Compositions and methods for treatment of melanoma

Abstract

Compositions and methods for treatment of melanoma arc described.

Description

Attorney Ref.2013237-1447 COMPOSITIONS AND METHODS FOR TREATMENT OF MELANOMA CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of U.S. Prov. Appln. No.63 / 661,410 filed June 18, 2024 and of U.S. Prov. Appln. No.63 / 804,771 filed May 13, 2025, the entire contents of both of which are hereby incorporated by reference in their entireties. BACKGROUND

[0002] Advanced or metastatic melanoma (unresectable Stage III and Stage IV) remains a lethal disease with a high proportion of patients being resistant to approved therapies. There are limited treatment options for patients who progress on targeted therapy or immunotherapy. Therefore, there is a high unmet medical need justifying the development of novel therapies for advanced melanoma patients who have failed existing therapies. SUMMARY

[0003] In one aspect, the present disclosure, among other things, provides a method of treating a subject suffering from melanoma, the method comprising administering to the subject a therapeutically effective amount of a cancer therapy comprising: (a) an RNA therapy comprising one or more RNA molecules that collectively encode (i) a New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen, (ii) a melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) a tyrosinase antigen, and (iv) a transmembrane phosphatase with tensin homology (TPTE) antigen; and (b) a PD-1 inhibitor, wherein the melanoma is a PD-1 / PD-L1 inhibitor-refractory / relapsed, unresectable Stage III or IV melanoma, thereby treating the subject.

[0004] In some embodiments, a method as described herein comprises treating a subject suffering from melanoma, the method comprising administering to the subject a therapeutically effective amount of a cancer therapy comprising: (a) an RNA therapy comprising one or more RNA molecules that collectively encode (i) a New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen, (ii) a melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) a tyrosinase antigen, and (iv) a transmembrane phosphatase with tensin homology (TPTE) antigen; and (b) a PD-1 inhibitor, wherein before administering the cancer therapy, the subject received a PD-1 / PD-L1 inhibitor therapy and the subject suffers from a PD-1 / PD-L1 inhibitor-refractory / relapsed, unresectable Stage III or IV melanoma. 12829787v1Attorney Ref.2013237-1447

[0005] In some embodiments, a method as described herein comprises administering the cancer therapy to a subject suffering from a PD-1 / PD-L1 inhibitor-refractory / relapsed, unresectable Stage III or IV melanoma.

[0006] In some embodiments, a method as described herein comprises administering the cancer therapy for at least 12, 24, 36, or 48 months.

[0007] In some embodiments, after administering the cancer therapy, about 15% to about 40% of the subjects exhibit a clinical response (e.g., a clinical response that is a complete response or a partial response).

[0008] In some embodiments, after administering the cancer therapy, a higher level of subjects exhibit a clinical response, relative to a control response level.

[0009] In some embodiments, a control response level is or comprises a level of control subjects exhibiting a clinical response following treatment with (i) the RNA therapy alone or (ii) the PD-1 inhibitor alone, wherein the control subjects suffer from a PD-1 / PD-L1 inhibitor-refractory / relapsed, unresectable Stage III or IV melanoma before the treatment.

[0010] In some embodiments, a level of subjects exhibiting a clinical response is a statistically significant higher level relative to the control response level.

[0011] In another aspect, the disclosure features a method of treating a population of subjects to a predetermined level of therapeutic efficacy, the method comprising: administering a cancer therapy to the population of subjects, wherein the subjects suffer from a PD-1 / PD-L1 inhibitor-refractory / relapsed, unresectable Stage III or IV melanoma, wherein the cancer therapy comprises: (a) an RNA therapy comprising one or more RNA molecules that collectively encode (i) a New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen, (ii) a melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) a tyrosinase antigen, and (iv) a transmembrane phosphatase with tensin homology (TPTE) antigen; and (b) a PD-1 inhibitor, and wherein the predetermined level of therapeutic efficacy is a clinical response (e.g., a clinical response that is a complete response or a partial response) in about 15% to about 40% of the subjects, thereby treating the population of subjects.

[0012] In another aspect, the disclosure features a method of selecting a subject for a cancer therapy, wherein after receiving a PD-1 / PD-L1 inhibitor therapy, if the subject suffers from a PD-1 / PD-L1 inhibitor-refractory / relapsed, unresectable Stage III or IV melanoma, the subject is selected for treatment with the cancer therapy, wherein the cancer therapy comprises administering to the subject a therapeutically effective amount of: (a) an RNA therapy comprising one or more RNA molecules that collectively encode (i) a New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen, (ii) a melanoma-associated 12829787v1Attorney Ref.2013237-1447 antigen A3 (MAGE-A3) antigen, (iii) a tyrosinase antigen, and (iv) a transmembrane phosphatase with tensin homology (TPTE) antigen; and (b) a PD-1 inhibitor.

[0013] In another aspect, the disclosure features a method of treating a subject, the method comprising: selecting a subject suffering from melanoma, and administering to the subject a therapeutically effective amount of a cancer therapy comprising: (a) an RNA therapy comprising one or more RNA molecules that collectively encode (i) a New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen, (ii) a melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) a tyrosinase antigen, and (iv) a transmembrane phosphatase with tensin homology (TPTE) antigen; and (b) a PD-1 inhibitor, wherein the melanoma is unresectable metastatic melanoma, thereby treating the subject.

[0014] In some embodiments, a melanoma is Stage III or IV melanoma.

[0015] In some embodiments, a subject has received prior therapy comprising a PD-1 or PD-L1 inhibitor.

[0016] In some embodiments, a subject is refractory or relapsed in response to the prior therapy.

[0017] In any of the aspects described herein, the subject received the PD-1 or PD-L1 inhibitor once every 3 weeks.

[0018] In any of the aspects described herein, the subject received the PD-1 or PD-L1 inhibitor once every 3 weeks for at least 12 weeks.

[0019] In any of the aspects described herein, the RNA therapy comprises about 1 ug to about 500 ug total RNA (e.g., about 7 ug to about 400 ug total RNA, about 10 ug to about 300 ug total RNA, or about 50 ug to about 100 ug total RNA).

[0020] In any of the aspects described herein, the RNA therapy comprises one or more pharmaceutical compositions comprising the one or more RNA molecules.

[0021] In any of the aspects described herein, the RNA therapy comprises a first pharmaceutical composition comprising an RNA molecule that encodes the NY-ESO-1 antigen, a second pharmaceutical composition comprising an RNA molecule that encodes the MAGE-A3 antigen, a third pharmaceutical composition comprising an RNA molecule that encodes the tyrosinase antigen, and a fourth pharmaceutical composition comprising an RNA molecule that encodes the TPTE antigen.

[0022] In any of the aspects described herein, the first, second, third, and fourth pharmaceutical compositions are administered concurrently.

[0023] In some embodiments, at least two of the pharmaceutical compositions are administered sequentially. 12829787v1Attorney Ref.2013237-1447

[0024] In any of the aspects described herein, the PD-1 inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody.

[0025] In any of the aspects described herein, the PD-1 inhibitor is cemiplimab.

[0026] In any of the aspects described herein, the cancer therapy comprises administering: (a) the RNA therapy once weekly for 6 weeks, followed by once every 3 weeks; and (b) the PD-1 inhibitor once every 3 weeks.

[0027] In any of the aspects described herein, the one or more RNA molecules comprises a 5’ cap or 5’ cap analogue.

[0028] In any of the aspects described herein, the one or more RNA molecules comprises a sequence encoding a signal peptide.

[0029] In any of the aspects described herein, the one or more RNA molecules comprise at least one non-coding regulatory element.

[0030] In any of the aspects described herein, the one or more RNA molecules comprises a poly-adenine tail.

[0031] In any of the aspects described herein, the poly-adenine tail is or comprises a modified adenine sequence.

[0032] In any of the aspects described herein, the one or more RNA molecules comprises at least one 5’ untranslated region (UTR) and / or at least one 3’ UTR.

[0033] In any of the aspects described herein, the one or more RNA molecules comprises 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 that encodes at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen; (v) at least one sequence that encodes tetanus toxoid P2, tetanus toxoid P16, or both; (vi) a sequence encoding an MHC class I trafficking domain; (vii) at least one 3’UTR; and (viii) a poly-adenine tail.

[0034] In any of the aspects described herein, the one or more RNA molecules comprise natural ribonucleotides.

[0035] In any of the aspects described herein, the one or more RNA molecules comprise modified or synthetic ribonucleotides.

[0036] In any of the aspects described herein, at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen are full-length, non- mutated antigens.

[0037] In any of the aspects described herein, all of the NY-ESO-1 antigen, the MAGE- A3 antigen, the tyrosinase antigen, and the TPTE antigen are full-length, non-mutated antigens. 12829787v1Attorney Ref.2013237-1447

[0038] In any of the aspects described herein, the RNA therapy comprises lipid particles.

[0039] In any of the aspects described herein, the lipid particles comprise liposomes.

[0040] In any of the aspects described herein, the lipid particles comprise cationic liposomes.

[0041] In any of the aspects described herein, the lipid particles comprise lipid nanoparticles.

[0042] In any of the aspects described herein, the subject is a human.

[0043] In any of the aspects described herein, the RNA therapy induces an immune response in the subject.

[0044] In any of the aspects described herein, the method further comprises determining a level of the immune response in the subject.

[0045] In any of the aspects described herein, the level of the immune response is a de novo immune response induced by the RNA therapy.

[0046] In any of the aspects described herein, the method further comprises determining a level of the immune response in the subject before and after administration of the RNA therapy.

[0047] In any of the aspects described herein, the method further comprises comparing the level of the immune response in the subject after administration of an RNA therapy with the level of an immune response in the subject before administration of the RNA therapy.

[0048] In any of the aspects described herein, the level of the immune response in the subject after administration of the RNA therapy is increased compared with the level of the immune response in the subject before administration of the RNA therapy.

[0049] In any of the aspects described herein, the immune response in the subject is an adaptive immune response.

[0050] In any of the aspects described herein, the immune response in the subject is a T- cell response.

[0051] In any of the aspects described herein, the T-cell response is or comprises a CD4+ response.

[0052] In some embodiments, any of the aspects described herein, the T-cell response is or comprises a CD8+ response. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Fig.1 depicts exemplary constructs encoding antigens used and described herein, including additional non-coding features such as 5’ UTR, secretory protein, 3’ UTR and poly(A) tail. 12829787v1Attorney Ref.2013237-1447

[0054] Fig.2 depicts the flow diagram of the trial (for Cohorts II to VII and expanded cohorts) Note: Cohort I received only five weekly vaccination cycles followed by one vaccination cycle two weeks later over a maximum time period of 43 to 47 days. CT = computed tomography; d = day; DTH = delayed-type hypersensitivity; FU = follow-up; MRI = magnetic resonance imaging; qPCR = quantitative polymerase chain reaction.

[0055] Fig.3 depicts an overall trial design as detailed in Example 3, herein. DEFINITIONS

[0056] About or approximately: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In general, those skilled in the art, familiar within the context, will appreciate the relevant degree of variance encompassed by "about" or "approximately" in that context. For example, in some embodiments, the term "approximately" or "about" may encompass a range of values that are 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 referred value.

[0057] Administering: As used herein, the term "administering" or "administration" typically refers to the administration of a composition to a subject to achieve delivery of an agent that is, or is included in, a composition to a target site or a site to be treated. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may be parenteral. In some embodiments, administration may be oral. In some particular embodiments, administration may be intravenous. In some particular embodiments, administration may be subcutaneous. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and / or periodic (e.g., individual doses separated by a common period of time) dosing. 12829787v1Attorney Ref.2013237-1447 In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time. In some embodiments, administration may comprise a prime- and-boost protocol. A prime-and-boost protocol can include administration of a first dose of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine) followed by, after an interval of time, administration of a second dose of a pharmaceutical composition (e.g., an immunogenic composition, e.g., a vaccine). In the case of an immunogenic composition, a prime-and-boost protocol can result in an increased immune response in a patient.

[0058] 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 immunoglobulin structural elements sufficient to confer specific binding. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain.

[0059] Exemplary antibody agents include, but are not limited to monoclonal antibodies or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements are humanized, primatized, chimeric, etc., as is known in the art. In many embodiments, the term "antibody agent" is used to refer to one or more of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, embodiments, an antibody agent utilized in accordance with the present disclosure is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc.); antibody fragments such as Fab fragments, Fab' fragments, F(ab')2 fragments, Fd' fragments, Fd fragments, and isolated complementarity determining regions (CDRs) or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals ("SMIPsTM"); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies; Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, 12829787v1Attorney Ref.2013237-1447 Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc.], or other pendant group [e.g., poly-ethylene glycol, etc.].

[0060] Associated with: Two events or entities are “associated” with one another, as that term is used herein, if the presence, level and / or form of one is correlated with that of the other. 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 incidence of and / or susceptibility of the disease, disorder, or condition (e.g., across a relevant population), or likelihood of responsiveness to a treatment.

[0061] Blood-derived sample: The term “blood-derived sample,” as used herein, 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, blood plasma (including, e.g., fresh frozen plasma), blood serum, blood fractions, plasma fractions, serum fractions, blood fractions comprising red blood cells (RBC), platelets, leukocytes, etc., and cell lysates including fractions thereof (for example, cells, such as red blood cells, white blood cells, etc., may be harvested and lysed to obtain a cell lysate). In some embodiments, a blood-derived sample that is used for characterization described herein is a plasma sample.

[0062] Cancer: The term “cancer” is used herein to generally refer to a disease or condition in which cells of a tissue of interest exhibit relatively abnormal, uncontrolled, and / or autonomous growth, so that they exhibit an aberrant growth phenotype characterized by a significant loss of control of cell proliferation. In some embodiments, cancer may comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and / or non-metastatic. In some embodiments, cancer may be characterized by a solid tumor. In some embodiments, cancer may be characterized by a hematologic tumor. In general, examples of different types of cancers known in the art include, for example, hematopoietic cancers including leukemias, lymphomas (Hodgkin’s and non-Hodgkin’s), myelomas and myeloproliferative disorders; sarcomas, melanomas, adenomas, carcinomas of solid tissue, squamous cell carcinomas of the mouth, throat, larynx, and lung, liver cancer, genitourinary cancers such as prostate, cervical, bladder, uterine, and endometrial cancer and renal cell carcinomas, bone cancer, pancreatic cancer, skin cancer, cutaneous or intraocular melanoma, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, head and neck cancers, ovarian cancer, breast cancer, glioblastomas, colorectal cancer, gastro- 12829787v1Attorney Ref.2013237-1447 intestinal cancers and nervous system cancers, benign lesions such as papillomas, and the like. In particular embodiments, a cancer can be a melanoma.

[0063] Cap: As used herein, the term “cap” refers to a structure comprising or essentially consisting of a nucleoside-5 '-triphosphate that is typically joined to a 5'-end of an uncapped RNA (e.g., an uncapped RNA having a 5'- diphosphate). In some embodiments, a cap is or comprises a guanine nucleotide. In some embodiments, a cap is or comprises a naturally- occurring RNA 5’ cap, including, e.g., but not limited to a 7- methylguanosine cap, which has a structure designated as "m7G." In some embodiments, a cap is or comprises a synthetic cap analog that resembles an RNA cap structure and possesses the ability to stabilize RNA if attached thereto, including, e.g., but not limited to anti-reverse cap analogs (ARCAs) known in the art). Those skilled in the art will appreciate that methods for joining a cap to a 5’ end of an RNA are known in the art. For example, in some embodiments, a capped RNA may be obtained by in vitro capping of RNA that has a 5' triphosphate group or RNA that has a 5' diphosphate group with a capping enzyme system (including, e.g., but not limited to vaccinia capping enzyme system or Saccharomyces cerevisiae capping enzyme system). Alternatively, a capped RNA can be obtained by in vitro transcription (IVT) of a single-stranded DNA template, wherein, in addition to the GTP, an IVT system also contains a dinucleotide cap analog (including, e.g., a m7GpppG cap analog or an N7-methyl, 2’-O- methyl -GpppG ARCA cap analog or an N7-methyl, 3'-O-methyl-GpppG ARCA cap analog) using methods known in the art.

[0064] Co-administration: As used herein, the term “co-administration” refers to use of a pharmaceutical composition described herein and an additional therapeutic agent (e.g., a chemotherapeutic agent described herein). The combined use of a pharmaceutical composition described herein and an additional therapeutic agent (e.g., a chemotherapeutic agent described herein) may be performed concurrently or separately (e.g., sequentially in any order). In some embodiments of a pharmaceutical composition described herein, a pharmaceutical composition described herein and an additional therapeutic agent (e.g., a chemotherapeutic agent described herein) be combined in one pharmaceutically-acceptable carrier, or they may be placed in separate carriers and delivered to a target cell or administered to a subject at different times. Each of these situations is contemplated as falling within the meaning of “co-administration” or “combination,” provided that a pharmaceutical composition described herein and an additional therapeutic agent (e.g., a chemotherapeutic agent described herein) are delivered or administered sufficiently close in 12829787v1Attorney Ref.2013237-1447 time that there is at least some temporal overlap in biological effect(s) generated by each on a target cell or a subject being treated.

[0065] Combination therapy: As used herein, the term “combination therapy” refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). 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 a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, “administration” of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition.

[0066] Comparable: As used herein, the term “comparable” refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison therebetween so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.

[0067] Complementary: As used herein, the term “complementary” is used in reference to oligonucleotide hybridization related by base-pairing rules. For example, the sequence “C-A- G-T” is complementary to the sequence “G-T-C-A.” Complementarity can be partial or total. Thus, any degree of partial complementarity is intended to be included within the scope of the term “complementary” provided that the partial complementarity permits oligonucleotide 12829787v1Attorney Ref.2013237-1447 hybridization. Partial complementarity is where one or more nucleic acid bases is not matched according to the base pairing rules. Total or complete complementarity between nucleic acids is where each and every nucleic acid base is matched with another base under the base pairing rules.

[0068] Contacting: As used interchangeably herein, the term “delivery,” “delivering,” or “contacting” refers to introduction of ssRNA(s) or a composition comprising the same into a target cell (e.g., cytosol of a target cell). A target cell can be cultured in vitro or ex vivo or be present in a subject (in vivo). Methods of introducing ssRNA(s) or a composition comprising the same into a target cell can vary with in vitro, ex vivo, or in vivo applications. In some embodiments, ssRNA(s) or a composition comprising the same can be introduced into a target cell in a cell culture by in vitro transfection. In some embodiments, ssRNA(s) or a composition comprising the same can be introduced into a target cell via delivery vehicles (e.g., lipid nanoparticles described herein). In some embodiments, ssRNA(s) or a composition comprising the same can be introduced into a target cell in a subject by administering a pharmaceutical composition described herein to a subject.

[0069] Detecting: The term “detecting” is used broadly herein to include appropriate means of determining the presence or absence of an entity of interest or any form of measurement of an entity of interest in a sample. Thus, “detecting” may include determining, measuring, assessing, or assaying the presence or absence, level, amount, and / or location of an entity of interest. Quantitative and qualitative determinations, measurements or assessments are included, including semi-quantitative. Such determinations, measurements or assessments may be relative, for example when an entity of interest is being detected relative to a control reference, or absolute. As such, the term “quantifying” when used in the context of quantifying an entity of interest can refer to absolute or to relative quantification. Absolute quantification may be accomplished by correlating a detected level of an entity of interest to known control standards (e.g., through generation of a standard curve). Alternatively, relative quantification can be accomplished by comparison of detected levels or amounts between two or more different entities of interest to provide a relative quantification of each of the two or more different entities of interest, i.e., relative to each other.

[0070] Disease: As used herein, the term “disease” refers to a disorder or condition that typically impairs normal functioning of a tissue or system in a subject (e.g., a human subject) and is typically manifested by characteristic signs and / or symptoms. In some embodiments, an exemplary disease is cancer. 12829787v1Attorney Ref.2013237-1447

[0071] Encode: As used herein, the term “encode” or “encoding” refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., mRNA) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or an ssRNA (e.g., an mRNA) encodes a polypeptide if transcription and translation of mRNA corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of an ssRNA encoding a tumor-associated antigen (TAA) refers to a coding strand, the nucleotide sequence of which is identical to the mRNA sequence of such a tumor-associated antigen. In some embodiments, a coding region of an ssRNA encoding a TAA refers to a non- coding strand of such a TAA, which may be used as a template for transcription of a gene or cDNA.

[0072] Epitope: As used herein, the term “epitope” includes any moiety that is specifically recognized by an immune system of a patient. For example, an epitope may be any moiety that is specifically recognized by a T cell, a B cell, an immunoglobulin (e.g., antibody or receptor), immunoglobulin (e.g., antibody or receptor), binding component or an aptamer. In some embodiments, an epitope is comprised of a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface- exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized).

[0073] Expression: As used herein, “expression” of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5’ cap formation, and / or 3’ end formation); (3) translation of an RNA into a polypeptide or protein; and / or (4) post-translational modification of a polypeptide or protein.

[0074] Five prime untranslated region: As used herein, the terms "five prime untranslated region" or "5' UTR" refer to a sequence of an mRNA molecule between a transcription start site and a start codon of a coding region of an RNA. In some embodiments, “5’ UTR” refers to a sequence of an mRNA molecule that begins at a transcription start site and ends one 12829787v1Attorney Ref.2013237-1447 nucleotide (nt) before a start codon (usually AUG) of a coding region of an RNA, e.g., in its natural context.

[0075] 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 to be “homologous” to one another if their sequences are at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polynucleotide molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as "hydrophobic" or “hydrophilic” amino acids, and / or as having “polar” or “non-polar” side chains. Substitution of one amino acid for another of the same type may often be considered a “homologous” substitution.

[0076] 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 between polypeptide molecules are considered to be “substantially identical” to one another 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. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence 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 a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that 12829787v1Attorney Ref.2013237-1447 position. The percent identity between the 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. The comparison of sequences and determination of percent identity between two sequences can 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, nucleic acid sequence comparisons made with the ALIGN program use a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0077] RECIST Standard: As used herein, the term “RECIST” or “RECIST standard” refers to Response Evaluation criteria for In Solid Tumors. For example, RECIST standards are as described in Eisenhauer et al. (European J. Cancer 45: 228-247 (2009)), which is herein incorporated by reference in its entirety). In some embodiments, a RECIST standard is RECIST 1.1. In some embodiments, a RECIST standard is iRECIST. For example, iRECIST standards are as described in Seymour, L. et al. (Lancet Oncol.18:3 e143-e152 (2017)), which is herein incorporated by reference in its entirety). In some embodiments, a RECIST standard is an “irRECIST standard,” which is an immune-related Response Evaluation Criteria for In Solid Tumors. For example, irRECIST standards are as described in Nishino et al. (Clin Cancer Res 19:3936-43 (2013)), which is herein incorporated by reference in its entirety). In some embodiments, an irRECIST standard is irRECIST 1.1. In some embodiments, a RECIST standard is an “imRECIST standard,” which is an immune-modified Response Evaluation Criteria for In Solid Tumors. For example, irRECIST standards are as described in Hodi et al. (J Clin Oncol 36:850-8 (2018)), which is herein incorporated by reference in its entirety).

[0078] 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 large but has not yet spread to another body part. In some embodiments, a locally advanced tumor is used to describe cancer that has grown outside the tissue or organ it started but has not yet spread to distant sites in the body of a subject. By way of example only, in some embodiments, locally advanced pancreatic cancer typically refers to stage III disease with tumor extension to adjacent organs (e.g., lymph nodes, liver, duodenum, superior 12829787v1Attorney Ref.2013237-1447 mesenteric artery, and / or celiac trunk) but no signs of metastatic disease; yet complete surgical excision with negative pathologic margins is not possible.

[0079] Nucleic acid / Polynucleotide: As used herein, the term “nucleic acid” refers to a polymer of at least 10 nucleotides or more. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and / or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5- iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2- aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6- O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a 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, 12829787v1Attorney Ref.2013237-1447 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 or more residues or nucleotides long.

[0080] Nucleic acid particle: A “nucleic acid particle” can be used to deliver nucleic acid to a target site of interest (e.g., cell, tissue, organ, and the like). A nucleic acid particle may be formed from at least one cationic or cationically ionizable lipid or lipid-like material, at least one cationic polymer such as protamine, or a mixture thereof and nucleic acid. Nucleic acid particles include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

[0081] Nucleotide: As used herein, the term “nucleotide” refers to its art-recognized meaning. When a number of nucleotides is used as an indication of size, e.g., of a polynucleotide, a certain number of nucleotides refers to the number of nucleotides on a single strand, e.g., of a polynucleotide.

[0082] Patient: As used herein, the term “patient” refers to any organism who is suffering 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, a patient is a human. In some embodiments, a patient is suffering from or susceptible to one or more diseases or disorders or conditions. In some embodiments, a patient displays one or more symptoms of a disease or disorder or condition. In some embodiments, a patient has been diagnosed with one or more diseases or disorders or conditions. In some embodiments, a disease or disorder or condition that is amenable to provided technologies is or includes cancer, or presence of one or more tumors. In some embodiments, a patient is receiving or has received certain therapy to diagnose and / or to treat a disease, disorder, or condition. In some embodiments, a patient is a cancer patient.

[0083] PD-1 / PD-L1 inhibitor-refractory / relapsed: The term “PD-1 / PD-L1 inhibitor- refractory / relapsed”, as used herein, refers to a tumor or cancer (e.g., melanoma) that previously demonstrated a therapeutic response to, but no longer responds to, treatment with a PD-1 / PD-L1 inhibitor such as cemiplimab (e.g., previously was but no longer is partially or completely alleviated, ameliorated, relieved, inhibited, prevented, delayed in onset, reduced in severity, and / or reduced in incidence of one or more symptoms or features of a tumor or cancer following treatment with a PD-1 / PD-L1 inhibitor such as cemiplimab).

[0084] Polypeptide: The term “polypeptide”, as used herein, typically has its art- recognized meaning of a polymer of at least three amino acids or more. Those of ordinary 12829787v1Attorney Ref.2013237-1447 skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional, biologically active, or characteristic fragments, portions or domains (e.g., fragments, portions, or domains retaining at least one activity) of such complete polypeptides. In some embodiments, polypeptides 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, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, polypeptides may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof (e.g., may be or comprise peptidomimetics).

[0085] Reference / Reference standard: As used herein, “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and / or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. In some embodiments, a reference or control is or comprises a set specification (e.g., acceptance criteria). Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and / or comparison to a particular possible reference or control.

[0086] Ribonucleotide: As used herein, the term “ribonucleotide” encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g. , replacement with modified bases, stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. 12829787v1Attorney Ref.2013237-1447 The term “ribonucleotide” also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.

[0087] Ribonucleic acid (RNA): As used herein, the term “RNA” refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded portions. In some embodiments, an RNA can comprise a backbone structure as described in the definition of “Nucleic acid / Polynucleotide” above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA). In some embodiments where an RNA is a mRNA. In some embodiments where an RNA is a mRNA, a RNA typically comprises at its 3’ end a poly(A) region. In some embodiments where an RNA is a mRNA, an RNA typically comprises at its 5’ end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, a RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and / or by chemical synthesis methods).

[0088] Selective or specific: The term “selective” or “specific”, when used herein in reference to an agent having an activity, is understood by those skilled in the art to mean that the agent discriminates between potential target entities, states, or cells. For example, in some embodiments, an agent is said to bind “specifically” to its target if it binds preferentially with that target in the presence of one or more competing alternative targets. In many embodiments, specific interaction is dependent upon the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is to be understood that specificity need not be absolute. In some embodiments, specificity may be evaluated relative to that of a target-binding moiety for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to that of a reference specific binding moiety. In some embodiments, specificity is evaluated relative to that of a reference non-specific binding moiety.

[0089] Specific binding: As used herein, the term “specific binding” refers to an ability to discriminate between possible binding partners in the environment in which binding is to occur. An antibody agent that interacts with one particular target when other potential targets are present is said to "bind specifically" to the target with which it interacts. In some embodiments, specific binding is assessed by detecting or determining degree of association between CDRs of an antibody agent and their partners; in some embodiments, specific binding is assessed by detecting or determining degree of dissociation of an antibody agent- 12829787v1Attorney Ref.2013237-1447 partner complex; in some embodiments, specific binding is assessed by detecting or determining ability of an antibody agent to compete an alternative interaction between its partner and another entity. In some embodiments, specific binding is assessed by performing such detections or determinations across a range of concentrations.

[0090] Subject: As used herein, the term “subject” refers to an organism to be administered with a composition described herein, 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, a subject is a human subject. In some embodiments, a subject is suffering from a disease, disorder, or condition (e.g., cancer). In some embodiments, a subject is susceptible to a disease, disorder, or condition (e.g., cancer). In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition (e.g., cancer). In some embodiments, a subject displays one or more non-specific symptoms of a disease, disorder, or condition (e.g., cancer). In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., cancer). In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., cancer). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and / or therapy is and / or has been administered.

[0091] Suffering from: An individual who is “suffering from” a disease, disorder, and / or condition has been diagnosed with and / or displays one or more symptoms of a disease, disorder, and / or condition.

[0092] Synthetic: As used herein, the term “synthetic” refers to an entity that is artificial, or that is made with human intervention, or that results from synthesis rather than naturally occurring. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule that is chemically synthesized, e.g., in some embodiments by solid-phase synthesis. In some embodiments, the term “synthetic” refers to an entity that is made outside of biological cells. For example, in some embodiments, a synthetic nucleic acid or polynucleotide refers to a nucleic acid molecule (e.g., an RNA) that is produced by in vitro transcription using a template.

[0093] Therapeutic agent: As used interchangeably herein, the phrase “therapeutic agent” or “therapy” refers to an agent or intervention that, when administered to a subject or a patient, has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect. In some embodiments, a therapeutic agent or therapy is any substance that can be used 12829787v1Attorney Ref.2013237-1447 to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. In some embodiments, a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to alleviate, relieve, inhibit, present, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0094] Three prime untranslated region: As used herein, the terms "three prime untranslated region" or "3' UTR" refer to a sequence of an mRNA molecule that begins following a stop codon of a coding region of an open reading frame sequence. In some embodiments, the 3' UTR begins immediately after a stop codon of a coding region of an open reading frame sequence, e.g., in its natural context. In other embodiments, the 3' UTR does not begin immediately after stop codon of the coding region of an open reading frame sequence, e.g., in its natural context.

[0095] Threshold level (e.g., acceptance criteria): As used herein, the term “threshold level” refers to a level that are used as a reference to attain information on and / or classify the results of a measurement, for example, the results of a measurement attained in an assay. For example, in some embodiments, a threshold level means a value measured in an assay that defines the dividing line between two subsets of a population (e.g. a batch that satisfy quality control criteria vs. a batch that does not satisfy quality control criteria). Thus, a value that is equal to or higher than the threshold level defines one subset of the population, and a value that is lower than the threshold level defines the other subset of the population. A threshold level can be determined based on one or more control samples or across a population of control samples. A threshold level can be determined prior to, concurrently with, or after the measurement of interest is taken. In some embodiments, a threshold level can be a range of values.

[0096] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and / or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject at a later-stage of disease, disorder, and / or condition. 12829787v1Attorney Ref.2013237-1447

[0097] Unresectable tumor: As used herein, the term “unresectable tumor” typically refers to a tumor that is unable to be removed by surgery. In some embodiments, an unresectable tumor refers to a tumor that involves and / or grows into an essential organ or tissue (including blood vessels that may not be reconstructable) and / or that is otherwise in a location that cannot readily be accessed without unreasonable risk of damage to one or more other critical or essential organs and / or tissues (including blood vessels). In some embodiments, an unresectable tumor refers to a tumor that cannot be resected by surgery without risk of damage to a patient, which is determined in sound medical judgement to outweigh benefit expected to be received for that patient by resection. In some embodiments, “unresectability” of a tumor refers to the likelihood of achieving a margin-negative (R0) resection. In the context of pancreatic cancer, encasement of major vessels by a tumor such as superior mesenteric artery (SMA) or celiac axis, portal vein occlusion, and the presence of celiac or para-aortic lymphadenopathy are generally acknowledged as findings that preclude R0 surgery. Those skilled in the art will understand parameters that determine whether a tumor is unresectable or not.

[0098] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are 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 I. Introduction

[0099] The present disclosure provides technologies for treating cancer including, e.g., advanced or metastatic, unresectable Stage III, Stage IV, cutaneous melanoma. Treatment options for patients with advanced or metastatic melanoma who have progressed on targeted therapy or immunotherapy may include high-dose interleukin (IL)-2 or other cytotoxic therapies (e.g., dacarbazine, carboplatin / paclitaxel, albumin-bound paclitaxel). These agents have modest response rates of less than 20% in the first- and second-line settings, but no data exist in post-PD-1 settings. Furthermore, little consensus exists regarding optimal standard 12829787v1Attorney Ref.2013237-1447 chemotherapy (NCCN 2021). The first promising results were shown for a c-kit-inhibitor with an ORR of 23.3% (Guo et al.2011) whereas the multi- kinase-inhibitor sorafenib targeting both the MAPK-cascade as well as the vascular endothelial growth factor and platelet-derived growth factor cascades did not improve median PFS when compared to placebo in a Phase III, randomized, double-blind, placebo- controlled trial in combination with carboplatin and paclitaxel (Hauschild et al.2009).

[0100] The tolerability of available treatment options currently precludes the use of adjuvant therapy in patients with stage IIB or IIC high-risk disease and partially also for patients with stage III disease.

[0101] Exemplary compositions described herein comprise TAAs: NY-ESO-1, tyrosinase, MAGE-A3, and TPTE. Among other reasons, these cancer vaccine targets were selected based on the following criteria: • Low or lack of expression in toxicity-relevant organs. • Expression in a substantial fraction of melanoma cells. • The ability to induce antigen-specific immune responses. • Tumor biological role.

[0102] Considering the clonal heterogeneity of cancer and limitation of clinically available samples (only one location), the present disclosure provides the recognition that it is likely that more than the observed rate of 92% of patients actually express at least one of the selected TAAs. In addition, in a substantial percentage of patients, several of these TAAs were found to be co-expressed. Therefore, the present disclosure provides the insight that a significant population of melanoma patients would be expected to develop poly-epitopic, vaccine-induced immune responses and to benefit from treatment with compositions described herein. As used herein, the term “BNT111” refers to a pharmaceutical composition comprising a NY-ESO-1 antigen, a tyrosinase antigen, a MAGE-A3 antigen, and a TPTE antigen.

[0103] In some embodiments, compositions described herein (e.g., BNT111) can prime, activate and / or expand CD4+ and CD8+ T cell specificities, and thus, generate a complementary pool of T cell specificities directed against non-mutant TAAs that are frequently expressed in human melanoma irrespective of the mutational burden of the tumor.

[0104] The liposome formulation of compositions described herein (e.g., BNT111) is designed to deliver the antigens into secondary lymphatic tissues and exploits antiviral innate and adaptive immune mechanisms for induction of highly potent antigen-specific T cell 12829787v1Attorney Ref.2013237-1447 responses. Intravenously administered compositions described herein (e.g., BNT111) can be delivered to secondary lymphatic tissues (e.g., spleen, lymph nodes, and bone marrow) and are rapidly taken up by antigen-presenting cells (APCs). The proteins translated from the RNA components of compositions described herein (e.g., BNT111) can be processed and presented on the patients’ individual set of both HLA-class I and HLA-class II molecules (Kranz et al.2016, which is incorporated herein by reference in its entirety). The close proximity of APCs to T cells in lymphatic tissues represents an ideal microenvironment for efficient priming and amplification of CD8+ and CD4+ T-cell responses (Zinkernagel et al. 1997, which is incorporated herein by reference in its entirety). Components of compositions described herein activate APCs via toll-like receptor signaling, which results in a pulsatile release of pro-inflammatory cytokines, such as IFN-α, IL-6, IFN-γ, and IP-10. Also, secretion of Type-I interferons concomitant to efficient 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 cognate CD4+ T cell help, is necessary for overcoming tolerance to self-antigens. Based on this dual mechanism of action, repeated administration compositions described herein (e.g., BNT111) allows potent priming and rapid amplification of antigen specific CD8+ T-cell responses.

[0105] Together with TAA expression data and the observed dual mechanism of action, the present disclosure provides the expectation that the majority of melanoma patients will develop de novo or intensified poly-epitopic, vaccine-induced, antigen-specific immune responses and derive benefit from treatment with compositions described herein.

[0106] Activation, expansion, and differentiation of naïve T cells is physiologically associated with induction of the immune-regulatory checkpoint molecule PD-1 (Sharpe and Pauken 2018, which is incorporated herein by reference in its entirety). Therefore, as discussed further herein, anti-PD-1 / anti-PD-L1 blockade will augment the activity of T cell responses induced by compositions herein (e.g., BNT111), as supported by clinical trial data in humans. One reason for treatment failure in patients treated with PD-1 / PD-L1 blockade has been the lack of pre-formed antigen-specific T lymphocytes recognizing relevant tumor antigens. In some embodiments, such T lymphocytes are elicited by compositions described herein (e.g., BNT111), which induce potent antigen-specific CD4+ and CD8+ T cell responses. These T cells not only execute direct anti-tumor activity by their cytotoxicity upon recognition of 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. 12829787v1Attorney Ref.2013237-1447

[0107] BNT111 is a fixed set of four liposome-formulated protein-encoding ribonucleic acids (RNAs; henceforward referred to as RNA-lipoplexes [RNA-LPX]). In some embodiments, the four RNAs are individually complexed with liposomes and administered separately via IV injections. In some embodiments, the RNA-LPX formulation is designed for specific targeting of antigen-presenting cells (APCs) in lymphoid organs.

[0108] The RNA components of BNT111 code for four tumor-associated antigens (TAAs), namely New York esophageal squamous cell carcinoma-1 (NY-ESO-1), tyrosinase, melanoma antigen A3 (MAGE-A3), and transmembrane phosphatase with tensin homology (TPTE). All target antigens are known to be immunogenic and, with the exception of TPTE, have already been extensively studied as TAAs in numerous diverse clinical settings (Chen et al.2005; Shackleton et al.2004; Slingluff et al.2003; Sanderson et al.2005; Marchand et al. 2003; Carrasco et al.2008; Brichard et al.2007; Tyagi et al.2009; Banchereau et al.2005; Toungouz et al.2001; Oshita et al.2012; Weide et al.2009; Wilgenhof et al.2011).

[0109] The RNA design is optimized for the induction of strong antigen-specific immune responses. The RNA sequence contains naturally occurring sequence elements at 3’ and 5’ untranslated regions that significantly increase the intracellular half-life and the translational efficiency of the molecule (Holtkamp et al.2006). The encoded proteins are flanked by natural peptide tags which improve the processing of the antigens resulting in efficient presentation of antigen-derived peptide epitopes on major histocompatibility complex (MHC) class I and II molecules (Kreiter et al.2008). These tags include a secretory signal peptide for translocation of the nascent polypeptide chain into the endoplasmic reticulum and the transmembrane and cytoplasmic domain of the MHC class I molecule. The synthetic cap structure (beta-S-ARCA) increases the resistance of the RNA molecules to degradation by extracellular and intracellular ribonucleases (Kuhn et al.2010). The TAAs are fused to the tetanus toxoid epitopes (P2P16), which are well- known universal T helper epitopes to break immune tolerance.

[0110] The liposome formulation of BNT111 is designed to deliver mRNA that encode the antigens into dendritic cells in lymphoid organs for induction of antigen-specific T cell responses. IV injected RNA-LPX home to secondary lymphatic tissues, e.g., spleen, lymph nodes, and bone marrow, and are rapidly taken up by resident professional APCs. Without wishing to be bound by theory, the proteins translated from the RNA components of the BNT111 vaccine are processed and presented on the patient’s individual set of both human leukocyte antigen (HLA)-class I and HLA-class II molecules (Kranz et al.2016). The close proximity of APCs to T cells in lymphoid tissues is the ideal microenvironment for efficient 12829787v1Attorney Ref.2013237-1447 priming and amplification of CD8+ and CD4+ T cell responses (Zinkernagel et al.1997). RNA-LPX activate APCs via toll-like receptor signaling, which results in a pulsatile release of pro-inflammatory cytokines, such as interferon (IFN)-α, IL-6, IFN-γ, and interferon gamma-induced protein 10 (IP-10). The secretion of type I IFN concomitant to efficient antigen presentation stimulates immune cells and directly inhibits regulatory T cells (Srivastava et al.2014) which, in combination with cognate CD4+ T cell help, can overcome tolerance to self-antigens. Based on this mechanism of action, repeated applications of RNA- LPX cancer vaccines can allow potent priming and fast amplification of antigen-specific CD8+ T cell responses.

[0111] RNA-LPX-based cancer vaccines have demonstrated a favorable safety and tolerability profile in different indications, different treatment settings (metastatic, post neo- adjuvant, adjuvant) and with different types of cancer vaccine antigens. II. Tumor Associated Antigens

[0112] In some embodiments, the present disclosure, among other things, provides one or more RNA molecules that encode antigens. In some embodiments, antigens are tumor associated antigens (TAA). The present disclosure provides the insight that 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, 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 with tensin homology (TPTE) antigen, or (v) a combination thereof. In some embodiments, one or more RNA molecules that encode antigens (e.g., TAA, e.g., a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and / or a TPTE antigen) can be used to induce poly-epitopic CD8+ and CD4+ T cell responses that lead to the killing of tumor cells, which express at least one of the targeted antigens.

[0113] In some embodiments, at least one of a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and a TPTE antigen are full-length, non-mutated antigens. In some embodiments, all of a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and a TPTE antigen are full-length, non-mutated antigens. In some embodiments, a NY-ESO-1 antigen, a MAGE-A3 antigen, and a TPTE antigen are full-length, non-mutated antigens. In some embodiments, a NY-ESO-1 antigen and a MAGE-A3 antigen are full-length, non- mutated antigens. In some embodiments, at least one of a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and a TPTE antigen is not a full-length antigen. For example, in 12829787v1Attorney Ref.2013237-1447 some embodiments, a tyrosinase antigen is not full-length, but only comprises a portion of tyrosinase. In some embodiments, a tyrosinase antigen comprises a signal peptide, a EGF- like domain, a CµA domain, a CµB domain, or a combination thereof. In some embodiments, a TPTE antigen is not full-length, but only comprises a portion of a TPTE antigen.

[0114] In some embodiments, after administration of one or more RNA molecules (e.g., one or more RNA molecules that collectively encode a (i) NY-ESO-1 antigen, (ii) a MAGE- A3 antigen, (iii) a tyrosinase antigen, (iv) a TPTE antigen, or (v) a combination thereof) at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen are expressed from dendritic cells in lymphoid tissues of the patient.

[0115] In some embodiments, at least one of a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and a TPTE antigen are present in the cancer (e.g., melanoma). In some embodiments, methods described herein include determining the presence and / or abundance (e.g., a level or amount) of at least one of a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and a TPTE antigens in a cancer of a patient. 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 is assessed for a presence and / or abundance (e.g., a level or amount) of one of a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and a TPTE antigens.

[0116] New York esophageal squamous cell carcinoma (NY-ESO-1) antigen is a member of the cancer testis antigen (CTA) gene family. Approximately 50% of all CTA genes form multigene families on the X chromosome and are referred to as CT-X genes. These CTAs are located in specific clusters along the chromosome with the highest density in the Xq24–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 commonly believed that NY-ESO-1 expression is largely restricted to testicular germ cells and placenta trophoblasts with no or low expression at the transcript or protein level in normal healthy adult somatic cells. NY-ESO-1 is expressed in various human cancers including melanoma) (Giavina- Bianchi et al. J. Immunol. Res.2015, which is incorporated herein by reference in its entirety). According to at least one report). NY-ESO-1 protein was detected in about 20% of invasive melanomas (Giavina-Bianchi).

[0117] In some embodiments, an RNA molecule of one or more RNA molecules as described herein encodes a New York esophageal squamous cell carcinoma (NY-ESO-1) antigen, or an immunogenic fragment thereof. In some embodiments, the single RNA molecule that encodes a NY-ESO-1 antigen is a full-length, non-mutated antigen. In some 12829787v1Attorney Ref.2013237-1447 embodiments, an RNA molecule of the one or more RNA molecules described herein encodes a NY-ESO-1 antigen that does not comprises an amino acid substitution associated with melanoma cancer progression (e.g., a wild type amino acid sequence of the NY-ESO-1 antigen).

[0118] In some embodiments, a NY-ESO-1 antigen comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 2. In some embodiments, the NY-ESO-1 antigen comprises or consists of an amino acid sequence of SEQ ID NO: 2. In some embodiments, the NY-ESO-1 antigen comprises or consists of the amino acid sequence of SEQ ID NO:2 and lacks 1, 2, 3, 4, or 5 amino acids at the N-terminus of SEQ ID NO:2. In some embodiments, the NY-ESO-1 antigen comprises or consists of the amino acid sequence of SEQ ID NO:2 and lacks 1, 2, 3, 4, or 5 amino acids at the C-terminus of SEQ ID NO:2. In some embodiments, the NY-ESO-1 antigen comprises or consists of the amino acid sequence of SEQ ID NO:2 and lacks 1, 2, 3, 4, or 5 amino acids at the N-terminus of SEQ ID NO:2 and lacks 1, 2, 3, 4, or 5 amino acids at the C-terminus of SEQ ID NO:2.

[0119] In some embodiments, a NY-ESO-1 antigen is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 1. In some embodiments, a NY-ESO-1 antigen is encoded by a nucleic acid sequence that comprises or consists of the nucleotide sequence of SEQ ID NO:1. In some embodiments, the NY-ESO-1 antigen is encoded by a nucleic acid that comprises or consists of the nucleotide sequence of SEQ ID NO:1 and lacks 3, 6, 9, 12, or 15 nucleotides at the 5’ end of SEQ ID NO:1. In some embodiments, the NY-ESO-1 antigen is encoded by a nucleic acid that comprises or consists of the nucleotide sequence of SEQ ID NO:1 and lacks 3, 6, 9, 12, or 15 nucleotides at the 3’ end of SEQ ID NO:1. In some embodiments, the NY-ESO-1 antigen is encoded by a nucleic acid that comprises or consists of the nucleotide sequence of SEQ ID NO:1 and lacks 3, 6, 9, 12, or 15 nucleotides at the 3’ end of SEQ ID NO:1 and lacks 3, 6, 9, 12, or 15 nucleotides at the 5’ end of SEQ ID NO:1.

[0120] A melanoma-associated antigen A3 (MAGE-A3) antigen is a member of the MAGEA gene family. The MAGEA genes are clustered at chromosomal location Xq28. They have been implicated in some hereditary disorders, such as dyskeratosis congenita. MAGE- A3 is proposed to enhance ubiquitin ligase activity of RING-type zinc finger-containing E3 ubiquitin-protein ligases and may enhance ubiquitin ligase activity of TRIM28 and stimulate p53 / TP53 ubiquitination by TRIM28. MAGE-A3 is also proposed to act through recruitment and / or stabilization of the Ubl-conjugating enzyme (E2) at the E3:substrate complex. MAGE- 12829787v1Attorney Ref.2013237-1447 A3 is recognized to play a role in embryonal development and is re-expressed in tumor transformation or aspects of tumor progression. In some embodiment, in vitro expression promotes cell viability in melanoma cell lines. MAGE-A3 antigen is known to be recognized by T cell when expressed on a melanoma.

[0121] 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, the 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 a MAGE-A3 antigen that does not comprises an amino acid substitution associated with melanoma cancer progression (e.g., a wild type amino acid sequence of the MAGE-A3 antigen).

[0122] In some embodiments, a MAGE-A3 antigen comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 4. In some embodiments, a MAGE-A3 antigen comprises or consists of an amino acid sequence of SEQ ID NO: 4. In some embodiments, the MAGE-A3 antigen comprises or consists of the amino acid sequence of SEQ ID NO:4 and lacks 1, 2, 3, 4, or 5 amino acids at the N-terminus of SEQ ID NO:4. In some embodiments, the MAGE-A3 antigen comprises or consists of the amino acid sequence of SEQ ID NO:4 and lacks 1, 2, 3, 4, or 5 amino acids at the C-terminus of SEQ ID NO:4. In some embodiments, the MAGE-A3 antigen comprises or consists of the amino acid sequence of SEQ ID NO:4 and lacks 1, 2, 3, 4, or 5 amino acids at the N-terminus of SEQ ID NO:4 and lacks 1, 2, 3, 4, or 5 amino acids at the C-terminus of SEQ ID NO:4.

[0123] In some embodiments, a MAGE-A3 antigen is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 3. In some embodiments, a MAGE-A3 antigen is encoded by a nucleic acid sequence that comprises or consists of the nucleotide sequence of SEQ ID NO: 3. In some embodiments, the MAGE-A3 antigen is encoded by a nucleic acid that comprises or consists of the nucleotide sequence of SEQ ID NO:3 and lacks 3, 6, 9, 12, or 15 nucleotides at the 5’ end of SEQ ID NO:3. In some embodiments, the MAGE-A3 antigen is encoded by a nucleic acid that comprises or consists of the nucleotide sequence of SEQ ID NO:3 and lacks 3, 6, 9, 12, or 15 nucleotides at the 3’ end of SEQ ID NO:3. In some embodiments, the MAGE-A3 antigen is encoded by a nucleic acid that comprises or consists of the nucleotide sequence of SEQ ID NO:3 and lacks 3, 6, 9, 12, or 15 nucleotides at the 3’ end of SEQ ID NO:3 and lacks 3, 6, 9, 12, or 15 nucleotides at the 5’ end of SEQ ID NO:3. 12829787v1Attorney Ref.2013237-1447

[0124] A tyrosinase antigen is encoded by the TYR gene and is a member of the of the tyrosinase family or proteins, which are widely distributed among animals. This gene encodes a melanosomal enzyme that belongs to the tyrosinase family and plays an important role in the melanin biosynthetic pathway. Tyrosinase is known to be expressed in numerous 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).

[0125] 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, an 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 a tyrosinase antigen that does not comprises an amino acid substitution associated with melanoma cancer progression (e.g., a wild type amino acid sequence of the tyrosinase antigen). In some embodiments, a tyrosinase antigen is not full-length, but only comprises a portion of tyrosinase. In some embodiments, a tyrosinase antigen comprises a signal peptide, a EGF-like domain, a CµA domain, a CµB domain, or a combination thereof.

[0126] In some embodiments, a tyrosinase antigen comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 6. In some embodiments, the tyrosinase antigen comprises or consists of an amino acid sequence of SEQ ID NO: 6. In some embodiments, the tyrosinase antigen comprises or consists of the amino acid sequence of SEQ ID NO:6 and lacks 1, 2, 3, 4, or 5 amino acids at the N-terminus of SEQ ID NO:6. In some embodiments, the tyrosinase antigen comprises or consists of the amino acid sequence of SEQ ID NO:6 and lacks 1, 2, 3, 4, or 5 amino acids at the C-terminus of SEQ ID NO:6. In some embodiments, the tyrosinase antigen comprises or consists of the amino acid sequence of SEQ ID NO:6 and lacks 1, 2, 3, 4, or 5 amino acids at the N-terminus of SEQ ID NO:6 and lacks 1, 2, 3, 4, or 5 amino acids at the C-terminus of SEQ ID NO:6.

[0127] In some embodiments, a tyrosinase antigen is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 5 or 26. In some embodiments, a tyrosinase antigen is encoded by a nucleic acid sequence that comprises or consists of the nucleotide sequence of SEQ ID NO: 5 or 26. In some embodiments, the tyrosinase antigen is encoded by a nucleic acid that comprises or consists of the nucleotide sequence of SEQ ID NO:5 and lacks 3, 6, 9, 12, or 15 nucleotides at the 5’ end of SEQ ID NO:5. In some embodiments, the tyrosinase antigen is encoded by a nucleic acid that comprises or consists of the nucleotide sequence of SEQ ID NO:5 and lacks 12829787v1Attorney Ref.2013237-1447 3, 6, 9, 12, or 15 nucleotides at the 3’ end of SEQ ID NO:5. In some embodiments, the tyrosinase antigen is encoded by a nucleic acid that comprises or consists of the nucleotide sequence of SEQ ID NO:5 and lacks 3, 6, 9, 12, or 15 nucleotides at the 3’ end of SEQ ID NO:5 and lacks 3, 6, 9, 12, or 15 nucleotides at the 5’ end of SEQ ID NO:5. In some embodiments, the tyrosinase antigen is encoded by a nucleic acid that comprises or consists of the nucleotide sequence of SEQ ID NO:26 and lacks 3, 6, 9, 12, or 15 nucleotides at the 5’ end of SEQ ID NO:26. In some embodiments, the tyrosinase antigen is encoded by a nucleic acid that comprises or consists of the nucleotide sequence of SEQ ID NO:26 and lacks 3, 6, 9, 12, or 15 nucleotides at the 3’ end of SEQ ID NO:26. In some embodiments, the tyrosinase antigen is encoded by a nucleic acid that comprises or consists of the nucleotide sequence of SEQ ID NO:26 and lacks 3, 6, 9, 12, or 15 nucleotides at the 3’ end of SEQ ID NO:26 and lacks 3, 6, 9, 12, or 15 nucleotides at the 5’ end of SEQ ID NO:26.

[0128] A transmembrane phosphatase with tensin homology (TPTE) antigen is a member of the cancer testis antigen (CTA) family. CTA antigen expression is highly tissue-restricted. TPTE is a transmembrane phosphatase with tensin homology which may play a role in the signal transduction pathways of endocrine or spermatogenic function of testis. TPTE mRNA expression in healthy adult tissues is confined to the testis, and transcript levels are below the detection limit of highly sensitive RT-PCR in all other normal tissue specimens (Simon P, et al. Functional TCR retrieval from single antigen specific human T cells reveals multiple novel epitopes. In Cancer Immunol Res.2(12): 1230–44 (2014), which is incorporated by reference herein in its entirety).

[0129] In some embodiments, an RNA molecule of one or more RNA molecules as described herein encodes a TPTE antigen, or an immunogenic fragment thereof. In some embodiments, a RNA molecule encodes a full length, non-mutated TPTE antigen. In some embodiments, a RNA molecule encodes a truncated TPTE antigen. In some embodiments, a 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 a TPTE antigen that does not comprises an amino acid substitution associated with melanoma cancer progression (e.g., a wild type amino acid sequence of the TPTE antigen).

[0130] In some embodiments, an RNA molecule of one or more RNA molecules as described herein encodes a TPTE antigen or an immunogenic fragment thereof as described in WO2005 / 026205, the entire content of which is incorporated herein by reference for the purposes described herein. 12829787v1Attorney Ref.2013237-1447

[0131] In some embodiments, a TPTE antigen comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 8 or 28. In some embodiments, a TPTE antigen comprises or consists of an amino acid sequence of SEQ ID NO: 8 or 28. In some embodiments, the TPTE antigen comprises or consists of the amino acid sequence of SEQ ID NO:8 and lacks 1, 2, 3, 4, or 5 amino acids at the N- terminus of SEQ ID NO:8. In some embodiments, the TPTE antigen comprises or consists of the amino acid sequence of SEQ ID NO:8 and lacks 1, 2, 3, 4, or 5 amino acids at the C- terminus of SEQ ID NO:8. In some embodiments, the TPTE antigen comprises or consists of the amino acid sequence of SEQ ID NO:8 and lacks 1, 2, 3, 4, or 5 amino acids at the N- terminus of SEQ ID NO:8 and lacks 1, 2, 3, 4, or 5 amino acids at the C-terminus of SEQ ID NO:8. In some embodiments, the TPTE antigen comprises or consists of the amino acid sequence of SEQ ID NO:28 and lacks 1, 2, 3, 4, or 5 amino acids at the N-terminus of SEQ ID NO:28. In some embodiments, the TPTE antigen comprises or consists of the amino acid sequence of SEQ ID NO:28 and lacks 1, 2, 3, 4, or 5 amino acids at the C-terminus of SEQ ID NO:28. In some embodiments, the TPTE antigen comprises or consists of the amino acid sequence of SEQ ID NO:28 and lacks 1, 2, 3, 4, or 5 amino acids at the N-terminus of SEQ ID NO:28 and lacks 1, 2, 3, 4, or 5 amino acids at the C-terminus of SEQ ID NO:28.

[0132] In some embodiments, a TPTE antigen is encoded by a nucleic acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to that of SEQ ID NO: 7 or 27. In some embodiments, a TPTE antigen is encoded by a nucleic acid sequence that comprises or consists of the nucleotide sequence of SEQ ID NO: 7 or 27. In some embodiments, the TPTE antigen is encoded by a nucleic acid that comprises or consists of the nucleotide sequence of SEQ ID NO:7 and lacks 3, 6, 9, 12, or 15 nucleotides at the 5’ end of SEQ ID NO:7. In some embodiments, the TPTE antigen is encoded by a nucleic acid that comprises or consists of the nucleotide sequence of SEQ ID NO:7 and lacks 3, 6, 9, 12, or 15 nucleotides at the 3’ end of SEQ ID NO:7. In some embodiments, the TPTE antigen is encoded by a nucleic acid that comprises or consists of the nucleotide sequence of SEQ ID NO:7 and lacks 3, 6, 9, 12, or 15 nucleotides at the 3’ end of SEQ ID NO:7 and lacks 3, 6, 9, 12, or 15 nucleotides at the 5’ end of SEQ ID NO:7. In some embodiments, the TPTE antigen is encoded by a nucleic acid that comprises or consists of the nucleotide sequence of SEQ ID NO:27 and lacks 3, 6, 9, 12, or 15 nucleotides at the 5’ end of SEQ ID NO:27. In some embodiments, the TPTE antigen is encoded by a nucleic acid that comprises or consists of the nucleotide sequence of SEQ ID NO:27 and lacks 3, 6, 9, 12, or 15 nucleotides at the 3’ end of SEQ ID NO:27. In some embodiments, the TPTE antigen is encoded by a nucleic acid that 12829787v1Attorney Ref.2013237-1447 comprises or consists of the nucleotide sequence of SEQ ID NO:27 and lacks 3, 6, 9, 12, or 15 nucleotides at the 3’ end of SEQ ID NO:27 and lacks 3, 6, 9, 12, or 15 nucleotides at the 5’ end of SEQ ID NO:27.

[0133] In some embodiments, exemplary nucleic acid sequence encoding TAAs as described herein and amino acid sequence of TAAs as described herein are provided in Table 1 below. Table 1: Sequences for TAAs SEQ ID Identifier Sequence NO: G G C T A C A C L L12829787v1Attorney Ref.2013237-1447 3 MAGEA3 ATGCCCCTTGAACAGCGCTCACAGCACTGCAAACCTGA full length GGAGGGCCTTGAAGCAAGGGGCGAAGCTCTGGGGTTG G G C T G G T T S S L F II12829787v1Attorney Ref.2013237-1447 LTQHFVQENYLEYRQVPGSDPACYEFLWGPRALVETSYVK VLHHMVKISGGPHISYPPLHEWVLREGEE T G T C C C A A C C C C G C G C T A C C12829787v1Attorney Ref.2013237-1447 AACGACCCCATCTTCCTGCTGCACCACGCCTTCGTGGAC AGCATCTTCGAGCAGTGGCTGCGGAGACACAGACCCCT T A C T T T A C A T G A G A C C A A12829787v1Attorney Ref.2013237-1447 CTTCCCTCCTCTGCCGACGTTGAATTTTGCCTCTCTCTGA CCCAGTACGAAAGCGGTAGCATGGACAAAGCCGCCAAT C C C C T A T L I W Q Q FI12829787v1Attorney Ref.2013237-1447 GTGTCCAGCTTCGCCTTCGGCCTGTTCGGCGTGTTCCTG GTGCTGCTGGACGTGACACTGATCCTGGCCGACCTGATC G A T C A T C C C G T C G A G C C C C T12829787v1Attorney Ref.2013237-1447 CCCAAGAACGAGCTGGACAACCTGCACAAGCAGAAGG CCAGAAGAATCTACCCCAGCGACTTCGCCGTGGAGATC C G C G A T C A T C C C G T C G A12829787v1Attorney Ref.2013237-1447 GATCTGTAGCACCGCCAAAGAGTCCCTGTACTACTTCGG CGAGCGGAGAACCGACAAGACCCACAGCGAGAAGTTC C C C C T C K I Q P P L F I12829787v1Attorney Ref.2013237-1447 28 TPTE MNESPDPTDLAGVIIELGPNDSPQTSEFKGATEEAPAKESP fragment HTSEFKGAARVSPISESVLARLSKFEVEDAENVASYDSKIK I Q P P L F I. xemp ary em o men s o s enco ng prov e umor-assoca e antigens A. T-Cell Epitope

[0134] In some embodiments, the present disclosure, among other things, provides a pharmaceutical composition including one or more RNA molecules that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, (iv) a TPTE antigen, or (v) a combination thereof; and a T cell epitope. In some embodiments, the present disclosure provides a first pharmaceutical composition including RNA molecules encoding a NY-ESO-1 antigen, a second pharmaceutical composition including RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition including RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical composition including RNA molecules encoding a TPTE antigen, where one or more of the RNA molecules additionally encode a T cell epitope.

[0135] As used herein, the term “T cell epitope” refers to a part or fragment of a protein that is recognized by a T cell when presented in the context of MHC molecules. The term “major histocompatibility complex” and the abbreviation “MHC” includes MHC class I and MHC class II molecules and relates to a complex of genes which is present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting cells or diseased cells in immune reactions, wherein the MHC proteins or 12829787v1Attorney Ref.2013237-1447 molecules bind peptide epitopes and present them for recognition by T cell receptors on T cells. The proteins encoded by the MHC are expressed on the surface of cells, and display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to a T cell. In the case of class I MHC / peptide complexes, binding peptides are typically about 8 to about 10 amino acids long although longer or shorter peptides may be effective. In the case of class II MHC / peptide complexes, binding peptides are typically about 10 to about 25 amino acids long and are in particular about 13 to about 18 amino acids long, whereas longer and shorter peptides may be effective.

[0136] 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, an CD4 epitope comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the CD4 epitope depicted as a “P2P16” domain in SEQ ID NOs: 11, 12, 15, 16, 19, 20, 23, or 24.

[0137] In some embodiments, a CD4 epitope comprises a tetanus toxoid P2, tetanus toxid P16, or both. In some embodiments, a tetanus toxoid P2 comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the CD4 epitope depicted as a “P2” domain in SEQ ID NOs: 11, 12, 15, 16, 19, 20, 23, or 24. In some embodiments, a tetanus toxoid P16 comprises or consists of an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the CD4 epitope depicted as a “P16” domain in SEQ ID NOs: 11, 12, 15, 16, 19, 20, 23, or 24.

[0138] In some embodiments, the present disclosure, among other things, provides a pharmaceutical composition including one or more RNA molecules that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, (iv) a TPTE antigen, or (v) a combination thereof. In some embodiments, a single RNA molecule can 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 can 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 can encode each of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen. B. CD4+ epitope:

[0139] In some embodiments, the present disclosure, among other things, provides a pharmaceutical composition including one or more RNA molecules that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, (iv) a TPTE 12829787v1Attorney Ref.2013237-1447 antigen, or (v) a combination thereof; and a CD4+ epitope. In some embodiments, the present disclosure provides a first pharmaceutical composition including RNA molecules encoding a NY-ESO-1 antigen, a second pharmaceutical composition including RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition including RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical composition including RNA molecules encoding a TPTE antigen, where one or more of the RNA molecules encode a CD4+ epitope. In some embodiments, a CD4+ epitope is delivered by the same RNA molecule(s) that collectively encode the tumor associated antigens described herein. In some embodiments, a CD4+ epitope is delivered by a separate RNA molecule. In some embodiments, a CD4+ epitope is or comprises a non-specific antigen (e.g., an antigen that is not associated with melanoma). In some embodiments, a CD4+ epitope is or comprises a non-specific antigen that provides an adjuvant effect. For example, in some embodiments, a CD4+ epitope can include, without limitation, a tetanus toxid antigenic polypeptide, for example in some embodiments, a tetanus toxid P2 polypeptide and / or a tetanus toxoid P16 polypeptide. C. MHC trafficking domain:

[0140] In some embodiments, an RNA molecule described herein comprises a sequence encoding an MHC trafficking domain. In some embodiments, an MHC trafficking domain is or comprises a transmembrane region and a cytoplasmic region of a chain of an MHC molecule (e.g., a MHC Class I molecule), for example, in some embodiments as described in the International Patent Publication Number WO 2005 / 038030, the contents of which are incorporated herein by reference in their entireties for the purposes described herein. In some embodiments, an MHC trafficking domain is or comprises a MHC Class I trafficking domain. In some embodiments, an MHC class I trafficking domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the MHC Class I trafficking domain depicted as a “MITD” domain in SEQ ID NOs: 11, 12, 15, 16, 19, 20, 23, or 24. In some embodiments, an MHC class I trafficking domain comprises an amino acid sequence that is identical to the amino acid sequence of the MHC Class I trafficking domain as depicted as a “MITD” domain in SEQ ID NOs: 11, 12, 15, 16, 19, 20, 23, or 24.

[0141] Signal peptide-encoding region: In some embodiments, an RNA molecule described herein comprises a sequence encoding a signal peptide. In some embodiments, inclusion of such a signal peptide is useful for increased processing and presentation of antigens. In some embodiments, a signal peptide is or comprises a secretion signal peptide. In 12829787v1Attorney Ref.2013237-1447 some embodiments, a secretion signal peptide may correspond to a sequence encoding a human MHC class I complex alpha chain or a fragment thereof. In some embodiments, a secretion signal peptide may corresponds to a 70-80 bp fragment coding for a secretory signal peptide, which in some embodiments can guide translocation of a nascent polypeptide chain into an endoplasmic reticulum. In some embodiments, a signal peptide comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of the signal peptide-encoding region depicted as “Sec” in SEQ ID NOs: 11, 12, 15, 16, 19, 20, 23, or 24. In some embodiments, a signal peptide comprises an amino acid sequence that is identical to the amino acid sequence of the signal peptide depicted as “Sec” in SEQ ID NOs: 11, 12, 15, 16, 19, 20, 23, or 24. In some embodiments, a signal peptide is linked to the N-terminus of an antigen included in an RNA molecule.

[0142] In some embodiments, an RNA molecule described herein comprises at least one non-coding sequence element. In some embodiments, such a non-coding sequence element is included in an RNA molecule to enhance RNA stability and / or translation efficiency. Examples of non-coding sequence elements include but are not limited to a 3’ untranslated region (UTR), a 5’ UTR, a cap structure, a poly adenine (polyA) tail, and any combinations thereof. D. UTRs (5’ UTRs and / or 3’UTRs):

[0143] In some embodiments, a provided RNA molecule comprises a nucleotide sequence that encodes a 5’UTR of interest and / or a 3’ UTR of interest. One of skill in the art will appreciate that untranslated regions (e.g., 3’ UTR and / or 5’ UTR) of an mRNA sequence can contribute to mRNA stability, mRNA localization, and / or translational efficiency.

[0144] In some embodiments, a provided RNA molecule can comprise a 5’ UTR nucleotide sequence and / or a 3’ UTR nucleotide sequence. In some embodiments, such a 5’ UTR sequence can be operably linked to a 3’ of a coding sequence (e.g., encompassing one or more coding regions). Additionally or alternatively, in some embodiments, a 3’ UTR sequence can be operably linked to 5’ of a coding sequence (e.g., encompassing one or more coding regions).

[0145] In some embodiments, 5' and 3' UTR sequences included in an RNA molecule described herein can consist of or comprise naturally occurring or endogenous 5' and 3' UTR sequences for an open reading frame of a gene of interest. Alternatively, in some embodiments, 5’ and / or 3’ UTR sequences included in an RNA molecule are not endogenous to a coding sequence (e.g., encompassing one or more coding regions); in some such embodiments, such 5’ and / or 3’ UTR sequences can be useful for modifying the stability 12829787v1Attorney Ref.2013237-1447 and / or translation efficiency of an RNA sequence transcribed. For example, a skilled artisan will appreciate that AU-rich elements in 3' UTR sequences can decrease the stability of mRNA. Therefore, as will be understood by a skilled artisan, 3' and / or 5’ UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.

[0146] For example, one skilled in the art will appreciate that, in some embodiments, a nucleotide sequence consisting of or comprising a Kozak sequence of an open reading frame sequence of a gene or nucleotide sequence of interest can be selected and used as a nucleotide sequence encoding a 5’ UTR. As will be understood by a skilled artisan, Kozak sequences are known to increase the efficiency of translation of some RNA transcripts, but are not necessarily required for all RNAs to enable efficient translation. In some embodiments, a provided RNA molecule can comprise a nucleotide sequence that encodes a 5' UTR derived from an RNA virus whose RNA genome is stable in cells. In some embodiments, various modified ribonucleotides (e.g., as described herein) can be used in the 3' and / or 5' UTRs, for example, to impede exonuclease degradation of the transcribed RNA sequence.

[0147] In some embodiments, a 5’ UTR included in an RNA molecule described herein may be derived from human α-globin mRNA combined with Kozak region.

[0148] In some embodiments, an RNA molecule may comprise one or more 3’UTRs. For example, in some embodiments, an RNA molecule may comprise two copies of 3'-UTRs derived from a globin mRNA, such as, e.g., alpha2-globin, alpha1-globin, beta-globin (e.g., a human beta-globin) mRNA. In some embodiments, two copies of 3’UTR derived from a human beta-globin mRNA may be used, e.g., in some embodiments which may be placed between a coding sequence of an RNA molecule and a poly(A)-tail, to improve protein expression levels and / or prolonged persistence of an mRNA. In some embodiments, a 3’UTR derived from a human beta-globin as described in WO 2007 / 036366, the contents of which are incorporated herein by reference in their entireties for the purposes described herein, may be included in an RNA molecule described herein.

[0149] In some embodiments, a 3’ UTR included in an 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 content of which is incorporated herein by reference for the purposes described herein. In some embodiments, a 3‘-UTR may be a combination of at least two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I). These were 12829787v1Attorney Ref.2013237-1447 identified by an ex vivo selection process for sequences that confer RNA stability and augment total protein expression (see WO 2017 / 060314, herein incorporated by reference). E. PolyA tail:

[0150] In some embodiments, a provided ssRNA can comprise a nucleotide sequence that encodes a polyA tail. A polyA tail is a nucleotide sequence comprising a series of adenosine nucleotides, which can vary in length (e.g., at least 5 adenine nucleotides) and can be up to several hundred adenosine nucleotides. In some embodiments, a 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, a polyA tail is a nucleotide sequence comprising at least 120 adenosine nucleotides. In some embodiments, a polyA tail as described in WO 2007 / 036366, the contents of which are incorporated herein by reference in their entireties for the purposes described herein, may be included in an RNA molecule described herein.

[0151] In some embodiments, a polyA tail is or comprises a polyA homopolymeric tail. In some embodiments, a polyA tail may comprise one or more modified adenosine nucleosides, including, but not limited to, cordiocipin and 8-azaadenosine.

[0152] In some embodiments, a polyA tail may comprise one or more non-adenosine nucleotides. In some embodiments, a polyA tail may be or comprise a disrupted or modified polyA tail as described in WO 2016 / 005324, the entire content of which is incorporated herein by reference for the purpose described herein. For example, in some embodiments, a polyA tail included in an RNA molecule described herein may be or comprise a modified polyA sequence comprising: a linker sequence; a first sequence of at least 20 A consecutive nucleotides, which is 5’ of the linker sequence; and a second sequence of at least 20 A consecutive nucleotides, which is 3’ of the linker sequence. In some embodiments, a 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 consecutive nucleotides, which is 5’ of the linker sequence; and a second sequence of at least 70 A consecutive nucleotides, which is 3’ of the linker sequence. F. 5’ cap:

[0153] In some embodiments, an RNA molecule described herein may comprise a 5’ cap, which may be incorporated into such an RNA molecule during transcription, or joined to such an RNA molecule post-transcription. In some embodiments, an RNA molecule may comprise 12829787v1Attorney Ref.2013237-1447 an anti-reverse cap analog (ARCA). In some embodiments, an RNA molecule may comprise a cap analog beta-S-ARCA(D1) (m27,2’-OGppspG).

[0154] In some embodiments, an RNA molecule may comprise an S-ARCA cap structure as disclosed in WO2011 / 015347 or in WO2008 / 157688, the entire contents of each of which are incorporated herein by reference for the purposes described herein.

[0155] In some embodiments, an RNA molecule may comprise a 5’ cap structure for co- transcriptional capping of mRNA. Examples of a cap structure for co-transcriptional capping are known in the art, including, e.g., as described in WO 2017 / 053297, the entire content of which is incorporated herein by reference for the purposes described herein. In some embodiments, a 5’ cap included in an RNA molecule described herein is or comprises m7G(5')ppp(5')(2'OMeA)pG. In some embodiments, a 5’ cap included in an RNA molecule described herein is or comprises a Cap1 structure [e.g., but not limited to m27,3’-OGppp(m 2’- 1O)ApG].

[0156] In some embodiments, one or more RNA molecules that collectively encodes a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof comprise natural ribonucleotides. In some embodiments, one or more RNA molecules that collectively encodes a 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 an RNA molecule to increase its stability and / or to decrease its cytotoxicity. For example, in some embodiments, at least one of A, U, C, and G ribonucleotide of an RNA molecule described herein may be replaced by a modified ribonucleotide. For example, in some embodiments, some or all of cytidine residues present in an RNA molecule may be replaced by a modified cytidine, which in some embodiments may be, e.g., 5-methylcytidine. Alternatively or additionally, in some embodiments, some or all of uridine residues present in an RNA molecule may be replaced by a modified uridine, which in some embodiments may be, e.g., pseudouridine, such as, e.g., 1-methylpseudouridine. In some embodiments, all uridine residues present in an RNA molecule is replaced by pseudouridine, e.g., 1- methylpseudouridine.

[0157] In some embodiments, the present disclosure, among other things, provides a pharmaceutical composition including one or more RNA molecules where an RNA molecule comprises 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 that encodes at least one of a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and a TPTE antigen; (v) at least one sequence that encodes a 12829787v1Attorney Ref.2013237-1447 CD4+ epitope; (vi) a sequence encoding an MHC trafficking domain; (vii) at least one 3’UTR; and (viii) a poly-adenine tail. For example, in some embodiments, a cap structure that is included in an RNA molecule described herein can be a cap structure that can increase the resistance of RNA molecules to degradation by extracellular and intracellular RNases and leads to higher protein expression. In some embodiments, an exemplary cap structure is or comprises beta-S-ARCA(D1) (m27,2’-OGppspG). In some embodiments, an exemplary 5’ UTR sequence element that is included in an RNA molecule described herein is or comprises a characteristic sequence from human α-globin and a Kozak consensus sequence. In some embodiments, an exemplary 3’ UTR sequence element that is included in an RNA molecule described herein may be or comprise two copies of 3’UTR derived from a human beta- globin, or a combination of two sequence elements (FI element) derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and a mitochondrial encoded 12S ribosomal RNA (called I). See, e.g., WO2007 / 036366 and WO 2017 / 060314, the entire contents of each of which is incorporated herein by reference for the purposes described herein. In some embodiments, a poly(A)-tail that is included in an RNA molecule described herein can be designed to enhance RNA stability and / or translational 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 including a stretch of 30 adenosine residues, followed by a 10 nucleotide linker sequence and another stretch of 70 adenosine residues (A30L70). G. Linker:

[0158] In some embodiments, at least one sequence that encodes a linker can be present in an RNA molecule to separate individual components present in the RNA molecule. For example, in some embodiments, at least one sequence that encodes a linker can be present between a coding region that encodes one or more tumor associated antigens as described herein and a sequence that encodes a CD4+ epitope. In some embodiments, at least one sequence that encodes a linker can be present between a sequence that encodes a CD4+ epitope and a sequence that encodes an MHC trafficking domain. In some embodiments, a sequence that encodes a linker may encode a peptide linker. In some embodiments, a peptide linker may be enriched in glycine and / or serine. In some embodiments, a peptide linker that is enriched in glycine and / or serine can comprise at least one amino acid that is not glycine or serine. In some embodiments, a peptide linker can have a length of 3 to 20 amino acids or 3 12829787v1Attorney Ref.2013237-1447 to 15 amino acids, or 3 to 10 amino acids. In some embodiments, a peptide linker can have a length of 10 amino acids.

[0159] In some embodiments, one or more RNA molecules described herein is or comprises one or more mRNAs.

[0160] In some embodiments, a pharmaceutical composition comprises (i) an RNA molecule encoding a NY-ESO-1 antigen as disclosed in Table 2 below; an RNA molecule encoding a MAGE-A3 antigen as disclosed in Table 2 below; an RNA molecule encoding a tyrosinase antigen as disclosed in Table 2 below; and an RNA molecule encoding a TPTE antigen as disclosed in Table 2 below. In some such embodiments, a pharmaceutical composition can be prepared by mixing RNA molecules each encoding a tumor associated antigen as described herein in a molar ratio of about 1:1:1:1. For example, in some embodiments, if total RNA dose is 100µg, then a pharmaceutical composition can be prepared to include 25µg NY-ESO-1 antigen encoding RNA, 25µg MAGE-A3 antigen encoding RNA, 25µg tyrosinase antigen encoding RNA, 25µg TPTE antigen encoding RNA. In some embodiments, this can be achieved by forming, e.g., NY-ESO-1 antigen lipid particles (e.g., NY-ESO-1 antigen lipoplexes or lipid nanoparticles), MAGE-A3 antigen lipid particles (e.g., MAGE-A3 antigen lipoplexes or lipid nanoparticles), tyrosinase antigen lipid particles (e.g., tyrosinase antigen lipoplexes or lipid nanoparticles), and TPTE antigen lipid particles (e.g., TPTE antigen lipoplexes or lipid nanoparticles). In some embodiments, the RNA-lipid particles can then be mixed. For example, mixing can be after RNA and lipid particles form RNA-lipid particles (e.g., RNA-lipoplexes or RNA-lipid nanoparticles). Table 2: Exemplary constructs of RNA molecules each encoding a tumor associated antigen described herein Encoded protein New York esophageal squamous cell carcinoma (NY-ESO-1) antigen12829787v1Attorney Ref.2013237-1447 Exemplary RNA beta-S-ARCA(D1)-hAg-Kozak-sec-GS-MAGEA3-GS-P2P16-GS- construct 1 (e.g., MITD-2hBg-A120peptide; UTR = untranslated region; hAg = human alpha-globin; P2P16 = tetanus toxoid- derived P2 and P16 helper epitopes; 2hBg = 2 copies of human beta-globin; A120 = polyA tail of 120 As in length; A30L70 = two contiguous segments of adenine nucleotides (one segment having a length of 30 As in length while another segment having a length of 70 As in length) separated by linker; FI = a combination of at least two sequence elements derived from the "amino terminal enhancer of split" (AES) mRNA (called F) and the mitochondrial encoded 12S ribosomal RNA (called I)) H. RBL001.1 & RBL001.3 (NY-ESO-1)

[0161] In some embodiments, an RNA molecule encoding a NY-ESO-1 antigen is or comprises the nucleotide sequence of RBL001.1 or RBL001.3. In some embodiments, an RNA molecule encoding a NY-ESO-1 antigen comprises a sequence that encodes a polypeptide having the amino acid sequence of RBL001.1 or RBL001.3. In the following, the 12829787v1Attorney Ref.2013237-1447 sequence alignment of RBL001.1 and RBL003.1 is given for both the nucleotide sequences of the full-length RNAs as well as for the translated proteins (with the amino acid positioned below the third nucleotide of the respective codon triplet). Sequence elements as illustrated in Fig.1 are displayed above the nucleotide sequences. Differences in the nucleotide and amino acid sequences are indicated by “*”. SEQ ID NO: 9 for RBL001.1 RNA; SEQ ID NO: 10 for RBL001.3 RNA; SEQ ID NO: 11 for RBL001.1 protein; SEQ ID NO: 12 for RBL001.3 protein. [ 5’-UTR ][ sec RBL001.1 (RNA) GGGCGAACTA GTATTCTTCT GGTCCCCACA GACTCAGAGA GAACCCGCCA CCATGAGAGT 60 RBL001.3 (RNA) GGGCGAACTA GTATTCTTCT GGTCCCCACA GACTCAGAGA GAACCCGCCA CCATGAGAGT 60 RBL001.1 (prot.) M R 2 RBL001.3 (prot.) M R 2 sec RBL001.1 (RNA) GACCGCCCCC AGAACCCTGA TCCTGCTGCT GTCTGGCGCC CTGGCCCTGA CAGAGACATG 120 RBL001.3 (RNA) GATGGCCCCC AGAACCCTGA TCCTGCTGCT GTCTGGCGCC CTGGCCCTGA CAGAGACATG 120 ** RBL001.1 (prot.) V T A P R T L I L L L S G A L A L T E T 22 RBL001.3 (prot.) V M A P R T L I L L L S G A L A L T E T 22 *sec ][ GS ][ antigen RBL001.1 (RNA) GGCCGGAAGC GGAGGATCT ATGCAGGCCG AGGGCAGAGG 159 RBL001.3 (RNA) GGCCGGAAGC GGCGGCTCTG GAGGAGGCGG CTCCGGAGGC ATGCAGGCCG AGGGCAGAGG 180 * * ********** ********** RBL001.1 (prot.) W A G S G G S M Q A E G R 35 RBL001.3 (prot.) W A G S G G S G G G G S G G M Q A E G R 42 * * * * * * * antigen RBL001.1 (RNA) AACAGGCGGC AGCACAGGCG ACGCAGATGG ACCAGGCGGC CCTGGAATCC CTGATGGCCC 219 RBL001.3 (RNA) AACAGGCGGC AGCACAGGCG ACGCAGATGG ACCAGGCGGC CCTGGAATCC CTGATGGCCC 240 RBL001.1 (prot.) G T G G S T G D A D G P G G P G I P D G 55 RBL001.3 (prot.) G T G G S T G D A D G P G G P G I P D G 62 antigenRBL001.1 (RNA) AGGCGGCAAT GCTGGGGGAC CAGGAGAAGC TGGCGCCACA GGCGGGAGAG GACCTAGAGG 279 RBL001.3 (RNA) AGGCGGCAAT GCTGGGGGAC CAGGAGAAGC TGGCGCCACA GGCGGGAGAG GACCTAGAGG 300 RBL001.1 (prot.) P G G N A G G P G E A G A T G G R G P R 75 RBL001.3 (prot.) P G G N A G G P G E A G A T G G R G P R 82 antigen RBL001.1 (RNA) AGCTGGAGCC GCTAGAGCTT CTGGACCTGG GGGAGGCGCC CCTAGAGGAC CACATGGAGG 339 RBL001.3 (RNA) AGCTGGAGCC GCTAGAGCTT CTGGACCTGG GGGAGGCGCC CCTAGAGGAC CACATGGAGG 360 RBL001.1 (prot.) G A G A A R A S G P G G G A P R G P H G 95 RBL001.3 (prot.) G A G A A R A S G P G G G A P R G P H G 102 12829787v1Attorney Ref.2013237-1447 antigen RBL001.1 (RNA) CGCTGCCAGC GGCCTGAATG GCTGCTGCAG ATGCGGCGCC AGAGGCCCTG AGAGCCGGCT 399 RBL001.3 (RNA) CGCTGCCAGC GGCCTGAATG GCTGCTGCAG ATGCGGCGCC AGAGGCCCTG AGAGCCGGCT 420 RBL001.1 (prot.) G A A S G L N G C C R C G A R G P E S R 115 RBL001.3 (prot.) G A A S G L N G C C R C G A R G P E S R 122 antigen RBL001.1 (RNA) GCTGGAATTC TACCTGGCCA TGCCCTTCGC CACCCCCATG GAAGCCGAGC TGGCCAGAAG 459 RBL001.3 (RNA) GCTGGAATTC TACCTGGCCA TGCCCTTCGC CACCCCCATG GAAGCCGAGC TGGCCAGAAG 480 RBL001.1 (prot.) L L E F Y L A M P F A T P M E A E L A R 135 RBL001.3 (prot.) L L E F Y L A M P F A T P M E A E L A R 142 antigen RBL001.1 (RNA) ATCCCTGGCT CAGGACGCTC CTCCTCTGCC TGTGCCCGGC GTGCTGCTGA AAGAATTCAC 519 RBL001.3 (RNA) ATCCCTGGCT CAGGACGCTC CTCCTCTGCC TGTGCCCGGC GTGCTGCTGA AAGAATTCAC 540 RBL001.1 (prot.) R S L A Q D A P P L P V P G V L L K E F 155 RBL001.3 (prot.) R S L A Q D A P P L P V P G V L L K E F 162 antigen RBL001.1 (RNA) CGTGTCCGGC AACATCCTGA CCATCAGACT GACAGCCGCC GATCACAGAC AGCTCCAGCT 579 RBL001.3 (RNA) CGTGTCCGGC AACATCCTGA CCATCAGACT GACAGCCGCC GATCACAGAC AGCTCCAGCT 600 RBL001.1 (prot.) T V S G N I L T I R L T A A D H R Q L Q 175 RBL001.3 (prot.) T V S G N I L T I R L T A A D H R Q L Q 182 antigen RBL001.1 (RNA) GAGCATCAGC TCTTGCCTGC AGCAGCTGAG CCTGCTGATG TGGATCACCC AGTGCTTTCT 639 RBL001.3 (RNA) GAGCATCAGC TCTTGCCTGC AGCAGCTGAG CCTGCTGATG TGGATCACCC AGTGCTTTCT 660 RBL001.1 (prot.) L S I S S C L Q Q L S L L M W I T Q C F 195 RBL001.3 (prot.) L S I S S C L Q Q L S L L M W I T Q C F 202 antigen ][ GS RBL001.1 (RNA) GCCCGTGTTC CTGGCCCAGC CACCCAGCGG ACAGAGAAGG GGCGGATCCC TGGGAGGCGG 699 RBL001.3 (RNA) GCCCGTGTTC CTGGCCCAGC CACCCAGCGG ACAGAGAAGG GGAGGATCCG GTGGTGGCGG 720 * * ** * RBL001.1 (prot.) L P V F L A Q P P S G Q R R G G S L G G 215 RBL001.3 (prot.) L P V F L A Q P P S G Q R R G G S G G G 222 * GS ][ P2P16 RBL001.1 (RNA) GGGAAGCGGC AAGAAGCAGT ACATCAAGGC CAACAGCAAG TTCATCGGCA TCACCGAGCT 759 RBL001.3 (RNA) CAGCGGCGGC AAGAAGCAGT ACATCAAGGC CAACAGCAAG TTCATCGGCA TCACCGAGCT 780 ** ** RBL001.1 (prot.) G G S G K K Q Y I K A N S K F I G I T E 235 RBL001.3 (prot.) G S G G K K Q Y I K A N S K F I G I T E 242 * * P2P16 RBL001.1 (RNA) GAAGAAGCTG GGAGGGGGCA AACGGGGAGG CGGCAAAAAG ATGACCAACA GCGTGGACGA 819 RBL001.3 (RNA) GAAGAAGCTG GGAGGGGGCA AACGGGGAGG CGGCAAAAAG ATGACCAACA GCGTGGACGA 840 RBL001.1 (prot.) L K K L G G G K R G G G K K M T N S V D 255 RBL001.3 (prot.) L K K L G G G K R G G G K K M T N S V D 262 P2P16 RBL001.1 (RNA) CGCCCTGATC AACAGCACCA AGATCTACAG CTACTTCCCC AGCGTGATCA GCAAAGTGAA 879 RBL001.3 (RNA) CGCCCTGATC AACAGCACCA AGATCTACAG CTACTTCCCC AGCGTGATCA GCAAAGTGAA 900 RBL001.1 (prot.) D A L I N S T K I Y S Y F P S V I S K V 275 RBL001.3 (prot.) D A L I N S T K I Y S Y F P S V I S K V 28212829787v1Attorney Ref.2013237-1447 P2P16 ][ GS RBL001.1 (RNA) CCAGGGCGCT CAGGGCAAGA AACTGGGCTC TAGCGGAGGG GGAGGCTCTC CTGGCGGGGG 939 RBL001.3 (RNA) CCAGGGCGCT CAGGGCAAGA AACTGGGCTC TAGCGGAGGG GGAGGCTCTC CTGGCGGGGG 960 RBL001.1 (prot.) N Q G A Q G K K L G S S G G G G S P G G 295 RBL001.3 (prot.) N Q G A Q G K K L G S S G G G G S P G G 302 GS ][ MITD RBL001.1 (RNA) ATCTAGCATC GTGGGAATTG TGGCAGGACT GGCAGTGCTG GCCGTGGTGG TGATCGGAGC 999 RBL001.3 (RNA) ATCTAGCATC GTGGGAATTG TGGCAGGACT GGCAGTGCTG GCCGTGGTGG TGATCGGAGC 1020 RBL001.1 (prot.) G S S I V G I V A G L A V L A V V V I G 315 RBL001.3 (prot.) G S S I V G I V A G L A V L A V V V I G 322 MITD RBL001.1 (RNA) CGTGGTGGCT ACCGTGATGT GCAGACGGAA GTCCAGCGGA GGCAAGGGCG GCAGCTACAG 1059 RBL001.3 (RNA) CGTGGTGGCT ACCGTGATGT GCAGACGGAA GTCCAGCGGA GGCAAGGGCG GCAGCTACAG 1080 RBL001.1 (prot.) A V V A T V M C R R K S S G G K G G S Y 335 RBL001.3 (prot.) A V V A T V M C R R K S S G G K G G S Y 342 MITD ][3’-UTR RBL001.1 (RNA) CCAGGCCGCC AGCTCTGATA GCGCCCAGGG CAGCGACGTG TCACTGACAG CC TGACT 1119 RBL001.3 (RNA) CCAGGCCGCC AGCTCTGATA GCGCCCAGGG CAGCGACGTG TCACTGACAG CCTAGTAACT 1140 *** * RBL001.1 (prot.) S Q A A S S D S A Q G S D V S L T A 353 RBL001.3 (prot.) S Q A A S S D S A Q G S D V S L T A 360 3’-UTR RBL001.1 (RNA) CGAGAGCTCG CTTTCTTGCT GTCCAATTTC TATTAAA GGTTCCTTT GTTCCCTAAG 1172 RBL001.3 (RNA) CGAGCTGGTA CTGCATGCAC GCAATGCTAG CTGCCCCTTT CCCGTCCTGG GTACCCCGAG 1200 ****** *** **** ****** ** ********** **** ** * ** 3’-UTR RBL001.1 (RNA) TCCAACTACT AAACTGGGGG ATATTATGAA GGGCCTTGAG CATCTGGATT CTGCCTAATA 1232 RBL001.3 (RNA) TCTCCCCCGA CCTCGGGTCC CAGGTATGCT CCCACCTCCA CCTGCCCCAC TCACCACCTC 1260 *** **** *** * *** **** ** **** * *** * ******* *** *** * 3’-UTR RBL001.1 (RNA) AAAAACATTT ATTTTCATTG CTGCGTCGAG AGCTCGCTTT CTTGCTGTCC AATTTCT 1289 RBL001.3 (RNA) TGCTAGTTCC AGACACCTCC CAAGCACGCA GCAATGCAGC TCAAAACGCT TAGCCTAGCC 1320 **** ** ** **** * ** ***** ** ***** *** ******** * * ******** 3’-UTR RBL001.1 (RNA) A TTAAAGGTTC CTTT GTTCCCTAA GTCCAACTAC TAAACTGGGG 1333 RBL001.3 (RNA) ACACCCCCAC GGGAAACAGC AGTGATTAAC CTTTAGCAAT AAACGAAAGT TTAACTAAGC 1380 ********** *** ***** ** ******* ** ** * * *** * **** * ** * 3’-UTR RBL001.1 (RNA) GATATTATGA AGGGCCTTGA GCATCTGGAT TCTGCCTAAT AAAAAACATT TATTTTCATT 1393 RBL001.3 (RNA) TATACTAACC CCAGGGTTGG TCAATTTCGT GCCAGCCACA CCGAGACCTG GTCCAGAGTC 1440 * * *** *** ** * * ** *** * *** * ** *** * * * ******** * 3’-UTR ][ poly(A) RBL001.1 (RNA) GCTGCGTCGA GAGCTAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1453 RBL001.3 (RNA) GCTAGCCGCG TCGCTAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAGCATA TGACT 1500 ******* ** ** * ** ******* poly(A) RBL001.1 (RNA) AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1513 RBL001.3 (RNA) AAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1550 ***** poly(A) ] RBL001.1 (RNA) AAAAAAAAAA AAAAA 1528 RBL001.3 (RNA) AAAAAAAAAA AAAAA12829787v1Attorney Ref.2013237-1447 I. RBL002.2 & RBL002.4 (TYR)

[0162] In some embodiments, an RNA molecule encoding a Tyrosinase antigen is or comprises the nucleotide sequence of RBL002.2 or RBL002.4. In some embodiments, an RNA molecule encoding a Tyrosinase antigen comprises a sequence that encodes a polypeptide having the amino acid sequence of RBL002.2 or RBL002.4. In the following, the sequence alignment of RBL002.2 and RBL002.4 is given for both the nucleotide sequences of the full-length RNAs as well as for the translated proteins (with the amino acid positioned below the third nucleotide of the respective codon triplet). Sequence elements as illustrated in Fig.1 are displayed above the nucleotide sequences. Differences in the nucleotide and amino acid sequences are indicated by “*”. SEQ ID NO: 13 for RBL002.2 RNA; SEQ ID NO: 14 for RBL002.4 RNA; SEQ ID NO: 15 for RBL002.2 protein; SEQ ID NO: 16 for RBL002.4 protein. [ 5’-UTR ][ antigen RBL002.2 (RNA) GGGCGAACTA GTATTCTTCT GGTCCCCACA GACTCAGAGA GAACCCGCCA CCATGCTTCT 60 RBL002.4 (RNA) GGGCGAACTA GTATTCTTCT GGTCCCCACA GACTCAGAGA GAACCCGCCA CCATGCTTCT 60 RBL002.2 (prot.) M L 2 RBL002.4 (prot.) M L 2 antigen RBL002.2 (RNA) GGCTGTGCTC TATTGCTTGC TGTGGTCCTT CCAGACTTCT GCCGGACACT TTCCCAGAGC 120 RBL002.4 (RNA) GGCTGTGCTC TATTGCTTGC TGTGGTCCTT CCAGACTTCT GCCGGACACT TTCCCAGAGC 120 RBL002.2 (prot.) L A V L Y C L L W S F Q T S A G H F P R 22 RBL002.4 (prot.) L A V L Y C L L W S F Q T S A G H F P R 22 antigen RBL002.2 (RNA) ATGTGTGTCA TCCAAAAACC TCATGGAGAA AGAATGCTGT CCTCCTTGGA GTGGTGATAG 180 RBL002.4 (RNA) ATGTGTGTCA TCCAAAAACC TCATGGAGAA AGAATGCTGT CCTCCTTGGA GTGGTGATAG 180 RBL002.2 (prot.) A C V S S K N L M E K E C C P P W S G D 42 RBL002.4 (prot.) A C V S S K N L M E K E C C P P W S G D 42 antigen RBL002.2 (RNA) ATCCCCATGT GGACAGCTCT CAGGCAGAGG ATCTTGCCAG AACATTCTGC TGAGCAATGC 240 RBL002.4 (RNA) ATCCCCATGT GGACAGCTCT CAGGCAGAGG ATCTTGCCAG AACATTCTGC TGAGCAATGC 240 RBL002.2 (prot.) R S P C G Q L S G R G S C Q N I L L S N 62 RBL002.4 (prot.) R S P C G Q L S G R G S C Q N I L L S N 62 antigen RBL002.2 (RNA) ACCTCTTGGC CCACAGTTTC CCTTTACCGG AGTAGATGAC AGAGAAAGTT GGCCCTCCGT 300 RBL002.4 (RNA) ACCTCTTGGC CCACAGTTTC CCTTTACCGG AGTAGATGAC AGAGAAAGTT GGCCCTCCGT 300 RBL002.2 (prot.) A P L G P Q F P F T G V D D R E S W P S 82 RBL002.4 (prot.) A P L G P Q F P F T G V D D R E S W P S 82 antigen RBL002.2 (RNA) GTTCTACAAC AGAACATGCC AATGCAGCGG CAATTTTATG GGATTTAACT GCGGAAATTG 360 RBL002.4 (RNA) GTTCTACAAC AGAACATGCC AATGCAGCGG CAATTTTATG GGATTTAACT GCGGAAATTG 360 RBL002.2 (prot.) V F Y N R T C Q C S G N F M G F N C G N 102 RBL002.4 (prot.) V F Y N R T C Q C S G N F M G F N C G N 102 12829787v1Attorney Ref.2013237-1447 antigen RBL002.2 (RNA) CAAATTTGGA TTTTGGGGCC CAAACTGTAC AGAAAGAAGA CTGCTCGTTA GAAGAAACAT 420 RBL002.4 (RNA) CAAATTTGGA TTTTGGGGCC CAAACTGTAC AGAAAGAAGA CTGCTCGTTA GAAGAAACAT 420 RBL002.2 (prot.) C K F G F W G P N C T E R R L L V R R N 122 RBL002.4 (prot.) C K F G F W G P N C T E R R L L V R R N 122 antigen RBL002.2 (RNA) CTTTGATCTG AGCGCACCCG AAAAAGACAA ATTCTTCGCT TATCTGACTC TCGCCAAACA 480 RBL002.4 (RNA) CTTTGATCTG AGCGCACCCG AAAAAGACAA ATTCTTCGCT TATCTGACTC TCGCCAAACA 480 RBL002.2 (prot.) I F D L S A P E K D K F F A Y L T L A K 142 RBL002.4 (prot.) I F D L S A P E K D K F F A Y L T L A K 142 antigen RBL002.2 (RNA) CACAATAAGC AGTGATTATG TCATTCCCAT TGGCACTTAT GGACAGATGA AAAATGGCTC 540 RBL002.4 (RNA) CACAATAAGC AGTGATTATG TCATTCCCAT TGGCACTTAT GGACAGATGA AAAATGGCTC 540 RBL002.2 (prot.) H T I S S D Y V I P I G T Y G Q M K N G 162 RBL002.4 (prot.) H T I S S D Y V I P I G T Y G Q M K N G 162 600 600 182 182 660 660 202 202 720 720 222 222 780 780 242 242 840 840 262 262RBL002.2 (RNA) GAGCCCTGCC TCTTTCTTCA GTTCTTGGCA GATTGTCTGT TCAAGATTGG AAGAATACAA 900 RBL002.4 (RNA) GAGCCCTGCC TCTTTCTTCA GTTCTTGGCA GATTGTCTGT TCAAGATTGG AAGAATACAA 900 RBL002.2 (prot.) L S P A S F F S S W Q I V C S R L E E Y 282 RBL002.4 (prot.) L S P A S F F S S W Q I V C S R L E E Y 282 antigen RBL002.2 (RNA) TTCCCATCAG TCCCTGTGTA ACGGAACACC AGAAGGACCT CTGAGAAGAA ACCCTGGCAA 960 RBL002.4 (RNA) TTCCCATCAG TCCCTGTGTA ACGGAACACC AGAAGGACCT CTGAGAAGAA ACCCTGGCAA 960 RBL002.2 (prot.) N S H Q S L C N G T P E G P L R R N P G 302 RBL002.4 (prot.) N S H Q S L C N G T P E G P L R R N P G 302 12829787v1Attorney Ref.2013237-1447 antigen RBL002.2 (RNA) TCATGACAAA AGCAGGACAC CTAGACTTCC CTCCTCTGCC GACGTTGAAT TTTGCCTCTC 1020 RBL002.4 (RNA) TCATGACAAA AGCAGGACAC CTAGACTTCC CTCCTCTGCC GACGTTGAAT TTTGCCTCTC 1020 RBL002.2 (prot.) N H D K S R T P R L P S S A D V E F C L 322 RBL002.4 (prot.) N H D K S R T P R L P S S A D V E F C L 322 antigen RBL002.2 (RNA) TCTGACCCAG TACGAAAGCG GTAGCATGGA CAAAGCCGCC AATTTCAGCT TTAGAAATAC 1080 RBL002.4 (RNA) TCTGACCCAG TACGAAAGCG GTAGCATGGA CAAAGCCGCC AATTTCAGCT TTAGAAATAC 1080 RBL002.2 (prot.) S L T Q Y E S G S M D K A A N F S F R N 342 RBL002.4 (prot.) S L T Q Y E S G S M D K A A N F S F R N 342 antigen RBL002.2 (RNA) CTTGGAAGGA TTTGCCTCAC CTCTGACAGG AATTGCTGAT GCTAGCCAGT CATCCATGCA 1140 RBL002.4 (RNA) CTTGGAAGGA TTTGCCTCAC CTCTGACAGG AATTGCTGAT GCTAGCCAGT CATCCATGCA 1140 RBL002.2 (prot.) T L E G F A S P L T G I A D A S Q S S M 362 RBL002.4 (prot.) T L E G F A S P L T G I A D A S Q S S M 362 382 382 402 402 422 422 442 442 462 462 482 48212829787v1Attorney Ref.2013237-1447 GS ][ P2P16 RBL002.2 (RNA) GGGAAGC GGCAAGAAGC AGTACATCAA GGCCAACAGC AAGTTCATCG GCATCACCGA 1557 RBL002.4 (RNA) CGGCAGCGGC GGCAAGAAGC AGTACATCAA GGCCAACAGC AAGTTCATCG GCATCACCGA 1560RBL002.2 (prot.) G G S G K K Q Y I K A N S K F I G I T 501 RBL002.4 (prot.) G G S G G K K Q Y I K A N S K F I G I T 502 * P2P16 RBL002.2 (RNA) GCTGAAGAAG CTGGGAGGGG GCAAACGGGG AGGCGGCAAA AAGATGACCA ACAGCGTGGA 1617 RBL002.4 (RNA) GCTGAAGAAG CTGGGAGGGG GCAAACGGGG AGGCGGCAAA AAGATGACCA ACAGCGTGGA 1620 RBL002.2 (prot.) E L K K L G G G K R G G G K K M T N S V 521 RBL002.4 (prot.) E L K K L G G G K R G G G K K M T N S V 522 P2P16 RBL002.2 (RNA) CGACGCCCTG ATCAACAGCA CCAAGATCTA CAGCTACTTC CCCAGCGTGA TCAGCAAAGT 1677 RBL002.4 (RNA) CGACGCCCTG ATCAACAGCA CCAAGATCTA CAGCTACTTC CCCAGCGTGA TCAGCAAAGT 1680 RBL002.2 (prot.) D D A L I N S T K I Y S Y F P S V I S K 541 RBL002.4 (prot.) D D A L I N S T K I Y S Y F P S V I S K 542 P2P16 ][ GS RBL002.2 (RNA) GAACCAGGGC GCTCAGGGCA AGAAACTGGG CTCTAGCGGA GGGGGAGGCT CTCCTGGCGG 1737 RBL002.4 (RNA) GAACCAGGGC GCTCAGGGCA AGAAACTGGG CTCTAGCGGA GGGGGAGGCT CTCCTGGCGG 1740 RBL002.2 (prot.) V N Q G A Q G K K L G S S G G G G S P G 561 RBL002.4 (prot.) V N Q G A Q G K K L G S S G G G G S P G 562 GS ][ MITD RBL002.2 (RNA) AGCCGTGGTG GCTACCGTGA TGTGCAGACG GAAGTCCAGC GGAGGCAAGG GCGGCAGCTA 1797 RBL002.4 (RNA) AGCCGTGGTG GCTACCGTGA TGTGCAGACG GAAGTCCAGC GGAGGCAAGG GCGGCAGCTA 1800 RBL002.2 (prot.) G G S S I V G I V A G L A V L A V V V I 581 RBL002.4 (prot.) G G S S I V G I V A G L A V L A V V V I 582 MITD RBL002.2 (RNA) AGCCGTGGTG GCTACCGTGA TGTGCAGACG GAAGTCCAGC GGAGGCAAGG GCGGCAGCTA 1857 RBL002.4 (RNA) AGCCGTGGTG GCTACCGTGA TGTGCAGACG GAAGTCCAGC GGAGGCAAGG GCGGCAGCTA 1860 RBL002.2 (prot.) G A V V A T V M C R R K S S G G K G G S 601 RBL002.4 (prot.) G A V V A T V M C R R K S S G G K G G S 602 MITD RBL002.2 (RNA) CAGCCAGGCC GCCAGCTCTG ATAGCGCCCA GGGCAGCGAC GTGTCACTGA CAGCC TG 1914 RBL002.4 (RNA) CAGCCAGGCC GCCAGCTCTG ATAGCGCCCA GGGCAGCGAC GTGTCACTGA CAGCCTAGTA 1920 *** * RBL002.2 (prot.) Y S Q A A S S D S A Q G S D V S L T A 620 RBL002.4 (prot.) Y S Q A A S S D S A Q G S D V S L T A 621 ][RBL002.2 (RNA) ACTCGAGAGC TCGCTTTCTT GCTGTCCAAT TTCTATTAAA GGTTCC TTTGTTCCCT 1970 RBL002.4 (RNA) ACTCGAGCTG GTACTGCATG CACGCAATGC TAGCTGCCCC TTTCCCGTCC TGGGTACCCC 1980 *** *** *** * *** ****** ********* ******* ** * * 3’-UTR RBL002.2 (RNA) AAGTCCAACT ACTAAACTGG GGGATATTAT GAAGGGCCTT GAGCATCTGG ATTCTGCCTA 2030 RBL002.4 (RNA) GAGTCTCCCC CGACCTCGGG TCCCAGGTAT GCTCCCACCT CCACCTGCCC CACTCACCAC 2040 * *** * ****** * ******* ****** * *** * **** ****** ** 3’-UTR RBL002.2 (RNA) ATAAAAAACA TTTATTTTCA TTGCTGCGTC GAGAGCTCGC TTTCTTGCTG TCCAATTTCT 2090 RBL002.4 (RNA) CTCTGCTAGT TCCAGACACC TCCCAAGCAC GCAGCAATGC AGCTCAAAAC GCTTAGCCTA 2100 * ***** ** ** **** * ** ***** ******* ********** * ** ***** 12829787v1Attorney Ref.2013237-1447 3’-UTR RBL002.2 (RNA) ATTAAAGG TTCCTTT GTTCCC TAAGTCCAAC TACTAAACTG 2131 RBL002.4 (RNA) GCCACACCCC CACGGGAAAC AGCAGTGATT AACCTTTAGC AATAAACGAA AGTTTAACTA 2160 ********** ****** ** ***** **** ***** ** * **** * * *** * * 3’-UTR RBL002.2 (RNA) GGGGATATTA TGAAGGGCCT TGAGCATCTG GATTCTGCCT AATAAAAAAC ATTTATTTTC 2191 RBL002.4 (RNA) AGCTATACTA ACCCCAGGGT TGGTCAATTT CGTGCCAGCC ACACCGAGAC CTGGTCCAGA 2220 * ** * ****** ** ** ** * ** * *** * ***** * * ******** 3’-UTR ][ poly(A) RBL002.2 (RNA) ATTGCTGCGT CGAGAGCTAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2251 RBL002.4 (RNA) GTCGCTAGCC GCGTCGCTAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAGC ATATGACT 2278 * * **** ***** ** * ** **** poly(A) RBL002.2 (RNA) AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2311 RBL002.4 (RNA) AA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2330 ******** poly(A) ] RBL002.2 (RNA) AAAAAAAAAA AAAAAAAA 2329 RBL002.4 (RNA) AAAAAAAAAA AAAAAAAA 2348

[0163] In some embodiments, an RNA molecule encoding a Tyrosinase antigen is or comprises the nucleotide sequence of SEQ ID NO:25: GGGCGAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACCATG CTTCTGGCTGTGCTCTATTGCTTGCTGTGGTCCTTCCAGACTTCTGCCGGACACTTT CCCAGAGCATGTGTGTCATCCAAAAACCTCATGGAGAAAGAATGCTGTCCTCCTT GGAGTGGTGATAGATCCCCATGTGGACAGCTCTCAGGCAGAGGATCTTGCCAGAA CATTCTGCTGAGCAATGCACCTCTTGGCCCACAGTTTCCCTTTACCGGAGTAGATG ACAGAGAAAGTTGGCCCTCCGTGTTCTACAACAGAACATGCCAATGCAGCGGCAA TTTTATGGGATTTAACTGCGGAAATTGCAAATTTGGATTTTGGGGCCCAAACTGTA CAGAAAGAAGACTGCTCGTTAGAAGAAACATCTTTGATCTGAGCGCACCCGAAA AAGACAAATTCTTCGCTTATCTGACTCTCGCCAAACACACAATAAGCAGTGATTAT GTCATTCCCATTGGCACTTATGGACAGATGAAAAATGGCTCAACTCCCATGTTCAA CGACATCAACATATACGATCTGTTTGTGTGGATGCATTACTACGTGAGTATGGATGC TCTGCTGGGTGGCTCCGAAATATGGAGGGATATAGATTTTGCACACGAAGCTCCTG CCTTCCTTCCATGGCATAGACTGTTCCTGCTGAGATGGGAACAAGAAATCCAGAA ACTTACAGGCGATGAAAACTTCACTATCCCTTATTGGGATTGGAGAGATGCTGAGA AATGCGATATCTGTACCGATGAATACATGGGTGGTCAACACCCAACCAACCCCAAT CTCCTGAGCCCTGCCTCTTTCTTCAGTTCTTGGCAGATTGTCTGTTCAAGATTGGA AGAATACAATTCCCATCAGTCCCTGTGTAACGGAACACCAGAAGGACCTCTGAGA AGAAACCCTGGCAATCATGACAAAAGCAGGACACCTAGACTTCCCTCCTCTGCCG ACGTTGAATTTTGCCTCTCTCTGACCCAGTACGAAAGCGGTAGCATGGACAAAGC CGCCAATTTCAGCTTTAGAAATACCTTGGAAGGATTTGCCTCACCTCTGACAGGAA 12829787v1Attorney Ref.2013237-1447 TTGCTGATGCTAGCCAGTCATCCATGCACAACGCTTTGCACATCTATATGAATGGAA CCATGAGTCAGGTTCAGGGATCTGCCAATGATCCTATTTTCCTGCTGCACCATGCAT TCGTGGACTCTATCTTTGAGCAGTGGCTTAGAAGACACAGACCATTGCAGGAAGT CTATCCAGAGGCCAACGCACCAATCGGCCATAATAGAGAAAGCTACATGGTACCCT TCATTCCTCTGTACAGAAATGGAGATTTCTTCATCAGCTCCAAAGACCTGGGCTAC GATTACTCATATCTGCAAGACAGTGATCCCGATAGCTTCCAAGACTACATTAAGTCT TATCTCGAACAGGCGAGCAGAATCTGGTCCTGGGGAGGATCCGGTGGTGGCGGCA GCGGCGGCAAGAAGCAGTACATCAAGGCCAACAGCAAGTTCATCGGCATCACCG AGCTGAAGAAGCTGGGAGGGGGCAAACGGGGAGGCGGCAAAAAGATGACCAAC AGCGTGGACGACGCCCTGATCAACAGCACCAAGATCTACAGCTACTTCCCCAGCG TGATCAGCAAAGTGAACCAGGGCGCTCAGGGCAAGAAACTGGGCTCTAGCGGAG GGGGAGGCTCTCCTGGCGGGGGATCTAGCATCGTGGGAATTGTGGCAGGACTGGC AGTGCTGGCCGTGGTGGTGATCGGAGCCGTGGTGGCTACCGTGATGTGCAGACGG AAGTCCAGCGGAGGCAAGGGCGGCAGCTACAGCCAGGCCGCCAGCTCTGATAGC GCCCAGGGCAGCGACGTGTCACTGACAGCCTAGTAACTCGAGCTGGTACTGCATG CACGCAATGCTAGCTGCCCCTTTCCCGTCCTGGGTACCCCGAGTCTCCCCCGACCT CGGGTCCCAGGTATGCTCCCACCTCCACCTGCCCCACTCACCACCTCTGCTAGTTC CAGACACCTCCCAAGCACGCAGCAATGCAGCTCAAAACGCTTAGCCTAGCCACAC CCCCACGGGAAACAGCAGTGATTAACCTTTAGCAATAAACGAAAGTTTAACTAAG CTATACTAACCCCAGGGTTGGTCAATTTCGTGCCAGCCACACCGAGACCTGGTCCA GAGTCGCTAGCCGCGTCGCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGCA TATGACTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO:25) J. RBL003.1 & RBL003.3 (MAGE-A3)

[0164] In some embodiments, an RNA molecule encoding a MAGE-A3 antigen is or comprises the nucleotide sequence of RBL003.1 or RBL003.3. In some embodiments, an RNA molecule encoding a MAGE-A3 antigen comprises a sequence that encodes a polypeptide having the amino acid sequence of RBL003.1 or RBL003.3. In the following, the sequence alignment of RBL003.1 and RBL003.3 is given for both the nucleotide sequences of the full-length RNAs as well as for the translated proteins (with the amino acid positioned below the third nucleotide of the respective codon triplet). Sequence elements as illustrated in Fig.1 are displayed above the nucleotide sequences. Differences in the nucleotide and amino acid sequences are indicated by “*”. SEQ ID NO: 17 for RBL003.1 RNA; SEQ ID NO: 18 12829787v1Attorney Ref.2013237-1447 for RBL003.3 RNA; SEQ ID NO: 19 for RBL003.1 protein; SEQ ID NO: 20 for RBL003.3 protein. [ 5’-UTR ][ sec RBL003.1 (RNA) GGGCGAACTA GTATTCTTCT GGTCCCCACA GACTCAGAGA GAACCCGCCA CCATGAGAGT 60 RBL003.3 (RNA) GGGCGAACTA GTATTCTTCT GGTCCCCACA GACTCAGAGA GAACCCGCCA CCATGAGAGT 60 RBL003.1 (prot.) M R 2 RBL003.3 (prot.) M R 2 sec RBL003.1 (RNA) GACCGCCCCC AGAACCCTGA TCCTGCTGCT GTCTGGCGCC CTGGCCCTGA CAGAGACATG 120 RBL003.3 (RNA) GATGGCCCCC AGAACCCTGA TCCTGCTGCT GTCTGGCGCC CTGGCCCTGA CAGAGACATG 120 ** RBL003.1 (prot.) V T A P R T L I L L L S G A L A L T E T 22 RBL003.3 (prot.) V M A P R T L I L L L S G A L A L T E T 22 * sec ][ GS ][ antigen RBL003.1 (RNA) GGCCGGAAGC CTGCAG GGAGGATCT ATGCCCCTTG AACAGCGCTC 165 RBL003.3 (RNA) GGCCGGAAGC GGCGGCTCTG GAGGAGGCGG CTCCGGAGGC ATGCCCCTTG AACAGCGCTC 180 ********** ********** **** *** RBL003.1 (prot.) W A G S L Q G G S M P L E Q R 37 RBL003.3 (prot.) W A G S G G S G G G G S G G M P L E Q R 42 * * * * * * * * * antigen RBL003.1 (RNA) ACAGCACTGC AAACCTGAGG AGGGCCTTGA AGCAAGGGGC GAAGCTCTGG GGTTGGTCGG 225 RBL003.3 (RNA) ACAGCACTGC AAACCTGAGG AGGGCCTTGA AGCAAGGGGC GAAGCTCTGG GGTTGGTCGG 240 RBL003.1 (prot.) S Q H C K P E E G L E A R G E A L G L V 57 RBL003.3 (prot.) S Q H C K P E E G L E A R G E A L G L V 62 antigenRBL003.1 (RNA) TGCACAAGCA CCCGCCACTG AGGAACAGGA AGCCGCGTCT AGCTCATCAA CCCTGGTTGA 285 RBL003.3 (RNA) TGCACAAGCA CCCGCCACTG AGGAACAGGA AGCCGCGTCT AGCTCATCAA CCCTGGTTGA 300 RBL003.1 (prot.) G A Q A P A T E E Q E A A S S S S T L V 77 RBL003.3 (prot.) G A Q A P A T E E Q E A A S S S S T L V 82 antigen RBL003.1 (RNA) AGTGACACTG GGCGAAGTGC CTGCTGCGGA GAGTCCAGAC CCTCCCCAGT CCCCTCAAGG 345 RBL003.3 (RNA) AGTGACACTG GGCGAAGTGC CTGCTGCGGA GAGTCCAGAC CCTCCCCAGT CCCCTCAAGG 360 RBL003.1 (prot.) E V T L G E V P A A E S P D P P Q S P Q 97 RBL003.3 (prot.) E V T L G E V P A A E S P D P P Q S P Q 102 antigen RBL003.1 (RNA) CGCTTCTAGC CTGCCTACCA CGATGAACTA CCCACTGTGG TCACAGAGCT ATGAGGACAG 405 RBL003.3 (RNA) CGCTTCTAGC CTGCCTACCA CGATGAACTA CCCACTGTGG TCACAGAGCT ATGAGGACAG 420 RBL003.1 (prot.) G A S S L P T T M N Y P L W S Q S Y E D 117 RBL003.3 (prot.) G A S S L P T T M N Y P L W S Q S Y E D 122 antigen RBL003.1 (RNA) TTCCAATCAA GAAGAAGAAG GCCCGTCTAC CTTCCCCGAT CTTGAGTCCG AGTTTCAGGC 465 RBL003.3 (RNA) TTCCAATCAA GAAGAAGAAG GCCCGTCTAC CTTCCCCGAT CTTGAGTCCG AGTTTCAGGC 480 RBL003.1 (prot.) S S N Q E E E G P S T F P D L E S E F Q 137 RBL003.3 (prot.) S S N Q E E E G P S T F P D L E S E F Q 142 12829787v1Attorney Ref.2013237-1447 antigen RBL003.1 (RNA) CGCTCTGTCC CGGAAGGTGG CAGAGCTCGT GCACTTTCTC CTGTTGAAGT ATCGAGCCCG 525 RBL003.3 (RNA) CGCTCTGTCC CGGAAGGTGG CAGAGCTCGT GCACTTTCTC CTGTTGAAGT ATCGAGCCCG 540 RBL003.1 (prot.) A A L S R K V A E L V H F L L L K Y R A 157 RBL003.3 (prot.) A A L S R K V A E L V H F L L L K Y R A 162 antigen RBL003.1 (RNA) GGAGCCTGTC ACTAAGGCCG AAATGCTGGG CTCTGTAGTG GGGAATTGGC AGTATTTCTT 585 RBL003.3 (RNA) GGAGCCTGTC ACTAAGGCCG AAATGCTGGG CTCTGTAGTG GGGAATTGGC AGTATTTCTT 600 RBL003.1 (prot.) R E P V T K A E M L G S V V G N W Q Y F 177 RBL003.3 (prot.) R E P V T K A E M L G S V V G N W Q Y F 182 antigen RBL003.1 (RNA) CCCCGTGATC TTCAGCAAAG CCTCCAGCAG CCTGCAATTG GTGTTCGGTA TTGAACTGAT 645 RBL003.3 (RNA) CCCCGTGATC TTCAGCAAAG CCTCCAGCAG CCTGCAATTG GTGTTCGGTA TTGAACTGAT 660 RBL003.1 (prot.) F P V I F S K A S S S L Q L V F G I E L 197 RBL003.3 (prot.) F P V I F S K A S S S L Q L V F G I E L 202 antigen RBL003.1 (RNA) GGAAGTAGAT CCGATTGGGC ATCTGTACAT CTTTGCGACA TGTCTGGGAC TGTCCTATGA 705 RBL003.3 (RNA) GGAAGTAGAT CCGATTGGGC ATCTGTACAT CTTTGCGACA TGTCTGGGAC TGTCCTATGA 720 RBL003.1 (prot.) M E V D P I G H L Y I F A T C L G L S Y 217 RBL003.3 (prot.) M E V D P I G H L Y I F A T C L G L S Y 222 antigen RBL003.1 (RNA) CGGACTGCTC GGGGATAACC AGATTATGCC GAAAGCCGGT CTGCTGATCA TAGTTCTCGC 765 RBL003.3 (RNA) CGGACTGCTC GGGGATAACC AGATTATGCC GAAAGCCGGT CTGCTGATCA TAGTTCTCGC 780 RBL003.1 (prot.) D G L L G D N Q I M P K A G L L I I V L 237 RBL003.3 (prot.) D G L L G D N Q I M P K A G L L I I V L 242 antigen RBL003.1 (RNA) CATCATTGCC AGAGAGGGAG ATTGTGCTCC AGAGGAGAAG ATCTGGGAGG AATTGTCTGT 825 RBL003.3 (RNA) CATCATTGCC AGAGAGGGAG ATTGTGCTCC AGAGGAGAAG ATCTGGGAGG AATTGTCTGT 840 RBL003.1 (prot.) A I I A R E G D C A P E E K I W E E L S 257 RBL003.3 (prot.) A I I A R E G D C A P E E K I W E E L S 262 antigen RBL003.1 (RNA) GCTGGAGGTC TTTGAGGGTA GGGAGGACAG CATTCTCGGC GATCCCAAGA AACTCCTGAC 885 RBL003.3 (RNA) GCTGGAGGTC TTTGAGGGTA GGGAGGACAG CATTCTCGGC GATCCCAAGA AACTCCTGAC 900 RBL003.1 (prot.) V L E V F E G R E D S I L G D P K K L L 277 RBL003.3 (prot.) V L E V F E G R E D S I L G D P K K L L 282 antigen RBL003.1 (RNA) CCAGCACTTT GTCCAGGAGA ACTACCTCGA ATACAGACAG GTTCCAGGCA GTGACCCTGC 945 RBL003.3 (RNA) CCAGCACTTT GTCCAGGAGA ACTACCTCGA ATACAGACAG GTTCCAGGCA GTGACCCTGC 960 RBL003.1 (prot.) T Q H F V Q E N Y L E Y R Q V P G S D P 297 RBL003.3 (prot.) T Q H F V Q E N Y L E Y R Q V P G S D P 302 antigen RBL003.1 (RNA) TTGCTACGAG TTCCTTTGGG GACCCCGTGC ATTGGTAGAG ACAAGCTATG TCAAAGTGCT 1005 RBL003.3 (RNA) TTGCTACGAG TTCCTTTGGG GACCCCGTGC ATTGGTAGAG ACAAGCTATG TCAAAGTGCT 1020 RBL003.1 (prot.) A C Y E F L W G P R A L V E T S Y V K V 317 RBL003.3 (prot.) A C Y E F L W G P R A L V E T S Y V K V 322 antigen RBL003.1 (RNA) GCACCATATG GTGAAGATAT CTGGAGGACC ACACATCAGT TACCCACCCC TTCATGAGTG 1065 RBL003.3 (RNA) GCACCATATG GTGAAGATAT CTGGAGGACC ACACATCAGT TACCCACCCC TTCATGAGTG 1080 RBL003.1 (prot.) L H H M V K I S G G P H I S Y P P L H E 337 RBL003.3 (prot.) L H H M V K I S G G P H I S Y P P L H E 342 12829787v1Attorney Ref.2013237-1447 antigen ][ GS ][ P2P16 RBL003.1 (RNA) GGTTCTGCGC GAAGGGGAGG AGGGAGGATC CCTGGGAGGC GGGGGAAGCG GCAAGAAGCA 1125 RBL003.3 (RNA) GGTTCTGCGC GAAGGGGAGG AGGGAGGATC CGGTGGTGGC GGCAGCGGCG GCAAGAAGCA 1140 357 362 377 382 397 402 417 422 437 442 457 462 477 482489 D S A Q G S D V S L T A 494 3’-UTR RBL003.1 (RNA) CTGTCCAATT TCTATTAAA GGTTCCT TTGTTCCCTA AGTCCAACTA CTAAACTGGG 1598 RBL003.3 (RNA) ACGCAATGCT AGCTGCCCCT TTCCCGTCCT GGGTACCCCG AGTCTCCCCC GACCTCGGGT 1620 ** ****** ********** ****** ** * ** *** ** ***** * * 3’-UTR RBL003.1 (RNA) GGATATTATG AAGGGCCTTG AGCATCTGGA TTCTGCCTAA TAAAAAACAT TTATTTTCAT 1658 RBL003.3 (RNA) CCCAGGTATG CTCCCACCTC CACCTGCCCC ACTCACCACC TCTGCTAGTT CCAGACACCT 1680 ****** ****** * * ** * ***** ***** *** ***** ** ** **** * 12829787v1Attorney Ref.2013237-1447 3’-UTR RBL003.1 (RNA) TGCTGCGTCG AGAGCTCGCT TTCTTGCTGT CCAATTTCT ATTAAAGGT 1706 RBL003.3 (RNA) CCCAAGCACG CAGCAATGCA GCTCAAAACG CTTAGCCTAG CCACACCCCC ACGGGAAACA 1740 ** ***** ******* * ********** ** ****** ********** ***** *** 3’-UTR RBL003.1 (RNA) TCCTTT GTTCCCT AAGTCCAACT ACTAAACTGG GGGATATTAT GAAGGGCCTT 1759 RBL003.3 (RNA) GCAGTGATTA ACCTTTAGCA ATAAACGAAA GTTTAACTAA GCTATACTAA CCCCAGGGTT 1800 **** ***** **** ** * **** * ** ** * ** ** * * ***** ** (A) 1819 1860poly(A) RBL003.1 (RNA) AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1879 RBL003.3 (RNA) AAAAAAAAAA AAAAAAAAAA AAAAAAAGCA TATGACT AAA AAAAAAAAAA 1910 ** * ** ***** ******* poly(A) ] RBL003.1 (RNA) AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAA 1936 RBL003.3 (RNA) AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAA 1967 K. RBL004.1 & RBL004.3 (TPTE)

[0165] In some embodiments, an RNA molecule encoding a TPTE antigen is or comprises the nucleotide sequence of RBL004.1 or RBL004.3. In some embodiments, an RNA molecule encoding a TPTE antigen comprises a sequence that encodes a polypeptide having the amino acid sequence of RBL004.1 or RBL004.3. In the following, the sequence alignment of RBL004.1 and RBL004.3 is given for both the nucleotide sequences of the full- length RNAs as well as for the translated proteins (with the amino acid positioned below the third nucleotide of the respective codon triplet). Sequence elements as illustrated in Fig.1 are displayed above the nucleotide sequences. Differences in the nucleotide and amino acid sequences are indicated by “*”. SEQ ID NO: 21 for RBL004.1 RNA; SEQ ID NO: 22 for RBL004.3 RNA; SEQ ID NO: 23 for RBL004.1 protein; SEQ ID NO: 24 for RBL004.3 protein. [ 5’-UTR ][ sec RBL004.1 (RNA) GGGCGAACTA GTATTCTTCT GGTCCCCACA GACTCAGAGA GAACCCGCCA CCATGAGAGT 60 RBL004.3 (RNA) GGGCGAACTA GTATTCTTCT GGTCCCCACA GACTCAGAGA GAACCCGCCA CCATGAGAGT 60 RBL004.1 (prot.) M R 2 RBL004.3 (prot.) M R 2 sec RBL004.1 (RNA) GACCGCCCCC AGAACCCTGA TCCTGCTGCT GTCTGGCGCC CTGGCCCTGA CAGAGACATG 120 RBL004.3 (RNA) GATGGCCCCC AGAACCCTGA TCCTGCTGCT GTCTGGCGCC CTGGCCCTGA CAGAGACATG 120 ** RBL004.1 (prot.) V T A P R T L I L L L S G A L A L T E T 22 RBL004.3 (prot.) V M A P R T L I L L L S G A L A L T E T 22 *12829787v1Attorney Ref.2013237-1447 sec ][ GS ][ antigen RBL004.1 (RNA) GGCCGGAAGC CTG CAG ATGAACGAGA GCCCCGACCC 156 RBL004.3 (RNA) GGCCGGAAGC GGCGGCTCTG GAGGAGGCGG CTCCGGAGGC ATGAACGAGA GCCCCGACCC 180 ********** ********** ********** RBL004.1 (prot.) W A G S L Q M N E S P D 34 RBL004.3 (prot.) W A G S G G S G G G G S G G M N E S P D 42 * * * * * * * * * * antigen RBL004.1 (RNA) TACAGATCTG GCCGGCGTGA TCATCGAGCT GGGACCCAAC GATAGCCCTC AGACCAGCGA 216 RBL004.3 (RNA) TACAGATCTG GCCGGCGTGA TCATCGAGCT GGGACCCAAC GATAGCCCTC AGACCAGCGA 240 RBL004.1 (prot.) P T D L A G V I I E L G P N D S P Q T S 54 RBL004.3 (prot.) P T D L A G V I I E L G P N D S P Q T S 62 antigen RBL004.1 (RNA)276 RBL004.3 (RNA) GTTCAAGGGG GCCACAGAGG AAGCCCCTGC CAAAGAGAGC CCCCACACCT CCGAGTTTAA 300 RBL004.1 (prot.) E F K G A T E E A P A K E S P H T S E F 74 RBL004.3 (prot.) E F K G A T E E A P A K E S P H T S E F 82 antigen RBL004.1 (RNA) GGGCGCTGCT CGGGTGTCCC CTATCAGCGA GAGCGTGCTG GCCCGGCTGA GCAAGTTCGA 336 RBL004.3 (RNA) GGGCGCTGCT CGGGTGTCCC CTATCAGCGA GAGCGTGCTG GCCCGGCTGA GCAAGTTCGA 360 RBL004.1 (prot.) K G A A R V S P I S E S V L A R L S K F 94 RBL004.3 (prot.) K G A A R V S P I S E S V L A R L S K F 102antigen RBL004.1 (RNA) 396 RBL004.3 (RNA) GGTGGAGGAC GCCGAGAACG TGGCCAGCTA CGACAGCAAG ATCAAGAAAA TCGTGCACAG 420 RBL004.1 (prot.) E V E D A E N V A S Y D S K I K K I V H 114 RBL004.3 (prot.) E V E D A E N V A S Y D S K I K K I V H 122 antigen RBL004.1 (RNA) CATCGTGTCC AGCTTCGCCT TCGGCCTGTT CGGCGTGTTC CTGGTGCTGC TGGACGTGAC 456 RBL004.3 (RNA) CATCGTGTCC AGCTTCGCCT TCGGCCTGTT CGGCGTGTTC CTGGTGCTGC TGGACGTGAC 480 RBL004.1 (prot.) S I V S S F A F G L F G V F L V L L D V 134 RBL004.3 (prot.) S I V S S F A F G L F G V F L V L L D V 142 antigen RBL004.1 (RNA) ACTGATCCTG GCCGACCTGA TCTTCACCGA CAGCAAGCTG TACATCCCCC TGGAATACCG 516 RBL004.3 (RNA) ACTGATCCTG GCCGACCTGA TCTTCACCGA CAGCAAGCTG TACATCCCCC TGGAATACCG 540 RBL004.1 (prot.) T L I L A D L I F T D S K L Y I P L E Y 154 RBL004.3 (prot.) T L I L A D L I F T D S K L Y I P L E Y 162 antigen RBL004.1 (RNA) GTCCATCAGC CTGGCCATTG CCCTGTTCTT TCTGATGGAC GTGCTGCTGC GGGTGTTCGT 576 RBL004.3 (RNA) GTCCATCAGC CTGGCCATTG CCCTGTTCTT TCTGATGGAC GTGCTGCTGC GGGTGTTCGT 600 RBL004.1 (prot.) R S I S L A I A L F F L M D V L L R V F 174 RBL004.3 (prot.) R S I S L A I A L F F L M D V L L R V F 182antigen RBL004.1 (RNA) GGAGCGGCGG CAGCAGTACT TCAGCGACCT GTTCAACATC CTGGACACCG CCATCATCGT 636 RBL004.3 (RNA) GGAGCGGCGG CAGCAGTACT TCAGCGACCT GTTCAACATC CTGGACACCG CCATCATCGT 660 RBL004.1 (prot.) V E R R Q Q Y F S D L F N I L D T A I I 194 RBL004.3 (prot.) V E R R Q Q Y F S D L F N I L D T A I I 20212829787v1Attorney Ref.2013237-1447 antigen RBL004.1 (RNA) GATTCTGCTG CTGGTGGATG TGGTGTACAT CTTCTTCGAC ATCAAGCTGC TGAGAAACAT 696 RBL004.3 (RNA) GATTCTGCTG CTGGTGGATG TGGTGTACAT CTTCTTCGAC ATCAAGCTGC TGAGAAACAT 720 RBL004.1 (prot.) V I L L L V D V V Y I F F D I K L L R N 214 RBL004.3 (prot.) V I L L L V D V V Y I F F D I K L L R N 222 antigen RBL004.1 (RNA) CCCCCGGTGG ACCCATCTGC TGCGGCTGCT GAGACTGATC ATCCTGCTGC GGATCTTCCA 756 RBL004.3 (RNA) CCCCCGGTGG ACCCATCTGC TGCGGCTGCT GAGACTGATC ATCCTGCTGC GGATCTTCCA 780 RBL004.1 (prot.) I P R W T H L L R L L R L I I L L R I F 234 RBL004.3 (prot.) I P R W T H L L R L L R L I I L L R I F 242 antigen RBL004.1 (RNA) CCTGTTCCAC CAGAAGCGGC AGCTGGAAAA GCTGATCAGA CGGCGGGTGT CCGAGAACAA 816 RBL004.3 (RNA) CCTGTTCCAC CAGAAGCGGC AGCTGGAAAA GCTGATCAGA CGGCGGGTGT CCGAGAACAA 840 RBL004.1 (prot.) H L F H Q K R Q L E K L I R R R V S E N 254 RBL004.3 (prot.) H L F H Q K R Q L E K L I R R R V S E N 262 antigen RBL004.1 (RNA) GCGGCGGTAC ACCAGGGACG GCTTCGACCT GGACCTGACC TACGTGACCG AGCGGATCAT 876 RBL004.3 (RNA) GCGGCGGTAC ACCAGGGACG GCTTCGACCT GGACCTGACC TACGTGACCG AGCGGATCAT 900 RBL004.1 (prot.) K R R Y T R D G F D L D L T Y V T E R I 274 RBL004.3 (prot.) K R R Y T R D G F D L D L T Y V T E R I 282 antigen RBL004.1 (RNA) TGCCATGAGC TTCCCCAGCA GCGGCAGACA GAGCTTCTAC CGGAACCCCA TCAAAGAAGT 936 RBL004.3 (RNA) TGCCATGAGC TTCCCCAGCA GCGGCAGACA GAGCTTCTAC CGGAACCCCA TCAAAGAAGT 960 RBL004.1 (prot.) I A M S F P S S G R Q S F Y R N P I K E 294 RBL004.3 (prot.) I A M S F P S S G R Q S F Y R N P I K E 302 antigen RBL004.1 (RNA) GGTGCGGTTC CTGGACAAGA AGCACCGGAA CCACTACCGG GTGTACAACC TGTGCAGCGA 996 RBL004.3 (RNA) GGTGCGGTTC CTGGACAAGA AGCACCGGAA CCACTACCGG GTGTACAACC TGTGCAGCGA 1020 RBL004.1 (prot.) V V R F L D K K H R N H Y R V Y N L C S 314 RBL004.3 (prot.) V V R F L D K K H R N H Y R V Y N L C S 322 antigen RBL004.1 (RNA) GCGGGCCTAC GACCCCAAGC ACTTCCACAA CCGGGTGGTG CGGATCATGA TCGACGACCA 1056 RBL004.3 (RNA) GCGGGCCTAC GACCCCAAGC ACTTCCACAA CCGGGTGGTG CGGATCATGA TCGACGACCA 1080 RBL004.1 (prot.) E R A Y D P K H F H N R V V R I M I D D 334 RBL004.3 (prot.) E R A Y D P K H F H N R V V R I M I D D 342 antigen RBL004.1 (RNA) CAACGTGCCC ACCCTGCACC AGATGGTGGT GTTCACCAAA GAAGTGAACG AGTGGATGGC 1116 RBL004.3 (RNA) CAACGTGCCC ACCCTGCACC AGATGGTGGT GTTCACCAAA GAAGTGAACG AGTGGATGGC 1140 RBL004.1 (prot.) H N V P T L H Q M V V F T K E V N E W M 354 RBL004.3 (prot.) H N V P T L H Q M V V F T K E V N E W M 362 antigen RBL004.1 (RNA) CCAGGACCTG GAAAACATCG TGGCCATCCA CTGCAAGGGC GGCACCGACA GAACCGGCAC 1176 RBL004.3 (RNA) CCAGGACCTG GAAAACATCG TGGCCATCCA CTGCAAGGGC GGCACCGACA GAACCGGCAC 1200 RBL004.1 (prot.) A Q D L E N I374 RBL004.3 (prot.) A Q D L E N I 382 antigen RBL004.1 (RNA) CATGGTGTGC GCCTTTCTGA TCGCCAGCGA GATCTGTAGC ACCGCCAAAG AGTCCCTGTA 1236 RBL004.3 (RNA) CATGGTGTGC GCCTTTCTGA TCGCCAGCGA GATCTGTAGC ACCGCCAAAG AGTCCCTGTA 1260 RBL004.1 (prot.) T M V C A F L I A S E I C S T A K E S L 394 RBL004.3 (prot.) T M V C A F L I A S E I C S T A K E S L 402 12829787v1Attorney Ref.2013237-1447 antigen RBL004.1 (RNA) CTACTTCGGC GAGCGGAGAA CCGACAAGAC CCACAGCGAG AAGTTCCAGG GCGTGGAGAC 1296 RBL004.3 (RNA) CTACTTCGGC GAGCGGAGAA CCGACAAGAC CCACAGCGAG AAGTTCCAGG GCGTGGAGAC 1320 RBL004.1 (prot.) Y Y F G E R R T D K T H S E K F Q G V E 414 RBL004.3 (prot.) Y Y F G E R R T D K T H S E K F Q G V E 422 antigen RBL004.1 (RNA) ACCCAGCCAG AAAAGATATG TGGCTTACTT CGCCCAGGTG AAGCACCTGT ACAACTGGAA 1356 RBL004.3 (RNA) ACCCAGCCAG AAAAGATATG TGGCTTACTT CGCCCAGGTG AAGCACCTGT ACAACTGGAA 1380 RBL004.1 (prot.) T P S Q K R Y V A Y F A Q V K H L Y N W 434 RBL004.3 (prot.) T P S Q K R Y V A Y F A Q V K H L Y N W 442 antigen RBL004.1 (RNA) CCTGCCCCCC AGACGGATTC TGTTCATCAA GCACTTCATC ATCTACAGCA TCCCCAGATA 1416 RBL004.3 (RNA) CCTGCCCCCC AGACGGATTC TGTTCATCAA GCACTTCATC ATCTACAGCA TCCCCAGATA 1440 RBL004.1 (prot.) N L P P R R I L F I K H F I I Y S I P R 454 RBL004.3 (prot.) N L P P R R I L F I K H F I I Y S I P R 462 antigen RBL004.1 (RNA) CGTGCGGGAC CTGAAGATCC AGATCGAGAT GGAAAAGAAA GTGGTGTTCA GCACCATCTC 1476 RBL004.3 (RNA) CGTGCGGGAC CTGAAGATCC AGATCGAGAT GGAAAAGAAA GTGGTGTTCA GCACCATCTC 1500 RBL004.1 (prot.) Y V R D L K I Q I E M E K K V V F S T I 474 RBL004.3 (prot.) Y V R D L K I Q I E M E K K V V F S T I 482 antigen RBL004.1 (RNA) CCTGGGCAAG TGCAGCGTGC TGGACAACAT CACCACCGAC AAGATCCTGA TCGACGTGTT 1536 RBL004.3 (RNA) CCTGGGCAAG TGCAGCGTGC TGGACAACAT CACCACCGAC AAGATCCTGA TCGACGTGTT 1560 RBL004.1 (prot.) S L G K C S V L D N I T T D K I L I D V 494 RBL004.3 (prot.) S L G K C S V L D N I T T D K I L I D V 502 antigen RBL004.1 (RNA) CGACGGCCTG CCCCTGTACG ACGACGTGAA GGTGCAGTTC TTCTACAGCA ACCTGCCCAC 1596 RBL004.3 (RNA) CGACGGCCTG CCCCTGTACG ACGACGTGAA GGTGCAGTTC TTCTACAGCA ACCTGCCCAC 1620 RBL004.1 (prot.) F D G L P L Y D D V K V Q F F Y S N L P 514 RBL004.3 (prot.) F D G L P L Y D D V K V Q F F Y S N L P 522 antigen RBL004.1 (RNA) CTACTACGAC AATTGCAGCT TCTACTTCTG GCTGCACACC AGCTTCATCG AGAACAACAG 1656 RBL004.3 (RNA) CTACTACGAC AATTGCAGCT TCTACTTCTG GCTGCACACC AGCTTCATCG AGAACAACAG 1680 RBL004.1 (prot.) T Y Y D N C S F Y F W L H T S F I E N N 534 RBL004.3 (prot.) T Y Y D N C S F Y F W L H T S F I E N N 542 antigen RBL004.1 (RNA) GCTGTACCTG CCCAAGAACG AGCTGGACAA CCTGCACAAG CAGAAGGCCA GAAGAATCTA 1716 RBL004.3 (RNA) GCTGTACCTG CCCAAGAACG AGCTGGACAA CCTGCACAAG CAGAAGGCCA GAAGAATCTA 1740 RBL004.1 (prot.) R L Y L P K N E L D N L H K Q K A R R I 554 RBL004.3 (prot.) R L Y L P K N E L D N L H K Q K A R R I 562 antigen RBL004.1 (RNA) CCCCAGCGAC TTCGCCGTGG AGATCCTGTT TGGCGAGAAG ATGACCAGCA GCGACGTGGT 1776 RBL004.3 (RNA) CCCCAGCGAC TTCGCCGTGG AGATCCTGTT TGGCGAGAAG ATGACCAGCA GCGACGTGGT 1800 RBL004.1 (prot.) Y P S D F A V E I L F G E K M T S S D V 574 RBL004.3 (prot.) Y P S D F A V E I L F G E K M T S S D V 582 12829787v1Attorney Ref.2013237-1447 antigen ][ GS ][ P2P16 RBL004.1 (RNA) GGCCGGCAGC GACGGATCCC TGGGAGGCGG GGGAAGCGGC AAGAAGCAGT ACATCAAGGC 1836 RBL004.3 (RNA) GGCCGGCAGC GGAGGATCCG GTGGTGGCGG CAGCGGCGGC AAGAAGCAGT ACATCAAGGC 1860 ********** * ****** ** ***** * ***** ********** ********** RBL004.1 (prot.) V A G S D G S L G G G G S G K K Q Y I K 594 RBL004.3 (prot.) V A G S G G S G G G G S G G K K Q Y I K 602 * * * * P2P16 RBL004.1 (RNA) CAACAGCAAG TTCATCGGCA TCACCGAGCT GAAGAAGCTG GGAGGGGGCA AACGGGGAGG 1896 RBL004.3 (RNA) CAACAGCAAG TTCATCGGCA TCACCGAGCT GAAGAAGCTG GGAGGGGGCA AACGGGGAGG 1920 RBL004.1 (prot.) A N S K F I G I T E L K K L G G G K R G 614 RBL004.3 (prot.) A N S K F I G I T E L K K L G G G K R G 622 P2P16 RBL004.1 (RNA) CGGCAAAAAG ATGACCAACA GCGTGGACGA CGCCCTGATC AACAGCACCA AGATCTACAG 1956 RBL004.3 (RNA) CGGCAAAAAG ATGACCAACA GCGTGGACGA CGCCCTGATC AACAGCACCA AGATCTACAG 1980 RBL004.1 (prot.) G G K K M T N S V D D A L I N S T K I Y 634 RBL004.3 (prot.) G G K K M T N S V D D A L I N S T K I Y 642 P2P16 ][ GS RBL004.1 (RNA) CTACTTCCCC AGCGTGATCA GCAAAGTGAA CCAGGGCGCT CAGGGCAAGA AACTGGGCTC 2016 RBL004.3 (RNA) CTACTTCCCC AGCGTGATCA GCAAAGTGAA CCAGGGCGCT CAGGGCAAGA AACTGGGCTC 2040 RBL004.1 (prot.) S Y F P S V I S K V N Q G A Q G K K L G 654 RBL004.3 (prot.) S Y F P S V I S K V N Q G A Q G K K L G 662GS][ MITD RBL004.1 (RNA) 2076 RBL004.3 (RNA) TAGCGGAGGG GGAGGCTCTC CTGGCGGGGG ATCTAGCATC GTGGGAATTG TGGCAGGACT 2100 RBL004.1 (prot.) S S G G G G S P G G G S S I V G I V A G 674 RBL004.3 (prot.) S S G G G G S P G G G S S I V G I V A G 682 MITD RBL004.1 (RNA) GGCAGTGCTG GCCGTGGTGG TGATCGGAGC CGTGGTGGCT ACCGTGATGT GCAGACGGAA 2136 RBL004.3 (RNA) GGCAGTGCTG GCCGTGGTGG TGATCGGAGC CGTGGTGGCT ACCGTGATGT GCAGACGGAA 2160 RBL004.1 (prot.) L A V L A V V V I G A V V A T V M C R R 694 RBL004.3 (prot.) L A V L A V V V I G A V V A T V M C R R 702 MITD RBL004.1 (RNA) GTCCAGCGGA GGCAAGGGCG GCAGCTACAG CCAGGCCGCC AGCTCTGATA GCGCCCAGGG 2196 RBL004.3 (RNA) GTCCAGCGGA GGCAAGGGCG GCAGCTACAG CCAGGCCGCC AGCTCTGATA GCGCCCAGGG 2220 RBL004.1 (prot.) K S S G G K G G S Y S Q A A S S D S A Q 714 RBL004.3 (prot.) K S S G G K G G S Y S Q A A S S D S A Q 722 MITD ][ 3’-UTR RBL004.1 (RNA) CAGCGACGTG TCACTGACAG CC TGACT CGAGAGCTCG CTTTCTTGCT GTCCAATTTC 2253 RBL004.3 (RNA) CAGCGACGTG TCACTGACAG CCTAGTAACT CGAGCTGGTA CTGCATGCAC GCAATGCTAG 2280 *** * ****** *** **** ****** ** RBL004.1 (prot.) G S D V S L T A 722 RBL004.3 (prot.) G S D V S L T A 730 3’-UTR RBL004.1 (RNA) TATTAAA GGTTCCTTT GTTCCCTAAG TCCAACTACT AAACTGGGGG ATATTATGAA 2309 RBL004.3 (RNA) CTGCCCCTTT CCCGTCCTGG GTACCCCGAG TCTCCCCCGA CCTCGGGTCC CAGGTATGCT 2340 ********** **** ** * ** *** **** *** * *** **** ** 3’-UTR RBL004.1 (RNA) GGGCCTTGAG CATCTGGATT CTGCCTAATA AAAAACATTT ATTTTCATTG CTGCGTCGAG 2369 RBL004.3 (RNA) CCCACCTCCA CCTGCCCCAC TCACCACCTC TGCTAGTTCC AGACACCTCC CAAGCACGCA 2400 **** * *** * ******* *** *** * **** ** ** **** * ** ***** ** 12829787v1Attorney Ref.2013237-1447 3’-UTR RBL004.1 (RNA) AGCTCGCTTT CTTGCTGTCC AATTTCT A TTAAAGGTTC CTTT 2411 RBL004.3 (RNA) GCAATGCAGC TCAAAACGCT TAGCCTAGCC ACACCCCCAC GGGAAACAGC AGTGATTAAC 2460 ***** *** ******** * * ******** ********** *** ***** ** ******* 3’-UTR RBL004.1 (RNA) GTTCCCTAA GTCCAACTAC TAAACTGGGG GATATTATGA AGGGCCTTGA GCATCTGGAT 2470 RBL004.3 (RNA) CTTTAGCAAT AAACGAAAGT TTAACTAAGC TATACTAACC CCAGGGTTGG TCAATTTCGT 2520 ** ** * * *** * **** * ** * * * *** *** ** * * ** *** 3’-UTR ][ poly(A) RBL004.1 (RNA) TCTGCCTAAT AAAAAACATT TATTTTCATT GCTGCGTCGA GAGCTAAAAA AAAAAAAAAA 2530 RBL004.3 (RNA) GCCAGCCACA CCGAGACCTG GTCCAGAGTC GCTAGCCGCG TCGCTAAAAA AAAAAAAAAA 2580 * *** * ** *** * * * ******** * ******* ** poly(A) RBL004.1 (RNA) AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 2590 RBL004.3 (RNA) AAAAAAAAAA AAAAAGCATA TGACT AAAAA AAAAAAAAAA AAAAAAAAAA 2630 ** * ** ******* ***** poly(A) ] RBL004.1 (RNA) AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAA 2635 RBL004.3 (RNA) AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAA 2675 IV. Exemplary manufacturing processes

[0166] Individual RNA molecules can be produced by methods known in the art. For example, in some embodiments, single-stranded RNAs can be produced by in vitro transcription, for example, using a DNA template. A plasmid DNA used as a template for in vitro transcription to generate an RNA molecule described herein is also within the scope of the present disclosure.

[0167] A DNA template is used for in vitro RNA synthesis in the presence of an appropriate RNA polymerase (e.g., a recombinant RNA-polymerase such as a T7 RNA- polymerase) with ribonucleotide triphosphates (e.g., ATP, CTP, GTP, UTP). In some embodiments, RNA molecules (e.g., ones described herein) can be synthesized in the presence of modified ribonucleotide triphosphates. By way of example only, in some embodiments, N1-methylpseudouridine triphosphate (m1ΨTP) can be used to replace uridine triphosphate (UTP). As will be clear to those skilled in the art, during in vitro transcription, an RNA polymerase (e.g., as described and / or utilized herein) typically traverses at least a portion of a single-stranded DNA template in the 3'→ 5' direction to produce a single- stranded complementary RNA in the 5'→ 3' direction.

[0168] In some embodiments where an RNA molecule comprises a polyA tail, one of those skill in the art will appreciate that such a polyA tail may be encoded in a DNA template, e.g., by using an appropriately tailed PCR primer, or it can be added to an RNA molecule after in vitro transcription, e.g., by enzymatic treatment (e.g., using a poly(A) polymerase such as an E. coli Poly(A) polymerase). 12829787v1Attorney Ref.2013237-1447

[0169] In some embodiments, those skilled in the art will appreciate that addition of a 5' cap to an RNA (e.g., mRNA) can facilitate recognition and attachment of the RNA to a ribosome to initiate translation and enhances translation efficiency. Those skilled in the art will also appreciate that a 5' cap can also protect an RNA product from 5' exonuclease mediated degradation and thus increases half-life. Methods for capping 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 such as, e.g., capping enzymes of vaccinia virus). In some embodiments, a cap may be introduced during in vitro transcription, along with a plurality of ribonucleotide triphosphates such that a cap is incorporated into an RNA molecule ssRNA during transcription (also known as co-transcriptional capping).

[0170] Following RNA transcription, a DNA template is digested. In some embodiments, digestion can be achieved with the use of DNase I under appropriate conditions.

[0171] In some embodiments, RNA molecules can be purified after in vitro transcription reaction, for example, to remove components utilized or formed in the course of the production, like, e.g., proteins, DNA fragments, and / or or nucleotides. Various nucleic acid purifications that are known in the art can be used in accordance with the present disclosure. In some embodiments, RNA molecules may be purified using magnetic bead-based purification, which in some embodiments may be or comprise magnetic bead-based chromatography. In some embodiments, RNA molecules may be purified using hydrophobic interaction chromatography (HIC) followed by diafiltration.

[0172] In some embodiments, dsRNA may be obtained as side product during in vitro transcription. In some such embodiments, a second purification step may be performed to remove dsRNA contamination. For example, in some embodiments, cellulose materials (e.g., microcrystalline cellulose) may be used to remove dsRNA contamination, for examples in some embodiments in a chromatographic format. In some embodiments, cellulose materials (e.g., microcrystalline cellulose) can be pretreated to inactivate potential RNase contamination, for example in some embodiments by autoclaving followed by incubation with aqueous basic solution, e.g., NaOH. In some embodiments, cellulose materials may be used to purify RNA molecules according to methods described in WO 2017 / 182524, the entire content of which is incorporated herein by reference.

[0173] In some embodiments, a batch of ssRNAs may be further processed by one or more steps of filtration and / or concentration. For example, in some embodiments, RNA molecules, for example, after removal of dsRNA contamination, may be further subject to 12829787v1Attorney Ref.2013237-1447 diafiltration, for example, to adjust the concentration of ssRNAs to a desirable RNA concentration and / or to exchange buffer to a drug substance buffer.

[0174] In some embodiments, RNA molecules may be processed through 0.2 μm filtration before they are filled into appropriate containers.

[0175] In some embodiments, RNA quality control may be performed and / or monitored at any time during production process of RNA molecules and / or compositions comprising the same. For example, in some embodiments, RNA quality control parameters may be assessed and / or monitored after each or certain steps of RNA molecules manufacturing process, e.g., after in vitro transcription, and / or each purification step.

[0176] In some embodiments, one or more assessments may be utilized during manufacture, or other preparation or use of RNA molecules (e.g., as a release test).

[0177] In some embodiments, one or more quality control parameters may be assessed to determine whether RNA molecules described herein meet or exceed pre-determined acceptance criteria (e.g., for subsequent formulation and / or release for distribution). In some embodiments, such quality control parameters may include, but are not limited to RNA integrity, RNA concentration, residual DNA template and / or residual dsRNA. Methods for assessing RNA quality are known in the art.

[0178] In some embodiments, a batch of RNA molecules may be assessed for one or more features to determine next action step(s). For example, a batch of single stranded RNAs can be designated for one or more further steps of manufacturing and / or formulation and / or distribution if RNA quality assessment indicates that such a batch of single stranded RNAs meet or exceed the acceptance criteria. Otherwise, an alternative action can be taken (e.g., discarding the batch) if such a batch of single stranded RNAs does not meet or exceed the acceptance criteria.

[0179] In some embodiments, a batch of RNA molecules with exemplary assessment results can be utilized for one or more further steps of manufacturing and / or formulation and / or distribution. V. RNA delivery technologies

[0180] Provided compositions (e.g., including one or more molecules of RNA encoding one or more TAAs) may be delivered for therapeutic applications described herein using any appropriate methods known in the art, including, e.g., delivery as naked RNAs, or delivery mediated by viral and / or non-viral vectors, polymer-based vectors, lipid-based vectors, nanoparticles (e.g., lipid nanoparticles, polymeric nanoparticles, lipid-polymer hybrid nanoparticles, etc.), and / or peptide-based vectors. See, e.g., Wadhwa et al. “Opportunities and 12829787v1Attorney Ref.2013237-1447 Challenges in the Delivery of mRNA-Based Vaccines” Pharmaceutics (2020) 102 (27 pages), the content of which is incorporated herein by reference, for information on various approaches that may be useful for delivery RNA molecules described herein.

[0181] In some embodiments, one or more RNA molecules can be formulated with lipid particles for delivery (e.g., in some embodiments by intravenous injection).

[0182] In some embodiments, lipid particles can be designed to protect RNA molecules (e.g., mRNA) from extracellular RNases and / or engineered for systemic delivery of the RNA to target cells (e.g., dendritic cells). In some embodiments, such lipid particles may be particularly useful to deliver RNA molecules (e.g., mRNA) when RNA molecules are intravenously administered to a subject in need thereof.

[0183] In some embodiments, lipid particles comprise liposomes. In some embodiments, lipid particles comprise cationic liposomes.

[0184] In some embodiments, lipid particles comprise lipid nanoparticles.

[0185] In some embodiments, lipid particles comprise lipoplexes.

[0186] In some embodiments, lipid particles comprise N,N,N trimethyl-2-3-dioleyloxy-1- propanaminium chloride (DOTMA), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine phospholipid (DOPE), or both. In some embodiments, lipid particles comprise at least one ionizable aminolipid. In some embodiments, lipid particles comprise at least one ionizable aminolipid and a helper lipid. In some embodiments, a helper lipid is or comprises a phospholipid. In some embodiments, a helper lipid is or comprises a sterol. In some embodiments, lipid particles comprises at least one polymer-conjugated lipid.

[0187] RNA lipoplex particles: In some embodiments, RNA molecules described herein may be delivered by liposomal formulations. In some embodiments, negatively charged RNA molecules described herein are complexed with cationic liposomes to form RNA lipoplex particles. In some embodiments, RNA molecules described herein are embedded in a (phospho)lipid bilayer structure within an RNA lipoplex particle. In some embodiments, cationic liposomes can comprise a cationic lipid or an ionizable aminolipid (e.g., ones as described herein) and optionally an additional or helper lipid (e.g., at least one neutral lipid as described herein) to form injectable particle formulations.

[0188] In some embodiments, RNA lipoplex particles may be prepared by mixing liposomes with RNA molecules described herein. In some embodiments, liposomes may be obtained by injecting a solution of lipids in ethanol into water or a suitable aqueous phase. In some embodiments, cationic liposomes are stabilized in an aqueous formulation, e.g., as described in WO 2016 / 046060, the entire content of which is incorporated herein by 12829787v1Attorney Ref.2013237-1447 reference for the purposes described herein. In some embodiments, cationic liposomes may be produced by a method, e.g., as described in WO 2019 / 077053, the entire content of which is incorporated herein by reference for the purposes described herein.

[0189] In some embodiments, spleen targeting RNA lipoplex particles that are useful for delivering RNA molecules described herein are described in WO 2013 / 143683, the entire content of which is incorporated herein by reference for the purposes described herein. In some embodiments, RNA molecules and positively charged liposomes are mixed such that cationic lipids and RNA are present at a charge ratio of 1.3:2. Such charge ratio is determined to effectively target RNA to the spleen.

[0190] In some embodiments, an RNA lipoplex particle comprises a cationic lipid or an ionizable aminolipid (e.g., ones described herein) and an RNA molecule described herein. In some embodiments, such an RNA lipoplex particle may further comprise an additional or helper lipid (e.g., ones described herein). Without wishing to be bound by theory, electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles.

[0191] In some embodiments where a cationic lipid or an ionizable aminolipid (e.g., ones described herein) and a helper lipid are used, such a cationic lipid or an ionizable aminolipid and such a helper lipid may be present in a molar ratio of 2:1. In some embodiments, a cationic lipid or an ionizable aminolipid may be or comprise DOTMA. In some embodiments, a helper lipid may be or comprise a neutral lipid. In some embodiments, a neutral lipid may be or comprise DOPE.

[0192] In some embodiments, RNA lipoplex particles are nanoparticles. In some embodiments, RNA lipoplex nanoparticles can have a particle size (e.g., Z-average) of about 100 nm to 1000 nm or about 200 nm to 900 nm or about 200 nm to 800 nm, or about 250 nm to about 700 nm.

[0193] RNA lipid nanoparticles: In some embodiments, RNA molecules described herein may be delivered by lipid nanoparticle formulations. In some embodiments, RNA lipid nanoparticles may be prepared by mixing lipids with RNA molecules described herein. In some embodiments, at least a portion of RNA molecules are encapsulated by lipid nanoparticles. In some embodiments, at least 90% or higher (including, e.g., at least 95%, 96%, 97%, 98%, 99%, or higher) of RNA molecules are encapsulated by lipid nanoparticles.

[0194] In various embodiments, lipid nanoparticles can have an average size (e.g., Z- average) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm. In some embodiments, lipid nanoparticles can have a 12829787v1Attorney Ref.2013237-1447 particle size (e.g., Z-average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, an average size of lipid nanoparticles is determined by measuring the particle diameter.

[0195] In certain embodiments, RNA molecules (e.g., mRNAs), when present in provided lipid nanoparticles, are resistant in aqueous solution to degradation with a nuclease.

[0196] In some embodiments, lipid nanoparticles are cationic lipid nanoparticles comprising one or more cationic lipids (e.g., ones described herein). In some embodiments, cationic lipid nanoparticles 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). A. Helper lipids

[0197] In some embodiments, a lipid particle for delivery of RNA molecules described herein comprises at least one helper lipid, which may be a neutral lipid, a positively charged lipid, or a negatively charged lipid. In some embodiments, a helper lipid is a lipid that are useful for increasing the effectiveness of delivery of lipid-based particles such as cationic lipid-based particles to a target cell. In some embodiments, a helper lipid may be or comprise a structural lipid with its concentration chosen to optimize particle size, stability, and / or encapsulation.

[0198] In some embodiments, a lipid particle for delivery of RNA molecules described herein comprises a neutral helper lipid. Examples of such neutral helper lipids include, but are not limited to phosphotidylcholines such as 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dimyristoyl-sn-glycero- 3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), l ,2- dioleoyl-sn-glycero-3-phosphocholine (DOPC), phophatidylethanolamines such as 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), sphingomyelins (SM), ceramides, cholesterol, steroids such as sterols and their derivatives. Neutral lipids may be synthetic or naturally derived. Other neutral helper lipids that are known in the art, e.g., as described in WO 2017 / 075531 and WO 2018 / 081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein, can also be used in lipid particles described herein. In some embodiments, a lipid particle for delivery of RNA molecules described herein comprises DSPC and / or cholesterol. 12829787v1Attorney Ref.2013237-1447

[0199] In some embodiments, a lipid particle for delivery of RNA molecules described herein comprises at least one helper lipids (e.g., ones described herein). In some such embodiments, a lipid particle may comprise DOPE. B. Cationic lipids

[0200] In some embodiments, a lipid particle for delivery of RNA molecules described herein comprises a cationic lipid. A cationic lipid is typically a lipid having a net positive charge, for example in some embodiments at a certain pH. In some embodiments, a cationic lipid may comprise one or more amine group(s) which bear a positive charge. In some embodiments, a cationic lipid may comprise a cationic, meaning positively charged, headgroup. In some embodiments, a cationic lipid may 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, a cationic lipid comprises a polar headgroup, which in some embodiments may comprise one or more amine derivatives such as primary, secondary, and / or tertiary amines, quaternary ammonium, various combinations of amines, amidinium salts, or guanidine and / or imidazole groups as well as pyridinium, piperizine and amino acid headgroups such as lysine, arginine, ornithine and / or tryptophan. In some embodiments, a polar headgroup of a cationic lipid comprises one or more amine derivatives. In some embodiments, a polar headgroup of a cationic lipid comprises a quaternary ammonium. In some embodiments, a headgroup of a cationic lipid may comprise multiple cationic charges. In some embodiments, a headgroup of a cationic lipid comprises one cationic charge. Examples of monocationic lipids include, but are not limited to l,2- dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1 ,2-di-O-octadecenyl- 3 - trimethylammonium propane (DOTMA) and / or 1 ,2-dioleoyl-3-trimethylammonium propane (DOTAP), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 2,3- di(tetradecoxy)propyl-(2-hydroxyethyl)-dimethylazanium bromide (DMRIE), didodecyl(dimethyl)azanium bromide (DDAB), l ,2-dioleyloxypropyl-3 -dimethyl - hydroxyethyl ammonium bromide (DORIE), 3P-[N-(N\N'-dimethylamino- ethane)carbamoyl]cholesterol (DC-Choi) and / or dioleyl ether phosphatidylcholine (DOEPC).

[0201] In some embodiments, a positively charged lipid structure described herein may also include one or more other components that may be typically used in the formation of vesicles (e.g. for stabilization). Examples of such other components includes, without being limited thereto, fatty alcohols, fatty acids, and / or cholesterol esters or any other pharmaceutically acceptable excipients which may affect the surface charge, the membrane fluidity and assist in the incorporation of the lipid into the lipid assembly. Examples of sterols 12829787v1Attorney Ref.2013237-1447 include cholesterol, cholesteryl hemisuccinate, cholesteryl sulfate, or any other derivatives of cholesterol. In some embodiments, a one cationic lipid comprises DMEPC and / or DOTMA. In some embodiments, a cationic lipid comprises DOTMA.

[0202] In some embodiments, a cationic lipid is ionizable such that it can exist in a positively charged form or neutral form depending on pH. For example, in some embodiments, a cationic lipid is an ionizable aminolipid. Such ionization of a cationic lipid can affect the surface charge of the lipid particle under different pH conditions, which in some embodiments may influence plasma protein absorption, blood clearance, and / or tissue distribution as well as the ability to form endosomolytic non-bilayer structures. Accordingly, in some embodiments, a cationic lipid may be or comprise a pH responsive lipid. In some embodiments a pH responsive lipid is a fatty acid derivative or other amphiphilic compound which is capable of forming a lyotropic lipid phase, and which has a pKa value between pH 5 and pH 7.5. This means that the lipid is uncharged at a pH above the pKa value and positively charged below the pKa value. In some embodiments, a pH responsive lipid may be used in addition to or instead of a cationic lipid for example by binding one or more RNA molecules to a lipid or lipid mixture at low pH. pH responsive lipids include, but are not limited to, 1,2- dioieyioxy-3 -dimethylamino-propane (DODMA).

[0203] In some embodiments, a lipid particle may comprise one or more cationic lipids as described in WO 2017 / 075531 (e.g., as presented in Tables 1 and 3 therein) and WO 2018 / 081480 (e.g., as presented in Tables 1-4 therein), the entire contents of each of which are incorporated herein by reference for the purposes described herein.

[0204] In some embodiments, a cationic lipid that may be useful in accordance with the present disclosure is an amino lipid comprising a titratable tertiary amino head group linked via ester bonds to at least two saturated alkyl chains, which ester bonds can be hydrolyzed easily to facilitate fast degradation and / or excretion via renal pathways. In some embodiments, such an amino lipid has an apparent pKa of about 6.0-6.5 (e.g., in one embodiment with an apparent pKa of approximately 6.25), resulting in an essentially fully positively charged molecule at an acidic pH (e.g., pH 5). In some embodiments, such an amino lipid, when incorporated in a lipid particle, can confer distinct physicochemical properties that regulate particle formation, cellular uptake, fusogenicity and / or endosomal release of RNA molecules. In some embodiments, introduction of an aqueous RNA solution to a lipid mixture comprising such an amino lipid at pH 4.0 can lead to an electrostatic interaction between the negatively charged RNA backbone and the positively charged cationic lipid. Without wishing to be bound by any particular theory, such electrostatic 12829787v1Attorney Ref.2013237-1447 interaction leads to particle formation coincident with efficient encapsulation of RNA drug substance. After RNA encapsulation, adjustment of the pH of the medium surrounding the resulting lipid nanoparticles to a more neutral pH (e.g., pH 7.4) results in neutralization of the surface charge of the lipid nanoparticles. When all other variables are held constant, such charge-neutral particles display longer in vivo circulation lifetimes and better delivery to hepatocytes compared to charged particles, which are rapidly cleared by the reticuloendothelial system. Upon endosomal uptake, the low pH of the endosome renders lipid nanoparticle comprising such an amino lipid fusogenic and allows the release of the RNA into the cytosol of the target cell.

[0205] Cationic lipids may 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. C. Polymer-conjugated lipids

[0206] In some embodiments, a lipid nanoparticle for use in delivery of RNA molecules described herein may comprise at least one polymer-conjugated lipid. A polymer-conjugated lipid is typically a molecule comprising a lipid portion and a polymer portion conjugated thereto.

[0207] In some embodiments, a polymer-conjugated lipid is a PEG-conjugated lipid. In some embodiments, a PEG-conjugated lipid is designed to sterically stabilize a lipid particle by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. In some embodiments, a PEG-conjugated lipid can reduce its association with serum proteins and / or the resulting uptake by the reticuloendothelial system when such lipid particles are administered in vivo.

[0208] Various PEG-conjugated lipids are known in the art and include, but are not limited to pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy- polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG), a pegylated phosphatidylethanolamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2' ,3 '-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as ω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or 2,3- di(tetradecanoxy)propyl-N-(ω methoxy(polyethoxy)ethyl)carbamate, and the like.

[0209] Certain PEG-conjugated lipids (also known as PEGylated lipids) were clinically approved with safety demonstrated in clinical trials. PEG-conjugated lipids are known to 12829787v1Attorney Ref.2013237-1447 affect cellular uptake, a prerequisite to endosomal localization and payload delivery. The pharmacology of encapsulated nucleic acid can be controlled in a predictable manner by modulating the alkyl chain length of a PEG-lipid anchor. In some embodiments, PEG- conjugated lipids may be designed and / or selected based on reasonable solubility characteristics and / or its molecular weight to effectively perform the function of a steric barrier. For example, in some embodiments, a PEGylated lipid does not show appreciable surfactant or permeability enhancing or disturbing effects on biological membranes. In some embodiments, PEG in such a PEG-conjugated lipid can be linked to diacyl lipid anchors with a biodegradable amide bond, thereby facilitating fast degradation and / or excretion. In some embodiments, a LNP comprising a PEG-conjugated lipid retain a full complement of a PEGylated lipid. In the blood compartment, such a PEGylated lipid dissociates from the particle over time, revealing a more fusogenic particle that is more readily taken up by cells, ultimately leading to release of the RNA payload.

[0210] In some embodiments, a lipid particle (e.g., a lipid nanoparticle) may comprise one or more PEG-conjugated lipids or pegylated lipids as described in WO 2017 / 075531 and WO 2018 / 081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein. For example, in some embodiments, a PEG-conjugated lipid that may be useful in accordance with the present disclosure can have a structure as described in WO 2017 / 075531, or athereof, wherein: R8 and R9 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 bonds; and w has a mean value ranging from 30 to 60. In some embodiments, R8 and R9 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In some embodiments, w has a mean value ranging from 43 to 53. In other embodiments, the average w is about 45.

[0211] In some embodiments, lipids that form lipid nanoparticles described herein comprise: a polymer-conjugated lipid; a cationic lipid; and a helper neutral lipid. In some such embodiments, total polymer-conjugated lipid may be present in about 0.5-5 mol%, about 12829787v1Attorney Ref.2013237-1447 0.7-3.5 mol%, about 1-2.5 mol%, about 1.5-2 mol%, or about 1.5-1.8 mol% of the total lipids. In some embodiments, total polymer-conjugated lipid may be present in about 1-2.5 mol% of the total lipids. 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.

[0212] In some embodiments involving a polymer-conjugated lipid, a cationic lipid, and a helper neutral lipid in lipid nanoparticles described herein, total cationic lipid is present in about 35-65 mol%, about 40-60 mol%, about 41-49 mol%, about 41-48 mol%, about 42-48 mol%, about 43-48 mol%, about 44-48 mol%, about 45-48 mol%, about 46-48 mol%, or about 47.2-47.8 mol% of the total lipids.

[0213] In some embodiments involving a polymer-conjugated lipid, a cationic lipid, and a helper neutral lipid in lipid nanoparticles described herein, total neutral lipid is present in about 35-65 mol%, about 40-60 mol%, about 45-55 mol%, or about 47-52 mol% of the total lipids. In some embodiments, total neutral lipid is present in 35-65 mol% of the total lipids. In some embodiments, total non-steroid neutral lipid (e.g., DPSC) is present in about 5-15 mol%, about 7-13 mol%, or 9-11 mol% of the total lipids. In some embodiments, total non- steroid neutral lipid is present in about 9.5, 10 or 10.5 mol% of the total lipids. In some embodiments, the molar ratio of the total cationic lipid to the non-steroid neutral lipid ranges from about 4.1: 1.0 to about 4.9: 1.0, from about 4.5: 1.0 to about 4.8: 1.0, or from about 4.7: 1.0 to 4.8: 1.0. In some embodiments, total steroid neutral lipid (e.g., cholesterol) is present in about 35- 50 mol%, about 39-49 mol%, about 40-46 mol%, about 40- 44 mol%, or about 40- 42 mol% of the total lipids. In certain embodiments, total steroid neutral lipid (e.g., cholesterol) is present in about 39, 40, 41, 42, 43, 44, 45, or 46 mol% of the total lipids. 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.

[0214] In some embodiments, a lipid composition comprising a cationic lipid, a polymer- conjugated lipid, and a neutral lipid can have individual lipids present in certain molar percents of the total lipids, or in certain molar ratios (relative to each other) as described in WO 2018 / 081480, the entire contents of each of which are incorporated herein by reference for the purposes described herein. VI. Pharmaceutical compositions

[0215] The present disclosure provides, among other things, pharmaceutical compositions for delivering antigens (e.g., TAA) to a patient. In some embodiments, a pharmaceutical composition comprises one or more RNA molecules encoding a NY-ESO-1 antigen, a 12829787v1Attorney Ref.2013237-1447 MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof; and lipid particles (e.g., lipoplexes or lipid nanoparticles). In some embodiments, a pharmaceutical composition comprises one or more RNA molecules collectively encoding a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, and a TPTE antigen; and lipid particles (e.g., lipoplexes or lipid nanoparticles). In some embodiments, a pharmaceutical composition comprises at least four populations of RNA-lipid particles (e.g., lipoplexes or lipid nanoparticles), wherein each RNA-lipid particle comprises an RNA molecule and a lipid particle, and wherein the RNA molecules of each of the four RNA lipid particles is different, e.g. each RNA encodes a distinct TAA as described herein. In some embodiments, a pharmaceutical composition comprises RNA molecules encoding a NY-ESO-1 antigen, RNA molecules encoding a MAGE-A3 antigen, RNA molecules encoding a tyrosinase antigen, and RNA molecules encoding a TPTE antigen. In some embodiments, the present disclosure provides separate pharmaceutical compositions, where each pharmaceutical composition individually comprises RNA molecules encoding a NY-ESO-1 antigen, RNA molecules encoding a MAGE-A3 antigen, RNA molecules encoding a tyrosinase antigen, or RNA molecules encoding a TPTE antigen, respectively. In some embodiments, the present disclosure provides a first pharmaceutical composition comprising RNA molecules encoding a NY-ESO-1 antigen, a second pharmaceutical composition comprising RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition comprising RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical composition comprising RNA molecules encoding a TPTE antigen.

[0216] In some embodiments, one or more RNA molecules may be formulated with lipid nanoparticles (e.g., ones described herein) for administration to a patient. Accordingly, in some embodiments, a pharmaceutical composition comprises one or more RNA molecules encoding a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof; and lipid particles (e.g., lipoplexes or lipid nanoparticles), wherein the one or more RNA molecules are encapsulated with the lipid particles (e.g., form an RNA- lipid particle). In some embodiments, an RNA-lipid particle is an RNA-lipoplex particle. In some embodiments, an RNA-lipid particle is an RNA-lipid nanoparticles. In some embodiments, the disclosure provides a first pharmaceutical composition comprising RNA molecules encoding a NY-ESO-1 antigen; a second pharmaceutical composition comprising RNA molecules encoding a MAGE-A3 antigen; a third pharmaceutical composition comprising RNA molecules encoding a tyrosinase antigen; a fourth pharmaceutical composition comprising RNA molecules encoding a TPTE antigen; and lipid particles (e.g., 12829787v1Attorney Ref.2013237-1447 lipoplexes or lipid nanoparticles), wherein the RNA molecules are encapsulated with the lipid particles (e.g., form an RNA-lipid particle). In some embodiments, an RNA-lipid particle is an RNA-lipoplex particle. In some embodiments, an RNA-lipid particle is an RNA-lipid nanoparticles.

[0217] In some embodiments, a pharmaceutical composition is administered as a monotherapy. In some embodiments, a pharmaceutical composition is administered as part of a combination therapy.

[0218] In some embodiments, a pharmaceutical composition comprises a first RNA molecule encoding a NY-ESO-1 antigen, a second RNA molecule encoding a MAGE-A3, a third RNA molecule encoding a tyrosinase antigen, and a fourth RNA molecule encoding a TPTE antigen, a first RNA molecule, a second RNA molecule, a third RNA molecule, and a fourth RNA molecule may be present in the pharmaceutical composition in about equimolar amounts (e.g., a molar ratio of about 1:1:1:1). In some embodiments, the present disclosure provides a first pharmaceutical composition comprising RNA molecules encoding a NY- ESO-1 antigen, a second pharmaceutical composition comprising RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition comprising RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical composition comprising RNA molecules encoding a TPTE antigen, and the pharmaceutical compositions are administered in about equimolar amounts (e.g., a molar ratio of 1:1:1:1).

[0219] In some embodiments, a concentration of total RNA (e.g., a total concentration of all of the one or more RNA molecules) in a pharmaceutical composition described herein is of about 0.01 mg / mL to about 0.5 mg / mL, or about 0.05 mg / mL to about 0.1 mg / mL.

[0220] Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference in its entirety) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure. 12829787v1Attorney Ref.2013237-1447

[0221] In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by the United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or the International Pharmacopoeia.

[0222] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and / or granulating agents, surface active agents and / or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and / or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and / or perfuming agents can be present in the composition, according to the judgment of the formulator.

[0223] General considerations in the formulation and / or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[0224] In some embodiments, pharmaceutical compositions provided herein may be formulated with one or more pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference in its entirety).

[0225] Pharmaceutical compositions described herein can be administered by appropriate methods known in the art. As will be appreciated by a skilled artisan, the route and / or mode of administration may depend on a number of factors, including, e.g., but not limited to stability and / or pharmacokinetics and / or pharmacodynamics of pharmaceutical compositions described herein.

[0226] In some embodiments, pharmaceutical compositions described herein are formulated for parenteral administration, which includes modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.

[0227] In some embodiments, pharmaceutical compositions described herein are formulated for intravenous administration. In some embodiments, pharmaceutically 12829787v1Attorney Ref.2013237-1447 acceptable carriers that may be useful for intravenous administration include sterile aqueous solutions or dispersions and sterile powders for preparation of sterile injectable solutions or dispersions.

[0228] In some particular embodiments, pharmaceutical compositions described herein are formulated for subcutaneous administration. In some particular embodiments, pharmaceutical compositions described herein are formulated for intramuscular administration.

[0229] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, dispersion, powder (e.g., lyophilized powder), microemulsion, lipid nanoparticles, or other ordered structure suitable to high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The 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 dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as 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 an agent that delays absorption, for example, monostearate salts and gelatin.

[0230] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.

[0231] In some embodiments, dispersions are 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) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0232] Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by 12829787v1Attorney Ref.2013237-1447 the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0233] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into pharmaceutical compositions described herein. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.

[0234] Formulations of pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing active ingredient(s) into association with a diluent or another excipient and / or one or more other accessory ingredients, and then, if necessary and / or desirable, shaping and / or packaging the product into a desired single- or multi-dose unit.

[0235] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of at least one RNA product produced using a system and / or method described herein.

[0236] Relative amounts of one or more RNA molecules encapsulated in LNPs, a pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition can vary, depending upon the subject to be treated, target cells, diseases or disorders, and may also further depend upon the route by which the composition is to be administered.

[0237] In some embodiments, pharmaceutical compositions described herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients (e.g., one or more RNA molecules encapsulated in lipid nanoparticles) in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular 12829787v1Attorney Ref.2013237-1447 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 materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0238] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian could start doses of active ingredients (e.g., one or more RNA molecules encapsulated in lipid nanoparticles) employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. For example, exemplary doses as described in the present Examples may be used in preparing pharmaceutically acceptable dosage forms.

[0239] In some embodiments, a pharmaceutical composition is formulated (e.g., for intravenous administration) to deliver a dose of about 1 µg to about 500 µg (or any of the subranges included therein) of total RNA, e.g., as described in the present Examples.

[0240] In some embodiments, a pharmaceutical composition described herein may further comprise one or more additives, for example, in some embodiments that may enhance stability of such a composition under certain conditions. Examples of additives may include but are not limited to salts, buffer substances, preservatives, and carriers. For example, in some embodiments, a pharmaceutical composition may further comprise a cryoprotectant (e.g., sucrose) and / or an aqueous buffered solution, which may in some embodiments include one or more salts, including, e.g., alkali metal salts or alkaline earth metal salts such as, e.g., sodium salts, potassium salts, and / or calcium salts.

[0241] Exemplary formulations include, but are not limited to those listed in Table 3. Table 3: Exemplary pharmaceutical composition formulations Exemplary Formula 1 Exemplary Formula 212829787v1Attorney Ref.2013237-1447 NaCl 6.50 1.20 Sucrose - 220.0tyrosinase antigen, and a fourth RNA molecule encoding a TPTE antigen.

[0243] In some embodiments, a pharmaceutical composition described herein can be frozen to allow long-term storage.

[0244] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and / or perform such modification with merely ordinary, if any, experimentation.

[0245] To ensure appropriate quality of useful components (e.g., one or more RNA molecules(s) collectively encoding (i) a New York esophageal squamous cell carcinoma (NY- ESO-1) antigen, (ii) a melanoma-associated antigen A3 (MAGE-A3) antigen, (iii) a tyrosinase antigen, (iv) a transmembrane phosphatase with tensin homology (TPTE) antigen, or (v) a combination thereof) in pharmaceutical compositions described herein, one or more quality assessments and / or criteria (e.g., RNA quality assessments) may be performed and / or monitored.

[0246] Among other things, the present disclosure provides methods of characterizing one or more features of one or more RNA molecules or composition described herein.

[0247] In some embodiments, RNA integrity assessment of one or more RNA molecules (e.g., in some embodiments a pharmaceutical composition comprising one or more RNA molecules collectively encoding a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof can be performed by adaptation of a capillary gel electrophoresis assay.

[0248] Additionally or alternatively, in some embodiments, RNA ratio of a pharmaceutical composition comprising one or more one or more RNA molecules each 12829787v1Attorney Ref.2013237-1447 encoding (a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof can be measured by droplet digital PCR.

[0249] Additionally or alternatively, in some embodiments, residual DNA template and residual dsRNA are measured as in-process controls with acceptance criteria on the level of the drug substance intermediates to ensure individual RNA quality before mixing to the drug substance, for example, before mixing two or more one or more RNA molecules each encoding different TAA or combinations of TAA (e.g., a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof).

[0250] Additionally or alternatively, in some embodiments, residual host cell DNA and / or host cell protein may be measured in compositions comprising RNA molecules. VII. Checkpoint inhibitors

[0251] In some embodiments, methods of the disclosure include use of one or more immune checkpoint inhibitors (also referred to as a “checkpoint inhibitor”) in combination with one or more RNA molecules described herein, e.g., one or more mRNA molecules described herein. In some embodiments, an exemplary immune checkpoint inhibitor may be or comprise an immune checkpoint inhibitor indicated for treatment of cancer (e.g., melanoma), including, for example, but not limited to a PD-1 inhibitor, a PD-L1 inhibitor, a PD-L2 inhibitor, a CTLA4 inhibitor, LAG-3, or a combination thereof. In some embodiments, an immune checkpoint inhibitor is an antibody. Checkpoint inhibitors can include, for example, without limitation, those listed in Table 4. Table 4: Exemplary immune checkpoint molecules and inhibitors of those checkpoint molecules Checkpoint Inhibitor12829787v1Attorney Ref.2013237-1447 Checkpoint Inhibitor Molecule ,12829787v1Attorney Ref.2013237-1447 Checkpoint Inhibitor Molecule )

[0252] In some embodiments, one or more RNA molecules that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen is used in combination with cemiplimab. Cemiplimab is a high-affinity IgG4P human antibody to the PD-1 receptor (PDCD1, CD279) that blocks PD-1 / PD-L1-mediated T cell inhibition. Cemiplimab (LIBTAYO®) is approved in several countries worldwide for the 12829787v1Attorney Ref.2013237-1447 treatment of advanced cutaneous squamous cell carcinoma (CSCC), basal cell carcinoma (BCC), cervical cancer, and non- small cell lung cancer (NSCLC). The clinical activity of cemiplimab has been shown in multiple cancer indications. VIII. Combination therapy

[0253] In some embodiments, one or more RNA molecules described herein are administered to a subject in combination with one or more additional therapies, e.g., one or more additional cancer therapies, e.g., one or more checkpoint inhibitors. For example, one or more RNA molecules that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen are administered to a subject in combination with cemiplimab. In some embodiments, combined administration of one or more RNA molecules described herein and a checkpoint inhibitor, e.g., cemiplimab, results in an improvement in melanoma or a symptom thereof to an extent that is greater than one produced by either the one or more RNA molecules or the checkpoint inhibitor alone. The difference between the combined effect and the effect of each agent alone can be a statistically significant difference.

[0254] In some embodiments, one or more RNA molecules described herein may be administered in combination with radiotherapy and / or autologous peripheral stem cell or bone marrow transplantation.

[0255] In some embodiments, one or more RNA molecules described herein may administered in combination with a signal transduction inhibitor. In some embodiments, a signal transduction inhibitor can include a BRAF inhibitor (e.g., vemurafenib or dabrafenib). In some embodiments, a signal transduction inhibitor can include a MEK inhibitor.

[0256] In some embodiments, one or more RNA molecules described herein may be administered in combination with a intralesional therapy (e.g., talimogene laherparepvec).

[0257] In some embodiments, one or more RNA molecules described herein may be administered in combination with a cytotoxic therapy (e.g., IL-2, dacarbazine, carboplatin / paclitaxel, albumin-bound paclitaxel). IX. Patient populations

[0258] Technologies provided herein can be useful for treatment of diseases or conditions associated with cancer. In some embodiments, technologies provided herein can be useful for treatment of diseases and conditions associated with an epithelial cancer.

[0259] One type of cancer for which technologies described herein can be useful in treating is melanoma. Melanoma is a malignant tumor of melanocytes. Melanomas can arise 12829787v1Attorney Ref.2013237-1447 in the skin, but they can also arise from mucosal surfaces or at other sites to which neural crest cells migrate, including the uveal tract. (Kuk et al.2016, which is incorporated herein by reference in its entirety). Mucosal and uveal melanomas differ significantly from cutaneous melanoma in incidence, prognostic factors, molecular characteristics, and treatment (van der Kooij et al.2019, which is incorporated herein by reference in its entirety).

[0260] In the United States, it is estimated that in 2021 approximately 106,110 patients will be diagnosed with melanoma of the skin and there will be approximately 7,180 deaths (Siegel et al.2021, which is incorporated herein by reference in its entirety). Although the age-standardized incidence rate of melanoma is lower when compared to non-melanoma skin cancer (3.4 vs.11.0 per 100,000 in 2020, respectively), it has a high mortality rate (Globocan 2020; Coricovac et al.2018, each of which is incorporated herein by reference in its entirety). Invasive melanoma represents about 1% of skin cancers, but results in the most deaths caused by skin cancers (ACS 2021, which is incorporated herein by reference in its entirety).

[0261] The outcome of melanoma can depend on the stage at presentation. The 5-year survival for patients with early stage disease (e.g., localized) is approximately 99% of patients and for patients with regional stage (e.g., with spread to lymph nodes) 66% of patients. However, the 5-year survival for patients with distant disease is only approximately 27% (SEER CRS 2021; Swetter et al.2021, each of which is incorporated herein by reference in its entirety).

[0262] In some embodiments, technologies provided herein can be useful for treatment of melanoma. In some embodiments, technologies provided herein can be useful for treatment of cutaneous melanoma. In some embodiments, technologies provided herein can be useful for treatment of advanced stage cancer (e.g., melanoma). Examples of advanced stage cancer include, without limitation, Stage II, Stage III or Stage IV. In some embodiments, technologies provided herein can be useful for treatment of diseases or conditions associated Stage IIIB, Stage IIIC, or Stage IV melanoma.

[0263] In some embodiments, technologies provided herein can be useful for treatment of patients (e.g., adult patients) with melanoma that is metastatic.

[0264] In some embodiments, technologies provided herein can be useful for treatment of patients (e.g., adult patients) with melanoma that is unresectable, e.g., in some embodiments where surgical resection is likely to result in severe morbidity.

[0265] In some embodiments, technologies provided herein can be useful for treatment of patients (e.g., adult patients) with melanoma that are locally advanced. Additionally or alternatively, in some embodiments, cancer in such patients may have progressed following 12829787v1Attorney Ref.2013237-1447 treatment or such cancer patients may have no satisfactory alternative therapy. In some embodiments, patients who are receiving a treatment described herein may have received other cancer therapy, e.g., but not limited to chemotherapy.

[0266] In some embodiments, technologies provided herein can be useful for treatment of advanced melanoma. In some embodiments, technologies provided herein can be useful for treatment of checkpoint-inhibitor (CPI)-experienced patients with unresectable melanoma. In some embodiments, a subject who is administered a pharmaceutical composition described herein may have received a prior anti-cancer therapy. Examples of prior anti-cancer therapies include but are not limited to chemotherapy, interferons and interleukins, monoclonal antibodies, protein kinase inhibitors, radiotherapy, immune checkpoint inhibitors, or combinations thereof. For example, in some embodiments, a subject who is administered a pharmaceutical composition described herein may have received an immune checkpoint inhibitor but did not experience tumor regression. In another example, in some embodiments, a subject who is administered a pharmaceutical composition described herein may have received an immune checkpoint inhibitor and experienced tumor regression. In another example, in some embodiments, a subject who is administered a pharmaceutical composition described herein has received an immune checkpoint inhibitor and experienced tumor regression for a duration, followed by a duration during which the subject experienced refractory melanoma. Examples of such immune checkpoint inhibitors include, but are not limited to a PD-1 inhibitor, a PDL-1 inhibitor, a CTLA-4 inhibitor, or a combination thereof. In some embodiments, an immune checkpoint inhibitor is an antibody (e.g., but not limited to, ipilumumab and nivolumab). Additional examples of checkpoint inhibitors are included in Table 4. In some embodiments, one or more RNA molecules that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen are administered to a subject suffering from PD-1 / PD-L1 inhibitor- refractory / relapsed, unresectable Stage III or IV melanoma. In some embodiments, one or more RNA molecules that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen are administered in combination with a checkpoint inhibitor (e.g., cemiplimab) to a subject suffering from PD-1 / PD-L1 inhibitor-refractory / relapsed, unresectable Stage III or IV melanoma.

[0267] In some embodiments, a patient who meets one or more of the disease-specific inclusion criteria as described in Example 3 are amenable to treatment described herein (e.g., receiving a provided pharmaceutical composition as monotherapy or as part of a combination therapy). In some embodiments, such a patient that is administered a treatment described 12829787v1Attorney Ref.2013237-1447 herein may further meets one or more of the other inclusive criteria as described in Example 3. For example, one or more RNA molecules that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen are administered in combination with a checkpoint inhibitor (e.g., cemiplimab) to a subject, where the subject (a) is (or is determined to be) suffering from a histologically confirmed unresectable stage III or IV metastatic cutaneous melanoma (e.g., measured by RECIST 1.1) after receiving prior treatment with an anti-PD-1 therapy (e.g., where anti-PD-1 therapy was administered at approved doses for at least 12 consecutive weeks; and / or wherein the progression of disease occurred while receiving treatment with approved anti-PD-1 therapy or within 6 months of discontinuing anti-PD-1 therapy, regardless of any intervening therapy); and optionally (b) the subject exhibits an Eastern Cooperative Oncology Group (ECOG) performance status (PS) ≤ 1 and / or the subject exhibits a serum lactate dehydrogenase (LDH) ≤ ULN.

[0268] In some embodiments, a cancer patient who has melanoma but meets one or more of the exclusion criteria as described in Example 3 is not administered a treatment described herein. X. Readouts of patients having been administered a pharmaceutical composition in combination with cemiplimab

[0269] In some embodiments, administering one or more RNA molecules that collectively encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof, in combination with cemiplimab induces an immune response. In some embodiments, the methods described herein further comprise determining a level of immune response in the patient administered the pharmaceutical composition in combination with cemiplimab. For example, in some embodiments, determining a level of the immune response in the patient occurs before and after administration of the pharmaceutical composition and cemiplimab.

[0270] In some embodiments, exploiting the enhanced glucose consumption of following administration of the pharmaceutical composition and cemiplimab can be performed by [18F]-fluoro-2-deoxy-2-d-glucose (FDG)-positron emission tomography (PET) / computerized tomography (CT) scans of the spleen can be carried following administration of the pharmaceutical composition. Without wishing to be bound by theory, the (FDG)-(PET) / (CT) scans are used to indicate targeting and at least transient activation of lymphoid tissue- resident immune cells. In some embodiments, a level of immune response in the patient is determined using an interferon-γ enzyme-linked immune absorbent spot (ELISpot) assay. In 12829787v1Attorney Ref.2013237-1447 some embodiments, a level of metabolic activity in the patient’s spleen is measured using positron emission tomography (PET), computerized tomography (CT) scans, magnetic resonance imaging (MRI), or a combination thereof. In some embodiments, a level of metabolic activity in the patient’s spleen is measured using positron emission tomography (PET) and computerized tomography (CT) scans. In some embodiments, a level of metabolic activity in the patient’s spleen is measured using positron emission tomography (PET) and magnetic resonance imaging (MRI).

[0271] In some embodiments, a level of the immune response is a de novo immune response induced by a pharmaceutical composition in combination with cemiplimab. In some embodiments, a de novo immune response is an immune response that has developed in response to a pharmaceutical composition. In some embodiments, a de novo immune response does not include a background or pre-existing level of the immune response.

[0272] In some embodiments, administering a pharmaceutical composition comprising one or more RNA molecules that collectively encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof, to the patient (e.g., patient is classified as having no evidence of disease at the time of administration) induces an adaptive immune response. For example, in some embodiments, an immune response in the patient is a T cell response, where the T cell response includes a CD4+ and / or CD8+ T cell response. In some embodiments, administering a pharmaceutical composition comprising one or more RNA molecules that collectively encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof, to the patient induces CD4+ and / or CD8+ T cell immunity.

[0273] In some embodiments, the methods described herein include determining a level of immune response in a patient by measuring an amount of one or more cytokines in the patient’s plasma. For example, 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 there) can be used to determine the level of the immune response in the 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.

[0274] In some embodiments, the methods described herein include measuring a number of cancer lesions in the patient. For example, in some embodiments, the methods described herein include measuring a number of cancer lesions in the patient before and after administration of the pharmaceutical composition. In some embodiments, administering a 12829787v1Attorney Ref.2013237-1447 pharmaceutical composition comprising one or more RNA molecules that collectively encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof, to the patient (e.g., patient is classified as having no evidence of disease at the time of administration) reduces the number of cancer lesions, as compared to number cancer lesions in the patient before administration of the pharmaceutical composition.

[0275] In some embodiments, the methods described herein include measuring a number of T cells induced by the pharmaceutical composition in the patient. For example, in some embodiments, the methods described herein include measuring the number of T cells induced by the pharmaceutical composition in the patient at a plurality of time points following administration of the pharmaceutical composition. In another example, the methods described herein include measuring the number of T cells induced by the pharmaceutical composition in the patient following administration of a first dose the pharmaceutical composition and following administration of a second dose the pharmaceutical composition. In some embodiments, the number of T cells induced by the pharmaceutical composition in the patient is greater following administration of the second dose of the pharmaceutical composition than following administration of the first dose of the pharmaceutical composition.

[0276] In some embodiments, the methods described herein include determining a phenotype of T cells induced by the pharmaceutical composition in the patient following administration of the pharmaceutical composition. For example, in some embodiments, following administration of the pharmaceutical composition, at least a subset of T cells induced by the pharmaceutical composition in the patient have a T-helper-1 phenotype. In some embodiments, the phenotype of the T cells induced by the pharmaceutical composition in the patient have a PD1+ effector memory phenotype. In some embodiments, the phenotype of the T cells induced by the pharmaceutical composition in the patient have a T-helper-1 and PD1+ effector memory phenotype.

[0277] In some embodiments, the methods described herein include, for a patient classified as having evidence of disease, measuring the size of one or more cancer lesions in the patient. For example, in some embodiments, the methods described herein include measuring the size of one or more cancer lesions in the patient before and after administration of the pharmaceutical composition. In some embodiments, administering a pharmaceutical composition comprising one or more RNA molecules that collectively encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof, to the patient maintains or reduces the size of one or more cancer lesions, as compared to size of one or more cancer lesions in the patient before administration of the 12829787v1Attorney Ref.2013237-1447 pharmaceutical composition. In other words, the size of one or more cancer lesions does not increase after administration of a pharmaceutical composition described herein.

[0278] In some embodiments, the methods described herein include, for a patient classified as having evidence of disease, monitoring a duration of progression-free survival. In some embodiments, the methods described herein include comparing the duration of progression-free survival of the patient with than a reference duration of progression-free survival. In some embodiments, a reference duration of progression-free survival is an average duration of progression-free survival of a plurality of comparable patients who have not received a pharmaceutical composition described herein. In some embodiments, duration of progression-free survival of the patient is longer in time than a reference duration of progression-free survival. In some embodiments, duration of progression-free survival of the patient is 1%, 5%, 10%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, or more, longer in time than a reference duration of progression-free survival.

[0279] In some embodiments, the methods described herein include, for a patient classified as having evidence of disease, measuring a duration of disease stabilization. In some embodiments, disease stabilization is determined by applying an irRECIST or RECIST 1.1 standard. In some embodiments, a method described herein comprises comparing the duration of disease stabilization of the patient to a reference duration of disease stabilization. In some embodiments, a reference duration of disease stabilization is an average duration of disease stabilization of a plurality of comparable patients who have not received the pharmaceutical composition. In some embodiments, a patient administered a pharmaceutical composition described herein exhibits an increased duration of disease stabilization compared to the reference duration of disease stabilization.

[0280] In some embodiments, the methods described herein include, for a patient classified as having evidence of disease, measuring a duration of tumor responsiveness. In some embodiments, tumor responsiveness is determined by applying an irRECIST or RECIST 1.1 standard. In some embodiments, a method described herein comprises comparing the duration of tumor responsiveness of the patient to a reference duration of tumor responsiveness. In some embodiments, a reference duration of tumor responsiveness is an average duration of tumor responsiveness of a plurality of comparable patients who have not received the pharmaceutical composition. In some embodiments, a patient administered a pharmaceutical composition described herein exhibits an increased duration of tumor responsiveness compared to the reference duration of tumor responsiveness. 12829787v1Attorney Ref.2013237-1447

[0281] In some embodiments, the methods described herein include, for a patient classified as having no evidence of disease, monitoring a duration of disease-free survival. In some embodiments, a method described herein comprises comparing the duration disease- free survival of the patient to a reference duration of disease-free survival. In some embodiments, a duration of disease-free survival in a patient administered a pharmaceutical composition described herein exhibits longer in time than a reference duration of disease-free survival. In some embodiments, a reference duration of disease-free survival is an average duration of disease-free survival of a plurality of comparable patients who have not received the pharmaceutical composition. In some embodiments, a patient administered a pharmaceutical composition described herein exhibits an increased duration of disease-free survival compared to the reference duration of disease-free survival.

[0282] In some embodiments, the methods described herein include, for a patient classified as having no evidence of disease, measuring a duration to disease relapse. In some embodiments, disease relapse is determined by applying an irRECIST or RECIST 1.1 standard. In some embodiments, methods described herein comprise comparing the duration to disease relapse of the patient to a reference duration to disease relapse. In some embodiments, a reference duration to disease relapse is an average duration to disease relapse of a plurality of comparable patients who have not received the pharmaceutical composition. In some embodiments, a patient administered a pharmaceutical composition described herein exhibits an increased duration to disease relapse compared to the reference duration to disease relapse. XI. Readouts of patients having been administered a pharmaceutical composition

[0283] In some embodiments, administering one or more RNA molecules that collectively encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof, induces an immune response. In some embodiments, the methods described herein further comprise determining a level of immune response in the patient administered the pharmaceutical composition. For example, in some embodiments, determining a level of the immune response in the patient occurs before and after administration of the pharmaceutical composition.

[0284] In some embodiments, exploiting the enhanced glucose consumption of following administration of the pharmaceutical composition can be performed by [18F]-fluoro-2-deoxy- 2-d-glucose (FDG)-positron emission tomography (PET) / computerized tomography (CT) scans of the spleen can be carried following administration of the pharmaceutical composition. Without wishing to be bound by theory, the (FDG)-(PET) / (CT) scans are used 12829787v1Attorney Ref.2013237-1447 to indicate targeting and at least transient activation of lymphoid tissue-resident immune cells. In some embodiments, a level of immune response in the patient is determined using an interferon-γ enzyme-linked immune absorbent spot (ELISpot) assay. In some embodiments, a level of metabolic activity in the patient’s spleen is measured using positron emission tomography (PET), computerized tomography (CT) scans, magnetic resonance imaging (MRI), or a combination thereof. In some embodiments, a level of metabolic activity in the patient’s spleen is measured using positron emission tomography (PET) and computerized tomography (CT) scans. In some embodiments, a level of metabolic activity in the patient’s spleen is measured using positron emission tomography (PET) and magnetic resonance imaging (MRI).

[0285] In some embodiments, a level of the immune response is a de novo immune response induced by a pharmaceutical composition. In some embodiments, a de novo immune response is an immune response that has developed in response to a pharmaceutical composition. In some embodiments, a de novo immune response does not include a background or pre-existing level of the immune response.

[0286] In some embodiments, administering a pharmaceutical composition comprising one or more RNA molecules that collectively encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof, to the patient (e.g., patient is classified as having no evidence of disease at the time of administration) induces an adaptive immune response. For example, in some embodiments, an immune response in the patient is a T cell response, where the T cell response includes a CD4+ and / or CD8+ T cell response. In some embodiments, administering a pharmaceutical composition comprising one or more RNA molecules that collectively encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof, to the patient induces CD4+ and / or CD8+ T cell immunity.

[0287] In some embodiments, the methods described herein include determining a level of immune response in a patient by measuring an amount of one or more cytokines in the patient’s plasma. For example, 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 there) can be used to determine the level of the immune response in the 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. 12829787v1Attorney Ref.2013237-1447

[0288] In some embodiments, the methods described herein include measuring a number of cancer lesions in the patient. For example, in some embodiments, the methods described herein include measuring a number of cancer lesions in the patient before and after administration of the pharmaceutical composition. In some embodiments, administering a pharmaceutical composition comprising one or more RNA molecules that collectively encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof, to the patient (e.g., patient is classified as having no evidence of disease at the time of administration) reduces the number of cancer lesions, as compared to number cancer lesions in the patient before administration of the pharmaceutical composition.

[0289] In some embodiments, the methods described herein include measuring a number of T cells induced by the pharmaceutical composition in the patient. For example, in some embodiments, the methods described herein include measuring the number of T cells induced by the pharmaceutical composition in the patient at a plurality of time points following administration of the pharmaceutical composition. In another example, the methods described herein include measuring the number of T cells induced by the pharmaceutical composition in the patient following administration of a first dose the pharmaceutical composition and following administration of a second dose the pharmaceutical composition. In some embodiments, the number of T cells induced by the pharmaceutical composition in the patient is greater following administration of the second dose of the pharmaceutical composition than following administration of the first dose of the pharmaceutical composition.

[0290] In some embodiments, the methods described herein include determining a phenotype of T cells induced by the pharmaceutical composition in the patient following administration of the pharmaceutical composition. For example, in some embodiments, following administration of the pharmaceutical composition, at least a subset of T cells induced by the pharmaceutical composition in the patient have a T-helper-1 phenotype. In some embodiments, the phenotype of the T cells induced by the pharmaceutical composition in the patient have a PD1+ effector memory phenotype. In some embodiments, the phenotype of the T cells induced by the pharmaceutical composition in the patient have a T-helper-1 and PD1+ effector memory phenotype.

[0291] In some embodiments, the methods described herein include, for a patient classified as having evidence of disease, measuring the size of one or more cancer lesions in the patient. For example, in some embodiments, the methods described herein include measuring the size of one or more cancer lesions in the patient before and after administration of the pharmaceutical composition. In some embodiments, administering a pharmaceutical 12829787v1Attorney Ref.2013237-1447 composition comprising one or more RNA molecules that collectively encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof, to the patient maintains or reduces the size of one or more cancer lesions, as compared to size of one or more cancer lesions in the patient before administration of the pharmaceutical composition. In other words, the size of one or more cancer lesions does not increase after administration of a pharmaceutical composition described herein.

[0292] In some embodiments, the methods described herein include, for a patient classified as having evidence of disease, monitoring a duration of progression-free survival. In some embodiments, the methods described herein include comparing the duration of progression-free survival of the patient with than a reference duration of progression-free survival. In some embodiments, a reference duration of progression-free survival is an average duration of progression-free survival of a plurality of comparable patients who have not received a pharmaceutical composition described herein. In some embodiments, duration of progression-free survival of the patient is longer in time than a reference duration of progression-free survival. In some embodiments, duration of progression-free survival of the patient is 1%, 5%, 10%, 15%, 18%, 20%, 25%, 30%, 35%, 40%, or more, longer in time than a reference duration of progression-free survival.

[0293] In some embodiments, the methods described herein include, for a patient classified as having evidence of disease, measuring a duration of disease stabilization. In some embodiments, disease stabilization is determined by applying an irRECIST or RECIST 1.1 standard. In some embodiments, a method described herein comprises comparing the duration of disease stabilization of the patient to a reference duration of disease stabilization. In some embodiments, a reference duration of disease stabilization is an average duration of disease stabilization of a plurality of comparable patients who have not received the pharmaceutical composition. In some embodiments, a patient administered a pharmaceutical composition described herein exhibits an increased duration of disease stabilization compared to the reference duration of disease stabilization.

[0294] In some embodiments, the methods described herein include, for a patient classified as having evidence of disease, measuring a duration of tumor responsiveness. In some embodiments, tumor responsiveness is determined by applying an irRECIST or RECIST 1.1 standard. In some embodiments, a method described herein comprises comparing the duration of tumor responsiveness of the patient to a reference duration of tumor responsiveness. In some embodiments, a reference duration of tumor responsiveness is an average duration of tumor responsiveness of a plurality of comparable patients who have 12829787v1Attorney Ref.2013237-1447 not received the pharmaceutical composition. In some embodiments, a patient administered a pharmaceutical composition described herein exhibits an increased duration of tumor responsiveness compared to the reference duration of tumor responsiveness.

[0295] In some embodiments, the methods described herein include, for a patient classified as having no evidence of disease, monitoring a duration of disease-free survival. In some embodiments, a method described herein comprises comparing the duration disease- free survival of the patient to a reference duration of disease-free survival. In some embodiments, a duration of disease-free survival in a patient administered a pharmaceutical composition described herein exhibits longer in time than a reference duration of disease-free survival. In some embodiments, a reference duration of disease-free survival is an average duration of disease-free survival of a plurality of comparable patients who have not received the pharmaceutical composition. In some embodiments, a patient administered a pharmaceutical composition described herein exhibits an increased duration of disease-free survival compared to the reference duration of disease-free survival.

[0296] In some embodiments, the methods described herein include, for a patient classified as having no evidence of disease, measuring a duration to disease relapse. In some embodiments, disease relapse is determined by applying an irRECIST or RECIST 1.1 standard. In some embodiments, methods described herein comprise comparing the duration to disease relapse of the patient to a reference duration to disease relapse. In some embodiments, a reference duration to disease relapse is an average duration to disease relapse of a plurality of comparable patients who have not received the pharmaceutical composition. In some embodiments, a patient administered a pharmaceutical composition described herein exhibits an increased duration to disease relapse compared to the reference duration to disease relapse. XII. Treatment

[0297] In some embodiments, pharmaceutical compositions described herein can be taken up by target cells (e.g., dendritic cells) for translation of antigen-encoding RNA thereby inducing CD4+ and CD8+ T cell immunity against the antigens.

[0298] Accordingly, another aspect of the present disclosure relates to methods of using pharmaceutical compositions described herein. For example, one aspect provided herein is a method comprising administering a provided pharmaceutical composition to a subject suffering from cancer. In some embodiments, a provided pharmaceutical composition is administered by intravenous injection or infusion. Examples of a cancer include but are not limited to a epithelial cancer, including, but not limited to, melanoma (e.g., cutaneous 12829787v1Attorney Ref.2013237-1447 melanoma, Stage IIIB, Stage IIIC, or Stage IV melanoma). A specific cancer is a PD-1 / PD- L1 inhibitor-refractory / relapsed, unresectable Stage III or IV melanoma.

[0299] Dosing schedule: Those skilled in the art are aware that cancer therapeutics are often administered using varying ranges of a pharmaceutical composition that can be administered in dosing cycles.

[0300] In some embodiments, one or more RNA molecules described herein that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen (e.g., a first pharmaceutical composition comprising RNA molecules encoding a NY-ESO-1 antigen, a second pharmaceutical composition comprising RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition comprising RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical composition comprising RNA molecules encoding a TPTE antigen) are administered using a prime-and-boost protocol.

[0301] In some embodiments, one or more RNA molecules described herein that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen (e.g., a first pharmaceutical composition comprising RNA molecules encoding a NY-ESO-1 antigen, a second pharmaceutical composition comprising RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition comprising RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical composition comprising RNA molecules encoding a TPTE antigen) are administered in one or more dosing cycles. In some embodiments, a checkpoint inhibitor described herein (e.g., cemiplimab) is administered in one or more dosing cycles.

[0302] In some embodiments, one dosing cycle is at least 6 or more days (including, e.g., at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, 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 21 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.

[0303] In some embodiments, one dosing cycle may involve multiple doses, e.g., according to a pattern such as, for example, a dose may be administered periodically within a 12829787v1Attorney Ref.2013237-1447 cycle, or a 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 within a cycle. In some embodiments, one dosing cycle may involve at least 2 doses, including, e.g., 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 combinations thereof. In some embodiments, one 3-week dosing cycle includes 3 doses of one or more RNA molecules described herein (administered concurrently or sequentially) that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen (e.g., a first pharmaceutical composition comprising RNA molecules encoding a NY-ESO-1 antigen, a second pharmaceutical composition comprising RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition comprising RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical composition comprising RNA molecules encoding a TPTE antigen), where one dose is administered once weekly for 3 weeks. In some embodiments, one 3-week dosing cycle includes (a) 3 doses of one or more RNA molecules described herein (administered concurrently or sequentially) that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen (e.g., a first pharmaceutical composition comprising RNA molecules encoding a NY-ESO-1 antigen, a second pharmaceutical composition comprising RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition comprising RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical composition comprising RNA molecules encoding a TPTE antigen), where one dose is administered once weekly for 3 weeks; and (b) one dose of checkpoint inhibitor (e.g., cemiplimab).

[0304] In some embodiments, multiple cycles may be administered. For example, in some embodiments, at least 2 cycles (including, e.g., at least 3 cycles, at least 4 cycles, at least 5 cycles, at least 6 cycles, at least 7 cycles, at least 8 cycles, at least 9 cycles, at least 10 cycles, or more) can be administered. In some embodiments, the number of dosing cycles to be administered may vary with types of treatment (e.g., monotherapy vs. combination therapy). In some embodiments, at least 2 dosing cycles may be administered. In some embodiments, a first dosing cycle can be different from a second dosing cycle. In some embodiments, a first dosing cycle may comprise 6-8 weekly and / or biweekly doses, and a second dosing cycle that follows the first dosing cycle may comprise at least one monthly dose. 12829787v1Attorney Ref.2013237-1447

[0305] In some embodiments, a first 3-week dosing cycle includes 3 doses of one or more RNA molecules described herein (administered concurrently or sequentially) that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen (e.g., a first pharmaceutical composition comprising RNA molecules encoding a NY-ESO-1 antigen, a second pharmaceutical composition comprising RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition comprising RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical composition comprising RNA molecules encoding a TPTE antigen) where one dose is administered once weekly for 3 weeks, and a second 3-week dosing cycle includes 3 doses of one or more RNA molecules described herein (administered concurrently or sequentially) that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen (e.g., a first pharmaceutical composition comprising RNA molecules encoding a NY-ESO-1 antigen, a second pharmaceutical composition comprising RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition comprising RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical composition comprising RNA molecules encoding a TPTE antigen) where one dose is administered once weekly for 3 weeks.

[0306] In some embodiments, a first 3-week dosing cycle includes (a) 3 doses of one or more RNA molecules described herein (administered concurrently or sequentially) that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen (e.g., a first pharmaceutical composition comprising RNA molecules encoding a NY-ESO-1 antigen, a second pharmaceutical composition comprising RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition comprising RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical composition comprising RNA molecules encoding a TPTE antigen) where one dose is administered once weekly for 3 weeks, and (b) one dose of checkpoint inhibitor (e.g., cemiplimab); and a second 3-week dosing cycle includes (c) 3 doses of one or more RNA molecules described herein (administered concurrently or sequentially) that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen (e.g., a first pharmaceutical composition comprising RNA molecules encoding a NY-ESO-1 antigen, a second pharmaceutical composition comprising RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition comprising RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical composition 12829787v1Attorney Ref.2013237-1447 comprising RNA molecules encoding a TPTE antigen) where one dose is administered once weekly for 3 weeks, and (d) one dose of checkpoint inhibitor (e.g., cemiplimab).

[0307] In some embodiments, a first 3-week dosing cycle includes 3 doses of one or more RNA molecules described herein (administered concurrently or sequentially) that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen (e.g., a first pharmaceutical composition comprising RNA molecules encoding a NY-ESO-1 antigen, a second pharmaceutical composition comprising RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition comprising RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical composition comprising RNA molecules encoding a TPTE antigen) where one dose is administered once weekly for 3 weeks, a second 3-week dosing cycle includes 3 doses of one or more RNA molecules described herein (administered concurrently or sequentially) that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen (e.g., a first pharmaceutical composition comprising RNA molecules encoding a NY-ESO-1 antigen, a second pharmaceutical composition comprising RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition comprising RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical composition comprising RNA molecules encoding a TPTE antigen) where one dose is administered once weekly for 3 weeks, and a third dosing cycle includes at least one dose of one or more RNA molecules described herein (administered concurrently or sequentially) that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen (e.g., a first pharmaceutical composition comprising RNA molecules encoding a NY-ESO-1 antigen, a second pharmaceutical composition comprising RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition comprising RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical composition comprising RNA molecules encoding a TPTE antigen) where each dose is administered once every 3 weeks.

[0308] In some embodiments, a first 3-week dosing cycle includes (a) 3 doses of one or more RNA molecules described herein (administered concurrently or sequentially) that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen (e.g., a first pharmaceutical composition comprising RNA molecules encoding a NY-ESO-1 antigen, a second pharmaceutical composition comprising RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition comprising RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical 12829787v1Attorney Ref.2013237-1447 composition comprising RNA molecules encoding a TPTE antigen) where one dose is administered once weekly for 3 weeks, and (b) one dose of checkpoint inhibitor (e.g., cemiplimab); a second 3-week dosing cycle includes (c) 3 doses of one or more RNA molecules described herein (administered concurrently or sequentially) that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen (e.g., a first pharmaceutical composition comprising RNA molecules encoding a NY-ESO-1 antigen, a second pharmaceutical composition comprising RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition comprising RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical composition comprising RNA molecules encoding a TPTE antigen) where one dose is administered once weekly for 3 weeks, and (d) one dose of checkpoint inhibitor (e.g., cemiplimab); and a third dosing cycle includes (e) at least one dose of one or more RNA molecules described herein (administered concurrently or sequentially) that collectively encode (i) a NY-ESO-1 antigen, (ii) a MAGE-A3 antigen, (iii) a tyrosinase antigen, and (iv) a TPTE antigen (e.g., a first pharmaceutical composition comprising RNA molecules encoding a NY-ESO-1 antigen, a second pharmaceutical composition comprising RNA molecules encoding a MAGE-A3 antigen, a third pharmaceutical composition comprising RNA molecules encoding a tyrosinase antigen, and a fourth pharmaceutical composition comprising RNA molecules encoding a TPTE antigen) where each dose is administered once every 3 weeks and (f) at least one dose of checkpoint inhibitor (e.g., cemiplimab) administered once every 3 weeks.

[0309] In some embodiments, there may be a “rest period” between cycles; in some embodiments, there may be no rest period between cycles. In some embodiments, there may be sometimes a rest period and sometimes no rest period between cycles.

[0310] In some embodiments, a rest period may have a length within a range of several days to several months. For example, in some embodiments, a rest period may have a length of at least 3 days or more, including, e.g., 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, a rest period may have a length of at least 1 week or more, including, e.g., at least 2 weeks, at least 3 weeks, at least 4 weeks, or more.

[0311] Dose: Dosage of pharmaceutical compositions described herein may vary with a number of factors including, e.g., but not limited to body weight of a subject to be treated, cancer types and / or cancer stages, and / or monotherapy or combination therapy. In some embodiments, a dosing cycle involves administration of a set number and / or pattern of doses. 12829787v1Attorney Ref.2013237-1447 For example, in some embodiments, a pharmaceutical composition described herein is administered at least one dose per dosing cycle, including, e.g., at least two doses per dosing cycle, at least three doses per dosing cycle, at least four doses per dosing cycle, or more.

[0312] In some embodiments, a dosing cycle involves administration of a set cumulative dose, e.g., over a particular period of time, and optionally via multiple doses, which may be administered, for example, at set interval(s) and / or according to a set pattern. In some embodiments, a set cumulative dose may be administered via multiple doses at set intervals such that there is at least some temporal overlap in biological and / or pharmacokinetics effects generated by such multiple doses on a target cell or on a subject being treated. In some embodiments, a set cumulative dose may be administered via multiple doses at set intervals such that biological and / or pharmacokinetics effects generated by such multiple doses on a target cell or on a subject being treated may be additive. By way of example only, in some embodiments, a set cumulative dose of X mg may be administered via two doses with each dose of X / 2 mg, wherein such two doses are administered sufficiently close in time such that biological and / or pharmacokinetics effects generated by each X / 2-mg dose on a target cell or on a subject being treated may be additive.

[0313] In some embodiments, each dose or a cumulative dose (e.g., for intravenous administration) is administered at a level such that the one or more RNA molecules that collectively encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof, is expected to achieve level (e.g., plasma level and / or tissue level) that is high enough to for translation and antigen presentation in an antigen- presenting cell (e.g., a dendritic cell or immature dendritic cell) that induces a CD4+ and CD8+ T cell immunity against the one or more antigens throughout a dosing cycle.

[0314] In some embodiments, the methods provided in Example 3 (e.g., Tables A and B) provide exemplary dosing schedules.

[0315] In some embodiments, dosing may be adjusted based on response of a subject receiving the therapy. For example, in some embodiments, dosing may involve administration of a higher dose followed later by administration of a lower dose if one or more parameters for safety pharmacology assessment indicates that the prior dose may not satisfy the medical safety requirement according to a physician. Without wishing to be bound by any particular theory, the present disclosure, among other things, provides an insight that a pharmaceutically guided dose escalation (PGDE) method may be applied to determine an appropriate dose of pharmaceutical compositions described herein. 12829787v1Attorney Ref.2013237-1447

[0316] Also provided herein is also a method of determining a dosing regimen of a pharmaceutical composition comprising the one or more RNA molecules that collectively encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof. For example, in some embodiments, such a method comprises steps of: (A) administering a pharmaceutical composition (e.g., ones described herein) to a subject suffering from a melanoma or a subject who has been classified as no evidence of disease under a pre-determined dosing regimen; (B) monitoring or measuring evidence of disease (e.g., tumor lesion size and / or metastases) of the subject periodically over a period of time; (C) evaluating the dosing regimen based on the monitoring or measuring results and / or outcomes. For example, a dose and / or dosage frequency can be increased if reduction in tumor size after the administration of a pharmaceutical composition (e.g., ones described herein) is not therapeutically relevant; or a dose and / or dosage frequency can be decreased if reduction in tumor size after the administration of a pharmaceutical composition (e.g., ones described herein) is therapeutically relevant, but adverse effect (e.g., toxicity effect) is shown in the subject. If reduction in tumor size after the administration of a pharmaceutical composition (e.g., ones described herein) is therapeutically relevant, and no adverse effect (e.g., toxicity effect) is shown in the subject, no changes is made to a dosage regimen.

[0317] In some embodiments, such a method of determining a dosing regimen of a pharmaceutical composition comprising the one or more RNA molecules that collectively encode a NY-ESO-1 antigen, a MAGE-A3 antigen, a tyrosinase antigen, a TPTE antigen, or a combination thereof; may be performed in a group of animal subjects (e.g., mammalian non- human subjects) each a bearing a human melanoma xenograft tumor. In some such embodiments, a dose and / or dosage frequency can be increased if less than 30% of the animal subjects exhibit reduction in tumor size after the administration of a pharmaceutical composition (e.g., ones described herein) and / or extent of reduction in tumor size exhibited by the animal subjects is not therapeutically relevant; or a dose and / or dosage frequency can be decreased if reduction in tumor size after the administration of a pharmaceutical composition (e.g., ones described herein) is therapeutically relevant, but significant adverse effect (e.g., toxicity effect) is shown in at least 30% of the animal subjects. If reduction in tumor size after the administration of a pharmaceutical composition (e.g., ones described herein) is therapeutically relevant, and no significant adverse effect (e.g., toxicity effect) is shown in the animal subjects, no changes is made to a dosage regimen.

[0318] Although the dosing regimens (e.g., dosing schedule and / or doses) provided herein are principally suitable for administration to humans, it will be understood by the 12829787v1Attorney Ref.2013237-1447 skilled artisan that dose equivalents can be determined for administration to animals of all sorts. The ordinarily skilled veterinary pharmacologist can design and / or perform such determination with merely ordinary, if any, experimentation.

[0319] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0320] The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the disclosure in any way. EXEMPLIFICATION Example 1: Main Analysis of Phase I Clinical Trial NCT02410733

[0321] A Phase I Clinical Trial (NCT02410733) was performed to assess the safety and tolerability of melanoma FixVac, its preliminary efficacy and progression-free survival; to investigate vaccine-induced antigen-specific immune responses; and to determine a phase II dose. As used herein, the term “FixVac” refers to a pharmaceutical composition comprising one or more RNA molecules as depicted in Fig.1 and lipid particles (e.g., lipoplexes or lipid nanoparticles). BNT111 is an embodiment of FixVac.

[0322] The trial design, including materials and methods, inclusion and exclusion criteria, and dosing regimen are described in detail in WO2023 / 006920, the contents of which are incorporated herein by reference in its entirety. 1. Introduction

[0323] BNT111 is a fixed set of four ribonucleic acid (RNA) drug products RBL001.1, RBL002.2, RBL003.1, and RBL004.1 which code for four tumor-associated antigens (TAAs), namely New York esophageal squamous cell carcinoma-1 (NY‑ESO-1), tyrosinase, melanoma-associated antigen A3 (MAGE-A3), and trans‑membrane phosphatase with tensin homology (TPTE). Each RNA was complexed with liposomes (to form RNA-lipoplexes, RNA-LPXs) and separately administered in four sequential injections.

[0324] During the trial, BNT111 was optimized with new drug products and RNA drug substance sequences. This Example is confined to patients who received the original 12829787v1Attorney Ref.2013237-1447 (‘precursor’) drug products (referred to herein as the ‘Lipo-MERIT vaccine’). Example 2 describes data from patients who received the optimized drug products of BNT111 during Extended Treatment.

[0325] An overall description of the trial is presented in the flow diagram in Fig.2. 2. Trial design

[0326] This was a multicenter, open-label, interventional first-in-human trial in patients with advanced melanoma. This Phase I trial included seven dose escalation cohorts and three expanded cohorts for pharmacodynamics studies and evaluation of clinical response or disease-free survival (DFS) for patients with evaluable disease. The intra-patient dose escalation was continued as planned or modified as decided by the investigator based on adverse events (AEs).

[0327] An overview of the dose escalation in Cohorts I to VII and expanded cohorts is provided in the tables below. 12829787v1Attorney Ref.2013237-1447 Table 5: Dose escalation Cohorts I to VII Cohort Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Cycle 7 Cycle 8 I 7.2 (1.8) 14.4 (3.6) 29 (7.2) 29 (7.2) 29 (7.2) 29 (7.2) n.a. n.a. 6) 5) 8) ) ) 0)Table 6: Dosing in Expanded Cohorts A, B, and C Expand -ed Cycle 1 Cycle 2 Cycle 3 Cycle 4 Cycle 5 Cycle 6 Cycle 7 Cycle 8 6) 5) ) ribonucleic acid (RNA). 3. Objectives Table 7: Trial objectives and endpoints Objectives Endpoints Primary Determine• Occurrence of dose-limiting toxicities (DLTs)Lipo-MERIT vaccination cycles • Occurrence of adverse events (AEs) reported by relationship, grade, and seriousness according to National Cancer Institute (NCI) Common Terminology12829787v1Attorney Ref.2013237-1447 Objectives Endpoints Criteria for Adverse Events, version 4.03 (CTCAE v403)po- vaccne ater repettve of patients with complete responses (CR) or partial vaccination cycles in patients with advanced res onses (PR) as best overall res onse accordin to in l o4. Trial treatments Table 8: Trial Treatments Therapeutic cancer BNT111 e n5. Trial population

[0328] Adult patients with malignant melanoma Stage IIIB to IIIC or Stage IV (American Joint Committee on Cancer [AJCC] 2009 melanoma classification) along with confirmed expression of at least one tumor-associated antigen (TAA) either New York esophageal squamous cell carcinoma-1 (NY-ESO-1), tyrosinase, melanoma-associated antigen A3 (MAGE-A3), or trans-membrane phosphatase with tensin homology (TPTE) were included. 12829787v1Attorney Ref.2013237-1447 6. Inclusion criteria

[0329] Patients meeting all the following inclusion criteria were eligible for trial entry: 1. Cohort I: Stage IV malignant melanoma (AJCC 2009 melanoma classification) 2. Cohorts II to VII and expanded cohorts: Stage IIIB to C, or Stage IV malignant melanoma (AJCC 2009 melanoma classification) 3. Expanded Cohort C only patients with Stage IV melanoma (AJCC 2009 melanoma classification) with measurable disease (at least one target lesion according irRECIST) [applicable for all patients after approval of protocol version 10.0 and higher] and with disease progression at the time of first treatment with IMP [applicable for all patients included after approval of protocol version 11.0]) 4. Therapy only for subjects not eligible or declining any other available approved therapy after all available treatment options have been transparently disclosed (to be documented) 5. Expression of either NY-ESO-1, tyrosinase, MAGE-A3, and / or TPTE confirmed by reverse transcriptase quantitative polymerase chain reaction analysis from FFPE 6. ≥ 18 years of age 7. Written informed consent 8. Eastern Cooperative Oncology Group – performance status (ECOG-PS) 0-1 9. Life expectancy ≥ 6 months 10. WBC ≥ 3x109 / L 11. Hemoglobin ≥ 9 g / dl 12. Platelet count ≥ 100,000 / mm³ 13. Alanine aminotransferase / Aspartate aminotransferase < 3.0 upper limit of normal (ULN) (except for patients with liver metastasis) 14. Negative pregnancy test (measured by β-hCG) for females of childbearing age 7. Exclusion criteria

[0330] Patients meeting at least one of the following exclusion criteria were not eligible for the trial entry: 1. Pregnancy or breastfeeding 2. Primary ocular melanoma 3. Concurrence of a second malignancy other than squamous or basal cell carcinoma, non-active prostate cancer, or cervical carcinoma in situ or non-active treated urothelial carcinoma 4. Brain metastases 12829787v1Attorney Ref.2013237-1447 • Patients with history of treated or inactive brain metastasis are eligible for treatment in Expanded Cohort C, provided they meet all of the following criteria: o measurable disease outside of the brain (in addition to inactive brain metastasis). o no ongoing requirement of corticosteroids as therapy for brain metastases. o with corticosteroids discontinued ≥1 week prior to Visit 2 (Day 1) with no ongoing symptoms attributable to brain metastases. o the screening brain radiographic imaging is ≥4 weeks since completion of radiotherapy. 5. Post-splenectomy patients 6. Known hypersensitivity to the active substance or to any of the excipients 7. A serious local infection (e.g., cellulitis, abscess) or systemic infection (e.g., pneumonia, septicemia) which requires systemic antibiotic treatment within 2 weeks prior to the first dose of trial medication 8. Positive test for acute or chronic active hepatitis B or C infection 9. Clinically relevant active autoimmune disease 10. Systemic immune suppression: • HIV disease • Use of chronic oral or systemic steroid medication (topical or inhalational steroids are permitted) • Other clinically relevant systemic immune suppression 11. Symptomatic congestive heart failure (NYHA 3 or 4) 12. Unstable angina pectoris 13. Radiotherapy and minor surgery within 14 d prior to the first trial treatment administration 14. Myelosuppressive chemotherapy within 14 d and after reconstitution of blood values prior to the first trial treatment administration 15. Ipilimumab within 28 d prior to the first trial treatment administration 16. Treatments with BRAF inhibitors, MEK inhibitors, or the combination of both, and anti-PD-1 antibodies within 14 d prior to the first administration of trial treatment (not applicable for patients with parallel treatment in Expanded Cohorts A, B, or C at the discretion of the investigator) 17. Interferon, major surgery, vaccination, and other investigational agents within 28 d or five half- lives depending on what gives the longer range before the first treatment 12829787v1Attorney Ref.2013237-1447 18. Approved BRAF inhibitors vemurafenib or dabrafenib, approved anti-PD-1 inhibitors nivolumab or pembrolizumab as well as approved MEK inhibitor trametinib, or the approved combination of BRAF-MEK inhibitors in patients in dose escalation cohorts. Concomitant treatment with approved BRAF inhibitors, approved anti-PD-1 antibodies or MEK inhibitor as well as the approved combination of BRAF-MEK inhibitors is allowed for patients included in the expanded cohorts, after analysis of safety data collected for the dose escalation cohorts and DSMB approval. Local radiation will be allowed as concurrent treatment for patients in the expanded cohorts as well − After approval of protocol version 10.0 and higher, only anti-PD-1 antibodies are allowed for treatment of patients in Expanded Cohort C 19. Fertile males and females who are unwilling to use a highly effective method of birth control (<1% per year, e.g., condom with spermicide, diaphragm with spermicide, birth control pills, injections, patches, or intrauterine device) during trial treatment and for at least 28 d (male patients) and 90 d (female patients of childbearing potential) after the last dose of trial treatment 20. Presence of a severe concurrent illness or other condition (e.g., psychological, family, sociological, or geographical circumstances) that does not permit adequate follow-up and compliance with the protocol 8. Discontinuation from the trial, trial assessments, or investigational medicinal product(s)

[0331] Criteria for patient discontinuation were as follows: 1. Withdrawal of consent 2. Progression of cancer that is not qualified as slow progressive disease. Slow progressive disease is defined as (i) lactate dehydrogenase levels not increasing 3. ≥ 2.0-fold ULN, (ii) ECOG-PS ≤ 2 and (iii) only a maximal increase of one Grade in the ECOG-PS from baseline assessment) except for patients who consent in writing to treatment beyond progression 4. Pregnancy of a female patient 5. Development of an exclusion criterion during the course of the trial, if safety reasons are concerned, at the discretion of the investigator 6. Occurrence of an intolerable (serious) adverse reaction 7. Occurrence of severe hypersensitivity at the discretion of investigator 8. Lost to follow-up after a minimum of three attempts to contact the patient 9. Significant deviation of the trial protocol, e.g., non-compliance 10. Death 12829787v1Attorney Ref.2013237-1447 9. Concomitant anti-cancer treatments

[0332] Concomitant treatment with approved proto-oncogene B-Raf (BRAF) inhibitors vemurafenib or dabrafenib, the MAPK kinase (MEK) inhibitor trametinib, or the programmed death-1 (PD-1) inhibitors nivolumab or pembrolizumab, or other approved combinations were allowed for patients included in the expanded cohorts. 10. Methodology

[0333] Adult patients with an established diagnosis of advanced melanoma (Stage IIIB to IIIC, IV) and confirmed expression of at least one of the antigens (NY-ESO-1, tyrosinase, MAGE-A3, or TPTE) continued with baseline assessments to confirm the eligibility for the trial. The sponsor then assigned the eligible patients to an open cohort.

[0334] In the main treatment phase, patients received repetitive vaccination cycles over a maximum period of 64 to 68 d except for patients in Cohort I where the maximum treatment period was 43 to 47 d.

[0335] The trial duration for an individual patient was approximately 5 months without continued treatment. There was a total of nine visits within the trial and three follow-up visits, except for patients in Cohort I where there were 7 visits. Only patients with measurable disease without disease progression, unacceptable toxicity, or withdrawal of consent at follow-up Visit 2 were eligible for continued treatment.

[0336] Upon informed consent, eligible patients entered the continued treatment phase and received additional vaccinations every 28 d until (confirmed) disease progression, unacceptable toxicity, or withdrawal of consent. In the continued treatment phase, visits were planned every 28 d and in the long-term follow-up, visits occurred every 3 months.

[0337] For patients who suffered disease progression, the possibility to continue the trial treatment was made at the discretion of the investigator, in consultation with the sponsor, if the patient probably derived benefit from the continuation of treatment and consented to the continuation after disease progression.

[0338] Assessments for safety (vital signs and physical examination, electrocardiogram, AEs / serious AEs [SAEs] including dose-limiting toxicities [DLTs], laboratory assessments) were performed at baseline and thereafter at pre-defined time points (initially weekly visits, followed by visits every second week) until follow-up Visit 2 (Day 90) and continued treatment visits (every fourth week). 12829787v1Attorney Ref.2013237-1447

[0339] An independent Data and Safety Monitoring Board (DSMB) assessed the progress of the trial and safety data.

[0340] Blood samples were taken at each visit prior to the vaccination and at 3 or 4 additional time points after the vaccination for the first 2 to 6 vaccinations (depending on the cohort), for further vaccinations, blood samples were taken prior to the vaccination. In the main treatment, the total volume of blood taken per patient was between 596 mL to 862 mL depending on the cohort in which the patient was included.

[0341] All the patients in the trial were followed for survival and subsequent anti-cancer therapy approximately every 3 months until death, loss to follow-up, trial termination by the sponsor, or trial termination by the patient. 11. Statistical methods

[0342] All statistical analyses were carried out using SAS®, version 9.4, or higher.

[0343] Due to the exploratory nature of the cohorts, no formal sample size calculation was performed.

[0344] Only data until the cut-off date (30 SEP 2021) was used for the analysis of the main treatment and continued treatment with BNT111.

[0345] During the trial, BNT111 was optimized with new drug products and RNA drug substance sequences. This report does not contain the data from the patients who received the optimized BNT111 entering Extended Treatment. Hence, individual cut-off dates were used for such patients as seen in the below table. Data of patients receiving optimized drug products of BNT111 during Extended Treatment are described in Example 2. 12829787v1Attorney Ref.2013237-1447 Table 9: Individual data cut-off date for patients entering Extended Treatment with optimized BNT111 patient IDaCut-off date 131 AUG 2021

[0346] Data sets were defined as follows: • Screened set: all patients who signed informed consent. • Modified intent to treat (mITT) set: all patients who were assigned to IMP and had a baseline and at least one evaluable on-treatment / post-treatment tumor response assessment. Patients with evaluable disease were defined as those who had radiologically evaluable disease at baseline (i.e., patients with documented target / index or non-target / non-index lesions at the baseline tumor assessment). • Safety set (SAF): all patients who received IMP. • Per protocol set: all patients of the safety set who fulfilled the following criteria: o The absence of any important protocol deviations. o The completion of a minimal exposure of at least three vaccination cycles. o Availability of a baseline and at least one on-treatment / post-treatment tumor assessment.

[0347] The primary analysis (occurrence of DLTs and AEs) was based on the safety set. All secondary efficacy analyses were based on the mITT set.

[0348] All data from the main treatment and the continued treatment / long-term follow-up were analyzed together.

[0349] AEs were coded using the Medical Dictionary for Regulatory Activities (MedDRA®) version 24.1 coding system to get a System Organ Class (SOC) and Preferred Term (PT) for each AE and graded for severity using Common Terminology Criteria for AEs (CTCAE) v4.03.

[0350] Continuous variables were summarized by cohort and disease evaluability and therapy (monotherapy, combination therapy with PD-1 inhibitors and BRAF / MEK inhibitors) and overall using descriptive statistics. Categorical variables were summarized by cohort and disease evaluability and 12829787v1Attorney Ref.2013237-1447 therapy and overall presenting absolute and relative frequencies (n and %) of patients in each category. Time-to-event-endpoints were analyzed using Kaplan-Meier methodology by cohort and disease evaluability and therapy. The median survival time and the first and third quartile (including two-sided 95% confidence limits according to Brookmeyer and Crowley) are presented for each cohort and disease evaluability and therapy and overall. Survival rates (including two-sided 95% confidence interval [CI] based on Greenwood's formula) as well as the number and percentage of patients with events, censored and under risk are displayed for selected time points. 12. Summary of results A. Patient disposition

[0351] Overall, until the cut-off date, 188 patients were screened in this trial. Of the 188 patients, 70 patients were screen failed. Among the 118 patients who were successfully screened, 115 patients were assigned to treatment, of whom, 89 completed the main treatment. The main reason for treatment discontinuation was due to progressive disease (n = 16).

[0352] Among the 115 patients, 85 did not qualify for continued treatment, the main reason being progressive disease (n = 46) and patients were without lesions (n = 23).

[0353] There were 71 patients who were treated with BNT111 monotherapy (59 patients completed main treatment and 12 patients discontinued), 38 patients with BNT111 and a PD-1 inhibitor (27 patients completed main treatment and 11 patients discontinued) and 6 patients with BNT111 and a BRAF / MEK inhibitor (3 patients completed main treatment and 3 patients discontinued). The main reason for discontinuation in all the three therapy subgroups defined by trial treatment (monotherapy, combination with a PD-1 inhibitor, combination with a BRAF / MEK inhibitor) was due to progressive disease. B. Demographics and other baseline characteristics

[0354] The overall mean age (±standard deviation) of the patients assigned to the treatment was 54.8 (±15) years. Of the 115 patients, most of them (71.3%, n = 82) were below 65 years of age. Overall, 53.9% of patients were males and 46.1% were females. The median age was higher in the BNT111 and PD-1 inhibitor subgroup as compared to the BNT111 monotherapy subgroup (58.5 years [min to max: 21 to 86] vs.54 years [min to max: 26 to 80]).

[0355] A larger proportion of patients in the BNT111 and PD-1 inhibitor subgroup had Stage IV melanoma when compared to the BNT111 monotherapy subgroup (97.4% vs.71.8%). Additionally, a larger proportion of patients in the BNT111 and PD-1 inhibitor subgroup had an Eastern Cooperative Oncology Group – performance status (ECOG-PS) of 1 (21.1% vs.12.7%). 12829787v1Attorney Ref.2013237-1447

[0356] In the evaluable disease group, median number of prior therapies received by patients were similar between the BNT111 monotherapy (4.5 [range: 1 to 15]) and BNT111 and PD-1 inhibitor therapy subgroup (5.5 [range: 0 to 21]). The overall median number of prior therapies received by patients in the BNT111 monotherapy group was 3 (range: 0 to 15) which was lower than those that received prior therapy in the BNT111 and PD-1 inhibitor therapy subgroup (5.5 [range: 0 to 21]). C. Efficacy

[0357] In the mITT population with evaluable disease, of the 36 patients who were treated with BNT111 monotherapy, 1 patient (2.8%) had a CR and 3 patients (8.3%) had PRs. For the patients who received BNT111 and a PD-1 inhibitor (N = 36), none had a complete response (CR) and 9 patients (25%) had partial response (PR). The objective response rate (ORR) in patients who received BNT111 and a PD-1 inhibitor was 25% (95% CI: 12.1 to 42.2) which was higher than in the BNT111 monotherapy subgroup with 11.1% (95% CI: 3.1 to 26.1).

[0358] In addition, post-hoc analysis in patients with progressive disease at baseline showed a similar ORR (22.2%; CI: 8.6, 42.3) in the BNT111 and PD-1 inhibitor therapy subgroup and in the BNT111 monotherapy subgroup (7.4%; CI: 0.9, 24.3) compared to the evaluable patients in the mITT set. There was no notable difference between the two therapy subgroups with respect to stable disease. There was no apparent relationship between BNT111 dose received and clinical response expressed as CR or PR.

[0359] The DoR was longer at 22.9 months when compared to 8.4 months in patients who received BNT111 and a PD-1 inhibitor therapy and BNT111 monotherapy, respectively. Numerical differences were noted in the disease control rate (DCR) across treatment groups with 44.4% and 36.1% in patients who received BNT111 and a PD-1 inhibitor therapy and BNT111 monotherapy, respectively. The DCR across treatment groups in patients with progressive disease was somewhat lower than in the mITT set and somewhat higher in patients with at least stable disease at baseline, however no significant difference was found with largely overlapping CIs for the DCR for all groups.

[0360] The patients who received BNT111 and a PD-1 inhibitor therapy had numerically longer duration of clinical benefit at 25.9 months than patients who received BNT111 monotherapy (11.3 months). However, for all assessments the 95% confidence intervals were very wide and overlapped between groups indicating the absence of a statistically significant difference.

[0361] In the sensitivity analysis, there were no notable difference in the median PFS time (2.8 months) between the treatment subgroups. For patients with at least stable disease at baseline, overall median PFS was 12.1 months (39.2 months in BNT111 monotherapy and 12.1 months in BNT111 and 12829787v1Attorney Ref.2013237-1447 PD-1 inhibitor therapy) indicating that this population had a positive effect on median PFS in both the therapy subgroups.

[0362] In the non-evaluable disease BNT111 monotherapy subgroup, the median DFS was 34.8 months. No patients were treated with BNT111 and a PD-1 inhibitor combination in non-evaluable disease group.

[0363] The median overall survival (OS) time for the overall mITT population was 31.2 months. The median OS for mITT evaluable disease group was 21.5 months and the median OS was not yet reached for non-evaluable disease patients. The median OS in patients including missing and non- missing tumor response (at least stable disease) at baseline was longer at 39.2 months indicating that this population had a positive impact on the median OS of the overall mITT population. D. Pharmacodynamics

[0364] Consistent with pre-clinical findings (see, e.g., Sahin et al.2020 and Kranz et al.2016), cytokine secretion was pulsatile, transient and self-limiting and concentrations reached peak values 6 h post-vaccination and then returned to pre-vaccination levels at 24 h to 48 h post-vaccination. The analysis of cytokine data showed a clear dose dependency of peak cytokine levels with BNT111 dose, particularly for IL-6, IFN alpha, IFN gamma, IL-12, IL-10, and IP-10. The combination of BNT111 with a PD-1 inhibitor did not impact on the cytokine induction profile nor did it differ between disease evaluability subgroups.

[0365] Overall, 94 patients were assessed for delayed-type hypersensitivity (DTH) reaction at both baseline and follow-up. Of these, 44 patients did not have redness at either timepoint, and 71 patients did not have induration at either timepoint. Thus, quantitative DTH reaction data was available for 50 patients only. De novo redness responses (not present at baseline, but present at follow-up) were seen in 25 patients. De novo induration responses were seen in 15 patients. Redness was noted in both baseline and follow-up visits in 17 patients, and induration in 4 patients. Of the 17 patients with redness at both the timepoints, 14 patients had an increased reaction to at least one antigen. Of the 4 patients with induration at both timepoints, all 4 patients had an increased reaction to at least one antigen. 12829787v1Attorney Ref.2013237-1447 E. Safety and tolerability

[0366] In the seven dose escalation cohorts (Cohorts I to VII), of the 20 DLT-evaluable patients, none experienced any AEs that were assessed as DLTs.

[0367] The most frequently observed related AEs were pyrexia, chills, headache, fatigue, and nausea, and the majority were transient and manageable by administration of antipyretics and analgesics. Some AEs were more frequent in the BNT111 and PD-1 inhibitor than in the BNT111 monotherapy subgroup including nausea, vomiting, and hypotension.

[0368] Additionally, patients in the BNT111 and PD-1 inhibitor subgroup experienced a higher frequency of Grade ≥3 AEs and SAEs than patients in the BNT111 monotherapy subgroup. Conclusions regarding the safety profile of BNT111 monotherapy compared to BNT111 and BRAF / MEK inhibitors are limited given the small number of patients treated with BNT111 and BRAF / MEK inhibitors in this trial.

[0369] The most common AEs observed in patients treated with BNT111 monotherapy were expected and correlated with the laboratory and biomarker changes observed in this and other clinical trials with RNA-LPX-based IMPs, namely the distinct range of pro-inflammatory cytokines released following RNA-LPX administration.

[0370] The overall safety profile of patients with evaluable disease and non-evaluable disease was comparable; however, there was a higher frequency of patients with related Grade ≥3 AEs in the BNT111 monotherapy subgroup with evaluable disease when compared to patients with non-evaluable disease. 13. Conclusion

[0371] Overall, the data presented in this report demonstrate that both BNT111 monotherapy and BNT111 combined with a PD-1 inhibitor have an acceptable safety profile. • The most frequent AEs were mostly transient and manageable flu-like AEs. • Combining BNT111 with a PD-1 inhibitor impacted the overall safety profile: SAEs and AEs of Grade ≥3 were more frequent in patients in the BNT111 and PD-1 inhibitor subgroup than in the BNT111 monotherapy subgroup. • During screening for inclusion of patients into this trial, the vast majority of tumour samples (92.1%) expressed at least one of the selected TAAs, with 78.2% of samples expressing more than one TAA. 12829787v1Attorney Ref.2013237-1447 • In the evaluable disease group, the ORR in patients who received BNT111 and a PD-1 inhibitor therapy was 25% which was higher than in the patients who received BNT111 monotherapy with 11.1%. The results for patients with progressive disease at baseline were similar. • Responses were noted at both 100 µg total RNA and lower dose (14.4 µg total RNA), however, response at 100 µg was seen not only by clinical activity but also by immunogenicity which were supported by consistent increase of immunogenicity markers. • Duration of response was longer with BNT111 and PD-1 inhibitor combination therapy (22.9 months) compared to BNT111 monotherapy (8.4 months) but the difference was not statistically significant. • Numerical differences were noted in the DCR across treatment groups with 44.4% and 36.1% in patients who received BNT111 and a PD-1 inhibitor therapy and BNT111 monotherapy, respectively. There were no substantial differences for the different BNT111 doses in regard to best overall response and DCR. o There was 1 patient with a CR (was partial remission at baseline) in the BNT111 monotherapy subgroup. o The frequency of PRs was higher with the combination therapy than with BNT111 monotherapy although the difference was not statistically significant. • Median PFS was 2.8 months across the therapy subgroups. There were no substantial differences for the different BNT111 doses in regard to best overall response and DCR. PFS at 6, 12, and 24 months was higher in the BNT111 and PD-1 inhibitor subgroup than in the BNT111 monotherapy subgroup; however, the difference was not statistically significant. Of note, overall median PFS was 12.1 months in patients with at least stable disease at baseline (39.2 months in BNT111 monotherapy and 12.1 months in BNT and a PD-1 inhibitor therapy) indicating that this population had a positive effect on median PFS in both the therapy subgroups. • In the non-evaluable disease patients receiving BNT111 monotherapy, the median DFS was 34.8 months. • The median OS time for the mITT population was 31.2 months while median OS in patients with at least stable disease at baseline was longer at 39.2 months, indicating that this population had a positive impact on the median OS time of the overall mITT population. • In line with the mode of action of RNA-LPX (activation of TLR-signaling in antigen- presenting cells) patients had BNT111 dose-dependent increases of plasma levels of a distinct spectrum of cytokines, namely IL-6, IFN alpha, IFN gamma, IL-12, IL-10 and IP-10. 12829787v1Attorney Ref.2013237-1447

[0372] In conclusion, BNT111 monotherapy had a favorable safety profile that was in line with the known mode of action of RNA-LPX. The combination of BNT111 with a PD-1 inhibitor led to quantitative and qualitative modification of the safety profile which may have been due to known toxicities of immune checkpoint inhibitors and longer duration of treatment. In addition, the patients in the BNT111 and PD-1 inhibitor group had substantially more anti-cancer therapies before entering the trial, as well as a higher tumor burden. With regards to efficacy, there were numerical differences between the BNT111 monotherapy and BNT111 and a PD-1 inhibitor combination. The combination subgroup had a numerically more favorable outcome with a higher ORR, DoR, DCR, and PFS at 6, 12, and 24 months. Example 2: Addendum to Main Analysis of Phase I Trial NCT02410733 1. Introduction

[0373] After completion of the main treatment phase (described in Example 1), the patients were offered to receive optional Continued Treatment with BNT111. As a part of the optional Continued Treatment, until the end of August 2021, the patients received the original (‘precursor’) investigational medicinal product (IMP) (referred as ‘Lipo-MERIT vaccine’).

[0374] The ribonucleic acid (RNA) drug substance (DS) sequences (coding and non-coding regions) of the original IMP were then optimized to obtain a more defined RNA product and to have more robust manufacturing capabilities considering larger RNA batch sizes that are required for later stage clinical development and market supply. In addition, the preparation process was subject to improvements for manufacturability and prolonging shelf-life. With this, the original drug substances RBL001.1, RBL002.2, RBL003.1 and RBL004.1 which code for four tumor- associated antigens (TAAs), namely New York esophageal squamous cell carcinoma-1 (NY-ESO-1), tyrosinase, melanoma-associated antigen A3 (MAGE-A3), and transmembrane phosphatase with tensin homology (TPTE) were substituted with RBL001.3, RBL002.4, RBL003.3 and RBL004.3, respectively, encoding the same TAAs. This optimized version of the original Lipo-MERIT vaccine is referred to in Example 2 as “BNT111”.

[0375] Upon additional consent, the patients were offered continuing trial therapy with the optimized BNT111 in Extended Treatment for a maximum of an additional 18 months. 2. Trial treatments Table 10: Trial Treatments Therapeutic cancer BNT111Attorney Ref.2013237-1447 Mode of administration The four drug substances were individually complexed (pre-mixed) with liposomes to reveal the drug products. The drug products were separately

[0376] Patients who were enrolled in the trial and were still on Continued Treatment were offered to participate in the Extended Treatment with BNT111 upon signing of an additional informed consent. 4. Methodology

[0377] The first vaccination cycle for the Extended Treatment with BNT111 was scheduled 28 days after the last vaccination of Continued Treatment with Lipo-MERIT vaccine (original IMP). The patients received vaccination cycles every 4 weeks at the same dose that they had received on the Continued Treatment. No new dose-limiting toxicity (DLT) period or dose escalation was planned for the Extended Treatment with BNT111.

[0378] In the Extended Treatment with BNT111, the patients continued with the same treatment: either monotherapy or in combination with the same programmed death-1 (PD-1) inhibitor as it was in the Continued Treatment with the Lipo-MERIT vaccine. The trial treatment was offered for 18 months, and the patients were followed-up for safety for 90 days after the last vaccination. 5. Statistical methods

[0379] All statistical analyses were performed using the SAS-software version 9.4 or higher.

[0380] At the start of Extended Treatment with BNT111, the individual patients received the first BNT111 vaccination (see below table) based on their previous Continued Treatment vaccination date. Of note, the Extended Treatment start dates served as individual cut-off dates for the respective patients in the main analysis report. 12829787v1Attorney Ref.2013237-1447 Table 11: Start date of BNT111 for individual patients in the Extended Treatment patient IDaExtended Treatment Start Dates 1 31 AUG 2021Activities (MedDRA®) version 26.0 coding system to get a System Organ Class (SOC) and Preferred Term (PT) for each AE and graded for severity using Common Terminology Criteria for AEs (CTCAE) v4.03. 6. Data sets

[0382] The Safety Set Extended Treatment (SAF ET) was defined as all patients who received the IMP (i.e., at least one dose of BNT111) in the Extended Treatment. The other data sets are defined in Example 1.

[0383] The AEs were analyzed only for the SAF ET set and were displayed separately for the Lipo- MERIT vaccine and BNT111. In addition, intra-patient comparison of laboratory data and vital signs were performed from the data collected for the Lipo-MERIT vaccine and BNT111 in the Extended Treatment.

[0384] Also, as a part of exploratory analysis, the sponsor also compared the occurrence of AEs between Lipo-MERIT vaccine and BNT111 in the SAF ET patients. 7. Summary of results A. Patient disposition

[0385] Overall, a total of 115 patients were enrolled into the trial. Among the 115 patients, 30 patients were qualified for Continued Treatment while the remaining patients were withdrawn for the main reason being progressive disease (n = 46) and patients without lesions (n = 23). Of the 30 qualified patients for Continued Treatment, 8 patients entered Extended Treatment, while the remaining patients did not qualify for the Extended Treatment for the main reason being progressive disease (n = 14). 12829787v1Attorney Ref.2013237-1447

[0386] All the 8 patients who entered the Extended Treatment had evaluable disease. Six patients continued to receive BNT111 and a PD-1 inhibitor therapy (4 patients received nivolumab and 2 patients received pembrolizumab) and 2 patients continued to receive BNT111 monotherapy as they did during the Continued Treatment. B. Demographics

[0387] The overall mean age of the 8 patients was 47 years. All the 8 patients were below 65 years of age. Six patients were male and two were females and all were Caucasians. The median body mass index of the 8 patients was 26.7 kg / m2(range: 20.31 to 52.52).

[0388] The tumor stage according to American Joint Committee on Cancer for all the patients at baseline was Stage IV disease. At baseline, prior to the start of the trial, 3 patients had partial response (PR) (one patient was on monotherapy while on optional Continued Treatment and 2 patients were on combination therapy with PD-1 inhibitor), 3 patients had stable disease (one patient was on monotherapy while on optional Continued Treatment and 2 patients were on combination therapy with PD-1 inhibitor), and 2 patients had progressive disease (both were on combination therapy with PD-1 inhibitor during optional Continued Treatment). C. Efficacy

[0389] Based on the updated efficacy results, among the patients who received BNT111 and a PD-1 inhibitor therapy in the mITT population with evaluable disease (N = 36), none had a CR and 10 patients (27.8%) had PR. One patient who was at stable disease at the time of main analysis data cut- off, 30 SEP 2021, had PR upon efficacy data update.

[0390] One patient (who received BNT111 and PD-1 inhibitor), had a late response (from stable disease to PR) after the start of Extended Treatment. Compared to the median response duration of 8.4 months in the BNT111 monotherapy subgroup at the time of data cut-off date of the main analysis report, 30 SEP 2021, the median response duration of the overall trial was longer in this subgroup (20.9 months). This was largely driven by one patient who had a longer DoR. There was no longer median time to response or median DoR after long term follow-up of the Extended Treatment patients.

[0391] The median PFS remained the same at 2.8 months and the median OS in the overall evaluable disease population was increased from 21.5 months (at the main analysis data cut-off) to 22.9 months. Maximum PFS was not reached during longer follow-up for evaluable disease and non- evaluable disease groups.

[0392] Similarly, the median OS time for the overall mITT population was also slightly increased from 31.2 months to 32.4 months. 12829787v1Attorney Ref.2013237-1447 D. Pharmacodynamics

[0393] An immune response against at least one of the four TAAs was detected in 74% (58 / 78) of patients after administration of 8 Lipo-MERIT vaccines. While both CD4+ and CD8+ responses were observed, the T cell responses were mainly dominated by CD8+ T cells. Most vaccine-induced T cell responses were observed against NY-ESO-1 (52 / 78, 67%).

[0394] Although some of the observed ex vivo T cell immune responses were pre-existing and amplified by vaccination, 95% (55 / 58) of patients had immune responses that were induced de novo by the vaccine.

[0395] When comparing the pharmacodynamics between the two IMP versions, both Lipo-MERIT vaccine and BNT111 resulted in increased cytokine concentrations in the blood post treatment. Additionally, for both IMP versions, an increase from baseline concentrations was detectable by 2 hours post treatment, with the cytokine peaks typically occurring between 4 to 6 hours post-treatment, representing similar cytokine kinetics between the two IMPs. E. Safety and tolerability

[0396] All 8 patients exposed to BNT111 in the Extended Treatment experienced at least one TEAE. The most frequently reported related TEAEs were pyrexia, chills, and headache. These events were mostly Grade 1 and 2 and usually occurred within the first 6 hours and, in most cases, resolved the same day. Based on the mechanism of action of BNT111, these symptoms were expected. Based on the data collected during BNT111 development, risks associated with transient cytokine increased which included mild-to-moderate, transient and manageable AEs (e.g., pyrexia, chills, headache, fatigue, nausea and vomiting).

[0397] All the 8 patients had received Lipo-MERIT vaccine before receiving BNT111 with identical TAAs. The AEs appeared to be more frequent when the patients received Lipo-MERIT vaccine than BNT111. The increased frequency and occurrence of these events can be attributed to the duration of IMP intake since the patients received Lipo-MERIT vaccine for a longer duration compared to BNT111. There was no correlation between the severe TEAEs and the IMP versions.

[0398] The only SAE considered to be related to IMP during Extended ...

Claims

1. Attorney Ref.2013237-1447 CLAIMS What is claimed is:

1. A method of treating a subject suffering from melanoma, the method comprising administering to the subject a therapeutically effective amount of a cancer therapy comprising: (a) an RNA therapy comprising one or more RNA molecules that collectively encode (i) a New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen, (ii) a melanoma- associated antigen A3 (MAGE-A3) antigen, (iii) a tyrosinase antigen, and (iv) a transmembrane phosphatase with tensin homology (TPTE) antigen; and (b) a PD-1 inhibitor, wherein the melanoma is a PD-1 / PD-L1 inhibitor-refractory / relapsed, unresectable Stage III or IV melanoma, thereby treating the subject.

2. A method of treating a subject suffering from melanoma, the method comprising administering to the subject a therapeutically effective amount of a cancer therapy comprising: (a) an RNA therapy comprising one or more RNA molecules that collectively encode (i) a New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen, (ii) a melanoma- associated antigen A3 (MAGE-A3) antigen, (iii) a tyrosinase antigen, and (iv) a transmembrane phosphatase with tensin homology (TPTE) antigen; and (b) a PD-1 inhibitor, wherein before administering the cancer therapy, the subject received a PD-1 / PD-L1 inhibitor therapy and the subject suffers from a PD-1 / PD-L1 inhibitor-refractory / relapsed, unresectable Stage III or IV melanoma.

3. The method of claim 1 or 2, comprising administering the cancer therapy to a subject suffering from a PD-1 / PD-L1 inhibitor-refractory / relapsed, unresectable Stage III or IV melanoma.

4. The method of any one of claims 1-3, comprising administering the cancer therapy for at least 12, 24, 36, or 48 months. 12829787v1 Attorney Ref.2013237-1447 5. The method of claim 4, wherein after administering the cancer therapy, about 15% to about 40% of the subjects exhibit a clinical response (e.g., a clinical response that is a complete response or a partial response).

6. The method of claim 4, wherein after administering the cancer therapy, a higher level of subjects exhibit a clinical response, relative to a control response level.

7. The method of claim 6, wherein the control response level is a level of control subjects exhibiting a clinical response following treatment with (i) the RNA therapy alone or (ii) the PD-1 inhibitor alone, wherein the control subjects suffer from a PD-1 / PD-L1 inhibitor- refractory / relapsed, unresectable Stage III or IV melanoma before the treatment.

8. The method of claim 6 or 7, wherein level of subjects exhibiting a clinical response is a statistically significant higher level relative to the control response level.

9. A method of treating a population of subjects to a predetermined level of therapeutic efficacy, the method comprising: administering a cancer therapy to the population of subjects, wherein the subjects suffer from a PD-1 / PD-L1 inhibitor-refractory / relapsed, unresectable Stage III or IV melanoma, wherein the cancer therapy comprises: (a) an RNA therapy comprising one or more RNA molecules that collectively encode (i) a New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen, (ii) a melanoma- associated antigen A3 (MAGE-A3) antigen, (iii) a tyrosinase antigen, and (iv) a transmembrane phosphatase with tensin homology (TPTE) antigen; and (b) a PD-1 inhibitor, and wherein the predetermined level of therapeutic efficacy is a clinical response (e.g., a clinical response that is a complete response or a partial response) in about 15% to about 40% of the subjects, thereby treating the population of subjects. 12829787v1 Attorney Ref.2013237-1447 10. A method of selecting a subject for a cancer therapy, wherein after receiving a PD-1 / PD-L1 inhibitor therapy, if the subject suffers from a PD- 1 / PD-L1 inhibitor-refractory / relapsed, unresectable Stage III or IV melanoma, the subject is selected for treatment with the cancer therapy, wherein the cancer therapy comprises administering to the subject a therapeutically effective amount of: (a) an RNA therapy comprising one or more RNA molecules that collectively encode (i) a New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen, (ii) a melanoma- associated antigen A3 (MAGE-A3) antigen, (iii) a tyrosinase antigen, and (iv) a transmembrane phosphatase with tensin homology (TPTE) antigen; and (b) a PD-1 inhibitor.

11. A method of treating a subject, the method comprising: selecting a subject suffering from melanoma, and administering to the subject a therapeutically effective amount of a cancer therapy comprising: (a) an RNA therapy comprising one or more RNA molecules that collectively encode (i) a New York esophageal squamous cell carcinoma 1 (NY-ESO-1) antigen, (ii) a melanoma- associated antigen A3 (MAGE-A3) antigen, (iii) a tyrosinase antigen, and (iv) a transmembrane phosphatase with tensin homology (TPTE) antigen; and (b) a PD-1 inhibitor, wherein the melanoma is unresectable metastatic melanoma, thereby treating the subject.

12. The method of claim 11, wherein the melanoma is Stage III or IV melanoma.

13. The method of claim 11 or 12, wherein the subject has received prior therapy comprising a PD-1 or PD-L1 inhibitor.

14. The method of claim 13, wherein the subject is refractory or relapsed in response to the prior therapy. 12829787v1 Attorney Ref.2013237-1447 15. The method of claim 14, wherein the subject received the PD-1 or PD-L1 inhibitor once every 3 weeks.

16. The method of claim 15, wherein the subject received the PD-1 or PD-L1 inhibitor once every 3 weeks for at least 12 weeks.

17. The method of any one of claims 1-16, wherein the RNA therapy comprises about 1 ug to about 500 ug total RNA (e.g., about 7 ug to about 400 ug total RNA, about 10 ug to about 300 ug total RNA, or about 50 ug to about 100 ug total RNA).

18. The method of any one of claims 1-17, wherein the RNA therapy comprises one or more pharmaceutical compositions comprising the one or more RNA molecules.

19. The method of any one of claims 1-18, wherein the RNA therapy comprises a first pharmaceutical composition comprising an RNA molecule that encodes the NY-ESO-1 antigen, a second pharmaceutical composition comprising an RNA molecule that encodes the MAGE-A3 antigen, a third pharmaceutical composition comprising an RNA molecule that encodes the tyrosinase antigen, and a fourth pharmaceutical composition comprising an RNA molecule that encodes the TPTE antigen.

20. The method of claim 19, wherein the first, second, third, and fourth pharmaceutical compositions are administered concurrently.

21. The method of claim 19, wherein at least two of the pharmaceutical compositions are administered sequentially.

22. The method of any one of claims 1-21, wherein the PD-1 inhibitor is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody.

23. The method of any one of claims 1-22, wherein the PD-1 inhibitor is cemiplimab. 12829787v1 Attorney Ref.2013237-1447 24. The method of any one of claims 1-23, wherein the cancer therapy comprises administering: (a) the RNA therapy once weekly for 6 weeks, followed by once every 3 weeks; and (b) the PD-1 inhibitor once every 3 weeks.

25. The method of any one of the preceding claims, wherein the one or more RNA molecules comprises a 5’ cap or 5’ cap analogue.

26. The method of any one of the preceding claims, wherein the one or more RNA molecules comprises a sequence encoding a signal peptide.

27. The method of any one of the preceding claims, wherein the one or more RNA molecules comprise at least one non-coding regulatory element.

28. The method of any one of the preceding claims, wherein the one or more RNA molecules comprises a poly-adenine tail.

29. The method of claim 28, wherein the poly-adenine tail is or comprises a modified adenine sequence.

30. The method of any one of the preceding claims, wherein the one or more RNA molecules comprises at least one 5’ untranslated region (UTR) and / or at least one 3’ UTR.

31. The method of any one of the preceding claims, wherein the one or more RNA molecules comprises 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 that encodes at least one of the NY-ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen; 12829787v1 Attorney Ref.2013237-1447 (v) at least one sequence that encodes tetanus toxoid P2, tetanus toxoid P16, or both; (vi) a sequence encoding an MHC class I trafficking domain; (vii) at least one 3’UTR; and (viii) a poly-adenine tail.

32. The method of any one of the preceding claims, wherein the one or more RNA molecules comprise natural ribonucleotides.

33. The method of any one of the preceding claims, wherein the one or more RNA molecules comprise modified or synthetic ribonucleotides.

34. The method of any one of the preceding claims, wherein at least one of the NY- ESO-1 antigen, the MAGE-A3 antigen, the tyrosinase antigen, and the TPTE antigen are full- length, non-mutated antigens.

35. The method of any one of the preceding claims, 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.

36. The method of any one of the preceding claims, wherein the RNA therapy comprises lipid particles.

37. The method of claim 36, wherein the lipid particles comprise liposomes.

38. The method of claim 36 or 37, wherein the lipid particles comprise cationic liposomes.

39. The method of any one of claims 36-38, wherein the lipid particles comprise lipid nanoparticles. 12829787v1 Attorney Ref.2013237-1447 40. The method of any one of the preceding claims, wherein the subject is a human.

41. The method of any one of the preceding claims, wherein the RNA therapy induces an immune response in the subject.

42. The method of any one of the preceding claims, further comprising determining a level of the immune response in the subject.

43. The method of claim 42, wherein the level of the immune response is a de novo immune response induced by the RNA therapy.

44. The method of any one of the preceding claims, further comprising determining a level of the immune response in the subject before and after administration of the RNA therapy.

45. The method of any one of the preceding claims, further comprising comparing the level of the immune response in the subject after administration of the RNA therapy with the level of the immune response in the subject before administration of the RNA therapy.

46. The method of any one of the preceding claims, wherein the level of the immune response in the subject after administration of the RNA therapy is increased compared with the level of the immune response in the subject before administration of the RNA therapy.

47. The method of any one of claims 41-46, wherein the immune response in the subject is an adaptive immune response.

48. The method of any one of claims 41-46, wherein the immune response in the subject is a T-cell response.

49. The method of claim 48, wherein the T-cell response is or comprises a CD4+ response. 12829787v1 Attorney Ref.2013237-1447 50. The method of claim 48, wherein the T-cell response is or comprises a CD8+ response. 12829787v1

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