Peptide vaccine

Abstract

The present disclosure relates to polypeptides useful for the prevention or treatment of cancer, in particular breast cancer, ovarian cancer and colorectal cancer, and pharmaceutical compositions comprising the polypeptides. The disclosure also relates to methods of inducing a cytotoxic T cell response or treating cancer in a subject by administering a pharmaceutical composition comprising the peptide, as well as methods of identifying companion diagnostics for treating a subject. The peptides comprise T cell epitopes that are immunogenic in a high percentage of patients.

Description

Technical Field

[0001] The present disclosure relates to polypeptides and vaccines for the prevention or treatment of cancer, particularly most breast, ovarian and colorectal cancers. Background Art

[0002] Cancer is killing millions of people worldwide because existing drugs are not yet effective in preventing or treating it. Current checkpoint inhibitor immunotherapies that reactivate existing immune responses can provide clinical benefit to a subset of cancer patients. Current cancer vaccines that induce new immune responses are poorly immunogenic and fail to benefit most patients.

[0003] A recent analysis of 63,220 unique tumors showed that due to the extensive inter-individual tumor genomic heterogeneity, cancer vaccines need to be generated specifically for each patient (Hartmaier et al. Genome Medicine 2017 9:16). Using existing technologies, it is currently not feasible to scale up HLA-specific cancer vaccines to large populations. Summary of the invention

[0004] In antigen presenting cells (APCs), protein antigens are processed into peptides. These peptides bind to human leukocyte antigen molecules (HLA) and are presented to the cell surface of T cells as peptide-HLA complexes. Different individuals express different HLA molecules, and different HLA molecules present different peptides. Therefore, according to the prior art, a peptide or a fragment of a larger polypeptide is identified as immunogenic to a particular human subject if it is presented by an HLA molecule expressed by the subject. In other words, the prior art describes immunogenic peptides as HLA-restricted epitopes. However, HLA-restricted epitopes induce T cell responses only in a subset of individuals expressing HLA molecules. Peptides that activate T cell responses in one individual are inactive in other individuals despite HLA allele matching. Therefore, it was previously unknown how individual HLA molecules present antigen-derived epitopes that positively activate T cell responses.

[0005] As provided herein, the multiple HLAs expressed by an individual need to present identical peptides to trigger T cell responses. The polypeptide antigen fragments that are immunogenic to a specific individual are those fragments that can be combined with multiple Class I (activation of cytotoxic T cells) or Class II (activation of helper T cells) HLAs expressed by the individual. For example, the inventors have found that the T cell epitopes that are present and combined with at least three HLA I types of the subject predict that the subject makes an immune response to the polypeptide.

[0006] Based on this discovery, the present inventors have identified T cell epitopes from certain breast cancer, ovarian cancer and / or colorectal cancer associated polypeptide antigens (cancer testis antigen (CTA)) that are capable of binding at least three HLA class I in a high proportion of individuals. These T cell epitopes or antigen fragments comprising T cell epitopes can be used to induce a specific immune response against tumor cells expressing these antigens and for treating or preventing cancer.

[0007] In a first aspect, the present disclosure provides a polypeptide comprising a fragment of up to 50 consecutive amino acids of:

[0008] (a) a colorectal cancer-associated antigen selected from TSP50, EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN, LEMD1, MAGE-A8, MAGE-A6 and MAGE-A3, wherein the fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 21 to 40 and 234 to 250;

[0009] (b) an ovarian cancer-associated antigen selected from PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN and AKAP-3, wherein the fragment comprises the amino acid sequence of any one of SEQ ID NOs: 272 to 301; and / or

[0010] (c) a breast cancer-associated antigen selected from PIWIL-2, AKAP-4, EpCAM, BORIS, HIWI, SPAG9, PLU-1, TSGA10, ODF-4, SP17, RHOXF-2, PRAME, NY-SAR-35, MAGE-A9, NY-BR-1, SURVIVIN, MAGE-A11, HOM-TES-85 and NY-ESO-1, wherein the above fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 1 to 20, 24 and 172 to 194.

[0011] In some specific cases, the present disclosure provides a polypeptide,

[0012] (a) is a fragment of a colorectal cancer-associated antigen selected from TSP50, EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN, MAGE-A8, MAGE-A6, MAGE-A3 and LEMD1, wherein the fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 21 to 40 and 234 to 250; or

[0013] (b) comprising or consisting of two or more fragments of one or more colorectal cancer associated antigens selected from TSP50, EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN, MAGE-A8, MAGE-A6, MAGE-A3 and LEMD1, wherein each fragment comprises a different amino acid sequence selected from any one of SEQ ID NOs: 21 to 40 and 234 to 250, optionally wherein the above fragments overlap or are arranged end to end in the polypeptide; or

[0014] (c) is a fragment of an ovarian cancer-associated antigen selected from PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN and AKAP-3, wherein the fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 272 to 301; or

[0015] (d) comprising or consisting of two or more fragments of one or more ovarian cancer-associated antigens selected from PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN and AKAP-3, wherein each fragment comprises a different amino acid sequence selected from any one of SEQ ID NOs: 272 to 301, optionally wherein the above-mentioned fragments overlap or are arranged end to end in the polypeptide; or

[0016] (e) is a fragment of a breast cancer-associated antigen selected from SPAG9, AKAP-4, BORIS, NY-SAR-35, NY-BR-1, SURVIVIN, MAGE-A11, PRAME, MAGE-A9, HOM-TES-85, PIWIL-2, EpCAM, HIWI, PLU-1, TSGA10, ODF-4, SP17, RHOXF-2, wherein the fragment comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 20, 24, and 172 to 194; or

[0017] (f) comprising or consisting of two or more fragments of one or more breast cancer associated antigens selected from SPAG9, AKAP-4, BORIS, NY-SAR-35, NY-BR-1, SURVIVIN, MAGE-A11, PRAME, MAGE-A9, HOM-TES-8, PIWIL-2, EpCAM, HIWI, PLU-1, TSGA10, ODF-4, SP17, RHOXF-2, wherein each fragment comprises a different amino acid sequence selected from any one of SEQ ID NOs: 1 to 20, 24 and 172 to 194; optionally, wherein the above-mentioned fragments overlap or are arranged end to end in the polypeptide.

[0018] In some specific cases, the polypeptide comprises or consists of a fragment of the following antigens:

[0019] (a) TSP50, EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN, MAGE-A8, MAGE-A6, MAGE-A3 and LEMD1;

[0020] (b) PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN and AKAP-3; and / or

[0021] (c) SPAG9, AKAP-4, BORIS, NY-SAR-35, NY-BR-1, SURVIVIN, MAGE-A11, PRAME, MAGE-A9, HOM-TES-8, PIWIL-2, EpCAM, HIWI, PLU-1, TSGA10, ODF-4, SP17, RHOXF-2;

[0022] Each fragment comprises a different amino acid sequence selected from SEQ ID NOs: 21 to 40 and SEQ ID NOs: 234 to 250; SEQ ID NOs: 272 to 301; and / or SEQ ID NOs: 1 to 20, 24 and SEQ ID NOs: 172 to 194.

[0023] In some cases, the polypeptide comprises or consists of one or more amino acid sequences selected from SEQ ID NOs:41-80, SEQ ID NOs:251-271, SEQ ID NOs:302-331, and SEQ ID NOs:196-233.

[0024] In some cases, the polypeptide comprises or consists of one or more amino acid sequences selected from SEQ ID NOs: 41-80, SEQ ID NOs: 195-233, SEQ ID NOs: 251-271, and SEQ ID NOs: 302-331, or selected from SEQ ID NOs: 81-142, 332-346, and 435-449.

[0025] In another aspect, the disclosure provides a panel of two or more polypeptides as described above, wherein each peptide comprises or consists of a different amino acid sequence selected from SEQ ID NOs: 21 to 40 and 234 to 250; or selected from SEQ ID NOs: 272 to 301; or selected from SEQ ID NOs: 1 to 20, 24, and 172 to 194; or selected from SEQ ID NOs: 1 to 40, 234 to 250, 272 to 301, and 172 to 194. In some cases, the panel of polypeptides comprises or consists of one or more peptides comprising or consisting of an amino acid sequence of SEQ ID NOs: 130, 121, 131, 124, 134, 126, and / or SEQ ID NOs: 435 to 449.

[0026] On the other hand, the present disclosure provides a pharmaceutical composition or kit having one or more of the above-mentioned polypeptides or peptide panels as an active ingredient, or having a polypeptide comprising at least two amino acid sequences as an active ingredient, wherein the above amino acid sequences are selected from SEQ ID NOs: 21 to 40 and 234 to 250; SEQ ID NOs: 272 to 301; and / or SEQ ID NOs: 1 to 20, 24 and 172 to 194; or selected from SEQ ID NOs: 130, 121, 131, 124, 134, 126 and / or 435 to 449.

[0027] In another aspect, the present disclosure provides a method of inducing an immune response (e.g., vaccination, providing immunotherapy in a subject, or inducing a cytotoxic T cell response), the method comprising administering to the subject a pharmaceutical composition, kit, or panel as described above. The method may be a method of treating cancer, such as breast cancer, ovarian cancer, or colorectal cancer.

[0028] In other aspects, the present invention provides

[0029] - a pharmaceutical composition, kit or panel as described above, for use in a method of inducing an immune response or in a method of treating cancer, optionally breast cancer, ovarian cancer or colorectal cancer; and

[0030] - Use of a peptide or peptide panel as described above for the preparation of a medicament for inducing an immune response or for treating cancer, optionally breast cancer, ovarian cancer or colorectal cancer.

[0031] In another aspect, the present disclosure provides a method for identifying a human subject who may have a cytotoxic T cell response to administration of the above-mentioned pharmaceutical composition, the method comprising:

[0032] (i) determining that the active ingredient polypeptide of the pharmaceutical composition comprises a sequence of a T cell epitope capable of binding to at least three HLA class I molecules of the subject; and

[0033] (ii) identifying a subject as likely to have a cytotoxic T cell response to administration of the pharmaceutical composition.

[0034] In another aspect, the present disclosure provides a method for identifying a subject who may be clinically responsive to the above-mentioned treatment methods, the method comprising:

[0035] (i) determining that the active ingredient polypeptide comprises two or more different amino acid sequences, each of which is

[0036] a. a T cell epitope capable of binding to at least three HLA class I molecules of a subject; and

[0037] b. a fragment of a cancer associated antigen expressed by a cancer cell of the subject; and

[0038] (ii) Identifying subjects who are likely to be clinically responsive to treatment.

[0039] In another aspect, the present disclosure provides a method of determining the likelihood that a particular human subject will have a clinical response to the treatment method of claim 10, wherein one or more of the following factors corresponds to a higher likelihood of a clinical response:

[0040] (a) a higher number of amino acid sequences and / or different amino acid sequences are present in the active ingredient polypeptide, each of which is a T cell epitope capable of binding to at least three HLA class I molecules of the subject;

[0041] (b) a higher number of target polypeptide antigens comprising at least one amino acid sequence

[0042] A. is contained in the active ingredient polypeptide; and

[0043] B. is a T cell epitope capable of binding to at least three HLA class I molecules of the subject;

[0044] Optionally wherein the target polypeptide antigen is expressed in the subject, further optionally wherein the target polypeptide antigen is present in one or more samples obtained from the subject;

[0045] (c) a higher probability that the subject expresses a target polypeptide antigen, optionally a threshold amount of the target polypeptide antigen and / or optionally a target polypeptide antigen that has been determined to comprise at least one amino acid sequence that

[0046] A. is contained in the active ingredient polypeptide; and

[0047] B. is a T cell epitope capable of binding to at least three HLA class I molecules of the subject;

[0048] and / or

[0049] (d) predicting that the subject expresses a higher amount of a target polypeptide antigen, optionally a higher amount of a target polypeptide antigen expressed by the subject with a threshold probability, and / or optionally having determined a target polypeptide antigen comprising at least one amino acid sequence that

[0050] A. is contained in the active ingredient polypeptide; and

[0051] B. is a T cell epitope capable of binding to at least three HLA class I molecules of the subject.

[0052] In some cases, the cancer-associated antigen can be TSP50, EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN, LEMD1, MAGE-A8, MAGE-A6, MAGE-A3, PIWIL-4, WT1, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, PRAME, HIWI, PLU-1, TSGA10, ODF-4, RHOXF-2, NY-SAR-35, MAGE-A9, NY-BR-1, MAGE-A11, HOM-TES-85, NY-ESO-1 and AKAP-3. In some cases, the above method includes the step of determining that the cancer cells of the subject express one or more cancer-associated antigens. The cancer-associated antigen can be present in one or more samples obtained from the subject.

[0053] In some cases, administration of the active ingredient polypeptide of the pharmaceutical composition or kit may be selected as a method of treating a subject. The subject may be further treated by administering a pharmaceutical composition or active ingredient polypeptide.

[0054] In another aspect, the present disclosure provides a method of treatment as described above, wherein the subject has been identified as likely to have a clinical response or to have a minimum likelihood above a threshold of a clinical response to treatment as described above.

[0055] In another aspect, the present disclosure provides a method of identifying a human subject who is likely to have no clinical response to a treatment as described above, the method comprising

[0056] (i) determining that the active ingredient polypeptide of the pharmaceutical composition does not contain two or more different amino acid sequences, each of which is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; and

[0057] (ii) Identifying subjects who are likely not to respond clinically to treatment.

[0058] The above method may include the step of determining the HLA class I genotype of the subject.

[0059] The present disclosure will now be described in more detail by way of example and not limitation and with reference to the accompanying drawings. When the present disclosure is given, many equivalent modifications and variations will be apparent to those skilled in the art. Therefore, the exemplary embodiments of the present invention set forth are considered to be illustrative and not restrictive. Various changes may 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 herein by reference in their entirety.

[0060] The present disclosure includes combinations of the described aspects and preferred features, except where such combinations are clearly impermissible or stated to be expressly avoided. As used in this specification and the appended claims, the singular forms "a", "an", and "an" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a peptide" includes two or more such peptides.

[0061] 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

[0062] Figure 1 : ROC curves of HLA-restricted PEPI biomarkers.

[0063] Figure 2 : ROC curves for the ≥1 PEPI3+ test to determine diagnostic accuracy.

[0064] Figure 3: Distribution of HLA class I PEPI3+ relative to CD8+ T cell responses measured by prior art assays in peptide libraries used in CD8+ T cell response assays. A: HLA class I restricted PEPI3+. The 90% overall percentage of agreement (OPA) in T cell responses and PEPI3+ peptides demonstrates the use of the disclosed peptides in predicting vaccine-induced T cell response sets in individuals. B: HLA class I restricted epitopes (PEPI1+). The OPA between the predicted epitope and CD8+ T cell response was 28% (not statistically significant). Darkest gray: true positive (TP), peptide and T cell response detected; light gray: false negative (FN), only T cell response detected; lightest gray: false positive (FP), only peptide detected; dark gray: true negative (TN): neither peptide nor T cell response was detected.

[0065] Figure 4 : Distribution of HLA class II PEPIs relative to CD4+ T cell responses measured by prior art assays in the peptide pool used in the assay. A: HLA class II restricted PEPI4+. The OPA between PEPI4+ and CD4+ T cell responses was 67% (p=0.002). B: HLA class II restricted epitopes. The OPA between HLA class II restricted epitopes and CD4+ T cell responses was 66% (not statistically significant). Darkest grey: true positive (TP), peptide and T cell responses were detected; light grey: false negative (FN), only T cell responses were detected; lightest grey: false positive (FP), only peptides were detected; dark grey: true negative (TN): neither peptides nor T cell responses were detected.

[0066] Figure 5 : Multiple HLA-binding peptides defining the HPV-16 LPV vaccine-specific T cell responder set in 18 VIN-3 and 5 cervical cancer patients. HLA class I-restricted PEPI3 counts (A and B) and HLA class II-restricted PEPI3 counts (C and D) derived from each patient's LPV antigen. Light gray: immune responders measured after vaccination in clinical trials; dark gray: immune non-responders measured after vaccination in clinical trials. Results show that ≥3 HLA class I-binding peptides predict CD8+ T cell reactivity, and ≥4 HLA class II-binding peptides predict CD4+ T cell reactivity.

[0067] Figure 6: Multiple HLA class I binding peptides defining the HPV vaccine-specific T cell response set for two patients. A: Four HPV antigens in the HPV vaccine. The box indicates the length of the amino acid sequence from the N-terminus to the C-terminus. B: Method for identifying multiple HLA binding peptides for two patients: The HLA sequences of the patients are labeled with the 4-digit HLA genotype of the patient ID. The positions of the first amino acids of 54 and 91 epitopes that can bind to patient 12-11 and patient 14-5 HLA (PEPI1+), respectively, can be indicated by lines. PEPI2 represents peptides selected from PEPI1+ that can bind to multiple HLAs of the patient (PEPI2+). PEPI3 represents peptides that can bind to ≥3 HLAs of the patient (PEPI3+). PEPI4 represents peptides that can bind to ≥4 HLAs of the patient (PEPI4+). PEPI5 represents peptides that can bind to ≥5 HLAs of the patient (PEPI5+). PEP16 represents peptides that can bind to 6 HLAs of the patient (PEP16). C: DNA vaccine-specific PEPI3+ group of two patients characterizing their vaccine-specific T cell responses.

[0068] Figure 7 : Correlation between ≥1 PEPI3+ scores and CTL response rates for peptide targets measured in clinical trials.

[0069] Figure 8 : Correlation between ≥1 PEPI3+ score and clinical immune response rate (IRR) of immunotherapy vaccines. Dashed line: 95% confidence band.

[0070] Fig. 9 : Relationship between ≥2PEPI3+ score and disease control rate (DCR) of immunotherapy vaccines. Dashed line: 95% confidence band.

[0071] Fig.10 : Peptide hotspot analysis example: PRAME antigen hotspots of 433 patients in the model population. The y-axis is the 433 patients in the model population, and the x-axis is the amino acid sequence of the PRAME antigen (CTA). Each data point represents the PEPI of ≥3 HLA class I representatives of a patient starting from the specified amino acid position. The two most common PEPIs of the PRAME antigen (called bestEPI) are highlighted in dark gray (peptide hotspot = PEPI hotspot).

[0072] Fig.11 :The CTA expression curve was calculated by analyzing the expression frequency data of tumor-specific antigens (CTA) in human breast cancer tissues. (Cell line data are not included.)

[0073] Fig.12: Distribution of breast cancer antigen expression based on calculation of multi-antigen response from expression frequencies of 10 different CTAs selected. A: Non-cumulative distribution of expected values ​​for the number of expressed antigens (AG50) calculated. This value indicates that 6.14 vaccine antigens will likely be expressed by breast tumor cells. B: Cumulative distribution curve for the minimum number of expressed antigens (CTA expression curve). This indicates that a minimum of 4 vaccine antigens will be expressed in breast cancer cells (AG95) with a probability of 95%.

[0074] Fig.13 : PEPI represents antigen: Distribution of breast cancer vaccine-specific CTA antigens (referred to as "APs") with ≥1 PEPI in the model population (n=433) of breast cancer vaccine. A: Non-cumulative distribution of APs, where the average number of APs is: AP50=5.30, meaning that on average almost 6 CTAs will have a PEPI in the model population. B: Cumulative distribution curve of the minimum number of APs in the model population (n=433). This indicates that at least one vaccine antigen has a PEPI (AP95=1) in 95% of the model population (n=433).

[0075] Fig.14 A: PEPI represents the distribution of antigens expressed within the model population (n=433) calculated using breast cancer CTA expression rates (breast cancer vaccine-specific CTA antigens expressed by tumors, where ≥1 PEPI is predicted, referred to as "AGP"). A: Non-cumulative distribution of AGP, where the expected value of the numerically represented CTA represented by PEPI is AGP50=3.37. AGP50 is a measure of the effectiveness of the disclosed breast cancer vaccine in attacking breast tumors in an unselected patient population. AGP50=3.37 means that at least 3 CTAs from the vaccine are likely to be expressed by breast tumor cells and present PEPI in the model population. B: The cumulative distribution curve of the minimum number of AGPs in the model population (n=433) shows that at least one vaccine CTA will present PEPI in 92% of the population, and the remaining 8% of the population may have no AGP at all (AGP95=0, AGP92=1).

[0076] Fig.15 :The CTA expression curve was calculated by analyzing the expression frequency data of tumor-specific antigens (CTA) in human colorectal cancer tissues. (Cell line data are not included.)

[0077] Fig.16: Distribution of colorectal cancer antigen expression based on calculation of multi-antigen response from expression frequencies of 7 different CTAs selected. A: Non-cumulative distribution of expected values ​​for the number of vaccine antigens expressed in colorectal cancer (AG50) was calculated. This value shows that colorectal tumor cells may express 4.96 vaccine antigens. B: Cumulative distribution curve of the minimum number of expressed antigens (CTA expression curve). This indicates that a minimum of 3 antigens will be expressed in colorectal cancer cells (AG95) with a probability of 95%.

[0078] Fig.17 : Distribution of PEPI representative antigens (colorectal cancer vaccine-specific CTA antigens where ≥1 PEPI was predicted, referred to as "AP") within a model population (n=433) of colorectal cancer. A: Non-cumulative distribution of APs, where the mean number of APs is: AP50=4.73, meaning that on average 5 CTAs will be represented by a PEPI in the model population. B: Cumulative distribution curve of the minimum number of APs in the model population (n=433). This indicates that in 95% of the model population (n=433) (AP95=2) PEPIs will represent 2 or more antigens.

[0079] Fig.18 A: PEPI represents the distribution of antigens expressed within the model population (n=433) calculated using colorectal cancer CTA expression rates (colorectal cancer vaccine-specific CTA antigens expressed by tumors where ≥1 PEPI is predicted, referred to as "AGP"). A: Non-cumulative distribution of AGPs where the expected value of the numerically represented CTA represented by the PEPI is AGP50=2.54. AGP50 is a measure of the effectiveness of published colorectal cancer vaccines in attacking colorectal tumors in an unselected patient population. AGP50=2.54 means that at least 2-3 CTAs from the vaccine are likely to be expressed by colorectal tumor cells and present PEPIs in the model population. B: Cumulative distribution curve of the minimum number of AGPs in the model population (n=433) showing that at least 1 vaccine CTA will be expressed and also present PEPIs in 93% of the population (AGP93=1).

[0080] Fig.19 : Schematic diagram showing exemplary positions of amino acids in overlapping HLA class I and HLA class II binding epitopes in a 30-mer peptide.

[0081] Fig. 20: Antigenicity of PolyPEP1018 CRC vaccine in the general human population. The antigenicity of PolyPEP1018 in subjects was determined by AP counts, which indicate the number of vaccine antigens that induced T cell responses in subjects. The AP counts of PolyPEP1018 were determined in each of the 433 subjects in the model population using the PEPI test, and then the AP50 counts of the model population were calculated. The AP50 of PolyPEP1018 in the model population was 4.73. The average number of immunogenic antigens (i.e., antigens with ≥1 PEPI) in PolyPEP1018 in the general population was 4.73. Abbreviation: AP = antigens with ≥1 PEPI. Left: Cumulative distribution curve. Right: Significance distribution curve.

[0082] Fig.21 : Efficacy of the PolyPEP1018 CRC vaccine in the general human population. If the PEPIs in the vaccine are presented by tumor cells, vaccine-induced T cells can recognize and kill tumor cells. The number of AGPs (antigens expressed with PEPIs) is an indicator of vaccine efficacy in an individual and depends on the efficacy and antigenicity of PolyPEP1018. The average number of immunogenic CTAs (i.e., APs [expressed antigens with ≥1 PEPI]) of PolyPEP1018 in the model population was 2.54. The probability that PolyPEP1018 would induce T cell responses against multiple antigens in subjects in the model population (i.e., mAGPs) was 77%.

[0083] Fig. 22 : The probability of vaccine antigen expression in patient XYZ's tumor cells. The probability of 5 of the 12 target antigens in the vaccine regimen being expressed in the patient's tumor is greater than 95%. Therefore, the 12 peptide vaccines can together induce an immune response against at least 5 ovarian cancer antigens with a probability of 95% (AGP95). The probability of each peptide inducing an immune response in patient XYZ is 84%. AGP50 is the mean (expected value) = 7.9 (which is a measure of the effectiveness of the vaccine in attacking patient XYZ's tumor).

[0084] Fig.23 : MRI findings of patient XYZ treated with personalized (PIT) vaccine. This patient with advanced, heavily pretreated ovarian cancer had an unexpected objective response following PIT vaccine treatment. These MRI findings suggest that the PIT vaccine combined with chemotherapy resulted in a significant reduction in tumor burden. The patient now continues PIT vaccine treatment.

[0085] Fig.24: The probability of vaccine antigen expression in patient ABC tumor cells. The probability of 4 of the 13 target antigens in the vaccine being expressed in the patient's tumor is greater than 95%. Therefore, the 12 peptide vaccines can together induce an immune response against at least 4 breast cancer antigens with a probability of 95% (AGP95). The probability of each peptide inducing an immune response in patient ABC is 84%. AGP50 is the mean (expected value) of the discrete probability distribution = 6.45 (it is a measure of the effectiveness of the vaccine in attacking patient ABC tumors).

[0086] Sequence Description

[0087] SEQ ID NOs: 1 to 20 list the 9-mer T cell epitopes described in Table 17.

[0088] SEQ ID NOs: 21 to 40 list the 9-mer T cell epitopes described in Table 20.

[0089] SEQ ID NOs: 41 to 60 list the 15-mer T cell epitopes described in Table 17.

[0090] SEQ ID NOs: 61 to 80 list the 15-mer T cell epitopes described in Table 20.

[0091] SEQ ID NOs: 81 to 111 list the breast cancer vaccine peptides described in Table 18a.

[0092] SEQ ID NOs: 112 to 142 list the colorectal cancer vaccine peptides described in Table 21a.

[0093] SEQ ID NOs: 143 to 158 list breast cancer, colorectal cancer and / or ovarian cancer associated antigens.

[0094] SEQ ID NOs: 159 to 171 list additional peptide sequences described in Table 10.

[0095] SEQ ID NOs: 172 to 194 list additional 9-mer T cell epitopes described in Table 17.

[0096] SEQ ID NOs: 195 to 233 list additional 15-mer T cell epitopes described in Table 17.

[0097] SEQ ID NOs: 234 to 250 list additional 9-mer T cell epitopes described in Table 20.

[0098] SEQ ID NOs: 251 to 271 list additional 15-mer T cell epitopes described in Table 20.

[0099] SEQ ID NOs: 272 to 301 list the 9-mer T cell epitopes described in Table 23.

[0100] SEQ ID NOs: 302 to 331 list the 15-mer T cell epitopes described in Table 23.

[0101] SEQ ID NOs: 332 to 346 list the ovarian cancer vaccine peptides described in Table 24.

[0102] SEQ ID NOs: 347 to 361 further list breast cancer, colorectal cancer and / or ovarian cancer associated antigens.

[0103] SEQ ID NOs: 362 to 374 list the personalized vaccine peptides designed for patient XYZ described in Table 36.

[0104] SEQ ID NOs: 375 to 386 list personalized vaccine peptides designed for ABC of the patients described in Table 39.

[0105] SEQ ID NOs: 387 to 434 list additional 9-mer T cell epitopes described in Table 32

[0106] SEQ ID NOs: 435 to 449 list additional breast cancer vaccine peptides described in Table 18a. DETAILED DESCRIPTION

[0107] HLA genotype

[0108] HLA is encoded by most polymorphic genes of human genome.Every person has maternal and paternal alleles of three kinds of HLA class I molecules (HLA-A*, HLA-B*, HLA-C*) and four kinds of HLA class II molecules (HLA-DP*, HLA-DQ*, HLA-DRB1*, HLA-DRB3* / 4* / 5*).In fact, each person expresses different combinations of 6 kinds of HLA class I molecules and 8 kinds of HLA class II molecules, and they present different epitopes from the same protein antigen.The function of HLA molecule is to regulate T cell response.But, it is not known how people's HLA regulates T cell activation so far.

[0109] The nomenclature used to represent the amino acid sequence of an HLA molecule is as follows: gene name*allele:protein number, which, for example, may look like: HLA-A*02:25. In this example, "02" refers to the allele. In most cases, the allele is defined by serotype C, which means that the proteins of a given allele will not react with each other in serological assays. Protein numbers (25 in the above example) are assigned consecutively as proteins are discovered. A new protein number is assigned to any protein with a different amino acid sequence (e.g., even one 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 appended to the HLA nomenclature, but such information is not necessary for the methods described herein.

[0110] 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 nomenclature as described above that minimally specifies the allele and protein number of each HLA gene. The HLA genotype may be determined using any suitable method. For example, the sequence may be determined by sequencing the HLA loci using methods and protocols known in the art. Alternatively, the individual's HLA group may be stored in a database and accessed using methods known in the art.

[0111] Some subjects may have two HLA alleles encoding the same HLA molecule (e.g., two copies of HLA-A*02:25 in the case of homozygosity). The HLA molecules encoded by these alleles bind all the same T cell epitopes. For the purposes of this disclosure, as used herein, "binding to at least two HLA molecules of a subject" includes binding to HLA molecules encoded by two identical HLA alleles in a single subject. In other words, "binding to at least two HLA molecules of a subject" and the like may be additionally expressed as "binding to HLA molecules encoded by at least two HLA alleles of a subject."

[0112] Peptides

[0113] The present disclosure relates to polypeptides derived from CTA that are immunogenic to a high proportion of the human population.

[0114] As used herein, the term "polypeptide" refers to a full-length protein, a portion of a protein, or a peptide characterized by a string of amino acids. As used herein, the term "peptide" refers to a short polypeptide comprising 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15 to 10, or 11, or 12, or 13, or 14, or 15, or 20, or 25, or 30, or 35, or 40, or 45, or 50, or 55, or 60 amino acids.

[0115] As used herein, the term "fragment" or "fragment of a polypeptide" refers to a string or sequence of amino acids that is generally of reduced length relative to the above or a reference polypeptide and comprises the same amino acid sequence as the reference polypeptide in the common portion. Where appropriate, such a fragment according to the present disclosure may be included in a larger polypeptide of which it is a component. In some cases, a fragment may comprise the full length of a polypeptide, such as an entire polypeptide, such as a 9 amino acid peptide, which is a single T cell epitope. In some cases, the fragments referred to herein may contain 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9 to 20, or 25, or 30, or 35, or 40, or 45, or 50 amino acids.

[0116] As used herein, the term "epitope" or "T cell epitope" refers to a continuous amino acid sequence contained within a protein antigen that has a binding affinity to (can bind to) one or more HLAs. The epitope is HLA and antigen specific (HLA epitope pair, predicted by known methods), but not subject specific. If a T cell response (cytotoxic T cell response or helper T cell response) can be induced in a specific human subject, then the epitope, T cell epitope, polypeptide, polypeptide fragment, or composition comprising a polypeptide or fragment thereof is immunogenic to the subject. In some cases, the helper T cell response is a Th1 type helper T cell response. In some cases, an epitope, T cell epitope, polypeptide, polypeptide fragment, or composition comprising a polypeptide or fragment thereof is "immunogenic" if it is more likely to induce a T cell response or immune response in a subject than a different T cell epitope (or in some cases two different T cell epitopes each) that can bind only one HLA molecule of the subject.

[0117] The terms "T cell response" and "immune response" are used interchangeably herein and refer to the activation of T cells and / or the induction of one or more effector functions following the identification of one or more HLA epitope binding pairs. In some cases, an "immune response" includes an antibody response because HLA class II molecules stimulate auxiliary responses involved in inducing persistent CTL responses and antibody responses. Effector functions include cytotoxicity, cytokine production, and proliferation. According to the present disclosure, a polypeptide epitope, T cell epitope, or fragment is immunogenic to a particular subject if it is able to bind to at least two, or in some cases at least three class I or at least two, or in some cases at least three or at least four class II HLAs of the subject.

[0118] For the purpose of this disclosure, we propose the term "human epitope" or "PEPI" to distinguish subject-specific epitopes from HLA-specific epitopes. "PEPI" is a polypeptide fragment consisting of a continuous amino acid sequence of a polypeptide, which is a T cell epitope capable of binding to one or more HLA class I molecules of a specific human subject. In other cases, PEPI is a polypeptide fragment consisting of a continuous amino acid sequence of a polypeptide, which is a T cell epitope capable of binding to one or more HLA class II molecules of a specific human subject. In other words, "PEPI" is a T cell epitope identified by the HLA group of a specific individual. In contrast to "epitopes", PEPIs are individual-specific because different individuals have different HLA molecules that each binds to different T cell epitopes. The subject-specificity of this PEPI allows the manufacture of personalized cancer vaccines.

[0119] As used herein, "PEPI1" refers to a peptide or polypeptide fragment that can bind to one HLA class I molecule (or, in a specific case, HLA class II molecule) of an individual. "PEPI1+" refers to a peptide or polypeptide fragment that can bind to one or more HLA class I molecules of an individual.

[0120] "PEPI2" refers to a peptide or polypeptide fragment that can bind to two HLA class I (or class II) molecules of an individual. "PEPI2+" refers to a peptide or polypeptide fragment that can bind to two or more HLA class I (or class II) molecules of an individual, i.e., a fragment identified according to the method of the present disclosure.

[0121] "PEPI3" refers to a peptide or polypeptide fragment that can bind to three HLA class I (or class II) molecules of an individual. "PEPI3+" refers to a peptide or polypeptide fragment that can bind to three or more HLA class I (or class II) molecules of an individual.

[0122] "PEPI4" refers to a peptide or polypeptide fragment that can bind to four HLA class I (or class II) molecules of an individual. "PEPI4+" refers to a peptide or polypeptide fragment that can bind to four or more HLA class I (or class II) molecules of an individual.

[0123] "PEPI5" refers to a peptide or polypeptide fragment that can bind to five HLA class I (or class II) molecules of an individual. "PEPI5+" refers to a peptide or polypeptide fragment that can bind to five or more HLA class I (or class II) molecules of an individual.

[0124] "PEPI6" refers to a peptide or polypeptide fragment that is capable of binding to all six HLA class I (or six HLA class II) molecules in an individual.

[0125] In general, the epitope presented by HLA class I molecules is about 9 amino acids in length, and the epitope presented by HLA class II molecules is about 15 amino acids in length. However, for the purposes of this disclosure, as long as the epitope can bind to HLA, the epitope length can be more or less than 9 (for HLA class I) or more or less than 15 (for HLA class II) amino acids. For example, the epitope length that can bind to class I HLA can be 7 or 8 or 9 to 9 or 10 or 11 amino acids. The epitope length that can bind to class II HLA can be 13 or 14 or 15 to 15 or 16 or 17 amino acids.

[0126] A given subject's HLA will present only a limited number of different peptides generated by processing a protein antigen in an APC to a T cell. As used herein, "display" or "presentation" when used in relation to an HLA refers to the binding between a peptide (epitope) and an HLA. In this regard, "displaying" or "presenting" a peptide is synonymous with "binding" a peptide.

[0127] Using techniques known in the art, epitopes that will bind to a known HLA can be determined. Any suitable method can be used, provided that the same method is used to determine the multiple HLA epitope binding pairs that are directly compared. For example, biochemical analysis can be used. A list of epitopes known to be bound by a given HLA can also be used. Prediction or modeling software can also be used to determine which epitopes can be bound by a given HLA. Examples are provided in Table 1. In some cases, if the IC50 or predicted IC50 of a T cell epitope is less than 5000nM, less than 2000nM, less than 1000nM, or less than 500nM, then the T cell epitope can bind to a given HLA.

[0128] Table 1 Example software for determining epitope-HLA binding

[0129]

[0130]

[0131] MHCBN, Institute of Microbial Technology, Chandigarh, India http: / / www.imtech.res.in / raghava / mhcbn /

[0132] SYFPEITHI of Heidelberg Biomedical Informatics http: / / www.syfpeithi.de /

[0133] AntiJen from the Edward Jenner Vaccine Institute http: / / www.ddg-pharmfac.net / antijen / AntiJen / antijenhomepage.htm

[0134] MIF Bioinformatics' MHC ligand EPIMHC database http: / / immunax.dfci.harvard.edu / epimhc /

[0135] IEDB Immune Epitope Database http: / / www.iedb.org /

[0136] In some embodiments, the peptides of the present disclosure may comprise or consist of one or more fragments of one or more CTAs. CTAs are not normally expressed outside of embryonic development in healthy cells. In healthy adults, CTA expression is limited to male germ cells that do not express HLA and cannot present antigens to T cells. Therefore, when expressed in cancer cells, CTAs are considered to be expressed neoantigens.

[0137] CTAs are good choices for cancer vaccine targets because their expression (i) is specific to tumor cells, (ii) is more frequent in metastatic tumors than in primary tumors, and (iii) is conserved among metastatic tumors in the same patient (Gajewski ed. Targeted Therapeutics in Melanoma. Springer New York. 2012)

[0138] The peptides disclosed herein may include, for example, SPAG9 (SEQ ID NO: 143), AKAP-4 (SEQ ID NO: 144), BORIS (SEQ ID NO: 145), NY-SAR-35 (SEQ ID NO: 146), NY-BR-1 (SEQ ID NO: 147), SURVIVIN (SEQ ID NO: 148), MAGE-A11 (SEQ ID NO: 149), PRAME (SEQ ID NO: 150), MAGE-A9 (SEQ ID NO: 151), HOM-TES-85 (SEQ ID NO: 152), PIWIL-2 (SEQ ID NO: 349), EpCAM (SEQ ID NO: 154), HIWI (SEQ ID NO: 350), PLU-1 (SEQ ID NO: 351), TSGA10 (SEQ ID NO: 351), ODF-4 (SEQ ID NO: 352), SP17 (SEQ ID NO: 354), RHOXF-2 (SEQ ID NO: 355), EpCAM (SEQ ID NO: 156), HIWI (SEQ ID NO: 357), PLU-1 (SEQ ID NO: 358), TSGA10 (SEQ ID NO: 359), ODF-4 (SEQ ID NO: 360), SP17 (SEQ ID NO: 361), RHOXF-2 (SEQ ID NO: 362), EpCAM (SEQ ID NO: 163), HIWI (SEQ ID NO: 364), PLU-1 (SEQ ID NO: 365), TSGA10 (SEQ ID NO: 366), ODF-4 (SEQ ID NO: 367), SP17 (SEQ ID NO: 368), RHOXF-2 (SEQ ID NO: 369), RHOXF-3 (SEQ ID NO: 370), RHOXF-3 (SEQ ID NO: 371), RHOXF-4 ( one or more breast cancer associated antigens selected from PIWIL-4 (SEQ ID NO: 357), WT1 (SEQ ID NO: 358), EpCAM (SEQ ID NO: 154), BORIS (SEQ ID NO: 145), AKAP-4 (SEQ ID NO: 144), OY-TES-1 (SEQ ID NO: 359), SP17 (SEQ ID NO: 354), PIWIL-2 (SEQ ID NO: 349), PIWIL-3 (SEQ ID NO: 360), SPAG9 (SEQ ID NO: 143), PRAME (SEQ ID NO: 150), HIWI (SEQ ID NO: 350), SURVIVIN (SEQ ID NO: 148) and AKAP-3 (SEQ ID NO: 361),and / or one or more colorectal cancer associated antigens selected from TSP50 (SEQ ID NO: 153), EpCAM (SEQ ID NO: 154), SPAG9 (SEQ ID NO: 143), CAGE1 (SEQ ID NO: 155), FBXO39 (SEQ ID NO: 156), SURVIVIN (SEQ ID NO: 148), MAGE-A8 (SEQ ID NO 157), MAGE-A6 (SEQ ID NO: 158), LEMD1 (SEQ ID NO: 348) and MAGE-A3 (SEQ ID NO: 347), or one or more fragments thereof, or consisting thereof. In some cases, the peptide comprises or consists of one or more amino acid sequences selected from SEQ ID NOs: 41 to 80, or selected from SEQ ID NOs: 41 to 80, 195 to 233, 251 to 271 and 302 to 331, which are optimized for T cell activation / binding to all HLA types in the entire population. ,

[0139] In some cases, the amino acid sequence is flanked at the N-terminus and / or C-terminus by additional amino acids that are not part of the target polypeptide antigen sequence, in other words, the additional amino acids are not the same sequence of consecutive amino acids adjacent to the selected fragment in the target polypeptide antigen. In some cases, the sequence is flanked at the N-terminus and / or C-terminus or between target polypeptide fragments by up to 41 or 35 or 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. In other cases, each polypeptide is either composed of a fragment of the target polypeptide antigen, or is composed of two or more such fragments arranged end to end in a single peptide (arranged end to end in a peptide) or overlapping (wherein two or more fragments contain partially overlapping sequences, for example, where two PEPIs in the same polypeptide are within 50 amino acids of each other).

[0140] When fragments of different polypeptides or fragments from different regions of the same polypeptide are linked together in an engineered peptide, a new epitope may be produced around a junction (join) or a joint (junction). Such a new epitope includes at least one amino acid from each fragment on both sides of a junction or a joint, and may be referred to as a connection amino acid sequence in this article. The new epitope can induce an undesirable T cell response (autoimmunity) for healthy cells. Peptides can be designed, or polypeptides can be screened to avoid, eliminate or minimize the new epitopes corresponding to protein fragments expressed in normal healthy human cells and / or can be combined with at least two, or in some cases at least three, or at least four HLA class I molecules of a subject, or in some cases at least two, or at least three, four or five HLA class II molecules. In some cases, the above-mentioned peptides are designed or the above-mentioned polypeptides are screened to eliminate polypeptides with connection new epitopes, which can be combined with at least two HLA class I molecules expressed by individual subjects of the group in human subjects exceeding a threshold percentage in the intended treatment group. In some cases, the threshold is 20%, or 15%, or 10%, or 5%, or 2%, or 1%, or 0.5% of the population. Alignment can be determined using known methods such as the BLAST algorithm. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0141] The presence of at least two polypeptide fragments (epitopes) that can bind to at least three HLA class I (≥2 PEPI3+) of an individual in a vaccine or immunotherapeutic composition can predict clinical response. In other words, if ≥2 PEPI3+ can be identified in the active ingredient polypeptide of the vaccine or immunotherapeutic composition, the individual may be a clinical responder. The at least two multiple HLA-binding PEPIs of the composition polypeptide can target a single antigen (e.g., a polypeptide vaccine comprising two multiple HLA-binding PEPIs derived from a single tumor-associated antigen targeted by the vaccine), or different antigens (e.g., a polypeptide vaccine comprising one multiple HLA-binding PEPI derived from one tumor-associated antigen and a second multiple HLA-binding PEPI derived from a different tumor-associated antigen).

[0142] Without wishing to be bound by theory, the inventors believe that one reason for the increased likelihood of clinical benefit from vaccines / immunotherapy comprising at least two multiple HLA-binding PEPIs is that diseased cell populations, such as cancer or tumor cells or cells infected with a virus or pathogen such as HIV, are often heterogeneous within and between affected subjects. For example, a particular cancer patient may or may not express or overexpress a particular cancer-associated target polypeptide antigen of a vaccine, or their cancer may comprise a heterogeneous population of cells, some of which (over)express the antigen and some of which do not. In addition, when a vaccine / immunotherapy comprises or targets a greater number of multiple HLA-binding PEPIs, the likelihood of developing resistance is reduced because patients are less likely to develop resistance to the composition through mutations in the target PEPIs.

[0143] Most vaccines and immunotherapeutic compositions currently target only a single polypeptide antigen. However, according to the present disclosure, in some cases, it is beneficial to provide a pharmaceutical composition that targets two or more different polypeptide antigens. For example, most cancers or tumors are heterogeneous, meaning that different cancers or tumor cells of a subject (over) express different antigens. Tumor cells of different cancer patients also express different combinations of tumor-associated antigens. The most likely effective anti-cancer immunogenic compositions are those that target multiple antigens expressed by tumors in individual human subjects or populations and therefore target more cancers or tumor cells.

[0144] The beneficial effects of combining multiple bestEPIs in a single treatment (administering one or more pharmaceutical compositions containing multiple PEPIs together) can be illustrated by the personalized vaccine polypeptides described in Examples 15 and 16 below. Exemplary CTA expression probabilities in ovarian cancer are as follows: BAGE: 30%; MAGE A9: 37%; MAGE A4: 34%; MAGE A10: 52%. If patient XYZ is treated with a vaccine containing only PEPIs from BAGE and MAGE A9, the probability of having mAGP (multiple expressed antigens with PEPIs) will be 11%. If patient XYZ is treated with a vaccine containing only PEPIs from MAGE A4 and MAGE A10 CTAs, the probability of having mAGP will be 19%. However, if the vaccine contains all 4 of these CTAs (BAGE, MAGE A9, MAGE A4, and MAGE A10), the probability of having mAGP will be 50%. In other words, for two PEPI treatments, the effect will be greater than the combined probability of mAGP (probability mAGP for BAGE / MAGE, plus probability mAGP for MAGE A4 and MAGE A10). Patient XYZs PIT vaccine described in Example 15 contained an additional 9 PEPIs, so the probability of having mAGP was greater than 99.95%.

[0145] Likewise, the probabilities of expression of exemplary CTAs in breast cancer are as follows: MAGE C2: 21%; MAGE A1: 37%; SPC1: 38%; MAGE A9: 44%. The probability of mAGP for patient ABC treated with a vaccine containing only MAGE C2: 21% and PEPIs from MAGE A1 is 7%. The probability of mAGP for patient ABC treated with a vaccine containing only SPC1: 38% and PEPIs from MAGE A9 is 11%. The probability of mAGP for patient ABC treated with a vaccine containing PEPIs from MAGE C2: 21%; MAGE A1: 37%; SPC1: 38%; MAGE A9 is 44% (44>7+11). The PIT vaccine for patient ABC described in Example 16 contains an additional 8 PEPIs, so the probability of having mAGP is over 99.93%.

[0146] Thus, in some cases, a polypeptide or polypeptide panel of the present disclosure or an active ingredient polypeptide of a pharmaceutical composition or kit of the present disclosure may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 fragments of one or more cancer associated antigens or multiple CTAs such as the CTAs discussed above. Each fragment comprises or consists of a different targeting epitope having a selected from SEQ ID NOs: 1 to 40; or selected from SEQ ID NOs: 1 to 20; or selected from SEQ ID NOs: 21 to 40; or selected from SEQ ID NOs: 1 to 20, 24 and 172 to 194; or selected from SEQ ID NOs: 21 to 40 and 234 to 250; or selected from SEQ ID NOs: 272 to 301; or selected from SEQ ID NOs: 1 to 40, 172 to 194 and 234 to 250; or selected from SEQ ID NOs: 21 to 40, 234 to 250 and 272 to 301; or selected from SEQ ID NOs: 1 to 20, 24, 172 to 194 and 272 to 301; or selected from SEQ ID NOs: 1 to 40, 172 to 194, 234 to 250 and 272 to 301; or selected from SEQ ID NOs: 1 to 40, 172 to 194, 234 to 250 and 272 to 301; NO: 41 to 60, 64 and 195 to 233; or selected from SEQ ID NO: 61 to 80 and 251 to 271; or selected from SEQ ID NO: 302 to 331; or selected from SEQ ID NO: 41 to 80, 195 to 233 and 251 to 271; or selected from SEQ ID NO: 61 to 80, 251 to 271 and 302 to 331; or selected from SEQ ID NO: 41 to 60, 64, 191 to 233 and 302 to 331; or selected from SEQ ID NO: 41 to 80, 195 to 233, 251 to 271 and 332 to 346; or selected from SEQ ID NO: 1 to 20, 24, 41 to 60, 64, 172 to 194 and 195 to 233; or selected from SEQ ID NO: 1 NO: 21 to 40, 61 to 80, 234 to 250 and 251 to 271; or selected from SEQ ID NO: 271 to 331; or selected from SEQ ID NO: 1 to 80, 172 to 194, 195 to 233, 234 to 250 and 251 to 271; or selected from SEQ ID NO: 21 to 40, 61 to 80, 234 to 250, 251 to 271, 272 to 301 and 302 to 331; or selected from SEQ ID NO:NO: 1 to 80, 172 to 233, 234 to 271 and 272 to 331; or selected from SEQ ID NO: 81 to 111 and 435 to 449; or selected from SEQ ID NO: 112 to 142; or selected from SEQ ID NO: 332 to 346; or selected from SEQ ID NO: 81 to 142; or selected from SEQ ID NO: 112 to 142 and 332 to 346; or selected from SEQ ID NO: 81 to 111, 435 to 449 and 332 to 346; or selected from SEQ ID NO: 81 to 142 and 332 to 346; or selected from SEQ ID NO: 41 to 60, 64, 81 to 111, 435 to 449 and 195 to 233; or selected from SEQ ID NO: 61 to 80, 112 to 142 and 251 to 271; or selected from SEQ ID NO: NO:302 to 346; or selected from SEQ ID NO:41 to 142, 195 to 233 and 251 to 271; or selected from SEQ ID NO:61 to 80, 112 to 142, 251 to 271 and 302 to 346; or selected from SEQ ID NO:41 to 60, 64, 81 to 111, 435 to 449, 195 to 233 and 302 to 346; or selected from SEQ ID NO:41 to 142, 195 to 233, 251 to 271 and 302 to 346; or selected from SEQ ID NO:1 to 20, 24, 41 to 60, 64, 81 to 111, 435 to 449 and 172 to 233; or selected from SEQ ID NO:21 to 40, 61 to 80, 112 to 142, or 234 to 271; or selected from SEQ ID NO: NO: 272 to 346; or selected from SEQ ID NO: 1 to 142 and 172 to 271; or selected from SEQ ID NO: 21 to 40, 61 to 80, 112 to 142 and 234 to 346; or selected from SEQ ID NO: 1 to 20, 24, 41 to 60, 64, 81 to 111, 435 to 449, 172 to 233 and 272 to 346; or selected from SEQ ID NO: 1 to 142 and 172 to 346; or selected from SEQ ID NO: 1 to 2, or 1 to 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10, or 11, or 12, or 13, or 14, or 15, or 16, or 17, or 18, or 19, or SEQ ID NO: NO: 20 to 21, or 20 to 22, or 23, or 24, or 25, or 26, or 27, or 28, or 29, or 30, or 31, or 32, or 33, or 34, or 35, or 36, or 37, or 38, or 39; or having an amino acid sequence selected from SEQ ID NO: 41 to 80, or SEQ ID NO: 41 to 60, or SEQ ID NO: 61 to 80; or SEQ ID41 to 42, or 41 to 43, or 41 to 44, or 41 to 45, or 41 to 46, or 41 to 47, or 41 to 48, or 41 to 49, or 50, or 51, or 52, or 53, or 54, or 55, or 56, or 57, or 58, or 59, SEQ ID NO: 60 to 61, or 60 to 62, or 60 to 63, or 60 to 64, or 60 to 65, or 60 to 66, or 60 to 67, or 60 to 68, or 60 to 69, or 60 to 70, or 60 to 71, or 60 to 72, or 60 to 73, or 60 to 74, or 60 to 75, or 60 to 76, or 60 to 77, or 60 to 78, or 60 to 79; or having a different amino acid sequence selected from SEQ ID NO: 91 to 92, or 91 to 93, or 93 to 94, or 95 to 96, or 97 to 98, or 99 to 100; NO: 81 to 142; or selected from SEQ ID NO: 81 to 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 105, 106, 107, 108, 109, 110, or 111; or selected from SEQ ID NO: 81 to 105; or selected from SEQ ID NO: 99, 100, 92, 93, 101, 103, 104, 105 and 98; or selected from SEQ ID NO: 112 to 142; or selected from SEQ ID NO: 112 to 142. NO: 112 to 113, 114, 115, 116, 117, 118, 119, 120, 121, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 or 142; or selected from SEQ ID NO: 112 to 134; or selected from SEQ ID NO: 121, 124, 126, 127, 130, 131, 132, 133 and 134; or selected from SEQ ID NO: or SEQ ID NO: 41 to 80; or SEQ ID NO: 41 to 60; or SEQ ID NO: 61 to 80; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID NO: 41 to 60; or SEQ ID41 to 42, or 41 to 43, or 41 to 44, or 41 to 45, or 41 to 46, or 41 to 47, or 41 to 48, or 41 to 49, or 50, or 51, or 52, or 53, or 54, or 55, or 56, or 57, or 58, or 59, SEQ ID NO: 60 to 61, or 60 to 62, or 60 to 63, or 60 to 64, or 60 to 65, or 60 to 66, or 60 to 67, or 60 to 68, or 60 to 69, or 60 to 70, or 60 to 71, or 60 to 72, or 60 to 73, or 60 to 74, or 60 to 75, or 60 to 76, or 60 to 77, or 60 to 78, or 60 to 79; or having a different amino acid sequence selected from SEQ ID NO: 91 to 92, or 91 to 93, or 93 to 94, or 95 to 96, or 97 to 98, or 99 to 100; NO: 81 to 142; or selected from SEQ ID NO: 81 to 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 105, 106, 107, 108, 109, 110, or 111; or selected from SEQ ID NO: 81 to 105; or selected from SEQ ID NO: 99, 100, 92, 93, 101, 103, 104, 105 and 98; or selected from SEQ ID NO: 112 to 142; or selected from SEQ ID NO: 112 to 142. NO: 112 to 113, 114, 115, 116, 117, 118, 119, 120, 121, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141 or 142; or selected from SEQ ID NO: 112 to 134; or selected from SEQ ID NO: 121, 124, 126, 127, 130, 131, 132, 133 and 134; or selected from SEQ ID NO: 130, 121, 131, 124, 134, 126; or selected from SEQ ID NO: 435 to 449; or having an amino acid sequence selected from SEQ ID NO: 435 to 449 NO: 12, 32, 19 and / or 39, and / or SEQ ID NO: 21, 41, 23 and / or 43 and / or SEQ ID NO: 172, 177, 195 and / or 203, and / or SEQ ID NO: 1, 41 and / or 197, and / or SEQ ID NO: 4, 44 and / or 201, and / or SEQ ID NO: 1, 4, 44, 197 and / or 201, and / or SEQ ID NO: 1, 41, 197, 184 and / or 212, and / or SEQ ID NO: 3, 43 and / or 200, and / or SEQ ID NO: 3, 43, 200, 7 and / or 47, and / or SEQ IDNO: 10, 50 and / or 220, and / or SEQ ID NO: 24, 64 and / or 202, and / or SEQ ID NO: 6, 46 and / or 209, and / or SEQ ID NO: 182, 210, 185 and / or 213, and / or SEQ ID NO: 14, 54, 225 and 226, and / or SEQ ID NO: 190, 218, 11, 51 and / or 219, and / or SEQ ID NO: 12, 224 and / or 52, and / or SEQ ID NO: 192, 227 and / or 228, and / or SEQ ID NO: 17, 229, 230 and / or 57, and / or SEQ ID NO: 21, 252, 61 and / or 253, and / or SEQ ID NO: 23, 63 and / or 256, and / or SEQ ID NO: NO: 21, 252, 61, 253, 23, 63 and / or 256, and / or SEQ ID NO: 237 and / or 238, and / or SEQ ID NO: 26 and / or 240, and / or SEQ ID NO: 242, 244, 263 and / or 265, and / or SEQ ID NO: 29, 69 and / or 259, and / or SEQ ID NO: 24, 64 and / or 255, and / or SEQ ID NO: 236, 257 and / or 258, and / or SEQ ID NO: 27, 67, 241 and / or 262, and / or SEQ ID NO: 252, 249 and / or 264, and / or SEQ ID NO: 35, 250 and / or 75, and / or SEQ ID NO: 252, 249, 264, 35, 250 and / or 75, and / or SEQ ID NO: NO: 36, 266 and / or 76, and / or SEQ ID NO: 36, 266, 76, 39 and / or 79, and / or SEQ ID NO: 38, 268 and / or 78, and / or SEQ ID NO: 38, 268, 78, 246 and / or 270, and / or SEQ ID NO: 245, 269, and / or 248, and / or SEQ ID NO: 245, 269, 248, 40 and / or 80, and / or SEQ ID NO: 272, 302, 281 and / or 311, and / or SEQ ID NO: 276, 306, 300 and / or 330, and / or SEQ ID NO: 276, 306, 289 and / or 319, and / or SEQ ID NO: 277, 307, 283 and / or 313, and / or SEQ ID NO: NO: 277, 307, 290 and / or 320, and / or SEQ IDNO:282, 312, 297 and / or 327, or other combinations of sequences of the present disclosure, which are located within 50 to 60 amino acids of each other in any one or more of the following sequences: SEQ ID NO:143 to 158 and 347 to 351; and / or SEQ ID NO:18, 19 and / or 20 and / or SEQ ID NO:34 to 40; and / or the peptides corresponding to the SEQ ID NOs shown in Tables 17, 20 and / or 23 having an N%*B% value of less than 12% or 13% or 14% or 17.6% or 17.8% or 18% or 20% or 21% or 22% or 22.2% or 24% or 25% or 27% or 28% or 30% or 31% or 31.5% or 32% or 32.5% or 35%. In some cases, the peptide panel comprises or consists of one or more polypeptides comprising or consisting of the amino acid sequence of SEQ ID NO: 130, 121, 131, 124, 134, 126 and / or SEQ ID NO: 435-449.

[0147] In some cases, the disclosure provides a panel or groups of any two or more peptides described above. For example, the panel may include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more such peptides. In some cases, the panel includes or consists of peptides that include or consist of the following: all or any combination of the amino acid sequences of SEQ ID NOs: 99, 100, 92, 93, 101, 103, 104, 105, and 98; or all or any combination of the amino acid sequences of SEQ ID NOs: 121, 124, 126, 127, 130, 131, 132, 133, and 134. In some cases, the panel comprises or consists of peptides comprising or consisting of all or any combination of the amino acid sequences of SEQ ID NOs: 130, 121, 131, 124, 134, 126 and / or SEQ ID NOs: 435-449.

[0148] Immunogenicity and pharmaceutical compositions, methods of treatment and administration

[0149] In some aspects, the disclosure relates to the above-mentioned pharmaceutical compositions, kits or polypeptide groups (panels) having one or more polypeptides as active ingredients. These can be used for inducing an immune response, treatment, vaccination or providing immunotherapy methods to a subject, and the pharmaceutical composition can be a vaccine or immunotherapy composition. This treatment includes administering one or more polypeptides or pharmaceutical compositions to the subject, and the polypeptides or pharmaceutical compositions together contain all active ingredient polypeptides in the treatment. A variety of polypeptides or pharmaceutical compositions can be administered together or sequentially, for example, all pharmaceutical compositions or polypeptides can be administered to the subject within 1 year, or 6 months, or 3 months, or 60 or 50 or 40 or 30 days.

[0150] The term "active ingredient" as used herein refers to a polypeptide intended to induce an immune response, and may include a polypeptide product of a vaccine or immunotherapy composition produced in vivo after administration to a subject. For DNA or RNA immunotherapy compositions, the polypeptide may be produced in vivo by cells of a subject to which the composition is administered. For cell-based compositions, the polypeptide may be processed and / or presented by cells of the composition, such as autologous dendritic cells or antigen presenting cells pulsed with the polypeptide or containing an expression construct encoding the polypeptide. The pharmaceutical composition may include polynucleotides or cells encoding one or more active ingredient polypeptides.

[0151] The composition / kit may optionally further include at least one pharmaceutically acceptable diluent, carrier or preservative and / or additional polypeptides that do not contain any PEPI. The polypeptide may be engineered or non-naturally occurring. The kit may include one or more separate containers, each containing one or more active ingredient peptides. The composition / kit may be a personalized medicine for preventing, diagnosing, alleviating, treating or curing an individual disease such as cancer.

[0152] In addition to one or more immunogenic peptides, the immunogenic or pharmaceutical compositions or kits described herein may also include pharmaceutically acceptable excipients, carriers, diluents, buffers, stabilizers, preservatives, adjuvants, or other substances well known to those skilled in the art. These substances are preferably non-toxic and preferably do not interfere with the pharmaceutical activity of the active ingredient. The pharmaceutical carrier or diluent can be, for example, an aqueous solution. The exact nature of the carrier or other substance may depend on the route of administration, such as oral, intravenous, cutaneous or subcutaneous, intranasal, intramuscular, intradermal, and intraperitoneal.

[0153] The pharmaceutical compositions of the present disclosure may include one or more "pharmaceutically acceptable carriers". These carriers are typically large, slowly metabolized macromolecules, such as proteins, sugars, polylactic acid, polyglycolic acid, polymeric amino acids, amino acid copolymers, sucrose (Paoletti et al., 2001, Vaccine, 19:2118), trehalose (WO 00 / 56365), lactose, and lipid aggregates (e.g., oil droplets or liposomes). These carriers are well known to those of ordinary skill in the art. The pharmaceutical composition may also contain a diluent, such as water, saline, glycerol, etc. In addition, auxiliary substances may be present, such as wetting agents or emulsifiers, pH buffer substances, etc. Sterile, pyrogen-free phosphate-buffered saline is a typical carrier (Gennaro, 2000, Remington: The Science and Practice of Pharmacy, 20 th edition,ISBN:0683306472).

[0154] Pharmaceutical compositions of the present disclosure can be lyophilized or aqueous forms, i.e. solutions or suspensions. This type of liquid formulation allows the compositions to be administered directly in their packaged form without the need for reconstitution in an aqueous medium, and is therefore ideal for injection. Pharmaceutical compositions can be present in vials, or they can be present in pre-filled syringes. Syringes can be with or without needles. Syringes include single doses, while vials can include single doses or multiple doses.

[0155] The liquid formulation of the present disclosure is also suitable for reconstitution of other drugs from lyophilized forms. When the pharmaceutical composition is used for such extemporaneous reconstitution, the present disclosure provides a kit that may include two vials, or may include a prefilled syringe and a vial, the contents of the syringe being used to reconstitute the contents of the vial prior to injection.

[0156] Pharmaceutical compositions of the present disclosure may include antimicrobial agents, particularly when packaged in multiple doses. Antimicrobial agents may be used, such as 2-phenoxyethanol or parabens (methyl paraben, ethyl paraben, propyl paraben). Any preservative is preferably present at a low level. Preservatives may be added exogenously and / or may be components of a large number of antigens that are mixed to form compositions (e.g., present in pertussis antigens as preservatives).

[0157] The pharmaceutical compositions of the present disclosure may include detergents, for example, Tween (polysorbate), DMSO (dimethyl sulfoxide), DMF (dimethylformamide). Detergents are typically present at low levels, such as <0.01%, but may also be used at higher levels, such as 0.01-50%.

[0158] Pharmaceutical compositions of the present disclosure may include a sodium salt (eg, sodium chloride) and free phosphate ions (eg, by using a phosphate buffer) in solution.

[0159] In certain embodiments, the pharmaceutical composition can be encapsulated in a suitable carrier to deliver the peptide to an antigen presenting cell or to increase stability. As will be appreciated by those skilled in the art, a variety of carriers are suitable for delivering the pharmaceutical composition of the present disclosure. Non-limiting examples of suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendritic polymers, and other phospholipid-containing systems. Methods for incorporating pharmaceutical compositions into delivery vehicles are known in the art.

[0160] In order to increase the immunogenicity of the composition, the pharmaceutical composition may include one or more adjuvants and / or cytokines.

[0161] Suitable adjuvants include aluminum salts such as aluminum hydroxide or aluminum phosphate, but may also be salts of calcium, iron or zinc, or may be an insoluble suspension of acylated tyrosine or acylated sugars, or may be cationically or anionically derivatized sugars, polyphosphazenes, biodegradable microspheres, monophosphoryl lipid A (MPL), lipid A derivatives (e.g., with reduced toxicity), 3-O-deacylated monophosphoryl lipid A (3D-MPL), Quil A, saponin, QS21, Freund's incomplete adjuvant (Difco Laboratories, Detroit, Michigan), Merck Adjuvant 65 (Merck, Rahway, New Jersey), AS-2 (SmithKline Beecham, Philadelphia, Pennsylvania), CpG oligonucleotides, bioadhesives and mucoadhesives, microparticles, liposomes, polyoxyethylene ether preparations, polyoxyethylene ester preparations, muramyl peptides, or imidazoquinolone compounds (e.g., imiquimod and its homologues). Human immunomodulators suitable for use as adjuvants in the present disclosure include cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), macrophage colony stimulating factor (M-CSF), tumor necrosis factor (TNF), granulocyte macrophage colony stimulating factor (GM-CSF) can also be used as adjuvants.

[0162] In some embodiments, the composition comprises an adjuvant selected from the group consisting of Montanide ISA-51 (Seppic, Fairfield, NJ, USA), QS-21 (Aquila Biopharmaceuticals, Lexington, MA, USA), granulocyte-macrophage colony stimulating factor (GM-CSF), cyclophosphamide, bacillus Calmette-Guérin (BCG), Corynbacterium parvum, levamisole, azimezone, disopyrone, dinitrochlorobenzene (DNCB), keyhole limpet hemocyanin (KLH), Freund's adjuvant (complete and incomplete), mineral gel, aluminum hydroxide, lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, and diphtheria toxin (DT).

[0163] For example, the cytokine may be selected from the group consisting of transforming growth factors (TGFs) such as, but not limited to, TGF-β and TGF-β, insulin-like growth factor-I and / or insulin-like growth factor-II, erythropoietin (EPO), osteoinductive factors such as, but not limited to, interferons such as, but not limited to, interferons-α, interferons-β and interferons-γ, colony stimulating factors such as, but not limited to, macrophage colony stimulating factor, granulocyte-macrophage colony stimulating factor and granulocyte colony stimulating factor. In some embodiments, the cytokine may be selected from the group consisting of nerve growth factors such as NGF-β, platelet growth factors, transforming growth factors such as, but not limited to, TGF-α and TGF-β, insulin-like growth factor-I and insulin-like growth factor-II, erythropoietin, osteoinductive factors such as, but not limited to, interferons (IFNs) such as, but not limited to, IFN-α, IFN-β and IFN-γ, colony stimulating factors such as, macrophage colony stimulating factor, granulocyte-macrophage colony stimulating factor and granulocyte colony stimulating factor. , such as but not limited to the group consisting of 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.

[0164] It is contemplated that the adjuvant or cytokine may 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. Alternatively, the concentration of the adjuvant or cytokine may be about 0.01 to 50%, preferably about 2% to 30%.

[0165] In certain aspects, the pharmaceutical compositions of the present disclosure are prepared by physically mixing the adjuvant and / or cytokine with the PEPI under appropriate sterile conditions according to known techniques to produce the final product.

[0166] Examples of suitable compositions and methods of administration of polypeptide fragments are provided in Esseku & Adeye (2011) and Van den Mooter G (2006). The preparation of vaccines and immunotherapy compositions is generally described in "Vaccine Design" ("The subunit and adjuvant approach" (eds Powell MF & Newman MJ (1995) Plenum Press New York). Fullerton describes encapsulation in liposomes in U.S. Pat. No. 4,235,877, which is also conceivable.

[0167] In some embodiments, the compositions disclosed herein are prepared as nucleic acid vaccines. In some embodiments, nucleic acid vaccines are DNA vaccines. In some embodiments, DNA vaccines or gene vaccines include plasmids with promoters and suitable transcription and translation control elements and nucleic acid sequences encoding one or more polypeptides of the present disclosure. In some embodiments, plasmids also include sequences that enhance, for example, expression levels, intracellular targeting or proteasome processing. In some embodiments, DNA vaccines include viral vectors containing nucleic acid sequences encoding one or more polypeptides of the present disclosure. In other aspects, the compositions disclosed herein include one or more nucleic acids encoding peptides that have been determined to have immunoreactivity with biological samples. For example, in some embodiments, the compositions include 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, and the peptides include T cell epitope fragments that can bind to at least three HLA class I molecules and / or at least three HLA class II molecules of the patient. In some embodiments, the peptides are derived from antigens expressed in cancer. In some embodiments, DNA or gene vaccines also encode immunomodulatory molecules to manipulate the immune response produced, such as enhancing vaccine efficacy, stimulating the immune system or reducing immunosuppression. Strategies to enhance the immunogenicity of DNA or gene vaccines include encoding of heterologous antigens, fusion of antigens with activated T cells or triggering association recognition molecules, priming with DNA vectors, then strengthening with viral vectors, and the use of immunomodulatory molecules. In some embodiments, DNA vaccines are introduced by needles, gene guns, aerosol syringes, patches, microneedles, friction and other forms. In some forms, DNA vaccines are incorporated into liposomes or other forms of nano antibodies. In some embodiments, DNA vaccines include 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 lipids and cholesterol nanoparticles, cationic lipids, cholesterol and PEG nanoparticles, dendritic polymer nanoparticles. In some embodiments, DNA vaccines are administered by inhalation or ingestion. In some embodiments, DNA vaccines are introduced into blood, thymus, pancreas, skin, muscle, tumor or other parts.

[0168] In some embodiments, the compositions disclosed herein are prepared as RNA vaccines. In some embodiments, RNA is a non-replicating mRNA or a self-amplifying RNA of viral origin. In some embodiments, non-replicating mRNA encodes peptides disclosed herein and contains 5' and 3' untranslated regions (UTR). In some embodiments, the self-amplifying RNA of viral origin not only encodes the peptides disclosed herein, but also encodes a viral replication mechanism capable of intracellular RNA amplification and a large amount of protein expression. In some embodiments, RNA is directly introduced into an individual. In some embodiments, RNA is chemically synthesized or transcribed in vitro. In some embodiments, mRNA is produced from a linear DNA template using T7, T3 or Sp6 phage RNA polymerase, and the resulting product contains an open reading frame, flanking UTR, 5' cap and poly (A) tail encoding the peptide disclosed herein. In some embodiments, various forms of 5' caps are added during or after the transcription reaction using vaccinia virus capping enzymes or by incorporating synthetic caps or anti-retrotranscription cap analogs. In some embodiments, the optimal length of the poly (A) tail is added to the mRNA directly from the encoding DNA template or by using a poly (A) polymerase. The RNA encodes one or more peptides comprising fragments of T cell epitopes capable of binding to at least three HLA class I molecules and / or at least three HLA class II molecules of the patient. In some embodiments, the fragment is derived from an antigen expressed in cancer. In some embodiments, the RNA includes signals that enhance stability and translation. In some embodiments, the RNA also includes non-natural nucleotides that increase the half-life or modified nucleosides that change the immunostimulatory spectrum. In some embodiments, the RNA is introduced by a needle, a gene gun, an aerosol syringe, a patch, a microneedle, friction, and other forms. In some forms, the RNA vaccine is incorporated into liposomes or other forms of nanobodies that promote cellular uptake of RNA and protect it from degradation.In some embodiments, the RNA vaccine comprises a delivery system selected from a transfection agent, 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 electroporated in vivo, protamine complexed mRNA, mRNA associated with positively charged oil-in-water cationic nanoemulsions, mRNA associated with chemically modified dendrimers and complexed with polyethylene glycol (PEG)-lipids. , mRNA complexed with protamine in PEG-lipid nanoparticles, mRNA associated with cationic polymers such as polyethyleneimine (PEI), mRNA associated with cationic polymers such as PEI and lipid components, mRNA associated with polysaccharide (e.g., chitosan) particles or gels, mRNA in cationic lipid nanoparticles (e.g., 1,2-dioleoyloxy-3-trimethylammonium propane (DOTAP) or dioleoylphosphatidylethanolamine (DOPE) lipids), mRNA complexed with cationic lipids and cholesterol, or mRNA complexed with cationic lipids / cholesterol and PEG-lipids. In some embodiments, RNA vaccines are administered by inhalation or ingestion. In some embodiments, RNA is introduced into the blood, thymus, pancreas, skin, muscle, tumor, or other sites, and / or by intradermal, intramuscular, subcutaneous, intranasal, intranodal, intravenous, intrasplenic, intratumoral, or other delivery routes.

[0169] The polynucleotide or oligonucleotide components can be naked nucleotide sequences or combined with cationic lipids, polymers or targeting systems. They can be delivered by any available technology. For example, the polynucleotide or oligonucleotide can be introduced by needle injection, preferably intradermal, subcutaneous or intramuscular. Alternatively, delivery devices such as particle-mediated gene delivery can be used to deliver the polynucleotide or oligonucleotide directly through the skin. The polynucleotide or oligonucleotide can be topically applied to the skin or mucosal surface, for example, by intranasal, oral or rectal administration.

[0170] The uptake of polynucleotide or oligonucleotide constructs can be enhanced by several known transfection techniques, such as those involving the use of transfection agents. Examples of these agents include cationic agents such as calcium phosphate and DEAE-dextran and lipid transfection agents such as lipofectam and transfectam. The dose of the polynucleotide or oligonucleotide administered can be varied.

[0171] Administration is generally a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prevention may be considered treatment), which is an amount sufficient to result in a clinical response or show a clinical benefit to the individual, such as preventing or delaying the onset of a disease or condition, ameliorating one or more symptoms, inducing or prolonging remission, or delaying relapse or recurrence.

[0172] The dosage can be determined according to various parameters, in particular, according to the substance used, the age, weight and condition of the individual to be treated, the route of administration, and the desired regimen. The amount of antigen in each dose is selected as the amount of inducing an immune response. The doctor can determine the route of administration and dosage required for any particular individual. The dosage can be provided as a single dose or can be provided as multiple doses, such as taken at regular intervals, such as 2, 3 or 4 doses per hour. Typically, peptides, polynucleotides or oligonucleotides are typically administered in the range of 1pg to 1mg, more typically 1pg to 10μg for particle-mediated delivery, and in the range of 1μg to 1mg, more typically 1μg to 100μg, more typically 5μg to 50μg for other routes. Typically, it is expected that each dose will contain 0.01mg to 3mg antigen. The optimal amount of a specific vaccine can be determined by studies involving observations of immune responses in subjects.

[0173] Examples of the above techniques and protocols can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams & Wilkins.

[0174] In some cases according to the present disclosure, more than one peptide or peptide composition is administered. Two or more pharmaceutical compositions can be administered together / simultaneously and / or at different times or in sequence. Therefore, the present disclosure includes multiple groups of pharmaceutical compositions and uses thereof. The use of a combination of different peptides that optionally target different antigens is important for overcoming the challenges of tumor genetic heterogeneity and individual HLA heterogeneity. The combined use of the peptides disclosed herein expands the group of individuals who can experience clinical benefits from vaccination. The combined use of the peptides disclosed herein expands the population of individuals who can experience clinical benefits from vaccination. The pharmaceutical composition of multiple PEPIs manufactured for a regimen can define a drug product.

[0175] The route of administration includes, but is not limited to, intranasal, oral, subcutaneous, intradermal and intramuscular. Subcutaneous administration is particularly preferred. Subcutaneous administration can be, for example, by injection into the sides and front of the abdomen, upper arm or thigh, the scapular region of the back or the upper abdominal gluteal region.

[0176] Compositions of the present disclosure can also be administered in one or more dosages and by other routes of administration. For example, these other routes include intradermal, intravenous, intravascular, intraarterial, intraperitoneal, intrathecal, intratracheal, intracardiac, intralobar, intramedullary, intrapulmonary and intravaginal. Depending on the desired duration of treatment, compositions according to the present disclosure can be administered once or multiple times, and can also be administered intermittently, such as monthly administration for several months or years, and administered with different dosages.

[0177] Solid dosage forms for oral administration include capsules, tablets, caplets, pills, powders, micropills and granules. In such solid dosage forms, the active ingredient is usually combined with one or more pharmaceutically acceptable excipients, examples of which are described in detail above. Oral preparations can also be administered as aqueous suspensions, elixirs or syrups. For this reason, the active ingredient can be combined with various sweeteners or flavorings, coloring agents and emulsifiers and / or suspending agents if needed, and diluents such as water, ethanol, glycerol and combinations thereof.

[0178] One or more compositions of the present invention may be administered alone or in combination with other pharmacological compositions or treatments (e.g., chemotherapy and / or immunotherapy and / or vaccines), or methods of treatment and uses according to the present invention may be performed. 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 disclosed herein.

[0179] In some cases, the treatment can be administered in combination with checkpoint blockade therapy / checkpoint inhibitors, co-stimulatory antibodies, cytotoxic or non-cytotoxic chemotherapy and / or radiotherapy, targeted therapy or monoclonal antibody therapy. Chemotherapy has been shown to sensitize tumors to specific cytotoxic T cell killing induced by vaccination (Ramakrishnan et al. J. Clin. Invest. 2010; 120 (4): 1111-1124). Examples of checkpoint inhibitors are CTLA-4 inhibitors, Ipilimumab and programmed cell death-1 / programmed cell death ligand-1 (PD-1 / PD-L1) signaling inhibitors, Nibolumab, Pembrolizumab, Atezolizumab and Durvalumab.Examples of chemotherapeutic agents include alkylating agents, including nitrogen mustards such as dichloromethyldiethylamine (HN2), cyclophosphamide, ifosfamide, melphalan (L-sarcosine protease), and chlorambucil; anthracyclines; epothilones; nitrosoureas such as carmustine (BCNU), lomustine (CCNU), semustine (methyl CCNU), and streptozotocin (streptozotocin); triazenes such as dacarbazine (DTIC); dimethyltriazene imidazole carboxamide; ethyleneimines / methylmelamines such as hexamethylmelamine, thiotepa; alkyl sulfonates such as busulfan; antimetabolites including phyllotoxin; Acid analogs such as methotrexate (methotrexate); alkylating agents, antimetabolites, pyrimidine analogs such as fluorouracil (5-fluorouracil; 5-FU), floxuridine (fluorodeoxyuridine; FUdR) and cytosine arabinoside (cytosine arabinoside); purine analogs and related inhibitors, such as 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 (cisplatin); is-DDP), oxaliplatin and carboplatin; anthracenediones such as mitoxantrone and anthracyclines; substituted ureas such as hydroxyurea; methylhydrazine derivatives including methylprocarbazine (N-methylhydrazine, MIH) and methylprocarbazine; adrenocortical suppressants such as mitotane (o,p′-DDD) and aminoglutethimide; paclitaxel and analogs / derivatives; hormones / hormone therapies and agonists / antagonists including adrenocortical steroid antagonists such as prednisone and its equivalents, dexamethasone and aminoglutethimide, progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate, estrogens such as diethylstilbestrol and 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 flutamide; natural products including vinca alkaloids such as vinblastine (VLB) and vincristine, epipodophyllotoxins such as etoposide and teniposide, antibiotics such as dactinomycin (actinomycin D), daunorubicin (daunorubicin; erythromycin), doxorubicin, bleomycin, plicamycin (mithramycin) and mitomycin (mitomycin C), enzymes such as L-asparaginase, and biological response modifiers such as interferon alphenome.

[0180] In some cases, the method of treatment is a method of vaccination or a method of providing immunotherapy. As used herein, "immunotherapy" is to prevent or treat a disease or condition by inducing or enhancing an immune response in an individual. In certain embodiments, immunotherapy refers to a therapy including administering one or more drugs to an individual to cause a T cell response. In a specific embodiment, immunotherapy refers to a therapy including administering or expressing a polypeptide containing one or more PEPIs to an individual to cause a T cell response, thereby identifying and killing cells that display one or more PEPIs bound to class I HLA on its cell surface. In another specific embodiment, immunotherapy includes administering one or more PEPIs to an individual to cause a cytotoxic T cell response for cells, which cells display tumor-associated antigens (TAA) or cancer testis antigens (CTA) containing one or more PEPIs on the cell surface. In another embodiment, immunotherapy refers to a therapy including administering or expressing a polypeptide containing one or more class II HLA-presented PEPIs to an individual to cause a helper T cell response, thereby providing costimulation to the cytotoxic T cells that identify and kill diseased cells, which display one or more PEPIs bound to class I HLA on their cell surfaces. In another specific embodiment, immunotherapy refers to a therapy that includes administering to an individual one or more drugs that reactivate existing T cells to kill target cells. The theory is that the cytotoxic T cell response will eliminate cells displaying one or more PEPIs, thereby improving the clinical condition of the individual. In some cases, immunotherapy can be used to treat tumors. In other cases, immunotherapy can be used to treat diseases or disorders based on intracellular pathogens.

[0181] In some cases, the disclosure relates to the treatment of cancer or the treatment of solid tumors. In some cases, treatment is for breast cancer, ovarian cancer or colorectal cancer. In other cases, treatment can be directed to any other cancer or solid tumor that expresses a target tumor-associated antigen of the peptides of the present invention as described herein, or any cancer that expresses such a target polypeptide antigen in some or a high percentage of subjects. Treatment can be directed to cancer or malignant or benign tumors of any cell, tissue or organ type. Cancer may or may not be metastatic. Exemplary cancers include carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors or blastomas. Cancer may or may not be hormone-related or dependent cancers (e.g., estrogen or androgen-related cancers).

[0182] Peptide and patient selection

[0183] Specific polypeptide antigens, particularly short peptides derived from these antigens commonly used for vaccination and immunotherapy, induce immune responses in only a portion of human subjects. The polypeptides disclosed herein are specifically selected to induce immune responses in a high proportion of the general population, but due to HLA genotype heterogeneity, they may not be effective in all individuals. HLA genotype population heterogeneity refers to the fact that the immune or clinical response rates to the vaccines described herein are different between different human subgroups. In some cases, the vaccines described herein are used to treat specific or target subgroups.

[0184] The present disclosure also provides a method of identifying a human subject (a potential responder) who is likely to have a cytotoxic T cell response to administration of a pharmaceutical composition comprising a peptide of the present disclosure or predicting the likelihood that a subject will have a cytotoxic T cell response.

[0185] As provided herein, multiple HLAs of individuals are generally required to present T cell epitopes to trigger T cell responses. The best predictor of cytotoxic T cell responses to a given polypeptide determined by the inventors is the presence of at least one T cell epitope (≥1 PEPI3+) presented by three or more HLA class I of an individual. Therefore, the presence of one or more T cell epitopes that can bind to at least three HLAs of a subject in the active ingredient peptide of a pharmaceutical composition indicates that the subject has a cytotoxic T cell response to administering the pharmaceutical composition. The subject may be an immune responder.

[0186] In some cases, the T cell epitope capable of binding to at least three HLA class I molecules of the subject has an amino acid sequence of SEQ ID NOs: 1 to 40, or any one of SEQ ID NOs: 1 to 40, 172 to 194, 234 to 250, and 272 to 301. In other cases, the T cell epitope may have different amino acid sequences within one or more peptides of the pharmaceutical composition.

[0187] The inventors have also found that the presence of at least two epitopes in a vaccine or immunotherapy composition that can bind to at least three HLAs of an individual can predict clinical response. In other words, if an individual has a total of ≥2 PEPI3+ in the active ingredient polypeptide of the vaccine or immunotherapy composition, and these PEPI3+ are derived from antigen sequences that are actually expressed in the individual (e.g., the target tumor cells of the individual express the target tumor-associated antigen), then the individual is a possible clinical responder (i.e., a clinically relevant immune responder).

[0188] Therefore, some aspects of the present disclosure relate to a method for identifying that a subject may have a clinical response to a method of treating a disease according to the present disclosure, or predicting that a subject will have a possibility of a clinical response. "Clinical response" or "clinical benefit" as used herein can be to prevent or delay the onset of a disease or condition, improve one or more symptoms, induce or prolong remission, or delay recurrence or recurrence or deterioration, or any other improvement or stabilization of a subject's disease state. Where appropriate, "clinical response" can be related to "disease control" or "objective response" defined in the "Response Evaluation Criteria for Solid Tumors" (RECIST) guidelines.

[0189] In some embodiments, the method includes determining that one or more cancer-associated antigens selected from SPAG9, AKAP-4, BORIS, NY-SAR-35, NY-BR-1, SURVIVIN, MAGE-A11, PRAME, MAGE-A9, HOM-TES-85, TSP50, EpCAM, CAGE1, FBXO39, MAGE-A8, and MAGE-A6 are expressed by cancer. For example, the expression of cancer-associated antigens can be detected in a sample (e.g., a tumor biopsy) obtained from a subject using methods known in the art.

[0190] The inventors have found that it is not enough for a vaccine or immunotherapy composition to target an antigen expressed by a patient's cancer or tumor cells, nor is it enough for the target sequence of the antigen to bind to the patient's HLA class I (HLA restricted epitope). The composition may be effective only in patients of three or more HLA classes that both express the target antigen and have a single T cell epitope that binds the target antigen. In addition, as described above, at least two epitopes that are combined with at least 3 HLAs of the patient are usually required to induce a clinically relevant immune response.

[0191] Therefore, the method further includes determining that the active ingredient peptide of the pharmaceutical composition comprises two or more different amino acid sequences, each amino acid sequence is a) a fragment of a cancer associated antigen expressed by a subject's cancer cell determined as described above; and b) is capable of binding to at least three HLA class I T cell epitopes of the subject.

[0192] In some cases, the T cell epitope capable of binding to at least three HLA class I of the subject has an amino acid sequence of SEQ ID NOs: 1 to 40, or any one of SEQ ID NOs: 1 to 40, 172 to 194, 234 to 250, and 272 to 301. In other cases, the T cell epitope may have different amino acid sequences within one or more peptides of the pharmaceutical composition.

[0193] In some cases, the likelihood that a subject has 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 cancer or tumor cells and / or without determining the subject's HLA class I genotype. The known frequencies of antigen expression in the disease (such as MAGE-A3 in tumor types such as breast cancer or colorectal cancer) and / or the known frequencies of HLA class I and class II genotypes of subjects in the target population (e.g., a general population, a diseased population) can be used instead. In addition, as identified and described herein, by combining peptides (BestEPI) of the PEPIs with the highest presentation frequency in the targeted population among multiple high-frequency expressed target antigens in the disease, a cancer vaccine regimen that is effective for a high proportion of patients can be designed. However, preselecting patients who are most likely to have a clinical response using the companion diagnostic method described herein will increase the clinical response rate in the treated patients.

[0194] The probability of a subject responding to treatment is increased by (i) presenting more multiple HLA-binding PEPIs in the active ingredient polypeptide; (ii) presenting PEPIs in more target polypeptide antigens; and (iii) expression of the target polypeptide antigen in the subject or the subject's diseased cells. In some cases, the expression of the target polypeptide antigen in the subject may be known, for example if the target polypeptide antigen is present in a sample obtained from the subject. In other cases, population expression frequency data, such as the probability of antigen expression in breast cancer, ovarian cancer, or colorectal cancer, can be used to determine the probability that a particular subject or a diseased cell of a particular subject (over) expresses a particular or any combination of target polypeptide antigens. Population expression frequency data may involve a population of subjects and / or disease matches or a population intended for treatment. For example, the frequency or probability of expression of a specific cancer-associated antigen in a specific cancer or a subject with a specific cancer (e.g., breast cancer) can be determined by detecting the antigen in a tumor (e.g., a breast cancer tumor sample). In some cases, such expression frequencies can be determined from published digital and scientific publications. In some cases, the method of the present invention includes a step of determining the expression frequency of a relevant target polypeptide antigen in a relevant population.

[0195] Disclosed are a series of pharmacodynamic biomarkers that predict the activity / effect of vaccines in individual human subjects as well as in populations of human subjects. The biomarkers have been developed specifically for cancer vaccines, but similar biomarkers can be used for other vaccines or immunotherapy compositions. Exemplary biomarkers are as follows.

[0196] AG95 - Vaccine efficacy: The number of antigens in a cancer vaccine that are expressed with 95% probability by a specific tumor type. AG95 is an indicator of vaccine efficacy and is independent of the immunogenicity of the vaccine antigens. AG95 is calculated based on tumor antigen expression rate data. These data can be obtained from experiments published in peer-reviewed scientific journals. Technically, AG95 is determined by the binomial distribution of antigens in the vaccine and takes into account all possible variations and expression rates.

[0197] PEPI3+ count - Immunogenicity of the vaccine in a subject: Vaccine-derived PEPI3+ is a personal epitope that binds to at least 3 HLAs of a subject and induces a T cell response. PEPI3+ can be determined using a PEPI3+ test in subjects with a known full 4-digit HLA genotype.

[0198] AP count - Antigenicity of the vaccine in the subject: The number of vaccine antigens that are PEPI3+. The vaccine contains sequences from target polypeptide antigens expressed by diseased cells. The AP count is the number of antigens in the vaccine that are PEPI3+. The AP count represents the number of antigens in the vaccine that can induce a T cell response in the subject. The AP count characterizes the vaccine-antigen-specific T cell response of the subject because it depends only on the subject's HLA genotype and is independent of the subject's disease, age, and medication. Correct values ​​are between 0 (no antigen presents PEPI) and the maximum number of antigens (all antigens present PEPI).

[0199] AP50 - Antigenicity of the vaccine in a population: The average number of vaccine antigens with PEPI in a population. AP50 is suitable for characterizing vaccine-antigen-specific T cell responses in a given population, as it depends on the HLA genotype of the subjects in the population.

[0200] AGP count - vaccine efficacy in subject: the number of vaccine antigens expressed in the tumor with PEPIs. The AGP count represents the number of tumor antigens that the vaccine recognized and induced a T cell response (hit the target). The AGP count depends on the vaccine-antigen expression rate in the subject's tumor and the subject's HLA genotype. Correct values ​​are between 0 (no PEPIs expressing antigen presentation) and the maximum number of antigens (all antigens are expressed and present PEPIs).

[0201] ·AGP50 - Vaccine efficacy for cancer in a population: The average number of vaccine antigens expressed in a specified tumor with PEPI (ie, AGP) in a population. AGP50 represents the average number of tumor antigens that can be identified by a vaccine-induced T cell response. AGP50 depends on the expression rate of the antigen in the tumor type shown and the immunogenicity of the antigen in the target population. AGP50 can assess the efficacy of vaccines in different populations and can be used to compare different vaccines in the same population. The calculation of AGP50 is similar to that for AG50, except that expression is weighted by the presence of PEPI3+ on the vaccine antigen expressed in the subject. In a theoretical population where each subject has a PEPI from each vaccine antigen, AGP50 will be equal to AG50. In another theoretical population, where no subject has a PEPI from any vaccine antigen, AGP50 will be 0. In general, the following statements are valid: 0≤AGP50≤AG50.

[0202] mAGP - Candidate biomarker for selecting likely responders: The probability that a cancer vaccine induces a T cell response against multiple antigens expressed in a given tumor. mAGP is calculated, for example, by the expression rate of the vaccine-antigen in the tumor and the presentation rate of the vaccine-derived PEPI in the subject. Technically, mAGP is the sum of the probabilities of multiple AGPs (≥2AGPs) based on the AGP distribution.

[0203] The results of the above predictions can be used to inform doctors about the decision of the subject's treatment. Therefore, in some cases, the method of the present disclosure predicts that the subject will have or may have a T cell response and / or clinical response for treatment as described herein, and the method further includes selecting the treatment for human subjects. In some cases, if the probability of the response of the subject's target predetermined number of target polypeptide antigens (optionally wherein the target polypeptide antigen is (predicted) expressed) is higher than a predetermined threshold, the subject is selected for treatment. 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 include executing the treatment regimen to human subjects. Alternatively, the method can predict that the subject will not have an immune response and / or clinical response, and further includes selecting different treatment regimens for the subject.

[0204] Other embodiments of the present disclosure: (1)

[0205] 1. A pharmaceutical composition comprising one or more peptides, wherein each peptide comprises a different amino acid sequence of any one of SEQ ID NOs: 112 to 142.

[0206] 2. The pharmaceutical composition according to item 1, comprising 2 or more peptides, 3 or more peptides, 4 or more peptides, 5 or more peptides, or 6 or more peptides.

[0207] 3. The pharmaceutical composition according to item 1, comprising two peptides, wherein each peptide comprises a different amino acid sequence of SEQ ID NO: 121 and 124.

[0208] 4. The pharmaceutical composition according to item 1, comprising four peptides, wherein each peptide comprises a different amino acid sequence of SEQ ID NO: 126, 130, 131 and 134.

[0209] 5. The pharmaceutical composition according to item 1, comprising six peptides, wherein each peptide comprises a different amino acid sequence of SEQ ID NO: 121, 124, 126, 130, 131 and 134.

[0210] 6. The pharmaceutical composition according to item 5, further comprising at least one additional peptide comprising a fragment of an antigen selected from TSP50, EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN, MAGE-A8 and MAGE-A6.

[0211] 7. The pharmaceutical composition according to item 5, further comprising one or more additional peptides, each of the one or more additional peptides comprising a different amino acid sequence of any one of SEQ ID NOs: 112-120, 122, 123, 125, 127-129, 132, 133 and 135-142.

[0212] 8. The pharmaceutical composition according to item 1, further comprising a pharmaceutically acceptable adjuvant, diluent, carrier, preservative or a combination thereof.

[0213] 9. The pharmaceutical composition according to item 8, wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosphamide, Bacillus Calmette-Guerin (BCG), Corynebacterium brevis, levamisole, azimezone, propirone, dinitrochlorobenzene (DNCB), keyhole limpet hemocyanin (KLH), Freund's adjuvant (complete), Freund's adjuvant (incomplete), mineral gel, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT) and combinations thereof.

[0214] 10. A pharmaceutical composition comprising one or more nucleic acid molecules encoding one or more peptides, wherein each peptide comprises a different amino acid sequence of any one of SEQ ID NOs: 112 to 142.

[0215] 11. A method of identifying and treating a human subject with cancer who is likely to have a clinical response to administration of a pharmaceutical composition according to item 1, comprising

[0216] (i) measuring a biological sample of a subject to determine the subject's HLA genotype;

[0217] (ii) determining that the pharmaceutical composition comprises two or more sequences that are T cell epitopes capable of binding to at least three HLA class I molecules of the subject;

[0218] (iii) determining the probability that the subject's tumor expresses one or more antigens corresponding to the T cell epitopes identified in step (ii) using the population expression data for each antigen to identify the likelihood that the subject will have a clinical response to administration of the pharmaceutical composition; and

[0219] (iv) administering the composition described in item 1 to the identified subject.

[0220] 12. The method according to item 11, wherein the subject has colorectal cancer.

[0221] 13. The method according to item 11, wherein the pharmaceutical composition comprises 2 or more peptides, 3 or more peptides, 4 or more peptides, 5 or more peptides, or 6 or more peptides.

[0222] 14. The method according to item 11, wherein the pharmaceutical composition comprises two peptides, wherein each peptide comprises a different amino acid sequence of SEQ ID NO: 121 and 124.

[0223] 15. The method according to item 11, wherein the pharmaceutical composition comprises four peptides, wherein each peptide comprises a different amino acid sequence of SEQ ID NO: 126, 130, 131 and 134.

[0224] 16. The method according to item 11, wherein the pharmaceutical composition comprises six peptides, wherein each peptide comprises a different amino acid sequence of SEQ ID NO: 121, 124, 126, 130, 131 and 134.

[0225] 17. The method according to item 11, wherein the pharmaceutical composition further comprises at least one additional peptide comprising a fragment of an antigen selected from TSP50, EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN, MAGE-A8 and MAGE-A6.

[0226] 18. The method of item 11, wherein the pharmaceutical composition further comprises one or more additional peptides, each of the one or more additional peptides comprising a different amino acid sequence of any one of SEQ ID NOs: 112 to 120, 122, 123, 125, 127 to 129, 132, 133, and 135 to 142.

[0227] 19. The method according to item 11, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant, diluent, carrier, preservative or a combination thereof.

[0228] 20. The method according to item 19, wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosphamide, Bacillus Calmette-Guerin (BCG), Corynebacterium brevis, levamisole, azimezone, propirone, dinitrochlorobenzene (DNCB), keyhole limpet hemocyanin (KLH), Freund's adjuvant (complete), Freund's adjuvant (incomplete), mineral gel, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT) and combinations thereof.

[0229] 21. The method of item 11, further comprising administering to the identified subject a chemotherapeutic agent, a checkpoint inhibitor, a targeted therapeutic agent, a radiotherapeutic agent, another immunotherapeutic agent, or a combination thereof.

[0230] 22. The method according to item 13, further comprising before the applying step,

[0231] (i) assaying a tumor sample from a subject to determine that three or more peptides of the pharmaceutical composition comprise two or more different amino acid sequences, each of which is

[0232] a. a fragment of a cancer associated antigen expressed by a cancer cell of the subject as determined in step (i); and

[0233] b. a T cell epitope capable of binding to at least three HLA class I molecules of the subject; and

[0234] (ii) confirming that the subject is likely to have a clinical response to treatment.

[0235] Other embodiments of the present disclosure——(2)

[0236] Breast cancer

[0237] 1. A pharmaceutical composition comprising one or more peptides, wherein each peptide comprises a different amino acid sequence of any one of SEQ ID NOs: 81 to 111 and 435 to 449.

[0238] 2. The pharmaceutical composition according to item 1, comprising 2 or more peptides, 3 or more peptides, 4 or more peptides, 5 or more peptides, 6 or more peptides, 7 or more peptides, 8 or more peptides, 9 or more peptides, 10 or more peptides, 11 or more peptides, or 12 or more peptides.

[0239] 3. The pharmaceutical composition according to item 1, comprising 9 peptides, wherein each peptide comprises a different amino acid sequence of SEQ ID NO: 92, 93, 98, 99 to 101 and 103 to 105.

[0240] 4. The pharmaceutical composition according to item 1, further comprising at least one additional peptide, wherein the additional peptide comprises an antigenic fragment selected from PIWIL-2, AKAP-4, EpCAM, BORIS, HIWI, SPAG9, PLU-1, TSGA10, ODF-4, SP17, RHOXF-2, PRAME, NY-SAR-35, MAGE-A9, NY-BR-1, SURVIVIN, MAGE-A11, HOM-TES-85 and NY-ESO-1.

[0241] 5. The pharmaceutical composition according to item 4, wherein the antigen fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 1 to 20, 24 and 172 to 194.

[0242] 6. The pharmaceutical composition according to item 4, wherein the antigen fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 41 to 60 and 195 to 233.

[0243] 7. The pharmaceutical composition according to item 1, further comprising a pharmaceutically acceptable adjuvant, diluent, carrier, preservative or a combination thereof.

[0244] 8. The pharmaceutical composition according to item 7, wherein the adjuvant is selected from the group consisting of: Montanide ISA-51, QS-21, GM-CSF, cyclophosphamide, Bacillus Calmette-Guerin (BCG), Corynebacterium brevis, levamisole, azimezone, propirone, dinitrochlorobenzene (DNCB), keyhole limpet hemocyanin (KLH), Freund's adjuvant (complete), Freund's adjuvant (incomplete), mineral gel, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT) and combinations thereof.

[0245] 9. A pharmaceutical composition comprising one or more nucleic acid molecules encoding one or more peptides, wherein each peptide comprises a different amino acid sequence of any one of SEQ ID NOs: 81 to 111 and 435 to 449.

[0246] 10. The pharmaceutical composition of item 9, wherein the one or more nucleic acid molecules encode 2 or more peptides, 3 or more peptides, 4 or more peptides, 5 or more peptides, 6 or more peptides, 7 or more peptides, 8 or more peptides, 9 or more peptides, 10 or more peptides, 11 or more peptides, or 12 or more peptides.

[0247] 11. The pharmaceutical composition according to item 9, wherein the one or more nucleic acid molecules encode 9 peptides, wherein each peptide comprises a different amino acid sequence of SEQ ID NO: 92, 93, 98, 99 to 101 and 103 to 105.

[0248] 12. The pharmaceutical composition according to item 9, wherein the one or more nucleic acid molecules encode at least one additional peptide, wherein the additional peptide comprises an antigenic fragment selected from PIWIL-2, AKAP-4, EpCAM, BORIS, HIWI, SPAG9, PLU-1, TSGA10, ODF-4, SP17, RHOXF-2, PRAME, NY-SAR-35, MAGE-A9, NY-BR-1, SURVIVIN, MAGE-A11, HOM-TES-85 and NY-ESO-1.

[0249] 13. The pharmaceutical composition according to item 12, wherein the antigen fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 1 to 20, 24 and 172 to 194.

[0250] 14. The pharmaceutical composition according to item 12, wherein the antigen fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 41 to 60 and 195 to 233.

[0251] 15. The pharmaceutical composition according to item 9, further comprising a pharmaceutically acceptable adjuvant, diluent, carrier, preservative or a combination thereof.

[0252] 16. The pharmaceutical composition according to item 15, wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosphamide, Bacillus Calmette-Guerin (BCG), Corynebacterium brevis, levamisole, azimezone, propirone, dinitrochlorobenzene (DNCB), keyhole limpet hemocyanin (KLH), Freund's adjuvant (complete), Freund's adjuvant (incomplete), mineral gel, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT) and combinations thereof.

[0253] 17. A method for identifying and treating a human subject with cancer who may have a clinical response to administration of a pharmaceutical composition according to item 1, the method comprising

[0254] (i) measuring a biological sample of a subject to determine the subject's HLA genotype;

[0255] (ii) determining that the pharmaceutical composition comprises two or more sequences that are T cell epitopes capable of binding to at least three HLA class I molecules of the subject;

[0256] (iii) determining the probability that the subject's tumor expresses one or more antigens corresponding to the T cell epitopes identified in step (ii) using the population expression data for each antigen to identify the likelihood that the subject will have a clinical response to administration of the pharmaceutical composition; and

[0257] (iv) administering the composition of item 1 to the identified subject.

[0258] 18. The method according to item 17, wherein the subject has breast cancer.

[0259] 19. The method of item 17, wherein the pharmaceutical composition comprises 2 or more peptides, 3 or more peptides, 4 or more peptides, 5 or more peptides, 6 or more peptides, 7 or more peptides, 8 or more peptides, 9 or more peptides, 10 or more peptides, 11 or more peptides, or 12 or more peptides.

[0260] 20. The method according to item 17, wherein the pharmaceutical composition comprises 9 peptides, wherein each peptide comprises a different amino acid sequence of SEQ ID NO: 92, 93, 98, 99 to 101 and 103 to 105.

[0261] 21. The method according to item 17, wherein the pharmaceutical composition further comprises at least one additional peptide, wherein the additional peptide comprises an antigenic fragment selected from PIWIL-2, AKAP-4, EpCAM, BORIS, HIWI, SPAG9, PLU-1, TSGA10, ODF-4, SP17, RHOXF-2, PRAME, NY-SAR-35, MAGE-A9, NY-BR-1, SURVIVIN, MAGE-A11, HOM-TES-85 and NY-ESO-1.

[0262] 22. The method according to item 21, wherein the antigenic fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 1 to 20, 24 and 172 to 194.

[0263] 23. The method according to item 21, wherein the antigenic fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 41 to 60 and 195 to 233.

[0264] 24. The method according to item 17, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant, diluent, carrier, preservative or a combination thereof.

[0265] 25. The pharmaceutical composition according to item 24, wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosphamide, Bacillus Calmette-Guerin (BCG), Corynebacterium brevis, levamisole, azimezone, propirone, dinitrochlorobenzene (DNCB), keyhole limpet hemocyanin (KLH), Freund's adjuvant (complete), Freund's adjuvant (incomplete), mineral gel, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT) and combinations thereof.

[0266] 26. The method of clause 17, further comprising administering to the identified subject a chemotherapeutic agent, a checkpoint inhibitor, a targeted therapy agent, a radiation therapy agent, another immunotherapeutic agent, or a combination thereof.

[0267] 27. The method according to item 17, further comprising before the applying step,

[0268] (iii) assaying a tumor sample from the subject to determine that the three or more peptides of the pharmaceutical composition comprise two or more different amino acid sequences, wherein each amino acid sequence is

[0269] a. a fragment of a cancer associated antigen expressed by a cancer cell of the subject as determined in step (i); and

[0270] b. a T cell epitope capable of binding to at least three HLA class I molecules of the subject; and

[0271] (iv) confirming that the subject is likely to have a clinical response to treatment.

[0272] 28. A method of identifying and treating a human subject with cancer that may be immune responsive to administration of a pharmaceutical composition according to item 1, comprising

[0273] (i) measuring a biological sample of a subject to determine the subject's HLA genotype;

[0274] (ii) determining that the pharmaceutical composition comprises one or more sequences that are T cell epitopes capable of binding to at least three HLA class I molecules of the subject; and

[0275] (iii) administering the composition of item 1 to the identified subject.

[0276] 29. A kit comprising:

[0277] a. A first pharmaceutical composition comprising one or more peptides, wherein each peptide comprises a different amino acid sequence of any one of SEQ ID NOs: 81 to 111 and 435 to 449; and

[0278] b. a different second pharmaceutical composition comprising one or more peptides, wherein each peptide comprises a different amino acid sequence of any one of SEQ ID NOs: 81 to 111 and 435 to 449.

[0279] 30. A pharmaceutical composition comprising: a nucleic acid molecule expressing two or more polypeptides, each polypeptide comprising a fragment of up to 50 consecutive amino acids of an antigen selected from PIWIL-2, AKAP-4, EpCAM, BORIS, HIWI, SPAG9, PLU-1, TSGA10, ODF-4, SP17, RHOXF-2, PRAME, NY-SAR-35, MAGE-A9, NY-BR-1, SURVIVIN, MAGE-A11, HOM-TES-85 and NY-ESO-1, wherein each fragment comprises a different amino acid sequence selected from any one of SEQ ID NOs: 1 to 20, 24 and 172 to 194.

[0280] 31. A pharmaceutical composition comprising one or more peptides, wherein each peptide comprises a different amino acid sequence of any one of SEQ ID NOs: 332 to 346.

[0281] 32. The pharmaceutical composition according to item 31, comprising 2 or more peptides, 3 or more peptides, 4 or more peptides, 5 or more peptides, 6 or more peptides, 7 or more peptides, 8 or more peptides, 9 or more peptides, 10 or more peptides, 11 or more peptides, 12 or more peptides, 13 or more peptides, 14 or more peptides, or 15 or more peptides.

[0282] 33. The pharmaceutical composition according to item 31, comprising 15 peptides, wherein each peptide comprises a different amino acid sequence of SEQ ID NO: 332 to 346.

[0283] 34. The pharmaceutical composition according to item 31, further comprising at least one additional peptide comprising an antigenic fragment selected from PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN, and AKAP-3.

[0284] 35. The pharmaceutical composition according to item 34, wherein the fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 272 to 301.

[0285] 36. The pharmaceutical composition according to item 34, wherein the fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 302 to 331.

[0286] 37. The pharmaceutical composition according to item 31, further comprising a pharmaceutically acceptable adjuvant, diluent, carrier, preservative or a combination thereof.

[0287] 38. The pharmaceutical composition according to item 37, wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosphamide, Bacillus Calmette-Guerin (BCG), Corynebacterium brevis, levamisole, azimezone, propirone, dinitrochlorobenzene (DNCB), keyhole limpet hemocyanin (KLH), Freund's adjuvant (complete), Freund's adjuvant (incomplete), mineral gel, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT) and combinations thereof.

[0288] 39. A pharmaceutical composition comprising one or more nucleic acid molecules encoding one or more peptides, wherein each peptide comprises a different amino acid sequence of any one of SEQ ID NOs: 332 to 346.

[0289] 40. The pharmaceutical composition of item 39, wherein the one or more nucleic acid molecules encode 2 or more peptides, 3 or more peptides, 4 or more peptides, 5 or more peptides, 6 or more peptides, 7 or more peptides, 8 or more peptides, 9 or more peptides, 10 or more peptides, 11 or more peptides, 12 or more peptides, 13 or more peptides, 14 or more peptides, or 15 or more peptides.

[0290] 41. The pharmaceutical composition according to item 39, wherein the one or more nucleic acid molecules encode 15 peptides, wherein each peptide comprises a different amino acid sequence of SEQ ID NO: 332 to 346.

[0291] 42. The pharmaceutical composition of item 39, wherein the one or more nucleic acid molecules encode at least one additional peptide, wherein the additional peptide comprises an antigenic fragment selected from PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN, and AKAP-3.

[0292] 43. The pharmaceutical composition according to item 42, wherein the fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 272 to 301.

[0293] 44. The pharmaceutical composition according to item 42, wherein the fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 302 to 331.

[0294] 45. The pharmaceutical composition according to item 39, further comprising a pharmaceutically acceptable adjuvant, diluent, carrier, preservative or a combination thereof.

[0295] 46. ​​The pharmaceutical composition according to item 45, wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosphamide, Bacillus Calmette-Guerin (BCG), Corynebacterium brevis, levamisole, azimezone, propirone, dinitrochlorobenzene (DNCB), keyhole limpet hemocyanin (KLH), Freund's adjuvant (complete), Freund's adjuvant (incomplete), mineral gel, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT) and combinations thereof.

[0296] 47. A method of identifying and treating a human subject with cancer who is likely to have a clinical response to administration of a pharmaceutical composition according to item 28, the method comprising

[0297] (i) measuring a biological sample of a subject to determine the subject's HLA genotype;

[0298] (ii) determining that the pharmaceutical composition comprises two or more sequences that are T cell epitopes capable of binding to at least three HLA class I molecules of the subject;

[0299] (iii) determining the probability that the subject's tumor expresses one or more antigens corresponding to the T cell epitopes identified in step (ii) using the population expression data for each antigen to identify the likelihood that the subject will have a clinical response to administration of the pharmaceutical composition; and

[0300] (iv) administering the composition of item 28 to the identified subject.

[0301] 48. The method of item 47, wherein the subject has ovarian cancer.

[0302] 49. The method of item 47, wherein the pharmaceutical composition comprises 2 or more peptides, 3 or more peptides, 4 or more peptides, 5 or more peptides, 6 or more peptides, 7 or more peptides, 8 or more peptides, 9 or more peptides, 10 or more peptides, 11 or more peptides, 12 or more peptides, 13 or more peptides, 14 or more peptides, or 15 or more peptides.

[0303] 50. The method according to item 47, wherein the pharmaceutical composition comprises 15 peptides, wherein each peptide comprises a different amino acid sequence of SEQ ID NO: 332 to 346.

[0304] 51. The method of item 47, wherein the pharmaceutical composition further comprises at least one additional peptide comprising an antigenic fragment selected from PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN, and AKAP-3.

[0305] 52. The method according to item 51, wherein the fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 272 to 301.

[0306] 53. A method according to item 51, wherein the fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 302 to 331.

[0307] 54. The method according to item 47, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant, diluent, carrier, preservative or a combination thereof.

[0308] 55. The pharmaceutical composition according to item 54, wherein the adjuvant is selected from the group consisting of Montanide ISA-51, QS-21, GM-CSF, cyclophosphamide, Bacillus Calmette-Guerin (BCG), Corynebacterium brevis, levamisole, azimezone, propirone, dinitrochlorobenzene (DNCB), keyhole limpet hemocyanin (KLH), Freund's adjuvant (complete), Freund's adjuvant (incomplete), mineral gel, aluminum hydroxide (Alum), lysolecithin, pluronic polyols, polyanions, oil emulsions, dinitrophenol, diphtheria toxin (DT) and combinations thereof.

[0309] 56. The method of item 47, further comprising administering to the identified subject a chemotherapeutic agent, a checkpoint inhibitor, a targeted therapy agent, a radiation therapy agent, another immunotherapeutic agent, or a combination thereof.

[0310] 57. The method according to item 47, further comprising before the applying step,

[0311] A tumor sample from a subject is assayed to determine whether the three or more peptides of the pharmaceutical composition comprise two or more different amino acid sequences, each of which is

[0312] a. a fragment of a cancer associated antigen expressed by a cancer cell of the subject as determined in step (i); and

[0313] b. a T cell epitope capable of binding to at least three HLA class I molecules of the subject; and

[0314] Confirm that the subject is likely to have a clinical response to treatment.

[0315] 58. A method of identifying and treating a human subject having cancer that may be immune responsive to administration of a pharmaceutical composition according to item 31, comprising

[0316] (i) measuring a biological sample of a subject to determine the subject's HLA genotype;

[0317] (ii) determining that the pharmaceutical composition comprises one or more sequences that are T cell epitopes capable of binding to at least three HLA class I molecules of the subject; and

[0318] (iii) administering the composition of item 31 to the identified subject.

[0319] 59. A kit comprising:

[0320] a. A first pharmaceutical composition comprising one or more peptides, wherein each peptide comprises a different amino acid sequence of any one of SEQ ID NOs: 332 to 346; and

[0321] b. a different second pharmaceutical composition comprising one or more peptides, wherein each peptide comprises a different amino acid sequence of any one of SEQ ID NOs: 332 to 346.

[0322] 60. A pharmaceutical composition comprising: a nucleic acid molecule expressing two or more polypeptides, each polypeptide comprising a fragment of up to 50 consecutive amino acids of an antigen selected from PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN, and AKAP-3, wherein each fragment comprises a different amino acid sequence selected from any one of SEQ ID NOs: 272 to 301.

[0323] Examples

[0324] Example 1 HLA epitope binding prediction method and validation

[0325] The prediction of binding between specific HLA and epitopes (9-mer peptides) was based on the Immune Epitope Database tool for epitope prediction (www.iedb.org).

[0326] The HLA I epitope binding prediction process was validated by comparison with HLA I epitope pairs experimentally determined in the laboratory. A dataset of HLA I epitope pairs reported in peer-reviewed publications or public immunology databases was compiled.

[0327] The agreement rate with the experimentally determined data set (Table 2) was determined. The binding HLA I epitope pairs of the data set were correctly predicted with a probability of 93%. Coincidentally, the non-binding HLA I epitope pairs were also correctly predicted with a probability of 93%.

[0328] Table 2 Analytical specificity and sensitivity of HLA epitope binding prediction methods

[0329]

[0330] The accuracy of predicting multiple HLA binding epitopes was determined. Based on the analytical specificity and sensitivity using a 93% probability of true positive and true negative predictions and a 7% (=100%-93%) probability of false positive and false negative predictions, the probability of multiple HLA binding epitopes in the human body can be calculated. The probability of multiple HLA binding to epitopes shows the relationship between the number of HLA binding epitopes and the expected minimum actual binding number. According to the PEPI definition, 3 is the expected minimum number of HLA binding epitopes (bold).

[0331] Table 3 Prediction accuracy of various HLA binding epitopes

[0332]

[0333] The validated HLA epitope binding prediction method was used to determine all HLA epitope binding pairs described in the Examples below.

[0334] Example 2: Multi-HLA epitope presentation predicts cytotoxic T lymphocyte (CTL) responses

[0335] It is determined that the CTL response is predictive of one or more epitope presentation of a polypeptide antigen by one or more HLA I in an individual.

[0336] Through the study of 71 cancer patients and 9 HIV-infected patients (Table 4), 1-7 The study was conducted as a retrospective analysis of 6 clinical trials conducted. Patients from these studies were treated with HPV vaccines, three different NY-ESO-1 specific cancer vaccines, an HIV-1 vaccine, and a CTLA-4 specific monoclonal antibody, Ipilimab, which was shown to reactivate CTLs against NY-ESO-1 antigens in melanoma patients. All of these clinical trials measured antigen-specific CD8+ CTL responses (immunogenicity) in study subjects after vaccination. In some cases, correlations between CTL responses and clinical responses were reported.

[0337] No patient was excluded from the retrospective study for any reason other than data availability. The 157 patient data sets (Table 4) were randomized with a standard random number generator to create two independent cohorts for training and evaluation studies. In some cases, the cohorts included multiple data sets from the same patient, resulting in a training cohort of 76 data sets from 48 patients and a test / validation cohort of 81 data sets from 51 patients.

[0338] Table 4 Summary of patient datasets

[0339]

[0340]

[0341] The CTL responses of the reported training data set were compared with the HLA I restriction maps of the vaccine antigen epitopes (9-mer). The antigen sequence and HLA I genotype of each patient were obtained from publicly available protein sequence databases or peer-reviewed publications, and the HLA I epitope binding prediction method was unaware of the patient's clinical CTL response data. The number of epitopes of each antigen that were predicted to bind to at least 1 (PEPI1+), or at least 2 (PEPI2+), or at least 3 (PEPI3+), or at least 4 (PEPI4+), or at least 5 (PEPI5+), or all 6 (PEP16+) HLA class I molecules of each patient was determined, and the number of HLA binding was used as a classifier for the reported CTL response. The true positive rate (sensitivity) and true negative rate (specificity) (the number of HLA bindings) of each classifier were determined from the training data set.

[0342] ROC analysis was performed for each classifier. In the ROC curve, the true positive rate (sensitivity) at different cutoff points is plotted as a function of the false positive rate (1-specificity) ( Figure 1 Each point on the ROC curve represents a sensitivity / specificity pair corresponding to a particular decision threshold (epitope (PEPI) count). The area under the ROC curve (AUC) is a measure of the degree to which the classifier can distinguish between two diagnostic groups (CTL responders or non-responders).

[0343] This analysis unexpectedly revealed that epitope presentation predicted by multiple class I HLAs of the subject (PEPI2+, PEPI3+, PEPI4+, PEPI5+, or PEP16) in each case better predicted CTL responses than epitope presentation by only one or more HLA class I molecules (PEPI1+, AUC=0.48, Table 5).

[0344] Table 5 Diagnostic value of PEPI biomarkers determined by ROC analysis

[0345]

[0346] By considering the antigenic epitopes that can be presented by at least 3 HLA class I molecules of the individual, the CTL response of the individual is best predicted (PEPI3+, AUC=0.65, Table 5). The threshold count of PEPI3+ (the number of antigen-specific epitopes presented by 3 or more HLA of the individual) that best predicts a positive CTL response is 1 (Table 6). In other words, at least 3 HLA class I molecules (≥1PEPI3+) of the subject present at least one antigen-derived epitope, and then the antigen can trigger at least one CTL clone, and the subject is a possible CTL responder. Using ≥1PEPI3+ threshold to predict possible CTL responders ("≥1PEPI3+ test") provides a diagnostic sensitivity of 76% (Table 12).

[0347] Table 6 Determination of ≥1 PEPI3+ threshold to predict possible CTL responders in the training dataset

[0348]

[0349] Example 3 ≥1 Validation of PEPI3+ test

[0350] A test cohort of 81 data sets from 51 patients was used to validate a ≥1 PEPI3+ threshold to predict antigen-specific CTL responses. For each data set in the test cohort, it was determined whether a ≥1 PEPI3+ threshold (at least one antigen-derived epitope presented by at least three class I HLAs of the individual) was met. This was compared with the experimentally determined CTL responses reported in the clinical trial (Table 7).

[0351] Clinical validation demonstrated that PEPI3+ peptides induce CTL responses in individuals with a probability of 84%. 84% is the same value determined in the analytical validation of PEPI3+ predictions, where the epitope was predicted to bind to at least 3 HLAs of the individual (Table 3). These data provide strong evidence that PEPI induces an immune response in individuals.

[0352] Table 7 Diagnostic performance characteristics ≥1 PEPI3+ test (n=81)

[0353]

[0354]

[0355] ROC analysis used PEPI3+ count as the cutoff value to determine the diagnostic accuracy ( Figure 2 ). AUC value = 0.73. For ROC analysis, an AUC of 0.7 to 0.8 is generally considered a reasonable diagnosis.

[0356] PEPI3+ counts of at least 1 (≥1 PEPI3+) best predicted CTL responses in the test dataset (Table 8). This result confirmed the thresholds determined during training (Table 5).

[0357] Table 8 Confirmation of ≥1 PEPI3+ threshold to predict likely CTL responders in the test / validation datasets.

[0358]

[0359] Example 4 ≥1 PEPI3+ test predicts CD8+ CTL reactivities

[0360] The ≥1 PEPI3+ test was compared with previously reported methods for predicting CTL responses to peptide antigens in specific human subjects.

[0361] HLA genotypes were determined from DNA samples of 28 patients with cervical cancer and VIN-3 who received HPV-16 synthetic long peptide vaccine (LPV) in two different clinical trials 8 9 10 The LPV consists of long peptides covering the HPV-16 viral oncoproteins E6 and E7. The amino acid sequence of the LPV was obtained from these publications. These publications also report the T cell responses of each vaccinated patient to the overlapping peptide pool of the vaccine.

[0362] For each patient, LPV epitopes (9-mer) presented by at least three of the patient's class I HLAs (PEPI3+) were identified and their distribution in the peptide library was determined. Peptides containing at least one PEPI3+ (≥1 PEPI3+) were predicted to induce CTL responses. Peptides not containing PEPI3+ were predicted not to induce CTL responses.

[0363] The ≥1 PEPI3+ test correctly predicted 489 of 512 negative CTL responses and 8 of 40 positive CTL responses measured after vaccination ( Figure 3 A). Overall, the concordance between ≥1 PEPI3+ test and experimentally determined CD8+ T cell reactivity was 90% (p<0.001).

[0364] For each patient, the distribution between peptide repertoires of epitopes presented by at least one of the patient's class I HLAs (≥1 PEPI1+, HLA-restricted epitope prediction, prior art methods) was also determined. PEPI1+ correctly predicted 116 of the 512 negative CTL responses and 37 of the 40 positive CTL responses measured after vaccination ( Figure 3 B) Overall, the concordance between HLA-restricted epitope predictions (≥1 PEPI1+) and CD8+ T cell reactivity was 28% (not significant).

[0365] Example 5 Prediction of HLA class II restricted CD4+ helper T cell epitopes

[0366] CD4+ helper T cell responses were studied in 28 cervical cancer and VIN-3 patients who received HPV-16 synthetic long peptide vaccine (LPV) in two different clinical trials (as detailed in Example 4) after LPV vaccination ( Figure 4 ). The sensitivity of the HLA class II restricted epitope prediction was 78%, as the prior art tool predicted 84 positive responses (positive CD4+ T cell reactivity to the peptide pool of the human DP allele) out of 107 cases (sensitivity = 78%). The specificity was 22%, as it could exclude 7 negative responses out of 31 cases. Overall, the concordance between the HLA restricted class II epitope prediction and CD4+ T cell reactivity was 66%, which was not statistically significant.

[0367] Example 6 ≥1 PEPI3+ test predicts T cell response to full-length LPV polypeptide

[0368] Using the same reported studies as Examples 4 and 5, a ≥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. The results were compared to experimentally determined responses. The test accurately predicted CD8+ T cell reactivity in 11 of the 15 VIN-3 patients with positive CD8+ T cell reactivity test results (sensitivity 73%, PPV 85%) and 2 of the 5 cervical cancer patients (sensitivity 40%, PPV 100%). CD4+ T cell reactivity (PEPI4+) was accurately predicted to be 100% ( Figure 5 ).

[0369] It was also observed that class I and class II HLA restricted PEPI3+ counts were associated with clinical benefit reported in LPV vaccinated patients. Patients with higher PEPI3+ counts had complete or partial responses after 3 months.

[0370] Example 7 Case Study

[0371] pGX3001 is a DNA vaccine based on HPV16 that contains full-length E6 and E7 antigens with a linker between them. pGX3002 is a DNA vaccine based on HPV18 that contains full-length E6 and E7 antigens with a linker between them. A phase II clinical trial investigated the T cell responses of 17 HPV-infected cervical cancer patients vaccinated with pGX3001 and pGX3002 (VGX-3100 vaccination) 1 .

[0372] Figures 5 to 6 The position of each epitope (9-mer) presented by at least 1 (PEPI1+), at least 2 (PEPI2+), at least 3 (PEPI3+), at least 4 (PEPI4+), at least 5 (PEPI5+) or all 6 (PEP16) class I HLAs of two exemplary patients (patients 12-11 and patients 14-5) within the full-length sequences of two HPV-16 and two HPV-18 antigens.

[0373] For the combined vaccine, the total PEPI1+ count for patient 12-11 was 54 (54 epitopes presented by one or more class I HLAs), and the PEPI1+ count for patient 14-5 was 91. Therefore, for the four HPV antigens, patient 14-5 had a higher PEPI1+ count than patient 12-11. PEPI1+ represents a different vaccine antigen-specific HLA-restricted epitope set for patients 12-11 and 14-5. Only 27 PEPI1+ were common in these two patients.

[0374] For the PEPI3+ count (the number of epitopes presented by three or more of the patient's class I HLAs), the results for patients 12-11 and 14-5 were reversed. Patient 12-11 had a PEPI3+ count of 8, including at least one PEPI3+ in each of the four HPV16 / 18 antigens. Patient 14-5 had a PEPI3+ count of 0.

[0375] The reported immune responses for these two patients matched the PEPI3+ counts but not the PEPI1+ counts. Patients 12-11 developed an immune response to each of the four antigens after vaccination, as measured by ELISpot, while patients 14-5 did not develop an immune response to any of the four antigens of the vaccine. A similar pattern was observed when comparing the PEPI1+ and PEPI3+ groups for all 17 patients in the trial. There was no correlation between the PEPI1+ counts and experimentally measured T cell responses reported in clinical trials. However, we observed a correlation between T cell immunity predicted by ≥1 PEPI3+ tests and reported T cell immunity. ≥1 PEPI3+ tests predict immune responders to HPV DNA vaccines.

[0376] In addition, the diversity of the patient PEPI3+ groups is similar to the diversity of T cell responses typically found in cancer vaccine trials. Similar to patient 14-5, patients 12-3 and 12-6 did not have PEPI3+, which predicted that the HPV vaccine could not trigger T cell immunity. All other patients had at least one PEPI3, which predicted the possibility that the HPV vaccine could induce T cell immunity. Eleven patients had multiple PEPI3+, predicting that the HPV vaccine might trigger a polyclonal T cell response. Patients 15-2 and 15-3 produced high levels of T cell immune responses to E6 of both HPVs, but had poor immune responses to E7. Other patients 15-1 and 12-11 had the same degree of response to E7 of HPV18 and HPV16, respectively.

[0377] Example 8 Designing model populations for in silico experiments and identifying candidate precision vaccine targets in large populations

[0378] A computer simulation human test group of 433 subjects has a complete 4-bit HLA I class genotype (2×HLA-A*xx:xx; 2×HLA-B*xx:xx; 2×HLA-C*xx:xx) and compiled demographic information. The model population has a total of 152 different HLA alleles, representing> 85% of the currently known allele G group.

[0379] A "large population" database containing 7189 subjects was also established, which was characterized by 4 HLA genotypes and demographic information. The large population had 328 different HLA class I alleles. The HLA allele distribution of the model population was significantly correlated with the large population (Table 9) (Pearson p < 0.001). Therefore, the 433 patient model population represents a 16-fold larger population.

[0380] The model population represents 85% of the human population in terms of HLA diversity and HLA frequency.

[0381] Table 9 Statistical analysis of HLA distribution in the “model population” and “large population”

[0382]

[0383] Example 9: In silico experiments based on identification of multiple HLA binding epitopes predicted T cell response rates in reported clinical trials

[0384] The aim of this study was to determine whether a model population, such as that described in Example 8, could be used to predict CTL reactivity rates of a vaccine, ie, for use in in silico efficacy trials.

[0385] Twelve peptide vaccines derived from cancer antigens were identified from peer-reviewed publications that induced T cell responses in subsets of subjects. These peptides have been studied in clinical trials that have enrolled a total of 172 patients. T cell responses induced by the vaccine peptides have been determined from blood samples and reported. The immune response rate (IRR) as the percentage of study subjects with a positive T cell response measured in the clinical trial was determined. Figure 7 ).

[0386] Table 10 Clinical trials with peptide vaccines

[0387] Peptide vaccines Source antigen Peptide length T cell assay Group (n) References MMNLMQPKTQQTYTYD JUP 16-mer Polymer staining 18 12 GRGSTTTNYLLDRDDYRNTSD ADA17 21-mer Polymer staining 18 12 LKKGAADGGKLDGNAKLNRSLK BAP31 22-mer Polymer staining 18 12 FPPKDDHTLKFLYDDNQRPYPP TOP2A 22-mer Polymer staining 18 12 RYRKPDYTLDDGHGLLRFKST Abl-2 21-mer Polymer staining 18 12 QRPPFSQLHRFLADALNT DDR1 18-mer Polymer staining 18 12 ALDQCKTSCALMQQHYDQTSCFSSP ITGB8 25-mer Polymer staining 18 12 STAPPAHGVTSAPDTRPAPGSTAPP MUC-1 25-mer proliferation 80 13 YLEPGPVTA gp100 9-mer Tetramer 18 14 MTPGTQSPFFLLLLLTVLTVV MUC-1 21-mer Cytotoxicity 10 15 SSKALQRPV Bcr-Abl 9-mer ELISPOT 4 16 RMFPNAPYL WT-1 9-mer Polymer staining 24 17 RMFPNAPYL(HLA-A*0201) WT-1 9-mer Cytokine staining 18 18

[0388] In each of the 433 subjects in the model population described in Example 8, 12 peptides were studied with a ≥1 PEPI3+ test. The "≥1 PEPI3+ score" for each peptide was calculated as the proportion of subjects in the model population with at least one vaccine-derived epitope that binds to at least three subject-specific HLA class I molecules (≥1 PEPI3+). If the corresponding clinical trial stratifies patients in the HLA allele-selected population, the model population is also screened for subjects with the corresponding allele (example: WT1, HLA-A*0201).

[0389] Experimentally determined trial-reported response rates were compared with ≥1 PEPI3+ scores. Overall percentage of agreement (OPA) was calculated based on paired data (Table 11). 2 =0.77) are linearly correlated ( Figure 7 ). This result suggests that the identification of peptides predicted to bind to multiple HLAs of an individual can be used to predict the outcome of in silico clinical trials.

[0390] Table 11 Comparison of PEPI3+ scores and CTL response rates of 12 peptide vaccines

[0391]

[0392] *Percentage of subjects in the model population with ≥1 vaccine-derived PEPI3+

[0393] Example 10 Prediction of T cell response rates reported in Clinical Trial II based on computer simulation experiments identifying multiple HLA binding epitopes

[0394] Nineteen clinical trials with published immune response rates (IRRs) for peptide- or DNA-based vaccines were identified (Table 12). These trials involved 604 patients and covered 38 vaccines derived from tumor and viral antigens. Vaccine antigen-specific CTL responses were measured in each study patient, and response rates in the clinical study population were calculated and reported.

[0395] Each vaccine peptide from the 19 clinical trials was tested with ≥1 PEPI3+ in each subject in the model population. The ≥1 PEPI3+ score for each peptide was calculated as the proportion of subjects in the model population with at least one vaccine-derived PEPI3+. The experimentally determined trial-reported response rates were compared with the PEPI scores as described in Example 9 (Table 13). A linear correlation was observed between the response rate and the ≥1 PEPI3+ score (R2 = 0.70) ( Figure 8 ). The results demonstrate that identification of peptides predicted to bind to multiple HLAs of an individual can predict T cell responses in a subject and that in silico experiments can predict the outcomes of clinical trials.

[0396] Table 12 Response rates published in clinical trials

[0397]

[0398] Table 13 There is a linear correlation between PEPI score and response rate (R 2 =0.7)

[0399]

[0400]

[0401] *Percentage of subjects in the model population with ≥1 vaccine-derived PEPI3+

[0402] Example 11 In silico simulations based on identification of multiple HLA binding epitopes in peptide vaccines predict reported clinical trial immune response rates

[0403] IMA901 is a therapeutic vaccine for renal cell carcinoma (RCC) containing 9 peptides derived from tumor-associated peptides (TUMAPs) that are naturally present in human cancer tissues. In two independent clinical studies (Phase I and Phase II), a total of 96 HLA-A*02+ subjects with advanced RCC were treated with IMA901. Each of the 9 peptides of IMA901 has been identified as an HLA-A2 restricted epitope in the prior art. Based on currently recognized standards, they are all strong candidate peptides for promoting T cell responses against renal cancer in test subjects, because their presence has been detected in renal cancer patients, and because test patients are specifically selected to have at least one HLA molecule capable of presenting each peptide.

[0404] For each subject in the model population, how many of the nine peptides of the IMA901 vaccine can bind to three or more HLAs were determined. Since each peptide in the IMA901 vaccine is a 9-mer, this corresponds to the PEPI3+ count. The results are compared with the immune response rates reported in Phase I and Phase II clinical trials (Table 14).

[0405] Table 14 Immune response rate of IMA901 in the model population and two clinical trials

[0406]

[0407] *Patients whose immune response was not assessed

[0408] Results from phase I and II studies have shown variability in immune responses to the same vaccine in different trial cohorts. However, overall, there was good agreement between response rates predicted by ≥2 PEPI3+ tests and reported clinical response rates.

[0409] In a retrospective analysis, clinical investigators of the above trial found that subjects who responded to multiple peptides of the IMA901 vaccine were significantly (p=0.019) more likely to experience disease control (stable disease, partial response) than subjects who responded to only one peptide or no response. Six of the eight subjects (75%) who responded to multiple peptides experienced clinical benefit in the trial, compared to 14% and 33% of 0 and 1 peptide responders, respectively. Randomized Phase II trials have confirmed that immune responses to multiple TUMAPs are associated with longer overall survival.

[0410] Since the presentation of PEPI accurately predicts the responders of TUMAP, the clinical responders of IMA901 are likely to be patients who can present ≥2 PEPIs from TUMAP. This subgroup accounts for only 27% of HLA-A*02 selected patients, and according to the clinical trial results, it is expected that 75% of this subgroup will experience clinical benefits. The same clinical results show that if patient selection is based on ≥3 PEPIs from TUMAP, 100% of patients will experience clinical benefits, although this group will only represent 3% of the patient population selected by HLA-A*02. These results show that in the patient population studied in the IMA901 clinical trial, the disease control rate (stable disease or partial response) is between 3% and 27%. In the absence of a complete response, only a portion of these patients can experience survival benefits.

[0411] These findings explain the lack of improved survival in the Phase III IMA901 clinical trial. These results also suggest that HLA-A*02 enrichment of the study population was insufficient to achieve the primary overall survival endpoint in the Phase III IMA901 trial. As noted by the IMA901 trial investigators, a companion diagnostic (CDx) needs to be developed to select possible responders to peptide vaccines. These findings also suggest that selecting patients with ≥2 TUMAP-specific PEPIs may provide sufficient enrichment to demonstrate significant clinical benefit of IMA901.

[0412] Example 12 In silico experiments based on identification of vaccine-derived multi-HLA binding epitopes predict reported experimental clinical response rates

[0413] The correlation between the ≥2PPI3+ score of the immunotherapy vaccine determined in the model population described in Example 8 and the reported disease control rate (DCR, proportion of patients with complete response and partial response and stable disease) determined in clinical trials was determined.

[0414] Seventeen clinical trials with peptide- and DNA-based cancer immunotherapy vaccines published in peer-reviewed scientific journals have published disease control rates (DCR) or objective response rates (ORR) (Table 15). These trials involved 594 patients and covered 29 tumor and viral antigens. DCR was determined according to the Response Evaluation Criteria in Solid Tumors (RECIST), which is the current standard for clinical trials, in which clinical response is based on the change in the largest cross-sectional dimension. 42,43,44 In the absence of available DCR data, objective response rate (ORR) data, also defined according to RECIST guidelines, were used.

[0415] Table 16 compares the ≥2PPI3+ score for each vaccine in the model population and the published DCR or ORR. The observed correlation between the predicted and measured DCR provides evidence that not only immunogenicity but also cancer vaccine efficacy depends on the individual's multiple HLA sequences (R 2 =0.76) Fig. 9 ).

[0416] Table 15 Clinical trials selected for disease control rate (DCR) prediction

[0417]

[0418] Table 16 Disease control rate (DCR) and MultiPEPI score (predicted DCR) in 17 clinical trials

[0419]

[0420]

[0421] Example 13 Breast cancer vaccine design for large populations and compositions

[0422] Taking into account the heterogeneity of tumor antigens and patient HLA, we used the above-mentioned PEPI3+ test to design peptides for breast cancer vaccines that were effective in a large proportion of patients.

[0423] As reported in peer-reviewed publications, breast cancer CTA is identified and graded based on the overall expression frequency of antigens found in breast cancer tumor specimens (Chen et al. Multiple Cancer / Testis Antigens ArePreferentially Expressed in Hormone-Receptor Negative and High-Grade Breast Cancers. Plos One 2011;6(3):e17876.; Kanojia et al. Sperm-Associated Antigen 9, a Novel Biomarker for Early Detection of Breast Cancer. Cancer Epidemiol Biomarkers Prev 2009;18(2):630–639.; Saini et al. A Novel Cancer Testis Antigen, A-Kinase Anchor Protein 4 (AKAP4) Is a Potential Biomarker for Breast Cancer. Plos One 2013;8(2):e57095).

[0424] Based on the graded expression rates, we selected the CTA with the highest expression frequency as the target antigen for breast cancer vaccine. Fig.11 shown.

[0425] To select immunogenic peptides from the target CTA, we used the PEPI3+ assay and the model population described in Example 8 to identify 9-mer epitopes (PEPI3+s) that were most frequently presented by at least 3 HLAs of individuals in the model population. We refer to these epitopes as "bestEPIs" herein. Fig.10 An illustrative example of "PEPI3+ hotspot" analysis and bestEPI identification of the PRAME antigen is shown.

[0426] We multiplied the frequency of expression of each CTA reported by the frequency of PEPI3+ hot spots in the model population to identify T cell epitopes (9-mers) that will induce immune responses against breast cancer antigens in the highest proportion of individuals (Table 17). Then we selected 15-mers (Table 17) containing each selected 9-mer. Using the method described in Example 19 below, 15-mers were selected to bind to most HLA class II alleles of most subjects. These 15-mers can induce CTL and helper T cell responses in the highest proportion of subjects.

[0427] Table 17 BestEPI list for selecting breast cancer peptides for vaccine compositions (9-mers are underlined). N%: frequency of antigen expression in colorectal cancer; B%: BestEPI frequency, i.e., the percentage of individuals with epitopes that bind at least 3 HLA class I molecules of the subject in the model population (433 subjects); HLAII**: the percentage of individuals with CD4+T cell-specific PEPI4+ in normal donors (n=400); N%*B%: N% multiplied by B%.

[0428]

[0429]

[0430] We then designed 31 30-mer peptides (Table 18a). Each 30-mer consists of two optimized 15-mer fragments, usually from CTAs of different frequencies, where the 15-mer fragments are arranged end to end, and each fragment contains one of the 9-mers (BestEPI) in Table 17. Nine of these 30-mer peptides were selected as a panel of peptides, called PolyPEPI915 (Table 18b). Fig.11 The expression frequencies of the 10 CTAs targeted by PolyPEPI915 are shown singly and in combination.

[0431] Table 18a 30-mer breast cancer vaccine peptides

[0432]

[0433]

[0434] Table 18b Selected breast cancer vaccine peptides for PolyPEPI915 panel / composition

[0435]

[0436] *Percentage of individuals with CD8+ T cell-specific PEPI3+ in the model population (n=433).

[0437] **Percentage of individuals with CD4+ T cell-specific PEPI4+ in normal donors (n=433).

[0438] Characterization of PolyPEPI915

[0439] Tumor heterogeneity can be addressed by including peptide sequences targeting multiple CTAs in vaccines or immunotherapy regimens. The PolyPEPI915 composition targets 10 different CTAs. Based on the antigen expression rates of these 10 CTAs, we simulated the predicted average number of expressed antigens in cancer cells (AG50) and the minimum number of expressed antigens with 95% probability (AG95). 95% of individuals express at least 4 of the 10 target antigens (AG95 = 4), such as Fig.12 The antigen expression curve is shown in .

[0440] The above AG values ​​characterize vaccines that are independent of the target patient population. They can be used to predict the likelihood that a specific cancer (e.g., breast cancer) expresses an antigen targeted by a specific vaccine or immunotherapy composition. AG values ​​are based on known tumor heterogeneity but do not take into account HLA heterogeneity.

[0441] The HLA heterogeneity of certain populations can be characterized from the perspective of immunotherapy or vaccine compositions by the number of antigens representing PEPI3+. These are vaccine-specific CTA antigens predicted to be ≥1 PEPI3+, referred to herein as "AP". The average number of antigens with PEPI3+ (AP50) shows how the vaccine induces an immune response against the antigens targeted by the composition (breast cancer vaccine-specific immune response). The PolyPEPI915 composition can induce an immune response against an average of 5.3 vaccine antigens (AP50 = 5.30), and 95% of the model population can induce an immune response against at least one vaccine antigen (AP95 = 1) ( Fig.13 ).

[0442] The vaccine can be further characterized by the AGP value, which refers to the antigen with PEPI. This parameter is a combination of the previous two parameters: (1) AG depends on the frequency of antigen expression in a specific tumor type, but not on the HLA genotype of individuals in the population, and (2) AP depends on the HLA genotype of individuals in the population, regardless of the frequency of antigen expression. AGP depends on the expression frequency of the vaccine antigen in the disease and the HLA genotype of individuals in the population.

[0443] Combining the data of breast cancer AG and AP in the model population, we determined the AGP value of PolyPEPI915, which represents the probability distribution of vaccine antigens that induce immune responses against antigens expressed in breast tumors. For PolyPEPI915, the AGP50 value in the model population was 3.37. AGP92 = 1, meaning that 92% of the subjects in the model population induced an immune response against at least one expressed vaccine antigen ( Fig.14 ).

[0444] Example 14 Patient Selection Using Companion Diagnostics for Breast Cancer Vaccines

[0445] The likelihood that a particular patient will have an immune response or clinical response to treatment with, for example, one or more cancer vaccine peptides as described above can be determined based on (i) identification of PEPI3+ within the vaccine peptide (a 9-mer epitope capable of binding to at least three HLAs of the patient); and / or (ii) determination of target antigen expression in the patient's cancer cells, for example as measured in a tumor biopsy. Ideally, both parameters are determined and the best combination of vaccine peptides is selected for treating the patient. However, if, for example, determination of expressed tumor antigens by biopsy is not possible, not recommended, or is unreliable due to biopsy error (i.e., a biopsy tissue sample obtained from a small portion or metastatic tumor does not represent the entire repertoire of CTAs expressed in the patient), PEPI3+ analysis alone can be used.

[0446] Example 15 Comparison of PolyPEPI915 with competing breast cancer vaccines

[0447] We used the in silico clinical trial model described above to predict immune response rates for competing breast cancer vaccines that have been studied in clinical trials (Table 19). Immune response rates ranged from 3% to 91%.

[0448] Single peptide vaccines were immunogenic in 3% to 23% of individuals. In contrast, peptides with amino acid sequences selected from SEQ ID No: 81 to 111 were immunogenic in 44% to 73% of individuals in the same cohort. This result represents a significant improvement in the immunogenicity of each peptide in PolyPEPI915.

[0449] The immune response rates of the competing combination peptide products ranged from 10% to 62%. The PolyPEPI915 combination product of the present invention was 96% in the model population and 93% in the breast cancer patient population, representing an improvement in immunogenicity.

[0450] Table 19 Predicted immune response rates of competing breast cancer vaccines

[0451]

[0452] *Proportion of subjects with ≥1 PEPI3+

[0453] Another improvement using the PolyPEPI915 vaccine is the lower chance of tumor escape. Each 30-mer peptide in PolyPEPI915 targets 2 tumor antigens. CTLs targeting more tumor antigens are more effective against heterologous tumor cells than CTLs targeting a single tumor antigen.

[0454] Another improvement is that the PolyPEPI915 vaccine is that individuals who may respond to vaccination can use the methods described herein to identify based on their HLA genotype (sequence) and optionally the antigen expression in their tumor. Pharmaceutical compositions containing the PolyPEPI vaccine will not be applied to such individuals, whose HLA cannot present any PEPI3 from the vaccine. During clinical trials, the number of mAGP or AGP in the PolyPEPI915 scheme is related to the duration of individual responses. It is most likely necessary to have a vaccine combination of>1AGP to destroy heterologous tumor cells. Pharmaceutical compositions containing PolyPEPI will not be applied to individuals whose HLA cannot present any PEPI3 from the vaccine.

[0455] Example 16 Colorectal cancer vaccine design and composition

[0456] We show another example of a colorectal vaccine composition using the same design approach described above. Taking into account the heterogeneity of tumor antigens and patient HLA, we used the above PEPI3+ test to design peptides for colorectal cancer vaccines that were effective in a large proportion of patients.

[0457] Colorectal cancer CTA is based on the overall expression frequency of antigens found in colorectal cancer tumor specimens as reported in peer-reviewed publications ( Fig.19). (Choi J, ChangH. The expression of MAGE and SSX, and correlation of COX2, VEGF, and survivin in colorectalcancer. Anticancer Res 2012.32(2):559-564.; Goossens-Beumer IJ, Zeestraten EC, Benard A, Christen T, Reimers MS, Keijzer R, Sier CF, Liefers GJ, Morreau H,PutterH,Vahrmeijer AL,van de Velde CJ,Kuppen PJ.Clinical prognostic value ofcombined analysis of Aldh1,Survivin,and EpCAM expression in colorectalcancer.Br J Cancer 2014.110(12):2935-2944.;Li M,Yuan YH,Han Y,Liu YX,Yan L,Wang Y,Gu J.Expression profile of cancer-testis genes in 121human colorectalcancer tissue and adjacent normal tissue. Clinical Cancer Res 2005.11(5):1809–1814).

[0458] Based on the graded expression rates, we selected the CTA with the highest expression frequency as the target antigen for the colorectal cancer vaccine. The expression rates of the selected breast cancer-specific CTAs are shown in Fig.15 shown.

[0459] To select immunogenic peptides from the most frequently expressed colorectal cancer CTAs, we used the PEPI3+ assay and the model population described in Example 8 to identify the “bestEPI”.

[0460] We multiplied the reported expression frequency of each CTA (N%) by the frequency of PEPI3+ hotspots in the model population (B%) to identify T cell epitopes (9-mers) that would induce immune responses against colorectal cancer antigens in the highest proportion of individuals (Table 20). We then selected 15-mers that included each selected 9-mer (Table 20). Using the methods described in Example 19 below, 15-mers were selected to bind to the majority of HLA class II alleles in the majority of subjects. These 15-mers can induce CTL and helper T cell responses in the highest proportion of subjects.

[0461] Table 20 BestEPI list for selecting colorectal cancer peptides for vaccine composition (9-mers are underlined). N%: frequency of antigen expression in colorectal cancer; B%: BestEPI frequency, i.e., the percentage of individuals with epitopes that bind at least 3 HLA class I molecules of the subject in the model population (433 subjects); HLAII**: the percentage of individuals with CD4+ T cell-specific PEPI4+ in normal donors (n=400); N%*B%: N% multiplied by B%.

[0462]

[0463]

[0464] We then designed 31 30-mer peptides (Table 21a). Each 30-mer consists of two optimized 15-mer fragments, usually from CTAs of different frequencies, and each 30-mer usually contains at least one high-frequency HLA class II binding PEPI. The 15-mer fragments are arranged end to end, and each fragment contains one of the 9-mers (BestEPI) in Table 20 above. Nine of these 30-mer peptides were selected as a panel of peptides, called PolyPEPI915 (Table 21b). Fig.15 The expression frequencies of the eight CTAs targeted by PolyPEPI915 are shown singly and in combination.

[0465] Table 21a 30-mer colorectal cancer vaccine peptides

[0466]

[0467] *Percentage of individuals with CD8+ T cell-specific PEPI3+ in the model population (n=433).

[0468] **Percentage of individuals with CD4+ T cell-specific PEPI4+ in normal donors (n=433).

[0469] Table 21b Selected colorectal cancer vaccine peptides for use in PolyPEPI915 compositions

[0470]

[0471] *Percentage of individuals with CD8+ T cell-specific PEPI3+ in the model population (n=433).

[0472] **Percentage of individuals with CD4+ T cell-specific PEPI4+ in normal donors (n=433).

[0473] Characterization of PolyPEP1015 Colorectal Cancer Vaccine

[0474] Tumor heterogeneity: PolyPEP1015 combination targets 8 different CTAs ( Fig.15 Based on the antigen expression rates of these 8 CTAs, AG50 = 5.22 and AG95 = 3 ( Fig.16 Patient heterogeneity: AP50 = 4.73 and AP95 = 2 (AP95 = 2) Fig.17 ). Tumor and patient heterogeneity: AGP50 = 3.16 and AGP95 = 1 (model population) ( Fig.18 ).

[0475] Example 17 Comparison of colorectal cancer vaccine peptides with competing colorectal cancer vaccines

[0476] We used the above-mentioned computer simulation clinical trial model to determine the T cell responder ratio of the CRC peptide vaccine of the prior art and the current development, and compared it with polyPEP1015 (Table 22). Our PEPI3+ test proved that the competitive vaccine can induce an immune response against one tumor antigen in a portion of subjects (2% to 77%). However, the multi-antigen (multi-PEPI) response assay for 2 competitive multi-antigen vaccines resulted in no or 2% responders. * In the case of 1 or 2, 3, 4 or 5 antigens of the vaccine composition, the percentage of responders is the ratio of subjects from the 1≥PEPI3+ model population with HLA I (CD8+T cell response). Since multi-PEPI responses are associated with clinical responses induced by tumor vaccines, it is unlikely that any competitive vaccine will show clinical benefits in 98% of patients. On the contrary, we predict multi-PEPI responses in 95% of subjects, indicating the possibility of clinical benefits in most patients.

[0477] Table 22 Predicted immune response rates of PolyPEP1015 and competing colorectal cancer vaccines

[0478]

[0479] Example 18 Ovarian cancer vaccine design and composition

[0480] Using essentially the same design approach as described in Examples 13 and 16 above, we designed ovarian cancer vaccine peptides using the PEPI3+ test.

[0481] We multiplied the frequency of expression of the reported CTAs associated with ovarian cancer (N%) by the frequency of PEPI3+ hotspots in the model population (B%) to identify T cell epitopes (9-mers) that would induce an immune response against ovarian cancer antigens in the highest proportion of individuals (Table 23). We then selected 15-mers that included each selected 9-mer (Table 23). Using the method described in Example 20 below, 15-mers were selected to bind to most HLA class II alleles in most subjects.

[0482] Table 23 BestEPI list for selecting ovarian cancer peptides for vaccine composition (underlined 9-mers). N%: frequency of antigen expression in colorectal cancer; B%: BestEPI frequency, i.e., the percentage of individuals with epitopes that bind at least 3 HLA class I molecules of the subject in the model population (433 subjects); HLAII**: the percentage of individuals with CD4+T cell-specific PEPI4+ in normal donors (n=400); N%*B%: N% multiplied by B%.

[0483]

[0484] We then designed 15 30-mer peptides (Table 24).

[0485] Table 24 30-mer ovarian cancer vaccine peptides

[0486]

[0487] *Percentage of individuals with CD8+ T cell-specific PEPI3+ in the model population (n=433).

[0488] **Percentage of individuals with CD4+ T cell-specific PEPI4+ in normal donors (n=400).

[0489] Example 19 Efficacy of the Design Approach Illustrated by the PolyPEP1018 Colorectal Cancer Vaccine

[0490] The PolyPEP1018 colorectal cancer (CRC) vaccine (PolyPEP1018) composition is a peptide vaccine intended for use as an additional immunotherapy to the standard of care CRC treatment option for patients identified as possible responders using a companion in vitro diagnostic test (CDx). Clinical trials are ongoing in the United States and Italy to evaluate PolyPEP1018 in patients with metastatic colorectal cancer. The product contains 6 peptides (6 of the 30-mer peptide PolyPEP1015 described in Examples 16 and 17) mixed with the adjuvant Montanide. These 6 peptides were selected to induce T cell responses against 12 epitopes of 7 cancer testis antigens (CTAs) most commonly expressed in CRC. These 6 peptides were optimized to induce persistent CRC-specific T cell responses. Companion diagnostics (CDx) can be used to select possible responding patients with T cell responses against multiple CTAs expressed in tumors. This example illustrates the precise process used to design PolyPEP1018. This method can be used to design vaccines for other cancers and diseases.

[0491] A. Selection of multi-antigen targets

[0492] The selection of tumor antigens is essential for the safety and efficacy of cancer vaccines. The characteristic of a good antigen is limited expression in normal tissues, thereby preventing autoimmunity. Several classes of antigens meet this requirement, including uniquely mutated antigens (e.g., p53), viral antigens (e.g., human papillomavirus antigens in cervical cancer), and differentiation antigens (e.g., CD20 expressed in B-cell lymphomas).

[0493] The inventors selected a variety of cancer testis antigens (CTAs) as target antigens because they are expressed in various types of tumor cells and testicular cells, but not in any other normal somatic tissues or cells. CTAs are ideal targets for vaccines for at least the following reasons:

[0494] Tumors with higher histological grade and later clinical stage often show higher CTA expression frequency

[0495] Only a subset of tumor cells express a certain CTA

[0496] Different cancer types show significant differences in CTA expression frequencies

[0497] Tumors that are positive for CTA often show simultaneous expression of more than one CTA

[0498] None of the CTAs appear to be cell surface antigens, so they are unique targets for cancer vaccines (they are not suitable targets for antibody-based immunotherapies)

[0499] In order to identify the target CTA of PolyPEP1018, the inventors constructed a CTA expression knowledge base. The knowledge base contains CTAs represented by CRCs graded by expression rate. Related studies conducted by the inventors (see Example 11) show that vaccines that induce CTL responses against multiple antigens expressed in tumor cells can benefit patients. Therefore, 7 CTAs with high expression rates in CRC were selected for inclusion in the development of PolyPEP1018. Details are listed in Table 25.

[0500] Table 25 Target CTAs in PolyPEP1018 CRC vaccine

[0501]

[0502] B. Precise targeting through vaccine design based on PEPI3+ biomarkers

[0503] As described above, the PEPI3+ biomarker predicts the vaccine-induced T cell response of the subject. The inventors have developed and validated a test for accurately identifying PEPIs from antigen sequences and HLA genotypes (Examples 1, 2, 3). The PEPI test algorithm is used to identify the best PEPI (bestEPI) from the 7 target CTAs included in the PolyPEP1018 CRC vaccine.

[0504] The dominant PEPIs identified using the methods described herein induced CTL responses in the highest proportion of subjects:

[0505] i. Identify all HLA class I binding PEPIs from 7 CTA targets in each of the 433 subjects in the model population;

[0506] ii. Identification of the dominant PEPIs (BestEPIs), which are the PEPIs present in the largest subpopulation.

[0507] Table 26 gives 12 dominant PEPIs derived from 7 CTAs in polyPEP1018. The PEPI% in the model population represents the proportion of 433 subjects with the indicated PEPI, i.e., the proportion of subjects in which the indicated PEPI can induce a CTL response. Although optimization steps were used during the identification process, there was a very high variability (18%-78%) in the dominant PEPIs that induced CTL responses.

[0508] Table 26 Advantages of CRC-specific HLA class I binding in PolyPEP1018

[0509]

[0510] The inventors optimized each dominant PEPI to bind to the majority of HLA class II alleles in the majority of subjects. This should enhance efficacy because it will induce CD4+ helper T cells, which can enhance CD8+ CTL responses and contribute to long-term sustained T cell responses. Figure 4 The examples shown demonstrate that PEPIs that bind ≥3 HLA class II alleles are most likely to activate helper T cells.

[0511] The 15-mer peptides selected using the methods described herein contain dominant PEPIs for HLA class I and class II binding. Therefore, these peptides can induce CTL and helper T cell responses in the highest proportion of subjects.

[0512] process:

[0513] 1. Identify the HLA class II genotypes of 400 normal donors*;

[0514] 2. Extend each 9-mer dominant PEPI on both sides with amino acids matching the source antigen (Table 20);

[0515] 3. IEDB algorithm predicted HLA class II PEPI in 400 normal donors;

[0516] 4. Select the subject 15-mer peptide with the highest proportion of HLA class II binding PEPIs;

[0517] 5. When linking two 15-mer peptides, ensure that there is one dominant HLA class II PEPI in each vaccine peptide.

[0518] Table 27 shows 12 optimized 15-mer peptides derived from 7 CTAs in PolyPEP1018. These peptides have different HLA class II binding characteristics. Despite this optimized personalized vaccine design, there is high variability (0% to 100%) in the PEPI generation ability (≥3 HLA binding) among these peptides.

[0519] Table 27 Antigen-specific HLA class II binding to PEPI in PolyPEP1018

[0520]

[0521] Compared with shorter peptides, 30-mer vaccine peptides have the following advantages:

[0522] (i) Multiple precisely selected tumor-specific immunogens: Each 30-mer contains two precisely selected cancer-specific immunogenic peptides that are capable of inducing CTL and helper T cell responses in the most relevant population (similar to the model population).

[0523] (ii) Ensure natural antigen presentation: 30-mer peptides can be considered as prodrugs: they are not biologically active themselves, but are processed into smaller peptides (9 to 15 amino acids long) so that they can be loaded into the HLA molecules of specialized antigen-presenting cells. Antigen presentation produced by long peptide vaccination reflects the physiological pathway presented in HLA class I and II molecules. In addition, long peptide processing in cells is more efficient than processing of large intact proteins.

[0524] (iii) Elimination of induced tolerance T cell responses: 9-mer peptides do not need to be processed by professional antigen presenting cells and thus bind exogenously to HLA class I molecules. Therefore, injected short peptides will bind in large quantities to HLA class I molecules on all nucleated cells with surface HLA class I. In contrast, peptides >20-mer long are processed by antigen presenting cells before binding to HLA class I. Therefore, vaccination with long peptides is less likely to lead to tolerance and will promote the desired anti-tumor activity.

[0525] (iv) Induces long-lasting T cell responses because it can stimulate helper T cell responses by binding to a variety of HLA class II molecules

[0526] (v) Practicality: GMP production, formulation, quality control and administration of a smaller number of peptides, each with all of the above characteristics, is more feasible than a larger number of peptides providing different characteristics.

[0527] Each 30-mer peptide in PolyPEP1018 consists of 2 HLA class I binding dominant PEPIs and at least one HLA class II strongly binding PEPI. Strong binding PEPIs bind to 4 HLA class II alleles in >50% of individuals. Therefore, the vaccine peptides are suitable for HLA class I and class II alleles of individual subjects in the general population (which is the relevant population for CRC vaccine design).

[0528] As described above, the high variability of HLA genotypes in subjects leads to high variability in T cell responses induced by PolyPEP1018. This proves that the co-development of CDx to determine possible responders is reasonable. PEP13+ and >2PEP13+ biomarkers can predict the immune response and clinical response of subjects vaccinated with PolyPEP1018 as described in Examples 11 and 12, respectively. These biomarkers will be used to co-develop CDx to predict possible responders to the PolyPEP1018 CRC vaccine.

[0529] Example 20 Analysis of the composition and immunogenicity of PolyPEP1018 CRC vaccine

[0530] The peptides selected for the PolyPEP1018 composition are shown in Table 28.

[0531] Table 28 Selected colorectal cancer vaccine peptides for PolyPEP1018 composition

[0532]

[0533] *Percentage of individuals with HLA class I binding PEPI3+ in the model population (n=433).

[0534] **Percentage of individuals with HLA class II binding PEPI3+ in the model population (n=433).

[0535] The peptides of PolyPEPI1018 are formulated into two mixtures, namely, MIX 1 containing the peptides of SEQ IDs: 1 30 and 131 and MIX 2 containing the peptides of SEQ IDs: 121, 124, 134 and 126. MIX 1 and MIX 2 may be administered sequentially.

[0536] Immunogenicity characterization

[0537] The inventors used the PEPI3+ test to characterize the immunogenicity of PolyPEPI018 in a cohort of 37 CRC patients with complete HLA genotype data. T cell responses to the same 9-mer peptides that will be used in clinical trials were predicted in each patient. These peptides represent 12 dominant PEP13+ in the PolyPEP1018 peptide. The 9-mer is shown in Table 26.

[0538] The specificity and sensitivity of PEPI3+ prediction depends on the actual number of HLAs predicted to bind to a specific epitope. Specifically, the inventors have determined that the probability of an HLA-restricted epitope inducing a T cell response in a subject is typically 4%, which explains the low sensitivity of the prior art prediction methods based on HLA-restricted epitope predictions. Applying the PEPI3+ method, the inventors determined the probability of PolyPEPI018 inducing T cells to respond specifically to each dominant PEPI3+ in 37 CRC patients. The results of this analysis are summarized in Table 29.

[0539] Table 29 Probability of dominant PEPIs among the six peptides of PolyPEP1018 in 37 CRC patients

[0540]

[0541]

[0542] Abbreviations: CRC = colorectal cancer; PEPI = personal epitope

[0543] Note: The percentage represents the probability of PolyPEPI1018-induced CD8+ T cell response.

[0544] In summary, these results indicate that the most immunogenic peptide in PolyPEP1018 is CRC-P8, which is predicted to bind >3HLA in most patients. The least immunogenic peptide CRC-P3 binds to >1HLA in many patients and has a 22% chance of inducing a T cell response. Since the bioassays used to detect T cell responses are not as accurate as PEPI3+, this calculation may be the most accurate characterization of T cell responses in CRC patients. Although MAGE-A8 and SPAG9 are immunogenic in the model population used for vaccine design, there is no MAGE-A8-specific PEPI3+ in 37 CRC patients, and only 1 patient (3%) has SPAG9-specific PEPI3+.

[0545] Tables 30 and 31 show further characterization of the predicted PolyPEP1018 response rates in the model population described in Example 8 and in 295 CRC patients with known HLA class I genotypes.

[0546] Table 30 PolyPEP1018 response rates in the model population (433 normal donors).

[0547]

[0548] Table 31 Response rate of PolyPEPI1018 in 295 CRC patients

[0549]

[0550]

[0551] Toxicity characterization - immunoBLAST

[0552] A method has been developed that can be performed on any antigen to determine its potential to induce a toxic immune response such as autoimmunity. The method is referred to herein as immunoBLAST. PolyPEP1018 contains 6 30-mer polypeptides. Each polypeptide consists of two 15-mer peptide fragments derived from an antigen expressed in CRC. New epitopes may be generated in the connecting region of the two 15-mer peptides and may induce an undesirable T cell response (autoimmunity) against healthy cells. This was assessed using the immunoBLAST method applied by the inventors.

[0553] 16-mer peptides were designed for each 30-mer component of PolyPEP1018. Each 16-mer contains 8 amino acids from the end of the first 15 residues of the 30-mer and 8 amino acids from the start of the second 15 residues of the 30-mer, thus accurately spanning the connecting region of the two 15-mers. These 16-mers were then analyzed using BLAST (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to identify cross-reactive regions of local similarity with human sequences, which compares protein sequences to sequence databases and calculates the statistical significance of matches. 8-mers in the 16-mer were selected as the detection length because this length represents the minimum length required for a peptide to form an epitope and is the distance between anchor points during HLA binding.

[0554] like Fig.19 As shown, the positions of amino acids in the polypeptide are numbered. The starting position of the potential 9-mer peptide that can bind to HLA and form a new epitope is 8 amino acids at positions 8–15. The starting position of the tumor antigen-derived peptide carried by the 15-mer that can form a drug-active epitope is 7+7=14 amino acids at positions 1–7 and 16–22. The proportion of potential new epitope-generating peptides is 36.4% (8 / 22).

[0555] The PEP13+ test is used to identify new epitopes and neoPEP1 in the 9-mer epitope of the connecting region. The risk of PolyPEP1018 inducing unwanted T cell responses was assessed in 433 subjects in the model population by determining the proportion of subjects with PEP1I3+ in the 9-mer of the connecting region. The results of the new epitope / neoPEPI analysis are summarized in Table 32. Among the 433 subjects in the model population, the average number of predicted epitopes that can be produced by intracellular processing is 40.12. New epitopes are often generated; 11.61 (28.9%) of the 40.12 epitopes are new epitopes. Most peptides can be identified as new epitopes, but the number of subjects presenting new epitopes is different.

[0556] The epitopes carried by PolyPEP1018 produced an average of 5.21PEP13+. These PEPIs can activate T cells in subjects. The amount of potential new PEPIs is much lower than that of new epitopes (3.7%). In some subjects, these neoPEPIs are unlikely to compete with PEPIs for T cell activation. Importantly, the activated neoPEPI-specific T cells have no targets on healthy tissues.

[0557] Table 32 Identification of potential new epitopes of PolyPEP1018

[0558]

[0559]

[0560] Abbreviations: CRC = colorectal cancer; HLA = human leukocyte antigen; PEPI = personal epitope

[0561] Each of the 30-mer peptides in PolyPEP1018 was released for clinical development because none of the 8-mers in the linker region matched any human protein other than the target CTA.

[0562] Activity / Efficacy Characterization

[0563] The inventors have developed pharmacodynamic biomarkers to predict the activity / efficacy of vaccines in individual human subjects as well as in populations of human subjects. These biomarkers accelerate the development of more effective vaccines and also reduce development costs. The inventors have the following tools:

[0564] Antigen Expression Knowledge Base: The inventors have collected data from experiments published in peer-reviewed scientific journals on tumor antigens expressed by tumor cells and organized by tumor type to create a database of CTA expression levels - the CTA Database (CTADB). As of April 2017, CTADB contains data from 145 CTAs from 41,132 tumor samples and is organized by the frequency of CTA expression in different types of cancer.

[0565] Computer simulation test population: The inventors also collected data on HLA genotypes of several different model populations. Each individual in the population has complete 4-digit HLA genotype data. The population is summarized in Table 33.

[0566] Table 33 Computer simulation test population

[0567]

[0568] Abbreviations: CRC = colorectal cancer; HLA = human leukocyte antigen

[0569] Using these tools (or perhaps equivalent databases or model populations), the following markers can be assessed:

[0570] AG95 - Vaccine efficacy: The number of antigens expressed with 95% probability by a specific tumor type in a cancer vaccine. AG95 is an indicator of vaccine efficacy and is independent of the immunogenicity of the vaccine antigens. AG95 is calculated based on tumor antigen expression rate data collected in the CTADB. Technically, AG95 is determined by the binomial distribution of the CTA, taking into account all possible variations and expression rates. In this study, AG95 is calculated by accumulating the probability of a certain number of expressed antigens with the widest range of antigens, where the sum of probabilities is less than or equal to 95%. The correct value is between 0 (95% probability of no expression expected) and the maximum number of antigens (95% probability of all antigens expressed).

[0571] · PEPI3+ count - Immunogenicity of the vaccine in the subject: Vaccine-derived PEPI3+ is a personal epitope that induces a T cell response in the subject. PEPI3+ can be determined using the PEPI3+ test in subjects with a known full 4-digit HLA genotype.

[0572] AP count - antigenicity of the vaccine in the subject: the number of vaccine antigens that have PEPI3+. Vaccines such as PolyPEP1018 contain sequences from antigens expressed in tumor cells. The AP count is the number of antigens in the vaccine that have PEPI3+. The AP count represents the number of antigens in the vaccine that can induce a T cell response in the subject. The AP count characterizes the vaccine-antigen-specific T cell response of the subject because it depends only on the subject's HLA genotype and is independent of the subject's disease, age, and drug treatment. The correct value is between 0 (no antigen presents PEPI) and the maximum number of antigens (all antigens present PEPI).

[0573] AP50 - Antigenicity of the vaccine in a population: The average number of vaccine antigens with PEPI in a population. AP50 is suitable for characterizing vaccine-antigen specific T cell responses in a given population, as it depends on the HLA genotype of the subjects in the population. Technically, AP counts are calculated in the model population, and the binomial distribution of the results is used to calculate AP50.

[0574] AGP count - Efficacy of the vaccine in a subject: the number of vaccine antigens expressed in the tumor with PEPIs. The AGP count represents the number of tumor antigens that the vaccine recognized and induced a T cell response (hit the target). The AGP count depends on the vaccine-antigen expression rate in the subject's tumor and the subject's HLA genotype. The correct value is between 0 (no PEPIs expressing antigen presentation) and the maximum number of antigens (all antigens are expressed and present PEPIs).

[0575] ·AGP50 - cancer vaccine efficacy in a population: the average number of vaccine antigens expressed in a specified tumor with PEPI (ie, AGP) in a population. AGP50 represents the average number of tumor antigens that can be identified by a vaccine-induced T cell response. AGP50 depends on the expression rate of the antigen in the tumor type shown and the immunogenicity of the antigen in the target population. AGP50 can evaluate the efficacy of vaccines in different populations and can be used to compare different vaccines in the same population. The calculation of AGP50 is similar to that for AG50, except that expression is weighted by the appearance of PEPI3+ on the vaccine antigen expressed in the subject. In a theoretical population where each subject has a PEPI from each vaccine antigen, AGP50 will be equal to AG50. In another theoretical population, where no subject has a PEPI from any vaccine antigen, AGP50 will be 0. In general, the following statements are valid: 0≤AGP50≤AG50.

[0576] mAGP - a candidate biomarker for selecting likely responders: The probability that a cancer vaccine induces a T cell response against multiple antigens expressed in a given tumor. mAGP is calculated from the expression rate of vaccine-antigens in CRC and the presentation rate of vaccine-derived PEPIs in subjects. Technically, mAGP is the sum of the probabilities of multiple AGPs (>2 AGPs) based on the AGP distribution.

[0577] Application of these markers to assess antigenicity and efficacy in individual CRC patients

[0578] Table 34 shows the antigenicity and efficacy of PolyPEP1018 in 37 CRC patients using AP and AGP50, respectively. As expected from the high variability of PolyPEP1018-specific T cell responses (see Table 29), AP and AGP50 have high variability. The most immunogenic antigen in PolyPEP1018 is FOXO39; every patient has PEPI3+. However, FOXO39 is only expressed in 39% of CRC tumors, indicating that 61% of patients will have FOXO39-specific T cell responses that do not recognize tumors. The weakest immunogenic antigen is MAGE-A8; although the antigen is expressed in 44% of CRC tumors, none of the 37 CRC patients had PEPI3+. These results indicate that antigen expression and immunogenicity can be considered when determining the efficacy of cancer vaccines.

[0579] AGP50 represents the average number of antigens expressed in CRC tumors with PEPI. Patients with higher AGP50 values ​​are more likely to respond to PolyPEP1018, as higher AGP50 values ​​indicate that the vaccine can induce T cell responses against more antigens expressed in CRC cells.

[0580] The last column in Table 34 shows the probability of mAGP (multiple AGPs; i.e., at least 2 AGPs) in each of the 37 CRC patients. The average mAGP in CRC patients was 66%, indicating that the probability of CRC patients inducing T cell responses against multiple antigens expressed in tumors was 66%.

[0581] Table 34 Antigenicity (AP counts), potency (AGP50 counts) and mAGP of PolyPEP1018 in 37 CRC patients

[0582]

[0583]

[0584] Abbreviations: CRC = colorectal cancer; PEPI = personal epitope; CTA = cancer testis antigen; AP = expressed antigen with ≥1 PEPI

[0585] These biomarkers have immediate utility in vaccine development and routine clinical practice as they do not require invasive biopsies. Antigen expression data can be obtained from obtained tumor samples and organized in a database. 4-digit HLA genotyping can be performed from saliva samples. It is a validated test for transplantation and paternity testing performed by certified laboratories worldwide. These assessments will allow drug developers and physicians to gain greater insight into the immunogenicity and activity of tumor responses and the potential emergence of resistance.

[0586] Application of these markers to assess antigenicity and efficacy of PolyPEPI1018 in populations

[0587] Antigenicity of PolyPEP1018 CRC vaccine in the general population

[0588] The antigenicity of PolyPEP1018 in the subjects was determined by AP counts, which indicate the number of vaccine antigens that induced a T cell response in the subjects. The AP count of PolyPEP1018 was determined in each of the 433 subjects in the model population using the PEPI test, and then the AP50 count of the model population was calculated.

[0589] like Fig. 20 As shown, the AP50 of PolyPEP1018 in the model population is 3.62. Therefore, the average number of immunogenic antigens (i.e., antigens with ≥1 PEPI) in PolyPEP1018 in the general population is 3.62.

[0590] Efficacy of PolyPEP1018 CRC vaccine in the general population

[0591] If the PEPI in the vaccine is presented by tumor cells, the vaccine-induced T cells can recognize and kill the tumor cells. The number of AGPs (expressed antigens with PEPI) is an indicator of vaccine efficacy in an individual and depends on the potency and antigenicity of PolyPEP1018. Fig.21 As shown, in the model population, the average number of immunogenic CTAs (ie, APs [expressed antigens with ≥1 PEPI]) in PolyPEP1018 was 2.54.

[0592] The probability of PolyPEP1018 inducing T cell responses against multiple antigens (ie, mAGP) in subjects in the model population was 77%.

[0593] Comparison of the activity of PolyPEP1018 CRC vaccine in different populations

[0594] Table 35 shows the comparison of immunogenicity, antigenicity and efficacy of PolyPEP1018 in different populations.

[0595] Table 35 Comparison of immunogenicity, antigenicity and efficacy of PolyPEP1018 in different subpopulations

[0596]

[0597] Abbreviations: CRC = colorectal cancer; PEPI = personal epitope; SD = standard deviation; AP = expressed antigen with ≥1 PEPI

[0598] The average of PEPI3+ and AP results showed that PolyPEPI018 was highly immunogenic and antigenic in all populations; PolyPEP1018 induced an average of 3.7–6.0 CRC-specific T cell clones against 2.9–3.7 CRC antigens. PolyPEP1018 immunogenicity was similar in CRC patients and the average population (p>0.05), and this similarity may be due to the small sample size of the CRC population.

[0599] Example 21 Personalized immunotherapy composition for treating ovarian cancer

[0600] This example describes the treatment of ovarian cancer patients with personalized immunotherapy compositions, wherein the compositions are specifically designed for the patients based on their HLA genotypes based on the disclosures described herein. This example and Example 22 below provide clinical data to support the principle of inducing cytotoxic T cell responses on multiple HLA binding epitopes in a subject, based on the disclosures described herein.

[0601] The HLA class I and class II genotypes of patient XYZ with metastatic ovarian adenocarcinoma were determined from a saliva sample.

[0602] To prepare a personalized medicine composition for patient XYZ, 13 peptides were selected, each of which met the following two criteria: (i) derived from an antigen expressed in ovarian cancer, as reported in a peer-reviewed scientific publication; and (ii) comprising a fragment of a T cell epitope capable of binding to at least three HLA class I molecules of patient XYZ (Table 36). In addition, each peptide was optimized to bind to the maximum number of HLA class II molecules of the patient.

[0603] Table 36 Personalized vaccine for ovarian cancer patient XYZ

[0604] Vaccine for XYZ Target Antigen Antigen expression 20-mer peptide Maximum number of HLA class I Maximum number of HLA class II POC01_P1 AKAP4 89％ NSLQKQLQAVLQWIAASQFN 3 5 POC01_P2 BORIS 82％ SGDERSDEIVLTVSNSNVEE 4 2 POC01_P3 SPAG9 76％ VQKEDGRVQAFGWSLPQKYK 3 3 POC01_P4 OY-TES-1 75％ EVESTPMIMENIQELIRSAQ 3 4 POC01_P5 SP17 69％ AYFESLLEKREKTNFDPAEW 3 1 POC01_P6 WT1 63％ PSQASSGQARMFPNAPYLPS 4 1 POC01_P7 HIWI 63％ RRSIAGFVASINEGMTRWFS 3 4 POC01_P8 PRAME 60％ MQDIKMILKMVQLDSIEDLE 3 4 POC01_P9 AKAP-3 58％ ANSVVSDMMVSIMKTLKIQV 3 4 POC01_P10 MAGE-A4 37％ REALSNKVDELAHFLLRKYR 3 2 POC01_P11 MAGE-A9 37％ ETSYEKVINYLVMLNAREPI 3 4 POC01_P12a MAGE-A10 52％ DVKEVDPTGHSFVLVTSLGL 3 4 POC01_P12b BAGE 30％ SAQLLQARLMKEESPVVSWR 3 2

[0605] According to the validation of the PEPI test shown in Table 3, the 11 PEPI3 peptides in the immunotherapy composition can induce T cell responses in XYZ with a probability of 84%, and the two PEPI4 peptides (POC01-P2 and POC01-P5) induce T cell responses with a probability of 98%. The T cell response targets 13 antigens expressed in ovarian cancer. The expression of these cancer antigens in patient XYZ was not tested. Instead, the probability of successful killing of cancer cells was determined based on the probability of antigen expression in the patient's cancer cells and the positive predictive value of ≥1 PEPI3+ test (AGP count). The AGP count predicts the effectiveness of the vaccine in the subject: the number of vaccine antigens expressed in the patient's tumor (ovarian adenocarcinoma) with PEPI. The AGP count represents the number of tumor antigens that the vaccine identifies and induces a T cell response (hitting the target) against the patient's tumor. The AGP count depends on the vaccine-antigen expression rate in the subject's tumor and the subject's HLA genotype. The correct value must be between 0 (no antigen-presenting PEPI is expressed) and the maximum number of antigens (all antigens are expressed and present PEPI).

[0606] The probability that patient XYZ will express one or more of the 12 antigens is Fig. 22 As shown. AGP95=5, AGP50=7.9, mAGP=100%, AP=13.

[0607] The pharmaceutical composition for patient XYZ can be composed of at least 2 of the 13 peptides (Table 36), because it is determined that presenting at least two polypeptide fragments (epitopes) that can bind to at least three HLAs (≥2 PEPI3+) of an individual in a vaccine or immunotherapy composition can predict clinical response. The above peptides are synthesized and dissolved in a pharmaceutically acceptable solvent and mixed with an adjuvant before injection. It is desirable that the patient receive personalized immunotherapy with at least two peptide vaccines, but it is more preferred to increase the likelihood of killing cancer cells and reduce the chance of recurrence.

[0608] To treat patient XYZ, 12 peptides were formulated into 4 x 3 / 4 peptides (POC01 / 1, POC01 / 2, POC01 / 3, POC01 / 4). One treatment cycle was defined as administration of all 13 peptides within 30 days.

[0609] Patient history:

[0610] Diagnosis: Metastatic ovarian adenocarcinoma

[0611] Age: 51

[0612] Family history: Colon and ovarian cancer (mother); breast cancer (grandmother)

[0613] Tumor pathology: BRCA1-185delAG, BRAF-D594Y, MAP2K1-P293S, NOTCH1-S2450N

[0614] 2011: First diagnosis of ovarian adenocarcinoma; Wertheim procedure and chemotherapy; lymph node dissection

[0615] 2015: Pericardial fat tissue transfer, resection

[0616] 2016: Liver metastases

[0617] 2017: Retroperitoneal and mesenteric lymphadenopathy; early peritoneal cancer with a small amount of ascites

[0618] Previous treatment:

[0619] 2012: Paclitaxel-Carboplatin (6×)

[0620] 2014: Kailai-Carboplatin (1×)

[0621] 2016-2017 (9 months): Lymparza (olaparib) 2 x 400 mg / day, oral

[0622] 2017: Hycamtin infusion 5 x 2.5 mg (3x a series / month)

[0623] PIT vaccine treatment started on April 21, 2017.

[0624] Table 37 Patient XYZ peptide treatment regimen

[0625]

[0626] Patients' tumor MRI findings (baseline April 15, 2016):

[0627] Disease is primarily confined to the liver and lymph nodes. Use of MRI limits detection of metastases to the lungs (pulmonary)

[0628] May 2016 to January 2017: Olaparib treatment

[0629] December 25, 2016 (before PIT vaccine treatment): Significant reduction in tumor burden with confirmation of the response obtained at FU2

[0630] January to March 2017 - TOPO (topoisomerase)

[0631] April 6, 2017: FU3 demonstrated regrowth of existing lesions and appearance of new lesions leading to disease progression

[0632] ·PIT started on April 21, 2017

[0633] July 21, 2017 (after the second cycle of PIT): FU4 showed continued growth of the lesion, general enlargement of the pancreas, abnormal parapancreatic signals, and increased ascites

[0634] July 26, 2017 – CBP + Gem + Avastin

[0635] On September 20, 2017 (after 3 cycles of PIT), FU5 showed reversal of lesion growth and improved pancreatic / parapancreatic signals. The results suggest pseudoprogression

[0636] On November 28, 2017 (after 4 cycles of PIT), FU6 showed the best response with elimination of non-target lesions

[0637] The MRI data of patient XYZ are shown in Table 38 and Fig.23 shown.

[0638] Table 38 Summary of lesion responses

[0639]

[0640] Example 22 Design of personalized immunotherapy composition for treating breast cancer

[0641] Determine the HLA class I and class II genotypes of metastatic breast cancer patient ABC from a saliva sample. In order to prepare a personalized pharmaceutical composition for patient ABC, select 12 peptides, each of which meets the following two criteria: (i) derived from antigens expressed in breast cancer, as reported in peer-reviewed scientific publications; and (ii) comprising a fragment of a T cell epitope of at least three HLA class I molecules that can bind to patient ABC (Table 39). In addition, optimize each peptide to bind to the maximum number of HLA class II of the patient. These 12 peptides target 12 breast cancer antigens. The probability that patient ABC expresses one or more of the 12 antigens is as follows: Fig.24 shown.

[0642] Table 39: 12 peptides for the treatment of breast cancer patients

[0643]

[0644]

[0645] Predicted efficacy: AGP95 = 4; the probability that the PIT vaccine induces a CTL response against the four CTAs expressed in BRC09 breast cancer cells is 95%. Other efficacy parameters: AGP50 = 6.3, mAGP = 100%, AP = 12.

[0646] Efficacy detected after the first vaccination with all 12 peptides: 83% reduction in tumor metabolic activity (PET CT data).

[0647] To treat patient ABC, the 12 peptides were formulated into 4 x 3 peptides (PBR01 / 1, PBR 01 / 2, PBR 01 / 3, PBR01 / 4). One treatment cycle was defined as administration of all 12 different peptide vaccines within 30 days.

[0648] Patient history:

[0649] Diagnosis: Bilateral metastatic breast cancer: the right breast is ER positive, PR negative, and Her2 negative; the left breast is ER, PR, and Her2 negative.

[0650] First diagnosis: 2013 (4 years before PIT vaccine treatment)

[0651] 2016: Extensive metastatic disease with involvement of supra- and infra-diaphragmatic lymph nodes. Multiple liver and lung metastases.

[0652] 2016–2017 Treatment: Etrozole, Ibance, and Zoladex

[0653] result:

[0654] March 7, 2017: Previous PIT vaccine treatment

[0655] Multifocal metastatic disease of the liver with true external compression of the common bile duct origin and massive dilatation of the intrahepatic bile ducts. Abdominal, hilar, and retroperitoneal adenopathy

[0656] May 26, 2017: 1 PIT cycle later

[0657] Efficacy tested: 8 tumor metabolic activity (PET CT) liver, lung lymph nodes and other metastases decreased by 3%. Safety tested: Skin reactions

[0658] Local inflammation at the injection site within 48 hours after vaccine administration

[0659] Follow-up:

[0660] BRC-09 was treated with 5 cycles of PIT vaccine. She felt well and she refused a PET CT scan in September 2017. She became symptomatic in November and a PET CT scan showed progressive disease, but she refused all treatment. In addition, her oncologist found that she had stopped taking Pablocyclib since the spring and summer. Patient ABC died in January 2018.

[0661] The combination of Pablocyclib and personalized vaccines may be responsible for the significant early responses observed after vaccine administration. Palbocyclib has been shown to improve the activity of immunotherapy by increasing CTA presentation of HLAs and reducing the proliferation of Tregs: (Goel et al. Nature. 2017: 471-475). PIT vaccines can be used as an add-on to existing technology treatments to achieve maximum efficacy.

[0662] Example 23 Personalized immunotherapy composition for treating patients with advanced metastatic breast cancer

[0663] Patient BRC05 was diagnosed with right-sided inflammatory breast cancer with extensive lymphangiogenic carcinoma. Inflammatory breast cancer (IBC) is a rare but aggressive form of locally advanced breast cancer. It is called inflammatory breast cancer because its main symptoms are swelling and redness (the breast often looks inflamed). Most inflammatory breast cancers are invasive ductal carcinomas (starting in the milk ducts). This type of breast cancer is associated with the expression of oncoproteins of high-risk human papillomaviruses. In fact, HPV16 DNA was diagnosed in this patient's tumor.

[0664] Patient phase 2011 (6 years before PIT vaccine treatment)

[0665] T4: Tumor of any size that extends directly to the chest wall and / or skin (ulcer or skin nodule)

[0666] pN3a: metastasis to ≥10 axillary lymph nodes (at least 1 tumor deposit ≥2.0 mm); or metastasis to the subclavian (level III axillary lymph node) nodes.

[0667] 14 vaccine peptides were designed and prepared for patient BRC05 (Table 40). Peptides PBRC05-P01-P10 were prepared for this patient based on population expression data. The last 3 peptides in Table 40 (SSX-2, MORC, MAGE-B1) were designed from antigens whose expression was measured directly in the patient's tumor.

[0668] Table 40 Vaccine peptides of patient BRC05

[0669]

[0670]

[0671] Note: Bold red indicates CD8 PEPI and underlined indicates the best binding CD4 allele.

[0672] T cell responses were detected in peripheral mononuclear cells 2 weeks after the first immunization with the mixture of peptides PBRC05_P1, PBRC05_P2, PBRC05_P3, PBRC05_P4, PBRC05_P5, PBRC05_P6, PBRC05_P7.

[0673] Table 41 Antigen-specific T cell responses: number of spots / 300,000 PBMCs

[0674] antigen Stimulants Experience 1 Experience 2 average value SPAG9 PBRC05_P1 2 1 1.5 AKAP4 PBRC05_P2 11 4 7.5 MAGE-A11 PBRC05_P3 26 32 29 NY-SAR-35 PBRC05_P4 472 497 484.5 FSIP1 PBRC05_P5 317 321 319 NY-BR-1 PBRC05_P6 8 12 10 MAGE-A9 PBRC05_P7 23 27 25 none Negative control (DMSO) 0 3 1.5

[0675] The results showed that a single immunization with 7 peptides induced effective T cell responses against 3 of the 7 peptides, demonstrating effective MAGE-A11, NY-SAR-35, FSIP1, and MAGE-A9 specific T cell responses. There were weak responses to AKAP4 and NY-BR-1, and no response to SPAG9.

[0676] References

[0677] 1 Bagarazzi etal. Immunotherapy against HPV16 / 18 generates potentTH1and cytotoxic cellular immune responses. Science Translational Medicine. 2012; 4(155):155ra138.

[0678] 2 Gudmundsdotter et al. Amplified antigen-specific immune responses in HIV-1 infected individuals in a double blind DNA immunization and therapyinterruption trial. Vaccine. 2011; 29(33):5558-66.

[0679] 3Bioley et al.HLA class I-associated immunodominance affects CTLresponsiveness to an ESO recombinant protein tumor antigen vaccine.ClinCancer Res.2009;15(1):299-306.

[0680] 4 Valmori et al.Vaccination with NY-ESO-1protein and CpG in Montanideinduces integrated antibody / Th1 responses and CD8 Tcells through cross-priming.Proceedings of the National Academy of Sciences of the United Statesof America.2007;104(21):8947-52.

[0681] 5 Yuan et al.Integrated NY-ESO-1antibody and CD8+T-cell responsescorrelate with clinical benefit in advanced melanoma patients treated withipilimumab.Proc Natl Acad Sci U S A.2011;108(40):16723-16728.

[0682] 6 Kakimi et al.A phase I study of vaccination with NY-ESO-1f peptidemixed with Picibanil OK-432 and Montanide ISA-51 in patients with cancersexpressing the NY-ESO-1antigen.Int J Cancer.2011;129(12):2836-46.

[0683] 7Wada et al. Vaccination with NY-ESO-1 overlapping peptides mixed with Picibanil OK-432 and montanide ISA-51 in patients with cancers expressing the NY-ESO-1 antigen. J Immunother. 2014;37(2):84-92.

[0684] 8 Welters et al. Induction of tumor-specific CD4+ and CD8+ T-cell immunity in cervical cancer patients by a human papillomavirus type 16 E6 and E7 long peptides vaccine. Clin. Cancer Res. 2008;14(1):178-87.

[0685] 9 Kenter et al. Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia. N Engl J Med. 2009;361(19):1838-47. 1 0 Welters etal. Success or failure of vaccination for HPV16-positive vulvar lesions correlates with kinetics and phenotype of induced T-cell responses. PNAS. 2010;107(26):11895-9.

[0686] 11 http: / / www.ncbi.nlm.nih.gov / projects / gv / mhc / main.fcgi?cmd=init The MHC database, NCBI. (Accessed March 7, 2016)

[0687] 12 Karkada et al. Therapeutic vaccines and cancer: focus on DPX-0907. Biologics. 2014;8:27-38.

[0688] 13 Butts et al.Randomized phase IIB trial of BLP25 liposome vaccine instage IIIB and IV non-small-cell lung cancer.J Clin Oncol.2005;23(27):6674-81.

[0689] 14 Yuan et al.Safety and immunogenicity of a human and mouse gp100 DNAvaccine in a phase I trial of patients with melanoma.Cancer Immun.2009;9:5.

[0690] 15 Kovjazin et al.ImMucin:a novel therapeutic vaccine with promiscuousMHC binding for the treatment of MUC1-expressing tumors.Vaccine.2011;29(29-30):4676-86.

[0691] 16 Cathcart et al.Amultivalent bcr-abl fusion peptide vaccination trialin patients with chronic myeloid leukemia.Blood.2004;103:1037-1042.

[0692] 17 Chapuis et al.Transferred WT1-reactive CD8+T cells can mediateantileukemic activity and persist in post-transplant patients.Sci TranslMed.2013;5(174):174ra27.

[0693] 18Keilholz et al.A clinical and immunologic phase 2trial of Wilmstumor gene product 1(WT1)peptide vaccination in patients with AML andMDS.Blood;2009;113(26):6541-8.

[0694] 19 Walter et al.Multipeptide immune response to cancer vaccine IMA901after single-dose cyclophosphamide associates with longer patientsurvival.NatMed.2012;18(8):1254-61.

[0695] 20 Phuphanich et al.Phase I trial of a multi-epitope-pulsed dendriticcell vaccine for patients with newly diagnosed glioblastoma.Cancer ImmunolImmunother.2013;62(1):125-35.

[0696] 21 Kantoff et al.Overall survival analysis of a phase II randomizedcontrolled trial of a Poxviral-based PSA-targeted immunotherapy in metastaticcastration-resistant prostate cancer.J Clin Oncol.2010;28(7):1099-105.

[0697] 22 Tagawa et al.Phase I study of intranodal delivery of a plasmid DNAvaccine for patients with Stage IV melanoma.Cancer.2003;98(1):144-54.

[0698] 23 Slingluff et al.Randomized multicenter trial of the effects ofmelanoma-associated helper peptides and cyclophosphamide on theimmunogenicity of a multipeptide melanoma vaccine.J Clin Oncol.2011;29(21):2924-32.

[0699] 24 Kaida et al.Phase 1trial of Wilms tumor 1(WT1)peptide vaccine andgemcitabine combination therapy in patients with advanced pancreatic orbiliary tract cancer.J Immunother.2011;34(1):92-9.

[0700] 25 Fenoglio etal.A multi-peptide,dual-adjuvant telomerase vaccine(GX301)is highly immunogenic in patients with prostate and renalcancer.Cancer Immunol Immunother;2013;62:1041–1052.

[0701] 26 Krug et al.WT1 peptide vaccinations induce CD4 and CD8 T cell immuneresponses in patients with mesothelioma and non-small cell lungcancer.CancerImmunol Immunother;2010;59(10):1467-79.

[0702] 27Slingluff et al. Clinical and immunologic results of a randomized phase II trial of vaccination using four melanoma peptides either administered in granulocyte-macrophage colony-stimulating factor in adjuvant or pulsed on dendritic cells. J Clin Oncol;2003;21(21):4016-26.

[0703] 28 Hodi et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med;2010;363(8):711-23. 29 Carmon et al. Phase I / II study exploring ImMucin, a pan-major histocompatibility complex, anti-MUC1 signal peptide vaccine, in multiple myeloma patients. Br J Hematol. 2014;169(1):44-56.

[0704] 30 http: / / www.merckgroup.com / en / media / extNewsDetail.html?newsId=EB4A4 6A2AC4A52E7C1257AD9001F3186&newsT ype=1 (Accessed on March 28, 2016)

[0705] 31Trimble etal.Safety,efficacy,and immunogenicity of VGX-3100,atherapeutic synthetic DNAvaccine targeting human papillomavirus 16and 18E6and E7 proteins for cervical intraepithelial neoplasia 2 / 3:a randomised,double-blind,placebo-controlled phase 2b trial.Lancet.2015;386(10008):2078-88.

[0706] 32 Cusi et al.Phase I trial of thymidylate synthase poly epitopepeptide(TSPP)vaccine in advanced cancer patients.Cancer Immunol Immunother;2015;64:1159–1173.

[0707] 33 Asahara etal.Phase I / II clinical trial using HLA-A24-restrictedpeptide vaccine derived from KIF20A for patients with advanced pancreaticcancer.J Transl Med;2013;11:291.

[0708] 34 Yoshitake etal.Phase II clinical trial of multiple peptidevaccination for advanced head and neckcancer patients revealed induction ofimmune responses and improved OS.Clin Cancer Res;2014;21(2):312-21.

[0709] 35Okuno etal.Clinical Trial of a 7-Peptide Cocktail Vaccine with OralChemotherapy for Patients with Metastatic Colorectal Cancer.Anticancer Res;2014;34:3045-305.

[0710] 36 Rapoport et al.Combination Immunotherapy after ASCT for MultipleMyeloma Using MAGE-A3 / Poly-ICLC Immunizations Followed by Adoptive Transferof Vaccine-Primed and Costimulated Autologous T Cells.Clin Cancer Res;2014;20(5):1355–1365.

[0711] 37 Greenfield et al.A phase I dose-escalation clinical trial of apeptidebased human papillomavirus therapeutic vaccine with Candida skin testreagent as a novel vaccine adjuvant for treating women with biopsy-provencervical intraepithelial neoplasia2 / 3.Oncoimmunol;2015;4:10,e1031439.

[0712] 38 Snyder et al.Genetic basis for clinical response to CTLA-4blockadein melanoma.N Engl J Med.2014;371(23):2189-99.

[0713] 39Van Allen et al.Genomic correlates of response to CTLA-4blockade inmetastatic melanoma.Science;2015;350:6257.

[0714] 40 Li et al.Thrombocytopenia caused by the development of antibodies tothrombopoietin.Blood;2001;98:3241-3248

[0715] 41 Takedatsu et al.Determination of Thrombopoietin-Derived PeptidesRecognized by Both Cellular and Humoral Immunities in Healthy Donors andPatients with Thrombocytopenia.2005;23(7):975-982

[0716] 42 Eisenhauer et al.New response evaluation criteria in solid tumors:revised RECIST guideline(version 1.1).Eur J Cancer;2009;45(2):228-47.

[0717] 43 Therasse etal.New guidelines to evaluate the response to treatmentin solid tumors:European Organization for Research and Treatment of Cancer,National Cancer Institute of the United States,National Cancer Institute ofCanada.J Natl Cancer Inst;2000;92:205-216.

[0718] 44Tsuchida&Therasse.Response evaluation criteria in solid tumors(RECIST):New guidelines.Med Pediatr Oncol.2001;37:1-3.

[0719] 45 Durie et al.International uniform response criteria for multiplemyeloma.Leukemia;2006;20:1467–1473.

Claims

1. A polypeptide comprising a fragment of up to 50 consecutive amino acids of: (a) a colorectal cancer-associated antigen selected from the group consisting of TSP50, EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN, LEMD1, MAGE-A8, MAGE-A6 and MAGE-A3, wherein the fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 21 to 40 and 234 to 250; (b) an ovarian cancer-associated antigen selected from the group consisting of PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN and AKAP-3, wherein the fragment comprises the amino acid sequence of any one of SEQ ID NOs: 272 to 301; and / or (c) a breast cancer-associated antigen selected from the group consisting of PIWIL-2, AKAP-4, EpCAM, BORIS, HIWI, SPAG9, PLU-1, TSGA10, ODF-4, SP17, RHOXF-2, PRAME, NY-SAR-35, MAGE-A9, NY-BR-1, SURVIVIN, MAGE-A11, HOM-TES-85 and NY-ESO-1, wherein the fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 1 to 20, 24 and 172 to 194; Optionally, wherein the fragment is flanked at the N-terminus and / or C-terminus by additional amino acids that are not part of the breast cancer, ovarian cancer or colorectal cancer associated antigen sequence.

2. The polypeptide of claim 1, wherein the polypeptide a. is a fragment of a colorectal cancer-associated antigen selected from TSP50, EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN, MAGE-A8, MAGE-A6, MAGE-A3 and LEMD1, wherein the fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 21 to 40 and 234 to 250; or b. comprising or consisting of two or more fragments of one or more colorectal cancer associated antigens selected from TSP50, EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN, MAGE-A8, MAGE-A6, MAGE-A3 and LEMD1, wherein each fragment comprises a different amino acid sequence selected from any one of SEQ ID NOs: 21 to 40 and 234 to 250, optionally wherein the fragments are arranged overlappingly or end to end in the polypeptide; or c. is a fragment of an ovarian cancer-associated antigen selected from PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN and AKAP-3, wherein the fragment comprises an amino acid sequence selected from any one of SEQ ID NOs: 272 to 301; or d. comprising or consisting of two or more fragments of one or more ovarian cancer-associated antigens selected from PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN and AKAP-3, wherein Each fragment comprises a different amino acid sequence selected from any one of SEQ ID NOs: 272 to 301, optionally wherein the fragments are arranged overlapping or end-to-end in the polypeptide; or e. A fragment of a breast cancer-associated antigen selected from SPAG9, AKAP-4, BORIS, NY-SAR-35, NY-BR-1, SURVIVIN, MAGE-A11, PRAME, MAGE-A9, HOM-TES-85, PIWIL-2, EpCAM, HIWI, PLU-1, TSGA10, ODF-4, SP17, RHOXF-2, wherein the fragment comprises an amino acid sequence of any one of SEQ ID NOs: 1 to 20, 24 and 172 to 194; or f. comprising or consisting of two or more fragments of one or more breast cancer-associated antigens selected from SPAG9, AKAP-4, BORIS, NY-SAR-35, NY-BR-1, SURVIVIN, MAGE-A11, PRAME, MAGE-A9, HOM-TES-8, PIWIL-2, EpCAM, HIWI, PLU-1, TSGA10, ODF-4, SP17, RHOXF-2, wherein Each fragment comprises a different amino acid sequence selected from any one of SEQ ID NOs: 1 to 20, 24 and 172 to 194; optionally, wherein the fragments are arranged overlappingly or end-to-end in the polypeptide.

3. The polypeptide according to claim 1 or 2, wherein the polypeptide comprises or consists of fragments of at least two different cancer-associated antigens, wherein The cancer-associated antigen is selected from: (a) TSP50, EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN, MAGE-A8, MAGE-A6, MAGE-A3 and LEMD1; (b) PIWIL-4, WT1, EpCAM, BORIS, AKAP-4, OY-TES-1, SP17, PIWIL-2, PIWIL-3, SPAG9, PRAME, HIWI, SURVIVIN and AKAP-3; and / or (c) SPAG9, AKAP-4, BORIS, NY-SAR-35, NY-BR-1, SURVIVIN, MAGE-A11, PRAME, MAGE-A9, HOM-TES-8, PIWIL-2, EpCAM, HIWI, PLU-1, TSGA10, ODF-4, SP17, RHOXF-2; Each fragment comprises a different amino acid sequence selected from SEQ ID NOs: 21 to 40 and 234 to 250; SEQ ID NOs: 272 to 301; and / or SEQ ID NOs: 1 to 20, 24 and 172 to 194.

4. The polypeptide according to any one of claims 1 to 3, comprising or consisting of one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 41 to 80, 251 to 271, 302 to 331 and 196 to 233.

5. The polypeptide according to any one of claims 1 to 4, comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 81 to 142, 332 to 346 and 435 to 449.

6. A panel of two or more polypeptides as described in any one of claims 1 to 5, wherein (a) each polypeptide comprises a different amino acid sequence selected from the group consisting of SEQ ID NOs: 21 to 40 and 234 to 250; or (b) each polypeptide comprises a different amino acid sequence selected from the group consisting of SEQ ID NOs: 272 to 301; or (c) each peptide comprises a different amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 20, 24 and 172 to 194; or (c) each peptide comprises a different amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 40, 234 to 250, 272 to 301 and 172 to 194.

7. The polypeptide panel according to claim 6, comprising six peptides having the amino acid sequences of SEQ ID NOs: 130, 121, 131, 124, 134, 126.

8. A pharmaceutical composition or kit comprising one or more polypeptides as described in any one of claims 1 to 5, or a panel of polypeptides as described in claim 6 or 7, or polypeptides comprising at least two amino acid sequences selected from SEQ ID NOs: 21 to 40 and 234 to 250; SEQ ID NOs: 272 to 301; and / or SEQ ID NOs: 1 to 20, 24 and 172 to 194 as active ingredients.

9. A method of providing immunotherapy or vaccination for inducing a cytotoxic T cell response in a subject, the method comprising administering to the subject the pharmaceutical composition of claim 8.

10. The method of claim 9, which is a method of treating cancer, optionally colorectal cancer, ovarian cancer or breast cancer.

11. A method for identifying a human subject who is likely to have a cytotoxic T cell response to administration of the pharmaceutical composition of claim 8, the method comprising: (i) determining that the active ingredient polypeptide of the pharmaceutical composition comprises a T cell epitope sequence capable of binding to at least three HLA class I molecules of the subject; as well as (ii) identifying the subject as likely to have a cytotoxic T cell response to administration of the pharmaceutical composition.

12. The method of claim 11, further comprising using population expression data for each antigen, the antigen (a) Selected from TSP50, EpCAM, SPAG9, CAGE1, FBXO39, SURVIVIN, LEMD1, MAGE-A8, MAGE-A6, MAGE-A3, PIWIL-4, WT1, BORIS, AKAP-4, OY-TES-1, SP17, P and (b) comprises an amino acid sequence, wherein the amino acid sequence is: i. a fragment of the active ingredient peptide of the pharmaceutical composition; and ii. a T cell epitope capable of binding to at least three HLA class I molecules of the subject, To determine the likelihood that the subject will have a cytotoxic T cell response targeted to one or more polypeptide antigens expressed by cancer cells of the subject.

13. A method for identifying a subject who may be clinically responsive to the treatment method of claim 10, the method comprising (i) determining that the active ingredient polypeptide of the pharmaceutical composition comprises two or more different amino acid sequences, each of which is a. a T cell epitope capable of binding to at least three HLA class I molecules of the subject; and b. a fragment of a cancer associated antigen expressed by a cancer cell of the subject, optionally wherein the cancer associated antigen is present in a sample obtained from the subject; and (ii) identifying said subject as being likely to be clinically responsive to said treatment method.

14. A method of determining the likelihood that a particular human subject will have a clinical response to the treatment method of claim 10, wherein one or more of the following factors corresponds to a higher likelihood of a clinical response: (a) a higher number of amino acid sequences and / or different amino acid sequences are present in the active ingredient polypeptide, each of which is a T-cell epitope capable of binding to at least three HLA classes I of the subject; (b) a higher number of target polypeptide antigens, comprising at least one amino acid sequence, wherein the amino acid sequence A. contained in the active ingredient polypeptide; B. is a T cell epitope capable of binding to at least three HLA classes I of the subject; Optionally, wherein the target polypeptide antigen is expressed in the subject; further optionally, wherein the target polypeptide antigen is present in one or more samples obtained from the subject; (c) the subject has a higher probability of expressing the target polypeptide antigen, optionally a threshold number of the target polypeptide antigen and / or optionally the target polypeptide antigen has been determined to comprise at least one amino acid sequence that is A. contained in the active ingredient polypeptide; and B. is a T cell epitope capable of binding to at least three HLA classes I of the subject; and / or (d) predicting that the subject expresses a higher amount of the target polypeptide antigen, optionally the subject expresses a higher amount of the target polypeptide antigen with a threshold probability, and / or optionally having determined that the target polypeptide antigen comprises at least one amino acid sequence, wherein the amino acid sequence is A. contained in the active ingredient polypeptide; B. is a T-cell epitope capable of binding to at least three HLA class I cells of the subject.

15. The method of claim 14, wherein the method comprises (i) identifying a polypeptide antigen targeted by the active ingredient polypeptide comprising an amino acid sequence, wherein the amino acid sequence A. contained in the active ingredient polypeptide; B. is a T cell epitope capable of binding to at least three HLA class I of the subject; (ii) using the population expression data for each antigen identified in step (i) to determine a probability that the subject expresses one or more antigens identified in step (i), wherein the one or more antigens together comprise at least two different amino acid sequences of step (i); and (iii) determining the probability that the subject will have a clinical response to administration of the pharmaceutical composition, kit or polypeptide panel, wherein a higher probability determined in step (ii) corresponds to a more likely clinical response.

16. The method of claim 15, wherein the at least two different amino acid sequences are contained in the amino acid sequences of two different polypeptide antigens targeted by the active ingredient polypeptide.

17. The method of any one of claims 13 to 16, further comprising selecting or recommending administration of the pharmaceutical composition as a treatment method for the subject, and optionally further treating the subject by administering the pharmaceutical composition.

18. The method of treatment of claim 10, wherein the subject is identified as likely to have a clinical response or as having a minimal likelihood of having a clinical response to the treatment that exceeds a threshold by the method of any one of claims 13 to 16.

19. The method of any one of claims 9, 10, 17, and 18, wherein the therapeutic agent is administered in combination with a chemotherapeutic agent, a targeted therapeutic agent, or a checkpoint inhibitor.

20. A method for identifying a human subject who is likely not to clinically respond to the treatment method of claim 10, the method comprising (i) determining that the active ingredient peptide of the pharmaceutical composition does not contain two or more different amino acid sequences, each of which is a T cell epitope capable of binding to at least three HLA class I molecules of the subject; and (iii) identifying that the subject is likely not to respond clinically to treatment.

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