Heterocritical neoepitope vaccines

Abstract

Compositions of peptides modified to enhance HLA binding or T cell recognition while preserving reactivity to target neoepitopes enhance the activation of neoantigen-specific T cells. The method of use involves treating cancer by administering one or more of these peptides, which are produced by computer modeling.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority under 35 U.S.C. 119(e) to U.S. Provisional Application No. 63 / 459,442, filed on April 14, 2023. The entire contents of U.S. Provisional Application No. 63 / 459,442 are incorporated herein by reference.

[0002] Description of Research Sponsored by the Federal Government This disclosure was made with government support under grants CA247886 and CA271470 awarded by the National Institutes of Health. The U.S. government retains certain rights in this invention.

Background Art

[0003] Background Many cancer neoantigens are poorly immunogenic because of the weak binding of neoepitopes to human leukocyte antigens (HLA), thereby limiting their presentation to cognate T cells. Modifying this peptide amino acid sequence can improve HLA binding or T - cell recognition while preserving reactivity to the target neoepitope, thereby enhancing the activation of neoantigen - specific T cells. These modified epitopes improve antigen immunogenicity and are called heteroclitic epitopes or altered peptide ligands (APLs).

Summary of the Invention

[0004] Summary The invention relates to the design and modeling of heterocritical epitope vaccine candidates tailored to diverse HLA subtypes. Therefore, in certain circumstances, a method for identifying heterocritical neoepitopes includes the following steps: modeling a peptide on human leukocyte antigen (HLA), wherein the modeling step includes identifying the positional sequence similarity of peptide epitopes that bind to one or more HLA haplotypes; selecting an HLA-peptide structure having greater sequence identity than a control HLA-peptide structure; modeling a heterocritical neoepitope within an HLA cleft by introducing an amino acid rotomer having the lowest energy confirmation at each position; verifying the resulting HLA-peptide neoepitope complex using root-mean-square deviation (Cα-RMSD); performing a high-resolution Monte Carlo simulation with minimal docking of the peptide and the HLA cleft to identify the lowest full-atom energy conformation; and identifying the heterocritical neoepitope. In certain embodiments, modeling of a peptide library onto human leukocyte antigens (HLAs) involves aligning the peptide sequence with the consensus binding motif of the HLA molecule. In certain embodiments, the consensus binding motif for an 8-mer, 9-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitope includes distinct anchor residue binding motifs subdivided into hydrophobic, hydrophilic, or neutral binding anchors. In certain embodiments, root mean square deviation analysis is performed to identify any unstable structural changes in the HLA cleft. In certain embodiments, an anchor energy optimized structure is selected to identify anchor residue modifications.In certain embodiments, peptides are constructed that contain anchor residue amino acid combinations for each HLA class and its subgroups. In certain embodiments, the HLA types include classes I, II, and their subgroups. In certain embodiments, the peptides are derived from tumor cells. In certain embodiments, the peptides contain tumor antigens. In some embodiments, heterocritical epitope vaccine candidates are selected based on one or more evaluations of the contact between the peptide and the HLA cleft residue, solvent-assessable surface area (SASA), peptide rigidity as a measure of CαRMSD for the top 10 predictive structural models for each peptide-HLA complex, surface hydrophobicity, electrostatic potential, and / or induction of maximum T cell expansion and activation compared to the parent peptide.

[0005] Methods for constructing and identifying heterocritical peptides are disclosed herein. In some embodiments, the method includes aligning a peptide sequence onto a parent sequence. In some embodiments, the parent sequence may be a control sequence. In some embodiments, the method includes identifying a sequence having high homology to the epitope of the parent sequence. In some embodiments, the method includes introducing an amino acid rotomer having the lowest energy confirmation into the sequence having high homology to the epitope of the parent sequence. In some embodiments, the method further includes quantifying the difference in structural features between the parent sequence and the sequence having high homology to the epitope of the parent sequence. In some embodiments, the features include contact between the peptide and an HLA cleft residue, solvent exposure area (SASA), peptide stiffness as a measure of CαRMSD for the top 10 predictive structural models for each peptide-HLA complex, surface hydrophobicity, and / or electrostatic potential. In some embodiments, the method further includes co-culturing sequences with high homology with HLA-matched lymphocytes in vitro to verify which heterocritic peptide induces the greatest T cell expansion and activation compared to the parent peptide when evaluated by an IFN-γ erythropote assay. In some embodiments, the method further includes a step of selecting / identifying the heterocritic peptide. In some embodiments, the heterocritic peptide contains at least 75%, 85%, 90%, 95%, 97%, 98%, or 99% sequence identity to one or more of SEQ ID NO: 1-838. In some embodiments, the heterocritic peptide contains SEQ IDNO:2, 3, 5~12, 14~21, 23~30, 32~39, 41~48, 50~57, 59~66, 68~75, 77~84, 86~93, 85~99, 101~105, 107~111, 113~117, 119~123, 125~129, 1 31~135, 137~141, 143~146, 148~152, 154~162, 164~168, 170~179, 181~185, 187~196, 198~202, 204~213, 215~219, 221~230, 232~236, 238 ~244, 246~252, 254~260, 262~268, 270~276, 278~284, 286~292, 294~300, 302~308, 310~316, 318~324, 326~332, 334~340, 342~348, 350~356, 358~364, 366~372, 374~380, 382~388, 390~396, 398~404, 406~412, 414~420, 422~428, 430~436, 438~443, 445~450, 452~457, 459~464 , 466~471, 473~478, 480~485, 487~492, 494~499, 501~506, 508~512, 514~518, 520~524, 526~530, 532~536, 538~542, 544~548, 550~554, 556~560, 562~566, 568~573, 585~580, 582~587, 589~594, 596~601, 603~607, 609~613, 615~619, 621~625, 627~631, 633~638, 340~644, 646 ~651, 653~657, 659~664, 666~670, 672~677, 679~683, 685~690, 692~696, 698~703, 705~711, 713~718, 720~726, 728~733, 735~741, 743~748, 750~756, 758~763, 765~771, 773~777, 779~783, 785~789, 791~795, 797~801, 803~807, 809~813, 815~819, 821~825, 827~831, 833, orThe sequence identity includes at least 75%, 85%, 90%, 95%, 97%, 98%, and 99% for any one or more of the 834. In certain embodiments, the heterocritic peptide is SEQ IDNO:2, 3, 5~12, 14~21, 23~30, 32~39, 41~48, 50~57, 59~66, 68~75, 77~84, 86~93, 85~99, 101~105, 107~111, 113~117, 119~123, 125~129, 131~ 135, 137~141, 143~146, 148~152, 154~162, 164~168, 170~179, 181~18 5, 187~196, 198~202, 204~213, 215~219, 221~230, 232~236, 238~244, 246~252, 254~260, 262~268, 270~276, 278~284, 286~292, 294~300, 302~308, 310~316, 318~324, 326~332, 334~340, 342~348, 350~356, 358~364, 366~372, 374~380, 382~388, 390~396, 398~404, 406~412, 414~420, 422~428, 430~436, 438~443, 445~450, 452~457, 459~464, 466~471, 4 73~478, 480~485, 487~492, 494~499, 501~506, 508~512, 514~518, 520~524, 526~530, 532~536, 538~542, 544~548, 550~554, 556~560, 562~566, 568~573, 585~580, 582~587, 589~594, 596~601, 603~607, 609~613, 615~619, 621~625, 627~631, 633~638, 340~644, 646~651, 653~657, 6 The heterocritic peptide includes one or more of the following SEQ IDs: 59-664, 666-670, 672-677, 679-683, 685-690, 692-696, 698-703, 705-711, 713-718, 720-726, 728-733, 735-741, 743-748, 750-756, 758-763, 765-771, 773-777, 779-783, 785-789, 791-795, 797-801, 803-807, 809-813, 815-819, 821-825, 827-831, 833, or 834. In certain embodiments, the heterocritic peptide has one or more conserved substitutions, SEQ IDThe heterocritic peptide comprises an amino acid sequence having at least 95% sequence identity with one or more of NO:1 to 838. In some embodiments, the heterocritic peptide comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:833, or SEQ ID NO:834.

[0006] In another aspect, a method for preparing a library of immunogenic peptides includes the following steps: modeling peptides on human leukocyte antigens (HLA), wherein the modeling step includes identifying the positional sequence similarity of peptide epitopes that bind to one or more HLA haplotypes; selecting HLA-peptide structures having greater sequence identity than a control HLA-peptide structure; modeling heterocritical neoepitopes within HLA clefts by introducing amino acid rotomers having the lowest energy confirmation at each position; verifying the resulting HLA-peptide complexes using root mean square deviation analysis (Cα-RMSD); performing high-resolution Monte Carlo simulations with minimal docking of the peptides and HLA clefts to identify the lowest total atomic energy conformation; and preparing a library of immunogenic peptides. In certain embodiments, the peptides include peptides isolated from tumor cells. In certain embodiments, the peptides include tumor antigens. In certain embodiments, the tumor antigen is the Kirsten rat sarcoma viral (KRAS) tumor antigen. In certain embodiments, the KRAS tumor antigen contains one or more mutations. In certain embodiments, the KRAS peptide sequence is positionally aligned with an 8-mer, 9-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitope that binds to an HLA molecule. In certain embodiments, the 8-mer, 9-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitopes contain distinct anchor residue binding motifs that are subdivided into hydrophobic, hydrophilic, or neutral binding anchors. In certain embodiments, the HLA-KRAS peptide containing the greatest sequence similarity is selected for modeling within the HLA cleft.In certain embodiments, modeling into HLA clefts involves introducing amino acid rotomers having the lowest energy confirmation at each position. In certain embodiments, root mean square deviation analysis is performed to identify any unstable structural changes in the HLA cleft. In certain embodiments, anchor energy optimized HLA-KRAS structures are selected to identify anchor residue modifications. In certain embodiments, a library of KRAS peptides containing anchor residue amino acid combinations for each HLA class and its subgroups is constructed. In certain embodiments, HLA types include classes I, II, and their subgroups. In certain embodiments, KRAS peptides include heterocritical epitopes. In some embodiments, immunogenic peptides are selected based on one or more assessments of the contact between the peptide and the HLA cleft residue, solvent exposure area (SASA), peptide stiffness, surface hydrophobicity, electrostatic potential as measures of CαRMSD for the top 10 predictive structural models for each peptide-HLA complex, and / or induction of maximum T cell expansion and activation compared to the parent peptide.

[0007] In certain embodiments, the heterocritical peptide sequence is positionally aligned with an epitope that binds to an HLA molecule. In some embodiments, the epitope is an 8-mer epitope, 9-mer epitope, 10-mer epitope, 11-mer epitope, 12-mer epitope, 13-mer epitope, 14-mer epitope, 15-mer epitope, 16-mer epitope, 17-mer epitope, 18-mer epitope, 19-mer epitope, 20-mer epitope, 21-mer epitope, 22-mer epitope, 23-mer epitope, 24-mer epitope, or 25-mer epitope.

[0008] In another context, a method for treating cancer involves administering a therapeutically effective amount of one or more peptides containing one or more heterocritical epitopes to a subject in need.

[0009] In another aspect, the cancer vaccine comprises one or more peptides, the peptide comprising at least one heterocritic neoepitope. In a particular embodiment, the peptide is a tumor antigen. In a particular embodiment, the tumor antigen comprises the Kirsten rat sarcoma virus (KRAS) tumor antigen. In a particular embodiment, the KRAS tumor antigen comprises one or more mutations. In a particular embodiment, the peptide comprising at least one heterocritic neoepitope is presented on the surface of antigen-presenting cells (APCs) via the HLA antigen presentation pathway to induce a tumor-specific immune response. In a particular embodiment, the peptide is immunogenic specifically against the target HLA type.

[0010] In another context, the expression vector encodes one or more peptides embodied herein.

[0011] In another aspect, isolated cells include an expression vector encoding one or more peptides as embodied herein. In certain embodiments, isolated cells include autologous cells, allogeneic cells, haplotype-compatible cells, haplotype-incompatible cells, haplotype-matched cells, xenocells, cell lines, or combinations thereof. In certain embodiments, isolated cells include T cells, B cells, natural killer (NK) cells, macrophages, dendritic cells, stem cells, induced pluripotent stem cells (iPSCs), or combinations thereof.

[0012] In another aspect, a method for treating cancer in a subject diagnosed with cancer includes the steps of: isolating cells from a biological sample subject; culturing the isolated cells with one or more peptides prepared by a method embodied herein; isolating T cells, NK cells, and / or antigen-presenting cells cultured with one or more peptides; expanding the T cells, NK cells, and / or antigen-presenting cells to produce a therapeutically effective composition of tumor antigen-specific T cells, NK cells, and / or antigen-presenting cells; and adoptively transferring tumor antigen-specific T cells, NK cells, and / or antigen-presenting cells into a subject, thereby treating the subject diagnosed with cancer. In certain embodiments, the peptides comprise one or more heterocritical neoepitopes.

[0013] In another aspect, one or more immunogenic peptides contain at least 75% sequence identity to one or more of SEQ ID NO:1-838. In certain embodiments, the immunogenic peptide contains one or more of SEQ ID NO:1-838. In certain embodiments, the immunogenic peptide contains an amino acid sequence having one or more conserved substitutions and having at least 95% sequence identity to one or more of SEQ ID NO:1-838. In some embodiments, the immunogenic peptide contains the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:833, or SEQ ID NO:834. In some embodiments, immunogenic peptides are selected based on one or more assessments of the contact between the peptide and the HLA cleft residue, solvent exposure area (SASA), peptide stiffness, surface hydrophobicity, electrostatic potential as measures of CαRMSD for the top 10 predictive structural models for each peptide-HLA complex, and / or induction of maximum T cell expansion and activation compared to the parent peptide.

[0014] definition Unless otherwise defined herein, scientific and technical terms used in connection with this application shall have the meanings generally understood by those skilled in the art to which this disclosure belongs. This disclosure is not limited to, and therefore should be understood to be, modified, the specific methods, protocols, and reagents described herein.The specificity of a specific chemical is Singleton et al., Dictionary of Microbiology and Molecular Biology 3rd ed., J. Wiley & Sons New York, NY (2001); March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 5th ed., J. Wiley & Sons New York, NY (2001); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012); Jon Lorsch (ed.) Laboratory Methods in Enzymology: DNA, Elsevier, (2013); Frederick M. Ausubel (ed.), Current Protocols in Molecular Biology (CPMB), John Wiley and Sons, (2014); John E. Coligan (ed.), Current Protocols in Protein Science (CPPS), John Wiley and Sons, Inc., (2005); Inc., (2003).Each of these provides a general guide to those skilled in the art for many of the terms used in this application.

[0015] Standard nomenclature is used for natural amino acids and their abbreviations. For example, L-alanine is represented by the three-letter abbreviation Ala or the one-letter abbreviation "A". When the "D" stereoisomer of alanine is shown, it is represented as D-Ala.

[0016] DNA bases follow a standard nomenclature, with cytosine, guanosine, adenine, and thymine represented by "C," "G," "A," and "T," and codons that code for DNA follow a standard genetic code. For example, the amino acid Leu is coded by TTA, TTG, CTT, CTC, CTA, or CTG, and Asp is coded by GAT or GAC.

[0017] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural form unless otherwise clearly specified by the context. Furthermore, the terms “including,” “includes,” “having,” “has,” and “with,” or their variants thereof, are intended to be inclusive, as with the term “comprising,” to the extent that they are used in detailed descriptions and / or claims.

[0018] The terms “about” or “approximately” mean that a particular value is within an acceptable margin of error as determined by those skilled in the art. This depends in part on how the value is measured or determined, i.e., on the limits of the measurement system. For example, “about” may mean within a range of 1 or a standard deviation greater than 1, according to convention in the art. Or, “about” may mean within 20%, 10%, 5%, or 1% of a given value or range. Or, particularly with respect to biological systems or biological processes, the term may mean within an order of five times a given value, or also within twice a given value. Where a particular value is described in this application and claims, unless otherwise specified, the term “about” should be assumed to mean that the particular value is within an acceptable margin of error. All numerical values, whether expressly indicated herein or not, are deemed to be qualified by the term “about.” Listing numerical ranges by endpoints includes all numbers within that range (for example, 1-5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5).

[0019] As used herein, “adjuvant” refers to a substance that enhances the body’s immune response to an antigen or vaccine and may be added to formulations containing immunizing agents. Adjuvants improve the immune response even after administration of only a single dose of vaccine. Adjuvants may include, for example, aluminum hydroxide and aluminum phosphate, saponins, e.g., Quil A, QS-21 (Cambridge Biotech Inc., Cambridge Mass.), GPI-0100 (Galenica Pharmaceuticals, Inc., Birmingham, Ala.), nonmetabolizable oils, mineral oils and / or vegetable oils and / or animal oils, polymers, carbomers, surfactants, natural organic compounds, plant extracts, carbohydrates, cholesterol, lipids, water-in-oil emulsions, oil-in-water emulsions, water-in-oil-in-water emulsions, HRA-3 (acryloacrylate crosslinked polymer), HRA-3 with cottonseed oil (CSO), or acryloacrylate polyol crosslinked polymer. The emulsion may be based in particular on light liquid paraffin oil (European Pharmacopoeia type); isoprenoid oils, e.g., squalane or squalene; alkenes, in particular oils resulting from oligomerization of isobutene or decene; esters of acids or alcohols containing linear alkyl groups, more specifically vegetable oils, ethyl oleate, propylene glycol di-(caprylate / caprate), glyceryl tri-(caprylate / caprate) or propylene glycol dioleate; or esters of branched fatty acids or alcohols, in particular esters of isostearate. The oil is used in combination with an emulsifier to form an emulsion.Emulsifiers include nonionic surfactants, in particular esters of sorbitan, mannide (e.g., anhydromannitol oleate), glycols, polyglycerols, propylene glycols, and esters of oleic acid, isostearic acid, ricinoleic acid, or hydroxystearic acid, as well as polyoxypropylene-polyoxyethylene copolymer blocks, in particular PLURONIC® trademark products, in particular L121. See Hunter et al., The Theory and Practical Application of Adjuvants (Ed. Stewart-Tull, DES) John Wiley and Sons, NY, pp 51-94 (1995) and Todd et al., Vaccine 15:564-570 (1997). In a preferred embodiment, the adjuvant is present in a concentration of about 0.01 to about 50 vol% relative to the final product, about 2 to 30 vol%, about 5 to about 25 vol%, about 7 to about 22 vol%, and about 10 to about 20 vol%. "Adjuvant-added" refers to a composition that incorporates or is combined with an adjuvant.

[0020] In descriptions and claims, a conjunctive list of elements or features may follow a phrase such as “at least one of” or “one or more of.” The term “and / or” may also appear in a list of two or more elements or features. Unless implicitly or explicitly contradicts the context in which such phrases are used, they are intended to mean either any of the enumerated elements or features individually, or any combination of any of the enumerated elements or features with any of the other enumerated elements or features. For example, the phrases “at least one of A and B,” “one or more of A and B,” and “A and / or B” are intended to mean “A only,” “B only,” or “A and B,” respectively. The same interpretation is intended for lists containing three or more items. For example, the phrases “at least one of A, B, and C,” “one or more of A, B, and C,” and “A, B, and / or C” are intended to mean “A only,” “B only,” “C only,” “A and B,” “A and C,” “B and C,” or “A, B and C,” respectively. Furthermore, the use of the term “based on” in the foregoing and in the claims is intended to mean “at least partially based on” features or elements not enumerated.

[0021] As used herein, the term “HLA-peptide structure” refers to a complex in which a peptide is bound to one or more HLA-haplotypes. As used herein, the term “amino acid” refers to natural and synthetic α, β, γ, and δ amino acids, including, but not limited to, amino acids found in proteins, namely glycine, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine. Alternatively, the amino acid may be alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutaminyl, aspartoyl, glutaroyl, lysinyl, arginine, histidinyl, β-alanyl, β-valinyl, β-leucinyl, β-isoleucinyl, β-prolinyl, β-phenylalaninyl, β-tryptophanyl, β-methioninyl, β-glycinyl, β-serinyl, β-threoninyl, β-cysteinyl, β-tyrosinyl, β-asparaginyl, β-glutaminyl, β-aspartoyl, β-glutaroyl, β-lysinyl, β-arginine, or a derivative of β-histidinyl. The amino acid may also be a non-natural amino acid. Examples of non-natural amino acids include, but are not limited to, D-amino acids (i.e., amino acids with the opposite chirality to the natural form), N-α-methylamino acids, C-α-methylamino acids, β-methylamino acids, and D- or L-β-amino acids. Other non-natural amino acids include, for example, β-alanine (β-Ala), norleucine (Nle), norvaline (Nva), homoarginine (Har), 4-aminobutyric acid (γ-Abu), 2-aminoisobutyric acid (Aib), 6-aminohexanoic acid (ε-Ahx), ornithine (orn), sarcosine, α-aminoisobutyric acid, 3-aminopropionic acid, 2,3-diaminopropionic acid (2,3-diaP), D- or L-phenylglycine, D-(trifluoromethyl)-phenylalanine, and Dp-fluorophenylalanine.When the term amino acid is used, it is considered to be a specific and independent disclosure of esters of α, β, γ, and δ glycine in D and L configurations, alanine, valine, leucine, isoleucine, methionine, phenylalanine, tryptophan, proline, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0022] As used herein, "cancer" means a disease, condition, trait, genotype, or phenotype characterized by uncontrolled cell proliferation or replication, as known in the art, including colorectal cancer, as well as, for example, leukemia, e.g., acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), and chronic lymphocytic leukemia; AIDS-related cancers, e.g., Kaposi's sarcoma; breast cancer; bone cancers, e.g., osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, giant cell tumor, adamantinoma, and chordoma; brain cancers, e.g., meningioma, gliablastoma, low-grade astrocytoma, oligodendrocytoma, pituitary tumor, Schwannoma, primary CNS lymphoma, and metastatic brain cancer; and various lymphomas, e.g., mantle cell lymphoma, non-Hodgkin lymphoma. Cancers of the head and neck including peritoneum, adenoma, squamous cell carcinoma, laryngeal cancer, gallbladder cancer and bile duct cancer, retinal cancers such as retinoblastoma, esophageal cancer, gastric cancer, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, lung cancer, bladder cancer, prostate cancer, lung cancer (including non-small cell lung cancer), pancreatic cancer, sarcoma, Wilms' tumor, cervical cancer, head and neck cancer, skin cancer, nasopharyngeal cancer, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adenocarcinoma, parotid gland cancer Cancer, endometrial sarcoma, multidrug-resistant cancer; and proliferative disorders and conditions, such as neovascularization associated with tumor angiogenesis, macular degeneration (e.g., wet / dry AMD), corneal neovascularization, diabetic retinopathy, neovascular glaucoma, myopic degeneration, and other proliferative disorders and conditions, such as restenosis and polycystic kidney disease, as well as other cancers or proliferative disorders, conditions, traits, genotypes, or phenotypes.

[0023] As used herein, the term “combination therapy” refers to a situation in which two or more different pharmaceutical agents are administered in overlapping regimens so that the subject is simultaneously exposed to them. When two or more different agents are used in combination therapy, they may be administered simultaneously or separately. This combination administration may include simultaneous administration of two or more agents in the same dosage form, simultaneous administration of different dosage forms, and separate administration. That is, two or more agents may be prescribed together in the same dosage form and administered simultaneously. Or, two or more agents may be administered simultaneously, with the agents present in separate formulations. Another option is that one or more additional agents may be administered immediately after the administration of the first agent. In separate dosing protocols, two or more agents may be administered with a difference of several minutes, several hours, or several days. The treatment of the subject also includes various combination therapies involving physical treatments, such as surgery and radiation-based treatments.

[0024] As used herein, the transitional term “comprising” is synonymous with “including,” “contains,” or “characterizes,” and is inclusive or open-ended, and does not exclude any additional elements or steps of the method that are not enumerated. As used herein, the term “comprising” may be interchangeable with the terms “contains,” or “including,” or, as used herein, with the term “having.” In contrast, the transitional phrase “consisting of” excludes any elements, steps, or components not specified in the claims. The transitional phrase “essentially consisting of” limits the claims to the specified material or step and any material or step that does not substantially affect the basic and novel features of the claimed disclosure.

[0025] "Diagnostic" or "diagnosed" means determining the presence or nature of a pathological condition. Diagnostic methods differ in terms of sensitivity and specificity. The "sensitivity" of a diagnostic assay is the percentage of diseased individuals that test positive (the percentage of "true positives"). Individuals with the disease that are not detected by this assay are "false negatives." Subjects that are not diseased and test negative in this assay are called "true negatives." The "specificity" of a diagnostic assay is 1 minus the false positive rate, and the "false positive" rate is defined as the proportion of disease-free individuals that test positive. A condition may not be definitively diagnosed by a particular diagnostic method, but it is sufficient if the method provides a positive indicator that aids in diagnosis.

[0026] A "disease" is an animal's health condition in which the animal is unable to maintain homeostasis, and in which the animal's health continues to deteriorate unless the disease goes into remission.

[0027] "Heterocritical epitope," "heterocritical analog," "heterocritical neoepitope," or "modified peptide ligand (APL)" are used synonymously herein and refer to modified versions (i.e., analogs) of endogenous peptide sequences that have been engineered to induce a potent immune response. Heterocritical epitopes have an increased ability or efficacy to stimulate specific T cells when measured by an increased response to a particular dose, or by requiring a smaller amount to achieve the same response, and thus derive benefits as vaccine components because these epitopes induce a more potent T cell response than those induced by natural epitopes. In some embodiments, the potent immune response involves improved HLA binding. In some embodiments, the potent immune response involves improved T cell recognition. In some embodiments, heterocritical neoepitopes are derived from tumor antigens or cancer neoantigens.

[0028] In this specification, "immunogenic peptide" or "antigenic peptide" is used synonymously and refers to a peptide or epitope that can be recognized by the immune system and induce an immune response. An immunogenic peptide or antigenic peptide may contain a motif that binds to an MHC molecule and induces a T cell response, or a motif that can be recognized by a B cell receptor on a B cell and induce antibody production.

[0029] "Immunogenic epitope" or "antigenic epitope" are used synonymously herein and refer to the recognition of a portion of an antigen by the immune system, such as antibodies, B cells, or T cells. In some embodiments, an epitope is a specific fragment of an antigen to which an antibody binds. Epitopes are usually non-self proteins, but in some cases, sequences originating from the host may be recognized.

[0030] As used herein, the term “immune cell” refers to any cell of the immune system involved in mediating the immune response. Non-exclusive examples of immune cells include T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, neutrophils, or combinations thereof. In some contexts, immune cells express CD3. In certain contexts, immune cells expressing CD3 are T cells (e.g., CD4). + T cells or CD8 + These are T cells. In some aspects, immune cells that can be targeted using a targeting moiety (e.g., anti-CD3) are naive CD4 cells. + This includes T cells. In some situations, immune cells have memory CD4 + Includes T cells. In some situations, immune cells are effectors of CD4 + Includes T cells. In some situations, immune cells are naive CD8 + This includes T cells. In some situations, immune cells have memory CD8 + Includes T cells. In some situations, immune cells are effectors of CD8 +Includes T cells. In some contexts, immune cells include γδT cells. In some contexts, immune cells are dendritic cells. In certain contexts, dendritic cells include plasmacytoid dendritic cells (pDCs), conventional dendritic cells 1 (cDC1), conventional dendritic cells 2 (cDC2), inflammatory monocyte-derived dendritic cells, Langerhans cells, dermal dendritic cells, lysozyme-expressing dendritic cells (LysoDCs), Kupffer cells, or any combination thereof.

[0031] As used herein, "lentivirus" refers to a genus of the family Retroviridae. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells. Lentiviruses can deliver a significant amount of genetic information to the host cell's DNA, making them one of the most efficient methods of gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0032] Parenteral administration of immunogenic compositions includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), intravitreous (ivi), intracisional (icm), or intrasternal injection or infusion.

[0033] The terms “patient,” “individual,” and “subject” are used synonymously herein and refer to the mammalian subject to be treated, with human patients preferred. In some cases, the methods of the present invention may be used in laboratory animals, veterinary applications, and in the development of animal models of diseases, including but not limited to rodents, including mice, rats, and hamsters, as well as primates.

[0034] The term "sequence identity percentage" is determined by comparing two optimally aligned sequences across a comparison window, where, for the two optimal alignments, some of the polynucleotide or polypeptide sequences within the comparison window may contain additions or deletions (i.e., gaps) compared to a reference sequence (without additions or deletions). In some embodiments, the percentage is calculated by determining the number of positions in both sequences where identical nucleic acid bases or amino acid residues occur, determining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage.

[0035] Therefore, “major histocompatibility complex (MHC) proteins or molecules,” “MHC molecules,” “MHC proteins,” or “HLA proteins” refer to proteins that can bind to peptides resulting from the proteolytic cleavage of protein antigens, represent potential T cell epitopes, transport them to the cell surface, and present them to specific cells, particularly cytotoxic T lymphocytes or T helper cells. In some embodiments, class I MHC molecules consist of a heavy chain and a light chain, and are approximately 8-11 amino acids long, but can bind to peptides of 9 or 10 amino acids if they have a suitable binding motif, and present these peptides to cytotoxic T lymphocytes. In some embodiments, the peptides that bind to class I MHC molecules originate from endogenous protein antigens. In some embodiments, the heavy chain of a class I MHC molecule is preferably an HLA-A, HLA-B, or HLA-C monomer, and the light chain is β-2-microglobulin (β2M).

[0036] As used herein, terms such as “prevent,” “preventing,” “prevention,” and “preventive therapy” refer to reducing the likelihood of developing a disease or condition in individuals who do not currently have the disease or condition but are at risk of developing it or are susceptible to developing it.

[0037] The terms “sample,” “patient sample,” and “biological sample” encompass a variety of sample types obtained from patients, individuals, or subjects and can be used in diagnostic assays, prognostic assays, and / or monitoring assays. Patient samples may be obtained from healthy subjects, patients with illness, or lung cancer patients. In certain embodiments, the “provided” sample may be obtained by the person (or machine) performing the assay, or obtained by another person (or machine) and transferred to the person (or machine) performing the assay. Furthermore, samples obtained from patients may be divided, and only a portion may be used for diagnosis. Furthermore, the sample or a portion of it may be stored under conditions that preserve the sample for later analysis. This definition particularly includes blood and other liquid samples of biological origin (including, but not limited to, peripheral blood, serum, plasma, umbilical cord blood, amniotic fluid, cerebrospinal fluid, urine, saliva, feces, and synovial fluid), solid tissue samples, e.g., biopsy specimens or tissue cultures or cells and their offspring derived therefrom. In certain embodiments, the sample includes cerebrospinal fluid. In certain embodiments, the sample includes blood samples. In other embodiments, the sample includes plasma samples. In yet another embodiment, serum samples are used. The definition of “sample” also includes samples that, after being obtained, have been manipulated and washed by any means, for example by centrifugation, filtration, precipitation, dialysis, chromatography, or treatment with reagents, and have been concentrated for a particular population of cells. These terms further encompass clinical samples and also include cells in culture, cell supernatants, tissue samples, organs, etc. Samples may also include fresh-frozen tissue blocks and / or formalin-fixed, paraffin-embedded tissue blocks, for example, blocks prepared from clinical or pathological biopsies for pathological analysis or immunohistochemical study.

[0038] The terms "treat", "treated", "treating", "treatment", etc. are intended to refer to reducing or alleviating a disorder and / or symptoms associated with the disorder (e.g., neoplasia or tumor). "Treating" may refer to administering therapy to a subject after the onset or suspected onset of cancer. "Treating" includes the concept of "alleviating", and "alleviating" refers to reducing the frequency or severity of any symptoms associated with cancer or the effects of other diseases, and / or the occurrence or recurrence of side effects associated with cancer therapy. The term "treating" also encompasses the concept of "managing". "Managing" refers to reducing the severity of a particular disease or disorder in a patient or delaying its recurrence, e.g., extending the period of remission in a patient who has had the disease. Treatment of a disorder or condition does not require the disorder, condition, or symptoms associated therewith to be completely eliminated, but is understood not to exclude the complete elimination of the disorder, condition, or symptoms associated therewith.

[0039] The term "therapeutic effect" refers to some degree of reduction of one or more symptoms of a disorder (e.g., neoplasia or tumor) or its associated pathology. As used herein, "therapeutically effective amount" refers to the amount of an agent effective in extending the viability of a patient having such a disorder, reducing one or more signs or symptoms of the disorder, preventing or delaying more than would be expected in the absence of such treatment, etc., when administered as a single dose or multiple doses to cells or a subject. "Therapeutically effective amount" is intended to qualify the amount necessary to achieve a therapeutic effect. A physician or veterinarian of ordinary skill in the art will determine the "therapeutically effective amount" of the pharmaceutical composition required (e.g., ED 50) can be easily determined and prescribed. For example, a physician or veterinarian may start the dose of the compound of the Disclosure used in a pharmaceutical composition at a level lower than the level required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0040] The terms "transfected," "transformed," or "transduced" refer to the process by which exogenous nucleic acids are introduced into or transferred to host cells. A "transfected," "transformed," or "transduced" cell is a cell that has been transfected, transformed, or transduced with exogenous nucleic acids. Transfected / transformed / transduced cells include primary target cells and their offspring.

[0041] As used herein, "unnatural amino acid," "non-natural amino acid," "modified amino acid," or "chemically modified amino acid" refers to any amino acid other than the 20 α-amino acids encoded by genes, modified amino acids, or amino acid analogs. Unnatural amino acids have side chain groups that distinguish them from natural amino acids, but unnatural amino acids may also be natural compounds other than the 20 proteinogenic α-amino acids. In addition to the side chain groups that distinguish unnatural amino acids from natural amino acids, unnatural amino acids may have an extended backbone such as a β-amino acid.

[0042] Non-specific examples of unnatural amino acids include selenocysteine, pyrrolicin, homocysteine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methylphenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azide-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, and phosphonoth Rosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, unnatural analogs of tyrosine amino acids; unnatural analogs of glutamine amino acids; unnatural analogs of phenylalanine amino acids; unnatural analogs of serine amino acids; unnatural analogs of threonine amino acids; alkyl, aryl, acyl, azide, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, selen, ester, thioic acid, borethine Amino acids that are substituted with borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino acid, or any combination thereof; amino acids with photoactivatable crosslinkers; spin-labeled amino acids; fluorescent amino acids; amino acids with novel functional groups; amino acids that interact covalently or asynchronously with other molecules; metal-bonded amino acids; metal-containing amino acids; radioactive amino acids; photocaged and / or photoisomerizable amino acids; amino acids containing biotin or biotin analogs; glycosylated amino acids or carbohydrate-modified amino acids; keto-containing amino acids; amino acids containing polyethylene glycol or polyether; heavy atom-substituted amino acids; chemically cleavable or photocleavable amino acids; amino acids with long side chains; amino acids containing toxic groups; sugar-substituted amino acids, e.g., sugar-substituted serine; carbon-bonded sugar-containing amino acids; redox-active amino acids; acids containing α-hydroxy acids; amino acids containing aminothio acids;This includes α,α-disubstituted amino acids; β-amino acids; and cyclic amino acids other than proline. In embodiments of helicase described herein, one or more amino acids of the helicase are substituted with one or more unnatural amino acids and / or one or more natural amino acids.

[0043] As used herein, a "variant" of a polypeptide refers to an amino acid sequence modified by one or more amino acid residues. Variants may have "conservative" changes, where the substituted amino acids have similar structural or chemical properties (e.g., exchange of leucine for isoleucine). More rarely, variants may have "non-conservative" changes (e.g., exchange of glycine for tryptophan). Similar minor variations may include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without loss of biological activity may be found using computer programs well known in the art, such as LASERGENE software (DNASTAR).

[0044] As used herein, the term “virus” includes any type of virus or viral vector. For example, adenovirus, adeno-associated virus (AAV), recombinant adeno-associated virus (rAAV), herpes simplex virus, lentivirus, retrovirus, alphavirus, flavivirus, rhabdovirus, measles virus, Newcastle disease virus, poxvirus, vaccinia virus, modified Ankara virus, vesicular stomatitis virus, picornavirus. In various embodiments, the virus is a chimeric virus, synthetic virus, recombinant virus, mosaic virus, or pseudotyped virus. In some embodiments, the AAV / rAAV disclosed herein may also be AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and / or AAV10 capsid.

[0045] The vaccines of this disclosure may induce remission of a disease as described herein. “Inducing remission” means reducing, suppressing, attenuating, decreasing, stopping, or stabilizing the onset or progression of a disease (e.g., neoplasia, tumor, autoimmune disease, infection, etc.).

[0046] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs derived from any species to which the compositions and methods disclosed herein are applicable. Accordingly, these terms include, but are not limited to, genes and gene products derived from humans and mice. When a gene or gene product from a particular species is disclosed, this disclosure is intended to be illustrative only and should not be construed as limiting unless explicitly indicated by the context in which this disclosure is made. Accordingly, for example, the genes or gene products disclosed herein relate in some embodiments to mammalian nucleic acid sequences and amino acid sequences and are intended to include homologous and / or orthologous genes and gene products derived from other animals, including, but not limited to, other mammals, fish, amphibians, reptiles, and birds. In preferred embodiments, the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides, and proteins are human. The term “gene” is also intended to include varieties.

[0047] Unless otherwise specified, the implementation of this disclosure will utilize techniques from chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture, and transgenic biology. For example, Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Sambrook et al., 1989, Molecular Cloning, 2nd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Sambrook and Russell, 2001, Molecular Cloning, 3rd Ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Ausubel et al., 1992), Current Protocols in Molecular Biology (John Wiley & Sons, with regular updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow and Lane, 1988, Antibodies, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Jakoby and Pastan, 1979; Nucleic Acid Hybridization (BD Hames & SJ Higgins eds. 1984); Transcription And Translation (BD Hames & SJ Higgins eds. 1984); Culture Of Animal Cells (RI Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B.Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors For Mammalian Cells (JH Miller and MP Calos eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (DM Weir and CC Blackwell, eds., 1986); Riott, Essential Immunology, 6th Edition, Blackwell Scientific Publications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986); See Westerfield, M., *The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio)* (4th Ed., Univ. of Oregon Press, Eugene, 2000).

[0048] Any composition or method provided herein may be combined with one or more of the other compositions and methods provided herein. [Brief explanation of the drawing]

[0049] Novel features of the present invention are described in detail in the appended claims. The features and advantages of the present invention will be further understood by referring to the following detailed description illustrating exemplary embodiments in which the principles of the present invention are utilized, and by referring to the appended drawings.

[0050] [Figure 1] Figure 1 shows a series of Word plots of the 9-mer peptide bond consensus sequences derived from each HLA allele. The data was obtained using the HLAthena browser. [Figure 2] Figure 2 shows the NetMHC predicted binding affinity for the parental KRAS G12D epitope (circled in a square) and all modified peptide ligand heterocritical epitopes across a group of HLAs. Gray stars indicate weakly binding epitopes. Black stars indicate strong binding agents. [Figure 3] Figure 3 shows a series of schematic diagrams illustrating the structural modeling of the KRAS G12D epitope GADGVGKSA on HLA-A*02:01. It shows rotomeral substitution of anchor amino acid residue 9 from alanine to leucine, rotomeral substitution of anchor amino acid residue 2 from alanine to leucine, and rotomeral substitution of anchor amino acid residue 6 from glycine to leucine. Contact is shown. HLA contact residues are shown. Black arrows indicate interactions with increased contact. [Figure 4A] Figures 4A and 4B show heatmaps and tables demonstrating HLA-specific mutant KRAS minimal epitope targets validated by mass-spec for heterocritical peptide design. Figure 4B shows an edited list of mutant KRAS minimal epitopes and HLA restriction confirmed by mass spectrometry. [Figure 4B] Please refer to the explanation in Figure 4A. [Figure 5A]Figures 5A–5C show tables, heatmaps, and plots demonstrating amino acid modifications of mKRAS epitopes predicted to improve HLA-A*02:01 binding affinity. Figure 5A shows the peptide binding consensus sequences of 10-mer (left) and 9-mer (right) epitopes to HLA-A*02:01. Amino acids are shaded according to their physiological properties. Figure 5B shows heatmaps of the predicted binding affinity (nM) of parental G12V 10-mer and 9-mer and APLs to HLA-A*02:01. Figure 5C shows an edited list of mutant 10-mer and 9-mer KRAS minimal epitopes (parents) constrained to HLA-A*02:01 and modified modified peptide ligands (APLs). [Figure 5B] Please refer to the explanation in Figure 5A. [Figure 5C] Please refer to the explanation in Figure 5A. [Figure 6A] Figures 6A–6C show tables, heatmaps, and plots demonstrating amino acid modifications of mKRAS epitopes predicted to improve HLA-A*03:01 binding affinity. Figure 6A shows the peptide binding consensus sequences of 10-mer (left) and 9-mer (right) epitopes to HLA-A*03:01. Amino acids are shaded according to their physiological properties. Figure 6B shows heatmaps of the predicted binding affinity (nM) of parental 10-mer (left - KRAS G12D, G12V, G12C) and 9-mer (right - KRAS G12R, G12V) and APLs to HLA-A*03:01. X indicates the location of the KRAS mutation. Figure 6C shows an edited list of mutant 10-mer and 9-mer KRAS minimal epitopes (parents) constrained to HLA-A*03:01 and their modified peptide ligands (APLs). X indicates the location of the KRAS mutation. [Figure 6B] Please refer to the explanation in Figure 6A. [Figure 6C] Please refer to the explanation in Figure 6A. [Figure 7A]Figures 7A–7C show tables, heatmaps, and plots demonstrating amino acid modifications of mKRAS epitopes predicted to improve HLA-A*11:01 binding affinity. Figure 7A shows the peptide binding consensus sequences of 10-mer (left) and 9-mer (right) epitopes to HLA-A*11:01. Amino acids are shaded according to their physiological properties. Figure 7B shows heatmaps of the predicted binding affinity (nM) of parental 10-mers (left - KRAS G12D, G12R, G12V, G12C) and 9-mers (right - KRAS G12D, G12R, G12V) to APL for HLA-A*11:01. X indicates the location of the KRAS mutation. Figure 7C shows an edited list of the HLA-A*11:01-bound mutant 10-mer and 9-mer KRAS minimal epitopes (parents) and their modified altered peptide ligands (APLs). X indicates the location of the KRAS mutation. [Figure 7B] Please refer to the explanation in Figure 7A. [Figure 7C] Please refer to the explanation in Figure 7A. [Figure 8A] Figures 8A–8C show tables, heatmaps, and plots demonstrating amino acid modifications of mKRAS epitopes predicted to improve HLA-A*30:01 binding affinity. Figure 8A shows the peptide binding consensus sequences of the 10-mer (left)F and 9-mer (right) epitopes to HLA-A*30:01. Amino acids are shaded according to their physiological properties. Figure 8B shows heatmaps of the predicted binding affinity (nM) of parental 10-mer (left - KRAS G12R, G12V) and 9-mer (right - KRAS G12R, G12V) and APLs to HLA-A*30:01. X indicates the location of the KRAS mutation. Figure 8C shows an edited list of mutant 10-mer and 9-mer KRAS minimal epitopes (parents) constrained to HLA-A*30:01 and their modified peptide ligands (APLs). X indicates the location of the KRAS mutation. [Figure 8B] Please refer to the explanation in Figure 8A. [Figure 8C] Please refer to the explanation in Figure 8A. [Figure 9A]Figures 9A–9C show tables, heatmaps, and plots demonstrating amino acid modifications of mKRAS epitopes predicted to improve HLA-A*68:01 binding affinity. Figure 9A shows the peptide binding consensus sequences of 10-mer (left) and 9-mer (right) epitopes to HLA-A*68:01. Amino acids are shaded according to their physiological properties. Figure 9B shows heatmaps of the predicted binding affinity (nM) of parental 10-mer (left - KRAS G12D G12C, G12R, G12V) and 9-mer (right - KRAS G12V) and APLs to HLA-A*68:01. X indicates the location of the KRAS mutation. Figure 9C shows an edited list of mutant 10-mer and 9-mer KRAS minimal epitopes (parents) constrained to HLA-A*68:01 and their modified altered peptide ligands (APLs). X indicates the location of the KRAS mutation. [Figure 9B] Please refer to the explanation in Figure 9A. [Figure 9C] Please refer to the explanation in Figure 9A. [Figure 10A] Figures 10A–10C show tables, heatmaps, and plots demonstrating amino acid modifications of mKRAS epitopes predicted to improve HLA-B*07:02 binding affinity. Figure 10A shows the peptide binding consensus sequences of 10-mer epitopes matched to HLA-B*07:02. Amino acids are colored according to their physiological properties. Figure 10B shows heatmaps of the predicted binding affinity (nM) of parental 10-mer KRAS G12D and G12R epitopes and APLs to HLA-B*07:02. X indicates the location of the KRAS mutation. Figure 10C shows an edited list of HLA-B*07:02-constrained mutant 10-mer KRAS minimal epitopes (parents) and modified modified peptide ligands (APLs). X indicates the location of the KRAS mutation. [Figure 10B] Please refer to the explanation in Figure 10A. [Figure 10C] Please refer to the explanation in Figure 10A. [Figure 11A]Figures 11A–11C show tables, heatmaps, and plots illustrating amino acid modifications of mKRAS epitopes predicted to improve HLA-C*01:02 binding affinity. Figure 11A shows the peptide binding consensus sequence of a 9-mer epitope matched to HLA-C*01:02. Amino acids are colored according to their physiological and chemical properties. Figure 11B shows a heatmap of the predicted binding affinity (nM) of the parent 9-mer KRAS G12V epitope and its APL to HLA-C*01:02. Figure 11C shows an edited list of known mutant 10-mer KRAS minimal epitopes (parents) and modified modified peptide ligands (APLs) constrained to HLA-C*01:02. [Figure 11B] Please refer to the explanation in Figure 11A. [Figure 11C] Please refer to the explanation in Figure 11A. [Figure 12A] Figures 12A–12C show tables, heatmaps, and plots illustrating amino acid modifications of mKRAS epitopes predicted to improve HLA-C*08:02 binding affinity. Figure 12A shows the peptide binding consensus sequences of 10-mer (left) and 9-mer (right) epitopes to HLA-C*08:02. Amino acids are colored according to their physiological and chemical properties. Figure 12B shows heatmaps of the predicted binding affinity (nM) of parental 10-mer (left-KRAS G12D) and 9-mer (right-KRAS G12D) and APLs to HLA-C*08:02. Figure 12C shows an edited list of mutant 10-mer and 9-mer KRAS minimal epitopes (parent) constrained to HLA-C*08:02 and their modified peptide ligands (APLs). [Figure 12B] Please refer to the explanation in Figure 12A. [Figure 12C] Please refer to the explanation in Figure 12A. [Figure 13A]Figure 13 shows tables, heatmaps, and plots demonstrating amino acid modifications of mKRAS epitopes predicted to improve HLA-C*03:03 binding affinity. Figure 13A shows the peptide binding consensus sequences of 10-mer (left) and 9-mer (right) epitopes to HLA-C*03:03. Amino acids are colored according to their physiological properties. Figure 13B shows heatmaps of the predicted binding affinity (nM) of parental 10-mer (left - KRAS G12V) and 9-mer (right - KRAS G12V) and APLs to HLA-C*03:03. Figure 13C shows an edited list of mutant 10-mer and 9-mer KRAS minimal epitopes (parent) constrained to HLA-C*03:03 and their modified altered peptide ligands (APLs). [Figure 13B] Please refer to the explanation in Figure 13A. [Figure 13C] Please refer to the explanation in Figure 13A. [Figure 14A] Figures 14A–14C show a series of heatmaps, schematics, and graphs illustrating the KRAS APL binding assay-MBL tetramer quick-switch assay. Figure 14A shows a schematic of the KRAS modified peptide ligand binding assay-tetramer binding assay for HLA-A*11:01 tetramers. HLA-A*11:01 tetramers were incubated with 10 mM KRAS parent epitope or APL at RT for 4 hours. Peptide loading was then quantified by flow cytometry analysis using an anti-exiting peptide antibody. Figure 14B shows peptide loading calculated as a percentage compared to (+) control peptide and (-) control without peptide. Figure 14C shows the NetMHC predicted binding affinity to HLA-A*11:01-specific KRAS peptides and APL tested using this assay. [Figure 14B] Please refer to the explanation in Figure 14A. [Figure 14C] Please refer to the explanation in Figure 14A. [Figure 15-1]Figure 15 shows a series of graphs illustrating the results obtained from the KRAS APL HLA-A*11:01 binding assay. Percentage binding of modified peptide ligands to the KRAS G12D, G12R, G12V, and G12C 10-mer and 9-mer parent epitopes. Percentage binding of each APL was calculated for each parent epitope. [Figure 15-2] Please refer to the explanation in Figure 15-1. [Figure 16A] Figures 16A–16D show schematic diagrams and a series of graphs demonstrating that the KRAS G12D modified peptide ligand stimulates a T cell response to the parent antigen in vitro. In vitro immunogenicity assays were performed against the KRAS G12D modified peptide ligand in healthy HLA-A*11:01 donors. Figure 16A shows a schematic of the in vitro T cell antigen stimulation and expansion assay. Healthy donor dendritic cells were generated by attaching monocytes to a flask and differentiated in 800 U / mL hGMCSF and 400 IU / mL hIL-4 for 7 days. The dendritic cells were then matured in hIL-1B, IL-6, TNFα, and PGE2 for 2 days. Matured dendritic cells were then collected and treated with 40 μg / mL parent or modified peptide ligand for 2 hours. Applied dendritic cells were washed twice with PBS and then co-cultured with autologous CD8+ T cells in the presence of hIL-15, hIL-6, and hIL-21. After 7 days, more peptide-applied dendritic cells were added to each co-culture. The following day, fresh hIL-2 and IL-7 were added. Finally, the dendritic cells were cultured, and T cells were collected after 7 days and stained with HLA-A*11:01 tetramers containing the parental epitope sequence. Figure 16B shows the flow cytometry gate (gated to the HLA-DR-CD45RO+ event) of HLA-A*11:01 KRAS G12D 10-mer+ T cells. Figure 16C shows the quantified population of HLA-A*11:01 G12D 10-mer+ T cells in control, parental epitope, or modified peptide ligand expansion. Figure 16D shows KRAS G12D-HLA-A*11:01 tetramer staining of ATVGADGVGK-enlarged T cells 7 days after the third peptide immunization. A control without tetramer staining was used as a negative control. [Figure 16B] Please refer to the explanation in Figure 16A. [Figure 16C] Please refer to the explanation in Figure 16A. [Figure 16D] Please refer to the explanation in Figure 16A. [Figure 17] Figure 17 shows schematic diagrams of the KRAS G12D parent and modified peptide ligand-HLA contact and surface structure. Structural modeling of the KRAS G12D 10-mer parent epitope (G12D) or modified peptide ligand in HLA-A*11:01. The upper row shows the contact between the 10-mer peptide and the HLA-binding cleft. The lower row shows the surface area of ​​the peptide-HLA complex. Measurements are shown in Figure 20. [Figure 18] Figure 18 shows a schematic diagram of the hydrophobicity measurements of the KRAS G12D parent and modified peptide ligand-HLA complexes. Hydrophobicity measurements of the KRAS G12D parent and modified peptide ligand-HLA complexes are shown. Surface lipophilicity values ​​are mapped. [Figure 19] Figure 19 shows a schematic diagram of electrostatic potential measurements of the KRAS G12D parent and modified peptide ligand. Electrostatic potential measurements of the KRAS G12D parent and modified peptide ligand-HLA complexes are shown. Surface electrostatic potential values ​​were mapped. [Figure 20] Figure 20 shows a table of structural feature measurements for the KRAS G12D 10-mer parent epitope and modified peptide ligand modeled under the HLA-A*11:01 condition. [Figure 21]Figures 21A–21F show a series of plots and schematic diagrams demonstrating that the engineered peptide increased contact points and solvent exposure area and reduced stiffness in a mouse PDAC model. Figure 21A shows that mice were vaccinated twice with PBS, 50 μg of APL, or 5 μg of the parent peptide, with a 7-day interval between doses. Splenocytes were re-stimulated overnight with APL or the parent peptide 7 days after the final vaccine administration, and IFNγ production was measured using the Elispot. A Tukey multiple comparison test was performed after a two-way ANOVA. Figure 21B shows in silico structural modeling of APL or the parent peptide in mouse H2-Kb (mouse MHC class I molecule H2-Kb, minimal epitope of peptide 44, parent or engineered amino acid residue). Structural features were analyzed from the parent or APL MHC model. The number of contacts between the MHC-binding cleft and the peptide (Figure 21C) or mutant residue (Figure 21D) is shown. Figure 21E shows Cα-RMSD, a measure of peptide stiffness. Figure 21F shows the solvent exposure area (SASA). Independent Student t-tests were performed (ns=P>0.05, *P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001). [Figure 22A]Figures 22A–22C demonstrate the superior binding and T cell response of the engineered antigens to KRAS G12D APL1-HLA*11:01 and immunogenicity. Figure 22A shows nonlinear regression curves and EC50 calculations for binding between HLA-A*11:01 and the parental KRAS G12D 10-mer peptide and seven engineered antigens. Figure 22B shows seeding of 1 × 10⁶ expanded CD8 T cells treated with no peptide, unrelated peptide, parental peptide, or APL onto an Erispot Capture plate coated with anti-human IFNγ antibody. T cells were stimulated overnight with the control or parental antigen, and IFNγ captures were read. Uncorrected Fischer's LSD tests were performed after two-way ANOVA. (ns=P>0.05, *P≦0.05, **P≦0.01, ***P≦0.001, ****P≦0.0001). Figure 22C shows the in vitro immunogenicity studies of the HLA-A*11:01 parental epitope and its engineered counterpart (APL1). Human monocyte-derived DCs (moDCs) differentiated from healthy donor PBMCs were treated with 100 μM parental or APL1 + β2-microglobulin at 37°C (4 hours). After peptide application, the moDCs were washed with PBS and co-cultured with 6 × 10⁶ autologous CD8 T cells (+IL-2, IL-7, IL-15, and IL-21) for 7 days. Additional immunization was performed on peptide-treated moDCs on days 7, 14, and 21. T cells were collected and stained with HLA-A*11:01 tetramers loaded with the parent peptide, or subjected to IFNγ erythropoiesis. [Figure 22B] Please refer to the explanation in Figure 22A. [Figure 22C] Please refer to the explanation in Figure 22A. [Figure 23-1]Figure 23 shows a series of plots demonstrating a greater T cell response in KRAS G12D APL4 compared to the parental epitope in different HLA*11:01 human donors. In vitro immunogenicity studies of the HLA-A*11:01 parental epitope and its engineered counterpart (APL4). Human monocyte-derived dendritic cells (moDCs) differentiated from healthy donor PBMCs were treated with 100 μM parental or APL4 + β2-microglobulin at 37°C (4 hours). After peptide application, moDCs were washed with PBS and co-cultured with 6 × 10⁶ autologous CD8 T cells (+ IL-2, IL-7, IL-15, and IL-21) for 7 days. Additional immunization was performed on days 7, 14, and 21 using peptide-treated moDCs. T cells were collected and stained with HLA-A*11:01 tetramers loaded with the parental peptide (left) or targeted for IFNγ erythropoiesis (right). [Figure 23-2] Please refer to the explanation in Figure 23-1. [Figure 24-1] Figure 24A shows structural models of the A11-KRAS G12D parent epitope (left), A11-KRAS G12D APL1, and A11-KRAS G12D APL4 in the HLA-A*11:01 context. For each peptide, the top 10 energetically favorable models are shown (n=10). Figure 24B shows the CaRMSD of the 10 mermaid peptides in each model, calculated compared to all other models for that epitope. A Tukey multiple comparison test was performed after a two-way ANOVA. Figure 24C shows the structural analysis of the peptide neoantigen residue (G12D) in contact with HLA cleft residues associated with stabilizing peptide-HLA interactions (from left to right: Arg114, Gln155, Gln70, TRP147, Thr73). [Figure 24-2] Please refer to the explanation in Figure 24-1. [Figure 25] Figure 25A shows the binding affinity for KRAS G12D APL in the HLA-A*03:01 state. Figure 25B shows the binding affinity for KRAS G12D APL in the HLA-B*07:02 state. [Modes for carrying out the invention]

[0051] Detailed explanation This disclosure relates to predicting optimal anchor modifications that improve HLA-specific HLA binding and epitope immunogenicity by utilizing the structural features of neoantigen epitopes that bind to diverse HLA subtypes. The result of this prediction is an optimized heterocritic peptide vaccine candidate tailored to the patient's specific HLA type. This technique is applicable to shared neoantigens such as mutant KRAS G12D, which have been shown to be low immunogenic in patients in the context of vaccination. In certain aspects, this disclosure provides KRAS G12D heterocritic epitope vaccine candidates designed and modeled for diverse HLA subtypes. Furthermore, the software described herein is applicable to individual patient neoantigen targets, in which case the algorithm can prioritize immunogenic neoantigen targets based on the structural features of the neoantigen representation and identify the optimal heterocritic peptide for the patient's HLA subtype. In some embodiments, the heterocritic peptides disclosed herein are selected based on one or more assessments of the contact between the peptide and the HLA cleft residue, solvent exposure area (SASA), peptide stiffness, surface hydrophobicity, electrostatic potential as measures of CαRMSD for the top 10 predictive structural models for each peptide-HLA complex, and / or induction of maximum T cell expansion and activation compared to the parent peptide.

[0052] Immune system and antigen presentation The immune system can be classified into two functional subsystems: the innate immune system and the adaptive immune system. The innate immune system is the first-line defense against infection, and most potential pathogens are rapidly neutralized by this system before they can cause, for example, a visible infection. The adaptive immune system reacts to molecular structures called antigens of invading organisms. There are two types of adaptive immune responses, including humoral and cellular immune responses. In humoral immune responses, antibodies secreted into the body fluid by B cells bind to pathogen-derived antigens, and the pathogens are eliminated through various mechanisms, such as complement-mediated lysis. In cellular immune responses, T cells are activated, which can destroy other cells. For example, if a disease-related protein is present in a cell, it is fragmented into peptides within the cell by proteolysis. Then, specific cellular proteins attach to the antigen or the thus formed peptide and transport them to the cell surface. On the cell surface, the antigen or the thus formed peptide is presented to the body's molecular defense mechanisms, particularly T cells. Cytotoxic T cells recognize these antigens and kill the cells that possess them.

[0053] Molecules that transport peptides and present them on the cell surface are called major histocompatibility complex (MHC) proteins. MHC proteins are classified into two types called MHC class I and MHC class II. The structures of the two MHC class proteins are very similar. However, they have very different functions. MHC class I proteins are present on the surface of almost all cells in the body, including most tumor cells. MHC class I proteins are loaded with antigens that usually originate from endogenous proteins, or pathogens present inside the cell, and are then presented to naive or cytotoxic T lymphocytes (CTLs). MHC class II proteins are present on the surface of dendritic cells, B lymphocytes, macrophages, and other antigen-presenting cells. These primarily present peptides processed from external antigen sources, i.e., from outside the cell, to T helper (Th) cells. Most peptides that bind to MHC class I proteins originate from cytoplasmic proteins produced in the organism's own healthy host cells and usually do not stimulate an immune response. Therefore, cytotoxic T lymphocytes that recognize class I MHC molecules presenting such self-peptides are either eliminated in the thymus (central tolerance) or eliminated or inactivated after release from the thymus, i.e., tolerated (peripheral tolerance). MHC molecules can stimulate an immune response when they present peptides to intolerant T lymphocytes. Cytotoxic T lymphocytes have both a T cell receptor (TCR) and a CD8 molecule on their surface. The T cell receptor can recognize and bind to peptides complexed with MHC class I molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor that can bind to a specific MHC / peptide complex.

[0054] Peptide antigens attach to MHC class I molecules within the endoplasmic reticulum via competitive affinity binding before being presented on the cell surface. Here, the affinity of individual peptide antigens is directly related to their amino acid sequence and the presence of specific binding motifs at defined positions within that sequence. If the sequences of such peptides are known, the immune system can be manipulated to combat diseased cells, for example, using peptide vaccines. The human leukocyte antigen (HLA) system is a gene complex that codes for major histocompatibility complex (MHC) proteins in humans.

[0055] This disclosure provides methods for predicting peptides that can bind to HLA alleles. These embodiments include a set of candidate peptide sequences and include the step of identifying one or more structural features that indicate that the binding pocket or cleft of the HLA allele will be occupied by the candidate peptide. This is done, for example, by identifying the positional sequence similarity of peptide epitopes that bind to one or more HLA haplotypes. An HLA-peptide structure having greater sequence identity than a control HLA-peptide structure is selected, and a machine learning algorithm models a heterocritical neoepitope in the HLA cleft by introducing an amino acid rotomer having the lowest energy confirmation at each position. These structural features are input into a machine learning algorithm model to simulate the occupation of one or more binding peptides and one or more unbinding peptides on the HLA binding pocket in the crystal structure of the HLA allele or a similar crystal structure of HLA alleles. The structural features can be extracted from an output model created during the simulation. The structural features can also be identified using a machine learning algorithm model that infers the occupation of one or more candidate peptides on the HLA binding pocket. The inference may also be based on a model trained using simulated models of peptides that have been verified to bind to HLA alleles and peptides that have been verified not to bind to HLA alleles.

[0056] Vaccine therapies (e.g., cancer and infection) rely on the precise selection of immunizing peptides to enhance the immune response (e.g., against tumor-specific neoepitopes or viral epitopes). The ability to predict which epitopes will be presented, given a patient's specific HLA allele complements, is a fundamental requirement for successful vaccine design. Furthermore, the ability to predict which epitopes will be presented, given the unique mutation accumulations in different tumors and a patient's specific HLA allele complements, is a fundamental requirement for successful cancer vaccine design.

[0057] In certain embodiments, an initial input of a candidate peptide or a set of candidate peptides, e.g., a KRAS peptide, is provided. In certain embodiments, the candidate peptide or set of candidate peptides may be obtained from a subject or group of subjects requiring an immune response or a modified immune response. In certain embodiments, the candidate peptides can be identified in a peptide sequence database (e.g., derived from sequencing of subjects in which the immunogenic composition has a specific state of usefulness).

[0058] In certain embodiments, the peptide sequence database includes HLA allele-binding peptides and non-binding peptides. For each HLA allele, candidate peptides can be isolated and sequenced to identify HLA-binding peptides. In certain embodiments, candidate peptides can be obtained by a) preparing a population of cells expressing one class I HLA allele or a pair of class II HLA alleles (one α chain and one β chain), b) isolating each HLA-peptide complex from the cells, c) isolating peptides from the HLA-peptide complexes, and d) sequencing the peptides.

[0059] A population of cells may express one class I HLA allele, one pair of class II HLA alleles, or one class I HLA allele and one pair of class II HLA alleles. Appropriate cell populations include, for example, class I-deficient cell lines expressing one HLA class I allele, class II-deficient cell lines expressing one pair of HLA class II alleles, or class I and class II-deficient cell lines expressing one HLA class I and / or one pair of class II alleles.

[0060] The cell population may be professional antigen-presenting cells such as macrophages, B cells, and dendritic cells. In certain embodiments, the cells are B cells or dendritic cells. In certain embodiments, the cells are tumor cells or cells derived from tumor cell lines. In certain embodiments, the cells are cells isolated from a patient.

[0061] In some embodiments, the cell population is further modified, for example, by increasing or decreasing the expression and / or activity of at least one gene. In certain embodiments, the gene encodes a member of the immunoproteasome. In some embodiments, the immunoproteasome is involved in the processing of HLA class I binding peptides and includes the LMP2(βii), MECL-1(β2i), and LMP7(β5i) subunits. In some embodiments, the immunoproteasome can also be induced by interferon-γ. Thus, in some embodiments, the cell population may be exposed to one or more cytokines, growth factors, or other proteins. In some embodiments, the cells are stimulated with inflammatory cytokines, such as interferon-γ, IL-Iβ, IL-6, and / or TNF-α. The cell population may also be subjected to various environmental conditions, such as stress (heat stress, oxygen deficiency, glucose starvation, DNA damaging agents, etc.). In some embodiments, cells are exposed to one or more chemotherapeutic drugs, radiation, targeted therapies, or immunotherapies. Therefore, the methods disclosed herein can be used to test the effects of various genes or conditions on HLA peptide processing and presentation. In certain embodiments, the conditions used are selected to match the conditions of the patient from whom the HLA peptide population is to be identified.

[0062] In some embodiments, any HLA allele can be expressed in a cell population. In some embodiments, there is interest in sequentially performing the methods provided herein on different HLA alleles so that the resulting datasets can be combined and used. In certain embodiments, the HLA alleles are selected to correspond to the genotype of interest. In certain embodiments, the HLA alleles are mutant HLA alleles, which may be non-native alleles or native alleles in patients with the disease. In some embodiments, the methods disclosed herein have the further advantage of identifying HLA-binding peptides for HLA alleles associated with various low-frequency disorders and alleles. Thus, in one method, the HLA alleles are present in the population at frequencies less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, and 1%. In some embodiments, the HLA alleles are present in the population at frequencies less than 1%.

[0063] In some embodiments, the method further includes the steps of isolating a peptide from an HLA-peptide complex and sequencing the peptide to identify a candidate input peptide. In some embodiments, the peptide is isolated from the complex by any method known to those skilled in the art, such as acid elution. In some embodiments, any suitable sequencing method can be used, including mass spectrometry, for example, liquid chromatography-mass spectrometry (LC-MS or LC-MS / MS, or alternatively HPLC-MS or HPLC-MS / MS).

[0064] In some embodiments, the HLA-allele-specific binding peptide sequence database includes at least 1,000 different binding peptide sequences. The methods disclosed herein may also be used to create a database containing HLA-allele-specific binding peptide sequences for multiple HLA-alleles, at least two different HLA-alleles, and at least five, at least 10, 15, 20, 30, or more different alleles.

[0065] In one aspect, this disclosure provides a plurality of HLA-allele-specific binding peptides or sequences thereof, where the peptides correspond to peptides presented by a particular HLA allele. More specifically, in some embodiments, the provided HLA-allele-specific binding peptide sequence database is obtained by carrying out the methods described herein.

[0066] Structural characteristics of peptides In one aspect, the present disclosure provides a method for identifying HLA-allele-specific binding peptides, comprising the step of analyzing structural features indicating peptide occupation on the binding pocket of an HLA allele. In a particular embodiment, the structural features are amino acid residues that can be fitted to a model of peptide occupation on the binding pocket of an HLA allele (e.g., increased hydrophobicity, exposed hydrophobic surface and charge determined by peptide conformation within the binding pocket, and the size and position of various amino acid side chains). In certain embodiments, structural features are energy features not encoded by the peptide sequence but by the modeled three-dimensional structure of peptide occupancy on the HLA allele binding pocket (e.g., energies of attractive, repulsive, and solvation; energies of side-chain and backbone hydrogen bonds; and energies and probabilities of side-chain and backbone conformations) (see, e.g., Alford RF, Leaver-Fay A, Jeliazkov JR, O'Meara MJ, DiMaio FP, Park H, et al. The Rosetta all-atom energy function for macromolecular modeling and design. J Chem Theory Comput. (2017) 13:3031-48; and Riley et al., Structure Based Prediction of Neoantigen Immunogenicity. Front Immunol. 2019 Aug. 28; 10:2047).

[0067] In certain embodiments, the binding pocket is determined by structural analysis. Non-restrictive structural analysis methods include X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy (Alberts B, Johnson A, Lewis J, et al. Molecular Biolog). YThis includes (Analyzing Protein Structure and Function). In certain embodiments, crystal structures are used to verify the binding pocket. Any HLA-allele crystal structure can be used to simulate the occupancy of the peptide in the binding pocket. In certain embodiments, the structure is obtained by creating a crystal structure of the HLA allele. In certain embodiments, the crystal structure of the HLA molecule is obtained from a database (e.g., Protein Data Bank (PDB, rcsb.org)). In certain embodiments, if the structure of the HLA allele of interest is neither available nor can be created, the structure of a similar HLA allele is used. In certain embodiments, the similar HLA alleles include the HLA allele with the greatest similarity between the amino acid sequences of the binding pocket. In some embodiments, the similarity of the binding pocket can be calculated as the sum of pairwise residue similarities according to a 20×20 amino acid similarity matrix. In certain embodiments, similar HLA alleles include HLA alleles that have similarity between their binding motifs (i.e., two alleles are considered similar if they bind to similar peptides). In certain embodiments, an HLA allele binds to two or more of the same peptides. In certain embodiments, similar HLA alleles include HLA alleles from the same class (e.g., HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, HLA-K, and HLA-L). In certain embodiments, similar HLA alleles include HLA alleles that have the greatest amino acid sequence identity with respect to the HLA allele of interest. In certain embodiments, similar HLA alleles include HLA alleles that have at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with respect to the HLA allele of interest.

[0068] In certain embodiments, the structural features described herein are identified by simulating a model of how a candidate peptide occupies an HLA binding pocket. In certain embodiments, the method includes the step of analyzing a simulation of peptide occupancy on the binding pocket of an HLA allele.

[0069] In certain embodiments, how a peptide occupies the HLA-binding pocket can be computationally simulated using Multiple Sequence Comparison by Log-Expectation (MUSCLE) peptide sequence alignment, for example, by taking into account the positional sequence similarity of candidate peptide epitopes, such as the KRAS peptide. All eight epitopes that bind to each HLA and have a crystalline structure, such as 8-mer, 9-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitopes, are available.

[0070] In certain embodiments, the immunogenic peptide contains approximately 5 to 50 amino acids. In certain embodiments, the immunogenic peptide contains approximately 7 to 45 amino acids. In certain embodiments, the immunogenic peptide contains approximately 8 to 40 amino acids. In certain embodiments, the immunogenic peptide contains approximately 9 to 35 amino acids. In certain embodiments, the immunogenic peptide contains approximately 9 to 30 amino acids. In certain embodiments, the immunogenic peptide contains approximately 9 to 25 amino acids.

[0071] In certain embodiments, the immunogenic peptide comprises about 5 to about 50 amino acids, including an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NO:1-838. In some embodiments, the immunogenic peptides disclosed herein include one amino acid sequence from SEQ ID NO: 1-838. In some embodiments, the immunogenic peptides disclosed herein include one amino acid sequence from SEQ ID NO: 1-838 having one or more conserved amino acid substitutions. In certain embodiments, the immunogenic peptide includes an amino acid sequence having at least 95% sequence identity to one or more of SEQ ID NO: 1-838 having one or more conserved substitutions. In certain embodiments, the immunogenic peptide includes SEQ ID NO: 2. In some embodiments, the immunogenic peptide includes SEQ ID NO: 3. In some embodiments, the immunogenic peptide includes SEQ ID NO: 833. In some embodiments, the immunogenic peptide includes SEQ ID NO: 834.

[0072] In a particular embodiment, the immunogenic peptide contains one or more sequences of five amino acids from SEQ ID NO: 1 to 838.In certain aspects, immunogenic peptides are SEQ ID NO:2, 3, 5~12, 14~21, 23~30, 32~39, 41~48, 50~57, 59~66, 68~75, 77~84, 86~93, 85~99, 101~105, 107~111, 113~117, 119~123, 125~129, 131~1 35, 137~141, 143~146, 148~152, 154~162, 164~168, 170~179, 181~185, 187~196, 198~202, 204~213, 215~219, 221~230, 232~236, 238~244, 246 ~252, 254~260, 262~268, 270~276, 278~284, 286~292, 294~300, 302~308, 310~316, 318~324, 326~332, 334~340, 342~348, 350~356, 358~364, 366~372, 374~380, 382~388, 390~396, 398~404, 406~412, 414~420, 422~428, 430~436, 438~443, 445~450, 452~457, 459~464, 466~471, 473~478 , 480~485, 487~492, 494~499, 501~506, 508~512, 514~518, 520~524, 526~530, 532~536, 538~542, 544~548, 550~554, 556~560, 562~566, 568~573, 585~580, 582~587, 589~594, 596~601, 603~607, 609~613, 615~619, 621~625, 627~631, 633~638, 340~644, 646~651, 653~657, 659~664, 66 It contains one or more consecutive five amino acids from among 6-670, 672-677, 679-683, 685-690, 692-696, 698-703, 705-711, 713-718, 720-726, 728-733, 735-741, 743-748, 750-756, 758-763, 765-771, 773-777, 779-783, 785-789, 791-795, 797-801, 803-807, 809-813, 815-819, 821-825, 827-831, 833, or 834.In a particular embodiment, the immunogenic peptide contains one or more sequences of six amino acids from SEQ ID NO: 1 to 838.In certain aspects, immunogenic peptides are SEQ ID NO:2, 3, 5~12, 14~21, 23~30, 32~39, 41~48, 50~57, 59~66, 68~75, 77~84, 86~93, 85~99, 101~105, 107~111, 113~117, 119~123, 125~129, 131~1 35, 137~141, 143~146, 148~152, 154~162, 164~168, 170~179, 181~185, 187~196, 198~202, 204~213, 215~219, 221~230, 232~236, 238~244, 246 ~252, 254~260, 262~268, 270~276, 278~284, 286~292, 294~300, 302~308, 310~316, 318~324, 326~332, 334~340, 342~348, 350~356, 358~364, 366~372, 374~380, 382~388, 390~396, 398~404, 406~412, 414~420, 422~428, 430~436, 438~443, 445~450, 452~457, 459~464, 466~471, 473~478 , 480~485, 487~492, 494~499, 501~506, 508~512, 514~518, 520~524, 526~530, 532~536, 538~542, 544~548, 550~554, 556~560, 562~566, 568~573, 585~580, 582~587, 589~594, 596~601, 603~607, 609~613, 615~619, 621~625, 627~631, 633~638, 340~644, 646~651, 653~657, 659~664, 66 It contains one or more consecutive six amino acids from among 6-670, 672-677, 679-683, 685-690, 692-696, 698-703, 705-711, 713-718, 720-726, 728-733, 735-741, 743-748, 750-756, 758-763, 765-771, 773-777, 779-783, 785-789, 791-795, 797-801, 803-807, 809-813, 815-819, 821-825, 827-831, 833, or 834.In a particular embodiment, the immunogenic peptide contains one or more sequences of seven amino acids from SEQ ID NO: 1 to 838.In certain aspects, immunogenic peptides are SEQ ID NO:2, 3, 5~12, 14~21, 23~30, 32~39, 41~48, 50~57, 59~66, 68~75, 77~84, 86~93, 85~99, 101~105, 107~111, 113~117, 119~123, 125~129, 131~1 35, 137~141, 143~146, 148~152, 154~162, 164~168, 170~179, 181~185, 187~196, 198~202, 204~213, 215~219, 221~230, 232~236, 238~244, 246 ~252, 254~260, 262~268, 270~276, 278~284, 286~292, 294~300, 302~308, 310~316, 318~324, 326~332, 334~340, 342~348, 350~356, 358~364, 366~372, 374~380, 382~388, 390~396, 398~404, 406~412, 414~420, 422~428, 430~436, 438~443, 445~450, 452~457, 459~464, 466~471, 473~478 , 480~485, 487~492, 494~499, 501~506, 508~512, 514~518, 520~524, 526~530, 532~536, 538~542, 544~548, 550~554, 556~560, 562~566, 568~573, 585~580, 582~587, 589~594, 596~601, 603~607, 609~613, 615~619, 621~625, 627~631, 633~638, 340~644, 646~651, 653~657, 659~664, 66 It contains one or more of seven consecutive amino acids, including 6-670, 672-677, 679-683, 685-690, 692-696, 698-703, 705-711, 713-718, 720-726, 728-733, 735-741, 743-748, 750-756, 758-763, 765-771, 773-777, 779-783, 785-789, 791-795, 797-801, 803-807, 809-813, 815-819, 821-825, 827-831, 833, or 834.In a particular embodiment, the immunogenic peptide contains one or more sequences of eight amino acids from SEQ ID NO: 1 to 838.In certain aspects, immunogenic peptides are SEQ ID NO:2, 3, 5~12, 14~21, 23~30, 32~39, 41~48, 50~57, 59~66, 68~75, 77~84, 86~93, 85~99, 101~105, 107~111, 113~117, 119~123, 125~129, 131~1 35, 137~141, 143~146, 148~152, 154~162, 164~168, 170~179, 181~185, 187~196, 198~202, 204~213, 215~219, 221~230, 232~236, 238~244, 246 ~252, 254~260, 262~268, 270~276, 278~284, 286~292, 294~300, 302~308, 310~316, 318~324, 326~332, 334~340, 342~348, 350~356, 358~364, 366~372, 374~380, 382~388, 390~396, 398~404, 406~412, 414~420, 422~428, 430~436, 438~443, 445~450, 452~457, 459~464, 466~471, 473~478 , 480~485, 487~492, 494~499, 501~506, 508~512, 514~518, 520~524, 526~530, 532~536, 538~542, 544~548, 550~554, 556~560, 562~566, 568~573, 585~580, 582~587, 589~594, 596~601, 603~607, 609~613, 615~619, 621~625, 627~631, 633~638, 340~644, 646~651, 653~657, 659~664, 66 It contains one or more consecutive eight amino acids, including 6-670, 672-677, 679-683, 685-690, 692-696, 698-703, 705-711, 713-718, 720-726, 728-733, 735-741, 743-748, 750-756, 758-763, 765-771, 773-777, 779-783, 785-789, 791-795, 797-801, 803-807, 809-813, 815-819, 821-825, 827-831, 833, or 834.In a particular embodiment, the immunogenic peptide contains one or more consecutive nine amino acids from SEQ ID NO: 1 to 838.In certain aspects, immunogenic peptides are SEQ ID NO:2, 3, 5~12, 14~21, 23~30, 32~39, 41~48, 50~57, 59~66, 68~75, 77~84, 86~93, 85~99, 101~105, 107~111, 113~117, 119~123, 125~129, 131~1 35, 137~141, 143~146, 148~152, 154~162, 164~168, 170~179, 181~185, 187~196, 198~202, 204~213, 215~219, 221~230, 232~236, 238~244, 246 ~252, 254~260, 262~268, 270~276, 278~284, 286~292, 294~300, 302~308, 310~316, 318~324, 326~332, 334~340, 342~348, 350~356, 358~364, 366~372, 374~380, 382~388, 390~396, 398~404, 406~412, 414~420, 422~428, 430~436, 438~443, 445~450, 452~457, 459~464, 466~471, 473~478 , 480~485, 487~492, 494~499, 501~506, 508~512, 514~518, 520~524, 526~530, 532~536, 538~542, 544~548, 550~554, 556~560, 562~566, 568~573, 585~580, 582~587, 589~594, 596~601, 603~607, 609~613, 615~619, 621~625, 627~631, 633~638, 340~644, 646~651, 653~657, 659~664, 66 It contains one or more consecutive nine amino acids, including 6-670, 672-677, 679-683, 685-690, 692-696, 698-703, 705-711, 713-718, 720-726, 728-733, 735-741, 743-748, 750-756, 758-763, 765-771, 773-777, 779-783, 785-789, 791-795, 797-801, 803-807, 809-813, 815-819, 821-825, 827-831, 833, or 834.

[0073] In certain embodiments, the immunogenic peptide contains approximately 7 to 45 amino acids, comprising an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 1 to 838. In certain embodiments, immunogenic peptides contain approximately 8 to 40 amino acids.

[0074] In certain embodiments, the immunogenic peptide contains about 9 to about 35 amino acids, which include an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 1 to 838.

[0075] In certain embodiments, the immunogenic peptide contains about 9 to about 30 amino acids, comprising an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 1 to 838.

[0076] In certain embodiments, the immunogenic peptide contains about 9 to about 25 amino acids, which include an amino acid sequence having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 1 to 838.

[0077] In certain embodiments, the mutant KRAS epitopes include G12V, G12D, G12C, G12R, G12A, G13D, or combinations thereof.

[0078] In certain embodiments, the KRAS variants of the present disclosure include amino acid sequences having at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any one of SEQ ID NO: 1 to 838.In some embodiments, the KRAS variants disclosed herein are SEQ ID NO:2, 3, 5~12, 14~21, 23~30, 32~39, 41~48, 50~57, 59~66, 68~75, 77~84, 86~93, 85~99, 101~105, 107~111, 113~117, 119~123, 125~129, 131~ 135, 137~141, 143~146, 148~152, 154~162, 164~168, 170~179, 181~18 5, 187~196, 198~202, 204~213, 215~219, 221~230, 232~236, 238~244, 2 46~252, 254~260, 262~268, 270~276, 278~284, 286~292, 294~300, 302~308, 310~316, 318~324, 326~332, 334~340, 342~348, 350~356, 358~364, 366~372, 374~380, 382~388, 390~396, 398~404, 406~412, 414~420, 422~428, 430~436, 438~443, 445~450, 452~457, 459~464, 466~471, 47 3-478, 480-485, 487-492, 494-499, 501-506, 508-512, 514-518, 520-524, 526-530, 532-536, 538-542, 544-548, 550-554, 556-560, 562-566, 568-573, 585-580, 582-587, 589-594, 596-601, 603-607, 609-613, 615-619, 621-625, 627-631, 633-638, 340-644, 646-651, 653-657, 659 Contains any one of the following amino acid sequences: ~664, 666~670, 672~677, 679~683, 685~690, 692~696, 698~703, 705~711, 713~718, 720~726, 728~733, 735~741, 743~748, 750~756, 758~763, 765~771, 773~777, 779~783, 785~789, 791~795, 797~801, 803~807, 809~813, 815~819, 821~825, 827~831, 833, or 834.In a particular embodiment, the KRAS variant contains an amino acid sequence having at least 95% sequence identity with respect to one or more of the SEQ ID NOs.In some embodiments, the KRAS variants disclosed herein have one or more conservative substitutions, SEQ ID NO:2, 3, 5~12, 14~21, 23~30, 32~39, 41~48, 50~57, 59~66, 68~75, 77~84, 86~93, 85~99, 101~105, 107~111, 113~117, 119~123, 125~129, 131~ 135, 137~141, 143~146, 148~152, 154~162, 164~168, 170~179, 181~18 5, 187~196, 198~202, 204~213, 215~219, 221~230, 232~236, 238~244, 2 46~252, 254~260, 262~268, 270~276, 278~284, 286~292, 294~300, 302~308, 310~316, 318~324, 326~332, 334~340, 342~348, 350~356, 358~364, 366~372, 374~380, 382~388, 390~396, 398~404, 406~412, 414~420, 422~428, 430~436, 438~443, 445~450, 452~457, 459~464, 466~471, 47 3-478, 480-485, 487-492, 494-499, 501-506, 508-512, 514-518, 520-524, 526-530, 532-536, 538-542, 544-548, 550-554, 556-560, 562-566, 568-573, 585-580, 582-587, 589-594, 596-601, 603-607, 609-613, 615-619, 621-625, 627-631, 633-638, 340-644, 646-651, 653-657, 659 Contains any one of the following amino acid sequences: ~664, 666~670, 672~677, 679~683, 685~690, 692~696, 698~703, 705~711, 713~718, 720~726, 728~733, 735~741, 743~748, 750~756, 758~763, 765~771, 773~777, 779~783, 785~789, 791~795, 797~801, 803~807, 809~813, 815~819, 821~825, 827~831, 833, or 834.In certain embodiments, the KRAS variant includes SEQ ID NO:2. In some embodiments, the KRAS variant includes SEQ ID NO:3. In some embodiments, the KRAS variant includes SEQ ID NO.833. In some embodiments, the KRAS variant includes SEQ ID NO.834.

[0079] In certain embodiments, KRAS and / or mKRAS peptides include one or more modified amino acids, unnatural amino acids, substituted amino acids, or combinations thereof. Therefore, SEQ ID NO:2, 3, 5~12, 14~21, 23~30, 32~39, 41~48, 50~57, 59~66, 68~75, 77~84, 86~93, 85~99, 101~105, 107~111, 113~117, 119~123, 125~129, 1 31~135, 137~141, 143~146, 148~152, 154~162, 164~168, 170~179, 181~185, 187~196, 198~202, 204~213, 215~219, 221~230, 232~236, 238 ~244, 246~252, 254~260, 262~268, 270~276, 278~284, 286~292, 294~300, 302~308, 310~316, 318~324, 326~332, 334~340, 342~348, 350~356, 358~364, 366~372, 374~380, 382~388, 390~396, 398~404, 406~412, 414~420, 422~428, 430~436, 438~443, 445~450, 452~457, 459~46 4, 466~471, 473~478, 480~485, 487~492, 494~499, 501~506, 508~512, 514~518, 520~524, 526~530, 532~536, 538~542, 544~548, 550~554, 556~560, 562~566, 568~573, 585~580, 582~587, 589~594, 596~601, 603~607, 609~613, 615~619, 621~625, 627~631, 633~638, 340~644, 6 46~651, 653~657, 659~664, 666~670, 672~677, 679~683, 685~690, 692~696, 698~703, 705~711, 713~718, 720~726, 728~733, 735~741, 743~748, 750~756, 758~763, 765~771, 773~777, 779~783, 785~789, 791~795, 797~801, 803~807, 809~813, 815~819, 821~825, 827~831, 833,KRAS and / or mKRAS peptides, containing any one of 834, further comprise one or more modified amino acids, non-natural amino acids, substituted amino acids, or combinations thereof. Non-limiting examples of non-natural amino acids include selenocysteine, pyrrolicin, homocysteine, O-methyl-L-tyrosine, L-3-(2-naphthyl)alanine, 3-methylphenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, tri-O-acetyl-GlcNAcβ-serine, L-DOPA, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, and p-acyl-L-phenylalanine. p-benzoyl-L-phenylalanine, L-phosphoserine, phosphonoserine, phosphonotyrosine, p-iodophenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, unnatural analogs of tyrosine amino acids; unnatural analogs of glutamine amino acids; unnatural analogs of phenylalanine amino acids; unnatural analogs of serine amino acids; unnatural analogs of threonine amino acids; alkyl, aryl, Acyl, azide, cyano, halo, hydrazine, hydrazide, hydroxyl, alkenyl, alkynyl, ether, thiol, sulfonyl, seleno, ester, thio acid, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, hydroxylamine, keto, or amino-substituted amino acids, or any combination thereof; fluorescent amino acids; amino acids with novel functional groups; amino acids that interact covalently or acovalently with other molecules; metal-bonded amino acids; metal-containing amino acids; radioactive amino acids; photocaged and / or photoisomerizable amino acids; amino acids containing biotin or biotin analogs; glycosylated amino acids or carbohydrate-modified amino acids; keto-containing amino acids; amino acids containing polyethylene glycol or polyether; heavy atom-substituted amino acids; chemically cleavable or photocleavable amino acids; amino acids with long side chains; sugar-substituted amino acids, e.g.,This includes sugar-substituted serines, carbon-bonded sugar-containing amino acids, redox-active amino acids, α-hydroxy acids, aminothio acids, α,α-disubstituted amino acids, β-amino acids, and cyclic amino acids other than proline.

[0080] Candidate therapeutic peptides In certain embodiments, the methods described herein are used to verify the effectiveness of neoantigen vaccines. In this regard, there is interest in determining which neoantigen peptides are likely to bind to the target HLA in order to function effectively as immunogenic peptides.

[0081] In some embodiments, the target-specific HLA allele or HLA genotype can be determined by any method known in the art.

[0082] One of the barriers to developing curative and tumor-specific immunotherapies is identifying and selecting highly specific and limited tumor antigens to evade autoimmunity. Tumor neoantigens arise as a result of genetic alterations within malignant cells (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.) and represent the most tumor-specific class of antigens. Neoantigens have been rarely used in cancer vaccines or immunogenic compositions due to technical difficulties in neoantigen identification, selection of optimized neoantigens, and production of neoantigens for use in vaccines or immunogenic compositions. These problems may be addressed by identifying mutations in neoplastic / tumor present at the DNA level in tumors but not in corresponding germline samples from a high proportion of subjects with cancer, analyzing the identified mutations by the methods embodied herein to create multiple neoantigen epitopes that are expressed within neoplastic / tumor and bind to a high proportion of patient HLA alleles, and synthesizing multiple neoantigen peptides for use in cancer vaccines or immunogenic compositions suitable for treating a high proportion of subjects with cancer.

[0083] In some embodiments, the therapeutic vaccines disclosed herein may involve (1) the identification of mutant peptides capable of binding to HLA molecules in a high proportion of individuals, and (2) the formulation of a drug as a long-peptide multi-epitope vaccine. In some embodiments, targeting as many mutant epitopes as possible allows for leveraging the immense capabilities of the immune system, preventing opportunities for immune evasion by downregulating specific immune target gene products, and compensating for known inaccuracies of epitope prediction approaches. In some embodiments, the synthetic peptides described herein provide a particularly useful means for efficiently preparing multiple immunogens and rapidly translating mutant epitope identification into an effective vaccine. In some embodiments, the peptides can be readily chemically synthesized and easily purified using reagents free from contaminating bacterial or animal substances. Small size allows for clear focus on mutant regions of proteins and also reduces irrelevant antigen competition with other components (unmutated proteins or viral vector antigens). In some embodiments, the vaccines disclosed herein may be used in combination with (3) a potent vaccine adjuvant. In some embodiments, an effective vaccine may require a potent adjuvant to initiate an immune response. In some embodiments, poly-ICLC, TLR3 agonists, and MDA5 and RIG3 RNA helicase domains have exhibited several desirable properties for vaccine adjuvants. These properties include induction of local and systemic activation of immune cells in vivo, production of stimulating chemokines and cytokines, and stimulation of antigen presentation by DCs. Furthermore, poly-ICLC has been shown to promote long-lasting CD4 in humans. + Response and CD8 + It is possible to induce a response.

[0084] In certain embodiments, a method for identifying the peptide best suited to prepare an immunogenic composition tailored to a subject includes the step of selecting a plurality of peptides from a particular set of peptides that can bind to the HLA protein of the subject. In some embodiments, a method for identifying a plurality of target-specific peptides to prepare a target-specific immunogenic composition is provided herein, wherein the subject has a tumor and the target-specific peptides are specific to the subject and the tumor of the subject. In certain embodiments, as provided by the method for identifying HLA binding described herein, each of the target-specific peptides comprises a different tumor neoepitope that is epitope-specific to the tumor of the subject and each binds to the HLA protein of the subject.

[0085] In certain embodiments, the cells used in the method for confirming HLA binding as described herein are antigen-presenting cells.

[0086] Neoantigen In certain embodiments, tumor antigens that bind to HLA alleles are identified by the methods described herein. In certain embodiments, the tumor antigen is a neoantigen. In a further aspect, the present disclosure provides a method for identifying a peptide comprising a tumor neoantigen, the method comprising the step of identifying a peptide that binds to an HLA allele in tumor cells derived from a patient's tumor, in accordance with a particular HLA allele.

[0087] In some embodiments, the tumor antigen includes KRAS, BRAF, FBWX7, FGFR3, IDH1, MUC4, NRAS, PIK3CA, PPP2R1A, PTEN, or TP53. In some embodiments, the tumor antigen includes one or more mutations. In some embodiments, one or more mutations include KRAS G12D, KRAS G12V, KRAS G12R, KRAS G12C, BRAF V600E, BRAF V600M, FBXW7 R465C, FBXW7 R465Q, FGFR3 S249Q, IDH1 R132C, MUC4 D3157N, NRAS Q61K, NRAS G545R, PIK3CA G545K, PIK3CA H1047R, PIK3CA R88Q, PPP2R1A P179R, PTEN R130G, PTEN R130Q, TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y.

[0088] In some embodiments, mutant epitopes are effective in inducing immune responses. In some embodiments, spontaneous tumor regression or long-term survival is associated with CD8 against mutant epitopes. +Correlates with T cell responses (Buckwalter and Srivastava P K. 「It is the antigen(s), stupid」 and other lessons from over a decade of vaccine therapy of human cancer. Seminars in immunology 20:296-300 (2008); Karanikas et al., High frequency of cytolytic T lymphocytes directed against a tumor-specific mutated antigen detectable with HLA tetramers in the blood of a lung carcinoma patient with long survival. Cancer Res. 61:3718-3724 (2001); Lennerz et al., The response of autologous T cells to a human melanoma is dominated by mutated neoantigens. Proc Natl Acad Sci USA. 102: 16013 (2005))."Immunoediting" can be tracked by changes in the expression of dominant mutant antigens in mice and humans (Matsushita et al., Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting. Nature 482:400 (2012); DuPage et al., Expression of tumor-specific antigens underlies cancer immunoediting. Nature 482:405 (2012); and Sampson et al., Immunologic escape after prolonged progression-free survival with epidermal growth factor receptor variant III peptide vaccination in patients with newly diagnosed glioblastoma. J Clin Oncol. 28:4722-4729 (2010)).

[0089] In some embodiments, each tumor contains multiple patient-specific mutations that alter the protein-coding content of a gene. Such mutations can produce modified proteins ranging from a single amino acid change (caused by a missense mutation) to frameshifts, the addition of long novel amino acid sequence regions due to misreading of stop codons, or translation of intron regions (novel open-reading frame mutations; neo-ORFs). In some embodiments, these mutant proteins are valuable targets for the host immune response against the tumor because, unlike native proteins, they do not undergo self-tolerant immunosuppression. In some embodiments, mutant proteins are more likely to be immunogenic and more specific to tumor cells compared to the patient's normal cells. Mutant proteins are sometimes called neoantigens. The terms “neoantigen” or “neoantigenous” refer to a class of tumor antigens resulting from tumor-specific mutations that alter the amino acid sequence of a genome-encoded protein.

[0090] Embodiments disclosed herein provide a method for identifying novel peptides, including but not limited to unannotated open reading frames (nuORFs), such as neoantigens, that can induce cancer-specific T cell responses. Genomic abnormalities in cancer cells give rise to mutant peptides (neoantigens) that are presented on human leukocyte antigen (HLA) molecules and recognized by T cells, thus inducing an immune response against cancer cells. In some embodiments, patients vaccinated with neoantigen-based peptides may show an expansion of neoantigen-specific T cells, making it a promising means of cancer treatment (Ott et al., An immunogenic personal neoantigen vaccine or patients with melanoma, Nature 2017 Jul. 13; 547(7662):217-221; Sahin et al., 2017 Personalized RNA mutanome vaccines mobilize polyspecific therapeutic immunity against cancer, Nature, vol. 547, 2017, pp. 222-226). In some embodiments, neoantigens can be predicted based on mutations detected by whole-genome sequencing (WES). In other embodiments, their expression levels are estimated using mRNA sequencing (RNA-seq).Ribosome profiling (Ribo-seq) has enabled the monitoring of mRNA translation and has been used to predict overtranslated novel unannotated ORFs (nuORFs) (Fields et al., 2015. A Regression-Based Analysis of Ribosome-Profiling Data Reveals a Conserved Complexity to Mammalian Translation, Mol Cell, vol. 60, pp. 816-827; Ji et al., 2015. Many IncRNAs, 5′UTRs, and pseudogenes are translated and some are likely to express functional proteins, ELIFE, vol. 4). Ribo-seq analysis of human fibroblasts infected with HSV-1 and HCMV has identified nuORFs that provide peptides presented on major histocompatibility complex class I (MHCI) (Erhard et al., 2018. Improved Ribo-seq enables identification of cryptic translation events, Nat. Methods, vol. 15, no. 5, pp. 363-366).

[0091] The methods disclosed herein can be used to select target-specific peptides presented by tumors for any neoplasia. “Neoplasia” means any disease caused by or resulting from an inappropriately high level of cell division, an inappropriately low level of apoptosis, or both. For example, cancer is an example of neoplasia. Examples of cancer include leukemia (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin’s disease, non-Hodgkin’s disease), Waldenström macroglobulinemia, heavy chain diseases, and solid tumors, such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma). Liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synoviomas, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma This includes, but is not limited to, carcinoma, renal cell carcinoma, hepatome, nile duct carcinoma, choriocarcinoma, seminoma, fetal cancer, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendrocyte, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma.

[0092] Vaccines and immunological compositions In certain embodiments, peptides identified pursuant to this disclosure are used in vaccines or immunological compositions to treat any disease or condition described herein (e.g., tumor, autoimmune, infection, transplant). The terms “vaccine” and “immunological composition” are used synonymously and in this context are intended to refer to a pooled sample of one or more antigenic peptides, e.g., at least one, at least two, at least three, at least four, at least five, or more antigenic peptides. “Vaccines” should be understood to include protective vaccines, which are compositions for inducing immunity to prevent and / or treat a disease (e.g., neoplastic / tumor). “Vaccines” should also be understood to include tolerizing vaccines, which are compositions for reducing immunity to prevent and / or treat a disease (e.g., autoimmune disease). Tolerizing vaccines may be formulated using antigenic epitopes that are specific to allergens or to autoimmune antigens identified pursuant to this disclosure. In some embodiments, protective vaccines may be formulated using pathogen-specific or cancer cell-specific antigenic epitopes. Accordingly, in some embodiments, vaccines may also be medical agents containing antigens and intended for use in humans or animals to produce specific protection and protective substances upon vaccination. The “vaccine composition” may include pharmaceutically acceptable excipients, carriers, or diluents.

[0093] In some embodiments, the vaccine may contain one or more peptides identified in accordance with this disclosure. For example, 1 to 10 peptides. The range provided herein is understood to be an abbreviated expression of all values ​​within this range. For example, the range 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. With respect to subranges, "nested subranges" extending from either endpoint of the range are particularly intended. For example, nested subranges of the exemplary range 1 to 50 may include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, and 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0094] In certain aspects, protective vaccines are used to treat cancer. Further examples of cancers and cancerous conditions that can be treated with the therapies described in this document include, but are not limited to, patients who have been diagnosed with cancer or who are at risk of developing cancer and who need to be treated. The subjects include solid tumors, e.g., breast, ovarian, prostate, lung, kidney, stomach, colon, testis, head and neck, pancreas, brain, melanoma, and other tumors of tissue organs, as well as hematological malignancies, e.g., lymphomas and leukemias including acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma, tumors of the brain and central nervous system (e.g., tumors of the meninges, brain, spinal cord, cranial nerves, and other parts of the CNS, e.g., gliablastoma or medulloblastoma); cancers of the head and / or neck, mammary gland tumors, tumors of the circulatory system (e.g., heart, mediastinum and pleura, as well as other intrathoracic organs, vascular tumors, and tumor-associated vascular tissue); tumors of the blood and lymphatic system (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt lymphoma, AIDS-associated lymphoma, malignant immunoproliferative disorders, multiple Myeloma, and malignant plasma cell neoplasms, lymphocytic leukemia, myeloid leukemia, acute lymphoblastic leukemia or chronic lymphocytic leukemia, monocytic leukemia, other leukemias of specific cell types, leukemias of unspecified cell types, unspecified malignant neoplasms of lymphoid tissue, hematopoietic tissue, and related tissues, e.g., diffuse large cell lymphoma, T-cell lymphoma, or cutaneous T-cell lymphoma); excretory systems (e.g., kidneys, Tumors of the renal pelvis, ureters, bladder, and other urinary organs; tumors of the gastrointestinal tract (e.g., esophagus, stomach, small intestine, colon, colorectum, sigmoid-rectal junction, rectum, anus, and anal canal); tumors of the liver and intrahepatic bile ducts, gallbladder, as well as tumors of the biliary tract, pancreas, and other parts of the digestive organs; tumors of the oral cavity (e.g., lips, tongue, gums, floor of the mouth, palate, parotid gland, salivary gland, tonsils, oropharynx, nasopharynx, piriform sinus). Tumors of the sinuses, hypopharynx, and other parts of the oral cavity; tumors of the reproductive system (e.g., vulva, vagina, cervix, uterus, ovaries, and other parts associated with the female reproductive organs, placenta, penis, prostate, testes, and other parts associated with the male reproductive organs); tumors of the airway (e.g., nasal cavity, middle ear, paranasal sinuses (accessory sinuses), larynx, trachea, bronchi, and lungs, e.g., small cell lung cancer and non-small cell lung cancer);Tumors of the skeletal system (e.g., bones and articular cartilage of the limbs, osteoarticular cartilage, and other sites); tumors of the skin (e.g., malignant melanoma of the skin, non-melanoma skin cancer, basal cell carcinoma of the skin, squamous cell carcinoma of the skin, mesothelioma, Kaposi's sarcoma); and tumors involving other tissues, including the peripheral and autonomic nervous systems, connective tissue and soft tissue, retroperitoneum and peritoneum, eyes, thyroid gland, adrenal gland, and other endocrine glands and related structures; secondary and unspecified malignant neoplasms of lymph nodes; secondary malignant neoplasms of the respiratory and digestive systems; and secondary malignant neoplasms of other sites. Accordingly, the population of subjects described herein may have any of the above cancer types. In other embodiments, the population of subjects may be all subjects with solid tumors, or all subjects with liquid tumors.

[0095] In some embodiments, cancers that can be treated with the therapies described herein may include, among other things, cases that are refractory to treatment with other chemotherapeutic agents. As used herein, the term “refractory” refers to cancers (and / or metastases thereof) that do not show an antiproliferative response, or only a weak antiproliferative response (e.g., do not show inhibition of tumor growth, or only weak inhibition of tumor growth) after treatment with another chemotherapeutic agent. These may also be cancers that cannot be satisfactorily treated with other chemotherapeutic agents. In some embodiments, refractory cancers include not only (i) cancers that have already failed with one or more chemotherapeutic agents during the treatment of the patient, but also (ii) cancers that can be shown to be refractory by other means, e.g., biopsy and culture in the presence of chemotherapeutic agents.

[0096] In some embodiments, the therapies described herein are also applicable to the treatment of patients who have not been treated before and who need to be treated.

[0097] In some embodiments, the therapies described herein may also be applicable to subjects who do not have detectable neoplasias but are at high risk of disease recurrence.

[0098] In some embodiments, the therapies described herein are also applicable when the subject has previously undergone autologous hematopoietic stem cell transplantation (AHSCT), particularly when the patient demonstrates residual disease after AHSCT. The post-AHSCT situation is characterized by a small volume of residual disease, the injection of immune cells to a state of homeostasis expansion, and the complete absence of standard relapse-delaying therapies. These characteristics offer a unique opportunity to delay disease relapse using neoplasm vaccines or immunogenic compositional compositions.

[0099] In vitro peptide / polypeptide synthesis The proteins or peptides disclosed herein can be prepared by any technique known to those skilled in the art, including expression of proteins, polypeptides, or peptides by standard molecular biological techniques, isolation of proteins or peptides from natural sources, in vitro translation, or chemical synthesis of proteins or peptides. In some embodiments, nucleotide sequences corresponding to various genes, as well as protein, polypeptide, and peptide sequences, have been previously disclosed and can be found in computerized databases known to those skilled in the art. One such database is the GenBank and GenPept databases of the National Center for Biotechnology Information, located on the National Institutes of Health website. The coding regions of known genes can be amplified and / or expressed using the techniques disclosed herein or in a manner known to those skilled in the art. Alternatively, various commercially available preparations of proteins, polypeptides, and peptides are known to those skilled in the art.

[0100] In some embodiments, peptides can be readily chemically synthesized using reagents free from bacterial or animal contaminants (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149-54, 1963). In certain embodiments, neoantigen peptides are prepared by (1) parallel solid-phase synthesis in a multi-channel instrument using homogeneous synthesis and cleavage conditions, (2) purification by RP-HPLC column and column stripping, rewashing without exchanging peptides, and (3) analysis with a limited set of the most informative assays. A Good Manufacturing Practices (GMP) footprint may be defined around a set of peptides tailored to individual patients, thus requiring a suite changeover procedure only when synthesizing peptides for different patients.

[0101] Alternatively, a nucleic acid (e.g., a polynucleotide) encoding the neoantigen peptide of this disclosure may be used to produce the neoantigen peptide in vitro. The polynucleotide may be a natural or stabilized form of polynucleotide, such as DNA, cDNA, PNA, CNA, RNA, single-stranded and / or double-stranded, or, for example, a polynucleotide having a phosphorothiate backbone, or a combination thereof, and may or may not contain introns, as long as it encodes a peptide. In some embodiments, in vitro translation is used to produce the peptide. Many exemplary systems are available to those skilled in the art (e.g., Retic Lysate IVT Kit, Life Technologies, Waltham, Mass.).

[0102] Expression vectors capable of expressing polypeptides can also be prepared. Expression vectors for different cell types are well known in the art and can be selected without excessive experimentation. Generally, DNA is inserted into an expression vector, such as a plasmid, in the correct orientation and with the correct reading frame for expression. If necessary, the DNA may be ligated to appropriate transcriptional and translational regulatory nucleotide sequences recognized by the desired host (e.g., bacteria), but generally, such regulation can be utilized within the expression vector. The vector is then introduced into host bacteria for cloning using standard techniques (see, for example, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0103] Expression vectors containing isolated polynucleotides, as well as host cells containing the expression vectors, are also intended. The neoantigen peptide may be provided in the form of an RNA or cDNA molecule encoding the desired neoantigen peptide. One or more neoantigen peptides of this disclosure may be encoded by a single expression vector.

[0104] In certain embodiments, the polynucleotide may also include a coding sequence for a tumor-specific neoantigen peptide, fused in the same reading frame with, for example, a polynucleotide that assists in the expression and / or secretion of a polypeptide from a host cell (e.g., a leader sequence that functions as a secretory sequence controlling the transport of polypeptides from the cell). The polypeptide having the leader sequence is a preprotein and may have a leader sequence that is cleaved by the host cell to form a mature form of the polypeptide.

[0105] In certain embodiments, it is possible to provide isolated nucleic acid molecules having a nucleotide sequence that is at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 96%), 97%, 98%, or 99% identical to the polynucleotide encoding the tumor-specific neoantigen peptide of the present disclosure. A polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with other nucleotides, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the amino-terminal or carboxyl-terminal position of the reference nucleotide sequence, or anywhere between these terminal positions, and may be scattered individually among nucleotides in the reference sequence, or scattered in one or more consecutive groups within the reference sequence.

[0106] In practice, whether any given nucleic acid molecule is at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, or 99% identical to a reference sequence can be determined using a conventionally known computer program, such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981) to find the best homology segment between two sequences. When using Bestfit or any other sequence alignment program to determine, for example, whether a particular sequence is 95% identical to a reference sequence in this disclosure, the identity percentage is calculated over the full length of the reference nucleotide sequence, and parameters are set so that a homology gap of up to 5% > the total number of nucleotides in the reference sequence is allowed.

[0107] Recombinant expression vectors may be used to amplify and express DNA encoding tumor-specific neoantigen peptides. A recombinant expression vector is a replicable DNA construct in which a synthetic DNA fragment or a DNA fragment derived from cDNA encoding a tumor-specific neoantigen peptide or a bioequivalent analog is functionally linked to an appropriate transcriptional or translational regulatory element derived from a mammalian gene, microbial gene, viral gene, or insect gene. In some embodiments, the transcription unit includes, as detailed herein, (1) a genetic element that plays a role in regulating gene expression, e.g., a transcription promoter or enhancer, (2) a structural or coding sequence that is transcribed into mRNA and translated into a protein, and (3) a set of appropriate transcriptional and translational start and termination sequences. Such regulatory elements may include operator sequences for controlling transcription. The ability to replicate in the host and selection genes that facilitate the recognition of the transformant, usually conferred by the origin of replication, can be further incorporated. DNA regions are functionally linked when these functions are related to each other. For example, the DNA of a signal peptide (secretion leader) can be functionally linked to polypeptide DNA if the polypeptide is expressed as a precursor involved in polypeptide secretion. A promoter can functionally ligate to a coding sequence if it controls the transcription of the coding sequence. Alternatively, a ribosome binding site can functionally ligate to a coding sequence if it is positioned so that the coding sequence is translated. Generally, functional ligation means continuity, and in the case of a secretion leader, it means continuity and being within the reading frame. Structural elements intended for use in yeast expression systems may include a leader sequence that enables extracellular secretion of the translated protein by the host cell. Alternatively, if the recombinant protein is expressed without a leader sequence or transport sequence, it may include an N-terminal methionine residue. This residue can optionally be cleaved later from the expressed recombinant protein to obtain the final product.

[0108] Expression vectors useful for eukaryotic hosts, particularly mammals or humans, include, for example, vectors containing expression regulatory sequences derived from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids, such as plasmids derived from Escherichia coli, including pCR1, pBR322, pMB9 and their derivatives, and plasmids with a broader host range, such as MI3 and filamentous single-stranded DNA phages.

[0109] Suitable host cells for polypeptide expression include prokaryotic cells, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include Gram-negative or Gram-positive organisms, such as Escherichia coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems may also be used. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are well known in the art (see Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985).

[0110] Various mammalian or insect cell culture systems are also advantageous for expressing recombinant proteins. Expression of recombinant proteins in mammalian cells is feasible because such proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells described by Gluzman (Cell 23:175, 1981), as well as other cell lines capable of expressing suitable vectors, including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), 293, HeLa, and BHK cell lines. Mammalian expression vectors may also include non-transcription elements, such as origins of replication, appropriate promoters and enhancers linked to the gene to be expressed, as well as other 5' or 3' adjacent non-transcription sequences, and 5' or 3' untranslated sequences, such as required ribosome binding sites, polyadenylation sites, splice donor and splice acceptor sites, and transcription termination sequences. Baculovirus systems for producing heterologous proteins in insect cells are outlined in Luckow and Summers, Bio / Technology 6:47 (1988).

[0111] Proteins produced by transformed hosts can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion-exchange column chromatography, affinity column chromatography, and sizing column chromatography), centrifugation, solubility differential chromatography, or any other standard technique for protein purification. Affinity tags, such as hexahistidine, maltose-binding domains, influenza coat sequences, and glutathione-S-transferase, can be attached to proteins to facilitate purification by passage on a suitable affinity column. Isolated proteins can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0112] In vivo peptide / polypeptide synthesis This disclosure also intends to use nucleic acid molecules, for example in the form of DNA / RNA vaccines, as vehicles for delivering neoantigen peptides / polypeptides to targets that need them, in vivo.

[0113] In certain embodiments, antigens may be administered to patients in need using plasmids. In some embodiments, these may be plasmids consisting of a potent viral promoter for in vivo transcription and translation of the gene of interest (or complementary DNA) (Mor, et al., (1995), Journal Immunol. 155 (4): 2039-2046). Occasionally, intron A may be included to improve mRNA stability and thus increase protein expression (Leitner et al. (1997), Journal Immunol. 159 (12): 6112-6119). Plasmids may also contain potent polyadenylation / transcription termination signals, such as bovine growth hormone or rabbit β-globulin polyadenylation sequences. Occasionally, multicistronic vectors are constructed to express multiple immunogens, or to express immunogens and immunostimulant proteins.

[0114] In some embodiments, methods for enhancing protein expression involve optimizing the codon usage frequency of pathogenic mRNA to suit eukaryotic cells. Another consideration is promoter selection. Such promoters may be the SV40 promoter or Roussarcoma virus (RSV). Plasmids may be introduced into animal tissue by a number of different methods. Two of the most commonly used approaches are injection of saline-dissolved DNA using standard subcutaneous needle and gene gun delivery. In some embodiments, the saline-dissolved injection may be performed intramuscularly (EVI) or intradermally (ID) into skeletal muscle, with the DNA delivered into the extracellular space. This can be assisted by electroporation by transiently damaging muscle fibers with a myotoxin, such as bupivacaine, or by using a hypertonic solution of saline or sucrose. The immune response to this delivery method may be influenced by many factors, including needle type, needle arrangement, injection speed, injection volume, muscle type, and the age, sex, and physiological state of the injected animal. In some embodiments, the peptides disclosed herein can be administered to DNA or RNA encoding them.

[0115] Alternative delivery methods may include aerosol delivery of naked DNA to mucosal surfaces such as the nasal and pulmonary mucosa, as well as topical administration of pDNA to the ocular and vaginal mucosa. Mucosal surface delivery has also been achieved using cationic liposome-DNA preparations, biodegradable microspheres, attenuated Shigella or Listeria vectors for oral administration to the intestinal mucosa, and recombinant adenovirus vectors. DNA or RNA may also be delivered to cells after the cell membrane has been lightly mechanically disrupted to make the cell temporarily permeable. Such light mechanical disruption of the membrane can be achieved by slowly pushing the cell through a small opening (Ex vivo Cytosolic Delivery of Functional Macromolecules to Immune Cells, Sharei et al., PLOS ONE DOI: 10.1371 / journal.pone.O1 18803 Apr. 13, 2015).

[0116] In certain embodiments, the neoplastic vaccine or immunogenic composition may comprise, for example, separate DNA plasmids encoding one or more neoantigen peptides / polypeptides as identified in accordance with this disclosure. As discussed herein, the precise selection of the expression vector may depend on the peptide / polypeptide to be expressed and is well within the scope of the art. The expected persistence of the DNA construct (e.g., in an episomal, non-replicating, non-integrated form in muscle cells) is expected to provide a long period of protection.

[0117] One or more antigenic peptides of this disclosure may be encoded and expressed in vivo using a virus-based system (e.g., an adenovirus system, an adeno-associated virus (AAV) vector, a poxvirus, or a lentivirus). In one embodiment, the novel vaccine or immunogenic composition may include a virus-based vector, such as an adenovirus, for use in human patients where such use is necessary (see, for example, Baden et al. First-in-human evaluation of the safety and immunogenicity of a recombinant adenovirus serotype 26 HIV-1 Env vaccine (IPCAVD 001). J Infect Dis. 2013 Jan. 15; 207(2):240-7). Plasmids that can be used for adeno-associated virus, adenovirus, and lentivirus delivery have been previously described. The peptides and polypeptides of this disclosure can also be expressed in vectors, such as nucleic acid molecules discussed herein, such as RNA or DNA plasmids, viral vectors, such as poxviruses, such as orthopoxvirus, avipoxvirus, or adenovirus, AAV, or lentivirus. In some embodiments, this approach may involve the use of a vector to express the nucleotide sequence encoding the peptides of this disclosure. When introduced into an acutely or chronically infected host or an uninfected host, the vector can express immunogenic peptides, thereby inducing a host CTL response.

[0118] Among the vectors that can be used in the implementation of this disclosure, it is possible to integrate them into the cell host genome using retroviral gene transfer methods, and as a result, the inserted transgenes are often expressed for a long period of time. In certain embodiments, the retrovirus is a lentivirus. Furthermore, high transduction efficiency has been observed in many different cell types and target tissues. By incorporating exogenous envelope proteins, the tropism of the retrovirus can be altered to expand the potential target population of target cells. Just as a lentivirus can infect only certain cell types, retroviruses can also be manipulated to conditionally express the inserted transgene. In some embodiments, cell type-specific promoters can be used to target expression in specific cell types. Lentiviral vectors are retroviral vectors (therefore, both lentiviral vectors and retroviral vectors may be used in the implementation of this disclosure). Furthermore, in some embodiments, lentiviral vectors can be transduced or infected into non-dividing cells and may be preferred because they can produce high viral titers. Therefore, the choice of retroviral gene transfer system may depend on the target tissue. Retroviral vectors consist of cis-terminal repeat sequences with the ability to package exogenous sequences up to 6–10 kb. A minimal cis-terminal repeat sequence (LTR) is sufficient for vector replication and packaging, and the vector is then used to incorporate the desired nucleic acid into target cells to achieve permanent expression. Widely used retroviral vectors that may be used in the practice of this disclosure include retroviral vectors based on mouse leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof.

[0119] Minimal non-primate lentiviral vectors, such as lentiviral vectors based on equine infectious anemia virus (EIAV), are also useful (see, for example, Balagaan, (2006) J Gene Med; 8: 275-285, published online on November 21, 2005, in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002 / jgm.845). These vectors may contain a cytomegalovirus (CMV) promoter that expresses a target gene. Therefore, this disclosure intends to include viral vectors, including retroviral vectors and lentiviral vectors, among the vectors useful in carrying out this disclosure.

[0120] Those skilled in the art can determine an appropriate dosage. An appropriate dosage of the virus can be determined empirically. Adenovirus vectors are also useful in the practice of this disclosure. One advantage is that recombinant adenoviruses can efficiently transfer and express recombinant genes in vitro and in vivo in various mammalian cells and tissues, resulting in high expression of the transferred nucleic acid. Furthermore, the ability to productively infect quiescent cells expands the usefulness of recombinant adenovirus vectors. Moreover, high expression levels ensure that the nucleic acid product is expressed to a level sufficient to produce an immune response (see, for example, U.S. Patent No. 7,029,848).

[0121] In some embodiments, the adenovirus vector used can be selected from the group consisting of Ad5, Ad35, Adl1, C6, and C7 vectors. The sequence of the adenovirus 5 ("Ad5") genome is publicly available (Chroboczek, J., Bieber, F., and Jacrot, B. (1992) The Sequence of the Genome of Adenovirus Type 5 and Its Comparison with the Genome of Adenovirus Type 2, Virology 186, 280-285). The Ad35 vector is described in U.S. Patents 6,974,695, 6,913,922, and 6,869,794. Adenovirus vectors with deletions in E1, E3, and / or E4 may also be used. Certain adenoviruses with mutations in the E1 region have an improved safety margin. This is because E1-deficient adenovirus variants are either replication-deficient or at least significantly attenuated in non-permissible cells. Adenoviruses with mutations in the E3 region may have enhanced immunogenicity by disrupting the mechanism by which adenoviruses downregulate MHC class I molecules. Adenoviruses with E4 mutations may have reduced the immunogenicity of the adenovirus vector due to suppression of late gene expression. Such vectors may be particularly useful when repeated revaccination using the same vector is desirable. Adenovirus vectors with deletions or mutations in E1, E3, E4, El and E3, as well as El and E4, can be used in accordance with this disclosure. Furthermore, "gutless" adenovirus vectors in which all viral genes are deleted can also be used in accordance with this disclosure. In some embodiments, such vectors require a helper virus for replication and require a specific human 293 cell line expressing both E1a and Cre, conditions that do not exist in the natural environment. Such "gutless" vectors are non-immunogenic, and therefore, they can be administered multiple times for vaccine reinoculation."Gutless" adenovirus vectors can be used to insert heterologous inserts / genes, such as the transgenes of this disclosure, and can also be used for the purpose of simultaneously delivering multiple heterologous inserts / genes.

[0122] In one embodiment, the viral vector is an adenovirus vector, an adeno-associated virus vector (AAV), or a derivative thereof. The adeno-associated virus vector includes AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, DJ, DJ / 8, or their pseudotypes. In one embodiment, the AAV may be AAV1, AAV2, AAV5, or any combination thereof. In some embodiments, the AAV can be selected with respect to the cells to be targeted. For example, to target brain or neuronal cells, AAV serotypes 1, 2, 5 or the hybrid capsid AAV1, AAV2, AAV5, or any combination thereof can be selected. AAV4 can be selected when targeting cardiac tissue. In some embodiments, AAV8 may be useful for delivery to the liver.

[0123] In another embodiment, the cellular immune response to a nascent vaccine or immunogenic composition can be effectively activated by expressing relevant antigens present in the vaccine or immunogenic composition in non-pathogenic microorganisms. Well-known examples of such microorganisms include, but are not limited to, Mycobacterium bovis (BCG), Salmonella, and Pseudomona (see U.S. Patent No. 6,991,797). In another embodiment, poxviruses are used in the nascent vaccine or immunogenic composition. These include orthopoxvirus, avipox, vaccinia, MVA, NYVAC, canarypox, ALVAC, fowlpox, and TROVAC (see, for example, Verardi et al., Hum Vaccin Immunother. 2012 July; 8(7):961-70; and Moss, Vaccine. 2013; 31(39): 4220-4222).

[0124] In some embodiments, poxviruses that may be used in the practice of this disclosure, such as Chordopoxvirinae (vertebrate poxviruses), such as orthopoxviruses and avidoxviruses, such as vaccinia viruses (e.g., Wyeth strain, WR strain (e.g., ATCC® VR-1354), Copenhagen strain, NYVAC, NYVAC.1, NYVAC.2, MVA, MVA-BN), canary poxviruses (e.g., Wheatley C93 strain, ALVAC), fowlpox viruses (e.g., FP9 strain, Webster strain, TROVAC), dovepox, pigeonpox, quailpox, and raccoon pox, in particular their synthetic or non-naturally occurring recombinants, their use, and methods for producing and using such recombinants may be found in the scientific literature and patent documents.

[0125] In one embodiment, recombinant viral particles of a vaccine or immunogenic composition are administered to a patient in need. The dose of expressed neoantigen may be several micrograms to several hundred micrograms, for example, 5 to 500 μg. The vaccine or immunogenic composition can be administered in any appropriate amount to achieve expression at these dose levels. The viral particles are approximately 10 3 The amount of pfu may be administered to patients who need it, or it may be introduced into cells by transfection. Therefore, the viral particles are preferably at least about 10 4 pfu~about 10 6 PFU is administered to patients who need it, or introduced into cells by infection or transfection. However, patients who need it should receive at least about 10 8 PFU or at least about 10 7 pfu~about 10 9 PFU can be administered. The dosage for NYVAC is applicable to ALVAC, MVA, MVA-BN, and Avipox, such as Canarypox and fowlpox.

[0126] Pharmaceutical composition / delivery method In certain embodiments, the pharmaceutical composition optionally comprises, in combination with a pharmaceutically acceptable carrier, excipient, or additive, an effective amount of one or more antigenic peptides described herein (including their pharmaceutically acceptable salts).

[0127] "Pharmacologically acceptable" means that it is approved or eligible for approval by a federal or state regulatory authority, or is listed in the United States Pharmacopeia or any other generally accepted pharmacopoeia for use in animals, including humans.

[0128] A "pharmaceutically acceptable excipient, carrier, or diluent" means an excipient, carrier, or diluent that can be administered to a subject together with a drug, does not destroy the pharmacological activity of the drug when administered in a dose sufficient to deliver a therapeutic dose of the drug, and is non-toxic.

[0129] The “pharmaceutically acceptable salts” of the pooled tumor-specific neoantigens listed herein may be acid salts or base salts that are generally considered in the art to be suitable for use in contact with human or animal tissue without excessive toxicity, irritation, allergic reactions, or other problems or complications. Such salts include basic residues, e.g., mineral and organic acid salts of amines, as well as acidic residues, e.g., alkali or organic salts of carboxylic acids. Certain pharmaceutical salts include acids, such as hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, sulfanilic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethylsulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, pamoic acid, succinic acid, fumaric acid, maleic acid, propionic acid, dihydroxymaleic acid, hydroiodic acid, phenylacetic acid, alkanic acid, such as acetic acid, HOOC-(CH2) n The salts include, but are not limited to, salts of -COOH, where n is 0 to 4, etc. Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those skilled in the art will recognize, from this disclosure and knowledge in the art, further pharmaceutically acceptable salts of the pooled tumor-specific neoantigens provided herein, including those enumerated by Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985). Generally, pharmaceutically acceptable acid or base salts can be synthesized from parent compounds containing a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or free base form of these compounds with a stoichiometric amount of a suitable base or acid in a suitable solvent.

[0130] When administered as a combination, the therapeutic agent, for example, the neoantigen peptide, may be prescribed as separate compositions to be given at the same time or at different times. Alternatively, the therapeutic agent may be given as a single composition.

[0131] The composition may be administered once daily, twice daily, once every two days, once every three days, once every four days, once every five days, once every six days, once every seven days, once every two weeks, once every three weeks, once every four weeks, once every two months, once every six months, or once a year. The dosing interval can be adjusted according to the individual needs of each patient. For longer dosing intervals, long-release formulations or depot formulations may be used.

[0132] Combination therapy This disclosure also intends to describe combinations of the compositions of this disclosure with other drugs and / or with other treatment regimens or modalities such as surgery. When the compositions of this disclosure are used in combination with known therapeutic agents, the combination may be administered sequentially (continuously or interrupted by periods without treatment), simultaneously, or as a mixture. For example, in the case of cancer, a chemotherapeutic agent may be administered as part of combination therapy.

[0133] In certain embodiments, KRAS and / or mKRAS peptides are administered in conjunction with cancer therapy. In some embodiments, the compositions disclosed herein are administered in conjunction with cancer therapy. In some embodiments, immunogenic peptides containing at least 90%, 95%, 98%, or 99% sequence identity to any one amino acid sequence of SEQ ID NO: 1-838 can be administered in conjunction with cancer therapy. In some embodiments, one or more immunogenic peptides containing at least 75% sequence identity to any one or more of SEQ ID NO: 1-838 can be administered in conjunction with cancer therapy. In certain embodiments, immunogenic peptides containing one or more SEQ ID NO: 1-838 can be administered in conjunction with cancer therapy. In certain embodiments, immunogenic peptides containing amino acid sequences having one or more conservative substitutions and having at least 95% sequence identity to any one or more of SEQ ID NO: 1-838 can be administered in conjunction with cancer therapy.In some cases, SEQ ID NO: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148-152, 154-162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232-236, 238-244, 246-252 , 254~260, 262~268, 270~276, 278~284, 286~292, 294~300, 302~308, 310~316, 318~324, 326~332, 334~340, 342~348, 350~356, 358~364, 366~372, 374~380, 382~388, 390~396, 398~404, 406~412, 414~420, 422~428, 430~436, 438~443, 445~450, 452~457, 459~464, 466~471, 473~478, 480~485 , 487~492, 494~499, 501~506, 508~512, 514~518, 520~524, 526~530, 532~536, 538~542, 544~548, 550~554, 556~560, 562~566, 568~573, 585~580, 582~587, 589~594, 596~601, 603~607, 609~613, 615~619, 621~625, 627~631, 633~638, 340~644, 646~651, 653~657, 659~664, 666~670, 672~67 7. Immunogenic peptides containing any one of the amino acid sequences 7, 679-683, 685-690, 692-696, 698-703, 705-711, 713-718, 720-726, 728-733, 735-741, 743-748, 750-756, 758-763, 765-771, 773-777, 779-783, 785-789, 791-795, 797-801, 803-807, 809-813, 815-819, 821-825, 827-831, 833, or 834 can be administered in conjunction with cancer therapy.In some embodiments, the immunogenic peptide comprises the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:833, or SEQ ID NO:834 and can be administered in conjunction with cancer therapy. As used herein, the term “cancer therapy” refers to therapies useful in the treatment of cancer. Examples of anticancer agents include, for example, agents used in surgery, chemotherapy, immunotherapy, proliferation inhibitors, cytotoxic agents, radiotherapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer, such as anti-HER-2 antibodies (e.g., HERCEPTIN®), anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (TARCEVA®)), This includes, but is not limited to, platelet-derived growth factor inhibitors (e.g., GLEEVEC® (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following target ErbB2, ErbB3, ErbB4, PDGFR-β, BlyS, APRIL, BCMA, or VEGF receptors, TRAIL / Apo2, and other bioactive and organic chemical agents. Combinations thereof are also intended for use with the methods described herein.

[0134] "Chemotherapy agents" are compounds useful in the treatment of cancer. Examples of chemotherapy agents include erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), fulvestrant (FASLODEX®, Astrazeneca), sutent (SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), PTK787 / ZK 222584 (Novartis), oxaliplatin (Eloxatin®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib (GSK572016, GlaxoSmithKline), and ronafarnib (SCH 66336), sorafenib (BAY43-9006, Bayer Labs.), and gefitinib (IRESSA®, Astrazeneca), AG1478, AG1571 (SU 5271; Sugen), alkylating agents, e.g., thiotepa and CYTOXAN® cyclosphosphamide; alkyl sulfonates, e.g., busulfan, improsulfan, and pigosulfan; aziridines, e.g., benzodopa, carbocone, meturedopa, and uredopa; altoretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomellamine, etc. Lenimine and methylamelamin; acetogenins (especially bullatacin and bullatacinone); camptothecin (including its synthetic analog topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogs adozcicsin, carzcicsin, and bizcicsin); cryptophycin (especially cryptophycin 1 and cryptophycin 8); drastatin;Duocalmycin (including synthetic analogs, KW-2189 and CB1-TM1); eryuterobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, e.g., chlorambucil, chlornafadin, chlorophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, novembichin, fenestrin, prednimustine, trophosphamide, uracil mustard; nitrosureas, e.g., carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics, e.g., engine antibiotics (e.g., calitiamycin, in particular calitiamycin γ1 and calitiamycin ω1 (Angew Chem.) Intl. Ed. Engl. (1994) 33:183-186); Dinemicins containing dynemicin A; Bisphosphonates, e.g., chlordronate; Esperamicin;Furthermore, neocardinostatin chromophore and related pigment protein enediin antibiotic chromophore), acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, kactinomycin, carabicin, kaminomycin, cardinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN (trademark), doxorubicin (morpholino-doxorubicin, cyanomorpholino -Doxorubicin (including 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, mycophenolate, nogaramycin, olibomycin, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidine, yubenimex, dinostatin, zolubicin; metabolism Antagonists, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamipurine, thioguanine; pyrimidine analogs, e.g., ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, phloxuridine; androgens, e.g., carsterone, dromostanolone propionate, epithiostanolone Lu, mepithiostan, testolactone; anti-adrenal agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folic acid; acegraton; aldofamide glycoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisantren; edatraxate; defofamine; demecoltin; diaziquan; elfornithine; eriptinium acetate; eposylone; etogluside; gallium nitrate; hydroxyurea; lentinan;Ronidynin; Mytansinoids, e.g., mytansin and ansamitocin; Mitoguazone; Mitoxantrone; Mopidanmol; Nitraerine; Pentostatin; Fenamet; Pirarubicin; Losoxantrone; Podophyllinic acid; 2-Ethylhydrazide; Procarbazine; PSK (Trademark) Polysaccharide Complex (JHS Natural Products, Eugene, Oreg.); Lazoxane; Rhizoxin; Sizofuran; Spirogermanium; Tenuazonic acid; Triadiquan; 2,2',2''-Trichlorotriethylamine; Trichothecenes (especially T-2 toxin, verracurin A, roridin A, and anguidine); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipopromane; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxoids, e.g., TAXOL® Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE® Cremophor-free Albumin-Modified Nanoparticle Formulations of Paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE® doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs, e.g., cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO);This includes retinoids, such as retinoic acid; capecitabine; and any pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0135] The definition of "chemotherapeutic agent" includes (i) anti-hormone agents that act to modulate or inhibit hormonal effects on tumors, such as anti-estrogens, as well as selective estrogen receptor modulators (SERMs) such as tamoxifen (including NOLVADEX® (tamoxifen)), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifen, LY117018, onapristone, and FARESTON® (toremifene); (ii) for example, 4 (5) Aromatase inhibitors that inhibit aromatase, an enzyme that regulates estrogen production in the adrenal gland, such as imidazole, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane), formestanie, fadrozol, RIVISOR® (borozol), FEMARA® (letrozole), and ARIMIIDEX® (anastrozole); (iii) Antiandrogens, such as flutamide, nilutamide, bicalutamide, leupro Lido and goserelin; as well as troxacitabine (1,3-dioxolane nucleoside cytosine analog); (iv) aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinase inhibitors; (vii) antisense oligonucleotides, in particular antisense oligonucleotides that inhibit gene expression in signaling pathways linked to abnormal cell proliferation, such as PKC-α, Ralf, and H-Ras; (viii) ribozymes, such as VEGF expression inhibitors (e.g., ANGIOZYME® (ribozyme)); (i) and HER2 expression inhibitors; (ix) vaccines, e.g., gene therapy vaccines, e.g., ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1 inhibitor; ABARELIX® rmRH; (x) anti-angiogenic agents, e.g., bevacizumab (AVASTIN®, Genentech); and (xi) pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0136] In various embodiments, cancer treatment is immunotherapy selected from a group including tumor-regressive viruses, bacteria, tumor-regressive bacteria or other bacterial compositions, Bacillus Calmette-Guerin (BCG), microbiome modulators, and / or Toll-like receptor (TLR) agonists. In various embodiments, TLR agonists are TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, and / or TLR13 agonists. In various embodiments, TLR agonists are derived from viruses, plants, bacteria, and / or are synthesized. In various embodiments, the immunotherapy is an interferon-stimulating (STING) pathway modulator.

[0137] Those skilled in the art of cancer immunotherapy will recognize that other supplemental immunotherapies, including but not limited to GM-CSF aimed at increasing the number of bone marrow-derived innate immune cells, low-dose cyclophosphamide or PI3K inhibitors (e.g., PI3Kδ inhibitors) aimed at eliminating T regulatory cells that inhibit innate and adaptive immunity, and 5FU (e.g., capecitabine), PI3K inhibitors, or histone deacetylase inhibitors aimed at eliminating inhibitory bone marrow-derived suppressor cells, may be added to the above regimens to further enhance efficacy. For example, PI3K inhibitors include, but are not limited to, LY294002, Perifosin, BKM120, Duvelisib, PX-866, BAY 80-6946, BEZ235, SF1126, GDC-0941, XL147, XL765, Palomid 529, GSK1059615, PWT33597, IC87114, TG100-15, CAL263, PI-103, GNE-477, CUDC-907, and AEZS-136. In some contexts, PI3K inhibitors are PI3Kδ inhibitors, such as, but are not limited to, Idelalisib, RP6530, TGR1202, and RP6503. Immunotherapy may also include the administration of interleukins, such as IL-2, or interferons, such as INFα.

[0138] In certain embodiments, KRAS and / or mKRAS peptides are administered together with one or more immune checkpoint modulators. In some embodiments, compositions disclosed herein can be administered together with immune checkpoint modulators. In some embodiments, immunogenic peptides containing at least 90%, 95%, 98%, 99%, or 100% sequence identity to any one amino acid sequence of SEQ ID NO: 1-838 can be administered together with a checkpoint modulator. In some embodiments, one or more immunogenic peptides containing at least 75% sequence identity to any one or more of SEQ ID NO: 1-838 can be administered together with an immune checkpoint modulator. In certain embodiments, immunogenic peptides containing one or more of SEQ ID NO: 1-838 can be administered together with an immune checkpoint modulator.In some cases, SEQ ID NO:2, 3, 5~12, 14~21, 23~30, 32~39, 41~48, 50~57, 59~66, 68~75, 77~84, 86~93, 85~99, 101~105, 107~111, 113~117, 119~123, 125~129, 131~135, 1 37~141, 143~146, 148~152, 154~162, 164~168, 170~179, 181~185, 187~1 96, 198~202, 204~213, 215~219, 221~230, 232~236, 238~244, 246~252, 25 4~260, 262~268, 270~276, 278~284, 286~292, 294~300, 302~308, 310~316, 318~324, 326~332, 334~340, 342~348, 350~356, 358~364, 366~372, 374~380, 382~388, 390~396, 398~404, 406~412, 414~420, 422~428, 430~436, 438~443, 445~450, 452~457, 459~464, 466~471, 473~478, 480~485, 487~4 92, 494~499, 501~506, 508~512, 514~518, 520~524, 526~530, 532~536, 538~542, 544~548, 550~554, 556~560, 562~566, 568~573, 585~580, 582~587, 589~594, 596~601, 603~607, 609~613, 615~619, 621~625, 627~631, 633~638, 340~644, 646~651, 653~657, 659~664, 666~670, 672~677, 679~683 Immunogenic peptides containing any one of the amino acid sequences 685-690, 692-696, 698-703, 705-711, 713-718, 720-726, 728-733, 735-741, 743-748, 750-756, 758-763, 765-771, 773-777, 779-783, 785-789, 791-795, 797-801, 803-807, 809-813, 815-819, 821-825, 827-831, 833, or 834 can be administered together with a checkpoint modulator.In certain embodiments, immunogenic peptides containing amino acid sequences having at least 95% sequence identity to one or more of SEQ ID NO:1-838 with one or more conservative substitutions can be administered with an immune checkpoint modulator. In some embodiments, immunogenic peptides containing amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:833, or SEQ ID NO:834 can be administered with an immune checkpoint modulator. Immune checkpoints refer to inhibitory immune system pathways responsible for maintaining self-tolerance and regulating the duration and amplitude of physiological immune responses. Examples of checkpoint inhibitors include, but are not limited to, inhibitors of PD-1, PD-L1, PD-L2, CTLA4, TIM-3, LAG-3, CEACAM-1, CEACAM-5, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, or TGFR-β.

[0139] The term "checkpoint inhibitor" refers to a drug that fine-tunes the immune response by downregulating or inhibiting the antitumor immune response, specifically CD4 + T cells and / or CD8 +This refers to a group of molecules on the surface of T cells. Immune checkpoint proteins are well known in the art and include, but are not limited to, CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, 2B4, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, BTLA, SIRPα (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, and A2aR (see, for example, WO2012 / 177624). "Immune checkpoint inhibitor therapy" refers to the use of drugs that inhibit immune checkpoint inhibitors. By inhibiting one or more immune checkpoint inhibitors, inhibitory signaling can be blocked or otherwise neutralized, thereby upregulating the immune response to more effectively treat cancer. Exemplary agents useful for inhibiting immune checkpoint inhibitors include antibodies, small molecules, peptides, peptidomimetics, native ligands, and derivatives of native ligands that bind to and / or inactivate or inhibit immune checkpoint proteins or their fragments; as well as RNA interference, antisense, and nucleic acid aptamers that can downregulate the expression and / or activity of immune checkpoint inhibitor nucleic acids or their fragments.Examples of agents for upregulating the immune response include antibodies against one or more immune checkpoint inhibitor proteins that block the interaction between one or more immune checkpoint inhibitor proteins and their native receptors; inactivated forms of one or more immune checkpoint inhibitor proteins (e.g., dominant-negative polypeptides); small molecules or peptides that block the interaction between one or more immune checkpoint inhibitor proteins and their native receptors; fusion proteins that bind to the native receptors (e.g., extracellular components of immune checkpoint inhibitor proteins fused to the Fc portion of an antibody or immunoglobulin); and nucleic acid molecules that block the transcription or translation of immune checkpoint inhibitor nucleic acids. Such agents can upregulate the immune response by directly blocking the interaction between one or more immune checkpoint inhibitors and their native receptors (e.g., antibodies) and thereby blocking inhibitory signaling. Alternatively, agents can upregulate the immune response by indirectly blocking the interaction between one or more immune checkpoint proteins and their native receptors and thereby blocking inhibitory signaling. For example, a soluble version of an immune checkpoint protein ligand, such as one in which a stabilized extracellular domain binds to its receptor, can indirectly reduce the effective concentration of the receptor required for binding to the appropriate ligand. In one embodiment, an anti-PD-1 antibody, an anti-PD-L1 antibody, and an anti-CTLA-4 antibody are used individually or in combination.

[0140] In some embodiments, such therapy involves blockade of programmed cell death 1 (PD-1). In some embodiments, such therapy involves treatment with a drug that interferes with PD-1-related interactions (e.g., with PD-L1). In some embodiments, such therapy involves administration of an antibody drug that specifically interacts with PD-1 or PD-L1. In some embodiments, such therapy involves administration of one or more of the following: nivolumab (BMS-936558, MDX-1106, ONO-4538, fully human immunoglobulin G4 (IgG4) monoclonal PD-1 antibody), pembrolizumab (MK-3475, humanized monoclonal IgG4 anti-PD-1 antibody), BMS-936559 (fully human IgG4 PD-L1 antibody), MPDL3280A (humanized modified IgG1 monoclonal PD-L1 antibody), and / or MEDI4736 (humanized modified IgG1 monoclonal PD-L1 antibody).

[0141] Each aspect disclosed herein is intended to be applicable to each of the other disclosed aspects. Accordingly, all combinations of the various elements described herein are within the scope of this disclosure.

[0142] This disclosure is further illustrated by the following embodiments. These embodiments should not be construed as limiting. All references, patents, and published patent applications, as well as the drawings and sequence listings, cited throughout this application are incorporated herein by reference. [Examples]

[0143] Examples Example 1: Heterocritical neoepitope vaccine derived from computational structural modeling A library of KRAS G12D heterocritic epitopes was constructed for structural analysis of a group of 18 HLAs representing >90% of the world's population. The consensus binding motif for each HLA epitope exhibited distinct anchor residue binding motifs subdivided into hydrophobic, hydrophilic, or neutral binding anchors (Figure 1). Major anchor residues were primarily located at positions 2, 3, 5, and 9. Some alleles had additional minor anchor residues at positions 1, 4, 6, and 7. A library of amino acid-modified peptides was constructed using the KRAS G12D 9 mere epitopes GADGVGKSA and VVGADGVGK, and all possible combinations of anchor residue amino acids for each HLA subgroup. As proof of concept, NetMHC predictions of heterocritic epitope binding affinity within each HLA subgroup were performed in silico, showing increased binding affinity for several epitopes for all HLA types (Figure 2, showing GADGVGKSA only). Consistent with previous reports, parent GADGVGKSA is HLA-C * Binding to 08:02 was not predicted. This highlights the limitations of using predicted binding affinity to identify neoantigen targets.

[0144] In order to demonstrate the structural modeling approach described herein, first, each peptide is HLA-A *The peptide-HLA interaction was investigated by modeling on 02:01 (Figure 3). Multiple Sequence Comparison by Log-Expectation (MUSCLE) peptide sequence alignment was performed. This takes into account the positional sequence similarity of the KRAS G12D epitope with all epitopes that bind to each HLA and have usable crystal structures. Using the HLA-peptide structure with the greatest sequence similarity, the KRAS neoepitope was modeled within the HLA cleft by introducing amino acid rotomers with the lowest energy confirmation at each position. To ensure that no major destabilizing structural changes occurred in the entire HLA cleft, the resulting HLA-KRAS neoepitope complex was structurally validated using root mean square deviation analysis (Cα-RMSD). The lowest total atomic energy conformation was identified by performing high-resolution Monte Carlo and minimization docking between the peptide and the HLA cleft. Using the energy-optimized structure, the respective anchor residue modifications found in the heterocritical epitope library were introduced (Figures 2 and 3). Using this approach, it was found that the number of contacts between the peptide and the HLA molecule increased after substituting the amino acid anchor residues at positions 2, 6, and 9. This demonstrated that structural differences in HLA-heterocritical peptide interactions can be detected (Figure 3).

[0145] To design heterotropic peptides, we verified the minimal epitope targets of HLA-specific mutant KRAS using mass spectrometry (Figures 4A-4B).

[0146] These neoepitope-HLA structural models were used to analyze the stereochemical changes between the parent peptide and the modified peptide. Structural parameters included contact between the peptide and HLA cleft residues, solvent exposure area (SASA), peptide stiffness as a measure of CαRMSD for the top 10 predicted structural models for each peptide-HLA complex, surface hydrophobicity, and electrostatic potential. These measurements were compared to the modeled parent neoepitope in each HLA molecule.

[0147] Using the neoepitope-HLA structure model, HLA-A * 02:01 (Figures 5A-5C), HLA-A * 03:01 (Figures 6A-6C), HLA-A * aa:01 (Figures 7A-7C), HLA-A * 30:01 (Figures 8A-8C), HLA-A * 68:01 (Figures 9A-9C), HLA-B * 07:02 (Figures 10A-10C), HLA-C * 01:02 (Figures 11A-11C), HLA-C * 08:02 (Figures 12A-12C), and HLA-C * For 03:03 (Figures 13A-13C), we predicted the amino acid modifications of mKRAS epitopes exhibiting improved HLA-binding affinity.

[0148] HLA-A * The predicted binding affinity (nM) of the parent G12V 10-mer and 9-mer and the modified peptide ligand (APL) of APL for 02:01 was obtained (Figure 5B).

[0149] HLA-A * The predicted binding affinity (nM) of the parent 10-mer (KRAS G12D, G12V, G12C) and 9-mer (KRAS G12R, G12V) and the modified peptide ligand of APL (APL) to 03:01 was obtained (Figure 6B).

[0150] HLA-A *The predicted binding affinity (nM) of the modified peptide ligands of APL (APL) and the parent 10-mer (KRAS G12D, G12R, G12V, G12C) and 9-mer (KRAS G12D, G12R, G12V) for 11:01 was obtained (Figure 7B).

[0151] HLA-A * The predicted binding affinity (nM) of the parent 10-mer (KRAS G12R, G12V) and 9-mer (KRAS G12R, G12V) and the modified peptide ligand of APL (APL) to 30:01 was obtained (Figure 8B).

[0152] HLA-A * The predicted binding affinity (nM) of the modified peptide ligand of APL (APL) for 10 parent mers (KRAS G12D G12C, G12R, G12V) and 9 mers (KRAS G12V) against 68:01 was obtained (Figure 9B).

[0153] HLA-B * The predicted binding affinity (nM) of the parent 10 mers (KRAS G12D and G12R) and the modified peptide ligand of APL (APL) to 07:02 was obtained (Figure 10B).

[0154] HLA-C * The predicted binding affinity (nM) of the parent 9-mer (KRAS G12V) and the modified peptide ligand of APL (APL) for 01:02 was obtained (Figure 11B).

[0155] HLA-C * The predicted binding affinity (nM) of the parent 10-mer (KRAS G12D) and 9-mer (KRAS G12D) and the modified peptide ligand of APL (APL) for 08:01 was obtained (Figure 12B).

[0156] HLA-C * The predicted binding affinity (nM) of the parent 10-mer (KRAS G12V) and 9-mer (KRAS G12V) and the modified peptide ligand of APL (APL) to 03:03 was obtained (Figure 13B).

[0157] HLA-A using the MBL tetramer quick switch assay * 11:01-KRAS APL binding was tested. HLA-A * The 11:01 tetramer was incubated with 10 mM KRAS parent epitope or APL at room temperature for 4 hours. Peptide loading was then quantified by flow cytometry using an anti-exiting peptide antibody. The %APL binding to the tetramer was calculated compared to the parent peptide binding (Figure 14A). HLA-A levels tested using this assay were determined using NetMHC prediction. * We predicted the binding affinity of 11:01-specific KRAS peptides and APL.

[0158] Figure 15 shows the percentage binding of KRAS G12D, G12R, G12V, and G12C 10-mer and 9-mer parent epitopes and modified peptide ligands. APLs with a higher binding percentage compared to their parents are shaded in gray.

[0159] The ability of KRAS G12D APL to stimulate a T cell response to the parent antigen was tested in vitro. In vitro immunogenicity assays were performed using HLA-A * 11:01 This study was performed on healthy donors using a modified KRAS G12D peptide ligand. Healthy donor dendritic cells were prepared by attaching monocytes to a flask and differentiated in 800 U / mL hGMCSF and 400 IU / mL hIL-4 for 7 days. The dendritic cells were then matured in hIL-1B, IL-6, TNFα, and PGE2 for 2 days. Matured dendritic cells were then collected and treated with 40 μg / mL parental or modified peptide ligand for 2 hours. The treated dendritic cells were washed twice with PBS and then autologous CD8 in the presence of hIL-15, hIL-6, and hIL-21. + T cells were co-cultured with dendritic cells. After 7 days, more peptide-treated dendritic cells were added to each co-culture. The following day, fresh hIL2 and IL7 were added. Finally, dendritic cells were cultured and T cells were collected after 7 days, and HLA-A cells containing the parental epitope sequence were collected. *Staining with 11:01 tetramer (Figure 16A). T cell expansion was confirmed using flow cytometry (Figure 16B). KRAS G12D-HLA-A in ATVGADGVGK-expanded T cells 7 days after the third peptide immunization. * 11:01 tetramer staining showed an increase in the CD3+KRAS G12D tetramer+ T cell population, indicating increased binding affinity. A control without tetramers was used as a negative control.

[0160] The contact and surface structures of the KRAS G12D parent and modified peptide ligand-HLA were confirmed (Figure 17). The hydrophobicity of the KRAS G12D parent and modified peptide ligand-HLA was measured (Figure 18). The electrostatic potential of the KRAS G12D parent and modified peptide ligand was measured. HLA-A * Figure 20 summarizes the structural characteristic measurements of the KRAS G12D 10-mer parent epitope and modified peptide ligand modeled under the 11:01 condition.

[0161] Modified peptide ligand (APL) Using the Panc02 mouse model, modified peptide ligands (APLs) were manipulated or included in mouse neoantigen-targeted vaccines (HL Kinkead et al., JCI Insight. 2018;3(20):e122857. Doi.org / 10.1172 / jci.insight.122857). Figures 21A-21F demonstrate that the manipulated peptides increased contact points and solvent exposure area and reduced stiffness in the mouse PDAC model. Mice were vaccinated twice, 7 days apart, with either PBS, 50 μg APL, or 5 μg parent peptide. Seven days after the final vaccine administration, splenocytes were re-stimulated overnight with APL or parent peptide, and IFNγ production was measured using an Erispot. A two-way ANOVA was followed by a Tukey multiple comparison test (Figure 21A). In silico structural modeling of APL or parent peptide in mouse H2-Kb (tan, mouse MHC class I molecule H2-Kb, blue, minimal epitope of peptide 44, orange, parent or engineered amino acid residue). The number of contacts between the MHC-binding cleft and the peptide (Figure 21C) or mutant residue (Figure 21D) is shown. Figure 21E shows Cα-RMSD, a measure of peptide stiffness. Figure 21F shows solvent exposure area (SASA). Independent Student t-tests were performed. (ns=P>0.05) * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001).

[0162] The results shown in Figures 22A to 22C indicate that the manipulated antigen's KRAS G12D APL1-HLA * It has been demonstrated that 11:01 binding and immunogenicity show excellent binding and T cell response. Figure 22A shows HLA-A * Figure 21B shows nonlinear regression curves and EC50 calculations for binding between 11:01 and the parent KRAS G12D 10-mer peptide and seven types of manipulated antigens. Figure 21B: 1 × 10⁶ peptides without peptide, unrelated peptide, parent peptide, or from APL1. 6Enlarged CD8 T cells were seeded onto Erispot Capture plates coated with anti-human IFNγ antibody. T cells were stimulated overnight with control or parent antigen, and IFNγ capture was read. Unmodified Fisher LSD tests were performed after two-way ANOVA. (ns=P>0.05) * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** (P ≤ 0.0001). CD8 T cells enlarged with APL1 showed an increase in IFNγ spots, indicating a superior T cell response compared to parental controls. Figure 21C shows HLA-A * 11:01 This shows the in vitro immunogenicity studies of the parent epitope and its manipulated counterpart (APL1). Human monocyte-derived dendritic cells (moDCs) differentiated from healthy donor PBMCs were treated with 100 μM parent or APL1 + β2-microglobulin (4 hours) at 37°C. After peptide application, the moDCs were washed with PBS and 6 × 10⁶ cells were extracted. 6 Individual autologous CD8 T cells (+IL-2, IL-7, IL-15, and IL-21) were co-cultured for 7 days. Additional immunization was performed on days 7, 14, and 21 using peptide-treated moDCs. T cells were collected and HLA-A cells loaded with the parent peptide were immunized. * 11:01 The cells were stained with a tetramer or were targeted for IFNγ erythropoiesis.

[0163] KRAS G12D APL4 has a different HLA * 11:01 human donors show a larger T cell response compared to parental epitopes (Figure 23). HLA-A * 11:01 In vitro immunogenicity study of parental epitope and its engineered counterpart (APL4). Human monocyte-derived dendritic cells (moDCs) differentiated from healthy donor PBMCs were treated with 100 μM parental or APL4 + β2-microglobulin at 37°C (4 hours). After peptide application, the moDCs were washed with PBS and 6 × 10⁶ cells were extracted. 6Individual autologous CD8 T cells (+IL-2, IL-7, IL-15, and IL-21) were co-cultured for 7 days. Additional immunization was performed on days 7, 14, and 21 using peptide-treated moDCs. T cells were collected and HLA-A cells loaded with the parent peptide were immunized. * T cells were stained with the 11:01 tetramer (left) or targeted for IFNγ erythropoiesis (right). T cells treated with APL1 and APL4 showed increased IFNγ spots, indicating a superior T cell response compared to parental controls. In vitro results suggest that the structural modeling methods described herein are effective for HLA-A * It was verified that KRAS APL, which has excellent binding affinity to 11:01, can be predicted.

[0164] KRAS G12D HLA-A * 11:01 Structural analysis of APL1 and APL4 identifies peptide rigidity and stabilizing HLA cleft interactions as features associated with enhanced peptide immunogenicity (Figures 24A-24C). Figure 24A shows HLA-A * Figure 24B shows the structural models of the A11-KRAS G12D parent epitope (left), A11-KRAS G12D APL1, and A11-KRAS G12D APL4 in the 11:01 situation. For each peptide, the top 10 energetically favorable peptide models are shown (n=10). The CaRMSD of the 10 mermaid peptides in each model was calculated compared to all other models for that epitope. A Tukey multiple comparison test was performed after a two-way ANOVA. Figure 24C shows the structural analysis of the peptide neoantigen residue (G12D) in contact with HLA cleft residues (from left to right: Arg114, Gln155, Gln70, TRP147, Thr73) associated with stabilizing peptide-HLA interactions. Quantitative analysis of G12D residue interactions with the top 10 structural models of parent G12D, APL1, or APL4, specifically with respect to their respective HLA cleft residues, demonstrates that APL4 possesses an altered HLA cleft binding pattern. A significantly high proportion of interactions with the Gln70 residue suggests more diverse stabilization interactions within the HLA cleft.

[0165] HLA-A * 03:01 and HLA-B * Changes in binding affinity to KRAS G12D APL under the conditions of 07:02 (Figures 25A and 25B). HLA-A * 03:01 and B * Designed an optimized APL for 07:02, K562-TAP1 KO - We tested the enhanced binding affinity of the peptide compared to the parent neoantigen in HLA-expressing cell lines. Cells were incubated overnight at 37C with gradually increasing peptide concentrations. Then, the cells were stained to check for HLA-ABC expression on the cell surface. For each peptide concentration, the median fluorescence intensity of HLA expression was quantified by repeating the test twice. The control peptides correspond to positive control peptides specific to each HLA. Nonlinear regression curves were fitted to each graph. The peptide sequences corresponding to APL1 and the parent are summarized in the table below. Modified residues are shown in italics.

[0166] (Table 1) Peptide sequences corresponding to APL1, APL4, and the parent sequence G12D (10-mer) TIFF2026514104000001.tif78161

[0167] (Table 2) APL and parent sequence (HLA-A * Peptide sequence corresponding to 02:01) TIFF2026514104000002.tif197161TIFF2026514104000003.tif105161

[0168] (Table 3) APL and parent sequence (HLA-A * Peptide sequence corresponding to 03:01) TIFF2026514104000004.tif197161

[0169] (Table 4) APL and parent sequence (HLA-A * Peptide sequence corresponding to 11:01) TIFF2026514104000005.tif165161

[0170] (Table 5) APL and parent sequence (HLA-A * Peptide sequence corresponding to 24:02) TIFF2026514104000006.tif165161

[0171] (Table 6) APL and parent sequence (HLA-A * Peptide sequence corresponding to 24:02) TIFF2026514104000007.tif69170

[0172] (Table 7) APL and parent sequence (HLA-A * Peptide sequence corresponding to 30:01) TIFF2026514104000008.tif154161

[0173] (Table 8) APL and parent sequence (HLA-A * Peptide sequence corresponding to 68:01) TIFF2026514104000009.tif132161

[0174] (Table 9) APL and parent sequence (HLA-B * Peptide sequence corresponding to 07:02) TIFF2026514104000010.tif62170

[0175] (Table 10) APL and parent sequence (HLA-C * Peptide sequence corresponding to 01:02) TIFF2026514104000011.tif57170

[0176] (Table 11) APL and parent sequence (HLA-B * Peptide sequence corresponding to 08:02) TIFF2026514104000012.tif136161

[0177] (Table 12) APL and parent sequence (HLA-C * Peptide sequence corresponding to 04:01) TIFF2026514104000013.tif157161

[0178] (Table 13) APL and parent sequence (HLA-C * Peptide sequence corresponding to 03:03) TIFF2026514104000014.tif121161

[0179] (Table 14) Parent G12 array TIFF2026514104000015.tif46128

[0180] overview In a pancreatic mouse model (Panc02), the modified peptide ligand ("APL") was demonstrated to possess distinct structural features, measured by in silico modeling, that enhance APL immunogenicity in mice compared to the parent peptide (Figures 21A-21F). These data support the manipulation of antigens using in silico structural modeling.

[0181] HLA11 * It has been shown that heterocritical peptides modeled for 01, such as G12D (10 mers), improved T cell response compared to the parent peptide (Figures 22A-22C and 23).

[0182] Other embodiments From the foregoing explanation, it is clear that changes and modifications may be made to the disclosures herein to suit various uses and conditions. It is understood that in order to carry out the methods disclosed herein, steps of a particular method may be rearranged, removed, or changed. Such embodiments are also within the scope of the following claims.

[0183] All references to sequences, patents, and publications herein are incorporated by reference to the same extent that each independent patent and publication is indicated in detail and individually.

Claims

1. A method for identifying heterocritical neoepitopes with improved immunogenicity, comprising the following steps: A step of modeling a peptide library on human leukocyte antigens (HLA) to form an HLA-peptide structure, wherein the modeling step includes identifying the positional sequence similarities of peptide epitopes that bind to one or more HLA haplotypes; (a) A step of selecting an HLA-peptide structure that has sequence identity similar to the parent epitope being modeled; (b) A step of modeling heterocritical neoepitopes within HLA clefts by introducing amino acid rotomers that have the lowest energy confirmation at each position; (c) Steps for creating HLA-heterocritical neoepitope complexes; (d) A step of verifying the HLA-heterocritical neoepitope complex using root mean square deviation analysis (Cα-RMSD); and (e) A step of performing a high-resolution Monte Carlo simulation with minimization docking of the heterocritical neoepitope and the HLA cleft in order to identify the lowest total atomic energy conformation. Includes, This will allow us to identify heterocritical neoepitopes with improved immunogenicity. The aforementioned method.

2. The method according to claim 1, wherein the modeling of the peptide on the HLA includes aligning the peptide sequence with the consensus binding motif of the HLA molecule.

3. The method according to claim 2, wherein the consensus binding motif includes a consensus binding motif for an 8-mer, 9-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitope.

4. The method according to claim 3, wherein the consensus binding motif for the 8-mer, 9-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitopes comprises separate anchor residue binding motifs that are subdivided into hydrophobic binding anchors, hydrophilic binding anchors, or neutral binding anchors.

5. The method according to any one of claims 1 to 4, wherein the root mean square deviation analysis is performed to identify unstable structural changes in the HLA cleft.

6. The method according to any one of claims 1 to 5, wherein an anchor energy optimized structure is selected to identify anchor residue modifications.

7. The method according to any one of claims 1 to 6, wherein peptides are produced that contain combinations of anchor residue amino acids for each HLA class and its subgroups.

8. The method according to any one of claims 1 to 7, wherein the HLA includes an HLA type, and the HLA type includes classes I, II, and their subgroups.

9. The method according to any one of claims 1 to 8, wherein the peptide is derived from tumor cells or their antigenic derivatives.

10. The method according to any one of claims 1 to 9, wherein the peptide comprises a tumor antigen.

11. The method according to claim 10, wherein the tumor antigen comprises Carsten rat sarcoma virus (KRAS), BRAF, FBWX7, FGFR3, IDH1, MUC4, NRAS, PIK3CA, PPP2R1A, PTEN, or TP53.

12. The method according to claim 11, wherein the tumor antigen comprises one or more mutations.

13. The method according to claim 12, wherein the one or more mutations are selected from KRAS G12D, KRAS G12V, KRAS G12R, or KRAS G12C.

14. The method according to claim 12, wherein one or more of the mutations are selected from BRAF V600E or BRAF V600M.

15. The method according to claim 12, wherein one or more mutations are FBXW7 R465C or FBXW7 R465Q.

16. The method according to claim 12, wherein one or more of the mutations are FGFR3 S249Q.

17. The method according to claim 12, wherein one or more of the mutations are IDH1 R132C.

18. The method according to claim 12, wherein one or more of the mutations are MUC4 D3157N.

19. The method according to claim 12, wherein one or more mutations are NRAS Q61K or NRAS G545R.

20. The method according to claim 12, wherein the one or more mutations are PIK3CA G545K, PIK3CA H1047R, or PIK3CA R88Q.

21. The method according to claim 12, wherein one or more of the mutations are PPP2R1A P179R.

22. The method according to claim 12, wherein the one or more mutations are PTEN R130G or PTEN R130Q.

23. The method according to claim 12, wherein the one or more mutations are TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y.

24. The method according to any one of claims 1 to 23, wherein the HLA is HLA-A, HLA-B, or HLA-C.

25. The aforementioned HLA-A is HLA-A * 02:01, HLA-A * 03:01, HLA-A * 11:01, HLA-A * 30:01, or HLA-A * The method according to claim 24, wherein the ratio is 68:

01.

26. The aforementioned HLA-B is HLA-B * The method according to claim 24, wherein the time is 07:

02.

27. where the HLA-C is HLA-C * 01:02, HLA-C * 03:03, HLA-C * 03:04, or HLA-C * 08:02, the method according to claim 24

28. The method according to any one of claims 1 to 27, wherein the sequence identity greater than the sequence identity similar to the parent epitope includes at least 85%, 90%, 95%, 97%, and 99% sequence identity.

29. A method for preparing a library of immunogenic peptides, comprising the following steps: A step of modeling a peptide onto human leukocyte antigens (HLA), wherein the modeling step includes identifying the positional sequence similarity of peptide epitopes that bind to one or more HLA haplotypes; A step of selecting an HLA-peptide structure that has sequence identity similar to the modeled parent epitope; A process of modeling heterocritical neoepitopes within HLA clefts by introducing amino acid rotomers with the lowest energy confirmation at each position in order to form HLA-heterocritical neoepitope complexes; A step of verifying the resulting HLA-heterocritical neoepitope complex using root mean square deviation analysis (Cα-RMSD); and A process of performing a high-resolution Monte Carlo simulation involving minimization docking of the heterocritical neoepitope and the HLA cleft in order to identify the lowest total atomic energy conformation. Includes, This allows for the creation of a library of immunogenic peptides. The aforementioned method.

30. The method according to claim 29, wherein the step of modeling the heterocritical neoepitope within the HLA cleft includes introducing an amino acid rotomer having the lowest energy confirmation at each position.

31. The method according to any one of claims 29 to 30, wherein the root mean square deviation analysis is performed to identify unstable structural changes in the HLA cleft.

32. The method according to any one of claims 29 to 31, wherein the peptide comprises a peptide isolated from tumor cells or their antigenic derivatives.

33. The method according to any one of claims 29 to 32, wherein the peptide comprises a tumor antigen.

34. The method according to claim 33, wherein the tumor antigen comprises Carsten rat sarcoma virus (KRAS), BRAF, FBWX7, FGFR3, IDH1, MUC4, NRAS, PIK3CA, PPP2R1A, PTEN, or TP53.

35. The method according to claim 34, wherein the tumor antigen comprises one or more mutations.

36. The method according to claim 35, wherein one or more mutations are selected from the group consisting of G12D, G12V, G12R, and G12C.

37. The method according to claim 35, wherein one or more of the mutations are selected from BRAF V600E or BRAF V600M.

38. The method according to claim 35, wherein one or more mutations are FBXW7 R465C or FBXW7 R465Q.

39. The method according to claim 35, wherein one or more of the mutations are FGFR3 S249Q.

40. The method according to claim 35, wherein one or more of the mutations are IDH1 R132C.

41. The method according to claim 35, wherein one or more of the mutations are MUC4 D3157N.

42. The method according to claim 35, wherein the one or more mutations are NRAS Q61K or NRAS G545R.

43. The method according to claim 35, wherein the one or more mutations are PIK3CA G545K, PIK3CA H1047R, or PIK3CA R88Q.

44. The method according to claim 35, wherein one or more of the mutations are PPP2R1A P179R.

45. The method according to claim 35, wherein the one or more mutations are PTEN R130G or PTEN R130Q.

46. The method according to claim 35, wherein one or more of the mutations are TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y.

47. The method according to any one of claims 29 to 46, wherein the KRAS peptide sequence is positionally aligned with an 8-mer, 9-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitope that binds to an HLA molecule.

48. The method according to claim 47, wherein the 8-mer, 9-mer, 11-mer, 12-mer, 13-mer, 14-mer, 15-mer, 16-mer, 17-mer, 18-mer, 19-mer, 20-mer, 21-mer, 22-mer, 23-mer, 24-mer, or 25-mer epitope comprises a separate anchor residue binding motif which is subdivided into a hydrophobic binding anchor, a hydrophilic binding anchor, or a neutral binding anchor.

49. The method according to any one of claims 29 to 48, wherein an HLA-KRAS peptide containing the greatest sequence similarity is selected for modeling within an HLA cleft.

50. The method according to any one of claims 29 to 49, wherein an anchor energy optimized HLA-KRAS structure is selected to identify anchor residue modifications.

51. The method according to any one of claims 29 to 50, wherein a library of KRAS peptides is prepared, comprising combinations of anchor residue amino acids for each HLA class and its subgroups.

52. The method according to claim 51, wherein the KRAS peptide comprises a heterocritical epitope.

53. The method according to claim 51, wherein contact with a population of immune cells of the KRAS peptide containing a heterocritical epitope results in a higher percentage expansion of the immune cell population compared to a corresponding peptide that does not contain the heterocritical epitope but is otherwise identical.

54. The method according to claim 51, wherein contact with a population of immune cells of the KRAS peptide containing a heterocritical epitope results in a higher activation of the immune cell population compared to a corresponding peptide that does not contain the heterocritical epitope but is otherwise identical.

55. The method according to claim 54, wherein the activation includes IFNg secretion.

56. The method according to any one of claims 29 to 55, wherein the HLA includes an HLA type, and the HLA type includes classes I, II, and their subgroups.

57. The method according to any one of claims 29 to 56, wherein the HLA is HLA-A, HLA-B, or HLA-C.

58. The aforementioned HLA-A is HLA-A * 02:01, HLA-A * 03:01, HLA-A * 11:01, HLA-A * 30:01, or HLA-A * The method according to claim 57, which is 68:

01.

59. The aforementioned HLA-B is HLA-B * The method according to claim 57, wherein the time is 07:

02.

60. The aforementioned HLA-C is HLA-C * 01:02, HLA-C * 03:03, HLA-C * 03:04, or HLA-C * The method according to claim 57, wherein the time is 08:

02.

61. A method for treating cancer, comprising the step of administering one or more peptides containing one or more heterocritical epitopes to a subject in need.

62. The method according to claim 61, wherein the peptide is prepared by the method described in any one of claims 1 to 59.

63. The method according to claim 61 or 62, wherein the peptide comprises an amino acid sequence having at least 75% sequence identity with any one or more of SEQ ID NO: 1 to 838.

64. The aforementioned peptides are SEQ ID NO: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148-152, 154-162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232-236, 238-244, 246 ~252, 254~260, 262~268, 270~276, 278~284, 286~292, 294~300, 302~308, 310~316, 318~324, 326~332, 334~340, 342~348, 350~356, 358~364, 366~372, 374~380, 382~388, 390~396, 398~404, 406~412, 414~420, 422~428, 430~436, 438~443, 445~450, 452~457, 459~464, 466~471, 473~47 8, 480-485, 487-492, 494-499, 501-506, 508-512, 514-518, 520-524, 526-530, 532-536, 538-542, 544-548, 550-554, 556-560, 562-566, 568-573, 585-580, 582-587, 589-594, 596-601, 603-607, 609-613, 615-619, 621-625, 627-631, 633-638, 340-644, 646-651, 653-657, 659-664, 6 The method according to claim 63, comprising one or more of 66-670, 672-677, 679-683, 685-690, 692-696, 698-703, 705-711, 713-718, 720-726, 728-733, 735-741, 743-748, 750-756, 758-763, 765-771, 773-777, 779-783, 785-789, 791-795, 797-801, 803-807, 809-813, 815-819, 821-825, 827-831, 833, or 834.

65. The method according to claim 63 or 64, wherein the peptide comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:

833.

66. The method according to claim 63 or 64, wherein the peptide comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:

834.

67. The method according to any one of claims 61 to 66, further comprising the step of administering an adjuvant to the subject.

68. The method according to any one of claims 61 to 67, further comprising the step of administering one or more therapeutic agents to the subject.

69. A cancer vaccine comprising one or more peptides, each containing at least one heterocritical neoepitope.

70. The cancer vaccine according to claim 69, wherein the peptide is a tumor antigen.

71. The cancer vaccine according to claim 70, wherein the tumor antigen comprises Carsten rat sarcoma virus (KRAS), BRAF, FBWX7, FGFR3, IDH1, MUC4, NRAS, PIK3CA, PPP2R1A, PTEN, or TP53.

72. The cancer vaccine according to claim 71, wherein the tumor antigen comprises one or more mutations.

73. The cancer vaccine according to claim 72, wherein one or more of the mutations are selected from KRAS G12D, KRAS G12V, KRAS G12R, or KRAS G12C.

74. The cancer vaccine according to claim 72, wherein one or more of the mutations are selected from BRAF V600E or BRAF V600M.

75. The cancer vaccine according to claim 72, wherein one or more of the mutations are FBXW7 R465C or FBXW7 R465Q.

76. The cancer vaccine according to claim 72, wherein one or more of the mutations are FGFR3 S249Q.

77. The cancer vaccine according to claim 72, wherein one or more of the mutations are IDH1 R132C.

78. The cancer vaccine according to claim 72, wherein one or more of the mutations are MUC4 D3157N.

79. The cancer vaccine according to claim 72, wherein one or more of the mutations are NRAS Q61K or NRAS G545R.

80. The cancer vaccine according to claim 72, wherein the one or more mutations are PIK3CA G545K, PIK3CA H1047R, or PIK3CA R88Q.

81. The cancer vaccine according to claim 72, wherein one or more of the mutations are PPP2R1A P179R.

82. The cancer vaccine according to claim 72, wherein one or more of the mutations are PTEN R130G or PTEN R130Q.

83. The cancer vaccine according to claim 72, wherein one or more of the mutations are TP53 R175H, TP53 R248Q, TP53 R273H, TP53 R248Q, TP53 R273H, TP53 R282W, or TP53 R241Y.

84. The cancer vaccine according to any one of claims 69 to 83, wherein the peptide is shown on the surface of antigen-presenting cells (APCs) via the HLA antigen presentation pathway, thereby inducing a tumor-specific immune response.

85. The cancer vaccine according to any one of claims 69 to 84, wherein the peptide is specifically immunogenic to the target HLA type.

86. The cancer vaccine according to claim 85, wherein the HLA is HLA-A, HLA-B, or HLA-C.

87. The aforementioned HLA-A is HLA-A * 02:01, HLA-A * 03:01, HLA-A * 11:01, HLA-A * 30:01, or HLA-A * A cancer vaccine according to claim 86, wherein the ratio is 68:

01.

88. The aforementioned HLA-B is HLA-B * A cancer vaccine according to claim 86, wherein the ratio is 07:

02.

89. The aforementioned HLA-C is HLA-C * 01:02, HLA-C * 03:03, HLA-C * 03:04, or HLA-C * The cancer vaccine according to claim 86, wherein the time interval is 08:

02.

90. The cancer vaccine according to any one of claims 69 to 89, wherein the peptide is prepared by the method described in any one of claims 1 to 60.

91. The cancer vaccine according to any one of claims 69 to 90, wherein the peptide comprises an amino acid sequence having at least 75% sequence identity with any one or more of SEQ ID NO: 1 to 838.

92. The cancer vaccine according to any one of claims 69 to 91, wherein the peptide comprises one or more of SEQ ID NO: 1 to 838.

93. The aforementioned peptides are SEQ ID NO: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148-152, 154-162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232-236, 238-244, 246-25 2, 254-260, 262-268, 270-276, 278-284, 286-292, 294-300, 302-308, 310-316, 318-324, 326-332, 334-340, 342-348, 350-356, 358-364, 366-372, 374-380, 382-388, 390-396, 398-404, 406-412, 414-420, 422-428, 430-436, 438-443, 445-450, 452-457, 459-464, 466-471, 473-478, 480-4 85, 487-492, 494-499, 501-506, 508-512, 514-518, 520-524, 526-530, 532-536, 538-542, 544-548, 550-554, 556-560, 562-566, 568-573, 585-580, 582-587, 589-594, 596-601, 603-607, 609-613, 615-619, 621-625, 627-631, 633-638, 340-644, 646-651, 653-657, 659-664, 666-670, 672 A cancer vaccine according to any one of claims 91 to 92, comprising one or more of the following: ~677, 679-683, 685-690, 692-696, 698-703, 705-711, 713-718, 720-726, 728-733, 735-741, 743-748, 750-756, 758-763, 765-771, 773-777, 779-783, 785-789, 791-795, 797-801, 803-807, 809-813, 815-819, 821-825, 827-831, 833, or 834.

94. The cancer vaccine according to claim 93, wherein the peptide comprises the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:

833.

95. The cancer vaccine according to claim 93, wherein the peptide comprises the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:

834.

96. An expression vector encoding one or more peptides prepared by the method described in any one of claims 1 to 60, or a peptide comprising one amino acid sequence from SEQ ID NO: 1 to 838.

97. The aforementioned peptides are SEQ ID NO: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148-152, 154-162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232-236, 238-244, 246- 252, 254-260, 262-268, 270-276, 278-284, 286-292, 294-300, 302-308, 310-316, 318-324, 326-332, 334-340, 342-348, 350-356, 358-364, 366-372, 374-380, 382-388, 390-396, 398-404, 406-412, 414-420, 422-428, 430-436, 438-443, 445-450, 452-457, 459-464, 466-471, 473-478, 4 80-485, 487-492, 494-499, 501-506, 508-512, 514-518, 520-524, 526-530, 532-536, 538-542, 544-548, 550-554, 556-560, 562-566, 568-573, 585-580, 582-587, 589-594, 596-601, 603-607, 609-613, 615-619, 621-625, 627-631, 633-638, 340-644, 646-651, 653-657, 659-664, 666-67 An expression vector according to claim 96, comprising any one amino acid sequence of 0, 672-677, 679-683, 685-690, 692-696, 698-703, 705-711, 713-718, 720-726, 728-733, 735-741, 743-748, 750-756, 758-763, 765-771, 773-777, 779-783, 785-789, 791-795, 797-801, 803-807, 809-813, 815-819, 821-825, 827-831, 833, or 834.

98. A method for treating cancer in a subject diagnosed with cancer, comprising the following steps: A step of isolating cells from the biological sample of the subject; A step of culturing the isolated cells together with one or more peptides prepared by the method described in any one of claims 1 to 60; A step of isolating T cells, NK cells, and / or antigen-presenting cells cultured with one or more peptides, and expanding the T cells, NK cells, and / or antigen-presenting cells, in order to produce a composition effective for the treatment of tumor antigen-specific T cells, NK cells, and / or antigen-presenting cells; A step of adoptively transferring tumor antigen-specific T cells, NK cells, and / or antigen-presenting cells to the target. Includes, Therefore, the subject diagnosed with the cancer is treated. The aforementioned method.

99. The method according to claim 96, wherein the peptide comprises one or more heterocritic neoepitopes.

100. A cell comprising one or more peptides prepared by the method described in any one of claims 1 to 60, or a peptide comprising one amino acid sequence from SEQ ID NO: 1 to 838.

101. The aforementioned peptides are SEQ ID NO: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148-152, 154-162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232-236, 238-244, 246- 252, 254-260, 262-268, 270-276, 278-284, 286-292, 294-300, 302-308, 310-316, 318-324, 326-332, 334-340, 342-348, 350-356, 358-364, 366-372, 374-380, 382-388, 390-396, 398-404, 406-412, 414-420, 422-428, 430-436, 438-443, 445-450, 452-457, 459-464, 466-471, 473-478, 480-485, 487-492, 494-499, 501-506, 508-512, 514-518, 520-524, 526-530, 532-536, 538-542, 544-548, 550-554, 556-560, 562-566, 568-573, 585-580, 582-587, 589-594, 596-601, 603-607, 609-613, 615-619, 621-625, 627-631, 633-638, 340-644, 646-651, 653-657, 659-664, 666- A cell according to claim 100, comprising any one amino acid sequence of 670, 672-677, 679-683, 685-690, 692-696, 698-703, 705-711, 713-718, 720-726, 728-733, 735-741, 743-748, 750-756, 758-763, 765-771, 773-777, 779-783, 785-789, 791-795, 797-801, 803-807, 809-813, 815-819, 821-825, 827-831, 833, or 834.

102. A pharmaceutical composition comprising one or more peptides prepared by the method described in any one of claims 1 to 60, a cancer vaccine described in any one of claims 69 to 95, cells described in claim 100 or 101, an expression vector described in claim 96 or 97, or a peptide comprising one amino acid sequence of any one of SEQ ID NO: 1 to 838, and a pharmaceutically acceptable excipient or carrier.

103. The aforementioned peptides are SEQ ID NO: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148-152, 154-162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232-236, 238-244, 246-2 52, 254-260, 262-268, 270-276, 278-284, 286-292, 294-300, 302-308, 310-316, 318-324, 326-332, 334-340, 342-348, 350-356, 358-364, 366-372, 374-380, 382-388, 390-396, 398-404, 406-412, 414-420, 422-428, 430-436, 438-443, 445-450, 452-457, 459-464, 466-471, 473-478, 48 0-485, 487-492, 494-499, 501-506, 508-512, 514-518, 520-524, 526-530, 532-536, 538-542, 544-548, 550-554, 556-560, 562-566, 568-573, 585-580, 582-587, 589-594, 596-601, 603-607, 609-613, 615-619, 621-625, 627-631, 633-638, 340-644, 646-651, 653-657, 659-664, 666-670 The pharmaceutical composition according to claim 102, comprising any one amino acid sequence of 672-677, 679-683, 685-690, 692-696, 698-703, 705-711, 713-718, 720-726, 728-733, 735-741, 743-748, 750-756, 758-763, 765-771, 773-777, 779-783, 785-789, 791-795, 797-801, 803-807, 809-813, 815-819, 821-825, 827-831, 833, or 834.

104. Use in the manufacture of a medical agent for treatment of a subject, one or more peptides prepared by the method of any one of claims 1 to 60, a cancer vaccine of any one of claims 69 to 95, an expression vector of claim 96 or 97, cells of claim 100 or 101, a pharmaceutical composition of claim 102 or 103, or a peptide comprising any one amino acid sequence of SEQ ID NO: 1 to 838.

105. The aforementioned peptides are SEQ ID NO: 2, 3, 5-12, 14-21, 23-30, 32-39, 41-48, 50-57, 59-66, 68-75, 77-84, 86-93, 85-99, 101-105, 107-111, 113-117, 119-123, 125-129, 131-135, 137-141, 143-146, 148-152, 154-162, 164-168, 170-179, 181-185, 187-196, 198-202, 204-213, 215-219, 221-230, 232-236, 238-244, 246- 252, 254-260, 262-268, 270-276, 278-284, 286-292, 294-300, 302-308, 310-316, 318-324, 326-332, 334-340, 342-348, 350-356, 358-364, 366-372, 374-380, 382-388, 390-396, 398-404, 406-412, 414-420, 422-428, 430-436, 438-443, 445-450, 452-457, 459-464, 466-471, 473-478, 480-485, 487-492, 494-499, 501-506, 508-512, 514-518, 520-524, 526-530, 532-536, 538-542, 544-548, 550-554, 556-560, 562-566, 568-573, 585-580, 582-587, 589-594, 596-601, 603-607, 609-613, 615-619, 621-625, 627-631, 633-638, 340-644, 646-651, 653-657, 659-664, 666- The use according to claim 104, comprising any one amino acid sequence of 670, 672-677, 679-683, 685-690, 692-696, 698-703, 705-711, 713-718, 720-726, 728-733, 735-741, 743-748, 750-756, 758-763, 765-771, 773-777, 779-783, 785-789, 791-795, 797-801, 803-807, 809-813, 815-819, 821-825, 827-831, 833, or 834.

106. A peptide containing an amino acid sequence having at least 75%, 80%, 90%, 95%, or 98% sequence identity with one or more of SEQ ID NO: 1 to 838.

107. The peptide according to claim 106, comprising an amino acid sequence having at least 75%, 80%, 90%, 95%, or 98% sequence identity with respect to SEQ ID NO:2 or SEQ ID NO:

833.

108. The peptide according to claim 107, comprising an amino acid sequence having at least 75%, 80%, 90%, 95%, or 98% sequence identity with respect to SEQ ID NO:3 or SEQ ID NO:

834.

109. SEQ ID NO:2, 3, 5~12, 14~21, 23~30, 32~39, 41~48, 50~57, 59~66, 68~75, 77~84, 86~93, 85~99, 101~105, 107~111, 113~117, 119~123, 125~129, 131~ 135, 137-141, 143-146, 148-152, 154-162, 164-168, 170-179, 181-18 5, 187-196, 198-202, 204-213, 215-219, 221-230, 232-236, 238-244, 246-252, 254-260, 262-268, 270-276, 278-284, 286-292, 294-300, 302-308, 310-316, 318-324, 326-332, 334-340, 342-348, 350-356, 358-364, 366-372, 374-380, 382-388, 390-396, 398-404, 406-412, 414-420, 422-428, 430-436, 438-443, 445-450, 452-457, 459-464, 466-471, 4 73-478, 480-485, 487-492, 494-499, 501-506, 508-512, 514-518, 520-524, 526-530, 532-536, 538-542, 544-548, 550-554, 556-560, 562-566, 568-573, 585-580, 582-587, 589-594, 596-601, 603-607, 609-613, 615-619, 621-625, 627-631, 633-638, 340-644, 646-651, 653-657, 6 Peptides containing the amino acid sequences 59-664, 666-670, 672-677, 679-683, 685-690, 692-696, 698-703, 705-711, 713-718, 720-726, 728-733, 735-741, 743-748, 750-756, 758-763, 765-771, 773-777, 779-783, 785-789, 791-795, 797-801, 803-807, 809-813, 815-819, 821-825, 827-831, 833, or 834.

110. The peptide according to claim 110, comprising the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:

833.

111. The peptide according to claim 110, comprising the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:

834.

112. The method according to any one of the claims, further comprising the step of quantifying the difference in structural characteristics between the modeled peptide and the parent epitope.

113. The method according to claim 112, wherein the features include contact between the peptide and the HLA cleft residue, solvent exposure area (SASA), peptide stiffness, surface hydrophobicity, or electrostatic potential as measures of CαRMSD for the top 10 predictive structural models for each peptide-HLA complex.

114. The method according to any one of claims 112 or 108, further comprising co-culturing heterocritic peptides and HLA-matched lymphocytes in vitro to verify which heterocritic peptide induces the greatest T cell expansion and activation compared to the parent peptide when evaluated by an IFN-γ erythropoiesis assay.

115. (a) one or more peptides prepared by the method according to any one of claims 1 to 68, a cancer vaccine according to any one of claims 69 to 95, an expression vector according to claim 96 or 97, cells according to claim 100 or 101, a pharmaceutical composition according to claim 102 or 103, or a peptide according to any one of claims 106 to 111, (b) Informational materials containing instructions for administering a certain dose of one or more peptides, the cancer vaccine, the cells, or the pharmaceutical composition in a dosage form. A kit that includes this.

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