo) responses: CD4 ⁺ or CD8 ⁺ (fig. 20 a); CD4 ⁺ (fig. 20 b); or the frequency of patients responding to CD8 ⁺ (fig. 20 c). The numbers in the bar segments represent the number of patients evaluated in each segment. Only patients treated with monotherapy are included. Unexpectedly, the samples of NED patients showed stronger vaccine-induced responses (e.g., CD4 ⁺ or CD8 ⁺ (fig. 20 a), CD4 ⁺ (fig. 20 b), or CD8 ⁺ (fig. 20 c)) than the samples of ED patients.

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

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

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

Figure 26a shows disease-free survival data for NED patients based on the number of events (e.g., death, recurrence, and new treatments initiated) and the number of deletions (censor). Figure 26b shows Kaplan-Meier summary of disease free survival data for NED patients.

Figures 27a to 27c show overall survival data for ED patients (figure 27 a), NED patients (figure 27 b) and ED and NED patient combinations (figure 27 c) based on the number of events (e.g. death, recurrence and new treatments initiated) and the number of deletions. Figures 27d to 27f show Kaplan-Meier summaries of total survival data for ED patients (figure 27 d), NED patients (figure 27 e) and ED and NED patient combinations (figure 27 f).

Fig. 28a to 28c show a summary of adverse events for ED patients (fig. 28 a), NED patients (fig. 28 b) and ED and NED patient combinations (fig. 28 c).

Conclusion: the immunogenicity and safety profile of BNT111 as monotherapy was comparable in ED and NED patients, and promising signs of clinical activity were observed in NED patients.

Example 16: pharmacologic and immune response following administration of BNT111

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

Cytokines, such as IFN-gamma, IFN-alpha, TNF-alpha, IP-10, IL-2, IL-6, IL-10 and IL-12 (p 70) were analyzed at various time points ranging from baseline (i.e., prior to vaccination) to as long as 36 days after vaccination and were frequently sampled during the first 48 hours after vaccination. Patients exhibit dose-dependent transient increases in plasma levels and elevated body temperature for different cytokine profiles. Cytokine release is pulsed and peaks about 2 to 6 hours after dosing and the value returns to baseline at 24 hours or earlier. IFN- α -based cytokines were observed to include IFN- γ and activation in the continuous IP-10 mode, and activation of IL-12, IL-6 and TNF- α.

In blood samples from 20 patients analyzed with IFN-gamma-ELISA spots (ELISPot) after in vitro amplification, T cell responses against at least one TAA were observed in each patient. It includes T cell specificity that is undetected at baseline and is induced de novo by the vaccine, as well as T cell specificity that is present at low levels at baseline and is expanded and amplified by the vaccine antigen.

Of 80 patients, IFN-. Gamma. -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 an ex vivo detectable level.

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

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 persisted for months. In some patients, an antigen-specific CD8 ⁺ T cell response was observed to account for more than 10% of all peripheral blood CD8 ⁺ T cells.

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

Example 17: efficacy data after administration of BNT111

This example provides an overview of preliminary efficacy data observed after administration of BNT 111.

Preliminary efficacy against BNT111 monotherapy, BNT111 with nivolumab or pembrolizumab, and BNT111 in combination with a BRAF/MEK inhibitor are given. Figure 42 provides details of the best overall response for each of these treatment groups, depending on the highest dose administered.

Of 115 patients, 75 (68%) patients with unresectable stage III or IV melanoma exhibited an evaluable disease at baseline, which included 4 patients with only non-target lesions. The subgroup efficacy analysis group comprised patients with an evaluable disease at baseline, who received at least one dose of BNT111 and had a baseline and at least one in-treatment/post-treatment tumor response assessment (n=75).

Efficacy of 36 patients receiving BNT111 monotherapy, 36 patients receiving BNT111 and either nivolumab or pembrolizumab, and three patients receiving BNT111 and a BRAF/MEK inhibitor are given. All 36 BNT111 monotherapy patients received previous treatment with the checkpoint inhibitor, and of patients treated with BNT111 in combination with the PD-1 inhibitor, 35/36 patients received previous treatment with the checkpoint inhibitor. Most patients suffer from progressive disease at the beginning of treatment.

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

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

Of three patients whose efficacy can be evaluated and treated with the BNT111 cancer vaccine in combination with a BRAF/MEK inhibitor, 1 (33.3%) achieved SD.

Fig. 43 depicts the optimal 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: security analysis

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

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

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

The overall safety profile of combination therapy with PD-1 inhibitors versus BNT111 monotherapy is comparable in terms of influenza-like symptoms (reactivities), such as fever, chill, tachycardia and headache. The most common TEAEs in the PD-1 combination treatment subgroup compared to BNT111 monotherapy were syncope (13% vs. 0%) and melanocyte nevi (melanocytic naevus) (13% vs. 3%).

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

In addition, differences in hypotension (24% vs.9%) between the PD-1 inhibitor combination subgroup relative to the BNT111 monotherapy subgroup were also noted. The number of joint pain was reported to be higher in the BNT111 monotherapy subset (31% vs. 11%). In the Lipo-MERIT phase I trial, dose limiting toxicity (dose-limiting toxicity, DLT) during the dose escalation period (highest dose administered from 7.2 μg to 400 μg total RNA) was not reported.

Drug-related deaths were not reported. 11/115 (8%) of the patients died within the main course of the trial, i.e. within 90 days after the last trial treatment. No mortality was considered to be associated with BNT 111. Most patients die due to disease progression and general physical health deterioration.

TEAE considered to be relevant to study drug is transient, mostly influenza-like symptoms, and adverse event generic term criteria (Common Terminology Criteria for ADVERSE EVENT, CTCAE) are grade 1 and grade 2.

Thirteen (11%) of 115 patients experienced treatment-related TESAE;30/115 (26%) patients experienced a treatment-related TEAE with CTAE grade > 3; 19/115 (17%) patients had treatment-related TEAE that resulted in cessation of permanent trial treatment, and 19/115 (17%) patients had treatment-related TEAE that resulted in a dose reduction. Table 7 provides an overview of TEAE by category.

Table 7: overview of the number and percentage of patients with at least one TEAE by subgroup ^{1 To the point of 4}, lipo-MERTT

/>

Date of data extraction 1/eCRF data extraction was 24 days 5 months up to 2021.

2 AE lacking the measures taken in the case of BNT111 are not conservatively considered dose-reduced. For an event, the entry is missing in eCRF: in one patient of the extended cohort C (bnt111+pd-1 inhibitor treatment group), CRP was elevated for one event (no med dra coding, CTCAE grade was not reported, and was considered irrelevant).

3 AE lacking causal relationships are not conservatively considered to be associated with BNT 111. This applies to the following events: a right flank pain event (MedDRA coding in eCRF has not been performed); in one patient of expanded cohort C (bnt111+pd-1 inhibitor treatment group), CTCAE grade was not provided in eCRF.

4 A patient who received BNT111 as monotherapy at the first recruitment and bnt111+braf/MEK at the second recruitment is shown in the table. Thus, there is a difference between the total number displayed and the sum of the individual treatments.

AE = adverse event; CRP = C reactive protein; CTCAE = adverse event generic term criteria; DLT = dose limiting toxicity; eCRF = electronic case report table; medDRA = supervision active medical dictionary (Medical Dictionary for Regulatory Activitie); MEK = mitogen activated protein kinase; PD-1 = programmed death 1; PT = preferential term; SAE = severe adverse event; te=appears in treatment; TEAE = adverse event occurring in treatment; TESAE = severe adverse events occurring in treatment.

Table 8 provides a summary of the frequency of severe adverse events (TESAE) occurring in the relevant treatment at the worst CTCAE scale, and table 9 provides a summary of the same data at the treatment subpopulations. Table 8: lipo-MERIT-number of patients with correlation TESAE ¹ of worst CTCAE grade per PT (n=115) ²

1 TESAE is defined as occurring 90 days after the start of administration of the study drug until after the last intake of the study drug. The table includes TESAE from two treatment groups for four double enrolled patients.

Date of 2 data extraction/eCRF data extraction was 24 days 5 months up to 2021.

AE = adverse event; PT = preferential term; TESAE = severe adverse events occurring in treatment.

Table 9: number of patients with associated TESAE treated with BNT111 monotherapy or PD-1 inhibitor combination therapy (n=115) ^{1 To the point of 4}

Date of data extraction 1/eCRF data extraction was 24 days 5 months up to 2021.

2 TESAE is defined as occurring 90 days after the start of administration of the study drug until after the last intake of the study drug.

3 Patients may have TESAE encoded in more than one preferred term.

4 A patient who received BNT111 as monotherapy at the first recruitment and bnt111+braf/MEK at the second recruitment is shown in the table. Thus, there is a difference between the total number displayed and the sum of the individual treatments.

PD-1 = programmed death 1; TESAE = severe adverse events occurring in treatment.

Notably, in the Lipo-MERIT trial, eight patients were still treated with BNT111 monotherapy (n=2) or with a combination of BNT111 and a PD-1 inhibitor (n=6). All of these eight patients who used multiple previous series of treatments were in the so-called "continued treatment" for a duration of 15 to 52 months. Initially, "continue treatment" was only provided with all IMP components (based on four precursors RNARBL001.1, RBL002.2, RBL003.1, and RBL 004.1) in stock. However, since these eight patients with a large number of pretreatments achieved at least stabilization of their disease or response (partial or complete remission according to irRECIST) and thus continued to obtain clinical benefit from the trial treatment, the trial did not cease, but instead continued to provide further trial treatment with the current BNT111 substance (so-called "prolonged treatment"). For the benefit of these patients, the trial is allowed to proceed.

Example 19: pharmacological data obtained in mice

Mice can be related species for assessing the primary and secondary pharmacological effects and 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, ranging from induction of cd4+ and/or cd8+ T cell responses to enhancement of immune responses and subsequent immunomodulatory effects of TLR triggering, cell activation and cytokine secretion. However, given the species specificity of BNT111 TAA and the unique MHC set of molecules that can present a large set of antigenic peptides in each patient, there is no relevant and conclusive mouse tumor model for human melanoma TAA encoded by BNT111, and pharmacodynamic studies in mice as a single agent or in combination with checkpoint blockade are not feasible for BNT 111. Thus, most major pharmacodynamic, mechanism of action and anti-tumor activity studies were performed in mice with RNA-LPX vaccines encoding model antigens.

Non-clinical studies conducted have shown that vaccination with RNA LPX induces DC maturation and activation of a subset of major lymphocytes in the spleen and release systemic cytokines in the first 3 to 6 hours, including ifnα, tnfα, IP-10 and IL 6 in response to TLR7 triggered by single stranded RNA in mice (Kranz et al 2016, incorporated herein by reference in its entirety). Transient leukopenia is consistent with ifnα peak levels and can be attributed to ifnα downstream effects.

Vaccination with RNA LPX in mice effectively de novo sensitizes and expands cytotoxic cd4+ and cd8+ T cells that target BNT111 encoded antigens NY ESO 1, tyrosinase, MAGE A3, TPTE and other melanoma-associated antigens or model antigens. After in vitro co-incubation, human DCs loaded with BNT111 RNA were able to stimulate IFN- γ production by RNA dose-dependent antigen-specific cd8+ T cells expressing the corresponding TCR.

Induced antigen-specific cd8+ T cells were shown to be able to infiltrate mouse tumors, and RNA LPX vaccination was associated with the polarization of the tumor microenvironment toward pro-inflammatory, cytotoxic and less immunosuppressive structures (contexture). RNA LPX vaccination has been shown to trigger release of antigen from tumors, which enables vaccine-induced tumor-specific T cells to be further expanded, even after treatment has ceased.

Tumor-infiltrating cd8+ T cells up-regulate expression of PD 1 in response to RNA LPX vaccination, and PD L1 is significantly expressed by the tumor. 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 acts synergistically in inhibiting tumor growth and improving survival by sensitizing PD 1/PD L1 blockade resistant mouse tumors to this therapeutic combination. Enhancement of vaccine-induced disruption of tolerance to autoantigens expressed by B16 melanoma by PD 1/PD L1 blocking further demonstrates the strong anti-tumor activity of RNA LPX vaccination in combination with PD 1/PD L1 blocking.

Table 10 summarizes non-clinical major pharmacodynamic studies with BNT 111.

Table 10: summary of non-clinical major pharmacodynamic studies of BNT111

/>

/>

DC = dendritic cells; 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 inducible protein 10; IV = intravenous; MHC = major histocompatibility complex; NK = natural killer cells; OVA = ovalbumin; PBMC = peripheral blood mononuclear cells; pDC = plasmacytoid DC; PD-1 = programmed death ligand 1; PD-l1=programmed death protein 1; SD = single dose; RD = repeat dose; TAM = tumor associated macrophages; TCR = T cell receptor; tg = transgene; 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. * Initial development (applied to Lipo-MERIT assays) was based on four prodrug products RBL001.1, RBL002.2, RBL003.1 and RBL004.1, which encode the same target, but with slightly improved translatability and stability, e.g. against RNA.

To further elucidate the main pharmacodynamics, mechanism of action and antitumor activity of BNT111 and provide basis 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-associated proteins 1 and 2) were applied.

Table 11: summary of BNT111 supportive non-clinical major pharmacodynamic studies

/>

/>

/>

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=transgene, til=tumor-infiltrating leukocyte, tlr=toll-like receptor, tme=tumor microenvironment, tnf=tumor necrosis factor, treg=cd ⁺CD25⁺FoxP3⁺ T regulatory cell, trp=tyrosinase-associated protein, wb=whole blood.

Reference to the literature

The following references may be referred to throughout this disclosure. Each of which is incorporated 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 cancer vaccine development.Sci.Transl.Med.8,334ps9(2016).

3.Coulie,P.G.,Van den Eynde,B.J.,van der Bruggen,P.&Boon,T.Turnout antigens recognized by T Iymphocytes：at the core of cancer immunotherapy.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 therapeutic gene delivery by expression-augmenting 3′UTRs identified by cellular library screening.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 defence 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 a rare 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 establishes protective 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 checkpoint blockade.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.Tyrosinase immunoreactivity 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 immunerelated tumor response assessment：does reducing the number of lesions impact response assessment in melanoma patients treated with ipilimumabJ.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 cancer vaccines 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.Cell surface antigens of human malignant melanoma：mixed hemadsorption assays for humoral immunity to cultured autologous melanoma cells.Proc.Natl Acad.Sci.USA 73,3278-3282(1976).

31.Brochet,X.,Lefranc,M.-P.&Giudicelli,V.IMGT/V-QUEST：the highly customized and integrated system for IG and TR standardized V-J and V-D-J sequence analysis.Nucleic Acids Res.36,W503-W508(2008).

32.Bolotin,D.A.et al.MiXCR：software for comprehensive adaptive immunity profiling.Nat.Methods 12,380-381(2015).

33.Shugay,M.et al.VDJtools：unifying post-analysis of T cell receptor repertoires.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-free isoform 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 dendritic cells derived from CD34+progenitors and activated with type I interferon.J Immunother.2005;28(5)：505-16.

39.Brichard VG,Lejeune D.GSK′s antigen-specific cancer immunotherapy programme：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 melanoma patient 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 Long Road from Experimental Models to Clinical Outcome：A Review.Int J Mol Sci.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 tumour 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 Cancer Staging 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 of imatinib mesylate in patients with metastatic melanoma harboring c-Kit mutation 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-alfa therapy.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 cell stimulatory 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 defence for cancer immunotherapy.Nature.2016;534(7607)：396-401.

53.Kreiter S,Selmi A,Diken M,et al.Increased antigen presentation efficiency by coupling antigens to MHC class I trafficking signals.J Immunol.2008;180(1)：309-18.

54.Kuk D,Shoushtari AN,Barker CA,et al.Prognosis of mucosal,uveal,acral,nonacral cutaneous,and unknow 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 cytokine phenotypes and antibody response following tetanus toxoid booster immunization.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 ClinicalPractice.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.Improving mRNA-Based Therapeutic Gene Delivery by Expression-Augmenting 3′UTRs Identified 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：Talimogene aherparepvec(TVEC),a first-in-class intratumoral oncolytic viral therapy.J Immunother Cancer 2016;4：53.

65.Sanderson K,Scotland R,Lee P,et al.(2005)：Autoimmunity in a phase I trial ora fully human anti-cytotoxic T-lymphocyte antigen-4 monoclonal antibody 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 Cancer Institute.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 peptide vaccination with adjuvant Flt3 ligand.Cancer Immun.2004;4：9.

68.Sharpe AH,Pauken KE.The diverse functions of the PD1 inhibitory pathway.In Nature reviews.Immunology.2018;18(3)：153-67.

69.Siegal RL，Miller KD，Fuchs HE，et al.Cancer statistics 2021.CA Cancer 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 four melanoma peptides either administered in granulocyte-macrophage colony-stimulating factor in adjuvant or pulsed on dendritic cells.J Clin Oncol.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).Melan oma：Cutaneous-NCCN Clinical Practice Guidelines in Oncology.Version 2.2021-February 19,2021.

73.Testori AAE,Chellino S,and van Akkooi ACJ.Adjuvant therapy for melanoma：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.J Leukoe 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 Lung Cancer.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 metastatic melanoma 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 J Med.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 localisation regulates immune responses in a dose-and time-dependent fashion：a geographical view of immune reactivity.Immunol Rev.1997;156：199-209.

Equivalent solution

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is to be understood that the invention encompasses all variations, combinations and permutations in which one or more of the limitations, elements, clauses, descriptive terms, etc. of one or more of the listed claims are introduced into another claim (or as related to any other claim) that is dependent on the same base claim, unless otherwise indicated or unless it would be apparent to one of ordinary skill in the art that contradiction or inconsistency would arise. Furthermore, it should also be understood that any embodiment or aspect of the invention may be explicitly excluded from the claims, regardless of whether a particular exclusion is set forth in the specification. The scope of the invention is not intended to be limited to the above description, but rather is set forth in the following claims.

Claims (145)

1. A method, the method comprising:

administering at least one dose of a pharmaceutical composition to a patient, the pharmaceutical composition comprising:

(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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and

(B) Lipid particles;

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

2. The method of claim 1, wherein no evidence of disease is determined by applying the solid tumor immune-related response assessment criteria (irRECIST) criteria or RECIST 1.1 criteria.

3. A method, the 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) 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 (TPTE) antigen having tensin homology, 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 evidence of disease or absence of evidence of disease is determined by applying the standard of evaluation of immune-related responses of solid tumors (irRECIST) or the standard of RECIST 1.1.

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 said 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 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 transport domain.

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

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 poly adenine tail.

17. The method of claim 16, wherein the polyadenylation 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) A5 'cap or 5' cap analogue;

(ii) At least one 5' UTR;

(iii) A signal peptide;

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

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

(vi) A sequence encoding an MHC class I transport domain;

(vii) At least one 3' UTR; and

(Viii) Poly adenine tails.

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 1to 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 particle comprises a cationic liposome.

28. The method of any one of claims 1 to 25, wherein the lipid particle comprises a lipid nanoparticle.

29. The method of any one of claims 1 to 28, wherein the lipid particle comprises N, N trimethyl-2-3-dioleyloxy-1-propanamine chloride (DOTMA), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine phospholipid (DOPE), or both.

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, no evidence of disease, or both.

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

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

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

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 at least 8 doses of the pharmaceutical composition to the patient within 10 weeks.

49. The method of claim 48, comprising administering a dose of the pharmaceutical composition to the patient weekly for a period of 6 weeks, and subsequently 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 a dose of said pharmaceutical composition to said patient monthly following said at least 8 doses.

51. The method of any one of claims 1 to 47, comprising administering a dose of the pharmaceutical composition to the patient 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 from 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 of 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 of 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 to the patient an immune checkpoint inhibitor.

69. The method of any one of claims 66 to 68, wherein the checkpoint inhibitor is or comprises the following: PD-1 inhibitors, PDL-1 inhibitors, CTLA4 inhibitors, lag-3 inhibitors, or combinations thereof.

70. The method of any one of claims 66 to 69, wherein the checkpoint inhibitor is or comprises an antibody.

71. The method of any one of claims 66 to 70, wherein the checkpoint inhibitor is or comprises the following: the inhibitors listed in table 4 herein.

72. The method of any one of claims 66 to 71, wherein the checkpoint inhibitor is or comprises the following: ipilimumab, nivolumab, pembrolizumab, avilamab, cimetidine Li Shan, atrazumab, devaluzumab, or combinations thereof.

73. The method of any one of claims 66-72, wherein the checkpoint inhibitor is or comprises ipilimumab.

74. The method of any one of claims 66-72, wherein the checkpoint inhibitor is or comprises 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 a level of immune response in the patient.

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

78. The method of claim 77, wherein said pharmaceutical composition induces an immune response level in said patient that is comparable to an immune response level in a second patient to whom said pharmaceutical composition has been administered, said 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 to the level of immune response in the patient prior to administration of the pharmaceutical composition.

82. The method of claim 81, wherein the level of immune response in the patient after administration of the pharmaceutical composition is increased compared to the level of immune response in the patient prior to 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 as compared to the level of immune response in the patient prior to 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 1-88, 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 1-89, 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 a level of metabolic activity in the spleen of the patient.

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

93. The method of claim 91 or 92, wherein the level of metabolic activity in the spleen of the patient 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-Inducible 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 cancer 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 administration of the pharmaceutical composition.

99. The method of claim 98, wherein there is less cancer lesion 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 a plurality of 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 administration of a first dose of the pharmaceutical composition and after administration of 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 after administration of the second dose of the pharmaceutical composition is greater 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 a 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 T cells in the patient induced by the pharmaceutical composition have a T helper-1 phenotype.

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

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

108. The method of any one of claims 3 to 107, further comprising, for a patient 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 less than the size of the at least one cancer lesion prior to 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 evidence of disease.

112. The method of claim 111, which compares 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 not receiving the pharmaceutical composition.

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

115. The method of any one of claims 3 to 114, further comprising measuring disease stability duration for patients classified as evidence of disease.

116.115, Wherein disease stability is determined by application of irRECIST or RECIST 1.1 criteria.

117. The method of claim 115 or 116, further comprising comparing the patient's disease stability duration to a reference disease stability duration.

118. The method of claim 117, wherein the reference disease-stability duration is the average disease-stability duration of a plurality of comparable patients who did not receive the pharmaceutical composition.

119. The method of claim 118, wherein the patient exhibits an increased duration of disease stability as compared to the reference duration of disease stability.

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

121.120, Wherein tumor responsiveness is determined by application of 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 response duration is an average tumor response duration of a plurality of comparable patients who did not receive the pharmaceutical composition.

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

125. The method of any one of claims 1 to 106, further comprising monitoring disease-free survival duration 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 increased disease-free survival duration as compared to the reference disease-free survival duration.

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

130.129, Wherein disease recurrence is determined by application of irRECIST or RECIST 1.1 criteria.

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

132. The method of claim 131, wherein the reference to the duration of disease recurrence is the average of a plurality of comparable patients not receiving the pharmaceutical composition to the duration of disease recurrence.

133. The method of claim 132, wherein the patient exhibits an increased duration of relapse to disease compared to the duration of relapse to disease referenced.

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) 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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and

(B) Lipid particles;

and wherein the patient is classified as having no evidence of disease, but has been previously diagnosed as having cancer.

135. A pharmaceutical composition for treating 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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and

(B) Lipid particles;

and wherein the patient is classified as having no evidence of disease, but has been previously diagnosed as having 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) 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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and

(B) Lipid particles;

and wherein the patient is classified as having no evidence of disease, but has been previously diagnosed as having 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) 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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and

(B) Lipid particles;

and wherein the patient is classified as having no evidence of disease, but has been previously diagnosed as having 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) 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 (TPTE) antigen having tensin homology, 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) 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 (TPTE) antigen having tensin homology, 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) 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 (TPTE) antigen having tensin homology, 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) 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 (TPTE) antigen having tensin homology, or (v) a combination thereof; and

(B) Lipid particles.

145. The use of claim 143 or 144, wherein the cancer is melanoma.