WO2025171109A1

WO2025171109A1 - Cancer vaccines expressing post-translational modified antigens and methods of use thereof

Info

Publication number: WO2025171109A1
Application number: PCT/US2025/014759
Authority: WO
Inventors: William V. Williams; Miguel LOPEZ-LAGO; Giuseppe Del Priore; Pravin KESARWANI; Vikas Bhardwaj; Xiaoyi Zheng
Original assignee: Briacell Therapeutics Corp USA
Current assignee: Briacell Therapeutics Corp USA
Priority date: 2024-02-07
Filing date: 2025-02-06
Publication date: 2025-08-14
Anticipated expiration: 2026-08-07

Abstract

The present disclosure provides modified human cancer cells that express post-translational modified antigens. The present disclosure also provides methods for using the modified human cancer cells of the present disclosure for treating a cancer in a subject. Methods for enhancing antigen immunogenicity through inducing enzymatic or non-enzymatic post-translational modification of antigens are also provided.

Description

CANCER VACCINES EXPRESSING POST-TRANSLATIONAL MODIFIED ANTIGENS AND METHODS OF USE THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 63/550,785, filed February 7, 2024, and U.S. Provisional Application No. 63/717,624, filed November 7, 2024, the disclosures of which are herein incorporated by reference in their entirety for all purposes.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. Said .xml copy, created on January 29, 2025 is named 102144-001210PC-1485893, and is 6,751 bytes in size.

BACKGROUND

[0003] Post-translational modifications (PTMs) are generated by adding small chemical groups to amino acid residues after the translation of proteins. PTMs alter protein structure, function, and localization and play a pivotal role in physiological and pathophysiological conditions.

[0004] Developing effective immune responses is essential in developing vaccines as well as in cancer immunotherapy. It is generally assumed that to be effective, a whole-cell immunotherapy needs to express immunogenic antigens. Thus, there is a need for improved antigen immunogenicity for cancer and related disease treatment. The present disclosure satisfies these needs and provides related advantages as well.

SUMMARY

[0005] In one aspect, the present disclosure provides a modified human cancer cell comprising a recombinant polynucleotide encoding an enzyme that induces a post-translational modification (PTM) of an antigen in the cell. In some embodiments, the enzyme comprises a citrullination enzyme, a cysteinylation enzyme, an acetylation enzyme, a hydroxylation enzyme, a phosphorylation enzyme, a methylation enzyme, a formylation enzyme, an oxidation enzyme, a hydroxylation enzyme, a ubiquitination enzyme, or a combination thereof.

[0006] In one aspect, the present disclosure provides a modified human cancer cell comprising an antigen, wherein the antigen comprises a non-enzymatic PTM. In some embodiments, the non-enzymatic PTM is induced by irradiation, inducing cellular senescence, a chemical reaction, a small molecule, a cell culture supplement, cell culture medium, oxidative stress, or a combination thereof.

[0007] In some embodiments, the antigen is an antigen of a pathogen, a tumor-specific antigen, a tumor-associated antigen, a neo-antigen, an allergen, an antigen that is a target of an autoimmune response, or a fragment thereof.

[0008] In some embodiments, the modified human cancer cell further comprises a recombinant polynucleotide encoding the antigen. In some embodiments, the PTM of the antigen results in the antigen being more immunogenetic in comparison to the antigen without the PTM.

[0009] In some embodiments, the PTM comprises phosphorylation, acetylation, ubiquitination, succinylation, methylation, malonylation, glycosylation, sumoylation, nitrosylation, glutathionylation, amidation, hydroxylation, palmitoylation, glutarylation, crotonylation, oxidation, myristoylation, sulfation, formylation, citrullination, prenylation, cysteinylation, deamidation, dehydration, or a combination thereof. In some embodiments, the PTM comprises cysteinylation and/or citrullination.

[0010] In some embodiments, the modified human cancer cell further comprises (a) one or more recombinant polynucleotides each encoding an allele of a human leukocyte antigen (HLA) class I gene; and/or (b) one or more recombinant polynucleotides each encoding an allele of an HLA class II gene. In some embodiments, one or more HLA alleles endogenous to the cell have been inactivated. In some embodiments, the HLA class I gene comprises an HLA- A gene, an HLA-B gene, an HLA-C gene, an HLA-E gene, an HLA-F gene, an HLA-G gene, a beta-2-microglobulin (B2M) gene, or a combination thereof. In some embodiments, the HLA class II gene comprises an HLA-DP gene, an HLA-DM gene, an HLA-DO gene, an HLA-DQ gene, an HLA-DR gene, or a combination thereof.

[0011] In some embodiments, the modified human cancer cell further comprises a recombinant polynucleotide encoding a cytokine. In some embodiments, the cytokine comprises a chemokine, an interferon, an interleukin, a tumor necrosis factor, or a combination thereof. In some embodiments, the cytokine comprises an early T cell activation antigen-1 (ETA-1), a lymphocyte-activating factor (LAF), an interleukin-1 family member (IL-la, IL-P, IL-IRa, IL-18, IL-33, IL-36Ra, IL-36a, IL-36P, IL-36y, IL-37, IL-38), an interleukin-2 (IL-2), an interleukin-3 (IL-3), an interleukin-4 (IL-4), an interleukin-5 (IL-5), an interleukin-6 (IL-6), an interleukin-7 (IL-7), an interleukin-8 (IL-8), an interleukin-9 (IL-9), an interleukin- 10 (IL- IO), an interleukin- 12 (IL-12), an interleukin- 13 (IL-13), an interleukin- 15 (IL-15), an interleukin- 17 (IL- 17), an interleukin- 18 (IL- 18), an interleukin-21 (IL-21), an interleukin-23 (IL-23), an interleukin-25 (IL-25), an interleukin-33 (IL-33), an interferon alpha (IFN-a), an interferon lambda 1 (IFN-LI (IL-29)), an interferon lambda 2 (IFN-X2 (IL-28A)), an interferon lambda 3 (IFN-X3 (IL-28B)), an interferon lambda 4 (IFN-X4), a granulocyte-macrophage colony-stimulating factor (GM-CSF), a macrophage CSF (CSF-1), a macrophage migration inhibitory factor (MIF), a CD40L molecule (CD40L), a RANTES molecule (RANTES), a monocyte chemoattractant protein (MCP-1), a monocyte inflammatory protein (MIP-la, MIP-

IP), a lymphotactin, a fractalkine, or a combination thereof. In some embodiments, the cytokine comprises GM-CSF.

[0012] In some embodiments, the modified human cancer cell further comprises a recombinant polynucleotide encoding a co-stimulatory molecule. In some embodiments, the co-stimulatory molecule comprises a CD86 molecule (CD86), CD80 molecule (CD80), 4-1BB ligand molecule (4-1BBL a.k.a CD137L), ICOS ligand molecule (ICOS-L), CD70 molecule (CD70 a.k.a. CD27L), CD40 molecule (CD40), 0X40 ligand molecule (OX40L), GITR ligand molecule (GITRL), TIM-4 molecule (TIM-4), LIGHT molecule (LIGHT), ICAM1 molecule (ICAM1), LFA3 molecule (LFA3), a CD30 molecule (CD30), or a combination thereof.

[0013] In some embodiments, the human cancer cell is a human cancer cell line. In some embodiments, the human cancer cell line is a breast cancer, prostate cancer, melanoma, or lung cancer cell line.

[0014] In some embodiments, the human cancer cell is a primary cancer cell. In some embodiments, the primary cancer cell is from a biopsy or a circulating cancer cell from a patient. In some embodiments, the patient has a breast cancer, prostate cancer, melanoma, or lung cancer.

[0015] In some embodiments, the modified human cancer cell described herein is a replication-incompetent modified human cancer cell. In some embodiments, the modified human cancer cell is rendered replication incompetent by irradiation, freeze-thawing, and/or mitomycin C treatment.

[0016] In another aspect, the present disclosure provides a composition comprising a modified human cancer cell described herein. In some embodiments, the composition comprising the modified human cancer cell described herein is formulated as a pharmaceutical composition and further comprising a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition further comprises a cryoprotectant.

[0017] In another aspect, the present disclosure provides a kit for treating a subject in need thereof with cancer comprising a pharmaceutical composition described herein. In some embodiments, the kit further comprises a therapeutically effective amount of IFN-a2b. The kit may comprise instructions for treating the subject using any of the methods described herein.

[0018] In another aspect, the present disclosure provides a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. In some embodiments, the method further comprises, prior to the administering step, (i) obtaining a sample from the subject; (ii) determining prevalent PTM(s) in the cell sample; and (iii) selecting a modified human cancer cell for administering to the subject, wherein the modified human cancer cell comprises the prevalent PTM(s). In some instances, the prevalent PTM(s) are enzymatic PTM(s) wherein the modified human cancer cell comprises a recombinant polynucleotide encoding an enzyme that induces the prevalent PTM(s). In other instances, the prevalent PTM(s) are non-enzymatic PTM(s) wherein the modified human cancer cell induces the prevalent PTM(s) by non- enzymatic means. In some embodiments, the non-enzymatic means comprises irradiation, inducing cellular senescence, a chemical reaction, a small molecule, a cell culture supplement, cell culture medium, oxidative stress, or a combination thereof. In some embodiments, the sample is a tumor biopsy or a liquid biopsy. In some embodiments, the liquid biopsy comprises circulating tumor cells (CTCs), circulating tumor DNA (ctDNA or cell free DNA), circulating RNA (cfRNA), exosomes, or a combination thereof. In some embodiments, the determining step comprises Next-generation sequencing (NGS) or immunopeptidome analysis of the sample. In some embodiments, the subject has a breast cancer, prostate cancer, melanoma, or lung cancer.

[0019] In another aspect, the present disclosure provides a method for enhancing the immunogenicity of an antigen in a human cancer cell, comprising inducing a post-translational modification (PTM) of the antigen in the cell, wherein the PTM enhances the immunogenicity of the antigen.

[0020] In some embodiments, the PTM comprises phosphorylation, acetylation, ubiquitination, succinylation, methylation, malonylation, glycosylation, sumoylation, nitrosylation, glutathionylation, amidation, hydroxylation, palmitoylation, glutarylation, crotonylation, oxidation, myristoylation, sulfation, formylation, citrullination, prenylation, cysteinylation, deamidation, dehydration, or a combination thereof. In some embodiments, the PTM comprises cysteinylation and/or citrullination. In some embodiments, the antigen is an antigen of a pathogen, a tumor-specific antigen, a tumor-associated antigen, a neo-antigen, an allergen, an antigen that is a target of an autoimmune response, or a fragment thereof. In some embodiments, the cell comprises a recombinant polynucleotide encoding the antigen.

[0021] In some embodiments, the cell comprises (a) one or more recombinant polynucleotides each encoding an allele of a human leukocyte antigen (HLA) class I gene; and/or (b) one or more recombinant polynucleotides each encoding an allele of an HLA class II gene. In some embodiments, the cell comprises a recombinant polynucleotide encoding a cytokine. In some embodiments, the cell comprises a recombinant polynucleotide encoding a co-stimulatory molecule. In some embodiments, the cell is a human cancer cell line. In some embodiments, the cell is a primary cancer cell. In some embodiments, the cell is a breast cancer cell, a prostate cancer cell, a melanoma cell, or a lung cancer cell.

[0022] In some embodiments, the PTM is induced by an enzyme. In some embodiments, the enzyme comprises a citrullination enzyme, a cysteinylation enzyme, an acetylation enzyme, a hydroxylation enzyme, a phosphorylation enzyme, a methylation enzyme, a formylation enzyme, an oxidation enzyme, a hydroxylation enzyme, a ubiquitination enzyme, or a combination thereof. In some embodiments, the cell comprises a recombinant polynucleotide encoding the enzyme. In some embodiments, the enzyme comprises a citrullination enzyme. In some embodiments, the enzyme comprises a cysteinylation enzyme.

[0023] In some embodiments, the PTM is a non-enzymatic PTM. In some embodiments, the non-enzymatic PTM is induced by irradiation, inducing cellular senescence, a chemical reaction, a small molecule, a cell culture supplement, cell culture medium, oxidative stress, or a combination thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1 depicts post-translational modifications of MHC class I and/or II peptides in the immunopeptidome of the genetically modified human breast cancer cell line Bria-IMT.

[0025] FIG. 2 depicts a workflow of an immunopeptidome analysis, detailing the processing and presentation of various categories of tumor antigens, including tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), cancer-testis antigens (CTAs), post-translationally modified (PTM) antigens, and unconventional antigens (UCAs) in the SV-BR-1 cell line.

[0026] FIG. 3 depicts a workflow of a Class II or Class I antigen/epitope mapping assay.

[0027] FIGS. 4A-4D depict identification of T cell epitopes by a CD 154 (Class II) or CD 137 (Class I) epitope mapping assay. The assay identified five peptides that induce an immunogenic response: (FIG. 4A) the pre-vaccination sample responded to a cysteinylated Desmoplakin MHC Class II peptide: QGSS(cys-mod)IAGIYNETTKQKLG (SEQ ID NO: 1); (FIG. 4B) the post-vaccination sample responded to two citrullinated Filaggrin MHC Class II peptides: KLAQYYESTCitKEN (SEQ ID NO: 2), top, and FKLAQYYESTCitKEN (SEQ ID NO: 3), bottom; (FIG. 4C) the post-vaccination sample responded to an MFGE8 MHC Class I peptide: GLQHWVPEL (SEQ ID NO: 4); and (FIG. 4D) the pre-vaccination sample responded to an MHC Class II peptide from COX7C: ATPFLVVRHQLLKT (SEQ ID NO: 5).

DETAILED DESCRIPTION

I. Introduction

[0028] Post-translation modifications (PTMs) add small chemical moieties or chemical modifications at individual amino acids in translated proteins. PTMs regulate protein stability, folding, function, and their interaction with other biomolecules. PTMs can correlate with tumor progression, growth, and survival by modifying the normal functions of the protein in tumor cells. In addition, PTMs are frequently involved in many diseases beside cancer.

[0029] Induction of effective immune responses with whole-cell immunotherapies is an effective approach to treat and prevent diseases such as cancer. It is generally assumed that to be effective, a cancer vaccine needs to express high immunogenic antigens co-expressed in patient tumor cells, and that antigen-presenting cells (APC) such as dendritic cells (DCs) need to cross-present such antigens following uptake of vaccine cell fragments.

[0030] The transformational technology central to the present disclosure is the development of whole-cell therapeutic vaccines expressing highly immunogenic antigens. In particular, the modified human cancer cells described herein comprise post-translational modified antigens which are bound to HLA class I and/or class II molecules. These post-translationally modified antigens can overcome tolerance and induce immune responses in various diseases and cancers. Therefore, in combination with the Bria-IMT™ platform technology, a high immunogenic cancer vaccine can be developed by inducing enzymatic or non-enzymatic post-translational modifications (PTMs) of antigens in modified human cancer cells. In certain aspects, the modified human cancer cells described herein express one or more enzymes that induce PTMs of one or more antigens. In certain aspects, the modified human cancer cells described herein further express one or more immunomodulatory cytokines, one or more co-stimulatory molecules, one or more human major histocompatibility complex-I (MHC-I) molecules, and one or more MHC-II molecules.

[0031] Described herein are compositions of modified human cancer cells or cell lines for targeted immunotherapy of cancers. Additionally, kits containing the “off-the-shelf’ cell lines and methods for preventing or treating cancer in a subject in need thereof are provided.

II. Definitions

[0032] Unless specifically indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this disclosure belongs. In addition, any method or material similar or equivalent to a method or material described herein can be used in the practice of the present disclosure. For purposes of the present disclosure, the following terms are defined.

[0033] The terms “a,” “an,” or “the” as used herein not only include aspects with one member, but also include aspects with more than one member. For instance, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the agent” includes reference to one or more agents known to those skilled in the art, and so forth.

[0034] The terms “about” and “approximately” shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Typical, exemplary degrees of error are within 20 percent (%), preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, and particularly in biological systems, the terms “about” and “approximately” may mean values that are within an order of magnitude, preferably within 5-fold and more preferably within 2-fold of a given value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated.

[0035] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, rats, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0036] As used herein, the term “administering” includes oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intratumoral, intradermal, intralymphatic, intrathecal, intranasal, or subcutaneous administration to a subject. Administration is by any route, including parenteral and transmucosal (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). Parenteral administration includes, e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial. Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, etc.

[0037] The term “treating” refers to an approach for obtaining beneficial or desired results including, but not limited to, a therapeutic benefit and/or a prophylactic benefit. By therapeutic benefit is meant any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. Therapeutic benefit can also mean to effect a cure of one or more diseases, conditions, or symptoms under treatment.

[0038] The term “effective amount” or “sufficient amount” refers to the amount of a modified cancer cell or other composition that is sufficient to effect beneficial or desired results. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific amount may vary depending on one or more of: the particular agent chosen, the target cell type, the location of the target cell in the subject, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, and the physical delivery system in which it is carried.

[0039] For the purposes herein, an effective amount is determined by such considerations as may be known in the art. The amount must be effective to achieve the desired therapeutic effect in a subject suffering from cancer. The desired therapeutic effect may include, for example, amelioration of undesired symptoms associated with cancer, prevention of the manifestation of such symptoms before they occur, slowing down the progression of symptoms associated with cancer, slowing down or limiting any irreversible damage caused by cancer, lessening the severity of or curing a cancer, or improving the survival rate or providing more rapid recovery from a cancer.

[0040] The effective amount depends, inter alia, on the type and severity of the disease to be treated and the treatment regime. The effective amount is typically determined in appropriately designed clinical trials (dose range studies) and the person versed in the art will know how to properly conduct such trials in order to determine the effective amount. As generally known, an effective amount depends on a variety of factors including the distribution profile of a therapeutic agent (e.g., a whole-cell cancer vaccine) or composition within the body, the relationship between a variety of pharmacological parameters (e.g., half-life in the body) and undesired side effects, and other factors such as age and gender, etc.

[0041] The term “pharmaceutically acceptable carrier” refers to a substance that aids the administration of an active agent to a cell, an organism, or a subject. “Pharmaceutically acceptable carrier” refers to a carrier or excipient that can be included in the compositions of the disclosure and that causes no significant adverse toxicological effect on the subject. Nonlimiting examples of pharmaceutically acceptable carriers include water, sodium chloride, normal saline solutions, lactated Ringer’s, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavors and colors, liposomes, dispersion media, microcapsules, cationic lipid carriers, isotonic and absorption delaying agents, and the like. The carrier may also be substances for providing the formulation with stability, sterility and isotonicity (e.g. antimicrobial preservatives, antioxidants, chelating agents and buffers), for preventing the action of microorganisms (e.g. antimicrobial and antifungal agents, such as parabens, chlorobutanol, sorbic acid and the like) or for providing the formulation with an edible flavor etc. In some instances, the carrier is an agent that facilitates the delivery of a modified cancer cell to a target cell or tissue. One of skill in the art will recognize that other pharmaceutical carriers are useful in the present disclosure.

[0042] The term “nucleic acid” or “nucleotide” as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA, RNA, and hybrids thereof. DNA may be in the form of, e.g., antisense molecules, plasmid DNA, DNA-DNA duplexes, pre-condensed DNA, PCR products, vectors (Pl, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA may be in the form of small interfering RNA (siRNA), Dicer-substrate dsRNA, small hairpin RNA (shRNA), asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2’- O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605- 2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)). “Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. “Bases” include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkylhalides.

[0043] The term “enzyme” or “PTM enzyme” as used herein refers to a protein that is capable of inducing a chemical or structural change to a translated protein or peptide. This class includes multiple types of enzymes, including but not limited to, citrullination enzymes, cysteinylation enzymes, acetylation enzymes, hydroxylation enzymes, phosphorylation enzymes, methylation enzymes, formylation enzymes, oxidation enzymes, hydroxylation enzymes, and ubiquitination enzymes.

[0044] The term “gene” means the segment of DNA involved in producing a polypeptide chain. The DNA segment may include regions preceding and following the coding region (leader and trailer) involved in the transcription/translation of the gene product and the regulation of the transcription/translation, as well as intervening sequences (introns) between individual coding segments (exons). [0045] The terms “vector” and “expression vector” refer to a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular polynucleotide sequence in a host cell. An expression vector may be part of a plasmid, viral genome, or nucleic acid fragment. Typically, an expression vector includes a polynucleotide to be transcribed, operably linked to a promoter. The term “promoter” is used herein to refer to an array of nucleic acid control sequences that direct transcription of a nucleic acid. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. Other elements that may be present in an expression vector include those that enhance transcription (e.g., enhancers) and terminate transcription (e.g., terminators). In the context of the present disclosure, co-expression of multiple genes (e.g., polynucleotides ending an HLA class I allele and/or an HLA class II allele) may be achieved by co-transfection of two or more vectors, the use of multiple or bidirectional promoters, or the creation of bicistronic or multi ci str onic vectors. Gene co-expression may be driven by using a plasmid with multiple, individual expression cassettes. Generally, each promoter creates unique mRNA transcripts for each gene that is expressed. Bicistronic or multici stronic vectors simultaneously express two or more separate proteins from the same mRNA. Bicistronic vectors may contain an Internal Ribosome Entry Site (IRES) to allow for initiation of translation from an internal region of the mRNA. Multicistronic vectors containing one or more self-cleaving 2A peptides are advantageous as they allow gene co-expression from the same cassette. In some instances, multicistronic vectors are preferred when only a portion of the plasmid is packaged for viral delivery, or the relative expression levels between two or more genes is important.

[0046] The terms “self-cleaving peptide” and “self-cleaving 2A peptide” refer to a short peptide that can produce equimolar levels of multiple genes from the same mRNA. These peptides were first discovered in picornaviruses. Self-cleaving peptides are thought to function by making the ribosome skip the synthesis of a peptide bond at the C-terminus of a 2A element, leading to separation between the end of the 2A sequence and the next peptide downstream. The “cleavage” occurs between the glycine and proline residues found at the C- terminus, meaning the upstream cistron typically has a few additional residues added to the end, while the downstream cistron typically starts with the proline. Non-limiting examples of self-cleaving peptides include T2A, P2A, E2A, and F2A. [0047] “Recombinant” refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism. For example, a recombinant polynucleotide (or a copy or complement of a recombinant polynucleotide) is one that has been manipulated using well known methods. A recombinant expression cassette comprising a promoter operably linked to a second polynucleotide (e.g., a coding sequence) can include a promoter that is heterologous to the second polynucleotide as the result of human manipulation (e.g., by methods described in Sambrook el al., Molecular Cloning - A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1989) or Current Protocols in Molecular Biology Volumes 1-3, John Wiley & Sons, Inc. (1994-1998)). A recombinant expression cassette (or expression vector) typically comprises polynucleotides in combinations that are not found in nature. For instance, human manipulated restriction sites or plasmid vector sequences can flank or separate the promoter from other sequences. A recombinant protein is one that is expressed from a recombinant polynucleotide, and recombinant cells, tissues, and organisms are those that comprise recombinant sequences (polynucleotide and/or polypeptide). A recombinant cell is one that has been modified (e.g., transfected or transformed), with a recombinant nucleotide, expression vector or cassette, or the like.

[0048] The term “amino acid” refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. Amino acids include naturally-occurring a-amino acids and their stereoisomers, as well as unnatural (non-naturally occurring) amino acids and their stereoisomers. “Stereoisomers” of a given amino acid refer to isomers having the same molecular formula and intramolecular bonds but different three-dimensional arrangements of bonds and atoms (e.g., an L-amino acid and the corresponding D-amino acid).

[0049] Naturally-occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O- phosphoserine. Naturally-occurring a-amino acids include, without limitation, alanine (Ala), cysteine (Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (He), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gin), serine (Ser), threonine (Thr), valine (Vai), tryptophan (Trp), tyrosine (Tyr), and their combinations. Stereoisomers of a naturally- occurring a-amino acids include, without limitation, D-alanine (D-Ala), D-cysteine (D-Cys), D-aspartic acid (D-Asp), D-glutamic acid (D-Glu), D-phenylalanine (D-Phe), D-histidine (D- His), D-isoleucine (D-Ile), D-arginine (D-Arg), D-lysine (D-Lys), D-leucine (D-Leu), D- methionine (D-Met), D-asparagine (D-Asn), D-proline (D-Pro), D-glutamine (D-Gln), D- serine (D-Ser), D-threonine (D-Thr), D-valine (D-Val), D-tryptophan (D-Trp), D-tyrosine (D- Tyr), and their combinations.

[0050] Unnatural (non-naturally occurring) amino acids include, without limitation, amino acid analogs, amino acid mimetics, synthetic amino acids, TV-substituted glycines, and N- methyl amino acids in either the L- or D-configuration that function in a manner similar to the naturally-occurring amino acids. For example, “amino acid analogs” can be unnatural amino acids that have the same basic chemical structure as naturally-occurring amino acids (i.e., a carbon that is bonded to a hydrogen, a carboxyl group, an amino group) but have modified side-chain groups or modified peptide backbones, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. “Amino acid mimetics” refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally-occurring amino acid. Amino acids may be referred to by either the commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0051] The terms “identity,” “substantial identity,” “similarity,” “substantial similarity,” “homology” and the related terms and expressions used in the context of describing amino acid sequences refer to a sequence that has at least 60% sequence identity to a reference sequence. Examples include at least: 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, sequence identity, as compared to a reference sequence using the programs for comparison of amino acid sequences, such as BLAST using standard parameters. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default (standard) program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. A “comparison window” includes reference to a segment of any one of the number of contiguous positions (from 20 to 600, usually about 50 to about 200, more commonly about 100 to about 150), in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known. Optimal alignment of sequences for comparison may be conducted, for example, by the local homology algorithm of Smith and Waterman, 1981, by the homology alignment algorithm of Needleman and Wunsch, 1970, by the search for similarity method of Pearson and Lipman, 1988, by computerized implementations of these algorithms (for example, BLAST), or by manual alignment and visual inspection.

[0052] Algorithms that are suitable for determining percent sequence identity and sequence similarity include BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, and Altschul et al., 1977, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positivevalued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits acts as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negativescoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=l, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (Henikoff and Henikoff, 1989). The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (Karlin and Altschul, 1993).

[0053] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms encompass amino acid chains of any length, including full-length proteins (z.e., alleles), wherein the amino acid residues are linked by covalent peptide bonds. As used herein, the amino acid sequence of a polypeptide is presented from the N-terminus to the C-terminus. In other words, when describing an amino acid sequence of a polypeptide, the first amino acid at the N-terminus is referred to as the “first amino acid.”

[0054] When used in the context of describing partners of a recombinant polypeptide, the term “heterologous” refers to the relationship of one polynucleotide fusion partner to the other polynucleotide fusion partner: the manner in which the fusion partners are present in the recombinant polynucleotide is not one that can be found in a polynucleotide naturally occurs or encoding a naturally occurring protein. A “heterologous polynucleotide” may encode a peptide containing modifications of a naturally occurring protein sequence or a portion thereof, such as deletions, additions, or substitutions of one or more amino acid residues.

[0055] The term “cancer” is intended to include any member of a class of diseases characterized by the uncontrolled growth of aberrant cells. The term includes all known cancers and neoplastic conditions, whether characterized as malignant, benign, recurrent, soft tissue, or solid, and cancers of all stages and grades including advanced, pre- and post- metastatic cancers. Examples of different types of cancer include, but are not limited to, gynecological cancers (e.g., ovarian, cervical, uterine, vaginal, and vulvar cancers); lung cancers (e.g., non-small cell lung cancer, small cell lung cancer, mesothelioma, carcinoid tumors, lung adenocarcinoma); breast cancers (e.g., triple-negative breast cancer, ductal carcinoma in situ, invasive ductal carcinoma, tubular carcinoma, medullary carcinoma, mucinous carcinoma, papillary carcinoma, cribriform carcinoma, invasive lobular carcinoma, inflammatory breast cancer, lobular carcinoma in situ, Paget’s disease, Phyllodes tumors); digestive and gastrointestinal cancers such as gastric cancer (e.g., stomach cancer), colorectal cancer, gastrointestinal stromal tumors (GIST), gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer, bile duct cancer, small intestine cancer, and esophageal cancer; thyroid cancer; gallbladder cancer; liver cancer; pancreatic cancer; appendix cancer; prostate cancer (e.g., prostate adenocarcinoma); renal cancer (e.g., renal cell carcinoma); cancer of the central nervous system (e.g., glioblastoma, neuroblastoma, medulloblastoma); skin cancer (e.g., melanoma); bone and soft tissue sarcomas (e.g., Ewing’s sarcoma); lymphomas; choriocarcinomas; urinary cancers (e.g., urothelial bladder cancer); head and neck cancers; and bone marrow and blood cancers (e.g., acute leukemia, chronic leukemia (e.g., chronic lymphocytic leukemia), lymphoma, multiple myeloma). As used herein, a “tumor” comprises one or more cancerous cells. [0056] The term “allele” refers to a particular form or variant of a gene. Alleles can result from, for example, nucleotide substitutions, additions, or deletions, or can represent a variable number of short nucleotide repeats. In the context of human leukocyte antigen (HLA) genes, HLA alleles are named by the World Health Organization Naming Committee for Factors of the HLA system. Under this system, an HLA gene name is followed by a series of numerical fields. At a minimum, two numerical fields are included. As a non-limiting example, HLA- A*02: 101 denotes a specific allele of the HLA-A gene. The first field, separated from the gene name by an asterisk, denotes an allele group. The second field, separated from the first field by a colon, denotes the specific HLA protein that is produced. In some instances, a longer name is used (e.g., HLA-A*02: 101 :01 :02N). In this example, the third numerical field denotes whether a synonymous DNA substitution is present within the coding region, and the fourth numerical field denotes differences between alleles that exist in the non-coding region. In some other instances, an HLA allele name contains a letter at the end. Under the HLA allele naming system, “N” denotes that the allele is a null allele (i.e., the allele produces a non-functional protein), “L” denotes that the allele results in lower than normal cell surface expression of the particular HLA protein, “S” denotes that the allele produces a soluble protein not found on the cell surface, “Q” denotes a questionable allele (i.e., an allele that nay not affect normal expression), “C” denotes that the allele produces a protein that is present in cell cytoplasm but is not present at the cell surface, and “A” denotes an allele that results in aberrant expression (i.e., it is uncertain whether the particular HLA protein is expressed). One of skill in the art will be familiar with the various gene alleles and their naming conventions.

[0057] The term “human leukocyte antigen (HLA)” refers to a gene complex that encodes human major histocompatibility complex (MHC) proteins, which are a set of cell surface proteins that are essential for recognition of foreign molecules by the adaptive immune system. The HLA complex is found within a 3 Mbp stretch of chromosome 6p21. Class I MHC proteins, which present peptides from inside the cell, are encoded by the HLA-A, HLA-B, HLA- C, HLA-E, HLA-F, and HLA-G genes. HLA-A, HLA-B, and HLA-C genes are more polymorphic, while HLA-E, HLA-F, and HLA-G genes are less polymorphic. HLA-K and HLA- L are also known to exist as pseudogenes. In addition, beta-2-microglobulin is an MHC class I protein, encoded by the (B2M) gene. Non-limiting examples of HLA-A nucleotide sequences are set forth under GenBank reference numbers NM_001242758 and NM_002116. A nonlimiting example of an HLA-B nucleotide sequence is set forth under GenBank reference number NM_005514. Non-limiting examples of HLA-C nucleotide sequences are set forth under GenBank reference numbers NM_OO 1243042 andNM_002117. Anon-limiting example of an HLA-E nucleotide sequence is set forth under GenBank reference number NM 005516. A non-limiting example of an HLA-F nucleotide sequence is set forth under GenBank reference number NM_018950. A non-limiting example of an HLA-G nucleotide sequence is set forth under GenBank reference number NM 002127. A non-limiting example of a B2M nucleotide sequence is set forth under GenBank reference number NM 004048.

[0058] Class II MHC proteins, which present antigens from the outside of the cell to T lymphocytes, are encoded by the HLA-DP, HLA-DM, HLA-DO, HLA-DQ, and HLA-DR genes. HLA-DM genes include HLA-DMA and HLA-DMB. HLA-DO genes include HLA-DOA and HLA-DOB. HLA-DP genes include HLA-DP A 1 AHLA-DPBL HLA-DQ genes include HLA- DQA1, HLA-DQA2, HLA-DQB1, and HLA-DQB2. HLA-DR genes include HLA-DRA, HLA- DRB1, HLA-DRB3, HLA-DRB4, and HLA-DRB5. Non-limiting examples of HLA-DMA and HLA-DMB nucleotide sequences are set forth under GenBank reference numbers NM 006120 and NM 002118, respectively. Non-limiting examples of HLA-DRA, HLA-DRB1, HLA- DRB3, HLA-DRB4, and HLA-DRB5 nucleotide sequences are set forth in GenBank reference numbers NM_01911, NM_002124, NM_022555, NM_021983, NM_002125, respectively.

[0059] The term “vaccine” refers to a biological composition that, when administered to a subject, has the ability to produce an acquired immunity to a particular pathogen or disease in the subject. Typically, one or more antigens, or fragments of antigens, that are associated with the pathogen or disease of interest are administered to the subject. Vaccines can comprise, for example, inactivated or attenuated organisms (e.g., bacteria or viruses), cells, proteins that are expressed from or on cells (e.g., cell surface proteins), proteins that are produced by organisms (e.g., toxins), or portions of organisms (e.g., viral envelope proteins). In some instances, cells are engineered to express proteins such that, when administered as a vaccine, they enhance the ability of a subject to acquire immunity to that particular cell type (e.g., enhance the ability of a subject to acquire immunity to a cancer cell). As used herein, the term “vaccine” or “wholecell cancer vaccine” includes but is not limited to the modified cancer cell(s) of the present disclosure.

[0060] The term “cytokine” refers to small proteins released by cells that have a specific effect on the interactions and communications between cells. Cytokines are generally known as lymphokines (e.g., cytokines made by lymphocytes), monokines (e.g., cytokines made by monocytes), or chemokines (e.g., cytokines made by one leukocyte and acting on other leukocytes). Cytokines may act on the cells that secrete them (e.g., autocrine action), on nearby cells (e.g., paracrine action), or on distant cells (e.g., endocrine action). In the context of the present disclosure, cytokines may comprise a chemokine, an interferon, an interleukin, and/or a tumor necrosis factor (TNF). As an example, cytokines may comprise an early T cell activation antigen- 1 (ETA-1), a lymphocyte-activating factor (LAF), an interleukin- 1 family member (IL-la, IL-0, IL-IRa, IL-18, IL-33, IL-36Ra, IL-36a, IL-36 , IL-36y, IL-37, IL-38), an interleukin-2 (IL-2), an interleukin-3 (IL-3), an interleukin-4 (IL-4), an interleukin-5 (IL-5), an interleukin-6 (IL-6), an interleukin-7 (IL-7), an interleukin-8 (IL-8), an interleukin-9 (IL-9), an interleukin- 10 (IL-10), an interleukin- 12 (IL-12), an interleukin- 13 (IL-13), an interleukin- 15 (IL- 15), an interleukin- 17 (IL- 17), an interleukin- 18 (IL- 18), an interleukin-23 (IL-21), an interleukin-23 (IL-23), an interleukin-25 (IL-25), an interleukin-33 (IL-33), a type I interferon family member (IFN-a, IFN-p, IFN-e, IFN-K, IFN-ro), a type II interferon family member ( IFN- y), a type III interferon family member (IFN-kl (IL-29), IFN-X2 (IL-28A), IFN- .3 (IL-28B), IFN-X4), a granulocyte-macrophage colony-stimulating factor (GM-CSF), a macrophage CSF (CSF-1), a macrophage migration inhibitory factor (MIF), a CD40L molecule (CD40L), a RANTES molecule (RANTES), a monocyte chemoattractant protein (MCP-1), a monocyte inflammatory protein (MIP-la, MIP-10), a lymphotactin, and/or a fractalkine.

[0061] The term “granulocyte macrophage colony-stimulating factor (GM-CSF)” refers to a monomeric glycoprotein also known as “colony stimulating factor (CSF2)” that is secreted by cells such as macrophages, T cells, mast cells, natural killer (NK) cells, endothelial cells, and fibroblasts. GM-CSF functions as a cytokine that affects a number of cell types, in particular macrophages and eosinophils. As part of the immune/inflammatory cascade, GM-CSF stimulates stem cells to produce granulocytes (i.e., neutrophils, eosinophils, and basophils) and monocytes. The monocytes subsequently mature into macrophages and dendritic cells after tissue infiltration. A non-limiting example of a CSF2 nucleotide sequence (the gene that encodes GM-CSF) in humans is set forth under GenBank reference number NM 000758.

[0062] The term “interferon” refers to a cytokine that is produced in response to infection or other inflammatory stimuli. Interferons are signaling proteins that are synthesized and released by host cells in response to a pathogen (e.g., viruses, bacteria, parasites, tumor cells). Interferons are classified into three subgroups: type I interferons, type II interferon (IFNy), and type III interferons. Functionally, these cytokines modulate immune cell function. Although type III interferons are structurally distinct from type I interferons, they have overlapping functions, and both signal through the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathway to induce transcription of interferon-stimulated genes (ISGs) and promote immune responses. (See e.g., Goel et al. (2021). Interferon lambda in inflammation and autoimmune rheumatic diseases. Nat Rev Rheumatol 17, 349-362). Type I interferon proteins include IFN-a, IFN-P, IFN-s, IFN-K, IFN-T, IFN-5, IFN-^, IFN-CO, and IFN- v. Interferon alpha proteins are produced by leukocytes and are mainly involved in the innate immune response. Genes that encode IFN-a proteins include IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21. Nonlimiting examples ofIFNAl, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21 human nucleotide sequences are set forth in Gene Bank reference numbers NM_024013, NM_000605, NM_021068, NM_002169, NM_021002, NM_021057, NM_002170, NM_002171, NM_006900, NM_002172, NM_002173, NM_021268, and NM_002175, respectively. As an example, the gene IFNA2 encodes IFN- a2a, IFN-a2b, and IFN-a2c variants. As used herein, the terms “IFN-a” and “IFN-a2” are used interchangeably, and they refer to interferon proteins IFN-a2a or IFN-a2b. Type III interferon proteins include interferon lambda 1 (IFN-X.1 (IL-29), interferon lambda 2 (IFN-A2 (IL-28A)), interferon lambda 3 (IFN-A.3 (IL-28B)), and interferon lambda 4 (IFN-X4). Interferon lambda family members signal through the common IL-10 receptor subunit 2 (IL-10R2). Human interferon lambda proteins are encoded by four IFNL genes, IFNL1 (IL29 IFNL2 (IL28A), IFNL3 (IL28B), and UN 1.4.

[0063] The term “co-stimulatory molecule” refers to a cell surface molecule that amplifies or counteracts the initial activating signals provided to T cells from the T cell receptor (TCR) following its interaction with an antigen/major histocompatibility complex (MHC). Costimulatory molecules generally may influence T cell differentiation and fate. Co-stimulatory molecules belong to three major families, namely the immunoglobulin (Ig) superfamily, the tumor necrosis factor (TNF) - TNF receptor (TNFR) superfamily, and the T cell Ig and mucin (TIM) domain family. See e.g., Rodriguez-Manzanet, Roselynn et al. “The costimulatory role of TIM molecules.” Immunological reviews vol. 229,1 (2009): 259-70.) Exemplary costimulatory molecules and ligands include, but are not limited to, CD28 and ligands B7-1 (CD80), CTLA-4, PDL-1, orB7-2 (CD86), CTLA-4 and ligands B 7-1 (CD80) orB7-2 (CD86), ICOS and ligand ICOS-L, CD27 and ligand CD70, CD30 and ligand CD30L, CD40 and ligand CD40L (a.k.a. CD 154), 0X40 and ligand OX40L, GITR and ligand GITRL, TIM-1 and ligands TIM-1, TIM-4, IgA, or phosphatidylserine (PtdSer), TIM-2 and ligands H-ferritin or semaphorin 4A (Sem4A), and TIM-4 and ligand phosphatidylserine (PtdSer). In the context of the present disclosure, co-stimulatory molecules may comprise a CD86 molecule (CD86), CD80 molecule (CD80), 4-1BB ligand molecule (4-1BBL a.k.a CD137L), ICOS ligand molecule (ICOS-L), CD70 molecule (CD70 a.k.a. CD27L), CD40 molecule (CD40), 0X40 ligand molecule (OX40L), GITR ligand molecule (GITRL), TIM-4 molecule (TIM-4), LIGHT molecule (LIGHT), ICAM1 molecule (ICAM1), LFA3 molecule (LFA3), CD30 molecule (CD30), and combinations thereof (See e.g., FIG. 5).

[0064] The term “tumor antigen” refers to an antigenic substance produced in tumor cells that may trigger an immune response in the host. Tumor antigens generally refer to tumor- associated antigen (TAAs) or tumor-specific antigens (TSAs). Typically, TSAs are found in cancer cells only and are not in healthy (e.g., non-cancerous) cells. TSAs may arise from oncogenic driver mutations that generate novel peptide sequences (e.g., neoantigens). A nonlimiting example of a TSA is alphafetoprotein (AFP) expressed in germ cell tumors and hepatocellular carcinoma. TAAs have elevated levels in tumor cells and may express at lower levels in healthy cells. A non-limiting example of a TAA is melanoma-associated antigen (MAGE) expressed in the testis along with malignant melanoma.

[0065] The term “survival” refers to a length of time following the diagnosis of a disease and/or beginning or completing a particular course of therapy for a disease (e.g., cancer). The term “overall survival” includes the clinical endpoint describing patients who are alive for a defined period of time after being diagnosed with or treated for a disease, such as cancer. The term “disease-free survival” includes the length of time after treatment for a specific disease (e.g., cancer) during which a patient survives with no sign of the disease (e.g., without known recurrence). In certain embodiments, disease-free survival is a clinical parameter used to evaluate the efficacy of a particular therapy, which is usually measured in units of 1 or 5 years. The term “progression-free survival” includes the length of time during and after treatment for a specific disease (e.g., cancer) in which a patient is living with the disease without additional symptoms of the disease. In some embodiments, survival is expressed as a median or mean value.

III. Detailed Description of the Embodiments

[0066] The present disclosure is based, in part, on the inventors’ discovery that modified human cancer cells (e.g., SV-BR-l-GM, a modified human breast tumor cell line secreting GM-CSF) containing multiple post-translational modified antigens which are bound to HLA class I and/or class II molecules. The inventors discovered that these post-translationally modified antigens can overcome tolerance and induce an immune response in autoimmune diseases. Therefore, in combination with the Bria-IMT™ platform technology, a highly immunogenic cancer vaccine can be developed by inducing enzymatic or non-enzymatic post- translational modifications (PTMs) of antigens in modified cancer cells, such as Bria-OTS cell lines previously developed by the inventors.

[0067] In one aspect, the present disclosure provides a modified human cancer cell comprising a recombinant polynucleotide encoding an enzyme that induces a post-translational modification (PTM) of an antigen in the cell. In some embodiments, the enzyme comprises a citrullination enzyme, a cysteinylation enzyme, an acetylation enzyme, a hydroxylation enzyme, a phosphorylation enzyme, a methylation enzyme, a formylation enzyme, an oxidation enzyme, a hydroxylation enzyme, a ubiquitination enzyme, or a combination thereof. In some embodiments, the modified human cancer cell comprises a recombinant polynucleotide encoding a citrullination enzyme that induces a PTM of an antigen in the cell. In some embodiments, the modified human cancer cell comprises a recombinant polynucleotide encoding a cysteinylation enzyme that induces a PTM of an antigen in the cell. Without being bound to any theory, the introducing an exogenous PTM enzyme into a cell advantageously allows the cell to produce more PTMs of one or more antigens, resulting in the one or more antigens being more immunogenetic in comparison to the cell without the exogenous PTM enzyme. In some embodiments, the modified human cancer cell comprising a recombinant polynucleotide encoding an enzyme that induces a PTM of an antigen further comprises a recombinant polynucleotide encoding the antigen. In some embodiments, the modified human cancer cell comprising a recombinant polynucleotide encoding a PTM enzyme further comprises (i) one or more recombinant polynucleotides each encoding an allele of a human leukocyte antigen (HLA) class I gene; and/or (ii) one or more recombinant polynucleotides each encoding an allele of an HLA class II gene. In some embodiments, one or more HLA alleles endogenous to the cell have been inactivated in the modified human cancer cell. In some embodiments, the modified human cancer cell comprising a recombinant polynucleotide encoding a PTM enzyme further comprises a recombinant polynucleotide encoding a cytokine. In some embodiments, the modified human cancer cell comprising a recombinant polynucleotide encoding a PTM enzyme further comprises a recombinant polynucleotide encoding a co- stimulatory molecule. In some embodiments, the modified human cancer cell comprises one or more recombinant polynucleotides encoding a) an enzyme that induces a PTM of an antigen in the cell, b) the antigen; c) an allele of an HLA class I gene and/or an allele of an HLA class II gene; d) a cytokine; and/or e) a co-stimulatory molecule. In some embodiments, the modified human cancer cell comprising a recombinant polynucleotide encoding a PTM enzyme that targets an antigen in the cell further comprises one or more recombinant polynucleotides encoding a) the antigen; b) an allele of an HLA class I gene and/or an allele of an HLA class II gene; c) a cytokine; and/or d) a co-stimulatory molecule. In particular embodiments, the modified human cancer cell comprising a recombinant polynucleotide encoding a PTM enzyme that targets an antigen in the cell further comprises a) a recombinant polynucleotide encoding the antigen; b) one or more recombinant polynucleotides each encoding an allele of a HLA class I gene and/or one or more recombinant polynucleotides each encoding an allele of an HLA class II gene; c) a recombinant polynucleotide encoding a cytokine; and/or d) a recombinant polynucleotide encoding a co- stimulatory molecule. In some embodiments, one or more HLA alleles endogenous to the cell have been inactivated in the modified human cancer cell.

[0068] In another aspect, the present disclosure provides a modified human cancer cell comprising an antigen, wherein the antigen comprises a non-enzymatic PTM. In some embodiments, the non-enzymatic PTM is induced by irradiation, inducing cellular senescence, a chemical reaction, a small molecule, a cell culture supplement, cell culture medium, oxidative stress, or a combination thereof. In some embodiments, the non-enzymatic PTM comprises cysteinylation. In some embodiments, the modified human cancer cell comprising an antigen with non-enzymatic PTMs further comprises a recombinant polynucleotide encoding the antigen. In some embodiments, the modified human cancer cell comprising an antigen with non-enzymatic PTMs further comprises (i) one or more recombinant polynucleotides each encoding an allele of an HLA class I gene; and/or (ii) one or more recombinant polynucleotides each encoding an allele of an HLA class II gene. In some embodiments, one or more HLA alleles endogenous to the cell have been inactivated in the modified human cancer cell. In some embodiments, the modified human cancer cell comprising an antigen with non-enzymatic PTMs further comprises a recombinant polynucleotide encoding a cytokine. In some embodiments, the modified human cancer cell comprising an antigen with non-enzymatic PTMs further comprises a recombinant polynucleotide encoding a co-stimulatory molecule. In some embodiments, the modified human cancer cell comprising an antigen with non-enzymatic PTMs further comprises one or more recombinant polynucleotides encoding a) the antigen; b) an allele of an HLA class I gene and/or an allele of an HLA class II gene; c) a cytokine; and/or d) a co-stimulatory molecule. In particular embodiments, the modified human cancer cell comprising an antigen with non-enzymatic PTMs further comprises a) a recombinant polynucleotide encoding the antigen; b) one or more recombinant polynucleotides each encoding an allele of an HLA class I gene and/or one or more recombinant polynucleotides each encoding an allele of an HLA class II gene; c) a recombinant polynucleotide encoding a cytokine; and/or d) a recombinant polynucleotide encoding a co-stimulatory molecule. In some embodiments, one or more HLA alleles endogenous to the cell have been inactivated in the modified human cancer cell.

[0069] In some embodiments, multiple recombinant polynucleotides encoding different PTM enzymes can be introduced into the same cell. In some instances, a recombinant polynucleotide encoding one PTM enzyme is introduced into the cell. In other instances, a recombinant polynucleotide encoding two or more PTM enzymes is introduced into the cell. In yet other instances, two or more recombinant polynucleotides each encoding at least one PTM enzyme are introduced into the same cell. In certain instances, multiple recombinant polynucleotides encoding two, three, four, five, six, or more PTM enzymes are introduced into the same cell.

[0070] In some embodiments, the recombinant polynucleotides are integrated into the genome of the cell. In other embodiments, the recombinant polynucleotides are present on one or more vectors in the cell. In some instances, all of the recombinant polynucleotides can be present on the same vector. In other instances, each recombinant polynucleotide can be present on a separate vector. In yet other instances, two, three, four, five, six, or more recombinant polynucleotides can be present on the same vector. Any number of combinations of recombinant polynucleotides on a single vector and any number of vectors in a cell is permitted. In some instances, recombinant polynucleotides encoding one or more PTM enzymes, one or more antigens, one or more HLA class I molecules and/or HLA class II molecules, one or more cytokines, and/or one or more co-stimulatory molecules can be present on the same vector in the cell. In other instances, recombinant polynucleotides encoding one or more PTM enzymes, one or more antigens, one or more HLA class I molecules and/or HLA class II molecules, one or more cytokines, and/or one or more co-stimulatory molecules can be present on different vectors in the same cell.

[0071] As a non-limiting example, recombinant polynucleotides encoding one or more PTM enzymes (e.g., one, two, three, four, or more PTM enzymes) can be present on the same vector. As another non-limiting example, recombinant polynucleotides encoding one or more antigens can be present on the same vector. As a non-limiting example, recombinant polynucleotides encoding two unique HLA class I alleles can be present on the same vector. As another nonlimiting example, recombinant polynucleotides encoding two unique HLA class II alleles can be present on the same vector. As a further non-limiting example, recombinant polynucleotides encoding one or more cytokines (e.g, GM-CSF, IFN-a, IL-12, and/or IL-7), immunomodulatory molecules (e.g. HLA-DRA), and/or one or more co-stimulatory molecules (e.g., CD40, CD80, CD86, and/or 4-1BBL) can be present on the same vector. In one particular embodiment, the vector comprises one or more recombinant polynucleotides encoding one or more cytokines, immunomodulatory molecules, and/or co-stimulatory molecules selected from GM-CSF, IFN-a, IL-12, IL-7, HLA-DRA, CD40, CD80, CD86, and 4-1BBL. In some embodiments, the expression vector comprises one or more recombinant polynucleotides each encoding at least one immunomodulatory molecule, co-stimulatory molecule, and/or cytokine selected from a) GM-CSF and IFN-a; b) GM-CSF; c) CD86 and IL-12; d) CD40, e) CD80 and an HLA-DRA allele; and f) IL-7 and 4-1BBL.

[0072] In some embodiments, the cell comprises: (a) a vector comprising recombinant polynucleotides encoding one or more PTM enzymes (e.g., one, two, three, four, or more PTM enzymes); (b) a vector comprising recombinant polynucleotides encoding two unique HLA class I alleles; (c) a vector comprising recombinant polynucleotides encoding two unique HLA class II alleles; and/or (d) one or more vectors (e.g, one, two, three, four, or more vectors) each comprising recombinant polynucleotides encoding one or more cytokines, immunomodulatory molecules, and/or co-stimulatory molecules (e.g., pairwise combinations of GM-CSF, IFN-a, CD40, CD80, CD86, IL-12, IL-7, HLA-DRA, and 4-1BBL). In particular embodiments, the cell comprises: (a) a vector comprising recombinant polynucleotides encoding one or more PTM enzymes (e.g., one, two, three, four, or more PTM enzymes); (b) a vector comprising recombinant polynucleotides encoding two unique HLA class I alleles; (c) a vector comprising recombinant polynucleotides encoding two unique HLA class II alleles; and/or (d) one or more vectors (e.g., one, two, three, four, or more vectors) each comprising recombinant polynucleotides encoding one or more cytokines, immunomodulatory molecules, and/or co- stimulatory molecules selected from GM-CSF, IFN-a, CD40, CD80, CD86, IL- 12, IL-7, HLA- DRA, and 4-1BBL. In another particular embodiments, the cell comprises: (a) a vector comprising recombinant polynucleotides encoding one or more PTM enzymes (e.g., one, two, three, four, or more PTM enzymes); (b) a vector comprising recombinant polynucleotides encoding two unique HLA class I alleles; (c) a vector comprising recombinant polynucleotides encoding two unique HLA class II alleles; and/or (d) one or more vectors e.g., one, two, three, four, or more vectors) each comprising recombinant polynucleotides encoding at least one immunomodulatory molecule, co-stimulatory molecule, and/or cytokine selected from a) GM- CSF and IFN-a; b) GM-CSF; c) CD86 and IL-12; d) CD40, e) CD80 and an HLA-DRA allele; and f) IL-7 and 4-1BBL. In other particular embodiments, the cell further comprises e) a vector comprising recombinant polynucleotides encoding one or more antigens.

A. Post-translational modifications (PTMs)

[0073] Posttranslational modifications (PTMs) are the covalent processes of amino acid modification after protein biosynthesis. Currently, there are > 700 PTMs in UniProt’s PTM Knowledgebase that describe the target protein, site, and cellular location of these modifications. PTMs can occur on the amino acid side chains or at the protein's C- or N- termini. PTMs alter protein structure, function, and localization and play a pivotal role in physiological and pathophysiological conditions. One site in the same protein may undergo one or more types of modifications. Similarly, one modulator can perform multiple roles.

[0074] In some embodiments, the PTM comprises phosphorylation, acetylation, ubiquitination, succinylation, methylation, malonylation, glycosylation, sumoylation, nitrosylation, glutathionylation, amidation, hydroxylation, palmitoylation, glutarylation, crotonylation, oxidation, myristoylation, sulfation, formylation, citrullination, prenylation, cysteinylation, deamidation, dehydration, or a combination thereof. All types of PTMs are known in the art. (See, e.g., Ramazi S, Zahiri J. Posttranslational modifications in proteins: resources, tools and prediction methods. Database (Oxford). 2021;2021 :baab012; Jennings EQ, Fritz KS, Galligan JJ. Biochemical genesis of enzymatic and non-enzymatic post-translational modifications. Mol Aspects Med. 2022;86: 101053; Li W, Li F, Zhang X, Lin HK, Xu C. Correction: Insights into the post-translational modification and its emerging role in shaping the tumor microenvironment. Signal Transduct Target Ther. 2022;7(l):31; Srivastava AK, Guadagnin G, Cappello P, Novelli F. Post-Translational Modifications in Tumor-Associated Antigens as a Platform for Novel Immuno-Oncology Therapies. Cancers (Basel). 2022; 15(1): 138; each of which is hereby incorporated by reference in its entirety). In some embodiments, the PTM comprises cysteinylation. In some embodiments, the PTM comprises citrullination.

[0075] In some embodiments, the modified human cancer cell disclosed herein comprises a PTM of an antigen. In some instances, the antigen in a natural setting (such as in an unmodified cell) does not comprises the PTM, wherein the antigen in the modified human cancer cell comprises such PTM, by either enzymatic or non-enzymatic means, resulting in the antigen in the modified human cancer cell being more immunogenetic in comparison to the antigen in the natural setting. In other instances, the antigen in a natural setting (such as in an unmodified cell) comprises the PTM, wherein the antigen in the modified human cancer cell comprises an induced PTM, by either enzymatic or non-enzymatic means, in comparison to the antigen in the natural setting, resulting in a higher immunogenicity of the induced PTM antigen in the modified human cancer cell.

[0076] As disclosed above, PTMs can be divided into two categories based on their biochemical origin, enzymatic PTMs and non-enzymatic PTMs.

1. Enzymatic PTMs

[0077] Enzymatic PTMs include both the covalent conjunction of some chemical groups to protein side chains through enzyme catalyzation and the cleavage of a protein backbone through proteases or autocatalytic cleavage at a specific peptide. PTM enzymes can be divided into three subtypes based on their functional specificity; the “writers” are responsible for adding substrates, the “readers” recognize modified proteins to initiate downstream signaling cascade, and the “erasers” are best known for their role in removing PTMs (see, Li W, Li F, Zhang X, Lin HK, Xu C. Correction: Insights into the post-translational modification and its emerging role in shaping the tumor microenvironment. Signal Transduct Target Ther. 2022;7(l):31).

[0078] As disclosed herein, any PTM enzyme can be introduced to the cell. In some embodiments, the PTM enzyme is a citrullination enzyme, a cysteinylation enzyme, an acetylation enzyme, a hydroxylation enzyme, a phosphorylation enzyme, a methylation enzyme, a formylation enzyme, an oxidation enzyme, a hydroxylation enzyme, or a ubiquitination enzyme. In some embodiments, the enzyme comprises a citrullination enzyme. In some embodiments, the enzyme comprises a cysteinylation enzyme. In other embodiments, the PTM enzyme is a combination of a citrullination enzyme, a cysteinylation enzyme, an acetylation enzyme, a hydroxylation enzyme, a phosphorylation enzyme, a methylation enzyme, a formylation enzyme, an oxidation enzyme, a hydroxylation enzyme, and/or a ubiquitination enzyme. In some embodiments, the modified human cancer cell comprises recombinant polynucleotide(s) encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) PTM enzymes. [0079] In some embodiments, the enzyme is a citrullination enzyme. In some embodiments, the citrullination enzyme comprises a peptidyl arginine deiminase. Examples of peptidyl arginine deiminase include but are not limited to peptidyl arginine deiminase 1, peptidyl arginine deiminase 2, peptidyl arginine deiminase 3, peptidyl arginine deiminase 4, and peptidyl arginine deiminase 6 (Table 1). In some embodiments, the enzyme is a cysteinylation enzyme. In some embodiments, the cysteinylation enzyme comprises a protein disulfide isomerase (PDI). PDIs catalyze the controlled formation and rearrangement of disulfide bonds for structural stabilization of nascent proteins. Each member of the PDI family contains at least one thioredoxin-like (Trx-like) domain with a thioredoxin fold. These Trx-like domains may be catalytically active (a-domain), possessing a CXXC motif, or inactive (b-domain). PDIs also possess an acidic C-terminal extension (c-domain) that terminates with an ER retention sequence (see, Bechtel TJ, Weerapana E. From structure to redox: The diverse functional roles of disulfides and implications in disease. Proteomics. 2017 Mar; 17(6)). Non-limiting examples of PDIs are listed in Table 2. In some embodiments, the enzyme is an acetylation enzyme. Non-limiting examples of acetylation enzymes are listed in Table 3. In some embodiments, the enzyme is a hydroxylation enzyme such as hypoxia-inducible factor prolyl hydroxylase (Table 4). In some embodiments, the enzyme is a phosphorylation enzyme. Nonlimiting examples of phosphorylation enzymes are listed in Table 5. In some embodiments, the enzyme is a methylation enzyme. Non-limiting examples of methylation enzymes are listed in Table 6. In some embodiments, the enzyme is a formylation enzyme such as mitochondrial methionyl-tRNA formyltransferase (Table 7). In some embodiments, the enzyme is an oxidation enzyme. Non-limiting examples of oxidation enzymes are listed in Table 8. In some embodiments, the enzyme is a hydroxylation enzyme. Non-limiting examples of hydroxylation enzymes are listed in Table 9. In some embodiments, the enzyme is a ubiquitination enzyme. In some embodiments, the ubiquitination enzyme comprises ubiquitin enzyme 1. In some embodiments, the ubiquitination enzyme comprises ubiquitin enzyme 2. In some embodiments, the ubiquitination enzyme comprises ubiquitin enzyme 3. Non-limiting examples of ubiquitination enzymes are listed in Table 10.

Table 1. Exemplary Citrullination Enzymes

Table 2. Exemplary Cysteinylation Enzymes (Protein Disulfide-Isomerases)

Table 3. Exemplary Acetylation Enzymes

Table 4. Exemplary Hydroxylation Enzymes

Table 5. Exemplary Phosphorylation Enzymes

Table 7. Exemplary Formylation Enzymes

Table 8. Exemplary Oxidation Enzymes

Table 9. Exemplary Hydroxylation Enzymes

Table 10. Exemplary Ubiquitinylation Enzymes

2. Non-enzymatic PTMs

[0080] Non-enzymatic PTMs are often generated between electrophilic metabolites and nucleophilic amino acids, and are regulated by secondary enzymatic processes (see, Jennings EQ, Fritz KS, Galligan JJ. Biochemical genesis of enzymatic and non-enzymatic post- translational modifications. Mol Aspects Med. 2022;86: 101053; and Jennings EQ, Ray JD, Zerio CJ, et al. Sirtuin 2 Regulates Protein LactoylLys Modifications. Chembiochem. 2021;22(12):2102-2106.) Non-limiting examples of non-enzymatic PTMs are cysteinylation, glycation, glycoxidation, nitrosylation, oxidation, succination, and lipoxidation.

[0081] As disclosed herein, the non-enzymatic PTM can be induced by irradiation, inducing cellular senescence, a chemical reaction, a small molecule, a cell culture supplement, cell culture medium, oxidative stress, or a combination thereof. In some embodiments, the non- enzymatic PTM comprises cysteinylation. In some embodiments, non-enzymatic cysteinylation can be induced by culturing cells with excess cysteine. In some embodiments, the non-enzymatic PTM comprises oxidation. In some embodiments, non-enzymatic oxidation can be increased by irradiating cells. In some embodiments, non-enzymatic oxidation can be increased by culturing cells in serum-free media. In some embodiments, non-enzymatic PTMs can be prompted by inducing cell senescence. In some embodiments, a small molecule, such as a senescence inducer, can be used to induce non-enzymatic PTMs in the cells disclosed herein. Such senescence inducers include but are not limited to doxorubicin (genotoxic), palbociclib (CDK4/6 inhibitor), and nutlin-3 A (p53 activator).

B. Modified Human Cancer Cells

[0082] In some embodiments, the modified human cancer cell comprises at least one recombinant polynucleotide encoding one or more PTM enzymes, variants thereof, or fragments thereof. For instance, the modified human cancer cell may comprise a recombinant polynucleotide encoding 1, 2, 3, 4 or more PTM enzymes driven by one or more promoters. In some embodiments, the enzyme comprises a citrullination enzyme, a cysteinylation enzyme, an acetylation enzyme, a hydroxylation enzyme, a phosphorylation enzyme, a methylation enzyme, a formylation enzyme, an oxidation enzyme, a hydroxylation enzyme, a ubiquitination enzyme, or a combination thereof. In some embodiments, the enzyme comprises a citrullination enzyme. In some embodiments, the enzyme comprises a cysteinylation enzyme.

[0083] In some embodiments, any one of the PTM enzymes, variants thereof, or fragments thereof may have a polynucleotide sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or higher identity or similarity with its counterpart wild-type sequence. In some embodiments, any one of the PTM enzymes, variants thereof, or fragments thereof may have a polynucleotide sequence having at most 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity or similarity with its counterpart wild-type sequence. In some embodiments, a polynucleotide encoding the modified PTM enzyme or codon-optimized PTM enzyme, a variant thereof, or a fragment thereof has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or higher identity or similarity with its counterpart wild-type sequence. In some embodiments, a polynucleotide encoding the modified PTM enzyme or codon-optimized PTM enzyme, a variant thereof, or a fragment thereof has at most 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity or similarity with its counterpart wild-type sequence. In some embodiments, a polynucleotide encoding the modified PTM enzyme or codon-optimized PTM enzyme, a variant thereof, or a fragment thereof has about 10% to 99%, about 30% to 80%, about 40% to 95%, about 60% to 85% identity or similarity with its counterpart wild-type sequence.

[0084] In some embodiments, the modified human cancer cell comprises at least one recombinant polynucleotide encoding one or more antigens. For instance, the modified human cancer cell may comprise a recombinant polynucleotide encoding 1, 2, 3, 4 or more antigens driven by one or more promoters. In some embodiments, the antigen is an antigen of a pathogen, a tumor-specific antigen, a tumor-associated antigen, a neo-antigen, an allergen, an antigen that is a target of an autoimmune response, or a fragment thereof.

[0085] In some embodiments, the modified human cancer cell comprises at least one recombinant polynucleotide encoding one or more HLA class I genes, a codon optimized version, a variant thereof, or a fragment thereof. For instance, the modified human cancer cell may comprise a recombinant polynucleotide encoding 1, 2, 3, 4 or more HLA class I genes driven by one or more promoters. In some embodiments, the modified human cancer cell further comprises at least one recombinant polynucleotide encoding one or more HLA class II genes, a codon optimized version, a variant, or a fragment thereof. For instance, the modified human cancer cell may comprise a recombinant polynucleotide encoding 1, 2, 3, 4 or more HLA class II genes driven by one or more promoters.

[0086] In some embodiments, the modified human cancer cell comprises one or more recombinant polynucleotides encoding one or more HLA class I genes selected from an HLA- A gene, an HLA-B gene, an HLA-C gene, an HLA-E gene, an HLA-F gene, an HLA-G gene, a beta-2-microglobulin (B2M) gene, or a combination thereof. In some embodiments, the modified human cancer cell comprises one or more recombinant polynucleotides encoding at least one HLA class I allele including, but not limited to, an HLA-A*01 :01 allele, HLA- A*68:01 allele, HLA-A*02:01 allele, HLA-A* 11:01 allele, HLA-A*03:01 allele, HLA- A*23:01 allele, HLA-A*24:02 allele, and/or HL A- A*33: 03 allele. In some embodiments, the modified human cancer cell comprises one or more recombinant polynucleotides encoding at least one HLA class I allele selected from an HLA-A*01 :01 allele and an HLA-A*68:01 allele, an HLA-A*02:01 allele and an HLA-A* 11 :01 allele, an HLA-A*03:01 allele and an HLA- A*23:01 allele, and/or an HLA-A*24:02 allele and an HLA-A*33:03 allele.

[0087] In some embodiments, the modified human cancer cell comprises one or more recombinant polynucleotides encoding one or more HLA class II genes selected from an HLA- DP gene, an HLA-DM gene, an HLA-DO gene, an HLA-DQ gene, an HLA-DR gene, or a combination thereof. In some embodiments, the modified human cancer cell comprises one or more recombinant polynucleotides encoding at least one HLA class II allele including, but not limited to, an HLA-DRB3*02:02 allele, HLA-DRB5*01:01 allele, HLA-DRB4*01:01 allele, HLA-DRB3*01:01 allele, HLA-DRB3*03:01 allele, HLA-DRB5*01:02 allele, and/or HLA- DRB5*02:02 allele. In some embodiments, the modified human cancer cell comprises one or more recombinant polynucleotides encoding at least one HLA class II allele selected from an HLA-DRB3 *02:02 allele and an HLA-DRB5*01:01 allele, an HLA-DRB4*01:01 allele and an HLA-DRB3*01:01 allele, an HLA-DRB3*03:01 allele and an HLA-DRB5*01:02 allele, and/or an HLA-DRB5*02:02 allele and an HLA-DRB3*01 :01 allele.

[0088] In some embodiments, one or more HLA alleles endogenous to the cell have been inactivated in the modified human cancer cell. In particular embodiments, the HLA-A*24:02 allele and the HLA-DRB3*0L01 allele endogenous to the cell have been inactivated in the modified human cancer cell.

[0089] In some embodiments, the modified human cancer cell comprises one or more recombinant polynucleotides encoding a cytokine, a variant thereof, or a fragment thereof. The cytokine can be a chemokine, an interferon, an interleukin, or a tumor necrosis factor. The cytokine can be selected from an early T cell activation antigen-1 (ETA-1), a lymphocyteactivating factor (LAF), an interleukin-1 family member (IL-la, IL-P, IL-IRa, IL-18, IL-33, IL-36Ra, IL-36a, IL-36P, fL-36y, IL-37, IL-38), an interleukin-2 (IL-2), an interleukin-3 (IL- 3), an interleukin-4 (IL-4), an interleukin-5 (IL-5), an interleukin-6 (IL-6), an interleukin-7 (IL- 7), an interleukin-8 (IL-8), an interleukin-9 (IL-9), an interleukin- 10 (IL- 10), an interleukin- 12 (IL-12), an interleukin- 13 (IL-13), an interleukin- 15 (IL-15), an interleukin- 17 (IL-17), an interleukin- 18 (IL-18), an interleukin-21 (IL-21), an interleukin-23 (IL-23), an interleukin-25 (IL-25), an interleukin-33 (IL-33), an interferon alpha (IFN-a), an interferon lambda 1 (IFN- A.1 (IL-29)), an interferon lambda 2 (IFN- 2 ( IL-28A)), an interferon lambda 3 (IFN-X3 (IL- 28B)), an interferon lambda 4 (IFN-k4), a granulocyte-macrophage colony-stimulating factor (GM-CSF), a macrophage CSF (CSF-1), a macrophage migration inhibitory factor (MIF), a CD40L molecule (CD40L), a RANTES molecule (RANTES), a monocyte chemoattractant protein (MCP-1), a monocyte inflammatory protein (MIP-la, MIP-ip), a lymphotactin, or a fractalkine. In particular embodiments, the cytokine comprises GM-CSF.

[0090] In some embodiments, the modified human cancer cell comprises one or more recombinant polynucleotides encoding a co-stimulatory molecule, a variant thereof, or a fragment thereof. The co-stimulatory molecule can be selected from at least one of a CD86 molecule (CD86), CD80 molecule (CD80), 4- IBB ligand molecule (4-1BBL, also known as TNFSF9 or CD137L), ICOS ligand molecule (ICOS-L), CD70 molecule (CD70 a.k.a. CD27L), CD40 molecule (CD40), 0X40 ligand molecule (OX40L), GITR ligand molecule (GITRL), TIM-4 molecule (TIM-4), LIGHT molecule (LIGHT), ICAM1 molecule (ICAM1), LFA3 molecule (LFA3), a CD30 molecule (CD30), and a combination thereof.

[0091] In some embodiments, the modified human cancer cell comprises one or more recombinant polynucleotides encoding a) an enzyme that induces a PTM of an antigen in the cell, b) the antigen; c) an allele of an HLA class I gene and/or an allele of an HLA class II gene; d) a cytokine; and/or e) a co-stimulatory molecule. In some embodiments, the modified human cancer cell comprising a recombinant polynucleotide encoding a PTM enzyme that targets on an antigen in the cell further comprises one or more recombinant polynucleotides encoding a) the antigen; b) an allele of an HLA class I gene and/or an allele of an HLA class II gene; c) a cytokine; and/or d) a co-stimulatory molecule. In some embodiments, one or more HLA alleles endogenous to the cell have been inactivated in the modified human cancer cell.

[0092] In some embodiments, the modified human cancer cell comprising a recombinant polynucleotide encoding a PTM enzyme is derived from a breast cancer cell line such as an SV-BR-1 cell line or an SV-BR-l-GM cell line as described in WO 2017/147600, which is incorporated herein in its entirety. In some embodiments, the modified human cancer cell comprising a recombinant polynucleotide encoding a PTM enzyme is derived from a breast cancer SV-BR-l-Bria-OTS cell line. In some embodiments, the modified human cancer cell comprising a recombinant polynucleotide encoding a PTM enzyme is derived from a prostate cancer cell line such as a PC3-Bria-OTS. In some embodiments, the modified human cancer cell comprising a recombinant polynucleotide encoding a PTM enzyme is derived from a melanoma cell line such as a SK-MEL-24-Bria-OTS cell line. In some embodiments, the modified human cancer cell comprising a recombinant polynucleotide encoding a PTM enzyme is derived from a lung cancer cell line such as a H2228-Bria-OTS cell line. The Bria-OTS cell lines described herein overexpress a combination of cytokines, immunomodulatory molecules, and co-stimulatory molecules comprising GM-CSF, IFN-a, CD80, CD86, IL-12, IL-7, HLA- DRA, and 4-1BBL, along with a unique combination of exogenous HLA-A and HLA- DRB3/4/5 alleles (e.g. a combination of an HLA-A*01 :01 allele, an HLA-A*68:01 allele, an HLA-DRB3*02:02 allele, and an HLA-DRB5*0L01 allele; a combination of an HLA- A*02:01 allele, an HLA-A* 11 :01 allele, an HLA-DRB4*01:01 allele, and an HLA- DRB3*01 :01 allele; a combination of an HLA-A*03:01 allele, an HLA-A*23:01 allele, an HLA-DRB3*03:01 allele, and an HLA-DRB5*01 :02 allele; or a combination of an HLA- A*24:02 allele, an HLA-A*33:03 allele, an HLA-DRB5*02:02 allele, and an HLA- DRB3*01 :01 allele), while certain endogenous HLA-A and HLA-DRB3 alleles (e.g., HLA- A*24:02 and HLA-DRB3*01 :01) have been inactivated. The Bria-OTS cell lines are described in WO 2023/167973, which is incorporated herein in its entirety. In some embodiments, the modified human cancer cell comprising a recombinant polynucleotide encoding a PTM enzyme is derived from a cancer cell line (e.g., a breast cancer, prostate cancer, melanoma, or lung cancer cell line) that overexpresses a combination of cytokines, immunomodulatory molecules, and co-stimulatory molecules comprising GM-CSF, IFN-a, CD80, CD86, IL-12, IL-7, HLA- DRA, and CD40, along with a unique combination of exogenous HLA-A and HLA-DRB3/4/5 alleles (e.g., a combination of an HLA-A*01 :01 allele, an HLA-A*68:01 allele, an HLA- DRB3*02:02 allele, and an HLA-DRB5*0L01 allele; a combination of an HLA-A*02:01 allele, an HLA-A*ll:01 allele, an HLA-DRB4*01:01 allele, and an HLA-DRB3*01:01 allele; a combination of an HLA-A*03:01 allele, an HLA-A*23:01 allele, an HLA-DRB3*03:01 allele, and an HLA-DRB5*01 :02 allele; or a combination of an HLA-A*24:02 allele, an HLA- A*33:03 allele, an HLA-DRB5*02:02 allele, and an HLA-DRB3*0L01 allele), while certain endogenous HLA-A and HLA-DRB3 alleles (e.g., HLA-A*24:02 and HLA-DRB3*0L01) have been inactivated.

[0093] In some embodiments, the expression of the PTM enzymes, HLA alleles, antigens, cytokines (e.g., GM-CSF, IL-12, IL-7, and/or IFN-a), immunomodulatory molecules (e.g. HLA-DRA), and/or co-stimulatory molecules (e.g., CD40, CD80, CD86, and/or 4-1BBL) are under the control of two or more different promoters. In some instances, the expression of each PTM enzyme, HLA allele, antigen, cytokine (e.g., GM-CSF, IL-12, IL-7, and/or IFN-a), immunomodulatory molecule (e.g. HLA-DRA), and/or co-stimulatory molecule (e.g., CD40, CD80, CD86, and/or 4-1BBL) is under the control of a separate promoter. In some embodiments, the expression of the PTM enzymes, HLA alleles, antigens, cytokines (e.g., GM- CSF, IL-12, IL-7, and/or IFN-a), immunomodulatory molecules (e.g. HLA-DRA), and/or co- stimulatory molecules (e.g., CD40, CD80, CD86, 4-1BBL) are under the control of a single promoter. In some instances, the PTM enzymes, HLA alleles, antigens, cytokines (e.g., GM- CSF, IL-12, IL-7, and/or IFN-a), immunomodulatory molecules (e.g. HLA-DRA), and/or co- stimulatory molecules (e.g., CD40, CD80, CD86, and/or 4-1BBL) are expressed as a polycistronic mRNA in a multicistronic vector. In particular instances, one or more cistrons are separated by internal ribosomal entry sites. In other instances, one or more cistrons are separated by a self-cleaving peptide e.g., a T2A, P2A, E2A, F2A).

[0094] In some embodiments, the modified human cancer cell is derived from a human cancer cell line. Any number of human cancer cells or cancer cell lines are suitable for use in the compositions and methods described herein, including, for example, clonal or non-clonal human cancer cells or cancer cell lines. Non-limiting examples of human cancer cell lines include the following cell lines and subclones thereof the Bria-OTS-1 (BC1), Bria-OTS-2 (BC2), Bria-OTS-3 (BC3), Bria-OTS-4 (BC4), Bria-OTS-1+, Bria-OTS-2+, Bria-OTS-3+, Bria-OTS-4+, SV-BR-1, SV-BR-l-GM, SVCT, MDA-MB-231, MDA-MB-157, ZR-75-30, ZR-75-1, Hs 578T, MCF7, T47D, MTSV1-7 CE1, 1-7HB2, VP303, VP267, and VP229 breast cancer cell lines; the PC-3, Bria-Pros-1+, Bria-Pros-2+, Bria-Pros-3+, Bria-Pros-4+, LNCaP (e.g., clone FGC), Shmac 5, P4E6, and VCaP prostate cancer cell lines; the NCI-H2228, Bria- Lung-1+, Bria- Lung-2+, Bria- Lung-3+, Bria- Lung-4+, SHP-77, COR-L23/R, COR- L23/5010, MOR/0.2R, NCI-H69/LX20, ChaGo-K-1, and Meta 7 lung cancer cell lines; the SK-MEL-24, Bria-Mel-1+, Bria- Mel-2+, Bria- Mel-3+, Bria- Mel-4+, melanoma cell line; the UM-UC-3, T24/83, ECV304, RT4, and HT 1197 bladder cancer cell lines; the MDST8, C170, GP5d, GP2d, and LS 123 colon cancer cell lines; the MFE-280 and MFE-296 endometrial cancer cell lines; the CAKI 2, A.704, G-402, ACHN, G-401, UM-RC-7, and RCC4plusVHL renal cancer cell lines; the SK-HEP-1, Hep 3B, PLC/PRF/5, Hep G2, and Huh- 7D12 liver cancer cell lines; the HL60, Eos-HL-60, JVM-13, Sci-1, and Ri-1 leukemia cell lines; the BHL-89, COR-L24, U937(CD59+), My-La CD8+, and HGC-27 lymphoma cell lines; the A375-C6, GR-M, VA-ES-BJ, MEWO, and COLO 818 skin cancer cell lines; the AsPC-1, HuP-T4, HuP-T3, BxPC-3, and CFPAC-1 pancreatic cancer cell lines; the 8505C, 8305C, FTC-238, TT, RO82-W-1, and KI thyroid cancer cell lines; the HeLa DH, HR5-CL11, HtTA-1, HR5, Xl/5, HeLa, C-4I, C-4 II, HeLa S3, Ca Ski, HeLa229, Hep2 (HeLa derivative), HeLa B, Bu25 TK- HeLa Ohio, and HeLa (AC-free) cervical cancer cell lines; the NB69, BE(2)-C, BE(2)-M17, SK-N-BE(2), and SK-N-DZ brain cancer cell lines; the OV7, OV17R, OV58, OV56, A2780ADR, A2780, COLO 720 E, SW 626, SK-OV-3, PA-1, 59M, OAW28, TO14, PEO23, and COV362 ovarian cancer cell lines; the IMR 32 abdominal cancer cell line; the SW 13 adrenal cortex cancer cell line; the TR146 buccal mucosa cancer cell line; the SK- GT-4 esophageal cancer cell line; the TE 671 embryonic cancer cell line; the FLYRD18 fibrosarcoma cell line; the 1411H germ cell tumor cell line; the MFM-223 mammary gland cancer cell line; the H-EMC-SS muscle cancer cell line; the Detroit 562 pharyngeal cancer cell line; the BeWo placental cancer cell line; the Mero-95 pleural cavity cancer cell line, the SW 837, SW 1463, CMT 93, HRT-18, and HRA-19 rectal cancer cell lines; the Y79, WERI, and RB247C retinal cancer cell line; the CHP-100 spinal cancer cell line; the KARPAS 1718 splenic lymphoma cell line; the AGS and KATO-III stomach cancer cell lines; the NTERA-2 clone DI testicular cancer cell line; the SCC-9, H357, H103, BICR 56, and PE/CA-PJ49 tongue cancer cell lines; the MES-SA/Dx-5, MES-SA, COLO 685, and COLO 684 uterine cancer cell lines; and the HMVII vaginal cancer cell line. In particular embodiments, the human cancer cell line is a breast cancer (e.g, SV-BR-1), prostate cancer (e.g, PC-3, LNCaP), melanoma e.g., SK-MEL-24), or lung cancer (e.g., NCLH2228) cell line. The cell lines described herein and others are available, for example, from Sigma-Aldrich (www.sigmaaldrich.com).

[0095] In other embodiments, the modified human cancer cell is derived from a primary cancer cell. For example, the primary cancer cell is obtained from a tumor biopsy, or a circulating cancer cell, from a subject who is to be treated for cancer prior to modification of the cancer cell. In some embodiments, the patient has a breast cancer, prostate cancer, melanoma, or lung cancer.

C. Expression Vectors

[0096] In some embodiments, the modified human cancer cell described herein contains one or more expression vectors for expressing the recombinant PTM enzymes. A wide variety of expression vectors may be used, such as retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, plasmids or transposons. Viral vectors that may be used, for example, include vectors based on HIV, SV40, EBV, HSV or BPV. The expression vectors may be replication-defective by design such that the viral vector is defective for one or more functions that are essential for viral genome replication or synthesis and assembly of viral particles. Many of the currently existing replication-defective viruses can carry large therapeutic genes, effectively transduce various types of cells, and provide long-term and stable expression of genes of interest.

[0097] Lentiviruses are a subset of retroviruses commonly used in research. Lentiviruses can transduce both dividing and non-dividing cells without a significant immune response. These viruses also integrate stably into the host genome, enabling long term transgene expression. A common lentivirus is the human immunodeficiency virus (HIV), which uses the envelope glycoproteins of other viruses to target a broad range of cell types. [0098] One safety feature of lentiviruses is that the components necessary to produce an infectious viral particle (a virion) are generally divided among multiple plasmids. For instance, an infectious viral particle may comprise plasmids that components of the viral capsid and envelope (typically called the packaging and envelope plasmids), and plasmid that encodes the viral genome (typically called the transfer plasmid). Common lentiviral packaging and envelope plasmids that can be used herein include, but are not limited to, pRSV-Rev, pMDLg/pRRE, psPAX2, pCMV delta R8.2, pMD2.G, pCMV-VSV-G, pCMV-dR8.2 dvpr, pCI-VSVG, pCPRDEnv, pLTR-RD114A, pLTR-G, pCD/NL-BH*DDD, psPAX2-D64V, pCEP4-tat, pHEF-VSVG, pNHP, pCAG-Eco, and pCAG-VSVG. Common lentiviral transfer plasmids that can be used herein include, but are not limited to, pLKO.l puro, pLKO.l - TRC cloning plasmid, pLKO.3G, Tet-pLKO-puro, pSico, pLJMl-EGFP, FUGW, pLVTHM, pLVUT-tTR-KRAB, pLL3.7, pLB, pWPXL, pWPI, EF.CMV.RFP, pLenti CMV Puro DEST, pLenti-puro, pLOVE, pULTRA, pLX301, plnducer20, pHIV-EGFP, Tet-pLKO-neo, pLV- mCherry, pCW57.1, pLionll, pSLIK-Hygro, and plnducerlO-mir-RUP-PheS.

[0099] There are multiple approaches to produce lentiviral vectors. (See Logan et al. “Factors influencing the titer and infectivity of lentiviral vectors.” Hum Gene Ther. 2004 Oct;15(10):976-88. doi: 10.1089/hum.2004.15.976. PMID: 15585113; Dull, T et al. “A third- generation lentivirus vector with a conditional packaging system.” Journal of virology vol. 72,11 (1998): 8463-71. doi: 10.1128/JVI.72.11.8463-8471.1998). Alternatively, lentiviral vectors may be purchased from commercial providers. In general, production of lentiviral vectors involves multiple steps including plasmid development and production, cell expansion, plasmid transfection, viral vector production, purification, fill and finish. See e.g., www.addgene.org/viral-vectors/lentivirus/; www.thermofisher.com/us/en/home/clinical/cell- gene-therapy/gene-therapy/lv-production-workflow.html).

[0100] The lentiviral vector may be designed to express one or more genes of interest simultaneously. Various molecular strategies are available, including the use of multiple promoters, signals of splicing, fusion of genes, cleavage factors and multi ci str onic vectors. (See e.g., review by Shaimardanova et al., “Production and application of multi ci stronic constructs for various human disease therapies.” Pharmaceutics 2019, 11, 580.)

[0101] Multi ci stronic vectors generally contain sequences encoding the nucleotide sequences of internal ribosome entry site (IRES) and self-cleaving 2A peptides. The use of IRES and self-cleaving 2A peptides allows simultaneous expression of two or more separate proteins from the same mRNA.

[0102] Self-cleaving 2A peptides are used for the production of multi ci str onic vectors due to their small size and self-cleavage ability. 2A peptides are composed of 16-20 amino acids and originate from viral RNA. Common 2A peptides used to produce multi ci str onic vectors are F2A (2A peptide derived from the foot-and-mouth disease virus), E2A (2A peptide derived from the equine rhinitis virus), P2A (2A peptide derived from the porcine teschovirus-1), and T2A (2 A peptide derived from the Thosea asigna virus).

[0103] In constructs with 2A peptide sequences, translation is initiated once and synthesis along mRNA occurs continuously. During translation, the first peptide breaks from the second peptide in the 2A region. 2A peptide cleavage site is located between glycine and proline. The cleavage process occurs inside the ribosome during protein synthesis. The formation of a normal peptide bond between the amino acids is inhibited only at the cleavage site, thus the cleavage does not affect the translation of the subsequent protein and synthesis continues without the dissociation of ribosome. Thus, translation of multiple genes are dependent of each other. The use of different 2A peptides may influence the expression levels of downstream protein. Combinations of the order of 2A peptide sequence may prevent gradual decrease in the gene expression from the first to the last in multicistronic constructs. For instance, the combination of 2A peptide sequences in the following order, namely T2A, P2A, and E2A, is optimal when creating multicistronic vectors containing four genes.

[0104] In some embodiments, the modified human cancer cells described herein are expressed using non-viral approaches. Exemplary methods include, but are not limited to, cationic lipids such as liposomes and lipoplexes, polymers or polyplexes and dendrimers, naked plasmids for direct delivery, electroporation, ultrasound and micro bubbles, magnetofections, inorganic molecules.

[0105] In one aspect, the present disclosure provides an expression vector for the modified human cancer cell described herein. The vector may comprise one or more recombinant polynucleotides encoding at least one PTM enzyme listed in any one of Tables 1-10, a variant thereof, or a fragment thereof. The recombinant polynucleotide may further comprise at least one HLA-A class I allele and at least one HLA-A class II allele, a variant thereof, or a fragment thereof. The recombinant polynucleotide may further comprise at least one co-stimulatory molecule. In some embodiments, the recombinant polynucleotide further comprises a sequence encoding one or more cytokines (e.g., GM-CSF, IFN-a2 including IFN-a2a and IFN-a2b). In some embodiments, the recombinant polynucleotide encoding the PTM enzymes, HLA-A class I alleles and HLA-A class II alleles, co-stimulatory molecules, antigens, and/or cytokines, each has a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or higher identity or similarity with their counterpart wild-type sequence; each has a sequence having at most 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity or similarity with their counterpart wild-type sequence; or each has a sequence having about 10% to 99%, about 30% to 80%, about 40% to 95%, about 60% to 85% identity or similarity with their counterpart wild-type sequence.

[0106] In various embodiments, the expression vector comprises one or more recombinant polynucleotides encoding one or more promoters for driving expression of the selected PTM enzymes, HLA alleles, co-stimulatory molecules, or adjuvant. The expression vector may contain recombinant polynucleotides encoding a MNDU3 promoter, an EFla promoter, or both. For example, the expression vector may contain a first recombinant polynucleotide encoding a MNDU3 promoter and a second recombinant polynucleotide encoding an EFla promoter. In particular embodiments, the expression vector contains a recombinant polynucleotide encoding a MNDU3 promoter and an EFla promoter.

[0107] In some embodiments, the expression vector comprises one or more recombinant polynucleotides encoding at least one PTM enzyme, including, but not limited to, a citrullination enzyme, a cysteinylation enzyme, an acetylation enzyme, a hydroxylation enzyme, a phosphorylation enzyme, a methylation enzyme, a formylation enzyme, an oxidation enzyme, a hydroxylation enzyme, a ubiquitination enzyme, a variant thereof, a fragment thereof, or a combination thereof. In some embodiments, the expression vector comprises one or more recombinant polynucleotides encoding at least one PTM enzyme selected from a citrullination enzyme, a cysteinylation enzyme, an acetylation enzyme, a hydroxylation enzyme, a phosphorylation enzyme, a methylation enzyme, a formylation enzyme, an oxidation enzyme, a hydroxylation enzyme, a ubiquitination enzyme, a variant thereof, a fragment thereof, or a combination thereof.

[0108] In some embodiments, the expression vector comprises one or more recombinant polynucleotides each encoding at least one immunomodulatory molecule, co-stimulatory molecule, and/or cytokine(e.g., two or more immunomodulatory molecules, co-stimulatory molecules, or cytokines). In some embodiments, the at least one immunomodulatory molecule, co-stimulatory molecule, and/or cytokine (e.g., two or more immunomodulatory molecules, co-stimulatory molecules, or cytokines) include, but not limited to, CSF2, IFN-a, CD86, IL- 12, CD40, CD80, HLA-DRA, IL-7, and/or 4-1BBL (also known as TNFSF9 or CD137L). In some embodiments, the expression vector comprises one or more recombinant polynucleotides each encoding at least one immunomodulatory molecule, co-stimulatory molecule, and/or cytokine (e.g., two or more immunomodulatory molecules, co-stimulatory molecules, and/or cytokines) selected from CSF2 and IFN-a; CSF2; CD86 and IL-12; CD40, CD80 and an HLA- DRA allele; or IL-7 and 4-1BBL.

[0109] In some embodiments, the expression vector comprises one or more recombinant polynucleotides each encoding at least one HLA class I allele including, but not limited to, an HLA-A*01:01 allele, HLA-A*68:01 allele, HLA-A*02:01 allele, HLA-A* 11 :01 allele, HLA- A*03:01 allele, HLA-A*23:01 allele, HLA-A*24:02 allele, and/or HLA-A*33:03 allele. In some embodiments, the expression vector comprises one or more recombinant polynucleotides each encoding at least one HLA class I allele selected from an HLA-A*0L01 allele and an HLA-A*68:01 allele, an HLA-A*02:01 allele and an HLA-A* 11 :01 allele, an HLA-A*03:01 allele and an HLA-A*23:01 allele, and/or an HLA-A*24:02 allele and an HLA-A*33:03 allele.

[0110] In some embodiments, the expression vector comprises one or more recombinant polynucleotides each encoding at least one HLA class II allele including, but not limited to, an HLA-DRB3 *02:02 allele, HLA-DRB5*01:01 allele, HLA-DRB4*01 :01 allele, HLA- DRB3*01 :01 allele, HLA-DRB3*03:01 allele, HLA-DRB5*01:02 allele, and/or HLA- DRB5*02:02 allele. In some embodiments, the expression vector comprises one or more recombinant polynucleotides each encoding at least one HLA class II allele selected from an HLA-DRB3 *02:02 allele and an HLA-DRB5*01:01 allele, an HLA-DRB4*01 :01 allele and an HLA-DRB3*01 :01 allele, an HLA-DRB3*03:01 allele and an HLA-DRB5*01:02 allele, and/or an HLA-DRB5*02:02 allele and an HLA-DRB3*01 :01 allele.

[OHl] In some embodiments, the expression vector is capable of expressing the at least one PTM enzyme, immunomodulatory molecule, co-stimulatory molecule, or cytokine, the at least one HLA class I allele, and/or the at least one HLA class II allele in a cancer cell line (e.g., a modified human cancer cell line). In some embodiments, the expression vector is capable of expressing one or more (e.g., at least two) PTM enzymes, immunomodulatory molecules, co- stimulatory molecules, or cytokines in a cancer cell line (e.g., a modified human cancer cell line); or the expression vector is capable of expressing one or more (e.g., at least two) HLA class I allele in a cancer cell line (e.g., a modified human cancer cell line); or the expression vector is capable of expressing one or more (e.g., at least two) HLA class II allele in a cancer cell line (e.g., a modified human cancer cell line). In various embodiments, one or more endogenous HLA alleles in the cancer cell line have been inactivated.

[0112] In some embodiments, the expression vector further comprises a recombinant polynucleotide encoding a cytokine, a chemokine, an interferon, an interleukin, and/or a tumor necrosis factor. In some embodiments, the recombinant polynucleotide encodes one of the cytokines selected from at least one of an early T cell activation antigen- 1 (ETA-1), a lymphocyte-activating factor (LAF), an interleukin-1 family member (IL-la, IL-P, IL-IRa, IL- 18, IL-33, IL-36Ra, IL-36a, IL-36P, fL-36y, IL-37, IL-38), an interleukin-2 (IL-2), an interleukin-3 (IL-3), an interleukin-4 (IL-4), an interleukin-5 (IL-5), an interleukin-6 (IL-6), an interleukin-7 (IL-7), an interleukin-8 (IL-8), an interleukin-9 (IL-9), an interleukin- 10 (IL- 10), an interleukin- 12 (IL-12), an interleukin- 13 (IL-13), an interleukin- 15 (IL-15), an interleukin- 17 (IL-17), an interleukin- 18 (IL-18), an interleukin-21 (IL-21), an interleukin-23 (IL-23), an interleukin-25 (IL-25), an interleukin-33 (IL-33), an interferon alpha (IFN-a), an interferon lambda 1 (IFN- 1 (IL-29)), an interferon lambda 2 (IFN- 2 (IL-28 A)), an interferon lambda 3 (IFN- 3 (IL-28B)), an interferon lambda 4 (IFN-A4), a granulocyte-macrophage colonystimulating factor (GM-CSF), a macrophage CSF (CSF-1), a macrophage migration inhibitory factor (MIF), a CD40L molecule (CD40L), a RANTES molecule (RANTES), a monocyte chemoattractant protein (MCP-1), a monocyte inflammatory protein (MIP-la, MIP-ip), a lymphotactin, and/or a fractalkine. In some embodiments, the cytokine comprises a GM-CSF. In some embodiments, the cytokine comprises an IFN-a, such as e.g., IFN-a2a or IFN-a2b.

[0113] In some embodiments, the expression vector further comprises a recombinant polynucleotide encoding a co-stimulatory molecule selected from at least one of CD86 molecule (CD86), CD80 molecule (CD80), 4-1BB ligand molecule (4-1BBL a.k.a CD137L), ICOS ligand molecule (ICOS-L), CD70 molecule (CD70 a.k.a. CD27L), CD40 molecule (CD40), 0X40 ligand molecule (OX40L), GITR ligand molecule (GITRL), TIM-4 molecule (TIM-4), LIGHT molecule (LIGHT), ICAM1 molecule (ICAM1), LFA3 molecule (LFA3), a CD30 molecule (CD30), and/or a combination thereof.

[0114] In some embodiments, the expression vector further comprises a recombinant polynucleotide encoding an antigen (e.g., an antigen of a pathogen, a tumor-specific antigen, a tumor-associated antigen, a neo-antigen, an allergen, an antigen that is a target of an autoimmune response, or a fragment thereof). [0115] In some embodiments, the expression vector comprises one or more recombinant polynucleotides encoding a) an enzyme that induces a PTM of an antigen in the cell, b) the antigen; c) an allele of an HLA class I gene and/or an allele of an HLA class II gene; d) a cytokine; and/or e) a co-stimulatory molecule. In some embodiments, the expression vector comprising a recombinant polynucleotide encoding a PTM enzyme that targets on an antigen in the cell further comprises one or more recombinant polynucleotides encoding a) the antigen; b) an allele of an HLA class I gene and/or an allele of an HLA class II gene; c) a cytokine; and/or d) a co-stimulatory molecule. In some embodiments, the cytokine is GM-CSF.

D. Compositions

[0116] In one aspect, the present disclosure provides a composition comprising a modified human cancer cell as described herein. In some embodiments, the modified human cancer comprises a recombinant polynucleotide encoding an enzyme that induces a post-translational modification (PTM) of an antigen in the cell. In some embodiments, the modified human cancer comprises a recombinant polynucleotide encoding the antigen. In some embodiments, the modified human cancer comprising (a) one or more recombinant polynucleotides each encoding an allele of a human leukocyte antigen (HLA) class I gene; and/or (b) one or more recombinant polynucleotides each encoding an allele of an HLA class II gene. In some embodiments, the modified human cancer comprises a recombinant polynucleotide encoding a cytokine. In some embodiments, the modified human cancer comprises a recombinant polynucleotide encoding a co-stimulatory molecule.

[0117] In one aspect, the present disclosure provides a composition comprising a modified human cancer cell as described herein, the modified human cancer cell comprising (A) one or more vectors each comprising a recombinant polynucleotide encoding at least one PTM enzyme selected from a citrullination enzyme, a cysteinylation enzyme, an acetylation enzyme, a hydroxylation enzyme, a phosphorylation enzyme, a methylation enzyme, a formylation enzyme, an oxidation enzyme, a hydroxylation enzyme, a ubiquitination enzyme, and/or a combination thereof; (B) one or more vectors each comprising a recombinant polynucleotide encoding at least one gene selected from CSF2, IFN-a2, CD86, IL-12, CD40, CD80, HLA- DRA, IL-7, and/or 4-1BBL (also known as TNFSF9 or CD137L); and/or (C) (i) one or more vectors each comprising a recombinant polynucleotide encoding at least one HLA class I genes and/or (ii) one or more vectors each comprising a recombinant polynucleotide encoding at least one HLA class II genes. In some embodiments, one or more HLA alleles endogenous to the human cancer cell have been inactivated.

[0118] In some embodiments, the composition comprises at least 10,000 cells, at least 100,000 cells, at least 1,000,000 cells, at least 1,250,000 cells, at least 1,500,000 cells, at least 2,000,000 cells, at least 2,500,000 cells, at least 3,000,000 cells, at least 3,500,000 cells, at least 4,000,000 cells, at least 4,500,000 cells, at least 5,000,000 cells, at least 10,000,000 cells, at least 12,500,000 cells, at least 15,000,000 cells, at least 20,000,000 cells, at least 25,000,000 cells, at least 30,000,000 cells, at least 35,000,000 cells, at least 40,000,000 cells, at least 45,000,000 cells, or at least 50,000,000 cells. In some embodiments, the composition comprises at least 1,000,000 cells. In some embodiments, the composition comprises at least 20,000,000 cells.

[0119] In some embodiments, the composition comprises at most 10,000 cells, at most 100,000 cells, at most 1,000,000 cells, at most 1,250,000 cells, at most 1,500,000 cells, at most 2,000,000 cells, at most 2,500,000 cells, at most 3,000,000 cells, at most 3,500,000 cells, at most 4,000,000 cells, at most 4,500,000 cells, at most 5,000,000 cells, at most 10,000,000 cells, at most 12,500,000 cells, at most 15,000,000 cells, at most 20,000,000 cells, at most 25,000,000 cells, at most 30,000,000 cells, at most 35,000,000 cells, at most 40,000,000 cells, at most 45,000,000 cells, or at most 50,000,000 cells. In some embodiments, the composition comprises at most 20,000,000 cells. In some embodiments, the composition comprises at most 40,000,000 cells.

[0120] In some embodiments, the composition comprises about 1,000,000 to about 50,000,000 cells, about 5,000,000 to about 35,000,000 cells, about 10,000,000 to about 25,000,000 cells, about 15,000,000 to about 20,000,000 cells, or about 35,000,000 to about 40,000,000 cells. In some embodiments, the composition comprises about 1,000,000 cells. In some embodiments, the composition comprises about 20,000,000 cells. In some embodiments, the composition comprises about 40,000,000 cells.

[0121] In another aspect, the present disclosure provides a pharmaceutical composition. In some embodiments, the pharmaceutical composition comprises any of the compositions described herein and a pharmaceutically acceptable carrier. For example, the pharmaceutical composition may comprise a modified human cancer cell or cell line comprising at least 1, 2, 3, 4, 5, or more recombinant polynucleotides encoding at least one PTM enzyme selected from a citrullination enzyme, a cysteinylation enzyme, an acetylation enzyme, a hydroxylation enzyme, a phosphorylation enzyme, a methylation enzyme, a formylation enzyme, an oxidation enzyme, a hydroxylation enzyme, a ubiquitination enzyme, and/or a combination thereof. In some embodiments, the PTM enzyme comprises a citrullination enzyme. In some embodiments, the PTM enzyme comprises a cysteinylation enzyme. Further, the modified human cancer cell or cell line may comprise one or more co-stimulatory molecules, antigens, and/or cytokine described herein. The at least 1, 2, 3, 4, 5, or more recombinant polynucleotides may comprise a heterologous sequence encoding, e.g., a co-stimulatory molecule, an antigen, a cytokine, or a 2A splicing peptide. Thus, the recombinant polynucleotide encoding the one or more HLA alleles, co-stimulatory molecule, antigen, and/or cytokine may be separated by a sequencing encoding a 2 A splicing peptide (e.g., T2A, P2A, E2A). Generally, at least 1, 2, 3, 4, 5, or more recombinant polynucleotides are cloned into an expression vector (e.g., a replication defective lentiviral vector) for synthesis of the PTM enzymes, HLA alleles, co-stimulatory molecule, antigen, and/or cytokine, and introduced into the modified human cancer cell or cell line. Accordingly, the modified human cancer cell or cell line provided in the pharmaceutical composition may have at least 1, 2, 3, 4, 5, or more expression vectors, each comprising at least 1, 2, 3, 4, 5 or more recombinant polynucleotides encoding the PTM enzymes, HLA alleles, co-stimulatory molecule, antigen, and/or cytokine.

[0122] In some embodiments, the pharmaceutical composition further comprises a cryoprotectant, an interferon alpha (e.g., IFN-a2a or IFN-a2b), and/or an interferon lambda family member (e.g., an interferon lambda 1 (IFN-LI (IL-29)), an interferon lambda 2 (IFN-X2 (IL-28A)), an interferon lambda 3 (IFN-X3 (IL-28B)), an interferon lambda 4 (IFN-Z4)). In some embodiments, the interferon alpha e.g., IFN-a2a or IFN-a2b) is expressed by a vector comprising a polynucleotide sequence of the IFNA2 gene in the modified cancer cell as described herein. In some embodiments, the interferon alpha is a pegylated IFN-a2a provided exogenously. In some embodiments, the pharmaceutical composition further comprises one or more excipients. In some embodiments, the pharmaceutical composition further comprises CryoStor CS10, CryoStor CS2, or CryoStor CS5 cry opreservation media. In particular embodiments, the pharmaceutical composition comprises cells cryopreserved in CryoStor CS10, CryoStor CS2, or CryoStor CS5 cryopreservation media.

[0123] In some embodiments, the pharmaceutical composition is formulated in a dosage form comprising a total number of modified cancer cell per dose for administration to a subject in need therefor. In some embodiments, the pharmaceutical composition is formulated as an “off-the-shelf’ product for self-administration to a subject in need thereof. In some embodiments, the pharmaceutical composition may have at least at least 10,000 cells, at least 100,000 cells, at least 1,000,000 cells, at least 1,250,000 cells, at least 1,500,000 cells, at least 2,000,000 cells, at least 2,500,000 cells, at least 3,000,000 cells, at least 3,500,000 cells, at least 4,000,000 cells, at least 4,500,000 cells, at least 5,000,000 cells, at least 10,000,000 cells, at least 12,500,000 cells, at least 15,000,000 cells, at least 20,000,000 cells, at least 25,000,000 cells, at least 30,000,000 cells, at least 35,000,000 cells, at least 40,000,000 cells, at least 45,000,000 cells, or at least 50,000,000 cells. In some embodiments, the pharmaceutical composition comprises at least 1,000,000 cells. In some embodiments, the pharmaceutical composition comprises at least 20,000,000 cells.

[0124] In some embodiments, the pharmaceutical composition comprises at most 10,000 cells, at most 100,000 cells, at most 1,000,000 cells, at most 1,250,000 cells, at most 1,500,000 cells, at most 2,000,000 cells, at most 2,500,000 cells, at most 3,000,000 cells, at most 3,500,000 cells, at most 4,000,000 cells, at most 4,500,000 cells, at most 5,000,000 cells, at most 10,000,000 cells, at most 12,500,000 cells, at most 15,000,000 cells, at most 20,000,000 cells, at most 25,000,000 cells, at most 30,000,000 cells, at most 35,000,000 cells, at most 40,000,000 cells, at most 45,000,000 cells, or at most 50,000,000 cells. In some embodiments, the pharmaceutical composition comprises at most 20,000,000 cells. In some embodiments, the pharmaceutical composition comprises at most 40,000,000 cells.

[0125] In some embodiments, the pharmaceutical composition comprises about 1,000,000 to about 50,000,000 cells, about 5,000,000 to about 35,000,000 cells, about 10,000,000 to about 25,000,000 cells, about 15,000,000 to about 20,000,000 cells, or about 35,000,000 to about 40,000,000 cells. In some embodiments, the pharmaceutical composition comprises about 1,000,000 cells. In some embodiments, the pharmaceutical composition comprises about 20,000,000 cells. In some embodiments, the pharmaceutical composition comprises about 40,000,000 cells.

[0126] In some embodiments, the pharmaceutical composition is formulated in the form of a suspension. The formulation of pharmaceutical compositions is generally known in the art (see, e.g., REMINGTON’SPHARMACEUTICALSCIENCES, 18TH ED., Mack Publishing Co., Easton, PA (1990)). Prevention against microorganism contamination can be achieved through the addition of one or more of various antibacterial and antifungal agents. In particular embodiments, the pharmaceutical composition is a liquid formulation comprising cells resuspended in Lactated Ringer’s solution. [0127] Pharmaceutical forms suitable for administration include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. Typical carriers include a solvent or dispersion medium containing, for example, water-buffered aqueous solutions (z.e., biocompatible buffers, non-limiting examples of which include Lactated Ringer’s solution and CryoStor cry opreservation media (e.g., CS2, CS5, and CS10, containing 2%, 5%, and 10%, respectively of DMSO; available from BioLife Solutions, Bothell, WA)), ethanol, polyols such as glycerol, propylene glycol, polyethylene glycol, suitable mixtures thereof, surfactants, or vegetable oils.

[0128] Sterilization can be accomplished by an art-recognized technique, including but not limited to addition of antibacterial or antifungal agents, for example, paraben, chlorobutanol, sorbic acid or thimerosal. Further, isotonic agents such as sugars or sodium chloride may be incorporated in the subject compositions.

[0129] Production of sterile injectable solutions containing modified cancer cell(s), and/or other composition(s) of the present disclosure can be accomplished by incorporating the compound(s) in the required amount(s) in the appropriate solvent with various ingredients enumerated above, as required, followed by sterilization. To obtain a sterile powder, the above sterile solutions can be vacuum-dried or freeze-dried as necessary.

[0130] In some embodiments, the modified cancer cell(s), and/or other composition(s) provided herein are formulated for administration, e.g., intradermal injection, intralymphatic injection, oral, nasal, topical, or parental administration in unit dosage form for ease of administration and uniformity of dosage. Unit dosage forms, as used herein, refers to physically discrete units suited as unitary dosages for the subjects, e.g., humans or other mammals to be treated, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. In some instances, more concentrated dosage forms may be prepared, from which the more dilute unit dosage forms may then be produced. The more concentrated dosage forms thus will contain substantially more than, e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times the amount of the modified cancer cell(s), and/or other composition(s).

[0131] In some embodiments, the modified cancer cell(s), and/or other composition(s) provided herein are formulated for administration e.g., one or more doses over a period of time. In some embodiments, the modified cancer cell(s), and/or other composition(s) are formulated for administration every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, the modified cancer cell(s), and/or other composition(s) are formulated for administration every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 12 months, every 18 months, or every 24 months.

[0132] A dose may include, for example, about 50,000 to 50,000,000 (e.g., about 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, 1,000,000, 1,500,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, 5,500,000, 6,000,000, 6,500,000, 7,000,000, 7,500,000, 8,000,000, 8,500,000, 9,000,000, 9,500,000, 10,000,000, 11,000,000, 12,000,000, 13,000,000, 14,000,000, 15,000,000, 16,000,000, 17,000,000, 18,000,000, 19,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000, 50,000,000, or more) modified human cancer cells. In some embodiments, a dose may contain about 1,000,000 modified human cancer cells. In some embodiments, a dose may contain about 5,000,000 modified human cancer cells. In some embodiments, a dose may contain about 10,000,000 modified human cancer cells. In some embodiments, a dose may contain about 20,000,000 modified human cancer cells.

[0133] A dose may also include, for example, at least about 5,000,000 to 100,000,000 (e.g., about 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000, 50,000,000, 55,000,000, 60,000,000, 65,000,000, 70,000,000, 75,000,000, 80,000,000, 85,000,000, 90,000,000, 95,000,000, 100,000,000, or more) modified human cancer cells. In some embodiments, a dose may include at least about 1,000,000 modified human cancer cells. In some embodiments, a dose may include at least about 5,000,000 modified human cancer cells. In some embodiments, a dose may include at least about 10,000,000 modified human cancer cells. In some embodiments, a dose may include at least about 20,000,000 modified human cancer cells.

[0134] A dose may alternatively include, for example, at least about 100,000,000 to 1,000,000,000 (e.g., about 100,000,000, 150,000,000, 200,000,000, 250,000,000, 300,000,000, 350,000,000, 400,000,000, 450,000,000, 500,000,000, 550,000,000, 600,000,000,

650,000,000, 700,000,000, 750,000,000, 800,000,000, 850,000,000, 900,000,000,

950,000,000, 1,000,000,000, or more) modified human cancer cells. [0135] In some embodiments, the modified human cancer cells are replication-incompetent. In some embodiments, the modified human cancer cells are rendered replication incompetent by irradiation, freeze-thawing, or mitomycin C treatment. In some instances, the modified human cancer cells are irradiated. As disclosed herein, the irradiation can be used to both trigger non-enzymatic PTMs and destroy replication function of the cells. The irradiation dose may be, for example, between about 2 and 2,000 Gy (e.g., about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, or 2,000 Gy). In some embodiments, the modified human cancer cells are irradiated with a dose of about 200 Gy. In some embodiments, the modified human cancer cells are irradiated with a dose of about 100 Gy. In some embodiments, the modified human cancer cells are rendered replication incompetent by freeze-thawing. In some embodiments, the modified human cancer cells are rendered replication incompetent by mitomycin C treatment.

[0136] Methods for preparing such dosage forms are known to those skilled in the art (see, e.g., REMINGTON’S PHARMACEUTICAL SCIENCES, supra). The dosage forms typically include a conventional pharmaceutical carrier or excipient and may additionally include other medicinal agents, carriers, adjuvants, diluents, tissue permeation enhancers, solubilizers, and the like. Appropriate excipients can be tailored to the particular dosage form and route of administration by methods well known in the art (see, e.g., REMINGTON’S PHARMACEUTICAL SCIENCES, supra).

[0137] Examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, saline, syrup, methylcellulose, ethylcellulose, hydroxypropylmethylcellulose, and polyacrylic acids such as Carbopols, e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc. The dosage forms can additionally include lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying agents; suspending agents; preserving agents such as methyl-, ethyl-, and propyl-hydroxy-benzoates (i.e., the parabens); pH adjusting agents such as inorganic and organic acids and bases; sweetening agents; and flavoring agents. The dosage forms may also comprise biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes. [0138] In some embodiments, the pharmaceutical composition for administration may be an oral delivery vehicle such as a capsule, cachet or tablet, each of which contains a predetermined amount of the composition to provide the correct incremental dose to the patient. Oral delivery vehicles may be useful, for example, in avoiding contact between the composition and the mouth and upper gastrointestinal tract. For oral administration, the therapeutically effective dose can be in the form of tablets, capsules, emulsions, suspensions, solutions, syrups, sprays, lozenges, powders, and sustained-release formulations. Suitable excipients for oral administration include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, and the like.

[0139] In some embodiments, the therapeutically effective dose takes the form of a pill, tablet, or capsule, and thus, the dosage form can contain, along with the modified cancer cell(s), and/or other composition(s) described herein, any of the following: a diluent such as lactose, sucrose, dicalcium phosphate, and the like; a disintegrant such as starch or derivatives thereof; a lubricant such as magnesium stearate and the like; and a binder such a starch, gum acacia, polyvinylpyrrolidone, gelatin, cellulose and derivatives thereof.

[0140] In some embodiments, a suitable carrier masks the composition, e.g., the modified cancer cell(s), and/or other composition(s) from the mouth and upper gastrointestinal (GI) tract and reduces or prevents local itching/ swelling reactions in these regions during administration. For example, a carrier may contain one or more lipid, polysaccharide or protein constituents. In some cases, the carrier is a food product.

[0141] For topical administration, the therapeutically effective dose can be in the form of emulsions, lotions, gels, foams, creams, jellies, solutions, suspensions, ointments, and transdermal patches. For administration by inhalation, the modified cancer cell(s), and/or other composition(s) described herein can be delivered as a dry powder or in liquid form via a nebulizer. Aerosol formulations can be placed into pressurized acceptable propellants such as dichlorodifluoromethane. For parenteral administration, the therapeutically effective dose can be in the form of sterile injectable solutions and sterile packaged powders. Preferably, injectable solutions are formulated at a pH of from about 4.5 to about 7.5.

[0142] The therapeutically effective dose can also be provided in a lyophilized form. Such dosage forms may include a buffer, e.g., bicarbonate, for reconstitution prior to administration, or the buffer may be included in the lyophilized dosage form for reconstitution with, e.g., water. The lyophilized dosage form may further comprise a suitable vasoconstrictor, e.g., epinephrine. The lyophilized dosage form can be provided in a syringe, optionally packaged in combination with the buffer for reconstitution, such that the reconstituted dosage form can be immediately administered to an individual.

[0143] In some embodiments, the therapeutically effective dose may further comprise other components, for example, anti-allergy drugs, such as antihistamines, steroids, bronchodilators, leukotriene stabilizers and mast cell stabilizers. Suitable anti-allergy drugs are well known in the art.

E. Methods for Treating Cancer

[0144] In another aspect, the present disclosure provides a method for treating cancer in a subject. In some embodiments, the method comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition of the present disclosure (e.g., a pharmaceutical composition comprising modified cancer cells of the present disclosure) described herein.

[0145] In some embodiments, the method further comprises, prior to the administering step, (i) obtaining a sample from the subject; (ii) determining prevalent PTM(s) in the cell sample; and (iii) selecting a modified human cancer cell for administering to the subject, wherein the modified human cancer cell comprises the prevalent PTM(s).

[0146] In some instances, the prevalent PTM(s) are enzymatic PTM(s) wherein the modified human cancer cell comprises a recombinant polynucleotide encoding an enzyme that induces the prevalent PTM(s). For example, when a subject is identified as having prevalent citrullination PTMs in one or more immunogenic antigens, a modified human cancer cell described herein expressing a citrullination enzyme can be administered to the subj ect to induce a robust antigen-specific immune response.

[0147] In other instances, the prevalent PTM(s) are non-enzymatic PTM(s) wherein the modified human cancer cell induces the prevalent PTM(s) by non-enzymatic means. In some embodiments, the non-enzymatic means comprises irradiation, inducing cellular senescence, a chemical reaction, a small molecule, a cell culture supplement, cell culture medium, oxidative stress, or a combination thereof. For example, when a subject is identified as having prevalent cysteinylation PTMs in one or more immunogenic antigens, a modified human cancer cell described herein can be administered to the subject and non-enzymatic cysteinylation can be induced by culturing the modified human cancer cell with cysteine (e.g., excess cysteine in the cell culture medium) to induce a robust antigen-specific immune response. As another example, when a subject is identified as having prevalent cysteinylation PTMs in one or more immunogenic antigens, a modified human cancer cell described herein expressing or overexpressing a protein disulfide isomerase (PDI) can be administered to the subject to induce a robust antigen-specific immune response.

[0148] In some embodiments, the sample is a tumor biopsy or a liquid biopsy. In some embodiments, the liquid biopsy comprises circulating tumor cells (CTCs), circulating tumor DNA (ctDNA or cell free DNA), circulating RNA (cfRNA), exosomes, or a combination thereof. In some embodiments, the determining step comprises Next-generation sequencing (NGS) or immunopeptidome analysis of the sample. In some embodiments, the subject has a breast cancer, prostate cancer, melanoma, or lung cancer.

[0149] In some embodiments, the method comprises administering to the subject an effective amount of the pharmaceutical composition intradermally in the upper back or thighs. Without being bound by any theories, the upper back and thighs are chosen for patient acceptability as these areas have less nerves in the skin and are thus less sensitive. Additionally, the draining lymph nodes in the proximity may convey antigens from breast tumors in the upper and lower torso, which are common sites for breast cancer metastases. The method may further comprise administering to the subject the pharmaceutical composition in an interval of every week, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, the method comprises administering to be subject the pharmaceutical composition in an interval of every month, every 2 months, every 3 months, every 4 months, every 5 months, every 6 months, every 12 months, every 18 months, or every 24 months. In some embodiments, the method comprises administering to the subject the pharmaceutical composition for at least 6 weeks, 12 weeks, 24 weeks, 36 weeks, 48 weeks, 52 weeks or longer. In some embodiments, the method comprises administering to the subject the pharmaceutical composition for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, 12 months, or longer. In some embodiments, the method comprises administering to the subject the pharmaceutical composition for not more than 6 weeks, 12 weeks, 24 weeks, 36 weeks, 48 weeks, or 52 weeks. In some embodiments, the method comprises administering to the subject the pharmaceutical composition for not more than 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, or 12 months. [0150] In some embodiments, the method comprises administering to the subject an effective amount of the pharmaceutical composition through oral administration, topical contact, administration as a suppository, intravenous, intraperitoneal, intramuscular, intralesional, intratumoral, intradermal, intralymphatic, intrathecal, intranasal, or subcutaneous administration. In some embodiments, administration of the effective amount of the pharmaceutical composition is performed by parenteral administration (e.g., intravenous, intramuscular, intra-arteriole, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial) or transmucosal administration (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or transdermal). In some embodiments, the method comprises the use of liposomal formulations, intravenous infusion, or transdermal patches.

[0151] In some embodiments, the method further comprises administering to the subject one or more doses of cyclophosphamide intravenously at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, or longer, prior to administering to the subj ect the pharmaceutical composition described herein. In some embodiments, the cyclophosphamide is administered at least about 2-3 days prior to administering to the subject the pharmaceutical composition described herein. In some embodiments, a low-dose of cyclophosphamide at about 100, 150, 200, 250, 300, or 450 mg/m² is administered to the subject.

[0152] In some embodiments, the method further comprises administering to the subject one or more doses of an interferon-alpha-2b (IFN-a2b), IFN-a2a, or a pegylated IFN-a2a intradermally at the inoculation site of the pharmaceutical composition described herein. In some embodiments, the method further comprises administering to the subject one or more doses of IFN-a2b, IFN-a2a or pegylated IFN-a2a intradermally not more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, or 84 hours following administering to the subject the pharmaceutical composition described herein. In some embodiments, the method further comprises administering to the subject one or more doses of IFN-a2b, IFN-a2a or pegylated IFN-a2a intradermally at about 1-4 hours, about 2-6 hours, about 8-12 hours, about 10-24 hours, about 20-48 hours, or about 60-72 hours following administering to the subject the pharmaceutical composition described herein. In some embodiments, the method further comprises administering to the subject one or more doses of IFN-a2b, IFN-a2a or pegylated IFN-a2a intradermally no later than 5, 10, 15, 20, 25, 30, 45, 50, 60, 72, or 84 hours after administering to the subject the pharmaceutical composition. In some embodiments, the method further comprises administering to the subject one or more doses of IFN-a2b, IFN-a2a or pegylated IFN-a2a intradermally no later than 1, 2, 3, 4, 5, or 6 days following administering to the subject the pharmaceutical composition. In some embodiments, the method further comprises administering to the subject one or more doses of IFN-a2b, IFN-a2a or pegylated IFN-a2a intradermally no later than about 1-6 days, 2-3 days, or 3-5 days following administering to the subject the pharmaceutical composition. In some embodiments, the method further comprises administering to the subject a first dose of IFN-a2b, IFN-a2a or pegylated IFN-a2a intradermally between 1 to 4 hours and a second dose of IFN-a2b, IFN-a2a or pegylated IFN-a2a intradermally between 1-3 days following administering to the subject the pharmaceutical composition. In some embodiments, the IFN-a2b administered is at a low- dose between about 1-20,000 IU, 100-15,000 IU, 5000-12,000 IU, or 9,000-11,000 IU. In some embodiments, the IFN-a2b administered is at dose of about 10,000 IU. In some embodiments, the IFN-a2a or pegylated IFN-a2a administered is at a low-dose between about 0.01-0.1 micrograms (mcg), 0.05 - 0.15 mcg, 0.06 - 0.12 mcg, or 0.09 -0.11 mcg. In some embodiments, the IFN-a2b administered is at dose of about 0.1 mcg.

[0153] In some embodiments, the method further comprises administering to the subject one or more additional therapies. Examples of suitable additional types include, but are not limited to, chemotherapy, immunotherapy, radiotherapy, hormone therapy, a differentiating agent, and a small-molecule drug. One of skill in the art will readily be able to select an appropriate additional therapy.

[0154] Chemotherapeutic agents that can be used in the present disclosure include but are not limited to alkylating agents (e.g., nitrogen mustards (e.g., mechlorethamine, chlorambucil, cyclophosphamide, ifosfamide, melphalan), nitrosoureas (e.g., streptozocin, carmustine (BCNU), lomustine), alkyl sulfonates (e.g., busulfan), triazines (e.g., dacarbazine (DTIC), temozlomide), ethylenimines (e.g., thiotepa, altretamine (hexamethylmelamine)), platinum drugs (e.g., cisplatin, carboplatin, oxalaplatin), antimetabolites (e.g., 5 -fluorouracil (5-FU), 6- mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed), anthracycline anti-tumor antibiotics (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin), non-anthracycline anti-tumor antibiotics (e.g., actinomycin-D, bleomycin, mitomycin-C, mitoxantrone), mitotic inhibitors (e.g., taxanes (e.g., paclitaxel, docetaxel), epothilones (e.g., ixabepilone), vinca alkaloids (e.g., vinblastine, vincristine, vinorelbine), estramustine, corticosteroids (e.g., prednisone, methylprednisolone, dexamethasone), L-asparaginase, bortezomib, and topoisomerase inhibitors. Combinations of chemotherapeutic agents can be used. [0155] Topoisomerase inhibitors are compounds that inhibit the activity of topoisomerases, which are enzymes that facilitate changes in DNA structure by catalyzing the breaking and rejoining of phosphodiester bonds in the backbones of DNA strands. Such changes in DNA structure are necessary for DNA replication during the normal cell cycle. Topoisomerase inhibitors inhibit DNA ligation during the cell cycle, leading to an increased number of single- and double-stranded breaks and thus a degradation of genomic stability. Such a degradation of genomic stability leads to apoptosis and cell death.

[0156] Topoisomerases are often divided into type I and type II topoisomerases. Type I topoisomerases are essential for the relaxation of DNA supercoiling during DNA replication and transcription. Type I topoisomerases generate DNA single-strand breaks and also religate said breaks to re-establish an intact duplex DNA molecule. Examples of inhibitors of topoisomerase type I include irinotecan, topotecan, camptothecin, and lamellarin D, which all target type IB topoisomerases.

[0157] Type II topoisomerase inhibitors are broadly classified as topoisomerase poisons and topoisomerase inhibitors. Topoisomerase poisons target topoisomerase-DNA complexes, while topoisomerase inhibitors disrupt enzyme catalytic turnover. Examples of type II topoisomerase inhibitors include amsacrine, etoposide, etoposide phosphate, teniposide, doxorubicin, and fluoroquinolones.

[0158] In some embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. In some instances, the topoisomerase inhibitor is a topoisomerase I inhibitor, a topoisomerase II inhibitor, or a combination thereof. In particular embodiments, the topoisomerase inhibitor is selected from the group consisting of doxorubicin, etoposide, teniposide, daunorubicin, mitoxantrone, amsacrine, an ellipticine, aurintricarboxylic acid, HU-331, irinotecan, topotecan, camptothecin, lamellarin D, resveratrol, genistein, quercetin, epigallocatechin gallate (EGCG), and a combination thereof. EGCG is one example of a plant-derived natural phenol that serves as a suitable topoisomerase inhibitor. In some instances, the topoisomerase inhibitor is doxorubicin.

[0159] Immunotherapy refers to any treatment that uses the subj ect’ s immune system to fight a disease (e.g., cancer). Immunotherapy methods can be directed to either enhancing or suppressing immune function. In the context of cancer therapies, immunotherapy methods are typically directed to enhancing or activating immune function. In some instances, an immunotherapeutic agent comprises a monoclonal antibody that targets a particular type or part of a cancer cell. In some cases, the antibody is conjugated to a moiety such as a drug molecule or a radioactive substance. Antibodies can be derived from mouse, chimeric, or humanized, as non-limiting examples. Non-limiting examples of therapeutic monoclonal antibodies include alemtuzumab, bevacizumab, cetuximab, daratumumab, ipilimumab (MDX-101), nivolumab, ofatumumab, panitumumab, pembrolizumab, retifanlimab, rituximab, tositumomab, and trastuzumab.

[0160] Immunotherapeutic agents can also comprise an immune checkpoint inhibitor, which modulates the ability of the immune system to distinguish between normal and “foreign” cells. Programmed cell death protein 1 (PD-1) and protein death ligand 1 (PD-L1) are common targets of immune checkpoint inhibitors, as disruption of the interaction between PD1 and PD- L1 enhance the activity of immune cells against foreign cells such as cancer cells. Examples of PD-1 inhibitors include pembrolizumab, retifanlimab and nivolumab. An example of a PD- L1 inhibitor is atezolizumab.

[0161] Another immune checkpoint target for the treatment of cancer is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), which is a receptor that downregulates immune cell responses. Therefore, drugs that inhibit CTLA-4 can increase immune function. An example of such a drug is ipilimumab, which is a monoclonal antibody that binds to and inhibits CTLA-4.

[0162] The term “radiotherapy” refers to the delivery of high-energy radiation to a subject for the treatment of a disease (e.g., cancer). Radiotherapy can comprise the delivery of X-rays, gamma rays, and/or charged particles. Radiotherapy can be delivered locally (e.g. to the site or region of a tumor), or systemically (e.g., a radioactive substance such as radioactive iodine is administered systemically and travels to the site of the tumor).

[0163] The term “hormone therapy” can refer to an inhibitor of hormone synthesis, a hormone receptor antagonist, or a hormone supplement agent. Inhibitors of hormone synthesis include but are not limited to aromatase inhibitors and gonadotropin releasing hormone (GnRH) analogs. Hormone receptor antagonists include but are not limited to selective receptor antagonists and antiandrogen drugs. Hormone supplement agents include but are not limited to progestogens, androgens, estrogens, and somatostatin analogs. Aromatase inhibitors are used, for example, to treat breast cancer. Non-limiting examples include letrozole, anastrozole, and aminoglutethimide. GnRH analogs can be used, for example, to induce chemical castration. Selective estrogen receptor antagonists, which are commonly used for the treatment of breast cancer, include tamoxifen, raloxifene, toremifene, and fulvestrant. Antiandrogen drugs, which bind to and inhibit the androgen receptor, are commonly used to inhibit the growth and survival effects of testosterone on prostate cancer. Non-limiting examples include flutamide, apalutamide, and bicalutamide.

[0164] The term “differentiating agent” refers to any substance that promotes cell differentiation, which in the context of cancer can promote malignant cells to assume a less stem cell-like state. A non-limiting example of an anti-cancer differentiating agent is retinoic acid.

[0165] Small molecule drugs generally are pharmacological agents that have a low molecular weight (i.e., less than about 900 daltons). Non-limiting examples of small molecule drugs used to treat cancer include bortezomib (a proteasome inhibitor), imatinib (a tyrosine kinase inhibitor), and seliciclib (a cyclin-dependent kinase inhibitor), and epacadostat (an indoleamine 2,3 -dioxygenase (IDO1) inhibitor).

[0166] In some embodiments, the method of treating cancer of the present disclosure further comprises selecting a whole-cell cancer vaccine for the subject according to a method of the present disclosure described herein. In particular embodiments, the subject has stage I, stage II, stage III, and/or stage IV cancer. In other embodiments, the cancer is transitioning between stages. In some embodiments, the subject has a pre-cancerous lesion. In some embodiments, the subject does not have cancer.

[0167] In some embodiments, treating the subject comprises inhibiting cancer cell growth, inhibiting cancer cell proliferation, inhibiting cancer cell migration, inhibiting cancer cell invasion, ameliorating or eliminating the symptoms of cancer, reducing the size (e.g., volume) of a cancer tumor, reducing the number of cancer tumors, reducing the number of cancer cells, inducing cancer cell necrosis, pyroptosis, oncosis, apoptosis, autophagy, or other cell death, or enhancing the therapeutic effects of a composition or pharmaceutical composition. In some embodiments, treating the subject results in an increased survival time. In some instances, overall survival is increased. In other instances, disease-free survival is increased. In some instances, progression-free survival is increased. In particular embodiments, treating the subject results in a reduction in tumor volume and/or increased survival time.

[0168] In particular embodiments, treating the subject enhances the therapeutic effects of an anti-cancer therapy such as a chemotherapeutic agent, an immunotherapeutic agent, radiotherapy, hormone therapy, a differentiating agent, and/or a small-molecule drug. [0169] Therapy such as modified cancer cell(s), composition(s), and pharmaceutical composition(s) of the present disclosure can be administered using routes, dosages, and protocols that will readily be known to one of skill in the art. Administration can be conducted once per day, once every two days, once every three days, once every four days, once every five days, once every six days, or once per week. Therapy can be administered 1, 2, 3, 4, 5, 6,

7, 8, 9, 10, 11, 12, 13, 14, or more times per week. In some cases, modified cancer cell(s), composition(s), and/or pharmaceutical composition(s) of the present disclosure are administered as a single dose, co-administered (e.g., administered in separate doses or by different routes, but close together in time), or administered separately (e.g., administered in different doses, including the same or different route, but separated by about 1, 2, 3, 4, 5, 6, 7,

8, 9, 10, 11, 12, or more hours). In cases where multiple doses are to be administered in the same day, or where a single dose comprises one or more components (e.g., the modified cancer cell(s) and IFNa are administered separately), administration can occur, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more times in a day.

[0170] In some cases, therapeutic administration can occur about once per week, about every two weeks, about every three weeks, or about once per month. In other cases, therapeutic administration can occur about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more times per month. Treatment can continue for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more weeks; about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months; or longer. At any time during treatment, the therapeutic plan can be adjusted as necessary. For example, depending on the response to modified cancer cell(s), compositions, or pharmaceutical composition(s) of the present disclosure, a different vaccine may be selected, one or more additional therapeutic agents or drugs may be chosen, or any aspect of the therapeutic plan can be discontinued. One of skill in the art will readily be able to make such decisions, which can be informed by, for example, the results of allele profile comparison, changes in the activity and/or number of an immune cell, and/or changes in the the presence or level of one or more biomarkers.

[0171] The modified cancer cell(s), composition(s), and pharmaceutical composition(s) of the present disclosure can be administered by any suitable route, including those described herein. In some embodiments, the administration is by intradermal or intralymphatic injection. In some embodiments, the whole-cell cancer vaccine (e.g., comprising modified cancer cells of the present disclosure) is given separately from interferon alpha (IFNa). In some instances, the IFNa is injected locally. IFNa can be given before and/or after the vaccine is administered. Timing of the separate injections can be any suitable interval, including those described herein.

[0172] One of skill in the art will readily be able to administer the number of appropriate modified cancer cells to include in a particular dose. A dose may include, for example, about 50,000 to 50,000,000 (e.g, about 50,000, 60,000, 70,000, 80,000, 90,000, 100,000, 110,000, 120,000, 130,000, 140,000, 150,000, 160,000, 170,000, 180,000, 190,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,000, 650,000, 700,000, 750,000, 800,000, 850,000, 900,000, 950,000, 1,000,000, 1,500,000, 2,000,000, 2,500,000, 3,000,000, 3,500,000, 4,000,000, 4,500,000, 5,000,000, 5,500,000, 6,000,000, 6,500,000, 7,000,000, 7,500,000, 8,000,000, 8,500,000, 9,000,000, 9,500,000, 10,000,000, 11,000,000, 12,000,000, 13,000,000, 14,000,000, 15,000,000, 16,000,000, 17,000,000, 18,000,000, 19,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000, 50,000,000, or more) modified cancer cells. In some embodiments a dose may contain about 1,000,000 modified cancer cells. In some embodiments a dose may contain about 5,000,000 modified cancer cells. In some embodiments a dose may contain about 10,000,000 modified cancer cells. In some embodiments a dose may contain about 20,000,000 modified cancer cells.

[0173] A dose may also include, for example, at least about 5,000,000 to 100,000,000 (e.g., about 5,000,000, 6,000,000, 7,000,000, 8,000,000, 9,000,000, 10,000,000, 15,000,000, 20,000,000, 25,000,000, 30,000,000, 35,000,000, 40,000,000, 45,000,000, 50,000,000, 55,000,000, 60,000,000, 65,000,000, 70,000,000, 75,000,000, 80,000,000, 85,000,000, 90,000,000, 95,000,000, 100,000,000, or more) modified cancer cells.

[0174] A dose may alternatively include, for example, at least about 100,000,000 to 1,000,000,000 (e.g., about 100,000,000, 150,000,000, 200,000,000, 250,000,000, 300,000,000, 350,000,000, 400,000,000, 450,000,000, 500,000,000, 550,000,000, 600,000,000,

650,000,000, 700,000,000, 750,000,000, 800,000,000, 850,000,000, 900,000,000,

950,000,000, 1,000,000,000, or more) modified cancer cells.

[0175] In some embodiments, the modified cancer cells are irradiated. The irradiation dose may be, for example, between about 2 and 2,000 Gy (e.g., about, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, or 2,000 Gy). In particular embodiments, the modified cancer cells are irradiated with a dose of about 100 Gy.

[0176] In some embodiments, treating the subject results in an increase in the presence or level of one or more biomarkers measured or detected in a sample obtained from the subject. In particular embodiments, treating the subject results in no change the presence or level of the one or more biomarkers.

[0177] In some embodiments, treating the subject results in an increase in the activity and/or number of one or more immune cells. In some instances, the increase is produced in one cell type. In other instances, the increase is produced in multiple cell types. In some embodiments, the cell in which the level of activity and/or number is increased is selected from the group consisting of a peripheral blood mononuclear cell (PBMC), a lymphocyte (e.g. T lymphocyte, B lymphocyte, NK cell), a monocyte, a dendritic cell, a macrophage, a myeloid-derived suppressor cell (MDSC), and a combination thereof. In particular embodiments, the level of activity and/or number of immune cell(s) is measured using methods of the present disclosure described herein.

[0178] In some embodiments, an increase in immune cell activity and/or number indicates that the subject should be administered one or more additional doses of the pharmaceutical composition (e.g., comprising modified cancer cells of the present disclosure). In some instances, a different vaccine is administered. One of skill in the art will recognize that an increase in immune cell activity and/or number will occur, in some instances, after 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more doses of the vaccine have been administered.

[0179] In some embodiments, a sample is obtained from the subject. In other embodiments, a sample is obtained from a different subject or a population of subjects. Samples can be used for the purposes of selecting an appropriate cancer vaccine of the present disclosure, monitoring the response to vaccine therapy, and/or predicting how the subject will respond to vaccine therapy. Samples obtained from a different subject and/or a population of subjects can be used, for example, to establish reference ranges to facilitate comparisons that are part of the methods of the present disclosure. Samples can be obtained at any time, including before and/or after administration of the modified cancer cell(s), pharmaceutical composition(s), and/or other composition(s) of the present disclosure. In some embodiments, the sample comprises whole blood, plasma, serum, cerebrospinal fluid, tissue, saliva, buccal cells, tumor tissue, urine, fluid obtained from a pleural effusion, hair, skin, or a combination thereof. In general, the sample can comprise any biofluid. In some instances, the sample comprises circulating tumor cells (CTCs). The sample can also be made up of a combination of normal and cancer cells. In particular embodiments, the sample comprises circulating tumor cells (CTCs). The sample can be obtained, for example, from a biopsy, from a surgical resection, and/or as a fine needle aspirate (FNA). Samples can be used to determine, measure, or detect HLA allele(s), immune cell activity and/or number, and/or biomarker(s), as described herein.

[0180] In some embodiments, the results of the immune cell activity and/or number measurement, and/or biomarker presence or level determinations are recorded in a tangible medium. For example, the results of assays (e.g., the activity level and/or number of immune cells, the presence or level (e.g., expression) of one or more biomarkers and/or a prognosis or diagnosis (e.g., of whether or not there is the presence of cancer, the prediction of whether the subject will respond to a vaccine, or whether the subject is responding to a vaccine) can be recorded, e.g., on paper or on electronic media (e.g., audio tape, a computer disk, a CD, a flash drive, etc.).

[0181] In other embodiments, the methods further comprise the step of providing the results of assays, prognosis, and/or diagnosis to the patient (i.e., the subject) and/or the results of treatment.

F. Methods for Enhancing Antigen Immunogenicity

[0182] In another aspect, the present disclosure provides a method for enhancing the immunogenicity of an antigen in a cell herein. In some embodiments, the method comprises inducing a post-translational modification (PTM) of the antigen in the cell of the present disclosure described, wherein the PTM enhances the immunogenicity of the antigen. In some instances, the antigen in a natural setting (such as in an unmodified cell) does not comprises the PTM, wherein the antigen in the modified human cancer cell comprises such PTM, by either enzymatic or non-enzymatic means, resulting in the antigen in the modified human cancer cell being more immunogenetic in comparison to the antigen in the natural setting. In other instances, the antigen in a natural setting (such as in an unmodified cell) comprises the PTM, wherein the antigen in the modified human cancer cell comprises an induced PTM, by either enzymatic or non-enzymatic means, in comparison to the antigen in the natural setting, resulting in a higher immunogenicity of the induced PTM antigen in the modified human cancer cell. In some embodiments, the antigen is an antigen of a pathogen, a tumor-specific antigen, a tumor-associated antigen, a neo-antigen, an allergen, an antigen that is a target of an autoimmune response, or a fragment thereof.

[0183] In some embodiments, the PTM comprises phosphorylation, acetylation, ubiquitination, succinylation, methylation, malonylation, glycosylation, sumoylation, nitrosylation, glutathionylation, amidation, hydroxylation, palmitoylation, glutarylation, crotonylation, oxidation, myristoylation, sulfation, formylation, citrullination, prenylation, cysteinylation, deamidation, dehydration, or a combination thereof. In particular embodiments, the PTM comprises cysteinylation and/or citrullination.

[0184] In some embodiments, the cell is a modified human cancer cell comprising at least one recombinant polynucleotide encoding one or more antigens. In some embodiments, the cell is a modified human cancer cell comprising (i) one or more recombinant polynucleotides each encoding an allele of a human leukocyte antigen (HLA) class I gene; and/or (ii) one or more recombinant polynucleotides each encoding an allele of an HLA class II gene. In some embodiments, the cell is a modified human cancer cell comprising one or more recombinant polynucleotides encoding a cytokine, a variant thereof, or a fragment thereof. In some embodiments, the cell is a modified human cancer cell comprising one or more recombinant polynucleotides encoding a co-stimulatory molecule, a variant thereof, or a fragment thereof. In some embodiments, the cell is a modified human cancer cell comprising at least one recombinant polynucleotide encoding one or more PTM enzymes, variants thereof, or fragments thereof. In some embodiments, the cell is a modified human cancer cell comprising a) at least one recombinant polynucleotide encoding one or more PTM enzymes, variants thereof, or fragments thereof.; b) at least one recombinant polynucleotide encoding one or more antigens; c) (i) one or more recombinant polynucleotides each encoding an allele of a human leukocyte antigen (HLA) class I gene; and/or (ii) one or more recombinant polynucleotides each encoding an allele of an HLA class II gene; d) one or more recombinant polynucleotides encoding a cytokine, a variant thereof, or a fragment thereof; and/or e) one or more recombinant polynucleotides encoding a co-stimulatory molecule, a variant thereof, or a fragment thereof. In some embodiments, one or more HLA alleles endogenous to the cell have been inactivated in the modified human cancer cell.

[0185] In some embodiments, the cell is a human cancer cell line. In some embodiments, the cell is a primary cancer cell. In some embodiments, the cell is a breast cancer cell, a prostate cancer cell, a melanoma cell, or a lung cancer cell. [0186] In some embodiments, the PTM is induced by an enzyme. In some embodiments, the enzyme comprises a citrullination enzyme, a cysteinylation enzyme, an acetylation enzyme, a hydroxylation enzyme, a phosphorylation enzyme, a methylation enzyme, a formylation enzyme, an oxidation enzyme, a hydroxylation enzyme, a ubiquitination enzyme, or a combination thereof. In some embodiments, the enzyme comprises a citrullination enzyme. In some embodiments, the enzyme comprises a cysteinylation enzyme. In some embodiments, the cell comprises a recombinant polynucleotide encoding one or more enzymes selected from Tables 1-10

[0187] In some embodiments, the PTM is a non-enzymatic PTM. In some embodiments, the non-enzymatic PTM is induced by irradiation, inducing cellular senescence, a chemical reaction, a small molecule, a cell culture supplement, cell culture medium, oxidative stress, or a combination thereof. In some embodiments, the non-enzymatic PTM comprises cysteinylation. In some embodiments, non-enzymatic cysteinylation can be induced by culturing cells with excess cysteine. In some embodiments, non-enzymatic oxidation can be increased by irradiating cells. In some embodiments, non-enzymatic oxidation can be increased by culturing cells in serum-free media. In some embodiments, non-enzymatic PTMs can be prompted by inducing cell senescence. In some embodiments, a small molecule, such as a senescence inducer, can be used to induce non-enzymatic PTMs in the cells disclosed herein. Such senescence inducers include but are not limited to doxorubicin (genotoxic), palbociclib (CDK4/6 inhibitor), and nutlin-3 A (p53 activator).

G. Kits

[0188] In another aspect, the present disclosure provides a kit for treating a subject with a cancer. In some embodiments, the kit comprises a modified cancer cell line, a composition, and/or a pharmaceutical composition of the present disclosure described herein. The kits are useful for treating any cancer, some non-limiting examples of which include breast cancer, ovarian cancer, cervical cancer, prostate cancer, pancreatic cancer, colorectal cancer, gastric cancer, lung cancer, skin cancer, liver cancer, brain cancer, eye cancer, soft tissue cancer, renal cancer, bladder cancer, head and neck cancer, mesothelioma, acute leukemia, chronic leukemia, medulloblastoma, multiple myeloma, sarcoma, and any other cancer described herein, including a combination thereof.

[0189] Materials and reagents to carry out the various methods of the present disclosure can be provided in kits to facilitate execution of the methods. As used herein, the term “kit” includes a combination of articles that facilitates a process, assay, analysis, or manipulation. In particular, kits of the present disclosure find utility in a wide range of applications including, for example, diagnostics, prognostics, therapy, and the like.

[0190] Kits can contain chemical reagents as well as other components. In addition, the kits of the present disclosure can include, without limitation, instructions to the kit user, apparatus and reagents for sample collection and/or purification, apparatus and reagents for product collection and/or purification, apparatus and reagents for administering modified cancer cell(s) or other composition(s) of the present disclosure, apparatus and reagents for determining the level(s) of biomarker(s) and/or the activity and/or number of immune cells, apparatus and reagents for detecting PTMs, sample tubes, holders, trays, racks, dishes, plates, solutions, buffers or other chemical reagents, suitable samples to be used for standardization, normalization, and/or control samples. Kits of the present disclosure can also be packaged for convenient storage and safe shipping, for example, in a box having a lid. For instance, the kits may be stored and shipped at room temperature, on wet ice or with cold packs, or frozen in the vapor phase of liquid nitrogen or in dry ice.

[0191] In some embodiments, the kits also contain negative and positive control samples for detection of PTMs, immune cell activity and/or number, and/or the presence or level of biomarkers. In some embodiments, the negative control samples are non-cancer cells, tissue, or biofluid obtained from the subject who is to be treated or is already undergoing treatment. In other embodiments, the negative control samples are obtained from individuals or groups of individuals who do not have cancer. In other embodiments, the positive control samples are obtained from the subject, or other individuals or groups of individuals, who have cancer. In some embodiments, the kits contain samples for the preparation of a titrated curve of one or more biomarkers in a sample, to assist in the evaluation of quantified levels of the activity and/or number of one or more immune cells and/or biomarkers in a biological sample.

IV. Examples

[0192] The present disclosure will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results. Example 1: Post-translational modifications in HLA antigens

[0193] This example illustrates an immunopeptidome analysis of SV-BR-l-GM that detected post-translational modifications (PTMs) in multiple antigens that are bound to Class I and/or Class II HLA molecules.

[0194] To identify and characterize MHC-I and Il-associated peptides displayed by proprietary SV-BR-l-GM, a GM-CSF-secreting breast tumor cell line, an immunopeptidome analysis was performed after cell lysis and immunoprecipitation (IP) using sequential immunoaffinity resins specific to MHC class I or II, and elution of peptides for identification by mass spectrometry.

Materials and Methods

[0195] Cell samples were prepared using the protocol shown below. The materials used in the assay are listed in Table 11.

[0196] Sample Preparation:

1. Cell pellets were thawed on ice, then lysed in 1 ml of lysis buffer per 50 million cells, incubating 30 minutes on ice.

2. Insoluble material was pelleted at 800 * g for 5 minutes.

3. Supernatant was centrifuged at 20,000 x g for 30 minutes at 4°C. a. *50 pL lysate was retained from Pre IP for ELISA.

4. Resin was washed and combined with clarified lysates and mixed by gentle rotation at 4°C overnight.

5. The next day, samples were centrifuged at 800 x g for 5 minutes at 4°C. a. *Total supernatant was immediately subjected to sequential MHC class II IP (Step 11).

6. Three washes (buffers 1-3) of the resin were performed, which consisted of the following: a . Add 2.5 ml of buffer to resin and vortex. b. Centrifuge 325 x g for 5 minutes at 4°C.

C. Discard the supernatant.

7. At wash #4, 0.75 ml of buffer 4 was added and the total volume was transferred to LoBind tubes. a. An additional 0.75 ml buffer was added and centrifuged 800 x g for 5 minutes at 4°C. b. Supernatant was discarded.

8. 1 ml of elution buffer was added to each tube and incubated at 37°C for 5 minutes.

9. Samples were centrifuged at 800 x g for 5 minutes at 4°C to elute.

10. Eluates (supernatant) were collected into new LoBind Eppendorf tubes and stored at -80°C.

11. Reserved flowthrough was then mixed with 100 pL of L243 sepharose and incubated overnight at 4°C with gentle rotation. 12. The mixture was centrifuged at 800 x g for 5 minutes and the supernatant was transferred to a new tube and 100 pL retained for ELISA (Post IP).

13. Flow through from L243 IP was mixed with 100 pL of IVA12 sepharose and incubated overnight at 4°C with gentle rotation.

14. Resin with captured HLA-DR was washed and peptides were eluted as described in steps 6-10.

15. IVA12 sepharose was washed and peptides were eluted as described in steps 5-10.

16. All samples were submitted for LC-MS/MS analysis.

Table 11. Materials used in the immunopeptidome analysis.

[0197] LC-MS/MS analysis was performed using the methods shown below.

Sample Preparation

[0198] Peptides were concentrated and desalted using solid-phase extraction (SPE) with Waters pHLB Cl 8 plate. Peptides were loaded directly and eluted using 80/20 acetonitrile/water (0.1% TFA). Eluted peptides were lyophilized and reconstituted in 0.1% TFA.

Mass Spectrometry

[0199] Peptides (50% per sample) were analyzed by nano LC/MS/MS using a Waters NanoAcquity system interfaced to a ThermoFisher Fusion Lumos mass spectrometer. Peptides were loaded on a trapping column and eluted over a 75 pm analytical column at 350 nL/min; both columns were packed with Luna Cl 8 resin (Phenomenex). A 2-hour gradient was employed. The mass spectrometer was operated using a custom data-dependent method, with MS performed in the Orbitrap at 60,000 FWHM resolution and sequential MS/MS performed using high resolution CID and EThcD in the Orbitrap at 15,000 FWHM resolution. All MS data were acquired from m/z 300- 800. A 3 second cycle time was employed for all steps. Data Processing

[0200] Raw files were searched using a local copy of PEAKS with the following parameters:

• Enzyme: None

• Database: Swissprot Human Fixed modification: None

• Variable modifications: Oxidation (M), Acetyl (Protein N-terminus) Mass values: Monoisotopic

• Peptide Mass Tolerance: 10 ppm Fragment Mass Tolerance: 0.02 Da Max Missed Cleavages: N/A

• PSM FDR: 1%

• Chimeric peptide: TRUE

[0201] Peptides were further analyzed for PTMs (PEAKS PTM) and mutations (SPIDER).

The peptide. csv file was exported.

Results

[0202] A total of 6,932 MHC class I peptides and 5, 197 MHC class II peptides were detected at the 1% PSM FDR based on forward/decoy database searching. An overview of MHC class I and II peptide totals and intensities are shown in Tables 12 and 13, respectively.

'able 12. MHC class I peptide totals and intensities.

'able 13. MHC class II peptide totals and intensities.

[0203] In addition, 279 MHC class I peptides and 525 MHC class II peptides were detected with post-translational modification (PTM). FIG. 1 illustrates a histogram of PTMs in MHC class I and/or II peptides, indicating cancer cells have multiple peptides which bear PTMs in their immunopeptidome and those post-translational modified peptides are potential targets of an immune response. Example 2: Determining which enzymes to be introduced into cancer cells

[0204] This example illustrates how to determine which enzyme to be introduced into a cancer cell line or a primary cancer cell.

[0205] In a given tumor type (e.g., breast cancer), the prevalent PTMs can be found in public databases of immunopeptidome (e.g., http://www.zhang-lab.org/caatlas/, See-. Yi X, Liao Y, Wen B, et al. caAtlas: An immunopeptidome atlas of human cancer. iScience. 2021;24(10): 103107.). Cancer antigen atlas (caAtlas, http://www.zhang-lab.org/caatlas/) provides a central resource for the selection and prioritization of MHC -bound peptides for in vitro HLA binding assay and immunogenicity testing, which can be used to characterize post- translationally modified antigens and their cancer-association. For example, in a given tumor type (e.g. breast cancer), caAtlas indicates a high proportion of tumors have their immunopeptidome peptides undergone acetylation. As such, an acetylation enzyme, such as an acetyltransferase, can be introduced into the specific cancer cell line.

[0206] Alternatively, one can search public databases of microarray or RNAseq data (see sites.broadinstitute.org/ccle/datasets and www.cancer.gov/ccg/research/genome- sequencing/tcga) to determine which enzymes that produce PTMs are widely expressed in the specific type of tumor in general. For example, in a given tumor type (e.g., lung cancer), it is found in the RNA expression database(s) that a high proportion of tumors express high levels of peptidyl-arginine deiminase (PAD), the enzyme that catalyzes citrullination, indicating the PAD can be a good candidate to be introduced into the specific cancer cell line.

[0207] Similarly, we can also determine the prevalent PTMs in an individual cancer patient by evaluating biopsy material, circulating tumor cells, or circulating tumor RNA. If one or more enzymes (e.g., PAD) that produce PTMs are widely expressed in the patient’s tumor, we can select those one or more enzymes (e.g., PAD) to be introduced into the cancer cell for cancer treatment. Alternatively, we can examine the prevalent PTMs in the patient’s tumor or in the immunopeptidome of the tumor to determine which enzyme(s) to be introduced into the cancer cell for cancer treatment. For example, if an immunopeptidome analysis of the biopsy material, circulating tumor cells, or circulating tumor RNA provides evidence that most antigens have undergone citrullination, we can introduce PAD into the cancer cell for cancer treatment. Example 3: Generation of modified cancer cells expressing enzymes that induce

PTMs

[0208] This example describes how to generate modified cancer cells expressing enzymes that induce PTMs of an antigen.

[0209] Once a decision has been made regarding which enzyme(s) to be introduced into a cancer cell, we can introduce the gene(s) encoding such enzyme(s) into the cell through transfection, transduction, or other methods of genetic engineering. For example, the cell can be stably transfected or transduced with the gene encoding the PAD enzyme. In one embodiment, the transduction can be done through lentivirus.

[0210] Parental cell line. Any kind of human cancer cell lines can be used as parental cell lines for generating modified cancer cell lines expressing enzymes that induces PTMs. Modified cancer cells lines, such as the Bria-OTS cell lines described herein, also can be used as parental cell lines for generating modified cancer cell lines expressing enzymes that induces PTMs. The Bria-OTS cell lines were derived from the breast cancer parent cell line, SV-BR- 1, which expresses multiple cancer-associated antigens and immune stimulating factors including Class II HLA molecules that directly activate CD4+ T cells to enhance the immune response. Generation of the initial SV-BR-1 cell line is described in WO 2017/147600, which is incorporated herein in its entirety. Generation of the Bria-OTS cell lines is described in WO 2023/167973, which is incorporated herein by reference in its entirety.

[0211] To prepare the therapeutic composition described herein, a vial from the master cell bank (MCB) can be thawed, grown in culture, harvested, and cryopreserved.

[0212] Production of lentivirus. To produce lentiviral vectors, HEK 293T cells (Clontech Lenti-X 293 cells) are cultured in DI OHG media, transfected using Minis TRANS-IT 293 transfection Reagent with DMEM media, with the VSV-G envelope plasmid, packaging plasmid and vector. On the fifth day, the conditioned media can be harvested, centrifuged, and the supernatant can be concentrated using Millipore Centricon Plus 70 PL-100, and then filtered using Costar 0.45 Spinx centrifuge filter tubes. Viral titer will be determined using the ABM LV900 Lentivirus Titer Kit with Mastermix R. Testing of the lentiviral vector can be conducted, for example, by determining titer, sequence, RCL, bioburden, and/or endotoxin.

[0213] Lentiviral transduction. Cells can be seeded in a 12-well plate at a density of 0.29 x 10⁶ cells/well and transduced at 150 MOI the next day. For transduction, the medium can be 1ml RPMI 1640 + 10% FBS + L-Glutamine+ 20 mg/mL Protamine Sulfate. Once the cells recovered and reattached, cells are lifted using 4 mL TrypLE Express for 5 minutes. Reaction will be stopped with 8 mL culture media. Cells are counted and cryopreserved using CS10 at approximately 8 x 10⁵ cells/vial.

[0214] Lentiviral transduced cells can be transferred into culture and expanded. Cells can be further expanded, and then single cell cloning was initiated by seeding at 2 cells/well in 96 well plates. Single cell clones were transferred to 48-well plates. ELISA shall be performed to identify positive clones (e.g., enhanced enzyme production), and positive clones will be transferred to a 12-well plate. For example, the PAD positive clone (enhanced PAD production) will be expanded and then cryopreserved using CS10 freezing medium.

[0215] A master cell bank (MCB) may be created for each cell line and tested. As an illustration, the cell lines will be propagated in T-25, T-75 and T-150 flasks using RPMI with 10% FBS medium. To confirm the enhanced production of PAD, randomly selected flasks will be incubated in antibiotic-free RPMI + 10% FBS for 72 hours. The supernatant will be collected, and the concentration of PAD will be determined by ELISA. To confirm the expression of specific enzyme (e.g., PAD), cells will be subjected to flow cytometry using enzyme-specific antibodies and RT-PCR using enzyme-specific primers.

Example 4: Safety and efficacy of modified human cancer cell lines in cancer patients

[0216] This example illustrates one embodiment of testing the safety and efficacy of modified human cancer cell lines (e.g., Bria-OTS cell lines with induced PTM antigens) in patients with advanced metastatic or locally recurrent breast cancer in a clinical trial. This study will provide preliminary data regarding the safety, tolerability, tumor response in patients with advanced (e.g., metastatic or locally recurrent) breast cancer. A brief summary of the clinical study design is provided below.

[0217] This is a Phase l/2a, open-label clinical study with patients assigned to treatment with a breast cancer immunotherapy cell line. The primary objective of the study is to evaluate the safety of cellular immunotherapy in patients with advanced breast cancer. The secondary objective of the study is to evaluate the tumor response to cellular immunotherapy in patients with advanced breast cancer. Additionally, the study may include objectives to evaluate progression-free (PFS) and overall survival (OS) in advanced breast cancer patients treated with cellular immunotherapy, to evaluate the immune responses elicited by cellular immunotherapy in patients with advanced breast cancer, to evaluate patient and tumor characteristics that may be predictive of responses to cellular immunotherapy in patients with advanced breast cancer, and/or to evaluate Quality of Life (QOL) in advanced breast cancer patients treated with cellular immunotherapy. The cell lines being studied are the Bria-OTS cell lines with induced post-translational modified antigens (e.g., a Bria-OTS cell line expressing PAD that induces citrullination of antigens). Patients will be treated with the cell line that most closely matches their HLA type, with at least one match required for a patient to be treated.

[0218] Additionally, to boost the immune response, patients are pretreated with low-dose cyclophosphamide that downregulates T regulatory-cell mechanisms 48-72 hours (2-3 days) prior to each vaccine inoculation. Low-dose pegylated interferon-alpha-2a (IFN-a2a) serves as an adjuvant and is given by intradermal injection at 0.1 mcg to the inoculation site about 1-4 hours and about 24-72 hours (1-3 days) following vaccine inoculation. Biological samples are collected at regular intervals per protocol, and stored in a repository.

[0219] As an illustration, in Part 1 (Phase 1) of the study, there will be intra-patient dose escalation with each patient treated with a single cell line. Once the dose of cells has been decided in Part 1, in Part 2 (Phase 2a) patients may be treated with either 1 or 2 cell lines, with the goal to have patients match the cell lines used at least at 2 HLA types, preferably one class II (HLA-DR) type and one class I (HLA-A, B, or C) type.

[0220] Patient population. Patients will be screened to assure they fulfill the enrollment criteria. Screening must be performed within 30 days of initiating therapy, and imaging studies must be performed within 2 weeks of initiating therapy. All patients will be women with histologically confirmed breast cancer with recurrent and/or metastatic lesions via investigational site, which has failed prior therapy. Patients with any of the 4 breast cancer subtypes will be eligible, i.e., luminal A (HR+/HER2-), triple-negative (HR-/HER2-), luminal B (HR+/HER2+), and HER2-enriched (HR-/HER2+), given they meet the required specifications of failed prior treatments. Patients with new or progressive breast cancer metastatic to the brain will also be eligible if their intracranial disease is stable and not lifethreatening as noted in the protocol synopsis.

[0221] Planned enrollment for both parts of the study is up to 48 patients, with 12-24 patients (at least 3 patients with each cell line) to be evaluated initially. If the cell lines are found to be safe in Part 1, then the expansion cohort of 24 patients (at least 4 patients per cell line) will be enrolled in Part 2.

[0222] Study treatment and dosage escalation. The Bria-OTS cell lines with induced post- translational modified antigens (e.g., a Bria-OTS cell line expressing PAD that induces citrullination of antigens) will be irradiated to render them replication incompetent prior to freezing in viable freezing media. They will be shipped to the clinical sites frozen and thawed on site for inoculation. Patients will be evaluated initially every week during the dose escalation phase, including all safety assessments.

[0223] The dosage form is formulated in suspension of irradiated cells. Patients will be administered with a therapeutically effective amount of the composition through intradermal injection in the upper back or thighs over a period of time. Table 14 illustrates dosing regimen for Phase 1 monotherapy phase.

Table 14. Dosing regimen for monotherapy phase

[0224] Patients will return 2±1 days following each inoculation for safety assessments and to measure the delay ed-type hypersensitivity (DTH) response during the monotherapy phase. Following the monotherapy phase the patients will be treated with the top dose (i.e., maximum tolerated dose [MTD] or a pharmacologically active dose), which was the cell dose safely tolerated every 3 weeks. During this phase, the patient will also receive cyclophosphamide 300 mg/m² 2-3 days prior to each cell line inoculation, and pegylated IFN-a2a 0.1 mcg into each inoculation site, 1-4 hours and 1-3 days following each cell line inoculation. The entire cycle will be repeated every 3 weeks.

[0225] Once at least 3 patients have been safely treated with each cell line as a single cell line, Part 2 (Phase 2a) of the study will commence. During Part 2, all patients will be treated with 1 or 2 cell lines, with the goal to have patients match the cell lines used at least at two HLA types, preferably one class II (HLA-DR) type and one class I (HLA-A, B or C) type. Treatment will be preceded by cyclophosphamide, 300 mg/m², 2-3 days prior to the BC cell line inoculations. Treatment will be with 20,000,000 cells (up to 4 intradermal injections in the upper back and thighs). They will the receive pegylated IFN-a2a 0.1 mcg into each inoculation site, 1-4 hours and 1-3 days following each cell line inoculation. Patients will return 2±1 days following each inoculation for safety assessments and to measure the DTH response. Treatment cycles will be repeated every 3 weeks.

[0226] Safety Assessments. Safety assessments will include adverse event reporting, physical examinations, vital signs, cardiac assessments (ECG), and safety laboratory assessments. Safety /Tolerability responses will be evaluated and graded according to the new NIH Common Toxicity Criteria, CTCAE Version 5.0.

[0227] Unexpected or early death or life-threatening toxicity (such as myocardial infarction, renal failure, or thrombo-embolic disease) will be reported immediately to the Sponsor, the Institutional Review Board, and to the FDA, as required by regulation.

Grade 4 or higher toxicity, at least possibly related to the experimental therapy, in 2 or more patients will pause patient accrual until additional review and approval by the IRB and FDA of any necessary changes in the protocol that may be indicated.

[0228] A DLT is defined as a grade 3 or higher adverse event, including autoimmune, hypersensitivity, and graft-versus-host reactions, at least possibly related to the BC cell line(s). The DLT observation period will be 2 weeks after the initial administration of the investigational agent. The maximum tolerated dose (MTD) will be defined as the dose level at which <30% of patients experience a DLT. That dose will be used subsequently in Part 2. If, during Part 2, that dose is subsequently determined not to be tolerated, the next lower dose may be used and so on until a dose level is found with <30% DLTs. Note that the MTD is determined for each cell line and may differ for the different BC cell lines. Any subject that experiences a DLT will be withdrawn from the study. If a tolerated dose level for a given cell line cannot be determined that cell line, further investigation with that cell line will be paused, the data reviewed and may resume only with protocol amendment, FDA consultation, and IRB approval. If a tolerated dose level cannot be determined for all of the BC cell lines, the study will be paused, the data reviewed and may resume only with protocol amendment, FDA consultation, and IRB approval.

[0229] Efficacy Assessments: Efficacy will be assessed using investigator determined clinical benefit (e.g. PFS) and the RECIST criteria. Baseline imaging studies will be performed within 4 weeks of treatment start. This includes the following assessments: computed tomography (CT) of chest, abdomen, pelvis; and if clinically indicated, isotope bone scan, PET scan, mammogram, ultrasound/MRI scans or other X-Rays as clinically indicated for disease assessment and/or at the discretion of the Investigator. Any baseline additional SOC imaging should be repeated no less frequently than the schedule of activities imaging assessment intervals.

[0230] Measurable disease: Requires such features so as to be accurately measurable (+/- 10%) in at least one dimension on CT (< 1.0 cm cuts), MRI, plain X-ray, or medical photographs. Measurable disease seen on images obtained by spiral CT must be > 1.0 cm. Ultrasound imaging will be permitted only for superficial lesions. Bone lesions will not be considered under these criteria.

[0231] Non-measurable disease: This includes bone lesions, effusions, poorly-demarcated pulmonary infiltrates, and lesions <1.0 cm by radiological imaging.

[0232] Objective status at examination: Target lesions are to be defined as measurable lesions, up to 5 sites per patient and no more than 2 sites in any one organ.

1. Measurements of target lesions must be provided at Screening, at 9-12 weeks following the initiation of Screening procedures, and at 9-12 week intervals throughout treatment.

2. Development of new lesions must be documented.

[0233] Responses are defined according to local investigator standard of care criteria and blinded central review using the iRECIST/RECIST vl.l criteria.

[0234] Statistical Methods: A variety of statistical analyses will be performed to assess the relationship between clinical response, immunological response, and possible prognostic factors. The appropriate statistical test will be determined by the statistician.

[0235] Multiple regression and/or Cox regression will be performed to identify factors predictive of response if the number of subjects entered into the study permits. This may include logistic regression when using response as the endpoint and Cox regression when using survival time. Other parametric and nonparametric tests will be used as appropriate to evaluate relationships of interest. For all tests, criterion for statistical significance will be set at p <0.05, two-tailed test.

[0236] The Principal Investigator will review clinical and laboratory data at the times of reimaging and restaging, or earlier as needed. Protocol deviations will be entered onto a form to be included in the regulatory binder. Missing data, patient absence or other non-compliance will be documented in a Note to File, copied to the IRB, and included in the annual report, unless safety concerns warrant prompt FDA filing as per the criteria for Serious Adverse Events.

Example 5: Identification of Antigenic Determinants in SV-BR-1 Derived Cellular Breast Cancer Vaccines

[0237] This example illustrates the identification of immunogenic epitopes in SV-BR-1 derived cellular breast cancer vaccines using a T-cell epitope mapping assay. Several key immunogenic peptides were detected, including those with post-translational modifications (PTMs), such as citrullination and cysteinylation.

Background

[0238] Identifying antigenic determinants is crucial for developing effective cancer vaccines. This study focuses on SV-BR-1 derived cellular breast cancer vaccines, aiming to delineate specific antigens that elicit an immune response. These vaccines rely on two key concepts: tumor cells display immunogenic antigens activating T-cells via cross-presentation, where host dendritic cells (DCs) process exogenous tumor antigens and present them on HLA molecules to activate T cells. Additionally, genetic engineering enhances their role as antigen-presenting cells, amplifying immune responses. Bria-IMT, the first version, is a genetically modified tumor cell line engineered to secrete granulocyte-macrophage colony-stimulating factor (GM- CSF). This vaccine has shown encouraging clinical outcomes and is currently being tested in a phase 3 clinical trial, demonstrating its potential in cancer immunotherapy. Bria-OTS+, an advanced version, enhances tumor cells' ability to present antigens by expressing cytokines, co-stimulatory factors, and HLA alleles.

Objectives

[0239] Therapeutic cancer vaccines are designed to stimulate the immune system by leveraging tumor antigens to generate an antitumor response. In our clinical trials, we have been focusing on SV-BR-1 -GM, a breast cancer cell line engineered to secrete GM-CSF, as a therapeutic vaccine. This vaccine has demonstrated encouraging clinical outcomes, both when used as a monotherapy (NCT03066947, completed) and in combination with checkpoint inhibitors (NCT03328026, ongoing). To further enhance the therapeutic efficacy of SV-BR-1- GM, we characterized the patient’s immune response to the vaccine. It is well-known that cancer vaccines can induce both humoral and cellular immune responses. However, predicting these responses, especially in the context of whole-cell vaccines like SV-BR-l-GM, presents unique challenges. One of the major difficulties lies in identifying which specific tumor antigens elicit an immune response in patients, as whole-cell vaccines present a wide array of antigens. Here, we present an analysis of antigen-specific T-cell responses using a T-cell epitope mapping assay in breast cancer patients.

Methods

[0240] Identification of Tumor Antigens with Immunopeptidomics Using SV-BR-1 Cells. FIG. 2 illustrates the workflow for an immunopeptidome analysis, detailing the processing and presentation of various categories of tumor antigens, including tumor- associated antigens (TAAs), tumor-specific antigens (TSAs), cancer-testis antigens (CTAs), post-translationally modified (PTM) antigens, and unconventional antigens (UCAs) in the SV- BR-1 cell line. TAAs are proteins that are overexpressed or dysregulated in tumor cells compared to normal tissues. TSAs result from tumor-specific somatic mutations, creating neoantigens unique to cancer cells. CTAs are typically restricted to immune-privileged germline tissues but are aberrantly expressed in tumors. PTM antigens arise from alterations in post-translational modifications, such as phosphorylation or glycosylation, leading to the presentation of novel tumor-specific epitopes. UCAs encompass antigens from non-coding regions, alternative reading frames, or proteins from the dark proteome, providing unique epitopes in tumor cells. These antigens are processed by the proteasome into peptide fragments, which are subsequently loaded onto MHC class I molecules and transported to the cell surface.

[0241] In the immunopeptidomics workflow, the cells are lysed, and MHC-peptide complexes are immunoprecipitated using HLA-specific antibodies MHC I (W6/32), HLA-DR (L243) and MHC II (IVA12). The peptides are then eluted and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), which identifies them based on their mass-to-charge ratio (m/z). This method provides a comprehensive profile of the tumor immunopeptidome, enabling the identification of antigens for potential therapeutic targeting.

[0242] Class I and II antigen/epitope mapping. FIG. 3 illustrates an exemplary workflow for Class I and Class II antigen/epitope mapping. CD154-expressing T cells (Class II) and CD137-expressing T cells (Class I) were enriched using magnetic beads and flow sorted with activation and memory markers. The sorted T cells were expanded as oligoclones in 96-well plates with feeder cells, PHA, and IL-2. They were then stimulated with peptide pools, and positive responses were restimulated with individual peptides to identify the specific antigenic peptide.

[0243] Patients and clinical data. The SV-BR-l-GM “monotherapy” study (NCT03066947; 2013-8) was a prospective phase 1-2 study of the SV-BR-l-GM regimen, administered every two weeks for the first two doses, then monthly. This regimen included low-dose cyclophosphamide (300 mg/m²) administered intravenously 48-72 hours before each SV-BR-l-GM treatment (~20 x 10⁶ cells) delivered intradermally. Interferon-alpha2b (10,000 IU) was injected into the SV-BR-l-GM inoculation sites 2 and 4 days after each dose. Cells samples were collected pre- and post- vaccination from each patient. Table 15 provides patient information and the results of SV-BR-l-GM treatment.

Table 15. Patients and clinical data response upon transitioning to combination therapy with pembrolizumab.

Results

[0244] Immunopeptidome Analysis of MHC Class I and II Bound Peptides in Bria-IMT Cells. A total of 4123 peptides were detected for class I MHC and 2193 peptides for class II in human Bria-IMT cells at the 1% PSM FDR based on forward/decoy database searching. An overview of MHC class I and II (including HLA-DR specific) peptide totals and intensities are shown in Table 16.

Table 16. MHC class I and II (including HLA-DR specific) peptide totals and intensities in human Bria-IMT cells. [0245] Identification and Prioritization of Candidate Immunogenic Peptides for Immunogenicity Testing. We prioritized peptides from two main sources. The first source was peptides presented by SV-BR-1 cells. These peptides were directly identified in the immunopeptidome analysis of SV-BR-1 cells, indicating natural processing and presentation by the tumor cells used in our vaccine. The second source was expressed peptides with known immunogenicity, including the peptides that were not detected in our immunopeptidome but confirmed to be expressed in SV-BR-1 cells through RNA-seq analysis. These peptides are known to be presented by breast cancer cells and have demonstrated immunogenicity in public databases, such as The Cancer Epitope Database and Analysis Resource (CEDAR) and CaAtlas (www.zhang-lab.org/caatlas/).

[0246] We selected candidate immunogenic peptides for immunogenicity testing using the following parameters:

• MHC Binding Affinity: We used NetMHCpan tools to predict peptides with high binding affinities to prevalent HLA alleles (IC50 < 500 nM for class I, < 1,000 nM for class II).

• Tumor Specificity: We focused on tumor-associated antigens (TAAs) and cancer-testis antigens (CTAs) unique to SV-BR-1 cells.

• Overexpression in Tumors: Prioritized peptides from proteins highly expressed in tumor cells, as confirmed by RNA-seq data.

• Unique PTMs: Peptides containing post-translational modifications specific to tumor cells were included to capture novel epitopes.

• Immunogenicity Evidence: Cross-referenced candidates with immunogenicity databases to select peptides with documented immune responses.

• HLA Matching: Selected peptides relevant to HLA types common in our patient cohort used for the analysis.

[0247] A total of 80 MHC class I peptides (10 of them carrying PTMs) and 50 class II peptides (18 of them carrying PTMs) were selected for further analysis.

[0248] Identification of T cell epitopes by CD154 (Class II) or CD137 (Class I) epitope mapping assay. Peripheral blood mononuclear cells (PBMCs) from vaccinated patients, collected both pre- and post-vaccination, were subjected to ex vivo stimulation with various peptide pools to assess immediate T-cell activation. Stimulation conditions included a DMSO control for background measurement, a positive control to confirm cell reactivity, and Class I or Class II peptide mega-pools designed to stimulate CD8+ (cytotoxic) or CD4+ (helper) T cells, respectively. Following initial ex vivo activation, T cells identified by activation markers - CD137 and CRTAM for CD8+ cells, and CD154 and CD69 for CD4+ cells - were analyzed via flow cytometry to determine peptide-specific responses. Subsequently, each Mega-pool was subdivided into smaller sub-pools, which were tested individually to reduce background noise and improve specificity in identifying immunogenic peptides. Individual peptides were then tested within these sub-pools, allowing for precise identification of specific epitopes that induce T-cell responses. To confirm that CD4+ T-cell responses were MHC Class II- dependent, HLA-DR, HLA-DQ, or HLA-DP blocking antibodies were introduced alongside individual peptides. We identified five peptides that induce an immunogenic response, as shown in FIGS. 4A-4D. (FIG. 4A) the pre-vaccination sample responded to a cysteinylated Desmoplakin MHC Class II peptide, QGSS(cys-mod)IAGIYNETTKQKLG (SEQ ID NO: 1); (FIG. 4B) the post-vaccination sample responded to two citrullinated Filaggrin MHC Class II peptides, KLAQYYESTCitKEN (SEQ ID NO: 2) and FKLAQYYESTCitKEN (SEQ ID NO: 3); (FIG. 4C) the post-vaccination sample responded to an MFGE8 MHC Class I peptide, GLQHWVPEL (SEQ ID NO: 4) ; and (FIG. 4D) the pre-vaccination sample responded to an MHC Class II peptide from C0X7C, ATPFLVVRHQLLKT (SEQ ID NO: 5). A total of one CD8+ and four CD4+ T cell epitopes were identified through the epitope mapping assay. In particular, the immunogenic Desmoplakin peptide QGSS(cys-mod)IAGIYNETTKQKLG (SEQ ID NO: 1) was cysteinylated, a post-translational modification where a cysteine residue forms a disulfide bond with an existing cysteine residue in the peptide. The two immunogenic Filaggrin peptides, KLAQYYESTCitKEN (SEQ ID NO: 2) and FKLAQYYESTCitKEN (SEQ ID NO: 3), each contained a citrulline residue.

Conclusions

[0249] We successfully identified immunogenic peptides in patients treated with the SV-BR- 1 -GM cellular vaccine, demonstrating the vaccine’ s ability to elicit a targeted immune response against tumor antigens. Key immunogenic peptides were detected include those with post- translational modifications (PTMs), such as citrullination and cysteinylation. This study underscores the advantage of using cellular cancer vaccines over RNA and peptide-based vaccines due to their ability to present a broad and diverse repertoire of antigens, including both conventional and unconventional types. Unlike RNA and peptide vaccines which deliver specific known antigens, cellular vaccines, such as whole tumor cells or antigen-presenting cells with tumor lysates, present a broad array of naturally occurring tumor antigens. This includes post-translational modifications (PTMs), alternative splicing variants, non-coding sequences, and peptides from pseudogenes. Such antigens often represent unique tumor features absent in healthy cells. Cellular vaccines can also display unknown, patient-specific neoantigens that are hard to predict with RNA or peptide vaccines. This diverse antigen presentation stimulates a robust, polyclonal immune response, engaging both CD8+ and CD4+ T cells against multiple tumor targets, reducing immune escape and potentially leading to more durable clinical outcomes.

[0250] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, patent applications, and sequence accession numbers cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A modified human cancer cell comprising a recombinant polynucleotide encoding an enzyme that induces a post-translational modification (PTM) of an antigen in the cell.

2. The modified human cancer cell of claim 1, wherein the enzyme comprises a citrullination enzyme, a cysteinylation enzyme, an acetylation enzyme, a hydroxylation enzyme, a phosphorylation enzyme, a methylation enzyme, a formylation enzyme, an oxidation enzyme, a hydroxylation enzyme, a ubiquitination enzyme, or a combination thereof.

3. A modified human cancer cell comprising an antigen, wherein the antigen comprises a non-enzymatic PTM.

4. The modified human cancer cell of claim 3, wherein the non-enzymatic PTM is induced by irradiation, inducing cellular senescence, a chemical reaction, a small molecule, a cell culture supplement, cell culture medium, oxidative stress, or a combination thereof.

5. The modified human cancer cell of any one of claims 1-4, wherein the antigen is an antigen of a pathogen, a tumor-specific antigen, a tumor-associated antigen, a neo-antigen, an allergen, an antigen that is a target of an autoimmune response, or a fragment thereof.

6. The modified human cancer cell of any one of claims 1-5, further comprising a recombinant polynucleotide encoding the antigen.

7. The modified human cancer cell of any one of claims 1-6, wherein the PTM of the antigen results in the antigen being more immunogenetic in comparison to the antigen without the PTM.

8. The modified human cancer cell of any one of claims 1-7, wherein the PTM comprises phosphorylation, acetylation, ubiquitination, succinylation, methylation, malonylation, glycosylation, sumoylation, nitrosylation, glutathionylation, amidation, hydroxylation, palmitoylation, glutarylation, crotonylation, oxidation, myristoylation, sulfation, formylation, citrullination, prenylation, cysteinylation, deamidation, dehydration, or a combination thereof.

9. The modified human cancer cell of claim 8, wherein the PTM comprises cysteinylation and/or citrullination.

10. The modified human cancer cell of any one of claims 1-9, further comprising (a) one or more recombinant polynucleotides each encoding an allele of a human leukocyte antigen (HLA) class I gene; and/or (b) one or more recombinant polynucleotides each encoding an allele of an HLA class II gene.

11. The modified human cancer cell of claim 10, wherein one or more HLA alleles endogenous to the cell have been inactivated.

12. The modified human cancer cell of claim 10 or 11, wherein the HLA class I gene comprises an HLA-A gene, an HLA-B gene, an HLA-C gene, an HLA-E gene, an HLA-F gene, an HLA-G gene, a beta-2-microglobulin (B2M) gene, or a combination thereof.

13. The modified human cancer cell of any one of claims 10-12, wherein the HLA class II gene comprises an HLA-DP gene, an HLA-DM gene, an HLA-DO gene, an HLA- DQ gene, an HLA-DR gene, or a combination thereof.

14. The modified human cancer cell of any one of claims 1-13, further comprising a recombinant polynucleotide encoding a cytokine.

15. The modified human cancer cell of claim 14, wherein the cytokine comprises a chemokine, an interferon, an interleukin, a tumor necrosis factor, or a combination thereof.

16. The modified human cancer cell of claim 14 or 15, wherein the cytokine comprises an early T cell activation antigen-1 (ETA-1), a lymphocyte-activating factor (LAF), an interleukin-1 family member (IL-la, IL-P, IL-IRa, IL-18, IL-33, IL-36Ra, IL-36a, IL-36P, IL-36y, IL-37, IL-38), an interleukin-2 (IL-2), an interleukin-3 (IL-3), an interleukin-4 (IL-4), an interleukin-5 (IL-5), an interleukin-6 (IL-6), an interleukin-7 (IL-7), an interleukin-8 (IL-8), an interleukin-9 (IL-9), an interleukin- 10 (IL- 10), an interleukin- 12 (IL- 12), an interleukin- 13 (IL-13), an interleukin- 15 (IL-15), an interleukin- 17 (IL-17), an interleukin- 18 (IL-18), an interleukin-21 (IL-21), an interleukin-23 (IL-23), an interleukin-25 (IL-25), an interleukin-33 (IL-33), an interferon alpha (IFN-a), an interferon lambda 1 (IFN-X1 (IL-29)), an interferon lambda 2 (IFN-X2 (IL-28A)), an interferon lambda 3 (IFN-X3 (IL-28B)), an interferon lambda 4 (IFN-X4), a granulocyte-macrophage colony-stimulating factor (GM-CSF), a macrophage CSF (CSF-1), a macrophage migration inhibitory factor (MIF), a CD40L molecule (CD40L), a RANTES molecule (RANTES), a monocyte chemoattractant protein (MCP-1), a monocyte inflammatory protein (MIP-la, MIP-ip), a lymphotactin, a fractalkine, or a combination thereof.

17. The modified human cancer cell of any one of claims 14-16, wherein the cytokine comprises GM-CSF.

18. The modified human cancer cell of any one of claims 1-17, further comprising a recombinant polynucleotide encoding a co-stimulatory molecule.

19. The modified human cancer cell of claim 18, wherein the co-stimulatory molecule comprises a CD86 molecule (CD86), CD80 molecule (CD80), 4- IBB ligand molecule (4-1BBL a.k.a CD137L), ICOS ligand molecule (ICOS-L), CD70 molecule (CD70 a.k.a. CD27L), CD40 molecule (CD40), 0X40 ligand molecule (OX40L), GITR ligand molecule (GITRL), TIM-4 molecule (TIM-4), LIGHT molecule (LIGHT), ICAM1 molecule (ICAM1), LFA3 molecule (LFA3), a CD30 molecule (CD30), or a combination thereof.

20. The modified human cancer cell of any one of claims 1-19, wherein the human cancer cell is a human cancer cell line.

21. The modified human cancer cell of claim 20, wherein the human cancer cell line is a breast cancer, prostate cancer, melanoma, or lung cancer cell line.

22. The modified human cancer cell of any one of claims 1-19, wherein the human cancer cell is a primary cancer cell.

23. The modified human cancer cell of claim 22, wherein the primary cancer cell is from a biopsy or a circulating cancer cell from a patient.

24. The modified human cancer cell of claim 23, wherein the patient has a breast cancer, prostate cancer, melanoma, or lung cancer.

25. A replication-incompetent modified human cancer cell of any one of claims 1-24.

26. The replication-incompetent modified human cancer cell of claim 25, wherein the modified human cancer cell is rendered replication incompetent by irradiation, freeze-thawing, or mitomycin C treatment.

27. A composition comprising a modified human cancer cell of any one of claims 1-24.

28. A pharmaceutical composition comprising the composition of claim 27 and a pharmaceutically acceptable carrier.

29. The pharmaceutical composition of claim 28, further comprising a cryoprotectant.

30. A kit for treating a subject with cancer comprising the pharmaceutical composition of claim 28 or 29.

31. A method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 28 or 29.

32. The method of claim 31, wherein prior to the administering step, further comprising:

(i) obtaining a sample from the subject;

(ii) determining prevalent PTM(s) in the sample; and

(iii) selecting a modified human cancer cell for administering to the subject, wherein the modified human cancer cell comprises a recombinant polynucleotide encoding an enzyme that induces the prevalent PTM(s).

33. The method of claim 32, wherein the sample is a tumor biopsy or a liquid biopsy.

34. The method of claim 33, wherein the liquid biopsy comprises circulating tumor cells (CTCs), circulating tumor DNA (ctDNA or cell free DNA), circulating RNA (cfRNA), exosomes, or a combination thereof.

35. The method of claim 32, wherein the determining step comprises nextgeneration sequencing (NGS) or immunopeptidome analysis of the sample.

36. The method of any one of claims 31-35, wherein the subject has a breast cancer, prostate cancer, melanoma, or lung cancer.

37. A method for enhancing immunogenicity of an antigen in a human cancer cell, comprising inducing a post-translational modification (PTM) of the antigen in the cell, wherein the PTM enhances the immunogenicity of the antigen.

38. The method of claim 37, wherein the PTM comprises phosphorylation, acetylation, ubiquitination, succinylation, methylation, malonylation, glycosylation, sumoylation, nitrosylation, glutathionylation, amidation, hydroxylation, palmitoylation, glutarylation, crotonylation, oxidation, myristoylation, sulfation, formylation, citrullination, prenylation, cysteinylation, deamidation, dehydration, or a combination thereof.

39. The method of claim 38, wherein the PTM comprises cysteinylation and/or citrullination.

40. The method of any one of claims 37, wherein the antigen is an antigen of a pathogen, a tumor-specific antigen, a tumor-associated antigen, a neo-antigen, an allergen, an antigen that is a target of an autoimmune response, or a fragment thereof.

41. The method of any one of claims 37-40, wherein the cell comprises a recombinant polynucleotide encoding the antigen.

42. The method of any one of claims 37-41, wherein the cell comprises (a) one or more recombinant polynucleotides each encoding an allele of a human leukocyte antigen (HLA) class I gene; and/or (b) one or more recombinant polynucleotides each encoding an allele of an HLA class II gene.

43. The method of any one of claims 37-42, wherein the cell comprises a recombinant polynucleotide encoding a cytokine.

44. The method of any one of claims 37-43, wherein the cell comprises a recombinant polynucleotide encoding a co-stimulatory molecule.

45. The method of any one of claims 37-44, wherein the cell is a human cancer cell line.

46. The method of any one of claims 37-44, wherein the cell is a primary cancer cell.

47. The method of any one of claims 37-46, wherein the cell is a breast cancer cell, a prostate cancer cell, a melanoma cell, or a lung cancer cell.

48. The method of any one of claims 37-47, wherein the PTM is induced by an enzyme.

49. The method of claim 48, wherein the enzyme comprises a citrullination enzyme, a cysteinylation enzyme, an acetylation enzyme, a hydroxylation enzyme, a phosphorylation enzyme, a methylation enzyme, a formylation enzyme, an oxidation enzyme, a hydroxylation enzyme, a ubiquitination enzyme, or a combination thereof.

50. The method of claim 48 or 49, wherein the cell comprises a recombinant polynucleotide encoding the enzyme.

51. The method of any one of claims 37-47, wherein the PTM is a non- enzymatic PTM.

52. The method of claim 51, wherein the non-enzymatic PTM is induced by irradiation, inducing cellular senescence, a chemical reaction, a small molecule, a cell culture supplement, cell culture medium, oxidative stress, or a combination thereof.