Process for preparing vaccine composition

Abstract

To provide polypeptides, polynucleic acids, and pharmaceutical compositions containing the polypeptides for use in the prevention or treatment of cancer.SOLUTION: The present disclosure relates to polypeptides, polynucleic acids, and pharmaceutical compositions containing the polypeptides for use in the prevention or treatment of cancer. The present disclosure also relates to a method of inducing a cytotoxic T cell response or treating cancer in a subject by administering a pharmaceutical composition containing a peptide and to a companion diagnostic method. The present disclosure also relates to a method of preparing a peptide or polynucleic acid for use in a method of inducing a T cell response to a target polypeptide, the method comprising identifying an epitope in an antigen that binds to multiple HLA alleles of the highest percentage of subjects in a target population.SELECTED DRAWING: None

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy (created on August 29, 2019) is named N414322WO_SL.txt and is 1,450,065 bytes in size.

[0002] Field The present disclosure relates to peptides and compositions for use in vaccines and immunotherapy, nucleic acids and vectors encoding such peptides, methods for designing and producing such peptides, methods for predicting whether an individual subject will respond to treatment with such peptides, subject-specific compositions comprising such peptides, and therapeutic methods using such peptides. [Background technology]

[0003] For decades, scientists have believed that chronic diseases were beyond the reach of the human body's natural defenses. Recently, however, the field of cancer immunotherapy has seen rapid advances, with remarkable tumor regressions observed in individuals treated with antibodies that block immune-suppressing molecules. These clinical findings indicate that reactivating pre-existing T cell responses can result in meaningful clinical benefits for individuals. These advances have renewed enthusiasm for the development of cancer vaccines that induce tumor-specific T cell responses.

[0004] Despite hopes, current immunotherapies are effective in only a small proportion of individuals. Furthermore, most cancer vaccine trials have failed to demonstrate statistically significant efficacy due to low rates of tumor regression and antitumor T cell responses in individuals. Similar failures have been reported in the HIV and allergy fields with therapeutic and prophylactic vaccines that attempt to harness T cell responses. Overcoming the clinical failures of immunotherapies and vaccines is needed. Summary of the Invention

[0005] In antigen-presenting cells (APCs), protein antigens are processed into peptides. These peptides bind to HLA molecules and are presented to T cells on the cell surface as peptide-HLA complexes. Different individuals express different HLA molecules, which present different peptides. The inventors have demonstrated that an epitope that binds to a single HLA class I allele expressed in a subject is essential, but not sufficient, to induce a tumor-specific T cell response. Instead, tumor-specific T cell responses are optimally activated when the epitope is recognized and presented by HLA molecules encoded by at least three HLA class I genes of an individual (International Applications PCT / EP2018 / 055231, PCT / EP2018 / 055232, PCT / EP2018 / 055230, European Patents 3370065 and 3369431).

[0006] Based on this finding, the inventors developed a method to design and prepare peptides that induce T cell responses in the highest percentage of subjects in a given target human population, and used this method to design a set of peptides for use in cancer therapy.

[0007] Accordingly, in a first aspect, the present disclosure provides a peptide that is up to 50 amino acids in length and comprises the amino acid sequence of any of SEQ ID NOs: 1-2786 and / or 5432-5931.

[0008] In a further aspect, the disclosure provides a polynucleic acid or vector encoding a peptide that is up to 50 amino acids in length and comprises the amino acid sequence of any of SEQ ID NOs: 1-2786 and / or 5432-5931.

[0009] In a further aspect, the disclosure provides a panel of two or more peptides or two or more polynucleic acids or vectors, each peptide comprising a peptide comprising a different amino acid sequence selected from SEQ ID NOs: 1-2786 and / or 5432-5931, or each polynucleic acid or vector encoding a peptide comprising a different amino acid sequence selected from SEQ ID NOs: 1-2786 and / or 5432-5931.

[0010] In a further aspect, the disclosure provides a pharmaceutical composition or kit comprising one or more of a peptide, a polynucleic acid, a vector, or a panel, optionally comprising at least one pharma- ceutically acceptable diluent, carrier, or preservative.

[0011] In a further aspect, the disclosure provides a method for predicting that a particular human subject will have a cytotoxic T cell response and / or a helper T cell response to administration of a peptide, polynucleic acid, or vector of a pharmaceutical composition or kit, comprising: (i) a. determining that one or more peptides or encoded peptides of the pharmaceutical composition or kit contain 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; and b. predicting that the subject will have a cytotoxic T cell response to administration of the pharmaceutical composition; or (ii) a. determining that one or more peptides or encoded peptides of the pharmaceutical composition or kit contain 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; and b. predicting that the subject will have a helper T cell response to administration of the pharmaceutical composition. The present invention provides a method comprising:

[0012] In a further aspect, the present disclosure provides a method of vaccinating, providing immunotherapy or inducing a cytotoxic T cell response in a subject comprising administering to said subject a peptide, polynucleic acid, or vector of the pharmaceutical composition, or kit.

[0013] In a further aspect, the present disclosure provides a method for producing a method for treating a cancer cell comprising: - a peptide, a polynucleic acid or a vector of said pharmaceutical composition or kit for use in a vaccination method for providing immunotherapy or for inducing a cytotoxic T cell response in a subject; and - Use of said peptide or polynucleic acid in the manufacture of a medicament for vaccination to provide immunotherapy or to induce a cytotoxic T cell response in a subject. to provide.

[0014] In a further aspect, the disclosure provides a method for preparing a pharmaceutical composition or kit for use in a method for treating cancer in a particular human subject, comprising: a. selecting two or more peptides or one or more polynucleic acids or vectors encoding at least two peptides, each peptide or encoded peptide comprising an amino acid sequence selected from SEQ ID NOs: 1-2786 and / or 5432-5931 that comprises T cell epitopes capable of binding to at least three HLA class I alleles and / or T cell epitopes capable of binding to at least three HLA class II alleles of a specific human subject; b. preparing a pharmaceutical composition or kit comprising two or more peptides selected in step a, or one or more polynucleotides or vectors; The present invention provides a method comprising:

[0015] In a further aspect, the disclosure provides a method for designing or preparing a peptide, or a polynucleic acid or vector encoding a peptide, or a panel of peptides, or one or more polynucleic acids or vectors encoding a panel of peptides, for use in a method of inducing a T cell response against a target polypeptide, comprising: (i) selecting or defining a model human population comprising a plurality of subjects, each defined by an HLA class I genotype and / or an HLA class II genotype; (ii) for each subject in the model population, (a) an amino acid sequence of the target polypeptide that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; (b) an amino acid sequence of the target polypeptide that is a T cell epitope capable of binding to at least three HLA class II molecules of the subject; (c) an amino acid sequence of the target polypeptide that comprises a T cell epitope capable of binding to at least three HLA class I molecules of the subject and a T cell epitope capable of binding to at least three HLA class II molecules of the subject; or (d) a. a T cell epitope capable of binding to at least three HLA class II molecules; and b. comprises an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject. The amino acid sequence of the target polypeptide Identifying; (iii) selecting a polypeptide fragment window length of 9 to 50 amino acids; (iv) (a) having the length selected in step (iii); and (b) comprising the amino acid sequence identified in any one of steps (ii)(a) to (d) in the highest percentage of subjects in the model population; identifying a fragment of the target polypeptide; (v) optionally testing the fragments identified in step (iv) against additional predefined criteria, rejecting the fragments if they do not satisfy the further predefined criteria, and repeating step (iv); (a) having the length selected in step (iii); and (b) comprising the amino acid sequence identified in step (iv) in the next highest percentage of subjects in the model population. identifying alternative fragments of the target polypeptide; (vi) optionally repeating step (iv) and optionally step (v) in one or more further rounds, wherein in each round further fragments of the target polypeptide are identified, and in each round removing a subject from the model population if any of the fragments selected in step (iv) of any of the previous rounds and not rejected in step (v) comprise the amino acid sequence identified in step (ii) for that subject; (vii) designing or preparing a peptide, a polynucleic acid or vector encoding a peptide, a panel of peptides, or one or more polynucleic acids or vectors encoding a panel of peptides, each peptide comprising one or more of the target polypeptide fragments identified in step (iv), (v) or (vi), and optionally the polypeptide fragments are flanked at the N- and / or C-terminus by additional amino acids that are not part of the sequence of the target polypeptide antigen; The present invention provides a method comprising:

[0016] In a further aspect, the disclosure provides a panel peptide, polynucleic acid, or vector designed and / or prepared according to the method, or comprising or encoding two or more peptides designed and / or prepared according to the method.

[0017] In a further aspect, the disclosure provides a panel of peptides, or one or more polynucleic acids or vectors encoding a panel of peptides, for use in a method of inducing a T cell response to one or more target polypeptides in a subject of a target human population, wherein each of the peptides, or encoded peptides, (a) 9 to 50 amino acids in length; and (b) comprising fragments of the one or more target polypeptides, the fragments being capable of inducing a phenotype in at least 10% of subjects in the human population to be treated; a. an amino acid sequence of a target polypeptide that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; b. an amino acid sequence of a target polypeptide that is a T cell epitope capable of binding to at least three HLA class II molecules of the subject; c. an amino acid sequence of a target polypeptide that comprises a T cell epitope capable of binding to at least three HLA class I molecules of the subject and a T cell epitope capable of binding to at least three HLA class II molecules of the subject; or d. i. a T cell epitope capable of binding to at least three HLA class II molecules; and ii. comprises an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject. Amino acid sequence of the target polypeptide The present invention provides a panel of peptides, or one or more polynucleic acids or vectors encoding the panel of peptides, comprising an amino acid sequence comprising:

[0018] In a further aspect, the present disclosure provides a pharmaceutical composition or kit comprising a panel of peptides, or one or more polynucleic acids or vectors encoding said panel of peptides, optionally comprising at least one pharma- ceutically acceptable diluent, carrier, or preservative.

[0019] In a further aspect, the present disclosure provides a method of vaccinating, providing immunotherapy or inducing a cytotoxic T cell response in a subject comprising administering to said subject a pharmaceutical composition, or a panel of peptides, polynucleic acids, or vectors of the kit.

[0020] The present disclosure will now be described in more detail, by way of example and not by way of limitation, with reference to the accompanying drawings. Many equivalent modifications and variations will be apparent to those skilled in the art given this disclosure. Accordingly, the exemplary embodiments of the present disclosure described are considered to be illustrative and not limiting. Various modifications can be made to the described embodiments without departing from the scope of the present disclosure. All documents cited herein, whether above or below, are expressly incorporated by reference in their entirety.

[0021] The present disclosure includes combinations of the described embodiments and preferred features unless expressly stated to be unacceptable or to be explicitly avoided. As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" includes two or more such peptides.

[0022] The section headings used herein are for convenience only and are not to be construed as limiting in any way. [Brief description of the drawings]

[0023] [Figure 1] ROC curve for HLA-restricted PEPI biomarker. [Diagram 2] ROC curve of ≧1 PEPI3+ test for determining diagnostic accuracy. AUC=0.73 classifies the fair diagnostic value of the PEPI biomarker. [Figure 3A]Distribution of HLA class I PEPI3+ compared to CD8+ T cell responses measured by state of the art assays among peptide pools used in CD8+ T cell response assays. A: HLA class I restricted PEPI3+. 90% overall concordance (OPA) between T cell responses and PEPI3+ peptides indicates that the peptides of the invention are useful for predicting the set of vaccine-induced T cell responses in individuals (p<0.001). True positive (TP), both peptide and T cell responses were detected (shaded); True negative (TN), neither peptide nor T cell responses were detected (shaded); False negative (FN), only T cell responses were detected; False positive (FP), only peptide was detected. [Figure 3B] Distribution of HLA class I PEPI3+ among peptide pools used in the CD8+ T cell response assay compared to CD8+ T cell responses measured by state of the art assays. B: Class I HLA restricted epitopes (PEPI1+). The OPA between predicted epitopes and CD8+ T cell responses was 25% (not statistically significant). True positive (TP), both peptide and T cell responses were detected (shaded); True negative (TN), neither peptide nor T cell responses were detected (shaded); False negative (FN), only T cell responses were detected; False positive (FP), only peptides were detected. [Figure 4] Correlation between CD4 peptides predicted by the PEPI test and T cell reactivity measured with peptide pools in patients treated with SLP vaccine. A: PEPI binding to ≥3 HLA class II alleles; B: epitopes binding to one HLA class II allele. Grey: true positive (TP) and true negative (TN) responses; White: false negative (FN) and false positive (FP) responses. TP: both peptide and T cell responses detected; TN: neither peptide nor T cell responses detected; FN: only T cell responses detected; FP: only peptide detected. [Diagram 5]Multiple HLA-binding peptides defining HPV-16 LPV vaccine-specific T cell response sets in 20 VIN-3 patients and 5 cervical cancer patients. PEPI counts were compared with clinical responses after treatment with LPV. Predicted CD8+ T cell responders according to PEPI for HLA class I (A) and predicted CD4+ T cell responders according to PEPI for HLA class II (B). Correlation between PEPI counts for HLA class I (C) and class II (D) and clinical response during a 3-month follow-up period in VIN-3 patients. Predicted T cell responders: PEPI counts ≥ 1. Grey columns, patients with HPV16 E6 and / or E7-specific T cell responses; dashed columns, patients without T cell responses. CR, complete clinical responders; PR, partial clinical responders; NR, clinical non-responders. [Figure 6] Multiple HLA class I binding peptides that define HPV vaccine-specific T cell response sets for two patients. A: Four HPV antigens in the HPV vaccine. Boxes represent the length of amino acid sequence from N-terminus to C-terminus. B: Process of identifying multiple HLA binding peptides for two patients: Patient HLA sequences are labeled as 4-digit HLA genotypes from patient ID. Lines indicate the positions of the first amino acids of 54 and 91 epitopes that can bind to HLA (PEPI1+) of patient 12-11 and patient 14-5, respectively. PEPI2 represents peptides selected from PEPI1+ that can bind to multiple HLA of the patient (PEPI2+). PEPI3 represents peptides that can bind to ≧3 HLA of the patient (PEPI3+). PEPI4 represents peptides that can bind to ≧4 HLA of the patient (PEPI4+). PEPI5 represents peptides that can bind to ≧5 HLA of the patient (PEPI5+). PEPI6 represents peptides that can bind to 6 HLA of the patient (PEPI6). C: DNA vaccine-specific PEPI3+ sets of two patients characterize the vaccine-specific T cell responses of the patients. [Figure 7] Probability of developing TSA targeted by the IMA901 vaccine. [Figure 8]HLA class I allele binding characteristics of TUMAP of IMA901 peptide vaccine against 2,915 common alleles. (A) And for class I genotype (6 alleles) of 51 HLA-A*02+ RCC patients. Percentages at the bottom indicate the proportion of HLAs that TUMAP can bind. Dark grey lines indicate the binding HLA alleles. (B) Probability indicates the proportion of patients that can present a specified number of TUMAP with three or more HLAs. AP indicates the number of antigens that can generate at least one PEPI. In this case, both antigens and predicted PEPIs are 9-mers (SEQ ID NOs: 5958-5966, respectively, in the order of appearance), so AP=TUMAP=PEPI. [Figure 9] Correlation between immune responses measured for any TUMAP and immune responses to antigens expressed on tumors (AGPs) [Figure 10A] Correlation studies between immune response rate (IRR) and PEPI score, between objective response rate (ORR) and multi-PEPI score, and between objective response rate (ORR) and multi-Ag PEPI score. A: Preliminary experiment to investigate the relationship between PEPI score and immune response rate of therapeutic vaccine (r2=0.7, p=0.001). Dark gray dashed lines indicate 95% confidence intervals, and gray dashed lines indicate trend lines. [Figure 10B] Correlation studies between immune response rate (IRR) and PEPI score, between objective response rate (ORR) and multi-PEPI score, and between objective response rate (ORR) and multi-Ag PEPI score. B: Plot of IRR-PEPI score. (r2=0.47, p=0.001). Dark grey dashed lines indicate 95% confidence intervals, and grey dashed lines indicate trend lines. [Figure 10C] Correlation studies between immune response rate (IRR) and PEPI score, between objective response rate (ORR) and multi-PEPI score, and between objective response rate (ORR) and multi-Ag PEPI score. C: Multi-PEPI score and clinical response rate of therapeutic vaccine (r2=0.75, p=0.001). Dark grey dashed lines indicate 95% confidence intervals, and grey dashed lines indicate trend lines. [Figure 10D]Correlation studies between immune response rate (IRR) and PEPI score, between objective response rate (ORR) and multi-PEPI score, and between objective response rate (ORR) and multi-Ag PEPI score. D: ORR plotted against multi-PEPI score (r2=0.12, p=0.124). Dark grey dashed lines indicate 95% confidence intervals, and grey dashed lines indicate trend lines. [Figure 10E] Correlation studies between immune response rate (IRR) and PEPI score, between objective response rate (ORR) and multi-PEPI score, and between objective response rate (ORR) and multi-Ag PEPI score. E: ORR plotted against multi-Ag PEPI score for vaccines containing multiple antigens (r2=0.64, p=0.009). Dark grey dashed lines indicate 95% confidence intervals, grey dashed lines indicate trend lines. [Figure 10F] Correlation studies between immune response rate (IRR) and PEPI score, between objective response rate (ORR) and multi-PEPI score, and between objective response rate (ORR) and multi-Ag PEPI score. F: ORR plotted against multi-PEPI score for vaccines containing multiple antigens (r2=0.87, p=0.0002). Dark grey dashed lines indicate 95% confidence intervals, grey dashed lines indicate trend lines. [Figure 10G] Correlation studies between immune response rate (IRR) and PEPI score, between objective response rate (ORR) and multi-PEPI score, and between objective response rate (ORR) and multi-Ag PEPI score. G: ORR plotted against multi-PEPI score in patients with target antigen-positive disease (r2=0.56, p=0.005). Dark grey dashed lines indicate 95% confidence intervals, light grey dashed lines indicate trend lines. [Figure 11] OBERTO Clinical Trial Design (NCT03391232) [Figure 12]Antigen expression in the CRC cohort of the OBERTO trial (n=10). A: Frequency of expression of PolyPEPI1018 source antigens based on 2391 biopsies. B: The PolyPEPI1018 vaccine design identified three of the seven TSAs as being expressed in CRC tumors with a probability of over 95%. C: On average, four out of ten patients had a pre-existing immune response to each target antigen, meaning that the TSA was actually expressed in the patient's tumor. D: Seven out of ten had a pre-existing immune response to at least one, and on average, three different TSAs. [Figure 13] The immunogenicity of PolyPEPI1018 in CRC patients confirms that the target antigen and target peptide were appropriately selected. Top: Target peptide selection and peptide design of the PolyPEPI1018 vaccine composition (SEQ ID NO: 5967). Two 15mers were selected from the CRC-specific CTA (TSA) to contain the 9mer PEPI3+ that predominates in a representative model population. Table: The PolyPEPI1018 vaccine was retrospectively tested during preclinical trials in a CRC cohort and proved immunogenic for at least one antigen in all tested individuals by generating PEPI3+. Clinical immune responses specific for at least one antigen were measured in 90% of patients, and there was also a multi-antigen immune response against at least two antigens in 90% of patients and at least three antigens in 80% of patients when tested with an IFNy fluorescent spot assay specifically measured for the peptides that comprise the vaccine. [Figure 14A] Clinical response to PolyPEPI1018 treatment. A: Swimmer plot of clinical response from the OBERTO trial (NCT03391232). [Figure 14B] Clinical response to PolyPEPI1018 treatment. B: Association between progression-free survival (PFS) and AGP counts. [Figure 14C] Clinical response to PolyPEPI1018 treatment. C: Correlation between tumor volume and AGP count. [Figure 15]Illustrated hotspot analysis. The analysis identifies hotspots in peptides of amino acid sequence PIVQNIQGQMVHQAISPRTLNAWVKVVEEK (SEQ ID NO: 5932) in samples from seven patients (Pat1 to Pat7). × indicates the location of a T cell epitope (9mer) (HLA class I-bound PEPI3+) that can bind to at least three HLA class I alleles. Light shading indicates a T cell epitope (15mer) (HLA class II-bound PEPI4+) that can bind to at least four HLA class II alleles. Dark shading indicates HLA class II-bound PEPI4+ that incorporates HLA class I-bound PEPI3+. The 20mer containing the HLA class I-bound PEPI3+ in the greatest number of seven patients is shown. The 20mer containing the HLA class II-bound PEPI4+ that incorporates the HLA class I-bound PEPI3+ in the greatest number of seven patients is shown as the first hotspot 20mer. This first hotspot may be selected in the first cycle of the method of the present disclosure. In the second cycle, Pat1, Pat2, and Pat4 may be ignored and a designated second hotspot is selected. [Figure 16] Distribution of hotspot amino acid sequence selections after 30 cycles. Selection of less than 30 peptides indicates that no further sequences fulfilling the HLA binding criteria (20mers containing HLA class II-binding PEPI4+ integrated with HLA class I-binding PEPI3+) could be identified in the model population. [Figure 17] The personalized vaccine process. The process consists of collecting saliva samples and collecting tumor samples for tumor pathology. Based on the determined patient's HLA genotype and the patient's tumor type, select 12 tumor- and patient-specific peptides, and prepare a personalized vaccine containing the selected 12 peptides. Then, the oncologist administers the vaccine to the patient. [Figure 18-1] A feasibility study of a "simulated" breast cancer clinical trial. This example demonstrates that >80% of patients could be treated with a "patient-specific" vaccine selected from a "warehouse" of 100 different peptides. [Figure 18-2] A feasibility study of a "simulated" breast cancer clinical trial. This example demonstrates that >80% of patients could be treated with a "patient-specific" vaccine selected from a "warehouse" of 100 different peptides. [Figure 19] Probability of vaccine antigens being expressed in patient A's tumor cells. There is a greater than 95% probability that 5 of the 13 target antigens in the vaccine regimen will be expressed in the patient's tumor. As a result, the 13 peptide vaccines combined have a 95% chance of inducing an immune response against at least 5 ovarian cancer antigens (AGP95). Each peptide has an 84% chance of inducing an immune response in patient A. The AGP50 is mean(expected)=7.9 (a measure of the vaccine's effectiveness in attacking patient A's tumor). [Figure 20] Treatment schedule for Patient A [Figure 21] T cell responses of patient A. A. Left: Vaccine peptide-specific T cell response (20mer). Right: CD8+ cytotoxic T cell response (9mer). The predicted T cell responses are confirmed by bioassay. [Figure 22] MRI findings from Patient A treated with personalized (PIT) vaccine. This patient with advanced, heavily pretreated ovarian cancer had an unexpected objective response after treatment with PIT vaccine. These MRI findings suggest that the PIT vaccine, in combination with chemotherapy, significantly reduced the patient's tumor volume, but not the normal cells in the tissue where the tumor arose. [Diagram 23] Probability of vaccine antigens being expressed in tumor cells of patient B, and treatment schedule for patient B. A: The probability that 4 of the 13 target antigens in the vaccine are expressed in the patient's tumor is over 95%. B: As a result, the 12 peptide vaccines combined have a 95% probability of inducing an immune response against at least 4 breast cancer antigens (AGP95). Each peptide has an 84% probability of inducing an immune response in patient B. AGP50=6.45; a measure of the effectiveness of the vaccine in attacking patient B's tumor. C: Treatment schedule for patient B. [Figure 24] T cell responses of patient A. Left: P vaccine peptide-specific T cell responses (20mer). Right: Kinetics of vaccine-specific CD8+ cytotoxic T cell responses (9mer). The predicted T cell responses are confirmed by bioassay. [Diagram 25] Treatment schedule for patient C. [Figure 26] T cell responses of patient C. A: Vaccine peptide-specific T cell response (20mer). B: Vaccine peptide-specific CD8+ T cell response (9mer). C-D: Kinetics of vaccine-specific CD4+ T cell and CD8+ cytotoxic T cell responses (9mer), respectively. After 14 months, there is a long-lasting immune response specific for both CD4 and CD8 T cells. [Figure 27] Treatment schedule for Patient D. [Figure 28] Immune responses of patient D to PIT treatment. A: CD4+ specific T cell response (20mer) and B: CD8+ T cell specific T cell response (9mer). 0.5-4 months refers to the time period from the last vaccination until the collection of PBMC samples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Array Description SEQ ID NOs: 1-2786 show "hot spot" sequences from the cancer antigens listed in Table 25A. SEQ ID NOs: 2787 to 5431 show "hot spot" sequences from the cancer antigens listed in Table 28. SEQ ID NOs: 5432-5931 show "hot spot" sequences from the cancer antigens listed in Table 25B. SEQ ID NO:5932 shows the amino acid sequence shown in FIG. SEQ ID NOs: 5933 to 5945 show the sequences of the personalized vaccine for patient A and are listed in Table 31. SEQ ID NOs: 5946-5957 show the sequences of the personalized vaccine for patient B and are listed in Table 33. It has been done. SEQ ID NOs: 5958 to 5966 show the sequences of the 9-mer shown in FIG. SEQ ID NO: 5967 shows the vaccine peptide of PolyPEPI1018 shown in FIG.

[0025] Detailed Description HLA genotype HLA is encoded by the most polymorphic genes in the human genome. Each human 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 * Each person has maternal and paternal alleles for HLA. In reality, each human expresses different combinations of six HLA class I and eight HLA class II molecules that present different epitopes from the same protein antigen.

[0026] The nomenclature used to name the amino acid sequences of HLA molecules is as follows: HLA-A * Gene name, such as 02:25 * Allele: Protein number. In this example, "02" refers to the allele. In most cases, alleles are defined by serotype, i.e., proteins of a given allele do not react with each other in serological assays. Protein numbers ("25" in the above example) are assigned consecutively as proteins are discovered. Any protein with a different amino acid sequence is assigned a new protein number (e.g., even a single amino acid change in the sequence is considered a different protein number). Further information about the nucleic acid sequence of a given locus may be added to the HLA designation, but such information is not required for the methods described herein.

[0027] An individual's HLA class I genotype or HLA class II genotype may refer to the actual amino acid sequence of each class I or class II HLA of the individual, or may refer to a designation that minimally names the allele and protein number of each HLA gene, as described above. In some embodiments, an individual's HLA genotype is obtained or determined by assaying a biological sample 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. In some embodiments, the biological sample is a buccal swab sample. The HLA genotype may be obtained or determined using any suitable method. For example, the sequence may be determined via sequencing the HLA locus using methods and protocols known in the art. In some embodiments, the HLA genotype is determined using sequence-specific primer (SSP) technology. In some embodiments, the HLA genotype is determined using sequence-specific oligonucleotide (SSO) technology. In some embodiments, HLA genotype is determined using sequence-based typing (SBT) technology. In some embodiments, HLA genotype is determined using next-generation sequencing. Alternatively, the HLA set of an individual can be stored in a database and accessed using methods known in the art.

[0028] HLA-epitope binding A given HLA of a subject presents only a limited number of different peptides generated by processing of protein antigens in APCs to T cells. As used herein, "display" or "presentation", when used in relation to HLA, refers to the binding between a peptide (epitope) and HLA. In this context, "displaying" or "presenting" a peptide is synonymous with "binding" to a peptide.

[0029] As used herein, the term "epitope" or "T cell epitope" refers to one or more It refers to a sequence of consecutive amino acids in a protein antigen that has binding affinity (is capable of binding) to the HLA of a subject. Epitopes are HLA and antigen specific (HLA-epitope pairs, predicted by known methods), but not subject specific.

[0030] The term "personal epitope" or "PEPI" as used herein distinguishes subject-specific epitopes from HLA-specific epitopes. "PEPI" is a polypeptide fragment consisting of a sequence of consecutive amino acids of a polypeptide that is a T cell epitope that can bind to one or more HLA class I molecules of a specific human subject. In other words, "PEPI" is a T cell epitope that is recognized by the HLA class I set of a specific individual. In contrast to "epitope", PEPI is individual-specific, because different individuals have different HLA molecules, each of which binds to a different T cell epitope. Where appropriate, "PEPI" may also refer to a polypeptide fragment consisting of a sequence of consecutive amino acids of a polypeptide that is a T cell epitope that can bind to one or more HLA class II molecules of a specific human subject.

[0031] "PEPI1," as used herein, refers to a peptide or polypeptide fragment that is capable of binding to one HLA class I molecule (or, in certain circumstances, an HLA class II molecule) of an individual. "PEPI1+" refers to a peptide or polypeptide fragment that is capable of binding to one or more HLA class I molecules of an individual.

[0032] "PEPI2" refers to a peptide or polypeptide fragment capable of binding to two HLA class I (or II) molecules of an individual. "PEPI2+" refers to a peptide or polypeptide fragment capable of binding to two or more HLA class I (or II) molecules of an individual, i.e., fragments identified according to the methods disclosed herein.

[0033] "PEPI3" refers to a peptide or polypeptide fragment capable of binding to three HLA class I (or II) molecules of an individual. "PEPI3+" refers to a peptide or polypeptide fragment capable of binding to three or more HLA class I (or II) molecules of an individual.

[0034] "PEPI4" refers to a peptide or polypeptide fragment capable of binding to four HLA class I (or II) molecules of an individual. "PEPI4+" refers to a peptide or polypeptide fragment capable of binding to four or more HLA class I (or II) molecules of an individual.

[0035] "PEPI5" refers to a peptide or polypeptide fragment capable of binding to five HLA class I (or II) molecules of an individual. "PEPI5+" refers to a peptide or polypeptide fragment capable of binding to five or more HLA class I (or II) molecules of an individual.

[0036] "PEPI6" refers to a peptide or polypeptide fragment capable of binding to all six HLA class I (or all six HLA class II) molecules of an individual.

[0037] Generally speaking, epitopes presented by HLA class I molecules are about 9 amino acids long. However, for purposes of this disclosure, epitopes may be longer or shorter than 9 amino acids long, as long as the epitopes can bind to HLA. For example, epitopes that can be presented (bound) by one or more HLA class I molecules may be 7 amino acids long, or 8 amino acids long, or 9 amino acids long, or 10 amino acids long, or 11 amino acids long. Table 1. Examples of software for determining epitope-HLA binding

[0038] The epitopes that bind to known HLA can be determined using techniques known in the art. Any suitable method can be used, as long as the method can be used to determine multiple HLA-epitope binding pairs that are directly compared. For example, biochemical analysis can be used. It is also possible to use a list of epitopes that are known to bind to a given HLA. It is also possible to use predictive or modeling software to determine which epitopes can bind to a given HLA. An example is provided in Table 1. In some cases, a T cell epitope can bind to a given HLA if the IC50 or predicted IC50 is less than 5000nM, less than 2000nM, less than 1000nM, or less than 500nM.

[0039] [Table 1]

[0040] HLA molecules control T cell responses. Until recently, it was thought that the triggering of an immune response to an individual epitope was determined by the recognition of that epitope by the product of a single HLA allele, i.e., an HLA-restricted epitope. However, HLA-restricted epitopes induce T cell responses in only a small proportion of individuals. Peptides that activate T cell responses in one individual are inactive in other individuals, despite matching HLA alleles. Thus, it was unclear how an individual's HLA molecules present epitopes derived from antigens that actively activate T cell responses.

[0041] The inventors have found that in order to trigger a T cell response, multiple HLA expressed by an individual must present the same peptide. Thus, a fragment (epitope) of a polypeptide antigen that is immunogenic for a particular individual (PEPI) is one that can bind to multiple class I (activating cytotoxic T cells) or class II (activating helper T cells) HLA expressed by that individual. This discovery is described in International Application Nos. PCT / EP2018 / 055231, PCT / EP2018 / 055232, PCT / EP2018 / 055230, European Patent Nos. 3370065 and 3369431.

[0042] peptide In some embodiments, the present disclosure provides a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1-2786 shown in Table 25A and / or SEQ ID NOs: 5432-5931 shown in Table 25B and / or SEQ ID NOs: 2787-5431 shown in Table 28. SEQ ID NOs: 1-5931 are each a 20-mer fragment of a TAA comprising at least one HLA class II-bound PEPI4+ and at least one HLA class I-bound PEPI3+ integrated into the HLA class II-bound PEPI4+ in subjects of a model population of 16,000 subjects.

[0043] The 20-mer fragments were identified as described herein to maximize the number of subjects in the model population that mount a T cell response to the administration of at least one peptide comprising one of the 20-mers for each TAA, against the corresponding TAA. Thus, a panel of peptides, or a suitable subselection thereof, each comprising a different one or more of the 20-mer fragments, represents an ideal panel of peptides for selecting peptides for use in vaccinating against or providing immunotherapy to treat cancer in individual human subjects.

[0044] In some cases, a peptide or panel peptide of the disclosure may comprise one or more of the sequences of SEQ ID NOs: 1-2786 and / or 2787-5431 and / or 5432-5931 (respectively), which are fragments of polypeptide antigens associated with one or more specific cancers or cancer types, such as those in Table 24, or any others described herein. Peptides from such panels may be selected to treat the corresponding cancer. In some cases, the polypeptide antigen may have a minimum expression rate in the cancer, such as being expressed in at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% of such cancers. In some cases, the polypeptide antigens may be those most frequently expressed in cancer, e.g., the 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 of the most commonly expressed antigens as listed in Table 24.

[0045] In some cases, the peptide or panel peptides may include peptides comprising sequences of SEQ ID NOs: 1-2786 and / or 2787-5431 and / or 5432-5931 that are fragments of a particular polypeptide antigen or family of polypeptide antigens, such as any of those described herein (respectively). Peptides from such panels may be selected to treat a corresponding cancer associated with expression of the antigen.

[0046] In some cases, the peptides or panel peptides may include peptides comprising sequences of SEQ ID NOs: 1-2786 and / or 2787-5431 and / or 5432-5931 identified by the inventors as described herein in the first 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 cycle (respectively) of the methods described herein. The peptides identified in the earlier cycles are those capable of inducing a T cell response against the corresponding target antigen in the highest percentage of subjects in the model population.

[0047] In some cases, the panel of peptides is a fragment of an amino acid sequence of Table 25 or Table 28, or of a cancer-associated TAA selected from those listed in Table 24, and / or is a fragment of an amino acid sequence of a TAAs associated with cancer selected from those listed in Table 25 or Table 28, or of a TAAs associated with cancer selected from those listed in Table 24, and / or is a fragment of an amino acid sequence of a TAAs associated with cancer selected from those listed in Table 24, and / or is a fragment of an amino acid sequence of a TAAs associated with cancer selected from those listed in Table 25 or Table 28, or of a cancer-associated TAA selected from those listed in Table 24, and / or is a fragment of an amino acid sequence of a TAAs associated with cancer ... The peptides include peptides comprising any 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40 or 50, 100, 200, 300, 400, or 500 of the amino acid sequences in Table 25 or Table 28 obtained in cycle 1, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0048] In some cases, the panel comprises or encodes at least two, or 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 amino acid sequences selected from SEQ ID NOs: 1-2786 and / or 2787-5431 and / or 5432-5931, each amino acid sequence comprising a T cell epitope capable of binding to at least three HLA class I alleles and / or a T cell epitope capable of binding to at least three HLA class II alleles of an individual human subject. Such a panel is an individualized, subject-specific selection of peptides that can be used to induce a T cell response in a particular subject.

[0049] In some cases, peptides of the disclosure may be up to 50, 45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 amino acids in length. The peptides comprise or consist of an amino acid sequence selected from any of SEQ ID NOs: 1-2786 and / or 2787-5431 and / or 5432-5931 that is a fragment of one or more TAAs set forth in Table 24. In some cases, the fragments may comprise or consist of longer fragments of the TAA of which the sequences of SEQ ID NOs: 1-2786 and / or 2787-5431 and / or 5432-5931 are a part. The term "fragment" or "fragment of a polypeptide" as used herein refers to a chain of amino acids or an amino acid sequence that is typically shorter in length than a reference polypeptide and that contains an identical amino acid sequence to the reference polypeptide over the common portion. Such a fragment according to the present disclosure may, where appropriate, be included in the larger polypeptide of which it is a component.

[0050] In some cases, a fragment having the amino acid sequence of any one of SEQ ID NOs: 1 to 2786, or a longer fragment of a TAA that includes the amino acid sequence of any one of SEQ ID NOs: 1 to 2786. is flanked at the N-terminus and / or C-terminus of the peptide by additional amino acids that are not part of the contiguous sequence of the TAA. In some cases, the sequence may be flanked at the N-terminus and / or C-terminus by up to 30, or 25, or 20, or 15, or 10, or 9, or 8, or 7, or 6, or 5, or 4, or 3, or 2, or 1 additional amino acids.

[0051] In some aspects, the disclosure provides a polynucleic acid or vector encoding one or more peptides, wherein the encoded peptide comprises any one of the amino acid sequences or panels thereof of SEQ ID NOs: 1-2786 and / or 2787-5431 and / or 5432-5931 and / or 2787-3997 as set forth in Tables 25 and 28. All disclosures herein relating to peptides comprising any of the amino acid sequences of SEQ ID NOs: 1-2786 and set forth in Table 25 (as well as methods and compositions related to such peptides) apply equally to polynucleic acids or vectors encoding one or more peptides comprising any of the amino acid sequences of SEQ ID NOs: 1-2786 and / or 2787-5431 and / or 5432-5931 and / or 2787-3997.

[0052] Methods for Designing and Producing Peptides In some embodiments, the present disclosure provides methods for designing and preparing one or more peptides, or polynucleotides or vectors encoding peptides, that can be optimally used to induce a T cell response against one or more given polypeptide antigens in a given target population of subjects.

[0053] Target Polypeptide As used herein, the term "polypeptide" refers to a peptide characterized as a full-length protein, a portion of a protein, or a chain of amino acids. As used herein, the term "peptide" refers to a short polypeptide. A peptide is typically 9 amino acids long, or 10 amino acids long, or 11 amino acids long, or 12 amino acids long, or 13 amino acids long, or 14 amino acids long, or 15 amino acids long, or 16 amino acids long, or 17 amino acids long, or 18 amino acids long, or 19 amino acids long, or 20 amino acids long, or 21 amino acids long, or 22 amino acids long, or 23 amino acids long, or 24 amino acids long, or 25 amino acids long, or 26 amino acids long, or 27 amino acids long, or 28 amino acids long, or 29 amino acids long, or 30 amino acids long, or 35 amino acids long, or 40 amino acids long, or 45 amino acids long, or 50 amino acids long. In some cases, the peptide is not a 9-mer or 15-mer. Short peptides may not be processed by antigen-presenting cells, and therefore bind exogenously to HLA molecules. Thus, injected short peptides can bind more to HLA molecules on all nucleated cells with surface HLA class I, which leads to tolerance. On the other hand, polypeptides are not processed as efficiently as longer peptides. Thus, in some cases, peptides may be about 20 or 25 amino acids to about 30 or 35 amino acids in length.

[0054] The method may include selecting one or more target polypeptide antigens. The target polypeptide antigen may be any polypeptide or fragment of a polypeptide against which it is desired to initiate a T cell response, such as a CD4+ T cell response or a CD8+ T cell response, in a subject of a target population. Typically, the target polypeptide is a polypeptide expressed by a pathogenic organism (e.g., a bacterium or parasite), a virus, a cancer cell, or other disease-associated cell. In some cases, the polypeptide may be present in a sample taken from a subject, such as a subject of a specific or target human population.

[0055] The polypeptide may be a tumor-specific antigen (TSA) and / or a cancer or tumor-associated antigen (TAA). A TAA is a protein expressed in cancer or tumor cells. Examples of TAAs include novel antigens (neo-antigens expressed during tumor development and altered from similar proteins in normal or healthy cells), products of oncogenes and tumor suppressor genes, overexpressed or aberrantly expressed cellular proteins (e.g., HER2, MUC1), antigens produced by oncogenic viruses (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 or in published scientific literature, or through publicly available databases such as the database of the Ludwig Institute for Cancer Research (www.cta.lncc.br / ), the Cancer Immunity database (cancerimmunity.org / peptide / ), and the TANTIGEN tumor T cell antigen database (cvc.dfci.harvard.edu / tadb / ). Exemplary TAAs are listed in Tables 2 and 22. TSAs are antigens that are produced by certain types of tumors that are not present in normal cells of the tissue in which the tumor originates. TSAs include shared antigens, neoantigens, and unique antigens. In some cases, a polypeptide is not expressed or is minimally expressed in normal healthy cells or tissues, but is expressed (in cells or tissues) at a high rate (high frequency) in subjects with a particular disease or condition, such as a type of cancer or cancer originating from a particular cell type or tissue. Alternatively, a polypeptide may be expressed at low levels in normal healthy cells, but expressed (overexpressed) at high levels in diseased (e.g., cancer) cells or subjects with a disease or condition.In some cases, the polypeptide is expressed in at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or more of such individuals or of a subject-matched human subpopulation or model or target population, or is expressed at a higher level compared to normal healthy cells or subjects. For example, the populations may be matched by ethnicity, geographic location, sex, age, disease, disease type or stage, genotype, and / or expression of one or more biomarkers. Expression frequencies (rates) may also be determined from published values ​​and scientific publications.

[0056] In some cases, the target polypeptide is a cancer testis antigen (CTA). CTA is typically not expressed in healthy cells past embryonic development. In healthy adults, CTA expression is limited to male germ cells that do not express HLA and cannot present antigens to T cells. Thus, CTA is considered an expressible neoantigen when expressed in cancer cells. CTA expression is (i) specific to tumor cells, (ii) more frequent in metastases than primary tumors, and (iii) conserved between metastases of the same patient (Gajewski ed. Targeted Therapeutics in Melanoma. Springer New York. 2012).

[0057] In some cases, the target polypeptide is associated with or expressed in a cancer cell, or a particular type of cancer cell, or a cancer of a particular cell type of tissue. In some cases, the cancer is a solid tumor. In some cases, the cancer is a carcinoma, sarcoma, lymphoma, leukemia, germ cell tumor, or blastoma. The cancer may be a hormone-related or dependent cancer (e.g., estrogen- or androgen-related cancer) or a non-hormone-related or dependent cancer. The tumor may be malignant or benign. The cancer may be metastatic or non-metastatic. The cancer may or may not be associated with a viral infection or viral oncogene. In some cases, the cancer is melanoma, lung cancer, renal cell carcinoma, colorectal cancer, bladder cancer, glioma, head and neck cancer, ovarian cancer, non-melanoma skin cancer, prostate cancer, kidney cancer, stomach cancer, liver cancer, cervical cancer. , esophageal cancer, non-Hodgkin's lymphoma, leukemia, pancreatic cancer, uterine cancer, lip cancer, oral cavity cancer, thyroid cancer, brain cancer, nervous system cancer, gallbladder cancer, laryngeal cancer, pharyngeal cancer, myeloma, nasopharyngeal cancer, Hodgkin's lymphoma, testicular cancer, breast cancer, stomach cancer, colorectal cancer, renal cell carcinoma, hepatocellular carcinoma, childhood cancer, and Kaposi's sarcoma.

[0058] The polypeptide may be a viral protein expressed in the cell. Examples include HPV16 E6, E7; HIV Tat, Rev, Gag, Pol, Env; HTLV-Tax, Rex, Gag, Env, human herpes virus proteins, dengue virus proteins. The polypeptide may be a parasite protein expressed in the cell, such as a malaria protein.

[0059] Non-limiting examples of suitable polypeptides include those listed in one or more of Tables 2-5.

[0060] [Table 2-1]

[0061]

Table 2-2

[0062]

Table 2-3

[0063]

Table 2-4

[0064]

Table 3

[0065]

Table 4

[0066]

Table 5-1

[0067]

Table 5-2

[0068]

Table 5-3

[0069]

Table 5-4

[0070] Model Group The method may include a step of selecting or defining a model human population. A suitable model population is a population related to a human population or subpopulation in which a peptide designed or prepared by the method is intended to induce a T cell response. This may also be referred to as a target population or a population of interest. The peptides designed or generated by the method or the encoded peptides are intended to be used in a method of inducing a T cell response against a target polypeptide in subjects of the population of interest. A relevant population is a population that is representative or similar to the population of interest. In some cases, the model population is representative of the whole human body. In other cases, the model population may be a disease and / or subject matched population (subpopulation), such as a subpopulation matched to the population of interest by ethnicity, geographic location, sex, age, disease or cancer, type or stage of disease or cancer, genotype, and / or expression of one or more biomarkers (e.g., women with BRCA mutations for breast cancer vaccines), and / or partially HLA genotype (e.g., subjects have one or more specific HLA alleles). In some cases, the intended population may be subjects with cancer or cancer type, such as any of those described herein. For example, the model population may have HLA class I and / or class II genomes representative of those found in the world population or a subpopulation matched with subjects and / or diseases. In some cases, the model population is representative of at least 70%, or 75%, or 80%, or 84%, or 85%, or 86%, or 90%, or 95% of the intended population by HLA diversity and / or HLA frequency. In some cases, the model population may include at least 100, or at least 200, or at least 300, or at least 400, or at least 500, or at least 1000, or at least 5000, or at least 10000, or at least 15000 subjects.

[0071] Each subject in the model population is minimally defined by an HLA class I or class II genotype, e.g., a complete four-digit HLA class I genotype. Data regarding the HLA genotype of the model population may be stored or recorded in or retrieved from a database, or may be an in silico model human population.

[0072] HLA binding criteria The method includes identifying, for each subject of the model population, an amino acid sequence within the target polypeptide that meets certain HLA binding criteria, such as comprising a T cell epitope capable of binding to multiple HLA class I and / or class II HLA molecules as described herein. For example, an amino acid sequence that comprises a T cell epitope capable of binding to at least three HLA class I alleles of the subject and / or a T cell epitope capable of binding to at least three or four HLA class II alleles of the subject is optimal for inducing a CD4+ T cell and / or CD8+ T cell response. In some cases, the HLA class I-binding T cell epitope and the HLA class II-binding T cell epitope may overlap. In some cases, the HLA class I-binding T cell epitope may be fully integrated into the sequence of the HLA class II-binding T cell epitope. In some cases, the multiple HLA class I and class II binding epitopes are within a minimal distance of each other, for example, both within a 50 amino acid fragment, a 45 amino acid fragment, a 40 amino acid fragment, a 35 amino acid fragment, a 30 amino acid fragment, or a 25 amino acid fragment of the target polypeptide.

[0073] The method includes selecting a polypeptide fragment window length, which defines a fragment length across the target polypeptide that is used to identify hotspots in which the maximum number of subjects in the model population have amino acid sequences that meet the HLA binding criteria. The polypeptide fragment window length can be between 9 amino acids and 50 amino acids.

[0074] Peptides containing hotspot sequences identified by the methods described herein may be particularly useful for inducing T cell responses in a high percentage of subjects in a population of interest. Thus, peptides containing such sequences may be designed or prepared according to the present disclosure and used in therapeutic methods. The peptide may consist of the amino acid sequence of a hotspot fragment of a target polypeptide, or may include the sequence of a longer fragment of a target polypeptide of which the hotspot sequence is a part. In some cases, the target polypeptide fragment may be flanked at the N-terminus and / or C-terminus of the peptide by additional amino acids that are not part of the contiguous sequence of the target polypeptide antigen. In some cases, the fragment may be flanked at the N-terminus and / or C-terminus by up to 30, or 25, or 20, or 15, or 10, or 9, or 8, or 7, or 6, or 5, or 4, or 3, or 2, or 1 additional amino acids.

[0075] In some cases, the methods of the present disclosure may be repeated in an iterative process to identify additional fragments of the target polypeptide antigen that meet the HLA binding criteria in subjects of the model population. In some cases, the methods described herein may be repeated for up to 50 cycles, 45 cycles, 40 cycles, 35 cycles, 30 cycles, 25 cycles, 20 cycles, 19 cycles, 18 cycles, 17 cycles, 16 cycles, 15 cycles, 14 cycles, 13 cycles, 12 cycles, 11 cycles, 10 cycles, 9 cycles, 8 cycles, 7 cycles, 6 cycles, 5 cycles, 4 cycles, 3 cycles, 2 cycles, or 1 cycle.

[0076] In some cases, the objective of the iterative process may be to identify the minimum number of peptides or hotspots that induce the desired T cell response (cytotoxic T cell response and / or helper T cell response) in the maximum number of subjects of the model or therapeutic intent population. In this case, it is desirable to remove from the model population those subjects in which the hotspots or peptides selected in any previous round already meet the desired criteria before repeating the method in further cycles. This iterative method may continue in some cases until no further sequences that meet the HLA binding criteria can be identified or until a predetermined number of cycles, number of hotspots, or a predetermined minimum coverage of the model or therapeutic intent population is reached.

[0077] In some cases, further predefined criteria may be applied to the hotspot selection process. If a particular hotspot sequence does not meet such additional criteria, the hotspot may be ignored and another amino acid sequence may be selected for which the next highest number of subjects in the model population meet the HLA binding criteria for the selected window length, until a sequence that meets the additional predefined criteria is reached. In the iterative process, subjects in the model population for which the selected sequence meets all the HLA binding criteria and other criteria must be removed from the model population before proceeding to the next cycle.

[0078] In one example, the additional predetermined criteria may relate to features of the peptide sequence that affect manufacturing feasibility, for example, in some cases, a peptide / hotspot sequence may be rejected if it contains a particular amino acid residue such as cysteine ​​or a particular amino acid motif, or if it has less than a minimum level of hydrophilicity.

[0079] The methods of the present disclosure can be used to provide peptides useful for inducing a T cell response against a given polypeptide, or an ideal set of peptides from which to select peptides for inducing a T cell response against one or more given polypeptides in a particular subject of a given human population.

[0080] In other cases, the method may be repeated for a set of polypeptides, e.g., a set of polypeptides associated with the same disease or condition, e.g., polypeptides expressed by the same pathogen or same type of pathogen or associated with the same cancer or same type of cancer, e.g., those disclosed herein. The method can then provide an ideal set of peptides for selecting peptides for treating a disease or condition in a particular subject of a given human population.

[0081] Peptide Panel In some cases, the present disclosure provides a panel of peptides or a panel of polynucleic acids or vectors encoding the panel of peptides. The panel may be suitable for use in a method of inducing a T cell response against one or more target polypeptides in subjects of a human population of interest. The human population of interest may be a population as described herein and may be defined by the distribution of HLA genotypes in subjects of the population of interest as described herein.

[0082] In some cases, the panel is a panel designed and / or prepared according to the methods described herein, in other cases, the panel comprises or encodes two or more peptides designed and / or prepared according to the methods described herein.

[0083] In other cases, the panel comprises or encodes two or more peptides, each peptide comprising a fragment of one or more target polypeptides, the fragments comprising sequences that satisfy any of the HLA binding criteria described herein in a high proportion of the therapeutic population. In some cases, a "high" percentage refers to at least 1%, at least 2%, at least 5%, at least 10%, at least 12%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93 %, at least 48%, at least 49%, or at least 50%, or more than 1%, 2%, 5%, 10%, 12%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%.

[0084] The peptides of the panel may have any of the peptide characteristics described herein, for example, each peptide may be 9-50 amino acids in length; may comprise a fragment of one or more target polypeptides that is 9-50 amino acids in length and satisfies HLA binding requirements; the target polypeptide fragment may be flanked at the N-terminus and / or C-terminus of the peptide by additional amino acids that are not part of the contiguous sequence of the target polypeptide antigen; and / or the target polypeptide(s) may be any described herein, e.g., any of those listed in Tables 2-5.

[0085] In some cases, the target polypeptide of each peptide of the panel may be the same; that is, each peptide comprises a different fragment of the target polypeptide, each of which meets the HLA binding requirements in a high percentage of the intended therapeutic population.The panel then represents a selection of peptides that can be used to induce T cell responses against the same target polypeptide in different HLA-matched subjects.In some cases, the fragments of the target polypeptide in the peptides of the panel do not overlap or do not comprise any common T cell epitopes or PEPIs.

[0086] In other cases, the panel may include peptides designed to induce T cell responses against different target polypeptides, i.e., selected fragments of a target polypeptide included in the peptides are from different target polypeptides. In some cases, the panel includes such fragments from 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, or 40 different target polypeptides.

[0087] The different target polypeptides may be any different polypeptides that are useful to target or that the different PEPIs described herein can selectively target. In some cases, the different target polypeptide antigens are non-homologous, non-paralogous, or have less than 95%, or less than 90%, or less than 85%, or less than 80%, or less than 75%, or less than 70%, or less than 60%, or less than 50% sequence identity over the entire length of each polypeptide.

[0088] In some cases, each of the different target polypeptides targeted by the peptides of the panel are expressed by or associated with the same disease, condition, pathogen, or cancer, such as any described herein. Such a panel of peptides may be ideal for use in treating a disease or condition in a subject in need thereof, particularly if the peptides are HLA / PEPI matched to a particular subject as described herein.

[0089] In some cases, one or more, or each, of the target polypeptides is present in a sample taken from a human subject, indicating that the polypeptide(s) is expressed in the subject, e.g., a cancer or tumor associated antigen, TSA or CTA expressed by cancer cells in the subject.

[0090] In some cases, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-four, or twenty-five or more, or each, of the target polypeptide antigens are TSAs and / or CTAs.

[0091] Polypeptides and Patient Selection The peptides described herein can be used to induce a T cell response or provide vaccination or immunotherapy in a subject in need thereof. Typically, more than one peptide is selected for treatment of a subject. Each peptide may be selected for treatment of a subject based on (i) the disease or condition being treated in the subject and / or (ii) the subject's HLA genotype.

[0092] Each peptide selected for treatment of a subject may comprise a fragment as described herein of a target polypeptide antigen associated with the disease or condition being treated in the subject or expressed by a target cell of the treatment, such as a cancer cell. The disease or condition and the target polypeptide antigen may be any as described herein. Typically, each peptide selected for treatment of a subject comprises a fragment as described herein of a different target polypeptide antigen. The target polypeptide antigen may be selected because it is known to be expressed by the target cell in the subject. For example, the target polypeptide antigen may have been detected in a sample obtained from the subject, such as a tumor biopsy. In other cases, the target polypeptide antigen may be selected based on its expression in the cells that are the target of the treatment, for example, the expression of a particular TAA in a cancer or a particular type of cancer, such as any of those described herein. Typically, the peptide selected for treatment of a subject is one that comprises a fragment as described herein of a polypeptide antigen associated with the condition that has the highest expression for the condition being treated. Furthermore, the fragment is typically predicted to induce a T cell response in a particular subject, as further described herein.

[0093] Polypeptide antigens, especially short peptides derived from polypeptide antigens that are commonly used in vaccination and immunotherapy, induce immune response in only a small number of human subjects.The polypeptides of the present disclosure are specially selected to induce immune response in a high proportion of the general population or in a high proportion of a given treatment target population.However, due to the heterogeneity of HLA genotypes, they may not be effective in all individuals or all subjects of the treatment target population.

[0094] In some cases, the present disclosure provides a method for predicting that a particular human subject will have a T cell response (cytotoxic and / or helper) to administration of any of the peptides, panels of peptides, or pharmaceutical compositions or kits described herein. As provided herein, in order to trigger a T cell response, presentation of a T cell epitope by multiple HLAs of an individual is generally required. The best predictor of a cytotoxic (CD8+) T cell response to a given polypeptide is the presence of at least one T cell epitope presented by at least three HLA class I alleles of the subject (≧1 PEPI3+). Similarly, the presence of at least one T cell epitope presented by at least three or four HLA class II alleles of the subject can predict a helper (CD4+) T cell response. If such a T cell epitope corresponds to a fragment of a target polypeptide antigen, such as any of the target polypeptide antigens described herein, the subject is predicted to mount a T cell response that, if present, targets cells of the subject that express the target polypeptide. Thus, in some cases, the method may be for predicting a T cell response in a subject to a target polypeptide antigen, such as any described herein.

[0095] The inventors further found that the presence of at least two T cell epitopes in a vaccine or immunotherapy composition that (i) correspond to one or more target polypeptide fragments and (ii) can bind to at least three HLA class I alleles of an individual predicts clinical response. For example, if an individual has a total of two or more PEPI3+ in the active ingredient peptide(s) of the vaccine or immunotherapy composition, and these PEPI3+ are derived from polypeptide antigens that are actually expressed by target cells in the individual (e.g., the target tumor cells of the individual express the target tumor-associated antigen), the individual is a likely clinical responder (i.e., a clinically relevant immune responder).

[0096] A "clinical response" or "clinical benefit" as used herein may be prevention or delay of onset of a disease or condition, amelioration of one or more symptoms, induction or prolongation of remission, or delay of recurrence or relapse or worsening, or any other improvement or stabilization in a subject's disease state. Where appropriate, a "clinical response" may correlate with "disease control" or "objective response" as defined by RECIST (Response Evaluation Criteria in Solid Tumors) guidelines.

[0097] Accordingly, some aspects of the present disclosure relate to methods of predicting that a particular human subject will have a clinical response to the administration of a peptide, nucleic acid, or vector of the therapeutic methods described herein, or the pharmaceutical compositions or pharmaceutical kits described herein.

[0098] In some cases, the method includes determining that an active ingredient peptide(s) for treating a subject comprises two or more different amino acid sequences, each of which a) is a fragment of a target polypeptide antigen expressed by a target cell of the subject (e.g., a polypeptide antigen detected in a biopsy); and b) is a T cell epitope capable of binding to at least three HLA class I of the subject.

[0099] In some cases, the likelihood that a subject will have a clinical response to a peptide vaccine or immunotherapy composition such as those described herein can be determined without knowing whether the target antigen is expressed in the subject's target cells, e.g., cancer cells, and / or without determining the subject's HLA class I genotype. Known frequencies of antigen expression in the disease (e.g., MAGE-A3 in tumor types such as gastric cancer) and / or known frequencies of subjects' HLA class I and class II genotypes in a target population (e.g., ethnic, general, disease populations) can be used instead.

[0100] The likelihood that a subject will respond to treatment is increased by (i) the presence of more multiple HLA-binding PEPIs in the active ingredient polypeptide, (ii) the presence of more PEPIs in the target polypeptide antigens, and (iii) the expression of the target polypeptide antigens in the subject or in the diseased cells of the subject. The probability that a target cell in a subject (over)expresses a particular or any combination of target polypeptide antigens can be determined using population expression frequency data (expression rate), for example, the expression probability of the antigens in gastric cancer. The population expression frequency data can be for a subject and / or a disease-matched population, or a treatment intended population. For example, the frequency or probability that a particular cancer-associated antigen is expressed in a particular cancer, such as breast cancer, or in subjects with a particular cancer, can be determined by detecting the antigen in tumor, e.g., breast cancer tumor samples. Such expression frequencies can also be determined from published values ​​and scientific publications. In some cases, the method of the present disclosure may include a step of determining the expression frequency of the relevant target polypeptide antigen in the relevant population.

[0101] Various pharmacodynamic biomarkers have been disclosed for predicting vaccine activity / efficacy in individual human subjects and populations of human subjects. These biomarkers are useful for predicting more effective These biomarkers can be used to expedite efficient vaccine development and reduce development costs, and to evaluate and compare different compositions.

[0102] ·AG95 - Vaccine Potency: The number of antigens in a cancer vaccine that are expressed by a particular tumor type with a 95% probability. AG95 is an index of vaccine potency and is independent of the immunogenicity of the vaccine antigens. AG95 is calculated from tumor antigen expression data. Such data can be obtained from experiments published in peer-reviewed scientific journals. Technically, AG95 is determined from a binomial distribution of antigens in the vaccine, taking into account all possible variations and expression rates.

[0103] PEPI3+ count - immunogenicity of the vaccine in a subject: PEPI3+ from the vaccine is a personal epitope that binds to at least three HLAs of the subject and induces a T cell response. PEPI3+ can be determined using the PEPI3+ test in subjects whose 4-digit HLA genotype is fully known.

[0104] AP count - antigenicity of the vaccine in the subject: the number of vaccine antigens with PEPI3+. The vaccine contains sequences from the target polypeptide antigens expressed by disease cells. AP count is the number of antigens in the vaccine that contain PEPI3+, and AP count represents the number of antigens in the vaccine that can induce a T cell response in the subject. AP count characterizes the vaccine antigen-specific T cell response of the subject, since it depends only on the subject's HLA genotype and is independent of the subject's disease, age, and medication. The exact value is between 0 (no antigens present PEPI) and the maximum number of antigens (all antigens present PEPI).

[0105] AP50 - antigenicity of a vaccine in a population: the average number of vaccine antigens with PEPI in a population. AP50 depends on the HLA genotype of the subjects in the population and is therefore suitable for characterizing vaccine antigen-specific T cell responses in a given population.

[0106] AGP Count - Vaccine efficacy in a subject: number of vaccine antigens expressed in tumors with PEPI. AGP Count indicates the number of tumor antigens that the vaccine recognizes and induces a T cell response against (hits on target). AGP Count depends on the vaccine antigen expression rate in the subject's tumor and on the subject's HLA genotype. The exact value is between 0 (no expressed antigens present PEPI) and the maximum number of antigens (all antigens expressed and present PEPI).

[0107] · AGP50 - efficacy of cancer vaccine in a population: the average number of vaccine antigens expressed in a given tumor with PEPI in the population (i.e., AGP). AGP50 indicates the average number of tumor antigens that can be recognized by the T cell response induced by the vaccine. AGP50 depends on the expression rate of the antigen in a given tumor type and the immunogenicity of the antigen in the target population. AGP50 can estimate the efficacy of a vaccine in different populations and can also be used to compare different vaccines in the same population. The computational calculation of AGP50 is similar to that used for AG50, except that the expression is weighted by the occurrence rate of PEPI3+ in the subjects in the expressed vaccine antigen. In a theoretical population where each subject has PEPI from each vaccine antigen, AGP50 will be equal to AG50. In another theoretical population where there are no subjects with PEPI from any vaccine antigen, AGP50 will be 0. In general, the following statement is valid: 0≦AGP50≦AG50.

[0108] · mAGP - a potential biomarker for selecting potential responders: the potential for a cancer vaccine to induce T cell responses against multiple antigens expressed in a given tumor. mAGP is mAGP is calculated from the expression rate of vaccine antigens in tumors and the presence of vaccine-derived PEPI in the subject. Technically, based on the AGP distribution, mAGP is calculated based on the expression rate of vaccine antigens in tumors and the presence of vaccine-derived PEPI in the subject. AGP) probabilities.

[0109] Such prediction results can be used to inform a physician's decision regarding the treatment of a subject. Thus, in some cases, the method of the present disclosure predicts that a subject will have or is likely to have a T cell response and / or clinical response to a treatment described herein, and the method further comprises selecting a treatment for each human subject. In some cases, a subject is selected for treatment if the likelihood of a response targeting a predetermined number of target polypeptide antigens, optionally expressed (predicted to be expressed), exceeds a predetermined threshold. In some cases, the number of target polypeptide antigens or epitopes is 2. In some cases, the number of target polypeptide antigens or epitopes is 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10. The method may further comprise administering the treatment to the human subject. Alternatively, the method may predict that a subject will not have an immune response and / or clinical response, and may further comprise selecting a different treatment for each subject.

[0110] Pharmaceutical compositions, methods of treatment, and modes of administration In some aspects, the present disclosure relates to a pharmaceutical composition or kit comprising one or more of the peptides, polynucleic acids, or vectors described herein. Such pharmaceutical compositions or kits can be used in methods of inducing an immune response, treating, vaccinating, or providing immunotherapy to a subject. The pharmaceutical composition or kit can be a vaccine or immunotherapy composition or kit. Such treatment can include administering to a subject the peptide, polynucleic acid, or vector of the pharmaceutical composition or kit.

[0111] The pharmaceutical compositions or kits described herein may contain, in addition to one or more peptides, nucleic acids, or vectors, pharma- ceutically acceptable excipients, carriers, diluents, buffers, stabilizers, preservatives, adjuvants, or other materials known to those skilled in the art. Such materials are preferably non-toxic and preferably do not interfere with the pharmaceutical activity of the active ingredient(s). The pharmaceutical carrier or diluent may be, for example, a water-containing solution. The exact nature of the carrier or other materials may depend on the route of administration, for example, oral, intravenous, cutaneous or subcutaneous, intranasal, intramuscular, intradermal, and intraperitoneal routes.

[0112] The pharmaceutical compositions of the present disclosure may contain one or more "pharmaceutical acceptable carriers". These are typically large, slowly metabolized macromolecules such as proteins, sugars, polylactic acids, polyglycolic acids, polymeric 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, glycerol, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present. Sterile pyrogen-free phosphate-buffered saline is an exemplary carrier (Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472).

[0113] The pharmaceutical compositions of the present disclosure may be lyophilized or in aqueous form, i.e., solution or suspension. This type of liquid formulation is ideal for injection, since the composition can be administered directly from the packaged form without the need for reconstitution with an aqueous medium. The pharmaceutical compositions may be provided in vials or in pre-filled syringes. The syringes may be supplied with or without needles. The syringes may contain a single dose. whereas a vial may contain a single dose or multiple doses.

[0114] The liquid formulations of the present disclosure are also suitable for reconstituting other medicaments from lyophilized forms.When the pharmaceutical composition is used for such immediate reconstitution, the present disclosure provides a kit that may include two vials or may include one pre-filled syringe and one vial, and the contents of the syringe are used to reconstitute the contents of the vial before injection.

[0115] The pharmaceutical compositions of the present disclosure may contain an antibacterial agent, especially when packaged in a multi-dose format. Antibacterial agents such as 2-phenoxyethanol or parabens (methylparaben, ethylparaben, propylparaben) may be used. Any preservatives are preferably present at low levels. Preservatives may be added externally and / or may be a component of the bulk antigens that are mixed to form the composition (e.g., present as a preservative in pertussis antigens).

[0116] Pharmaceutical compositions of the present disclosure may also include detergents, such as Tween (polysorbate), DMSO (dimethylsulfoxide), DMF (dimethylformamide), etc. Detergents are generally present at low levels, e.g., <0.01%, but may be used at higher levels, e.g., 0.01-50%.

[0117] Pharmaceutical compositions of the present disclosure may contain sodium salts (e.g., sodium chloride) and free phosphate ions in solution (e.g., through the use of phosphate buffers).

[0118] In certain embodiments, the pharmaceutical composition may be encapsulated in a suitable vehicle to deliver the peptide to antigen-presenting cells or to enhance stability. As will be appreciated by those skilled in the art, a variety of vehicles are suitable for delivering the pharmaceutical composition of the present disclosure. Non-limiting examples of suitable structured fluid delivery systems include nanoparticles, liposomes, microemulsions, micelles, dendrimers, and other phospholipid-containing systems. Methods for incorporating pharmaceutical compositions into delivery vehicles are known in the art.

[0119] The pharmaceutical composition may also include one or more adjuvants and / or cytokines to enhance the immunogenicity of the composition.

[0120] Suitable adjuvants include aluminum salts, such as aluminum hydroxide or aluminum phosphate, but may also be calcium, iron, or zinc salts, or insoluble suspensions of acylated tyrosine or acylated sugars, or saccharides derivatized with cations or anions, polyphosphazenes, biodegradable microspheres, monophosphoryl lipid A (MPL), lipid A derivatives (e.g., those with reduced toxicity), 3-O-deacylated MPL [3D-MPL], Kir A, saponin, QS21, Freund's incomplete adjuvant (Difco Laboratories, Detroit, Mich.), Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ), AS-2 (Smith-Kline The adjuvant may be any of the following: erythrocyte colony stimulating factor (M-CSF), tumor necrosis factor (TNF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M ... can.

[0121] In some embodiments, the composition comprises an adjuvant selected from the group consisting of Montanide ISA-51 (Seppic, Inc., Fairfield, NJ, United States of America), QS-21 (Aquila Biopharmaceuticals, Inc., Lexington, Mass, United States of America), GM-CSF, cyclofosamide, Bacillus Calmette-Guerin (BCG), Corynbacterium parvum, levamisole, azimezone, isoprinisone, dinitrochlorobenzene (DNCB), keyhole limpet hemocyanin (KLH), Freund's adjuvant (complete and incomplete), mineral gel, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, and diphtheria toxin (DT).

[0122] By way of example, the cytokine may be selected from the group consisting of transforming growth factors (TGFs), such as, but not limited to, TGF-α and TGF-β; insulin-like growth factor-I and / or insulin-like growth factor-II; erythropoietin (EPO); osteoinductive factors; interferons, such as, but not limited to, interferon-α, -β, and -γ; colony-stimulating factors (CSFs), such as, but not limited to, macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF). In some embodiments, the cytokine is a nerve growth factor, such as NGF-β; platelet growth factor; transforming growth factor (TGF), such as, but not limited to, TGF-α and TGF-β; insulin-like growth factor-I and insulin-like growth factor-II; erythropoietin (EPO); osteoconductive factors; interferons (IFN), such as, but not limited to, IFN-α, IFN-β, and IFN-γ; macrophage-CSF (M-CSF), granulocyte-macrophage-CSF (GM-CSF), and granulocyte-C. The antibody is selected from the group consisting of colony stimulating factors (CSF) such as SF (G-CSF); interleukins (Il) such as, but not limited to, 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; IL-13, IL-14, IL-15, IL-16, IL-17, IL-18; LIF; Kit-ligand or FLT-3; angiostatin; thrombospondin; endostatin; tumor necrosis factor (TNF); and LT.

[0123] It is expected that the adjuvant or cytokine can be added in an amount of about 0.01 mg to about 10 mg per dose, preferably about 0.2 mg to about 5 mg per dose, or alternatively, the adjuvant or cytokine may be at a concentration of about 0.01 to 50%, preferably about 2% to 30%.

[0124] In certain aspects, pharmaceutical compositions of the present disclosure are prepared by physically mixing the adjuvants and / or cytokines with PEPI under appropriate sterile conditions according to known techniques to produce the final product.

[0125] Examples of suitable compositions and methods of administration of polypeptide fragments are provided in Esseku and Adeyeye (2011) and Van den Mooter G. (2006). Preparation of vaccine and immunotherapy compositions is described in Vaccine Design ("The subunit and adjuvant approach" (eds These are generally described in Powell MF & Newman MJ (1995) Plenum Press New York. Similarly envisaged encapsulation within liposomes is described in U.S. Patent No. 4,235,877 to Fullerton.

[0126] 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 or genetic vaccine comprises a plasmid that includes a promoter and appropriate transcriptional and translational control elements, and a nucleic acid sequence that encodes one or more polypeptides of the present disclosure. In some embodiments, the plasmid also includes sequences for enhancing, for example, expression levels, intracellular targeting, or proteasomal processing. In some embodiments, the DNA vaccine comprises a viral vector that includes a nucleic acid sequence that encodes one or more polypeptides of the present disclosure. In additional aspects, the compositions disclosed herein comprise one or more nucleic acids that encode peptides that are determined to be immunoreactive to a biological sample. For example, in some embodiments, the composition comprises one or more nucleotide sequences encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more peptides that contain fragments that are T cell epitopes capable of binding to at least three HLA class I molecules and / or at least three or four HLA class II molecules of a patient. In some embodiments, the peptides are derived from antigens expressed in the cancer. In some embodiments, the DNA or genetic vaccine also encodes an immunomodulatory molecule to manipulate the resulting immune response, such as to enhance vaccine efficacy, stimulate the immune system, or reduce immunosuppression. Strategies to enhance the immunogenicity of DNA or gene vaccines include encoding heterologous versions of the antigen, fusing the antigen to molecules that activate T cells or trigger associative recognition, priming with a DNA vector followed by boosting with a viral vector, and utilizing immune-modulating molecules. In some embodiments, DNA vaccines are delivered by needle, gene gun, aerosol injector, with a patch, through a microneedle, by abrasion, among other forms.In some forms, the DNA vaccine is incorporated into liposomes or other forms of nanobodies. In some embodiments, the DNA vaccine comprises 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; 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.

[0127] In some embodiments, the compositions disclosed herein are prepared as RNA vaccines. In some embodiments, the RNA is non-replicating mRNA or self-amplifying RNA derived from a virus. In some embodiments, the non-replicating mRNA encodes a peptide disclosed herein and contains 5' and 3' untranslated regions (UTRs). In some embodiments, the self-amplifying RNA derived from a virus encodes not only a peptide disclosed herein, but also the viral replication machinery that allows intracellular RNA amplification and expression of large amounts of protein. In some embodiments, the RNA is directly introduced into an individual. In some embodiments, the RNA is chemically synthesized or transcribed in vitro. In some embodiments, the mRNA is generated from a linear DNA template using T7, T3, or Sp6 phage RNA polymerase, and the resulting product contains an open reading frame encoding a 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 using vaccinia virus capping enzyme or by incorporating a synthetic cap or anti-reverse cap analog. In some embodiments, the coding DNA template is used directly or with poly(A) polymerase. A poly(A) tail of optimal length is added to the mRNA by. The RNA may encode one or more peptides, including fragments that are T cell epitopes capable of binding to at least three HLA class I molecules and / or at least three or four HLA class II molecules of the patient. In some embodiments, the fragments are derived from antigens expressed in the cancer. In some embodiments, the RNA includes signals to enhance stability and translation. In some embodiments, the RNA also includes non-natural nucleotides to extend half-life or modified nucleotides to alter the immunostimulatory profile. In some embodiments, the RNA is introduced by needle, gene gun, aerosol injector, with a patch, through a microneedle, by abrasion, among other forms. In some forms, the RNA vaccine is incorporated into liposomes or other forms of nanobodies, which facilitate cellular uptake of the RNA and protect the RNA from degradation.In some embodiments, the RNA vaccine is a combination 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; dendrimer nanoparticles; and / or naked mRNA; naked mRNA by in vivo electroporation; mRNA complexed with protamine; mRNA associated with positively charged oil-in-water cationic nanoemulsions; mRNA associated with chemically modified dendrimers and complexed with polyethylene glycol (PEG)-lipids. A; mRNA complexed with protamine in a PEG-lipid nanoparticle; mRNA associated with a cationic polymer such as polyethylenimine (PEI); mRNA associated with a cationic polymer such as PEI and a lipid component; mRNA associated with a polysaccharide (e.g., chitosan) particle or gel; mRNA in a cationic lipid nanoparticle (e.g., 1,2-dioleoyloxy-3-trimethylammoniumpropane (DOTAP) or dioleoylphosphatidylethanolamine (DOPE) lipid); mRNA complexed with a cationic lipid and cholesterol; or mRNA complexed with a cationic lipid, cholesterol, and a PEG-lipid. 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, intrasplenic, intratumoral, or other delivery route.

[0128] The polynucleotide or oligonucleotide components may be naked nucleotide sequences or may be combined with cationic lipids, polymers, or targeting systems. They may be delivered by any available technique. 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 a delivery device such as particle-mediated gene delivery. Polynucleotides or oligonucleotides may be administered topically to the skin, or to mucosal surfaces, for example, by intranasal, oral, or rectal administration.

[0129] Uptake of polynucleotide or oligonucleotide constructs can be enhanced by several known transfection techniques, including those involving the use of transfection agents. Examples of these agents include cationic agents, such as calcium phosphate and DEAE-dextran, and lipofection agents, such as lipofectam and transfectam. The dosage of polynucleotide or oligonucleotide administered can be varied.

[0130] Typically, a "prophylactically effective amount" or a "therapeutically effective amount" (although in some cases prevention may be considered treatment) is administered, which is an amount sufficient to produce a clinical response or show clinical benefit to the individual, e.g., to prevent the onset of a disease or condition. The amount is an amount effective to cause or delay, ameliorate one or more symptoms, induce or prolong remission, or delay recurrence or relapse.

[0131] The dose can be determined depending on various parameters, especially the substance used; the age, weight, and condition of the individual to be treated; the route of administration; and the regimen required. The amount of antigen in each dose is selected as an amount that induces an immune response. The physician can determine the route of administration and the dosage required for any particular individual. The dose can be provided as a single dose or as multiple doses, taken at regular intervals, for example, two, three, or four doses every 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 1 μg to 1 mg for other routes, more typically 1 to 100 μg, more typically 5 to 50 μg. Generally, each dose is expected to contain 0.01 to 3 mg of antigen. The optimal amount of a particular vaccine can be ascertained by studies involving observation of immune responses in subjects.

[0132] Examples of the above mentioned techniques and protocols can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0133] In some cases, the treatment method may include administering more than one peptide, polynucleic acid, or vector to the subject. These may be administered together / simultaneously and / or at different times or sequentially. To overcome the challenges of tumor genetic heterogeneity and individual HLA heterogeneity, it may be important to use a combination of different peptides, optionally targeting different antigens. The peptides of the present disclosure may be used in combination to expand the population that can experience clinical benefits from vaccination. Multiple pharmaceutical compositions of PEPIs manufactured for use in one regimen may define a formulation. In some cases, different peptides, polynucleic acids, or vectors of a single treatment may be administered to a subject within a period of, for example, one year, or six months, or three months, or 60 days, or 50 days, or 40 days, or 30 days.

[0134] Routes of administration include, but are not limited to, intranasal, oral, subcutaneous, intradermal, and intramuscular. Subcutaneous administration is particularly preferred. Subcutaneous administration may be performed, for example, by injection into the abdomen, the lateral and anterior parts of the upper arms or thighs, the scapular region of the back, or the upper ventral and dorsal gluteal regions.

[0135] The composition of the present disclosure may also be administered in one or more doses and by other administration routes.For example, such other routes include intradermal, intravenous, intravascular, intraarterial, intraperitoneal, intrathecal, intratracheal, intracardiac, intralobular, intramedullary, intrapulmonary, and intravaginal.Depending on the desired duration of treatment, the composition of the present disclosure may be administered once or several times, and intermittently, for example, monthly, for several months or years, with different dosages.

[0136] The solid dosage form for oral administration includes capsules, tablets, caplets, pills, powders, pellets, and granules.In such solid dosage forms, active ingredient is usually combined with one or more pharma-ceutically acceptable excipients, the examples of which are detailed above.Also, oral agents can be administered as aqueous suspensions, elixirs, or syrups.In these cases, active ingredient can be combined with various sweeteners or flavorings, colorings, and if necessary, emulsifiers and / or suspending agents, as well as diluents such as water, ethanol, glycerin, and combinations thereof.

[0137] Alone or in combination with other pharmacological compositions or treatments, e.g., chemotherapy and / or immunotherapy. One or more compositions of the present disclosure may be administered or methods and uses for treatment according to the present disclosure may be practiced in combination with immunotherapy and / or vaccines. The other therapeutic compositions or treatments may be, for example, one or more of those discussed herein, and may be administered simultaneously or sequentially (before or after) with the compositions or treatments of the present disclosure.

[0138] In some cases, treatment may be administered in combination with checkpoint blockade therapy / checkpoint inhibitors, costimulatory antibodies, cytotoxic or non-cytotoxic chemotherapy and / or radiation therapy, targeted therapy, or monoclonal antibody therapy. Chemotherapy has been demonstrated to sensitize tumors to killing by 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 including nitrogen mustards, e.g., mechlorethamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcolysin), and chlorambucil; anthracyclines; epothilones; nitrosoureas, e.g., carmustine (BCNU), lomustine (CCNU), semustine (methyl-CCNU), and streptozotocin (streptozotocin); triazenes, e.g., , decarbazine (DTIC; dimethyltriazenoimidazole-carboxamide; ethylenimine / methylmelamines, e.g., hexamethylmelamine, thiotepa; alkylsulfonates, e.g., busulfan; antimetabolites, including folic acid analogs, e.g., methotrexate (amethopterin); alkylating agents, antimetabolites, pyrimidine analogs, e.g., fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluoxetine; fluoxetine; fluoxetine; 6-mercaptopurine (6-mercaptopurine; 6-MP), thioguanine (6-thioguanine; TG), and pentostatin (2'-deoxycoformycin); epipodophyllotoxins; enzymes, such as L-asparaginase; biological response modifiers, such as IFNα, IL-2, G-CSF, and GM-CSF; Platinum coordination complexes such as cisplatin (cis-DDP), oxaliplatin, and carboplatin; anthracenediones such as mitoxantrone and the anthracyclines; 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 analogs / derivatives;Corticosteroid antagonists such as prednisone and equivalents, dexamethasone, and aminoglutethimide, progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate, estrogens such as diethylstilbestrol and ethinyl estradiol equivalents, antiestrogens such as tamoxifen, androgens including testosterone propionate and fluoxymesterone / equivalents, antiandrogens such as flutamide, gonadotropin releasing hormone analogs, and leuprolide, and nonsteroidal antiandrogens such as fluoxetine, Hormones / hormonal therapies and agonists / antagonists, including thamides; vinca alkaloids, such as vinblastine (VLB) and vincristine, epipodophyllotoxins, such as etoposide and teniposide, antibiotics, such as dactinomycin (actinomycin D), daunorubicin (daunomycin; rubidomycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C), enzymes, such as L-asparaginase, and biological response modifiers, such as interferon alphenome;

[0139] In some cases, the method of treatment is a method of vaccination or a method of providing immunotherapy. As used herein, "immunotherapy" refers to the treatment of a disease or condition by inducing or enhancing an immune response in an individual. In certain embodiments, immunotherapy refers to a treatment method that includes administering one or more drugs to an individual to induce a T cell response. In an embodiment, immunotherapy refers to a treatment comprising administering or expressing a polypeptide containing one or more PEPIs to an individual to induce a T cell response to recognize and kill cells that display one or more PEPIs on their cell surface in association with class I HLA. In another specific embodiment, immunotherapy refers to a treatment comprising administering one or more PEPIs to an individual to induce a cytotoxic T cell response against cells that display a tumor-associated antigen (TAA), tumor-specific antigen (TSA), or cancer-testis antigen (CTA) on their cell surface that contains the one or more PEPIs. In another embodiment, immunotherapy refers to a treatment comprising administering or expressing a polypeptide containing one or more PEPIs presented by class II HLA to an individual to induce a T helper response to provide costimulation to cytotoxic T cells that recognize and kill disease cells that display one or more PEPIs on their cell surface in association with class I HLA. In yet another specific embodiment, immunotherapy refers to a treatment comprising administering one or more drugs to an individual that reactivate existing T cells to kill target cells. Theoretically, the cytotoxic T cell response will eliminate the cells that display one or more PEPIs, thereby improving the clinical condition of the individual.In some instances, immunotherapy may be used to treat tumors.In other instances, immunotherapy may be used to treat diseases or disorders that are based on intracellular pathogens.

[0140] In some cases, the disclosure relates to the treatment of cancer or any particular type of cancer described herein, in other cases, the disclosure relates to the treatment of viral, bacterial, fungal, or parasitic infections, or any other disease or condition that can be treated by immunotherapy.

[0141] Further embodiments of the present disclosure 1. A pharmaceutical composition comprising two or more different peptides, each peptide being up to 50 amino acids in length and comprising the amino acid sequence of any of SEQ ID NOs: 1 to 2786 and / or 5432 to 5931.

[0142] 2. The pharmaceutical composition according to item 1, comprising at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 different peptides, each peptide being up to 50 amino acids in length and comprising any of the amino acid sequences of SEQ ID NOs: 1 to 2786 and / or 5432 to 5931.

[0143] 3. A pharmaceutical composition comprising one or more polynucleic acids or vectors encoding two or more peptides, each peptide being up to 50 amino acids in length and comprising the amino acid sequence of any of SEQ ID NOs: 1 to 2786 and / or 5432 to 5931.

[0144] 4. The pharmaceutical composition according to item 2, comprising at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 polynucleic acids or vectors.

[0145] 5. The pharmaceutical composition according to item 1 or 3, wherein each peptide or the encoded peptide comprises at least one amino acid sequence selected from one of the following groups: (a) a sequence listed in Table 25A and / or 25B that is shown in Table 25A or 25B to be a fragment of a breast cancer associated antigen listed in Table 24; (b) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a lung cancer associated antigen listed in Table 24; (c) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a prostate cancer associated antigen listed in Table 24; (d) a sequence listed in Table 25A and / or Table 25B, which is shown in Table 25A or 25B to be a fragment of a colorectal cancer associated antigen listed in Table 24; (e) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a bladder cancer associated antigen listed in Table 24; (f) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of an ovarian cancer associated antigen listed in Table 24; (g) a sequence listed in Table 25A and / or Table 25B, which is shown in Table 25A or 25B to be a fragment of a pancreatic cancer associated antigen listed in Table 24; (h) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a brain cancer associated antigen listed in Table 24; (i) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a leukemia associated antigen listed in Table 24; (j) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a lymphoma associated antigen listed in Table 24; (k) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a hepatocellular carcinoma associated antigen listed in Table 24; (l) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a melanoma associated antigen listed in Table 24; (m) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a thyroid cancer associated antigen listed in Table 24; (n) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a childhood cancer associated antigen listed in Table 24; (o) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a gastric cancer associated antigen listed in Table 24; (p) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a kidney cancer associated antigen listed in Table 24; (q) a sequence listed in Table 25A and / or Table 25B, which is shown in Table 25A or 25B to be a fragment of a head and neck cancer associated antigen listed in Table 24; and (r) a sequence listed in Table 25A and / or Table 25B, which is shown in Table 25A or 25B to be a fragment of a cervical cancer associated antigen listed in Table 24.

[0146] 6. The pharmaceutical composition according to any one of items 1 to 5, further comprising a pharma- ceutically acceptable adjuvant, diluent, carrier, preservative, or a combination thereof.

[0147] 7. a. a first pharmaceutical composition comprising one or more peptides, each peptide having a different amino acid sequence selected from any one of SEQ ID NOs: 1 to 2786 and / or 5432 to 5931; b. a second, different pharmaceutical composition comprising one or more peptides, each peptide comprising a different amino acid sequence of any one of SEQ ID NOs: 1 to 2786 and / or 5432 to 5931; Kit including:

[0148] 8. The kit according to item 7, further comprising an insert.

[0149] 9. A method for inducing a cytotoxic T cell response and / or a helper T cell response in a subject of a target population, comprising administering a pharmaceutical composition according to any one of items 1 to 7.

[0150] 10. Prior to the administration step: a. determining that each peptide or encoded peptide of the pharmaceutical composition contains 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; and b. predicting that the subject will have a cytotoxic T cell response to each peptide, each encoded peptide, polynucleic acid, or vector of the pharmaceutical composition. 10. The method of claim 9, further comprising determining whether the subject is likely to have a clinical response to administration of the pharmaceutical composition by:

[0151] 11. A method for vaccinating, providing immunotherapy or inducing a cytotoxic T cell response in a subject, comprising administering to said subject a pharmaceutical composition according to any one of items 1 to 6.

[0152] 12. The peptide, polynucleic acid, or vector of the pharmaceutical composition is a. determining that each peptide or encoded peptide of the pharmaceutical composition contains 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. predicting that the subject will have a cytotoxic T cell response to each peptide, encoded peptide, polynucleic acid, or vector of the pharmaceutical composition; 12. The method according to item 11, wherein the method is predicted to induce a cytotoxic T cell response and / or a helper T cell response in the subject using a method comprising:

[0153] 13. The method according to item 12, wherein the peptide or encoded peptide of the pharmaceutical composition is a fragment of two or more different cancer-associated antigens selected from those listed in Table 22.

[0154] 14. The method according to any one of items 9 to 14, which is a method for treating cancer, optionally bladder cancer, brain cancer, breast cancer, colorectal cancer, gastric cancer, hepatocellular carcinoma, leukemia, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, childhood cancer, thyroid cancer, or prostate cancer.

[0155] 15. A method for designing or preparing a peptide, or one or more polynucleic acids or vectors encoding a peptide, or a panel of peptides, or one or more polynucleic acids or vectors encoding a panel of peptides, for use in a method of inducing a T cell response against a target polypeptide, comprising: (i) selecting or defining a model human population comprising a plurality of subjects, each defined by an HLA class I genotype and / or an HLA class II genotype; (ii) for each subject in the model population, (a) an amino acid sequence of the target polypeptide that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; (b) an amino acid sequence of the target polypeptide that is a T cell epitope capable of binding to at least four HLA class II molecules of the subject; (c) an amino acid sequence of the target polypeptide that comprises T cell epitopes capable of binding to at least three HLA class I molecules of the subject and T cell epitopes capable of binding to at least four HLA class II molecules of the subject; or (d) a. a T cell epitope capable of binding to at least four HLA class II molecules; and b. comprises an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject. The amino acid sequence of the target polypeptide Identifying; (iii) selecting a polypeptide fragment window length of 9 to 50 amino acids; (iv) (c) having the length selected in step (iii); and (d) comprising the amino acid sequence identified in any one of steps (ii)(a) to (d) in the highest percentage of subjects in the model population; identifying a fragment of the target polypeptide; (v) optionally testing the fragments identified in step (iv) against additional predefined criteria, rejecting the fragments if they do not satisfy the further predefined criteria, and repeating step (iv); (a) having the length selected in step (iii); and (b) comprising the amino acid sequence identified in step (iv) in the next highest percentage of subjects in the model population. identifying alternative fragments of the target polypeptide; (vi) optionally repeating step (iv) and optionally step (v) in one or more further rounds, wherein in each round further fragments of the target polypeptide are identified, and in each round removing a subject from the model population if any of the fragments selected in step (iv) of any of the previous rounds and not rejected in step (v) comprise the amino acid sequence identified in step (ii) for that subject; (vii) designing or preparing a peptide, a polynucleic acid or vector encoding a peptide, a panel of peptides, or one or more polynucleic acids or vectors encoding a panel of peptides, each peptide comprising one or more of the target polypeptide fragments identified in step (iv), (v) or (vi), and optionally the polypeptide fragments are flanked at the N- and / or C-terminus by additional amino acids that are not part of the sequence of the target polypeptide antigen; The present invention provides a method comprising:

[0156] 16. The method of claim 15, wherein the target polypeptide is expressed by a pathogenic organism, a virus, or a cancer cell, or is a cancer-testis antigen, and optionally the target polypeptide is selected from an antigen listed in any of Tables 2-5.

[0157] 17. The method according to claim 15 or 16, further comprising selecting two or more peptides, polynucleic acids, or vectors designed or prepared according to the method according to claim 15 or 16 for use in a method of vaccinating, providing immunotherapy, or inducing a cytotoxic and / or helper T cell response in a subject, optionally wherein each of the two or more peptides or encoded peptides is (a) a fragment of a polypeptide expressed by a pathogenic organism, a virus, or a cancer cell; and (b) a T cell epitope capable of binding to at least three HLA class I molecules of the subject or a T cell epitope capable of binding to at least four HLA class II molecules of the subject. Contains an amino acid sequence; The method further comprises administering to the subject one or more peptides, polynucleic acids, or vectors.

[0158] 18. A pharmaceutical composition comprising a panel of peptides, polynucleic acids or vectors designed and / or prepared according to the method of item 15 or item 16, or comprising or encoding two or more peptides designed and / or prepared according to the method of item 15 or item 16.

[0159] 19. T cell responses to one or more target polypeptides in subjects of a target human population 2. A pharmaceutical composition comprising a panel of peptides, or one or more polynucleic acids or vectors encoding a panel of peptides, for use in a method for inducing an answer to a question related to a disease, wherein each of the peptides or encoded peptides is (a) 9 to 50 amino acids in length; and (b) comprising fragments of the one or more target polypeptides, the fragments being capable of inducing a phenotype in at least 10% of subjects in the human population to be treated; a. an amino acid sequence of a target polypeptide that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; b. an amino acid sequence of a target polypeptide that is a T cell epitope capable of binding to at least four HLA class II molecules of the subject; c. an amino acid sequence of a target polypeptide that comprises a T cell epitope capable of binding to at least three HLA class I molecules of the subject and a T cell epitope capable of binding to at least four HLA class II molecules of the subject; or d. i. a T cell epitope capable of binding to at least four HLA class II molecules; and ii. comprises an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject. Amino acid sequence of the target polypeptide 2. A pharmaceutical composition comprising an amino acid sequence comprising:

[0160] 20. The pharmaceutical composition according to item 18 or 19, further comprising a pharma- ceutically acceptable adjuvant, diluent, carrier, preservative, or a combination thereof.

[0161] 21. A method for vaccinating, providing immunotherapy or inducing a cytotoxic T cell response in a subject, comprising administering to said subject a pharmaceutical composition according to any of items 18 to 20.

[0162] 22. One or more or each of the peptides or encoded peptides of the pharmaceutical composition is (a) a fragment of a polypeptide expressed by a pathogenic organism, a virus, or a cancer cell; and (b) a T cell epitope capable of binding to at least three HLA class I molecules of the subject or a T cell epitope capable of binding to at least four HLA class II molecules of the subject. 22. The method of claim 21, comprising an amino acid sequence. EXAMPLES

[0163] Example 1 – HLA-epitope binding prediction process and validation Binding prediction between specific HLA and epitopes (9-mer peptides) was based on the Immune Epitope Database tool for epitope prediction (www.iedb.org).

[0164] We validated the HLA I-epitope binding prediction process by comparison with HLA class I-epitope pairs determined by laboratory experiments. A dataset of HLA I-epitope pairs reported in peer-reviewed publications or public immunological databases was compiled.

[0165] The concordance rate with the experimental dataset was calculated (Table 6). The bound HLA I-epitope pairs of the dataset were correctly predicted with a probability of 93%. I-epitope pairs were also correctly predicted with a probability of 93%.

[0166] [Table 6]

[0167] The accuracy of the prediction of multiple HLA-binding epitopes was also determined (Table 7). Based on the specificity and sensitivity of the analysis, using a probability of 93% for both true positive and true negative predictions, and a probability of 7% (=100%-93%) for false positive and false negative predictions, the probability of the presence of multiple HLA-binding epitopes in humans can be calculated. The probability of multiple HLA binding to one epitope shows the relationship between the number of HLA binding to the epitope and the expected minimum number of actual bindings. According to the PEPI definition, the expected minimum number of HLA binding to an epitope is 3 (bold).

[0168] [Table 7]

[0169] A validated HLA-epitope binding prediction process was used to determine all HLA-epitope binding pairs described in the Examples below.

[0170] Example 2 – Epitope presentation by multiple HLA predicts cytotoxic T lymphocyte (CTL) responses

[0171] This study examines whether the presentation of one or more epitopes of a polypeptide antigen by one or more HLA class I molecules of an individual predicts a CTL response.

[0172] This study was performed by retrospective analysis of six clinical trials conducted on 71 cancer and nine HIV-infected patients (Table 8). Patients in these trials 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 these clinical trials, antigen-specific CD8+ CTL responses (immunogenicity) were measured in the study subjects after vaccination. In some cases, correlations between CTL responses and clinical responses were observed. Relationships were reported.

[0173] No patients were excluded from the retrospective study for any reason other than data availability. The 157-patient dataset (Table 8) was randomized with a standard random number generator to create two independent cohorts for training and validation studies. In some cases, the cohorts contained multiple datasets from the same patient, resulting in a training cohort of 76 datasets from 48 patients and a testing / validation cohort of 81 datasets from 51 patients.

[0174] [Table 8]

[0175] The reported CD8+ T cell responses of the training dataset were compared to the HLA class I restriction profile of the epitopes (9mers) of the vaccine antigens. The antigen sequences and HLA class I genotypes of 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 clinical CD8+ T cell response data of patients whose CD8+ T cells were IFN-y producing CTLs specific for the vaccine peptides (9mers). The number of epitopes from each antigen predicted to bind to at least one (PEPI1+), or at least two (PEPI2+), or at least three (PEPI3+), or at least four (PEPI4+), or at least five (PEPI5+), or all six (PEPI6) HLA class I molecules of each patient was determined, and the number of bound HLAs was used as a classifier of the reported CTL response. The true positive rate (sensitivity) and true negative rate (specificity) were determined separately from the training data set for each classifier (number of HLAs combined).

[0176] A receiver operating characteristic (ROC) analysis was performed for each classifier. The ROC curves plotted the true positive rate (sensitivity) as a function of the false positive rate (1-specificity) for various cutoff points (Figure 1). Each point on the ROC curve represents the sensitivity corresponding to a particular discrimination threshold (epitope (PEPI) count). / specificity pair. The area under the ROC curve (AUC) is a measure of how well a classifier can distinguish between two diagnostic groups (CTL responders or non-responders).

[0177] This analysis unexpectedly revealed that the predictive value of epitope presentation by a subject's multiple class I HLA (PEPI2+, PEPI3+, PEPI4+, PEPI5+, or PEPI6) was in each case a better predictor of CD8+ T cell or CTL response than simply epitope presentation by one or more HLA class I (PEPI1+, AUC=0.48, Table 9).

[0178] [Table 9]

[0179] The CTL response of an individual is best predicted by considering the epitopes of the antigen that can be presented by at least three HLA class I alleles of the individual (PEPI3+, AUC=0.65, Table 9). The threshold count of PEPI3+ (the number of antigen-specific epitopes presented by three or more HLAs of an individual) that best predicted positive CTL response was 1 (Table 10). That is, if an epitope from at least one antigen is presented by at least three HLA class I of a subject (≧1 PEPI3+), the antigen can trigger at least one CTL clone, and the subject is a likely CTL responder. Using a threshold of ≧1 PEPI3+ to predict likely CTL responders ("≧1 PEPI3+ test"), a true positive rate (diagnostic sensitivity) of 76% was obtained (Table 10).

[0180] [Table 10]

[0181] Example 3 – Retrospective validation of the ≥1 PEPI3+ threshold as a novel biomarker for the PEPI test

[0182] In a retrospective analysis, a test cohort of 81 data sets from 51 patients was used to validate the ≧1 PEPI3+ threshold for predicting antigen-specific CD8+ T cell or CTL responses. For each data set in the test cohort, it was determined whether the ≧1 PEPI3+ threshold was met (at least one epitope from the antigen is presented by at least three class I HLAs of the individual). This was compared to the experimentally determined CD8+ T cell responses (CTL responses) reported in clinical trials (Table 11).

[0183] This retrospective validation demonstrated that PEPI3+ peptides were associated with CD8+ expression in individuals with 84% probability of infection. + T cell responses (CTL responses) in individuals. 84% is the same value as that determined by analytical validation of PEPI3+ predictions, epitopes that bind to at least three HLAs in individuals (Table 7). These data provide strong evidence that PEPI induces immune responses in individuals.

[0184] [Table 11]

[0185] The diagnostic accuracy was determined by ROC analysis using the PEPI3+ count as the cutoff value (Figure 2). The AUC value was 0.73. In ROC analysis, an AUC of 0.7 to 0.8 is generally considered to be a fair diagnostic value.

[0186] In the test data set, a PEPI3+ count of at least 1 (≧1 PEPI3+) best predicted CTL responses (Table 12), a result that confirmed the thresholds determined during training (Table 9).

[0187] [Table 12]

[0188] Example 4 – Clinical validation of the ≧1 PEPI3+ threshold as a novel biomarker for the PEPI test Vaccine design based on the PEPI3+ biomarker was first tested in a Phase I clinical trial in patients with metastatic colorectal cancer (mCRC) in the OBERTO Phase I / II clinical trial (NCT03391232). The study evaluated the safety, tolerability, and immunogenicity of single or multiple doses of PolyPEPI1018 as an add-on to maintenance therapy in subjects with mCRC. PolyPEPI1018 is a peptide vaccine that contains 12 unique epitopes derived from seven conserved TSAs that are frequently expressed in mCRC (WO2018158455A1). These epitopes were designed to bind at least three self-HLA alleles, which are more likely to induce a T cell response than epitopes presented by a single HLA (see Examples 2 and 3). Vaccine (dose: 0.2 mg / peptide) was administered to mCRC patients in the first-line setting immediately after transition to maintenance therapy with fluoropyrimidine and bevacizumab. Vaccine-specific T cell responses were predicted by first identifying PEPI3+ in silico (using the patient's complete HLA genotype and CRC-specific antigen expression rates) and then measuring by ELISpot after one vaccination cycle (phase I part of the trial).

[0189] 70 data sets from 10 patients (phase 1 cohort and OBERTO trial data sets) were used to prospectively validate the PEPI3+ biomarker as predictive of antigen-specific CTL responses. For each data set, the predicted PEPI3+ was determined in silico and compared to the vaccine-specific immune response measured by ELISPOT assay from the patient's blood. The diagnostic properties thus determined (positive predictive value, negative predictive value, overall concordance rate) were then compared to the retrospective validation results described in Example 3.

[0190] The overall concordance rate was 64% and the positive predictive value was high at 79%, which means that patients predicted as PEPI3+ have a 79% probability of generating a CD8+ T cell specific immune response against the antigen analyzed. The clinical trial data correlated significantly with the retrospective trial results (p=0.01), providing evidence that the PEPI3+ calculation using the PEPI test predicts antigen-specific T cell responses based on the patient's complete HLA genotype (Table 13).

[0191] [Table 13]

[0192] Example 5 - ≧1 PEPI3+ test predicts CD8+ T cell reactivity Supporting data are available showing that ≧1 PEPI3+ correlates with clinical immunogenicity data, whereas state-of-the-art single HLA-specific epitope definition does not correlate with vaccine-specific immunogenicity.

[0193] Calculation of > 1 PEPI3+ was compared to state of the art methods for predicting a particular human subject's CTL response to peptide antigens.

[0194] The HLA genotypes of 28 cervical cancer and VIN-3 patients who received an HPV-16-specific synthetic long peptide vaccine (LPV) in two separate clinical trials were determined from DNA samples. LPV consists of a long peptide that encompasses the oncoproteins E6 and E7 of the HPV-16 virus. The amino acid sequence of LPV was obtained from MJ 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 14, 178-187 (2008), GG Kenter, et al. Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N Engl J Med 361, 1838-1847 (2009), MJ Welters, et al. Success or failure of vaccination for HPV16-positive vulvar lesions correlates with kinetics and phenotype of induced T-cell responses. Proc Natl Acad Sci USA 107, 11895-11899 (2010). These publications also reported on the T cell responses of each vaccinated patient to the pool of overlapping peptides in the vaccine. Twenty-five patients (20 with VIN-3 and 5 with cervical cancer) had available immune response data, and 25 had available clinical response data.

[0195] For each patient, at least three LPV epitopes (9mers) (PEPI3+) presented by the patient's class I HLA were identified and their distribution within the peptide pool was determined. Peptides containing at least one PEPI3+ (≧1 PEPI3+) were predicted to induce CD8+ T cell responses. Peptides without PEPI3+ were predicted not to induce CD8+ T cell responses.

[0196] A PEPI3+ threshold of ≧1 correctly predicted 529 of 555 negative CD8+ T cell responses (95% true negative (TN) rate) and 9 of 45 positive CD8+ T cell responses (20% true positive (TP) rate) measured after vaccination (Figure 3A). Overall, the concordance between the PEPI3+ threshold of ≧1 and the experimentally determined CD8+ T cell reactivity was 90% (p<0.001). For each patient, the distribution of epitopes presented by at least one patient's class I HLA (≧1 PEPI1+, HLA-restricted epitope prediction, prior art method) in the peptide pool was also determined. 42 HLA class I binding epitopes predicted 45 CD8+ T cell responses (TP rate 93%). In contrast, only 105 of 555 negative T-cell responses were excluded by HLA-binding epitopes (TN rate 19%) (Figure 3B). Overall, the concordance rate between single HLA class I allele-binding epitopes and CD8+ T-cell responses was 25%, which was not statistically significant.

[0197] Example 6 – Prediction of HLA class II restricted CD4+ helper T cell epitopes CD4+ T helper responses after HPV-16 synthetic long peptide vaccine (LPV) vaccination were examined in 28 cervical cancer and VIN-3 patients who received LPV in two different clinical trials (detailed in Example 5) (Figure 4). The TP rate for prediction of HLA class II restricted epitopes was 95%, because state-of-the-art tools predicted positive responses (positive CD4+ T cell reactivity to peptide pools for a person's HLA class II alleles) in 112 of 117 cases. Of the 33 negative T cell responses, 0 could be excluded, resulting in a TN rate of 0%. Overall , the concordance rate between PEPI predictions of HLA-restricted class II and CD4+ T-cell reactivity was 75% (not significant).

[0198] HLA class II binding PEPI3+ predicted 86 of 117 positive CD4+ T cell responses (TP rate 73%) and excluded 17 of 33 negative T cell responses (TN rate 52%). Overall, the concordance between HLA class II PEPI3+ and CD4+ T cell responses was 69% (p=0.005) (Figure 4A).

[0199] Example 7 - A ≧1 PEPI3+ test predicts T cell responses to full-length LPV polypeptides Using the same study reported in Examples 5 and 6, the ≧1 PEPI3+ test was used to predict patient CD8+ and CD4+ T cell responses to the full-length E6 and E7 polypeptide antigens of the LPV vaccine. Results were compared to reported experimentally determined responses. The test correctly predicted CD8+ T cell reactivity (PEPI3+) in 11 of 15 VIN-3 patients (sensitivity 70%, PPV 85%) and 2 of 5 cervical cancer patients (sensitivity 40%, PPV 100%) who had a positive CD8+ T cell reactivity test result (Figure 5A). CD4+ T cell reactivity (PEPI3+) was correctly predicted in 100% of both VIN-3 and cervical cancer patients (Figure 5B).

[0200] We also observed that class I and class II HLA-restricted PEPI3+ counts correlated with the reported clinical benefit for patients vaccinated with LPV. Patients with higher PEPI3+ counts had either a complete or partial response already after 3 months. We also found a correlation between the number of PEPI and clinical response for HLA class II PEPIs, but not for HLA class I PEPIs, in patients with VIN-3, confirming the results of a post-hoc analysis of the clinical trial (Figures 5C and 5D).

[0201] Example 8 – Case study, correlation between PEPI3+ and vaccine-specific immunogenicity

[0202] "Vaccine-1" is an HPV16-based DNA vaccine containing full-length E6 and E7 antigens with a linker between them. "Vaccine-2" is an HPV18-based DNA vaccine containing full-length E6 and E7 antigens with a linker between them (Figure 6A). In a phase 2 clinical trial, T cell responses were examined in 17 HPV-infected cervical cancer patients who were vaccinated with both "Vaccine-1" and "Vaccine-2" (vaccine-3 vaccination, Bagarazzi et al. Science Translational Medicine.2012;4(155):155ra138.).

[0203] Figure 6B shows the location of each epitope (9-mer) within the full-length sequences of two HPV-16 and two HPV-18 antigens for two exemplary patients (patient 12-11 and patient 14-5) that is 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 HLAs in these patients.

[0204] Patient 12-11 had an overall PEPI1+ count of 54 for the combined vaccine (54 epitopes presented by one or more class I HLAs). Patient 14-5 had a PEPI1+ count of 91. Thus, patient 14-5 has a higher PEPI1+ count than patient 12-11 for the four HPV antigens. PEPI1+ represents a different set of vaccine antigen-specific HLA-restricted epitopes for patients 12-11 and 14-5. Only 27 PEPI1+ epitopes were common between these two patients.

[0205] For the PEPI3+ count (number of epitopes presented by 3 or more patient class I HLAs), the results for patients 12-11 and 14-5 were reversed: patient 12-11 had a PEPI3+ count of 8, containing at least one PEPI3+ in each of the four HPV16 / 18 antigens, whereas patient 14-5 had a PEPI3+ count of 0 (Figure 6C).

[0206] The reported immune responses of these two patients were consistent with the PEPI3+ counts but not with the PEPI1+ counts. Patient 12-11 developed an immune response to each of the four antigens after vaccination as measured by ELISpot, whereas patient 14-5 did not develop an immune response to any of the vaccine's four antigens. A similar pattern was observed when comparing the PEPI1+ and PEPI3+ sets of all 17 patients in the trial. There was no correlation between the PEPI1+ counts and the experimentally determined T cell responses reported from the clinical trial. However, there was a correlation between the T cell immunity predicted by a ≥1 PEPI3+ test and the reported T cell immunity. A ≥1 PEPI3+ test predicted immune responders to the HPV DNA vaccine.

[0207] Furthermore, the diversity of the PEPI3+ sets in the patients was similar to the diversity of T cell responses typically seen in cancer vaccine trials. Patients 12-3 and 12-6 did not have PEPI3+, as did patient 14-5, predicting that the HPV vaccine would not be able to trigger T cell immunity. All other patients had at least one PEPI3, predicting that the HPV vaccine may be able to trigger T cell immunity. Eleven patients had multiple PEPI3+, predicting that the HPV vaccine would likely trigger a polyclonal T cell response. Patients 15-2 and 15-3 were able to mount extensive T cell immunity against E6 of both HPVs, but had poor immunity against E7. The other patients, 15-1 and 12-11, had similar magnitude responses against E7 of HPV18 and HPV16, respectively.

[0208] Example 9 -Design of model populations to conduct in silico trials on large populations and identify candidate precision vaccine targets Complete 4-digit HLA class I genotype (2 × HLA-A * xx:xx;2×HLA-B * xx:xx;2×HLA-C * We compiled an in silico human clinical trial cohort of 433 subjects with genotype (HLA genotype: genotype xx:xx) and demographic information. The model population has mixed ethnicity subjects with a total of 152 different HLA alleles representing >85% of the currently known allele G groups.

[0209] We also established a "big population" database containing 7,189 subjects characterized by 4-digit HLA genotype and demographic information. The big population has 328 different HLA class I alleles. The HLA allele distribution of the model population was significantly correlated with the big population (Table 14) (Pearson p<.001). Thus, the model population of 433 patients represents a 16-fold larger population. The model population represents 85% of the human population when expressed by HLA diversity in addition to HLA frequency.

[0210] [Table 14]

[0211] Example 10 – In silico clinical trials based on identification of multiple HLA-binding epitopes in the multi-peptide vaccine IMA901 predict immune response rates in reported clinical trials Probability of targeting multiple antigens in tumors of RCC patients IMA901 is a therapeutic vaccine for renal cell carcinoma (RCC) that contains nine peptides derived from tumor-associated antigens (TUMAPs). TUMAPs have been proven to be antigens that are naturally presented in human cancer tissues and commonly overexpressed in a subset of patients with a given cancer entity (Table 15). The probability of TSA expression in subjects treated with the IMA901 vaccine was estimated using data available from the scientific literature (Figure 7). Bayes' rule was used, assuming that the expression probability follows a beta distribution.

[0212] We defined AG50 as the number of TSAs (AGs) in a cancer vaccine that a particular tumor type has a 50% chance of expressing. AG50 modeling of cancer vaccines assumes that each AG exerts an effect proportional to the expression rate of the AG in the tumor type (if each AG in the vaccine is immunogenic).

[0213] For the IMA901 vaccine targeting nine antigens (nine TUMAPs), the AG50 value was 4.7, which means that about half of the antigens were overexpressed in 50% of the patients' tumors. Furthermore, the probability of targeting two expressed antigens was 100%, and for three antigens it was 96%. These results suggest the high efficacy of the IMA901 vaccine based on the selection of target antigens.

[0214] [Table 15]

[0215] Probability of inducing immune responses against multiple antigens in RCC patients' tumors A total of 96 patients with advanced RCC were randomly assigned to HLA-A * 02+ subjects were treated with IMA901 (Walter Se t al,Multipeptide immune response to cancer vaccine IMA901 after single-dose cyclophosphamide associates with longer patient survival,Nature Medicine,(2012),18,1254-1261). Each of the nine peptides in IMA901 binds to HLA-A * Based on currently accepted criteria, all nine peptides are strong candidates for boosting T cell responses against renal cancer because their presence has been detected in renal cancer patients and each peptide is present in at least one HLA molecule (HLA-A, HLA-B, HLA-C, HLA-D, HLA-E, HLA-F, HLA-G, HLA-H ... * Despite this limitation, the immune response rates measured for at least one peptide of the vaccine in the Phase I and Phase II clinical trials were 74% and 64%, respectively. Analysis of the HLA binding properties of each TUMAP in IMA901 by in silico prediction showed that eight of the nine TUMAPs had many HLA-A binding sites. * 02 allele, supporting the identification process (Figure 8). However, we found that each TUMAP was able to bind to many other HLA-B alleles. * and HLA-C * We found that it was able to bind to the allele (Figure 8A).

[0216] Complete 4-digit HLA genotypes were not available for subjects enrolled in the IMA901 clinical trial, so HLA-A genotypes from another clinical trial were used. *Genotype data from 51 RCC subjects selected in 02 were used to characterize the immunogenicity of the IMA901 vaccine (see: Chowell D, Morris LGT, Grigg CM, Weber JK, Samstein RM, et al. Patient HLA class I genotype influences cancer response to checkpoint blockade immunotherapy. Science. 2018;359(6375):582-587.). As shown in Figure 8B, only a few TUMAPs are able to bind to multiple HLAs in the same subject. The most immunogenic peptide in this context was MET-001, which was able to generate PEPIs in 35% of RCC patients. However, consistent with Figure 8A, CCN-001 was unable to generate PEPIs in any patient; CCN-001 was unable to generate PEPIs in any patient; * Based on Figure 8A, MUC-001 could theoretically bind to other alleles (both HLA-B and HLA-C), but these alleles were not present in patients in our model population, so this peptide was not able to generate PEPI.

[0217] The immunogenicity of the IMA901 vaccine as determined in two clinical trials was compared with the PEPI response rate determined using the PEPI test in our RCC model population. 67% (CI95 53-78%) had an immune response to at least one peptide of the IMA901 vaccine. * Thirty-three percent (CI95 22-47%) of 02+ subjects did not have the three HLAs that bind to any TUMAP. Interestingly, IMA901 did not bind to HLA-A or HLA-B in Phase I and II clinical trials, respectively. *PEPI testing did not induce T cell responses in 25% and 36% of subjects selected in IMA901 02. Furthermore, PEPI testing predicted that 30% (CI95 19-43%) of subjects would have one PEPI against one TUMAP and 37% (CI95 25-51%) would have two or more PEPI against at least two IMA901 peptides, which is consistent with the average 40% and 27% immune responses against one or more TUMAPs in both clinical trials (Table 16). The difference in immunogenicity seen in the three cohorts can be explained by differences in HLA genotypes of the study subjects and potential errors in measuring T cell responses and determining PEPI by PEPI testing (see Example 1). The results of phase I and II trials show variability in immune response rates of the same vaccine in different clinical trial cohorts. However, the agreement between PEPI response rates and immunogenicity of peptide vaccines is consistent with the host It is determined by the HLA sequence.

[0218] [Table 16]

[0219] Similar to AG50, we define AP50 as the average number of antigens with PEPIs in the vaccine, which indicates the extent to which the vaccine can induce an immune response against the antigens targeted by the composition (cancer vaccine-specific immune response). Thus, AP depends on the HLA heterogeneity of the analyzed population, and not on the expression of antigens in the tumor. The IMA901 composition can induce an immune response against an average of 1.06 vaccine antigens (AP50=1.06), i.e., HLA-A * In the RCC model population selected in 02, an immune response against at least one vaccine antigen can be induced, a result far less than the designed immunogenicity challenge (HLA-matched patients treated with 9 peptides).

[0220] Comparison of immunogenicity and clinical responses of TUMAP with IMA901 peptide vaccine The immune response induced by vaccine against a single antigen may not be sufficient for clinical activity because a given antigen may not be expressed in patients.Therefore, the inventors have defined the immune response targeting the expressed antigen as AGP, taking into account both the immunogenicity and expression probability of vaccine antigen in tumors as presented above.AGP depends on the antigen (AG) expression rate in applicable tumors and the HLA genotype of the subject that can make PEPI (P) in the test population.

[0221] Therefore, the correlation between immune responses to various numbers of antigens (TUMAPs) and immune responses to antigens with high probability of expression (AGPs) was investigated. It was found that the immune response induced by one peptide (one TUMAP) corresponds to 0.98 AGP, i.e., the probability that the immune response induced by any peptide of the IMA901 vaccine targets the antigen expressed in the tumor is 98% (Figure 9). However, the immune response induced by two or three TUMAPs corresponds to only 1.44 and 2.21 AGPs, respectively. The 0.35 AGP, corresponding to 0 TUMAPs, shows the cumulative error of PEPI test prediction (see Example 1).

[0222] To characterize the efficacy of cancer vaccines, we define AGP50, which is a parameter that indicates the number of antigens that vaccine-induced CTL can recognize in tumors with a 50% probability.Computer calculation is similar to AG50, but in addition to expression, it also considers the occurrence of PEPI presentation in specific vaccine antigens.The AGP50 of IMA901 vaccine for RCC model population is 1.10.

[0223] In a retrospective analysis, IMA901 clinical trial investigators found that significantly more subjects who responded to multiple TUMAPs of IMA901 experienced disease control (DC, stable disease, or partial response) compared to subjects who did not respond or responded to only one TUMAP (Table 17). Because the presence of PEPI accurately predicted responders to TUMAP, we investigated the relationship between disease control rate and AGPs in the subpopulation of TUMAP responders. Similar to the investigators, we used our RCC model population to analyze the proportion of patients likely to have an immune response to expressed antigens (i.e., ≧1 AGP) for subpopulations predicted to have an immune response to 0, 1, or 2 TUMAPs. Interestingly, the proportion of patients with 1AGP is similar to the proportion of patients with disease control in the subpopulations: 33% of patients had disease control, whereas 47% (CI95 23-67%) had 1AGP, and in the subgroup with immune responses to the two TUMAPs, significantly more patients had disease control and AGP, 75% vs. 90% (CI95 70, 97%), respectively. These results suggest that only patients with an immune response to at least one expressed tumor antigen are likely to experience clinical benefit. Moreover, the proportion of patients with 1AGP in our RCC model population is similar to the disease control rates in phase I and II trials conducted with the IMA901 vaccine (Table 17).

[0224] [Table 17]

[0225] Analysis of IMA901 vaccine efficacy in multiple populations As shown in Table 18, an AG50 value of 4.7 was observed for the IMA901 vaccine, suggesting high efficacy based on the selection of the target antigen. However, the efficacy of the vaccine was not confirmed in the unselected general population and in the HLA-A *In both unselected RCC model populations and HLA-A 02 selected subjects, the AP50 for IMA901 was only 0.75 and 1.12, respectively. * Similar results were obtained for the population selected in 02. The results were consistent with HLA-A * Although the higher abundance of .02 demonstrated improved antigenicity of IMA901, it did not guarantee the immunogenicity of the vaccine. As a result, the AGP50 values, which indicate the efficacy of the vaccine, were low in each population.

[0226] [Table 18]

[0227] Example 11 – In silico drug discovery based on identification of multiple HLA-binding epitopes predicts reported T cell response rates in clinical trials The objective of this study was to determine whether a model population such as that described in Example 9 could be used to predict vaccine CTL reactivity rates, i.e., to be used in in silico efficacy trials, and whether correlations could be made between clinical outcomes in vaccine trials and PEPI.

[0228] Published clinical trial results were collected from studies with therapeutic vaccines, which included 1,790 subjects in 64 clinical trials treated with 42 therapeutic vaccines covering 61 different antigens (Table 19). The same vaccines used in these clinical trials were used in an in silico clinical trial with a model population of 433 subjects with human leukocyte antigen (HLA) genotype (described in Example 9). No subjects were excluded for reasons other than data availability. IRR was defined as the proportion of subjects in the study population who had a T cell response induced by the study vaccine. ORR was defined as the proportion of subjects in the study population who had an objective response (complete response and partial response) after vaccination. In the in silico clinical trial, the proportion of subjects with PEPI (personal epitope that binds to the subject's three HLA alleles), multiple PEPIs, and PEPIs in multiple antigens were computed to obtain PEPI score, multi-PEPI score, and multi-AgPEPI score, respectively. Immune response rates and objective response rates (IRR and ORR) from published clinical trials were compared with the PEPI score, multi-PEPI score, and multi-AgPEPI score. All reported and calculated scores are summarized in Table 20. [Table 19]

[0229] [Table 20-1]

[0230] [Table 20-2]

[0231] We investigated the correlation between a PEPI3+ score of ≥1 and immune response rate in a previous study of 12 cancer antigen-derived peptide vaccines that induced T cell responses in a subset of 172 subjects from 19 clinical trials identified from peer-reviewed publications. We compared the experimental response rates reported from the clinical trials with a PEPI3+ score of ≥1 and found a linear correlation (R 2 = 0.70) was found (p = 0.001) (Figure 10A). The correlation between ≥ 1 PEPI3+ score and immune response rate was then confirmed by analyzing 59 clinical trials involving 1,343 subjects treated with 40 different vaccines. Each vaccine was analyzed by comparing the published IRR from the clinical trials with the PEPI score from the model population (Figure 10B). The correlation between the IRR and PEPI score was significant (r 2 = 0.465 and p = 0.001). This result demonstrated that the PEPI score determined by in silico clinical trials in MP accurately predicted the IRR observed in clinical trials.

[0232] To test whether polyclonal T cell responses increase the likelihood of tumor shrinkage, we compared ORR and multi-PEPI scores. In preliminary studies, we analyzed the relationship between clinical response (either ORR or DCR) and multi-PEPI scores in 17 clinical trials conducted with peptide and DNA-based immunotherapy vaccines. The results of these studies demonstrated a significant correlation between clinical response rate and multi-PEPI scores (r 2 = 0.75, p < 0.001). To confirm these findings, ORR data were collected and analyzed from 27 clinical trials with 21 different vaccines involving 600 subjects (Error! Reference not found). Multi-PEPI score was calculated as the proportion of subjects with more than one PEPI from the study vaccine in the model population. The results of this experiment demonstrated that ORR did not correlate with multi-PEPI score (Error! Reference not found).

[0233] Previous studies have shown that T cell responses to multiple antigens are associated with longer progression-free survival and The results suggested that the multi-Ag PEPI score correlated with overall survival. Therefore, we hypothesized that inducing T cell responses against multiple tumor antigens would increase the chances of tumor shrinkage. To test this hypothesis, we collected and analyzed ORR data from 10 clinical trials with 9 different vaccines involving 263 subjects treated with multi-antigen targeted vaccines. Multi-Ag PEPI score was calculated as the proportion of subjects with vaccine-specific PEPI in at least two antigens. The results of this experiment showed a strong correlation between ORR and multi-Ag PEPI score (r 2 = 0.64, p = 0.01) and between ORR and multi-PEPI score (r 2 = 0.88, p = 0.001) (Error!Reference not found and F, respectively). These results suggest that T cell responses to multiple tumor antigens may recognize a larger population of tumor cells, thereby increasing the likelihood of tumor shrinkage.

[0234] In a second analysis, we investigated whether PEPI-specific T cell responses against antigens expressed in the target tumor would increase the likelihood of tumor shrinkage. A total of 15 clinical trials enrolled subjects with target antigen-positive disease, and 11 trials did not preselect subjects based on antigen expression. The proportion of subjects who achieved an objective response was significantly higher in CTs that included target antigen-positive subjects compared to CTs that did not preselect (21.0% vs. 3.6%, respectively, p=0.03).

[0235] The correlation between ORR and the multi-PEPI score was statistically significant in subjects with confirmed expression of the target antigen (r 2 = 0.56, p = 0.005) (Figure 10G). These results highlight the importance of the presence of cognate PEPI in the tumor and that its presence increases the likelihood of tumor shrinkage.

[0236] This study demonstrated that the association between subjects' HLA genotypes and PEPI was the most important factor in predicting clinical response to vaccines, and also demonstrated that the PEPI score could predict clinical outcomes of therapeutic vaccines.

[0237] Example 12 – Study design and preliminary safety data of the OBERTO Phase I / II clinical trial The OBERTO trial is a Phase I / II study of PolyPEPI1018 vaccine and CDx for the treatment of metastatic colorectal cancer (NCT03391232). The study design is shown in Figure 11.

[0238] Registration Criteria Histologically confirmed metastatic adenocarcinoma of the colon or rectum -At least one measurable reference lesion according to RECIST 1.1 Demonstrated PR or stable disease during first-line treatment with one systemic chemotherapy regimen and one biologic therapy regimen Maintenance therapy with a fluoropyrimidine (5-fluorouracil or capecitabine) plus the same biologic agent (bevacizumab, cetuximab, or panitumumab) used during induction is planned to begin before the first day of treatment with the investigational drug -Have had your last CT scan less than 3 weeks prior to the first day of treatment Subject Dropouts and Discontinuations If during the first study period (12 weeks) a patient experiences disease progression and requires initiation of second-line treatment, the patient will be dropped out of the study. If, during the second part of the study (after the second dose), a patient experiences disease progression and requires initiation of second-line treatment, they will remain in the study, receive their third vaccination as scheduled, and complete follow-up. - Transient localized erythema and edema at the vaccination site were observed as expected. A flu-like syndrome with mild fever and fatigue was also observed in some patients. These reactions are already known for peptide vaccination and are usually associated with the mechanism of action, since fever and flu-like syndrome may be the result and sign of the induction of an immune response (known as a typical vaccine reaction in childhood vaccinations). Only one serious adverse event (SAE) “probably related” to the vaccine was recorded (Table 21). There was one non-vaccine related dose-limiting toxicity (DLT) (syncope).

[0239] Safety results are summarized in Table 21.

[0240] [Table 21]

[0241] Example 13 – Expression frequency-based target antigen selection during vaccine design and its clinical validation for mCRC Common tumor antigens allow precise targeting of all tumor types, including those with low mutational burden. Population expression data collected so far from 2,391 CRC biopsies represent the variability of antigen expression in CRC patients worldwide (Figure 12A).

[0242] PolyPEPI1018 is a peptide vaccine that we designed to contain 12 unique epitopes derived from seven conserved testis-specific antigens (TSAs) that are highly expressed in mCRC. In our model, by selecting TSAs that are highly expressed in CRC, targets are accurately identified and tumor biopsies are unnecessary. We calculated that the probability that three of the seven TSAs are expressed in each tumor is greater than 95%. (Figure 12B)

[0243] A phase I study evaluated the safety, tolerability, and immunogenicity of PolyPEPI1018 as an add-on to maintenance therapy in subjects with metastatic colorectal cancer (mCRC) (NCT03391232) (see also Example 4).

[0244] Immunogenicity measurements demonstrated pre-existing immune responses and indirectly confirmed the expression of target antigens in patients. Vaccine-induced T cell responses were confirmed by measuring immunogenicity with enrichment fluorescent spot assay (ELISPOT) from PBMC samples isolated at various time points before vaccination and after a single subsequent immunization with PolyPEPI1018; PBMC samples were stimulated in vitro with vaccine-specific peptides (9mer and 30mer) to determine vaccine-induced T cell responses above baseline. At least two patients, an average of four, had pre-existing CD8+ T cell responses against each target antigen (Figure 12C). Seven out of ten patients had pre-existing immune responses against at least one antigen (average 3) (Figure 12D). These results provide evidence that target selection was appropriate, since CD8+ T cell responses against CRC-specific target TSAs in patients analyzed before PolyPEPI1018 vaccination were sufficient to detect the presence of the target antigens in the target antigens. This is because antigen expression is confirmed. Targeting actual (expressed) TSA is a prerequisite for an effective tumor vaccine.

[0245] Example 14 – Preclinical and clinical immunogenicity of PolyPEPI1018 vaccine demonstrates proper peptide selection The PolyPEPI1018 vaccine contains six 30mer peptides, each designed by conjugating two immunogenic 15mer fragments (each containing a 9mer PEPI, so that there are two PEPIs in each 30mer, intentionally) derived from seven TSAs (Figure 13). These antigens are frequently expressed in CRC tumors based on the analysis of 2,391 biopsies (Figure 12).

[0246] Preclinical immunogenicity results calculated for the model population (n=433) and CRC cohort (n=37) yielded predicted immunogenicity of 98% and 100% based on the prediction of the PEPI test, which was clinically proven in the OBERTO trial (n=10), where immune responses to at least one antigen were measured in 90% of patients. More interestingly, 90% of patients had vaccine peptide-specific immune responses to at least two antigens, and 80% had CD8+ T cell responses to three or more different vaccine antigens, providing evidence that appropriate target antigens were selected during the design of PolyPEPI1018. CD4+ T cell-specific and CD8+ T cell-specific clinical immunogenicity are detailed in Table 22. High immune response rates were observed for both effector T cells and memory effector T cells, and immune responses were boosted or de novo induced by the vaccine in 9 out of 10 patients for both CD4+ T cells and CD8+ T cells. Furthermore, the percentage of CRC-reactive polyfunctional CD8+ and CD4+ T cells increased 2.5- and 13-fold, respectively, in PBMCs of vaccinated patients.

[0247] [Table 22]

[0248] Example 15 – Clinical response to PolyPEPI1018 treatment The OBERTO clinical trial (NCT03391232), further described in Examples 4, 12, 13 and 14, was analyzed for preliminary objective tumor response rates (RECIST 1.1) (Figure 14). Of the 11 vaccinated patients on maintenance therapy, 5 had stable disease (SD) at the time of preliminary analysis (12 weeks), 3 experienced unexpected tumor responses (partial responses, PR) observed during treatment (maintenance therapy + vaccination) and 3 had progressive disease (PD) according to RECIST 1.1 criteria. Stable disease was achieved as best response in 69% of patients on maintenance therapy (capecitabine and bevacizumab). Patient 020004 was treated after 12 weeks. The efficacy of the treatment persisted and patient 010004 was suitable for definitive surgery as the treatment response was sustained over a long period of time. After the third vaccination, the patient had no evidence of disease and was therefore a complete responder, as shown in the swimmer plot in Figure 14.

[0249] After one vaccination, the ORR was 27% and the DCR was 63%; in patients who received at least two doses (of three), two of five had an ORR (40%) and the DCR was as high as 80% (SD+PR+CR in four of five) (Table 23).

[0250] [Table 23]

[0251] Based on data from five patients who received multiple doses of the PolyPEPI1018 vaccine in the OBERTO-101 clinical trial, preliminary data suggest that higher AGP counts (>2) are associated with longer PFS and increased rates of tumor size reduction (Figures 14B and C).

[0252] Example 16 - Selection of peptides for the treatment of cancer Based on the findings of the role of PEPI in T cell activation described herein, a method for designing peptides for treating cancer was developed, specifically, peptides were designed to stimulate T cell responses against known tumor-associated antigens in the largest number of human subjects.

[0253] We selected 192 TSAs known to be expressed in one or more of 19 cancer indications (Table 24). Data on the expression rate of TSAs in various cancer indications, when available in peer-reviewed publications, were used to rank the TSAs by frequency of expression in each indication. The ranking order of TSAs differs for each indication.

[0254] [Table 24-1]

[0255] [Table 24-2]

[0256] [Table 24-3]

[0257] [Table 24-4]

[0258] [Table 24-5]

[0259] [Table 24-6]

[0260] [Table 24-7]

[0261] [Table 24-8]

[0262] A model population containing 15,693 subjects was used, with up to 500 male and 500 female subjects from each of a wide range of ethnicities. For each subject, the complete six HLA class I and DQ&DRB1 class II alleles were available. The number of HLA class II bindings was replicated to simulate the entire genome.

[0263] For each of the 15,693 subjects, we identified all 15-mer amino acid sequences in each TSA that met the following HLA-binding criteria: (i) were predicted to bind to at least four HLA class II alleles in the subject (HLA class II-binding PEPI4+); and (ii) contained a 9-mer amino acid sequence predicted to bind to at least three HLA class I alleles in the subject (HLA class I-binding PEPI3+).

[0264] Hotspots in the amino acid sequence of each TSA were identified, which are 20-mers including 15-mers that meet the HLA binding criteria for the largest number of subjects in the population of 15,693 subjects. The hotspot analysis is shown in FIG.

[0265] The hotspot analysis was repeated for an additional 29 cycles or until no further sequences could be identified that met the HLA binding criteria. The hotspot sequences were screened against the criteria of manufacturing feasibility. Any hotspot sequences that contained cysteine ​​residues or had a calculated hydrophilicity value of less than 33% were rejected, and instead another hotspot sequence containing a 15mer that met the HLA binding criteria for the next highest number of subjects was selected.

[0266] In each cycle, subjects who met the HLA binding criteria for any hotspot sequence selected in any previous cycle were excluded. In this way, the hotspots selected maximized the number of subjects in the population in which hotspot sequences predicted to induce both CD4+ and CD8+ T cell responses were selected in each cycle. The hotspot sequences selected in each cycle and the TSAs of which they are fragments are shown in Table 25. A total of 3286 hotspot sequences were selected. Figure 16 shows the distribution of hotspot sequence selection across the 192 CTAs.

[0267] [Table 25A-1]

[0268]

Table 25A-2

[0269]

Table 25A-3

[0270]

Table 25A-4

[0271]

Table 25A-5

[0272]

Table 25A-6

[0273]

Table 25A-7

[0274]

Table 25A-8

[0275]

Table 25A-9

[0276]

Table 25A-10

[0277]

Table 25A-11

[0278]

Table 25A-12

[0279]

Table 25A-13

[0280]

Table 25A-14

[0281]

Table 25A-15

[0282]

Table 25A-16

[0283]

Table 25A-17

[0284]

Table 25A-18

[0285]

Table 25A-19

[0286]

Table 25A-20

[0287]

Table 25A-21

[0288]

Table 25A-22

[0289]

Table 25A-23

[0290]

Table 25A-24

[0291]

Table 25A-25

[0292]

Table 25A-26

[0293]

Table 25A-27

[0294]

Table 25A-28

[0295]

Table 25A-29

[0296]

Table 25A-30

[0297]

Table 25A-31

[0298]

Table 25A-32

[0299]

Table 25A-33

[0300]

Table 25A-34

[0301]

Table 25A-35

[0302]

Table 25A-36

[0303]

Table 25A-37

[0304]

Table 25B-1

[0305]

Table 25B-2

[0306]

Table 25B-3

[0307]

Table 25B-4

[0308] [Table 25B-5]

[0309] [Table 25B-6]

[0310] [Table 25B-7]

[0311] Example 17 –Personalized selection of peptides to treat cancer Effective immunotherapy for cancer patients stimulates T cell responses (ideally CD4+ and CD8+ T cell responses) that target TAAs expressed by the cancer cells of a particular patient.

[0312] For the treatment of a particular cancer patient, peptides containing one or more of the 3286 hotspot amino acid sequences can be selected based on: (i) the type of cancer (selecting peptides containing hotspot sequences that are fragments of TSA associated with the patient's cancer); (ii) expression of TSA or expression rate of TSA (sample the patient's cancer cells and select peptides containing hotspot sequences that are fragments of TSA that are actually expressed in the patient's cancer cells; or select peptides containing hotspot sequences that are fragments of TSA that are most frequently expressed in the patient's cancer type); (iii) the patient's HLA genotype (selecting peptides containing fragments of TSA that contain amino acid sequences that are T cell epitopes (HLA class I binding PEPI3+) that can bind to at least three HLA class I alleles of the patient (ideally also containing amino acid sequences that are T cell epitopes (HLA class II binding PEPI2 / 3 / 4 / 5+) that can bind to multiple HLA class II alleles of the patient).

[0313] To illustrate the options for selecting peptides containing hotspot amino acid sequences to treat individual patients, we identified HLA class I-binding PEPI3+ at hotspot sequences in three colorectal cancer patients, one ovarian cancer patient, and three breast cancer patients with known HLA genotypes. Only hotspot sequences that are fragments of related TSAs are considered.The number of such hotspot sequences that contain PEPI3+ and the number of antigens that can be targeted using selected peptides that contain hotspot amino acid sequences are shown in Table 26.As expected, more hotspots that contain PEPI3+ were identified, and more TSAs could be targeted using the hotspot sequences identified after more cycles of the method described in Example 16. [Table 26]

[0314] Table 27 shows the amino acid sequences of peptides / hotspots that are fragments of breast cancer-associated TSAs identified only in the first cycle of the method described in Example 16, which are predicted to induce T cell responses in the three breast cancer patients in Table 26.

[0315] [Table 27-1]

[0316] [Table 27-2]

[0317] Example 18 –Selection of polynucleic acids for treating cancer Some peptides that are difficult to manufacture and use as peptide vaccines can be used in vaccines and immunotherapy when delivered to patients as polynucleic acids or vectors that encode the peptides.To optimally design a set of cancer therapeutic peptides encoded by nucleic acids or vectors for administration to patients, the method of Example 16 was repeated, except that hotspot sequences that do not meet the manufacturing feasibility requirements of peptides were not eliminated.Table 28 shows the hotspot sequences identified in the first 20 cycles and the TSAs of which they are fragments.

[0318] [Table 28-1]

[0319] [Table 28-2]

[0320] [Table 28-3]

[0321] [Table 28-4]

[0322] [Table 28-5]

[0323] [Table 28-6]

[0324] [Table 28-7]

[0325]

Table 28-8

[0326]

Table 28-9

[0327]

Table 28-10

[0328]

Table 28-11

[0329]

Table 28-12

[0330]

Table 28-13

[0331]

Table 28-14

[0332]

Table 28-15

[0333]

Table 28-16

[0334]

Table 28-17

[0335]

Table 28-18

[0336]

Table 28-19

[0337]

Table 28-20

[0338]

Table 28-21

[0339]

Table 28-22

[0340]

Table 28-23

[0341]

Table 28-24

[0342]

Table 28-25

[0343]

Table 28-26

[0344]

Table 28-27

[0345]

Table 28-28

[0346] [Table 28-29]

[0347] [Table 28-30]

[0348] [Table 28-31]

[0349] [Table 28-32]

[0350] [Table 28-33]

[0351] Example 19 –Process for personalized vaccination The process of personalized vaccination consists of three main steps as shown in Figure 17. First, during the consultation with the oncologist, the patient submits a saliva sample and a tumor sample (biopsy) for tumor pathology testing. The second step is to match the vaccine peptides with the patient's unique genetic code: based on the determined patient's HLA genotype and the determined patient's tumor type, 12 tumor- and patient-specific peptides are selected for each patient from the hotspot sequences listed in Table 25. The vaccine is then prepared and after fill-finish + QC release, the vaccine vials are shipped to the clinical site. At the clinic, the oncologist administers the vaccine to the patient as the third step of the process. The production of personalized vaccines is performed under GMP conditions. Vaccine selection and preparation can take place within 6-8 weeks.

[0352] The eligibility criteria for personalized vaccination are as follows: More than 12 immunogenic peptides (12 PEPIs) derived from 12 or more cancer-specific antigens (expression rate in disease (ER) ≥ 10%), and AGP ≥ 3 (the predicted number of antigens expressed in the patient's tumor) ·PEPI PANEL is in stock at the Warehouse.

[0353] Example 20 - Feasibility Study for a "Simulated" Breast Cancer Clinical Trial Here we describe a model where the peptide set ("warehouse") consists of 100 immunogenic peptides derived from a breast cancer specific TSA (Figure 18). These 100 peptides represent the peptide set for vaccine selection in this example. In this stock, 100 mg of each peptide is available, which corresponds to 25 peptide doses (for vaccination).

[0354] During the feasibility study, 509 HLA genotyped breast cancer subjects were screened, identifying 82 (16%) patients who were not eligible because fewer than 12 peptides were found. This means that 82% of patients could be treated with a "patient-specific" vaccine from a panel of 100 peptides. However, if three doses per patient are intended during treatment, the amount (100 mg each) is sufficient for only 32% of patients, and therefore 267 (52%) patients who do not have enough peptides in the warehouse must also be excluded from the feasibility study. As a result, 160 patients are enrolled and can be treated with three consecutive vaccine doses. Manufacturing (GMP) can be performed within about 6 months.

[0355] Example 21 - Vaccine Selection for Breast Cancer Patients (Patient C in Example 22) and Colorectal Cancer Patients (Patient D in Example 22) Here, two examples of the vaccine selection process from the peptides listed in Table 25 are presented.

[0356] The PIT vaccine for Patient C described in Example 22 was designed using a fully personalized design process and individually manufactured and proved to be highly immunogenic: 11 of 12 vaccine peptides (92%) induced CD8+ T cell responses and 11 / 12 (92%) generated CD4+ T cell specific immune responses.

[0357] Based on the HLA genotype and tumor pathology report (breast cancer) of patient C, the patient matching process was performed according to Example 16, resulting in 116 of 3286 sequences selected from 38 breast cancer-specific TSAs (according to the selection criteria of expression rate (ER) ≥ 10%). These 116 peptides can be used for vaccine selection for patient C and contain PIT vaccine sequences. Three examples of randomly selected 12 peptides from the 116 peptides are shown in Table 25 along with the predicted AGP numbers.

[0358] The predicted AGP value for the PIT vaccine in patient C was 6.45, meaning that at least six vaccine-specific TSAs were likely to be expressed in the patient's tumor and would be targeted by an immune response at least 50% of the time. (AGP95 was 4, meaning that PIT vaccine peptides from at least four different TSAs were likely to be expressed in the patient's tumor and would be targeted by an immune response in patient C at least 95% of the time.)

[0359] [Table 29]

[0360] A similar analysis was performed for patient D. During the patient matching process according to Example 19, based on the HLA genotype and tumor pathology report (colorectal cancer, CRC) data of patient D, 136 of the 3286 sequences (from 37 of the 53 CRC-specific TSAs) were selected from Table 25. These 136 peptides can be used for vaccine selection for patient D. Three examples of random selection of 13 peptides from the 136 peptides are shown in Table 30 with the calculated AGP numbers. Since patient D had a PIT vaccine consisting of 13 peptides, the random selection was also performed to a set of 13 peptides.

[0361] The PIT vaccine for patient D described in Example 22 was designed using a fully personalized design process and manufactured individually and proved to be highly immunogenic: 13 of 13 vaccine peptides (100%) induced CD8+ T cell responses and 7 / 13 (54%) generated CD4+ T cell specific immune responses.

[0362] The predicted AGP value for the PIT vaccine in patient D was 6.60, which is the predicted AGP value for at least six Vaccine-specific TSAs are likely to be expressed in the patient's tumor and are at least 50% likely to be targeted by an immune response. (AGP95 is 4, which means PIT vaccine peptides from at least 4 different TSAs are likely to be expressed in the patient's tumor and are at least 95% likely to be targeted by an immune response in patient D.)

[0363] [Table 30]

[0364] Example 22 –Personalized immunotherapy (PIT) design and treatment for ovarian, breast, and colorectal cancer This example provides proof-of-concept data from four metastatic cancer patients treated with a personalized immunotherapeutic vaccine composition to support the principle on which the present disclosure is based in part, that binding of epitopes by multiple HLA of a subject induces a cytotoxic T cell response.

[0365] POC01-Composition for the treatment of ovarian cancer using PIT (Patient A) This example is based on the disclosure herein and is based on the patient's HLA genotype. We describe the treatment of ovarian cancer patients with personalized immunotherapy compositions designed specifically for the patient.

[0366] The HLA class I and class II genotypes of a patient with metastatic ovarian adenocarcinoma (Patient A) were determined from a saliva sample.

[0367] To generate a personalized pharmaceutical composition for patient A, 13 peptides were selected, each of which fulfills two criteria: (i) is derived from an antigen expressed in ovarian cancer that has been reported in peer-reviewed scientific publications, and (ii) contains a fragment that is a T cell epitope capable of binding to at least three HLA class I of patient A (Table 31). Furthermore, each peptide is optimized to bind to the maximum number of HLA class II of the patient.

[0368] [Table 31]

[0369] The eleven PEPI3 peptides in this immunotherapy composition can induce a T cell response in patient A with a probability of 84% and two PEPI4 peptides (POC01-P2 and POC01-P5) with a probability of 98% according to the validation of the PEPI test shown in Table 7. The T cell response targets 13 antigens expressed in ovarian cancer. The expression of these cancer antigens in patient A was not tested. Instead, the probability of successful cancer cell killing was determined based on the probability of antigen expression in the patient's cancer cells and the positive predictive value (AGP count) of a PEPI3+ test of ≧1. The AGP count predicts the efficacy of the vaccine in a subject: the number of vaccine antigens expressed in the patient's tumor (ovarian adenocarcinoma) with PEPI. The AGP count indicates the number of tumor antigens that the vaccine recognizes and induces a T cell response (hits the target) against the patient's tumor. The AGP count depends on the expression rate of the vaccine antigen in the subject's tumor and the subject's HLA genotype. The exact value is between 0 (no expressed antigens presenting PEPI) and the maximum number of antigens (all antigens expressed and presenting PEPI).

[0370] The probability that patient A expresses one or more of the 13 antigens is shown in Figure 19. AGP95 (AGP with 95% probability) = 5, AGP50 (mean of the discrete probability distribution - expected value -) = 7.9, mAGP (probability that AGP is at least 2) = 100%, AP = 13.

[0371] The pharmaceutical composition for Patient A may be composed of at least two of the 13 peptides (Table 31), because it has been determined that 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+) predicts clinical response. The peptides are synthesized, dissolved in a pharma- ceutical acceptable solvent, mixed with an adjuvant, and then injected. It is desirable for the patient to receive personalized immunotherapy with at least two peptide vaccines, but more is preferable to increase the probability of killing cancer cells and reduce the chance of recurrence.

[0372] For the treatment of patient A, the 13 peptides were formulated as 4x3 or 4 peptides (POC01 / 1, POC01 / 2, POC01 / 3, POC01 / 4). One treatment cycle is defined as the administration of all 13 peptides within a 30 day period. Patient History: Diagnosis: Metastatic ovarian adenocarcinoma Age:51 Family medical history: colon and ovarian cancer (mother), breast cancer (grandmother) Tumor pathology: 2011: Initially diagnosed with ovarian adenocarcinoma; Wertheim surgery and chemotherapy; lymph node removal 2015: Metastasis to pericardial adipose tissue, resection 2016: Liver metastasis 2017: Progression in retroperitoneum and mesenteric lymph nodes; primary peritoneal carcinomatosis with small amount of ascites. Prior treatment: 2012: Paclitaxel-carboplatin (6x) 2014: Caelyx-Carboplatin (1x) 2016-2017 (9 months): Lymparza (olaparib) 2 × 400 mg / day, orally 2017: Hycamtin inf. 5 x 2.5 mg (3 x 1 seria / month) PIT vaccine treatment was started on April 21, 2017. Figure 20. 2017-2018: Patient A received 8 cycles of vaccination as add-on therapy and was still alive 17 months (528 days) after the start of treatment. During this interval, after the third and fourth vaccine treatments, the patient experienced a partial response as the best response. The patient died in October 2018.

[0373] Interferon (IFN)-γ ELISPOT bioassays confirmed the predicted T cell responses of patient A to the 13 peptides. Positive T cell responses (defined as >5-fold above control or >3-fold above control and >50 spots) were detected for all 13 20-mer peptides and all 13 9-mer peptides with the sequence of PEPI for each peptide capable of binding to the highest HLA class I allele in patient A ( FIG. 21 ).

[0374] Patient Tumor MRI Findings (Baseline April 15, 2016) (BL: Baseline for Tumor Response Assessment in Figure 22) Disease was primarily confined to the liver and lymph nodes. The use of MRI is limited to the detection of pulmonary (intrapulmonary) metastases. May 2016 to January 2017: Olaparib treatment (FU1: Follow-up observation 1 in Figure 22) December 25, 2016 (before PIT vaccine treatment). The tumor volume was dramatically reduced, and confirmation of response was obtained at (FU2: follow-up observation 2 in FIG. 22). January to March 2017 - TOPO Protocol (Topoisomerase) April 6, 2017 (FU3 in Figure 22) Existing lesions re-grow, resulting in new tumors leading to disease progression. New lesions were evident: peritoneal carcinomatosis with increased ascites, progressive liver tumors and lymph node April 21, 2017 PIT started July 26, 2017 (after PIT of the second cycle): (FU4 in Figure 22) Progression / suspected progression Rapid progression, significant pleural and ascites in lymph nodes, liver, retroperitoneum, and chest. Carboplatin, gemcitabine, and Avastin were initiated. September 20, 2017 (after 3 cycles of PIT): (FU5 in Figure 22) Partial response Complete remission in the pleural area / pleural effusion and ascites Remission in the liver, retroperitoneal region, and lymph nodes These findings suggest pseudoprogression. November 28, 2017 (after 4 cycles of PIT): (FU6 in Figure 22) Partial response Complete remission in the thoracic region. Remission in the liver, retroperitoneal region, and lymph nodes. April 13, 2018: Progress Complete response in thoracic and retroperitoneal regions. Progression in liver centers and lymph nodes. June 12, 2018: Stable disease Complete response in thoracic and retroperitoneal regions. Minimal regression in central liver and lymph nodes. July 2018: Progress October 2018: Patient A dies Partial MRI data for patient A are shown in Table 32 and FIG.

[0375] [Table 32]

[0376] Design, safety, and immunogenicity of PBRC01, a personalized immunotherapy composition for treating metastatic breast cancer (Patient B) The HLA class I and class II genotypes of metastatic breast cancer patient B were determined from a saliva sample. To create a personalized pharmaceutical composition for patient B, 12 peptides were selected, each of which meets two criteria: (i) is derived from an antigen expressed in breast cancer, reported in peer-reviewed scientific publications, and (ii) contains a fragment that is a T cell epitope that can bind to at least three HLA class I of patient B (Table 33). Furthermore, each peptide is optimized to bind to the maximum number of HLA class II of the patient. The 12 peptides target 12 breast cancer antigens. The probability that patient B expresses one or more of the 12 antigens is shown in Figure 23.

[0377] [Table 33]

[0378] Expected benefits: AGP95=4; 95% probability that the PIT vaccine will induce a CTL response against the four TSAs expressed on breast cancer cells from patient B. Additional efficacy parameters: AGP50=6.45, mAGP=100%, AP=12.

[0379] For the treatment of patient B, 12 peptides were formulated as 4x3 peptides (PBR01 / 1, PBR01 / 2, PBR01 / 3, PBR01 / 4). One treatment cycle is defined as the administration of all 12 different peptide vaccines within 30 days (Figure 23C). Patient History: 2013: Diagnosis: Breast cancer; CT scan and bone scan ruled out metastatic disease. 2014: Double mastectomy, followed by chemotherapy 2016: Widespread metastatic disease with lymph node metastases both above and below the diaphragm. Multiple liver and lung metastases. Treatment: 2013-2014: Adriamycin-cyclophosphamide and paclitaxel 2017: Letrozole, palbociclib, and gozorelin, and the PIT vaccine 2018: The patient's condition worsened and he died in January PIT vaccine treatment was started on April 7, 2017. Treatment schedule and The main characteristics of the disease are shown in Table 34.

[0380] [Table 34]

[0381] Eight to twelve vaccine peptides were predicted with 95% confidence to induce T cell responses in patient B. Peptide-specific T cell responses were measured in all available PBMC samples using an interferon (IFN)-γ ELISPOT bioassay (Figure 24). The results confirmed the prediction: nine peptides showed a positive reaction, proving that T cells could recognize tumor cells of patient B expressing FISP1, BORIS, MAGE-A11, HOM-TES-85, NY-BR-1, MAGE-A9, SCP1, MAGE-A1, and MAGE-C2 antigens. Some tumor-specific T cells were present after the first vaccination and boosted with additional treatments (e.g., MAGE-A1), others were induced after the boost (e.g., MAGE-A9). Such a broad range of tumor-specific T cell responses is remarkable in patients with advanced cancer.

[0382] Patient B's medical history and results March 7, 2017: Pre-PIT vaccine treatment Multimetastatic disease of the liver with true extrinsic compression of the origin of the common bile duct and massive dilatation of the entire intrahepatic bile duct. Adenopathy of the peritoneum, porta hepatis, and retroperitoneum. March 2017: Treatment started - letrozole, palbociclib, gosorelin, and PIT vaccine May 2017: Drug withdrawal May 26, 2017: After one cycle of PIT Metabolic activity of the tumor decreased by 83% (PET CT) Liver, lungs, lymph nodes, and other metastases. June 2017: Normalization of neutrophil counts, as confirmed by the patient, indicates discontinuation of palbociclib Tumor marker rebound delayed by 4 months March-May 2017: CEA and CA remained elevated consistent with the patient's anti-cancer treatment outcome (Ban, Future Oncol 2018) June-September 2017: CEA and CA decreased consistent with delayed response to immunotherapy Quality of life February to March 2017: Hospitalized for illness and jaundice April to October 2017: Very good November 2017: Worsening condition (tumor escape?) January 2018: Patient B dies.

[0383] The immunogenicity results are summarized in FIG.

[0384] Patient Clinical Outcome Measurements: One month prior to initiation of PIT vaccination treatment, PET CT confirmed extensive DFG avid disease with lymph node metastases both above and below the diaphragm (Table 34). The patient had progressive multiple liver, multifocal bone, and lung metastases, as well as retroperitoneal adenopathy. The patient's intrahepatic enzymes were elevated consistent with damage caused by liver metastases, with elevated bilirubin and jaundice. The patient was administered letrozole, palbociclib, and gozorelin as anticancer treatment. Two months after initiation of PIT vaccination, the patient began to feel very well, and her quality of life had normalized. Indeed, the patient's PET CT showed significant morphological regression in liver, lung, bone, and lymph node metastases. At the supradiaphragmatic stage, metabolic adenopathy was not identifiable.

[0385] The combination of pablocilib and the personalized vaccine was likely responsible for the remarkable early response observed after vaccination. Pablocilib has been shown to improve the activity of immunotherapy by increasing HLA presentation of TSA and decreasing Treg proliferation (Goel et al. Nature. 2017:471-475). The treatment results of patient B suggest that maximum efficacy can be achieved using the PIT vaccine as an add-on to state-of-the-art therapy.

[0386] Patient B's tumor biomarkers were tracked to separate the effect of state-of-the-art therapy from that of the PIT vaccine. Tumor markers remained unchanged for the first 2-3 months of treatment, then declined rapidly, suggesting a delayed effect typical of immunotherapy (Table 34). Furthermore, by the time the tumor biomarkers declined, the patient had already voluntarily discontinued treatment, confirmed by an increase in neutrophil count.

[0387] After the fifth PIT treatment, the patient experienced symptoms. Tumor markers and liver enzyme levels were elevated again. Thirty-three days after the last PIT vaccination, the patient's PET CT showed significant metabolic progression in liver, peritoneum, skeleton, and left adrenal sites, confirming the laboratory findings. Isolated recurrence in distant metastases could be due to potential immune resistance; possibly caused by downregulation of both HLA expression impairing tumor recognition by PIT-induced T cells. However, PET CT detected complete regression of metabolic activity in all axillary and mediastinal axillary supradiaphragmatic targets (Table 34). These localized tumor responses could explain the known delayed and sustained responses to immunotherapy, since it is unlikely that these tumor sites will not recur after discontinuation of anticancer drug treatment.

[0388] Personalized immunotherapy composition for the treatment of a patient with metastatic breast cancer (Patient C) To treat a patient with metastatic breast cancer (Patient C), a PIT vaccine with a similar design to that described for Patient A and Patient B was prepared. The PIT vaccine contained 12 PEPIs. The predicted efficacy of the PIT vaccine is AGP=4. The treatment schedule of the patient is shown in Figure 25.

[0389] Tumor Pathology Primary tumor in 2011. HER2-, ER+, sentinel node negative 2017 Multiple bone metastases: ER+, cytokeratin 7+, cytokeratin 20-, C A125-, TTF1-, CDX2- treatment 2011 Wide local excision, negative sentinel nodes; radiation therapy 2017~ Anticancer therapy (Tx): letrozole (2.5mg / day), denosumab; Radiation (Rx): 1 bone PIT vaccine (3 cycles) as an add-on to standard treatment

[0390] Bioassays confirmed positive T cell responses (defined as >5-fold above control or >3-fold above control and >50 spots) to 11 of 12 20-mer peptides from the PIT vaccine and 11 of 12 9-mer peptides with the sequence of PEPI of each peptide capable of binding to the patient's highest HLA class I allele (Figure 26). Long-lasting memory T cell responses were detected 14 months after the last vaccination (Figure 26C-D).

[0391] Treatment outcomes The clinical results of the treatment of Patient C are shown in Table 35. Patient C has a partial response and evidence of healing of bone metastases.

[0392] [Table 35]

[0393] The immune response is shown in Figure 26. Expected immunogenicity, PEPI=12 (CI95%[8,12] Immunogenicity detected: 11 (20mer) and 11 (9mer) antigen-specific T cell responses after three PIT vaccinations (Fig. 26A,B). PIT vaccine-specific immune responses could still be detected 4.5, 11, or 14 months after the last vaccination (Fig. 26C,D).

[0394] Personalized immunotherapy composition for the treatment of a patient with metastatic colorectal cancer (Patient D) Tumor Pathology February 2017: Surgery for mCRC (MSS) with liver metastasis, primary tumor (sigmoid colon) pT3 pN2b(8 / 16)M1. KRAS G12D, TP53-C135Y, KDR-Q472H, MET-T1010I mutations. SATB2 expression. EGFR wt, PIK3CA-I391M (non-driver). 2017 (June) Partial liver resection: KRAS-G12D(35G>A)NRAS wt, May 2018: Second resection: SATB2 expression, lung metastasis 3→21 treatment 2017 FOLFOX-4 (oxaliplatin, calcium folinate, 5-FU) → Allergic reaction during the second treatment DeGramont (5-FU + Calcium folinate) June 2018 → FOLFIRI + ramucirumab, twice weekly; chemoembolization 2018 (October): PIT vaccination (13 patient-specific peptides, administered four times) as an add-on to standard treatment.

[0395] The patient treatment schedule is shown in FIG.

[0396] Treatment outcomes The patient's general condition was good, and pulmonary progression was confirmed by CT scan 8 months later. Using 9-mer and 20-mer peptides for stimulation, both PIT-induced and pre-existing T cell responses were measured by enriched fluorescent spots from PBMCs (FIG. 28).

[0397] A summary of immune response rates and immunogenicity results demonstrates that the vaccine is appropriately designed for target antigen selection and induction of multiple peptide-targeted immune responses, both CD4+ and CD8+ specific.

[0398] [Table 36]

[0399] The present invention includes the following. [Item 1] A peptide having a length of up to 50 amino acids and comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 2786 and / or 5432 to 5931. [Item 2] A polynucleic acid or vector encoding the peptide according to Item 1. [Item 3] A panel of two or more types of peptides according to Item 1 or two or more types of polynucleotides or vectors according to Item 2, wherein each peptide comprises a peptide having a different amino acid sequence selected from SEQ ID NOs: 1 to 2786 and / or 5432 to 5931, or each polynucleic acid or vector encodes a peptide having a different amino acid sequence selected from SEQ ID NOs: 1 to 2786 and / or 5432 to 5931. [Item 4] The panel of peptides, polynucleic acids, or vectors according to item 3, wherein each peptide or encoded peptide comprises at least one amino acid sequence selected from one of the following groups: (a) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a breast cancer associated antigen listed in Table 24; (b) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a lung cancer associated antigen listed in Table 24; (c) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a prostate cancer associated antigen listed in Table 24; (d) a sequence listed in Table 25A and / or Table 25B, which is shown in Table 25A or 25B to be a fragment of a colorectal cancer associated antigen listed in Table 24; (e) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a bladder cancer associated antigen listed in Table 24; (f) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of an ovarian cancer associated antigen listed in Table 24; (g) a sequence listed in Table 25A and / or Table 25B, which is shown in Table 25A or 25B to be a fragment of a pancreatic cancer associated antigen listed in Table 24; (h) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a brain cancer associated antigen listed in Table 24; (i) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a leukemia associated antigen listed in Table 24; (j) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a lymphoma associated antigen listed in Table 24; (k) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a hepatocellular carcinoma associated antigen listed in Table 24; (l) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a melanoma associated antigen listed in Table 24; (m) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a thyroid cancer associated antigen listed in Table 24; (n) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a childhood cancer associated antigen listed in Table 24; (o) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a gastric cancer associated antigen listed in Table 24; (p) a sequence listed in Table 25A and / or Table 25B that is shown in Table 25A or 25B to be a fragment of a kidney cancer associated antigen listed in Table 24; (q) a sequence listed in Table 25A and / or Table 25B, which is shown in Table 25A or 25B to be a fragment of a head and neck cancer associated antigen listed in Table 24; (r) a sequence listed in Table 25A and / or Table 25B, which is shown in Table 25A or 25B to be a fragment of a cervical cancer associated antigen listed in Table 24. [Item 5] A pharmaceutical composition or kit comprising one or more peptides described in item 1, one or more polynucleic acids or vectors described in item 2, or a panel of peptides, polynucleic acids, or vectors described in items 3 or 4, optionally comprising at least one pharma- ceutically acceptable diluent, carrier, or preservative. [Item 6] The pharmaceutical composition or kit according to Item 5, comprising one or more polynucleic acids or vectors according to Item 2 encoding at least two types of peptides according to Item 1 or at least two types of peptides according to Item 1, wherein each peptide or each encoded peptide comprises an amino acid sequence selected from SEQ ID NOs: 1 to 2786 and / or 5432 to 5931, the amino acid sequence comprising T cell epitopes capable of binding to at least three HLA class I alleles and / or T cell epitopes capable of binding to at least three HLA class II alleles of an individual human subject. [Item 7] A method for predicting that a particular human subject will have a CD8+ T cell response and / or a CD4+ T cell response to each peptide or encoded peptide of the pharmaceutical composition or kit according to Item 5, comprising: (i) a. determining that each peptide or encoded peptide contains 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; and b. predicting that the subject will have a CD8+ T cell response to each peptide of the pharmaceutical composition, or each peptide, polynucleic acid, or vector of the kit; and / or (ii) a. determining that each peptide or encoded peptide contains 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; and b. predicting that the subject will have a CD4+ T cell response to each peptide of the pharmaceutical composition, or each peptide, polynucleic acid, or vector of the kit. The method includes: [Item 8] The method according to Item 7, further comprising administering the pharmaceutical composition to the subject. [Item 9] A method for vaccinating, providing immunotherapy, or inducing a cytotoxic T cell response in a subject, comprising administering to the subject a peptide, polynucleic acid, or vector of the pharmaceutical composition or kit described in Item 5 or 6. [Item 10] The method described in Item 9, wherein the peptide, polynucleic acid, or vector of the pharmaceutical composition or kit is predicted to induce a cytotoxic T cell response and / or a helper T cell response in the subject using the method described in Item 7. [Item 11] The method according to any one of Items 8 to 10, which is a method for treating cancer, optionally bladder cancer, brain cancer, breast cancer, colorectal cancer, gastric cancer, hepatocellular carcinoma, leukemia, lung cancer, lymphoma, melanoma, ovarian cancer, pancreatic cancer, childhood cancer, thyroid cancer, prostate cancer, kidney cancer, head and neck cancer, esophageal cancer, and cervical cancer. [Item 12] A method for preparing a pharmaceutical composition or kit for use in a method for treating cancer in a specific human subject, comprising: a. selecting one or more polynucleic acids or vectors according to item 2 encoding two or more peptides according to item 1 or at least two peptides according to item 1, wherein each peptide or encoded peptide comprises an amino acid sequence selected from SEQ ID NOs: 1 to 2786 and / or 5432 to 5931, the amino acid sequence comprising T cell epitopes capable of binding to at least three HLA class I alleles and / or T cell epitopes capable of binding to at least three HLA class II alleles of the specific human subject; b. preparing a pharmaceutical composition or kit comprising two or more peptides selected in step a, or one or more polynucleic acids or vectors; The method includes: [Item 13] A method for designing or preparing a peptide, or a polynucleic acid or vector encoding a peptide, or a panel of peptides, or one or more polynucleic acids or vectors encoding a panel of peptides, for use in a method for inducing a T cell response against a target polypeptide, comprising: (i) selecting or defining a model human population comprising a plurality of subjects, each defined by an HLA class I genotype and / or an HLA class II genotype; (ii) for each subject in the model population, (a) an amino acid sequence of the target polypeptide that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; (b) an amino acid sequence of the target polypeptide that is a T cell epitope capable of binding to at least three HLA class II molecules of the subject; (c) an amino acid sequence of the target polypeptide that comprises a T cell epitope capable of binding to at least three HLA class I molecules of the subject and a T cell epitope capable of binding to at least three HLA class II molecules of the subject; or (d) a. a T cell epitope capable of binding to at least three HLA class II molecules; and b. comprises an amino acid sequence that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject. The amino acid sequence of the target polypeptide Identifying; (iii) selecting a polypeptide fragment window length of 9 to 50 amino acids; (iv) (a) having the length selected in step (iii); and (b) comprising the amino acid sequence identified in any one of steps (ii)(a) to (d) in the highest percentage of subjects in the model population; identifying a fragment of the target polypeptide; (v) optionally testing the fragments identified in step (iv) against additional predefined criteria, rejecting said fragments if they do not satisfy the further predefined criteria, and repeating step (iv); (a) having the length selected in step (iii); and (b) comprising the amino acid sequence identified in step (iv) in the next highest percentage of subjects in the model population. identifying alternative fragments of the target polypeptide; (vi) optionally repeating step (iv) and optionally step (v) in one or more further rounds, wherein in each round further fragments of said target polypeptide are identified, and in each round removing a subject from said model population if any of the fragments selected in step (iv) of any of the previous rounds and not rejected in step (v) comprise the amino acid sequence identified in step (ii) for that subject; (vii) a peptide, a polynucleic acid or a vector encoding a peptide, a peptide or designing or preparing one or more polynucleic acids or vectors encoding a panel of peptides, each peptide comprising one or more of the target polypeptide fragments identified in step (iv), (v) or (vi), and optionally wherein the polypeptide fragments are flanked at the N- and / or C-terminus by additional amino acids which are not part of the sequence of the target polypeptide antigen. The method includes: [Item 14] The method according to Item 13, wherein the target polypeptide is expressed by a pathogenic organism, a virus, or a cancer cell, or is a cancer-testis antigen, and optionally the target polypeptide is selected from the antigens listed in any of Tables 2 to 5. [Item 15] The method further comprises selecting one or more peptides, polynucleic acids, or vectors designed or prepared according to the method of items 13 or 14 for use in a method of vaccinating, providing immunotherapy, or inducing a cytotoxic and / or helper T cell response in a subject, wherein optionally each of the one or more peptides or encoded peptides is (a) a fragment of a polypeptide expressed by a pathogenic organism, a virus, or a cancer cell; and (b) a T cell epitope capable of binding to at least three HLA class I molecules of the subject or a T cell epitope capable of binding to at least three HLA class II molecules of the subject. 15. The method according to item 13 or 14, comprising an amino acid sequence. [Item 16] The method according to Item 15, further comprising administering the one or more peptides, polynucleic acids, or vectors to the subject. [Item 17] A panel of peptides, polynucleic acids, or vectors designed and / or prepared according to the method described in Item 13 or 14, or comprising or encoding two or more peptides designed and / or prepared according to the method described in Item 13 or 14, or a panel of peptides, polynucleic acids, or vectors selected according to the method described in Item 15. [Item 18] A panel of peptides, or one or more polynucleic acids or vectors encoding a panel of peptides, for use in a method of inducing a T cell response to one or more target polypeptides in a subject of a target human population, wherein each of the peptides or encoded peptides is (a) 9 to 50 amino acids in length; and (b) comprising fragments of said one or more target polypeptides, said fragments being capable of inducing a phenotype in at least 10% of subjects in the human population to be treated; a. an amino acid sequence of a target polypeptide that is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; b. an amino acid sequence of a target polypeptide that is a T cell epitope capable of binding to at least three HLA class II molecules of the subject; c. an amino acid sequence of a target polypeptide that comprises a T cell epitope capable of binding to at least three HLA class I molecules of the subject and a T cell epitope capable of binding to at least three HLA class II molecules of the subject; or d. i. a T cell epitope capable of binding to at least three HLA class II molecules; and ii. capable of binding to at least three HLA class I molecules of the subject Contains an amino acid sequence that is a T cell epitope Amino acid sequence of the target polypeptide A panel of peptides, or one or more polynucleic acids or vectors encoding a panel of peptides, comprising an amino acid sequence comprising: [Item 19] A pharmaceutical composition or kit comprising a panel of peptides described in Item 17 or 18, or one or more polynucleic acids or vectors encoding the panel of peptides, optionally comprising at least one pharma- ceutically acceptable diluent, carrier, or preservative. [Item 20] A method for vaccinating, providing immunotherapy, or inducing a cytotoxic T cell response in a subject, comprising administering to the subject a panel of peptides, polynucleic acids, or vectors of the pharmaceutical composition or kit described in Item 19. [Item 21] One or more of the peptides or the encoded peptides, or each of the peptides, (a) a fragment of a polypeptide expressed by a pathogenic organism, a virus, or a cancer cell; and (b) a T cell epitope capable of binding to at least three HLA class I molecules of the subject or a T cell epitope capable of binding to at least three HLA class II molecules of the subject. 21. The method of claim 20, comprising an amino acid sequence.

Claims

[Claim 1] A peptide that is up to 50 amino acids in length and comprises the amino acid sequence of any of SEQ ID NOs: 1-2786 and / or 5432-5931.

Images