Compositions and methods for personalized neoplasia vaccines

Abstract

To provide methods of making a personalized neoplasia vaccine for a subject diagnosed as having a neoplasia.SOLUTION: The method comprises: identifying a plurality of mutations in a neoplasia; analyzing the plurality of mutations to identify a subset of at least five neo-antigenic mutations predicted to encode neo-antigenic peptides, the neo-antigenic mutations being selected from the group consisting of missense mutations, neoORF mutations, and any combination thereof; and producing, based on the identified subset, a personalized neoplasia vaccine.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Description of Rights to Inventions Made Based on Federal Government Sponsored Research This study was supported by the following grants from the National Institutes of Health Grant Numbers: NIH / NCI-1R01CA155010-02 and NHLBI-5R01HL103532-03. The federal government has certain rights in this invention.

[0002] Cross - References to Related Applications This application claims the benefit of and priority to U.S. Provisional Patent Application No. 61 / 809,406, filed on April 7, 2013, and U.S. Provisional Patent Application No. 61 / 869,721, filed on August 25, 2013, the contents of which are hereby incorporated by reference herein.

[0003] The present invention relates to personalized strategies for treating neoplasms. More particularly, the present invention relates to the identification and use of a patient-specific pool of tumor-specific neoantigens in personalized tumor vaccines for treating a subject.

Background Art

[0004] Approximately 1.6 million Americans are diagnosed with neoplasms each year, and it is estimated that in 2013, approximately 580,000 people in the United States will die from this disease. In the past few decades, the detection, diagnosis, and treatment of neoplasms have improved significantly, resulting in a significant increase in survival rates for many types of neoplasms. However, only about 60% of people diagnosed with neoplasms are still alive 5 years after the start of treatment, and thus neoplasms are the second leading cause of death in the United States.

[0005] Currently, there are numerous existing cancer therapies, including ablation techniques (e.g., surgical procedures, cryogenic / thermal treatments, ultrasound, radiofrequency, and radiation) and chemical techniques (e.g., pharmaceuticals, cytotoxic / chemotherapeutic agents, monoclonal antibodies, and various combinations thereof). Unfortunately, such treatments often involve serious risks, toxic side effects, and extremely high costs, and in addition, their effectiveness is also uncertain.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0007] Regarding cancer therapies that attempt to target cancerous cells by the patient's own immune system (e.g., cancer vaccines), there is increasing interest because such therapies can reduce / eliminate some of the above-mentioned drawbacks. Cancer vaccines typically consist of tumor antigens and immune-stimulating molecules (e.g., cytokines or TLR ligands), which work together to induce antigen-specific cytotoxic T cells, causing the T cells to target and destroy tumor cells. Current cancer vaccines typically contain common tumor antigens, which are natural proteins that are selectively expressed or overexpressed in tumors found in many individuals (i.e., proteins encoded by the DNA of all normal cells in an individual). Such common tumor antigens are useful for identifying specific types of tumors, but they are not ideal as immunogens for targeting T cell responses to specific tumor types because they are subject to an immune-suppressing self-tolerance effect. Therefore, a method for identifying more effective tumor antigens is needed.

Means for Solving the Problems

[0008] The present invention relates to strategies for personalized neobiological therapy, and more particularly, to the identification and use of personalized cancer vaccines consisting essentially of a pool of tumor-specific and patient-specific neoantigens for treating a subject's tumor. As described below, the present invention, at least in part, can identify all or substantially all of the mutant neoantigens uniquely present in an individual patient's neoplasm / tumor by using whole genome / exome sequencing, and by analyzing this group of mutant neoantigens, can identify a specific optimized subset of neoantigens to be used as a personalized neobiological vaccine for treating the patient's neoplasm / tumor, based on the discovery.

[0009] In one aspect, the present invention provides a method for manufacturing a personalized neobiological vaccine for a subject diagnosed with a neoplasm, the method comprising identifying a plurality of mutations in the neoplasm, and analyzing the plurality of mutations to identify a subset of at least five neoantigen mutations predicted to encode neoantigen peptides, wherein the neoantigen mutations are selected from the group consisting of missense mutations, neo-ORF mutations, and any combination thereof, and making a personalized neobiological vaccine based on the identified subset.

[0010] In certain embodiments, the present invention provides that the step of identifying further comprises sequencing the genome, transcriptome, or proteome of the neoplasm. In another embodiment, the analyzing step is a step of determining one or more features associated with a subset of at least five neoantigen mutations predicted to encode neoantigen peptides, wherein the features are selected from the group consisting of molecular weight, cysteine content, hydrophilicity, hydrophobicity, charge, and binding affinity; and further including a step of ranking each of the neoantigen mutations within the identified subset of at least five neoantigen mutations based on the determined features. In one embodiment, the neoantigen mutations ranked up to the top 5 to up to the top 30 are included in the personalized neoplasm vaccine. In another embodiment, the neoantigen mutations are ranked according to the order shown in FIG. 8.

[0011] In one embodiment, the personalized neoplasm vaccine includes at least about 20 neoantigen peptides corresponding to the neoantigen mutations. In another embodiment, the personalized neoplasm vaccine includes one or more DNA molecules having the ability to express at least about 20 neoantigen peptides corresponding to the neoantigen mutations. In another embodiment, the personalized neoplasm vaccine includes one or more RNA molecules having the ability to express at least 20 neoantigen peptides corresponding to the neoantigen mutations.

[0012] In an embodiment, the personalized neoplasm vaccine includes a neoORF mutation predicted to encode a neoORF polypeptide with a Kd ≤ 500 nM. In another embodiment, the personalized neoplasm vaccine includes a missense mutation predicted to encode a polypeptide with a Kd ≤ 150 nM, and its native cognate protein has a Kd ≥ 1000 nM or ≤ 150 nM.

[0013] In another embodiment, at least about 20 neoantigen peptides range from about 5 to about 50 amino acids in length. In another embodiment, at least about 20 neoantigen peptides range from about 15 to about 35 amino acids in length. In another embodiment, at least about 20 neoantigen peptides range from about 18 to about 30 amino acids in length. In another embodiment, at least about 20 neoantigen peptides range from about 6 to about 15 amino acids in length. In yet another embodiment, at least about 20 neoantigen peptides are 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

[0014] In one embodiment, the personalized neoplasm vaccine further comprises an adjuvant. In other embodiments, the adjuvant is poly ICLC, 1018 ISS, aluminum salts, A mplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IM P321, IS patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONT AK, PepTel.RTM, vector systems, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulo n, badimezan, and / or AsA404 (DMXAA). In a preferred embodiment, the adjuvant is poly ICLC.

[0015] In another aspect, the present invention includes a method of treating a subject diagnosed with a neoplasm with an individualized neoplasm vaccine, the method comprising: identifying a plurality of mutations in the neoplasm; analyzing the plurality of mutations to identify a subset of at least five neoantigen mutations predicted to encode expressed neoantigen peptides, wherein the neoantigen mutations are selected from the group consisting of missense mutations, neo-ORF mutations, and any combination thereof; producing an individualized neoplasm vaccine based on the identified subset; and administering the individualized neoplasm vaccine to the subject, thereby treating the neoplasm.

[0016] In another embodiment, the identifying step may further comprise sequencing the genome, transcriptome, or proteome of the neoplasm. In yet another embodiment, the analyzing step comprises determining one or more characteristics associated with a subset of at least five neoantigen mutations predicted to encode expressed neoantigen peptides, wherein the characteristics are selected from the group consisting of molecular weight, cysteine content, hydrophilicity, hydrophobicity, charge, and binding affinity; and ranking each of the neoantigen mutations within the identified subset of at least five neoantigen mutations based on the determined characteristics.

[0017] In one embodiment, the top 5 to top 30 ranked neoantigen mutations are included in the individualized neoplasm vaccine. In another embodiment, the neoantigen mutations are ranked according to the order shown in FIG. 8.

[0018] In one embodiment, the individualized neoplasm vaccine comprises at least 20 neoantigen peptides corresponding to the neoantigen mutations. In another embodiment, the individualized neoplasm vaccine comprises one or more DNA molecules capable of expressing at least 20 neoantigen peptides corresponding to the neoantigen mutations.

[0019] In one embodiment, the personalized neoplasm vaccine comprises one or more RNA molecules capable of expressing at least 20 neoantigenic peptides corresponding to neoantigenic mutations. In one embodiment, the personalized neoplasm vaccine comprises a neoORF mutation predicted to encode a neoORF polypeptide with a Kd≦500 nM.

[0020] In another embodiment, the personalized neoplasm vaccine comprises a polypeptide having a Kd≦150 nM. wherein the native cognate protein has a Kd of > 1000 nM or < 150 nM.

[0021] In one embodiment, the at least 20 neo-antigenic peptides are in the range of about 5 to about 50 amino acids in length. In one embodiment, the at least 20 neo-antigenic peptides are in the range of about 15 to about 35 amino acids in length. In one embodiment, the at least 20 neo-antigenic peptides are in the range of about 18 to about 30 amino acids in length. In one embodiment, the at least 20 neo-antigenic peptides are in the range of about 6 to about 15 amino acids in length. In one embodiment, the at least 20 neo-antigenic peptides are 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

[0022] In one embodiment, the administering step further comprises splitting the generated vaccine into two or more subpools; and injecting each of the subpools into a different site in the patient. In one embodiment, each of the subpools injected into the different sites contains neo-antigen peptides such that the number of individual peptides in the subpool targeting any single patient HLA is one, or as few as possible between two or more.

[0023] In one embodiment, the administering step further includes the step of dividing the prepared vaccine into two or more sub-pools, and each sub-pool contains at least 5 neoantigen peptides selected to optimize the interactions within the pool.

[0024] In one embodiment, the optimization includes reducing the negative interactions between the neoantigen peptides in the same pool. In another aspect, the present invention includes an individualized neoplasm vaccine prepared by the method described above.

[0025] Definitions To assist in the understanding of the present invention, several terms and phrases are defined below: Unless specifically stated or otherwise apparent from the context, as used herein, the term "about" is understood to be within the normal tolerance range in the art, for example, within 2 standard deviations of the mean value. About can be understood to be within 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the recited value. Unless otherwise apparent from the context, all numerical values provided herein are modified by the term about.

[0026] "Agent" means any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof. "Improve" means reducing, suppressing, attenuating, decreasing, stopping, or stabilizing the onset or progression of a disease (e.g., neoplasm, tumor, etc.).

[0027] "Modification" means a change (increase or decrease) in the expression level or activity of a gene or polypeptide when detected by standard methods known in the art as described herein. As used herein, modification includes a change of 10% in the expression level, preferably a change of 25% in the expression level, more preferably a change of 40%, and most preferably a change of 50% or more in the expression level.

[0028] "Analog" means a molecule that is not identical but has similar functional or structural characteristics. For example, a tumor-specific neoantigen polypeptide analog retains the biological activity of the corresponding naturally occurring tumor-specific neoantigen polypeptide while having specific biochemical modifications that enhance the function of the analog compared to the naturally occurring polypeptide. Such biochemical modifications can increase the protease resistance, membrane permeability, or half-life of the analog, for example, without changing ligand binding. The analog may include non-natural amino acids. Such biochemical modifications can increase the protease resistance, membrane permeability, or half-life of the analog, for example, without changing ligand binding. The analog may include non-natural amino acids.

[0029] The term "combination therapy" encompasses the administration of a pooled sample of neoantigens specific to a neoplasm / tumor and one or more additional therapeutic agents as part of a specific therapeutic regimen intended to result in a beneficial (additive or synergistic) effect from the co-action of the therapeutic agents. The beneficial effects of the combination include, but are not limited to, pharmacokinetic or pharmacodynamic co-actions resulting from the combination of the therapeutic agents. Administration of these therapeutic agents in combination is typically carried out over a defined period (usually minutes, hours, days, or weeks depending on the selected combination). "Combination therapy" is intended to encompass sequential administration of these therapeutic agents (i.e., each therapeutic agent is administered at a different time point), as well as substantially simultaneous administration of these therapeutic agents, or at least two of the therapeutic agents. Substantially simultaneous administration can be achieved by administering to the subject, for example, a single capsule having a defined ratio of each therapeutic agent or in single capsules for each of the plurality of therapeutic agents. For example, one combination of the present invention may include a pooled sample of tumor-specific neoantigens and at least one additional therapeutic agent (e.g., a chemotherapeutic agent, an anti-angiogenic agent, an immunosuppressive agent, an anti-inflammatory agent, etc.) at the same or different time points, or they can be formulated as a single co-formulated pharmaceutical composition containing two compounds. As another example, a combination of the present invention (e.g., a pooled sample of tumor-specific neoantigens and at least one additional therapeutic agent) may be formulated as separate pharmaceutical compositions that can be administered at the same or different time points. Sequential or substantially simultaneous administration of each therapeutic agent can be achieved by any suitable route including, but not limited to, oral, intravenous, subcutaneous, intramuscular, direct absorption through mucosal tissues (e.g., nose, mouth, vagina, and rectum), and ocular routes (e.g., intravitreal, intraocular, etc.). The therapeutic agents can be administered by the same route or by different routes. For example, one component of a particular combination may be administered by intravenous injection while one or more other components of the combination may be administered orally. These components may be administered in any therapeutically effective order.

[0030] The term "combination" encompasses a group of compounds or non-pharmacological therapies useful as part of combination therapy. In the present disclosure, "comprises", "comprising", "containing", "having", etc. can have the meanings assigned to them in the United States Patent Law, and can mean "includes", "including", etc.; "consisting essentially of" or "consists essentially" likewise has the meaning assigned to it in the United States Patent Law. This term is open-ended and allows the presence of elements other than those described, provided that the basic or novel features of the described elements are not changed by the presence of other elements, except for prior art embodiments.

[0031] "Control" means a standard or reference condition. "Disease" means any pathological condition or disorder that impairs or interferes with the normal function of cells, tissues, or organs.

[0032] "Effective amount" means the amount necessary to improve the symptoms of a disease (e.g., neoplasm / tumor) compared to an untreated patient. The effective amount of one or more active compounds used in the practice of the present invention for the treatment of a disease will vary depending on the method of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is referred to as an "effective" amount.

[0033] "Fragment" means a portion of a polypeptide or nucleic acid molecule. This portion preferably contains at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of the reference nucleic acid molecule or polypeptide. Fragments can contain 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides or amino acids. ​​

[0034] "Hybridization" means a hydrogen bond, which may be a Watson-Crick, Hoogsteen or reverse Hoogsteen hydrogen bond, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair by forming a hydrogen bond.

[0035] "Inhibitory nucleic acid" means a double-stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that, when administered to mammalian cells, results in a decrease (e.g., 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule or its ortholog, or at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of some or all of the nucleic acids detailed herein.

[0036] "Isolated polynucleotide" means a nucleic acid (e.g., DNA) that does not contain the gene from which the nucleic acid molecule of the present invention is derived in the natural genome of an organism - or in the genomic DNA of a neoplasm / tumor derived from an organism. Thus, this term includes, for example, recombinant DNA incorporated into a vector; incorporated into a self-replicating plasmid or virus; or incorporated into the genomic DNA of a prokaryotic or eukaryotic organism (e.g., DNA encoding a neo-ORF, read-through, or indel-derived polypeptide identified in a patient's tumor); or recombinant DNA that exists as a separate molecule independent of other sequences (e.g., cDNA or genomic or cDNA fragments produced by PCR or restriction endonuclease digestion). In addition, this term includes RNA molecules transcribed from DNA molecules, as well as recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

[0037] "Isolated polypeptide" means a polypeptide of the present invention that has been separated from the components with which it is naturally associated. Typically, a polypeptide is isolated when it contains less than 60% by weight of the proteins and naturally occurring organic molecules with which it is naturally associated. Preferably, this preparation is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight of the polypeptide of the present invention. The isolated polypeptides of the present invention can be obtained, for example, by extraction from natural sources, by expressing recombinant nucleic acids encoding such polypeptides; or by chemically synthesizing the protein. Purity can be measured by any suitable method, such as by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0038] "Ligand" should be understood to mean a molecule having a structure complementary to the structure of a receptor and having the ability to form a complex with that receptor. According to the present invention, a ligand means a peptide or peptide fragment having a suitable length and a suitable binding motif in its amino acid sequence, and thus this peptide or peptide fragment should be understood to have the ability to form a complex with a protein of MHC class I or MHC class II.

[0039] "Mutation", for the purposes of this manuscript, means a DNA sequence found in a patient's tumor DNA sample that is not found in the corresponding normal DNA sample of the same patient. "Mutation" can also refer to an RNA sequence pattern of a patient that cannot be attributed to a predicted mutation based on known information about an individual gene and that is reasonably considered to be a novel mutation in the splicing pattern of one or more genes that are specifically modified in the patient's tumor cells. For example, it can also refer to a novel mutation in the splicing pattern of one or more genes that are specifically modified in the patient's tumor cells.

[0040] "Neoantigen" or "neoantigenic" means a class of tumor antigens generated by one or more tumor-specific mutations that modify the amino acid sequence of a genomically encoded protein.

[0041] As used herein, the term "neoplasm" means any disease caused by or resulting from unduly high levels of cell division, unduly low levels of apoptosis, or both. For example, cancer is an example of a neoplasm. Examples of cancers include, without limitation, leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute monocytic leukemia, acute monoblastic 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 disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, 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, renal cell carcinoma, hepatocellular carcinoma, bile duct (nile duct) carcinoma, choriocarcinoma, seminoma, embryonal carcinoma , Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). Lymphoproliferative disorders are also considered proliferative diseases.

[0042] Unless specifically recited or apparent from the context, as used herein, the term "or" is understood to be inclusive. Unless specifically recited or apparent from the context, as used herein, the terms "a," "an," and "the" are understood to be either singular or plural.

[0043] The term "patient" or "subject" refers to an animal that is the subject of treatment, observation, or experiment. By way of example only, subjects include, but are not limited to, mammals such as, but not limited to, humans or non-human mammals such as non-human primates, cows, horses, dogs, sheep, or cats.

[0044] "Pharmaceutically acceptable" means approved or approvable by a federal or state government regulatory agency for use in animals, including humans, or included in the United States Pharmacopeia or other generally recognized pharmacopeia.

[0045] "Pharmaceutically acceptable excipient, carrier or diluent" refers to an excipient, carrier or diluent that can be administered to a subject together with a drug and that is non-toxic and does not impair the pharmacological activity of the drug even when administered in a dose sufficient to deliver a therapeutic amount of the drug.

[0046] A "pharmaceutically acceptable salt" of a pooled tumor-specific neoantigen as described herein may be an acidic or basic salt that is generally considered in the art to be suitable for use in contact with human or animal tissue without undue toxicity, irritation, allergic reaction, or other problems or complications. Such salts include inorganic salts and organic acid salts of basic residues such as amines, and alkali salts or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutical salts include, but are not limited to, 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, hydroxymaleic acid, hydroiodic acid, phenylacetic acid, alkanoic acids such as acetic acid, HOOC-(CH2) nExamples of salts of acids include -COOH (where n is from 0 to 4). Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those skilled in the art will recognize additional pharmaceutically acceptable salts of the pooled tumor-specific neoantigens provided herein, including those described in Remington’s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985). In general, pharmaceutically acceptable acidic or basic salts can be synthesized from the parent compound containing a basic or acid moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting these compounds in the form of the free acid or free base with a stoichiometric amount of the appropriate base or acid in a suitable solvent.

[0047] As used herein, the terms "prevent," "preventing," "prevention," "preventive treatment," etc. refer to reducing the likelihood of the onset of a disease or condition in a subject who does not have the disease or condition but is at risk of or predisposed to developing it.

[0048] "Primer set" means a set of oligonucleotides that can be used, for example, in PCR. The primer set can consist of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, or more primers.

[0049] "The protein or molecule of the major histocompatibility complex (MHC)", "MHC molecule", "MHC protein" or "HLA protein" should be understood to mean, in particular, a peptide resulting from the proteolytic cleavage of a protein antigen and corresponding to a potential T cell epitope, and having the ability to bind such peptides and transport them to the cell surface where they are presented to specific cells, in particular naive T cells, cytotoxic T lymphocytes or T helper cells. The major histocompatibility complex in the genome is a genetic region where gene products are expressed on the cell surface and contains genetic regions important for the binding and presentation of endogenous and / or exogenous antigens and thus for the regulation of immunological processes. The major histocompatibility complex is classified into two groups of genes encoding different proteins: MHC class I and MHC class II molecules. These two MHC class molecules are specialized for different antigen sources. MHC class I molecules typically present, but are not limited to, endogenously synthesized antigens, such as viral proteins and tumor antigens. MHC class II molecules present protein antigens derived from exogenous sources, such as bacterial products. The cell biology and expression patterns of these two MHC classes are adapted to these different roles.

[0050] MHC class I molecules consist of a heavy chain and a light chain and have the ability to bind peptides of about 8 - 11 amino acids, but usually 9 or 10 amino acids, and present them to naive and cytotoxic T lymphocytes when the peptide has a suitable binding motif. The peptides bound by MHC class I molecules typically, but not exclusively, are derived from endogenous protein antigens. The heavy chain of MHC class I molecules is preferably an HLA-A, HLA-B or HLA-C monomer, and the light chain is β-2-microglobulin.

[0051] Class II MHC molecules consist of an α-chain and a β-chain and have the ability to bind peptides of about 15 to 24 amino acids and present them to T helper cells when the peptides have a suitable binding motif. Peptides bound by class II MHC molecules usually are derived from extracellular or foreign protein antigens. The α-chain and β-chain are, specifically, HLA-DR, HLA- DQ and HLA-DP monomers.

[0052] The ranges provided herein are to be understood as being shorthand for all values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-ranges 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, as well as all decimal values intervening between the aforementioned integers, e.g., 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9, etc. With respect to sub-ranges, “nested sub-ranges” extending from either endpoint of the range are specifically contemplated. For example, nested sub-ranges of the illustrative range of 1 to 50 may include, in one direction, 1 to 10, 1 to 20, 1 to 30, and 1 to 40, or, in the other direction, 50 to 40, 50 to 30, 50 to 20, and 50 to 10.

[0053] "Receptor" should be understood to mean a biomolecule or molecular class having ligand-binding ability. A receptor can function to transmit information in a cell, cell formation, or organism. A receptor includes at least one receptor unit, and often includes two or more receptor units, where each receptor unit can consist of a protein molecule, specifically a glycoprotein molecule. A receptor has a structure that complements the structure of a ligand and can form a complex with the ligand as a binding partner. After binding to a ligand on the surface of a cell, signal information can be transmitted by a change in the three-dimensional structure of the receptor. According to the present invention, a receptor can refer to a ligand, specifically a peptide or peptide fragment of a suitable length, and specific proteins of MHC class I and II having the ability to form a receptor / ligand complex.

[0054] "Receptor / ligand complex" should also be understood to mean "receptor / peptide complex" or "receptor / peptide fragment complex", specifically peptide-presenting or peptide-fragment-presenting MHC molecules of class I or class II.

[0055] "Reduce" means a negative change of at least 10%, 25%, 50%, 75%, or 100%. "Reference" means a standard or control condition.

[0056] A "reference array" is a defined array used as the basis for array comparison. The reference array may be a subset of a particular array, or the whole of it; for example, it may be a segment of a full-length cDNA or genomic sequence, or a complete cDNA or genomic sequence. For polypeptides, the length of the reference polypeptide sequence can generally be at least about 10 to 2,000 amino acids, 10 to 1,500, 10 to 1,000, 10 to 500, or 10 to 100. Preferably, the length of the reference polypeptide sequence is at least about 10 to 50 amino acids, more preferably at least about 10 to 40 amino acids, even more preferably about 10 to 30 amino acids, about 10 to 20 amino acids, about 15 to 25 amino acids, or about 20 amino acids. For nucleic acids, the length of the reference nucleic acid sequence can generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer close to or between them.

[0057] "Specifically binds" means a compound or antibody that recognizes and binds to the polypeptide of the present invention, but does not substantially recognize and bind to other molecules in a sample, for example, in a biological sample. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecule encoding the polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to the endogenous nucleic acid sequence, but can typically exhibit substantial identity. A polynucleotide having "substantial identity" with an endogenous sequence typically has the ability to hybridize with at least one strand of a double-stranded nucleic acid molecule. "Hybridize" means to pair between complementary polynucleotide sequences (for example, the genes described herein) or a portion thereof under various stringency conditions to form a double-stranded molecule (see, for example, Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152: 399; Kimmel, A. R. (1987) Methods Enzymol. 152: 507).

[0058] For example, stringent salt concentrations can typically be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be achieved when no organic solvent, such as formamide, is present, while high stringency hybridization can be achieved in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions can typically include a temperature of at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Various additional parameters, such as hybridization time, the concentration of detergents, such as sodium dodecyl sulfate (SDS), and whether to include carrier DNA, are well known to those skilled in the art. By combining these various conditions as needed, various stringency levels can be achieved. In a preferred embodiment, hybridization can be performed at 30°C in 750 mM Na Cl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization can be performed at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In the most preferred embodiment, hybridization can be performed at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA . Useful variations on these conditions will be readily apparent to those skilled in the art

[0059] For many applications, the washing steps following hybridization may also differ in terms of stringency. The washing stringency conditions can be defined by salt concentration and temperature. As noted above, the washing stringency can be increased by decreasing the salt concentration or increasing the temperature. For example, a stringent salt concentration for the washing step can preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the washing step can generally include a temperature of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In a preferred embodiment, the washing step can be carried out at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step can be carried out at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step can be carried out at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations on these conditions will be readily apparent to those skilled in the art.Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196: 180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72: 3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0060] "Substantially identical" means a polypeptide or nucleic acid molecule that exhibits at least 50% identity with a reference amino acid sequence (e.g., any one of the amino acid sequences described herein) or nucleic acid sequence (e.g., any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and even more preferably 90%, 95% or even 99% identical at the amino acid or nucleic acid level to the sequence used for comparison.

[0061] Sequence identity is typically determined using sequence analysis software (e.g., Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). ーcenter (University of Wisconsin Biotechnology Center), 1710 Unive rsity Avenue, Madison, Wis. 53705's sequence analysis softw measured using the ALIGN, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs. Such software matches identical or similar sequences by assigning a degree of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method of determining the degree of identity, the BLAST program may be used with a probability score of e -3 ~e -100 probability score.

[0062] A "T cell epitope" is to be understood to mean a peptide sequence that can be bound in the form of a peptide-presenting MHC molecule or MHC complex by a class I or II MHC molecule and can then be recognized and bound by a naive T cell, cytotoxic T lymphocyte, or T helper cell in this form.

[0063] As used herein, the terms "treating," "treated," "treating," "treatment," etc. refer to reducing or ameliorating a disorder and / or symptoms associated therewith (e.g., a neoplasm or tumor). It will be understood that treating a disorder or condition does not necessarily require complete disappearance of the associated disorder, condition, or symptoms, although this is not excluded.

[0064] The term "therapeutic effect" refers to the degree of reduction of one or more of the symptoms of a disorder (e.g., a neoplasm or tumor) or any of the associated pathology. A "therapeutically effective amount," as used herein, refers to the amount of a drug that, when administered to a cell or subject in a single or multiple doses, is effective in extending the survival of a patient having such a disorder, reducing, preventing or delaying one or more of the signs or symptoms of the disorder, to a greater extent than would be expected in the absence of such treatment. A "therapeutically effective amount" is intended to determine the adequacy of the amount required to achieve a therapeutic effect. A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the "therapeutically effective amount" of the required pharmaceutical composition (e.g., ED 50 ). For example, a physician or veterinarian may start with a dosage of the compound of the invention used in the pharmaceutical composition at a level lower than the dosage required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0065] The pharmaceutical composition typically must provide a dosage of the compound of about 0.0001 mg to about 200 mg per kg of body weight per day. For example, the dosage for systemic administration to a human patient can range from 0.01 - 10 μg / kg, 20 - 80 μg / kg, 5 - 50 μg / kg, 75 - 150 μg / kg, 100 - 500 μg / kg, 250 - 750 μg / kg, 500 - 1000 μg / kg, 1 - 10 mg / kg, 5 - 50 mg / kg, 25 - 75 mg / kg, 50 - 100 mg / kg, 100 - 250 mg / kg, 50 - 100 mg / kg, 250 - 500 mg / kg, 500 - 750 mg / kg, 750 - 1000 mg / kg, 1000 - 1500 mg / kg, 1500 - 2000 mg / kg, 5 mg / kg, 20 mg / kg , 50 mg / kg, 100 mg / kg, 200 mg / kg. Pharmaceutical dosage unit forms are prepared to provide from about 0.001 mg to about 5000 mg, for example from about 100 to about 2500 mg, of the compound or combination of essential ingredients per dosage unit form.

[0066] "Vaccine" should be understood to mean a composition that elicits immunity for the prevention and / or treatment of diseases (e.g., neoplasms / tumors). Thus, a vaccine is a drug that contains an antigen and is intended to be used to generate specific protective and prophylactic substances by vaccination in humans or animals.

[0067] In the description of the list of chemical groups in any definition of a variable group herein, the definition of the variable group as any single group or combination of the listed groups is included. In the description of embodiments regarding variable groups or aspects herein, the embodiments as any single embodiment or the embodiments in combination with any other embodiment or part thereof are included.

[0068] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein. Reading the following detailed description in conjunction with the following drawings will better understand the above and other features and advantages of the present disclosure:

Brief Description of the Drawings

[0069]

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Mode for Carrying Out the Invention

[0070] The present invention relates to an individualized strategy for treating a neoplasm, more particularly a tumor, by administering to a subject (e.g., a mammal such as a human) a therapeutically effective amount of a pharmaceutical composition (e.g., a cancer vaccine) comprising a plurality of neoplasia / tumor-specific neoantigens. As described in more detail below, the present invention is based, at least in part, on the discovery that whole genome / exome sequencing can be used to identify all or substantially all of the mutant neoantigens uniquely present in the neoplasm / tumor of an individual patient, and that by analyzing this population of mutant neoantigens, a specific optimized subset of neoantigens can be identified that can be used as an individualized cancer vaccine for treating the patient's neoplasm / tumor. For example, as shown in FIG. 1, by sequencing the neoplasm / tumor DNA and normal DNA of each patient to identify tumor-specific mutations and determining the patient's HLA allotype, a population of neoplasm / tumor-specific neoantigens can be identified. The population of neoplasm / tumor-specific neoantigens and their cognate native antigens are then subjected to bioinformatics analysis using a validated algorithm to predict which tumor-specific mutations create epitopes that can bind to the patient's HLA allotype, and more particularly which tumor-specific mutations create epitopes that can bind more effectively to the patient's HLA allotype compared to the cognate native antigen. Based on this analysis, a plurality of peptides corresponding to a subset of those mutations can be designed and synthesized for each patient and pooled together for use as a cancer vaccine when immunizing the patient. The peptide neoantigens may be combined with an adjuvant (e.g., poly ICLC) or another anti-neoplastic agent. Without being bound by theory, these neoantigens are expected to avoid central thymic tolerance (thus allowing for a more potent anti-tumor T cell response) while reducing the potential for autoimmunity (e.g., by avoiding targeting of normal self-antigens).

[0071] 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 front line of defense against infection, and the most potentially capable pathogens are rapidly neutralized by this system before they can cause an infection that can be recognized, for example. The adaptive immune system responds to the molecular structures of invading organisms, called antigens. There are two types of adaptive immune responses, including the humoral immune response and the cell-mediated immune response. In the humoral immune response, antibodies secreted into the body fluids by B cells bind to pathogen-derived antigens, leading to the elimination of pathogens through various mechanisms, such as complement-mediated lysis. In the cell-mediated immune response, T cells with the ability to destroy other cells are activated. For example, when disease-related proteins are present in cells, those proteins are fragmented into peptides by proteolysis within the cells. Next, specific cellular proteins attach to these antigens or peptides thus formed and transport them to the cell surface, where these antigens or peptides are presented to the body's molecular defense mechanisms, specifically T cells. Cytotoxic T cells recognize these antigens and kill the cells that have such antigens.

[0072] The molecule that transports and presents peptides to the cell surface is the major histocompatibility complex (MHC) is referred to as a protein. MHC proteins are classified into two types called MHC class I and MHC class II. The structures of these 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 many tumor cells. MHC class I proteins are usually loaded with antigens derived from endogenous proteins or pathogens present within the cell, and then it is presented to naive or cytotoxic T lymphocytes (CTLs). MHC class II proteins are present on dendritic cells, B lymphocytes, macrophages, and other antigen-presenting cells. MHC class II proteins mainly present peptides processed from an external antigen source, i.e., outside the cell, to T helper (Th) cells. Most of the peptides that bind to MHC class I proteins are derived from cytoplasmic proteins that occur in the healthy host cells of the organism itself and usually do not stimulate an immune response. Therefore, cytotoxic T lymphocytes that recognize such self-peptide-presenting MHC molecules of class I are removed in the thymus (central tolerance) or removed or inactivated, i.e., tolerized (peripheral tolerance) after being released from the thymus. MHC molecules have the ability to stimulate an immune response when presenting peptides to non-tolerized T lymphocytes. Cytotoxic T lymphocytes have both a T cell receptor (TCR) and a CD8 molecule on their surface. The T cell receptor has the ability to recognize and bind to peptides complexed with MHC class I molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor with the ability to bind to a specific MHC / peptide complex.

[0073] Peptide antigens attach to MHC class I molecules by competitive affinity binding within the endoplasmic reticulum before being presented on the cell surface. Here, the affinity of an individual peptide antigen is directly related to its amino acid sequence and the presence of specific binding motifs at defined positions within the amino acid sequence. If the sequence of such a peptide is known, it is possible to manipulate the immune system against diseased cells, for example, using peptide vaccines.

[0074] One of the significant obstacles hindering the development of curative and tumor-specific immunotherapeutic agents is the identification and selection of highly specific and restricted tumor antigens to avoid autoimmunity. Tumor neoantigens resulting from genetic changes (e.g., inversions, translocations, deletions, missense mutations, splice-site mutations, etc.) within malignant cells correspond to the most tumor-specific antigen class. Neoantigens have rarely been used in cancer vaccines because their identification, selection of optimized neoantigens, and generation of neoantigens for use in vaccines are technically difficult. According to the present invention, these problems can be addressed as follows: · Identifying all or almost all mutations in a neoplasm / tumor at the DNA level using whole-genome, whole-exome (e.g., only captured exons), or RNA sequencing of the tumor relative to the corresponding germline sample of each patient; · Analyzing the identified mutations with one or more peptide-MHC binding prediction algorithms to generate a plurality of candidate neoantigen T cell epitopes that are expressed within the neoplasm / tumor and can bind to the patient's HLA alleles; and · Synthesizing a plurality of candidate neoantigen peptides selected from the set of all neo-ORF peptides and predicted binding peptides for use in a cancer vaccine.

[0075] For example, to convert sequencing information into a therapeutic vaccine, the following is included: (1) Prediction of personal mutant peptides that can bind to an individual's HLA molecules. To efficiently select which specific mutations should be utilized as immunogens, identification of the patient's HLA type and the ability to predict which mutant peptides can efficiently bind to the patient's HLA alleles are required. In recent years, the accuracy of prediction algorithms for major HLA-A and -B alleles has improved with neural network-based learning methods using validated binding and non-binding peptides.

[0076] (2) Formulating the drug as a multi-epitope vaccine of long peptides. In practice By targeting as many mutant epitopes as possible, a great deal of the immune system's capabilities are harnessed, the opportunity for immune escape is blocked by downregulating specific immunotargeted gene products, and the known inaccuracies of epitope prediction methods are compensated for. Synthetic peptides provide a particularly useful means for efficiently preparing multiple immunogens and rapidly turning the identification of mutant epitopes into an effective vaccine. Peptides can be easily chemically synthesized using reagents that do not contain contaminating bacteria or animal substances and can be easily purified. Due to their small size, it is possible to clearly focus on the mutant regions of proteins, and there is also a reduction in irrelevant antigen competition from other components (non-mutant proteins or viral vector antigens).

[0077] (3)Combination with a potent vaccine adjuvant. For an effective vaccine, it is necessary for a potent adjuvant to elicit an immune response. As described below, the TLR3 agonist and poly ICLC, which is the RNA helicase domain of MDA5 and RIG3, exhibit some of the properties desirable for a vaccine adjuvant. These properties include the induction of local and systemic activation of immune cells in vivo, the production of stimulatory chemokines and cytokines, and the stimulation of antigen presentation by DCs. Furthermore, poly ICLC can induce durable CD4 + and CD8 + responses. Importantly, significant similarities in the upregulation of transcriptional and signaling pathways were observed between subjects vaccinated with poly ICLC and volunteers who had received the highly effective live-attenuated yellow fever vaccine. Furthermore, in a recent phase 1 study, more than 90% of ovarian cancer patients immunized with poly ICLC in combination with the NY-ESO-1 peptide vaccine (in addition to Montanide) showed induction of CD4 + and CD8 + T cells and an antibody response to the peptide. At the same time, poly ICLC has been extensively tested in more than 25 clinical trials to date and exhibits a relatively safe toxicity profile.

[0078] The above advantages of the present invention are described in further detail below. Identification of Tumor - Specific Neoantigen Mutations The present invention is based, at least in part, on the ability to identify all or substantially all mutations (e.g., translocations, inversions, large and small deletions and insertions, missense mutations, splice site mutations, etc.) within a neoplasm / tumor. Specifically, these mutations are present in the genome of the subject's neoplasm / tumor cells, but not in the normal tissue from which the subject is derived. Such mutations are particularly interesting when they result in a change that gives rise to a protein (e.g., a neoantigen) having a modified amino acid sequence unique to the patient's neoplasm / tumor. For example, useful mutations can include: (1) nonsynonymous mutations that result in different amino acids in a protein; (2) read-through mutations in which a stop codon is modified or deleted, resulting in the translation of a longer protein having a novel tumor-specific sequence at the C-terminus; (3) splice site mutations in which an intron enters the mature mRNA, resulting in a unique tumor-specific protein sequence; (4) chromosomal rearrangements (i.e., gene fusions) that result in a chimeric protein having a tumor-specific sequence at the junction of two proteins; (5) frameshift mutations or deletions that result in a novel open reading frame having a novel tumor-specific protein sequence, etc. Peptides or mutant polypeptides having mutations caused, for example, by splice site, frameshift, read-through, or gene fusion mutations in tumor cells can be identified by sequencing the DNA, RNA, or protein of the tumor relative to normal cells.

[0079] Also within the scope of the present invention are personal neoantigen peptides derived from common tumor driver genes, and previously identified tumor-specific mutations may further be included. For example, known common tumor driver genes and tumor mutations in common tumor driver genes can be referenced at (www)sanger.ac.uk / cosmic on the World Wide Web.

[0080] Currently, with several concepts, direct sequence information is being obtained in parallel from millions of individual molecules of DNA or RNA. Real-time single molecule sequencing-by-synthesis technology relies on the detection when fluorescent nucleotides are incorporated into the nascent DNA strand complementary to the template being sequenced. In one method, oligonucleotides 30-50 bases in length are covalently immobilized to a glass coverslip at the 5' end. These immobilized strands serve two functions. First, when the template has a structure with a capture tail complementary to the oligonucleotide bound to the surface, these strands act as capture sites for the target template strand. These strands also serve as primers for template-directed primer extension that forms the basis of sequence readout. The capture primer functions as a defined site for sequencing using multiple cycles of dye-linker synthesis, detection, and chemical cleavage to remove the dye. Each cycle consists of addition of polymerase / labeled nucleotide mixture, rinse, imaging, and cleavage of the dye. In an alternative method, polymerase is modified with a fluorescent donor molecule and immobilized on a slide glass while each nucleotide is color-coded with an acceptor fluorescent moiety attached to the γ-phosphate. This system detects the interaction between the fluorescent tag-labeled polymerase and the fluorescently modified nucleotide as the nucleotide is incorporated into the new strand. Other sequencing-by-synthesis technologies also exist.

[0081] Preferably, any suitable sequencing-by-synthesis platform can be used to identify mutations. Currently, four major sequencing-by-synthesis platforms: the Genome Sequencer from Roche / 454 Life Sciences, the HiSeq Analyzer from Illumina / Solexa, Applied BioSystems' SOLiD system, and the Heliscope system from Helicos Biosciences are available. Sequencing-by-synthesis platforms are also available from Pacific Biosciences and VisiGe They are also described by n Biotechnologies. Each of these platforms can be used in the method of the present invention. In some embodiments, a plurality of nucleic acid molecules to be sequenced are attached to a support (e.g., a solid support). To immobilize the nucleic acid on the support, a capture sequence / universal priming site can be added to the 3' and / or 5' ends of the template. The nucleic acid may be attached to the support by hybridizing the capture sequence with a complementary sequence covalently attached to the support. The capture sequence (also referred to as a universal capture sequence) is a nucleic acid sequence that can act as a universal primer and is complementary to the sequence attached to the support.

[0082] As an alternative to the capture sequence, a member of a coupling pair (e.g., an antibody / antigen, receptor / ligand, or avidin-biotin pair as described, for example, in US Patent Application Publication No. 2006 / 0252077) may be linked to each fragment that will be captured on a surface coated with the respective second member of the coupling pair. After capture, the sequence can be analyzed by single molecule detection / sequencing as described, for example, in the Examples and US Patent No. 7,283,337, including, for example, template-dependent sequencing-by-synthesis. In sequencing-by-synthesis, the molecules attached to the surface are exposed to a plurality of labeled nucleotide triphosphates in the presence of polymerase. The sequence of the template is determined by the order of the labeled nucleotides incorporated at the 3' end of the growing strand. This can be done in real-time or in a step-and-repeat manner. For real-time analysis, different optical labels are incorporated into each nucleotide and the incorporated nucleotides can be stimulated using a plurality of lasers.

[0083] To obtain a nucleic acid sample for use in the sequencing method described herein, any cell type or tissue may be utilized. In a preferred embodiment, the DNA or RNA sample is obtained from a neoplasm / tumor or a body fluid obtained by known techniques (e.g., venipuncture), such as blood or saliva. Alternatively, the nucleic acid test can be performed on a dry sample (e.g., hair or skin). This can be done.

[0084] To detect the presence of a specific mutation or allele in an individual's DNA or RNA, various methods are available. Advancements in this field have led to accurate, simple, and inexpensive large-scale SNP genotyping. More recently, for example, various DNA "chip" technologies have been described, including dynamic allele-specific hybridization (DASH), microplate array diagonal gel electrophoresis (MADGE), pyrosequencing, oligonucleotide-specific ligation, the TaqMan system, and Affymetrix SNP chips. These methods typically require amplification of the target genetic region by PCR. According to other newly developed methods based on the creation of small signal molecules by invasive cleavage and subsequent mass spectrometry or immobilized padlock probes and rolling circle amplification, the need for PCR may ultimately be eliminated. Some of the methods known in the art for detecting specific single nucleotide polymorphisms are summarized below. It is understood that the method of the present invention includes any available method. PCR-based detection means may include simultaneous multiplex amplification of multiple markers. For example, in the art, it is well known to select PCR primers such that PCR products are generated that do not overlap in size and can be analyzed simultaneously.

[0085] PCR-based detection means may include simultaneous multiplex amplification of multiple markers. For example, in the art, it is well known to select PCR primers such that PCR products are generated that do not overlap in size and can be analyzed simultaneously.

[0086] Alternatively, it is possible to amplify different markers with primers that are labeled differently and thus can be detected separately. Of course, with hybridization-based detection means, differential detection of multiple PCR products in a sample is possible. Other techniques that enable multiplex analysis of multiple markers are known in the art.

[0087] Several methods have been developed to facilitate the analysis of single nucleotide polymorphisms in genomic DNA or cellular RNA. In one embodiment, single nucleotide polymorphisms can be detected using special exonuclease-resistant nucleotides as disclosed, for example, in U.S. Patent No. 4,656,127. According to this method, a primer complementary to the allele sequence immediately 3' to the polymorphic site is made capable of hybridizing to a target molecule obtained from a particular animal or human. If the polymorphic site on the target molecule contains a nucleotide complementary to the particular exonuclease-resistant nucleotide derivative present, said derivative will be incorporated at the end of the hybridized primer. By such incorporation, the primer becomes resistant to exonuclease and can thus be detected. Since the identity of the exonuclease-resistant derivative in the sample is known, the finding that the primer is exonuclease-resistant reveals that the nucleotide present at the polymorphic site of the target molecule was complementary to that of the nucleotide derivative used in the reaction. This method has the advantage that it does not require determination of large amounts of irrelevant sequence data.

[0088] In another embodiment of the present invention, solution-based methods are used to determine the identity of nucleotides at polymorphic sites. Cohen et al. (French Patent No. 2,650,840; International Publication No. 1991 / 02087 pamphlet). As in the method of U.S. Patent No. 4,656,127, a primer complementary to the allele sequence immediately 3' to the polymorphic site can be used. This method determines the identity of the nucleotides at the site using labeled dideoxynucleotide derivatives, which will be incorporated at the end of the primer when complementary to the nucleotide at the polymorphic site.

[0089] An alternative method known as Genetic Bit Analysis or GBA® is described in International Publication No. 1992 / 15712 pamphlet). GBA® uses a mixture of a labeled terminator and a primer complementary to the sequence 3' to the polymorphic site. Thus, the labeled terminator incorporated depends on and is complementary to the nucleotide present at the polymorphic site of the target molecule being evaluated. In contrast to the method of Cohen et al. (French Patent No. 2,650,840; International Publication No. 1991 / 02087 pamphlet), the GBA® method is preferably a heterogeneous assay in which the primer or target molecule is immobilized on a solid phase.

[0090] In recent years, several primer-induced nucleotide incorporation procedures for assaying DNA polymorphic sites have been described (Komher, J. S. et al., Nucl. Acids. Res. 17: 7779-7784 (1989); Sokolov, B. P., Nucl. Acids Res. 18: 3671 (1990); Syvanen, A.-C, et al., Genomics 8: 684-692 (1990); Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.) 88: 1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat. 1: 159-164 (1992); Ugozzoli, L. et al., GATA 9: 107-112 (1992); Nyren, P. et al., Anal. Biochem. 208: 171-175 (1993)). These methods all rely on the discrimination between bases at polymorphic sites by labeling and are different from GBA® in that they rely on the incorporation of deoxynucleotides. In such a format, since the signal is proportional to the number of incorporated deoxynucleotides, polymorphisms occurring in runs of the same nucleotide can produce signals proportional to the length of the run (Syvanen, A.-C, et al., Amer. J. Hum. Genet. 52: 46-59 (1993)).

[0091] An alternative method for identifying tumor-specific neoantigens is direct protein sequencing. Protein sequencing of enzyme digests using multidimensional MS techniques (MSn), including tandem mass spectrometry (MS / MS), can also be used for the identification of the neoantigens of the present invention. Such proteomic techniques allow for rapid and highly automated analysis (see, for example, K. Gevaert and J. Vandekerckhove, Electrophoresis 21: 1145-1154 (2000)). It is further contemplated that within the scope of the present invention, the proteome of a patient's tumor can be analyzed using high-throughput de novo sequencing methods for unknown proteins to identify expressed neoantigens. For example, expressed neoantigens can be identified using metashotgun protein sequencing (see, for example, Guthals et al. (2012) "Assembly of metacontigs by See "Shotgun Protein Sequencing with Meta-contig Assembly," Molecular and Cellular Proteomics 11 (10): 1084-96.

[0092] Tumor-specific neoantigens can also be identified by identifying neoantigen-specific T cell responses using MHC multimers. For example, high-throughput analysis of neoantigen-specific T cell responses in patient samples may be performed using MHC tetramer-based screening techniques (see, e.g., Hombrink et al. (2011) "High-Throughput Identification of Potential Minor Histocompatibility Antigens by MHC Tetramer-Based Screening: Feasibility and Limitations", 6(8): 1-11; Hadrup et al. (2009) "Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers", Nature Methods, 6(7): 520-26; van Rooij et al. (2013) "Tumor exome analysis reveals neoantigen-specific T-cell reactivity in an Ipilimumab-responsive melanoma", Journal of Clinical Oncology, 31: 1-4; and Heemskerk et al. (2013) "The cancer antigenome", EMBO Journal, 32(2): 194-203). Within the scope of the present invention, such tetramer-based screening techniques can be used for the initial identification of tumor-specific neoantigens or for assessing which neoantigens a patient may have already been exposed to, and thus can be used as a secondary screening protocol to facilitate the selection of candidate neoantigens for the vaccines of the present invention.

[0093] Design of Tumor - Specific Neoantigens The present invention further comprises isolated peptides (e.g., neoantigen peptides comprising tumor-specific mutations identified by the methods of the present invention, peptides comprising known tumor-specific mutations, and mutant polypeptides or fragments thereof identified by the methods of the present invention). These peptides and polypeptides are referred to herein as "neoantigen peptides" or "neoantigen polypeptides". The term "peptide" as used herein typically refers to a continuous series of residues, typically L-amino acids, joined to one another by peptide bonds between the α-amino and α-carboxyl groups of adjacent amino acids, and is used synonymously with "mutant peptide" and "neoantigen peptide" and "wild-type peptide". The polypeptide or peptide may be of various lengths and may include at least a small region (an "epitope") predicted to bind to the patient's HLA molecules as well as additional adjacent amino acids extending in both the N-terminal and C-terminal directions. The polypeptide or peptide may be in its neutral (uncharged) form or in the form of a salt and may or may not include modifications such as glycosylation, side-chain oxidation, or phosphorylation, provided that the modifications do not destroy the biological activity of the polypeptide as described herein.

[0094] ​In certain embodiments, the size of at least one neoantigen peptide molecule can include, but is not limited to, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or more amino acid residues, and any range derivable therefrom. In a specific embodiment, the neoantigen peptide molecule is 50 amino acids or less. In a preferred embodiment, the neoantigen peptide molecule is equal to about 20 to about 30 amino acids.

[0095] Longer peptides can be designed in several ways. For example, if the HLA binding region (e.g., "epitope") is predicted or known, the longer peptide can consist of any of the following: individual binding peptides with an extension of 0 - 10 amino acids towards the N-terminus and C-terminus of each corresponding gene product. The longer peptide can also consist of some or all of the continuations of the binding peptides each having an extension sequence. In another example, if a long (more than 10 residues) neoepitope sequence is found to be present in a tumor (e.g., due to a frameshift, read-through or intron introduction resulting in a novel peptide sequence) by sequencing, the longer peptide can consist of the entire stretch of the novel tumor-specific amino acids. In any of the examples, the use of the longer peptide requires endogenous processing by professional antigen-presenting cells such as dendritic cells and can result in more effective antigen presentation and induction of a T cell response. In some cases, it may be desirable or preferable to modify the extension sequence so as to improve the biochemical properties (such as solubility or stability) of the polypeptide, or to improve the likelihood of efficient proteasomal processing of the peptide (Zhang et al (2012) "Aminopeptidase substrate preference affects HIV epitope presentation and predicts immune escape patterns in HIV-infected individuals". J. Immunol 188: 5924-34; Hearn et al (2010) "Characterizing the specificity and co-operation of aminopeptidases in the cytosol and ER during MHC Class I antigen presentation". J. Immunol 184(9): 4725-32; Wiemerhaus et al (2012) "Peptidases trimming MHC Class I ligands". Curr Opin Immunol 25: 1-7). Peptidases trimming MHC Class I ligands). Curr Opin Immunol 25: 1-7).

[0096] Neoantigen peptides and polypeptides can bind to HLA proteins. In preferred embodiments neoantigen peptides and polypeptides can bind to HLA proteins with a higher affinity compared to the corresponding native / wild-type peptides. A neoantigen peptide or polypeptide can have an IC 50 of less than about 1000 nM, less than about 500 nM, less than about 250 nM, less than about 200 nM, less than about 150 nM, less than about 100 nM, or less than about 50 nM.

[0097] In preferred embodiments, the neoantigen peptides and polypeptides of the present invention do not induce an autoimmune response and / or do not induce immune tolerance upon administration to a subject. The present invention also provides a composition comprising a plurality of neoantigen peptides. In some embodiments, the composition comprises at least 5 or more neoantigen peptides. In some embodiments, the composition contains at least about 6, about 8, about 10, about 12, about 14, about 16, about 18, or about 20 distinct peptides. In some embodiments, the composition contains at least 20 distinct peptides. According to the present invention, two or more of the distinct peptides can be derived from the same polypeptide. For example, when a preferred neoantigen mutation encodes a neoORF polypeptide, two or more of the neoantigen peptides can be derived from that neoORF polypeptide. In one embodiment, two or more neoantigen peptides derived from a neoORF polypeptide can include an array tiled across the polypeptide (e.g., the neoantigen peptides can include a series of overlapping neoantigen peptides spanning a portion or all of the neoORF polypeptide). Without being bound by theory, each peptide is thought to have its own epitope; thus, a tiled array across one neoORF polypeptide can give rise to polypeptides targeted to different HLA molecules. The neoantigen peptides can be derived from any protein-coding gene. Exemplary polypeptides from which the neoantigen peptides can be derived can be referred to, for example, in the COSMIC database (at (www)sanger.ac.uk / cosmic on the World Wide Web). COSMIC curates comprehensive information on somatic mutations in human cancers. The peptides can contain tumor-specific mutations. In some aspects, the tumor-specific mutations are in common driver genes or are driver mutations common to a particular cancer type. For example, common driver mutation peptides can include, but are not limited to: SF3B1 polypeptide, MYD88 polypeptide, TP53 polypeptide, ATM polypeptide, Abl polypeptide, FBXW7 polypeptide, DDX3X polypeptide, MAPK1 polypeptide, or GNB1 polypeptide.

[0098] Neoantigen peptides, polypeptides, and analogs can be further modified to include additional chemical moieties that are not normally part of the protein. Such derivatized moieties can improve the solubility, biological half-life, absorption, or binding affinity of the protein. Such moieties can also reduce or eliminate any undesirable side effects of the protein, etc. An overview of these moieties can be found in Remington’s Pharmaceutical Sciences, 20 th ed., Mack Publishing Co., Easton, PA (2000).

[0099] For example, neoantigen peptides and polypeptides having the desired activity can be modified to increase or at least maintain substantially all of the biological activity of the unmodified peptide that binds to the desired MHC molecule and activates the appropriate T cells while providing certain desired attributes, such as improved pharmacological properties, as needed. For example, neoantigen peptides and polypeptides can be subjected to various changes such as conservative or non-conservative substitutions, where such changes can provide certain advantages in their use, such as improved MHC binding. Such conservative substitutions can include replacing one amino acid residue with another that is biologically and / or chemically similar, for example, replacing one hydrophobic residue with another, or one polar residue with another. The effect of single amino acid substitutions can also be explored using D-amino acids. Such modifications are described, for example, in Merrifield, Science 232: 341-347 (1986); Barany & Merrifield, The Peptides, Gr oss & Meienhofer, eds. (N.Y., Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, III., Pierce), 2 nd Ed. (1984). It can be prepared using well-known peptide synthesis procedures as described.

[0100] Neoantigen peptides and polypeptides may also be modified by extending or shortening the amino acid sequence of the compound, for example, by addition or deletion of amino acids. Neoantigen peptides, polypeptides, or analogs may also be modified by altering the order or composition of specific residues. Those skilled in the art will understand that certain amino acid residues essential for biological activity, such as those at important contact sites or conserved residues, generally cannot be modified without adversely affecting biological activity. Non-essential amino acids need not be limited to those naturally occurring in proteins, such as L-α-amino acids, or their D-isomers, but may equally include non-natural amino acids such as β-γ-δ-amino acids, as well as many derivatives of L-α-amino acids.

[0101] Typically, a neoantigen polypeptide or peptide can be optimized by determining the effects of charge, hydrophobicity, etc. on MHC binding using a series of peptides with single amino acid substitutions. For example, a series of positively charged (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acid substitutions may be made along the length of the peptide, and various sensitivity patterns to different MHC molecules and T cell receptors will emerge. In addition, multiple substitutions using small relatively neutral moieties such as Ala, Gly, Pro, or similar residues may be used. Such substitutions may be homo-oligomers or hetero-oligomers. The number and type of residues substituted or added depend on the required spacing between essential contact points and the specific functional attributes required (e.g., hydrophobicity and hydrophilicity). An increase in binding affinity to MHC molecules or T cell receptors compared to the affinity of the parent peptide can also be achieved by such substitutions. In any case, such substitutions must use amino acid residues or other molecular fragments selected to avoid steric and charge hindrances that may inhibit binding.

[0102] Amino acid substitutions are typically single residue substitutions. Substitutions, deletions, insertions, or any combination thereof may be used in combination to arrive at the final peptide. Substitution variants are those in which at least one residue of the peptide has been removed and a different residue inserted in its place.

[0103] Neoantigen peptides and polypeptides may be modified to provide desired attributes. For example, by ligation with a sequence containing at least one epitope capable of inducing a T helper cell response, the ability of the peptide to induce CTL activity can be enhanced. Particularly preferred immunogenic peptide / T helper conjugates are linked by a spacer molecule. The spacer typically consists of a relatively small neutral molecule such as an amino acid or amino acid mimic that is substantially uncharged under physiological conditions. The spacer is typically selected from, for example, Ala, Gly, or other neutral spacers of nonpolar or neutral polar amino acids. It will be understood that in some cases this spacer need not be composed of the same residues and may thus be a hetero- or homo-oligomer. When present, the spacer is usually at least 1 or 2 residues and more usually may be 3 - 6 residues. Alternatively, the peptide may be linked to the T helper peptide without a spacer.

[0104] The neoantigen peptide can be linked to the T helper peptide either directly or via a spacer, at either the amino or carboxy terminus of the peptide. The amino terminus of either the neoantigen peptide or the T helper peptide may be acylated. Exemplary T helper peptides include tetanus toxoid 830 - 843, influenza 307 - 319, malaria sporozoite circumsporozoite 382 - 398 and 378 - 389.

[0105] Production of Tumor - Specific Neoantigens The present invention is based, at least in part, on the ability to present a pool of tumor-specific neo-antigens to the immune system of a patient. Those skilled in the art will appreciate that there are various ways to produce such tumor-specific neo-antigens. In general, such tumor-specific neo-antigens can be produced either in vitro or in vivo. The tumor-specific neo-antigens may be produced in vitro as peptides or polypeptides, which may then be formulated into personalized neoplasia vaccines and administered to a subject. As described in more detail below, such in vitro production may be performed by various methods known to those skilled in the art, such as, for example, peptide synthesis or expression of the peptide / polypeptide from DNA or RNA molecules in any of a variety of bacterial, eukaryotic, or viral recombinant expression systems, followed by purification of the expressed peptide / polypeptide. Alternatively, the tumor-specific neo-antigens may be produced in vivo by introducing into a subject a molecule (e.g., DNA, RNA, viral expression system, etc.) encoding the tumor-specific neo-antigen, followed by expression of the encoded tumor-specific neo-antigen. In Vitro Peptide / Polypeptide Synthesis The protein or peptide may be produced by any technique known to those of skill in the art, including expression of the protein, polypeptide or peptide by standard molecular biology techniques, isolation of the protein or peptide from a natural source, or chemical synthesis of the protein or peptide. Nucleotide and protein, polypeptide and peptide sequences corresponding to various genes have been disclosed and can be found in computerized databases known to those of skill in the art. One such database is the National Center for Biotechnology Information at the National Institutes of Health website. and the Genbank and GenPept databases at the National Institute for Biotechnology Information. The coding regions of known genes can be amplified and / or expressed using the techniques disclosed herein or as would be known to one of skill in the art. Alternatively, a variety of commercially available protein, polypeptide and peptide preparations are known to those of skill in the art.

[0106] Peptides can be easily chemically synthesized using reagents that do not contain contaminating bacteria or animal substances (Merrifield RB: "Solid phase peptide synthesis. I. The synthesis of a tetrapeptide" . J. Am. Chem. Soc. 85: 2149-54, 1963).

[0107] A further aspect of the present invention provides a nucleic acid (e.g., polynucleotide) encoding the neoantigen peptide of the present invention, which can be used to produce the neoantigen peptide in vitro. The polynucleotide can be, for example, DNA, cDNA, PNA, CNA, RNA, either single-stranded and / or double-stranded, or a polynucleotide in natural or stabilized form, such as a polynucleotide having a phosphorothiate backbone etc., or a combination thereof, and may or may not contain introns as long as it encodes the peptide. Yet another aspect of the present invention provides an expression vector having the ability to express the polypeptide according to the present invention. Expression vectors for various cell types are well known in the art and can be selected without performing more experiments than necessary. Generally, DNA is inserted into an expression vector such as a plasmid in the appropriate orientation and 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 such control is generally available in the expression vector. The vector is then introduced into a host bacterium for cloning using standard techniques (see, e.g., Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

[0108] The invention further encompasses variants and equivalents that are substantially homologous to the identified tumor-specific neoantigens described herein. These may contain, for example, conservative substitution mutations, i.e., substitutions of one or more amino acids by similar amino acids. For example, a conservative substitution refers to substituting an amino acid with another amino acid within the same general class, e.g., substituting one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. The intended conservative amino acid substitutions are well known in the art.

[0109] The invention also includes expression vectors containing isolated polynucleotides, as well as host cells containing such expression vectors. Also contemplated within the scope of the invention is that neoantigen peptides in the form of RNA or cDNA molecules encoding the desired neoantigen peptides may be provided. The invention also provides that one or more neoantigen peptides of the invention may be encoded by a single expression vector. The invention also provides that one or more neoantigen peptides of the invention may be encoded and expressed in vivo using a virus-based system (e.g., an adenovirus system).

[0110] The term "polynucleotide encoding a polypeptide" encompasses polynucleotides containing only the coding sequence of the polypeptide as well as polynucleotides containing additional coding and / or non-coding sequences. The polynucleotides of the invention may be in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; it may be double-stranded or single-stranded, and in the case of single-stranded, it may be the coding strand or the non-coding strand (antisense strand).

[0111] ​In embodiments, the polynucleotide may comprise a coding sequence of a tumor-specific neoantigen peptide fused in the same reading frame with, for example, a polynucleotide that aids in the expression and / or secretion of a polypeptide from a host cell (e.g., a leader sequence that functions as a secretion sequence for controlling the transport of a polypeptide from the cell). A polypeptide having a leader sequence is a preprotein and may have a leader sequence that is cleaved by the host cell to form the mature form of the polypeptide.

[0112] In embodiments, the polynucleotide may comprise a coding sequence of a tumor-specific neoantigen peptide fused in the same reading frame with, for example, a marker sequence that enables the purification of the encoded polypeptide and then allows it to be incorporated into an individualized neoplasm vaccine. For example, the marker sequence may be a hexahistidine tag supplied by the pQE-9 vector that provides purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence may be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used. Further tags include, but are not limited to, calmodulin tag, FLAG tag, Myc tag, S tag, SBP tag, Softag 1, Softag 3, V5 tag, Xpress tag, Isopeptag, SpyTag, biotin carboxyl carrier protein (BCCP) tag, GST tag, fluorescent protein tag (e.g., green fluorescent protein tag), maltose binding protein tag, Nus tag, Strep tag, thioredoxin tag, TC tag, Ty tag, and the like.

[0113] In embodiments, the polynucleotide may comprise one or more coding sequences of tumor-specific neoantigen peptides fused in the same reading frame to create a single concatenated neoantigen peptide construct having the ability to produce multiple neoantigen peptides.

[0114] In an embodiment, the present invention provides a polynucleotide encoding a tumor-specific neoantigen peptide of the present invention, which has 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 an isolated nucleic acid molecule having a nucleotide sequence.

[0115] A polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence is intended to be identical to the nucleotide sequence of the reference sequence, except that the nucleotide sequence of the polynucleotide may contain up to 5 point mutations per 100 nucleotides of the reference 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 replaced with another nucleotide, or up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may be scattered individually among the nucleotides in the reference nucleotide sequence, at the amino-terminal or carboxy-terminal positions of the reference nucleotide sequence, or anywhere between those terminal positions, or may exist as one or more contiguous clusters within the reference sequence.

[0116] In practice, whether any particular nucleic acid molecule is at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identical to a reference sequence is determined using the Bestfit program (Wisconsin Sequence Analysis Package, version It can be determined by conventional methods using well-known computer programs such as the UNIX (registered trademark) version of Wisconsin Package, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 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 determining whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention using Bestfit or any other sequence alignment program, the parameter settings are such that the percentage of identity is calculated over the entire length of the reference nucleotide sequence, and a homology gap of up to 5% of the total number of nucleotides in the reference sequence is allowed.

[0117] The isolated tumor-specific neoantigen peptides described herein can be produced in vitro (e.g., in a laboratory) by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing DNA sequences encoding isolated polypeptide sequences and expressing those sequences in suitable transformed hosts. In some embodiments, the DNA sequences are constructed using recombinant techniques by isolating or synthesizing the DNA sequences encoding the wild-type proteins of interest. Optionally, mutations can be introduced into the sequences by site-directed mutagenesis to provide functional analogs thereof. See, for example, Zoeller et al., Proc. Nat’l. Acad. Sci. USA 81: 5662-5066 (1984) and U.S. Patent No. 4,588,585. See also U.S. Patent No. 4,588,585.

[0118] In an embodiment, the DNA sequence encoding the polypeptide of interest can be constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and by selecting codons preferred by the host cell that produces the recombinant polypeptide of interest. Standard methods can be applied for the synthesis of the isolated polynucleotide sequence encoding the isolated polypeptide of interest. For example, a gene can be constructed by reverse translation using the complete amino acid sequence. Further, DNA oligomers containing nucleotide sequences encoding specific isolated polypeptides can be synthesized. For example, several small oligonucleotides encoding a portion of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0119] After assembly (e.g., by synthesis, site-directed mutagenesis, or another method), the polynucleotide sequence encoding the specific isolated polypeptide of interest can be inserted into an expression vector and optionally operably linked to expression control sequences appropriate for protein expression in the desired host. The adequacy of the assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, to achieve high expression levels of the transfected gene in the host, the gene can be operably linked to transcriptional and translational expression control sequences that function in the selected expression host.

[0120] A recombinant expression vector may be used to amplify and express DNA encoding a tumor-specific neoantigen peptide. A recombinant expression vector is a replicable DNA construct having a synthetic or cDNA-derived DNA fragment encoding a tumor-specific neoantigen peptide or a biologically equivalent analog operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. The transcription unit generally includes the assembly of (1) one or more genetic elements having a regulatory role in gene expression, such as a transcriptional promoter or enhancer, (2) a structural or coding sequence that is transcribed into mRNA and translated into protein, and (3) appropriate transcriptional and translational start and termination sequences, as described in more detail below. Such regulatory elements may include operator sequences that control transcription. A replication ability in a host (usually conferred by an origin of replication), and a selectable gene for facilitating the recognition of transformants may be further incorporated. DNA regions are operably linked when they are functionally related to each other. For example, the DNA of a signal peptide (secretory leader) is operably linked to the DNA of a polypeptide when it is expressed as a precursor involved in the secretion of the polypeptide; a promoter is operably linked to a coding sequence when it controls the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence when it is in a position enabling translation. Generally, being operably linked means being adjacent, and in the case of a secretory leader, means being adjacent and in the reading frame. The structural elements intended for use in a yeast expression system 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 contain an N-terminal methionine residue. Optionally, cleavage of this residue following the expressed recombinant protein may result in the final product.

[0121] The selection of the expression control sequence and the expression vector may depend on the selection of the host. A wide variety of expression host / vector combinations can be used. Examples of expression vectors useful for eukaryotic hosts include vectors containing expression control sequences derived from, for example, SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Examples of expression vectors useful for bacterial hosts include known bacterial plasmids, such as plasmids derived from Escherichia coli containing pCR 1, pBR322, pMB9, and their derivatives, and plasmids with a broader host range, such as M13 and filamentous single-stranded DNA phages. Plasmids and their derivatives derived from Escherichia coli (E. coli) containing pCR 1, pBR322, pMB9, and filamentous single-stranded DNA phages with a broader host range, such as M13, are included.

[0122] Suitable host cells for the expression of polypeptides include prokaryotic cells, yeast cells, insect cells, or higher eukaryotic cells under the control of an appropriate promoter. Prokaryotes include gram-negative or gram-positive organisms, such as Escherichia coli (E. coli) or Bacilli. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems can also be used. Cloning and expression vectors suitable for use in bacterial, fungal, yeast, and mammalian cell hosts are well known in the art (see Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., 1985).

[0123] Various mammalian or insect cell culture systems are also advantageously used to express recombinant proteins. Expression of recombinant proteins in mammalian cells can be carried out because such proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include those by Gluzman (Cell 23: 175, 1981). Plasmids and their derivatives derived from Escherichia coli (E. coli) containing pCR 1, pBR322, pMB9, and filamentous single-stranded DNA phages with a broader host range, such as M13, are included. The COS-7 line of monkey kidney cells described, as well as other cell lines having the ability to express an appropriate vector, such as L cells, C127, 3T3, Chinese hamster ovary (CHO), HeLa, and BHK cell lines, can be mentioned. Mammalian expression vectors can include non-transcribed elements, such as an origin of replication, suitable promoters and enhancers linked to the gene to be expressed, as well as other 5' or 3' flanking non-transcribed sequences, and 5' or 3' untranslated sequences, such as an essential ribosome binding site, a polyadenylation site, splice donor and acceptor sites, and a transcription termination sequence. The baculovirus system for producing heterologous proteins in insect cells has been reviewed by Luckow and Summers, Bio / Technology 6: 47 (1988) .

[0124] The protein produced by the transformed host can be purified by any suitable method. Such standard methods can include chromatography (e.g., ion exchange, affinity, and size exclusion column chromatography, etc.), centrifugation, solubility differences, or any other standard protein purification technique. Binding an affinity tag, such as hexahistidine, maltose binding domain, influenza coat sequence, glutathione-S-transferase, etc., to the protein can enable easy purification by passing it through an appropriate affinity column. The isolated protein can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0125] For example, the supernatant from a system that secretes a recombinant protein into the culture medium is first passed through a commercially available protein concentration filter, such as Amicon or Millipore Pelli It can be concentrated using a tangential flow filtration unit. After the concentration step, the concentrate can be added to a suitable purification matrix. Alternatively, an anion exchange resin, such as a matrix or substrate having pendant diethylaminoethyl (DEAE) groups, can be used. The matrix may be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl groups or carboxymethyl groups. Finally, the cancer stem cell protein-Fc composition can be further purified using one or more reverse phase high performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium, such as silica gel having pendant methyl groups or other aliphatic groups. A homogeneous recombinant protein can also be provided by using some or all of the aforementioned purification steps in various combinations.

[0126] Recombinant proteins produced in bacterial cultures can be isolated, for example, by first extracting from cell pellets, followed by concentrating one or more times, salting out, and performing water-soluble ion exchange or size exclusion chromatography steps. High performance liquid chromatography (HPLC) may be used in the final purification step. The microbial cells used for the expression of recombinant proteins can be disrupted by any convenient method including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents.

[0127] In vivo peptide / polypeptide synthesis The present invention also contemplates the use of nucleic acid molecules as a vehicle for delivering neoantigen peptides / polypeptides to a subject in vivo, for example, in the form of DNA / RNA vaccines (see, for example, International Publication No. WO 2012 / 159643 and International Publication No. WO 2012 / 159754, which are hereby incorporated by reference in their entirety).

[0128] In one embodiment, the personalized neoplasm vaccine can include a separate DNA plasmid encoding one or more neoantigen peptides / polypeptides as identified, for example, according to the present invention. As discussed above, the exact choice of expression vector will depend on the peptide / polypeptide to be expressed and is well within the skill of the art. The expected persistence of the DNA construct (e.g., episomal, non-replicating, non-integrating form in muscle cells) is expected to result in an increased protection period.

[0129] In another embodiment, the personalized neoplasm vaccine can include an RNA or cDNA molecule encoding the neoantigen peptide / polypeptide of the present invention. In another embodiment, the personalized neoplasm vaccine can include a virus-based vector for use in a human patient, such as, for example, an adenovirus system (see, e.g., Baden et al., "First-in-human evaluation of the safety and immunogenicity of a recombinant adenovirus serotype 26 HIV-1 Env vaccine (IPCAVD 001)", which is hereby incorporated by reference in its entirety). J Infect Dis. 2013 Jan 15; 207(2): 240-7).

[0130] Pharmaceutical Compositions / Delivery Methods The present invention also relates to a pharmaceutical composition comprising an effective amount of one or more compounds according to the present invention (including pharmaceutically acceptable salts thereof), optionally in combination with a pharmaceutically acceptable carrier, excipient or additive.

[0131] "Pharmaceutically acceptable derivative or prodrug" means any pharmaceutically acceptable salt, ester, salt of an ester, or other derivative of a compound of the present invention that has the ability to provide (directly or indirectly) the compound of the present invention upon administration to a recipient. Particularly preferred derivatives and prodrugs are those that increase the bioavailability of the compound of the present invention (e.g., by enabling easier absorption into the bloodstream of a compound administered orally or intravitreally) when such a compound is administered to a mammal, or those that enhance the delivery of the parent compound to a biological compartment (e.g., the retina) compared to the parent species.

[0132] The tumor-specific neoantigen peptides of the present invention can be administered as a single pharmaceutically active agent, but may also be used in combination with one or more other agents and / or adjuvants. When administered in combination, the therapeutic agents may be formulated as separate compositions administered at the same or different times, or the therapeutic agents may be administered as a single composition.

[0133] The tumor-specific neoantigen peptides of the present invention may be administered by injection, orally, parenterally, by inhalation spray, rectally, vaginally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. As used herein, the term parenteral includes intralymphatic, subcutaneous, intravenous, intramuscular, intrasternal, infusion methods, intraperitoneal, ocular or intravitreal, intracapsular, buccal, percutaneous, intranasal, intracranial including intracerebral and intradural, intra-articular including ankle joint, knee joint, hip joint, shoulder joint, elbow joint, wrist joint, directly into a tumor, and in the form of suppositories.

[0134] The pharmaceutically active compounds of the present invention can be processed according to conventional pharmaceutical methods for preparing pharmaceutical agents for administration to patients, including humans and other mammals. The modification of the active compound affects the solubility, bioavailability and metabolic rate of the active species, and thus can result in the control of the delivery of the active species. This can be easily evaluated by preparing derivatives by known methods that are well within the scope of those skilled in the art and testing their activities.

[0135] Pharmaceutical compositions based on these chemical compounds contain the above-described tumor-specific neoantigen peptides in a therapeutically effective amount for the treatment of the diseases and conditions (e.g., neoplasms / tumors) described herein, optionally in combination with pharmaceutically acceptable additives, carriers and / or excipients. Those skilled in the art will recognize that the therapeutically effective amount of one or more compounds according to the present invention may vary depending on the infectious disease or condition to be treated, its severity, the treatment regimen employed, the pharmacokinetics of the drug used, and the patient (animal or human) being treated.

[0136] To prepare the pharmaceutical compositions according to the present invention, one or more therapeutically effective amounts of the compounds according to the present invention are preferably thoroughly mixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques such that a dosage is produced. The carrier can take a wide variety of forms depending on, for example, the desired form of preparation for administration, among others, ocular, oral, topical or parenteral, such as gels, creams, ointments, lotions and timed-release implantable formulations. When preparing a pharmaceutical composition as an oral dosage form, any conventional pharmaceutical medium can be used. Thus, for liquid oral formulations such as suspensions, elixirs and solutions, suitable carriers and additives can be used, including water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents and the like. For solid oral formulations such as powders, tablets, capsules, and for solid formulations such as suppositories, suitable carriers and additives can be used, including starches, sugar carriers such as dextrose, mannitol, lactose and related carriers, diluents, granulating agents, lubricants, binders, disintegrants and the like. If desired, tablets or capsules may be enteric-coated or may be sustained-release by standard techniques.

[0137] The active compound is present in a pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to the patient a therapeutically effective amount for the desired indication without causing significant toxic effects to the patient being treated.

[0138] Oral compositions may generally contain an inert diluent or an edible carrier. The oral compositions may be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound or its prodrug derivative may be admixed with excipients and used in the form of tablets, troches, or capsules. Binding agents having pharmaceutical compatibility, and / or auxiliary agent materials may be included as part of the composition.

[0139] Tablets, pills, capsules, troches, etc. may contain any of the following ingredients, or compounds of a similar nature: binding agents such as microcrystalline cellulose, tragacanth gum or gelatin; excipients such as starch or lactose, dispersing agents such as alginic acid or corn starch; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier such as a fatty oil. In addition, the dosage unit form may contain various other materials that modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or enteric solvents.

[0140] Formulations of the invention suitable for oral administration may be provided as discrete units, such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil emulsion and as a bolus, etc.

[0141] Tablets may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by mixing the active ingredient in free-flowing form, such as a powder or granules, with a binder, lubricant, inert diluent, preservative, surfactant, or dispersing agent and compressing the mixture in a suitable machine. Molded tablets may be prepared by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine. Tablets may optionally be coated or scored and formulated to provide for the sustained or controlled release of the active ingredient therein. Methods for formulating such sustained or controlled release compositions of pharmaceutically active ingredients are known in the art and are described in several issued U.S. patents, including, but not limited to, U.S. Patent Nos. 3,870,790; 4,226,859; 4,369,172; 4,842,866; and 5,705,190, the disclosures of which are hereby incorporated by reference in their entirety. Coatings can be used to deliver the compound to the intestine (see, for example, U.S. Patent Nos. 6,638,534; 5,541,171; 5,217,720; and 6,569,457, and the references cited therein).

[0142] The active compound or a pharmaceutically acceptable salt thereof may also be administered as a component of elixirs, suspensions, syrups, lozenges, chewing gums, etc. Syrups may contain, in addition to the active compound, sucrose or fructose as a sweetening agent, and certain preservatives, dyes, and colorings and flavorings.

[0143]

[0144] ​Solutions or suspensions for ocular, parenteral, intradermal, subcutaneous, or topical application may contain the following components: a sterile diluent, such as water for injection, physiological saline solution, fixed oil, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffering agent, such as acetate, citrate, or phosphate, and an agent for adjusting tonicity, such as sodium chloride or dextrose.

[0145] In one embodiment, the active compound is prepared with a carrier that can protect the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic-co-glycolic acid (PLGA) may be used. The method for preparing such formulations will be apparent to those skilled in the art.

[0146] Those skilled in the art will recognize that, in addition to tablets, other dosage forms can be formulated to provide sustained or controlled release of the active ingredient. Such dosage forms include, but are not limited to, capsules, granules, and gelcaps.

[0147] Liposome suspensions can also be a pharmaceutically acceptable carrier. This can be prepared by methods known to those skilled in the art. For example, liposome formulations can be prepared by dissolving the appropriate one or more lipids in an inorganic solvent and then evaporating the solvent to leave a thin film of dried lipid on the surface of the container. Next, an aqueous solution of the active compound is introduced into the container. Then, the container is swirled by hand to free the lipid material from the sides of the container and disperse the lipid aggregates, thereby forming a liposome suspension. Other preparation methods well known to those skilled in the art can also be used in this aspect of the present invention.

[0148] The pharmaceutical preparation may conveniently be provided in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient with one or more pharmaceutical carriers or one or more excipients. In general, the pharmaceutical preparation is prepared by uniformly and thoroughly bringing the active ingredient into association with a liquid carrier, or by micronizing a solid carrier, or by both, and then, if necessary, shaping the product.

[0149] Preparations and compositions suitable for topical administration in the mouth include flavored bases, usually lozenges containing the ingredient in sucrose and acacia or tragacanth; troches containing an inert base such as gelatin and glycerin, or sucrose and acacia, and the active ingredient; and mouthwashes containing the ingredient to be administered in a suitable liquid carrier.

[0150] Preparations suitable for topical administration to the skin may be provided as ointments, creams, gels and pastes containing the ingredient to be administered in a pharmaceutically acceptable carrier. A preferred topical delivery system is a transdermal patch containing the ingredient to be administered.

[0151] Preparations for rectal administration may be provided as suppositories with a suitable base containing, for example, cocoa butter or a salicylate. Preparations suitable for nasal administration when the carrier is solid include, for example, a powder having a particle size in the range of 20 to 500 microns, which is administered by the method of insufflation, i.e., by rapid inhalation through the nasal passages from a container of powder held to the nose. Suitable preparations when the carrier is liquid are, for example, a liquid for administration as a nasal spray or as a nasal drops, containing an aqueous or oily solution of the active ingredient.

[0152] Preparations suitable for vaginal administration may be provided as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing, in addition to the active ingredient, a carrier as is known to be appropriate in the art.

[0153] Parenteral formulations can be enclosed in glass or plastic ampoules, disposable syringes or multi-dose vials. When administered intravenously, preferred carriers include, for example, physiological saline or phosphate-buffered saline (PBS).

[0154] For parenteral formulations, the carrier usually may contain sterile water or an aqueous sodium chloride solution, but may also contain other ingredients including those that aid dispersion. Naturally, when sterile water is used and maintained sterile, the composition and the carrier will also be sterilized. Injectable suspensions may also be prepared, in which case suitable liquid carriers, suspending agents, etc. may be used.

[0155] Formulations suitable for parenteral administration may include aqueous and non-aqueous sterile injection solutions that may contain antioxidants, buffers, bacteriostatic agents and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions that may contain suspending and thickening agents. These formulations may be provided in unit-dose or multi-dose containers, such as sealed ampoules and vials, and may be stored in a freeze-dried state that requires only the addition of a sterile liquid carrier, such as water for injection, immediately prior to use. Ready-to-prepare injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the types described heretofore.

[0156] Administration of the active compound can range from continuous administration (intravenous infusion) to several oral administrations per day (e.g., Q.I.D.), and among the several routes of administration, particularly include oral, topical, intraocular or peribulbar, parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may include a penetration enhancer), buccal and suppository administration, including intraocular or peribulbar routes.

[0157] The application of the therapeutic agent of the subject matter may be local and thus may be administered to the site of interest. To provide the composition of the subject matter to the site of interest, various techniques can be used, such as injection, use of a catheter, trocar, projectile, Pluronic® gel, stent, sustained release drug polymer, or other devices that provide internal access. If an organ or tissue is accessible because it has been removed from a patient, such organ or tissue may be placed in a media bath containing the composition of the subject matter, the composition of the subject matter may be applied to the organ, or by any convenient method it may be applied.

[0158] Tumor-specific neoantigen peptides can be administered by a device suitable for the controlled and sustained release of a composition effective in achieving the desired local or systemic physiological or pharmacological effect. This method includes positioning a sustained release drug delivery system in the region where drug release is desired and moving the drug from the device to the desired treatment area.

[0159] Tumor-specific neoantigen peptides may be used in combination with at least one other known therapeutic agent, or a pharmaceutically acceptable salt of said agent. Examples of known therapeutic agents that can be used in combination therapy include, but are not limited to, corticosteroids (e.g., cortisone, prednisone, dexamethasone), non-steroidal anti-inflammatory drugs (NSAIDs) (e.g., ibuprofen, celecoxib, aspirin, indomethicin, naproxen), alkylating agents such as busulfan, cisplatin, mitomycin C, and carboplatin; anti-mitotic agents such as colchicine, vinblastine, paclitaxel, and docetaxel; topo I inhibitors such as camptothecin and topotecan; topo II inhibitors such as doxorubicin and etoposide; and / or RNA / DNA antimetabolites such as 5-azacytidine, 5-fluorouracil, and methotrexate; DNA antimetabolites such as 5-fluoro-2'-deoxy-uridine, ara-C, hydroxyurea, and thioguanine; antibodies such as Herceptin® and Rituxan®.

[0160] In addition to the components detailed above, it must be understood that the formulations of the present invention may include other conventional agents in the art, taking into account the formulation type in question. For example, those suitable for oral administration may include flavoring agents.

[0161] In certain pharmaceutical forms, compounds in prodrug form may be preferred. Those skilled in the art will recognize how to readily modify the compound to be in prodrug form to facilitate delivery of the active compound to the target site in the host organism or patient. Those skilled in the art will also be able to utilize the preferred pharmacokinetic parameters of the prodrug form, where applicable, in delivering the compound to the target site in the host organism or patient to maximize the intended effect of the compound.

[0162] Preferred prodrugs include derivatives in which a group that enhances water solubility or active transport across the intestinal membrane is added to the structure of the formulations described herein. For example, Alexander, J. et al. Journal of Medicinal Chemistry 1988, 31, 318-322; Bundgaard, H. Design of Prodrugs; Elsevier: Amsterdam, 1985; pp 1-92; Bundgaard, H.; Nielsen, N. M. Journal of Medicinal Chemistry 1987, 30, 451-454; Bundgaard, H. A Textbook of Drug Design and Development; Harwood Academic Publ.: Switzerland, 1991; pp 113-191; Digenis, G. A. et al. Handbook of Experimental Pharmacology 1975, 28, 86-112; Friis, G. J.; Bundgaard, H. A Textbook of Drug Design and Development; 2 ed.; Overseas Publ.: Amsterdam, 1996; pp 351-385; Pitman, I. H. Medicinal Research Reviews 1981, 1, 189-214. The prodrug form may itself be active or may be such that upon metabolism after administration it provides an active therapeutic agent in vivo.

[0163] The pharmaceutically acceptable salt form can be the compound according to the invention in a preferred chemical form for inclusion in the pharmaceutical compositions according to the invention. The compound or its derivatives may be provided in the form of a pharmaceutically acceptable salt, including the prodrug form of these agents. As used herein, the term pharmaceutically acceptable salt or complex means that it retains the desired biological activity of the parent compound and is limited to normal cells Refers to suitable salts or complexes of the active compounds according to the present invention that exhibit the resulting toxic effects. Non-limiting examples of such salts are, inter alia, (a) acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.) and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, and polyglutamic acid; (b) among others, base addition salts formed with metal cations such as zinc, calcium, sodium, potassium, etc.

[0164] The compounds herein are commercially available or can be synthesized. As will be understood by those skilled in the art, further methods for synthesizing the compounds of the formulas herein will be apparent to those skilled in the art. In addition, various synthetic steps may be carried out in a different order or sequence to obtain the desired compounds. Synthetic chemical transformations and protecting group methodologies (protection and deprotection) useful in the synthesis of the compounds described herein are known in the art and are described, for example, in R. Larock, Comprehensive Organic Transformations, 2nd. Ed., Wiley-VCH Publishers (1999); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd. Ed., John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser’s Reagents for Organic Synthesis, John Wiley and Sons (1999); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and those described in their subsequent editions are included.

[0165] Additional agents that may be included with the tumor-specific neoantigen peptides of the present invention may contain one or more asymmetric centers and can thus exist as racemates and racemic mixtures, single enantiomers, individual diastereomers, and mixtures of diastereomers. All such isomeric forms of these compounds are expressly included in the present invention. The compounds of the present invention may also be represented in multiple tautomeric forms, and in such cases, the present invention expressly includes all tautomeric forms of the compounds described herein (for example, alkylation of a ring system can result in alkylation at multiple sites, and the present invention expressly includes all of such reaction products). All such isomeric forms of such compounds are expressly included in the present invention. All crystalline forms of the compounds described herein are expressly included in the present invention.

[0166] Preferred unit dosage formulations contain the daily dose or unit of the component to be administered, the daily sub-doses as described above, or appropriate proportions thereof. The dosage regimen for treating a disorder or disease with the tumor-specific neoantigen peptides of the present invention and / or the compositions of the present invention is based on various factors including the type of disease, the age, weight, sex, medical condition, severity of the pathology, route of administration, and the particular compounds used. Accordingly, the dosage regimen can vary widely but can be determined routinely using standard methods.

[0167] The amount and dosage regimen administered to a subject can depend on many factors, such as the method of administration, the nature of the condition being treated, the weight of the subject being treated, and the judgment of the prescribing physician. The amount of the compound contained in the formulation having therapeutic activity according to the present invention is an amount effective for the treatment of a disease or condition. Generally, the therapeutically effective amount of the present compound in the dosage form is usually from slightly less than about 0.025 mg / kg / day to about 2.5 g / kg / day, preferably from about 0.1 mg / kg / day to about 100 mg / kg / day or considerably more, depending on the compound used, the condition or infection being treated, and the route of administration. However, exceptions to this dosage range can be contemplated by the present invention. In its most preferred form, the compound according to the present invention is administered in an amount in the range of about 1 mg / kg / day to about 100 mg / kg / day. The dosage of the compound can depend on the condition being treated, the particular compound, and other clinical factors such as the patient's weight and condition and the route of administration of the compound. It should be understood that the present invention has application for use in both humans and domestic animals.

[0168] For oral administration to humans, dosages of about 0.1 to 100 mg / kg / day, preferably about 1 to 100 mg / kg / day are generally sufficient. When drug delivery is systemic rather than local, this dosage range generally results in effective blood level concentrations of the active compound in the patient in the range of less than about 0.04 to about 400 μg / cc of blood or more.

[0169] The compound is advantageously administered in any suitable unit dosage form, including but not limited to those containing from 0.001 to 3000 mg, preferably from 0.05 to 500 mg of active ingredient per unit dosage form. Oral dosages of 10 to 250 mg are usually convenient.

[0170] The concentration of the active compound in the pharmaceutical composition may depend on the absorption, distribution, inactivation, and excretion rates of the drug as well as other factors known to those skilled in the art. It should be noted that the dosage value may also vary depending on the severity of the condition to be alleviated. Further, for any particular subject, the specific dosage regimen must be adjusted over time according to the individual needs and the professional judgment of the caregiver or supervisor administering the composition, and it is to be understood that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition. The active ingredient may be administered once or divided into multiple smaller doses and administered at various time intervals.

[0171] In certain embodiments, the compound is administered once daily; in other embodiments, the compound is administered twice daily; in yet other embodiments, the compound is administered 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 needs of the individual patient. Extended-release or depot formulations may be used for longer dosing intervals.

[0172] The compounds of the present invention can be used for the treatment of acute diseases and disease states and may also be used for the treatment of chronic conditions. In certain embodiments, the compounds of the present invention are administered over a period of 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years, 10 years, or more than 15 years; or, for example, any period of days, months, or years where the lower end of the range is between 14 days and 15 years and the upper end of the range is between 15 days and 20 years (e.g., 4 weeks to 15 years, 6 months to 20 years). In some cases, it may be advantageous for the compounds of the present invention to be administered over the patient's lifetime. In a preferred embodiment, the patient is monitored to confirm the progression of the disease or disorder and the dosage is adjusted accordingly. In a preferred embodiment, the treatment according to the present invention is effective for at least 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years, 10 years, 15 years, 20 years, or over the lifetime of the subject.

[0173] The present invention provides a pharmaceutical composition containing at least one tumor-specific neoantigen described herein. In embodiments, the pharmaceutical composition contains a pharmaceutically acceptable carrier, excipient, or diluent, which includes any pharmaceutical that does not cause the development of an immune response harmful to the subject receiving administration of the composition itself and can be administered without undue toxicity. As used herein, the term "pharmaceutically acceptable" means approved by a regulatory authority of the federal or state government for use in mammals, more particularly humans, or listed in the United States Pharmacopeia, the European Pharmacopeia, or other generally recognized pharmacopeias. These compositions can be useful for the treatment and / or prevention of viral infections and / or autoimmune diseases.

[0174] A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is provided in Remington It is provided in Remington: The Science and Practice of Pharmacy (21st ed., Lippincott Williams & Wilkins) and Goodman & Gilman's The Pharmacological Basis of Therapeutics (17th ed., McGraw-Hill), which are incorporated herein by reference. The formulation of the pharmaceutical composition must be suitable for the method of administration. In embodiments, the pharmaceutical composition is suitable for administration to humans and may be sterile, particulate matter-free, and / or pyrogen-free.

[0175] Pharmaceutically acceptable carriers, excipients, or diluents include, but are not limited to, physiological saline, buffered physiological saline, dextrose, water, glycerol, ethanol, sterile isotonic buffered aqueous solutions, and combinations thereof.

[0176] Wetting agents, emulsifying agents, and lubricants such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening agents, flavoring agents, and fragrances, preservatives, and antioxidants may also be present in the composition.

[0177] Examples of pharmaceutically acceptable antioxidants include, but are not limited to: (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0178] In embodiments, the pharmaceutical composition is provided in solid forms such as lyophilized powders suitable for reconstitution, liquid solutions, suspensions, emulsions, tablets, pills, capsules, sustained release formulations, or powders.

[0179] In an embodiment, the pharmaceutical composition is in liquid form and is provided, for example, within a sealed container that indicates the quantity and concentration of the active ingredient in the pharmaceutical composition. In related embodiments, the pharmaceutical composition in liquid form is provided in a hermetically sealed container.

[0180] The method of formulating the pharmaceutical composition of the present invention is conventional and well-known in the art (see Remington and Remington’s). One of ordinary skill in the art can readily formulate a pharmaceutical composition having the desired characteristics (e.g., route of administration, biocompatibility, and release profile).

[0181] The method of preparing the pharmaceutical composition includes the step of associating the active ingredient with a pharmaceutically acceptable carrier and optionally one or more auxiliary components. The pharmaceutical composition can be prepared by uniformly and thoroughly associating the active ingredient with a liquid carrier, or by micronizing a solid carrier, or by both, and then, if necessary, shaping the product. Further methodologies regarding the preparation of pharmaceutical compositions, including the preparation of multilayer dosage forms, are described in Ansel’s Pharmaceutical Dosage Forms and Drug Delivery Systems (9th ed., Lippincott Williams & Wilkins) (incorporated herein by reference).

[0182] Pharmaceutical compositions suitable for oral administration can be in the form of capsules, cachets, pills, tablets, lozenges (using flavored bases, usually sucrose and acacia or tragacanth), powders, granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or as water-in-oil or oil-in-water liquid emulsions, or as elixirs or syrups, or as troches (using inert bases such as gelatin and glycerin, or sucrose and acacia) and / or as mouthwashes, each containing one or more of the compounds described herein as one or more active ingredients, derivatives thereof, or salts It contains a predetermined amount of a pharmaceutically acceptable salt or prodrug. The active ingredient may also be administered as a bolus, lozenge, or paste.

[0183] For solid dosage forms for oral administration (e.g., capsules, tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, excipients, or diluents, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia, etc.; (3) humectants, such as glycerol; (4) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) dissolution inhibitors, such as paraffin; (6) absorption promoters, such as quaternary ammonium compounds; (7) wetting agents, such as acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical composition may also contain buffering agents. Solid compositions of the same type can also be prepared using fillers and excipients in soft and hard gelatin capsules, such as lactose or milk sugar, and high molecular weight polyethylene glycol, etc.

[0184] Tablets may be prepared by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropylmethylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or sodium cross-linked carboxymethylcellulose), a surfactant, and / or a dispersant. Molded tablets can be prepared by molding a mixture of powdered active ingredient moistened with an inert liquid diluent using a suitable machine.

[0185] Tablets, and other solid dosage forms such as dragees, capsules, pills, and granules, may optionally be scored or prepared with coatings and shells such as enteric coatings and other coatings well known in the art.

[0186] In some embodiments, it is desirable to delay the absorption of the compound from subcutaneous or intramuscular injection in order to extend the effect of the active ingredient. This can be achieved by using a liquid suspension of a crystalline or amorphous substance that is poorly water-soluble. At this time, the absorption rate of the active ingredient depends on its dissolution rate, and in turn the dissolution rate can depend on the crystal size and crystal form. Alternatively, the absorption delay of the parenterally administered active ingredient is achieved by dissolving or suspending the compound in an oily medium. In addition, sustained absorption of injectable pharmaceutical dosage forms can be brought about by incorporating agents that delay absorption such as aluminum monostearate and gelatin.

[0187] Controlled-release parenteral compositions may be in the form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oily solutions, oil suspensions, emulsions, or the active ingredient may be incorporated into one or more biocompatible carriers, liposomes, nanoparticles, implants or infusion devices.

[0188] Materials used in the preparation of microspheres and / or microcapsules include biodegradable / bioerodible polymers such as polyglactin, poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutamine) and poly(lactic acid).

[0189] Biocompatible carriers that can be used when formulating a controlled-release parenteral preparation include carbohydrates such as dextran and proteins such as albumin, lipoproteins or antibodies. Materials used for implants can be non-biodegradable, for example, polydimethylsiloxane, or biodegradable, for example, poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(orthoester) and the like.

[0190] In an embodiment, one or more active ingredients are administered by aerosol. This is achieved by preparing an aqueous aerosol containing the compound, a liposome formulation, or solid particles. A non-aqueous (e.g., fluorocarbon propellant) suspension may be used. The pharmaceutical composition can also be administered using a sonic nebulizer that minimizes the exposure of the drug to shear that can cause degradation of the compound.

[0191] Typically, an aqueous aerosol is prepared by formulating an aqueous solution or suspension of one or more active ingredients together with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary depending on the requirements of the particular compound, but typically include non-ionic surfactants (Tween, Pluronic, or polyethylene glycol), innocuous proteins such as serum albumin, sorbitan esters, amino acids such as oleic acid, lecithin, glycine, buffers, salts, saccharides or sugar alcohols. Aerosols are generally prepared from isotonic solutions.

[0192] Dosage forms for topical or transdermal administration of one or more active ingredients include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The one or more active ingredients can be mixed under aseptic conditions with a pharmaceutically acceptable carrier and, optionally, any preservative, buffer or propellant.

[0193] Transdermal patches suitable for use in the present invention are disclosed in Transdermal Drug Delivery: Developmental Issues and Research Initiatives (Marcel Dekker Inc., 1989) and U.S. Patent Nos. 4,743,249, 4,906,169, 5,198,223, 4,816,540, 5,422,119, 5,023,084, which are incorporated herein by reference. The transdermal patch can also be any transdermal patch well-known in the art, including a trans-scrotal patch. The pharmaceutical composition in such a transdermal patch can contain one or more absorption promoters or skin penetration enhancers well-known in the art (see, for example, U.S. Patent Nos. 4,379,454 and 4,973,468, which are incorporated herein by reference). The transdermal therapeutic system used in the present invention can be based on iontophoresis, diffusion, or a combination of these two effects.

[0194] The transdermal patch has the further advantage of providing controlled delivery of one or more active ingredients to the body. Such dosage forms can be prepared by dissolving or dispersing the one or more active ingredients in a suitable medium. Absorption promoters can also be used to increase the flux of the active ingredient through the skin. The rate of such flux can be controlled either by providing a rate-limiting membrane or by dispersing the one or more active ingredients in a polymeric matrix or gel.

[0195] Such pharmaceutical compositions may be in the form of creams, ointments, lotions, liniments, gels, hydrogels, solutions, suspensions, sticks, sprays, pastes, plasters and other types of transdermal drug delivery systems. This composition may also contain pharmaceutically acceptable carriers or excipients, such as emulsifiers, antioxidants, buffers, preservatives, humectants, penetration enhancers, chelating agents, gel formers, ointment bases, fragrances, and skin protectants.

[0196] Examples of emulsifiers include, but are not limited to, naturally occurring gums such as acacia gum or tragacanth gum, naturally occurring phosphatides such as soy lecithin and sorbitan monooleate derivatives.

[0197] Examples of antioxidants include, but are not limited to, butylated hydroxyanisole (BHA), ascorbic acid and its derivatives, tocopherol and its derivatives, and cysteine.

[0198] Examples of preservatives include, but are not limited to, parabens such as methyl or propyl p-hydroxybenzoate and benzalkonium chloride. Examples of humectants include, but are not limited to, glycerin, propylene glycol, sorbitol and urea.

[0199] Examples of penetration enhancers include, but are not limited to, propylene glycol, DMSO, triethanolamine, N,N-dimethylacetamide, N,N-dimethylformamide, 2-pyrrolidone and its derivatives, tetrahydrofurfuryl alcohol, propylene glycol, propylene glycol monolaurate or diethylene glycol monoethyl or monomethyl ether containing methyl laurate, eucalyptol, lecithin, Transcutol® and Azone®.

[0200] Examples of chelating agents include, but are not limited to, sodium EDTA, citric acid and phosphoric acid. Examples of gelling agents include, but are not limited to, carbopol, cellulose derivatives, bentonite, alginates, gelatin, and polyvinylpyrrolidone.

[0201] In addition to one or more active ingredients, the ointments, pastes, creams, and gels of the present invention may contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0202] Powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances. Sprays may further contain conventional propellants such as chlorofluorocarbons, and volatile unsubstituted hydrocarbons such as butane and propane.

[0203] Injectable depot forms are prepared by forming a microcapsule matrix of one or more compounds of the present invention in a biodegradable polymer such as polylactide - polyglycolide. The release rate of the compound can be controlled depending on the ratio of the compound to the polymer and the nature of the specific polymer used. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by encapsulating the drug in liposomes or microemulsions that are compatible with living tissues.

[0204] Subcutaneous implants are well known in the art and are suitable for use in the present invention. The subcutaneous implantation method is preferably non-irritating and mechanically elastic. The implant may be of the matrix type, reservoir type, or a hybrid thereof. In a matrix type device, the carrier material may be porous or non-porous, solid or semi-solid, and permeable or impermeable to one or more active compounds. The carrier material may be biodegradable or may erode slowly after administration. In some cases, the matrix is non-degradable, but instead relies on the diffusion of the active compound through a matrix in which the carrier material degrades. An alternative subcutaneous implant method utilizes a reservoir device, where one or more active compounds are surrounded by a rate-limiting membrane, e.g., a membrane having zero-order kinetics independent of component concentration. Devices consisting of a matrix surrounded by a rate-limiting membrane are also suitable for use.

[0205] Both reservoir type and matrix type devices may contain polydimethylsiloxane, e.g., Silastic (trademark), or other silicone rubbers. The matrix material may be insoluble polypropylene, polyethylene, polyvinyl chloride, ethyl vinyl acetate, polystyrene, and polymethacrylate, as well as glycerol esters of the palmitostearic acid glycerol, stearic acid glycerol, and behenic acid glycerol types. The material may be a hydrophobic or hydrophilic polymer and may optionally contain solubilizing agents.

[0206] The subcutaneous implant device may be a delayed release capsule made of any suitable polymer, as described, for example, in U.S. Patent Nos. 5,035,891 and 4,210,644, which are hereby incorporated by reference.

[0207] Generally, it is possible to apply at least four different techniques to provide rate control of release and skin permeation of a pharmaceutical compound. These techniques are as follows: membrane-based suppression systems, adhesive diffusion control systems, matrix dispersion systems, and microreservoir systems. It is understood that controlled release transdermal and / or topical compositions can be achieved by suitably combining these techniques.

[0208] In a membrane-based suppression system, the active ingredient is present in a reservoir that is completely encapsulated within a shallow compartment formed from a drug-impermeable laminate such as a metal-plastic laminate and a rate-limiting polymeric membrane, such as a microporous or non-porous polymeric membrane, for example an ethylene-vinyl acetate copolymer. The active ingredient is released through the rate-limiting polymeric membrane. In the drug reservoir, the active ingredient can either be dispersed within a solid polymer matrix or suspended within a non-leachable viscous liquid medium such as a silicone fluid. A thin layer of an adhesive polymer is affixed to the outer surface of the polymeric membrane to achieve intimate contact between the transdermal system and the skin surface. The adhesive polymer is preferably a polymer that is low in allergenicity and compatible with the active drug substance.

[0209] In an adhesive diffusion control system, the reservoir of the active ingredient is formed by directly dispersing the active ingredient within an adhesive polymer and then applying, for example by solvent casting, an adhesive containing the active ingredient onto a flat sheet with a substantially drug-impermeable metal-plastic backing to form a thin drug reservoir layer.

[0210] A matrix dispersion system is characterized in that a reservoir of the active ingredient is formed by substantially uniformly dispersing the active ingredient within a hydrophilic or lipophilic polymer matrix. The drug-containing polymer is then formed into a disc having a substantially well-defined surface area and a controlled thickness. An adhesive polymer is applied along the perimeter to form a strip of adhesive around the disc.

[0211] The microreservoir system can be considered a combination of a reservoir system and a matrix dispersion system. In this case, the reservoir of the active substance is formed by first suspending a drug solid in an aqueous solution of a water-soluble polymer and then dispersing this drug suspension in a lipophilic polymer to form a very large number of non-leachable microspherical drug reservoirs.

[0212] Any of the above-described controlled release, long-term release, and sustained release compositions can be formulated to release the active ingredient in about 30 minutes to about 1 week, about 30 minutes to about 72 hours, about 30 minutes to 24 hours, about 30 minutes to 12 hours, about 30 minutes to 6 hours, about 30 minutes to 4 hours, and about 3 hours to 10 hours. In embodiments, the effective concentration of one or more active ingredients persists in the subject's body for 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours, or more after administration of the pharmaceutical composition to the subject.

[0213] Dosage When the agents described herein are administered to humans or animals as pharmaceuticals, they can be administered by themselves or as a pharmaceutical composition containing the active ingredient in combination with a pharmaceutically acceptable carrier, excipient, or diluent.

[0214] The actual dosage level and the time course of administration of the active ingredient in the pharmaceutical composition of the present invention can be varied for a particular patient, composition, and method of administration so as to achieve an amount of the active ingredient effective to achieve the desired therapeutic response without being toxic to the patient. Generally, the agents or pharmaceutical compositions of the present invention are administered in an amount sufficient to reduce or eliminate symptoms associated with viral infections and / or autoimmune diseases.

[0215] Exemplary dosage ranges include 0.01 mg to 250 mg per day, 0.01 mg to 100 mg per day, 1 mg to 100 mg per day, 10 mg to 100 mg per day, 1 mg to 10 mg per day, and 0.01 mg to 10 mg per day. The preferred dosage of the drug is the maximum amount that the patient can tolerate and that does not cause severe or unacceptable side effects. In embodiments, the drug is administered at a concentration of about 10 μg to about 100 mg per kg of body weight per day, about 0.1 to about 10 mg / kg per day, or about 1.0 mg to about 10 mg / kg of body weight per day.

[0216] In embodiments, the pharmaceutical composition contains an amount of the drug in the range of 1 to 10 mg, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg. In embodiments, a therapeutically effective dosage results in a drug concentration in serum of about 0.1 ng / ml to about 50 - 100 μg / ml. The pharmaceutical composition typically must provide a dosage of about 0.001 mg to about 2000 mg of the compound per kg of body weight per day. For example, the dosage for systemic administration to a human patient can be in the range of 1 - 10 μg / kg, 20 - 80 μg / kg, 5 - 50 μg / kg, 75 - 150 μg / kg, 100 - 500 μg / kg, 250 - 750 μg / kg, 500 - 1000 μg / kg, 1 - 10 mg / kg, 5 - 50 mg / kg, 25 - 75 mg / kg, 50 - 100 mg / kg, 100 - 250 mg / kg, 50 - 100 mg / kg, 250 - 500 mg / kg, 500 - 750 mg / kg, 750 - 1000 mg / kg, 1000 - 1500 mg / kg, 1500 - 2000 mg / kg, 5 mg / kg, 20 mg / kg, 50 mg / kg, 100 mg / kg, 500 mg / kg, 1000 mg / kg, 1500 mg / kg, or 2000 mg / kg. Pharmaceutical dosage unit forms are prepared to provide a combination of about 1 mg to about 5000 mg, for example, about 100 to about 2500 mg of the compound or essential ingredient per dosage unit form.

[0217] In an embodiment, a drug of about 50 nM to about 1 μM is administered to a subject. In related embodiments, a drug of about 50 - 100 nM, 50 - 250 nM, 100 - 500 nM, 250 - 500 nM, 250 - 750 nM, 500 - 750 nM, 500 nM - 1 μM, or 750 nM - 1 μM is administered to a subject.

[0218] Determination of an effective amount is well within the ability of one of ordinary skill in the art, particularly in view of the detailed disclosure provided herein. Generally, an effective or efficacious amount of a drug is determined by first administering a low dose of the drug and then incrementally increasing the administered dose or dosage until the desired effect (e.g., reduction or elimination of symptoms associated with a viral infection or autoimmune disease) is observed in the subject with minimal or acceptable toxic side effects. Methods applicable to determining appropriate dosages and dosing schedules for administration of the pharmaceutical compositions of the present invention are described, for example, in Goodman and Gilman’s The Pharmacological Basis of Therapeutics, Goodman et al., eds., 11th Edition, McGraw-Hill 2005, and Remington: The Science and Practice of Pharmacy, 20th and 21st Editions, Gennaro and University of the Sciences in Philadelphia, Eds., Lippencott Williams & Wilkins (2003 and 2005) (each of which is incorporated herein by reference). Combination Therapy The tumor-specific neoantigen peptides and pharmaceutical compositions described herein can also be administered in combination with another therapeutic molecule. The therapeutic molecule can be any compound that reduces a neoplasm or its symptoms. Examples of such compounds include, but are not limited to, chemotherapeutic agents, anti-angiogenic agents, checkpoint blockade antibodies, or other molecules that reduce immunosuppression.

[0219] Tumor-specific neoantigen peptides can be administered before, during, or after the administration of a further therapeutic agent. In embodiments, the tumor-specific neoantigen peptides are administered before the first administration of the further therapeutic agent. In embodiments, the tumor-specific neoantigen peptides are administered after the first administration of the further therapeutic agent (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or more). In embodiments, the tumor-specific neoantigen peptides are administered simultaneously with the first administration of the further therapeutic agent.

[0220] Vaccine In exemplary embodiments, the present invention relates to immunogenic compositions, e.g., vaccine compositions having the ability to generate a specific T cell response. The vaccine composition comprises mutant neoantigen peptides and mutant neoantigen polypeptides corresponding to tumor-specific neoantigens identified by the methods described herein.

[0221] Suitable vaccines may preferably contain a plurality of tumor-specific neoantigen peptides. In certain embodiments, the vaccine can comprise 1 to 100 sets of peptides, more preferably 1 to 50 such peptides, even more preferably 10 to 30 sets of peptides, and even more preferably 15 to 25 peptides. According to another preferred embodiment, the vaccine can comprise about 20 peptides, more preferably 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, or 30 different peptides, even more preferably 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 different peptides, and most preferably 18, 19, 20, 21, 22, 23, 24, or 25 different peptides.

[0222] In one embodiment of the present invention, different tumor-specific neoantigen peptides and / or polypeptides are selected for use in a neoplasm vaccine such that an immune attack against the patient's neoplasm / tumor is most likely to occur. Without being bound by theory, it is believed that including a diverse range of tumor-specific neoantigen peptides can result in a broad scale immune attack against the neoplasm / tumor. In one embodiment, the selected tumor-specific neoantigen peptide / polypeptide is encoded by a missense mutation. In a second embodiment, the selected tumor-specific neoantigen peptide / polypeptide is encoded by a combination of a missense mutation and a neo-ORF mutation. In a third embodiment, the selected tumor-specific neoantigen peptide / polypeptide is encoded by a neo-ORF mutation.

[0223] In one embodiment where the selected tumor-specific neoantigen peptide / polypeptide is encoded by a missense mutation, the peptide and / or polypeptide is selected based on its ability to associate with the patient's specific MHC molecule. Peptides / polypeptides derived from neo-ORF mutations can also be selected based on their ability to associate with the patient's specific MHC molecule, but can also be selected even if they are predicted not to associate with the patient's specific MHC molecule. The vaccine composition has the ability to generate a specific cytotoxic T cell response and / or a specific helper T cell response.

[0224] The vaccine composition may further comprise an adjuvant and / or a carrier. Examples of useful adjuvants and carriers are provided below. The peptides and / or polypeptides in the composition can associate with a carrier, for example a protein having the ability to present the peptide to T cells or an antigen presenting cell such as a dendritic cell (DC).

[0225] ​An adjuvant is any substance that, when mixed with a vaccine composition, increases the immune response to a mutant peptide or is otherwise modified. A carrier is a scaffold structure capable of associating with a neoantigen peptide, such as a polypeptide or a polysaccharide. Optionally, the adjuvant is conjugated covalently or non-covalently to the peptide or polypeptide of the present invention.

[0226] The ability of an adjuvant to increase the immune response to an antigen typically manifests as a marked increase in the immune-mediated response or a reduction in disease symptoms. For example, an increase in humoral immunity typically manifests as a marked increase in the titer of antibodies produced against the antigen, and an increase in T cell activity typically manifests as an increase in cell proliferation, or cytotoxicity, or cytokine secretion. An adjuvant can also alter the immune response, for example, by changing a primary humoral response or a Th2 response to a primary cellular response or a Th1 response.

[0227] Suitable adjuvants include, but are not limited to, 1018 ISS, aluminum salts , Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, O NTAK, PepTel.RTM. vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, QS21 stimulon from Aquila derived from saponin (Aquila Biotech, Worcester, Mass., USA), mycobacteria Examples include A extracts, synthetic bacterial cell wall mimetics, and other proprietary adjuvants such as Ribi's Detox, Quil or Superfos. Several immunological adjuvants specific for dendritic cells (e.g., MF59) and their formulations have been previously described (Dupuis M, et al., Cell Immunol. 1998; 186(1): 18-27; Allison A C; Dev Biol Stand. 1998; 92: 3-11). Cytokines may also be used. Some cytokines have been directly associated with affecting dendritic cell migration to lymphoid tissues (e.g., TNF-α), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Patent No. 5,849,589, which is specifically incorporated herein by reference in its entirety) and acting as immune adjuvants (e.g., IL-12) (Gabrilovich D I, et al., J Immunother Emphasis Tumor Immunol. 1996 (6): 414-418).

[0228] Toll-like receptors (TLRs) can also be used as adjuvants and are important members of a family of pattern recognition receptors (PRRs) that recognize conserved motifs shared by many microorganisms termed "pathogen-associated molecular patterns" (PAMPs). Recognition of these "danger signals" activates multiple elements of the innate and adaptive immune systems. TLRs are expressed by cells of the innate and adaptive immune systems such as dendritic cells (DCs), macrophages, T and B cells, mast cells, and granulocytes and are localized in various intracellular compartments such as the cell membrane, lysosomes, endosomes, and endolysosomes. Different TLRs recognize different PAMPs. For example, TLR4 is activated by LPS contained in the bacterial cell wall and TLR9 is activated by unmethylated bacterial or viral CpG DNA 、and TLR3 is activated by double-stranded RNA. TLR ligand binding triggers the activation of one or more intracellular signaling pathways, ultimately leading to the production of many major molecules associated with inflammation and immunity, particularly the transcription factors NF-κB and type I interferons. TLR-mediated DC activation enables enhanced DC activation, phagocytosis, activation and upregulation of co-stimulatory markers such as CD80, CD83, and CD86, the migration of DCs to draining regional lymph nodes, and the expression of CCR7 that promotes antigen presentation to T cells, as well as increased secretion of cytokines such as type I interferons, IL-12, and IL-6. All of these downstream events are critically important for the induction of the adaptive immune response.

[0229] Among the most promising cancer vaccine adjuvants currently in clinical development are the TLR9 agonist CpG and the synthetic double-stranded RNA (dsRNA) TLR3 ligand poly ICLC. In preclinical studies, poly ICLC has been found to be the most potent TLR adjuvant due to its induction of inflammatory cytokines and lack of stimulation of IL-10, as well as the maintenance of high levels of co-stimulatory molecules in DCs, compared to LPS and CpG. Furthermore, poly ICLC was recently directly compared to CpG in non-human primates (rhesus macaques) as an adjuvant to a protein vaccine consisting of human papillomavirus (HPV) 16 capsomers (Stahl-Hennig C, Eisenblatter M, Jasny E, et al. 「Synthetic double-stranded RNAs are adjuvants for the induction of T helper 1 and humoral immune responses to human papillomavirus in rhesus macaques)」. PLoS pathogens. Apr 2009; 5(4)). rhesus macaques)」. PLoS pathogens. Apr 2009; 5(4))。

[0230] CpG immunostimulatory oligonucleotides have also been reported to enhance the effect of adjuvants in vaccine settings. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system via Toll-like receptors (TLRs), primarily TLR9. TLR9 activation induced by CpG enhances antigen-specific humoral and cellular responses to a variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly, this enhances dendritic cell maturation and differentiation and results in enhanced activation of Th1 cells and potent cytotoxic T lymphocyte (CTL) generation even in the absence of CD4 T cell help. The Th1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA) that normally promote a Th2 bias. CpG oligonucleotides exhibit even higher adjuvant activity when formulated or co-administered with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions or similar formulations that are particularly required to induce a strong response when the antigen is relatively weak. CpG oligonucleotides also accelerate the immune response and make it possible to reduce the antigen dose by about two orders of magnitude with an antibody response equivalent to that to a complete-dose vaccine without CpG in some experiments (Arthur M. Krieg, Nature Reviews, Drug Discovery, 5, Jun. 2006, 471-484). U.S. Patent No. 6,406,705 B1 describes that an antigen-specific immune response is induced by the combined use of CpG oligonucleotides, non-nucleic acid adjuvants and antigens. A commercially available CpG TLR9 antagonist is dSLIM (double Stem Loop Immunomodulator) by Mologen (Berlin, Germany), which is the present invention It is a preferred component of the pharmaceutical composition of the invention. Other TLR-binding molecules, such as RNA-binding TLR7, TLR8 and / or TLR9, may also be used.

[0231] Xanthenone derivatives, such as vadimezan or AsA404 (also known as 5,6-dimethylaxanthenone-4-acetic acid (DMXAA)), etc., can also be used as adjuvants according to embodiments of the present invention. Alternatively, such derivatives may also be administered, for example, via systemic or intratumoral delivery in parallel with the vaccine of the present invention and can stimulate immunity at the tumor site. Without being bound by theory, such xanthenone derivatives are thought to act by stimulating interferon (IFN) production by stimulation of the IFN gene (STING) receptor (see, for example, Conlon et al. (2013) "Mouse STING binds and signals in response to the vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid, but human STING does not", Journal of Immunology, 190: 5216-25 and Kim et al. (2013) "Anticancer flavonoids are mouse-selective STING agonists", 8: 1396-1401)).

[0232] ​Other examples of useful adjuvants include, but are not limited to, chemically modified CpGs (e.g., CpR, Idera), poly(I:C) (e.g., poly i:CI2U), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies, such as cyclophosphamide, sunitinib, bevacizumab, celecoxib, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL-999, CP-5476 32, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which can act therapeutically and / or as adjuvants. The amounts and concentrations of adjuvants and additives useful in the context of the present invention can be readily determined by those skilled in the art without undue experimentation. Further adjuvants include colony stimulating factors such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim).

[0233] Poly ICLC is a synthetically prepared double-stranded RNA consisting of poly I and poly C strands with an average length of about 5000 nucleotides, stabilized against heat denaturation and hydrolysis by serum nucleases by the addition of polylysine and carboxymethyl cellulose. This compound activates the RNA helicase domains of TLR3 and MDA5, both members of the PAMPs family, and causes activation of DC and natural killer (NK) cells and production of a "natural mixture" of type I interferons, cytokines, and chemokines. Furthermore, poly ICLC exerts a more direct, broad-host targeted anti-infective effect, and possibly an anti-tumor effect, mediated by the two IFN-inducible ribozyme systems 2'5'-OAS and P1 / eIF2a kinase (also known as PKR(4-6)), as well as RIG-I helicase and MDA5.

[0234] In rodents and non-human primates, poly ICLC enhances T cell responses to viral antigens, cross-priming, and tumor-specific, virus-specific, and self-antigen-specific CD8 +Enhanced induction of T cells has been shown. In recent studies in non-human primates, poly ICLC has been found to be essential for the generation of antibody responses and T cell immunity against DC-targeted or non-targeted HIV Gag p24 protein, highlighting its effectiveness as a vaccine adjuvant.

[0235] In human subjects, transcriptional analysis of serial whole blood samples showed similar gene expression profiles among eight healthy human volunteers who received a single subcutaneous dose of poly ICLC, with a maximum of 212 gene expression differences between these eight subjects compared to four placebo recipients. Notably, when comparing poly ICLC gene expression data to prior data from volunteers immunized with the highly effective yellow fever vaccine YF17D, numerous canonical transcription and signaling pathways were shown to be similarly upregulated at peak time points, including those of the innate immune system.

[0236] Recently, immunological analyses were reported for patients with ovarian, fallopian tube, and primary peritoneal cancer in complete clinical remission after the second or third treatment in a phase 1 study of subcutaneous vaccination with synthetic overlapping long peptides (OLPs) derived from the cancer-testis antigen NY-ESO-1 alone, or in combination with Montanide-ISA-51, or in combination with 1.4 mg of poly ICLC and Montanide. Addition of poly ICLC and Montanide significantly enhanced the generation of NY-ESO-1-specific CD4 + and CD8 + T cells and antibody responses compared to OLP alone or OLP and Montanide.

[0237] The vaccine composition according to the present invention may contain two or more different adjuvants. Furthermore, the present invention encompasses therapeutic compositions containing any adjuvant substance, including any of the above or combinations thereof. It is also contemplated that the peptide or polypeptide and the adjuvant may be administered separately in any suitable order.

[0238] The carrier can exist independently of the adjuvant. The function of the carrier can be, for example, to confer stability, increase biological activity, or increase the half-life in serum. Furthermore, the carrier can assist in the presentation of the peptide to T cells. The carrier can be any suitable carrier known to those skilled in the art, such as a protein or an antigen-presenting cell. The carrier protein can be, but is not limited to, keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones such as insulin or palmitic acid. For human immunization, the carrier can be a physiologically acceptable carrier that is acceptable and safe for humans. However, tetanus toxoid and / or diphtheria toxoid are suitable carriers in one embodiment of the present invention. Alternatively, the carrier may be dextran, such as sepharose.

[0239] Cytotoxic T cells (CTLs) recognize antigens in the form of peptides bound to MHC molecules rather than intact foreign antigens themselves. The MHC molecules themselves are located on the cell surface of antigen-presenting cells. Thus, activation of CTLs is possible only when a trimolecular complex of peptide antigen, MHC molecule, and APC is present. Correspondingly, CTLs can enhance the immune response not only when only peptides are used for activation of CTLs, but also when APCs having their respective MHC molecules are added. Thus, in some embodiments, the vaccine composition according to the present invention further contains at least one antigen-presenting cell.

[0240] Antigen-presenting cells (or stimulatory cells) typically have MHC class I or II molecules on their surface and, in one embodiment, substantially lack the ability to load selected antigens onto the MHC class I or II molecules themselves. As described in more detail below, selected antigens can be readily loaded onto MHC class I or II molecules in vitro.

[0241] Preferably, the antigen-presenting cell is a dendritic cell. More preferably, the dendritic cell is an autologous dendritic cell pulsed with the neoantigen peptide. This peptide may be any suitable peptide that elicits an appropriate T cell response. T cell therapies using autologous dendritic cells pulsed with peptides derived from tumor-associated antigens are disclosed in Murphy et al. (1996) The Prostate 29, 371-380 and Tjua et al. (1997) The Prostate 32, 272-278.

[0242] Thus, in one embodiment of the invention, a vaccine composition containing at least one antigen-presenting cell is pulsed with or loaded with one or more peptides of the invention. Alternatively, peripheral blood mononuclear cells (PBMCs) isolated from a patient may be loaded with the peptide ex vivo and infused back into the patient. As an alternative, the antigen-presenting cell contains an expression construct encoding the peptide of the invention. The polynucleotide may be any suitable polynucleotide, preferably having the ability to transduce dendritic cells and thus result in the presentation of the peptide and induction of immunity. Therapy The invention further provides a method of inducing a neoplasm / tumor-specific immune response in a subject, a method of vaccinating against a neoplasm / tumor, and a method of treating and / or alleviating the symptoms of cancer in a subject by administering to the subject the neoantigen peptide or vaccine composition of the invention.

[0243] According to the invention, the cancer vaccine described above can be used in patients diagnosed with having cancer or at risk of developing cancer. In one embodiment, the patient may have a solid tumor, such as breast, ovarian, prostate, lung, kidney, stomach, colon, testicular, head and neck, pancreatic, brain, melanoma, and other tumors and hematological tumors of tissues and organs, such as lymphoma and leukemia, such as acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T cell lymphocytic leukemia, and B cell lymphoma.

[0244] The peptide or composition of the present invention is administered in an amount sufficient to induce a CTL response. The neoantigen peptide, polypeptide or vaccine composition of the present invention can be administered alone or in combination with other therapeutic agents. The therapeutic agent can be, for example, a chemotherapeutic agent or a biotherapeutic agent, radiation, or immunotherapy. Any suitable treatment for a particular cancer can be administered. Examples of chemotherapeutic agents or biotherapeutic agents include, but are not limited to, aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin alpha, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol®), pilocarpine, prochloroperazine, rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine and vinorelbine tartrate. For the treatment of prostate cancer, a preferred chemotherapeutic agent that can be used in combination with anti-CTLA-4 is paclitaxel (Taxol®).

[0245] In addition, an anti-immunosuppressant or immunostimulant may be further administered to the subject. For example, an anti-CTLA antibody or anti-PD-1 or anti-PD-L1 is further administered to the subject. Blockade of CTLA-4 or PD-1 / PD-L1 by an antibody can enhance the immune response against cancerous cells in a patient. Specifically, CTLA-4 blockade has been shown to be effective when following a vaccination protocol (Hodi et al 2005).

[0246] The optimal amount and optimal dosing regimen of each peptide to be included in the vaccine composition can be determined by those skilled in the art without performing more experiments than necessary. For example, the peptide or its variant can be prepared for intravenous (i.v.) injection, subcutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, intramuscular (i.m.) injection. Preferred methods of peptide injection include s.c, i.d., i.p., i.m., and i.v. Preferred methods of DNA injection include i.d., i.m., s.c, i.p. and i.v. For example, 1 to 5 A dose of 00 mg, 50 μg to 1.5 mg, preferably 10 μg to 500 μg of the peptide or DNA may be administered, which may depend on each peptide or DNA. Doses in this range have been successfully used in past studies (Brunsvig P F, et al., Cancer Immunol Immunother. 2006; 55(12): 1553-1564; M. Staehler, et al., ASCO meeting 2007; Abstract No 3017). Other administration methods of the vaccine composition are known to those skilled in the art.

[0247] The pharmaceutical composition of the present invention can be made such that the selection, number and / or amount of the peptide present in the composition is tissue, cancer, and / or patient-specific. For example, the exact selection of the peptide can be guided by the expression pattern of the parent protein in a given tissue so as to avoid side effects. The selection can depend on the specific type of cancer, the disease state, the previous treatment regimen, the patient's immune state, and, of course, the patient's HLA-haplotype. Furthermore, the vaccine according to the present invention can contain components tailored to the individual according to the individual needs of a particular patient. Examples include varying the amount of peptide according to the expression of relevant neoantigens in a particular patient, personal allergies or other unwanted side effects resulting from treatment, and the adjustment of secondary treatment after the first treatment round or scheme.

[0248] The pharmaceutical composition containing the peptide of the present invention can be administered to an individual already suffering from cancer. In a therapeutic application, the composition is administered to the patient in an amount sufficient to induce an effective CTL response against the tumor antigen and to cure or at least partially arrest the symptoms and / or complications. The amount sufficient to achieve this is defined as a "therapeutically effective dose". The amount effective for this use may depend, for example, on the peptide composition, the method of administration, the stage and severity of the disease being treated, the patient's weight and general health, as well as the judgment of the prescribing physician, but generally ranges from about 1.0 μg to about 50,000 μg of peptide for a 70 kg patient for the initial immunization (for therapeutic or prophylactic administration), followed by a boost dosage, or ranges from about 1.0 μg to about 10,000 μg of peptide, and the boost regimen continues over a period of weeks to months, depending on the patient's response and condition and, optionally, by measuring the specific CTL activity in the patient's blood. It should be noted that the peptides and compositions of the present invention can generally be used in severe medical conditions, i.e., life-threatening or potentially life-threatening situations, particularly when the cancer has metastasized. In a therapeutic use, administration should be initiated as early as possible after detection or surgical resection of the tumor. Thereafter, the boost dosage continues at least until the symptoms have substantially remitted and for a subsequent period.

[0249] A pharmaceutical composition for therapeutic treatment (e.g., a vaccine composition) is intended for parenteral, topical, nasal, oral, or local administration. Preferably, the pharmaceutical composition is administered parenterally, for example, intravenously, subcutaneously, intradermally, or intramuscularly. The composition may be administered to the surgical resection site to induce a local immune response against the tumor. The present invention provides a composition for parenteral administration comprising a solution of a peptide, and the vaccine composition is dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. Various aqueous carriers, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, etc., can be used. These compositions can be sterilized by conventional well-known sterilization techniques or can be sterile filtered. The resulting aqueous solution can be packaged for use as is or can be lyophilized, and the lyophilized preparation is combined with a sterile solution before administration. The composition may contain pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, tonicity agents, wetting agents, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc., as required to approximate physiological conditions.

[0250] The concentration of the peptide of the present invention in the pharmaceutical formulation varies widely, i.e., usually from less than about 0.1% to at least about 2% to 20% - 50% or more by weight, and can be selected mainly according to the detailed administration method selected, depending on the fluid volume, viscosity, etc.

[0251] The liposome suspension containing the peptide can be administered intravenously, locally, topically, etc., at different doses depending, inter alia, on the administration method, the peptide being delivered, and the disease stage being treated. To target immune cells, a ligand, such as an antibody or a fragment thereof specific for the cell surface determinant of the desired immune system cells, can be incorporated into the liposome.

[0252] For solid compositions, for example, conventional or nanoparticle non-toxic solid carriers including pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. can be used. For oral administration, pharmaceutically acceptable non-toxic compositions are formed by admixing commonly used excipients, such as any of the carriers already listed, and approximately 10% to 95% of the active ingredient, i.e., one or more peptides of the present invention, more preferably at a concentration of 25% to 75%.

[0253] For aerosol administration, the immunogenic peptide is preferably provided in a micronized form together with a surfactant and a propellant. Typical proportions of the peptide are from 0.01% to 20% by weight, preferably 1% to 10%. The surfactant is of course non-toxic and is preferably soluble in the propellant. Representative examples of such agents are fatty acids containing 6 to 22 carbon atoms, such as caproic acid, octanoic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric acid and oleic acid, etc., and esters or partial esters with aliphatic polyhydric alcohols or their cyclic anhydrides. Mixed esters, such as mixed or natural glycerides, may be used. The surfactant can account for 0.1% to 20% of the composition by weight, preferably 0.25 to 5%. The remainder of the composition is a normal propellant. A carrier may also be included as required, such as lecithin for nasal delivery. The peptides and polypeptides of the present invention can be readily chemically synthesized using reagents that do not contain bacterial or animal substances (Merrifield RB: "Solid phase peptide synthesis. I. The synthesis of a tetrapeptide". J. Am. Chem. Soc. 85: 2149-54, 1963).

[0254]

[0255] For treatment or immunization, the nucleic acid encoding the peptide of the present invention and optionally one or more of the peptides described herein may also be administered to a patient. Many methods are conveniently used to deliver nucleic acids to patients. For example, nucleic acids can be delivered directly as "naked DNA". This technique is described, for example, in Wolff et al., Science 247: 1465-1468 (1990) and U.S. Patent Nos. 5,580,859 and 5,589,466 which are described in the specification. Nucleic acids may also be administered using, for example, the ballistic delivery as described in U.S. Patent No. 5,204,253. Particles composed of DNA alone may be administered. Alternatively, the DNA may be adhered to particles such as gold particles.

[0256] Also, nucleic acids complexed with cationic compounds, such as cationic lipids, can be delivered. Lipid-mediated gene delivery methods are described, for example, in WO 96 / 18372; WO 93 / 24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Patent No. 5,279,833; WO 91 / 06309; and Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987).

[0257] RNA encoding the peptide of interest can also be used for delivery (see, for example, Kiken et al, 2011; Su et al, 2011). The peptides and polypeptides of the present invention may also be attenuated viruses such as vaccinia or fowlpox It can also be expressed by a host. This approach involves the use of vaccinia virus as a vector for expressing the nucleotide sequence encoding the peptide of the present invention. When introduced into an acutely or chronically infected host or a non-infected host, the recombinant vaccinia virus expresses the immunogenic peptide and thus induces a host CTL response. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacillus Calmette-Guerin). The BCG vector is described in Stover et al. (Nature 351: 456-460 (1991)). The present invention A variety of other vectors useful for the therapeutic administration or immunization of the peptides of the present invention, such as Salmonella typhi vectors, will be apparent to those skilled in the art from the description herein.

[0258] In a preferred means of administering the nucleic acid encoding the peptide of the present invention, a minigene construct encoding multiple epitopes is used. To create a DNA sequence (minigene) encoding a CTL epitope selected for expression in human cells, the amino acid sequence of the epitope is reverse-translated. A human codon usage table is used as a guide for codon selection for each amino acid. These DNA sequences encoding the epitopes are directly adjacent, creating a continuous polypeptide sequence. Additional elements may be incorporated into the minigene design to optimize expression and / or immunogenicity. Examples of amino acid sequences that can be reverse-translated and included in the minigene sequence include: helper T lymphocyte, epitope, leader (signal) sequence, and endoplasmic reticulum retention signal. In addition, the MHC presentation of the CTL epitope can be improved by including synthetic (e.g., polyalanine) or naturally occurring flanking sequences adjacent to the CTL epitope.

[0259] The mini gene sequence is converted into DNA by assembling oligonucleotides encoding the plus and minus strands of the mini gene. Overlapping oligonucleotides (30 - 100 bases in length) are synthesized, phosphorylated, purified, and annealed under appropriate conditions using well-known techniques. The ends of the oligonucleotides are ligated using T4 DNA ligase. This synthetic mini gene encoding the CTL epitope polypeptide can then be cloned into a desired expression vector.

[0260] To ensure expression in target cells, standard regulatory sequences well-known to those skilled in the art are included in the vector. Several vector elements are required: a promoter containing a downstream cloning site for mini gene insertion; a polyadenylation signal for efficient transcription termination; an origin of replication for Escherichia coli (E. coli); and an E. coli selectable marker (e.g., ampicillin or kanamycin resistance). For this purpose, numerous promoters can be used, such as the human cytomegalovirus (hCMV) promoter. For other suitable promoter sequences, see U.S. Patent Nos. 5,580,859 and 5,589,466.

[0261] To optimize mini gene expression and immunogenicity, additional vector modifications may be desirable. In some cases, an intron is required for efficient gene expression, and one or more synthetic or naturally occurring introns can be incorporated into the transcription region of the mini gene. To increase mini gene expression, it may also be considered to include an mRNA stabilization sequence. Recently, it has been proposed that immunostimulatory sequences (ISS or CpG) play a role in the immunogenicity of DNA vaccines. These sequences can be included outside the mini gene coding sequence in the vector if they are found to enhance immunogenicity.

[0262] In some embodiments, a bicistronic expression vector can be used that allows for the production of an epitope encoded by a mini-gene and a second protein included to enhance or reduce immunogenicity. Advantageously, a protein or polypeptide that can enhance the immune response when co-expressed Examples of proteins or polypeptides include cytokines (e.g., IL2, IL12, GM-CSF), cytokine-inducing molecules (e.g., LeIF) or costimulatory molecules. Helper (HTL) epitopes may be linked to an intracellular targeting signal and expressed separately from the CTL epitope. This may allow for the induction of HTL epitopes into different cell compartments than the CTL epitope. If necessary, this may facilitate more efficient entry of HTL epitopes into the MHC class II pathway and thus an improvement in CTL induction. In contrast to CTL induction, co-expression of an immunosuppressive molecule (e.g., TGF-β) may be beneficial in certain diseases to specifically reduce the immune response.

[0263] After the expression vector is selected, the mini-gene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into a suitable Escherichia coli (E. coli) strain and the DNA is prepared using standard techniques. The orientation and DNA sequence of the mini-gene as well as all other elements included in the vector are confirmed using restriction enzyme mapping and DNA sequence analysis. Bacterial cells with the correct plasmid can be stored as a master cell bank and a working cell bank.

[0264] Purified plasmid DNA can be prepared for injection using various formulations. The simplest of these is the reconstitution of lyophilized DNA in sterile phosphate-buffered saline (PBS). Various methods have been described and new technologies may become available. As mentioned above, nucleic acids are conveniently formulated with cationic lipids. In addition, glycolipids, fusogenic liposomes, peptides, and compounds collectively referred to as protective, interactive, non-condensing (PINC) can be complexed to purify plasmid DNA and affect variables such as stability, muscle dispersion, or transport to specific organs or cell types.

[0265] Target cell sensitization can be used as a functional assay for the expression of CTL epitopes encoded by mini-genes and MHC class I presentation. Plasmid DNA is introduced into mammalian cell lines suitable as targets for standard CTL chromium release assays. The transfection method used may depend on the final formulation. Electroporation may be used for "naked" DNA, while cationic lipids allow for direct in vitro transfection. Co-transfecting a plasmid expressing green fluorescent protein (GFP) can enable enrichment of transfected cells using fluorescence-activated cell sorting (FACS). These cells are then labeled with chromium-51 and used as target cells for epitope-specific CTL lines. 51 Cytolysis detected by Cr release indicates that MHC presentation of the CTL epitope encoded by the mini-gene has occurred.

[0266] In vivo immunogenicity is a second approach to testing the function of mini-gene DNA formulations. Transgenic mice expressing appropriate human MHC molecules are immunized with the DNA formulation. The dose and route of administration depend on the formulation (e.g., IM for DNA in PBS, IP for lipid-complexed DNA). Twenty-one days after immunization, splenocytes are harvested and restimulated for one week in the presence of peptides encoding each test epitope. These effector cells (CTLs) are assayed using standard techniques for the lysis of chromium-51-labeled target cells loaded with the peptide. Lysis of target cells sensitized by MHC loading of the peptide corresponding to the epitope encoded by the mini-gene demonstrates the function of the DNA vaccine regarding in vivo induction of CTLs.

[0267] Peptides can also be used to induce CTLs ex vivo. The resulting CTLs can be used to treat chronic tumors in patients who do not respond to other conventional therapies or who would not respond to peptide vaccine treatment approaches. Ex vivo CTL responses to specific tumor antigens are induced by incubating patient CTL precursors (CTLps) with antigen-presenting cells (APCs) from a source and appropriate peptides. During an appropriate incubation time (typically 1 to 4 weeks), CTLps are activated, mature into effector CTLs and expand, and then the cells are infused back into the patient, where they destroy their specific target cells (i.e., tumor cells). To optimize in vitro conditions for the generation of specific cytotoxic T cells, the cultures of stimulator cells are maintained in an appropriate serum-free medium.

[0268] Cells to be activated, such as precursor CD8 + cells, are incubated with stimulator cells after adding to the stimulator cell culture an amount of antigen peptide sufficient to become loaded onto human class I molecules expressed on the surface of the stimulator cells. In the present invention, a sufficient amount of the peptide means about 200, and preferably 200 or more, human class I MH It is an amount capable of expressing the C molecule on the surface of each stimulating cell. Preferably, the stimulating cells are incubated with a peptide at >2 μg / ml. For example, the stimulating cells are incubated with a peptide at >3, 4, 5, 10, 15 μg / ml, or more.

[0269] Next, resting cells or precursor CD8 + cells are incubated with appropriate stimulating cells for a period sufficient to activate the CD8 + cells under culture conditions. Preferably, the CD8 + cells are activated in an antigen-specific manner. Resting cells or precursor CD8 + (effector) cells and the ratio of stimulating cells can vary from individual to individual and can further depend on variables such as the compliance of the individual's lymphocytes with the culture conditions and the nature and severity of the disease state or other conditions such as the treatment modalities within the described scope. However, preferably, the lymphocyte:stimulating cell ratio is in the range of about 30:1 to 300:1. The effector / stimulating culture can be maintained for the time required to stimulate a therapeutically usable or effective number of CD8 + cells.

[0270] For CTL induction in vitro, specific recognition of peptides bound to allelic-specific MHC class I molecules on APCs is required. For the stimulation of CTLs, particularly in the primary immune response, the number of specific MHC / peptide complexes per APC is critically important. Even a small number of peptide / MHC complexes per cell is sufficient to make cells susceptible to lysis by CTLs or to stimulate a secondary CTL response, but a significantly larger number of MHC / peptide complexes is required to successfully activate CTL precursors (pCTLs) during the primary response. Loading peptides onto empty major histocompatibility complex molecules on cells enables the induction of a primary cytotoxic T lymphocyte response.

[0271] Since mutant cell lines do not exist for each human MHC allele, it is advantageous to use a technique that removes endogenous MHC-associated peptides from the surface of APCs and then loads the resulting empty MHC molecules with the desired immunogenic peptides. Using non-transformed (non-tumorigenic), non-infected cells, preferably autologous cells of the patient, as APCs is desirable for the design of CTL induction protocols for the development of ex vivo CTL therapy. The present application discloses a method of stripping endogenous MHC-associated peptides from the surface of APCs and subsequently loading the desired peptides.

[0272] Stable MHC class I molecules are trimeric complexes formed by the following elements: 1) a peptide, usually 8-10 residues; 2) a transmembrane heavy chain polymorphic protein chain having a peptide binding site in its a1 and a2 domains; and 3) a non-covalently associated non-polymorphic light chain, p2 microglobulin. Removal of the bound peptide from the complex and / or dissociation of p2 microglobulin renders the MHC class I molecule non-functional and destabilized, resulting in rapid degradation. All MHC class I molecules isolated from PBMCs have endogenous peptides bound to them. Thus, the first step is to remove all endogenous peptides bound to MHC class I molecules on APCs without causing their degradation, after which exogenous peptides can be added to them.

[0273] As two possible ways to remove the peptides bound to MHC class I molecules, lowering the culture temperature from 37°C to 26°C overnight to destabilize p2 microglobulin and using weak acid treatment to strip endogenous peptides from the cells can be mentioned. By these methods, the previously bound peptides are released into the extracellular environment, enabling new foreign peptides to bind to the empty class I molecules. The low-temperature incubation method can efficiently bind foreign peptides to the MHC complex, but it is necessary to incubate overnight at 26°C, which can slow down the metabolic rate of the cells. Also, cells that do not actively synthesize MHC molecules (e.g., resting PBMC) may not produce a large amount of empty surface MHC molecules depending on the low-temperature procedure.

[0274] Harsh acid stripping involves trifluoroacetic acid, peptide extraction at pH 2, or acid denaturation of immunoprecipitated class I-peptide complexes. Since it is important to remove endogenous peptides while maintaining the optimal metabolic state that is crucial for the viability of APCs and antigen presentation, these methods are not feasible for CTL induction. Weakly acidic solutions at pH 3 such as glycine or citrate-phosphate buffer have been used for the identification of endogenous peptides and the identification of tumor-associated T cell epitopes. This treatment is particularly effective in that only MHC class I molecules are destabilized (and associated peptides are released), while other surface antigens, including MHC class II molecules, remain intact. Most importantly, treatment of cells with a weakly acidic solution does not affect the viability or metabolic state of the cells. Stripping of endogenous peptides is performed at 4°C for 2 minutes, and since APCs are in a state where they can immediately perform their function after being loaded with the appropriate peptides, weak acid treatment is rapid. This technique is utilized herein for the generation of peptide-specific APCs that give rise to primary antigen-specific CTLs. The resulting APCs are peptide-specific CD8 + Efficient in the induction of CTLs.

[0275] Activated CD8 +The cells can be effectively separated from the stimulating cells using one of various known methods. For example, the stimulating cells, the peptides loaded on the stimulating cells, or CD8 + monoclonal antibodies specific to the cells (or segments thereof) can be utilized to bind to their appropriate complementary ligands. The antibody-tagged molecules can then be extracted from the stimulating effector cell mixture by appropriate means, such as well-known immunoprecipitation or immunoassay methods.

[0276] activated CD8 + The effective amount of the cytotoxicity of the cells can vary between in vitro use and in vivo use, and also depending on the amount and type of the cells that are the ultimate targets of these killer cells. The amount can also vary depending on the condition of the patient and must be determined by an expert considering all appropriate factors. However, preferably, compared to about 5×10 6 ~5×10 7 cells used in mice, for adult humans, about 1×10 6 ~about 1×10 12 , more preferably about 1×10 8 ~about 1×10 11 , even more preferably about 1×10 9 ~about 1×10 10 activated CD8 + cells are utilized.

[0277] Preferably, as discussed above, the activated CD8 + cells are recovered from the cell culture before administering the CD8 + cells to the individual to be treated. However, unlike other currently proposed treatment modalities, it is important to note that this method uses a non-tumorigenic cell culture system. Thus, when complete separation of the stimulating cells and the activated CD8 + cells is achieved, there is no inherent risk associated with the administration of a small number of stimulating cells, while the administration of mammalian tumor-promoting cells can be extremely dangerous.

[0278] Methods of reintroducing cellular components are known in the art, and procedures such as those exemplified in U.S. Patent No. 4,844,893 to Honsik et al. and U.S. Patent No. 4,690,91 to Rosenberg No. 5. For example, administration of activated CD8 + cells by intravenous injection is appropriate.

[0279] CD8 + Cell activity may be enhanced using CD4 + cells. Many immune-based anti-cancer therapies may be more effective when targeting a patient's tumor using both CD8 + and CD4 + T lymphocytes, so the identification of CD4 + T cell epitopes for tumor antigens is of interest. CD4 + cells have the ability to enhance the CD8 T cell response. In many studies in animal models, it has been clearly demonstrated that results are favorable when both CD4 + and CD8 + T cells are involved in the anti-tumor response (see, for example, Nishimura et al. (1999) "The individual roles of antigen-specific T helper type 1 (TH1) and Th2 cells in tumor eradication in vivo). Distinct role of antigen-specific T helper type 1 (TH1) and Th2 cells in tumor eradication in vivo). J Ex Med 190: 617-27). Universal CD4 + T cell epitopes applicable to the development of therapies for different types of cancer have been identified (see, for example, Kobayashi et al. (2008) Current Opinion in Immunology 20: 221-27). For example, an HLA-DR-restricted helper peptide derived from tetanus toxoid is used in a melanoma vaccine to CD4 +T cells are non-specifically activated (see, e.g., Slingluff et al. (2007) "Immunologic and Clinical Outcomes of a Randomized Phase II Trial of Two Multipeptide Vaccines for Melanoma in the Adjuvant Setting", Clinical Cancer Research 13(21): 6386-95). Within the scope of the present invention, it is contemplated that such CD4 cells may be applicable at three different levels of tumor specificity: 1) a broad level where universal CD4 + epitopes (e.g., tetanus toxoid) are used to enhance CD8 + cells; 2) an intermediate level where natural tumor-associated CD4 + epitopes are used to enhance CD8 + cells; and 3) a patient-specific level where neoantigen CD4 + epitopes are used to enhance CD8 + cells in a patient-specific manner. +

[0280] CD8 +Cell-mediated immunity can also be induced by neoantigen-loaded dendritic cell (DC) vaccines. DCs are potent antigen-presenting cells that can initiate T cell immunity and can be used as cancer vaccines when loaded with one or more peptides of interest, for example, by direct peptide injection. For example, patients newly diagnosed with metastatic melanoma have been shown to be immunized with autologous peptides using CD40L / IFN-γ-activated mature DCs pulsed with three HLA-A*0201-restricted gp100 melanoma antigen-derived peptides using an IL-12p70-producing patient DC vaccine (see, e.g., Carreno et al (2013) "L-12p70-producing patient DC vaccine elicits Tc1-polarized immunity", Journal of Clinical Investigation, 123(8): 3383-94 and Ali et al. (2009) "In situ regulation of DC subsets and T cells mediates tumor regression in mice", Cancer Immunotherapy, 1(8): 1-10). It is contemplated that within the scope of the present invention, neoantigen-loaded DCs can be prepared by stimulating DCs using the synthetic TLR3 agonist polyinosinic·polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly ICLC). Poly ICLC is a potent individual maturation stimulus for human DCs as evaluated by upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon γ-induced protein 10 (IP-10), interleukin 1 (IL-1), and type I interferon (IFN), and minimal interleukin 10 (IL-10) production. DCs can be distinguished from cryopreserved peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis, which can be isolated by Ficoll gradient centrifugation and frozen in aliquots.

[0281] As an example, the following 7-day activation protocol can be used. On day 1, thaw PBMCs and plate them in a tissue culture flask. Incubate at 37°C for 1 - 2 hours in a tissue culture incubator, and then select monocytes that adhere to the plastic surface. After the incubation, wash away the lymphocytes and culture the adhered monocytes for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage colony-stimulating factor (GM-CSF) to differentiate them into immature DCs. On day 6, pulse the immature DCs with keyhole limpet hemocyanin (KLH) protein, which serves as a control regarding the quality of the vaccine and can boost the immunogenicity of the vaccine. DCs mature upon stimulation and are loaded with peptide antigens and incubated overnight. On day 7, wash the cells and freeze them using a rate-controlled freezer in 1 ml aliquots containing 4 - 20 × 10 cells. Before injecting the DCs into the patient, a lot release test for the DC batch can be performed to meet minimal specifications (see, for example, Sabado et al. (2013) "Preparation of tumor antigen-loaded mature dendritic cells for immunotherapy", J. Vis Exp. Aug 1; (78). doi: 10.3791 / 50085). 6

[0282] ​A DC vaccine can be incorporated into a scaffold system to facilitate delivery to a patient. Treatment of a patient's neoplasm with a DC vaccine differentiates resident immature DCs by releasing factors that mobilize host dendritic cells and locally providing an adjuvant (e.g., danger signal) while the antigen is released, and promotes the release of activated antigen-loaded DCs to lymph nodes (or desired site of action), where the DCs can interact with T cells to generate a potent cytotoxic T lymphocyte response against cancer neoantigens, and a biomaterial system can be utilized. An implantable biomaterial may be used to specifically generate a potent cytotoxic T lymphocyte response against a neoplasm in a patient. These biomaterial resident dendritic cells can then be activated by their exposure to danger signals that mimic infection in accordance with the release of antigen from the biomaterial. The activated dendritic cells then migrate from the biomaterial to the lymph nodes and induce a cytotoxic T effector response. This approach has previously been demonstrated to cause regression of established melanoma in preclinical studies using lysates prepared from tumor biopsies (e.g., Ali et al. (2209) "D C subsets and in situ regulation of T cells mediate tumor regression in mice", Cancer Immunotherapy 1(8): 1-10; Ali et al. (2009) "Infection-mimicking materials to program dendritic cells in situ in situ)”. Nat Mater 8: 151-8), such vaccines are currently being tested in a Phase I clinical trial recently initiated at the Dana-Farber Cancer Institute. This approach has also been shown to result in glioblastoma regression and the induction of a potent memory response that prevents relapse, using a C6 rat glioma model. 24. In the current proposal, the ability of such implantable biomatrix vaccine delivery scaffolds to amplify and maintain tumor-specific dendritic cell activation may result in a more robust anti-tumor immune priming compared to what can be achieved by conventional subcutaneous or intranodal vaccine administration.

[0283] In the practice of the present invention, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology are used, which are well within the scope of those skilled in the art. Such techniques are described in detail in references such as “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Wei, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the present invention and can thus be considered in the preparation and practice of the present invention. Techniques particularly useful for detailed embodiments are discussed in the next section.

Example

[0284] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the assays, screening, and treatment methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0285] Example 1: Cancer Vaccine Test Protocol The above-described compositions and methods can be tested in 15 patients with high-risk melanoma (complete resection stages IIIB, IIIC, and IVM1a,b) according to the general flow process shown in FIG. 2. Patients can receive a series of priming vaccinations with a mixture of individualized tumor-specific peptides and poly-ICLC over a 4-week period, followed by two boosts during the maintenance period. All vaccinations can be delivered subcutaneously. The vaccine can be evaluated with respect to safety, tolerability, immune response, and clinical effect in patients, as well as the feasibility of vaccine production and initiation of vaccination within an appropriate time frame. The first cohort consists of 5 patients, and after sufficient safety has been demonstrated, an additional cohort of 10 patients can be enrolled (see, for example, FIG. 3 showing the methodology of a first population study). Peripheral blood is extensively monitored for peptide-specific T cell responses, and patients can be followed for up to 2 years to assess disease recurrence.

[0286] As described above, in both animals and humans, there is substantial evidence that mutant epitopes are effective in inducing an immune response and that cases of spontaneous tumor regression or long-term survival correlate with CD8 + T cell responses to mutant epitopes (Buckwalter and Srivastava PK. “It's an Antigen, Darn It” and Vaccine Therapy of Human Cancer "It is the antigen(s), stupid" and other lessons from over a decade of vaccitherapy of human cancer). Seminars in immunology 20: 296-300 (2008); Karanikas et al, "High frequency of cytolytic T lymphocytes specific for tumor-specific mutated antigens detectable by HLA tetramers in the blood of long-term surviving lung cancer patients." Cancer Res. 61: 3718-3724 (2001); Lennerz et al, "The response of autologous T cells to a human melanoma is dominated by mutated neo-antigens." Proc Natl Acad Sci USA. 102: 16013 (2005)) and the ability to track "immune editing" in response to alterations in the expression of dominant mutated antigens in mice and humans (Matsushita et al, "Cancer exome analysis reveals a T-cell-dependent mechanism of cancer immunoediting 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).

[0287] Next-generation sequencing can now rapidly reveal the presence of individual mutations in each tumor, most commonly single amino acid changes (e.g., missense mutations; Fig. 4A) and, less frequently, frameshift insertions / deletions / gene fusions, read-through mutations at stop codons, and novel stretches of amino acids generated by the translation of inappropriately spliced introns (e.g., neoORFs; Fig. 4B). NeoORFs are particularly beneficial as immunogens because their entire sequence is completely novel to the immune system and thus resembles viral or bacterial foreign antigens. Thus, neoORFs are: (1) highly specific to the tumor (i.e., not expressed in any normal cells); (2) able to bypass central tolerance, thereby increasing the precursor frequency of neoantigen-specific CTLs. For example, recently, the ability to utilize similar foreign sequences in therapeutic anticancer vaccines with peptides derived from human papillomavirus (HPV) has been demonstrated It has been achieved. Approximately 50% of 19 patients with pre-neoplastic viral-induced diseases who received 3 to 4 vaccinations with a mixture of HPV peptides derived from the viral oncogenes E6 and E7 maintained a complete remission for over 24 months (Kenter et al, "Vaccination against HPV-16 Oncoproteins for Vulvar Intraepithelial Neoplasia," NEJM 361: 1838 (2009)). V-16 Oncoproteins for Vulvar Intraepithelial Neoplasia)」NEJM 361: 1838 (2009)).

[0288] Sequencing techniques have revealed that each tumor contains multiple patient-specific mutations that alter the protein-coding content of the gene. Such mutations can create modified proteins that range from single amino acid changes (caused by missense mutations) to the addition of long regions of novel amino acid sequences resulting from frameshifts, readthrough of stop codons, or translation of intron regions (novel open reading frame mutations; neoORF). These mutant proteins, unlike the native proteins, are not subject to the immunosuppressive effects of self-tolerance and are thus useful targets for the host immune response against tumors. Therefore, mutant proteins are even more likely to be immunogenic and are also more specific for tumor cells compared to the patient's normal cells.

[0289] Using recently improved algorithms that predict which missense mutations give rise to strong binding peptides to the patient's cognate MHC molecules, a set of peptides representing the optimal mutant epitopes (both neoORF and missense) for each patient can be identified, prioritized, and up to 20 or more peptides can be prepared for immunization (Zhang et al, "Machine learning competition in immunology - prediction of HLA class I-binding peptides"). "Competition in immunology - Prediction of HLA class I binding peptides)" J Immunol Methods 374: 1 (2011); Lundegaard et al, "Prediction of epitopes using neural network based methods" J Immunol Methods 374: 26 (2011)). Peptides approximately 20 - 35 amino acids in length are synthesized because such "long" peptides have been shown to undergo efficient internalization, processing and cross - presentation in professional antigen - presenting cells such as dendritic cells and to induce CTLs in humans (Melief and van der Burg, "Immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines" Nature Rev Cancer 8: 351 (2008)). In addition to a potent and specific immunogen, an effective immune response requires a powerful adjuvant to activate the immune system (Speiser and Romero, "Molecularly defined vaccines for cancer immunotherapy, and protective T cell immunity" Seminars in Immunol 22: 144 (2010)) ).

[0290] ​​. For example, Toll-like receptors (TLRs) have emerged as powerful sensors of microbial and viral pathogen "danger signals" that effectively induce the innate immune system and then the adaptive immune system (Bhardwaj and Gnjatic, "TLR Agonists: Are They Good Adjuvants?" Cancer J. 16: 382-391 (2010)). Among TLR agonists, poly ICLC (a synthetic double-stranded RNA mimic) is one of the most potent activators of bone marrow-derived dendritic cells. In human volunteer trials, poly ICLC has been shown to be safe and to induce a gene expression profile equivalent to that induced by one of the most potent live attenuated viral vaccines, yellow fever vaccine YF-17D, in peripheral blood cells (Caskey et al, "Synthetic double-stranded RNA induces innate immune responses similar to a live viral vaccine in humans" J Exp Med 208: 2357 (2011)). Hilbertol (registered trademark), a GMP formulation of poly ICLC prepared by Oncovir, Inc, can be utilized as an adjuvant. AGONISTS: Are They Good Adjuvants?)」Cancer J. 16: 382-391 (2010))。TLR a mong agonists, poly ICLC (a synthetic double-stranded RNA mimic) is one of the most potent activators of bone marrow-derived dendritic cells. In human volunteer trials, poly ICLC has been shown to be safe and to induce a gene expression profile equivalent to that induced by one of the most potent live attenuated viral vaccines, yellow fever vaccine YF-17D, in peripheral blood cells (Caskey et al, "Synthetic double-stranded RNA induces innate immune responses similar to a live viral vaccine in humans" J Exp Med 208: 2357 (2011)). Oncovir, Inc has prepared a GMP formulation of poly ICLC called Hil bertol (registered trademark) that can be used as an adjuvant. tonol(registered trademark) can be used as an adjuvant.

[0291] Example 2: Target Patient Population Patients with stage IIIB, IIIC and IVM1a,b melanoma have a significantly high risk of disease recurrence and death even if the disease is completely surgically resected ((Balch et al, "Final Version of 2009 AJCC Melanoma Staging and Classification" J Clin Oncol 27: 6199-6206 (2009) The only systemic adjuvant therapy available to this patient population is interferon-α (IFNα), which offers measurable but marginal benefit and is associated with significant, often dose-limiting, toxicity (Kirkwood et al., "Interferon-α in High-Risk Excised Cutaneous Melanoma"). Adjuvant serum alpha-2b: Eastern Cooperative Oncology Group trial EST 1684 (Interferon alfa-2b Adjuvant Therapy of High-Risk Resected Cutaneous Melanoma: The Eastern Cooperative Oncology Group Trial EST 1684) J Clin Oncol 14: 7-17 (1996); Kirkwood et al, "High- and Low-dose Interferon Alpha-2b in High-Risk Melanoma: First Analysis of Intergroup Trial E1690 / S9111 / C9190" Trial E1690 / S9111 / C9190) J Clin Oncol 18: 2444-2458 (2000). These patients These patients are not immunocompromised, either from previous cancer-targeted therapies or from active cancer, and therefore represent an excellent patient population in which to evaluate the safety and immunological impact of a vaccine. Finally, the current standard of care for these patients does not mandate any treatment after surgery, allowing an 8-10 week window for a vaccine formulation.

[0292] The target population can be patients with cutaneous melanoma with clinically detectable, histologically confirmed lymph nodes (local or distant) or in-transit metastases that have been completely resected and are disease-free (many of stage IIIB (patients with ulcerated primary tumors with micrometastatic lymph nodes (T1-4b, N1a or N2a) may be excluded as sufficient tumor tissue for sequencing and cell line generation is required), all of stage IIIC, and stage IVM1a, b). These can be patients with a first diagnosis of early-stage melanoma or patients with disease recurrence after a previous diagnosis.

[0293] Tumor excision: For the purpose of rendering the patient disease-free of melanoma, the patient may undergo complete excision of their primary melanoma (if not already removed) and all local metastatic disease. After sufficient tumor has been excised for pathological evaluation, the remaining tumor tissue is placed in a sterile medium in a sterile container and prepared for disaggregation. A portion of the tumor tissue is used for whole exome and transcriptome sequencing and cell line generation, and the remaining tumor may be frozen.

[0294] Normal tissue collection: Normal tissue samples (blood or sputum samples) may be collected for whole exome sequencing. Patients with clinically evident regional metastatic disease or completely resected distant lymph node, skin, or lung metastatic disease (but no unresectable distant or visceral metastatic disease) may be identified and enrolled in the study. Enrollment of patients prior to surgery is required to obtain fresh tumor tissue for melanoma cell line establishment (thereby generating target cells for in vitro cytotoxicity assays as part of the immune monitoring program).

[0295] Example 3: Dosage and Schedule For all patients who meet all pre-treatment criteria, vaccination may be initiated as soon as possible after the study drug has arrived and meets acceptance criteria. There are four separate study drugs for each patient, each of which may contain 5 out of 20 patient-specific peptides. Immunization may generally proceed according to the schedule shown in Figure 5.

[0296] Patients can be treated in the outpatient department. Immunization on each treatment day consists of four 1-ml subcutaneous injections, and each injection can be given in a separate limb in order to target different regions of the lymphatic system and reduce antigen competition. If the patient has undergone complete axillary or inguinal lymph node dissection, the vaccine can be administered as an alternative to the right or left diaphragm. Each injection consists of one of the four test drugs for the patient, and the same test drug can be injected into the same limb for each cycle. The composition of each 1-ml injection is as follows: 0.75 ml of test drug containing 300 μg each of five patient-specific peptides 0.25 ml (0.5 mg) of 2 mg / ml poly ICLC (Hiltonol®) During the induction / priming phase, patients can be immunized on days 1, 4, 8, 15, and 22. During the maintenance phase, patients can receive booster doses at weeks 12 and 24.

[0297] Blood samples can be taken at multiple time points: before (baseline; two samples on different days); day 15 during priming vaccination; 4 weeks (week 8) after induction / priming vaccination; before (week 12) and after (week 16) the first boost; before (week 24) and after (week 28) the second boost. For each sample, 50 - 150 ml of blood can be taken (except at week 16). The primary immunological endpoint is at week 16, and thus patients can undergo leukapheresis (unless specifically instructed otherwise based on the evaluation of the patient and the physician).

[0298] Example 4: Immune Monitoring The immune strategy is a "prime-boost" approach that includes an initial series of closely spaced immunizations to induce an immune response, followed by a rest period to establish memory T cells. Booster immunizations follow this, and the T cell response 4 weeks after this boost is expected to yield the strongest response and can be a primary immunological endpoint. Initially, a global immune response can be monitored by stimulating peripheral blood mononuclear cells with a pool of overlapping 15mer peptides (11aa overlap) containing all immune epitopes in an ex vivo ELISPOT assay for 18 hours starting from this point. Baseline responses to this peptide pool can be established by evaluating pre-vaccination samples. Additional PBMC samples can be evaluated as needed to examine the kinetics of the immune response to the entire peptide mixture. For patients showing responses significantly above baseline, the pool of all 15mers can be deconvoluted to determine which specific immune peptides were immunogenic. Additionally, several further assays can be performed on an individual basis for appropriate samples: · Use the pool of all 15mers or sub-pools as stimulating peptides in an intracellular cytokine staining assay to identify and quantify antigen-specific CD4 + , CD8 + , central memory, and effector memory populations · Similarly, use these pools to evaluate the pattern of cytokines secreted by these cells and determine the T H 1 to T H 2 phenotypes · Use extracellular cytokine staining and flow cytometry of unstimulated cells to quantify Tregs and myeloid-derived suppressor cells (MDSCs) · If successful in establishing a melanoma cell line from responding patients and identifying activating epitopes, a cytotoxicity assay of T cells can be performed using mutant peptides and corresponding wild-type peptides PBMC of the primary immunological endpoint can be evaluated for "epitope spreading" as shown in Figure 6 by using known melanoma tumor-associated antigens as stimulants and including some additional identified mutated epitopes that were not selected among the immunogens. Perform immunohistochemistry on tumor samples to quantify CD4 + , CD8 + , MDSC, and Treg infiltrating populations.

[0299] Example 5: Clinical Efficacy in Patients with Metastatic Disease Vaccine treatment of metastatic disease patients is complicated by the need for an effective treatment for active cancer and the resulting lack of a treatment break window for vaccine preparation. Furthermore, these cancer treatments can impair the patient's immune system and may in some cases prevent the induction of an immune response. With these considerations in mind, the timing of vaccine preparation can be selected in settings where it is compatible with other standard treatment approaches for a specific patient population in terms of time and / or in settings where such standard treatments can be reliably combined with immunotherapy approaches. There are two types of settings that can be explored: 1. Combination with checkpoint blockade: Checkpoint blockade antibodies have emerged as effective immunotherapies for metastatic melanoma (Hodi et al, "Improved Survival with Ipilimumab in Patients with Metastatic Melanoma)" NEJM 363: 711-723 (2010)), non-small cell lung cancer (NSCLC), and renal cell carcinoma (Topalian et al, "Safety, Activity, and Immune Correlates of Anti-PD-1 Antibody in Cancer )」In other disease settings, including Brahmer et al, 「Safety and Activity of Anti-PD-L1 Antibody in Patients with Advanced Cancer」 NEJM 366: 2455-2465 (2012); 「Anti-PD-L1 Antibody and Multicytokine Therapy in Advanced Melanoma」 NEJM 366: 2443-2454 (2012), it has been actively explored. Although the mechanism of action has not been clarified, both the reversal of local immunosuppression and the enhancement of the immune response are possible explanations. When a potent vaccine is integrated to induce an immune response with checkpoint blockade antibodies, synergy can be brought about, as observed in multiple animal studies (van Elsas et al 「Combined immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and granulocyte / macrophage colony-stimulating factor (GM-CSF)-producing vaccine induces rejection of subcutaneous and metastatic tumors with autoimmune depigmentation」 J Exp Med 190: 35-366 (1999); Li et al, It has been actively explored in other disease settings. Although the mechanism of action has not been clarified, both the reversal of local immunosuppression and the enhancement of the immune response are possible explanations. When a potent vaccine is integrated to induce an immune response with checkpoint blockade antibodies, synergy can be brought about, as observed in multiple animal studies (van Elsas et al 「Combined immunotherapy of B16 melanoma using anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) and granulocyte / macrophage colony-stimulating factor (GM-CSF)-producing vaccine induces rejection of subcutaneous and metastatic tumors with autoimmune depigmentation」 J Exp Med 190: 35-366 (1999); Li et al, 「Anti-programmed death-1 synergizes with granulocyte macrophage colony-stimulating factor-secreting tumor cell immunotherapy providing therapeutic benefit to mice with established tumors」 Clin Cancer Res 15: 1623-1634 (2009); Pardoll, D. M. 「The blockade of immune checkpoints in cancer immunotherapy」 Nature Reviews Cancer 12: 252-264 (2012); Curran et al. 「PD-1 and the blockade of immune checkpoints in cancer immunotherapy」 Nature Reviews Cancer 12: 252-264 (2012); Curran et al. 「PD-1 and "PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors". Proc Natl Acad Sci USA. 2010 Mar 2; 107(9): 4275-80; Curran et al.「 Tumor vaccines expressing flt3 ligand synergize with ctla-4 blockade to reject preimplanted tumors". Cancer Res. 2009 Oct 1; 69(19): 7747-55). As shown in Figure 7, patients can immediately start checkpoint blockade therapy while the vaccine is being prepared, and after preparation, vaccine administration is integrated with antibody therapy; and 2. Combination with standard treatment regimens that exhibit beneficial immune properties 2. Combination with standard treatment regimens that exhibit beneficial immune properties a) Patients with renal cell carcinoma (RCC) presenting with metastatic disease typically undergo surgical debulking, followed generally by systemic treatment with one of the approved tyrosine kinase inhibitors (TKIs), such as sunitinib, pazopanib, and sorafenib. Among the approved TKIs, sunitinib has been shown to increase TH1 responsiveness and decrease Tregs and myeloid-derived suppressor cells (Finke et al, "Sunitinib reverses Type-1 immune suppression and decreases T-regulatory cells in renal cell carcinoma patients," Clin Can Res 14: 6674-6682 (2008); Terme et al, "VEGFA-VEGFR pathway blockade inhibits tumor-induced regulatory T cell proliferation in colorectal cancer," (Cancer Research Author Manuscript published Online (2102)). By enabling immediate treatment of patients with an approved therapeutic agent that does not compromise the immune system, a window is provided for the preparation of the vaccine, and a synergistic effect with the vaccine therapeutic agent can be achieved. In addition, cyclophosphamide (CTX) has been shown to have an inhibitory effect on Treg cells in multiple animal and human studies, and more recently, a single dose of CTX prior to vaccination has been shown to improve the survival of RCC patients who responded to the vaccine (Walter et al, "Single-dose cyclo phosphamide enhances the efficacy of a therapeutic cancer vaccine in patients with metastatic renal cell carcinoma," J Clin Oncol 30: 1943-1949 (2012)). Since patients can be immediately treated with an approved therapeutic agent that does not compromise the immune system, a window is provided for the preparation of the vaccine, and a synergistic effect with the vaccine therapeutic agent can be achieved. In addition, cyclophosphamide (CTX) has been shown to have an inhibitory effect on Treg cells in multiple animal and human studies, and more recently, a single dose of CTX prior to vaccination has been shown to improve the survival of RCC patients who responded to the vaccine (Walter et al, "Single-dose cyclo Multipeptide immune responses to the cancer vaccine IMA901 after phosphamide are associated with longer patient survival (Nature Medicine 18: 1254-1260 (2012)). Both of these immune potentiation approaches are being utilized in a recently completed Phase 3 trial of a native peptide vaccine in RCC (Clinical Trials.gov, NCT01265901 IMA901 in patients receiving sunitinib for advanced / metastatic renal cell carcinoma)); Patients Receiving Sunitinib for Advanced / Metastatic Renal Cell Carcinoma)); b) Alternatively, standard treatment for glioblastoma (GBM) involves surgery, recovery and follow-up radiation and low-dose temozolomide (TMZ), followed by the initiation of standard-dose TMZ after a 4-week rest period. This standard treatment provides a window for vaccine preparation, followed by the initiation of vaccination and then the initiation of standard-dose TMZ. Interestingly, in a study of metastatic melanoma, vaccination with a peptide vaccine during standard-dose TMZ treatment showed increased immunoresponsiveness as measured compared to vaccination alone, suggesting an additional synergistic benefit (Kyte et al, "Telomerase peptide vaccination combined with temozolomide: A clinical trial in stage IV melanoma patients" Clin Cancer Res 17: 4568 (2011)).

[0300] Example 6: Vaccine Preparation The patient's tumor tissue can be surgically resected, the tumor tissue can be dissociated, and a part used for DNA and RNA extraction and the establishment of patient-specific melanoma cell lines can be isolated. Whole exome sequencing (for example, by using the Illumina HiSeq platform) is performed using the DNA and / or RNA extracted from the tumor tissue, and HLA typing information can be determined. Within the scope of the present invention, it is contemplated that missense or neo ORF neoantigen peptides can be directly identified by protein-based techniques (for example, mass spectrometry).

[0301] Bioinformatics analysis can be performed as follows. Sequence analysis of exome and RNA-SEQ fastQ files can utilize existing bioinformatics pipelines that have been widely used and validated in large-scale projects such as TCGA for many patient samples (for example, Chapman et al, 2011; Stransky et al, 2011; Berger et al, 2012). There are two consecutive analysis categories: data processing and cancer genome analysis.

[0302] Data processing pipeline: The Picard data processing pipeline (picard.sourceforge.net / ) was developed by the sequencing platform. Raw data extracted from the sequencer for each tumor and normal sample (for example, Illumina) is subjected to the following processing using various modules of the Picard pipeline: (i) Quality recalibration: The original base quality scores reported by the Illumina pipeline can be recalibrated based on the read cycle, lane, flow cell tile, base in question, and preceding bases.

[0303] (ii) Alignment: Read pairs can be aligned to the human genome (hg19) using BWA (Li and Durbin, 2009). ​(iii) Duplicate marking: PCR and optical duplicates can be identified based on read pair mapping positions and marked in the final bam file.

[0304] The output of Picard is a bam file (Li et al, 2009) (samtools. sourceforge.net / SAM1.pdf), which stores the nucleotide sequences, quality scores, and alignment details of all reads for a given sample.

[0305] Cancer mutation detection pipeline: The tumor bam file and the corresponding normal bam file from the Picard pipeline can be analyzed as follows: 1. Quality Control (i) By comparing the initial SNP fingerprinting performed at dozens of sites for the sample with the pileup of exome sequencing at those sites, sample mix-ups during sequencing can be detected.

[0306] (ii) By first comparing the insert size distributions of the lanes corresponding to the same library for both the tumor and normal samples, and discarding lanes with different distributions, tumor / normal mix-ups within the sample can be identified. Applying bioinformatics analysis to the tumor and corresponding normal exome samples can yield DNA copy number profiles. Tumor samples should also have more copy number variations compared to the corresponding normal samples. Discard lanes corresponding to normal samples with flat profiles, which is similar to discarding tumor lanes that do not match the profiles of other lanes from the same tumor sample.

[0307] (iii) Tumor purity and ploidy can be estimated based on the copy number profiles created by bioinformatics. (iv) Use ContEst (Cibulskis et al, 2011) to detect cross-sample contamination of the sample The level can be determined.

[0308] 2. Local Realignment around the Putative Indel True somatic and germline indels for the reference genome often result in misalignments as well as missense mutations and miscalls of indels. This can be corrected by using the GATK IndelRealigner module (available at (www)b roadinstitute.org / gatk) (McKenna et al, 2010; Depristo et al, 2011) to perform local realignment of all reads mapped around the putative indel and comprehensively evaluate them to ensure the consistency and correctness of indel calls.

[0309] 3. Identification of Somatic Single - Nucleotide Variations (SSNVs) Somatic base pair substitutions can be identified by analyzing a patient's tumor and corresponding normal sample using a Bayesian statistical framework called MuTect (Cibulskis et al, 2013). In the preprocessing step, the overwhelming majority of low-quality bases or reads with mismatches to the genome are filtered out. Next, Mutect calculates two log odds (LOD) scores, which encapsulate the confidence in the presence and absence of variants in the tumor sample and normal sample, respectively. In the post-processing step, candidate mutations are experimentally filtered by various criteria to account for artifacts of capture, sequencing, and alignment. For example, one such filter tests the agreement between the distribution of the orientations of reads with a mutation and the overall distribution of the orientations of reads mapped to the locus to ensure no strand bias. The final set of mutations is then annotated by the Oncotator tool with several fields including genomic region, codon, cDNA, and protein changes.

[0310] 4. Identification of Somatic Small Insertions and Deletions Using the local realignment output from Section 2.2, candidate somatic and germline indels can be predicted based on the evaluation of reads supporting variants in tumor bam alone or in both tumor and normal bams. Further filtering can be performed based on the number and distribution of mismatches and base quality scores (McKenna et al, 2010, DePristo et al, 2011). All indels can be manually inspected using the Integrated Genomics Vi ewer (Robinson et al, 2011) (available at (www)broadinstitute.org / igv on the World Wide Web) to ensure high-fidelity calls.

[0311] 5. Gene Fusion Detection The first step of the gene fusion detection pipeline is the alignment of tumor RNA-Seq reads against a library of known gene sequences and the subsequent mapping of this alignment to genomic coordinates. Genomic mapping helps to collapse multiple read pairs that map to different transcript variants sharing exons to a common genomic position. The bam file with aligned DNA can be queried for read pairs where the two mates map to two different coding regions that are either on different chromosomes or, if on the same chromosome, at least 1MB apart. It may also be required that the paired ends aligned in each gene are in the orientation that matches the coding --> coding 5’->3’ orientation of the (putative) fusion mRNA transcript. A list of gene pairs with at least two such "chimeric" read pairs can be enumerated as the initial list of putative events for further refinement. Next, all unaligned reads can be extracted from the original bam file with the constraint that their mate was initially aligned, and mapped to one of the genes of the gene pairs obtained as described above. Next, an attempt can be made to align all such reads that were not initially aligned to a custom "reference" made at all possible exon-exon junctions (full length, from boundary to boundary, coding 5’->3’ orientation) between the discovered gene pairs. If one of such reads that was not initially aligned is (uniquely) mapped to the junction between an exon of gene X and an exon of gene Y and its mate is actually mapped to one of genes X or Y, such a read can be marked as a "fusion" read. A gene fusion event can be called if there are no excessive number of mismatches around the exon:exon junction and there is at least one fusion read with correct relative orientation to its mate with at least 10bp coverage in either gene. Gene fusions between highly homologous genes (e.g., HLA family) are likely to be errors and can be removed by filtering.

[0312] 6. Estimation of Clonality The clonality of mutations can be estimated using bioinformatics analysis. For example, the ABSOLUTE algorithm (Carter et al, 2012; Landau et al, 2013) can be used to estimate tumor purity, ploidy, absolute copy number, and the clonality of mutations. The probability density distribution of the allele frequency of each mutation can be created and subsequently converted to the cancer cell fraction (CCF) of the mutation. Mutations can be classified as clonal or subclonal based on whether the posterior probability that their CCF exceeds 0.95 is greater than or less than 0.5, respectively.

[0313] 7. Quantification of Expression The TopHat suite (Langmead et al, 2009) can be used to align RNA-Seq reads of tumor bam and corresponding normal bam to the hg19 genome. The quality of the RNA-Seq data can be evaluated by the RNA-SeQC (DeLuca et al, 2012) package. Next, the RSEM tool (Li et al, 2011) can be used to estimate gene and isoform expression levels. The reads per million mapped reads per kilobase and τ estimated values can be used to rank the neoantigens identified in each patient as described in other sections. can be prioritized.

[0314] 8. Verification of Mutations in RNA - Seq The mutations identified by the analysis of whole exome data (Section 2.3) can be evaluated for their presence in the corresponding RNA-Seq tumor bam file of the patient. For each mutant locus, a power calculation based on the beta-binomial distribution can be performed to ensure that there is at least 80% power to detect it in the RNA-Seq data. Mutations identified by capture can be considered verified if there are at least two reads with the mutation at a site with appropriate power.

[0315] Selection of tumor-specific mutation-containing epitopes: Using the neural network-based algorithm netMHC, provided and maintained by the Center for Biological Sequence Analysis, Technical University of Denmark, Netherlands, all missense mutations and neoORFs can be analyzed for the presence of mutation-containing epitopes. This group of algorithms has been evaluated as the top epitope prediction algorithms based on a competition recently completed among a series of related methods (reference). For multiple different human HLA A and B alleles, the algorithm was trained using a neural network-based approach utilizing over 100,000 measured binding and non-binding interactions. The accuracy of the algorithm was evaluated by performing predictions from mutations found in CLL patients with known HLA allotypes. The included allotypes were A0101, A0201, A0310, A1101, A2402, A6801, B0702, B0801, B1501. Predictions were made using netMHCpan in mid-2011 for all 9mer and 10mer peptides across each mutation. Based on these predictions, 74 9mer peptides and 63 10mer peptides (most having predicted affinities less than 500 nM) were synthesized and binding affinities were measured using a competitive binding assay (Sette).

[0316] The predictions of these peptides were repeated in March 2013 using each of the latest versions of the netMHC servers (netMHCpan, netMHC, and netMHCcons). These three algorithms were the top algorithms among the 20 groups used in the 2012 competition (Zhang et al). Next, the measured binding affinities were evaluated for each of the new predictions. For each set of predicted and measured values, the % of correct predictions for each range, as well as the number of samples, were obtained. The definition of each range is as follows: The measured binding affinities were evaluated for each of the new predictions. For each set of predicted and measured values, the % of correct predictions for each range, as well as the number of samples, were obtained. The definition of each range is as follows:

[0317] The predictions of these peptides were repeated in March 2013 using each of the latest versions of the netMHC servers (netMHCpan, netMHC, and netMHCcons). These three algorithms were the top algorithms among the 20 groups used in the 2012 competition (Zhang et al). Next, for each of the new predictions, the measured binding affinities were evaluated. For each set of predicted and measured values, the % of correct predictions for each range, as well as the number of samples, were obtained. The definition of each range is as follows: For each set of predicted and measured values, the % of correct predictions for each range, as well as the number of samples, were obtained. The definition of each range is as follows: It is predicted to have an affinity of 0 - 150: below 150 nM, and it is measured to have an affinity of 150 nM or less.

[0318] 0 - 150 * : It is predicted to have an affinity of 150 nM or less, and it is measured to have an affinity of 500 nM or less. 151 - 500 nM: It is predicted to have an affinity higher than 150 nM but 500 nM or less, and it is measured to have an affinity of 500 nM or less.

[0319] FN(>500 nM): False negative - It is predicted to have an affinity higher than 500 nM, but it is measured to have an affinity of 500 nM or less. For the 9 - mer peptides (Table 1), there was little difference between the algorithms, and the 151 - 500 nM range of netMHC cons was slightly higher, but due to the small number of samples it was judged not to be significant.

[0320]

Table 1

[0321] Similarly for the 10 - mer peptides (Table 2), there was little difference between the algorithms, but netMHC produced significantly more false positives compared to netMHCpan or netMMHCcons. However, the 10 - mer prediction accuracy was slightly lower in the 0 - 150 nM and 0 - 150 * nM ranges and significantly lower in the 151 - 500 nM range compared to the 9 - mer.

[0322]

Table 2

[0323] For the 10 - mer, in the 151 - 500 nM range, the accuracy regarding the conjugate is less than 50%, so only the prediction in the 0 - 150 nM range can be used. The number of samples for any individual HLA allele was too small to draw any conclusions regarding the accuracy of the prediction algorithms for the various alleles. The data for the largest available subset (0-150 * nM; 9mer) are shown in Table 3 as an example.

[0324]

Table 3

[0325] For HLA C alleles, there is little available data to judge the accuracy of prediction, so only the predictions of HLA A and B alleles can be used (Zhang et al). The evaluation of melanoma sequence information and peptide binding prediction was performed using information from the TCGA database. Information from 220 melanoma samples from various patients revealed that on average there were approximately 450 missense and 5 neo-ORFs per patient. Twenty patients were randomly selected and the predicted binding affinities of all missense mutations were calculated using netMHC (Lundegaard et al "Prediction of epitopes using neural network based methods" ​」(J Immunol Methods 374: 26 (2011)). Since the HLA allotypes of these patients were unknown, the number of predicted binding peptides per allotype was adjusted based on the frequency of the respective allotype (bone marrow registry dataset of the expected disease-predisposed population [Caucasians for melanoma] in the geographical area), and the number of predicted actionable mutant epitopes per patient was determined. For each of these mutant epitopes (MUT), the corresponding native (WT) epitope binding was also predicted. Using single peptides of predicted missense conjugates with Kd ≤ 500 nM and a WT / MUT Kd ratio greater than 5-fold, and overlapping peptides spanning the full length of each neoORF, 80% (16 out of 20) of the patients were predicted to have at least 20 peptides suitable for vaccination. One quarter of the patients could have neoORF peptides constituting about half to all of the 20 peptides. Thus, melanoma has a mutation load sufficient to expect that a high proportion of patients generate a sufficient number of immunogenic peptides.

[0326] Example 7: Prioritization of Immunopeptides Peptides for immunization can be prioritized based on several criteria: neoORF vs. missense, predicted Kd of the mutant peptide, comparability of the predicted affinity of the native peptide compared to the mutant peptide, whether the mutation occurs in a cancer driver gene or in a related pathway, and RNA-Seq read counts (see, e.g., Figure 8).

[0327] As shown in Figure 8, peptides derived from segments of neoORF mutations predicted to bind (Kd < 500 nM) can be given the highest priority based on the lack of tolerance to these completely novel sequences and their excellent tumor specificity.

[0328] The same class of missense mutations predicted to not bind the native peptide (Kd > 1000 nM) and to bind the mutant peptide with strong / moderate affinity (Kd < 150 nM) can be given the next highest priority. This class (Group I considered above) corresponds to approximately 20% of the T cell responses observed in nature.

[0329] The third highest priority can be given to the subset of the Group II class that binds more strongly (< 150 nM) considered above. This class accounts for approximately two-thirds of the T cell responses observed in nature.

[0330] All of the remaining peptides derived from neo-ORF mutations can be given the fourth priority. Despite being predicted not to bind, these are included based on the known false negative rate, potential binding to HLA-C, potential for the presence of class II epitopes, and high value of utilizing completely foreign antigens.

[0331] The fifth priority can be given to the subset of Group II with low predicted binding affinity (150 - 500 nM). This class accounts for approximately 10% of the T cell responses observed in nature.

[0332] As the predicted affinity decreases, a higher stringency can be applied to the expression level. Within each grouping, peptides can be ranked based on their binding affinity (e.g., the lowest Kd can have the highest priority). Within a given grouping of missense mutations, a higher priority can be assigned to cancer driver mutations. A library of approximately 12.6 million unique 9- and 10-mer normal human peptidomes curated from all known human protein sequences (HG19) has been created. Prior to the final selection, missense mutations and any potential predicted epitopes derived from all neoORF regions can be screened against this library, and exact matches can be excluded. As discussed below, specific peptides predicted to have deleterious biochemical properties can be removed or modified.

[0333] According to the techniques herein, the RNA level can be analyzed to assess neoantigen expression. For example, RNA-Seq read counts can be used as a proxy to estimate neoantigen expression. However, currently, there is no available information to evaluate the minimum required RNA expression level needed to induce cell lysis in tumor cells. Even the expression level from the "pioneer" translation of messages destined for nonsense-mediated decay may be sufficient for target generation. Thus, the techniques herein first set a wide tolerance limit for the RNA level that can vary inversely with the priority group. As the predicted affinity decreases, a higher stringency can be applied to the expression level. Those skilled in the art will understand that such limits can be adjusted as additional information becomes available.

[0334] Due to their novelty and excellent tumor specificity, neo-ORFs are highly valuable as targets, and thus can be utilized even when no mRNA molecules detectable by RNA-Seq are present (rank 1) if they have predicted binding epitopes (Kd ≤ 500 nM). Regions of neo-ORFs without predicted binding epitopes (>500 nM) can generally be utilized only if a certain level of RNA expression is detected (rank 4). All missense mutations with strong to moderate predicted MHC binding affinities (≤150 nM) can generally be utilized, except when there are no RNA-Seq reads (ranks 2 and 3). Missense mutations with lower predicted binding affinities (150 - ≤500 nM) are likely to be utilized only if a moderately high level of RNA expression is detected (rank 5).

[0335] Oncogenic drivers may correspond to the high-priority group. For example, missense mutations within a given grouping range can have a higher priority. This approach is based on the observed downregulation of genes targeted by immune pressure (e.g., immune editing). In contrast to other immune targets where the downregulation may not have a harmful effect on cancer cell growth, the persistent expression of oncogenic driver genes is essential for cancer cell survival and thus can block immune escape pathways. Exemplary oncogenic drivers are listed in Table 3-1 (e.g., Vogelstein et al; gene assignments for GOTERM_BP gene ontology terms - biological functions, on the World Wide Web (www)geneontology.org; gene assignments for BIOCARTA signal transduction pathways, on the World Wide Web (www)biocarta.com; gene assignments for pathways according to the KEGG KEGG pathway database on the World Wide Web (www)genome.jp / krgg / pathway.html; gene assignments for pathways and gene interactions according to the REACTOME REACTOME pathway, on the World Wide Web (www)reactome.org). See (www)reactome.org on the World Wide Web for gene assignments for pathways and gene interactions according to the REACTOME REACTOME pathway.) (Refer to (www)reactome.org on the World Wide Web for gene assignments for pathways and gene interactions according to the REACTOME REACTOME pathway.)

[0336]

Table 4-1

[0337]

Table 4-2

[0338]

Table 4-3

[0339]

Table 4-4

[0340] Example 8: Peptide Preparation and Formulation The GMP neoantigen peptides for immunization were chemically synthesized according to FDA regulations, Merrifield RB: “Solid phase peptide synthesis I. The synthesis of a tetrapeptide”. J. Am. Chem. Soc. 85: 2149-54, 1963). Three development runs of each of about 20 - 30mer peptides of 20 were carried out. Each run was carried out in the same facility and the same equipment as used in the GMP runs was utilized using draft GMP batch records. Each run successfully produced >50 mg of each peptide and they were tested by all the release tests currently planned (e.g., appearance, identity by MS, purity by RP-HPLC, content by nitrogen element, and TFA content by RP-HPLC) and conformed to the target specifications as appropriate. The products were also produced within the time frame expected for this part of the process (about 4 weeks). The lyophilized bulk peptides are being subjected to long-term stability testing, which can be evaluated at various time points up to 12 months.

[0341] Using materials from these runs, the planned dissolution and mixing procedures have been tested. Briefly, each peptide can be dissolved at a high concentration (50 mg / ml) in 100% DMSO and diluted to 2 mg / ml in an aqueous solvent. Initially, PBS was expected to be used as the diluent; however, salting out of a few peptides resulted in visible turbidity. D5W (5% dextrose in water) has been shown to be much more effective; 37 out of 40 peptides were successfully diluted to a clear solution. The only problematic peptides are extremely hydrophobic peptides. The predicted biochemical properties of the planned immunopeptides may be evaluated and the synthetic plan accordingly changed (using shorter peptides, shifting the synthetic region in the N-terminal or C-terminal direction around the predicted epitope, or potentially utilizing alternative peptides). Ten separate peptides in DMSO / D5W were subjected to two freeze / thaw cycles and complete recovery was shown. Two individual peptides were dissolved in DMSO / D5W and placed under stability at two temperatures (-20 °C and -80 °C). These peptides will be evaluated up to six months (RP-HPLC, MS and pH). To date, both peptides are stable at the 12-week time point and further time points will be evaluated at 24 weeks.

[0342] As shown in Figure 9, the design of the dosage form process is to prepare four pools of patient-specific peptides, each consisting of five peptides. An RP-HPLC assay has been prepared and is considered suitable for the evaluation of these peptide mixtures. This assay achieves good resolution of multiple peptides within a single mixture and can also be used for the quantification of individual peptides.

[0343] Bio-burden can be reduced using membrane filtration (0.2 μm pore size) and final filtration sterilization can be carried out. Initially, four different appropriate sized filter types were evaluated and the Pall, PES filter (4612) was selected. To date, four different mixtures of five different peptides each have been prepared and sequentially filtered through two PES filters individually. The recovery rate of each individual peptide was evaluated using RP-HPLC assay. For 18 out of 20 peptides, the recovery rate after two filtrations was over 90%. For two extremely hydrophobic peptides, the recovery rate was less than 60% when evaluated on a small scale, but almost completely recovered (87 and 97%) when the scale was increased. As described above, a method can be taken to limit the hydrophobic nature of the selected sequences.

[0344] GMP neoantigen peptides for immunization can be chemically synthesized according to FDA regulations, Merrifield RB: “Solid phase peptide synthesis I. The synthesis of a tetrapeptide”. J. Am. Chem. Soc. 85: 2149-54, 1963) and can be prepared.

[0345] Example 9: Endpoint Evaluation The primary immunological endpoint of this study can be the evaluation of the T cell response measured by ex vivo IFN-γ ELISPOT. IFN-γ secretion occurs as a result of the recognition or mitogenic stimulation of cognate peptides by CD4 and / or CD8 + T cells. Since the 20-30mer peptides used for vaccination need to be processed by antigen-presenting cells into smaller peptides, in vivo, a large number of different CD4 + and CD8 + and CD8 +Without being bound by theory, the combination of personalized neo-antigenic peptides, which are novel to the immune system and therefore not subject to the immunosuppressive effects of self-tolerance, with the potent immune adjuvant poly-ICLC may result in potent CD4 T cell-mediated immune responses. + and / or CD8 + It is anticipated that IFN-γ ELISPOT will be able to induce a T cell response. It is therefore anticipated that T cell responses will be detectable ex vivo, i.e., there will be no need to expand epitope-specific T cells in vitro through short-term culture. Patients may be initially evaluated using the complete peptide immunogen pool as a stimulant in an ELISPOT assay. For patients who demonstrate a robust positive response, the exact immunogenic peptide or peptides may be determined in follow-up analysis. IFN-γ ELISPOT detects T cell activity ex vivo and specifically In addition to the analysis of the magnitude of T cell responses and determinant mapping in peripheral blood monocytes, other aspects of the immune response induced by the vaccine are critically important and can be evaluated. These evaluations can be performed using ex vivo IFN-γ ELISPOT responses in screening assays. These may be performed in patients presenting with melanoma. These include assessment of T cell subsets (Th1 vs. Th2, T effector vs. memory cells), analysis of the presence and abundance of regulatory cells, e.g., regulatory T cells or myeloid derived suppressor cells, and cytotoxicity assays if patient-specific melanoma cell lines are successfully established.

[0346] Example 10: Peptide Synthesis GMP peptides may be synthesized by standard solid phase synthetic peptide chemistry and purified by RP-HPLC. Each individual peptide may be analyzed by a variety of qualified assays to assess appearance (visual), purity (RP-HPLC), identity (by mass spectrometry), quantity (elemental nitrogen), and trifluoroacetate counter ion (RP-HPLC) and release.

[0347] The individualized neoantigen peptides can be composed of up to 20 distinct peptides unique to each patient. Each peptide can be a linear polymer of about 20 to about 30 L-amino acids linked by standard peptide bonds. The amino terminus may be a primary amine (NH2-), and the carboxy terminus is a carbonyl group (-COOH). The standard 20 amino acids commonly found in mammalian cells are utilized (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine). The molecular weight of each peptide varies based on its length and sequence and is calculated for each peptide.

[0348] The individualized neoantigen peptides may be supplied as a box containing 2 ml Nunc Cryo vials with color-coded caps, with each vial containing a 1.5 ml frozen DMSO / D5W solution containing up to 5 peptides at a concentration of about 400 μg / ml. There can be 10 to 15 vials for each of the 4 groups of peptides. The vials should be stored at -80 °C until use. Ongoing stability tests support the storage temperature and duration.

[0349] Storage and Stability: The individualized neoantigen peptides are stored frozen at -80 °C. The thawed and sterile-filtered in-process intermediates and final mixtures of the individualized neoantigen peptides and poly-ICLC can be kept at room temperature but must be used within 4 hours. Compatibility: The individualized neoantigen peptides can be mixed with one-third volume of poly-ICLC immediately before use and.

[0350] Example 11: Administration After mixing with the individualized neoantigen peptide / polypeptide, the vaccine (e.g., peptide + poly-ICLC) will be administered subcutaneously.

[0351] Preparation of individualized neoantigen peptide / polypeptide pools: Peptides can be combined together into four pools of up to five peptides each. The selection criteria for each pool can be based on the specific MHC alleles to which the peptides are predicted to bind.

[0352] Pool composition: The composition of the pools can be selected based on the specific HLA alleles to which each peptide is predicted to bind. The four pools can be injected into anatomical sites that drain into separate lymph node basins. This approach was selected to potentially reduce antigen competition between peptides that bind to the same HLA allele as much as possible, and it involves a broad subset of the patient's immune system in the generation of an immune response. For each patient, peptides predicted to bind to up to four different HLA A and B alleles can be identified. Some neo-ORF-derived peptides are not associated with any specific HLA allele. The method of distributing peptides into different pools can be to spread each set of peptides associated with a specific HLA allele across as many of the four pools as possible. There is a very high likelihood that there will be situations where there are more than four predicted peptides for a given allele, and in such cases, it may be necessary to assign two or more peptides associated with a specific allele to the same pool. Neo-ORF peptides not associated with any specific allele can be randomly assigned to the remaining slots. An example is shown below. Some neo-ORF-derived peptides are not associated with any specific HLA allele. The method of distributing peptides into different pools can be to spread each set of peptides associated with a specific HLA allele across as many of the four pools as possible. There is a very high likelihood that there will be situations where there are more than four predicted peptides for a given allele, and in such cases, it may be necessary to assign two or more peptides associated with a specific allele to the same pool. Neo-ORF peptides not associated with any specific allele can be randomly assigned to the remaining slots. An example is shown below.

[0353] [Table 5]

[0354] Whenever possible, peptides predicted to bind to the same MHC allele can be placed in separate pools. Some neo-ORF peptides may be predicted not to bind to any of the patient's MHC alleles. However, these peptides can still be utilized, mainly because they are completely novel and thus not subject to the immunosuppressive effects of central tolerance and are therefore likely to be immunogenic. Neo-ORF peptides also have a dramatically reduced potential for autoimmunity because there are no equivalent molecules in any normal cells. In addition, there are false negatives from the prediction algorithm, and there is a possibility that this peptide may contain an HLA class II epitope (HLA class II epitopes are not predicted with high confidence based on the current algorithm). All peptides not identified with a specific HLA allele can be randomly assigned to individual pools. The amount of each peptide is based on a final dose of 300 μg of each peptide per injection. The final dose of each peptide per injection is 300 μg.

[0355] For each patient, four separate pools of five synthetic peptides (named "A", "B", "C", and "D") are prepared by the manufacturer and stored at -80°C. On the day of immunization, a complete vaccine consisting of one or more peptide components and poly-ICLC can be prepared in a laminar flow biosafety cabinet in the research pharmacy. Each vial (A, B, C, and D) can be thawed at room temperature and transferred into the biosafety cabinet for the remaining steps. 0.75 ml of each peptide pool can be withdrawn from the vial and placed in separate syringes. Separately, four 0.25 ml (0.5 mg) aliquots of poly-ICLC can be withdrawn and placed in separate syringes. Next, the contents of the syringe containing each peptide pool can be gently mixed by transferring between the syringes with the 0.25 ml aliquot of poly-ICLC. All 1 ml of the mixture can be used for injection. These four preparations can be named "Test Agent A", "Test Agent B", "Test Agent C", and "Test Agent D".

[0356] Injection: For each immunization, each of the four test drugs can be subcutaneously injected into one limb. For each individual test drug, it can be administered to the same limb for each immunization throughout the treatment period (i.e., test drug A can be injected into the left arm on days 1, 4, 8, etc., and test drug B can be injected into the right arm on days 1, 4, 8, etc.). Alternative anatomical sites for patients in the state after complete axillary or inguinal lymph node dissection are the left and right diaphragms, respectively.

[0357] The vaccine can be administered according to a prime / boost schedule. The priming dose of the vaccine can be administered on days 1, 4, 8, 15, and 22 as shown above. During the boost period, the vaccine can be administered on day 85 (week 13) and day 169 (week 25).

[0358] All patients who receive at least one dose of vaccine administration can potentially be evaluated for toxicity. If patients receive all vaccinations during the induction period and the first vaccination (boost) during the maintenance period, those patients can potentially be evaluated for immunological activity.

[0359] Example 12: Pharmacodynamic Testing The immunization strategy is a "prime-boost" approach that includes an initial series of closely spaced immunizations to induce an immune response and a subsequent rest period to establish memory T cells. This is followed by booster immunizations, and it is expected that the T cell response 4 weeks after this boost (16 weeks after the first vaccination) will produce the strongest response and can be the primary immunological endpoint. Immunological monitoring is carried out step by step as outlined below, and the intensity and quality of the induced immune response can be characterized. As shown in Schema B and specified in the test calendar, peripheral blood can be collected at two separate time points (baseline) before the first vaccination and at various time points thereafter, and PBMCs can be frozen. After a complete set of samples for each of the induction and maintenance periods has been collected, immunological monitoring of a given patient can be carried out. If sufficient tumor tissue is available, a portion of the tumor can be used to establish an autologous melanoma cell line for use in a cytotoxic T cell assay.

[0360] Example 13: Screening Ex Vivo IFN - γ ELISPOT For each patient, a set of screening peptides can be synthesized. The screening peptides are 15 amino acids long (sometimes 16mers or 17mers can be used), overlapping by 11 amino acids, covering the full length of each peptide or the full length of the neo-ORF for neo-ORF-derived peptides. The complete set of patient-specific screening peptides are pooled together at approximately equal concentrations, and a portion of each peptide can also be kept individually. The purity of the peptide pool is determined by the low background 5-fold increase in the number of peptides in ex vivo IFN-γ ELISPOT established. This can be confirmed by testing PBMCs from healthy donors. Initially, PBMCs obtained at baseline and at week 16 (primary immunological endpoint) were analyzed using overlapping 15mer peptides. The mice may be stimulated with the complete pool of peptides (11 amino acid overlap) for 18 hours to examine the overall response to the peptide vaccine. Subsequent assays may utilize PBMCs harvested at other time points as indicated. Ex vivo IFN-γ ELISPOT assays If no response is identified using the primary immunological endpoint, PBMCs can be stimulated with peptide pools for longer periods (up to 10 days) and analyzed again.

[0361] Example 14: Epitope Deconvolution in Follow - up Ex Vivo IFN - γ ELISPOT Assay Epitope Deconvolution Ex vivo IFN-γ ELISP induced by overlapping peptide pools OT response (at least 55 spot-forming units / 10 6 After a positive result (defined as at least a three-fold increase over PBMC or baseline) was observed, the peptide pools were deconvoluted into subpools based on the immunizing peptide and analyzed by ex vivo IFN-γ ELISA. By repeating the POT assay, the specific immunogenic peptides that elicit this response may be identified. By using overlapping 8 - 10mer peptides derived from the activating peptide, attempts can be made to accurately characterize the stimulating epitope. Further assays can be performed on an individual case - by - case basis for appropriate samples. For example, · Use the entire 15mer pool or sub - pools as stimulating peptides in an intracellular cytokine staining assay to identify and quantify antigen - specific CD4 + 、CD8 + 、central memory and effector memory populations. · Similarly, use these pools to evaluate the pattern of cytokines secreted by these cells and determine the T H 1 - to - T H 2 phenotypes. · Use extracellular cytokine staining and flow cytometry of unstimulated cells to quantify Treg and myeloid - derived suppressor cells (MDSC).

[0362] · If a melanoma cell line has been successfully established from a responding patient and the activating epitope has been identified, a cytotoxicity assay of T cells can be performed using mutant peptides and corresponding wild - type peptides. · Evaluate "epitope spreading" by using PBMC of primary immunological endpoints with known melanoma tumor - associated antigens as stimulants and several additional identified mutant epitopes that were not selected among the immunogens. CD4 + 、CD8 + 、Perform immunohistochemistry of tumor samples to quantify MDSC and Treg infiltration populations.

[0363] Example 15: Pipeline for Systematic Identification of Tumor Neoantigens Using recent sequencing technologies and advances in peptide epitope prediction, we developed a two-step pipeline to systematically discover candidate tumor-specific HLA-binding neoantigens. As shown in Figure 10, this approach begins with tumor DNA sequencing (e.g., by either whole exome sequencing (WES) or whole genome sequencing (WGS)) in parallel with the corresponding normal DNA, and nonsynonymous somatic mutations are comprehensively identified (see, e.g., Lawrence et al. 2013; Cibulski et al. 2012). Next, candidate tumor-specific mutant peptides resulting from tumor mutations that have the potential to bind to an individual's class I HLA protein and thus be presented to CD8 T cells can be predicted using a prediction algorithm such as NetMHCpan (see, e.g., Lin 2008; Zhang 2011). Candidate peptide antigens were further evaluated based on experimental verification of their binding to HLA and the expression of cognate mRNA in autologous leukemic cells. the potential to bind, and thus + be presented to CD8

[0364] We applied this pipeline to a large dataset of sequenced CLL samples (see, e.g., Wang et al. 2011). From 91 cases sequenced by either WES or WGS, a total of 1838 nonsynonymous mutations were identified in the protein-coding regions It was found that this corresponded to an average somatic mutation rate of 0.72 (±0.36 s.d.) megabase pairs, with a range of 0.08 - 2.70, and an average of 20 nonsynonymous mutations per patient (range, 2 - 76) (see, e.g., Wang et al. 2011). Three general classes of mutations were identified that give rise to regions of amino acid change and thus may be expected to generate immunologically recognizable epitopes. The most abundant class included missense mutations that caused single amino acid (aa) changes and corresponded to 90% of the somatic mutations in CLL. In 91 samples, 99% contained missense mutations, and 69% had 10 - 25 missense mutations (see, e.g., FIG. 2A). The other two classes of mutations, frameshift and splice site mutations (mutations at exon - intron junctions), together generate longer stretches of novel amino acid sequences (neo - open reading frames, or neoORFs) that are specific to the tumor and may result in a greater number of neoantigen peptides per given alteration compared to missense mutations. However, consistent with data from other cancer types, neoORF - generating mutations were approximately 10 - fold less frequent in CLL compared to missense mutations (see, e.g., FIGS. 2B - 2C). Given the prevalence of missense mutations, subsequent experimental studies focused on the analysis of neoepitopes generated by missense mutations.

[0365] Example 16: Somatic Missense Mutations Generate Neoepitopes Predicted to Bind to Individual HLA Class I Alleles T cell recognition of peptide epitopes by the T cell receptor (TCR) requires the presentation of peptides bound within the binding groove of HLA molecules on the surface of antigen - presenting cells. A recent comparative study among more than 30 available class I prediction algorithms has shown that NetMHCpan consistently functions with high sensitivity and specificity across HLA alleles (see, e.g., Zhang et al. 2011).

[0366] The yuan was tested against a set of 33 known mutant epitopes identified in the literature based on their functional activity (i.e., the ability to stimulate an anti-neoplastic cytolytic T cell response) or characterized as minor histocompatibility antigens, and it was determined whether this algorithm could correctly predict binding for the 33 known mutant epitopes (see, for example, Tables 4 and 5). Tables 4 and 5 show the HLA-peptide binding affinities of known functionally induced immunogenic mutant epitopes between human cancers using NetMHCpan. Table 4 shows epitopes derived from missense mutations (NSCLC: non-small cell lung cancer; MEL: melanoma; CLL: chronic lymphocytic leukemia; RCC: renal clear cell carcinoma; BLD: bladder cancer; NR: not reported;). Yellow: IC 50 <150 nM, green: IC 50 150 - 500 nM and gray: IC 50 >500 nM.

[0367]

Table 6-1

[0368]

Table 6-2

[0369] Table 5 shows epitopes derived from minor histocompatibility antigens (MM: multiple myeloma; HM: hematological malignancies; B-ALL: B cell acute lymphocytic leukemia).

[0370]

Table 7

[0371] Among all possible 9mers and 10mers of the tiling, NetMHCpan identified all 33 functionally validated mutant epitopes as the best binding peptides among the possible options for a given mutation. The median predicted binding affinity (IC 50 ) for each of the known reported HLA restricting elements of the 33 mutant epitopes was 32 nM (range, 3 - 11, 192 nM). By setting the predicted IC 50 cutoffs at 150 and 500 nM, 82% and 91% respectively of the functionally validated peptides were captured (see, for example, Tables 4 and 5 and Figure 12A).

[0372] Based on its high sensitivity and specificity, NetMHCpan was then applied to 31 of 91 CLL cases for which HLA typing information was available. By convention, peptides with IC <150 nM were considered strong to moderate binders, IC 50 150 - 500 nM were considered weak binders, and IC 50 >500 nM were considered non - binders (see, for example, Cai et al. 2012). For all 91 CLL cases, a median of 10 strong 50 binding peptides (range, 2 - 40) and 12 moderate to weak binding peptides (range, 2 - 41) were found. Overall, a median of 22 peptides (range, 6 - 81) per case were predicted to have IC <500 nM (see, for example, Figure 12B and Table 6). Specifically, Table 6 shows the number and affinity distribution of peptides predicted from 31 CLL cases with available HLA typing. Patients expressing the 8 most common HLA - A, HLA - B alleles in the white population are shown in gray. 50

[0373]

Table 8

[0374] Example 17: More than Half of the Predicted HLA - Binding Neoepitopes Showed Direct Binding to HLA Proteins In Vitro ​As shown in Table 7, the IC 50 nM scores created by HLA-peptide binding prediction were verified using a competitive MHC I allele binding assay, focusing on class I-A and -B alleles. For this purpose, 112 mutant peptides (9- or 10-mer mutant peptides) with predicted IC scores of less than 500 nM identified from 4 CLL cases (patients 1-4) were synthesized. The experimental results correlated with the binding prediction. Experimental binding (defined as IC 50 <500 nM) was confirmed in 76.5% and 36%, respectively, of the peptides predicted to be <150 nM or 150-500 nM IC 50 (see, for example, Fig. 12C). Overall, approximately 54.5% (61 / 112) of the predicted peptides were experimentally verified to be complexes with individual HLA alleles. Overall, as shown in (Fig. 13), the prediction of 9-mer peptides was more sensitive, being able to experimentally verify 60% and 44.5%, respectively, of the predicted peptides (IC 50 <500 nM), compared to 10-mer peptides. 50

[0375]

Table 9-1

[0376]

Table 9-2

[0377]

Table 9-3

[0378]

Table 9-4

[0379]

Table 9-5

[0380] Example 18: Neoantigens are Expressed in CLL Tumors CTL responses to epitopes can be useful only if the gene encoding the epitope is expressed in the target cells. Twenty-six out of 31 patient samples that were sequenced and typed for HLA were subjected to genome-wide expression profiling (see, e.g., Brown et al. 2012). The expression levels of 347 genes with mutations in the CLL samples were classified as having low / no (lowest quartile), moderate (middle two quartiles), or high (highest quartile) expression. As shown in Figure 12D, 80% of the 347 mutated genes (or 79% of 180 mutations with predicted HLA binding) were expressed at moderate to high expression levels. A similarly high frequency of expression was observed among a subset of 221 mutated genes with predicted class I binding epitopes (88.6%).

[0381] RNA levels were determined based on reads per gene product and could be ranked by quartiles. "H" - upper quartile; "M" - middle two quartiles; "L" - lowest quartile (excluding genes without reads); "-" - no detectable reads. Higher stringency can be applied to expression levels as predicted affinity decreases. Neo-ORFs with predicted binders were also utilized in cases where no mRNA molecules were detectable by RNA-Seq. Currently, there is no data available to evaluate to what extent (if any) a minimum expression level in tumor cells is required for a neo-ORF to be useful as a target for activated T cells. Even the expression level of the "pioneer" translation of messages destined for nonsense-mediated decay mechanisms can be sufficient for target generation ((Chang YF, Imam JS, Wilkinson MF: "The nonsense-mediated decay RNA surveillance pathway). Annual Review of Biochemistry 76: 51-74, 2007). Therefore , due to its novelty and excellent tumor specificity, the neo-ORF has high value as a target, so even if its expression is low or undetectable at the RNA level, the neo-ORF can be utilized as an immunogen.

[0382] Example 19: T Cells Targeting Candidate Neoepitopes were Detected in CLL Patient 1 after HSCT The setting after allogeneic hematopoietic stem cell transplantation (HSCT) in CLL was analyzed to determine whether an immune response against the predicted mutant peptides could occur in patients. T cell reconstitution from healthy donors after HSCT can overcome the host's endogenous immune deficiency and also enable priming against leukemia cells in the host in vivo. The analysis focused on two patients who both received unrelated dose-reduced preconditioned allogeneic HSCT for advanced CLL and achieved sustained remission lasting more than 4 years after HSCT (see, for example, Table 8). Post-transplant T cells were collected at 7 years (Patient 1) and 4 years (Patient 2) after transplantation.

[0383] Table 8 shows the clinical characteristics of CLL patients 1 and 2. Both patients achieved ongoing sustained remission lasting more than 7 (Patient 1) and 4 years (Patient 2) after HSCT. M: male; HSCT: hematopoietic stem cell transplantation; RIC: reduced-intensity conditioning; Flu / Bu: fludarabine / busulfan; GvHD: graft-versus-host disease; URD: unrelated donor; Mis: missense; FS: frameshift.

[0384]

Table 10

[0385] For the patient (Patient 1), 25 missense mutations were identified by WES. Overall, 30 peptides from 13 mutations were predicted to bind to the individual's HLA (IC 50 <150 peptides of 13; IC 50Peptide 1 at 150 - 500 nM (7 peptides). As shown in Figure 14A, HLA binding was confirmed for 14 peptides derived from 9 mutations by experimental verification of peptide prediction. All 30 predicted HLA-binding peptides were selected for the T cell priming assay and grouped into 5 pools of 6 peptides / pools (see, for example, Table 9). Peptides with comparable predicted binding scores were grouped into the same pool.

[0386] Table 9 provides an overview of the peptides derived from the missense mutations of Patient 1 included in the peptide pools for the T cell stimulation assay. In Patient 1, ICs that bind to HLA-A and -B alleles 50 All of the predicted peptides at <500 nM were used. Five mutant peptide pools containing 6 peptides / pools listed in descending order of predicted binding affinity for MHC class I alleles. The corresponding experimental HLA-peptide binding affinities, wild-type peptides, and their predicted IC 50 scores are included in the rightmost column.

[0387]

Table 11 - 1

[0388]

Table 11 - 2

[0389] The neoantigen responsiveness of T cells was tested by expanding T cells using autologous antigen-presenting cells (APCs) pulsed with candidate neoantigen peptide pools (once a week for 4 weeks). As shown in Figure 14B, the responsiveness in the IFN-γ ELISPOT assay was for the pool It was detected against 2, but not against irrelevant peptides (Tax peptides). By deconvolution of the pool, it was revealed that the mutant (mut) ALMS1 and C6orf89 peptides in pool 2 are immunogenic. ALMS1 plays a role in ciliary function, cell quiescence, a...

Claims

**Claim 1** A method for manufacturing an individualized cancer vaccine for a subject diagnosed with having cancer, comprising: identifying a plurality of mutations in the cancer; identifying a subset of at least five neoantigen mutations predicted to encode neoantigen peptides by analyzing the plurality of mutations, wherein the neoantigen mutations are selected from the group consisting of missense mutations, neo ORF mutations, and any combination thereof; producing an individualized cancer vaccine based on the identified subset; and a method comprising the above steps. **Claim 2** The method according to claim 1, wherein the step of identifying further comprises: sequencing the genome, transcriptome, or proteome of the cancer. **Claim 3** The method according to claim 1, wherein the step of analyzing further comprises: determining one or more characteristics associated with the subset of at least five neoantigen mutations predicted to encode neoantigen peptides, wherein the characteristics are selected from the group consisting of molecular weight, cysteine content, hydrophilicity, hydrophobicity, charge, and binding affinity; and ranking each of the neoantigen mutations within the identified subset of at least five neoantigen mutations based on the determined characteristics. **Claim 4** The method according to claim 3, wherein the neoantigen mutations ranked from top 5 to top 30 are included in the individualized cancer vaccine. **Claim 5** The method according to claim 3, wherein the neoantigen mutations are ranked according to the order shown in FIG.

8. **Claim 6** The method according to claim 4, wherein the individualized cancer vaccine comprises at least about 20 neoantigen peptides corresponding to the neoantigen mutations. **Claim 7** The method according to claim 4, wherein the individualized cancer vaccine comprises one or more DNA molecules capable of expressing at least about 20 neoantigen peptides corresponding to the neoantigen mutations. **Claim 8** The method according to claim 4, wherein the individualized cancer vaccine comprises one or more RNA molecules capable of expressing at least 20 neoantigen peptides corresponding to the neoantigen mutations. **Claim 9** ​ ​ The method according to claim 1, wherein the individualized neoplastic vaccine comprises a neo-ORF mutation predicted to encode a neo-ORF polypeptide with a Kd ≤ 500 nM.

10. The method according to claim 1, wherein the individualized neoplastic vaccine comprises a missense mutation predicted to encode a polypeptide with a Kd ≤ 150 nM, and its native cognate protein has a Kd ≥ 1000 nM or ≤ 150 nM.

11. The method according to claim 6, wherein the at least about 20 neoantigen peptides range from about 5 to about 50 amino acids in length.

12. The method according to claim 6, wherein the at least about 20 neoantigen peptides range from about 15 to about 35 amino acids in length.

13. The method according to claim 6, wherein the at least about 20 neoantigen peptides range from about 18 to about 30 amino acids in length.

14. The method according to claim 6, wherein the at least about 20 neoantigen peptides range from about 6 to about 15 amino acids in length.

15. The method according to claim 6, wherein the at least about 20 neoantigen peptides are 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

16. The method according to claim 1, wherein the individualized neoplastic vaccine further comprises an adjuvant.

17. The method according to claim 1, wherein the adjuvant is selected from the group consisting of poly ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS patch, ISS, ISCOMATRIX, Juvlmune, Lipovac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM, vector systems, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulon, badimezan, and AsA404 (DMXAA).

18. The method according to claim 17, wherein the adjuvant is poly ICLC.

19. A method of treating a subject diagnosed with a neoplasm with an individualized neoplasm vaccine, identifying a plurality of mutations in the neoplasm; identifying, by analyzing the plurality of mutations, a subset of at least five neoantigen mutations predicted to encode expressed neoantigen peptides, wherein the neoantigen mutations are selected from the group consisting of missense mutations, neo ORF mutations, and any combination thereof; producing an individualized neoplasm vaccine based on the identified subset; administering the individualized neoplasm vaccine to the subject, thereby treating the neoplasm comprising the method.

20. The identifying step further comprises sequencing the genome, transcriptome, or proteome of the neoplasm The method according to claim 19.

21. The analyzing step further comprises determining one or more characteristics associated with the subset of at least five neoantigen mutations predicted to encode expressed neoantigen peptides, wherein the characteristics are selected from the group consisting of molecular weight, cysteine content, hydrophilicity, hydrophobicity, charge, and binding affinity; ranking each of the neoantigen mutations within the identified subset of at least five neoantigen mutations based on the determined characteristics The method according to claim 19.

22. The method according to claim 21, wherein the neoantigen mutations ranked from top 5 to top 30 are included in the individualized neoplasm vaccine.

23. The method according to claim 21, wherein the neoantigen mutations are ranked according to the order shown in FIG.

8.

24. The method according to claim 22, wherein the individualized neoplasm vaccine comprises at least 20 neoantigen peptides corresponding to the neoantigen mutations.

25. The method according to claim 22, wherein the individualized neoplasm vaccine comprises one or more DNA molecules capable of expressing at least 20 neoantigen peptides corresponding to the neoantigen mutations.

26. The method according to claim 22, wherein the individualized neoplastic vaccine comprises one or more RNA molecules having the ability to express at least 20 neoantigen peptides corresponding to the neoantigen mutations.

27. The method according to claim 19, wherein the individualized neoplastic vaccine comprises a neo-ORF mutation predicted to encode a neo-ORF polypeptide with a Kd ≤ 500 nM.

28. The method according to claim 19, wherein the individualized neoplastic vaccine comprises a missense mutation predicted to encode a polypeptide with a Kd ≤ 150 nM, and its native cognate protein has a Kd ≥ 1000 nM or ≤ 150 nM.

29. The method according to claim 24, wherein the at least 20 neoantigen peptides range from about 5 to about 50 amino acids in length.

30. The method according to claim 24, wherein the at least 20 neoantigen peptides range from about 15 to about 35 amino acids in length.

31. The method according to claim 24, wherein the at least 20 neoantigen peptides range from about 18 to about 30 amino acids in length.

32. The method according to claim 24, wherein the at least 20 neoantigen peptides range from about 6 to about 15 amino acids in length.

33. The method according to claim 24, wherein the at least 20 neoantigen peptides are 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length.

34. The step of administering comprises the step of dividing the prepared vaccine into two or more sub-pools, and the step of injecting each of the sub-pools into different sites of the patient The method according to claim 16, further comprising.

35. For each of the sub-pools injected into different sites, the number of individual peptides in the sub-pool targeting any single patient HLA is 1, or the number of neoantigen peptides is as small as possible while being 2 or more. The method according to claim 34.

36. The step of administering further comprises the step of dividing the prepared vaccine into two or more sub-pools, and each sub-pool comprises at least 5 neoantigen peptides selected to optimize the interaction within the pool. The method according to claim 31.

37. The method according to claim 36, wherein the optimization comprises reducing the negative interaction between the neoantigen peptides in the same pool.

38. The method according to claim 19, wherein the administering step further comprises a step of delivering a dendritic cell (DC) vaccine, and one or more of the at least five neoantigen mutations predicted to encode an expressed neoantigen peptide are loaded onto the DC.

39. An individualized neoplasm vaccine prepared by the method according to claim 1.

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