Platform for identifying personalized immunogenic peptides

Abstract

The present disclosure relates to methods for identifying fragments of a polypeptide that are immunogenic to a particular human subject, methods for preparing personalized pharmaceutical compositions comprising such polypeptide fragments, pharmaceutical compositions specific to a human subject comprising such polypeptide fragments, and methods of treatment using such compositions. The methods include identifying fragments of a polypeptide that bind to multiple HLAs in a subject. [Selection diagram] None

Description

Technical Field

[0001] field The present disclosure relates to a method for predicting whether a polypeptide is immunogenic to a specific human subject, a method for identifying a fragment of a polypeptide that is immunogenic to a specific human subject, a method for preparing a personalized or precision pharmaceutical composition or kit comprising such a polypeptide fragment, a human subject-specific pharmaceutical composition comprising such a polypeptide fragment, and a method of treatment using such a composition.

Background Art

[0002] background For decades, scientists have hypothesized that chronic diseases are beyond the reach of the natural defenses of the human individual. However, in recent years, the field of cancer immunotherapy has been accelerated by the significant tumor regression observed in individuals treated with antibodies that inhibit immunosuppressive molecules. From these clinical findings, it has been shown that the reactivation of existing T cell responses can provide significant clinical benefits to individuals. These advances have renewed the enthusiasm for the development of cancer vaccines that induce tumor-specific T cell responses.

[0003] Despite its promise, current immunotherapies are effective only in some individuals. In addition, due to the low tumor regression rate and anti-tumor T cell responses in individuals, many cancer vaccine clinical trials have not been able to show statistically significant efficacy. Similar failures have also been reported for therapeutic and preventive vaccines that have attempted to include T cell responses in the fields of HIV and allergy. There is a need to overcome the clinical failures of immunotherapy and vaccines.

Summary of the Invention

[0004] overview In antigen-presenting cells (APCs), protein antigens are processed into peptides. These peptides bind to human leukocyte antigen molecules (HLA) and are presented on the cell surface as peptide-HLA complexes to T cells. Different individuals express different HLA molecules, and different HLA molecules present different peptides. Therefore, according to advanced technologies in this field, a peptide, or a fragment of a larger polypeptide, is identified as immunogenic to a specific human subject when presented by the HLA molecule expressed by that subject. In other words, advanced technologies in this field describe immunogenic peptides as HLA-restricted epitopes. However, HLA-restricted epitopes induce a T cell response only in some individuals expressing that HLA molecule. A peptide that activates a T cell response in one individual is inactive in other individuals, regardless of HLA allele matching. Therefore, it was unknown how an individual's HLA molecule presents an antigen-derived epitope that actively activates a T cell response.

[0005] As provided herein, multiple HLAs expressed by an individual must present the same peptide to elicit a T cell response. Therefore, a polypeptide antigen fragment that is immunogenic to a particular individual can bind to multiple class I (cytotoxic T cell activating) or class II (helper T cell activating) HLAs expressed by that individual.

[0006] Accordingly, in a first embodiment, the present disclosure provides a method for predicting whether a polypeptide or polypeptide fragment is immunogenic to a particular human subject, the method comprising the following steps: (i) The polypeptide (a) an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules of the subject; or, (b) an amino acid sequence which is a T cell epitope capable of binding to at least two HLA class II molecules of the subject, A step of determining whether or not it includes; and, (ii) A. The polypeptide is immunogenic to the subject if it contains at least one sequence that satisfies the requirements of step (i); or B. If the polypeptide does not contain at least one sequence that satisfies the requirements of step (i), then the polypeptide is not immunogenic to the subject. The process of making predictions, Includes.

[0007] This disclosure also provides a method for identifying polypeptide fragments as immunogenic to specific human subjects, the method comprising the following steps (i) The polypeptide (a) an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules of the subject; or, (b) an amino acid sequence which is a T cell epitope capable of binding to at least two HLA class II molecules of the subject, The process of determining whether to include; and (ii) A step of identifying the sequence as a polypeptide fragment that is immunogenic to the subject, Includes.

[0008] In some embodiments, the method of the present disclosure includes the step of determining or obtaining the HLA class I genotype and / or HLA class II genotype of a particular human subject.

[0009] A particular polypeptide antigen may contain one or more fragments that are T cell epitopes capable of binding to multiple HLAs in a particular individual. The group formed by combining all such fragments characterizes the individual's antigen-specific T cell response set, where the amino acid sequence of each fragment characterizes the specificity of its respective activated T cell clone.

[0010] Therefore, depending on the case, this method is repeated until all fragments of polypeptides that are T cell epitopes capable of binding to at least two HLA class I cells and / or at least two HLA class II cells of the subject are identified. This method characterizes the subject's immune response to the polypeptides.

[0011] The Disclosure further provides a method for treating a human subject in need of treatment, comprising the step of administering to the subject a polypeptide, pharmaceutical composition, or polypeptide kit or polypeptide panel comprising a polypeptide fragment identified or selected by any of the above methods; the use thereof in a method for treating a relevant human subject; and the use thereof in the manufacture of a pharmaceutical for the treatment of a relevant subject.

[0012] A polypeptide fragment determined to be immunogenic to a specific human subject by the method described above can be used to prepare an immunogenic composition specific to that human subject.

[0013] Accordingly, in a further embodiment, the present disclosure provides a method for designing or preparing a human subject-specific pharmaceutical composition or kit or panel of polypeptides for use in a method of treating a particular human subject, the method being: (i) A step of selecting a polypeptide fragment, wherein the fragment has been identified as immunogenic to the subject by the method described above; (ii) If the fragment selected in step (i) is an HLA class I binding epitope, the step of optionally selecting a longer fragment of the polypeptide, wherein the longer fragment is a. Includes the fragment selected in step (i); and b. A T cell epitope capable of binding to at least three or most likely HLA class II molecules of the subject; (iii) A step of selecting a first sequence of up to 50 consecutive amino acids of the polypeptide, wherein the consecutive amino acids include the amino acid sequence of the fragment selected in step (i) or the amino acid sequence of the longer fragment selected in step (ii); (iv) Repeating steps (i) through (iii) to select a second amino acid sequence of up to 50 consecutive amino acids of a polypeptide that is the same as or different from the first amino acid sequence; (v) optionally repeating steps (i) through (iii) to select one or more additional amino acid sequences of up to 50 consecutive amino acid sequences of polypeptides that are the same as or different from the first and second amino acid sequences; and, (vi) A step of designing or preparing a subject-specific pharmaceutical composition, kit, or panel of polypeptides having as an active ingredient one or more polypeptides having all of the amino acid sequences selected in a preceding step, wherein one or more sequences, or each of them, are flanked at the N-terminus and / or C-terminus by additional amino acids that are not part of the polypeptide sequence. Includes.

[0014] Depending on the case, each peptide may consist of either one of the selected amino acid sequences, or two or more selected amino acid sequences that are arranged end-to-end or overlapping within a single peptide.

[0015] The Disclosure further provides a panel of human subject-specific pharmaceutical compositions, kits, or polypeptides for use in methods of treating specific human subjects in need of treatment, wherein the compositions, kits, or panels comprise, as active ingredients, a first and a second peptide, and optionally one or more additional peptides, each peptide comprising an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules and / or at least two HLA class II molecules of the subject, wherein the amino acid sequences of the T cell epitopes of the first, second, and optional additional peptides are distinct from one another, and the pharmaceutical compositions or kits optionally comprise at least one pharmaceutically acceptable diluent, carrier, or preservative.

[0016] The Disclosure further provides a panel of human subject-specific pharmaceutical compositions, kits, or polypeptides for use in a method of treating a particular human subject in need of treatment, the composition or kit comprising as an active ingredient a polypeptide comprising a first region, a second region, and optionally one or more additional regions, each region comprising an amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules and / or at least two HLA class II molecules of the subject, wherein the amino acid sequences of the T cell epitopes in the first, second, and optional additional regions are distinct from one another, and the pharmaceutical composition or kit optionally comprises at least one pharmaceutically acceptable diluent, carrier, or preservative.

[0017] The Disclosure further provides a method for designing or preparing a polypeptide for inducing an immune response in a specific human subject, the method comprising the steps of: selecting an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules or at least three HLA class II molecules of the subject; and designing or preparing a polypeptide comprising the selected amino acid sequence.

[0018] In a further manner, this disclosure is: - A method or treatment for inducing an immune response, comprising the step of administering to a human subject in need thereof a human subject-specific pharmaceutical composition, polypeptide, kit, or panel as described above, wherein the composition, kit, or polypeptide panel is specific to the subject; - Human subject-specific immunogenic compositions, kits, or panels, as described above, for use in methods or treatments for inducing an immune response in specific human subjects; and, - In the manufacture of pharmaceuticals, the use of a polypeptide of a pharmaceutical composition, kit, or panel specific to a human subject, as described above, wherein the pharmaceutical is intended to induce an immune response in the specific subject or to be used in the treatment of the specific subject. To provide.

[0019] In a further manner, this disclosure is: (a) A storage module configured to store data including the subject's class I and / or class II HLA genotype and the amino acid sequence of one or more test polypeptides; and (b) A computational module configured to identify and / or quantify the amino acid sequences in one or more test polypeptides that can bind to multiple HLA class I molecules of the subject and / or to multiple HLA class II molecules of the subject. We provide a system that includes this.

[0020] This disclosure provides a method for treating a human subject in need of treatment, the method comprising administering to the subject a polypeptide, a polypeptide panel, a pharmaceutical composition, or a polypeptide of an active ingredient of a kit described above, wherein the subject is determined to express at least three HLA class I molecules and / or at least three HLA class II molecules that can be bound to the polypeptide or to one or more polypeptides of the pharmaceutical composition or kit.

[0021] Herein, the present disclosure will be described in more detail by reference to the accompanying drawings, not as an exemplification but as an illustration. Given this disclosure, many equivalent modifications and variations will be obvious to those skilled in the art. Accordingly, the exemplary aspects of the described disclosure are illustrative and not restrictive. Various modifications can be made to the described aspects without departing from the scope of the present disclosure. All documents referenced herein, whether preceding or succeeding, are expressly incorporated in their entirety by reference.

[0022] This disclosure includes combinations of the described embodiments and preferred features, unless such combination is clearly unacceptable or explicitly stated to be avoided. As used herein and in the appended claims, the singular “a,” “an,” and “the” include plural references unless explicitly stated otherwise. Thus, for example, a reference to “a peptide” includes two or more such peptides.

[0023] In this specification, section headings are used for convenience only and should not be construed as limiting. [Brief explanation of the drawing]

[0024] [Figure 1] ROC curves of HLA-restricted PEPI biomarkers. [Figure 2] ROC curves for one or more PEPI3+ trials to determine the accuracy of the diagnosis. [Figure 3]Distribution of HLA class I PEPI3+ compared to CD8+ T cell response, measured by a state-of-the-art assay, among the peptide pools used in the CD8+ T cell response assay. A: HLA class I-restricted PEPI3+. An overall percentage of agreement (OPA) of 90% between the T cell response and the PEPI3+ peptide indicates the usefulness of the disclosed peptide for predicting the vaccine-induced T cell response set of an individual. B: Class I HLA-restricted epitope (PEPI3+). The OPA between the predicted epitope and the CD8+ T cell response was 28% (not statistically significant). Darkest gray: True positive (TP), both peptide and T cell response detected; Light gray: False negative (FN), only T cell response detected; Lightest gray: False positive (FP), only peptide detected; Dark gray: True negative (TN): Neither peptide nor T cell response detected. [Figure 4] Distribution of HLA class II PEPI compared to CD4+ T cell response, as measured by the assay, among peptide pools used in the latest technology assay. A: HLA class II-restricted PEPI4+. OPA between PEPI4+ and CD4+ T cell response was 67% (p=0.002). B: Class II HLA-restricted epitope. OPA between class II HLA-restricted epitope and CD4+ T cell response was 66% (not statistically significant). Darkest gray: True positive (TP), both peptide and T cell response detected; Light gray: False negative (FN), only T cell response detected; Lightest gray: False positive (FP), only peptide detected; Dark gray: True negative (TN): Neither peptide nor T cell response detected. [Figure 5]Multiple HLA-binding peptides defining the HPV-16LPV vaccine-specific T-cell response set in 18 VIN-3 and 5 cervical cancer patients. HLA class I-restricted PEPI3 counts (A and B) and HLA class II-restricted PEPI3 counts (C and D) derived from each patient's LPV antigen. Light gray: immune responders measured after vaccination in the clinical trial; dark gray: immune non-responders measured after vaccination in the clinical trial. The results show that three or more HLA class I-binding peptides predict CD8+ T-cell responsiveness, and four or more HLA class II-binding peptides predict CD4+ T-cell responsiveness. [Figure 6] Multiple HLA class I binding peptides that define the HPV vaccine-specific T cell response sets of two patients. A: Four HPV antigens in the HPV vaccine. Boxes represent the length of the amino acid sequence from the N-terminus to the C-terminus. B: Identification process of multiple HLA binding peptides for two patients: Patient HLA sequences labeled as the 4-digit HLA genotype from the right of the patient ID. Lines indicate the positions of the first amino acids of 54 and 91 epitopes that can bind to the HLA (PEPI1+) of patients 12-11 and 14-5, respectively. PEPI2 represents a peptide selected from PEPI1+ that can bind to multiple HLAs of the patient (PEPI2+). PEPI3 represents a peptide that can bind to three or more HLAs of the patient (PEPI3+). PEPI4 represents a peptide that can bind to four or more HLAs of the patient (PEPI4+). PEPI5 represents a peptide that can bind to five or more HLAs of the patient (PEPI5+). PEPI6 represents a peptide that can bind to six or more HLAs in a patient (PEPI6+). C: DNA vaccine-specific PEPI3+ sets from two patients characterize T cell responses specific to those vaccines. [Figure 7] Correlation between PEPI3+ scores of 1 or higher and CTL response rates for peptide targets determined in clinical trials. [Figure 8] Correlation between a PEPI3+ score of 1 or greater and the clinical immune response rate (IPR) for immunotherapy vaccines. Dashed line: 95% confidence interval. [Figure 9]Correlation between immunotherapy vaccines, two or more PEPI3+ scores, and disease control rate (DCR). Dotted line: 95% confidence interval. [Figure 10] HLA trials in IPI responders. Overall survival (OS) of melanoma patients treated with ipilimumab. Data from four independent clinical trials: HLA responders (black line) and HLA non-responders (gray line). Statistical analysis: Cox proportional hazards survival regression. A: Trial 1: 18 HLA responders and 30 HLA non-responders; B: Trial 2: 24 HLA responders and 20 HLA non-responders; C: Trial 3: 6 HLA responders and 11 HLA non-responders; D: Trial 4: 13 HLA responders and 38 HLA non-responders. [Figure 11] Multiple HLA-binding peptides in mutant neoantigens. A: Correlation between mutational load and neoantigen load (neoantigens are neoepitopes according to van Allen) and B: Correlation between PEPI3+ load and clinical benefit (min-Q1-median-Q3-max). [Figure 12] HLA map of rindopepimutt on HLA alleles of subjects in a model population. [Figure 13] Probability of vaccine antigen expression in tumor cells of XYZ patients. In vaccine therapy, there is a greater than 95% probability that 5 out of 12 target antigens will be expressed in the patient's tumor. As a result, using the 12 peptide vaccines together can induce an immune response against at least 5 ovarian cancer antigens with a 95% probability (AGP95). The probability that each peptide will induce an immune response in the patient's XYZ is 84%. AGP50 is the mean (expected value) of 7.9 (this is a measure of the vaccine's effectiveness in attacking tumors in XYZ patients). [Figure 14] These are MRI findings of patient XYZ treated with a personalized (PIT) vaccine. This late-stage, largely pre-treated uterine cancer patient exhibited an unexpected objective response after PIT vaccine treatment. These MRI findings suggest that PIT vaccine, combined with chemotherapy, significantly reduced the patient's tumor burden. The patient is currently continuing PIT vaccine treatment. [Figure 15] The probability of vaccine antigen expression in tumor cells of ABC patients. In the vaccine, there is a greater than 95% probability that four of the 13 target antigens will be expressed in the patient's tumor. As a result, using the 12 peptide vaccines together can induce an immune response against at least four breast cancer antigens with a 95% probability (AGP95). The probability that each peptide will induce an immune response in ABC patients is 84%. AGP50 is the mean (expected value) of the discrete probability distribution and is 6.45 (this is a measure of the vaccine's effectiveness in attacking tumors in ABC patients). [Figure 16] A schematic diagram showing the typical positions of amino acids that overlap between HLA class I-binding epitopes and HLA class II-binding epitopes in a 30-mer peptide. [Modes for carrying out the invention]

[0025] Array description Sequence IDs 1-13 represent additional peptide sequences listed in Table 17. Sequence IDs 14-26 represent personalized vaccine peptides designed for patient XYZ as listed in Table 26. Sequence IDs 27-38 represent personalized vaccine peptides designed for patient A, B, and C as listed in Table 29. Sequence IDs 39-86 represent further 9mer T cell epitopes as described in Table 33.

[0026] Detailed explanation HLA genotype HLA is encoded by the most polymorphic gene in the human genome. Each person has three HLA class I molecules (HLA-A * HLA-B * HLA-C * ) and four HLA class II molecules (HLA-DP * HLA-DQ * HLA-DRB1 * HLA-DRB3 * / 4 * / 5* ) has maternal and paternal alleles. In fact, each person expresses different combinations of six HLA class I molecules and eight HLA class II molecules that present different epitopes from the same protein antigen. The function of HLA molecules is to control the T cell response. However, even with the latest knowledge, it was unclear how human HLA controls T cell activation.

[0027] The nomenclature used to specify the amino acid sequence of HLA molecules is as follows: gene name * Allele: a protein number, for example, HLA-A * 02:25. In this example, "02" refers to the allele. In most cases, alleles are defined by serotype, meaning that the proteins of a given allele will not react with each other in other serological assays. The protein number (in the above example, "25") is assigned sequentially when the protein is discovered. New protein numbers are assigned to all proteins with different amino acid sequences (for example, even a change in one amino acid in the sequence is considered a different protein number). Additional information about the nucleic acid sequence of a given locus may be added to the HLA nomenclature, but such information is not required for the methods described herein.

[0028] An individual's HLA class I or HLA class II genotype may refer to the actual amino acid sequence of each class I or class II HLA in the individual, or it may refer to a nomenclature that specifies, at a minimum, the alleles and protein numbers of each HLA gene, as described above. In some embodiments, an individual's HLA genotype is obtained or determined by assaying a biological sample derived from the individual. The biological sample typically contains the subject's DNA. The biological sample may be, for example, a blood, serum, plasma, saliva, urine, breath, cell, or tissue sample. In some embodiments, the biological sample is a saliva sample. Depending on the embodiment, the biological sample may be a buccal swab sample. HLA genotypes can be obtained or determined using any suitable method. For example, sequences can be determined by sequencing of HLA loci using methods and protocols known in the art. In some embodiments, HLA genotypes are determined using sequence-specific primer (SSP) techniques. In some embodiments, HLA genotypes are determined using sequence-specific oligonucleotide (SSO) techniques. In some embodiments, HLA genotypes are determined using sequence-based typing (SBT) techniques. In some embodiments, HLA genotypes are determined using next-generation sequencing. Alternatively, an individual's HLA set may be stored in a database and accessed using methods known in the art.

[0029] HLA epitope binding A given HLA of a subject will present a limited number of different peptides to T cells, which are produced by the processing of protein antigens within the APC. As used herein, "display" or "present," when used in relation to HLA, refers to the binding of a peptide (epitope) to HLA. In this regard, "displaying" or "presenting" a peptide is synonymous with "binding" a peptide.

[0030] As used herein, the terms “epitope” or “T-cell epitope” refer to a sequence of amino acids contained within a protein antigen that has binding affinity to (can bind to) one or more HLAs. Epitopes are HLA and antigen-specific (as predicted by known methods, HLA-epitope pairs), but not subject-specific. An epitope, T-cell epitope, polypeptide, polypeptide fragment, or composition comprising a polypeptide or its fragment is “immunogenic” to a particular human subject if it can induce a T-cell response (cytotoxic T-cell response or helper T-cell response) in that subject. In some cases, the helper T-cell response is a Th1-type helper T-cell response. In some cases, an epitope, T cell epitope, polypeptide, polypeptide fragment, or composition containing a polypeptide or its fragment is immunogenic to a particular human subject if it is more likely to induce a T cell response or immune response in that subject than any different T cell epitope (or possibly two different T cell epitopes) that can bind to just one HLA molecule in that subject.

[0031] The terms “T cell response” and “immune response” are used interchangeably herein and refer to the activation of T cells following recognition of one or more HLA-epitope binding pairs, and / or the induction of one or more effector functions. In some cases, “immune response” includes antibody responses, as HLA class II molecules stimulate helper responses associated with the induction of both prolonged CTL responses and antibody responses. Effector functions include cytotoxicity, cytokine production, and proliferation. According to this disclosure, an epitope, T cell epitope, or polypeptide fragment is immunogenic to a particular subject if it can bind to at least two, or possibly at least three, class I, or at least two, or possibly at least three, or at least four class II HLAs in that subject.

[0032] For the purposes of this disclosure, we have coined the terms “personal epitope” or “PEPI” to distinguish between HLA-specific epitopes and subject-specific epitopes. A “PEPI” is a polypeptide fragment consisting of a sequence of amino acids of a polypeptide that is a T cell epitope capable of binding to one or more HLA class I molecules of a particular human subject. In other cases, a “PEPI” is a polypeptide fragment consisting of a sequence of amino acids of a polypeptide that is a T cell epitope capable of binding to one or more HLA class II molecules of a particular human subject. In other words, a “PEPI” is a T cell epitope recognized by the HLA set of a particular individual. In contrast to “epitopes,” PEPIs are individual-specific because different individuals have different HLA molecules that bind to different T cell epitopes, each.

[0033] As used herein, "PEPI1" refers to a peptide or polypeptide fragment that can bind to one HLA class I molecule (or, in certain cases, an HLA class II molecule) in an individual. "PEPI1+" refers to a peptide or polypeptide fragment that can bind to one or more HLA class I molecules in an individual.

[0034] "PEPI2" refers to a peptide or polypeptide fragment that can bind to two HLA class I (or II) molecules in an individual. "PEPI2+" refers to a peptide or polypeptide fragment that can bind to two or more HLA class I (or II) molecules in an individual, i.e., a fragment identified according to the methods disclosed herein.

[0035] "PEPI3" refers to a peptide or polypeptide fragment that can bind to three HLA class I (or II) molecules in an individual. "PEPI3+" refers to a peptide or polypeptide fragment that can bind to three or more HLA class I (or II) molecules in an individual.

[0036] "PEPI4" refers to a peptide or polypeptide fragment that can bind to four HLA class I (or II) molecules in an individual. "PEPI4+" refers to a peptide or polypeptide fragment that can bind to four or more HLA class I (or II) molecules in an individual.

[0037] "PEPI5" refers to a peptide or polypeptide fragment that can bind to five HLA class I (or II) molecules in an individual. "PEPI5+" refers to a peptide or polypeptide fragment that can bind to five or more HLA class I (or II) molecules in an individual.

[0038] "PEPI6" refers to a peptide or polypeptide fragment that can bind to all six HLA class I (or six HLA class II) molecules in an individual.

[0039] Generally speaking, epitopes presented by HLA class I molecules are approximately 9 amino acids long, and epitopes presented by HLA class II molecules are approximately 15 amino acids long. However, for the purposes of this disclosure, epitopes may have more or fewer amino acids than 9 (in the case of HLA class I) or more or fewer than 15 amino acids (in the case of HLA class II), as long as they can bind to HLA. For example, an epitope that can bind to class I HLA may have an amino acid length between 7, 8, or 9 and 9, 10, or 11. An epitope that can bind to class II HLA may have an amino acid length between 13, 14, or 15 and 15, 16, or 17.

[0040] Therefore, the disclosures herein include, for example, methods for predicting whether a polypeptide is immunogenic to a particular human subject, or methods for identifying a polypeptide fragment as immunogenic to a particular human subject, and such methods include (i) The polypeptide a. A sequence of 7 to 11 consecutive amino acids that can bind to at least two HLA class I molecules of the subject; or, b. A sequence of 13 to 17 consecutive amino acids that can bind to at least two HLA class II molecules of the subject, A step of determining whether or not to include; and (ii) Predicting that the polypeptide is immunogenic to the subject if it contains at least one sequence that satisfies the requirements of step (i); or predicting that the polypeptide is not immunogenic to the subject if it does not contain at least one sequence that satisfies the requirements of step (i); or identifying a sequence of amino acids as a fragment of a polypeptide that is immunogenic to the subject. Includes.

[0041] It is possible to determine epitopes that will bind to known HLAs using techniques known in the art. Any suitable method can be used, provided that the same method is used to determine pairs that bind to multiple HLA-epitopes that are directly compared. For example, biochemical analysis can be used. It is also possible to use a list of epitopes known to bind to a given HLA. Predictive or modeling software can also be used to determine which epitopes can bind to a given HLA. An example is shown in Table 1. In some cases, T cell epitopes can bind to a given HLA if they have an IC50 or predicted IC50 of less than 5000 nM, less than 2000 nM, less than 1000 nM, or less than 500 nM.

[0042] [Table 1]

[0043] As provided herein, the presentation of T cell epitopes by multiple HLAs of an individual is generally required to evoke a T cell response. Accordingly, the method of the present invention comprises determining whether a polypeptide has a sequence that is a T cell epitope capable of binding to at least two HLA class I molecules or at least two HLA class II (PEPI2+) molecules of a particular human subject.

[0044] The best predictor of a cytotoxic T cell response to a given polypeptide is the presence of at least one T cell epitope presented by three or more HLA class I molecules in an individual (≧1 PEPI3+). Therefore, the method may include a step of determining whether the polypeptide has a sequence that is a T cell epitope capable of binding to at least three HLA class I molecules in a particular human subject. The method may also include a step of determining whether the polypeptide has a sequence that is a T cell epitope capable of binding to exactly three HLA class I molecules in a particular human subject. A helper T cell response may be predicted by the presence of at least one T cell epitope presented by three or more (≧1 PEPI3+) or four or more (≧1 PEPI4+) HLA class II molecules in an individual. Therefore, the method may also include a step of determining whether the polypeptide has a sequence that is a T cell epitope capable of binding to at least three HLA class II molecules in a particular human subject. In other cases, the method includes determining whether the polypeptide has a sequence that is a T cell epitope capable of binding to at least four HLA class II cells of a particular human subject. In other cases, the method includes determining whether the polypeptide has a sequence that is a T cell epitope capable of binding to just three and / or just four HLA class II cells of a particular human subject.

[0045] In some cases, this disclosure may be used to predict whether a polypeptide / fragment will induce both a cytotoxic T cell response and a helper T cell response in a particular human subject. The polypeptide / fragment comprises both an amino acid sequence that is a T cell epitope capable of binding to multiple HLA class I molecules of the subject and an amino acid sequence that is a T cell epitope capable of binding to multiple HLA class II molecules of the subject. The HLA class I binding epitopes and HLA class II binding epitopes may overlap completely or partially. In some cases, fragments of such polypeptides may be identified by the steps of selecting an amino acid sequence that is a T cell epitope capable of binding to multiple (e.g., at least two or at least three) HLA class I molecules of the subject, and then screening one or more longer fragments of the polypeptide that are extended at the N-terminus and / or C-terminus to bind to one or more HLA class II molecules of the subject.

[0046] Some subjects may have two HLA alleles encoding the same HLA molecule (for example, in the case of isozygotes, HLA-A * (Two copies of 02:25). The HLA molecules encoded by these alleles bind to all of the same T cell epitopes. For the purposes of this disclosure, as used herein, “binding to at least two HLA molecules of a subject” includes binding to HLA molecules encoded by two identical HLA alleles in one subject. In other words, “binding to at least two HLA molecules of a subject” could also be expressed as “binding to HLA molecules encoded by at least two HLA alleles of a subject.”

[0047] polypeptide antigen This specification describes methods for predicting whether a polypeptide is immunogenic to a particular human subject, and for identifying a polypeptide fragment that is immunogenic to a particular human subject. As used herein, the term “polypeptide” refers to a full-length protein, a part of a protein, or a peptide characterized as a string of amino acids. As used herein, the term “peptide” refers to a short polypeptide containing amino acids between 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, and 10, or 11, or 12, or 13, or 14, or 15, or 20, or 25, or 30, or 35, or 40, or 45, or 50.

[0048] As used herein, the terms “fragment” or “polypeptide fragment” refer to a string of amino acids or amino acid sequences that is typically shorter in length than a reference polypeptide and contains the same amino acid sequence as the reference polypeptide in a common portion. Such fragments as provided herein may be contained in a larger polypeptide that is a component, where appropriate. In some cases, such fragments may include the full length of the polypeptide, for example, the entire polypeptide (e.g., a 9-amino acid peptide) as a single T cell epitope.

[0049] In some cases, the polypeptide is expressed by pathogenic organisms (e.g., bacteria or parasites), viruses, or cancer cells, or is associated with autoimmune diseases or autoimmune responses or disease-associated cells, or is an allergen, or is an antigen that is a component of a pharmaceutical or pharmaceutical composition such as a vaccine or immunotherapy composition, or is a polypeptide consisting of all or part of such antigen. In some cases, the method of the present disclosure includes a first step of identifying or selecting a suitable polypeptide, for example, a polypeptide further described below.

[0050] Polypeptides or antigens may be expressed in cells, or in particular in affected cells of a subject (e.g., tumor-associated antigens, viruses, polypeptides expressed by intracellular bacteria or parasites, or in vivo products of vaccines or immunotherapy compositions), or may be obtained from the environment (e.g., food, allergens, or drugs). Polypeptides or antigens may be present in samples taken from specific human subjects. Both polypeptide antigens and HLAs can be precisely defined by amino acid sequences or nucleotide sequences, and can be sequenced using methods known in the art.

[0051] The polypeptide or antigen may be a cancer or tumor-associated antigen (TAA). TAAs are proteins expressed in cancer or tumor cells. Cancer or tumor cells may be present in the sample obtained from the subject. Examples of TAAs include neoantigens expressed during tumorigenesis, products of oncogenes and tumor suppressor genes, overexpressed or abnormally expressed intracellular proteins (e.g., HER2, MUC1), antigens produced by oncoviruses (e.g., EBV, HPV, HCV, HBV, HTLV), cancer-testis antigens (CTAs) (e.g., MAGE family, NY-ESO), and cell type-specific differentiation antigens (e.g., MART-1). TAA sequences can be found experimentally, in published scientific papers, or through publicly available databases (e.g., the Ludwig Cancer Institute database (www.cta.lncc.br / ), the Cancer Immunity Database (cancerimmunity.org / peptide / ), and the TANTIGEN Tumor T Cell Antigen Database (cvc.dfci.harvard.edu / tadb / )).

[0052] In some cases, polypeptides or antigens may not be expressed or may be expressed at minimal levels in normal, healthy cells or tissues, but may be expressed at high rates (high frequency) (in those cells or tissues) in subjects with certain diseases or conditions, such as certain types of cancer or tumors (e.g., breast cancer, ovarian cancer, or melanoma), derived from specific cell types or tissues. Further examples of non-limited cancers include non-melanoma skin, lung, prostate, kidney, bladder, stomach, liver, cervix, esophagus, non-Hodgkin lymphoma, leukemia, pancreas, uterine body, lips, oral cavity, thyroid, brain, nervous system, gallbladder, larynx, pharynx, myeloma, nasopharyngeal, Hodgkin lymphoma, testis, and Kaposi's sarcoma. Alternatively, polypeptides may be expressed at low levels in normal, healthy cells but at high levels (overexpression) in subjects with pathological (e.g., cancerous) cells or diseases or conditions. Depending on the circumstances, the polypeptide may be expressed, or expressed at a higher level compared to normal, healthy cells or subjects, in at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of a human subpopulation compatible with such individual or subject. For example, the subpopulation may be compatible with the subject by ethnicity, geographical location, sex, age, disease, type or stage of disease, genotype, or expression of one or more biomarkers.

[0053] In some cases, the frequency of expression can be determined from published figures or scientific papers. In some cases, the method of this disclosure includes a step of identifying or selecting such polypeptides.

[0054] Polypeptides may be associated with or highly (overexpressed) cancer cells or solid tumors. Exemplary cancers include carcinoma, sarcoma, lymphoma, leukemia, germ cell tumor, or blastoma. Cancers may or may not be hormone-related or hormone-dependent (e.g., estrogen or androgen-related cancers). Tumors may or may not be malignant. Cancers may or may not be metastatic.

[0055] In some cases, polypeptides are cancer-testicular antigens (CTAs). CTAs typically do not appear after embryonic development in healthy cells. In healthy adults, CTA expression is limited to male germ cells that do not express HLA and cannot present antigens to T cells; therefore, when expressed in cancer cells, CTAs are considered to be expressible neoantigens. CTA expression is (i) specific to tumor cells, (ii) more frequent in metastatic tumors than in primary tumors, and (iii) conserved among metastatic tumors in the same patient (Gajewski ed. Targeted Therapeutics in Melanoma. Springer New York. 2012).

[0056] Polypeptides are expressed by cells of an individual (e.g., cancer cells), but may be mutant neoantigens that have been altered from similar proteins in normal or healthy cells. The methods of this disclosure may include steps of identifying a polypeptide that is a mutant neoantigen or a polypeptide that is a mutant neoantigen in a particular human subject, or identifying a neoepitope. For example, a neoantigen may be present in a sample obtained from a subject. Mutant neoantigens or neoepitopes can be used to target disease-associated cells, such as cancer cells, that express neoantigens containing the neoantigen or neoepitope. Mutations in polypeptides expressed by cells, for example, cells in a sample taken from a subject, can be detected, for example, by sequencing, but most do not induce an immune response to neoantigen-expressing cells. Currently, the identification of mutant neoantigens that induce an immune response relies on the prediction of mutant HLA-restricted epitopes and further in vitro testing of the immunogenicity of the predicted epitopes in blood samples from an individual. This process is inaccurate, time-consuming, and expensive.

[0057] As provided herein, the identification of mutable epitopes (neoepitopes) that bind to multiple HLA molecules reliably defines mutable neoantigens. Thus, in some cases according to this disclosure, a polypeptide is a mutable neoantigen, and immunogenic fragments of the polypeptide contain (or consist of) neoantigen-specific mutations.

[0058] Polypeptides may also be viral proteins expressed within cells. Examples include HPV16 E6, E7; HIV Tat, Rev, Gag, Pol, Env; HTLV-Tax, Rex, Gag, Env; human herpesvirus proteins; and dengue virus proteins. Polypeptides may also be parasitic proteins expressed within cells, such as malaria proteins.

[0059] Polypeptides may be active ingredients in pharmaceutical compositions such as vaccines or immunotherapy compositions, or optionally, candidate active ingredients for novel pharmaceutical compositions. As used herein, the term “active ingredient” refers to a polypeptide intended to induce an immune response and may include polypeptide products of vaccines or immunotherapy compositions produced in vivo after administration to a subject. In DNA or RNA immunotherapy compositions, polypeptides may be produced in vivo by the cells of the subject to whom the composition is administered. In cell-based compositions, polypeptides may be processed and / or presented by cells of the composition, e.g., autologous dendritic cells or antigen-presenting cells pulsed with the polypeptide or containing an expression construct encoding the polypeptide. Pharmaceutical compositions may include polynucleotides or cells encoding one or more active ingredient polypeptides.

[0060] In other cases, the polypeptide may be a targeted polypeptide antigen of a pharmaceutical, vaccine, or immunotherapy composition. The polypeptide is a targeted polypeptide antigen when the composition is intended or designed to induce an immune response (e.g., a cytotoxic T cell response) that targets or is directed towards the polypeptide. A targeted polypeptide antigen is typically a polypeptide expressed by a pathogen, virus, or infected cell, such as a cancer cell. The targeted polypeptide antigen may be a TAA or CTA.

[0061] Currently, more than 200 clinical trials are researching cancer vaccines that contain tumor antigens.

[0062] Polypeptides may be allergens that enter an individual's body, for example, through the skin, lungs, or oral route.

[0063] Non-exclusive examples of suitable polypeptides include one or more of those listed in Tables 2-7.

[0064] Genetic sequences can be obtained from sequencing of biological material. Sequencing can be performed by any suitable method for determining DNA and / or RNA and / or amino acid sequences. This disclosure utilizes both HLA genotypes and amino acid sequences. However, methods for identifying HLA genotypes from an individual's genetic sequence, and methods for obtaining amino acid sequences from DNA or RNA sequence data, are not the subject of this disclosure.

[0065] [Table 2-1]

[0066] [Table 2-2]

[0067] [Table 2-3]

[0068] Table 2-4

[0069] Table 2-5

[0070] Table 2-6

[0071] Table 3

[0072] Table 4-1

[0073] Table 4-2

[0074] Table 5-1

[0075] Table 5-2

[0076] Table 5-3

[0077] Table 5-4

[0078] Table 5-5

[0079] Table 6-1

[0080] Table 6-2

[0081] Table 6-3

[0082] Table 6-4

[0083] Table 6-5

[0084] Table 6-6

[0085] Table 6-7

[0086] Table 7-1

[0087] Table 7-2

[0088] [Table 7-3]

[0089] [Table 7-4]

[0090] Prediction of an individual's immunological response to polypeptide antigens. Certain polypeptide antigens induce an immune response in only a small percentage of human subjects. Currently, there are no diagnostic tests that can predict whether a polypeptide antigen is likely to induce an immune response in a given individual. In particular, there is a need for tests that can predict whether a person is an immune responder to a vaccine or immunotherapy composition.

[0091] According to this disclosure, a polypeptide antigen-specific T cell response in an individual is defined by the presence within the polypeptide of one or more fragments that can be presented by multiple HLA class I or multiple HLA class II molecules in the individual.

[0092] Where applicable, this disclosure provides a method for predicting whether a subject will exhibit an immune response to the administration of a polypeptide, and an immune response is predicted if the polypeptide is immunogenic according to any method described herein. A cytotoxic T cell response is predicted if the polypeptide contains at least one amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules of the subject. A helper T cell response is predicted if the polypeptide contains at least one amino acid sequence that is a T cell epitope capable of binding to at least two HLA class II molecules of the subject. A cytotoxic T cell response is not predicted if the polypeptide does not contain any amino acid sequence that is a T cell epitope capable of binding to at least two HLA class I molecules of the subject. A helper T cell response is not predicted if the polypeptide does not contain any amino acid sequence that is a T cell epitope capable of binding to at least two HLA class II molecules of the subject.

[0093] In some cases, the polypeptide is the active ingredient of the pharmaceutical composition, and the method includes predicting the development or generation of anti-drug antibodies (ADAs) against the polypeptide. The pharmaceutical composition may be a drug selected from those listed in Table 8. According to this disclosure, ADA development will occur if the polypeptide of the active ingredient is recognized by multiple HLA class II molecules of a subject, resulting in a helper T cell response that supports an antibody response against the active ingredient, or to that extent. The presence of such an epitope (PEPI) may predict the development of ADA in a subject. The method may further include selecting or recommending the administration of a pharmaceutical composition to a particular human subject for the treatment of that subject, which is predicted to induce little or no ADA, or further including administering the composition to the subject at the discretion of the subject. In other cases, the method predicts that a pharmaceutical composition will induce an unacceptable ADA, and the method further includes selecting or recommending a different treatment or therapy. The polypeptide may be a checkpoint inhibitor. The method may include predicting whether a subject will respond to treatment with a checkpoint inhibitor.

[0094] [Table 8]

[0095] Furthermore, currently, there are no studies that can predict the likelihood (likelihood) that an individual will show a clinical response to or obtain clinical benefit from a vaccine or immunotherapy composition. This is important because T-cell responses measured in cohorts of individuals currently participating in clinical trials of vaccines or immunotherapies show little correlation with clinical responses. In other words, the subpopulation of clinical responders is substantially smaller than the subpopulation of immune responders. Therefore, in order to enable the individualization of vaccines and immunotherapies, it is important to predict not only the likelihood of an immune response in a particular subject, but also whether the drug-induced immune response will be clinically effective (e.g., able to kill cancer cells or cells infected with pathogens or pathogens).

[0096] The inventors have found that the presence of at least two polypeptide fragments (epitopes) capable of binding to at least three HLA class I individuals (two or more PEPI3+) in a vaccine or immunotherapy composition predicts a clinical response. In other words, if two or more PEPI3+ can be identified within the active ingredient polypeptide of a vaccine or immunotherapy composition, the individual is likely to be a clinical responder. As used herein, “clinical response” or “clinical benefit” may be prevention or delay of the onset of a disease or condition, improvement of one or more symptoms, induction or prolongation of remission, delay of relapse, recurrence or exacerbation, or any other improvement or stabilization in the subject’s disease state. Where appropriate, “clinical response” may correlate with “disease control” or “objective response” as defined in the Response Evaluation Criteria In Solid Tumors (RECIST) guidelines.

[0097] Therefore, in some cases, this disclosure provides a method for predicting whether a subject will show a clinical response to administration of a pharmaceutical composition, such as a vaccine or immunotherapy composition, which comprises one or more polypeptides as an active ingredient. The method includes determining whether one or more polypeptides together contain at least two different sequences, each of which is a T cell epitope capable of binding to at least two, or possibly at least three, HLA class I molecules of the subject; and predicting that the subject will show a clinical response to administration of the pharmaceutical composition if the one or more polypeptides together contain at least two different sequences, each of which is a T cell epitope capable of binding to at least two, or possibly at least three, HLA class I molecules of the subject; or predicting that the subject will not show a clinical response to administration of the pharmaceutical composition if the one or more polypeptides together contain one or fewer sequences that are T cell epitopes capable of binding to at least two, or possibly at least three, HLA class I molecules of the subject.

[0098] For the purposes of this method, two T cell epitopes are "different" from each other if they have different sequences, or in some cases if they have the same sequence repeated in the target polypeptide antigen. In some cases, different T cell epitopes in the target polypeptide antigen do not overlap with each other.

[0099] In some cases, all of one or more polypeptides or polypeptide fragments of an active ingredient that are immunogenic to a particular human subject are identified using the methods described herein. Identification of at least one polypeptide fragment that is a T cell epitope capable of binding to at least two or at least three HLA class I molecules in a subject predicts that the polypeptide will or is likely to induce a cytotoxic T cell response in the subject. Identification of at least one polypeptide fragment that is a T cell epitope capable of binding to at least two, at least three, or at least four HLA class II molecules in a subject predicts that the polypeptide will or is likely to induce a helper T cell response in the subject. Identification of no polypeptide fragment that is a T cell epitope capable of binding to at least two or at least three HLA class I molecules in a subject predicts that the polypeptide will not or is unlikely to induce a cytotoxic T cell response in the subject. The identification of no polypeptide fragments that are T cell epitopes capable of binding to at least two, at least three, or at least four HLA class II molecules in a subject predicts that the polypeptide will not induce or is unlikely to induce a helper T cell response in the subject. The identification of at least two polypeptide fragments of one or more active ingredients of a vaccine or immunotherapy composition (each fragment being a T cell epitope capable of binding to at least two, or at least three HLA class I molecules in a subject) predicts that the subject is more likely to show or will show a clinical response to the composition. The identification of fewer than two polypeptide fragments that are T cell epitopes capable of binding to at least two, or at least three HLA class I molecules in a subject predicts that the subject is less likely to show or will not show a clinical response to the composition.

[0100] While we do not wish to be bound by theory, one reason why the potential for clinical benefit from vaccines / immunotherapies containing at least two or more HLA-conjugated PEPIs increases is that affected cell populations, such as cancer or tumor cells or cells infected with viruses or pathogens such as HIV, are often heterogeneous both within and between affected subjects. For example, certain cancer patients may or may not express a specific cancer-associated target polypeptide antigen of a vaccine, or those cancers may contain heterogeneous cell populations, some of which (over)express the antigen and others which do not. In addition, the likelihood of developing resistance to the composition via mutations in the targeted PEPI decreases, so the likelihood of resistance decreases when more HLA-conjugated PEPIs are included in or targeted by the vaccine / immunotherapy.

[0101] Therefore, the likelihood that a subject will respond to treatment increases with (i) the presence of more HLA-binding PEPIs in the active ingredient polypeptide; (ii) the presence of more PEPIs in the target polypeptide antigen; and (iii) the (over)expression of the target polypeptide antigen in the subject or in the subject's affected cells. In some cases, for example, if the target polypeptide antigen is present in a sample obtained from the subject, the expression of the target polypeptide antigen in the subject may be known. In other cases, the probability that a particular subject, or the affected cells of a particular subject, (over)express a particular target polypeptide antigen or any combination thereof may be determined using population expression frequency data. Population expression frequency data may be relevant to the subject- and / or disease-appropriate population or intent-to-treat population. For example, the frequency or probability of expression of a particular cancer-associated antigen in a particular cancer (e.g., breast cancer) or in a subject with a particular cancer can be determined by detecting the antigen in a tumor (e.g., a breast cancer tumor sample). In some cases, such expression frequencies may be determined from published figures and scientific papers. In some cases, the method of the present invention includes the step of determining the frequency of expression of a relevant target polypeptide antigen in a relevant population.

[0102] Various pharmacodynamic biomarkers have been disclosed that predict the activity / efficacy of vaccines in individual human subjects and in populations of human subjects. While these biomarkers have been developed particularly for cancer vaccines, similar biomarkers can be used for other vaccines or immunotherapy compositions. These biomarkers can facilitate the development of more effective vaccines, reduce development costs, and be used to evaluate and compare various compositions. Examples of biomarkers are listed below.

[0103] • AG95 - Vaccine efficacy: The number of antigens in a cancer vaccine that express a specific tumor type with a 95% probability. AG95 is an indicator of vaccine efficacy and does not depend on the immunogenicity of the vaccine antigen. AG95 is calculated from tumor antigen expression rate data. Such data can be obtained from experiments published in peer-reviewed scientific journals. Technically, AG95 is determined from the binomial distribution of antigens in the vaccine, taking into account all possible variations and expression rates.

[0104] • PEPI3+ count - Immunogenicity of the vaccine in subjects: PEPI3+ derived from the vaccine is a personal epitope that binds to at least three HLAs in a subject and induces a T-cell response. PEPI3+ can be determined using a PEPI3+ test in subjects whose complete four-digit HLA genotype is known.

[0105] AP Count - Antigenicity of the vaccine in the subject: The number of vaccine antigens that possess PEPI3+. The vaccine contains a sequence derived from a target polypeptide antigen expressed by infected cells. The AP count is the number of antigens in the vaccine that contain PEPI3+, and represents the number of antigens in the vaccine that can induce a T cell response in the subject. The AP count depends only on the subject's HLA genotype and does not depend on the subject's disease, age, or medication, thus characterizing the subject's vaccine antigen-specific T cell response. The correct value is between 0 (no PEPI is presented by the antigen) and the maximum number of antigens (all antigens present PEPI).

[0106] • AP50 - Vaccine antigenicity within a population: The average number of vaccine antigens with PEPI within a population. AP50 is suitable for characterizing vaccine-antigen-specific T cell responses within a given population because it depends on the HLA genotype of subjects within the population.

[0107] • AGP Count - Vaccine Efficacy in Subjects: This is the number of vaccine antigens expressed in tumors using PEPI. The AGP count indicates the number of tumor antigens that the vaccine recognizes and that induce a T-cell response to (hit the target). The AGP count depends on the vaccine-antigen expression rate in the subject's tumors and the subject's HLA genotype. The correct value is between 0 (no PEPI presentation due to expressed antigens) and the maximum number of antigens (all antigens are expressed and present PEPI).

[0108] AGP50 - Effectiveness of cancer vaccine in a population: The average number of vaccine antigens (i.e., AGPs) expressed in tumors indicated by PEPI within the population. AGP50 represents the average number of tumor antigens that can be recognized by the vaccine-induced T-cell response. AGP50 depends on the expression rate of the antigen in the indicated tumor type and the immunogenicity of the antigen in the target population. AGP50 can be used to assess the effectiveness of vaccines in different populations and to compare different vaccines in the same population. The calculation of AGP50 is similar to that used for AG50, except that the expression of expressed vaccine antigens is weighted by the presence of PEPI3+ in the subject. In a theoretical population where each subject has a PEPI derived from each vaccine antigen, AGP50 is equal to AG50. In other theoretical populations where no subject has a PEPI derived from any vaccine antigen, AGP50 is 0. Generally, the following statement is valid: 0 ≤ AGP50 ≤ AG50.

[0109] • mAGP - A candidate biomarker for selecting high-probability responders: The possibility that a cancer vaccine may induce a T-cell response to multiple antigens expressed in the indicated tumor. mAGP is calculated from the expression rate of vaccine antigens (e.g., vaccine antigens in the tumor) and the presence of vaccine-derived PEPI in the subject. Technically, based on the AGP distribution, mAGP is the sum of the probabilities of multiple AGPs (two or more AGPs).

[0110] The results of the predictions described above may be used to inform a physician's decision regarding the treatment of a subject. Accordingly, in some cases, the polypeptide is, for example, an active ingredient in a vaccine or immunotherapy composition, and the method of this disclosure predicts whether a subject will exhibit, is likely to exhibit, or exhibits an immune response and / or clinical response to a treatment involving administration of the active ingredient polypeptide to a subject, or the threshold of the minimum likelihood of exhibiting such an immune response. The method further includes selecting a treatment for a particular human subject, or a vaccine or immunotherapy composition for the treatment of a particular human subject. Furthermore, methods of treatment using subject-specific pharmaceutical compositions, kits, or panels of polypeptides, each containing one or more polypeptides as active ingredients, are also provided, wherein the pharmaceutical composition, kit, or panel of polypeptides has been determined to have a threshold of the minimum likelihood of inducing a clinical response in a subject, where the likelihood of response is determined using the method described herein. Depending on the circumstances, the minimum threshold may be defined by one or more pharmacodynamic biomarkers described herein, such as the minimum PEPI3+ count (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more PEPI3+), the minimum AGP count (e.g., AGP = at least 2, or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more), and / or the minimum mAGP (e.g., AGP = at least 2, or at least 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more). For example, depending on the circumstances, a subject may be selected for treatment if the probability of a targeted response is greater than a predetermined threshold (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more) when the predetermined number of target polypeptide antigens (optionally, whether or not the target polypeptide antigen is expressed) exceeds a predetermined threshold. Alternatively, the method may further include predicting that one or more polypeptides of the composition will not induce a T cell response and / or a clinical response in the subject, and selecting a different treatment for a particular human subject.

[0111] Prediction of autoimmune or adverse immune responses to polypeptide antigens HLA differences can affect the probability of developing autoimmune diseases, conditions, or responses. The methods of this disclosure may be used to identify polypeptides or polypeptide fragments that are immunogenic and / or associated with autoimmune diseases or autoimmune responses. The methods may include determining whether a polypeptide contains an amino acid sequence that is a T cell epitope capable of binding to at least three, or at least four, or at least five HLA class I cells in a subject; or, in other cases, a sequence that is a T cell epitope capable of binding to at least four, or at least five, or at least six HLA class II cells in a subject; and identifying the polypeptide or the sequence as immunogenic or associated with or related to autoimmune diseases or autoimmune responses in the subject.

[0112] HLA differences can also affect the probability that a subject will experience immunotoxicity from a drug or polypeptide administered to them. An adverse immune response may occur if the polypeptide administered to a subject contains a fragment that corresponds to an antigen expressed in the subject's normal, healthy cells and includes an amino acid that is a T-cell epitope capable of binding to multiple HLA class I molecules in the subject. Therefore, in some cases according to this disclosure, the method is used to identify an adverse immunogenic region or fragment of a polypeptide, or to identify a subject who is likely to experience immunotoxicity to the administration of one or more polypeptides or fragments thereof. The polypeptide may be an active ingredient in a vaccine or immunotherapy composition.

[0113] The method may include determining whether the polypeptide contains a sequence that is a T cell epitope capable of binding to at least two, or in other cases at least three, HLA class I molecules of the subject. The method may also include determining whether the polypeptide contains a sequence that is a T cell epitope capable of binding to at least four, or at least five, HLA class I molecules of the subject; or whether it contains an amino acid sequence that is a T cell epitope capable of binding to at least four, or at least five, or at least six, or at least seven, HLA class II molecules of the subject. The method may further include identifying such sequence as having adverse immunogenicity to the subject, or predicting an adverse immune response in the subject. In other cases, such amino acid sequence is not identified, and the method further includes predicting no adverse immune response in the subject. The method may further include selecting or recommending, for the treatment of a subject, the administration of one or more polypeptides or pharmaceutical compositions that do not induce immunotoxicity or are predicted to have low immunotoxicity, and further treating the subject by administering such polypeptides at an optional level. This disclosure also provides a method for treating a subject in need of treatment by administering a polypeptide or composition relating to the subject.

[0114] In some cases, the methods described herein further include mutating a polypeptide predicted to be immunogenic in a particular human subject, or a polypeptide predicted to be immunogenic in a certain proportion of subjects in a human population. Methods are also provided for reducing the immunogenicity of a polypeptide identified as immunogenic in a particular human subject or a certain proportion of a human population using any one of the methods described herein. The polypeptide may be mutated to reduce the number of PEPIs in the polypeptide, or to reduce the number of HLA class I or class II molecules in the subject or the population that bind to fragments of the polypeptide identified as immunogenic in a certain proportion of the subject or the population. The mutation may reduce or prevent adverse immune responses, or increase efficacy by preventing the development of ADA in a subject or a certain proportion of the population. Mutant polypeptides may be further selected or recommended for the treatment of a subject or subjects in the population. A subject may be further treated by administration of a mutated polypeptide. The disclosure also provides a method for treating a subject in need of treatment by administering such a mutated polypeptide.

[0115] Predicting an individual's response to treatment with checkpoint inhibitors. Typically, some or all tumor-induced tumor-specific T cell clones are inactive or poorly functioning in patients with metastatic cancer. Inactive tumor-specific T cells cannot kill tumor cells. Some of these inactive T cells can be reactivated by checkpoint inhibitors (such as ipilimumab), e.g., monoclonal antibodies that recognize checkpoint molecules (e.g., CTLA-4, PD-1, Lag-3, Tim-3, TIGIT, BTLA). According to this disclosure, treating a subject with a checkpoint inhibitor would be effective only if the expressed cancer antigen is adequately recognized by the individual's HLA, i.e., if an epitope exists in the cancer or disease-related antigen recognized by multiple, preferably at least three, HLA class I molecules of the subject. Therefore, in some cases, the methods of this disclosure may be used to identify one or more subsets of T cell clones that can be reactivated by checkpoint inhibitors, or to predict likely responders to checkpoint inhibitor (immuno) therapy.

[0116] Accordingly, the present disclosure may provide a method for predicting whether a subject will respond to cancer with a checkpoint inhibitor. The method may include the step of identifying or selecting one or more polypeptides or polypeptide fragments that are related to the disease or condition being treated, or related to achieving an immune or clinical response to treatment with a checkpoint inhibitor. The polypeptide may be a tumor-associated and / or mutagenic antigen. The polypeptide may be present in a sample obtained from a subject. The polypeptide may be frequently (over) expressed in the subject and / or disease-appropriate populations. The polypeptide may consist of or comprise PEPI(PEPI3+) identified in subjects known to have responded positively to checkpoint inhibitors. The polypeptide may consist of or comprise amino acid sequences stored or recorded in a database, or extracted from a database.

[0117] Depending on the circumstances, the method may include determining whether the polypeptide contains a sequence that is a T cell epitope capable of binding to multiple HLA class I molecules of the subject. Depending on the circumstances, the presence of at least two, or at least three, four, five, six, seven, or eight different such amino acid sequences may be determined, and / or the presence of at least two, or at least three, four, or five different target polypeptide antigens capable of binding to at least two, or possibly at least three, or at least four HLA class II molecules of the subject, may be determined. Depending on the circumstances, the method may include determining whether the polypeptide contains a sequence that is a T cell epitope capable of binding to at least two, or possibly at least three, or at least four HLA class II molecules of the subject. If the above requirements are met, a response to treatment with a checkpoint inhibitor can be predicted. If the above requirements are not met, no response or no clinical response can be predicted.

[0118] This disclosure provides a method for identifying polypeptide fragments or T cell epitopes within polypeptides that may be targeted by the immune response of a subject after treatment with a checkpoint inhibitor, or that will be targeted by T cells reactivated by treatment with a checkpoint inhibitor.

[0119] The method may further include selecting, recommending, and / or administering a checkpoint inhibitor to a subject predicted to respond, or selecting, recommending, and / or administering a different treatment to a subject predicted not to respond to a checkpoint inhibitor. In other cases, the disclosure provides a method for treating a human subject in need of treatment, the method comprising administering a checkpoint inhibitor to the subject, the subject being predicted to respond to the administration of the checkpoint inhibitor by the method described herein.

[0120] Examples of checkpoint inhibitors include, but are not limited to, PD-1 inhibitors, PD-L1 inhibitors, Lag-3 inhibitors, Tim-3 inhibitors, TIGIT inhibitors, BTLA inhibitors, and CTLA-4 inhibitors. Costimulatory antibodies send positive signals via immunoregulatory receptors, which include, but are not limited to, ICOS, CD137, CD27, OX-40, and GITR. In one embodiment, the checkpoint inhibitor is a CLTA-4 inhibitor.

[0121] Design and preparation of pharmaceutical compositions for individual human subjects In some embodiments, this disclosure provides methods for designing or preparing polypeptides or polynucleic acids encoding polypeptides for inducing an immune response, a cytotoxic T cell response, or a helper T cell response in a particular human subject. This disclosure also provides human subject-specific drugs, immunogenic compositions, or pharmaceutical compositions, kits, or panels of peptides, methods for designing or preparing them, compositions that may be obtained by such methods, and their use in methods for inducing an immune response, a cytotoxic T cell response, or a helper T cell response in a subject, or in methods for providing treatment, vaccination, or immunotherapy to a subject. A pharmaceutical composition, kit, or panel of peptides has one or more polypeptides as active ingredients, the polypeptides together comprising two or more T cell epitopes (PEPIs) that can bind to multiple HLA class I or multiple HLA class II molecules of the subject, which are immunogenic to the subject as described herein or have been identified as immunogenic to the subject by the methods described herein.

[0122] The composition / kit may further optionally comprise at least one pharmaceutically acceptable diluent, carrier, or preservative and / or additional polypeptides that do not contain any PEPIs. The polypeptides may be modified or non-naturally occurring. The kit may comprise one or more separate containers, each containing one or more active ingredient peptides. The composition / kit may be a personalized pharmaceutical product for the prevention, diagnosis, mitigation, treatment, or cure of an individual disease such as cancer.

[0123] Typically, each PEPI is a fragment of a target polypeptide antigen, and a polypeptide containing one or more PEPIs is a target polypeptide antigen for treatment, vaccination, or immunotherapy. The method may include a step of identifying one or more suitable target polypeptide antigens. Typically, each target polypeptide antigen will be associated with the same disease or condition, pathogen or group of pathogens or viruses, or type of cancer.

[0124] For each PEPI, the composition, kit, or panel may contain a sequence of up to 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, or 9 consecutive amino acids of a target polypeptide antigen such as the polypeptides described herein, or the method may include selecting such an amino acid sequence, wherein the sequence of amino acids includes the amino acid sequence of the PEPI.

[0125] In some cases, the amino acid sequence may have additional amino acids adjacent to the N-terminus and / or C-terminus that are not part of the continuous sequence of the target polypeptide antigen. In some cases, the sequence may have up to 41 or 35 or 30 or 25 or 20 or 1 additional amino acids adjacent to the N-terminus and / or C-terminus, or present between target polypeptide fragments. Otherwise, each polypeptide may consist of fragments of the target polypeptide antigen, or consist of two or more fragments that are arranged end-to-end within a single peptide (arranged consecutively from end to end), or overlap (two or more fragments contain partially overlapping sequences, for example, two PEPI within the same polypeptide being within 50 amino acids of each other).

[0126] When fragments of different polypeptides, or fragments derived from different regions of the same polypeptide, are linked together within a modified peptide, a neoepitope may be formed around the joint or junction. Such a neoepitope comprises at least one amino acid derived from each fragment on either side of the joint or junction, and may be referred to herein as the junctional amino acid sequence. Neoepitopes can induce undesirable T cell responses (autoimmunity) against healthy cells. Peptides may be designed or screened to avoid or eliminate neoepitopes corresponding to protein fragments expressed in normal, healthy human cells, and / or neoepitopes that can bind to at least two, possibly at least three, or at least four HLA class I molecules of a subject, or possibly at least two, possibly at least three, or four, or five HLA class II molecules of a subject. The methods of this disclosure may be used to identify or screen for neoepitopes as described herein. Alignment may be determined using known methods such as the BLAST algorithm. The software for performing BLAST analysis is available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0127] At least two of the HLA-binding PEPIs of the composition polypeptide may both target a single antigen (e.g., a polypeptide vaccine containing two HLA-binding PEPIs derived from a single antigen targeted by the vaccine / immunotherapy (e.g., tumor-associated antigen)) or they may target different antigens (e.g., a peptide vaccine containing a first set of HLA-binding PEPIs derived from one antigen (e.g., tumor-associated antigen) and a second set of HLA-binding PEPIs derived from a different antigen (e.g., tumor-associated antigen)).

[0128] In some cases, the active ingredient polypeptide may contain all or at least 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 or more different PEPIs together, or the method may include selecting such PEPIs. PEPIs may be fragments of one or more different target polypeptide antigens. By identifying specific fragments of each target polypeptide antigen that are immunogenic to a particular subject, it is possible to incorporate multiple such fragments, optionally derived from multiple different target polypeptide antigens, into a single active ingredient polypeptide or multiple active ingredient polypeptides for use in combination or to maximize the number of T cell clones that can be activated by one or more polypeptides of a particular length.

[0129] Currently, most vaccines and immunotherapy compositions target only a single polypeptide antigen. However, according to this disclosure, it is beneficial to provide a pharmaceutical composition or polypeptide active ingredient that targets, in some cases, two or more different polypeptide antigens. For example, most cancers and tumors are heterogeneous, meaning that different cancer or tumor cells in a subject (over)express different antigens. Tumor cells in different cancer patients also express various combinations of tumor-associated antigens. The most likely effective anti-cancer immunogenic compositions are those that target multiple antigens expressed by tumors (and therefore more cancer or tumor cells in individual human subjects or in a population).

[0130] The beneficial effects of combining multiple PEPIs in a single treatment (administration of one or more pharmaceutical compositions containing multiple PEPIs) can be illustrated by the individualized vaccine polypeptides described in Examples 17 and 18 below. The exemplary CTA expression probabilities in ovarian cancer are as follows: BAGE: 30%; MAGE A9: 37%; MAGE A4: 34%; MAGE A10: 52%. When patient XYZ is treated with a vaccine containing PEPIs only for BAGE and MAGE A9, the probability of having mAGP (multiple expressed antigens with PEPIs) is 11%. When patient XYZ is treated with a vaccine containing PEPIs only for MAGE A4 and MAGE A10, the probability of having multiAGP is 19%. However, when the vaccine contains all four of these CTAs (BAGE, MAGE A9, MAGE A4, and MAGE A10), the probability of having mAGP is 50%. In other words, the effect is higher than the combined probability of mAGP for both PEPI treatments (probability of mAGP for BAGE / MAGE + probability of mAGP for MAGE A4 and MAGE A10). The PIT vaccine of patient XYZ described in Example 17 contains an additional nine PEPIs, and therefore the probability of having mAGP is over 99.95%.

[0131] Similarly, the probabilities of exemplary CTA expression in breast cancer are as follows: MAGE C2: 21%; MAGE A1: 37%; SPC1: 38%; MAGE A9: 44%. Treatment of patients ABC with a vaccine containing PEPI only in MAGE C2: 21% and MAGE A1 shows a 7% probability of mAGP. Treatment of patients ABC with a vaccine containing PEPI only in SPC1: 38% and MAGE A9 shows an 11% probability of mAGP. Treatment of patients ABC with a vaccine containing PEPI in MAGE C2: 21%; MAGE A1: 37%; SPC1: 38%; MAGE A9 has a 44% (44 > 7 + 11) probability of mAGP. The PIT vaccine for patients ABC described in Example 18 further contains eight PEPIs and therefore has a probability of having mAGP of over 99.93%.

[0132] Therefore, in some cases, the PEPI of the active ingredient polypeptide is derived from two or more different target polypeptide antigens, for example, different antigens associated with a particular disease or condition (e.g., different cancer or tumor-associated antigens or antigens expressed by a target pathogen). In some cases, the PEPI is derived from all or at least 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 or more different target polypeptide antigens. The different target polypeptide antigens may be any different polypeptides that are useful for targeting or that can be selectively targeted by different PEPI3+. The different target polypeptide antigens may be non-homologs or non-paralogs, or have less than 95%, 90%, 85%, 80%, 75%, 70%, 60%, or 50% sequence identity across the full length of each polypeptide. The different polypeptides are not shared by any PEPI3+. Alternatively, if PEPI3+ is not shared with other polypeptide antigens targeted by the active ingredient polypeptide, then PEPI3+ originates from a different target polypeptide antigen.

[0133] In some cases, one or more immunogenic polypeptide fragments are derived from polypeptides present in a sample taken from a specific human subject. This is because the polypeptide is expressed in the subject, for example, exhibiting cancer or tumor-associated antigens or carcinometrizygous antigens expressed in the subject's cancer cells. In some cases, one or more or each polypeptide is a subject's mutagenic neoantigen or an expressible neoantigen. One or more or each fragment may contain neoantigen-specific mutations. Because mutagenic neoantigens are subject-specific, compositions targeting one or more neoantigen-specific mutations are individualized with respect to both those specific diseases and specific HLA sets.

[0134] In other cases, one or more immunogenic polypeptide fragments are not normally expressed or are expressed only minimally in normal, healthy cells or tissues, but are expressed at a high frequency in subjects with a particular disease or condition, as described above, or are derived from a target polypeptide antigen expressed in the affected cells of such subjects. The method may include identifying or selecting such target polypeptide antigens. In some cases, two or more immunogenic polypeptide fragments / PEPIs are derived from different cancer or tumor-associated antigens that are expressed at high frequency (overexpressed) in subjects with cancer, obtained from a certain type of cancer or a specific cell type or tissue. In some cases, the immunogenic polypeptide fragments are derived from all or at least 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, or 40 different cancer or tumor-associated polypeptides. In some cases, one or more polypeptides, each or at least one, at least two, at least three, at least four, at least five, at least six, or at least seven polypeptides, are selected from the antigens listed in any one of Tables 2-7.

[0135] In some cases, one or more of the target polypeptide antigens are cancer testicular antigens (CTAs). In some cases, an immunogenic polypeptide fragment / PEPI is derived from at least one, or at least two, three, four, five, six, seven, eight, nine, ten, eleven, twelfth, thirteen, twelve, thirteen, four, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, fifteen Each of the following is a CTA: one, two, three, four, or five CTAs; or derived from six or more different polypeptide antigens, with one, two, three, four, five, or six of them at random, or at least one, two, three, four, five, or six CTAs; or derived from seven or more different polypeptide antigens, with one, two, three, four, five, six, or seven of them at random, or at least one, two, three, four, five, six, or seven CTAs; or derived from eight or more different polypeptide antigens, with one, two, three, four, five, six, seven, or eight of them at random, or at least one, two, three, four, five, six, seven, or eight CTAs. In some cases, one or more of the target polypeptide antigens are expressed by bacteria, viruses, or parasites.

[0136] In some cases, one or more polypeptide fragments may comprise an amino acid sequence that is a T cell epitope capable of binding to at least two or at least three HLA class I cells in a subject, and one or more polypeptide fragments may comprise an amino acid sequence that is a T cell epitope capable of binding to at least two, at least three, or at least four HLA class II cells in a subject, with the HLA class I and HLA class II binding fragments being optionally redundant. Compositions prepared in this manner may induce both cytotoxic T cell responses and helper T cell responses in specific human subjects.

[0137] Immunogenicity and pharmaceutical composition, treatment method and administration method In some aspects, this disclosure relates to the above-mentioned pharmaceutical compositions, kits, or panels of polypeptides having one or more polypeptides as active ingredients. These may be used in methods of providing an immune response induction, treatment, vaccination, or immunotherapy to a subject, and the pharmaceutical composition may be a vaccine or immunotherapy composition. Such treatment involves administering to a subject a pharmaceutical composition comprising one or more polypeptides or all of the polypeptides of the active ingredients of the treatment together. Multiple polypeptides or pharmaceutical compositions may be administered together or sequentially, for example, all pharmaceutical compositions or polypeptides may be administered to a subject over a period of one year, six months, three months, 60 days, 50 days, 40 days, or 30 days.

[0138] The immunogenic or pharmaceutical compositions or kits described herein may contain, in addition to one or more immunogenic peptides, pharmaceutically acceptable excipients, carriers, diluents, buffers, stabilizers, preservatives, adjuvants, or other materials well known to those skilled in the art. Such materials are preferably non-toxic and preferably do not interfere with the pharmaceutically active ingredient. The pharmaceutically active carrier or diluent may be, for example, a water-containing solution. The exact properties of the carrier or other material may depend on the route of administration, e.g., oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intradermal, and intraperitoneal routes.

[0139] The pharmaceutical compositions of this disclosure may contain one or more "pharmaceutically acceptable carriers." These are typically large, slowly metabolized polymers such as proteins, sugars, polylactic acid, polyglycolic acid, high molecular weight amino acids, amino acid copolymers, sucrose (Paoletti et al., 2001, Vaccine, 19:2118), trehalose (WO 00 / 56365), lactose, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those skilled in the art. The pharmaceutical compositions may also contain diluents such as water, saline, or glycerol. Furthermore, auxiliary substances such as wetting agents or emulsifiers and pH buffers may be present. Sterile, pyrogen-free phosphate-buffered saline is a typical carrier (Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20th edition, ISBN:0683306472).

[0140] The pharmaceutical compositions of this disclosure may be lyophilized or in aqueous form, i.e., solutions or suspensions. This type of liquid formulation allows the composition to be administered directly from the form in which it is packaged without the need for reconstitution in an aqueous medium, and is therefore ideal for injection. The pharmaceutical compositions may be supplied in vials or in pre-filled syringes. Syringes may be supplied with or without needles. Syringes may contain a single dose, while vials may contain a single dose or multiple doses.

[0141] The liquid formulations of this disclosure are also suitable for reconstituting other drugs from lyophilized forms. When the pharmaceutical composition is used for such immediate reconstitution, this disclosure provides a kit which may include two vials, or one pre-filled syringe and one vial, the contents of which are used to reconstitute the contents of the vial before injection.

[0142] The pharmaceutical compositions of this disclosure may contain antimicrobial agents, particularly when packaged in a multi-dose form. Antimicrobial agents such as 2-phenoxyethanol or parabens (methyl, ethyl, propylparaben) may be used. Any preservatives are preferably present at low levels. Preservatives may be added externally and / or may be components of the bulk antigen that forms the mixed composition (e.g., present as a preservative in pertussis antigen).

[0143] The pharmaceutical compositions of this disclosure may contain a detergent, such as Tween (polysorbate), DMSO (dimethyl sulfoxide), or DMF (dimethylformamide). The detergent is generally present at low levels, such as less than 0.01%, but may be used at higher levels, such as 0.01 to 50%.

[0144] The pharmaceutical compositions of this disclosure may contain a sodium salt (e.g., sodium chloride) and free phosphate ions in solution (e.g., by using a phosphate buffer).

[0145] In certain embodiments, the pharmaceutical composition may be encapsulated in a suitable vehicle for either delivering the peptide to antigen-presenting cells or for increasing its stability. As will be understood by those skilled in the art, a variety of media are suitable for delivering the pharmaceutical compositions of this disclosure. Not limited examples of well-constructed fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers, and other phospholipid-containing systems. Methods for incorporating pharmaceutical compositions into delivery vehicles are known in the art.

[0146] To increase the immunogenicity of the composition, the pharmaceutical composition may contain one or more adjuvants and / or cytokines.

[0147] Suitable adjuvants include aluminum salts such as aluminum hydroxide or aluminum phosphate, but may also be salts of calcium, iron, or zinc, or an insoluble suspension of acylated tyrosine or acylated sugar, or cationic or anionic derivatized sugars, polyphosphazenes, biodegradable microspheres, monophosphoryl lipid A (MPL), lipid A derivatives (e.g., with reduced toxicity), 3-O-deacetylated MPL [3D-MPL], quil A, saponins, QS21, Freund's incomplete adjuvant (Difco Laboratories, Detroit, Michigan), Merck adjuvant 65 (Merck and Company, Inc., Loway, New Jersey), AS-2 (Smith-Kline). The adjuvants may also be CpG oligonucleotides, bioadhesives and mucosal adhesives, microparticles, liposomes, polyoxyethylene ether preparations, polyoxyethylene ester preparations, muramil peptides, or imidazoquinolone compounds (e.g., imiquimod and its homologs). Human immunomodulators suitable for use as adjuvants in this disclosure include cytokines such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), macrophage colony-stimulating factor (M-CSF), tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF).

[0148] In some embodiments, the composition comprises an adjuvant selected from the group consisting of Montanide ISA-51 (Seppic, Inc., Fairfield, New Jersey, USA), QS-21 (Aquilia Biopharmaceuticals, Inc., Lexington, Massachusetts, USA), GM-CSF, cyclophosamide, Bacillus calmette-Guéran (BCG), Corynebacterium parvum, levamisol, azimezone, isoprinizone, dinitrochlorobenzene (DNCB), keyhole limpet hemocyanin (KLH), Freund's adjuvant (complete or incomplete), mineral gel, aluminum hydroxide (Alum), lysolecithin, pluronic polyol, polyanion, peptide, oil emulsion, dinitrophenol, and diphtheria toxin (DT).

[0149] For example, cytokines may be selected from the group consisting of transforming growth factors (TGF) such as TGF-α and TGF-β (but not limited to these); insulin-like growth factor-I and / or insulin-like growth factor-II; erythropoietin (EPO); bone induction factors; interferons (IFN) such as interferon-α, -β, and -γ (but not limited to these); colony-stimulating factors (CSF) such as macrophage-CSF (M-CSF) (but not limited to these); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF). In some embodiments, cytokines may be nerve growth factors such as NGF-β; platelet growth factor; transforming growth factors (TGF) such as TGF-α and TGF-β (but not limited to these); insulin-like growth factors Selected from the group consisting of Factor-I and insulin-like growth factor-II; erythropoietin (EPO); bone induction factor; interferons such as (but not limited to) IFN-α, IFN-β, IFN-γ; colony-stimulating factors (CSF) such as macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF); interleukins such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; interleukins such as (but not limited to) IL-13, IL-14, IL-15, IL-16, IL-17, IL-18; LIF; kit-ligant or FLT-3; angiostatin; thrombospondin; endostatin; tumor necrosis factor (TNF); and LT.

[0150] It is expected that an adjuvant or cytokine may be added in a dose of approximately 0.01 mg to approximately 10 mg per dose, preferably approximately 0.2 mg to approximately 5 mg per dose. Alternatively, the adjuvant or cytokine may be at a concentration of approximately 0.01 to 50%, preferably approximately 2% to 30%.

[0151] In certain embodiments, the pharmaceutical compositions of the present disclosure are prepared by physically mixing an adjuvant and / or cytokine with PEPI under appropriate sterile conditions according to known techniques to produce the final product.

[0152] Examples of suitable compositions and administration methods for polypeptide fragments are provided in Esseku and Adeyeye (2011) and Van den Mooter G. (2006). The preparation of vaccine and immunotherapy compositions is generally described in Vaccine Design, “The subunit and adjuvant approach” (Powell MF & Newman MJ eds. (1995) Plenum Press New York). Similarly conceivable encapsulation in liposomes is described in Fullerton, U.S. Patent 4,235,877.

[0153] In some embodiments, the compositions disclosed herein are prepared as nucleic acid vaccines. In some embodiments, the nucleic acid vaccine is a DNA vaccine. In some embodiments, the DNA vaccine or gene vaccine comprises a plasmid having a promoter, as well as appropriate transcription and translational regulatory elements, and a nucleic acid sequence encoding one or more polypeptides of the Disclosure. In some embodiments, the plasmid also comprises sequences for enhancing, for example, expression levels, intracellular targeting, or proteasome processing. In some embodiments, the DNA vaccine comprises a viral vector comprising a nucleic acid sequence encoding one or more polypeptides of the Disclosure. In additional embodiments, the compositions disclosed herein comprise one or more nucleic acids encoding peptides determined to be immune-responsive to a biological sample. For example, in some embodiments, the composition comprises one or more nucleic acid sequences encoding peptides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more, which include a fragment that is a T cell epitope capable of binding to at least three HLA class I molecules and / or at least three HLA class II molecules of a patient. In some embodiments, the peptides are derived from antigens expressed in cancer. In some embodiments, DNA or gene vaccines also encode immunomodulatory molecules for manipulating the immune response, resulting in enhanced vaccine efficacy, stimulation of the immune system, or reduced immunosuppression. Strategies for enhancing the immunogenicity of DNA or gene vaccines include encoding heterologous versions of antigens, fusing antigens to molecules that activate T cells or induce associative recognition, priming with DNA vectors followed by boosting with viral vectors, and the use of immunomodulatory molecules. In some embodiments, DNA vaccines are delivered in various forms, including, in patches, via microneedles, and by abrasion, particularly by needles, gene guns, and aerosol injectors. In some forms, DNA vaccines are incorporated into liposomes or other forms of nanobodies.In some embodiments, the DNA vaccine may be a delivery system selected from the group consisting of transfection agents; protamine; protamine liposomes; polysaccharide particles; cationic nanoemulsions; cationic polymers; cationic polymer liposomes; cationic nanoparticles; cationic lipid and cholesterol nanoparticles; cationic lipid, cholesterol and PEG nanoparticles; and dendrimer nanoparticles. In some embodiments, the DNA vaccine is administered by inhalation or ingestion. In some embodiments, the DNA vaccine is introduced into the blood, thymus, pancreas, skin, muscle, tumor, or other site.

[0154] In some embodiments, the compositions disclosed herein are prepared as RNA vaccines. In some embodiments, the RNA is non-replicating mRNA or self-amplified RNA derived from a virus. In some embodiments, the non-replicating mRNA encodes the peptide disclosed herein and includes 5' and 3' untranslated regions (UTRs). In some embodiments, the self-amplified RNA derived from a virus encodes not only the peptide disclosed herein but also a viral replication mechanism that enables intracellular RNA amplification and abundant protein expression. In some embodiments, the RNA is introduced directly into an organism. In some embodiments, the RNA is chemically synthesized or transcribed in vitro. In some embodiments, the mRNA is prepared from a linear DNA template using T7, T3, or Sp6 phage RNA polymerase, and the resulting product includes an open reading frame encoding the peptide disclosed herein, adjacent UTRs, a 5' cap, and a poly(A) tail. In some embodiments, various versions of the 5' cap are added during or after the transcription reaction by using a vaccinia virus capping enzyme or by incorporating a synthetic cap or an anti-reverse cap analog. In some embodiments, a poly(A) tail of optimal length is added to the mRNA, either directly from the encoding DNA template or by using poly(A) polymerase. The RNA encodes one or more peptides, each containing a fragment that is a T cell epitope capable of binding to at least three HLA class I and / or at least three HLA class II molecules in the patient. In some embodiments, the fragment is derived from an antigen expressed in cancer. In some embodiments, the RNA contains signals to enhance stability and translation. In some embodiments, the RNA also contains non-native nucleotides to increase half-life or modified nucleosides to alter the immunostimulatory profile. In some embodiments, the RNA is introduced by abrasion, via microneedles, in patches, and in particular by needles, gene guns, aerosol injectors, among many other forms.In some forms, RNA vaccines are incorporated into liposomes or other forms of nanobodies that promote the intracellular uptake of RNA and protect it from degradation. In some embodiments, the RNA vaccine may include transfection agents; protamine; protamine liposomes; polysaccharide particles; cationic nanoemulsions; cationic polymers; cationic polymer liposomes; cationic nanoparticles; cationic lipid and cholesterol nanoparticles; cationic lipid, cholesterol and PEG nanoparticles; dendrimer nanoparticles; and / or naked mRNA; naked mRNA obtained by in vivo electroporation; mRNA complexed with protamine; mRNA associated with positively charged oil-in-water cationic nanoemulsions; and mRNA associated with chemically modified dendrimers and complexed with polyethylene glycol (PEG) lipids. A delivery system selected from the group consisting of: A; mRNA complexed with protamine in PEG-lipid nanoparticles; mRNA associated with cationic polymers such as polyethyleneimine (PEI); mRNA associated with cationic polymers such as PEI and lipid components; mRNA associated with polysaccharide (e.g., chitosan) particles or gels; mRNA in cationic lipid nanoparticles (e.g., 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP) or dioleoylphosphatidylethanolamine (DOPE) lipids); mRNA complexed with cationic lipids and cholesterol; or mRNA complexed with cationic lipids, cholesterol and PEG-lipids. In some embodiments, the RNA vaccine is administered by inhalation or ingestion. In some embodiments, the RNA is introduced into the blood, thymus, pancreas, skin, muscle, tumor, or other site, and / or by intradermal, intramuscular, subcutaneous, intranasal, intranodal, intravenous, intrapancreatic, intratumoral, or other delivery routes.

[0155] The polynucleotide or oligonucleotide components may be naked nucleotide sequences or may be combined with cationic lipids, polymers, or targeting systems. They can be delivered by any available technology. For example, polynucleotides or oligonucleotides may be introduced by needle injection, preferably intradermally, subcutaneously, or intramuscularly. Alternatively, polynucleotides or oligonucleotides may be delivered directly across the skin using delivery devices such as particle-mediated gene delivery. Polynucleotides or oligonucleotides may be administered topically to the skin or muscle surface, for example, by nasal, oral, or rectal administration.

[0156] The incorporation of polynucleotide or oligonucleotide constructs may be enhanced by several known transfection techniques, such as those involving the use of transfection agents. Examples of such agents include cationic agents, such as calcium phosphate and DEAE-dextran, and lipofectants, such as lipofectams and transfectams. The dosage of the administered polynucleotide or oligonucleotide can be modified.

[0157] Typically, the dose is a “prophylactically effective dose” or a “therapeutically effective dose” (where prevention may be considered treatment), which is sufficient to produce a clinical response in the individual or to demonstrate a clinical benefit, such as preventing or delaying the onset of a disease or condition, improving one or more symptoms, inducing or prolonging remission, or delaying relapse or recurrence.

[0158] The dosage is determined by various parameters, particularly the substance used; the age, weight, and condition of the individual being treated; the route of administration; and the required therapeutic dose. The amount of antigen in each dose is selected as the amount that induces an immune response. A physician may determine the required route of administration and dosage for any particular individual. The dosage may be provided as a single dose or as multiple doses, for example, at regular intervals, e.g., two, three, or four doses per hour. Typically, peptides, polynucleotides, or oligonucleotides are administered in the range of 1 pg to 1 mg, more typically 1 pg to 10 μg, for particle-mediated delivery, and in the range of 1 μg to 1 mg, more typically 1 to 100 μg, more typically 5 to 50 μg, for other routes. Generally, each dose is expected to contain 0.01 to 3 mg of antigen. The optimal dose for a particular vaccine can be determined by studies observing the immune response of subjects.

[0159] Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 20th edition, 2000, published by Lippincott, Williams & Wilkins.

[0160] In some cases of this disclosure, multiple peptides or compositions of peptides are administered. Two or more pharmaceutical compositions may be administered together / simultaneously and / or at different times or sequentially. Thus, this disclosure includes sets of pharmaceutical compositions and their use. The use of combinations of different peptides, optionally peptides targeting different antigens, is important to overcome the challenges of genetic heterogeneity of tumors and HLA heterogeneity of individuals. Multiple pharmaceutical compositions of PEPIs manufactured for use in a particular therapeutic method may define a formulation.

[0161] Routes of administration include, but are not limited to, intranasal, oral, subcutaneous, intradermal, and intramuscular administration. Subcutaneous administration is particularly preferred. Subcutaneous administration may be by injection into, for example, the abdomen, the lateral and anterior aspects of the upper arm or thigh, the scapular region of the back, or the buttock region on both the upper abdominal and dorsal sides.

[0162] Those skilled in the art will recognize that the compositions of the present disclosure can be administered in one or more doses and by other routes of administration. For example, such other routes include intradermal, intravenous, intravascular, intraarterial, intraperitoneal, intrathecal, intratracheal, intracardiac, intralobar, intraboneal, intrapulmonary, and vaginal administration. Depending on the duration of treatment desired, the compositions of the present disclosure may be administered once or several times, intermittently, for example, once a month, in different doses.

[0163] Solid formulations for oral administration include capsules, tablets, caplets, pills, powders, pellets, and granules. In such solid formulations, the active ingredient is usually combined with one or more pharmaceutically acceptable excipients, examples of which are detailed above. Oral formulations may also be administered as aqueous suspensions, elixirs, or syrups. In these, the active ingredient may be combined with various sweeteners or flavorings, colorants, and, if necessary, emulsifiers and / or suspending agents, as well as diluents such as water, ethanol, glycerin, and combinations thereof.

[0164] One or more compositions of the Disclosure may be administered, and the methods and uses for treatment according to the Disclosure may be performed alone or in combination with other pharmaceutical compositions or treatments, such as chemotherapy and / or immunotherapy and / or vaccines. Other therapeutic compositions or treatments may be one or more of those discussed herein, and may be administered concurrently with or sequentially (before or after) the compositions or treatments of the Disclosure.

[0165] In some cases, treatment may be combined with checkpoint inhibitors, costimulatory antibodies, cytotoxic or non-cytotoxic chemotherapy, and / or radiotherapy, targeted therapy, or monoclonal antibody therapy. Chemotherapy has been shown to sensitize tumors to tumor-specific cytotoxic T cells induced by vaccination (Ramakrishnan et al. J Clin Invest. 2010; 120(4):1111-1124). Examples of chemotherapeutic agents include alkylating agents such as mechloretamine (NH2), cyclophosphamide, ifosfamide, melphalan (L-sarcolicin), and nitrogen mustards such as chlorambucil; anthracyclines; epothilone; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), and streptozosin (streptozotocin); triazenes such as dacarbazine (DTIC; dimethyltriazenoimidazole-carboxamide); ethyleneimine / methylmelamines such as hexamethylmelanin and thiotepa; alkyl sulfonates such as busulfan; antimetabolites including folic acid analogs such as methotrexate (ametopterin); alkylating agents, antimetabolites, fluorouracil (5-fluorouracil; 5-FU), floxyuridine (fluorodeoxyuridine) Pyrimidine analogs such as siuridine (FUdR) and cytarabine (cytosine arabinoside); purine analogs and related inhibitors such as mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2'-deoxycoformycin); enzymes such as epipodophyllotoxin and L-asparaginase; biological response moduloents such as INFα, IL-2, G-CSF, and GM-CSF; platinum coordination complexes such as cisplatin (cic-DDP), oxaliplatin, and carboplatin; anthracendions such as mitoxantrone and anthracycline; substituted ureas such as hydroxyurea; procarbazine (N-methylhydrazine, MIH) and methylhydrazine derivatives including procarbazine; adrenal cortical inhibitors such as mitotane (o,p'-DDD) and aminoglutethimide; taxol and its analogs / derivatives;Adrenocorticosteroid antagonists such as prednisone and its equivalents, dexamethasone and aminoglutethimide; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens such as diethylstilbestrol and ethinylestradiol equivalents; antiestrogens such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-related hormone analogs and leuprolide; and nonsteroidal anti-inflammatory drugs such as flutamide. Examples include hormones / hormone therapy agents containing throgens and agonists / antagonists; vinca alkaloids such as vinblastine (VLB) and vincristine; epipodophyllotoxins such as etoposide and teniposide; antibiotics such as dactinomycin (actinomycin C), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mitramycin), and mitomycin (mitomycin D); enzymes such as L-asparaginase; and natural products containing biological response moduliants such as interferon alfenone.

[0166] Depending on the context, this treatment method may be a method of vaccination or a method of providing immunotherapy. As used herein, “immunotherapy” is the treatment of a disease or condition by inducing or enhancing the immune response of an individual. In certain embodiments, immunotherapy refers to a therapy comprising administering one or more drugs to an individual to induce a T cell response. In certain embodiments, immunotherapy refers to a therapy comprising administering or expressing polypeptides containing one or more PEPIs to an individual to induce a T cell response that recognizes and kills cells that present one or more PEPIs on their cell surface in conjunction with class I HLA. In other specific embodiments, immunotherapy refers to a therapy comprising administering one or more PEPIs to an individual to induce a cytotoxic T cell response against cells that present tumor-associated antigens (TAAs) or cancer-testicular antigens (CTAs) containing one or more PEPIs on their cell surface. In other embodiments, immunotherapy refers to a therapy that involves administering to an individual a polypeptide containing one or more PEPIs presented by class II HLA, or expressing such polypeptides, thereby inducing a T helper response and providing co-stimulation to cytotoxic T cells that recognize and kill infected cells that, together with class I HLA, present one or more PEPIs on their cell surface. In yet another specific embodiment, immunotherapy refers to a therapy that involves administering to an individual one or more drugs to reactivate existing T cells and kill target cells. The theory is that the cytotoxic T cell response eliminates cells presenting one or more PEPIs, thereby improving the clinical condition of the individual. In some cases, immunotherapy may be used to treat tumors. In other examples, immunotherapy may be used to treat diseases or disorders based on intracellular pathogens.

[0167] Depending on the circumstances, this disclosure relates to the treatment of cancer or solid tumors. The treatment may be for any type of cancer or malignant or benign tumor of any cell, tissue, or organ. The cancer may be metastatic or not. Exemplary cancers include carcinoma, sarcoma, lymphoma, leukemia, germ cell tumor, or blastoma. The cancer may be hormone-related or dependent (e.g., estrogen or androgen-related cancer).

[0168] In other cases, this disclosure relates to infections caused by viruses, bacteria, fungi, or parasites, or to any other disease or condition that can be treated by immunotherapy.

[0169] system This disclosure provides a system comprising a storage module configured to store data including a subject's class I and / or class II HLA genotype and the amino acid sequences of one or more test polypeptides; and a computation module configured to identify and / or quantify the amino acid sequences in one or more test polypeptides that can bind to multiple HLAs of the subject. The system may be for obtaining data from at least one sample from at least one subject. The system may include an HLA genotyping module for determining the subject's class I and / or class II HLA genotype. The storage module may be configured to store data output from the genotyping module. The HLA genotyping module can receive a biological sample obtained from a subject and determine the subject's class I and / or class II HLA genotype. Typically, the sample contains the subject's DNA. The sample may be, for example, a blood, serum, plasma, saliva, urine, breath, cell, or tissue sample. The system may further include an output module configured to display the sequence of one or more fragments of one or more polypeptides predicted to be immunogenic to a subject, or the prediction of any output described herein, or the selection or recommendation of any treatment, or the value of any pharmacodynamic biomarker described herein.

[0170] Further embodiments of this disclosure 1. A human subject-specific pharmaceutical composition for treating a disease or disorder in a specific human subject, (a) at least two distinct polypeptides, each having a length of 10 to 50 amino acids, comprising T cell epitopes that bind to at least three HLA class I molecules and / or at least three HLA class II molecules of the subject, wherein the T cell epitopes of the at least two polypeptides are distinct from each other; and (b) Pharmacologically acceptable adjuvants, A pharmaceutical composition specific to human subjects, including [the specified ingredient].

[0171] 2. A human subject-specific pharmaceutical composition according to item 1, comprising at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve different polypeptides.

[0172] 3. A pharmaceutical composition specific to the human subject of item 1, comprising 3 to 40 different polypeptides.

[0173] 4. A pharmaceutical composition specific to the human subject of item 1, wherein the T cell epitopes that bind to at least three HLA class I molecules of the subject contain 7 to 11 amino acids, and / or the T cell epitopes that bind to at least three HLA class II molecules contain 13 to 17 amino acids.

[0174] 5. At least two different polypeptide epitopes are derived from a single antigen. A pharmaceutical composition specific to human subjects of item 1.

[0175] 6. At least two different polypeptide epitopes are derived from two or more antigens. A pharmaceutical composition specific to human subjects as described in item 1.

[0176] 7. A pharmaceutical composition specific to a human subject as described in item 5, wherein the antigen is an antigen expressed by cancer cells, a neoantigen expressed by cancer cells, a cancer-related antigen, a tumor-related antigen, or an antigen expressed by a target pathogen, an antigen expressed by a virus, an antigen expressed by a bacterium, an antigen expressed by a fungus, an antigen associated with an autoimmune disease, or an allergen.

[0177] 8. A pharmaceutical composition specific to the human subject described in item 7, wherein cancer cells are derived from the human subject.

[0178] 9. A pharmaceutical composition specific to the human subject in item 5, wherein the antigen is selected from the antigens listed in Tables 2 to 7.

[0179] 10. A pharmaceutical composition specific to a human subject of item 1, further comprising up to 10 amino acids, wherein at least two different polypeptides are adjacent to a T cell epitope, which is part of a contiguous sequence adjacent to an epitope in a corresponding antigen.

[0180] 11. A pharmaceutical composition specific to a human subject of item 1, further comprising up to 10 amino acids, wherein at least two different polypeptides are adjacent to a T cell epitope that is not part of a contiguous sequence adjacent to an epitope in the corresponding antigen.

[0181] 12. A human subject-specific pharmaceutical composition according to item 1, wherein at least two polypeptides are arranged end-to-end or overlapping within a joined polypeptide.

[0182] 13. A human subject-specific pharmaceutical composition according to item 12, comprising two or more different conjugated polypeptides, wherein the two or more different conjugated polypeptides comprise different epitopes from one another.

[0183] 14. The conjugated polypeptides are screened, and all neoepitopes spanning the junction between the two polypeptides are substantially removed, and the polypeptides are... (i) A fragment of human polypeptide expressed in the healthy cells of the subject; (ii) A T cell epitope capable of binding to at least two HLA class I molecules of the subject; or, (iii) Satisfying both requirements (i) and (ii), A pharmaceutical composition specific to human subjects as described in item 13.

[0184] 15. At least two polypeptides, (i) Equivalent to a fragment of human polypeptide expressed in healthy cells; or (ii) A T cell epitope that corresponds to a fragment of human polypeptide expressed in healthy cells and is capable of binding to at least two HLA class I molecules of the subject, A pharmaceutical composition specific to the human subject of item 1, which does not contain any amino acid sequence.

[0185] 16. A pharmaceutical composition specific to a human subject according to item 1, further comprising a pharmaceutically acceptable diluent, carrier, preservative, or combination thereof.

[0186] 17. A pharmaceutical composition specific to human subjects of item 1, wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, Bacillus calmette-Guélain (BCG), Corynebacterium parvum, Lebamisol, azimezone, isoprinisone, dinitrochlorobenzene (DNCB), keyhole limpet hemocyanin (KLH), Freund's adjuvant (complete), Freund's adjuvant (incomplete), mineral gel, aluminum hydroxide (Alum), lysolecithin, pluronic polyol, polyanion, oil emulsion, dinitrophenol, diphtheria toxin (DT), and combinations thereof.

[0187] 18. A kit comprising one or more separate containers, each container being: (i) one or more polypeptides having a length of 10 to 50 amino acids, comprising an amino acid sequence that is a T cell epitope that binds to at least three HLA class I molecules and / or at least three HLA class II molecules of the subject; and (ii) pharmaceutically acceptable adjuvants, diluents, carriers, preservatives, or combinations thereof A kit that includes this.

[0188] 19. A kit of item 18 comprising at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve different polypeptides, wherein the amino acid sequences of the T cell epitopes of each different polypeptide are different from one another.

[0189] 20. The kit of item 18, which further includes the accompanying documentation.

[0190] 21. A pharmaceutical composition comprising a nucleic acid molecule expressing two or more polypeptides, each polypeptide being 10 to 50 amino acids long and comprising T cell epitopes that bind to at least three HLA class I molecules and / or at least three HLA class II molecules of a subject, wherein each of the two or more polypeptides comprises a different T cell epitope, and the polypeptides do not contain amino acid sequences that are adjacent to each other in the corresponding antigen.

[0191] 22. The pharmaceutical composition of item 21, wherein the nucleic acid molecule expresses at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve different polypeptides, each polypeptide being 10 to 50 amino acids long and comprising an amino acid sequence that is a T cell epitope bound to at least three HLA class I molecules and / or at least three HLA class II molecules of the subject, the amino acid sequences of the T cell epitopes of each different polypeptide being different from one another.

[0192] 23. A human subject-specific pharmaceutical composition for treating a disease or disorder in a particular human subject, the pharmaceutical composition comprising at least one different polypeptide, each comprising a first region and a second region. (i) A first region of 10-50 amino acids in length comprises an amino acid sequence which is a T cell epitope that binds to at least three HLA class I molecules and / or at least three HLA class II molecules of the subject, (ii) The second region, 10 to 50 amino acids in length, comprises an amino acid sequence that is a T cell epitope that binds to at least three HLA class I molecules and / or at least two HLA class II molecules of the subject, The amino acid sequences of the T cell epitopes in the first and second regions of at least three different polypeptides contain different sequences. A pharmaceutical composition specific to human subjects.

[0193] 24. A human subject-specific pharmaceutical composition according to item 23, comprising at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve different polypeptides.

[0194] A pharmaceutical composition specific to the human subject of item 23, comprising 25.2 to 40 different polypeptides.

[0195] 26. A human subject-specific pharmaceutical composition according to item 23, wherein the T cell epitopes that bind to at least three HLA class I molecules of the subject contain 7 to 11 amino acids, and / or the T cell epitopes that bind to at least three HLA class II molecules contain 13 to 17 amino acids.

[0196] 27. A pharmaceutical composition specific to a human subject of item 23, wherein the epitopes in the first and second regions are derived from a single antigen.

[0197] 28. A pharmaceutical composition specific for a human subject according to item 23, wherein the epitopes of the first and second regions are derived from two or more different antigens.

[0198] 29. The antigen is an antigen expressed by cancer cells, a neoantigen expressed by cancer cells, a cancer-related antigen, a tumor-related antigen, or an antigen expressed by a target pathogenic organism, an antigen expressed by a virus, an antigen expressed by bacteria, an antigen expressed by fungi, an antigen associated with an autoimmune disease, or an allergen. A pharmaceutical composition specific for a human subject according to item 27.

[0199] 30. A pharmaceutical composition specific for a human subject according to item 29, wherein the cancer cells are derived from the subject.

[0200] 31. A pharmaceutical composition specific for a human subject according to item 27, wherein the antigen is selected from the antigens listed in Tables 2 to 7.

[0201] 32. The polypeptide is screened and substantially all neoepitopes spanning the junction between the two regions are removed, and the two regions are: (i) corresponding to a fragment of a human polypeptide expressed in healthy cells of the subject; (ii) a T cell epitope capable of binding to at least two HLA class I molecules of the subject; or, (iii) satisfying both requirements of (i) and (ii). A pharmaceutical composition specific for a human subject according to item 23.

[0202] 33. At least one polypeptide is: (i) corresponding to a fragment of a human polypeptide expressed in healthy cells; or (ii) corresponding to a fragment of a human polypeptide expressed in healthy cells and being a T cell epitope capable of binding to at least two HLA class I molecules of the subject. A pharmaceutical composition specific for a human subject according to item 23, which does not contain any amino acid sequence.

[0203] 34. A human subject-specific pharmaceutical composition according to item 23, further comprising a pharmaceutically acceptable adjuvant, diluent, carrier, preservative, or combination thereof.

[0204] 35. A pharmaceutical composition specific to human subjects of item 34, wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, Bacillus calmette-Guélain (BCG), Corynebacterium parvum, Lebamisol, azimezone, isoprinisone, dinitrochlorobenzene (DNCB), keyhole limpet hemocyanin (KLH), Freund's adjuvant (complete), Freund's adjuvant (incomplete), mineral gel, aluminum hydroxide (Alum), lysolecithin, pluronic polyol, polyanion, oil emulsion, dinitrophenol, diphtheria toxin (DT), and combinations thereof.

[0205] 36. A method for preparing a human subject-specific pharmaceutical composition for use in a treatment method for a specific human subject, the method being: (i) A step of selecting a polypeptide fragment, wherein the fragment is identified as immunogenic to the subject as follows: a) A step of determining whether the fragment contains the following amino acid sequence; 1) An amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; or 2) An amino acid sequence that is a T cell epitope capable of binding to at least three HLA class II molecules of the subject; or 3) Satisfying both requirements (1) and (2); and b) the step of identifying the sequence as a polypeptide fragment that is immunogenic to the subject; and, (ii) A step of selecting a first sequence of up to 50 consecutive amino acids of the polypeptide, wherein the consecutive amino acids include the amino acid sequence of the fragment selected in step (i); and, (iii) A step of preparing a pharmaceutical composition specific to a human subject, having one or more polypeptides as active ingredients, together with all of the amino acid sequences selected in the preceding step. A method for preparing a pharmaceutical composition specific to a human subject, including [a specific compound].

[0206] 37. The method of claim 36, further comprising repeating steps (i) to (ii) before the preparation step, and selecting a second amino acid of up to 50 consecutive amino acids of a polypeptide which is the same as or different from the first amino acid sequence.

[0207] 38. The method of claim 37, further comprising the step of repeating steps (i) to (ii) one or more times before the preparation step to select one or more additional amino acid sequences of up to 50 consecutive amino acids of a polypeptide that is the same as or different from the first and second amino acid sequences.

[0208] 39. The method of item 36, wherein, prior to the preparation step, if the fragment selected in step (i) is an HLA class I binding epitope, the step of selecting a longer fragment of the polypeptide, the longer fragment further comprising the fragment selected in step (i); and capable of binding to at least three HLA class II molecules of the subject.

[0209] 40. The method of item 36, wherein each polypeptide comprises one of the selected amino acid sequences, or comprises two or more selected amino acid sequences arranged end-to-end or overlapping within a single conjugated polypeptide.

[0210] 41. Any neoepitope formed at the junction between any two of the selected amino acid sequences and positioned end-to-end within a single junctioned polypeptide is screened, and the following amino acid sequences are selected: (i) Equivalent to a fragment of human polypeptide expressed in healthy cells; (ii) a T cell epitope capable of binding to at least two HLA class I molecules of the subject; or, (iii) the method of item 36, wherein substantially all polypeptides containing both requirements of (i) and (ii) are removed.

[0211] 42. One or more polypeptides are screened, and the following amino acid sequences, (i) corresponding to a fragment of a human polypeptide expressed in healthy cells; or (ii) corresponding to a fragment of a human polypeptide expressed in healthy cells and being a T cell epitope capable of binding to at least two HLA class I molecules of the subject, the method of item 36, wherein the polypeptides containing are removed.

[0212] 43. The method of item 36, further comprising determining the HLA class I genotype and HLA class II genotype derived from a biological sample of the subject before step (i).

[0213] 44. The method of item 43, wherein the determination of the HLA class I genotype and HLA class II genotype is performed by sequence-based typing (SBT) method.

[0214] 45. The method of item 43, wherein the determination of the HLA class I genotype and HLA class II genotype is performed by sequencing, next-generation sequencing, sequence-specific primer (SSP) method, or sequence-specific oligonucleotide (SSO) method.

[0215] 46. The method of item 43, wherein the biological sample is blood, serum, plasma, saliva, buccal swab, urine, exhaled breath, cells, or tissue sample.

[0216] 47. The method of item 43, wherein the biological sample is saliva or buccal swab.

[0217] 48. A method for treating cancer in a specific human subject who needs treatment, The process includes administering a pharmaceutical composition containing at least one polypeptide to a specific human subject. Each of the at least one polypeptides, having a length of 10 to 50 amino acids, comprises a first amino acid sequence that is a T cell epitope that binds to at least three HLA class I molecules and / or at least three HLA class II molecules of the subject. A method in which each T cell epitope of at least one polypeptide is derived from an antigen that is specific to cancer.

[0218] 49. The method of item 48, wherein the composition comprises at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve different polypeptides, the amino acid sequences of each T cell epitope of the different polypeptides being different from one another, and the composition is derived from one or more antigens expressed by cancer cells derived from the subject.

[0219] 50. The method of item 48, wherein the composition comprises 2 to 40 different polypeptides.

[0220] 51. The method of item 48, wherein the T cell epitopes that bind to at least three HLA class I molecules of the subject contain 7 to 11 amino acids, and / or the T cell epitopes that bind to at least three HLA class II molecules of the subject contain 13 to 17 amino acids.

[0221] 52. The method of item 48, wherein the composition comprises at least two different polypeptides, the epitopes of the amino acid sequences of at least two different polypeptides are derived from a single antigen.

[0222] 53. The method of item 48, wherein the composition comprises at least two different polypeptides, the epitopes of at least two different polypeptides derived from two or more different antigens.

[0223] 54. The method of paragraph 48, wherein one or more antigens are neoantigens, cancer-associated antigens, or tumor-associated antigens expressed by cancer cells.

[0224] 55. The method of item 48, wherein one or more antigens are selected from the antigens listed in Table 2.

[0225] 56. The method of item 48, further comprising up to 10 amino acids adjacent to a T cell epitope, wherein at least one different polypeptide is part of a contiguous sequence adjacent to the epitope in the corresponding antigen.

[0226] 57. The method of item 48, further comprising up to 10 amino acids adjacent to a T cell epitope, wherein at least one different polypeptide is not part of a contiguous sequence adjacent to the epitope in the corresponding antigen.

[0227] 58. The method of item 48, wherein the composition comprises at least two different polypeptides, the two polypeptides being joined together, with their ends aligned or overlapping within the polypeptide.

[0228] 59. The method of claim 58, comprising two or more different conjugated polypeptides, wherein the two or more different conjugated polypeptides comprise different epitopes from one another.

[0229] 60. The conjugated polypeptides were screened, and substantially all neoepitopes spanning the junction between the two polypeptides were removed, and the polypeptides were, (i) A fragment of human polypeptide expressed in the healthy cells of the subject; (ii) A T cell epitope capable of binding to at least two HLA class I molecules of the subject; or, (iii) Satisfying both requirements (i) and (ii), The method described in item 59.

[0230] 61. At least one polypeptide, (i) Equivalent to a fragment of human polypeptide expressed in healthy cells; or (ii) A T cell epitope that corresponds to a fragment of human polypeptide expressed in healthy cells and is capable of binding to at least two HLA class I molecules of the subject, The method of item 48, which does not include any amino acid sequence.

[0231] 62. The method of item 48, further comprising a pharmaceutically acceptable adjuvant, diluent, carrier, preservative, or combination thereof.

[0232] 63. The method of item 62, wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, Bacillus calmette-Guélain (BCG), Corynebacterium parvum, Lebamisol, azimezone, isoprinisone, dinitrochlorobenzene (DNCB), keyhole limpet hemocyanin (KLH), Freund's adjuvant (complete), Freund's adjuvant (incomplete), mineral gel, aluminum hydroxide (Alum), lysolecithin, pluronic polyol, polyanion, oil emulsion, dinitrophenol, diphtheria toxin (DT), and combinations thereof.

[0233] 64. The method of item 48, further comprising the step of administering chemotherapy, targeted therapy, radiotherapy, checkpoint inhibitors, other immunotherapies, or a combination thereof.

[0234] 65. A human subject-specific pharmaceutical composition for treating a disease or disorder in a particular human subject, comprising (a) a polypeptide having a length of 10 to 50 amino acids and comprising a T cell epitope that binds to at least three HLA class I molecules and / or at least three HLA class II molecules of the subject; and (b) a pharmaceutically acceptable adjuvant.

[0235] 66. A human subject-specific pharmaceutical composition of item 65, comprising at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, or at least twelve different polypeptides, each different polypeptide having a length of 10 to 50 amino acids, comprising a T cell epitope that binds to at least three HLA class I molecules and / or at least three HLA class II molecules of the subject, wherein the amino acid sequences of the T cell epitopes of each different polypeptide are different from one another.

[0236] 67.2 A pharmaceutical composition specific to human subjects of item 66, comprising 40 different polypeptides.

[0237] 68. A human subject-specific pharmaceutical composition according to item 65, wherein the T cell epitopes that bind to at least three HLA class I molecules of the subject contain 7 to 11 amino acids, and / or the T cell epitopes that bind to at least three HLA class II molecules contain 13 to 17 amino acids.

[0238] 69. A pharmaceutical composition specific to a human subject of item 66, comprising at least two different polypeptides, wherein the epitopes of at least two different polypeptides are derived from a single antigen.

[0239] 70. A pharmaceutical composition specific to a human subject according to item 66, comprising at least two different polypeptides, wherein the epitopes of at least two different polypeptides are derived from two or more different antigens.

[0240] 71. A human subject-specific pharmaceutical composition according to item 69, wherein the antigen is an antigen expressed by cancer cells, a neoantigen expressed by cancer cells, a cancer-associated antigen, a tumor-associated antigen, or an antigen expressed by a target pathogen, an antigen expressed by a virus, an antigen expressed by a bacterium, an antigen expressed by a fungus, an antigen associated with an autoimmune disease, or an allergen.

[0241] 72. A human subject-specific pharmaceutical composition according to item 71, wherein the cancer cells are derived from the subject.

[0242] 73. A pharmaceutical composition specific to the human subject of item 69, wherein the antigen is selected from the antigens listed in Tables 2 to 7.

[0243] 74. A human subject-specific pharmaceutical composition according to claim 69, comprising at least two different polypeptides, the two polypeptides arranged end-to-end or overlapping within a conjugated polypeptide.

[0244] 75. A pharmaceutical composition specific to human subjects of item 65, wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosamide, Bacillus calmette-Guélain (BCG), Corynebacterium parvum, Lebamisol, azimezone, isoprinisone, dinitrochlorobenzene (DNCB), keyhole limpet hemocyanin (KLH), Freund's adjuvant (complete), Freund's adjuvant (incomplete), mineral gel, aluminum hydroxide (Alum), lysolecithin, pluronic polyol, polyanion, oil emulsion, dinitrophenol, diphtheria toxin (DT), and combinations thereof.

[0245] 76. A human subject-specific pharmaceutical composition of item 65, comprising at least two different polypeptides, wherein at least two of the polypeptides are arranged end-to-end or overlapping within a conjugated polypeptide.

[0246] 77. A human subject-specific pharmaceutical composition according to claim 76, comprising two or more different conjugated polypeptides, wherein the two or more different conjugated polypeptides comprise different epitopes from one another.

[0247] 78. The conjugated polypeptides were screened, and substantially all neoepitopes spanning the junction between the two polypeptides were removed, and the polypeptides were, (i) A fragment of human polypeptide expressed in the subject's healthy cells; (ii) A T cell epitope capable of binding to at least two HLA class I molecules of the subject; or, (iii) Satisfying both requirements (i) and (ii), A pharmaceutical composition specific to human subjects as described in item 77.

[0248] 79. At least two polypeptides, (i) Equivalent to a fragment of human polypeptide expressed in healthy cells; or (ii) A T cell epitope that corresponds to a fragment of human polypeptide expressed in healthy cells and is capable of binding to at least two HLA class I molecules of the subject, A pharmaceutical composition specific to the human subject of item 66, which does not contain any amino acid sequence.

[0249] 80. (a)(i) A first polypeptide having a length of 10 to 50 amino acids and comprising a T cell epitope that binds to at least three HLA class I molecules and / or at least three HLA class II molecules of the subject; and (ii) a pharmaceutical composition specific to a first human subject, comprising a pharmaceutically acceptable adjuvant; (b) (i) a second polypeptide having a length of 10 to 50 amino acids and comprising a T cell epitope that binds to at least three HLA class I molecules and / or at least three HLA class II molecules of the subject; and (ii) a pharmaceutical composition specific to a second human subject, comprising a pharmaceutically acceptable adjuvant. Includes, The first and second polypeptides are kits containing different T cell epitopes.

[0250] 81. The kit of item 77, wherein the first composition and / or the second composition comprises one or more additional polypeptides, each additional polypeptide being 10 to 50 amino acids long and comprising an amino acid sequence which is a T cell epitope that binds to at least three HLA class I molecules and / or at least three HLA class II molecules of the subject, the amino acid sequences comprising different T cell epitopes. [Examples]

[0251] Example 1: Prediction process and validation of HLA-epitope binding The predicted binding between specific HLAs and epitopes (9-mer peptides) is based on the immunoepitope database tool for epitope prediction (www.iedb.org).

[0252] The predictive process for HLA I epitope binding was validated by comparing it with HLA I epitope pairs determined by laboratory experiments. The dataset followed HLA I epitope pairs reported in peer-reviewed publications or public immunological databases.

[0253] The percentage of agreement with the experimentally determined dataset (Table 9) was calculated. The bound HLA I epitope pairs in the dataset were correctly predicted with a probability of 93%. Coincidentally, the unbound HLA I epitope pairs were also correctly predicted with a probability of 93%.

[0254] [Table 9]

[0255] The accuracy of predicting multiple HLA-binding epitopes was measured. Based on the specificity and sensitivity of the analysis, using 93% probabilities for both true positive and true negative predictions, and 7% (=100%-93%) probabilities for false positive and false negative predictions, the probability of multiple HLA-binding epitopes existing in humans can be calculated. The probability that multiple HLAs bind to an epitope represents the relationship between the number of HLAs that bind to the epitope and the predicted minimum number of actual bindings. For each definition of PEPI, 3 is the predicted minimum number of HLAs that bind to the epitope (bold).

[0256] [Table 10]

[0257] Using the validated HLA-epitope binding prediction process, all HLA-epitope binding pairs described in the following examples were determined.

[0258] Example 2: Epitope presentation by multiple HLAs predicts cytotoxic T lymphocyte (CTL) response. We determined that the presentation of one or more epitopes of polypeptide antigens by one or more HLA I cells in an individual predicts a CTL response.

[0259] The study involved a retrospective analysis of six clinical trials conducted on 71 cancer patients and 9 HIV-infected patients (Table 11). 1-7 Patients in these studies were treated with HPV vaccine, three different NY-ESO-1 specific cancer vaccines, one HIV-1 vaccine, and a CTLA-4 specific monoclonal antibody (ipilimumab) that has been shown to reactivate CTLs against the NY-ESO-1 antigen in melanoma patients. In all of these clinical trials, antigen-specific CD8+ CTL response (immunogenicity) was measured in study subjects after vaccination. In some cases, a correlation between CTL response and clinical response was reported.

[0260] Patients were not excluded from the retrospective study for any reason other than data availability. 157 patient datasets (Table 11) were randomized using a standard random number generator to create two independent cohorts for training and evaluation studies. In some cases, the cohorts contained multiple datasets from the same patient, resulting in a training cohort with 76 datasets from 48 patients and a trial / validation cohort with 81 datasets from 51 patients.

[0261] [Table 11]

[0262] The reported CTL responses from the training dataset were compared to the HLA I restriction profile of the vaccine antigen epitope (9mer). Antigen sequences and HLA I genotypes for each patient were obtained from publicly available protein sequence databases or peer-reviewed publications, and the HLA I-epitope binding prediction process was blinded to the patients' clinical CTL response data. The number of epitopes from each antigen predicted to bind to at least one (PEPI1+), at least two (PEPI2+), at least three (PEPI3+), at least four (PEPI4+), at least five (PEPI5+), or all six (PEPI6) HLA class I molecules was determined for each patient, and the number of bound HLAs was used as a classifier for the reported CTL response. The true positive rate (sensitivity) and true negative rate (specificity) were determined individually from the training dataset for each classifier (number of HLA bindings).

[0263] ROC analysis was performed for each classification index. In the ROC curve, the true positive rate (sensitivity) was plotted as a function of the false positive rate (1-specificity) for various cutoff points (Figure 1). Each point on the ROC curve represents a sensitivity / specificity pair corresponding to a specific decision threshold (epitope (PEPI) count). The area under the ROC curve (AUC) is the criterion for how well the classification index can distinguish between the two diagnostic groups (CTL responders or non-responders).

[0264] Analysis revealed that predicted epitope presentation by multiple HLA classes in subjects (PEPI2+, PEPI3+, PEPI4+, PEPI5+, or PEPI6) was a better predictive indicator in all cases than epitope presentation by only one or more HLA class I (PEPI1+, AUC=0.48, Table 12).

[0265] [Table 12]

[0266] Individual CTL responses were best predicted by considering the antigen epitopes that could be presented by at least three HLA class I cells in the individual (PEPI3+, AUC=0.65, Table 12). The PEPI3+ threshold count (number of antigen-specific epitopes presented by three or more HLA cells in the individual) that best predicted a positive CTL response was 1 (Table 13). In other words, if at least one antigen-derived epitope is presented by at least three HLA class I cells in the subject (≧1 PEPI3+), then that antigen is likely to cause at least one CTL clone, and the subject is likely to be a CTL responder. By using the PEPI3+ threshold of 1 or more to predict likely CTL responders ("≧1 PEPI3+ test"), a diagnostic sensitivity of 76% was provided (Table 13).

[0267] [Table 13]

[0268] Example 3: Verification of one or more PEPI3+ tests We used a study cohort of 81 datasets from 51 patients to validate the threshold of 1 or greater PEPI3+ for predicting antigen-specific CTL responses. For each dataset in the study cohort, we determined whether the threshold of 1 or greater PEPI3+ was met (at least one antigen-derived epitope presented by at least three class I HLAs in the individual). This was compared to experimentally determined CTL responses reported from clinical trials (Table 14).

[0269] Clinical validation demonstrated that the PEPI3+ peptide induces a CTL response in individuals with an 84% probability. This 84% ​​probability is consistent with the value determined in the PEPI3+ predictive analysis and is an epitope that binds to at least three HLA cells in the individual (Table 10). These data provide strong evidence that PEPI induces an immune response in individuals.

[0270] [Table 14]

[0271] Using the PEPI3+ count as the cutoff value, ROC analysis determined the accuracy of the diagnosis (Figure 2). The AUC value was 0.73. In ROC analysis, an AUC of 0.7 to 0.8 is generally considered to represent a fair diagnosis.

[0272] A count of at least 1 PEPI3+ (≥1 PEPI3+) best predicted the CTL response in the test dataset (Table 15). This result confirmed the threshold determined during training (Table 12).

[0273] [Table 15]

[0274] Example 4: One or more PEPI3+ tests predict CD8+ CTL responsiveness. One or more PEPI3+ trials were compared to previously reported methods for predicting the CTL response of specific human subjects to peptide antigens.

[0275] The HLA genotypes of 28 patients with cervical cancer and VIN-3 who received the HPV-16 synthetic long peptide vaccine (LPV) in two different clinical trials were determined from DNA samples. 8 8 9 10 LPV consists of long peptides that cover the oncogeneic proteins E6 and E7 of the HPV-16 virus. The amino acid sequences of LPV were obtained from these publications. The publications also report the T-cell response of each vaccinated patient to a pool of duplicated vaccine peptides.

[0276] For each patient, we identified LVP epitopes (9mers) presented by at least three patient class I HLA (PEPI3+) and determined their distribution across peptide pools. Peptides containing at least one PEPI3+ (≧1 PEPI3+) were predicted to induce a CTL response. Peptides not containing PEPI3+ were predicted not to induce a CTL response.

[0277] One or more PEPI3+ tests correctly predicted 489 of 512 negative CTL responses measured after vaccination and 8 of 40 positive CTL responses (Figure 3A). Overall, the agreement between one or more PEPI3+ tests and the experimentally determined CD8+ T cell response was 90% (p<0.001).

[0278] For each patient, the distribution of epitopes across the peptide pools presented by at least one patient's class I HLA (≥1 PEPI1+, prediction of HLA-restricted epitopes, prior art methods) was also determined. ≥1 PEPI1+ correctly predicted 116 of 512 negative CTL responses and 37 of 40 positive CTL responses measured after vaccination (Figure 3B). Overall, the agreement between prediction of HLA-restricted epitopes (≥1 PEPI1+) and CD8+ T cell responsiveness was 28% (not statistically significant).

[0279] Example 5: Prediction of HLA class II-restricted CD4+ helper T cell epitopes We investigated the CD4+ T helper response after LPV vaccination in 28 cervical cancer and VIN-3 patients who received HPV-16 synthetic long peptide vaccine (LPV) in two different clinical trials (detailed in Example 4). The sensitivity for predicting HLA class II restricted epitopes was 78%, as the latest tools predicted 84 out of 107 positive responses (positive CD4+ T cell responsiveness to the peptide pool for human DP alleles) (sensitivity = 78%). The specificity was 22%, as 7 out of 31 negative responses could be excluded. Overall, the agreement between HLA-restricted class II epitope prediction and CD4+ T cell responsiveness was 66%, which was not statistically significant.

[0280] Example 6 One or more PEPI3+ tests predict the T cell response to full-length LPV polypeptides. Using the same tests reported in Examples 4 and 5, one or more PEPI3+ tests were used to predict patients' CD8+ and CD4+ T-cell responses to the full-length E6 and E7 polypeptide antigens of the LPV vaccine. The results were compared with experimentally determined responses and reported. The tests correctly predicted CD8+ T-cell responsiveness (PEPI3+) in 11 out of 15 VIN-3 patients showing positive CD8+ T-cell responsiveness test results (sensitivity 73%, PPV 85%), and CD8+ T-cell responsiveness in 2 out of 5 cervical cancer patients (sensitivity 40%, PPV 100%). CD4+ T-cell responsiveness (PEPI4+) was correctly predicted 100% in both VIN-3 and cervical cancer patients (Figure 5).

[0281] Class I and Class II HLA-restrictive PEPI3+ counts were also observed to correlate with the reported clinical benefit in patients vaccinated against LVP. Patients with higher PEPI3+ counts showed either a full or partial response by 3 months.

[0282] Example 7: Case Study pGX3001 is an HPV16-based DNA vaccine containing full-length E6 and E7 antigens with a linker in between. pGX3002 is an HPV18-based DNA vaccine containing full-length E6 and E7 antigens with a linker in between. In Phase II clinical trials, both pGX3001 and pGX3002 vaccines were administered (VGX-3100 vaccination). 1 We investigated the T-cell response in 17 HPV-infected patients with cervical cancer.

[0283] Figures 5-6 show the locations of each epitope (9mer) presented by at least one (PEPI1+), at least two (PEPI2+), at least three (PEPI3+), at least four (PEPI4+), at least five (PEPI5+), or all six (PEPI6) class I HLA in two exemplary patients (patients 12-11 and patients 14-5) within the full-length sequences of two HPV-16 and two HPV-18 antigens in these patients.

[0284] Patients 12-11 showed a total of 54 PEPI1+ counts for the combination vaccine (54 epitopes presented by one or more class I HLAs). Patients 14-5 showed 91 PEPI1+ counts. Therefore, patients 14-5 showed higher PEPI1+ counts for four HPV antigens than patients 12-11. PEPI1+ represents different vaccine antigen-specific HLA-restricted epitope sets in patients 12-11 and 14-5. Only 27 PEPI1+ counts were common between these two patients.

[0285] For PEPI3+ counts (the number of epitopes presented by class I HLA in three or more patients), the results for patients 12-11 and 14-5 were reversed. Patients 12-11 showed a PEPI3+ count of 8, with at least one PEPI3+ present in each of the four HPV16 / 18 antigens. Patients 14-5 showed a PEPI3+ count of 0.

[0286] The reported immune responses of these two patients were consistent with PEPI3+ counts, not PEPI1+ counts. Patients 12-11 developed immune responses to each of the four antigens after vaccination, as measured by ELISpot, while patients 14-15 did not develop immune responses to any of the four antigens of the vaccine. A similar pattern was observed when comparing the sets of PEPI1+ and PEPI3+ for all 17 patients in the trial. There was no correlation between PEPI1+ counts and experimentally determined T-cell responses reported from clinical trials. However, a correlation was observed between T-cell immunity predicted by one or more PEPI3+ trials and reported T-cell immunity. One or more PEPI3+ trials predicted immune responders to the HPV DNA vaccine.

[0287] Furthermore, the diversity of the patients' PEPI3+ sets was similar to the diversity of T-cell responses typically seen in cancer vaccine trials. Patients 12-3 and 12-6 were similar to patients 14-5 and did not have any PEPI3+ that predicted the HPV vaccine would not evoke T-cell immunity. All other patients had at least one PEPI3+ that predicted the HPV vaccine would likely evoke T-cell immunity. Eleven patients had multiple PEPI3+ that predicted the HPV vaccine would likely evoke a polyclonal T-cell response. Patients 15-2 and 15-3 were able to initiate higher T-cell immunity against E6 of both HPVs, but lower immunity against E7. The other patients, 15-1 and 12-11, showed similarly large responses to E7 of HPV18 and HPV16, respectively.

[0288] Example 8: Design of a model population for conducting in silico trials to identify accurate vaccine target candidates in large populations. Complete 4-digit HLA class I genotype (2xHLA-A * xx:xx;2xHLA-B * xx:xx;2xHLA-C * An in silicohuman trial cohort of 433 subjects with xx:xx) and demographic information. This model population consists of ethnically mixed subjects with a total of 152 different HLA alleles representing over 85% of the currently known G allele group.

[0289] A database of a "Big Population" was also established, containing 7,189 subjects characterized by four-digit HLA genotypes and demographic information. This Big Population possesses 328 distinct HLA class I alleles. The distribution of HLA alleles in the model population was significantly correlated with that of the Big Population (Table 16) (Pearson p<0.001). Therefore, the patient model population of 433 individuals represents a population 16 times larger.

[0290] The model population represents 85% of humanity, as determined by HLA diversity and frequency.

[0291] [Table 16]

[0292] Example 9: In silico testing based on the identification of multiple HLA-binding epitopes predicts T cell response rates in reported clinical trials. The objective of this study was to determine whether a model population, such as the one described in Example 8, could be used to predict the CTL response rate of a vaccine (for example, in an in silico efficacy trial).

[0293] Twelve peptide vaccines derived from cancer antigens that induced T-cell responses in subpopulations of subjects were identified from peer-reviewed publications. These peptides were investigated in clinical trials involving a total of 172 patients (from four ethnic groups). T-cell responses induced by the vaccine peptides were determined and reported from blood samples. The immune response rate was determined as the percentage of study subjects showing a positive T-cell response as measured in clinical trials (Figure 7).

[0294] [Table 17]

[0295] In each of the 433 subjects in the model population described in Example 8, 12 peptides were investigated in one or more PEPI3+ tests. The "PEPI3+ score of 1 or higher" for each peptide was calculated as the proportion of subjects in the model population who had at least one vaccine-derived epitope that could bind to at least three HLA class I (PEPI3+ of 1 or higher) specific to the subject. If the corresponding clinical trial stratified the population by selecting patients based on HLA alleles, the model population was also filtered for subjects with each respective allele (e.g., WT1, HLA-A * 0201).

[0296] The experimentally determined response rates reported from the trials were compared with PEPI3+ scores of 1 or higher. The overall agreement rate (OPA) was calculated based on paired data (Table 18). A linear correlation was also found between PEPI3+ scores of 1 or higher and the response rate (R 2 =0.77) (Figure 7). This result indicates that identifying peptides predicted to bind to multiple HLAs in an individual is useful for predicting clinical trial outcomes in silico.

[0297] [Table 18]

[0298] Example 10: In silico testing based on the identification of multiple HLA-binding epitopes predicts the T cell response rate reported in Clinical Trial II. We identified 19 clinical trials using published immune response rates (IRRs) conducted with peptide or DNA-based vaccines (Table 19). These trials involved 604 patients (from 9 ethnic groups) and covered 38 vaccines derived from tumor and viral antigens. Vaccine antigen-specific CTL responses were measured in each trial patient, and response rates for the clinical trial population were calculated and reported.

[0299] Each vaccine peptide from 19 clinical trials was investigated in one or more PEPI3+ trials in each subject of the model population. A PEPI3+ score of 1 or higher for each peptide was calculated as the proportion of subjects in the model population with at least one vaccine-derived PEPI3+ score. The experimentally determined response rates reported from the trials were compared to the PEPI scores as in Example 9 (Table 20). A linear correlation (R) was found between the response rate and a PEPI3+ score of 1 or higher. 2 A value of 0.70 was observed (Figure 8). This result confirms that by identifying peptides predicted to bind to multiple HLAs in an individual, it is possible to predict the subject's T cell response and thus predict the outcome of clinical trials using in silico testing.

[0300] [Table 19]

[0301] [Table 20]

[0302] Example 11: In silico testing based on the identification of multiple HLA-binding epitopes in multiple peptide vaccines predicts the immune response rate of reported clinical trials. IMA901 is a therapeutic vaccine for renal cell carcinoma (RCC) containing nine peptides derived from tumor-associated peptides (TUMAPs) naturally present in human cancer tissue. It was developed from HLA-A in a total of 96 patients with advanced RCC. *O2+ subjects were treated with IMA901 in two independent clinical trials (Phase I and Phase II). Each of the nine peptides in IMA901 was identified in prior art as an HLA-A2-restricted epitope. Based on currently accepted standards, their presence has been detected in kidney cancer patients, and since the study subjects were specifically selected to have at least one HLA molecule capable of presenting each of the peptides, they are all potent candidate peptides to boost the T-cell response to kidney cancer in the study subjects.

[0303] For each subject in the model population, the number of peptides from the nine peptides of the IMA901 vaccine that could bind to three or more HLAs was determined. Since each peptide of the IMA901 vaccine is 9mer, this corresponds to a PEPI3+ count. The results were compared to the immune response rates reported in Phase I and Phase II clinical trials (Table 21).

[0304] [Table 21]

[0305] The results from Phase I and Phase II studies demonstrate variability in immune responses to the same vaccine across different trial cohorts. However, overall, there was good agreement between the response rates predicted by two or more PEPI3+ trials and the reported clinical response rates.

[0306] In a retrospective analysis, clinical researchers in the above trials found that subjects who responded to multiple peptides of the IMA901 vaccine were significantly more likely to experience disease control (stable disease, partial response) than subjects who responded to only one peptide or did not respond at all (p=0.019). Six of the eight subjects who responded to multiple peptides (75%) experienced clinical benefit in the trial, in contrast to 14% and 33% of subjects who responded to zero or one peptide, respectively. A randomized phase II trial confirmed that immune responses to multiple TUMAPs were associated with longer overall survival.

[0307] The presence of PEPI accurately predicted responders to TUMAP; therefore, clinical responders to IMA901 are likely patients who can present two or more PEPIs derived from TUMAP. This subpopulation is HLA-A * Only 27% of patients selected in 02 were included, and according to the results of the clinical trial, 75% of this subpopulation are predicted to experience clinical benefit. The results of the same clinical trial also indicated that if patient selection was based on three or more PEPIs derived from TUMAP, this population would be HLA-A * Even if this represents only 3% of the patient population selected in 02, it suggests that 100% of patients experience clinical benefit. These results suggest that the disease control rate (stable disease or partial response) is between 3% and 27% in the patient population studied in the IMA901 clinical trial. In the absence of a complete response, only a fraction of these patients experience a survival benefit.

[0308] These results explain the lack of survival improvement in the Phase III clinical trial of IMA901. Furthermore, these results suggest that the HLA-A of the study population is not the same. *The enrichment of patient 02 was also demonstrated to be insufficient to reach the primary overall survival endpoint in the Phase III trial of IMA901. As the IMA901 trial researchers pointed out, there is a need to develop companion diagnostics (CDx) to select patients who are more likely to respond to the peptide vaccine. These results also suggest that the selection of patients with two or more TUMAP-specific PEPIs may provide sufficient enrichment to demonstrate a significant clinical benefit of IMA901.

[0309] Example 12: In silico testing based on the identification of multiple HLA-binding epitopes derived from the vaccine predicts the reported experimental clinical response rate. The correlation between PEPI3+ scores of 2 or higher for immunotherapy vaccines determined in the model population described in Example 8 and the reported disease control rates (DCR, the proportion of patients with complete response, partial response, and stable disease) determined in clinical trials was investigated.

[0310] Seventeen clinical trials using peptide- and DNA-based immunotherapy vaccines with published disease control rates (DCR) or objective response rates (ORR) were identified from peer-reviewed scientific journals (Table 22). These trials involved 594 patients (from 5 ethnic groups) and covered 29 tumor and viral antigens. DCR was determined according to the Response Evaluation Criteria for Solid Tumors (RECIST), the current standard for clinical trials, but clinical response is based on the greatest change in cross-sectional dimensions. 42、43、44 When DCR data was unavailable, objective response rate (ORR) data, as defined according to RECIS guidelines, was used.

[0311] Table 23 compares PEPI3+ scores of 2 or higher for each vaccine in the model population with published DCR or ORR. A correlation was observed between predicted and measured DCR, providing further evidence that not only the immunogenicity but also the efficacy of cancer vaccines depends on multiple HLA sequences in the individual (R2 = 0.76) (Figure 9).

[0312] [Table 22-1]

[0313] [Table 22-2]

[0314] [Table 23]

[0315] Example 13: A set of multiple HLA-binding peptides derived from tumor antigens predicts responders to the checkpoint inhibitor immunotherapy ipilimumab. We determined whether the survival benefit of melanoma patients treated with the checkpoint inhibitor ipilimumab could be predicted by the number of melanoma-specific PEPI3+ receptors potentially expressed in the patients' tumors.

[0316] Eighty melanoma-associated antigens (TAAs) were identified from PEPI3+ (IPI-PEPI panel: 627 PEPIs), and a panel of PEPI3+ was selected that was shared by melanoma patients treated with ipilimumab who had long-term clinical benefit, and was absent in patients who did not have long-term clinical benefit. These PEPI3+s are reactivated by ipilimumab and define specific T cells that attack the patient's tumor cells. Patients with specific HLA sequences that can present more melanoma-specific PEPIs have more T cells that are reactivated by ipilimumab and are more likely to benefit from ipilimumab immunotherapy.

[0317] The clinical benefit from ipilimumab treatment was determined for 160 patients from four independent clinical trial cohorts. Two cohorts were derived from trials CA184-007 (10 mg / kg ipilimumab) and CA184-002 (3 mg / kg ipilimumab), and two cohorts were derived from published clinical trial datasets of 10 mg / kg and 3 mg / kg ipilimumab.5、38、39 .

[0318] We predicted 80 melanoma antigen-derived epitopes bound to all six HLA class I identities in each patient, and then calculated the number of melanoma-specific PEPI3+ identities bound to at least three class I HLA identities in each patient (4,668 PEPIs). Each patient with at least one of the 627 PEPIs qualified as a responder. The IPI-PEPI panel predicted overall survival for both 10 mg / kg and 3 mg / kg ipilimumab. The results were highly significant and consistent across four independent cohorts (Figure 10).

[0319] Example 14: Multiple HLA-binding epitopes define a patient's mutagenic neoantigen. The ability of PEPI3+ to identify neoantigens from mutations was determined. PEPI3+ was used in 110 melanoma patients treated with ipilimumab, based on publicly available exome mutation data. 39 The determination was made using the following method. From exome mutation data, 9,502 antigenic mutations were obtained from 110 patients (Figure 11A). There was considerable variability in the median weighting for non-synonymous mutations per sample, ranging from 309 (29-4,738) in the clinical benefit cohort and 147 (7-5,854) in the cohort with minimal or no clinical benefit. Based on the epitope prediction results, these mutations had 211 (8-1,950) and 56 (2-3,444) neoepitopes in the clinical benefit cohort and the cohort with minimal or no clinical benefit, respectively.

[0320] We identified the variant PEPI3+ neoepitopes from the published mutations (Figure 11B and Table 24). These mutations resulted in a median of 16 PEPI neoepitopes and 6 PEPI neoepitopes in the clinical benefit cohort and the cohort with minimal or no clinical benefit, respectively.

[0321] The results indicate that PEPI defines a mutant neoantigen derived from a genetically modified protein expressed within an individual. Such a neoantigen is a PEPI3+ peptide that can activate T cells in the patient's body. If genetic modification occurs within the tumor cells of an individual that produces PEPI3+, this PEPI3+ can induce a T cell response. These PEPI3+-containing peptides can be included in drugs (e.g., vaccines, T-cell therapies) to induce an immune response against the individual's tumor.

[0322] [Table 24]

[0323] Example 15: In silico testing based on the identification of multiple HLA-binding epitopes predicts the reported cellular immune response rate to vaccines targeting mutant antigens. Epidermal growth factor receptor variant III (EGFRvIII) is a tumor-specific mutation widely expressed in glioblastoma multiforme (GBM) and other neoplasms. The mutation splits the codon and generates a novel glycine at the fusion junction. 1、2 This mutation includes an 801 bp in-frame deletion from the extracellular domain of EGFR. This mutation encodes a constitutively active tyrosine kinase that increases tumorigenesis and tumor cell migration and enhances resistance to radiation and chemotherapy. 3、4、5、6、7、8、9 This insertion creates tumor-specific epitopes not found in normal adult tissue, making EGFRvIII a suitable target candidate for anti-tumor immunotherapy. 10 Rindopepimut is a 13-amino acid peptide vaccine (LEEKKGNYVVTDHC) in which an additional C-terminal cysteine ​​residue is attached to the EGFRvIII mutation. 11 .

[0324] In a Phase II clinical trial, a peptide conjugated with keyhole limpet hemocyanin (KLH) was administered to newly diagnosed EGFRvIII-expressing GBM patients. The first three vaccinations were given every other week, starting four weeks after completion of radiation therapy. Subsequent vaccinations were given monthly until radiographic evidence of tumor progression or death was obtained. All vaccinations were administered intradermally in the groin. Immunological evaluation showed that only 3 out of 18 patients developed a cellular immune response as assessed by the DTH response test.

[0325] An in silico study was conducted on 433 subjects from a model population possessing the rindopepimut sequence. Of the 433 subjects, four were PEPI3+, confirming the low immunogenicity found in the Phase II study (Table 25).

[0326] [Table 25]

[0327] The HLA map of rindpepimut on the HLA alleles of subjects in the model population (Figure 12) shows that only a small fraction of the HLA-A and HLA-C alleles can bind to the vaccine epitope, which explains the absence of PEPI3+ in the in silico cohort.

[0328] In a recent Phase III clinical trial, 745 patients were enrolled and further demonstrated ineffectiveness when randomly assigned to either a treatment arm of lindopepimut and temozolomide (n=371) or a control and temozolomide (n=374). 12 Following the interim analysis, the trial was terminated due to ineffectiveness. The analysis showed no significant difference in overall survival: the median overall survival was 20.1 months (95% CI 18.5-22.1) in the lindopepimut group compared to 20.0 months (18.1-21.9) in the control group (HR 1.01, 95% CI 0.79-1.30; p=0.93).

[0329] References for Example 15 1 Bigner et al. Characterization of the epidermal growth factor receptor in human glioma cell lines and xenografts. Cancer Res 1990;50: 8017-22. 2 Libermann et al. Amplification, enhanced expression and possible rearrangement of EGF receptor gene in primary human brain tumours of glial origin. Nature 1985;313: 144-7. 3 Chu et al. Receptor dimerization is not a factor in the signalling activity of a transforming variant epidermal growth factor receptor (EGFRvIII). Biochem J 1997; 324: 855-61. 4 Batra et al. Epidermal growth factor ligand-independent, unregulated, cell-transforming potential of a naturally occurring human mutant EGFRvIII gene. Cell Growth Differ 1995;6: 1251-9. 5 Nishikawa et al. A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. PNAS 1994; 91: 7727-31. 6 Lammering et al. Inhibition of the type III epidermal growth factor receptor variant mutant receptor by dominant-negative EGFR-CD533 enhances malignant glioma cell radiosensitivity. Clin Cancer Res 2004; 10: 6732-43. 7 Nagane et al. A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis. Cancer Res 1996; 56: 5079-86. 8 Lammering et al. Radiation-induced activation of a common variant of EGFR confers enhanced radioresistance. Radiother Oncol 2004; 72: 267-73. 9 Montgomery et al. Expression of oncogenic epidermal growth factor receptor family kinases induces paclitaxel resistance and alters β-tubulin isotype expression. J Biol Chem 2000; 275: 17358-63. 10 Humphrey et al. Anti-synthetic peptide antibody reacting at the fusion junction of deletion-mutant epidermal growth factor receptors in human glioblastoma. PNAS 1990; 87: 4207-11. 11 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. 12 Weller at al. Rindopepimut with temozolomide for patients with newly diagnosed, EGFRvIII-expressing glioblastoma (ACT IV): a randomized, double-blind, international phase 3 trial. Lancet Oncol 2017; 18(10): 1373-1385.

[0330] Example 16: Multiple HLA-binding peptides in an individual can predict immunotoxicity. Thrombopoietin (TPO) is a highly immunogenic protein drug that causes toxicity in many patients. Using state-of-the-art technology, EpiVax / Genentech identified a class II HLA-restricted epitope and found that the most immunogenic region of TPO is located at the C-terminus (U.S. Patent Application Publication No. 20040209324A1).

[0331] This disclosure reveals that multiple class II HLA-binding epitopes (PEPI3+) derived from TPO were determined in US subjects with 400 HLA class II genotypes identified. Most of the PEPI3+ peptides in these individuals were located between amino acids 1-165 within the N-terminal region of TPO. In some subjects, the C-terminus of PEPI3+ was sporadically identified. However, these results differed from those of state-of-the-art technology.

[0332] The published literature supports the disclosed results and provides experimental evidence for the immunotoxic region located at the N-terminus of TPO. 40、41 In many individuals treated with TPO drugs, anti-drug antibodies (ADAs) against this region of the drug were generated. These antibodies not only negated the therapeutic effect of the drug but also induced systemic adverse events, namely antibody-dependent cytotoxicity (ADCC) and immunotoxicity such as complement-dependent cytotoxicity associated with thrombocytopenia, neutropenia, and anemia. These data demonstrate that the identification of multiple HLA-binding peptides in an individual predicts the immunotoxicity of TPO. Therefore, this disclosure is useful for identifying the toxic immunogenic region of a drug, identifying subjects likely to experience immunotoxicity from a drug, identifying the region of polypeptide drugs that may be targeted by ADAs, and identifying subjects likely to experience ADAs.

[0333] Example 17: Personalized immunotherapy composition for the treatment of ovarian cancer This example describes the treatment of an ovarian cancer patient with a personalized immunotherapy composition, the composition being specifically designed for the patient based on the patient's HLA genotype as disclosed herein. This example and subsequent Example 19 provide clinical data supporting the principle of epitope binding by multiple HLAs of a subject for inducing a cytotoxic T cell response on which this disclosure is based.

[0334] HLA class I and class II genotypes were determined from saliva samples of XYZ individuals with metastatic ovarian adenocarcinoma.

[0335] To create personalized pharmaceutical compositions for patient XYZ, thirteen peptides were selected that each met the following two criteria: (i) derived from antigens expressed in ovarian cancer as reported in peer-reviewed scientific publications; and (ii) containing fragments that are T cell epitopes capable of binding to at least three HLA class I cells in patient XYZ (Table 26). In addition, each peptide was optimized to bind to the maximum number of HLA class II cells in the patient.

[0336] [Table 26]

[0337] According to the validation of the PEPI test shown in Table 10, the 11 PEPI3 peptides in this immunotherapy composition can induce a T cell response in XYZ with an 84% probability, and the 2 PEPI4 peptides (POC01-P2 and POC01-P5) can induce a T cell response in XYZ with a 98% probability. The T cell response targeted 13 antigens expressed in ovarian cancer. The expression of these cancer antigens in the patient's XYZ was not tested. Instead, the probability of successfully killing cancer cells was determined based on the probability of antigen expression in the patient's cancer cells and the positive predictive value of the 1 PEPI3+ test (AGP count). The AGP count predicts the efficacy of the vaccine in the subject: the number of vaccine antigens expressed in the tumors (ovarian adenocarcinoma) of patients with PEPI. The AGP count indicates the number of tumor antigens that the vaccine recognizes and induces a T cell response against the patient's tumor (hitting the target). The AGP count depends on the expression rate of vaccine antigens in the subject's tumor and the subject's HLA genotype. The correct value must be between 0 (no PEPI is presented due to expressed antigens) and the maximum number of antigens (all antigens are expressed and PEPI is presented).

[0338] Figure 13 shows the probability that patient XYZ will express one or more of the 12 antigens. AGP95=5, AGP50=7.9, mAGP=100%, AP=13.

[0339] The presence of at least two polypeptide fragments (epitopes) capable of binding to at least three HLAs of an individual in a vaccine or immunotherapy composition (2 PEPI3+) has been determined to predict a clinical response; therefore, a pharmaceutical composition for patient XYZ may consist of at least two of 13 peptides (Table 26). The peptides are synthesized, dissolved in a pharmaceutically acceptable solvent, and mixed with an adjuvant before injection. While it is desirable for patients to receive personalized immunotherapy with at least two peptide vaccines, it is more preferable to increase the probability of killing cancer cells and reduce the likelihood of recurrence.

[0340] For the treatment of patient XYZ, 12 peptides were formulated as 4×3 / 4 peptides (POC01 / 1, POC01 / 2, POC01 / 3, POC01 / 4). One treatment cycle was defined as administering all 13 peptides within 30 days.

[0341] Patient's medical history: Diagnosis: Metastatic ovarian adenocarcinoma Age: 51 Family medical history: Colon cancer and ovarian cancer (mother), breast cancer (grandmother) Tumor pathology BRCAI-185delAG, BRAF-D594Y, MAP2K1-P293S, NOTCHI-S2450N • 2011: First diagnosis of ovarian adenocarcinoma; Wertheim surgery and chemotherapy; lymph node dissection. • 2015: Metastasis in pericardial adipose tissue, resection 2016: Liver metastasis • 2017: Progression of retroperitoneal and mesenteric lymph nodes; early peritoneal cancer with a small amount of ascites. Past treatments • 2012: Paclitaxel-Carboplatin (6x) • 2014: Caelyx-Carboplatin (1x) • 2016-2017 (9 months): Lymparza (olaparib) 2 x 400 mg / day, orally • 2017: Highcamtin injection 5 x 2.5 mg (3 x 1 series / month) PIT vaccine treatment started on April 21, 2017.

[0342] [Table 27]

[0343] Patient tumor MRI findings (reference date: April 15, 2016) The disease was primarily limited to the liver and lymph nodes. The use of MRI limits the detection of lung (pulmonary) metastases. • May 2016 - January 2017: Olaparib treatment • December 25, 2016 (before PIT vaccine treatment): Confirmation of the response obtained with FU2 showed a dramatic reduction in tumor volume. • January-March 2017: TOPO protocol (topoisomerase) April 6, 2017: FU3 showed regrowth of existing lesions and the emergence of new lesions leading to disease progression. ·April 21, 2017: PIT starts • July 21, 2017 (after the second cycle of PIT): FU4 showed abnormal para-pancreatic signaling with continued lesion growth, general spleen enlargement, and increased ascites. July 26, 2017: CBP + Gem + Avastin • September 20, 2017 (after the third cycle of PIT): FU5 showed a reversal of lesion growth and improvement in pancreatic / parapancreatic signaling. This finding suggests pseudo-progression. • November 28, 2017 (after the 4th cycle of PIT): FU6 showed the best response, along with resolution of non-target lesions. The patient's XYZ MRI data are shown in Table 28 and Figure 14.

[0344] [Table 28]

[0345] Example 18: Design of a personalized immunotherapy composition for the treatment of breast cancer The HLA class I and class II genotypes of metastatic breast cancer patients A, B, and C were determined from saliva samples. To create personalized pharmaceutical compositions for patients A, B, and C, twelve peptides were selected, each meeting the following two criteria: (i) derived from antigens expressed in breast cancer as reported in peer-reviewed scientific publications; and (ii) containing fragments that are T cell epitopes capable of binding to at least three HLA class I antigens in patients A, B, and C (Table 29). Furthermore, each peptide was optimized to bind to the maximum number of HLA class II antigens in the patients; the twelve peptides target 12 breast cancer antigens. The probability that patients A, B, and C will express one or more of the twelve antigens is shown in Figure 15.

[0346] [Table 29]

[0347] Expected effect: AGP95=4; PIT vaccine has a 95% chance of inducing a CTL response to four CTAs expressed in BRC09 breast cancer cells. Additional potency parameters: AGP50=6.3, mAGP=100%, AP=12.

[0348] Efficacy detected after the initial vaccination of all 12 peptides: 83% reduction in tumor metabolic activity (PET CT data)

[0349] For the treatment of patient ABC, 12 peptides were formulated as 4x3 peptides (PBR01 / 1, PBR01 / 2, PBR01 / 3, PBR01 / 4). One treatment cycle was defined as administering all 12 different peptide vaccines within 30 days.

[0350] Patient's medical history Diagnosis: Bilateral metastatic breast cancer: Right breast is ER-positive, PR-negative, and Her2-negative; left breast is ER-positive, PR-negative, and Her2-negative. First diagnosis: 2013 (4 years before PIT vaccine treatment) 2016: Extensive metastatic disease with lymph node metastasis both above and below the diaphragm. Multiple liver and lung metastases. Treatment in 2016-2017: Etrozole, Ibrance (palbociclib), and Zoladex

[0351] result March 7, 2017: Before PIT vaccine treatment Multiple metastatic hepatic disease involving true exogenous compression of the common bile duct origin and extensive dilation of the entire intrahepatic bile duct. Adenopathy of the abdominal cavity, hepatic hilum, and retroperitoneum. May 26, 2017: After one PIT cycle Detected efficacy: 83% reduction in tumor metabolic activity (PET-CT) in the liver, lung lymph nodes, and other metastases. Detected safety : Skin response Local inflammation at the injection site within 48 hours after vaccine administration.

[0352] Follow-up investigation: RBC-09 was treated with 5 cycles of the PIT vaccine. She was feeling very well and refused a PET-CT scan in September 2017. In November, she developed symptoms, and a PET-CT scan showed progressive disease, but she refused all treatment. In addition, her oncologist found that she had not taken palbociclib since spring / summer. Patient ABC died in January 2018.

[0353] The combination of pablocyclib and a personalized vaccine was likely responsible for the significant early response observed after vaccine administration. Palbociclib has been shown to improve the activity of immunotherapy by increasing HLA-mediated CTA presentation and reducing Treg proliferation (Goel et al. Nature. 2017:471-475). PIT vaccines may be used as an add-on to modern therapies for maximum efficacy.

[0354] Example 19: Personalized immunotherapy composition for the treatment of patients with advanced metastatic breast cancer. Patient BRC05 was diagnosed with inflammatory breast cancer (IBC) with extensive lymphangitis carcinomatose in the right breast. Inflammatory breast cancer (IBC) is rare but is an aggressive form of locally advanced breast cancer. It is called inflammatory breast cancer because its main symptoms are swelling and redness (the breast often appears inflamed). Most inflammatory breast cancers are invasive tubular carcinomas (originating in the milk ducts). This type of breast cancer is associated with the expression of high-risk human papillomavirus oncoproteins. 1 In fact, HPV16 DNA was detected in this patient's tumor.

[0355] Patient stage in 2011 (6 years before PIT vaccine treatment) T4: Tumors of any size (ulcers or skin nodules) with direct extension to the chest wall and / or skin. pN3a: Metastasis to 10 or more axillary lymph nodes (at least one tumor deposition greater than 2.0 mm; or metastasis to subclavian lymph nodes (level III axillary lymph nodes)).

[0356] Fourteen vaccine peptides were designed and prepared for patient RBC05 (Table 30). Based on population expression data, peptides RBRC05-P01-P10 were created for this patient. The last three peptides in Table 29 (SSX-2, MORC, MAGE-BI) were designed from antigens whose expression was directly measured in this patient's tumor.

[0357] [Table 30]

[0358] T cell responses were measured by measuring peripheral mononuclear cells two weeks after initial vaccination with a mixture of peptides PBRC05_P1, PBRC05_P2, PBRC05_P3, PBRC05_P4, PBRC05_P5, PBRC05_P6, and PBRC05_P7.

[0359] [Table 31]

[0360] The results showed that a single immunization with seven peptides induced a potent T cell response to three of the seven peptides, exhibiting strong, specific T cell responses to MAGE-A11, NY-SAR-35, FSIPI, and MAGE-A9. There were weak responses to AKAP4 and NY-BR-1, and no response to SPAG9.

[0361] Example 20: Personalized immunotherapy composition for the treatment of patients with early metastatic breast cancer. Medical history: Left chest wall removed in 2011 due to neoplasm. Treatment: Aromatase inhibitors and lumbar spine radiation therapy (osseal mets).

[0362] In 2017, prior to PIT vaccine treatment, metastatic lesions were observed in the ventral bow of the right fifth rib and the right third rib. Recurrent malignancies need to be ruled out in the left breast. A metastatic malignant tumor with right axillary lymph node involvement may be present in the right breast.

[0363] [Table 32]

[0364] The patient received two cycles of the PIT vaccine.

[0365] Example 21: Characterization of Toxicity - immunoBLAST We have developed a method for applying any antigen to determine the potential for inducing harmful immune responses, such as autoimmunity. This method is described herein. liver It's called BLAST.

[0366] PolyPEPI1018 contains six 30-mer polypeptides. Each polypeptide consists of two 15-mer peptide fragments derived from antigens expressed in CRCs. Neoepitopes can be generated at the linkage region of the two 15-mer peptides, potentially inducing an undesirable T-cell response (autoimmunity) against healthy cells. liver We evaluated this using the BLAST methodology.

[0367] We designed 16-mer peptides for each component of the 30-mer of polyPEPI1018. Each 16-mer contains eight amino acids from the terminal end of the first 15 residues of the 30-mer and eight amino acids from the starting end of the second 15 residues of the 30-mer, thus extending precisely across the connecting region of the two 15-mers. We then analyzed these 16-mers and identified cross-reactivity regions partially similar to human sequences using BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), which calculates the statistical significance of match by comparing the protein sequence to a sequence database. Since 8mer represents the minimum length required for a peptide to form an epitope and is the distance between anchor points during HLA binding, we selected 8mers from the 16-mers as the test length.

[0368] As shown in Figure 16, the amino acid positions within the polypeptide were numbered. The starting positions of the potential 9-mer peptides that can bind to HLA and form neoepitopes are the 8 amino acids from positions 8 to 15. The starting positions of the tumor antigen-derived peptides contained in the 15-mer that can form pharmaceutically active epitopes are the 7+7=14 amino acids at positions 1 to 7 and positions 16-22. The ratio of potential neoepitope-forming peptides is 36.4% (8 / 22).

[0369] Using the PEPI3+ test, neoepitopes and neo-PEPI were identified within the 9-mer epitope of the connection region. The risk of poly-PEPI1018 inducing an unwanted T-cell response was assessed in a model population of 433 subjects by determining the proportion of subjects with PEPI3+ in the 9-mer of the connection region. The results of the neoepitope / neo-PEPI analysis are summarized in Table 33. Among the 433 subjects in the model population, the mean expected number of epitopes that could be generated by intracellular processing was 40.12. Neoepitopes were frequently generated; 11.61 (28.9%) of the 40.12 epitopes were neoepitopes. While most peptides could be identified as neoepitopes, the number of subjects presenting neoepitopes varied.

[0370] The epitopes possessed by polyPEPI1018 create an average of 5.21 PEPI3+ cells. These PEPIs can activate the subjects' T cells. The amount of potential neoPEPIs was significantly less than that of neoepitopes (3.7%). In some subjects, these neoPEPIs may slightly compete with PEPI for T cell activation. Importantly, the activated neoPEPI-specific T cells did not have targets in healthy tissue.

[0371] [Table 33-1] [Table 33-2]

[0372] References 1Bagarazzi et al. Immunotherapy against HPV16 / 18 generates potent TH1 and cytotoxic cellular immune responses. Science Translational Medicine. 2012; 4(155):155ra138. 2 Gudmundsdotter et al. Amplified antigen-specific immune responses in HIV-1 infected individuals in a double blind DNA immunization and therapy interruption trial. Vaccine. 2011; 29(33):5558-66. 3 Bioley et al. HLA class I - associated immunodominance affects CTL responsiveness to an ESO recombinant protein tumor antigen vaccine. Clin Cancer Res. 2009; 15(1):299-306. 4 Valmori et al. Vaccination with NY-ESO-1 protein and CpG in Montanide induces integrated antibody / Th1 responses and CD8 T cells through cross-priming. Proceedings of the National Academy of Sciences of the United States of America. 2007; 104(21):8947-52. 5Yuan et al. Integrated NY-ESO-1 antibody and CD8+ T-cell responses correlate with clinical benefit in advanced melanoma patients treated with ipilimumab.Proc Natl Acad Sci U S A. 2011;108(40):16723-16728. 6 Kakimi et al. A phase I study of vaccination with NY-ESO-1f peptide mixed with Picibanil OK-432 and Montanide ISA-51 in patients with cancers expressing the NY-ESO-1 antigen.Int J Cancer. 2011;129(12):2836-46. 7Wada et al. Vaccination with NY-ESO-1 overlapping peptides mixed with Picibanil OK-432 and montanide ISA-51 in patients with cancers expressing the NY-ESO-1 antigen. J Immunother. 2014;37(2):84-92. 8 Welters et al. Induction of tumor-specific CD4+ and CD8+ T-cell immunity in cervical cancer patients by a human papillomavirus type 16 E6 and E7 long peptides vaccine. Clin. Cancer Res. 2008; 14(1):178-87. 9Kenter et al. Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N Engl J Med. 2009; 361(19):1838-47. 10 Welters et al. Success or failure of vaccination for HPV16-positive vulvar lesions correlates with kinetics and phenotype of induced T-cell responses. PNAS. 2010; 107(26):11895-9. 11 http: / / www.ncbi.nlm.nih.gov / projects / gv / mhc / main.fcgi?cmd=init The MHC database, NCBI (Accessed Mar 7, 2016). 12 Karkada et al. Therapeutic vaccines and cancer: focus on DPX-0907. Biologics. 2014;8:27-38. 13 Butts et al. Randomized phase IIB trial of BLP25 liposome vaccine in stage IIIB and IV non-small-cell lung cancer. J Clin Oncol. 2005;23(27):6674-81. 14 Yuan et al.Safety and immunogenicity of a human and mouse gp100 DNA vaccine in a phase I trial of patients with melanoma.Cancer Immun. 2009;9:5. 15Kovjazin et al. ImMucin: a novel therapeutic vaccine with promiscuous MHC binding for the treatment of MUC1-expressing tumors. Vaccine. 2011;29(29-30):4676-86. 16 Cathcart et al. Amultivalent bcr-abl fusion peptide vaccination trial in patients with chronic myeloid leukemia.Blood. 2004;103:1037-1042. 17 Chapuis et al. Transferred WT1-reactive CD8+ T cells can mediate antileukemic activity and persist in post-transplant patients. Sci Transl Med. 2013;5(174):174ra27. 18 Keilholz et al. A clinical and immunologic phase 2 trial of Wilms tumor gene product 1 (WT1) peptide vaccination in patients with AML and MDS. Blood; 2009; 113(26):6541-8. 19 Walter et al. Multipeptide immune response to cancer vaccine IMA901 after single-dose cyclophosphamide associates with longer patient survival. Nat Med. 2012;18(8):1254-61. 20Phuphanich et al. Phase I trial of a multi-epitope-pulsed dendritic cell vaccine for patients with newly diagnosed glioblastoma. Cancer Immunol Immunother. 2013;62(1):125-35. 21 Kantoff et al. Overall survival analysis of a phase II randomized controlled trial of a Poxviral-based PSA-targeted immunotherapy in metastatic castration-resistant prostate cancer. J Clin Oncol. 2010;28(7):1099-105. 22 Tagawa et al. Phase I study of intranodal delivery of a plasmid DNA vaccine for patients with Stage IV melanoma. Cancer. 2003;98(1):144-54. 23 Slingluff et al. Randomized multicenter trial of the effects of melanoma-associated helper peptides and cyclophosphamide on the immunogenicity of a multipeptide melanoma vaccine.J Clin Oncol. 2011;29(21):2924-32. 24Kaida et al. Phase 1 trial of Wilms tumor 1 (WT1) peptide vaccine and gemcitabine combination therapy in patients with advanced pancreatic or biliary tract cancer. J Immunother. 2011;34(1):92-9. 25 Fenoglio et al. A multi-peptide, dual-adjuvant telomerase vaccine (GX301) is highly immunogenic in patients with prostate and renal cancer. Cancer Immunol Immunother; 2013; 62:1041-1052. 26 Krug et al. WT1 peptide vaccinations induce CD4 and CD8 T cell immune responses in patients with mesothelioma and non-small cell lung cancer. Cancer Immunol Immunother; 2010; 59(10):1467-79. 27 Slingluff et al. Clinical and immunologic results of a randomized phase II trial of vaccination using four melanoma peptides either administered in granulocyte-macrophage colony-stimulating factor in adjuvant or pulsed on dendritic cells. J Clin Oncol; 2003; 21(21):4016-26. 28Hodi et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med; 2010;363(8):711-23. 29 Carmon et al. Phase I / II study exploring ImMucin, a pan-major histocompatibility complex, anti-MUC1 signal peptide vaccine, in multiple myeloma patients. Br J Hematol. 2014; 169(1):44-56. 30 1572253780046_54.html?newsId=EB4A46A2AC4A52E7C1257AD9001F3186&newsType=1 (Accessed Mar 28, 2016) 31 Trimble et al. Safety, efficacy, and immunogenicity of VGX-3100, a therapeutic synthetic DNA vaccine targeting human papillomavirus 16 and 18 E6 and E7 proteins for cervical intraepithelial neoplasia 2 / 3: a randomised, double-blind, placebo-controlled phase 2b trial. Lancet. 2015;386(10008):2078-88. 32 Cusi et al. Phase I trial of thymidylate synthase poly epitope peptide (TSPP) vaccine in advanced cancer patients. Cancer Immunol Immunother; 2015; 64:1159-1173. 33 Asahara et al. Phase I / II clinical trial using HLA-A24-restricted peptide vaccine derived from KIF20A for patients with advanced pancreatic cancer. J Transl Med; 2013;11:291. 34 Yoshitake et al. Phase II clinical trial of multiple peptide vaccination for advanced head and neck cancer patients revealed induction of immune responses and improved OS. Clin Cancer Res; 2014;21(2):312-21. 35 Okuno et al. Clinical Trial of a 7-Peptide Cocktail Vaccine with Oral Chemotherapy for Patients with Metastatic Colorectal Cancer. Anticancer Res; 2014; 34: 3045-305. 36 Rapoport et al. Combination Immunotherapy after ASCT for Multiple Myeloma Using MAGE-A3 / Poly-ICLC Immunizations Followed by Adoptive Transfer of Vaccine-Primed and Costimulated Autologous T Cells. Clin Cancer Res; 2014; 20(5): 1355-1365. 37Greenfield et al. A phase I dose-escalation clinical trial of a peptidebased human papillomavirus therapeutic vaccine with Candida skin test reagent as a novel vaccine adjuvant for treating women with biopsy-proven cervical intraepithelial neoplasia 2 / 3. Oncoimmunol; 2015; 4:10, e1031439. 38 Snyder et al. Genetic basis for clinical response to CTLA-4 blockade in melanoma. N Engl J Med. 2014; 371(23):2189-99. 39 Van Allen et al. Genomic correlates of response to CTLA-4 blockade in metastatic melanoma. Science; 2015; 350:6257. 40 Li et al. Thromcytopenia caused by the development of antibodies to thrombopoietin, Blood; 2001; 98:3241-3248 41 Takedatsu et al. Determination of Thrombopoietin-Derived Peptides Recognized by Both Cellular and Humoral Immunities in Healthy Donors and Patients with Thrombocytopenia. 2005; 23(7): 975-982 42Eisenhauer et al. New response evaluation criteria in solid tumors: revised RECIST guideline (version 1.1). Eur J Cancer; 2009; 45(2):228-47. 43 Therasse et al. New guidelines to evaluate the response to treatment in solid tumors: European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst; 2000; 92:205-216. 44 Tsuchida & Therasse. Response evaluation criteria in solid tumors (RECIST): New guidelines. Med Pediatr Oncol. 2001; 37:1-3. 45 Durie et al. International uniform response criteria for multiple myeloma. Leukemia; 2006;20:1467-1473.

Claims

1. A method for identifying a polypeptide fragment as immunogenic to a specific human subject, or for predicting whether a polypeptide or polypeptide fragment is immunogenic to a specific human subject, (i) The polypeptide (a) an amino acid sequence consisting of nine consecutive amino acids that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; or (b) A T cell epitope consisting of 15 consecutive amino acids that can bind to at least three HLA class II molecules of the subject, A step of determining whether or not to include; and (ii) (I) The step of identifying the sequence as a polypeptide fragment that is immunogenic to the subject; or (II) A. If the polypeptide contains at least one sequence that satisfies the requirements of step (i), the polypeptide is immunogenic to the subject; or B. If the polypeptide does not contain at least one sequence that satisfies the requirements of step (i), a step of predicting that the polypeptide is not immunogenic to the subject. A method that includes this.

2. Step (i) includes determining that the polypeptide contains an amino acid sequence that is a T cell epitope consisting of nine consecutive amino acids and capable of binding to at least three HLA class I molecules of the subject, and identifying a fragment of the polypeptide that is a T cell epitope consisting of fifteen consecutive amino acids and capable of binding to at least one HLA class II molecule of the subject, wherein the HLA class II binding epitope contains an amino acid sequence of an HLA class I binding T cell epitope. The method according to claim 1.

3. The polypeptide is (a) A substance expressed by a pathogen, virus, or cancer cell, associated with an autoimmune disease, or an allergen, or a component of a pharmaceutical composition, (b) Select from the antigens listed in Tables 2 to 6, (c) an antigen or neoantigen expressed by cancer cells, (d) It is a mutagenic neoantigen, (e) Identifying the polypeptide and / or fragment as immunogenic or associated with an autoimmune disease or autoimmune response in the subject by determining that the polypeptide consists of nine consecutive amino acids and contains an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules in the subject, The method according to claim 1 or claim 2.

4. The method according to any one of claims 1 to 3, wherein all fragments of a polypeptide that is a T cell epitope consisting of nine consecutive amino acids and capable of binding to at least three HLA class I molecules, and / or all fragments of a polypeptide that is a T cell epitope consisting of fifteen consecutive amino acids and capable of binding to at least three HLA class II molecules of the subject are identified.

5. The method according to claim 4, wherein the above method is repeated for each polypeptide that is an active ingredient of a specific pharmaceutical composition.

6. The process further includes a step of predicting whether a subject exhibits a cytotoxic T cell response or a helper T cell response in response to the administration of one or more polypeptides, or a pharmaceutical composition containing one or more polypeptides as active ingredients. A. A cytotoxic T cell response is predicted if the polypeptide consists of nine consecutive amino acids and includes at least one amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; B. A helper T cell response is predicted if the polypeptide consists of 15 consecutive amino acids and includes at least one amino acid sequence that is a T cell epitope capable of binding to at least three HLA class II molecules of the subject; C. If the polypeptide consists of nine consecutive amino acids and does not contain any amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject, a cytotoxic T cell response is not expected; or, D. If the polypeptide consists of 15 consecutive amino acids and does not contain any amino acid sequence that is a T cell epitope capable of binding to at least three HLA class II molecules of the subject, a helper T cell response is not expected. The method according to any one of claims 1 to 5.

7. The method according to claim 6, (I) The subject is predicted to exhibit a cytotoxic T cell response and / or a helper T cell response, and the method further includes the step of determining the likelihood that the subject will exhibit a cytotoxic T cell response and / or a helper T cell response targeting a polypeptide antigen expressed in the subject, The method is, (i) (a) (1) A T cell epitope consisting of nine consecutive amino acids and capable of binding to at least three HLA class I molecules of the subject, or (2) A T cell epitope consisting of fifteen consecutive amino acids and capable of binding to at least three HLA class II molecules of the subject; and (b) The amino acid sequence of the polypeptide is A step of identifying one or more polypeptide antigens containing an amino acid sequence, and (ii) Using population frequency data for one or more polypeptide antigens identified in step (i), determine the likelihood that the subject will exhibit a cytotoxic T cell response and / or helper T cell response targeting the polypeptide antigens expressed in the subject. including, or (II) The polypeptide is a component of a pharmaceutical composition, and the method comprises the step of determining the likelihood that a subject will develop anti-drug antibodies (ADAs) after administration of the polypeptide, wherein a predicted helper T cell response corresponds to a higher likelihood of ADAs, and an unpredicted helper T cell response corresponds to a lower likelihood of ADAs. method.

8. (I) A step of predicting whether a subject will show a clinical response to administration of a pharmaceutical composition or panel of polypeptides comprising one or more polypeptides as active ingredients, the step of determining whether the polypeptides of the one or more active ingredients together contain at least two different amino acid sequences that are T cell epitopes, each consisting of nine consecutive amino acids and capable of binding to at least three HLA class I molecules of the subject; and A. If one or more active ingredient polypeptides each consist of nine consecutive amino acids and together contain at least two different sequences that are T cell epitopes capable of binding to at least three HLA class I molecules of the subject, the subject will show a clinical response to administration of the pharmaceutical composition or panel of polypeptides; or B. If one or more active ingredient polypeptides consist of nine consecutive amino acids and together contain one or fewer sequences that are T cell epitopes capable of binding to at least three HLA class I molecules of the subject, the subject will not show a clinical response to administration of the pharmaceutical composition or the polypeptide panel. This includes a step of predicting and / or, (II) The step of determining the likelihood that a particular human subject will show a clinical response to administration of a pharmaceutical composition or panel of polypeptides comprising one or more polypeptides as active ingredients, (1) One or more of the following factors; (a) The presence of more amino acid sequences and / or different amino acid sequences in the polypeptide of the active ingredient, each consisting of nine consecutive amino acids and capable of binding to at least three HLA class I cells of the subject, which are T cell epitopes; (b) A larger number of target polypeptide antigens, A. Contained in the polypeptide of the active ingredient; and, B. A T cell epitope consisting of nine consecutive amino acids, wherein (i) it can bind to at least three HLA class I cells of the subject, or (ii) it can bind to at least three HLA class I cells of the subject, and the target polypeptide antigen is expressed in the subject, or the target polypeptide antigen is present in one or more samples obtained from the subject. A larger number of target polypeptide antigens containing at least one amino acid sequence that is both of the above, (c) A higher probability that the subject will express the target polypeptide antigen, A higher probability that the subject will express a threshold number of the target polypeptide antigen, and / or A. Contained in the polypeptide of the active ingredient; and B. A T cell epitope consisting of nine consecutive amino acids and capable of binding to at least three HLA class I cells of the subject. A higher probability that the subject expresses a target polypeptide antigen determined to contain at least one amino acid sequence that is both of the above; and / or, (d) A number of target polypeptide antigens that are predicted to be expressed by the subject, or a number of target polypeptide antigens that are expressed by the subject with threshold probability, and / or A. Contained in the polypeptide of the active ingredient; and B. A T cell epitope consisting of nine consecutive amino acids and capable of binding to at least three HLA class I cells of the subject. A larger number of target polypeptide antigens that have been determined to contain at least one amino acid sequence, This corresponds to a higher probability of clinical response, and / or, (2) The above method is (i) Which polypeptide antigen is targeted by the polypeptide of the active ingredient? A. Contained in the polypeptide of the active ingredient; and B. A T cell epitope consisting of nine consecutive amino acids and capable of binding to at least three HLA class I cells of the subject. A process to identify whether it contains the amino acid sequence of both of the above, (ii) Using population frequency data for each antigen identified in step (i), determine the probability that the subject expresses one or more antigens identified in step (i) that together contain at least two different amino acid sequences from step (i); and (iii) A step of determining the likelihood that the subject will show a clinical response to administration of the pharmaceutical composition or panel of polypeptides, wherein a higher probability determined in step (ii) corresponds to a higher likelihood of a clinical response. including and / or (3) One or more of the following factors: (a) The presence of more amino acid sequences and / or different amino acid sequences in the polypeptide of the active ingredient, each of which is a T cell epitope consisting of 15 consecutive amino acids, each capable of binding to at least three HLA class II cells of the subject; (b) A. Contained in the polypeptide of the active ingredient; and B. (i) A T cell epitope consisting of 15 consecutive amino acids that can bind to at least three HLA class II cells of the subject or (II) A T cell epitope consisting of 15 consecutive amino acids that can bind to at least three HLA class II cells of the subject, wherein the target polypeptide antigen is expressed in the subject or present in one or more samples obtained from the subject. A larger number of target polypeptide antigens containing at least one amino acid sequence that is both of the above; (c) i. A. Contained in the polypeptide of the active ingredient; and B. A T cell epitope consisting of nine consecutive amino acids and capable of binding to at least three HLA class I cells of the subject. At least one amino acid sequence which is both of the above, and ii. A. Contained in the polypeptide of the active ingredient; and B. A T cell epitope consisting of 15 consecutive amino acids that can bind to at least three HLA class II cells of the subject. At least one amino acid sequence that is both of the above, A larger number of target polypeptide antigens, including; (d) A. Contained in the polypeptide of the active ingredient; and, B. A T cell epitope consisting of 15 consecutive amino acids and capable of binding to at least three HLA class II cells of the subject. A higher probability than the subject expressing a target polypeptide antigen determined to contain at least one amino acid sequence that is both of the above, or a higher probability than the subject expressing a threshold number of the target polypeptide antigen; (e) i. A. Contained in the polypeptide of the active ingredient; and B. A T cell epitope consisting of nine consecutive amino acids and capable of binding to at least three HLA class I cells of the subject. At least one amino acid sequence which is both of the above, and ii. A. Contained in the polypeptide of the active ingredient; and B. A T cell epitope consisting of 15 consecutive amino acids and capable of binding to at least three HLA class II cells of the subject. At least one amino acid sequence that is both of the above The probability that the subject expresses a threshold number of the target polypeptide antigens determined to include the target polypeptide antigens is higher than the probability that the subject expresses a threshold number of the target polypeptide antigens; (f) A larger number of target polypeptide antigens than are predicted to be expressed by the subject, or a number that is predicted to be expressed with threshold probability by the subject, A. Contained in the polypeptide of the active ingredient; and B. A T cell epitope consisting of 15 consecutive amino acids that can bind to at least three HLA class II cells of the subject. A larger number of target polypeptide antigens determined to contain at least one amino acid sequence that is both of the above; and / or (g) A larger number of target polypeptide antigens than are predicted to be expressed by the subject, or the subject expresses them with threshold probability, i. A. Contained in the polypeptide of the active ingredient; and B. A T cell epitope consisting of nine consecutive amino acids and capable of binding to at least three HLA class I cells of the subject. At least one amino acid sequence which is both of the above, and ii. A. Contained in the active ingredient polypeptide; and B. Capable of binding to at least three HLA class II cells of the subject, It is a T cell epitope consisting of 15 consecutive amino acids. A larger number of target polypeptide antigens determined to contain at least one amino acid sequence that is both of the above; However, to better address the higher probability of clinical response, The method according to any one of claims 1 to 7.

9. (i) Repeating the method for one or more further panels of pharmaceutical compositions or polypeptides, and ranking the panels of compositions or polypeptides by their likelihood of inducing a clinical response in the subject, and / or (II) The step of predicting whether administration of the polypeptide, pharmaceutical composition, or panel of polypeptides will induce an adverse immune response in the subject, (a) The polypeptide is i. Capable of binding to at least three HLA class I cells in the subject; and ii. A fragment of human polypeptide expressed in healthy cells, It contains at least one amino acid sequence, and a harmful immune response is predicted; or (b) The polypeptide is A. Capable of binding to at least three HLA class I cells in the subject; and B. A fragment of human polypeptide expressed in healthy cells, It contains no amino acid sequence and is not expected to cause a harmful immune response. The method according to claim 8.

10. A method according to any one of claims 1 to 5, further comprising the step of predicting whether the subject will show a clinical response to administration of a checkpoint inhibitor to treat cancer, A step of determining whether one or more cancer-associated antigens together contain at least two different amino acid sequences that are T cell epitopes, each consisting of nine consecutive amino acids and capable of binding to at least three HLA class I antigens of the subject, and A. If one or more cancer-associated antigens together contain at least two different sequences that are T-cell epitopes, each consisting of nine consecutive amino acids and capable of binding to at least three HLA class I molecules of the subject, the subject will show a clinical response to administration of a checkpoint inhibitor; or, B. If one or more cancer-associated antigens include one or fewer sequences that consist of nine consecutive amino acids and are T-cell epitopes capable of binding to at least three HLA class I molecules of the subject, the subject will not have a clinical response to the administration of a checkpoint inhibitor. A method that includes a prediction step.

11. The method according to claim 10, The process further includes determining the likelihood that the subject will show a clinical response to the administration of a checkpoint inhibitor for the treatment of cancer. (i) A step of selecting a plurality of polypeptide antigens related to the type of cancer of the subject; (ii) The step of identifying which of the cancer-associated antigens contains an amino acid sequence that is a T cell epitope consisting of nine consecutive amino acids and capable of binding to at least three HLA class I molecules of the subject; and (iii) A step of determining the likelihood that a subject will show a clinical response to administration of a checkpoint inhibitor for treating cancer, using population expression data for each cancer-related antigen identified in step (iii), wherein a high probability of the subject expressing one or more cancer-related antigens identified in step (ii), each consisting of nine consecutive amino acids and including at least two amino acid sequences that are T cell epitopes capable of binding to at least three HLA class I molecules of the subject, corresponds to a higher likelihood of the subject showing a clinical response. Methods that include...

12. A method for designing or preparing a pharmaceutical panel of pharmaceutical compositions or polypeptides specific to a particular human subject for use in a method for treating or preventing cancer or infection by a virus or pathogen in a particular human subject, (i) A step of selecting a polypeptide fragment that is an antigen expressed by a pathogen, virus, or cancer cell, wherein the fragment has been identified as immunogenic to a subject by the method of any one of claims 1 to 11; (ii) A step of selecting a first sequence of up to 50 consecutive amino acids of the polypeptide, wherein the consecutive amino acids include the amino acid sequence of the fragment selected in step (i); (iii) Repeating steps (i) and (ii), a second amino acid sequence of up to 50 consecutive amino acids of a polypeptide identical or different from the first amino acid sequence, wherein the fragment of the first amino acid sequence selected in step (i) and identified by any method of claims 1 to 11 has an amino acid sequence different from the fragment of the second amino acid sequence selected in step (i) and identified by any method of claims 1 to 11; (iv) A step of designing or preparing a subject-specific pharmaceutical composition or pharmaceutical panel of polypeptides having as active ingredients one or more polypeptides having all the amino acid sequences selected in a preceding step. Methods that include...

13. A method according to claim 12, The step (i) selects a fragment that is an HLA class I binding epitope consisting of nine consecutive amino acids, and further includes the step of selecting a longer fragment of the polypeptide. Longer fragments, a. Including the fragment selected in step (i); and b. A T-cell epitope consisting of 15 consecutive amino acids that can bind to at least three or most likely HLA class II molecules of the subject, and In step (ii), the consecutive amino acids include the aforementioned fragment or a longer fragment selected in step (i). method.

14. (v) Repeating steps (i) through (iii) to select one or more additional amino acid sequences of up to 50 consecutive amino acid sequences of polypeptides that are identical or different to the first and second amino acid sequences; and (vi) A step of designing or preparing a subject-specific pharmaceutical composition or pharmaceutical panel of polypeptides having as active ingredients one or more polypeptides having all the amino acid sequences selected in a preceding step. The method according to claim 12 or claim 13, including the method described in claim 12.

15. Each polypeptide consists of either one of the selected amino acid sequences, or contains two or more selected amino acid sequences arranged end-to-end or overlapping within a single peptide, or consists of two or more selected amino acid sequences. The method according to any one of claims 12 to 14.

16. The method according to claim 15, All neoepitopes formed at the junction between any two selected amino acid sequences, which are arranged end-to-end within a single polypeptide, (i) Corresponding to a fragment of human polypeptide expressed in healthy cells; (ii) A T cell epitope consisting of nine consecutive amino acids that can bind to at least three HLA class I molecules of the subject; or (iii) Satisfying both requirements of (i) and (ii), Polypeptides containing neoepitope amino acid sequences are screened for removal, and / or (b) (i) Equivalent to a fragment of human polypeptide expressed in healthy cells; or (ii) A T cell epitope that corresponds to a fragment of human polypeptide expressed in healthy cells, consists of nine consecutive amino acids, and can bind to at least three HLA class I molecules of the subject, One or more polypeptides are screened to remove polypeptides containing amino acid sequences. method.

17. (a) A storage module configured to store data including the subject's class I and / or class II HLA genotype and the amino acid sequence of one or more test polypeptides; and (b) A system comprising a computational module configured to identify and / or quantify the amino acid sequence of one or more test polypeptides, wherein (i) an epitope consisting of nine consecutive amino acids and capable of binding to at least three HLA class I molecules of the subject, or (ii) an epitope consisting of fifteen consecutive amino acids capable of binding to at least three HLA class II molecules of the subject.

18. The system according to claim 17, further, (c) (i) prediction of whether one or more polypeptides are immunogenic to a subject; or sequences of one or more fragments of one or more polypeptides that are predicted to be immunogenic to a subject; (ii) Prediction of whether an individual will exhibit an immune response to the administration of one or more polypeptides or one or more pharmaceutical compositions containing one or more polypeptides as active ingredients; (iii) Predicting whether a subject will show a clinical response to a treatment method comprising administering to a subject one or more pharmaceutical compositions containing one or more polypeptides as active ingredients; (iv) The likelihood that a subject will show a clinical response to administration of one or more pharmaceutical compositions containing one or more polypeptides as active ingredients; (v) Prediction of whether administration of one or more polypeptides or one or more pharmaceutical compositions comprising one or more polypeptides will induce an adverse immune response in a subject; (vi) Prediction that one or more polypeptides are associated with autoimmune disease in the subject; (vii) Prediction of whether the subject will show a clinical response to administration of a checkpoint inhibitor; or (viiii) Recommendation on whether a subject should be treated by administration of one or more polypeptides and / or one or more pharmaceutical compositions. Output module configured to display A system that further includes this.

Images