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

Abstract

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

Description

[0001] Cross-application of related applications

[0002] 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 incorporated herein by reference in their entirety for all purposes.

[0003] sequence list

[0004] This application includes a sequence list, which has been filed with this application and is incorporated herein by reference in its entirety. The .xml copy, created on January 29, 2025, is named 102144-001210PC-1485893 and has a size of 6,751 bytes. Background Technology

[0005] Post-translational modifications (PTMs) are produced by adding small chemical groups to amino acid residues after protein translation. PTMs alter protein structure, function, and localization, and play a crucial role under physiological and pathophysiological conditions.

[0006] Developing effective immune responses is crucial for vaccine development and cancer immunotherapy. Whole-cell immunotherapy is generally considered to be effective only when it expresses immunogenic antigens. Therefore, there is a need to improve the immunogenicity of antigens for the treatment of cancer and related diseases. This disclosure addresses these needs and provides relevant advantages. Summary of the Invention

[0007] In one aspect, this disclosure provides modified human cancer cells comprising recombinant polynucleotides encoding enzymes that induce post-translational modifications (PTMs) of cellular antigens. In some embodiments, said enzymes include citrullinated lactamases, cysteine ​​lactamases, acetyltransferases, hydroxylases, phosphorylases, methyltransferases, formylates, oxidases, hydroxylases, ubiquitinases, or combinations thereof.

[0008] In one aspect, this disclosure provides modified human cancer cells comprising antigens, wherein the antigens comprise non-enzymatic PTMs. In some embodiments, the non-enzymatic PTMs are induced by irradiation, induced cell senescence, chemical reactions, small molecules, cell culture supplements, cell culture media, oxidative stress, or a combination thereof.

[0009] In some implementations, the antigen is a fragment of one or more of the following: pathogen antigen, tumor-specific antigen, tumor-associated antigen, neoantigen, allergen, antigen that is a target of an autoimmune response.

[0010] In some embodiments, the modified human cancer cells also contain recombinant polynucleotides encoding antigens. In some embodiments, the PTM of the antigen results in the antigen being more immunogenic compared to antigens without PTM.

[0011] In some embodiments, PTM includes phosphorylation, acetylation, ubiquitination, succinylation, methylation, malonylation, glycosylation, SUMOylation, nitrosylation, glutathioneylation, amidation, hydroxylation, palmitoylation, glutarylation, crotonylation, oxidation, myristylation, sulfation, formylation, citrullination, prenylation, cysteineation, deamidation, dehydration, or combinations thereof. In some embodiments, PTM includes cysteineation and / or citrullineation.

[0012] In some embodiments, the modified human cancer cells further comprise (a) one or more recombinant polynucleotides encoding alleles of human leukocyte antigen (HLA) class I genes; and / or (b) one or more recombinant polynucleotides encoding alleles of HLA class II genes. In some embodiments, one or more endogenous HLA alleles have been inactivated. In some embodiments, HLA class I genes include HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, β-2-microglobulin (B2M) genes, or combinations thereof. In some embodiments, HLA class II genes include HLA-DP, HLA-DM, HLA-DO, HLA-DQ, HLA-DR genes, or combinations thereof.

[0013] In some embodiments, the modified human cancer cells also contain recombinant polynucleotides encoding cytokines. In some embodiments, the cytokines include chemokines, interferons, interleukins, tumor necrosis factor, or combinations thereof. In some implementations, cytokines include early T-cell activation antigen-1 (ETA-1), lymphocyte activating factor (LAF), interleukin-1 family members (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36γ, IL-37, IL-38), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-17 (IL-17), and interleukin-18. Interleukin-21 (IL-21), Interleukin-23 (IL-23), Interleukin-25 (IL-25), Interleukin-33 (IL-33), Interferon-α (IFN-α), Interferon-λ1 (IFN-λ1 (IL-29)), Interferon-λ2 (IFN-λ2 (IL-28A)), Interferon-λ3 (IFN-λ3 (IL-28B)), Interferon-λ4 (IFN-λ4), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Macrophage CSF (CSF-1), Macrophage migration inhibitory factor (MIF), CD40L molecule (CD40L), RANTES molecule (RANTES), Monocyte chemotactic protein (MCP-1), Monocyte inflammatory protein (MIP-1α, MIP-1β), Lymphocyte chemotactic factor, Fractal chemotactic factor (fractalkine), or a combination of the above. In some implementations, the cytokine includes GM-CSF.

[0014] In some embodiments, the modified human cancer cells also contain recombinant polynucleotides encoding co-stimulatory molecules. In some embodiments, the co-stimulatory molecules include CD86 molecules (CD86), CD80 molecules (CD80), 4-1BB ligand molecules (4-1BBL, also known as CD137L), ICOS ligand molecules (ICOS-L), CD70 molecules (CD70, also known as CD27L), CD40 molecules (CD40), OX40 ligand molecules (OX40L), GITR ligand molecules (GITRL), TIM-4 molecules (TIM-4), LIGHT molecules (LIGHT), ICAM1 molecules (ICAM1), LFA3 molecules (LFA3), CD30 molecules (CD30), or combinations thereof.

[0015] In some implementations, the human cancer cells are human cancer cell lines. In some implementations, the human cancer cell lines are breast cancer cell lines, prostate cancer cell lines, melanoma cell lines, or lung cancer cell lines.

[0016] In some implementations, the human cancer cells are primary cancer cells. In some implementations, the primary cancer cells are derived from a patient's biopsy or circulating cancer cells. In some implementations, the patient has breast cancer, prostate cancer, melanoma, or lung cancer.

[0017] In some embodiments, the modified human cancer cells described herein are non-replicating modified human cancer cells. In some embodiments, the modified human cancer cells are made non-replicating through irradiation, freeze-thaw cycles, and / or treatment with mitomycin C.

[0018] On the other hand, this disclosure provides compositions comprising human cancer cells containing the modifications described herein. In some embodiments, the compositions comprising human cancer cells containing the modifications described herein are formulated as pharmaceutical compositions, and the pharmaceutical compositions further comprise a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical compositions further comprise a cryoprotectant.

[0019] On the other hand, this disclosure provides a kit product for treating patients in need of cancer, comprising the pharmaceutical compositions described herein. In some embodiments, the kit product further comprises a therapeutically effective amount of IFN-α2b. The kit product may include instructions for use in treating the patient using any of the methods described herein.

[0020] On the other hand, this disclosure provides a method for treating cancer in a subject, the method comprising administering a therapeutically effective amount of the pharmaceutical composition described herein to the subject. In some embodiments, prior to the administration step, the method further comprises (i) obtaining a sample from the subject; (ii) identifying a prevalent PTM in the cell sample; and (iii) selecting modified human cancer cells for administration to the subject, wherein the modified human cancer cells contain a prevalent PTM. In some examples, the prevalent PTM is an enzymatic PTM, wherein the modified human cancer cells contain a recombinant polynucleotide encoding an enzyme that induces the prevalent PTM. In other examples, the prevalent PTM is a non-enzymatic PTM, wherein the modified human cancer cells induce the prevalent PTM via a non-enzymatic method. In some embodiments, the non-enzymatic method includes irradiation, induced cell senescence, chemical reactions, small molecules, cell culture supplements, cell culture media, 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 includes circulating tumor cells (CTCs), circulating tumor DNA (ctDNA or cell-free DNA), circulating RNA (cfRNA), exosomes, or a combination thereof. In some implementations, the identification steps include next-generation sequencing (NGS) or immunopeptidome analysis of the sample. In some implementations, the subject has breast cancer, prostate cancer, melanoma, or lung cancer.

[0021] On the other hand, this disclosure provides a method for enhancing the immunogenicity of antigens in human cancer cells, including inducing post-translational modifications (PTMs) of antigens in cells, wherein the PTMs enhance the immunogenicity of the antigens.

[0022] In some embodiments, the PTM includes phosphorylation, acetylation, ubiquitination, succinylation, methylation, malonylation, glycosylation, SUMOylation, nitrosylation, glutathioneylation, amidation, hydroxylation, palmitoylation, glutarylation, crotonylation, oxidation, myristylation, sulfation, formylation, citrullination, isoprenelation, cysteineation, deamidation, dehydration, or a combination thereof. In some embodiments, the PTM includes cysteineation and / or citrullination. In some embodiments, the antigen is a fragment of a pathogen antigen, a tumor-specific antigen, a tumor-associated antigen, a neoantigen, an allergen, an antigen that is a target of an autoimmune response, or a combination thereof. In some embodiments, the cell contains a recombinant polynucleotide encoding said antigen.

[0023] 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, prostate cancer cell, melanoma cell, or lung cancer cell.

[0024] In some embodiments, PTM is induced by an enzyme. In some embodiments, the enzyme includes citrullinated lactamase, cysteine ​​lactamase, acetyltransferase, hydroxylase, phosphorylase, methyltransferase, formylate, oxidase, hydroxylase, ubiquitinase, or a combination thereof. In some embodiments, the cell contains a recombinant polynucleotide encoding the enzyme. In some embodiments, the enzyme includes citrullinated lactamase. In some embodiments, the enzyme includes cysteine ​​lactamase.

[0025] In some implementations, the PTM is a non-enzymatic PTM. In some implementations, the non-enzymatic PTM is induced by irradiation, induced cell senescence, chemical reactions, small molecules, cell culture supplements, cell culture media, oxidative stress, or a combination thereof. Attached Figure Description

[0026] Figure 1 Post-translational modifications of MHC class I and / or class II peptides in the immunopeptidome of the genetically modified human breast cancer cell line Bria-IMT were depicted.

[0027] Figure 2 The workflow of immunopeptidomics analysis is described, and the processing and presentation of various tumor antigens in the SV-BR-1 cell line are detailed. These tumor antigens include tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), cancer testis antigens (CTAs), post-translational modification (PTM) antigens, and unconventional antigens (UCAs).

[0028] Figure 3 The workflow for class II or class I antigen / epitope mapping assays is described.

[0029] Figures 4A-4D T cell epitopes identified by CD154 (class II) or CD137 (class I) epitope mapping assay were depicted. This assay identified five peptides that induce immunogenic responses: ( Figure 4APre-vaccination samples responded to cysteine-modified desmoplakin MHC class II peptide QGSS (cys-mod) IAGIYNETTKQKLG (SEQ ID NO:1); Figure 4B The samples received a vaccine that responded to two citrullinated filaggrin MHC class II peptides: KLAQYYESTCitKEN (SEQ ID NO: 2, top) and FKLAQYESTCitKEN (SEQ ID NO: 3, bottom). Figure 4C The vaccine-prescribed samples responded to the MFGE8 MHC class I peptide GLQHWVPEL (SEQ ID NO: 4); and ( Figure 4D The pre-vaccination sample responded to the MHC class II peptide ATPFLVVRHQLLKT (SEQ ID NO: 5) from COX7C. Detailed Implementation

[0030] 1. Introduction

[0031] Post-translational modifications (PTMs) involve the addition of small chemical moieties or chemical modifications to individual amino acids in translated proteins. PTMs regulate protein stability, folding, function, and interactions with other biomolecules. PTMs can be associated with tumor progression, growth, and survival by altering the normal function of proteins in tumor cells. Furthermore, PTMs are frequently involved in many diseases beyond cancer.

[0032] Inducing an effective immune response using whole-cell immunotherapy is an effective method for treating and preventing diseases such as cancer. It is generally believed that cancer vaccines are effective only if they express highly immunogenic antigens co-expressed in the patient's tumor cells, and antigen-presenting cells (APCs), such as dendritic cells (DCs), need to cross-present these antigens after taking up vaccine cell fragments.

[0033] The core transformative technology of this disclosure is the development of whole-cell therapeutic vaccines expressing highly immunogenic antigens. Specifically, the modified human cancer cells described herein contain post-translational modified antigens that bind to HLA class I and / or II molecules. These post-translational modified antigens are capable of overcoming tolerance and inducing immune responses in various diseases and cancers. Therefore, by incorporating the Bria-IMT™ platform technology, highly immunogenic cancer vaccines can be developed by inducing enzymatic or non-enzymatic post-translational modifications (PTMs) of antigens in modified human cancer cells. In some aspects, the modified human cancer cells described herein express one or more enzymes that induce PTMs of one or more antigens. In some aspects, the modified human cancer cells described herein also 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.

[0034] This article describes compositions of modified human cancer cells or cell lines for targeted immunotherapy of cancer. Furthermore, kits comprising "off-the-shelf" cell lines and methods for preventing or treating cancer in individuals of need are also provided.

[0035] II. Definition

[0036] Unless otherwise specifically stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Furthermore, any methods or materials similar to or equivalent to those described or made herein may be used in the practice of this disclosure. The following terms are defined for the purposes of this disclosure.

[0037] As used herein, the terms “a,” “an,” or “the” include not only aspects of a single member but also aspects of more than one member. For example, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” include plural indicators. Thus, for example, reference to “a cell” includes a plurality of such cells, and reference to “the pharmaceutical agent” includes reference to one or more pharmaceutical agents known to those skilled in the art, etc.

[0038] The terms “about” and “approximately” generally refer to the acceptable degree of error of a measured quantity given the nature or precision of the measurement. Typical exemplary degrees of error are within 20% of a given value or range of values, preferably within 10%, and more preferably within 5%. Optionally, particularly in biological systems, the terms “about” and “approximately” may refer to values ​​within an order of magnitude, preferably within 5 times the given value, and more preferably within 2 times. Unless otherwise stated, the numerical values ​​given herein are approximate, i.e., the terms “about” or “approximately” can be inferred when not explicitly stated.

[0039] The terms “object,” “individual,” and “patient” are used interchangeably herein and refer to a vertebrate, preferably a mammal, and more preferably a human. Mammals include, but are not limited to, mice, rats, apes, humans, farm animals, sporting animals, and pets. Tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro are also included.

[0040] As used herein, the term "application" includes oral administration, local contact administration, administration as a suppository, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intratumoral administration, intradermal administration, intralymphatic administration, intrathecal administration, intranasal administration, or subcutaneous administration to the subject. Administration can be via any route, including parenteral and transmucosal administration (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or percutaneous). Parenteral administration includes, for example, intravenous administration, intramuscular administration, intra-arterial administration, intradermal administration, subcutaneous administration, intraperitoneal administration, intraventricular administration, and intracranial administration. Other delivery methods include, but are not limited to, the use of liposomal formulations, intravenous infusion, and transdermal patches.

[0041] The term "treatment" refers to a method of achieving a beneficial or desired outcome, including but not limited to therapeutic and / or preventative benefits. A therapeutic benefit means any treatment-related improvement or effect in the treatment of one or more diseases, conditions, or symptoms. A therapeutic benefit can also mean the achievement of a cure for one or more diseases, conditions, or symptoms in the treatment.

[0042] The terms "effective amount" or "sufficient amount" refer to the amount of modified cancer cells or other compositions sufficient to achieve a beneficial or desired outcome. Therapeutic effective amounts can vary depending on one or more of the following factors: the subject and disease condition, the subject's weight and age, the severity of the disease condition, the method of administration, etc., all of which can be readily determined by those skilled in the art. Specific amounts can vary depending on one or more of the following factors: the specific agent selected, the target cell type, the location of the target cells in the subject, the administration regimen followed, whether it is administered in combination with other compounds, the timing of administration, and the physical delivery system used to carry it.

[0043] For the purposes of this document, the effective amount is determined by considerations that may be known in the art. This amount must be effective to achieve the desired therapeutic effect in cancer patients. The desired therapeutic effect may include, for example, improving undesirable symptoms associated with cancer, preventing the manifestation of such symptoms before their onset, slowing the progression of cancer-related symptoms, slowing or limiting any irreversible damage caused by cancer, reducing the severity of cancer or curing cancer, or improving survival or providing a faster cancer recovery.

[0044] Effective doses depend particularly on the type and severity of the disease to be treated and the treatment regimen. Effective doses are typically determined in properly designed clinical trials (dose range studies), and those skilled in the art will know how to correctly conduct such trials to determine the effective dose. It is well known that effective doses depend on a variety of factors, including the distribution profile of the therapeutic agent (e.g., whole-cell cancer vaccines) or the composition in vivo, the relationship between various pharmacological parameters (e.g., half-life in vivo) and undesirable side effects, and other factors such as age and sex.

[0045] The term "pharmaceutically acceptable carrier" refers to a substance that facilitates the administration of an active agent to cells, organisms, or objects. "Pharmaceutically acceptable carrier" refers to a carrier or excipient that can be included in the compositions of this disclosure and has no significant adverse toxicological effects on the object. Non-limiting examples of pharmaceutically acceptable carriers include water, sodium chloride, physiological saline solutions, lactated Ringer's solution, plain sucrose, plain glucose, binders, fillers, disintegrants, lubricants, coating agents, sweeteners, flavoring agents and coloring agents, liposomes, dispersion media, microencapsulations, cationic lipid carriers, isotonic agents, and absorption delay agents. A carrier can also be a substance used to provide stability, sterility, and isotonicity of the formulation (e.g., antimicrobial preservatives, antioxidants, chelating agents, and buffers), to prevent the action of microorganisms (e.g., antimicrobial and antifungal agents, such as parabens, chlorobutanol, sorbic acid, etc.), or to provide an edible flavor to the formulation. In some examples, the carrier is an agent that facilitates the delivery of modified cancer cells to target cells or tissues. Those skilled in the art will recognize that other drug carriers may be used in this disclosure.

[0046] As used herein, the term "nucleic acid" or "nucleotide" refers to a polymer containing at least two single-stranded or double-stranded deoxyribonucleotides or ribonucleotides, and includes DNA, RNA, and their hybrids. DNA can be in the following forms: antisense molecules, plasmid DNA, DNA-DNA duplexes, precondensed DNA, PCR products, vectors (P1, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations thereof. RNA can be in the following forms: small interfering RNA (siRNA), Dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric 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 bonds, which are synthetic, naturally occurring, or non-natural, and have binding properties similar to a reference nucleic acid. Examples of such analogues include, but are not limited to, thiophosphates, aminophosphates, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs). Unless specifically defined, the term includes nucleic acids containing known natural nucleotide analogues that have similar binding properties to a reference nucleic acid. Unless otherwise stated, specific nucleic acid sequences also implicitly include variants of conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences, as well as explicitly indicated sequences. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is substituted with a mixture of bases 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)). A “nucleotide” comprises a sugar, deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together by phosphate groups. A “base” includes purines and pyrimidines, including natural compounds such as adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs; and synthetic derivatives of purines and pyrimidines, including but not limited to modifications that place new reactive groups, such as, but not limited to, amines, alcohols, thiols, carboxylic acid esters, and alkyl halides.

[0047] As used herein, the term "enzyme" or "PTM enzyme" refers to a protein or peptide that can induce chemical or structural changes in translation. This class includes a variety of enzymes, including but not limited to citrullinated enzymes, cysteine ​​lactamases, acetyltransferases, hydroxylases, phosphorylases, methyltransferases, formylates, oxidases, hydroxylases, and ubiquitinases.

[0048] The term "gene" refers to a segment of DNA that is involved in the production of a polypeptide chain. This DNA segment may include regions before and after (leader and tail) coding regions involved in the transcription / translation of the gene product and the regulation of transcription / translation, as well as intercalation sequences (introns) between individual coding segments (exons).

[0049] The terms “vector” and “expression vector” refer to nucleic acid constructs generated recombinantly or synthetically, having a specific set of nucleic acid elements that allow transcription of specific polynucleotide sequences in a host cell. Expression vectors can be part of a plasmid, a viral genome, or a nucleic acid fragment. Typically, an expression vector comprises a polynucleotide to be transcribed, operatively linked to a promoter. As used herein, the term “promoter” refers to an array of nucleic acid control sequences that direct nucleic acid transcription. As used herein, for example, in the case of a polymerase type II promoter (TATA element), the promoter comprises an essential nucleic acid sequence located close to the transcription start site. The promoter may also optionally include distal enhancer or repressor elements, which may be located up to several thousand base pairs from the transcription start site. Other elements that may be present in an expression vector include elements that enhance transcription (e.g., enhancers) and terminate transcription (e.g., terminators). In the context of this disclosure, co-expression of multiple genes (e.g., polynucleotides terminating HLA class I alleles and / or HLA class II alleles) can be achieved by co-transfection of two or more vectors, using multiple or bidirectional promoters, or creating bicistronic or polycistronic vectors. Gene co-expression can be driven by using plasmids with multiple individual expression cassettes. Typically, each promoter creates a unique mRNA transcript for each expressed gene. Bicistronic or polycistronic vectors simultaneously express two or more different proteins from the same mRNA. Bicistronic vectors may contain internal ribosome entry sites (IRES) to allow translation to begin from an internal region of the mRNA. Polycistronic vectors containing one or more self-cleaving 2A peptides are advantageous because they allow co-expression of genes from the same cassette. In some examples, polycistronic vectors are preferred when only a portion of the plasmid is packaged for viral delivery, or when the relative expression levels between two or more genes are important.

[0050] The terms "autocleaving peptide" and "autocleaving 2A peptide" refer to short peptides capable of producing equimolar levels of multiple genes from the same mRNA. These peptides were first discovered in picornaviruses. Autocleaving peptides are thought to function by causing the ribosome to skip the synthesis of the peptide bond at the C-terminus of the 2A element, resulting in separation between the end of the 2A sequence and the next downstream peptide. The "cleavage" occurs between glycine and proline residues found at the C-terminus, meaning that the upstream cistron typically has several additional residues added to the end, while the downstream cistron typically begins with proline. Non-limiting examples of autocleaving peptides include T2A, P2A, E2A, and F2A.

[0051] “Recombinant” refers to a genetically modified polynucleotide, polypeptide, cell, tissue, or organism. For example, a recombinant polynucleotide (or a copy or complementary sequence of a recombinant polynucleotide) is a polynucleotide manipulated using well-known methods. A recombinant expression cassette containing a promoter operatively linked to a second polynucleotide (e.g., a coding sequence) may include a promoter heterologous to the second polynucleotide as a result of human manipulation (e.g., as described in Sambrook). et al. , Molecular Cloning - A Laboratory Manual (Methods described in *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)). Recombinant expression cassettes (or expression vectors) typically comprise polynucleotides in combinations not found in nature. For example, artificially manipulated restriction sites or plasmid vector sequences can side-mount promoters or separate promoters from other sequences. Recombinant proteins are proteins expressed from recombinant polynucleotides, and recombinant cells, tissues, and organisms are those containing recombinant sequences (polynucleotides and / or polypeptides). Recombinant cells are cells modified (e.g., transfected or transformed) using recombinant nucleotides, expression vectors, or expression cassettes.

[0052] The term "amino acid" refers to any monomeric unit that can be incorporated into a peptide, polypeptide, or protein. Amino acids include naturally occurring α-amino acids and their stereoisomers, as well as non-natural (non-naturally occurring) amino acids and their stereoisomers. A "stereoisomer" of a given amino acid is an isomer that has the same molecular formula and intramolecular bonds, but differs in the three-dimensional arrangement of the bonds and atoms (e.g., L-amino acids and their corresponding D-amino acids).

[0053] Naturally occurring amino acids are those encoded by the genetic code, as well as those modified later, such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Naturally occurring α-amino acids include, but are not limited to, alanine (Ala), cysteine ​​(Cys), aspartic acid (Asp), glutamic acid (Glu), phenylalanine (Phe), glycine (Gly), histidine (His), isoleucine (Ile), arginine (Arg), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (Pro), glutamine (Gln), serine (Ser), threonine (Thr), valine (Val), tryptophan (Trp), tyrosine (Tyr), and combinations thereof. Naturally occurring stereoisomers of α-amino acids include, but are not limited to, 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 combinations thereof.

[0054] Non-natural (not naturally occurring) amino acids include, but are not limited to, L- or D-configured amino acid analogs, amino acid mimics, synthetic amino acids, N-substituted glycine, and N-methyl amino acids that function in a manner similar to naturally occurring amino acids. For example, an "amino acid analog" can be a non-natural amino acid that has the same basic chemical structure as a naturally occurring amino acid (i.e., the carbon atom bound to a hydrogen, carboxyl group, or amino group) but has modified side chain groups or a modified peptide backbone, such as homoserine, ortholeucine, methionine sulfoxide, and methionine methylsulfonium. An "amino acid mimic" is a compound that has a different structure from the general chemical structure of an amino acid but functions in a manner similar to naturally occurring amino acids. Amino acids can be represented by well-known three-letter symbols or by single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee.

[0055] The terms “identity,” “substantial identity,” “similarity,” “substantial similarity,” “homology,” and related terms and expressions used in the context of describing amino acid sequences refer to sequences that have at least 60% sequence identity with a reference sequence. Examples include sequences that, when compared to a reference sequence, show 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 using a program for comparing amino acid sequences (e.g., BLAST with standard parameters). For sequence comparisons, one sequence is typically used as a reference sequence for comparison with the test sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and subsequence coordinates are specified if necessary, along with the sequence algorithm program parameters. Default (standard) program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence relative to the reference sequence based on the program parameters. The “comparison window” includes a segment that refers to any one of the number of consecutive positions (from 20 to 600, typically from about 50 to about 200, more typically from about 100 to about 150), whereby, after optimal alignment of two sequences, the sequence can be compared with a reference sequence of the same number of consecutive positions. Methods for sequence alignment used for comparison are well known. For example, optimal alignment of sequences for comparison can be performed using the local homology algorithm of Smith and Waterman, 1981, the homology alignment algorithm of Needleman and Wunsch, 1970, the similarity search method of Pearson and Lipman, 1988, computerized implementations of these algorithms (e.g., BLAST), or manual alignment and visual inspection.

[0056] Algorithms suitable for determining sequence identity percentages and sequence similarity include BLAST and BLAST 2.0, which are described in Altschul. et al. , 1990 and Altschul et al., 1977. The software used for BLAST analysis is publicly available from the National Center for Biotechnology Information (NCBI) website. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence. When compared with words of the same length in the database sequence, the short words match or satisfy a certain positive threshold score T. T is called the neighborhood word score threshold. These initial neighborhood word hits serve as seeds to initiate a search for longer HSPs containing them. These word hits are then extended in both directions along each sequence until the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for non-matching residues; always < 0). For amino acid sequences, the cumulative score is calculated using a score matrix. Extension of word hits in each direction is stopped when: the cumulative alignment score decreases by an amount X from its maximum value; the cumulative score becomes zero or lower due to the accumulation of one or more negatively scored residue alignments; or the end of any 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 a default word size (W) of 28, expectation (E) of 10, M = 1, and N = -2, and compares two strands. For amino acid sequences, the BLASTP program uses a default word size (W) of 3, expectation (E) of 10, and the BLOSUM 62 scoring matrix (Henikoff and Henikoff, 1989). The BLAST algorithm also performs statistical analysis on the similarity between two sequences (Karlin and Altschul, 1993).

[0057] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The term applies to amino acid polymers, where one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids, as well as to both naturally occurring and non-naturally occurring amino acid polymers. As used herein, the term includes amino acid chains of any length, including full-length proteins (i.e., alleles), where 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 the amino acid sequence of a polypeptide, the first amino acid located at the N-terminus is referred to as the “first amino acid.”

[0058] When used in the context of describing the mating bodies of recombinant polypeptides, the term "heterologous" refers to the relationship between one polynucleotide fusion mating body and another: the fusion mating body is present in the recombinant polynucleotide in a manner that cannot be found in naturally occurring polynucleotides or polynucleotides encoding naturally occurring proteins. A "heterologous polynucleotide" can encode a peptide containing a modification of a naturally occurring protein sequence or a portion thereof, such modification being, for example, the deletion, addition, or substitution of one or more amino acid residues.

[0059] The term "cancer" is intended to include any member of a class of diseases characterized by the uncontrolled growth of abnormal cells. This term includes all known cancers and tumor conditions, regardless of whether they are malignant, benign, recurrent, soft tissue, or solid, and all stages and grades of cancer, including advanced, pre-metastatic, and post-metastatic cancers. Examples of different types of cancer include, but are not limited to, gynecological cancers (e.g., ovarian cancer, cervical cancer, uterine cancer, vaginal cancer, and vulvar cancer); 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 tumor); and digestive and gastrointestinal cancers, such as gastric cancer. Cancer, colorectal cancer, gastrointestinal stromal tumor (GIST), gastrointestinal carcinoid tumors, colon cancer, rectal cancer, anal cancer, bile duct cancer, small bowel cancer, and esophageal cancer; thyroid cancer; gallbladder cancer; liver cancer; pancreatic cancer; appendix cancer; prostate cancer (e.g., prostate adenocarcinoma); kidney cancer (e.g., renal cell carcinoma); central nervous system cancers (e.g., glioblastoma, neuroblastoma, medulloblastoma); skin cancers (e.g., melanoma); bone and soft tissue sarcomas (e.g., Ewing sarcoma); lymphoma; choriocarcinoma; urinary system 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, “tumor” includes one or more cancer cells.

[0060] The term "allele" refers to a specific form or variant of a gene. For example, alleles can arise from nucleotide substitutions, additions, or deletions, or they can represent a variable number of short nucleotide repeats. In the context of the human leukocyte antigen (HLA) gene, HLA alleles are named by the World Health Organization HLA Systematic Factor Nomenclature Committee. In this system, the HLA gene name is followed by a series of numeric fields. At least two numeric fields are included. As a non-limiting example, HLA-A... 02:101 indicates a specific allele of the HLA-A gene. The first field, separated from the gene name by an asterisk, represents the allele genome. The second field, separated from the first field by a colon, represents the specific HLA protein produced. In some examples, a longer name (e.g., HLA-A) is used. 02:101:02N). In this example, the third numeric field indicates the presence of synonymous DNA substitution in the coding region, and the fourth numeric field indicates the differences between alleles present in the non-coding region. In some other examples, HLA allele names include a letter at the end. In the HLA allele nomenclature system, "N" indicates that the allele is a null allele (i.e., the allele produces a nonfunctional protein), "L" indicates that the allele causes lower surface expression of a specific HLA protein than normal cells, "S" indicates that the allele produces a soluble protein not found on the cell surface, "Q" indicates a problematic allele (i.e., an allele that may not affect normal expression), "C" indicates that the allele produces a protein present in the cytoplasm but not on the cell surface, and "A" indicates an allele causing aberrant expression (i.e., it is uncertain whether a specific HLA protein is expressed). Those skilled in the art will be familiar with various gene alleles and their nomenclature conventions.

[0061] The term "human leukocyte antigen (HLA)" refers to the gene complex encoding the human major histocompatibility complex (MHC) protein, a group of cell surface proteins essential for the adaptive immune system to recognize foreign molecules. The HLA complex is located within a 3 Mbp segment of chromosome 6p21. Class I MHC proteins, which present peptides from within the cell, are... HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and HLA-G Gene encoding. HLA-A , HLA-B , HLA-C Higher genetic polymorphism, and HLA-E , HLA- F , HLA-G Lower genetic polymorphism. (Known) HLA-K and HLA-L It also exists as a pseudogene. Furthermore, β-2-microglobulin is an MHC class I protein, composed of ( B2M Gene encoding. HLA-A Non-restrictive examples of nucleotide sequences are listed under GenBank reference numbers NM_001242758 and NM_002116. HLA-B Non-restrictive examples of nucleotide sequences are listed under GenBank reference number NM_005514. HLA-C Non-restrictive examples of nucleotide sequences are listed under GenBank reference numbers NM_001243042 and NM_002117.HLA-E Non-restrictive examples of nucleotide sequences are listed under GenBank reference number NM_005516. HLA- F Non-restrictive examples of nucleotide sequences are listed under GenBank reference number NM_018950. HLA-G Non-restrictive examples of nucleotide sequences are listed under GenBank reference number NM_002127. B2M Non-restrictive examples of nucleotide sequences are listed under GenBank reference number NM_004048.

[0062] Class II MHC proteins that present antigens to T lymphocytes from outside the cell are... HLA-DP, HLA-DM, HLA-DO, HLA- DQ and HLA-DR Gene encoding. HLA-DM Genes include HLA-DMA and HLA-DMB . HLA-DO Genes include HLA-DOA and HLA- DOB . HLA-DP Genes include HLA-DPA1 and HLA-DPB1 . 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 . HLA-DMA and HLA-DMB Non-restrictive examples of nucleotide sequences are listed under GenBank reference numbers NM_006120 and NM_002118, respectively. HLA-DRA, HLA-DRB1, HLA-DRB3, HLA-DRB4 and HLA-DRB5 Non-restrictive examples of nucleotide sequences are listed under GenBank reference numbers NM_01911, NM_002124, NM_022555, NM_021983, and NM_002125.

[0063] The term "vaccine" refers to a biological composition that, when administered to a subject, has the ability to induce acquired immunity against a specific pathogen or disease in the subject. Typically, one or more antigens or fragments of antigens associated with the target pathogen or disease are administered to the subject. Vaccines may include, for example, inactivated or attenuated organisms (e.g., bacteria or viruses), cells, proteins expressed in or on cells (e.g., cell surface proteins), proteins produced by an organism (e.g., toxins), or a part of an organism (e.g., viral envelope proteins). In some examples, cells are engineered to express proteins such that, when administered as a vaccine, they enhance the subject's ability to acquire immunity against a specific cell type (e.g., enhance the subject's ability to acquire immunity against cancer cells). As used herein, the term "vaccine" or "whole-cell cancer vaccine" includes, but is not limited to, the modified cancer cells of this disclosure.

[0064] The term "cytokine" refers to small proteins released by cells that have specific effects on cell-cell interactions and communication. Cytokines are commonly referred to as lymphokines (e.g., cytokines produced by lymphocytes), monocytes (e.g., cytokines produced by monocytes), or chemokines (e.g., cytokines produced by one leukocyte and acting on other leukocytes). Cytokines can act on cells that secrete them (e.g., autocrine action), neighboring cells (e.g., paracrine action), or distant cells (e.g., endocrine action). In the context of this disclosure, cytokines may include chemokines, interferons, interleukins, and / or tumor necrosis factor (TNF). For example, cytokines may include early T cell activation antigen-1 (ETA-1), lymphocyte activating factor (LAF), interleukin-1 family members (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36γ, IL-37, IL-38), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8); interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), interleukin-17 (IL-17), and interleukin-18. Interleukin-18 (IL-18), Interleukin-23 (IL-21); Interleukin-23 (IL-23), Interleukin-25 (IL-25), Interleukin-33 (IL-33), Type I interferon family members (IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-ω), Type II interferon family members (IFN-γ), Type III interferon family members (IFN-λ1 (IL-29), IFN-λ2 (IL-28A), IFN-λ3 (IL-28B), IFN-λ4), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Macrophage CSF (CSF-1), macrophage migration inhibitory factor (MIF), CD40L molecule (CD40L), RANTES molecule (RANTES), monocyte chemotactic protein (MCP-1), monocyte inflammatory protein (MIP-1α, MIP-1β), lymphocyte chemotactic factor and / or fractal chemotactic factor.

[0065] The term "granulocyte-macrophage colony-stimulating factor (GM-CSF)" refers to a monomeric glycoprotein, also known as "colony-stimulating factor (CSF2)," secreted by cells such as macrophages, T cells, mast cells, natural killer (NK) cells, endothelial cells, and fibroblasts. GM-CSF acts as a cytokine, influencing multiple cell types, particularly macrophages and eosinophils. As part of an immune / inflammatory cascade, GM-CSF stimulates stem cells to produce granulocytes (i.e., neutrophils, eosinophils, and basophils) and monocytes. Monocytes subsequently mature into macrophages and dendritic cells upon tissue infiltration. In humans... CSF2 Non-restrictive examples of nucleotide sequences (encoding the gene for GM-CSF) are listed under GenBank reference number NM_000758.

[0066] The term "interferon" refers to cytokines produced in response to infection or other inflammatory stimuli. Interferons are signaling proteins synthesized and released by host cells in response to pathogens (e.g., viruses, bacteria, parasites, tumor cells). Interferons are classified into three subgroups: type I interferons, type II interferons (IFNγ), and type III interferons. These cytokines functionally regulate immune cell function. Although type III interferons differ structurally from type I interferons, they have overlapping functions, and both induce the transcription of interferon-stimulated genes (ISGs) and promote immune responses through the Janus kinase (JAK) signaling and activator of transcription (STAT) pathways. (See, for example, Goel et al. (2021). Interferonlambda in inflammation and autoimmune rheumatic diseases.) Nat Rev Rheumatol 17, 349–362). Type I interferon proteins include IFN-α, IFN-β, IFN-ε, IFN-κ, IFN-τ, IFN-δ, IFN-ζ, IFN-ω, and IFN-v. Interferon-α protein is produced by leukocytes and is primarily involved in the innate immune response. Genes encoding IFN-α protein include IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17 and IFNA21 . IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21Non-restrictive examples of human nucleotide sequences are listed in GeneBank 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. For example, genes... IFNA2 It encodes IFN-α2a, IFN-α2b, and IFN-α2c variants. The terms “IFN-α” and “IFN-α2” used herein are used interchangeably and refer to either the interferon protein IFN-α2a or IFN-α2b. Type III interferon proteins include interferon λ1 (IFN-λ1(IL-29)), interferon λ2 (IFN-λ2(IL-28A)), interferon λ3 (IFN-λ3(IL-28B)), and interferon λ4 (IFN-λ4). Members of the interferon λ family signal through a common IL-10 receptor subunit 2 (IL-10R2). Human interferon λ protein consists of four... IFNL Gene coding, that is IFNL1 (IL29), IFNL2 (IL28A), IFNL3 (IL28B) and IFNL4 .

[0067] The term "co-stimulatory molecule" refers to cell surface molecules that amplify or counteract the initial activation signal provided to T cells by the T cell receptor (TCR) after the interaction of the T cell receptor (TCR) with the antigen / major histocompatibility complex (MHC). Co-stimulatory molecules can often influence T cell differentiation and fate. Co-stimulatory molecules belong to three major families: the immunoglobulin (Ig) superfamily, the tumor necrosis factor (TNF)-TNF receptor (TNFR) superfamily, and the T cell Ig and mucin (TIM) domain family. (See, for example, Rodriguez-Manzanet, Roselynn et al. "The costimulatory role of TIM molecules.") Immunological reviewsvol. 229,1 (2009): 259-70). Exemplary costimulatory molecules and ligands include, but are not limited to, CD28 and ligand B7-1 (CD80), CTLA-4, PDL-1 or B7-2 (CD86), CTLA-4 and ligand B7-1 (CD80) or B7-2 (CD86), ICOS and ligand ICOS-L, CD27 and ligand CD70, CD30 and ligand CD30L, CD40 and ligand CD40L (also known as CD154), OX40 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 this disclosure, co-stimulatory molecules may include CD86 molecules (CD86), CD80 molecules (CD80), 4-1BB ligand molecules (4-1BBL, also known as CD137L), ICOS ligand molecules (ICOS-L), CD70 molecules (CD70, also known as CD27L), CD40 molecules (CD40), OX40 ligand molecules (OX40L), GITR ligand molecules (GITRL), TIM-4 molecules (TIM-4), LIGHT molecules (LIGHT), ICAM1 molecules (ICAM1), LFA3 molecules (LFA3), CD30 molecules (CD30), and combinations thereof (see, for example, Figure 5).

[0068] The term "tumor antigen" refers to an antigenic substance produced in tumor cells that may trigger an immune response in the host. Tumor antigens are generally referred to as tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs). Typically, TSAs are present only in cancer cells and not in healthy (e.g., non-cancerous) cells. TSAs can be caused by oncogenic driver mutations that produce novel peptide sequences (e.g., neoantigens). A non-limiting example of TSA is alpha-fetoprotein (AFP), which is expressed in germ cell tumors and hepatocellular carcinoma. TAAs are expressed at elevated levels in tumor cells, while they can be expressed at lower levels in healthy cells. A non-limiting example of TAA is melanoma-associated antigen (MAGE), which is expressed in the testes and in malignant melanoma.

[0069] The term "survival" refers to the length of time following a disease diagnosis and / or the initiation or completion of a specific treatment course for a disease (e.g., cancer). The term "overall survival" includes clinical endpoints describing patients who survive within a defined time period following the diagnosis or treatment of a disease (e.g., cancer). The term "disease-free survival" includes the length of time a patient survives without signs of disease (e.g., no known recurrence) after treatment for a specific disease (e.g., cancer). In some embodiments, disease-free survival is a clinical parameter used to evaluate the efficacy of a specific treatment, typically measured in 1-year or 5-year units. The term "progression-free survival" includes the length of time a patient survives with and without additional symptoms of a disease during and after treatment for a specific disease (e.g., cancer). In some embodiments, survival is expressed as median or mean.

[0070] III. Detailed Description of the Implementation Plan

[0071] This disclosure is partly based on the inventors' discovery that modified human cancer cells (e.g., SV-BR-1-GM, modified human breast tumor cell lines secreting GM-CSF) contain a variety of post-translational modified antigens that bind to HLA class I and / or II molecules. The inventors have found that these post-translational modified antigens can overcome tolerance and induce immune responses in autoimmune diseases. Therefore, by combining the Bria-IMT™ platform technology, highly immunogenic cancer vaccines can be developed by inducing enzymatic or non-enzymatic post-translational modifications (PTMs) of antigens in modified cancer cells (e.g., the Bria-OTS cell line previously developed by the inventors).

[0072] In one aspect, this disclosure provides modified human cancer cells comprising recombinant polynucleotides encoding enzymes that induce post-translational modifications (PTMs) of antigens in cells. In some embodiments, said enzymes include citrullinated enzymes, cysteine ​​lactamases, acetyltransferases, hydroxylases, phosphorylases, methyltransferases, formylates, oxidases, hydroxylases, ubiquitinases, or combinations thereof. In some embodiments, the modified human cancer cells comprise recombinant polynucleotides encoding citrullinated enzymes that induce PTMs of antigens in cells. In some embodiments, the modified human cancer cells comprise recombinant polynucleotides encoding cysteine ​​lactamases that induce PTMs of antigens in cells. Without being bound by any theory, introducing exogenous PTM enzymes into cells advantageously allows the cells to produce multiple PTMs of one or more antigens, resulting in said one or more antigens being more immunogenic than cells lacking exogenous PTM enzymes. In some embodiments, the modified human cancer cells comprising recombinant polynucleotides encoding enzymes that induce PTMs of antigens also comprise recombinant polynucleotides encoding antigens. In some embodiments, the modified human cancer cells containing recombinant polynucleotides encoding PTM enzymes further comprise: (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 endogenous HLA alleles have been inactivated in the modified human cancer cells. In some embodiments, the modified human cancer cells containing recombinant polynucleotides encoding PTM enzymes further comprise recombinant polynucleotides encoding cytokines. In some embodiments, the modified human cancer cells containing recombinant polynucleotides encoding PTM enzymes further comprise recombinant polynucleotides encoding co-stimulatory molecules. In some embodiments, the modified human cancer cells comprise one or more recombinant polynucleotides encoding: a) an enzyme that induces PTM of antigens in cells; b) an 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, human cancer cells modified with recombinant polynucleotides encoding PTM enzymes targeting antigens in cells also contain one or more recombinant polynucleotides encoding: a) antigens; b) alleles of HLA class I genes and / or HLA class II genes; c) cytokines; and / or d) co-stimulatory molecules.In certain embodiments, the modified human cancer cells comprising a recombinant polynucleotide encoding a PTM enzyme targeting an antigen in the cell further comprise: 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 endogenous HLA alleles have been inactivated in the modified human cancer cells.

[0073] In another aspect, this disclosure provides modified human cancer cells containing an antigen, wherein the antigen comprises a non-enzymatic PTM. In some embodiments, the non-enzymatic PTM is induced by irradiation, induced cellular senescence, chemical reaction, small molecules, cell culture supplements, cell culture medium, oxidative stress, or a combination thereof. In some embodiments, the non-enzymatic PTM comprises cysteine ​​oxidation. In some embodiments, the modified human cancer cells containing the antigen having a non-enzymatic PTM further contain a recombinant polynucleotide encoding said antigen. In some embodiments, the modified human cancer cells containing the antigen having a non-enzymatic PTM further contain: (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 endogenous HLA alleles have been inactivated in the modified human cancer cells. In some embodiments, the modified human cancer cells containing the antigen having a non-enzymatic PTM further contain a recombinant polynucleotide encoding a cytokine. In some embodiments, the modified human cancer cells containing the antigen having a non-enzymatic PTM further contain a recombinant polynucleotide encoding a co-stimulatory molecule. In some embodiments, the modified human cancer cells containing an antigen having a non-enzymatic PTM further comprise 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 specific embodiments, the modified human cancer cells containing an antigen having a non-enzymatic PTM further comprise: 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 endogenous HLA alleles have been inactivated in the modified human cancer cells.

[0074] In some implementations, multiple recombinant polynucleotides encoding different PTM enzymes may be introduced into the same cell. In some examples, a recombinant polynucleotide encoding one PTM enzyme is introduced into the cell. In other examples, recombinant polynucleotides encoding two or more PTM enzymes are introduced into the cell. In still other examples, two or more recombinant polynucleotides, each encoding at least one PTM enzyme, are introduced into the same cell. In some examples, multiple recombinant polynucleotides encoding two, three, four, five, six, or more PTM enzymes are introduced into the same cell.

[0075] In some embodiments, the recombinant polynucleotide is integrated into the genome of the cell. In other embodiments, the recombinant polynucleotide is present on one or more vectors in the cell. In some examples, all recombinant polynucleotides may be present on the same vector. In other examples, each recombinant polynucleotide may be present on a separate vector. In still other examples, two, three, four, five, six, or more recombinant polynucleotides may be present on the same vector. A single vector in the cell and any number of combinations of recombinant polynucleotides on any number of vectors are permitted. In some examples, 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 may be present on the same vector in the cell. In other examples, 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 may be present on different vectors in the same cell.

[0076] As a non-limiting example, recombinant polynucleotides encoding one or more PTM enzymes (e.g., one, two, three, four, or more PTM enzymes) may be present on the same vector. As another non-limiting example, recombinant polynucleotides encoding one or more antigens may be present on the same vector. As a non-limiting example, recombinant polynucleotides encoding two distinct HLA class I alleles may be present on the same vector. As another non-limiting example, recombinant polynucleotides encoding two distinct HLA class II alleles may be present on the same vector. As another non-limiting example, recombinant polynucleotides encoding one or more cytokines (e.g., GM-CSF, IFN-α, 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) may 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 the following: GM-CSF, IFN-α, 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 the following: a) GM-CSF and IFN-α; b) GM-CSF; c) CD86 and IL-12; d) CD40; e) CD80 and HLA-DRA alleles; and f) IL-7 and 4-1BBL.

[0077] In some embodiments, the cell comprises: (a) a vector containing a recombinant polynucleotide encoding one or more PTM enzymes (e.g., one, two, three, four or more PTM enzymes); (b) a vector containing a recombinant polynucleotide encoding two distinct HLA class I alleles; (c) a vector containing a recombinant polynucleotide encoding two distinct HLA class II alleles; and / or (d) one or more vectors (e.g., one, two, three, four or more vectors) each containing a recombinant polynucleotide encoding one or more cytokines, immunomodulatory molecules and / or costimulatory molecules (e.g., paired combinations of GM-CSF, IFN-α, CD40, CD80, CD86, IL-12, IL-7, HLA-DRA and 4-1BBL). In a particular embodiment, the cell comprises: (a) a vector containing a recombinant polynucleotide encoding one or more PTM enzymes (e.g., one, two, three, four or more PTM enzymes); (b) a vector containing a recombinant polynucleotide encoding two distinct HLA class I alleles; (c) a vector containing a recombinant polynucleotide encoding two distinct HLA class II alleles; and / or (d) one or more vectors (e.g., one, two, three, four or more vectors) each containing a recombinant polynucleotide encoding one or more cytokines, immunomodulatory molecules and / or costimulatory molecules selected from GM-CSF, IFN-α, CD40, CD80, CD86, IL-12, IL-7, HLA-DRA and 4-1BBL. In another specific embodiment, the cell comprises: (a) a vector containing a recombinant polynucleotide encoding one or more PTM enzymes (e.g., one, two, three, four, or more PTM enzymes); (b) a vector containing a recombinant polynucleotide encoding two distinct HLA class I alleles; (c) a vector containing a recombinant polynucleotide encoding two distinct HLA class II alleles; and / or (d) one or more vectors (e.g., one, two, three, four, or more vectors) each containing a recombinant polynucleotide encoding at least one immunomodulatory molecule, costimulatory molecule, and / or cytokine selected from: a) GM-CSF and IFN-α; b) GM-CSF; c) CD86 and IL-12; d) CD40; e) CD80 and HLA-DRA alleles; f) IL-7 and 4-1BBL. In another specific embodiment, the cell further comprises e) a vector containing a recombinant polynucleotide encoding one or more antigens.

[0078] A. Post-translation modification (PTM)

[0079] Post-translational modifications (PTMs) are covalent processes of amino acid modification following protein biosynthesis. Currently, UniProt's PTM knowledge base contains over 700 PTMs, describing their target proteins, sites, and cellular locations. PTMs can occur on amino acid side chains or at the C- or N-terminus of proteins. PTMs alter protein structure, function, and localization, playing a crucial role under physiological and pathophysiological conditions. A single site within the same protein can undergo one or more types of modification. Similarly, a single regulator can play multiple roles.

[0080] In some embodiments, PTMs include phosphorylation, acetylation, ubiquitination, succinylation, methylation, malonylation, glycosylation, SUMOylation, nitrosylation, glutathioneylation, amidation, hydroxylation, palmitoylation, glutarylation, crotonylation, oxidation, myristylation, sulfation, formylation, citrullination, isoprenelation, cysteineation, deamidation, dehydration, or combinations thereof. All types of PTMs are known in the art. (See, e.g., Ramazi S, Zahiri J. Posttranslational modifications in proteins: resources, tools and predictionmethods. 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 Immuno-Oncology Therapies. Cancers (Basel). 2022;15(1):138; Each of the above references is incorporated herein by reference in its entirety. In some embodiments, PTM includes cysteine. In some embodiments, PTM includes citrulline.

[0081] In some embodiments, the modified human cancer cells disclosed herein contain PTMs of antigens. In some examples, antigens in the natural environment (e.g., antigens in unmodified cells) do not contain PTMs, wherein the antigens in the modified human cancer cells contain such PTMs by enzymatic or non-enzymatic methods, resulting in antigens in the modified human cancer cells being more immunogenic than antigens in the natural environment. In other examples, antigens in the natural environment (e.g., in unmodified cells) contain PTMs, wherein the antigens in the modified human cancer cells contain induced PTMs by enzymatic or non-enzymatic methods, resulting in induced PTM antigens in the modified human cancer cells being more immunogenic than antigens in the natural environment.

[0082] As disclosed above, PTMs can be divided into two categories based on their biochemical origin: enzymatic PTMs and non-enzymatic PTMs.

[0083] 1. Enzymatic PTM

[0084] Enzymatic proteolytic metaplasia (PTM) involves the covalent conjugation of chemical groups to protein side chains via enzymatic catalysis, as well as the cleavage of the protein backbone by proteases or autocatalytic cleavage on specific peptides. PTM enzymes can be classified into three subtypes based on their functional specificity: “writers” are responsible for adding substrates, “readers” recognize modified proteins to initiate downstream signaling cascades, and “erasers” are best known for their role in removing PTM (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(1):31).

[0085] As disclosed herein, any PTM enzyme can be introduced into cells. In some embodiments, the PTM enzyme is a citrullase, cysteine ​​lactamase, acetyltransferase, hydroxylase, phosphorylase, methyltransferase, formylate, oxidase, hydroxylase, or ubiquitinase. In some embodiments, the enzyme includes a citrullase. In some embodiments, the enzyme includes a cysteine ​​lactamase. In other embodiments, the PTM enzyme is a combination of a citrullase, cysteine ​​lactamase, acetyltransferase, hydroxylase, phosphorylase, methyltransferase, formylate, oxidase, hydroxylase, and / or ubiquitinase. In some embodiments, the modified human cancer cells comprise a recombinant polynucleotide encoding one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) PTM enzymes.

[0086] In some embodiments, the enzyme is a citrullinated enzyme. In some embodiments, the citrullinated enzyme includes a peptidyl arginine deiminase. Examples of peptidyl arginine deiminases 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 cysteine ​​chemotransferase. In some embodiments, the cysteine ​​chemotransferase includes a protein disulfide isomerase (PDI). PDIs catalyze the controlled formation and rearrangement of disulfide bonds for the structural stability of nascent proteins. Each member of the PDI family contains at least one thioredoxin-like (Trx-like) domain having a thioredoxin fold. These Trx-like domains may be catalytically active (a domain) with a CXXC motif; or inactive (b domain). PDI also has an acidic C-terminal extension (c-domain) ending at the ER-retained 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 PDI are listed in Table 2. In some embodiments, the enzyme is an acetyltransferase. Non-limiting examples of acetyltransferases are listed in Table 3. In some embodiments, the enzyme is a hydroxylase, such as hypoxia-inducible factor prolyl hydroxylase (Table 4). In some embodiments, the enzyme is a phosphorylase. Non-limiting examples of phosphorylases are listed in Table 5. In some embodiments, the enzyme is a methyltransferase. Non-limiting examples of methyltransferases are listed in Table 6. In some embodiments, the enzyme is a formylate, such as mitochondrial methionyl-tRNA formyltransferase (Table 7). In some embodiments, the enzyme is an oxidase. Non-limiting examples of oxidases are listed in Table 8. In some embodiments, the enzyme is a hydroxylase. Non-limiting examples of hydroxylases are listed in Table 9. In some embodiments, the enzyme is a ubiquitinase. In some embodiments, the ubiquitinase includes ubiquitinase 1. In some embodiments, the ubiquitinase includes ubiquitinase 2. In some embodiments, the ubiquitinase includes ubiquitinase 3. Non-limiting examples of ubiquitinases are listed in Table 10.

[0087] Table 1. Exemplary citrullinated enzymes

[0088] Table 2. Exemplary cysteine ​​chemotransferases (protein disulfide isomerases)

[0089] Table 3. Exemplary acetyltransferases

[0090] Table 4. Exemplary hydroxylases

[0091] Table 5. Exemplary phosphorylases

[0092] Table 6. Exemplary methyltransferases

[0093] Table 7. Exemplary Formylates

[0094] Table 8. Exemplary oxidases

[0095] Table 9. Exemplary hydroxylases

[0096] Table 10. Exemplary ubiquitinating enzymes

[0097] 2. Non-enzymatic PTM

[0098] Non-enzymatic post-translational modifications (PTMs) are typically 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 include cysteine, saccharification, sugar oxidation, nitrosation, oxidation, succinate, and lipoxidation.

[0099] As disclosed herein, non-enzymatic PTMs can be induced by irradiation, induced cellular senescence, chemical reactions, small molecules, cell culture supplements, cell culture media, oxidative stress, or a combination thereof. In some embodiments, the non-enzymatic PTMs comprise cysteine. In some embodiments, non-enzymatic cysteine ​​can be induced by culturing cells with an excess of cysteine. In some embodiments, the non-enzymatic PTMs comprise 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 a serum-free medium. In some embodiments, non-enzymatic PTMs can be promoted by inducing cellular senescence. In some embodiments, small molecules, such as senescence inducers, 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 (a CDK4 / 6 inhibitor), and nutlin-3A (a p53 activator).

[0100] B. Modified human cancer cells

[0101] In some embodiments, the modified human cancer cells comprise at least one recombinant polynucleotide encoding one or more PTM enzymes, variants thereof, or fragments thereof. For example, the modified human cancer cells 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 citrullinated lactamase, cysteine ​​lactamase, acetyltransferase, hydroxylase, phosphorylase, methyltransferase, formylate, oxidase, hydroxylase, ubiquitinase, or a combination thereof. In some embodiments, the enzyme comprises citrullinated lactamase. In some embodiments, the enzyme comprises cysteine ​​lactamase.

[0102] In some embodiments, any one of the PTM enzymes, their variants, or fragments thereof may have a polynucleotide sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or higher identity or similarity with its corresponding wild-type sequence. In some embodiments, any one of the PTM enzymes, their variants, or fragments thereof may have a polynucleotide sequence that has at most 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity or similarity with its corresponding wild-type sequence. In some embodiments, the polynucleotide encoding a modified PTM enzyme or a codon-optimized PTM enzyme, its variants, or fragments thereof has at least 70%, 75%, 80%, 85%, 90%, 95%, 99%, or higher identity or similarity with its corresponding wild-type sequence. In some embodiments, the polynucleotide encoding a modified PTM enzyme or a codon-optimized PTM enzyme, its variants, or fragments thereof has up to 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity or similarity to its corresponding wild-type sequence. In some embodiments, the polynucleotide encoding a modified PTM enzyme or a codon-optimized PTM enzyme, its variants, or fragments thereof has about 10% to 99%, about 30% to 80%, about 40% to 95%, or about 60% to 85% identity or similarity to its corresponding wild-type sequence.

[0103] In some embodiments, the modified human cancer cells contain at least one recombinant polynucleotide encoding one or more antigens. For example, the modified human cancer cells may contain a recombinant polynucleotide encoding antigens 1, 2, 3, 4, or more, driven by one or more promoters. In some embodiments, the antigen is a fragment of one or more of the following: a pathogen antigen, a tumor-specific antigen, a tumor-associated antigen, a neoantigen, an allergen, an antigen that is a target of an autoimmune response, or a pathogen-specific antigen.

[0104] In some embodiments, the modified human cancer cells contain at least one recombinant polynucleotide encoding one or more HLA class I genes, their codon-optimized versions, variants thereof, or fragments thereof. For example, the modified human cancer cells may contain recombinant polynucleotides encoding HLA class 1, 2, 3, 4, or more HLA class I genes driven by one or more promoters. In some embodiments, the modified human cancer cells also contain at least one recombinant polynucleotide encoding one or more HLA class II genes, their codon-optimized versions, variants thereof, or fragments thereof. For example, the modified human cancer cells may contain recombinant polynucleotides encoding HLA class 1, 2, 3, 4, or more HLA class II genes driven by one or more promoters.

[0105] In some embodiments, the modified human cancer cells comprise one or more recombinant polynucleotides encoding one or more HLA class I genes, wherein the one or more HLA class I genes are selected from HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, β-2-microglobulin (B2M) genes, or combinations thereof. In some embodiments, the modified human cancer cells comprise one or more recombinant polynucleotides encoding at least one HLA class I allele, wherein the at least one HLA class I allele includes, but is not limited to, HLA-A. 01:01 Alleles, HLA-A 68:01 allele, HLA-A 02:01 Alleles, HLA-A 11:01 alleles, HLA-A 03:01 Alleles, HLA-A 23:01 alleles, HLA-A 24:02 alleles and / or HLA-A 33:03 allele. In some embodiments, the modified human cancer cells contain one or more recombinant polynucleotides encoding at least one HLA class I allele, said at least one HLA class I allele being selected from HLA-A. 01:01 Alleles and HLA-A 68:01 allele, HLA-A 02:01 Alleles and HLA-A 11:01 alleles, HLA-A 03:01 Alleles and HLA-A 23:01 allele, and / or HLA-A 24:02 alleles and HLA-A 33:03 alleles.

[0106] In some embodiments, the modified human cancer cells comprise one or more recombinant polynucleotides encoding one or more HLA class II genes, wherein the one or more HLA class II genes are selected from HLA-DP genes, HLA-DM genes, HLA-DO genes, HLA-DQ genes, HLA-DR genes, or combinations thereof. In some embodiments, the modified human cancer cells comprise one or more recombinant polynucleotides encoding at least one HLA class II allele, wherein the at least one HLA class II allele includes, but is not limited to, HLA-DRB3. 02:02 allele, HLA-DRB5 01:01 allele, HLA-DRB4 01:01 allele, HLA-DRB3 01:01 allele, HLA-DRB3 03:01 Alleles, HLA-DRB5 01:02 Alleles and / or HLA-DRB5 02:02 allele. In some embodiments, the modified human cancer cells comprise one or more recombinant polynucleotides encoding at least one HLA class II allele, said at least one HLA class II allele being selected from HLA-DRB3. 02:02 alleles and HLA-DRB5 01:01 allele, HLA-DRB4 01:01 Alleles and HLA-DRB3 01:01 allele, HLA-DRB3 03:01 Alleles and HLA-DRB5 01:02 alleles, and / or HLA-DRB5 02:02 alleles and HLA-DRB3 01:01 allele.

[0107] In some embodiments, one or more endogenous HLA alleles in the cells have been inactivated in the modified human cancer cells. In a specific embodiment, endogenous HLA-A... 24:02 alleles and HLA-DRB3 The 01:01 allele has been inactivated in modified human cancer cells.

[0108] In some implementations, the modified human cancer cells contain one or more recombinant polynucleotides encoding cytokines, their variants, or fragments thereof. The cytokines can be chemokines, interferons, interleukins, or tumor necrosis factor. Cytokines can be selected from early T cell activating antigen-1 (ETA-1), lymphocyte activating factor (LAF), interleukin-1 family members (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36γ, IL-37, IL-38), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), and interleukin-17. Interleukin-17, Interleukin-18 (IL-18), Interleukin-21 (IL-21), Interleukin-23 (IL-23), Interleukin-25 (IL-25), Interleukin-33 (IL-33), Interferon-α (IFN-α), Interferon-λ1 (IFN-λ1 (IL-29)), Interferon-λ2 (IFN-λ2 (IL-28A)), Interferon-λ3 (IFN-λ3 (IL-28B)), Interferon-λ4 (IFN-λ4), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Macrophage CSF (CSF-1), Macrophage migration inhibitory factor (MIF), CD40L molecule (CD40L), RANTES molecule (RANTES), Monocyte chemotactic protein (MCP-1), Monocyte inflammatory protein (MIP-1α, MIP-1β), Lymphocyte chemotactic factor or fractal chemotactic factor. In a particular implementation, the cytokine includes GM-CSF.

[0109] In some embodiments, the modified human cancer cells comprise one or more recombinant polynucleotides encoding a co-stimulatory molecule, a variant thereof, or a fragment thereof. The co-stimulatory molecule may be selected from at least one of the following: CD86 molecule (CD86), CD80 molecule (CD80), 4-1BB ligand molecule (4-1BBL, also known as TNFSF9 or CD137L), ICOS ligand molecule (ICOS-L), CD70 molecule (CD70, also known as CD27L), CD40 molecule (CD40), OX40 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.

[0110] In some embodiments, the modified human cancer cells contain one or more recombinant polynucleotides encoding: a) an enzyme that induces PTM for antigens in cells; b) an antigen; c) an allele of HLA class I genes and / or an allele of HLA class II genes; d) cytokines; and / or e) co-stimulatory molecules. In some embodiments, the modified human cancer cells containing recombinant polynucleotides encoding PTM enzymes targeting antigens in cells also contain one or more recombinant polynucleotides encoding: a) an antigen; b) an allele of HLA class I genes and / or an allele of HLA class II genes; c) cytokines; and / or d) co-stimulatory molecules. In some embodiments, one or more endogenous HLA alleles in the cells have been inactivated in the modified human cancer cells.

[0111] In some embodiments, the human cancer cells modified with recombinant polynucleotides encoding PTM enzymes are derived from breast cancer cell lines, such as the SV-BR-1 or SV-BR-1-GM cell lines described in WO 2017 / 147600 (which is incorporated herein in its entirety). In some embodiments, the human cancer cells modified with recombinant polynucleotides encoding PTM enzymes are derived from the breast cancer SV-BR-1-Bria-OTS cell line. In some embodiments, the human cancer cells modified with recombinant polynucleotides encoding PTM enzymes are derived from prostate cancer cell lines, such as PC3-Bria-OTS. In some embodiments, the human cancer cells modified with recombinant polynucleotides encoding PTM enzymes are derived from melanoma cell lines, such as the SK-MEL-24-Bria-OTS cell line. In some embodiments, the human cancer cells modified with recombinant polynucleotides encoding PTM enzymes are derived from lung cancer cell lines, such as the H2228-Bria-OTS cell line. The Bria-OTS cell line described in this article overexpresses a combination of cytokines, immunomodulatory molecules, and co-stimulatory molecules including GM-CSF, IFN-α, CD80, CD86, IL-12, IL-7, HLA-DRA, and 4-1BBL, as well as unique combinations of exogenous HLA-A and HLA-DRB3 / 4 / 5 alleles (e.g., HLA-A). 01:01 Alleles, HLA-A 68:01 allele, HLA-DRB3 02:02 alleles and HLA-DRB5 01:01 allele combination; HLA-A 02:01 Alleles, HLA-A 11:01 allele, HLA-DRB4 01:01 Alleles and HLA-DRB3 01:01 allele combination; HLA-A 03:01 Alleles, HLA-A 23:01 allele, HLA-DRB3 03:01 Alleles and HLA-DRB5 01:02 Allele combination; or HLA-A 24:02 allele, HLA-A 33:03 allele, HLA-DRB5 02:02 alleles and HLA-DRB3 01:01 allele combination), while certain endogenous HLA-A and HLA-DRB3 alleles (e.g. HLA-A) 24:02 and HLA-DRB3 01:01) has been inactivated. The Bria-OTS cell line is described in WO2023 / 167973, which is incorporated herein in its entirety. In some embodiments, human cancer cells containing modifications of recombinant polynucleotides encoding PTM enzymes are derived from cancer cell lines (e.g., breast cancer cell lines, prostate cancer cell lines, melanoma cell lines, or lung cancer cell lines) that overexpress a combination of cytokines, immunomodulatory molecules, and co-stimulatory molecules including GM-CSF, IFN-α, CD80, CD86, IL-12, IL-7, HLA-DRA, and CD40, as well as a unique combination of exogenous HLA-A and HLA-DRB3 / 4 / 5 alleles (e.g., HLA-A 01:01 Alleles, HLA-A 68:01 allele, HLA-DRB3 02:02 alleles and HLA-DRB5 01:01 allele combination; HLA-A 02:01 Alleles, HLA-A 11:01 allele, HLA-DRB4 01:01 Alleles and HLA-DRB3 01:01 allele combination; HLA-A 03:01 Alleles, HLA-A 23:01 allele, HLA-DRB3 03:01 Alleles and HLA-DRB5 01:02 Allele combination; or HLA-A 24:02 allele, HLA-A 33:03 allele, HLA-DRB5 02:02 alleles and HLA-DRB3 01:01 allele combination), while certain endogenous HLA-A and HLA-DRB3 alleles (e.g. HLA-A) 24:02 and HLA-DRB3 (01:01) has been deactivated.

[0112] In some embodiments, the expression of PTM enzymes, HLA alleles, antigens, cytokines (e.g., GM-CSF, IL-12, IL-7, and / or IFN-α), immunomodulatory molecules (e.g., HLA-DRA), and / or co-stimulatory molecules (e.g., CD40, CD80, CD86, and / or 4-1BBL) is controlled by two or more different promoters. In some cases, the expression of each PTM enzyme, HLA allele, antigen, cytokine (e.g., GM-CSF, IL-12, IL-7, and / or IFN-α), immunomodulatory molecule (e.g., HLA-DRA), and / or co-stimulatory molecule (e.g., CD40, CD80, CD86, and / or 4-1BBL) is controlled by a separate promoter. In some embodiments, the expression of PTM enzymes, HLA alleles, antigens, cytokines (e.g., GM-CSF, IL-12, IL-7, and / or IFN-α), immunomodulatory molecules (e.g., HLA-DRA), and / or co-stimulatory molecules (e.g., CD40, CD80, CD86, 4-1BBL) is controlled by a single promoter. In some examples, the expression of PTM enzymes, HLA alleles, antigens, cytokines (e.g., GM-CSF, IL-12, IL-7, and / or IFN-α), immunomodulatory molecules (e.g., HLA-DRA), and / or co-stimulatory molecules (e.g., CD40, CD80, CD86, and / or 4-1BBL) as polycistronic mRNAs in a polycistronic vector. In certain examples, one or more cistrons are separated by an internal ribosome entry site. In other examples, one or more cistrons are separated by a self-cleaving peptide (e.g., T2A, P2A, E2A, F2A).

[0113] In some embodiments, the modified human cancer cells are derived from human cancer cell lines. Any number of human cancer cells or cancer cell lines are suitable for the compositions and methods described herein, including, for example, cloned or non-cloned human cancer cells or cancer cell lines. Non-limiting examples of human cancer cell lines include the following cell lines and their subclones: 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-1-GM, SVCT, MDA-MB-231, MDA-MB-157, ZR-75-30, ZR-75-1, Hs578T, MCF7, T47D, MTSV1-7 CE1, 1-7HB2, VP303, VP267, and VP229 breast cancer cell lines; PC-3, Bria-Pros-1+, Bria-Pros-2+, Bria-Pros-3+, Bria-Pros-4+, and LNCaP. (e.g., cloned FGC), Shmac 5, P4E6 and VCaP prostate cancer cell lines; 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; SK-MEL-24, Bria-Mel-1+, Bria-Mel-2+, Bria-Mel-3+, Bria-Mel-4+ melanoma cell lines; UM-UC-3, T24 / 83, ECV304, RT4 and HT 1197 bladder cancer cell lines; MDST8, C170, GP5d, GP2d and LS 123 colon cancer cell lines; MFE-280 and MFE-296 endometrial cancer cell lines; CAKI 2, A.704, G-402, ACHN, G-401, UM-RC-7, and RCC4plusVHL renal cancer cell lines; SK-HEP-1, Hep 3B, PLC / PRF / 5, Hep G2, and Huh-7D12 liver cancer cell lines; HL60, Eos-HL-60, JVM-13, Sci-1, and Ri-1 leukemia cell lines; BHL-89, COR-L24, U937 (CD59+), My-La CD8+, and HGC-27 lymphoma cell lines; A375-C6, GR-M, VA-ES-BJ, MEWO, and COLO818 skin cancer cell lines;AsPC-1, Hup-T4, Hup-T3, BxPC-3 and CFPAC-1 pancreatic cancer cell lines; 8505C, 8305C, FTC-238, TT, RO82-W-1 and K1 thyroid cancer cell lines; HeLa DH, HR5-CL 11, HtTA-1, HR5, X1 / 5, HeLa, C-4I, C-4 II, HeLa S3, Ca Ski, HeLa 229, Hep2 (HeLa derivative), HeLa B, Bu25 TK-HeLaOhio and HeLa (AC-free) cervical cancer cell lines; NB69, BE(2)-C, BE(2)-M17, SK-N-BE(2), SK-N-DZ brain cancer cell lines; OV7, OV17R, OV58, OV56, A2780ADR, A2780, COLO 720 E, SW 626, SK-OV-3, PA-1, 59M, OAW28, TO14, PEO23, COV362 ovarian cancer cell lines; IMR 32 abdominal cancer cell line; SW 13 adrenocortical cancer cell line; TR146 oral mucosal cancer cell line; SK-GT-4 esophageal cancer cell line; TE 671 embryonic cancer cell line; FLYRD 18 fibrosarcoma cell line; 1411 H germ cell tumor cell line; MFM-223 breast cancer cell line; H-EMC-SS muscle cancer cell line; Detroit 562 pharyngeal cancer cell line; BeWo placental cancer cell line; Mero-95 pleural cancer cell line; SW 837, SW1463, CMT 93, HRT-18, HRA-19 rectal cancer cell line; Y 79, WERI, RB247C retinal cancer cell line; CHP-100 spinal cancer cell line; KARPAS The cell lines described herein include: 1718 splenic lymphoma cell line; AGS and KATO-III gastric cancer cell lines; NTERA-2 clone D1 testicular cancer cell line; SCC-9, H357, H103, BICR 56, and PE / CA-PJ 49 tongue cancer cell lines; MES-SA / Dx-5, MES-SA, COLO 685, and COLO 684 uterine cancer cell lines; and HMVII vaginal cancer cell line. In certain embodiments, the human cancer cell lines are 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., NCI-H2228) cell lines. The cell lines described herein and other cell lines may be obtained, for example, from Sigma-Aldrich (www.sigmaaldrich.com).

[0114] In other embodiments, the modified human cancer cells are derived from primary cancer cells. For example, the primary cancer cells are obtained from a tumor biopsy or circulating cancer cells derived from a subject of cancer to be treated prior to the modification of the cancer cells. In some embodiments, the patient has breast cancer, prostate cancer, melanoma, or lung cancer.

[0115] C. Expression vehicle

[0116] In some embodiments, the modified human cancer cells described herein comprise one or more expression vectors for expressing recombinant PTM enzymes. A variety of expression vectors can be used, such as retroviral vectors, lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, plasmids, or transposons. Viral vectors that can be used include, for example, vectors based on HIV, SV40, EBV, HSV, or BPV. By design, the expression vector can be replication-deficient, such that one or more functions essential for viral genome replication or viral particle synthesis and assembly are defective. Many existing replication-deficient viruses can carry large therapeutic genes, efficiently transduce various cell types, and provide long-term and stable expression of target genes.

[0117] Lentivirals are a subset of retroviruses commonly used in research. Lentivirals can transduce both dividing and non-dividing cells without a significant immune response. These viruses also stably integrate into the host genome, enabling long-term transgenic expression. A common lentivirus is the human immunodeficiency virus (HIV), which utilizes envelope glycoproteins of other viruses to target a wide range of cell types.

[0118] A safety feature of lentiviruses is that the components required to produce infectious viral particles (viral particles) are typically distributed among multiple plasmids. For example, an infectious viral particle may include plasmids that serve as components of the viral capsid and envelope (often referred to as packaging and envelope plasmids) and plasmids that encode the viral genome (often referred to as transfer plasmids). Common lentiviral packaging and envelope plasmids that can be used in this paper 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, and pCD / NL-BH. DDD, psPAX2-D64V, pCEP4-tat, pHEF-VSVG, pNHP, pCAG-Eco and pCAG-VSVG. Common lentiviral transfer plasmids that can be used in this study include, but are not limited to, pLKO.1 puro, pLKO.1 – TRC clone plasmid, pLKO.3G, Tet-pLKO-puro, pSico, pLJM1-EGFP, FUGW, pLVTHM, pLVUT-tTR-KRAB, pLL3.7, pLB, pWPXL, pWPI, EF.CMV.RFP, pLenti CMV Puro DEST, pLenti-puro, pLOVE, pULTRA, pLX301, plinducer20, pHIV-EGFP, Tet-pLKO-neo, pLV-mCherry, pCW57.1, pLionII, pSLIK-Hygro, and pInducer10-mir-RUP-PheS.

[0119] There are several methods for generating lentiviral vectors. (See Logan et al. “Factors influencing thetiter 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 can be purchased from commercial vendors. Typically, the production of lentiviral vectors involves multiple steps, including plasmid development and production, cell amplification, plasmid transfection, viral vector production, purification, filling, and completion. (See, for example, www.addgene.org / viral-vectors / lentivirus / ; www.thermofisher.com / us / en / home / clinical / cell-gene-therapy / gene-therapy / lv-production-workflow.html).

[0120] Lentiviral vectors can be programmed to simultaneously express one or more target genes. A variety of molecular strategies are available, including the use of multiple promoters, splicing signals, gene fusions, cleavage factors, and polycistronic vectors. (See, for example, review Shaimardanova et al., “Production and application of multicistronic constructs for various human disease therapies.” Pharmaceutics 2019, 11, 580).

[0121] Polycistronic vectors typically contain nucleotide sequences encoding an internal ribosome entry site (IRES) and a self-cleaving 2A peptide. Using IRES and the self-cleaving 2A peptide allows for the simultaneous expression of two or more separate proteins from the same mRNA.

[0122] Self-cleaving 2A peptides are used to generate polycistronic vectors due to their small size and self-cleaving ability. 2A peptides consist of 16-20 amino acids and are derived from viral RNA. Common 2A peptides used to generate polycistronic vectors are F2A (derived from foot-and-mouth disease virus), E2A (derived from equine rhinitis virus), P2A (derived from porcine chezinvirus-1), and T2A (derived from *Thosea asigna* virus).

[0123] In constructs containing the 2A peptide sequence, translation is initiated once and continues along the mRNA synthesis pathway. During translation, the first peptide cleaves from the second peptide in the 2A region. The 2A peptide cleavage site is located between glycine and proline. The cleavage process occurs within the ribosome during protein synthesis. Normal peptide bond formation between amino acids is inhibited only at the cleavage site; therefore, cleavage does not affect the translation of subsequent proteins, and synthesis continues without ribosome dissociation. Consequently, the translation of multiple genes is interdependent. Using different 2A peptides may affect the expression levels of downstream proteins. The combination of 2A peptide sequence sequences can prevent a gradual decrease in gene expression from the first to the last in a polycistronic construct. For example, when creating a polycistronic vector containing four genes, the optimal combination of 2A peptide sequences is T2A, P2A, and E2A.

[0124] In some implementations, non-viral methods are used to express the modified human cancer cells described herein. Exemplary methods include, but are not limited to, cationic lipids, such as liposomes and lipid complexes, polymers or polymer complexes and dendritic polymers, naked plasmids for direct delivery, electroporation, sonication and microbubbling, magnetic transfection, and inorganic molecules.

[0125] In one aspect, this disclosure provides expression vectors for modified human cancer cells as described herein. The vector may contain one or more recombinant polynucleotides encoding at least one PTM enzyme, a variant thereof, or a fragment thereof listed in any of Tables 1-10. The recombinant polynucleotide may also contain at least one HLA-A1 allele and at least one HLA-AII allele, a variant thereof, or a fragment thereof. The recombinant polynucleotide may also contain at least one co-stimulatory molecule. In some embodiments, the recombinant polynucleotide further contains a sequence encoding one or more cytokines (e.g., GM-CSF, IFN-α2 including IFN-α2a and IFN-α2b). In some embodiments, the recombinant polynucleotides encoding PTM enzymes, HLA-A class I and HLA-A class II alleles, co-stimulatory molecules, antigens, and / or cytokines each have a sequence that has at least 70%, 75%, 80%, 85%, 90%, 95%, or 99% higher identity or similarity to their corresponding wild-type sequences; each has a sequence that has at most 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity or similarity to their corresponding wild-type sequences; or each has a sequence that has about 10% to 99%, about 30% to 80%, about 40% to 95%, or about 60% to 85% identity or similarity to their corresponding wild-type sequences.

[0126] In various embodiments, the expression vector comprises one or more recombinant polynucleotides encoding one or more promoters for driving the expression of a selected PTM enzyme, HLA allele, co-stimulatory molecule, or adjuvant. The expression vector may contain a recombinant polynucleotide encoding the MNDU3 promoter, the EF1α promoter, or both. For example, the expression vector may contain a first recombinant polynucleotide encoding the MNDU3 promoter and a second recombinant polynucleotide encoding the EF1α promoter. In a particular embodiment, the expression vector contains recombinant polynucleotides encoding both the MNDU3 promoter and the EF1α promoter.

[0127] In some embodiments, the expression vector comprises one or more recombinant polynucleotides encoding at least one PTM enzyme, said at least one PTM enzyme including, but not limited to, citrullinated lactamases, cysteine ​​lactamases, acetyltransferases, hydroxylases, phosphorylases, methyltransferases, formylates, oxidases, hydroxylases, ubiquitinases, variants thereof, fragments thereof, or combinations thereof. In some embodiments, the expression vector comprises one or more recombinant polynucleotides encoding at least one PTM enzyme, said at least one PTM enzyme being selected from citrullinated lactamases, cysteine ​​lactamases, acetyltransferases, hydroxylases, phosphorylases, methyltransferases, formylates, oxidases, hydroxylases, ubiquitinases, variants thereof, fragments thereof, or combinations thereof.

[0128] In some embodiments, the expression vector comprises one or more recombinant polynucleotides, each encoding at least one immunomodulatory molecule, costimulatory molecule, and / or cytokine (e.g., two or more immunomodulatory molecules, costimulatory molecules, or cytokines). In some embodiments, the at least one immunomodulatory molecule, costimulatory molecule, and / or cytokine (e.g., two or more immunomodulatory molecules, costimulatory molecules, or cytokines) includes, but is not limited to, CSF2, IFN-α, 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, costimulatory molecule, and / or cytokine (e.g., two or more immunomodulatory molecules, costimulatory molecules, or cytokines), wherein the at least one immunomodulatory molecule, costimulatory molecule, and / or cytokine is selected from CSF2 and IFN-α; CSF2; CD86 and IL-12; CD40, CD80, and HLA-DRA alleles; or IL-7 and 4-1BBL.

[0129] In some embodiments, the expression vector contains one or more recombinant polynucleotides, each encoding at least one HLA class I allele, including but not limited to HLA-A. 01:01 Alleles, HLA-A 68:01 allele, HLA-A 02:01 Alleles, HLA-A 11:01 alleles, HLA-A 03:01 Alleles, HLA-A 23:01 alleles, HLA-A 24:02 alleles and / or HLA-A 33:03 alleles. In some embodiments, the expression vector contains one or more recombinant polynucleotides, each encoding at least one HLA class I allele selected from HLA-A. 01:01 Alleles and HLA-A 68:01 allele, HLA-A 02:01 Alleles and HLA-A 11:01 alleles, HLA-A 03:01 Alleles and HLA-A 23:01 allele, and / or HLA-A 24:02 alleles and HLA-A 33:03 alleles.

[0130] In some embodiments, the expression vector contains one or more recombinant polynucleotides, each encoding at least one HLA class II allele, including but not limited to HLA-DRB3. 02:02 allele, HLA-DRB5 01:01 allele, HLA-DRB4 01:01 allele, HLA-DRB3 01:01 allele, HLA-DRB3 03:01 Alleles, HLA-DRB5 01:02 Alleles and / or HLA-DRB5 02:02 alleles. In some embodiments, the expression vector contains one or more recombinant polynucleotides, each encoding at least one HLA class II allele selected from HLA-DRB3. 02:02 alleles and HLA-DRB5 01:01 allele, HLA-DRB4 01:01 Alleles and HLA-DRB3 01:01 Alleles, HLA-DRB3 03:01 Alleles and HLA-DRB5 01:02 alleles, and / or HLA-DRB5 02:02 alleles and HLA-DRB3 01:01 allele.

[0131] In some embodiments, the expression vector is capable of expressing at least one PTM enzyme, an immunomodulatory molecule, a co-stimulatory molecule or cytokine, at least one HLA class I allele, and / or 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 alleles 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 alleles 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.

[0132] In some implementations, the expression vector also contains recombinant polynucleotides encoding cytokines, chemokines, interferons, interleukins, and / or tumor necrosis factor. In some embodiments, the recombinant polynucleotide encodes one of a cytokine selected from at least one of the following: early T-cell activation antigen-1 (ETA-1), lymphocyte activating factor (LAF), interleukin-1 family members (IL-1α, IL-β, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36γ, IL-37, IL-38), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), and interleukin-17. Interleukin-17 (IL-18), Interleukin-21 (IL-21), Interleukin-23 (IL-23), Interleukin-25 (IL-25), Interleukin-33 (IL-33), Interferon-α (IFN-α), Interferon-λ1 (IFN-λ1 (IL-29)), Interferon-λ2 (IFN-λ2 (IL-28A)), Interferon-λ3 (IFN-λ3 (IL-28B)), Interferon-λ4 (IFN-λ4), Granulocyte-macrophage colony-stimulating factor (GM-CSF), Macrophage CSF (CSF-1), Macrophage migration inhibitory factor (MIF), CD40L molecule (CD40L), RANTES molecule (RANTES), Monocyte chemotactic protein (MCP-1), Monocyte inflammatory protein (MIP-1α, MIP-1β), Lymphocyte chemotactic factor and / or fractal chemotactic factor. In some embodiments, the cytokine includes GM-CSF. In some embodiments, the cytokine includes IFN-α, such as, for example, IFN-α2a or IFN-α2b.

[0133] In some embodiments, the expression vector further comprises a recombinant polynucleotide encoding a co-stimulatory molecule selected from at least one of the following: CD86 molecule (CD86), CD80 molecule (CD80), 4-1BB ligand molecule (4-1BBL, also known as CD137L), ICOS ligand molecule (ICOS-L), CD70 molecule (CD70, also known as CD27L), CD40 molecule (CD40), OX40 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 / or combinations thereof.

[0134] In some implementations, the expression vector also includes a recombinant polynucleotide encoding an antigen (e.g., an antigen of a pathogen, a tumor-specific antigen, a tumor-associated antigen, a neoantigen, an allergen, an antigen that is a target of an autoimmune response, or a fragment thereof).

[0135] In some embodiments, the expression vector comprises one or more recombinant polynucleotides encoding: a) an enzyme that induces PTM of an antigen in cells; b) an 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 targeting an antigen in cells further comprises one or more recombinant polynucleotides encoding: a) an 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.

[0136] D. Composition

[0137] In one aspect, this disclosure provides compositions comprising human cancer cells containing the modifications described herein. In some embodiments, the modified human cancer cells comprise recombinant polynucleotides encoding an enzyme that induces post-translational modification (PTM) of antigens in the cell. In some embodiments, the modified human cancer cells comprise recombinant polynucleotides encoding antigens. In some embodiments, the modified human cancer cells comprise (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 cells comprise recombinant polynucleotides encoding cytokines. In some embodiments, the modified human cancer cells comprise recombinant polynucleotides encoding co-stimulatory molecules.

[0138] In one aspect, this disclosure provides compositions comprising modified human cancer cells as described herein, said modified human cancer cells comprising (A) one or more vectors each comprising a recombinant polynucleotide encoding at least one PTM enzyme selected from citrullinated nucleotides, cysteine ​​nucleotides, acetyltransferases, hydroxylases, phosphorylases, methyltransferases, formylates, oxidases, hydroxylases, ubiquitinases, and / or combinations thereof; (B) one or more vectors each comprising a recombinant polynucleotide encoding at least one gene selected from CSF2, IFN-α2, 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 gene and / or (ii) one or more vectors each comprising a recombinant polynucleotide encoding at least one HLA class II gene. In some embodiments, one or more endogenous HLA alleles of the human cancer cells have been inactivated.

[0139] 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, The composition contains 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 contains at least 1,000,000 cells. In some embodiments, the composition contains at least 20,000,000 cells.

[0140] In some embodiments, the composition comprises up to 10,000 cells, up to 100,000 cells, up to 1,000,000 cells, up to 1,250,000 cells, up to 1,500,000 cells, up to 2,000,000 cells, up to 2,500,000 cells, up to 3,000,000 cells, up to 3,500,000 cells, up to 4,000,000 cells, and up to 4,500,000 cells. The composition may contain up to 5,000,000 cells, up to 10,000,000 cells, up to 12,500,000 cells, up to 15,000,000 cells, up to 20,000,000 cells, up to 25,000,000 cells, up to 30,000,000 cells, up to 35,000,000 cells, up to 40,000,000 cells, up to 45,000,000 cells, or up to 50,000,000 cells. In some embodiments, the composition comprises up to 20,000,000 cells. In some embodiments, the composition comprises up to 40,000,000 cells.

[0141] 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.

[0142] On the other hand, this disclosure provides pharmaceutical compositions. 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 modified human cancer cells or cell lines containing at least 1, 2, 3, 4, 5, or more recombinant polynucleotides encoding at least one PTM enzyme selected from citrullinated chemiluminases, cysteine ​​chemiluminases, acetyltransferases, hydroxylases, phosphorylases, methyltransferases, formylates, oxidases, hydroxylases, ubiquitinases, and / or combinations thereof. In some embodiments, the PTM enzyme comprises a citrullinated chemiluminase. In some embodiments, the PTM enzyme comprises a cysteine ​​chemiluminase. Furthermore, the modified human cancer cells or cell lines may comprise one or more co-stimulatory molecules, antigens, and / or cytokines described herein. The at least 1, 2, 3, 4, 5, or more recombinant polynucleotides may comprise heterologous sequences encoding, for example, co-stimulatory molecules, antigens, cytokines, or 2A splice peptides. Therefore, recombinant polynucleotides encoding one or more HLA alleles, co-stimulatory molecules, antigens, and / or cytokines can be isolated by sequencing encoding 2A splice peptides (e.g., T2A, P2A, E2A). Typically, at least 1, 2, 3, 4, 5, or more recombinant polynucleotides are cloned into expression vectors (e.g., replication-defective lentiviral vectors) for the synthesis of PTM enzymes, HLA alleles, co-stimulatory molecules, antigens, and / or cytokines, and introduced into modified human cancer cells or cell lines. Thus, modified human cancer cells or cell lines provided in pharmaceutical compositions can have at least 1, 2, 3, 4, 5, or more expression vectors, each containing at least 1, 2, 3, 4, 5, or more recombinant polynucleotides encoding PTM enzymes, HLA alleles, co-stimulatory molecules, antigens, and / or cytokines.

[0143] In some embodiments, the pharmaceutical composition further comprises a cryoprotectant, interferon α (e.g., IFN-α2a or IFN-α2b), and / or a member of the interferon λ family (e.g., interferon λ1 (IFN-λ1 (IL-29)), interferon λ2 (IFN-λ2 (IL-28A)), interferon λ3 (IFN-λ3 (IL-28B)), interferon λ4 (IFN-λ4)). In some embodiments, interferon α (e.g., IFN-α2a or IFN-α2b) is composed of a cryoprotectant, interferon α (e.g., IFN-α2a or IFN-α2b), and / or a member of the interferon λ family (e.g., interferon λ1 (IFN-λ1 (IL-29)), interferon λ2 (IFN-λ2 (IL-28A)), interferon λ3 (IFN-λ3 (IL-28B)), or interferon λ4 (IFN-λ4)). IFNA2Vectors carrying the polynucleotide sequence of the gene are expressed in cancer cells modified as described herein. In some embodiments, interferon α is exogenously provided PEGylated IFN-α2a. 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 cryopreservation medium. In specific embodiments, the pharmaceutical composition comprises cells cryopreserved in CryoStor CS10, CryoStor CS2, or CryoStor CS5 cryopreservation medium.

[0144] In some embodiments, the pharmaceutical composition is formulated as a dosage form comprising a total number of modified cancer cells per dose for administration to a subject in need. In some embodiments, the pharmaceutical composition is formulated as an "off-the-shelf" product for self-administration to a subject in need. In some embodiments, the pharmaceutical composition may have 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, or at least 4,500,000 cells. 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.

[0145] In some embodiments, the pharmaceutical composition comprises up to 10,000 cells, up to 100,000 cells, up to 1,000,000 cells, up to 1,250,000 cells, up to 1,500,000 cells, up to 2,000,000 cells, up to 2,500,000 cells, up to 3,000,000 cells, up to 3,500,000 cells, up to 4,000,000 cells, or up to 4,500,000 cells. The pharmaceutical composition may contain up to 5,000,000 cells, up to 10,000,000 cells, up to 12,500,000 cells, up to 15,000,000 cells, up to 20,000,000 cells, up to 25,000,000 cells, up to 30,000,000 cells, up to 35,000,000 cells, up to 40,000,000 cells, or up to 50,000,000 cells. In some embodiments, the pharmaceutical composition comprises up to 20,000,000 cells. In some embodiments, the pharmaceutical composition comprises up to 40,000,000 cells.

[0146] 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.

[0147] In some embodiments, the pharmaceutical composition is formulated as a suspension. The formulations of the pharmaceutical composition are generally known in the art (see, for example, Remington’s Pharmaceutical Sciences (18th Ed., Mack Publishing Co., Easton, PA (1990)). Microbial contamination can be prevented by adding one or more of various antibacterial and antifungal agents. In a particular embodiment, the pharmaceutical composition is a liquid formulation comprising cells resuspended in lactated Ringer's solution.

[0148] Suitable drug forms for administration include sterile aqueous solutions or dispersions and sterile powders for immediate preparation of sterile injectable solutions or dispersions. Typical carriers include solvents or dispersion media comprising, for example, aqueous solutions buffered with water (i.e., biocompatible buffers, non-limiting examples of which include lactated Ringer's solution and CryoStor cryopreservation media (e.g., CS2, CS5, and CS10, which contain 2%, 5%, and 10% DMSO, respectively); which are available from BioLifeSolutions, Bothell, WA), ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol), suitable mixtures thereof, surfactants, or vegetable oils.

[0149] Sterilization can be achieved through recognized techniques, including but not limited to the addition of antibacterial or antifungal agents, such as parabens, chlorobutanol, sorbic acid, or thimerosal. Additionally, isotonic agents, such as sugars or sodium chloride, can be incorporated into the subject composition.

[0150] As needed, sterile injectable solutions containing modified cancer cells and / or other compositions of this disclosure can be produced by incorporating the required amounts of the compound into a suitable solvent having the various components listed above, followed by sterilization. To obtain a sterile powder, the above-mentioned sterile solution can be vacuum-dried or freeze-dried as needed.

[0151] In some embodiments, the modified cancer cells and / or other compositions provided herein are formulated for administration in unit dosage forms, such as intradermal injection, intralymphatic injection, oral, nasal, topical, or parenteral administration, to facilitate administration and uniform dosing. As used herein, a unit dosage form refers to a physically discrete unit suitable as a single dose for a subject (e.g., a human or other mammal to be treated), each unit containing a predetermined amount of active substance calculated to produce the desired therapeutic effect, bound to a desired drug carrier. In some cases, a more concentrated dosage form can be prepared, from which more dilute unit dosage forms can then be derived. Thus, a more concentrated dosage form will substantially contain amounts of modified cancer cells and / or other compositions greater than, for example, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times.

[0152] In some embodiments, the modified cancer cells and / or other compositions provided herein are formulated for administration over a period of time, e.g., one or more doses. In some embodiments, the modified cancer cells and / or other compositions are formulated for administration once weekly, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, the modified cancer cells and / or other compositions are formulated for administration once monthly, 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.

[0153] Dosage may include, for example, approximately 50,000 to 50,000,000 (e.g., approximately 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). 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 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 The dose may contain 0, 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, the dose may contain about 1,000,000 modified human cancer cells. In some embodiments, the dose may contain about 5,000,000 modified human cancer cells. In some embodiments, the dose may contain about 10,000,000 modified human cancer cells. In some embodiments, the dose may contain about 20,000,000 modified human cancer cells.

[0154] The dosage 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). The dose may contain at least about 1,000,000 modified human cancer cells. In some embodiments, the dose may contain at least about 5,000,000 modified human cancer cells. In some embodiments, the dose may contain at least about 10,000,000 modified human cancer cells. In some embodiments, the dose may contain at least about 20,000,000 modified human cancer cells.

[0155] Optionally, the dosage may include, for example, at least about 100,000,000 to 1,000,000,000. (For example, approximately 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.

[0156] In some embodiments, the modified human cancer cells are non-replicating. In some embodiments, the modified human cancer cells are rendered non-replicating by irradiation, freeze-thaw cycles, or treatment with mitomycin C. In some cases, the modified human cancer cells are irradiated. As disclosed herein, irradiation can be used to trigger non-enzymatic PTM and disrupt cellular replication. The irradiation dose can, for example, be about 2 to 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 Gy). The doses are 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 non-replicating by freeze-thaw cycles. In some embodiments, the modified human cancer cells are rendered non-replicating by treatment with mitomycin C.

[0157] The methods used to prepare such dosage forms are known to those skilled in the art (see, for example, REMINGTON’S PHARMACEUTICAL SCIENCES (Ibid.). The dosage form typically contains conventional drug carriers or excipients, and may additionally contain other agents, carriers, adjuvants, diluents, tissue penetration enhancers, solubilizers, etc. Suitable excipients can be tailored to specific dosage forms and routes of administration using methods known in the art (e.g., see [link to relevant documentation]). REMINGTON’S PHARMACEUTICAL SCIENCES (Same as above).

[0158] Examples of suitable excipients include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth gum, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, brine, syrup, methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, and polyacrylic acid, such as carbopol, e.g., Carbopol 941, Carbopol 980, Carbopol 981, etc. The dosage form may further include lubricants such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifiers; suspending agents; preservatives such as methylparaben, ethylparaben, and propylparaben (i.e., paraben esters); pH adjusters such as inorganic acids, organic acids, inorganic bases, and organic bases; sweeteners; and flavoring agents. The dosage form may also contain biodegradable polymer beads, dextran, and cyclodextrin inclusion complexes.

[0159] In some embodiments, the pharmaceutical composition for administration may be an oral delivery medium, such as capsules, pouches, or tablets, each containing a predetermined amount of the composition to provide the patient with the correct incremental dose. For example, an oral delivery medium may be used to avoid contact between the composition and the mouth and upper digestive tract. For oral administration, the therapeutically effective dose may 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-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, gelatin, sucrose, magnesium carbonate, etc.

[0160] In some implementations, the therapeutically effective dose is in the form of pills, tablets, or capsules. Therefore, in addition to the modified cancer cells and / or other compositions described herein, the dosage form may also contain any of the following: diluents, such as lactose, sucrose, dicalcium phosphate, etc.; disintegrants, such as starch or its derivatives; lubricants, such as magnesium stearate; binders, such as starch, gum arabic, polyvinylpyrrolidone, gelatin, cellulose, and their derivatives.

[0161] In some embodiments, a suitable carrier masks the composition, such as modified cancer cells and / or other compositions, from the oral cavity and upper gastrointestinal tract (GI) and reduces or prevents local itching / swelling reactions in these areas during application. For example, the carrier may contain one or more lipid, polysaccharide, or protein components. In some cases, the carrier is a food product.

[0162] For topical application, the therapeutically effective dose may be in the form of emulsions, lotions, gels, foams, creams, gels, solutions, suspensions, ointments, and transdermal patches. For inhalation administration, the modified cancer cells and / or other compositions described herein may be delivered in dry powder or liquid form via a nebulizer. Aerosol formulations may be placed in a pressurizable propellant, such as dichlorodifluoromethane. For parenteral administration, the therapeutically effective dose may be in the form of sterile injectable solutions and sterile packaged powders. Preferably, the injectable solutions are formulated at a pH of about 4.5 to about 7.5.

[0163] Therapeutic doses can also be provided in lyophilized form. Such dosage forms may include a buffer solution, such as bicarbonate, for reconstitution before administration, or said buffer solution may be included in a lyophilized dosage form for reconstitution, such as with water. The lyophilized dosage form may also contain a suitable vasoconstrictor, such as epinephrine. The lyophilized dosage form may be provided in a syringe, optionally packaged in combination with a buffer solution for reconstitution, so that the reconstituted dosage form can be immediately administered to an individual.

[0164] In some implementations, the therapeutically effective dose may also include other components, such as anti-allergy medications, including antihistamines, steroids, bronchodilators, leukotriene stabilizers, and mast cell stabilizers. Suitable anti-allergy medications are well known in the art.

[0165] E. Methods of treating cancer

[0166] On the other hand, this disclosure provides a method for treating cancer in a subject. In some embodiments, the method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition of this disclosure (e.g., a pharmaceutical composition comprising modified cancer cells of this disclosure).

[0167] In some implementations, the method further includes: (i) obtaining a sample from the subject prior to the application step; (ii) identifying a prevalent PTM in the cell sample; and (iii) selecting a modified human cancer cell for application to the subject, wherein the modified human cancer cell contains a prevalent PTM.

[0168] In some examples, the ubiquitous PTM is an enzymatic PTM, wherein the modified human cancer cells contain a recombinant polynucleotide encoding an enzyme that induces the ubiquitous PTM. For example, when a subject is identified as having a ubiquitous citrullinated PTM in one or more immunogenic antigens, modified human cancer cells expressing citrullinated lipases as described herein can be administered to the subject to induce a robust antigen-specific immune response.

[0169] In other examples, the ubiquitous PTM is a non-enzymatic PTM, wherein the modified human cancer cells induce the ubiquitous PTM via a non-enzymatic method. In some embodiments, the non-enzymatic method includes irradiation, induction of cell senescence, chemical reactions, small molecules, cell culture supplements, cell culture media, oxidative stress, or a combination thereof. For example, when a subject is identified as having a ubiquitous cysteine-modified PTM in one or more immunogenic antigens, the modified human cancer cells described herein can be administered to the subject, and non-enzymatic cysteine ​​modification can be induced by culturing the modified human cancer cells with cysteine ​​(e.g., excess cysteine ​​in cell culture media) to induce a robust antigen-specific immune response. As another example, when a subject is identified as having a ubiquitous cysteine-modified PTM in one or more immunogenic antigens, the modified human cancer cells expressing or overexpressing a protein disulfide isomerase (PDI) described herein can be administered to the subject to induce a robust antigen-specific immune response.

[0170] In some embodiments, the sample is a tumor biopsy or a liquid biopsy. In some embodiments, the liquid biopsy includes circulating tumor cells (CTCs), circulating tumor DNA (ctDNA or cell-free DNA), circulating RNA (cfRNA), exosomes, or a combination thereof. In some embodiments, the identifying steps include next-generation sequencing (NGS) or immunopeptidome analysis of the sample. In some embodiments, the subject has breast cancer, prostate cancer, melanoma, or lung cancer.

[0171] In some embodiments, the method includes administering an effective amount of the drug composition intradermally to the subject's upper back or thigh. Without being bound by any theoretical constraints, the upper back and thigh are chosen for patient acceptability because these areas of the skin have fewer nerves and are therefore less sensitive. Furthermore, adjacent draining lymph nodes may transmit antigens from breast tumors in the upper and lower trunks, which are common sites of breast cancer metastasis. The method may also include administering the drug composition to the subject at intervals of weekly, every 2 weeks, every 3 weeks, every 4 weeks, every 5 weeks, or every 6 weeks. In some embodiments, the method includes administering the drug composition to the subject at intervals of monthly, 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 includes administering the drug composition to the subject for at least 6 weeks, 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks, 52 weeks, or longer. In some embodiments, the method includes administering the pharmaceutical composition to a subject for a duration of 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 includes administering the pharmaceutical composition to a subject for a duration of no more than 6 weeks, 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks, or 52 weeks. In some embodiments, the method includes administering the pharmaceutical composition to a subject for a duration of no more than 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, 10 months, or 12 months.

[0172] In some embodiments, the method includes administering an effective amount of the pharmaceutical composition to a subject by means of: oral administration, local contact, administration as a suppository, intravenous administration, intraperitoneal administration, intramuscular administration, intralesional administration, intratumoral administration, intradermal administration, intralymphatic administration, intrathecal administration, intranasal administration, or subcutaneous administration. In some embodiments, the effective amount of the pharmaceutical composition is administered via parenteral administration (e.g., intravenous administration, intramuscular administration, intra-arterial administration, intradermal administration, subcutaneous administration, intraperitoneal administration, intraventricular administration, and intracranial administration) or transmucosal administration (e.g., buccal, sublingual, palatal, gingival, nasal, vaginal, rectal, or percutaneous administration). In some embodiments, the method includes using a liposomal formulation, intravenous infusion, or transdermal patch.

[0173] In some embodiments, the method further includes intravenous administration of one or more doses of cyclophosphamide to a subject for at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, or longer prior to administration of the pharmaceutical composition described herein. In some embodiments, cyclophosphamide is administered to the subject for at least about 2-3 days prior to administration of the pharmaceutical composition described herein. In some embodiments, about 100, 150, 200, 250, 300, or 450 mg / m² is administered to the subject. 2 Low-dose cyclophosphamide.

[0174] In some embodiments, the method further includes intradermal administration of one or more doses of interferon-α-2b (IFN-α2b), IFN-α2a, or PEGylated IFN-α2a to a subject at the injection site of the pharmaceutical composition described herein. In some embodiments, the method further includes intradermal administration of one or more doses of IFN-α2b, IFN-α2a, or PEGylated IFN-α2a to a subject no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 24, 48, 60, 72, or 84 hours after administration of the pharmaceutical composition described herein. In some embodiments, the method further includes intradermal administration of one or more doses of IFN-α2b, IFN-α2a, or PEGylated IFN-α2a to a subject 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 after administration of the pharmaceutical composition described herein. In some embodiments, the method further includes intradermal administration of one or more doses of IFN-α2b, IFN-α2a, or PEGylated IFN-α2a to a subject no later than 5, 10, 15, 20, 25, 30, 45, 50, 60, 72, or 84 hours after administration of the pharmaceutical composition to the subject. In some embodiments, the method further includes intradermal administration of one or more doses of IFN-α2b, IFN-α2a, or PEGylated IFN-α2a to a subject no later than 1, 2, 3, 4, 5, or 6 days after administration of the pharmaceutical composition to the subject. In some embodiments, the method further includes intradermal administration of one or more doses of IFN-α2b, IFN-α2a, or PEGylated IFN-α2a to a subject no later than about 1-6 days, 2-3 days, or 3-5 days after administration of the pharmaceutical composition to the subject. In some embodiments, the method further includes intradermal administration of a first dose of IFN-α2b, IFN-α2a, or PEGylated IFN-α2a to a subject within 1 to 4 hours after administration of the pharmaceutical composition, and intradermal administration of a second dose of IFN-α2b, IFN-α2a, or PEGylated IFN-α2a to a subject 1 to 3 days after administration of the pharmaceutical composition. In some embodiments, the administered IFN-α2b is a low dose of about 1-20,000 IU, 100-15,000 IU, 5,000-12,000 IU, or 9,000-11,000 IU. In some embodiments, the administered dose of IFN-α2b is about 10,000 IU. In some embodiments, the applied IFN-α2a or PEGylated IFN-α2a is in low doses of 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 applied IFN-α2b is in doses of about 0.1 mcg.

[0175] In some embodiments, the method further includes administering one or more additional treatments to the subject. Examples of suitable additional types include, but are not limited to, chemotherapy, immunotherapy, radiation therapy, hormone therapy, differentiating agents, and small molecule drugs. Those skilled in the art will be able to readily select appropriate additional treatments.

[0176] Chemotherapeutic agents that can be used in this disclosure include, but are not limited to, alkylating agents (e.g., nitrogen mustards (e.g., nitrogen mustard, chlorambucil, cyclophosphamide, ifosfamide, melphalan), nitrosoureas (e.g., streptozotocin, carmustine (BCNU), lomustine), alkyl sulfonates (e.g., busulfan), and triazines (e.g., dacarbazine). DTIC), temozolomide), ethyleneimines (e.g., thiotepa, hexamethylmelamine), platinum-based drugs (e.g., cisplatin, carboplatin, oxaliplatin), antimetabolites (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, fluorouridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed), anthracycline antitumor antibiotics (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin), non-anthracycline antitumor antibiotics (e.g., thiorubicin, doxorubicin, epirubicin, idarubicin), non-anthracycline antitumor antibiotics (e.g., thiorubicin, temozolomide, ethyleneimine, thiotepa, hexamethylmelamine, thiotepa), platinum-based drugs (e.g., cisplatin, carboplatin, oxaliplatin), antimetabolites (e.g., 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed), anthracycline antitumor antibiotics (e.g., daunorubicin, doxorubicin, epirubicin, idarubicin), non-anthracycline antitumor antibiotics (e.g., thiotepa, hexamethylmelamine, oxorubicin, temozolomide, thiotepa, hexamethylmelamine, oxorubicin, temozolomide, thiotepa, oxorubic Actinomycin-D, bleomycin, mitomycin-C, mitoxantrone), mitotic inhibitors (e.g., taxanes (e.g., paclitaxel, docetaxel), epothilones (e.g., ixabepilone), vinblastine alkaloids (e.g., vincristine, vinorelbine), estramustine, corticosteroids (e.g., prednisone, methylprednisolone, dexamethasone), L-asparaginase, bortezomib, and topoisomerase inhibitors. Combinations of chemotherapeutic agents can be used.

[0177] Topoisomerase inhibitors are compounds that inhibit the activity of topoisomerases, enzymes that promote changes in DNA structure by catalyzing the breaking and rejoining of phosphodiester bonds in the DNA backbone. These changes in DNA structure are essential for DNA replication during the normal cell cycle. Topoisomerase inhibitors inhibit DNA joining during the cell cycle, leading to an increase in the number of single-strand and double-strand breaks, thereby causing a degradation of genome stability. This degradation of genome stability results in apoptosis and cell death.

[0178] Topoisomerases are generally classified into type I and type II topoisomerases. Type I topoisomerases are crucial for the relaxation of DNA supercoiling during DNA replication and transcription. Type I topoisomerases produce single-strand breaks in DNA and also rejoin those breaks to rebuild complete double-stranded DNA molecules. Examples of inhibitors of type I topoisomerases include irinotecan, topotecan, camptothecin, and lamellarin D, all of which target type IB topoisomerases.

[0179] Type II topoisomerase inhibitors are broadly classified into topoisomerase toxins and topoisomerase inhibitors. Topoisomerase toxins target the topoisomerase-DNA complex, while topoisomerase inhibitors disrupt the enzyme's catalytic conversion. Examples of type II topoisomerase inhibitors include amsacrine, etoposide, etoposide phosphate, teniposide, doxorubicin, and fluoroquinolones.

[0180] In some embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. In some examples, the topoisomerase inhibitor is a combination of topoisomerase I inhibitors, topoisomerase II inhibitors, or more thereof. In specific embodiments, the topoisomerase inhibitor is selected from doxorubicin, etoposide, teniposide, daunorubicin, mitoxantrone, acridine, roserine, ginsenoside tricarboxylic acid, HU-331, irinotecan, topotecan, camptothecin, spirotinic acid D, resveratrol, genistein, quercetin, epigallocatechin gallate (EGCG), or a combination thereof. EGCG is an example of a plant-derived natural phenol as a suitable topoisomerase inhibitor. In some examples, the topoisomerase inhibitor is doxorubicin.

[0181] Immunotherapy is any treatment that uses a subject's immune system to fight a disease, such as cancer. Immunotherapy methods can target either enhancing or suppressing immune function. In the context of cancer treatment, immunotherapy methods typically target enhancing or activating immune function. In some examples, immunotherapeutic agents include monoclonal antibodies that target a specific type or portion of cancer cells. In some cases, antibodies are partially conjugated to substances such as drug molecules or radioactive materials. As a non-limiting example, antibodies can be derived from mice, chimeric, or humanized antibodies. Non-limiting examples of therapeutic monoclonal antibodies include alemtuzumab, bevacizumab, cetuximab, daratumumab, ipilimumab (MDX-101), nivolumab, ofatumumab, panitumumab, pembrolizumab, relefanliumab, rituximab, tositumomab, and trastuzumab.

[0182] Immunotherapy agents can also include immune checkpoint inhibitors, which modulate the immune system's ability to distinguish between normal cells and "foreign" cells. Programmed cell death protein 1 (PD-1) and protein death ligand 1 (PD-L1) are common targets of immune checkpoint inhibitors because disruption of the interaction between PD1 and PD-L1 enhances 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.

[0183] Another immune checkpoint target used to treat cancer is cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), a receptor that downregulates immune cell responses. Therefore, drugs that inhibit CTLA-4 can enhance immune function. An example of such a drug is ipilimumab, a monoclonal antibody that binds to and inhibits CTLA-4.

[0184] The term "radiotherapy" refers to the delivery of high-energy radiation to a subject to treat a disease, such as cancer. Radiation therapy can include the delivery of X-rays, gamma rays, and / or charged particles. Radiation therapy can be delivered locally (e.g., at the site or area of ​​a tumor) or systemically (e.g., by systemic administration of radioactive materials, such as radioactive iodine, to the tumor site).

[0185] The term "hormone therapy" can refer to hormone synthesis inhibitors, hormone receptor antagonists, or hormone supplements. Hormone synthesis inhibitors 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 anti-androgen drugs. Hormone supplements include, but are not limited to, progestins, 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 commonly used to treat breast cancer include tamoxifen, raloxifene, toremifene, and fulvestrant. Anti-androgen drugs that bind to and inhibit androgen receptors are commonly used to suppress the growth and survival effects of testosterone in prostate cancer. Non-limiting examples include flutamide, apalutamide, and bicalutamide.

[0186] The term "differentiation agent" refers to any substance that promotes cell differentiation; in the context of cancer, such a substance can encourage malignant cells to exhibit a less stem cell-like state. A non-limiting example of an anti-cancer differentiation agent is retinoic acid.

[0187] Small molecule drugs are typically agents with low molecular weights (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), seliciclib (a cyclin-dependent kinase inhibitor), and epacadostat (an indoleamine 2,3-dioxygenase (IDO1) inhibitor).

[0188] In some embodiments, the method of treating the cancer of this disclosure further includes selecting a whole-cell cancer vaccine for the subject according to the methods of this disclosure described herein. In certain embodiments, the subject has stage I, II, III, and / or IV cancer. In other embodiments, the cancer transitions between stages. In some embodiments, the subject has precancerous lesions. In some embodiments, the subject does not have cancer.

[0189] In some embodiments, the therapeutic target includes inhibiting cancer cell growth, inhibiting cancer cell proliferation, inhibiting cancer cell migration, inhibiting cancer cell invasion, improving or eliminating cancer symptoms, reducing the size (e.g., volume) of cancerous tumors, reducing the number of cancerous tumors, reducing the number of cancer cells, inducing cancer cell necrosis, pyroptosis, metastasis, apoptosis, autophagy, or other cell death, or enhancing the therapeutic effect of the composition or pharmaceutical composition. In some embodiments, the therapeutic target results in increased survival time. In some cases, overall survival is increased. In other examples, disease-free survival is increased. In some examples, progression-free survival is increased. In specific embodiments, the therapeutic target results in tumor volume reduction and / or increased survival time.

[0190] In a specific implementation scheme, the treatment subject enhances the therapeutic effect of anticancer treatment, which is, for example, chemotherapy agents, immunotherapy agents, radiotherapy, hormone therapy, differentiation agents, and / or small molecule drugs.

[0191] Treatment with modified cancer cells, compositions, and pharmaceutical compositions such as those disclosed herein can be administered using routes, dosages, and regimens readily known to those skilled in the art. Administration can be once daily, every two days, every three days, every four days, every five days, every six days, or weekly. Treatment can be administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times, eleven times, twelve times, thirteen times, fourteen times, or more weekly. In some cases, the modified cancer cells, compositions, and / or pharmaceutical compositions of this disclosure are administered as a single dose, co-administered (e.g., administered at a single dose or via a different route, but close in time), or administered separately (e.g., administered at different doses, including via the same or different routes, but spaced about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more hours apart). In cases where multiple doses are administered on the same day, or where a single dose contains one or more components (e.g., administration of modified cancer cells and IFNa separately), administration can occur in one day, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more times.

[0192] In some cases, treatment administration may occur approximately once a week, approximately once every two weeks, approximately once every three weeks, or once a month. In other cases, treatment administration may occur approximately 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 may last approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more; approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more; or longer. At any time during treatment, the treatment plan may be adjusted as needed. For example, depending on the response to the modified cancer cells, composition, or pharmaceutical composition of this disclosure, different vaccines may be selected, one or more additional therapeutic agents or drugs may be selected, or any aspect of the treatment plan may be discontinued. Those skilled in the art will be able to readily make such decisions, for example, by understanding the results of allele profile comparisons, changes in the activity and / or number of immune cells, and / or changes in the presence or level of one or more biomarkers.

[0193] The modified cancer cells, compositions, and pharmaceutical compositions of this disclosure can be administered via any suitable route, including those described herein. In some embodiments, administration is by intradermal or intralymphatic injection. In some embodiments, a whole-cell cancer vaccine (e.g., containing cancer cells modified by this disclosure) is administered separately from interferon alpha (IFNa). In some examples, IFNa is injected locally. IFNa can be given before and / or after vaccination. The timing of separate injections can be at any suitable interval, including those described herein.

[0194] Those skilled in the art will be able to readily administer an appropriate amount of modified cancer cells contained in a specific dose. The dose may contain, 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, 400,000,000). 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, 13,000,0 The dose may contain 0, 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, the dose may contain about 1,000,000 modified cancer cells. In some embodiments, the dose may contain about 5,000,000 modified cancer cells. In some embodiments, the dose may contain about 10,000,000 modified cancer cells. In some embodiments, the dose may contain about 20,000,000 modified cancer cells.

[0195] The dosage 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.

[0196] Optionally, the dosage may contain, for example, at least about 100,000,000 to 1,000,000,000. (For example, approximately 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.

[0197] In some implementations, the modified cancer cells are irradiated. The irradiation dose can be, for example, from about 2 to 2000 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 Gy). The doses are 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 a particular embodiment, the modified cancer cells are irradiated with a dose of approximately 100 Gy.

[0198] In some implementations, the treatment of the subject results in an increase in the presence or level of one or more biomarkers measured or detected in samples obtained from the subject. In certain implementations, the treatment of the subject results in no change in the presence or level of one or more biomarkers.

[0199] In some embodiments, the treatment results in an increase in the activity and / or number of one or more immune cells. In some examples, the increase occurs in one cell type. In other examples, the increase occurs in multiple cell types. In some embodiments, the cells with increased activity levels and / or numbers are selected from peripheral blood mononuclear cells (PBMCs), lymphocytes (e.g., T lymphocytes, B lymphocytes, NK cells), monocytes, dendritic cells, macrophages, myeloid-derived suppressor cells (MDSCs), and combinations thereof. In specific embodiments, the activity level and / or number of immune cells are measured using the methods described herein.

[0200] In some embodiments, an increase in the activity and / or number of immune cells indicates that one or more additional doses of the pharmaceutical composition (e.g., cancer cells containing modifications of this disclosure) should be administered to the subject. In some examples, different vaccines are administered. Those skilled in the art will recognize that, in some examples, an increase in the activity and / or number of immune cells will occur after administration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more doses of the vaccine.

[0201] In some embodiments, samples are obtained from the subject. In other embodiments, samples are obtained from different subjects or groups of subjects. Samples can be used for the purposes of selecting an appropriate cancer vaccine of this disclosure, monitoring response to vaccine treatment, and / or predicting a subject's response to vaccine treatment. For example, samples obtained from different subjects and / or groups of subjects can be used to establish reference ranges to facilitate comparisons as part of the methods of this disclosure. Samples can be obtained at any time, including before and / or after administration of the modified cancer cells, pharmaceutical compositions, and / or other compositions of this disclosure. In some embodiments, samples include whole blood, plasma, serum, cerebrospinal fluid, tissue, saliva, buccal cells, tumor tissue, urine, fluid obtained from pleural effusion, hair, skin, or a combination thereof. Typically, the sample can include any biological fluid. In some examples, the sample includes circulating tumor cells (CTCs). The sample may also consist of a combination of normal cells and cancer cells. In a particular embodiment, the sample includes circulating tumor cells (CTCs). Samples can be obtained, for example, from biopsy, surgical resection, and / or as fine-needle aspiration (FNA). As described in this article, samples can be used to identify, measure or detect HLA alleles, immune cell activity and / or quantity and / or biomarkers.

[0202] In some implementations, the results of measurements of immune cell activity and / or quantity, and / or determination of the presence or level of biomarkers, are recorded in a tangible medium. For example, the results of the measurements (e.g., the level and / or quantity of immune cell activity, the presence or level (e.g., expression) of one or more biomarkers and / or prognosis or diagnosis (e.g., the presence of cancer, prediction of whether a subject will respond to a vaccine, or whether a subject will respond to a vaccine) can be recorded on, for example, paper or electronic media (e.g., audio tape, computer disk, CD, flash drive, etc.).

[0203] In other embodiments, the method further includes the step of providing the patient (i.e., the subject) with the measurement results, prognostic results, and / or diagnostic results and / or treatment results.

[0204] F. Methods for enhancing antigen immunogenicity

[0205] On the other hand, this disclosure provides methods for enhancing the immunogenicity of antigens in cells described herein. In some embodiments, the method includes inducing post-translational modifications (PTMs) of antigens in the cells of this disclosure, wherein the PTMs enhance the immunogenicity of the antigens. In some examples, antigens in the natural environment (e.g., antigens in unmodified cells) do not contain PTMs, wherein antigens in modified human cancer cells contain such PTMs by enzymatic or non-enzymatic methods, resulting in antigens in modified human cancer cells being more immunogenic than antigens in the natural environment. In other examples, antigens in the natural environment (e.g., in unmodified cells) contain PTMs, wherein antigens in modified human cancer cells contain induced PTMs by enzymatic or non-enzymatic methods, resulting in induced PTM antigens in modified human cancer cells being more immunogenic than antigens in the natural environment. In some embodiments, the antigen is a fragment of one or more of the following: a pathogen antigen, a tumor-specific antigen, a tumor-associated antigen, a neoantigen, an allergen, an antigen that is a target of an autoimmune response.

[0206] In some embodiments, PTM includes phosphorylation, acetylation, ubiquitination, succinylation, methylation, malonylation, glycosylation, SUMOylation, nitrosylation, glutathioneylation, amidation, hydroxylation, palmitoylation, glutarylation, crotonylation, oxidation, myristylation, sulfation, formylation, citrullination, isoprenelation, cysteineation, deamidation, dehydration, or combinations thereof. In specific embodiments, PTM includes cysteineation and / or citrullination.

[0207] In some embodiments, the cells are modified human cancer cells comprising at least one recombinant polynucleotide encoding one or more antigens. In some embodiments, the cells are modified human cancer cells 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 cells are modified human cancer cells comprising one or more recombinant polynucleotides encoding a cytokine, a variant thereof, or a fragment thereof. In some embodiments, the cells are modified human cancer cells comprising one or more recombinant polynucleotides encoding a co-stimulatory molecule, a variant thereof, or a fragment thereof. In some embodiments, the cells are modified human cancer cells comprising at least one recombinant polynucleotide encoding one or more PTM enzymes, a variant thereof, or a fragment thereof. In some embodiments, the cells are modified human cancer cells 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 fragment thereof; and / or e) one or more recombinant polynucleotides encoding a co-stimulatory molecule, a variant thereof, or fragment thereof. In some embodiments, one or more endogenous HLA alleles in the cells have been inactivated in the modified human cancer cells.

[0208] In some embodiments, the cells are human cancer cell lines. In some embodiments, the cells are primary cancer cells. In some embodiments, the cells are breast cancer cells, prostate cancer cells, melanoma cells, or lung cancer cells.

[0209] In some embodiments, PTM is induced by an enzyme. In some embodiments, the enzyme includes citrullinated nucleotides, cysteine ​​nucleotides, acetyltransferases, hydroxylases, phosphorylases, methyltransferases, formylates, oxidases, hydroxylases, ubiquitinases, or combinations thereof. In some embodiments, the enzyme includes citrullinated nucleotides. In some embodiments, the enzyme includes cysteine ​​nucleotides. In some embodiments, the cell contains a recombinant polynucleotide encoding one or more enzymes selected from Tables 1-10.

[0210] In some embodiments, the PTM is a non-enzymatic PTM. In some embodiments, the non-enzymatic PTM is induced by irradiation, induced cellular senescence, chemical reactions, small molecules, cell culture supplements, cell culture media, oxidative stress, or a combination thereof. In some embodiments, the non-enzymatic PTM includes cysteine ​​oxidation. In some embodiments, non-enzymatic cysteine ​​oxidation can be induced by culturing cells with an excess of 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 a serum-free medium. In some embodiments, the non-enzymatic PTM can be induced by inducing cellular senescence. In some embodiments, small molecules, such as senescence inducers, can be used to induce non-enzymatic PTM in the cells disclosed herein. Such senescence inducers include, but are not limited to, doxorubicin (genotoxic), palbociclib (CDK4 / 6 inhibitor), and nutlin-3A (p53 activator).

[0211] G. Package Products

[0212] On the other hand, this disclosure provides kit products for treating cancer subjects. In some embodiments, the kit products comprise modified cancer cell lines, compositions, and / or pharmaceutical compositions as described herein. The kit products can be used to treat any cancer, some of which, as non-limiting examples, 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, kidney cancer, bladder cancer, head and neck cancer, mesothelioma, acute leukemia, chronic leukemia, medulloblastoma, multiple myeloma, sarcoma, and any other cancer described herein, including combinations thereof.

[0213] Materials and reagents for performing the various methods of this disclosure can be provided in kits to facilitate the execution of the methods. As used herein, the term "kit" includes a combination of articles that are easy to handle, measure, analyze, or manipulate. In particular, the kits of this disclosure are found to be useful in a wide range of applications, including, for example, diagnosis, prediction, and treatment.

[0214] The kit may contain chemical reagents and other components. Furthermore, the kit of this disclosure may include, but is not limited to, instructions for users of the kit, apparatus and reagents for sample collection and / or purification, apparatus and reagents for product collection and / or purification, apparatus and reagents for administering the modified cancer cells or other compositions of this disclosure, apparatus and reagents for determining the level of biomarkers and / or the activity and / or quantity of immune cells, apparatus and reagents for detecting PTM, sample tubes, holders, trays, supports, culture dishes, plates, solutions, buffers or other chemical reagents, and suitable samples for standardization, normalization and / or control samples. For ease of storage and safe transport, the kit of this disclosure may also be packaged, for example, in a box with a lid. For example, the kit may be stored and transported at room temperature, on moist ice or in a refrigerated bag, or frozen in liquid nitrogen gas phase or dry ice.

[0215] In some embodiments, the kit product also includes negative and positive control samples for detecting PTM, immune cell activity and / or quantity, and / or the presence or level of biomarkers. In some embodiments, the negative control sample is a non-cancerous cell, tissue, or biological fluid obtained from a subject to be treated or who has undergone treatment. In other embodiments, the negative control sample is obtained from an individual who has never had cancer or a group of individuals. In other embodiments, the positive control sample is obtained from a subject who has cancer or another individual or a group of individuals. In some embodiments, the kit product includes a sample for preparing titration curves for one or more biomarkers in the sample to help evaluate the quantitative levels of the activity and / or quantity of one or more immune cells and / or biomarkers in the biological sample.

[0216] IV. Examples

[0217] This disclosure will be described in more detail by way of specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the invention in any way. Those skilled in the art will readily recognize various non-critical parameters that can be changed or modified to produce substantially the same results.

[0218] Example 1: Post-translational modification of HLA antigens

[0219] This example illustrates an immunopeptidome analysis of SV-BR-1-GM, which detected post-translational modifications (PTMs) in multiple antigens that bind to class I and / or class II HLA molecules.

[0220] To identify and characterize MHC-I and MHC-II related peptides displayed by the proprietary SV-BR-1-GM (a breast cancer cell line that secretes GM-CSF), immunopeptidomic analysis was performed using MHC I or MHC II class-specific sequential immunoaffinity resins after cell lysis and immunoprecipitation (IP), and the eluted peptides were identified by mass spectrometry.

[0221] Materials and methods

[0222] Cell samples were prepared using the protocol shown below. The materials used in the assay are listed in Table 11.

[0223] Sample preparation: 1. Thaw the cell pellet on ice, then lyse at 1 ml lysis buffer per 50 million cells and incubate on ice for 30 minutes.

[0224] 2. Centrifuge the insoluble material at 800×g for 5 minutes to precipitate it.

[0225] 3. Centrifuge the supernatant at 20,000 × g for 30 minutes at 4°C.

[0226] a. Reserve 50 µL of lysis buffer before IP for ELISA.

[0227] 4. Wash the resin and combine it with the clarified pyrolysis product, then gently rotate overnight at 4°C to mix.

[0228] 5. The next day, centrifuge the sample at 800 × g for 5 minutes at 4°C.

[0229] a. Immediately perform sequential MHC Class II IP on the total supernatant (step 11).

[0230] 6. The resin is washed three times (with buffer 1-3), which consists of the following steps: a. Add 2.5 ml of buffer solution to the resin and vortex.

[0231] b. Centrifuge at 325 × g for 5 minutes at 4°C.

[0232] c. Discard the supernatant.

[0233] 7. In Wash #4, add 0.75 ml of Buffer 4 and transfer the total volume to a LoBind tube.

[0234] a. Add an additional 0.75 ml of buffer and centrifuge at 800 × g for 5 minutes at 4°C.

[0235] b. Discard the supernatant.

[0236] 8. Add 1 ml of elution buffer to each tube and incubate at 37°C for 5 minutes.

[0237] 9. Elute the sample by centrifuging at 800 × g for 5 minutes at 4°C.

[0238] 10. Collect the eluent (supernatant) into a new LoBind Eppendorf tube and store at -80°C.

[0239] 11. Then mix the retained flowthrough with 100 µL of L243 agarose and incubate overnight at 4°C with gentle rotation.

[0240] 12. Centrifuge the mixture at 800 xg for 5 minutes, transfer the supernatant to a new tube, and reserve 100 µL for ELISA (after IP).

[0241] 13. Mix the flow-through solution from L243 IP with 100 µL of IVA12 agarose and incubate overnight at 4°C with gentle rotation.

[0242] 14. As described in steps 6-10, wash the resin containing the captured HLA-DR and elute the peptide.

[0243] 15. As described in steps 5-10, wash the IVA12 agarose and elute the peptides.

[0244] 16. All samples are provided for LC-MS / MS analysis.

[0245] Table 11. Materials used for immunopeptidome analysis.

[0246]

[0247] Perform LC-MS / MS analysis using the methods shown below.

[0248] Sample preparation

[0249] Peptides were concentrated and desalted using solid-phase extraction (SPE) on a Waters µHLB C18 plate. The peptides were loaded directly and eluted with 80 / 20 acetonitrile / water (0.1% TFA). The eluted peptides were lyophilized and reconstituted in 0.1% TFA.

[0250] Mass spectrometry analysis

[0251] Peptides (50% per sample) were analyzed by nano LC / MS / MS using a Waters NanoAcquity system connected to a ThermoFisher Fusion Lumos mass spectrometer. Peptides were loaded onto capture columns and eluted at 350 nL / min on a 75 µm analysis column; both columns were packed with Luna C18 resin (Phenomenex). A 2-hour gradient was used. The mass spectrometer was operated using a custom data correlation method, with MS performed in Orbitrap at 60,000 FWHM resolution, and sequential MS / MS performed in Orbitrap at 15,000 FWHM resolution using high-resolution CID and EThCD. All MS data were taken from m / z 300–800. All steps used a 3-second cycle time.

[0252] Data processing

[0253] Search for the original file using a local copy of PEAKS with the following parameters: Enzymes: None Database: SWISSProt Personal Fixed Modifiers: None Variable modifications: oxidation (M), acetyl (N-terminus of protein) Quality value: single isotope Peptide quality tolerance: 10 ppm; Fragmentation quality tolerance: 0.02 Da; Maximum Missed Cleavages: N / A PSM FDR: 1% Chimeric peptide: TRUE Further analysis of the peptide's PTM (PEAKS PTM) and mutations (SPIDER) was performed. The peptide.csv file was exported.

[0254] result

[0255] Based on a positive / decoy database search, a total of 6,932 MHC class I peptides and 5,197 MHC class II peptides were detected at 1% PSM FDR. An overview of the total amount and intensity of MHC class I and II peptides is shown in Tables 12 and 13, respectively.

[0256] Table 12. Total amount and intensity of MHC class I peptides.

[0257]

[0258] Table 13. Total amount and intensity of MHC class II peptides.

[0259]

[0260] In addition, 279 MHC class I peptides and 525 MHC class II peptides were detected by post-translational modification (PTM). Figure 1 Histograms of PTMs in MHC class I and / or class II peptides are shown, indicating that cancer cells have multiple PTM-carrying peptides in their immune peptidomite, and that these post-translational modified peptides are potential targets for immune responses.

[0261] Example 2: Identifying the enzyme to be introduced into cancer cells

[0262] This example illustrates how to determine which enzymes to introduce into cancer cell lines or primary cancer cells.

[0263] In a given tumor type (e.g., breast cancer), ubiquitous PTMs can be found in public databases of immunopeptidomes (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). The Cancer Antigen Atlas (caAtlas, http: / / www.zhang-lab.org / caatlas / ) provides a core resource for the selection and prioritization of MHC-binding peptides for in vitro HLA binding assays and immunogenicity testing, and can be used to characterize post-translational modification antigens and their cancer associations. For example, in a given tumor type (e.g., breast cancer), caAtlas indicates a high proportion of tumors that have undergone acetylation of immunopeptidome peptides. Therefore, acetyltransferases, such as acetyltransferases, can be introduced into specific cancer cell lines.

[0264] Alternatively, public databases of microarray or RNAseq data (see sites.broadinstitute.org / ccle / datasets and www.cancer.gov / ccg / research / genome-sequencing / tcga) can be searched to determine which enzymes that produce PTMs are typically widely expressed in specific types of tumors. For example, in a given tumor type (e.g., lung cancer), a high proportion of tumors expressing high levels of peptidyl arginine deiminase (an enzyme that catalyzes citrullination) found in RNA expression databases indicates that PADs may be good candidates for introduction into specific cancer cell lines.

[0265] Similarly, we can identify prevalent PTMs in an individual cancer patient by evaluating biopsy material, circulating tumor cells, or circulating tumor RNA. If one or more enzymes that produce PTMs (e.g., PADs) are widely expressed in the patient's tumor, we can selectively introduce these enzymes (e.g., PADs) into the cancer cells for cancer therapy. Alternatively, we can examine the prevalent PTMs in the patient's tumor or the tumor's immunopeptidome to determine which enzymes to introduce into the cancer cells for cancer therapy. For example, if an immunopeptidome analysis of biopsy material, circulating tumor cells, or circulating tumor RNA provides evidence that most antigens have undergone citrullination, we can introduce PADs into the cancer cells for cancer therapy.

[0266] Example 3: Generation of cancer cells modified with enzymes that induce PTM

[0267] This example describes how to generate modified cancer cells that express PTMs that induce antigens.

[0268] Once the specific enzymes to introduce into cancer cells are determined, genes encoding these enzymes can be introduced into the cells via transfection, transduction, or other genetic engineering methods. For example, cells can be stably transfected or transduced using genes encoding the PAD enzyme. In one implementation, transduction can be accomplished using lentiviruses.

[0269] Parental cell lines. Any type of human cancer cell line can be used as a parental cell line to generate modified cancer cell lines expressing enzymes that induce PTM. Modified cancer cell lines, such as the Bria-OTS cell line described herein, can also be used as parental cell lines to generate modified cancer cell lines expressing enzymes that induce PTM. The Bria-OTS cell line is derived from the breast cancer parental cell line SV-BR-1, which expresses multiple cancer-associated antigens and immunostimulatory factors, including class II HLA molecules that directly activate CD4+ T cells to enhance the immune response. The generation of the initial SV-BR-1 cell line is described in WO 2017 / 147600, which is incorporated herein by reference in its entirety. The generation of the Bria-OTS cell line is described in WO 2023 / 167973, which is incorporated herein by reference in its entirety.

[0270] To prepare the therapeutic composition described herein, vials from the master cell bank (MCB) can be thawed, grown in a culture medium, harvested, and cryopreserved.

[0271] Lentiviral generation. To generate the lentiviral vector, HEK 293T cells (Clontech Lenti-X 293 cells) were cultured in D10HG medium and transfected with DMEM medium, VSV-G envelope plasmid, packaging plasmid, and vector using the Mirus TRANS-IT 293 transfection reagent. On day 5, the conditioned medium was harvested, centrifuged, and the supernatant was concentrated using a Millipore Centricon Plus 70 PL-100 centrifuge tube and then filtered using a Costar 0.45 Spinx centrifuge filter. The viral titer was determined using the ABM LV 900 Lentivirus Titer Kit with MasterMixR. The lentiviral vector was tested, for example, by determining the titer, sequence, RCL, bioload, and / or endotoxin.

[0272] Lentiviral transduction. Cells can transduce at a rate of 0.29 × 10⁻⁶. 6 Cells were seeded at a density of 100 cells / well in 12-well plates and transduced the next day at 150 MOI. For transduction, the medium could be 1 ml RPMI 1640 + 10% FBS + L-glutamine + 20 mg / mL proteinamine sulfate. Once cells recovered and reattached, they were digested with 4 mL TrypLE Express for 5 minutes. The reaction was terminated with 8 mL of medium. Cells were counted and treated with CS10 at approximately 8 × 10⁶ cells / well. 5 Individual cells / vial cryopreservation.

[0273] Lentivirally transduced cells can be transferred to cultures and amplified. Cells can be further amplified, and single-cell clones can be initiated by seeding 2 cells / well in 96-well plates. Single-cell clones are then transferred to 48-well plates. ELISA is performed to identify positive clones (e.g., enhanced enzyme production), and positive clones are transferred to 12-well plates. For example, PAD-positive clones (enhanced PAD production) will be amplified and then cryopreserved using CS10 cryopreservation medium.

[0274] Master cell banks (MCBs) can be created and tested for each cell line. As an example, cell lines will be propagated in T-25, T-75, and T-150 culture flasks using RPMI medium containing 10% FBS. To confirm the generation of PAD enhancement, randomly selected culture flasks will be incubated with 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 enzymes (e.g., PAD), cells will be subjected to flow cytometry using enzyme-specific antibodies, and RT-PCR will be performed using enzyme-specific primers.

[0275] Example 4: Safety and efficacy of modified human cancer cell lines in cancer patients

[0276] This embodiment illustrates one implementation of testing the safety and efficacy of modified human cancer cell lines (e.g., the Bria-OTS cell line with induced PTM antigen) in patients with advanced metastatic or locally recurrent breast cancer in a clinical trial. This study will provide preliminary data on safety, tolerability, and tumor response in patients with advanced (e.g., metastatic or locally recurrent) breast cancer. A brief overview of the clinical study design is provided below.

[0277] This is a phase 1 / 2a, open-label clinical study in which patients are assigned to receive cell line therapy for breast cancer immunotherapy. The primary objective of this study is to evaluate the safety of cell line therapy in patients with advanced breast cancer. A secondary objective is to evaluate tumor response to cell line therapy in patients with advanced breast cancer. Additionally, this study may include the following objectives: evaluating progression-free survival (PFS) and overall survival (OS) in patients with advanced breast cancer treated with cell line therapy; evaluating the immune response induced by cell line therapy in patients with advanced breast cancer; evaluating patient and tumor characteristics that may predict response to cell line therapy in patients with advanced breast cancer; and / or evaluating quality of life (QOL) in patients with advanced breast cancer treated with cell line therapy. The cell line under investigation is the Bria-OTS cell line with an induced post-translational modification antigen (e.g., the Bria-OTS cell line expressing PAD, which induces citrullination of the antigen). Patients will be treated with the cell line most closely matched to their HLA type; at least one match is required for each patient to be treated.

[0278] In addition, to enhance the immune response, patients were pretreated with a low dose of cyclophosphamide 48–72 hours (2–3 days) prior to each vaccination, which downregulates T regulatory cell mechanisms. A low dose of pegylated interferon-α-2a (IFN-α2a) was administered as an adjuvant, at a dose of 0.1 mcg via intradermal injection at the injection site approximately 1–4 hours and approximately 24–72 hours (1–3 days) post-vaccination. Biosamples were collected periodically according to the protocol and stored in a repository.

[0279] As an example, in Part 1 (Phase 1) of the study, the in-patient dose for each patient treated with a single cell line will be escalated. Once the cell dose is determined in Part 1, in Part 2 (Phase 2a), patients may be treated with one or two cell lines, with the aim of matching patients with cell lines using at least two HLA types, preferably one class II (HLA-DR) type and one class I (HLA-A, HLA-B, or HLA-C) type.

[0280] Patient Population. Patients will be screened to ensure they meet the inclusion criteria. Screening must be conducted within 30 days of starting treatment and imaging studies must be performed within 2 weeks of starting treatment. All patients are women with recurrent and / or metastatic breast cancer, histologically confirmed by the research center, who have failed previous treatment. Patients with any of the four breast cancer subtypes will be eligible: Luminal A (HR+ / HER2-), triple-negative (HR- / HER2-), Luminal B (HR+ / HER2+), and HER2-enriched (HR- / HER2+), provided they meet the criteria for previous treatment failure. As described in the protocol summary, patients with newly diagnosed or progressively metastatic breast cancer to the brain will also be eligible if their intracranial disease is stable and not life-threatening.

[0281] The study plans to enroll up to 48 patients in two parts, with 12–24 patients (at least 3 patients per cell line) for initial evaluation. If the cell lines are found to be safe in the first part, an expanded cohort of 24 patients (at least 4 patients per cell line) will be recruited in the second part.

[0282] The study investigated treatment and dose escalation. Bria-OTS cell lines carrying induced post-translational modification antigens (e.g., Bria-OTS cell lines expressing PAD, which induces citrullination of the antigen) were irradiated to prevent replication before being frozen in viable cryogenic media. They were then transported to a clinical research center for freezing and thawing for inoculation. During the dose escalation phase, patients were initially evaluated weekly, including all safety assessments.

[0283] The dosage form was formulated as a suspension for irradiated cells. A therapeutically effective amount of the composition was administered to the patient over a period of time via intradermal injection into the upper back or thigh. Table 14 shows the dosing regimen for the Phase 1 monotherapy phase.

[0284] Table 14. Dosing regimens during monotherapy phase

[0285] During the monotherapy phase, patients will undergo a safety reassessment and delayed-type hypersensitivity (DTH) measurement 2 ± 1 days after each inoculation. Following the monotherapy phase, patients will receive the maximal dose (i.e., the maximum tolerated dose [MTD] or pharmacologically active dose), which is a safely tolerated cell dose every 3 weeks. During this phase, patients will also receive 300 mg / m² 2–3 days prior to each cell line inoculation. 2Cyclophosphamide was administered, and 0.1 mcg of polyethylene glycol-modified IFN-α was given at each inoculation site 1–4 hours and 1–3 days after each cell line inoculation. The entire cycle was repeated every 3 weeks.

[0286] Once at least three patients have been safely treated with each cell line as a single cell line, Phase 2 (Phase 2a) will begin. During Phase 2, all patients will receive treatment with one or two cell lines, with the aim of matching patients with cell lines using at least two HLA types, preferably one class II (HLA-DR) type and one class I (HLA-A, HLA-B, or HLA-C) type. Two to three days prior to BC cell line inoculation, a 300 mg / m² dose will be administered. 2 Cyclophosphamide treatment. Treatment will use 20,000,000 cells (up to 4 intradermal injections in the upper back and thigh). Patients will receive 0.1 mcg of PEGylated IFN-α2a at each injection site 1–4 hours and 1–3 days after each cell line inoculation. Patients will be reassessed for safety and DTH response measured 2 ± 1 days after each inoculation. Treatment cycles will be repeated every 3 weeks.

[0287] Safety assessment. The safety assessment will include adverse event reporting, physical examination, vital signs, cardiac assessment (ECG), and laboratory safety evaluation. Safety / tolerability responses will be evaluated and graded according to the new NIH Universal Toxicity Criteria, CTCAE version 5.0.

[0288] As required by regulations, any unexpected or premature death or life-threatening toxicity (such as myocardial infarction, kidney failure, or thromboembolic disease) must be reported immediately to the sponsor, the agency review board, and the FDA.

[0289] Patient recruitment will be suspended if at least grade 4 or higher toxicity, which is likely related to the experimental treatment, occurs in two or more patients until the IRB and FDA conduct an additional review and approval of any necessary changes to the protocol that may be indicated.

[0290] DLT is defined as a grade 3 or higher adverse event that is likely associated with at least the BC cell line, including autoimmune reactions, hypersensitivity reactions, and graft-versus-host disease. The DLT observation period is 2 weeks after the first administration of the study agent. The maximum tolerated dose (MTD) is defined as the dose level at which <30% of patients experience DLT. This dose will be used in the subsequent Part 2. If, during Part 2, the dose is subsequently determined to be intolerable, a next lower dose may be used, and so on, until a dose level at which less than 30% DLT is found. Note that the MTD is determined for each cell line and may vary for different BC cell lines. Any subject experiencing DLT will be withdrawn from the study. If a tolerated dose level cannot be determined for a given cell line, further study of that cell line will be paused, data reviewed, and will only resume after protocol revision, FDA consultation, and IRB approval. If a tolerated dose level cannot be determined for all BC cell lines, the study will be paused, data reviewed, and will only resume after protocol revision, FDA consultation, and IRB approval.

[0291] Efficacy assessment: Efficacy will be assessed using investigator-determined clinical benefit (e.g., PFS) and RECIST criteria. Baseline imaging studies will be performed within 4 weeks of treatment initiation. This includes the following assessments: computed tomography (CT) scans of the chest, abdomen, and pelvis; and, if clinically indicated, isotope bone scans, PET scans, mammograms, ultrasound / MRI scans, or other X-ray examinations, based on clinical indications from disease assessment and / or the investigator's discretion. Any additional baseline SOC imaging should be repeated at a frequency no less than the schedule of active imaging assessment intervals.

[0292] Measurable disease: This requires that at least one dimension of such features be accurately measurable (+ / -10%) on CT (≤1.0 cm incision), MRI, conventional X-ray, or medical imaging. Measurable diseases seen on images obtained from spiral CT must be ≥1.0 cm. Ultrasound imaging is permitted only for superficial lesions. Bone lesions are not considered based on these criteria.

[0293] Unmeasurable disease: This includes bone lesions, exudates, poorly defined lung infiltrates, and radiographic lesions < 1.0 cm.

[0294] Objective conditions of the examination: target lesions are defined as measurable lesions, with a maximum of 5 sites per patient and no more than 2 sites per organ.

[0295] 1. Measurement of target lesions must be provided at screening time, 9-12 weeks after the start of the screening program, and at 9-12 week intervals throughout the treatment process.

[0296] 2. The development of new lesions must be recorded.

[0297] The response was defined using the iRECIST / RECIST v1.1 criteria, based on the local investigator’s standard of care and blinded center review.

[0298] Statistical methods: Various statistical analyses will be performed to assess the relationship between clinical response, immune response, and potential prognostic factors. Appropriate statistical tests will be determined by the statistician.

[0299] If the number of participants in the study allows, multiple regression and / or Cox regression will be performed to identify factors predicting response. This may include logistic regression using response as the endpoint and Cox regression using survival time. Other parametric and nonparametric tests will be used appropriately to evaluate the relationship of interest. For all tests, the criterion for statistical significance will be set at p < 0.05, using two-tailed tests.

[0300] The principal investigator will review clinical and laboratory data as early as possible during re-imaging and re-staging, or as needed. Protocol deviations will be entered into a form that will be included in the regulatory binder. Missing data, patient absences, or other non-compliance will be recorded in the document notes, copied to the IRB, and included in the annual report, unless a safety issue requires timely submission to the FDA based on the criteria for serious adverse events.

[0301] Example 5: Identification of antigenic determinants in SV-BR-1-derived cell-derived breast cancer vaccines

[0302] This example illustrates the identification of immunogenic epitopes in an SV-BR-1-derived cellular breast cancer vaccine using T-cell epitope mapping assays. Several key immunogenic peptides were detected, including peptides with post-translational modifications (PTMs), such as citrullination and cysteine.

[0303] background

[0304] Identifying antigenic determinants is crucial for developing effective cancer vaccines. This study focuses on SV-BR-1-derived cellular breast cancer vaccines, aiming to characterize specific antigens that elicit an immune response. These vaccines rely on two key concepts: tumor cells present immunogenic antigens that activate T cells via cross-presentation, and host dendritic cells (DCs) process exogenous tumor antigens and present them on HLA molecules to activate T cells. Furthermore, genetic engineering enhances their role as antigen-presenting cells, thereby amplifying the immune response. Bria-IMT (version 1) is a genetically modified tumor cell line engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF). This vaccine has shown encouraging clinical results and is currently undergoing a phase 3 clinical trial, demonstrating its potential in cancer immunotherapy. Bria-OTS+ (upgraded version) enhances the ability of tumor cells to present antigens by expressing cytokines, co-stimulatory factors, and HLA alleles.

[0305] Purpose

[0306] Therapeutic cancer vaccines are designed to stimulate the immune system by utilizing tumor antigens to generate an anti-tumor response. In our clinical trials, we have focused on SV-BR-1-GM (an engineered breast cancer cell line that secretes GM-CSF) as a therapeutic vaccine. This vaccine has shown encouraging clinical results when used as monotherapy (NCT03066947, completed) and in combination with a checkpoint inhibitor (NCT03328026, ongoing). To further enhance the therapeutic efficacy of SV-BR-1-GM, we characterized the immune response of patients to the vaccine. Cancer vaccines are known to induce humoral and cellular immune responses. However, predicting these responses, especially in the context of whole-cell vaccines such as SV-BR-1-GM, presents unique challenges. One of the main difficulties lies in identifying which specific tumor antigens elicit an immune response in patients, as whole-cell vaccines provide a broad range of antigens. Here, we used T-cell epitope mapping assays to analyze antigen-specific T-cell responses in breast cancer patients.

[0307] method

[0308] Identification of tumor antigens using the immunopeptide genome of SV-BR-1 cells. Figure 2The workflow of immunopeptidomics analysis is illustrated, detailing the processing and presentation of various tumor antigens in the SV-BR-1 cell line, including tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), cancer testis antigens (CTAs), post-translational modification (PTM) antigens, and unconventional antigens (UCAs). TAAs are proteins that are overexpressed or dysregulated in tumor cells compared to normal tissues. TSAs result from tumor-specific somatic mutations, producing novel antigens specific to tumor cells. CTAs are typically confined to immune-privileged germline tissues but are aberrantly expressed in tumors. PTM antigens originate from alterations in post-translational modifications, such as phosphorylation or glycosylation, leading to novel tumor-specific epitopes. UCAs include antigens from non-coding regions, alternative reading frames, or proteins from the dark proteome, providing unique epitopes in tumor cells. These antigens are processed into peptide fragments by the proteasome, subsequently loaded onto MHC class I molecules, and transported to the cell surface.

[0309] In this immunopeptidomics workflow, cells are lysed, and MHC-peptide complexes are immunoprecipitated using HLA-specific antibodies MHCI (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 peptides 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 targets.

[0310] Mapping of class I and class II antigens / epitopes. Figure 3 An exemplary workflow for class I and II antigen / epitope mapping is illustrated. T cells expressing CD154 (class II) and T cells expressing CD137 (class I) are enriched using magnetic beads and then flow-cytometry-sorted using activation and memory markers. The sorted T cells are expanded into oligoclonal cells in 96-well plates containing feeder cells, PHA, and IL-2. They are then stimulated with a peptide pool and restimulated with individual peptides to identify specific antigenic peptides.

[0311] Patient and clinical data. The SV-BR-1-GM “monotherapy” study (NCT03066947; 2013-8) was a prospective phase 1-2 study of the SV-BR-1-GM regimen, with the first two doses administered every two weeks, followed by monthly administration. This regimen included approximately 20 x 10 mg of SV-BR-1-GM per treatment. 6A low-dose cyclophosphamide (300 mg / m²) was administered intravenously 48–72 hours prior to intradermal delivery of individual cells. Interferon-α2b (10,000 IU) was injected at the SV-BR-1-GM inoculation site 2 and 4 days after each administration. Cell samples were collected from each patient before and after vaccination. Table 15 provides patient information and outcomes of SV-BR-1-GM treatment.

[0312] Table 15. Patient and Clinical Data In the monotherapy study, patient 06-001 had stable disease, but experienced partial remission after switching to combination therapy with pembrolizumab.

[0313] result

[0314] Immunopeptiographic analysis of MHC class I and II binding peptides in Bria-IMT cells. Based on a positive / decoy database search, a total of 4123 peptides of class I MHC and 2193 peptides of class II MHC were detected in human Bria-IMT cells under 1% PSM FDR. An overview of the total amount and intensity of MHC class I and II peptides (including HLA-DR specific) is shown in Table 16.

[0315] Table 16. Total amount and intensity of MHC class I and II (including HLA-DR specific) peptides in human Bria-IMT cells.

[0316]

[0317] Identification and prioritization of candidate immunogenic peptides for immunogenicity testing. We prioritized peptides from two primary sources. The first source was peptides presented by SV-BR-1 cells. These peptides were directly identified in immunopeptidome analysis of SV-BR-1 cells, indicating that they were naturally processed and presented by the tumor cells used in our vaccine. The second source consisted of peptides expressed with known immunogenicity, including peptides not detected in our immunopeptidome but confirmed to be expressed in SV-BR-1 cells by RNA-seq analysis. These peptides are known to be presented by breast cancer cells and their immunogenicity has been demonstrated in public databases such as the Cancer Epitope Database and Analysis Resource (CEDAR) and CaAtlas (www.zhang-lab.org / caatlas / ).

[0318] We use the following parameters to select candidate immunogenic peptides for immunogenicity testing: MHC binding affinity: We used the NetMHCpan tool to predict peptides with high binding affinity to ubiquitous HLA alleles (IC50 ≤ 500 nM for class I; IC50 ≤ 1,000 nM for class II).

[0319] Tumor specificity: We focused on tumor-associated antigens (TAAs) and cancer testis antigens (CTAs) specific to SV-BR-1 cells.

[0320] Overexpression in tumors: preferential sequencing of peptides that are highly expressed in tumor cells, as confirmed by RNA-seq data.

[0321] Unique PTM: Contains peptides with post-translational modifications specific to tumor cells to capture novel epitopes.

[0322] Immunogenicity evidence: Candidates are cross-referenced with immunogenicity databases to select peptides with recorded immune responses.

[0323] HLA matching: Peptides associated with common HLA types in our patient cohort were selected for analysis.

[0324] A total of 80 MHC class I peptides (10 of which carry PTM) and 50 class II peptides (18 of which carry PTM) were selected for further analysis.

[0325] T cell epitopes were identified by mapping for CD154 (class II) or CD137 (class I) epitopes. Peripheral blood mononuclear cells (PBMCs) from vaccinated patients, collected before and after vaccination, were in vitro stimulated with various peptide pools to assess immediate T cell activation. Stimulation conditions included a DMSO control for background measurements, a positive control confirming cell reactivity, and class I or class II peptide giant pools designed to stimulate CD8+ (cytotoxic) or CD4+ (helper) T cells, respectively. After initial in vitro activation, T cells identified by activation markers (CD137 and CRTAM for CD8+ cells, and CD154 and CD69 for CD4+ cells) were analyzed by flow cytometry to determine peptide-specific responses. Subsequently, each giant pool was subdivided into smaller sub-pools, which were tested individually to reduce background noise and improve the specificity for identifying immunogenic peptides. Individual peptides were then detected in these sub-pools to precisely identify specific epitopes that induce T cell responses. To confirm that the CD4+ T cell response is MHC class II dependent, HLA-DR, HLA-DQ, or HLA-DP blocking antibodies were introduced along with individual peptides. We identified five peptides that induced immunogenic responses, such as... Figures 4A-4D As shown. Figure 4A Pre-vaccination samples responded to cysteine-modified desmosome plaque MHC class II peptide QGSS(cys-mod)IAGIYNETTKQKLG (SEQ ID NO:1); Figure 4B The samples received a vaccine that responded to two citrullinated filaggrin MHC class II peptides, KLAQYYESTCitKEN (SEQ ID NO: 2) and FKLAQYYESTCitKEN (SEQ ID NO: 3). Figure 4C The vaccine-prescribed samples responded to the MFGE8 MHC class I peptide GLQHWVPEL (SEQ ID NO: 4); and ( Figure 4D Pre-vaccination samples responded to the COX7C-derived MHC class II peptide ATPFLVVRHQLLKT (SEQ ID NO: 5). A total of one CD8+ and four CD4+ T cell epitopes were identified by epitope mapping assays. Specifically, the immunogenic desmosome plaque peptide QGSS(cys-mod)IAGIYNETTKQKLG (SEQ ID NO: 1) was cysteine-modified, a post-translational modification in which a cysteine ​​residue forms a disulfide bond with an existing cysteine ​​residue in the peptide. Both immunogenic filaggrin peptides, KLAQYYESTCitKEN (SEQ ID NO: 2) and FKLAQYYESTCitKEN (SEQ ID NO: 3), contain citrulline residues.

[0326] in conclusion

[0327] We successfully identified immunogenic peptides in patients treated with the SV-BR-1-GM cell vaccine, demonstrating its ability to elicit targeted immune responses against tumor antigens. Key immunogenic peptides detected included peptides with post-translational modifications (PTMs), such as citrullination and cysteine. This study highlights the advantages of using cellular cancer vaccines over RNA and peptide-based vaccines because they offer a broad and diverse repertoire of antigens, including both conventional and unconventional types. Unlike RNA and peptide vaccines that deliver specific known antigens, cellular vaccines, such as whole tumor cells or antigen-presenting cells with tumor lysates, present a broad range of naturally occurring tumor antigens. This includes post-translational modifications (PTMs), optional splice variants, non-coding sequences, and peptides derived from pseudogenes. Such antigens often represent unique tumor features not present in healthy cells. Cellular vaccines can also present unknown patient-specific neoantigens that are difficult to predict with RNA or peptide vaccines. This diverse antigen presentation stimulates robust polyclonal immune responses, enabling CD8+ and CD4+ T cells to target multiple tumor targets, reducing immune escape, and potentially leading to more durable clinical outcomes.

[0328] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and various modifications or changes will occur to those skilled in the art, which will be included within the spirit and scope of this application and the appended claims. All publications, patents, patent applications, and serial number referenced herein are incorporated herein by reference in their entirety for all purposes.

Claims

1. Modified human cancer cells containing recombinant polynucleotides encoding enzymes that induce post-translational modifications (PTMs) of antigens in the cells.

2. The modified human cancer cell according to claim 1, wherein the enzyme comprises citrullinated enzyme, cysteine ​​esterase, acetyltransferase, hydroxylase, phosphorylase, methyltransferase, formylate, oxidase, hydroxylase, ubiquitinase or a combination thereof.

3. Modified human cancer cells containing antigens, wherein the antigens contain non-enzymatic PTM.

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

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

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

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

8. The modified human cancer cell according to any one of claims 1-7, wherein the PTM comprises phosphorylation, acetylation, ubiquitination, succinylation, methylation, malonylation, glycosylation, SUMOylation, nitrosylation, glutathioneylation, amidation, hydroxylation, palmitoylation, glutarylation, crotonylation, oxidation, myristylation, sulfation, formylation, citrullination, isopreneylation, cysteineation, deamidation, dehydration, or a combination thereof.

9. The modified human cancer cell of claim 8, wherein the PTM comprises cysteine ​​and / or citrulline.

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

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

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

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

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

15. The modified human cancer cell according to claim 14, wherein the cytokine comprises chemokines, interferons, interleukins, tumor necrosis factor, or a combination thereof.

16. The modified human cancer cell according to claim 14 or 15, wherein the cytokines comprise early T-cell activation antigen-1 (ETA-1), lymphocyte activating factor (LAF), interleukin-1 family members (IL-1α, IL-2, IL-3, IL-1Ra, IL-18, IL-33, IL-36Ra, IL-36α, IL-36β, IL-36γ, IL-37, IL-38), interleukin-2 (IL-2), interleukin-3 (IL-3), interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-7 (IL-7), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-10 (IL-10), interleukin-12 (IL-12), interleukin-13 (IL-13), interleukin-15 (IL-15), and interleukin-17. Interleukin-17, Interleukin-18, Interleukin-21, Interleukin-23, Interleukin-25, Interleukin-33, Interferon-α, Interferon-λ1, Interferon-λ2, Interferon-λ3, Interferon-λ4, Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Macrophage CSF (CSF-1), Macrophage Migration Inhibitor Factor (MIF), CD40L, RANTES, Monocyte Chemokines (MCP-1), Monocyte Inflammatory Proteins (MIP-1α, MIP-1β), Lymphocyte Chemokines, Fractal Chemokines, or combinations thereof.

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

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

19. The modified human cancer cell according to claim 18, wherein the co-stimulatory molecule comprises CD86 molecule (CD86), CD80 molecule (CD80), 4-1BB ligand molecule (4-1BBL, also known as CD137L), ICOS ligand molecule (ICOS-L), CD70 molecule (CD70, also known as CD27L), CD40 molecule (CD40), OX40 ligand molecule (OX40L), GITR ligand molecule (GITRL), TIM-4 molecule (TIM-4), LIGHT molecule (LIGHT), ICAM1 molecule (ICAM1), LFA3 molecule (LFA3), CD30 molecule (CD30) or a combination thereof.

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

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

22. The modified human cancer cell according to 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 derived from a biopsy or circulating cancer cell from a patient.

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

25. The non-replicating modified human cancer cell according to any one of claims 1-24.

26. The non-replicating modified human cancer cells according to claim 25, wherein the modified human cancer cells are made non-replicating by irradiation, freeze-thaw treatment or mitomycin C treatment.

27. A composition comprising any one of the modified human cancer cells according to 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 person suffering from 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 application step, the method further comprises: (i) Obtain a sample from the object; (ii) Identify the PTMs that are prevalent in the sample; as well as (iii) Selecting modified human cancer cells for application to the object, wherein the modified human cancer cells contain a recombinant polynucleotide encoding an enzyme that induces the ubiquitous PTM.

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 (CTC), 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 next-generation sequencing (NGS) or immunopeptidome analysis of the sample.

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

37. A method for enhancing the immunogenicity of an antigen in human cancer cells, comprising inducing a post-translational modification (PTM) of the antigen in the cells, 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, glutathioneylation, amidation, hydroxylation, palmitoylation, glutarylation, crotonylation, oxidation, myristylation, sulfation, formylation, citrullination, isopreneylation, cysteineation, deamidation, dehydration, or a combination thereof.

39. The method of claim 38, wherein the PTM comprises cysteine ​​and / or citrulline.

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

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

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

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

45. The method according to any one of claims 37-44, wherein the cells are human cancer cell lines.

46. ​​The method according to any one of claims 37-44, wherein the cells are primary cancer cells.

47. The method according to any one of claims 37-46, wherein the cells are breast cancer cells, prostate cancer cells, melanoma cells, or lung cancer cells.

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

49. The method according to claim 48, wherein the enzyme comprises citrullinated enzyme, cysteine ​​lactamase, acetyltransferase, hydroxylase, phosphorylase, methyltransferase, formylate, oxidase, hydroxylase, ubiquitinase, or a combination thereof.

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

51. The method according to 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, induced cell senescence, chemical reaction, small molecule, cell culture supplement, cell culture medium, oxidative stress, or a combination thereof.

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