Combination therapy using neoantigen vaccines

Abstract

The present invention provides vaccines or immunogenic compositions administered in combination with other agents, such as checkpoint blocking inhibitors, for treating or preventing tumors in a subject.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 368,963, filed on July 20, 2022, which is incorporated herein by reference in its entirety. Background Art

[0003] Cancer immunotherapy is the use of the immune system to treat cancer. Immunotherapy takes advantage of the fact that cancer cells often have molecules on their surface that can be detected by the immune system, called tumor antigens, which are usually proteins or other macromolecules (e.g., carbohydrates). Active immunotherapy instructs the immune system to attack tumor cells by targeting tumor antigens. Passive immunotherapy enhances existing anti-tumor responses and includes the use of monoclonal antibodies, lymphocytes, and cytokines. Tumor vaccines typically consist of tumor antigens and immunostimulatory molecules (e.g., adjuvants, cytokines, or TLR ligands), which work together to induce antigen-specific cytotoxic T cells (CTLs) that recognize and lyse tumor cells. One of the key obstacles to developing curative and tumor-specific immunotherapies is the identification and selection of highly specific and restrictive tumor antigens to avoid autoimmunity.

[0004] Tumor neoantigens generated due to genetic changes in malignant cells (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.) represent the most tumor-specific class of antigens and can be patient-specific or shared. Tumor neoantigens are specific to tumor cells because the mutations and their corresponding proteins are only present in tumors. They also avoid central tolerance and are therefore more likely to be immunogenic. Therefore, tumor neoantigens provide excellent targets for immune recognition, including both humoral and cellular immunity. Therefore, there is still a need to develop additional cancer therapeutics. Summary of the invention

[0005] Provided herein is a method for treating or preventing cancer in a human subject in need thereof, the method comprising administering to the human subject in need thereof: (a) a first component comprising: (i) a polypeptide comprising a cancer-specific neo-epitope of a protein expressed by a cancer cell of the cancer, (ii) a polynucleotide encoding the polypeptide of (i), (iii) one or more APCs comprising the polypeptide of (i) or the polynucleotide of (ii), and (iv) a T cell receptor (TCR) specific for a complex comprising an HLA protein expressed by the human subject and a T cell receptor comprising the cancer neo-epitope. (a) a human subject having a tumor-specific neo-epitope expressed by a cancer cell, or (b) a human subject having a tumor-specific neo-epitope expressed by a cancer cell, or (c) a human subject having a tumor-specific neo-epitope expressed by a cancer cell, or (d) a human subject having a tumor-specific neo-epitope expressed by a cancer cell, or (e) a human subject having a tumor-specific neo-epitope expressed by a cancer cell, or (v) a human subject having a TCR containing (iv); and (b) a second component comprising an anti-cancer agent, which is an antibody or an antigen-binding portion thereof that specifically binds to a programmed death protein-1 (PD-1) receptor and inhibits PD-1 activity; and (c) a third component comprising a platinum-based chemotherapy; wherein the human subject: (i) has not previously received systemic treatment for metastatic disease, (ii) has not previously received immunotherapy with an anti-PD-1 antibody, and (iii) has not previously received immunotherapy with an anti-PD-L1 antibody.

[0006] In some embodiments, the method comprises administering to the human subject a combination of the second component and the third component prior to administering the first component.

[0007] In some embodiments, the method includes administering a combination of the second component and the third component to the human subject for a period of 12 weeks before administering the first component. In some embodiments, the production of the first component occurs during the period of 12 weeks of administering the combination of the second component and the third component. In some embodiments, the method includes administering the first component for a period of 12 weeks after the period of 12 weeks of administering the combination of the second component and the third component. In some embodiments, the period of 12 weeks of administering the first component after the period of 12 weeks of administering the combination of the second component and the third component includes administering the first component at four separate anatomical sites of the human subject. In some embodiments, the period of 12 weeks of administering the first component after the period of 12 weeks of administering the combination of the second component and the third component includes administering five priming doses of the first component and two booster doses of the first component. In some embodiments, the period of 12 weeks of administering the first component after the period of 12 weeks of administering the combination of the second component and the third component includes administering a priming dose of the first component once a week on the 1st and 4th days and then in the 13th, 14th and 15th weeks; and administering booster doses in the 19th and 23rd weeks. In some embodiments, the second component is applied to the human subject during the 12-week period of applying the first component. In some embodiments, the second component is applied to the human subject after the 12-week period of applying the first component and the second component. In some embodiments, the second component is applied to the human subject for a period of at least 28 weeks after the 12-week period of applying the first component and the second component. In some embodiments, the second component is applied to the human subject for a period of 80 weeks after the 12-week period of applying the first component and the second component. In some embodiments, the second component is applied to the human subject for a total period of at least 52 weeks or about 103 weeks or about 104 weeks. In some embodiments, the third component is not applied to the human subject during or after the application of the first component. In some embodiments, the third component is not applied to the human subject after the 12-week period of applying the combination of the second component and the third component before applying the first component.

[0008] In some embodiments, the human subject has a KRAS mutation, a TP53 mutation, and / or a KEAP1 mutation. In some embodiments, the cancer-specific neo-epitopes of the first component do not include KRAS neo-epitopes, TP53 neo-epitopes, and / or KEAP1 neo-epitopes.

[0009] In some embodiments, the cancer is lung cancer.

[0010] In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC).

[0011] In some embodiments, the NSCLC has squamous histology.

[0012] In some embodiments, the NSCLC has non-squamous histology.

[0013] In some embodiments, the NSCLC is metastatic NSCLC.

[0014] In some embodiments, the first component comprises a polypeptide comprising a cancer-specific neo-epitope of a protein expressed by cancer cells of the cancer.

[0015] In some embodiments, the first component comprises an adjuvant.

[0016] In some embodiments, the adjuvant comprises poly I:poly C.

[0017] In some embodiments, the cancer-specific neo-epitopes include at least two different cancer-specific neo-epitopes of proteins expressed by cancer cells of the cancer.

[0018] In some embodiments, the cancer-specific neo-epitopes include up to twenty different cancer-specific neo-epitopes of proteins expressed by cancer cells of the cancer.

[0019] In some embodiments, the method comprises comparing: (i) a nucleic acid sequence obtained by whole genome sequencing or whole exome sequencing of a cancer cell from a single subject with (ii) a nucleic acid sequence obtained by whole genome sequencing or whole exome sequencing of a non-cancerous cell from a single subject. In some embodiments, the method comprises identifying a plurality of cancer-specific nucleic acid sequences unique to the cancer cells of the human subject based on the comparison.

[0020] In some embodiments, the method comprises predicting or calculating the binding affinity of the cancer-specific neo-epitope sequence encoded by the identified plurality of cancer-specific nucleic acid sequences to the protein encoded by the HLA allele of the human subject by performing an HLA peptide binding analysis using a program executed on a computer system.

[0021] In some embodiments, the method comprises selecting an IC for a protein predicted or calculated to be encoded by an HLA allele of the human subject. 50 At least two cancer-specific neo-epitope sequences at less than 500 nM or 150 nM or less.

[0022] In some embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, cross-competes with nivolumab for binding to human PD-1.

[0023] In some embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, comprises a heavy chain constant region of human IgG1 or IgG4 isotype.

[0024] In some embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, is a chimeric antibody, a humanized antibody, or a human monoclonal antibody, or a portion thereof.

[0025] In some embodiments, the anti-PD-1 antibody is pembrolizumab.

[0026] The method of claim 35, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered once every 2 weeks, every 3 weeks, or every 4 weeks at a dose ranging from 0.1 to 10.0 mg / kg body weight.

[0027] In some embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, is administered once every 3 weeks at a dose of 5 or 10 mg / kg body weight.

[0028] In some embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, is administered once every 2 weeks at a dose of 3 mg / kg body weight.

[0029] In some embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, is administered via intravenous infusion on day 1 of every 3-week cycle at a dose of 200 mg.

[0030] In some embodiments, the platinum-based chemotherapy is platinum-based dual chemotherapy (PT-DC).

[0031] In some embodiments, the PT-DC is a combination of pemetrexed and carboplatin.

[0032] In some embodiments, the carboplatin is administered at a dose that achieves an area under the free carboplatin plasma concentration versus time curve (AUC) of 5.

[0033] In some embodiments, the pemetrexed is administered at a dose of 500 mg / m^2.

[0034] In some embodiments, the PT-DCs are administered simultaneously with the anti-PD-1 antibody, or an antigen-binding portion thereof, for 4 doses of the anti-PD-1 antibody, or an antigen-binding portion thereof, followed by repeated administration of the anti-PD-1 antibody, or an antigen-binding portion thereof, alone.

[0035] In some embodiments, the methods promote epitope spreading.

[0036] In some embodiments, the methods promote epitope spreading of epitopes that are distinct from any of the cancer-specific neo-epitopes.

[0037] In some embodiments, the epitope that is different from any of the cancer-specific neo-epitopes comprises a KRAS neo-epitope, a TP53 neo-epitope, and / or a KEAP1 neo-epitope.

[0038] In some embodiments, the KRAS neo-epitope comprises a G12C or G12V mutation.

[0039] In some embodiments, the median progression-free survival (PFS) of a first population of human subjects suffering from the cancer treated with the first, second, and third components is longer than the median PFS of a second population of subjects suffering from the cancer treated with the second and / or third components but not the first component.

[0040] In some embodiments, the overall response rate (ORR) of a first population of human subjects suffering from the cancer treated with the first, second, and third components is higher than the ORR of a second population of subjects suffering from the cancer treated with the second and / or third components but not the first component.

[0041] In some embodiments, the percentage of subjects in a first population of human subjects having the cancer treated with the first, second, and third components who have a progression-free survival (PFS) of at least 9 months is greater than the percentage of subjects in a second population of human subjects having the cancer treated with the second and / or third components but not the first component who have a PFS of at least 9 months.

[0042] In some embodiments, the percentage of subjects in a first population of human subjects having the cancer treated with the first, second, and third components who have a progression-free survival (PFS) of at least 12 months is greater than the percentage of subjects in a second population of human subjects having the cancer treated with the second and / or third components but not the first component who have a PFS of at least 12 months.

[0043] In some embodiments, the median overall survival (OS) of a first population of human subjects suffering from the cancer treated with the first, second, and third components is longer than the median OS of a second population of subjects suffering from the cancer treated with the second and / or third components but not the first component.

[0044] In some embodiments, the percentage of subjects achieving complete remission, partial remission, prolonged disease stabilization, or disease stabilization for 6 months or longer (CBR) in a first population of human subjects suffering from the cancer treated with the first, second, and third components is higher than the CBR in a second population of subjects suffering from the cancer treated with the second and / or third components but not the first component.

[0045] In some embodiments, the reduction in tumor size in a first population of human subjects suffering from the cancer treated with the first, second, and third components is greater than the reduction in tumor size in a second population of subjects suffering from the cancer treated with the second and / or third components but not the first component.

[0046] In some embodiments, the level of tumor-infiltrating CD4+ T cells in a first population of human subjects treated with the first, second, and third components is higher than the level of tumor-infiltrating CD4+ T cells in a second population of subjects treated with the second and / or third components but not the first component.

[0047] In some embodiments, the levels of effector cells and cytotoxic CD4+T cells generated in a first population of human subjects treated with the first, second, and third components are higher than the levels of effector cells and cytotoxic CD4+T cells generated in a second population of subjects treated with the second and / or third components but not the first component. In some embodiments, the method increases the level of CD4+T cells specific for cancer-specific neo-epitopes that upregulate the expression of ZEB2, PDCD1, TOX, TIGT, CXCR3, ITGB1, GZMA, and / or ICOS.

[0048] In some embodiments, the method increases CD4+ / CD62L specific for cancer-specific neoepitopes. hi / Levels of CD69+ / CD27+ / CCR7+T cells.

[0049] In some embodiments, the method increases the levels of CD4+ / NKG7+ / CCL4+ / CCL5+ / GNLY+ / LAG3+ T cells specific for a cancer-specific neoepitope.

[0050] In some embodiments, prior to administering the first component, the second component, and / or the third component, the human subject is identified as having a PD-L1 positive cancer.

[0051] In one aspect, provided herein is a method for treating or preventing cancer in a human subject in need thereof, the method comprising administering to the human subject in need thereof: (a) a first component comprising: (i) a polypeptide comprising a cancer-specific neo-epitope of a protein expressed by a cancer cell of the cancer, (ii) a polynucleotide encoding the polypeptide of (i), (iii) one or more APCs comprising the polypeptide of (i) or the polynucleotide of (ii), (iv) a T cell receptor (TCR) specific for a complex comprising an HLA protein expressed by the human subject and a cancer-specific neo-epitope of a protein expressed by a cancer cell having the cancer neo-epitope, or (v) a T cell receptor (TCR) specific for a complex comprising an HLA protein expressed by the human subject and a cancer-specific neo-epitope of a protein expressed by a cancer cell having the cancer neo-epitope. v) a T cell containing the TCR of (iv); and (a) a second component comprising an anticancer agent, which is an antibody or an antigen-binding portion thereof that specifically binds to a programmed death protein-1 (PD-1) receptor and inhibits PD-1 activity; and (b) a third component comprising a platinum-based chemotherapy; wherein: (i) the method promotes epitope expansion of epitopes that are different from any of the cancer-specific neoepitopes; (ii) the median progression-free survival (PFS) of a first population of human subjects with the cancer treated with the first, second and third components is longer than that of a second population of human subjects with the cancer treated with the second and / or third components but not the first component. (iii) the overall response rate (ORR) of a first population of human subjects with the cancer treated with the first, second and third components is higher than the ORR of a second population of subjects with the cancer treated with the second component and / or the third component but not the first component; (iv) the percentage of subjects with at least 9 or 12 months progression-free survival (PFS) in the first population of human subjects with the cancer treated with the first, second and third components is higher than the percentage of subjects with at least 9 or 12 months PFS in the second population of subjects with the cancer treated with the second component and / or the third component but not the first component (v) a first population of human subjects with the cancer treated with the first, second, and third components has a longer median overall survival (OS) than a second population of subjects with the cancer treated with the second and / or third components but not the first component; (vi) a percentage of subjects achieving complete remission, partial remission, prolonged disease stabilization, or disease stabilization for 6 months or longer (CBR) in a first population of human subjects with the cancer treated with the first, second, and third components is higher than a CBR in a second population of subjects with the cancer treated with the second and / or third components but not the first component;(vii) a reduction in tumor size in a first population of human subjects with the cancer treated with the first, second, and third components is greater than a reduction in tumor size in a second population of subjects with the cancer treated with the second and / or third components but not the first component; (viii) the level of CD4+ T cells infiltrating tumors in the first population of human subjects treated with the first, second, and third components is greater than the level of CD4+ T cells infiltrating tumors in the second population of subjects treated with the second and / or third components but not the first component; (ix) a reduction in tumor size in a second population of human subjects treated with the first, second, and third components is greater than a reduction in tumor size in a second population of subjects with the cancer treated with the second and / or third components but not the first component; The method further comprises: (i) increasing the level of effector cells and cytotoxic CD4+ T cells generated in a first population of human subjects treated with the second component and / or the third component but not the first component compared to the level of effector cells and cytotoxic CD4+ T cells generated in a second population of subjects treated with the second component and / or the third component but not the first component; (x) the method increases the level of CD4+ T cells specific for a cancer-specific neo-epitope that upregulates the expression of ZEB2, PDCD1, TOX, TIGT, CXCR3, ITGB1, GZMA and / or ICOS; (xi) the method increases CD4+ / CD62L specific for a cancer-specific neo-epitope; hi / CD69+ / CD27+ / CCR7+ T cells; and / or (xii) the method increases the level of CD4+ / NKG7+ / CCL4+ / CCL5+ / GNLY+ / LAG3+ T cells specific for cancer-specific neoepitopes.

[0052] In some embodiments, prior to administering the first component, the second component, and / or the third component, the human subject is identified as having a PD-L1 positive cancer.

[0053] In some embodiments, the human subject has not previously received systemic treatment for metastatic disease, has not previously received immunotherapy with an anti-PD-1 antibody, and / or has not previously received immunotherapy with an anti-PD-L1 antibody.

[0054] In some embodiments, provided herein is a method for treating or preventing a tumor in a human subject in need thereof, the method comprising administering to a subject in need thereof: a first component comprising (i) a peptide comprising a neo-epitope of a protein, (ii) a polynucleotide encoding the peptide, (iii) one or more APCs comprising the peptide or the polynucleotide encoding the peptide, or (iv) a T cell receptor (TCR) specific for the neo-epitope complexed with an HLA protein; and a second component comprising at least two therapeutic agents, e.g., the anti-PD-L1 monoclonal antibody pembrolizumab, carboplatin, and / or pemetrexed.

[0055] In some embodiments, the first component comprises a tumor vaccine or an immunogenic composition.

[0056] In some embodiments, the first component further comprises an adjuvant. In some embodiments, the adjuvant is polyICLC.

[0057] In some embodiments, the first component comprises a tumor vaccine or immunogenic composition containing a neoantigenic peptide, wherein the peptide comprises at least two, at least three, at least four, or at least five peptides. In some embodiments, the peptide comprises up to 15, up to 20, up to 25, or up to 30 peptides. In some embodiments, the length of the peptide is 5 to 50 amino acids. In some embodiments, the length of the peptide is 14 to 35 amino acids. In some embodiments, the neoepitope of each peptide is unique.

[0058] In some embodiments, the first component further comprises a pH adjuster. In some embodiments, the first component further comprises a pharmaceutically acceptable carrier.

[0059] In some embodiments, the subject has a tumor selected from the following: non-Hodgkin lymphoma (NHL), clear cell renal cell carcinoma (ccRCC), melanoma, sarcoma, leukemia or bladder cancer, colon cancer, brain cancer, breast cancer, head and neck cancer, endometrial cancer, lung cancer, ovarian cancer, pancreatic cancer or prostate cancer. In some embodiments, the tumor is a metastatic melanoma. In some embodiments, the subject has no detectable tumor, but is at high risk for disease recurrence. In embodiments, the cancer is selected from: adrenal cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, glioblastoma, head and neck cancer, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, ovarian cancer, pancreatic cancer, melanoma, gastric cancer, uterine body endometrial cancer and uterine carcinosarcoma. In embodiments, the cancer is selected from the group consisting of prostate cancer, bladder cancer, squamous cell lung cancer, NSCLC, breast cancer, head and neck cancer, lung adenocarcinoma, GBM, glioma, CML, AML, supratentorial ependymoma, acute promyelocytic leukemia, solitary fibrous tumor, and crizotinib-resistant cancer. In embodiments, the cancer is selected from the group consisting of CRC, head and neck cancer, gastric cancer, squamous cell lung cancer, lung adenocarcinoma, prostate cancer, bladder cancer, gastric cancer, renal cell carcinoma, and uterine cancer. In embodiments, the cancer is selected from the group consisting of melanoma, squamous cell lung cancer, DLBCL, uterine cancer, head and neck cancer, uterine cancer, liver cancer, and CRC. In embodiments, the cancer is selected from the group consisting of lymphoid cancer, Burkitt's lymphoma, neuroblastoma, prostate adenocarcinoma, colorectal adenocarcinoma, uterine / endometrial adenocarcinoma, MSI+, endometrial serous carcinoma, endometrial carcinosarcoma-malignant mesodermal mixed tumor, glioma, astrocytoma, GBM, acute myeloid leukemia associated with MDS, chronic lymphocytic leukemia-small lymphocytic lymphoma, myelodysplastic syndrome, acute myeloid leukemia, breast luminal NS carcinoma, chronic myeloid leukemia, pancreatic ductal carcinoma, chronic myelomonocytic leukemia, myelofibrosis, myelodysplastic syndrome, prostate adenocarcinoma, essential thrombocythemia, and myeloid myoblastoma. In embodiments, the cancer is selected from the group consisting of colorectal cancer, uterine cancer, endometrial cancer, and gastric cancer. In embodiments, the cancer is selected from the group consisting of cervical cancer, head and neck cancer, anal cancer, gastric cancer, Burkitt's lymphoma, and nasopharyngeal carcinoma. In embodiments, the cancer is selected from the group consisting of: bladder cancer, colorectal cancer, and gastric cancer. In embodiments, the cancer is selected from the group consisting of: lung cancer, CRC, melanoma, breast cancer, NSCLC, and CLL. In embodiments, the subject is a partial responder or non-responder to checkpoint inhibitor therapy.In embodiments, the cancer is selected from the group consisting of bladder urothelial carcinoma (BLCA), invasive breast cancer (BRCA), breast cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), chronic lymphocytic leukemia (CLL), colorectal cancer (CRC), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), prostate cancer, skin melanoma (SKCM), stomach adenocarcinoma (STAD), thyroid adenocarcinoma (THCA) and uterine body endometrioid carcinoma (UCEC). In embodiments, the cancer is selected from the group consisting of colorectal cancer, uterine cancer, endometrial cancer, gastric cancer and Lynch syndrome. In embodiments, the cancer is an MSI+ cancer.

[0060] In some embodiments, the first component is administered before the second component. In some embodiments, the second component is administered before the first component. In some embodiments, the first component and the second component are administered on the same day. In some embodiments, the second component is administered before the first component. In some embodiments, the administration of pembrolizumab is initiated before the administration of the first component is initiated. In some embodiments, the administration of pembrolizumab is initiated before the administration of the carboplatin or pemetrexed is initiated. In some embodiments, the administration of pembrolizumab is initiated before the administration of carboplatin or pemetrexed is initiated. In some embodiments, pembrolizumab is administered on the same day as the first administration of the first component. In some embodiments, carboplatin is initiated on the same day as the first administration of the first component. In some embodiments, nivolumab is continued to be administered every 12-36 weeks or longer after the first administration of pembrolizumab. In some embodiments, nivolumab is continued to be administered every 2 weeks, every 3 weeks, every 4 weeks, every 6 weeks, or every 8 weeks after the first administration of pembrolizumab. In some embodiments, the administration of inhibitors (such as checkpoint inhibitors or chemotherapeutic agents) is initiated after tumor resection. In some embodiments, the first component is administered in a prime-boost dosing regimen.

[0061] In some embodiments, the first component is administered as a priming at week 1, week 2, week 3, or week 4. In some embodiments, the first component is administered as a boost at month 2, month 3, month 4, or month 5. In some embodiments, the first component is administered as a boost at week 19, week 20, week 21, week 22, week 23, or week 24.

[0062] In some embodiments, the peptides are administered at an average dose level of about 300-500 μg / ml per peptide. In some embodiments, the total dose of peptide administered is 4-8 mg. In some embodiments, pembrolizumab is administered at a dose of 200-260 mg.

[0063] In some embodiments, the first component and / or the second component is administered intravenously or subcutaneously.

[0064] In some embodiments, the dose of the peptide is divided into at least 2, at least 3, at least 4, or at least 5 sub-doses. In some embodiments, each sub-dose of the peptide comprises at least 4 or at least 5 peptides.

[0065] In some embodiments, each peptide is administered at a dose of 200-400 μg. In some embodiments, each sub-dose is administered at a different site in the subject.

[0066] In some embodiments, the method further comprises administering one or more additional agents. In some embodiments, the additional agents are selected from the group consisting of: chemotherapeutic agents, anti-angiogenic agents, and agents that reduce immunosuppression.

[0067] In some aspects, provided herein is a method of treating or preventing cancer in a human subject in need thereof who has been treated with pembrolizumab, the method comprising administering to the subject one or more chemotherapeutic agents at a dose of 1%-95% of the dose of the chemotherapeutic agent normally administered in a monotherapy regimen.

[0068] In some aspects, provided herein is a method of treating or preventing cancer in a human subject in need thereof who has been treated with pembrolizumab at a dose that is 1%-95% of the dose of pembrolizumab normally administered in a monotherapy regimen, the method comprising administering to the subject one or more chemotherapeutic drugs, e.g., at a dose lower than their normal dose as a monotherapy.

[0069] In some aspects, provided herein is a method of treating or preventing cancer in a human subject in need thereof who has been treated with pembrolizumab, the method comprising administering to the subject: carboplatin at a dose of 1%-95% of the dose normally administered in a monotherapy regimen; and pemetrexed at a dose lower than the dose normally administered in a monotherapy regimen.

[0070] In some embodiments, the method further comprises administering to the subject at least five peptides, each peptide comprising a unique neo-epitope of a protein, at a dose of 100-500 μg of each peptide.

[0071] In some aspects, provided herein is a composition comprising: a first component comprising (i) a peptide containing a neo-epitope of a protein, (ii) a polynucleotide encoding the peptide, (iii) one or more APCs containing the peptide or the polynucleotide encoding the peptide, or (iv) a T cell receptor (TCR) specific for the neo-epitope complexed with an HLA protein; and a second component comprising at least two inhibitors, wherein the at least two inhibitors include: pembrolizumab and a platinum-based chemotherapeutic agent or pembrolizumab and pemetrexed, or pembrolizumab and a platinum-based chemotherapeutic agent and pemetrexed.

[0072] Other aspects and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other and different embodiments, and its several details are capable of modification in various obvious aspects, all without departing from the present disclosure. Therefore, the drawings and descriptions are to be regarded as illustrative rather than restrictive in nature.

[0073] Incorporation by reference

[0074] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference, as if each individual publication, patent, or patent application was specifically and individually incorporated herein by reference. To the extent that publications and patents or patent applications incorporated by reference conflict with the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such conflicting material. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] The novel features of the present disclosure are particularly set forth in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by referring to the following detailed description and the accompanying drawings (also referred to herein as "Figures" and "FIG."), which set forth illustrative embodiments using the principles of the present disclosure, wherein:

[0076] Figure 1A-Figure 1C : NEO-PV-01 vaccine generation, clinical study design, and patient handling.

[0077] Figure 1A Schematic diagram of sequencing patient tumors, predicting neoantigens restricted to class I MHC molecules, and generating synthetic long peptides for inclusion in personalized neoantigen vaccines.

[0078] Figure 1B Treatment with pembrolizumab and pemetrexed plus carboplatin was initiated at week 0; NEO-PV-01 was then administered between weeks 12 and 24, with pembrolizumab continued for up to 2 years.

[0079] Figure 1C Study Patient Disposition. Seventeen of the 38 patients (45%) in the ITT set terminated the study prematurely without receiving the vaccine due to the following reasons: inability to produce the vaccine due to insufficient tumor and / or insufficient amount of neoantigen (10 patients), adverse events (2 patients), patient withdrawal of consent (2 patients), disease progression, investigator decision, or administration of concomitant medication prohibited by the study (1 patient each).

[0080] Figure 2A-2E : Response rate and durability after treatment with NEO-PV-01 plus chemotherapy and anti-PD-1.

[0081] Figure 2A .Best radiographic change (%) in the sum of target lesions for each patient who received at least one dose of vaccine (VAX pool). Dark shaded narrow bars represent the best change before NEO-PV-01; light shaded wider bars represent the best overall change in the study for patients who received at least 1 dose of NEO-PV-01 plus anti-PD-1. Red indicates progressive disease, gray indicates stable disease, and blue indicates partial response.

[0082] Figure 2B Radiographic changes (%) of target lesions after initiation of pembrolizumab treatment per patient (same colors as in a). Thirty of the 38 ITT patients with at least one post-baseline RECIST assessment were included.

[0083] Figure 2C A swim-lane plot summarizing all patients in the study. Each bar represents a subject in the study, and the length of the bar represents the duration of the study.

[0084] Figure 2D .Kaplan-Meier estimates of PFS (top) and OS (bottom) for the ITT (left) and vaccinated (right) patient sets.

[0085] Figure 2E Measurement of ctDNA in peripheral blood of subsets of patients, as measured by the percent change in mean tumor molecules per mL of blood; open circles indicate undetectable ctDNA levels (left). Example of quantification of individual mutant ctDNA molecules detected in non-PFS-9 patients (blue) and PFS-9 patients (green).

[0086] Figure 3A-Figure 3F : Correlations with clinical response were observed in the tumor microenvironment (TME) before treatment, including T cell infiltration, MHC class II expression, and TCR diversity.

[0087] Figure 3ACD4+ (top) and CD8+ (bottom) T cells / mm outside (left) or inside (right) tumors in patient tumor biopsy material obtained by multiplex IHC at pre-treatment time points 2 Correlation with PFS (months). Pearson correlation coefficients (R) and associated p-values ​​are shown.

[0088] Figure 3B .Representative images of IHC analysis of CD4+ and CD8+ T cells in patient tumor biopsy material from one non-PFS-9 patient (2L3) and one PFS-9 patient (2L5) stained with DAPI (blue), CD3 (red), CD4 (green), CD8 (white), and PanCK (cyan) are shown. White arrows indicate CD8+ T cells surrounding the tumor area, and yellow arrows indicate CD4+ T cells infiltrating into the TME. (Scale bar, 50 μM).

[0089] Figure 3C Correlation between HLA class II gene expression in tumor biopsies at pre-treatment time points and PFS (months). Pearson correlation coefficients (R) and associated p-values ​​are shown.

[0090] Figure 3D .Representative images of MHC class II expression obtained by multiplex IHC in patient tumor biopsy material from one non-PFS-9 patient (2L3) and one PFS-9 patient (2L5) stained with HLA-DR / DP / DQ (red), PanCK (green), DAPI (blue), CD11c (white), and CD14 (yellow) are shown. Individual channel images of patient 2L5 (for HLA-DR / DP / DQ, CD14, CD11c, and PanCK) are shown below the multiplex images. White arrows indicate HLA-DR / DP / DQ+CD11c+CD14+ cells, and yellow arrows indicate HLA-DR / DP / DQ+CD11c-CD14+ cells (scale bar, 50 μm).

[0091] Figure 3E Analysis of Shannon entropy (left) and unique amino acid (UniAA) counts (right) in tumor biopsies at pre-treatment time points and correlation with PFS (months). Pearson correlation coefficients (R) and associated p-values ​​are shown.

[0092] Figure 3F .Analysis of Shannon entropy (left) and unique amino acid counts (right) in tumor biopsy material at pre-treatment (week 0), pre-vaccine (week 12), and post-vaccine (week 24) time points is shown, with PFS-9 patients represented in green and non-PFS-9 patients represented in blue.

[0093] Figure 4A-4E: NEO-PV-01 plus chemotherapy and anti-PD-1 induce neoantigen-reactive T cell responses that are specific for neoepitopes, persistent, and show cytotoxic potential.

[0094] Figure 4A . Percentage of all NEO-PV-01 vaccination peptides that elicited IFNγ responses in serial PBMCs at the indicated time points.

[0095] Figure 4B The table summarizes the overall immune response detected in the 13 patients analyzed, as well as the percentage characterized as a CD4+ or CD8+ response.

[0096] Figure 4C Specificity of immune responses to mutant peptides (green) versus wild-type peptide (red) over a range of peptide concentrations as measured by IFNγ ELISpot assay. Representative responses of patients 2L7 (IM13 and IM04) and 2L3 (IM03 and IM15) are shown.

[0097] Figure 4D Durability of immune responses induced by IM peptides as measured by IFNγ ELISpot assay in PBMCs collected at week 52 after initiation of chemotherapy plus anti-PD-1 therapy. Data are presented as stacked bar graphs for individual patients, with responses detected at both week 20 and week 52 shown in light green and responses detected only at week 20 shown in dark green.

[0098] Figure 4E .The cytotoxic potential of the immune response generated by NEO-PV-01, as measured by surface expression of the marker CD107a combined with intracellular IFNγ expression at post-vaccine time points. Representative flow cytometry plots for patient 2L7 are shown on the left, comparing peptide recall (bottom) with DMSO recall (top) in the ex vivo (left) and 5-day stimulation (right) assay designs. The data are summarized on the right and presented as stacked bar graphs for individual patients, with positive (cytotoxic potential) responses shown in dark green and negative (no cytotoxic potential) responses shown in light green. The table below summarizes the detected responses for all patients analyzed and classifies the responses as CD4+, CD8+, or both. Aggregate data are presented as mean + / - SEM.

[0099] Figure 5A-5E : In the majority of patients analyzed, NEO-PV-01 plus chemotherapy and anti-PD-1 induced epitope-expanded responses, in which mutKRAS responses were observed.

[0100] Figure 5AEpitope expansion was measured in 13 patients. Post-vaccine PBMCs were tested for reactivity to a panel of 10-25 predicted neoantigenic peptides not included in the vaccine by IFNγ ELISpot assay. Responses characterized as epitope expansion were detected only at the post-vaccine time point (week 20) ​​and not at the pre-vaccine time point (week 10).

[0101] Figure 5B .T cell responses to individual non-immune (NIM) peptides at post-vaccination time points are shown for 9 patients. NIM peptides that did not elicit reactivity post-vaccination are not shown. Generation of immune responses to each NIM peptide was tested using overlapping assay peptides, and the assay peptides that generated the greatest response in either the ex vivo assay or the 5-day assay are shown. Responses with * represent ex vivo responses.

[0102] Figure 5C Four patients elicited a mutKRAS-specific epitope expansion response as determined by IFNγ ELISpot assay. The PFS-9 status of each patient is indicated below the graph, and the specific G12 mutation is indicated above the graph. Responses with * represent ex vivo responses.

[0103] Figure 5D . Specificity of the immune response as measured by IFNγ ELISpot assay to mutant peptide (solid line) versus wild-type peptide (dashed line) over a range of peptide concentrations for each of 4 patients where an epitope-expanding response to mutKRAS was observed.

[0104] Figure 5E .Surface expression of the cytolytic marker CD107a (x-axis) and intracellular expression of IFNγ (y-axis) are shown by FACS analysis of the mutKRAS-specific epitope expansion response observed in patient 2L15. The control (DMSO) is depicted on the left side of the single figure, and the NIM peptide is depicted on the right side. The positive in this assay is determined as >1.5-fold stimulation compared to the DMSO control in the double positive gate. The parental gate is shown below the FACS plot pair.

[0105] Figure 6A-6H :After vaccination, neoantigen-specific CD4+ T cell responses share an activated effector phenotype in the periphery.

[0106] Fig. 6A Table summarizes five patients analyzed using a combination of multimer-based sorting of neoantigen-specific CD4+ T cells (representative flow cytometry panel on the right), CITE-Seq, TCRSeq, and gene expression analysis.

[0107] Figure 6BUnsupervised clustering (left) and heatmap (right) of normalized gene expression for cluster analysis of tetramer+ and tetramer- CD4+ T cell samples from five patients.

[0108] Figure 6C Separate unsupervised clustering plots for visualization of tetramer- cells (top) and tetramer+ cells (bottom) from five patients.

[0109] Fig.6D .Comparison of the proportion of each UMAP cluster between tetramer+ and tetramer- populations.

[0110] Fig. 6E .Measurement of the Gini coefficient (as an indicator of TCR repertoire clonality) for both tetramer+ and tetramer- populations. Each point represents a single patient.

[0111] Fig. 6F Measurement of clonotypes covering the top 30% of the TCR repertoire of tetramer+CD4+ T cells detected by single-cell TCR sequencing using bulk TCR-seq data at pre-treatment, pre-vaccine, and post-vaccine time points.

[0112] Figure 6G Multiplex IHC was performed per mm at pre-treatment, pre-vaccine, and post-vaccine time points (if available). 2 Measurement of CD3+CD4+ T cell numbers in tumor biopsies. Aggregate data are presented as mean + / - SEM.

[0113] Figure 6H Representative IHC images of patient 2L7 (PFS-9) stained with DAPI (blue), CD3 (red), CD4 (yellow), and PanCK (green) at pre-treatment (left), pre-vaccine (middle), and post-vaccine (right) time points are shown. (Scale bar, 50 μm). Longitudinal biopsies were obtained from the same lung lesion in this patient.

[0114] Figure 7A-7C : Radiographic responses in vaccinated patients were compared with pretreatment tumor PD-L1 levels, and longitudinal tracking of individual ctDNA molecules from each patient measured the abundance of predicted neoantigen genes and genes included in the personalized Signatera pool.

[0115] Fig. 7A Radiographic response in tumors of patients who received at least one dose of NEO-PV-01 versus pre-treatment PD-L1 levels. PD-L1 levels are shown below the bars in the waterfall graph. Scoring was based on the percentage PD-L1 on tumor cells as follows: <1% was indicated as -, 1-<50% was indicated as +, and ≥50% was indicated as ++.

[0116] Figure 7B Serial ctDNA measurements were performed in 17 patients for 16 variants predicted to have high-quality neoepitopes based on an in-house bioinformatics algorithm.

[0117] Figure 7C .16 selected somatic target mutations selected by Natera variant calling method.

[0118] Figure 8A-8B : NEO-PV-01 plus chemotherapy and anti-PD-1 induces durable T cell reactivity against multiple vaccine neoepitopes.

[0119] Fig. 8A .T cell responses to individual immunization (IM) peptides are shown for 9 patients. Immune peptides that did not elicit reactivity are not shown. Generation of immune responses to each IM peptide was tested using overlapping assay peptides, and the assay peptides that generated the greatest response for each IM peptide are shown in the pre- and post-vaccine time points. Each bar corresponds to an IM peptide and the corresponding assay peptide that generated the greatest response in either the ex vivo assay or the 5-day assay. Immune peptides marked in red on the x-axis elicit pre-vaccine responses.

[0120] Figure 8B .T cell responses were quantified as in Panel A, with additional analysis at 52 weeks after initiation of chemotherapy plus pembrolizumab for 4 patients. The inset of patient 2L11 was enlarged to visualize responses to IM17 and IM07. Immunizing peptides marked in red on the x-axis elicited pre-vaccine responses, and those marked in green elicited responses detected only at the 52-week time point. Aggregate data are presented as mean + / - SEM.

[0121] Figure 9A-9B : NEO-PV-01 plus chemotherapy and anti-PD-1 induce cytotoxic CD4+ and CD8+ T cell responses. Surface expression of the cytolytic marker CD107a (x-axis) and intracellular expression of IFNγ (y-axis) were shown by FACS analysis of 56 IM peptides in 12 patients. The control (DMSO) is depicted on the left side of the individual graphs, and the IM peptides are depicted on the right side. Only IM peptides that are positive in this assay (>1.5-fold stimulation compared to the DMSO control in the double positive gate) are shown. The parental gate is shown below each FACS plot pair, including those classified as CD4+ T cell responses ( Fig. 9A ) or CD8+ T cell response ( Fig. 9B )’s response.

[0122] Figure 10A- Fig. 10E : NEO-PV-01 plus chemotherapy and anti-PD-1 induces epitope-expanding responses that are cytotoxic and durable.

[0123] Figure 10A. The cytotoxic potential of epitope expansion response, as measured by surface expression of marker CD107a after vaccine time point combined with intracellular IFNγ expression. Data are presented as stacked bar graphs of individual patients, where positive (cytotoxic potential) responses are shown in dark green, and negative (no cytotoxic potential) responses are shown in light green. The following table summarizes the detected responses of all patients analyzed, and the responses are classified as CD4+, CD8+, or both.

[0124] Fig. 10B .The control (DMSO) is depicted on the left side of the individual graphs, and the non-immune (NIM) peptide is depicted on the right side. Only NIM peptides that were positive in this assay (>1.5-fold stimulation compared to the DMSO control in the double positive gate) are shown. The parental gate is shown below each FACS plot pair.

[0125] Fig. 10C .Distribution of PFS (months) for patients with or without KRAS mutations in pre-treatment tumor biopsy (ITT set). Vaccinated patients are shown as "+" and unvaccinated patients are shown as "·". The four patients in whom an epitope expansion response to mutKRAS was observed are shown in green. All other patients are shown in black. The box plots represent the 25%, 50%, and 75% percentiles, and the whiskers extend to the 95% confidence interval values. The p-values ​​are derived from a two-tailed Student's t-test.

[0126] Fig. 10D .Shows the T cell response of 3 patients to single non-immune (NIM) peptides at pre-vaccine (week 10), post-vaccine (week 20) ​​and week 52 time points. NIM peptides that did not cause reactivity are not shown. The generation of immune responses to each NIM peptide was tested using overlapping assay peptides, and the assay peptides that generated the maximum response for each NIM peptide were shown at these three time points. Each bar corresponds to a NIM peptide and the corresponding assay peptide that generated the maximum response in an in vitro assay or a 5-day assay. NIM peptides marked in red on the x-axis cause pre-vaccine responses, and those marked in green cause responses detected only at the week 52 time point. The illustration of patient 2L16 was enlarged to visualize the responses to NIM101 and NIM12.

[0127] Fig. 10E Persistence of NIM peptide-induced immune responses as measured by IFNγ ELISpot assay in PBMCs collected at week 52 after initiation of chemotherapy plus anti-PD-1 therapy. Data are presented as stacked bar graphs for individual patients, with responses detected at both week 20 and week 52 shown in light green and responses detected only at week 20 shown in dark green. Aggregate data are presented as mean + / - SEM.

[0128] Figures 11A-11G :The phenotype of neoantigen-reactive CD4+ effector cells is different from other effector cells.

[0129] Fig.11A Table summarizes 5 patients analyzed using multimeric sorting of neoantigen-specific CD4+ T cells in combination with the corresponding mutant and wild-type peptide sequences (shown for the responses analyzed). NA = Not Applicable.

[0130] Fig. 11B . Comparison of antibody (rows 1, 3, and 5) and RNA (rows 2, 4, and 6) expression levels of genes included in the CITE antibody panel.

[0131] Fig. 11C . Unsupervised clustering of tetramer- and tetramer+ samples, where normalized expression of selected RNA markers was used for clustering.

[0132] Fig.11D In the unsupervised clustering of tetramer+ and tetramer-CD4+ T cell samples, effector cells (described as CD45RO+CD45RA-CD62L lo CCR7 lo ) are identified in blue. All other cells are shown in pink.

[0133] Fig.11E (Top) Unsupervised clustering of effector cells only (both tetramer+ and tetramer-) yields 5 unique clusters. (Bottom) Separate unsupervised clustering plots to visualize tetramer- cells and tetramer+ cells among all effector cells.

[0134] Fig.11F . Unsupervised clustering of effector cells by individual patient.

[0135] Fig.11G . Heatmap of normalized gene expression for each of the 5 unique clusters identified based on clustering of all effector cells.

[0136] Fig.12 :Neoantigen-specific CD4+T cell responses were detected by MHC class II tetramer staining, and effector and central memory phenotypes were displayed by flow cytometry. At the time point after vaccination, CD4+T cells from 8 patients were analyzed for MHC class II tetramers in 27 peptide-MHC combinations. The leftmost figure of each patient depicts the tetramer+ population with green circles (quantified by the total tetramer+ cell number and the percentage of the main CD4+ population). For a subset of patients, additional typing was performed by flow cytometry to characterize cells as initial, effector, effector memory or central memory based on CD45RA and CD62L expression (rightmost figure).

[0137] Figure 13A-13I Neoantigen-specific CD4+ T cells displayed different phenotypes among patients but showed a consistent phenotype when clones targeting a single epitope were compared, and neoantigen-reactive TCRs were functional after vaccination, and tumors showed accumulation of peripherally expanded TCRs.

[0138] Fig.13A The table summarizes the TCR clones covering the top 30% repertoire for each patient in the single-cell TCRseq data and the corresponding frequencies of tetramer+CD4+ T cell clones in the peripheral blood of that patient at the post-vaccination time point.

[0139] Fig. 13B . Unsupervised clustering of tetramer+ samples only, with corresponding gene and protein expression markers specific to each cluster.

[0140] Fig. 13C . Unsupervised clustering of tetramer+ samples depicting the normalized expression of selected genes used for clustering.

[0141] Fig.13D . Heatmap of normalized gene expression based on clustering of tetramer+ samples only.

[0142] Fig.13E . Unsupervised clustering of re-clustered tetramer+ samples by individual patients.

[0143] Fig.13F Heatmap represents the abundance of cells of each clonotype in the tetramer+CD4+ T cell cluster. The most expanded clones were defined as those that encompassed the top 30% of the TCR repertoire for each patient assayed by single-cell TCR-Seq.

[0144] Figure 13G .CD4+TCR clones identified by single cell TCRseq as specifically recognizing the corresponding immunopeptide epitope in the context of matched MHC class II alleles were cloned into Jurkat cell lines (performed separately for each patient shown). Each of the 4 clonal Jurkat cell lines was co-cultured with matched patient APCs and the corresponding IM peptide at different peptide concentrations, and IL-2 secretion in the supernatant was measured as a readout of recognition of the peptide:MHC complex by the TCR. Reactivity with the corresponding wild-type sequence was also tested, except for patient 2L11 / IM18 (where the mutation was a frameshift mutation, where no wild-type peptide was available).

[0145] Fig.13H Multiplex IHC was performed per mm at pre-treatment, pre-vaccine, and post-vaccine time points (if available). 2Measurement of the number of CD3+CD8+ T cells in tumor biopsies. Aggregate data are presented as mean + / - SEM.

[0146] Fig.13I .TCR sequencing was performed on selected post-vaccination tumor biopsy samples where corresponding pre-treatment or pre-vaccine biopsy material was also available. TCRs found only in the post-vaccination biopsy were then cross-referenced with TCRs found in the peripheral blood. Only TCRs found to be increased in the peripheral blood at the post-vaccination time point were visualized. TCRs amplified in the periphery after vaccination are shown in red, and TCRs detected only in the periphery at the post-vaccination time point are shown in blue. DETAILED DESCRIPTION

[0147] New immunotherapeutics and their uses based on the discovery of neoantigens generated by mutational events unique to individual tumors are described herein. Thus, the disclosure described herein provides peptides, polynucleotides encoding these peptides, and peptide binders that can be used, for example, to stimulate an immune response to tumor-associated antigens or neoepitopes to produce immunogenic compositions or cancer vaccines for the treatment of disease.

[0148] The following description and examples describe the embodiments of the present disclosure in detail. It should be understood that the present disclosure is not limited to the specific embodiments described herein, and can therefore be changed. Those skilled in the art will recognize that the present disclosure has many variations and modifications, which are all encompassed within its scope.

[0149] All terms are intended to be understood as understood by those skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0150] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0151] Although various features of the present disclosure may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although for clarity the present disclosure may be described herein in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0152] The following definitions supplement those in the art and are for current applications, and are not attributed to any related or unrelated circumstances, for example, any commonly owned patent or application. Although any methods and materials similar or equivalent to the methods and materials described herein can be used to test the practice of the present disclosure, preferred materials and methods are described herein. Therefore, the terms used herein are only used for the purpose of describing specific embodiments and are not intended to be limiting.

[0153] I. definition

[0154] The terms used herein are only used for the purpose of describing a particular situation and are not intended to be limiting. In this application, unless otherwise specifically stated, the use of the singular includes the plural. As used herein, the singular forms "a", "an", and "the" are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0155] In some embodiments of the application, the use of "or" means "and / or" unless otherwise stated. As used herein, the terms "and / or" and "any combination thereof" and their grammatical equivalents may be used interchangeably. In some embodiments, these terms may express that any combination is specifically contemplated. For illustrative purposes only, the following phrases "A, B, and / or C" or "A, B, C, or any combination thereof" may mean "A alone; B alone; C alone; A and B; B and C; A and C; and A, B, and C." The term "or" may be used in conjunction or separately, unless the context specifically mentions separate use.

[0156] In some embodiments, the term "about" or "approximately" can mean within an acceptable error range for a particular value determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., subject to the limitations of the measurement system. For example, in some embodiments, according to the practice in the art, "about" can mean within 1 standard deviation or greater than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, within 5 times, or within 2 times. When describing a particular value in the present application and claims, unless otherwise indicated, it should be assumed that the term "about" means within an acceptable error range for the particular value.

[0157] As used in this specification and claims, the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended, and do not exclude additional unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

[0158] References in the specification to "some embodiments," "embodiments," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in conjunction with the embodiments is included in at least some embodiments of the present disclosure, but not necessarily all embodiments. To aid in understanding the present disclosure, a number of terms and phrases are defined below.

[0159] In some embodiments, the "major histocompatibility complex" or "MHC" can be a gene cluster that plays a role in controlling the cellular interactions responsible for the physiological immune response. In the human body, the MHC complex is also called the human leukocyte antigen (HLA) complex. For a detailed description of the MHC and HLA complexes, see Paul, Fundamental Immunology, 3rd edition, Raven Press, New York (1993). "Proteins or molecules of the major histocompatibility complex (MHC)", "MHC molecules", "MHC proteins" or "HLA proteins" can be understood to mean proteins that are capable of binding to peptides generated by proteolytic cleavage of protein antigens and representing potential lymphocyte epitopes (e.g., T cell epitopes and B cell epitopes), transporting these epitopes to the cell surface and presenting them there to specific cells, particularly cytotoxic T lymphocytes, T helper cells or B cells. The major histocompatibility complex in the genome includes genetic regions whose gene products can be expressed on the cell surface and are important for binding and presenting endogenous and / or exogenous antigens and thus regulating immune processes. The major histocompatibility complex can be divided into two gene groups encoding different proteins, MHC class I and MHC class II. The cellular biology and expression patterns of the two MHC classes are thought to adapt to these different roles.

[0160] "Human leukocyte antigen" or "HLA" may, in some embodiments, be a human class I or class II major histocompatibility complex (MHC) protein (see, e.g., Stites et al., Immunology, 8th ed., Lange Publishing, Los Altos, Calif. (1994)).

[0161] As used herein, "polypeptide", "peptide" and their grammatical equivalents may refer to a polymer of amino acid residues in some embodiments. A "mature protein" may be full-length, and it optionally includes glycosylation or other modifications typical for the protein in a given cellular environment. The polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) may contain synthetic amino acids that replace one or more naturally occurring amino acids. Such synthetic amino acids may be known in the art, and may include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine, β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, dihydroindole -2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N', N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α, γ-diaminobutyric acid, α, β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine. The present disclosure also contemplates that the expression of the polypeptides described herein in engineered cells can be associated with post-translational modification of one or more amino acids of the polypeptide construct. Non-limiting examples of post-translational modifications may include phosphorylation, acylation (including acetylation and formylation), glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation (including methylation and ethylation), ubiquitination, addition of pyrrolidone carboxylic acid, formation of disulfide bridges, sulfation, myristoylation, palmitoylation, prenylation, farnesylation, geranylation, glycosylphosphatidylinositolization, lipidation, and iodination. The term "polypeptide" or "peptide" may also mean a polypeptide that has been separated from components that naturally accompany it. Typically, when the polypeptide is at least 60% by weight, it can be separated from proteins and naturally occurring organic molecules that are naturally associated with it. In some embodiments, the preparation may be at least 75%, at least 90%, or at least 99% by weight of the polypeptide. An isolated polypeptide may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemical synthesis of the protein. Purity can be measured by any suitable method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0162] In some embodiments, an "immunogenic" peptide or "immunogenic" epitope or "peptide epitope" can be a peptide comprising an allele-specific motif such that the peptide will bind to an HLA molecule and induce a cell-mediated or humoral response, e.g., cytotoxic T lymphocytes (CTLs (e.g., CD8+ )), helper T lymphocytes (Th (e.g., CD4 + Thus, the immunogenic peptides described herein are capable of binding to appropriate HLA molecules and then inducing a CTL (cytotoxic) response or a HTL (and humoral) response to the peptides.

[0163] In some embodiments, the term "neoantigen" or "neoantigenicity" may refer to a class of tumor antigens generated by one or more tumor-specific mutations that change the amino acid sequence of a protein encoded by the genome. Neoantigens include, but are not limited to, tumor antigens that are generated, for example, by substitutions of protein sequences, frameshift mutations, polypeptide fusions, in-frame deletions, insertions, expression of endogenous retroviral polypeptides, and tumor-specific overexpression of polypeptides.

[0164] In some embodiments, the terms "neoantigen peptide" and "neoantigenic peptide" are used interchangeably with "peptide" in this specification, and may refer to a series of residues (typically L-amino acids) linked to each other, typically through peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. Similarly, in some embodiments, the term "polypeptide" may be used interchangeably with "mutant polypeptide", "neoantigen polypeptide" and "neoantigenic polypeptide" in this specification to refer to a series of residues (e.g., L-amino acids) linked to each other, typically through peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. In some embodiments, the polypeptide or peptide may be of various lengths, in its neutral (uncharged) form or salt form, and may be free of modifications (such as glycosylation, side chain oxidation or phosphorylation) or contain such modifications, provided that the modification does not destroy the biological activity of the polypeptide as described herein. In some embodiments, the peptide or polypeptide as used herein may include at least one flanking sequence. In some embodiments, the term "flanking sequence" as used herein may refer to a fragment or region of a neoantigenic peptide that is not part of a neoepitope. In some embodiments, the term "residue" may refer to an amino acid residue or an amino acid mimetic residue that is incorporated into a peptide or protein via an amide bond or an amide bond mimetic, or a nucleic acid (DNA or RNA) that may encode an amino acid or an amino acid mimetic.

[0165] In some embodiments, "neoplasia" can mean any disease caused or produced by inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. For example, cancer is an example of a neoplasm. Examples of cancer include, but are not limited to, leukemias (e.g., acute leukemias, acute lymphocytic leukemias, acute myeloid leukemias, acute myeloblastic leukemias, acute promyelocytic leukemias, acute myelomonocytic leukemias, acute monocytic leukemias, acute erythroleukemias, chronic leukemias, chronic myeloid leukemias, chronic lymphocytic leukemias), polycythemia vera, lymphomas (e.g., Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain diseases, and solid tumors (such as sarcomas and carcinomas (e.g., fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, osteoblastic sarcoma, chordoma, angiosarcoma, endothelial cell sarcoma, lymphangiosarcoma, lymphangioendothelial cell sarcoma, synovioma, mesothelioma, sarcomas ... The diseases described herein include, but are not limited to, leiomyoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). Lymphoproliferative disorders are also considered proliferative diseases.

[0166] In some embodiments, the term "tumor vaccine" may refer to a pooled sample of neoplasia / tumor-specific neoantigens (e.g., at least two, at least three, at least four, at least five or more neoantigenic peptides). In some embodiments, "vaccine" should be understood to mean a composition used to generate immunity for the prevention and / or treatment of a disease (e.g., a tumor / tumor). Therefore, a vaccine may be a medicament comprising an antigen and intended for use in humans or animals to generate specific defenses and protective substances through vaccination. In some embodiments, a "vaccine composition" or "tumor vaccine composition" may include a pharmaceutically acceptable excipient, carrier, or diluent.

[0167] In some embodiments, immune checkpoints can affect inhibitory pathways that slow down or terminate immune responses and prevent uncontrolled activity of immune cells from causing excessive tissue damage. "Checkpoint inhibitors" can refer to any small molecule chemical compound, antibody, nucleic acid molecule or polypeptide or fragment thereof, which can inhibit the inhibitory pathway, thereby allowing a wider range of immune activity. In certain embodiments, checkpoint inhibitors are inhibitors of programmed death protein-1 (PD-1) pathways, such as anti-PD1 antibodies, such as but not limited to nivolumab. In other embodiments, checkpoint inhibitors are anti-cytotoxic T lymphocyte-associated antigen (CTLA-4) antibodies. In other embodiments, checkpoint inhibitors target CD28 CTLA4 (g superfamily, such as BTLA, LAG3, ICOS, PDL1 or KIR, Page et al., Annual Review of Medicine 65: 27 (2014)) another member. In some cases, targeted checkpoint inhibitors are completed with inhibitory antibodies or similar molecules. In other cases, targeted checkpoint inhibitors are completed with agonists for targets.

[0168] In other embodiments, the inhibitor targets a member of the TNF superfamily, such as CD40, OX40, CD137, GITR, CD27, or TIM-3. In some cases, targeting a member of the TNF superfamily is accomplished with an inhibitory antibody or similar molecule. In other cases, targeting a member of the TNF superfamily can be accomplished with an agonist directed to the target; examples of this type include stimulatory targets CD40, OX40, and GITR.

[0169] The term "combination" may include in some embodiments the administration of a vaccine or immunogenic composition (e.g., a pooled sample of tumor / tumor-specific neoantigens) and one or more inhibitors (such as checkpoint inhibitors or chemotherapeutic agents) as part of a treatment regimen that is intended to provide a beneficial (additive or synergistic) effect through the combined action of one or more of these therapeutic agents. The combination may also include one or more additional agents, such as, but not limited to, chemotherapeutic agents, anti-angiogenic agents, and agents that reduce immunosuppression. The beneficial effects of the combination include, but are not limited to, pharmacokinetic and pharmacodynamic co-actions caused by the combination of therapeutic agents. The combined administration of these therapeutic agents may typically be performed within a defined time period (e.g., minutes, hours, days, or weeks, depending on the selected combination).

[0170] "Combination therapy" may be intended to include administering these therapeutic agents in a sequential manner, that is, wherein each therapeutic agent may be administered at different times, and administering these therapeutic agents or at least two of these therapeutic agents in a substantially simultaneous manner. Substantially simultaneous administration may be accomplished, for example, by administering to the subject a single capsule of each therapeutic agent having a fixed ratio or a plurality of single capsules for each of these therapeutic agents. For example, a combination of the present disclosure may include a pooled sample of tumor-specific neoantigens and inhibitors (such as checkpoint inhibitors or chemotherapeutics) administered at the same or different times, or the composition may be formulated into a single, co-formulated pharmaceutical composition comprising the two compounds. As another example, a combination of the present disclosure (e.g., a pooled sample of tumor-specific neoantigens and inhibitors (such as checkpoint inhibitors (e.g., anti-PD-L1 antibodies) and / or chemotherapeutics)) may be formulated into a separate pharmaceutical composition that may be administered at the same or different times. As used herein, the term "simultaneously" may refer to the simultaneous administration of one or more agents. For example, in certain embodiments, a vaccine or immunogenic composition and an inhibitor (such as a checkpoint inhibitor or chemotherapeutic) are administered simultaneously. At the same time, it includes concurrent administration, i.e., administration during the same time period. In certain embodiments, one or more medicaments can be administered simultaneously within the same hour, or simultaneously on the same day. The sequential administration or substantially simultaneous administration of each therapeutic agent can be achieved by any appropriate approach, including but not limited to oral route, intravenous route, subcutaneous route, intramuscular route, direct absorption by mucosal tissue (e.g., nose, mouth, vagina and rectum) and ocular route (e.g., intravitreal, intraocular, etc.). Therapeutic agents can be administered by the same approach or by different approaches. For example, a component of a particular combination can be administered by intravenous injection, and one or more other components of the combination can be administered orally. These components can be administered in any therapeutically effective order. The phrase "combination" includes a group of compounds or non-drug therapies that can be used as a part of a combination therapy.

[0171] The term "pharmaceutically acceptable" may mean approved or approvable by a regulatory agency of the Federal or a state government or listed in the US Pharmacopeia or other generally recognized pharmacopeia, for use in animals, including humans. Examples of cancer can include, without limitation, leukemias (e.g., acute leukemias, acute lymphocytic leukemias, acute myeloid leukemias, acute myeloblastic leukemias, acute promyelocytic leukemias, acute myelomonocytic leukemias, acute monocytic leukemias, acute erythroleukemias, chronic leukemias, chronic myeloid leukemias, chronic lymphocytic leukemias), polycythemia vera, lymphomas (e.g., Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors (such as sarcomas and carcinomas (e.g., fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, osteoblastic sarcoma, chordoma, angiosarcoma, endothelial cell sarcoma, lymphangiosarcoma, lymphangioendothelial cell sarcoma, synovioma, mesothelioma, The term "proliferative disease" refers to a type of cancer that is not a monoclonal antibody. The term "proliferative disease" refers to a type of cancer that is not a monoclonal antibody. The term "proliferative disease" refers to a type of cancer that is not a monoclonal antibody. The term "proliferative disease" refers to a type of cancer that is not a monoclonal antibody. The term "proliferative disease" refers to a type of cancer that is not a monoclonal antibody. The term "proliferative disease" refers to a type of cancer that is not a monoclonal antibody.

[0172] A "pharmaceutically acceptable excipient, carrier or diluent" may refer to an excipient, carrier or diluent that can be administered to a subject together with a pharmaceutical agent and that does not destroy the pharmacological activity of the pharmaceutical agent and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the pharmaceutical agent.

[0173] A "pharmaceutically acceptable salt" of the tumor-specific neoantigens described herein may in some embodiments be an acid salt or a base salt that is generally considered in the art to be suitable for contact with human or animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications. Such salts include mineral salts and organic acid salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutically acceptable salts include, but are not limited to, acid salts such as hydrochloride, phosphate, hydrobromide, malate, glycolate, fumarate, sulfate, sulfamate, sulfanilate, format, toluenesulfonate, methanesulfonate, benzenesulfonate, edisylate, 2-hydroxyethylsulfonate, nitrate, benzoate, 2-acetoxybenzoate, citrate, tartrate, lactate, stearate, salicylate, glutamate, ascorbate, pamoate, succinate, fumarate, maleate, propionate, hydroxymaleate, hydroiodide, phenylacetate, alkanoate (such as acetate, HOOC-(CH2)n-COOH salt, wherein n is 0-4), etc. Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those of ordinary skill in the art will recognize from this disclosure and the knowledge in the art additional pharmaceutically acceptable salts for the tumor-specific neoantigens provided herein, including those listed by Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, PA, page 1418 (1985). In general, pharmaceutically acceptable acid or base salts can be synthesized from a parent compound containing a basic or acidic moiety by any conventional chemical method. In short, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of an appropriate base or acid in an appropriate solvent.

[0174] As used herein, in some embodiments, the terms "prevent", "preventing", "prophylactic treatment", and the like may refer to reducing the probability of a subject developing a disease or condition, wherein the subject does not have the disease or condition but is at risk of or susceptible to the disease or condition.

[0175] The term "prime / boost" or "prime / boost dosing regimen" may refer to the continuous administration of a vaccine or immunogenic composition or immune composition. A priming administration (priming) may be the administration of a first vaccine or immunogenic composition or immune composition type, and may include one, two, or more administrations. A booster administration may be the second administration of a vaccine or immunogenic composition or immune composition type, and may include one, two, or more administrations, and may, for example, include or consist essentially of annual administrations. In certain embodiments, the administration of a tumor vaccine or immunogenic composition may employ a priming / boost dosing regimen.

[0176] The scope provided herein can be understood as the abbreviation of all values ​​in the described scope.For example, 1 to 50 scopes should be understood to include any number, combination of numbers or subranges from the following: 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49 or 50, and all intermediate decimal values ​​between the above-mentioned integers, for example, 1.1,1.2,1.3,1.4,1.5,1.6,1.7,1.8 and 1.9.About subrange, " nested subrange " extending from any end point of scope is particularly considered. For example, nested sub-ranges of the exemplary range of 1 to 50 may include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in the other direction.

[0177] "Receptor" may be understood to mean a biological molecule or grouping of molecules that is capable of binding a ligand. Receptors may be used to transmit information in cells, cell structures or organisms. Receptors comprise at least one receptor unit and often contain two or more receptor units, wherein each receptor unit may consist of a protein molecule, in particular a glycoprotein molecule. Receptors may have a structure that is complementary to the structure of a ligand and may complex with a ligand as a binding partner. Signal transduction information may be transmitted by conformational changes after the receptor binds to a ligand on the cell surface. According to the present disclosure, a receptor may refer to a specific protein of MHC class I and class II that is capable of forming a receptor / ligand complex with a ligand, in particular a peptide or peptide fragment of a suitable length.

[0178] The term "subject" refers to an animal that can be used as a treatment, observation or experimental object. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or non-human mammal, such as a non-human primate, a cow, a horse, a dog, a sheep or a cat.

[0179] The terms "treat", "treated", "treating", "treatment", and the like may be intended to refer to the reduction or improvement of a condition (e.g., a tumor or tumour) and / or symptoms associated therewith. "Treatment" may refer to the administration of a combination therapy to a subject after the onset or suspected onset of cancer. "Treatment" may include the concept of "relief", which refers to reducing the frequency or severity of the occurrence or recurrence of any symptom or other adverse effect associated with cancer and / or side effects associated with cancer therapy. The term "treatment" may also encompass the concept of "management", which refers to reducing the severity of a patient's particular disease or condition or delaying its recurrence, for example, prolonging the period of remission for a patient suffering from the disease. It will be understood that, although it cannot be excluded, the treatment of a condition or illness does not require the complete elimination of the condition, illness, or symptoms associated therewith.

[0180] The term "therapeutic effect" may refer to a certain degree of relief of one or more symptoms of a disease (e.g., a tumor or a tumour) or its associated pathology. "Therapeutically effective amount" as used herein may refer to the amount of an effective agent that exceeds the expected condition in the absence of such treatment, such as extending the viability of a patient with such a disease, reducing one or more signs or symptoms of the disease, preventing or delaying, etc., when a single or multiple doses are administered to a cell or object. "Therapeutically effective amount" may be intended to limit the amount required to achieve a therapeutic effect. A physician or veterinarian with ordinary skills in the art can easily determine and prescribe a "therapeutically effective amount" (e.g., ED50) of the desired pharmaceutical composition. For example, a physician or veterinarian can begin administering a compound of the present disclosure used in a pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0181] An "adverse event" or AE may generally refer to any unfortunate medical occurrence in a patient to whom a drug is administered that is not necessarily causally related to the treatment. An AE may be any unfavorable and unintentional sign (e.g., including abnormal laboratory results), symptom, or illness that is transiently associated with the use of an investigational drug, whether or not it is considered related to the study treatment. This includes any new occurrence or pre-existing condition that increases in severity or frequency since the administration of the study treatment. An abnormal laboratory value or test result may constitute an AE only if it causes clinical signs or symptoms, is considered clinically significant, or requires therapy.

[0182] The progression of the cancer being studied cannot be considered an AE unless the patient's care team considers it to be drug-related. "Adverse drug reactions (ADRs)" can be defined as all harmful and unexpected responses of a drug associated with any dose. There is at least a reasonable possibility of a causal relationship between the drug and the AE, i.e., such a relationship cannot be ruled out. An expected AE may be an AE listed or characterized in the applicable product information (e.g., current IB). An unexpected AE may be an AE whose nature, severity, or frequency is not identified, as described in the applicable product information (e.g., current IB). An unexpected ADR may be an ADR whose nature or severity is inconsistent with the applicable product information. ADRs that are more specific or more severe than those described in the IB may also be considered unexpected. A “serious adverse event” or SAE can be any AE occurring at any dose, regardless of causality: results in death; is life-threatening (life-threatening means that the patient is at immediate risk of death as a result of the reaction that occurs, i.e., reactions that could hypothetically result in death if they occurred in a more severe form cannot be included); requires hospitalization of the patient or prolongation of an existing hospitalization (if the patient had a pre-existing condition or disease prior to study enrollment, a hospitalization and / or surgical procedure that was planned to occur during the study but was planned prior to study enrollment cannot be considered an AE, provided that it did not worsen in an unexpected manner during the study (e.g., surgery was performed earlier than planned)); results in persistent or significant disability / incapacity (disability can be defined as a significant disruption in a person’s ability to carry out normal life functions); can be a congenital anomaly / birth defect or a major medical event, defined as an event that does not result in death, is life-threatening, or requires hospitalization but that, based on appropriate medical judgment, can be considered a SAE when it could endanger one or more patients and could require medical or surgical intervention to prevent one of the outcomes listed in the definition of an SAE. Examples of such medical events include allergic bronchospasm requiring intensive treatment in the emergency department or at home, blood dyscrasias or seizures that do not result in the patient being hospitalized, or the development of drug dependence or abuse.

[0183] Each patient can be carefully monitored for any AEs from the signing of the consent form until 30 days after treatment cessation. This information can be obtained in the form of non-leading questions (e.g., “How do you feel?”) as well as signs and symptoms detected during each examination, observations by the investigator, and spontaneous reports by the patient. All AEs (serious and non-serious) reported spontaneously by the patient and / or in response to the investigator's open-ended questions or revealed by observation, physical examination or other diagnostic procedures can be recorded on the appropriate page of the eCRF. Where possible, signs and symptoms indicating common underlying pathology can be recorded as a composite event. All SAEs occurring from the signing of the ICF until 90 days after the last dose of nivolumab or 30 days after the last dose of nivolumab (if the patient is starting a new anticancer therapy) should be reported by the investigator to the sponsor assigned to participate in the study within 1 working day after the time point of the SAE being perceived (see below). If the sponsor cannot be contacted for any reason, other physicians may be contacted. All SAEs can be reported regardless of whether they are considered to be causally related to the study treatment. An SAE form may be completed and the information collected may include the patient number, a narrative description of the event, and the investigator's assessment of the event's severity and relatedness to study treatment. The sponsor or CRO may request a sample of follow-up information on SAEs.

[0184] The present disclosure relates to methods of treating neoplasms, more particularly tumors, by administering to a subject a tumor vaccine or immunogenic composition comprising a plurality of tumor / tumor-specific neoantigens and at least one inhibitor, such as a checkpoint inhibitor or a chemotherapeutic agent.

[0185] Human tumors can contain a large number of unique deoxyribonucleic acid (DNA) mutations that result in changes in the amino acid sequence of the encoded protein. These new protein sequences, often referred to as neoantigens, range from single amino acid changes (caused by missense mutations) to long regions where new amino acid sequences are added due to frameshifts, read-through of stop codons, or translation of intronic regions (new open reading frames [neoORFs]). Tumor neoantigens are generated primarily due to mutations in the tumor. Therefore, they are extremely tumor-specific and are not limited by the immunosuppressive effects of self-tolerance.

[0186] The immune response to neoantigens can depend largely on the ability of major histocompatibility complex (MHC) molecules to effectively bind small peptides (epitopes) containing altered amino acid sequences and present them to T cells. Such epitopes can be synthetically generated and used in vaccines to initiate antigen-specific T cell responses targeting tumor cells expressing mutant proteins.

[0187] The binding of peptides to MHC can be used as a surrogate for the immunogenicity of a given peptide sequence. Advanced algorithms for predicting the binding of peptides to MHC have been developed using a large amount of peptide binding data to different MHC molecules (Lundegaard, 2011). These algorithms can be used to predict with high accuracy whether a specific peptide sequence will bind to MHC and with what affinity. Using these algorithms, protein sequences containing tumor-encoded mutations (both missense and neoORF) can be evaluated in silico for binding to specific MHC molecules.

[0188] In some embodiments, for example, if the subject suffers from cancer, the subject may comprise a mutant epitope comprising an altered amino acid sequence. In one embodiment, the mutant epitope is determined by sequencing the genome and / or exome of tumor tissue and healthy tissue from a cancer patient using next generation sequencing technology. In another embodiment, a gene selected based on its mutation frequency and ability to act as a neoantigen is sequenced using next generation sequencing technology. Next generation sequencing is applicable to genome sequencing, genome resequencing, transcriptome analysis (RNA-Seq), DNA-protein interactions (ChiP sequencing) and epigenomic characterization (de Magalhaes JP, Finch CE, Janssens G (2010). "Next-generation sequencing in aging research: emerging applications, problems, pitfalls and possible solutions". Ageing Research Reviews 9(3): 315-323; Hall N (Can 2007). "Advanced sequencing technologies and their wider impact in microbiology". J. Exp. Biol. 209(Pt 9): 1518-1525; Church GM (January 2006). "Genomes for ail". Sci. Am. 294(1): 46-54; ten Bosch JR, Grody WW (2008). "Keeping Up with the Next Generation". The Journal of Molecular Diagnostics 10(6):484-492; Tucker T, Marra M, Friedman JM (2009). "Massively Parallel Sequencing: The Next Big Thingin Genetic Medicine". The American Journal of Human Genetics 85(2):142-154).

[0189] Next generation sequencing can now rapidly reveal the presence of discrete mutations, such as coding mutations in individual tumors, most commonly single amino acid changes (e.g., missense mutations), as well as less common novel amino acid segments generated by frameshift insertions / deletions / gene fusions, readthrough mutations in stop codons, and translation of improperly spliced ​​introns (e.g., neoORFs). NeoORFs are particularly valuable as immunogens because their entire sequence is novel to the immune system and thus resembles viral or bacterial exogenous antigens. Thus, neoORFs: (1) are highly specific to tumors (i.e., not expressed in any normal cells); and (2) can bypass central tolerance, thereby increasing the precursor frequency of neoantigen-specific CTLs. For example, the ability to utilize similar exogenous sequences in therapeutic anticancer vaccines or immunogenic compositions has recently been demonstrated with peptides derived from human papillomavirus (HPV). Among 19 patients with proneoplastic virus-induced disease who received 3-4 vaccinations with a mixture of HPV peptides derived from the viral oncogenes E6 and E7, -50% maintained complete remission for >24 months (Kenter et al., Vaccination against HPV-16 Oncoproteins for Vulvar Intraepithelial Neoplasia NEJM 361:1838 (2009)).

[0190] Sequencing techniques have shown that each tumor contains a variety of patient-specific mutations that alter the protein-coding content of genes. Such mutations produce altered proteins ranging from single amino acid changes (caused by missense mutations) to long regions that add new amino acid sequences due to frameshifts, read-through of stop codons, or translation of intronic regions (new open reading frame mutations; neoORFs). Unlike natural proteins, these mutant proteins are valuable targets for the host's immune response to tumors; they are not limited by the immunosuppressive effects of self-tolerance. Therefore, compared with the patient's normal cells, mutant proteins are more likely to be immunogenic and more specific to tumor cells.

[0191] An alternative method for identifying tumor-specific neoantigens is direct protein sequencing. Protein sequencing of enzymatic digests using multidimensional MS techniques (MSn), including tandem mass spectrometry (MS / MS), can also be used to identify neoantigens of the present disclosure. Such proteomics methods allow for rapid, highly automated analysis (see, e.g., Gevaert and J. Vandekerckhove, Electrophoresis 21: 1145-1154 (2000)). It is further contemplated within the scope of the present disclosure that high-throughput methods for de novo sequencing of unknown proteins can be used to analyze the proteome of a patient's tumor to identify expressed neoantigens. For example, meta shotgun protein sequencing can be used to identify expressed neoantigens (see, e.g., Gutilais et al. (2012) Shotgun Protein Sequencing with Meta-contig Assembly, Molecular and Cellular Proteomics 11 (30): 3084-96).

[0192] Tumor-specific neoantigens can also be identified using MHC multimers to identify neoantigen-specific T cell responses. For example, MHC tetramer-based screening techniques can be used for high-throughput analysis of neoantigen-specific T cell responses in patient samples (see, e.g., Hombrink et al. (2011) High-Throughput Identification of Potential Minor Histocompatibility Antigens by MHC Tetramer-Based Screening: Feasibility and Limitations 6(8): 1-11; Hadrup et al. (2009) Parallel detection of antigen-specific T-cell. responses by multidimensional encoding of MHC multimers, Nature Methods, 6(7): 520-26; van Rooij et al. (2013) Tumor exome analysis reveals neoantigen-specific T-cell reactivity in an Ipilimumab-responsive melanoma, Journal of Clinical Oncology, 31: 1-4; and Heemskerk et al. (2013) The cancer antigenome, EMBO Journal, 32(2): 194-203). Such tetramer-based screening techniques can be used for the primary identification of tumor-specific neoantigens, or alternatively as a secondary screening approach to assess what neoantigens a patient may have been exposed to, thereby aiding in the selection of candidate neoantigens for use in the present disclosure.

[0193] In one embodiment, sequencing data obtained from determining whether a mutation exists in a cancer patient is analyzed to predict personalized mutant peptides that can bind to an individual's HLA molecules. In one embodiment, a computer is used to analyze the data. In another embodiment, the sequence data is analyzed for the presence of neoantigens. In one embodiment, neoantigens are determined by their affinity for MHC molecules. Effective selection of which specific mutations to use as immunogens requires the ability to identify the patient's HLA type and predict which mutant peptides will effectively bind to the patient's HLA alleles. Recently, a neural network-based learning method using validated binding and non-binding peptides has improved the accuracy of the prediction algorithm for major HLA-A and HLA-B alleles. By utilizing recently improved algorithms to predict which missense mutations produce peptides that bind strongly to the patient's cognate MHC molecules, a collection of peptides representing the best mutant epitopes (both neoORF and missense) for each patient can be identified and prioritized (Zhang et al., Machine learning competition in immunology-Prediction of HLA class I binding peptides J Immunol Methods 374:1 (2011); Lundegaard et al., Prediction of epitopes using neural network based methods J Immunol Methods 374:26 (2011)).

[0194] Targeting as many mutant epitopes as possible utilizes the tremendous capacity of the immune system, prevents the opportunity of immune escape by downregulating specific immune targeting gene products, and compensates for the known inaccuracies of epitope prediction methods. Synthetic peptides provide a particularly useful means for effectively preparing multiple immunogens and quickly converting the identification of mutant epitopes into effective vaccines or immunogenic compositions. Peptides can be easily chemically synthesized and purified using reagents that do not contain contaminating bacteria or animal substances. Small size can clearly focus on the mutant region of the protein, and also reduces irrelevant antigenic competition with other components (unmutated proteins or viral vector antigens).

[0195] In one embodiment, the pharmaceutical formulation is a multi-epitope vaccine or immunogenic composition of a long peptide. Such "long" peptides undergo effective internalization, processing and cross-presentation in professional antigen presenting cells (such as dendritic cells), and have been shown to induce CTL in humans (Melief and van der Burg, Immunotherapy of established (pre) malignant disease by synthetic long peptide vaccines Nature Rev Cancer 8: 351 (2008)). In one embodiment, at least one peptide is prepared for immunization. In some embodiments, 20 or more peptides are prepared for immunization. In one embodiment, the length of the neoantigenic peptide is in the range of about 5 to about 50 amino acids. In another embodiment, a peptide with a length of about 15 to about 35 amino acids is synthesized. In some embodiments, the length of the neoantigenic peptide is in the range of about 20 to about 35 amino acids.

[0196] In some embodiments, a personalized cancer vaccine consisting of up to 20 synthetic peptides of approximately 14 to 35 amino acids in length is provided, which are obtained from the mutant tumor DNA (neoantigens) of an individual patient. Since these mutations are not expressed in the patient's normal cells, they are specific targets expressed only on tumor cells.

[0197] Unlike most previously used cancer vaccines, this neoantigen peptide vaccine is based on the generation of a novel unique product for each individual patient or cancer phenotype. The degree of possible tumor mutations and the wide range of human leukocyte antigen (HLA) haplotypes in patients make it extremely unlikely that any 2 patients will receive the same vaccine.

[0198] The generation of neoantigens can begin with whole exome DNA and RNA sequencing of tumor and normal tissue samples and the HLA-A, HLA-B, and HLA-C genotype of the subject. These data can then be used to identify coding sequence mutations that have occurred in the subject's tumor. In some cases, these mutations can include single amino acid missense mutations, fusion proteins, and neoORFs that can vary in length from 1 amino acid to up to hundreds of amino acids. Long peptides of 14-35 residues in length can then be specifically designed based on the specific mutations identified in individual tumors. The vaccine can then consist of a mixture of the following peptides, which are predicted to induce a response in CD4+ and / or CD8+ T cells. In order to predict which peptides are most likely to induce this immune response, multiple filters can be applied to the entire collection of long peptides covering the subject's tumor mutation group. The main criterion is the HLA binding affinity of the mutant epitope compared to its native protein. Epitope selection algorithms can be used to identify mutation-containing epitopes that are predicted to bind to each subject's MHC class I molecule (Lundegaard, 2011). Other key criteria can include RNA expression, mutation type (e.g., missense vs. neoORF), the likelihood that the mutation is an oncogenic driver, and the physical location of one or more mutant residues on the peptide. Up to 35 peptides can be selected and prioritized for synthesis. Thereafter, up to 20 synthetic peptides can be mixed in up to 4 pools for injection, with up to 5 peptides per pool. Each of the 4 pools can be injected into a subject.

[0199] Ⅱ. Generation of tumor-specific neoantigens

[0200] The present disclosure is based, at least in part, on the ability to present a pool of tumor-specific neoantigens to the patient's immune system. A person skilled in the art will understand, based on the present disclosure and the knowledge in the art, that there are a variety of methods for producing such tumor-specific neoantigens. Typically, such tumor-specific neoantigens can be produced in vitro or in vivo. Tumor-specific neoantigens can be produced in vitro in the form of peptides or polypeptides, which can then be formulated into personalized tumor vaccines or immunogenic compositions and administered to a subject. As described in further detail herein, such in vitro production can be performed by various methods known to those skilled in the art, for example, peptide synthesis or expression of peptides / polypeptides from DNA or RNA molecules in any of a variety of bacterial, eukaryotic or viral recombinant expression systems, followed by purification of the expressed peptides / polypeptides. Alternatively, tumor-specific neoantigens can be produced in vivo by introducing molecules encoding tumor-specific neoantigens (e.g., DNA, RNA, viral expression systems, etc.) into a subject, followed by expression of multiple encoded tumor-specific neoantigens. Methods for producing neoantigens in vitro and in vivo are also further described herein, as are methods for pharmaceutical compositions and delivery combination therapies.

[0201] A. In vitro peptide / peptide synthesis

[0202] Protein or peptide can be prepared by any technology known to those skilled in the art, including expression of protein, polypeptide or peptide by standard molecular biology techniques, separation of protein or peptide from natural sources, in vitro translation, or chemical synthesis of protein or peptide. Nucleotide and protein, polypeptide and peptide sequences corresponding to various genes have been previously disclosed and can be found in computerized databases known to those of ordinary skill in the art. Exemplary databases can be found in the National Center for Biotechnology Information, Genbank and GenPept databases located at the website of the National Institutes of Health. The coding region of known genes can be amplified and / or expressed using technology disclosed herein or technology known to those of ordinary skill in the art. Alternatively, various commercial preparations of proteins, polypeptides and peptides are known to those skilled in the art.

[0203] Peptides can be easily chemically synthesized using reagents that do not contain contaminating bacteria or animal substances (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a trapeptide. J. Am. Chem. Soc. 85: 2149-54, 1963). In certain embodiments, neoantigenic peptides are prepared by: (1) parallel solid phase synthesis on a multichannel instrument using uniform synthesis and cleavage conditions; (2) purification on a P-HPLC column with column stripping; and re-washing between peptides, but not replacement; followed by (3) analysis with a limited set of the most informative assays. A good manufacturing practice (GMP) footprint can be determined around a collection of peptides for an individual patient, so only kit changes to the procedure are required between peptide syntheses for different patients.

[0204] Alternatively, nucleic acids (e.g., polynucleotides) encoding neoantigenic peptides of the present disclosure can be used to produce neoantigenic peptides in vitro. The polynucleotides can be, for example, single-stranded and / or double-stranded forms of DNA, cDNA, PNA, CNA, RNA, polynucleotides or natural or stable forms (e.g., polynucleotides with a thiophosphate backbone), or a combination thereof, and as long as it encodes a peptide, it may or may not contain introns. In one embodiment, in vitro translation is used to produce peptides. There are many exemplary systems that can be used by those skilled in the art (e.g., Retic Lysate IVT kit, Life Technologies, Waltham, MA).

[0205] Expression vectors capable of expressing polypeptides can also be prepared. The expression vectors of different cell types are well known in the art, and can be selected without too many experiments. Usually, DNA is inserted into an expression vector (such as a plasmid) with appropriate direction and correct reading frame for expression, and if necessary, DNA can be connected to the appropriate transcription and translation regulation control nucleotide sequence identified by the desired host (e.g., bacteria), although this control is usually available in the expression vector. Then using standard techniques, the vector is introduced into the host bacteria for cloning (see, for example, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0206] Also contemplated are expression vectors comprising isolated polynucleotides and host cells comprising the expression vectors. Neoantigenic peptides can be provided in the form of RNA or cDNA molecules encoding the desired neoantigenic peptides. One or more neoantigenic peptides of the present disclosure can be encoded by a single expression vector.

[0207] The term "polynucleotide encoding a polypeptide" encompasses polynucleotides that include only the coding sequence of the polypeptide as well as polynucleotides that include additional coding and / or non-coding sequences. The polynucleotide may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and if a single strand may be a coding strand or a non-coding (antisense) strand, the DNA may be double-stranded or single-stranded.

[0208] In an embodiment, the polynucleotide may comprise a coding sequence for a tumor-specific neoantigenic peptide fused in the same reading frame to a polynucleotide that facilitates, for example, expression and / or secretion of the polypeptide from a host cell (e.g., a leader sequence that functions as a secretory sequence for controlling transport of the polypeptide from the cell). A polypeptide with a leader sequence is a preprotein and can allow the leader sequence to be cleaved by a host cell to form a mature form of the polypeptide.

[0209] In an embodiment, the polynucleotide may be included in the same reading frame with, for example, a marker sequence that allows purification of the encoded polypeptide, and the encoded polypeptide can then be incorporated into a personalized tumor vaccine or immunogenic composition. For example, in the case of a bacterial host, the marker sequence can be a hexameric histidine tag supplied by a pQE-9 vector to provide purification of a mature polypeptide fused to the marker, or when a mammalian host (e.g., COS-7 cells) is used, the marker sequence can be a hemagglutinin (HA) tag derived from an influenza virus hemagglutinin protein. Additional tags include, but are not limited to, calmodulin tags, FLAG tags, Myc tags, S tags, SBP tags, Softag 1, Softag 3, V5 tags, Xpress tags, Isopeptag, SpyTag, biotin carboxyl carrier protein (BCCP) tags, GST tags, fluorescent protein tags (e.g., green fluorescent protein tags), maltose binding protein tags, Nus tags, Strep tags, thioredoxin tags, TC tags, Ty tags, etc.

[0210] In an embodiment, the polynucleotide may comprise the coding sequence of one or more tumor-specific neoantigenic peptides fused in the same reading frame to create a single concatemerized neoantigenic peptide construct capable of producing multiple neoantigenic peptides.

[0211] In certain embodiments, an isolated nucleic acid molecule having a nucleotide sequence can be provided that is at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, or at least 96%, 97%, 98% or 99% identical to a polynucleotide encoding a tumor-specific neoantigenic peptide of the present disclosure.

[0212] A polynucleotide having a nucleotide sequence that is at least, for example, 95% "identical" to a reference nucleotide sequence means that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may include up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence that is at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the amino or carboxyl terminal positions of the reference nucleotide sequence or anywhere between these terminal positions, interspersed individually between the nucleotides of the reference sequence or in one or more consecutive groups within the reference sequence.

[0213] In fact, whether any particular nucleic acid molecule is at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments at least 95%, 96%, 97%, 98% or 99% identical to a reference sequence can be routinely determined using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711). Bestfit uses the local homology algorithm of Smit and Waterman, Advances in Applied Mathematics 2:482-489 (1981) to find the best homology segment between two sequences. When Bestfit or any other sequence alignment program is used to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present disclosure, the parameters are set so that the percent identity is calculated over the entire length of the reference nucleotide sequence, and homology gaps in the reference sequence are allowed to account for up to 5% of the total number of nucleotides.

[0214] The isolated tumor-specific neoantigenic peptides described herein can be produced in vitro (e.g., in a laboratory) by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing DNA sequences encoding isolated polypeptide sequences and expressing these sequences in suitable transformed hosts. In some embodiments, recombinant technology is used to construct DNA sequences by isolating or synthesizing DNA sequences encoding wild-type target proteins. Optionally, the sequence can be mutated by site-specific mutagenesis to provide its functional analogs. See, for example, Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81: 5662-5066 (1984) and U.S. Patent No. 4,588,585.

[0215] In an embodiment, an oligonucleotide synthesizer is used to construct a DNA sequence encoding a target polypeptide by chemical synthesis. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and by selecting those codons preferred in a host cell (producing the target recombinant polypeptide therein). The isolated polynucleotide sequence of the isolated target polypeptide of the synthetic coding separation can be applied using standard methods. For example, a complete amino acid sequence can be used to construct a back-translated gene. In addition, a DNA oligomer containing a nucleotide sequence encoding a specific isolated polypeptide can be synthesized. For example, the small oligonucleotides of the parts of several desired polypeptides can be synthesized and then connected. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0216] Once assembled (e.g., by synthesis, site-directed mutagenesis or another method), the polynucleotide sequence encoding the specific isolated polypeptide of interest is inserted into an expression vector and operably linked to an expression control sequence suitable for protein expression in a desired host. Correct assembly can be confirmed by nucleotide sequencing, restriction mapping and expression of the biologically active polypeptide in a suitable host. As is well known in the art, in order to obtain a transfected gene with a high expression level in a host, the gene can be operably linked to transcription and translation expression control sequences that work in a selected expression host.

[0217] Recombinant expression vectors can be used to amplify and express DNA encoding tumor-specific neoantigen peptides. Recombinant expression vectors are replicable DNA constructs having synthetic or cDNA-derived DNA fragments encoding tumor-specific neoantigen peptides or bioequivalent analogs, which are operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral or insect genes. Transcription units typically include one or more genetic elements that have a regulatory effect in gene expression, such as transcriptional promoters or enhancers, (2) structural sequences or coding sequences that are transcribed into mRNA and translated into proteins, and (3) appropriate transcriptional and translational initiation and termination sequences, as described in detail herein. Such regulatory elements may include an operator sequence that controls transcription. In addition, selection genes that are usually conferred by the origin of replication to replicate in the host and promote the recognition of transformants may be incorporated. When DNA regions are functionally related to each other, they are operably linked. For example, the DNA of a signal peptide (secretory leader) is operably linked to the DNA of a polypeptide if it is expressed as a precursor that participates in the secretion of the polypeptide; a promoter is operably linked to a coding sequence if it controls the transcription of a sequence; or a ribosome binding site is operably linked to a coding sequence if its position allows translation. Usually, operably linked means continuous, and in the case of a secretory leader, operably linked means continuous and in the reading frame. Structural elements intended for use in yeast expression systems include leader sequences that allow host cells to secrete translated proteins to the extracellular space. Alternatively, in the case of recombinant proteins expressed without using a leader or transport sequence, the protein may include an N-terminal methionine residue. Subsequently, the residue may optionally be cut from the expressed recombinant protein to provide a final product.

[0218] Expression vectors useful for eukaryotic hosts, especially mammals or humans include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids, such as plasmids from Escherichia coli, including pCR 1, pBR322, pMB9, and derivatives thereof; broader host range plasmids, such as M1 3, and filamentous single-stranded DNA phages.

[0219] Suitable host cells for expressing polypeptides include prokaryotes, yeasts, insects or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram-negative or gram-positive organisms, such as Escherichia coli or bacillus. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems can also be used. Suitable cloning and expression vectors for bacterial, fungal, yeast and mammalian cell hosts are well known in the art (see Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, Y., 1985).

[0220] Various mammalian or insect cell culture systems are also advantageously used to express recombinant proteins. Expression of recombinant proteins can be carried out in mammalian cells because such proteins are usually correctly folded, appropriately modified and fully functional. Examples of suitable mammalian host cell lines include the COS-7 system of monkey kidney cells described by Gluzman (Cell 23:175, 1981), and other cell lines capable of expressing suitable vectors, including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), 293, HeLa and BHK cell lines. Mammalian expression vectors can include non-transcriptional elements, such as replication origin, suitable promoters and enhancers connected to the gene to be expressed, and other 5' or 3' flanking non-transcribed sequences and 5' or 3' non-translated sequences (such as necessary ribosome binding sites, polyadenylation sites, splicing donors and acceptor sites) and transcription termination sequences. Luckow and Summers, Bio / Technology 6:47 (1988) reviewed the baculovirus system for producing heterologous proteins in insect cells.

[0221] The protein produced by the transformed host can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange chromatography, affinity chromatography, and size fractionation column chromatography, etc.), centrifugation, differential solubility, or by any other standard techniques for protein purification. Affinity tags such as hexahistidine, maltose binding domain, influenza capsid sequence, glutathione-S-transferase, etc. can be attached to jl protein to allow protein to be purified more easily after passing through an appropriate affinity column. Such techniques such as proteolysis, nuclear magnetic resonance, and x-ray crystallography can also be used to physically characterize the separated protein.

[0222] For example, commercially available protein concentration filters (e.g., Amicon or Millipore Pellicon ultrafiltration units) can be used to concentrate the supernatant from the system, which secretes the recombinant protein into the culture medium. After the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin can be used, such as a matrix or substrate with a diethylaminoethyl (DEAE) group of a side joint. The matrix can be acrylamide, agarose, dextran, cellulose or other types commonly used in protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps using hydrophobic RP-HPLC media (e.g., silica gel with a methyl or other aliphatic group of a side joint) can be used to further purify the cancer stem cell protein-Fc composition. Some or all of the above purification steps in different combinations can also be used to provide a homogeneous recombinant protein.

[0223] Recombinant proteins produced in bacterial cultures can be isolated, for example, by initial extraction from a cell pellet followed by one or more concentration, salting out, aqueous ion exchange, or size exclusion chromatography steps. High performance liquid chromatography (HPLC) can be used for final purification steps. Microbial cells used for recombinant protein expression can be disrupted by any convenient method, including freeze-thaw cycles, sonication, mechanical disruption, or use of cell lysing agents.

[0224] B. In vivo peptide / polypeptide synthesis

[0225] The present disclosure also contemplates the use of nucleic acid molecules as vehicles for delivering neoantigenic peptides / polypeptides to subjects in need in vivo, for example, in the form of DNA / RNA vaccines (see, e.g., WO 2012 / 159643 and WO 2012 / 159754, which are incorporated herein by reference in their entireties).

[0226] In one embodiment, neoantigens can be administered to patients in need by using plasmids. These plasmids are usually composed of strong viral promoters for driving in vivo transcription and translation of the target gene (or complementary DNA) (Mor et al., (1995). The Journal of Immunology 155 (4): 2039-2046). Intron A can sometimes be included to improve mRNA stability and thereby increase protein expression (Leitner et al., (1997). The Journal of Immunology 159 (12): 6112-6119). Plasmids also include strong polyadenylation / transcription termination signals, such as bovine growth hormone or rabbit β-globin polyadenylation sequences (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42:343-410; Robinson et al., (2000). Adv. Virus Res. Advances in Virus Research 55:1-74; Bohm et al., (1996). Journal of Immunological Methods 193(i):29-40). Polycistronic vectors are sometimes constructed to express more than one immunogen, or to express an immunogen and an immunostimulatory protein (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54:129-88).

[0227] Since the plasmid is the "vehicle" for expressing the immunogen, it is critical to optimize the vector design for maximum protein expression (Lewis et al., (1999), Advances in Vims Research (Academic Press) 54: 129-88). One way to enhance protein expression is by optimizing the codon usage of the pathogenic mRNA of eukaryotic cells. Another consideration is the choice of promoter. Such promoters can be the SV40 promoter or Rous sarcoma virus (RSV).

[0228] Plasmids can be introduced into animal tissues by a variety of different methods. The two most popular methods are DNA injected in saline using a standard subcutaneous needle, and gene gun delivery. The construction of DNA vaccine plasmids and the schematic overview of the subsequent delivery to the host by these two methods are shown in Scientific American (Weiner et al., (1999) Scientific American 281 (I): 34-41). Saline injection is usually performed in skeletal muscle by intramuscular (IM), or intradermal (ID), where DNA is delivered to the extracellular space. This can be done by electroporation by temporarily damaging muscle fibers with myotoxins (such as bupivacaine); or by using a hypertonic solution of saline or sucrose to assist (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42: 343-410). The immune response to this delivery method can be influenced by many factors, including needle type, needle alignment, injection speed, injection volume, muscle type, and the age, sex, and physiological condition of the injected animal (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42:343-410).

[0229] Gene gun delivery is another commonly used delivery method, which uses compressed helium as a promoter to ballistically accelerate plasmid DNA (pDNA) adsorbed onto gold or tungsten microparticles into target cells (Alarcon et al., (1999), Adv, Parasitol. Advances in Parasitology 42:343-410; Lewis et al., (1999), Advances in Virus Research (Academic Press) 54:12.9-88).

[0230] Alternative delivery methods can include aerosol instillation of naked DNA on mucosal surfaces, such as nasal and lung mucosa (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88), and topical application of pDNA to the eye and vaginal mucosa (Lewis et al., (1999) Advances in Vims Research (Academic Press) 54: 129-88). Mucosal surface delivery has also been achieved using cationic liposome-DNA formulations, biodegradable microspheres, attenuated Shigella or Listeria vectors for oral administration to the intestinal mucosa, and recombinant adenoviral vectors.

[0231] The delivery method determines the dose of DNA required to produce an effective immune response. Saline injection requires varying amounts of DNA from 10 μg-1 mg, while gene gun delivery requires as little as 1 / 100 to 1 / 1000 of the DNA compared to intramuscular saline injection to produce an effective immune response. Typically, 0.2 μg-20 μg is required, but amounts as low as 16 ng have been reported. These amounts vary from species to species, for example, mice require as little as about 1 / 10 of the DNA compared to primates. Saline injection requires more DNA because the DNA is delivered to the extracellular space of the target tissue (usually muscle), where the DNA must overcome physical barriers (such as the basement membrane and extensive connective tissue, to name a few) before being taken up by the cells, whereas gene gun delivery bombards the DNA directly into the cells, thereby reducing "loss" (see, e.g., Sedegah et al., (1994). Proceedings of the National Academy of Sciences of the United States of America 91(21):9866-9870; Daheshia et al., (1997). The Journal of Immunology 159(4):1945-1952; Chen et al., (1998). The Journal of Immunology 160(5):2425-2432; Sizemore (1995) Science 270(5234):299-302; Fynan et al., (1993) Proc. Natl. Acad. Sci. USA 90(24):11478-82).

[0232] In one embodiment, the tumor vaccine or immunogenic composition can include a separate DNA plasmid encoding one or more neoantigenic peptides / polypeptides, such as those identified according to the present disclosure. As discussed herein, the precise selection of expression vectors can depend on the peptide / polypeptide to be expressed, and is well within the skill of the skilled person. It is expected that the expected persistence of the DNA construct (e.g., free form, non-replicating form, non-integrated form in muscle cells) provides increased protection duration.

[0233] One or more neoantigenic peptides of the present disclosure can be encoded and expressed in vivo using a virus-based system (e.g., an adenovirus system, an adenovirus-associated virus (AAV) vector, a poxvirus, or a lentivirus). In one embodiment, a tumor vaccine or immunogenic composition can include a virus-based vector, such as an adenovirus, for a human patient in need (see, e.g., Baden et al. First-in-human evaluation of the safety and immunogenicity of arecombinant adenovirus serotype 26 HIV-1Env vaccine (1PCAVD 001). J Infect Dis. January 15, 2013; 207 (2): 240-7, incorporated herein by reference in its entirety). Plasmids that can be used for adeno-associated virus, adenovirus, and lentivirus delivery have been previously described (see, e.g., U.S. Patent Nos. 6,955,808 and 6,943,019 and U.S. Patent Application No. 20080254008, incorporated herein by reference).

[0234] In the vector that can be used to practice the present disclosure, it is possible to integrate into the host genome of the cell using a retroviral gene transfer method (usually resulting in long-term expression of the inserted transgene). In some embodiments, the retrovirus is a lentivirus. In addition, high transduction efficiency has been observed in many different cell types and target tissues. The tropism of the retrovirus can be changed by incorporating exogenous envelope proteins and amplifying target populations of potential target cells. Retroviruses can also be engineered to allow conditional expression of the inserted transgene so that only certain cell types are infected by the lentivirus. Cell type-specific promoters can be used to target expression in specific cell types. Lentiviral vectors are retroviral vectors (so both lentiviral vectors and retroviral vectors can be used to practice the present disclosure). In addition, lentiviral vectors are able to transduce or infect non-dividing cells, and generally produce high viral titers. Therefore, the selection of retroviral gene transfer systems depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats that have packaging capabilities for exogenous sequences up to 6-10kb. The minimal cis-acting LTR is sufficient to replicate and package the vector, which is then used to integrate the desired nucleic acid into the target cell to provide permanent expression. Widely used retroviral vectors that can be used to practice the present disclosure include those based on murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., (1992) J. Virol. 66: 2731-2739; Johann et al., (1992) J. Virol. 66: 1635-1640; Sommnerfelt et al., (1990) Virol. 176: 58-59; Wilson et al., (1998) J. Virol. 63: 2374-2378; Miller et al., (1991) J. Virol. 65: 2220-2224; PCT / US94 / 05700). Zo et al. administered approximately 10 μl of a titer of 1 x 10 9 Transducing units (TU) / ml of recombinant lentivirus. These types of dosages can be adapted or extrapolated to use of the retroviral or lentiviral vectors of the present disclosure.

[0235] Also useful in the practice of the present disclosure are minimal non-primate lentiviral vectors, such as lentiviral vectors based on equine infectious anemia virus (EIAV) (see, e.g., Balagaan, (2006) J Gene Med; 8:275-285, published online on November 21, 2005 at Wiley InterScience (interscience.wiley.com). DOI: 1002 / jgm.845). The vector may have a cytomegalovirus (CMV) promoter driving expression of the target gene. Thus, the present disclosure contemplates one or more vectors useful in practicing the present disclosure: viral vectors, including retroviral vectors and lentiviral vectors.

[0236] Also useful in practicing the present disclosure are adenoviral vectors. One advantage is the ability of recombinant adenoviruses to efficiently transfer and express recombinant genes in a variety of mammalian cells and tissues in vitro and in vivo, which results in high expression of the transferred nucleic acid. In addition, the ability to productively infect quiescent cells increases the utility of recombinant adenoviral vectors. In addition, high expression levels ensure that the product of the nucleic acid will be expressed to a level sufficient to generate an immune response (see, e.g., U.S. Patent No. 7,029,848, incorporated herein by reference).

[0237] In the embodiments herein, delivery is via an adenovirus, which may contain at least 1 x 10 5 In embodiments herein, the dose may be at least about 1 x 10 6 particles (e.g., about 1 x 10 6 -1x 10 2 particles), at least about 1x 10 7 particles, at least about 1x 10 8 particles (e.g., about 1 x 10 8 -1x 10 11 particles or about 1x 10 8 -1x 10 12 particles), or at least about 1 x 10 9 particles (e.g., about 1 x 10 9 -lx 10 10 particles or about 1x 10 9 -1x 10 12 particles), or even at least about 1 x 10 10 particles (e.g., about 1 x 10 10 -1x 10 12 Alternatively, the dose contains no more than about 1 x 10 14particles, no more than about 1x 10 13 particles, no more than about 1x 10 12 particles, no more than about 1x 10 11 particles or no more than about 1x 10 10 particles (e.g., no more than about 1 x 10 9 particles). Thus, a dose may contain a single dose of adenoviral vector having, for example, about 1 x 10 6 Particle units (pu), about 2x 10 6 pu, about 4x 10 6 pu, about 1x 10 7 pu, about 2x 10 7 pu, about 4x 10 7 pu, about 1x 10 8 pu, about 2x 10 8 pu, about 4x 10 8 pu, about 1x 10 9 pu, about 2x 10 9 pu, about 4x 10 9 pu, about 1x 10 10 pu, about 2x10 10 pu, about 4x 10 10 pu, about 1x 10 11 pu, about 2x 10 11 pu, about 4x 10 11 pu, about 1x 10 12 pu, about 2x 10 12 pu, or about 4x 10 12 See, e.g., the adenoviral vectors in U.S. Pat. No. 8,454,972 B2, issued Jun. 4, 2013 to Nabel et al., incorporated herein by reference, and the dosages at col. 29, lines 36-58 thereof. In embodiments herein, the adenovirus is delivered via multiple doses.

[0238] In terms of in vivo delivery, AAV has advantages over other viral vectors due to its lower toxicity, and the possibility of causing insertional mutagenesis is lower because it will not integrate into the host genome. The packaging limit of AAV is 4.5 or 4.75Kb. Constructs greater than 4.5 or 4.75Kb result in a significant reduction in virus production. There are many promoters that can be used to drive the expression of nucleic acid molecules. AAV ITR can be used as a promoter, and is conducive to eliminating the need for additional promoter elements. For ubiquitous expression, the following promoters can be used: CMV, CAG, CBh, PGK, SV40, ferritin heavy chain or light chain, etc. For brain expression, the following promoters can be used: synaptic protein I for all neurons, CaMKIIα for excitatory neurons, GAD67 or GAD65 or VGAT for GABA neurons, etc. Promoters for driving RNA synthesis can include: Pol III promoters, such as U6 or HI. The use of Pol II promoters and intron boxes can be used to express guide RNA (gRNA).

[0239] As for AAV, the AAV can be AAV1, AAV2, AAV5, or any combination thereof. The AAV can be selected for the cells to be targeted; for example, AAV serotype 1, 2, 5, or hybrid capsid AAV1, AAV2, AAV5, or any combination thereof can be selected to target brain or neuronal cells; and AAV4 can be selected to target cardiac tissue. AAV8 can be used for delivery to the liver. The above promoters and vectors can be used alone.

[0240] In the embodiments herein, delivery is via AAV. It is believed that a therapeutically effective dose of AAV for in vivo delivery to humans is in the range of about 20 to about 50 ml of saline solution containing about 1 x 10 10 About 1x 10 50 Functional AAV / ml solution. The dosage can be adjusted to balance the therapeutic benefit with any side effects. In the embodiments herein, the AAV dosage is generally about 1 x 10 5 Up to 1x 10 50 Genome AAV, about 1x 10 8 Up to 1x 10 20 Genome AAV, about 1x 10 10 Up to 1x 10 16 genome, or about 1x 10 11 About 1x 10 16 The human dose can be about 1x 10 13These concentrations can be delivered in about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of carrier solution. In some embodiments, the AAV is used at a titer of about 2 x 10 13 Viral genomes / ml, and each striatal hemisphere of the mouse received a 500 nanoliter injection. Other effective doses can be easily determined by a person of ordinary skill in the art through routine experiments to establish dose response curves. (See, for example, U.S. Patent No. 8,404,658B2, which is incorporated herein by reference in its entirety).

[0241] In another embodiment, effective activation of the cellular immune response to tumor vaccines or immunogenic compositions can be achieved by expressing the relevant neoantigens in the vaccine or immunogenic composition in a non-pathogenic microorganism. Well-known examples of such microorganisms are Mycobacterium bovis BCG, Salmonella, and Pseudomonas (see U.S. Pat. No. 6,991,797, which is incorporated herein by reference in its entirety).

[0242] In another embodiment, poxviruses are used in tumor vaccines or immunogenic compositions. These poxviruses include orthopoxviruses, fowlpox, cowpox, MVA, NYVAC, canarypox, ALVAC, fowlpox, TROVAC, etc. (see, for example, Verardi et al., Hum Vaccin Immunother. 2012 July; 8(7): 961-70; and Moss, Vaccine. 2013: 31(39): 4220-4222). Poxvirus expression vectors were described in 1982 and were rapidly and widely used in vaccine development and research in many fields. The advantages of vectors include simple construction, the ability to accommodate large amounts of exogenous DNA, and high expression levels.

[0243] In another embodiment, vaccinia virus is used in tumor vaccines or immunogenic compositions to express neoantigens. (See, e.g., Rolph et al., Recombinant viruses as vaccines and immunological tools. Curr Opin Immunol 9: 517-524, 1997). Recombinant vaccinia virus is able to replicate in the cytoplasm of infected host cells, so the target polypeptide can induce an immune response. In addition, poxviruses have been widely used as vaccine or immunogenic composition carriers because they can target the encoded antigens by directly infecting immune cells (particularly antigen presenting cells) for processing through the major histocompatibility complex class I pathway, and also because of their own adjuvant capabilities.

[0244] In another embodiment, ALVAC is used as a vector in a tumor vaccine or immunogenic composition. ALVAC is a canarypox virus that can be modified to express foreign transgenes and has been used as a vaccination approach against prokaryotic and eukaryotic antigens (Horig H, Lee DS, Conkright W, et al. Phase I clinical trial, of arecombinant canarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule. Cancer Immunol Immunother 2000; 49: 504-14; von Mehren M, Arlen P, Tsang KY, et al. Pilot study of a dual generecombinant avipox vaccine containing both carcinoembryonic antigen (CEA) and B7.1 transgenes in patients with recurrent CEA-expressing adenocarcinomas. Clin Cancer Res 2000; 6: 2219-28; Musey L, Ding Y, Elizaga M, et al. HIV-1 vaccination administered intramuscularly can induce both, systemic and mucosal T cell immunity in HIV-1-uninfected individuals. J Immunol 2003;171:1094-101; Paoletti E. Applications of pox virus vectors to vaccination:anupdate. Proc Natl Acad Sci USA 1996;93:11349-53; U.S. Patent No. 7,255,862).In a Phase I clinical trial, ALVAC virus expressing the tumor antigen CEA showed an excellent safety profile and resulted in increased CEA-specific T cell responses in selected patients; however, no objective clinical responses were observed (Marshall JL, Hawkins MJ, Tsang KY, et al. Phase I study in cancer patients of a replication-defectiveavipox recombinant, vaccine that expresses human carcinoembryonic antigen. J Clin Oncol 1999; 17: 332-7).

[0245] In another embodiment, the modified Ankara cowpox (MVA) virus can be used as a viral vector for a neoantigen vaccine or immunogenic composition. MVA is a member of the Orthopoxviridae family and is generated by continuous passage of the Ankara strain of cowpox virus (CVA) in chicken embryonic fibroblasts for about 570 times (for a review, see Mayr, A., et al., Infection 3, 6-14, 1975). As a result of these passages, the resulting MVA virus contains 3.1 kilobases less genomic information than CVA and is highly host cell restricted (Meyer, H. et al., J. Gen. Virol. 72, 103 1 -1038, 1991). MVA is characterized by its extreme attenuation, i.e., reduced virulence or infectivity, but still has excellent immunogenicity. When tested in various animal models, MVA has been shown to be non-toxic even in immunosuppressed individuals. In addition, -HER2 is a candidate immunotherapy designed to treat HER-2 positive breast cancer and is currently in clinical trials. (Mandl et al. Cancer Immunol Immunother. Jan 2012; 61(1):19-29). Methods for making and using recombinant MVA have been described (see, e.g., U.S. Pat. Nos. 8,309,098 and 5,185,146, which are incorporated herein in their entirety).

[0246] In another embodiment, the modified Copenhagen strain of vaccinia virus NYVAC and NYVAC variants are used as vectors (see U.S. Pat. No. 7,255,862; PCT WO 95 / 30018; U.S. Pat. Nos. 5,364,773 and 5,494,807, which are incorporated herein by reference in their entireties).

[0247] In one embodiment, the recombinant viral particles of a vaccine or immunogenic composition are administered to a patient in need thereof. The dose of the expressed neoantigen can range from a few micrograms to several hundred micrograms, for example 5 to 500 mu.g. The vaccine or immunogenic composition can be administered in any suitable amount to achieve expression at these dosage levels. The viral particles can be administered in an amount of about at least 10 3.5 pfu can be administered to a patient in need thereof or transfected into a cell; thus, the viral particles can be administered in an amount of at least about 10 4 pfu to about 10 6 pfu is administered to a patient in need thereof or to an infected cell or transfected into a cell; however, a patient in need thereof may be administered at least about 10 8 pfu, so that the amount administered can be at least about 10 7 pfu to about 10 9 The doses for NYVAC are applicable to ALVAC, MVA, MVA-BN, and avian pox (such as canary pox and fowl pox).

[0248] III. Adjuvant for vaccine or immunogenic composition

[0249] Toll-like receptors (TLRs) are important members of the pattern recognition receptor (PRR) family expressed by cells of the innate and adaptive immune systems. TLRs recognize conserved motifs common to many microorganisms (called pathogen-associated molecular patterns (PAMPs)). Different TLRs recognize different PAMPs, and TLR ligand binding leads to activation of inflammatory signaling cascades (including nuclear factor kappa light chain (NF-κB) transcription factors and type I interferons (IFNs) of activated B cells). Toll-like receptor-mediated activation of APCs (such as dendritic cells (DCs)) leads to increased expression of MHC and T cell co-stimulatory molecules, and can help promote the initiation of peptide-specific T cell responses.

[0250] Non-limiting examples of cancer vaccine adjuvants include the TLR9 agonist 5'-C-phospho-G-3' (CpG) and synthetic double-stranded RNA (dsRNA) TLR3 ligand polyinosinic-polycytidylic-polylysine carboxymethylcellulose (adjuvant) (polyICLC) (polyinosinic:polycytidylic acid). CpG is a synthetic dinucleotide and pICLC is a synthetic dsRNA stabilized with polylysine and carboxymethylcellulose.

[0251] PolyICLC is a synthetic, dsRNA "host-targeting" therapeutic virus mimetic and PAMP with broad innate and adaptive immune adjuvant function. PolyICLC exerts its function through TLR3, melanoma differentiation-associated protein 5 (MDA5), and several nuclear and cytoplasmic enzyme systems involved in antiviral and antitumor host defense (oligoadenylate synthetase, dsRNA-dependent protein kinase R [PKR], retinoic acid-inducible gene 1 [RIG-1] helicase, and MDA5).

[0252] Stimulation with polyICLC results in activation of DCs and natural killer (NK) cells and production of a natural cocktail of type I IFNs, cytokines, and chemokines (Meylan, 2006). This adjuvant has been shown to induce local and systemic activation of immune cells in vivo, produce stimulatory chemokines and cytokines, and stimulate antigen presentation by DCs. In preclinical studies, polyICLC appears to be an effective TLR adjuvant due to its induction of proinflammatory cytokines, lack of stimulation of interleukin-10 (IL-10), and maintenance of high levels of co-stimulatory molecules in DCs (Bogunovic, 2011). Furthermore, polyICLC was directly compared with CpG as an adjuvant for a protein vaccine composed of human papillomavirus (HPV) 16 capsids in nonhuman primates and was found to be effective in inducing HPV-specific TLRs. H 1 (T helper cell 1) immune response is more effective (Stahl-Hennig, 2009).

[0253] PolyICLC can induce persistent CD4+ and CD8+ responses in humans. Striking similarities were seen in the upregulation of transcriptional and signal transduction pathways between patients vaccinated with polyICLC and volunteers receiving a highly effective, replication-competent yellow fever vaccine (Okada, 2011). In a recent Phase 1 study, >90% of patients with ovarian cancer immunized with a combination of polyICLC and NY-ESO-1 peptide vaccines showed induction of CD4+ and CD8+ T cells and antibody responses to the peptides (Sabbatini, 2012). Without being bound by theory, it is expected that these neoantigens will bypass central thymic tolerance (thereby allowing stronger anti-tumor T cell responses) while reducing the likelihood of autoimmunity (e.g., by avoiding targeting normal self-antigens). An effective immune response advantageously includes a strong adjuvant for activating the immune system (Speiser and Romero, Molecularly defined vaccines for cancer immunotherapy, and protective T cell immunity Seminars in Immunol 22: 144 (2010)). For example, Toll-like receptors (TLRs) have become strong sensors of "danger signals" from microbial and viral pathogens, effectively inducing the innate immune system and, in turn, the adaptive immune system (Bhardwaj and Gnjatic, TLR AGONISTS: Are They Good Adjuvants? Cancer J. 16: 382-391 (2010)). Among TLR agonists, polyICLC (a synthetic double-stranded RNA mimetic) is one of the most potent activators of myeloid-derived dendritic cells. In human volunteer studies, polyICLC has been shown to be safe and to induce a gene expression profile in peripheral blood cells that is comparable to that induced by one of the most potent live attenuated viral vaccines, the yellow fever vaccine YF-17D (Caskey et al., Synthetic double-stranded RNA induces innate immune responses similar to a live viral vaccine in humans J Exp Med 208: 2357 (2011)). In some embodiments, a GMP formulation of polyICLC prepared by Oncovir, Inc. is used. As adjuvant. In other embodiments, other adjuvants described herein are contemplated. For example, oil-in-water, water-in-oil, or multiphase W / O / W; see, for example, US 7,608,279 and Aucouturier et al., Vaccine 19 (2001), 2666-2672, and references cited therein.

[0254] IV. Immune checkpoint modulators

[0255] Immune checkpoints are key signaling pathways in the immune system that maintain self-tolerance and regulate the duration and magnitude of physiological immune responses. Under normal conditions, these pathways prevent excessive effector activity of T cells. Two important examples of this pathway are the cell surface receptors CTLA-4 and PD-1 (Teft, 2006; Keir, 2008). In some cases, tumors express or overexpress inhibitory immune checkpoint pathways as a primary mechanism of immune escape. Since many immune checkpoints are initiated by ligand-receptor interactions, these signals can be easily blocked by antibodies or modulated by recombinant forms of ligands or receptors (Pardoll, 2012).

[0256] Immune checkpoint proteins are important targets for pharmacological blockade (Teft, 2006; Keir, 2008), and significant clinical responses have been observed following treatment with antibodies that block PD-1 and CTLA-4 (see, e.g., Brahmer, 2010; Robert, 2011; Topalian, 2012; Powles, 2014; Topalian, 2014; Brahmer, 2015; Le, 2015; Robert, 2015; Reck, 2016; Langer, 2017). Thus, the present disclosure features novel combinations of tumor vaccines or immunogenic compositions with anti-PD-1 antibodies in exemplary embodiments.

[0257] PD-1 receptor refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed on many cell types (including Treg, activated B cells and natural killer (NK) cells), and is mainly expressed on previously activated T cells in vivo, and binds to two ligands PD-L1 and PD-L2. The endogenous ligands PD-L1 and PD-L2 of PD1 are expressed in activated immune cells and non-hematopoietic cells (including tumor cells). As used herein, PD-1 means including human PD-1 (hPD-1), variants, isoforms and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under GENBANK accession number U64863. Programmed death protein ligand-1 (PD-L1) is one of the two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), which downregulates T cell activation and cytokine secretion after binding to PD-1. As used herein, PD-L1 includes human PD-L1 (hPD-L1), variants, isoforms and species homologs of hPD-L1, and analogs having at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found under GENBAN accession number Q9NZQ7. Tumors have been shown to escape immune surveillance by expressing PD-L1 / L2, thereby inhibiting tumor-infiltrating lymphocytes via PD-1 / PD~L1,2 interactions (Dong et al. Nat. Med. 8:793-800.2002).

[0258] In some embodiments, the anti-PD-1 antibody is nivolumab. Bristol-Myers Squibb Company, NY) is a human immunoglobulin G4 (IgG4) mAb that binds to the programmed death protein 1 (PD-1) receptor and blocks its interaction with PD-L1 and programmed death protein ligand 2 (PD-L2), thereby reversing the PD-1 pathway-mediated inhibition of immune responses, including anti-tumor immune responses. Binding of PD-L1 and PD-L2 to the PD-1 receptor expressed on T cells inhibits T cell proliferation and cytokine production. PD-1 ligands are upregulated in some tumors, and signaling through this pathway can contribute to the inhibition of active T cell immune surveillance of tumors. The antibodies of the present disclosure include, but are not limited to, all anti-PD-1 and anti-PD-L1 Abs disclosed in U.S. Patent Nos. 8,008,449 and 7,943,743, respectively. Other anti-PD-1 mAbs have been described, for example, in U.S. Pat. Nos. 7,488,802 and 8,168,757, and anti-PD-L1 mAbs have been described, for example, in U.S. Pat. Nos. 7,635,757 and 8,217,149 and U.S. Publication No. 2009 / 0317368. U.S. Pat. No. 8,008,449 exemplifies seven anti-PD-1 HuMAbs: 1708, 2D3, 4M, 5C4 (also referred to herein as nivolumab or BMS-936558), 4A11, 7D3, and 5F4.

[0259] In addition to CTLA-4 and PD-1 / PD-L1, many other immunomodulatory targets have been preliminarily identified, many of which are being studied in clinical trials with corresponding therapeutic antibodies. Page et al. (Annu. Rev. Med. 2014.65) details the targets of antibody immunomodulators in Figure 1, which are incorporated herein by reference.

[0260] The present disclosure features, in exemplary aspects, novel combinations of tumor vaccines or immunogenic compositions and one or more inhibitors of the PD-L1 pathway. In some embodiments, the inhibitor of the PD-1 pathway is an anti-PD-L1 antibody, such as pembrolizumab.

[0261] The present disclosure is characterized in other exemplary aspects by a new combination of a tumor vaccine or immunogenic composition with an anti-PD-L1 antibody pembrolizumab and one or more chemotherapeutic agents. In one embodiment, one or more chemotherapeutic agents include a platinum-based anticancer therapeutic agent. Platinum-based anticancer agents may be selected from carboplatin, dicycloplatin, oxaliplatin, satraplatin and nedaplatin for treating cancer in combination with a tumor vaccine or immunogenic composition and pembrolizumab or a pharmaceutically acceptable salt or solvate thereof. In certain preferred embodiments, the platinum-based anticancer agent is carboplatin. In some embodiments, one or more chemotherapeutic agents include a first chemotherapeutic agent carboplatin and a second chemotherapeutic agent. In some embodiments, the second chemotherapeutic agent is an antimetabolite. In some embodiments, the antimetabolite is pemetrexed. In some embodiments, the new combination further comprises an adjuvant.

[0262] In some embodiments, PD-L1 inhibitors such as pembrolizumab can be administered once or more than once with neoantigen administration. In some dosage regimens, the PD-L1 inhibitor can be administered once as a priming dose at the beginning of the vaccination period, followed by one, two, three, four, five or more booster doses during and / or after the neoantigen vaccine dose. In some embodiments, a pembrolizumab dose is administered before the neoantigen vaccine is administered. In some embodiments, a pembrolizumab dose is administered more than once before the neoantigen vaccine is administered. In some embodiments, a pembrolizumab dose is administered after the neoantigen vaccine is administered. In some embodiments, a pembrolizumab dose is administered more than once after the neoantigen vaccine is administered. In some embodiments, two, three, four, five or more pembrolizumab doses are administered after the neoantigen vaccine is administered. In some embodiments, a nivolumab dose is administered before and after the neoantigen vaccine is administered.

[0263] In some embodiments, the adjuvant is Hiltolol. In some embodiments, the adjuvant is polyICLC. In some embodiments, Hiltolol and polyICLC are used.

[0264] It is increasingly recognized that somatic mutations in tumor cells can lead to the presentation of neoantigens that can be recognized by the host immune system and directly lead to tumor cell killing (Ott and Wu 2019; Ott et al. 2017; Keskin et al. 2019; Hu et al. 2021). Many clinical trials have attempted to exploit this mechanism by inducing tumor control using personalized neoantigen-based vaccination, especially in the setting of solid tumors with high neoantigen burden, with some promising success in the adjuvant setting (Ott et al. 2017; Keskin et al. 2019; Sahin et al. 2017; Hilf et al. 2019). The inventors previously reported a similar personalized neoantigen vaccine trial in combination with anti-PD1 in patients with melanoma, non-small cell lung cancer (NSCLC), and bladder urothelial carcinoma (TCC), which demonstrated safety and feasibility, as well as the ability to deeply characterize the immune response generated to the vaccine peptides (Ott et al. 2020).

[0265] Treatment of patients with NSCLC through the use of immunotherapeutic treatment modalities has been a relatively recent development with encouraging effects on the prognosis of patients diagnosed with the disease. Several key studies have approved immune checkpoint inhibitors (ICIs), including the PD-1 inhibitor pembrolizumab, as monotherapy or in combination with standard of care (SOC) chemotherapy regimens (Gettinger et al. 2015; Gandhi et al. 2018). Studies evaluating the combination of pembrolizumab plus chemotherapy have resulted in an extension of progression-free survival (PFS) from 4.9 to 9.0 months in non-squamous NSCLC and from 4.8 to 6.4 months in squamous NSCLC. (Gandhi et al. 2018; Gadgeel et al. 2020)(Borghaei et al. 2020). However, despite these successes, it is clear that additional treatment options are urgently needed to further prolong PFS in this patient population. Given the incomplete success observed in the setting of ICIs alone or in combination with chemotherapy, there is a strong rationale for incorporating neoantigen-based vaccination strategies to further enhance antitumor immune responses in a targeted manner.

[0266] The addition of a neoantigen vaccine to a regimen of chemotherapy and pembrolizumab provides a promising strategy for inducing and expanding tumor-specific CD4 + and CD8 + We have reported the use of a neoantigen vaccine in combination with anti-PD-1 as a therapy for advanced NSCLC (Ott et al. 2020), with robust generation of CD4 + and CD8 +In addition, the combination of neoantigen vaccines and tyrosine kinase inhibitors was recently studied in a small study of 16 patients with advanced NSCLC carrying EGFR mutations (Li et al. 2021). Both studies showed that this approach of adding neoantigen vaccines is feasible, safe, and leads to additional tumor-specific immunity.

[0267] In some embodiments, the subject has a tumor selected from the following: non-Hodgkin lymphoma (NHL), clear cell renal cell carcinoma (ccRCC), melanoma, sarcoma, leukemia or bladder cancer, colon cancer, brain cancer, breast cancer, head and neck cancer, endometrial cancer, lung cancer, ovarian cancer, pancreatic cancer or prostate cancer. In some embodiments, the tumor is a metastatic melanoma. In some embodiments, the subject has no detectable tumor, but is at high risk for disease recurrence. In embodiments, the cancer is selected from: adrenal cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, glioblastoma, head and neck cancer, renal chromophobe cell carcinoma, renal clear cell carcinoma, renal papillary carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, ovarian cancer, pancreatic cancer, melanoma, gastric cancer, uterine body endometrial cancer and uterine carcinosarcoma. In embodiments, the cancer is selected from the group consisting of prostate cancer, bladder cancer, squamous cell lung cancer, NSCLC, breast cancer, head and neck cancer, lung adenocarcinoma, GBM, glioma, CML, AML, supratentorial ependymoma, acute promyelocytic leukemia, solitary fibrous tumor, and crizotinib-resistant cancer. In embodiments, the cancer is selected from the group consisting of CRC, head and neck cancer, gastric cancer, squamous cell lung cancer, lung adenocarcinoma, prostate cancer, bladder cancer, gastric cancer, renal cell carcinoma, and uterine cancer. In embodiments, the cancer is selected from the group consisting of melanoma, squamous cell lung cancer, DLBCL, uterine cancer, head and neck cancer, uterine cancer, liver cancer, and CRC. In embodiments, the cancer is selected from the group consisting of lymphoid cancer, Burkitt's lymphoma, neuroblastoma, prostate adenocarcinoma, colorectal adenocarcinoma, uterine / endometrial adenocarcinoma, MSI+, endometrial serous carcinoma, endometrial carcinosarcoma-malignant mesodermal mixed tumor, glioma, astrocytoma, GBM, acute myeloid leukemia associated with MDS, chronic lymphocytic leukemia-small lymphocytic lymphoma, myelodysplastic syndrome, acute myeloid leukemia, breast luminal NS carcinoma, chronic myeloid leukemia, pancreatic ductal carcinoma, chronic myelomonocytic leukemia, myelofibrosis, myelodysplastic syndrome, prostate adenocarcinoma, essential thrombocythemia, and myeloid myoblastoma. In embodiments, the cancer is selected from the group consisting of colorectal cancer, uterine cancer, endometrial cancer, and gastric cancer. In embodiments, the cancer is selected from the group consisting of cervical cancer, head and neck cancer, anal cancer, gastric cancer, Burkitt's lymphoma, and nasopharyngeal carcinoma. In embodiments, the cancer is selected from the group consisting of: bladder cancer, colorectal cancer, and gastric cancer. In embodiments, the cancer is selected from the group consisting of: lung cancer, CRC, melanoma, breast cancer, NSCLC, and CLL. In embodiments, the subject is a partial responder or non-responder to checkpoint inhibitor therapy. In embodiments, the subject is a partial responder or non-responder to CD40 agonist therapy.In embodiments, the cancer is selected from the group consisting of bladder urothelial carcinoma (BLCA), invasive breast cancer (BRCA), breast cancer, cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), chronic lymphocytic leukemia (CLL), colorectal cancer (CRC), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney renal papillary cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), prostate cancer, skin melanoma (SKCM), stomach adenocarcinoma (STAD), thyroid adenocarcinoma (THCA) and uterine body endometrioid carcinoma (UCEC). In embodiments, the cancer is selected from the group consisting of colorectal cancer, uterine cancer, endometrial cancer, gastric cancer and Lynch syndrome. In embodiments, the cancer is an MSI+ cancer.

[0268] Ⅴ. Pharmaceutical composition / delivery method

[0269] The present disclosure also relates to pharmaceutical compositions comprising an effective amount of one or more compounds according to the present disclosure (including pharmaceutically acceptable salts thereof), optionally in combination with a pharmaceutically acceptable carrier, excipient or additive.

[0270] When administered as a combination, the therapeutic agents (i.e., tumor vaccine or immunogenic composition and one or more inhibitors (such as one or more checkpoint inhibitors or chemotherapeutic drugs)) can be formulated as separate compositions that are given at the same time or different times, or the therapeutic agents can be given as a single composition.

[0271] The composition can be administered once a day, twice a day, once every two days, once every three days, once every four days, once every five days, once every six days, once every seven days, once every two weeks, once every three weeks, once every four weeks, once every two months, once every six months, or once a year. The dosing interval can be adjusted according to the needs of the individual patient. For longer administration intervals, a time-delayed release or depot formulation can be used.

[0272] The compositions of the present disclosure can be used to treat acute diseases and disease conditions, and can also be used to treat chronic conditions. In particular, the compositions of the present disclosure can be used in methods of treating or preventing neoplasms.

[0273] In certain embodiments, the compounds of the present disclosure are administered for a period of more than two weeks, three weeks, one month, two months, three months, four months, five months, six months, one year, two years, three years, four years or five years, ten years or fifteen years; or, for example, any time period range in days, months or years, wherein the lower end of the range is any time period between 14 days and 15 years, and the upper end of the range is between 15 days and 20 years (e.g., 4 weeks and 15 years, 6 months and 20 years). In some cases, it may be advantageous to administer the compounds of the present disclosure for the rest of the patient's life. In some embodiments, the patient is monitored to check the progress of the disease or illness, and the dosage is adjusted accordingly. In some embodiments, treatment according to the present disclosure is effective for at least two weeks, three weeks, one month, two months, three months, four months, five months, six months, one year, two years, three years, four years or five years, ten years, fifteen years, twenty years, or for the rest of the subject's life.

[0274] As described herein, in certain embodiments, administration of the inhibitor is initiated before administration of the tumor vaccine or immunogenic composition is initiated. In other embodiments, administration of the inhibitor is initiated after administration of the tumor vaccine or immunogenic composition is initiated. In other embodiments, administration of the inhibitor is initiated simultaneously with administration of the tumor vaccine or immunogenic composition.

[0275] Inhibitors (such as checkpoint inhibitors or chemotherapeutic drugs) can be administered every 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks or longer after the first administration of inhibitors (such as checkpoint inhibitors or chemotherapeutic drugs). It should be understood that the 1st week means including the 1st-7th day, the 2nd week means including the 8th-14th day, the 3rd week means including the 15th-21st day, and the 4th week means including the 22nd-28th day. When administration is described as being performed at weekly intervals, this means an interval of about 7 days, but in any given week, the administration day can be one or more days before or after the planned day.

[0276] In certain embodiments, the administration of inhibitors, such as checkpoint inhibitors or chemotherapeutic drugs, is suspended within one week before the administration of tumor vaccines or immunogenic compositions. In other embodiments, the administration of inhibitors, such as checkpoint inhibitors, is suspended during the administration of tumor vaccines or immunogenic compositions.

[0277] Surgical resection uses surgery to remove abnormal tissue in cancer, such as mediastinal tumors, neurogenic tumors or germ cell tumors or thymomas. In other embodiments, tumor vaccines or immunogenic compositions are administered 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks or longer after tumor resection. In some embodiments, tumor vaccines or immunogenic compositions are administered 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks or 12 weeks after tumor resection.

[0278] The initiation / boosting scheme refers to the continuous administration of a vaccine or an immunogenic composition or an immune composition. In certain embodiments, the administration of a tumor vaccine or an immunogenic composition is in the initiation / boosting dosing scheme, for example, a tumor vaccine or an immunogenic composition is administered at the 1st, 2nd, 3rd or 4th week as an initiation, and a tumor vaccine or an immunogenic composition is administered at the 2nd, 3rd or 4th month as a booster. In another embodiment, different initiation-boosting strategies are used to cause a larger cytotoxic T cell response (see Schneider et al., Induction of CD8+Tcells using heterologous prime-boostimmunization strategies, Immunological Reviews Vol. 170, No. 1, pp. 29-38, August 1999). In another embodiment, DNA encoding a neoantigen is used to induce immunity, followed by protein boosting. In another embodiment, immunity is induced using a protein, followed by boosting immunity with a virus encoding a neoantigen. In another embodiment, immunity is induced using a virus encoding a neoantigen, and another virus is used to boost immunity. In another embodiment, immunity is induced using a protein, and DNA is used to boost immunity. In some embodiments, a DNA vaccine or immunogenic composition is used to induce a T cell response, and a recombinant viral vaccine or immunogenic composition is used to enhance the response. In some embodiments, a viral vaccine or immunogenic composition is co-administered with a protein or DNA vaccine or immunogenic composition to serve as an adjuvant for the protein or DNA vaccine or immunogenic composition. The patient can then be boosted with a viral vaccine or immunogenic component, a protein or DNA vaccine or an immunogenic composition (see Hutchings et al., Combination of protein and viral vaccines induces potent cellular and humoral immune responses and enhanced protection from murine malaria challenge. Infect Immun, December 2007; 75(12):5S19-26. Epub October 1, 2007).

[0279] As used herein, the term "fixed intermittent dosing regimen" refers to a repetitive preplanned cycle of drug administration in which the drug is administered on one or more consecutive days ("days on"), followed by one or more consecutive rest days in which the drug is not administered ("days off").

[0280] In some embodiments, the cycle is regular, because in each cycle, the pattern of the administration day and the withdrawal day is the same. In some embodiments, the cycle is irregular, because from one cycle to the next cycle, the pattern of the administration day and the withdrawal day is different. However, in some embodiments, each of the repeated cycles is planned in advance, because it is not determined by merely responding to the occurrence of one or more adverse events. In some embodiments, the composition comprising the first component and / or the second component is repeatedly administered for one to ten cycles, for example, one cycle, two cycles, three cycles, four cycles, five cycles, six cycles, seven cycles, eight cycles, nine cycles or ten cycles.

[0281] In some embodiments, the cycle includes 3 to 60 days. In some embodiments, the cycle includes 7 to 50 days, such as 7 to 30 days, 7 to 21 days or 7 to 14 days. In some embodiments, the cycle consists of 7 days.

[0282] In some embodiments, the fixed intermittent dosing regimen includes administering an effective amount of the composition comprising the first component and / or the second component for 1 to 5 consecutive days (such as 2 to 5 consecutive days), followed by a repetitive cycle of 6 to 2 days (such as 5 to 2 days of rest). In some embodiments, the fixed intermittent dosing regimen includes administering an effective amount of the composition comprising the first component and / or the second component for 5 consecutive days, followed by a repetitive cycle of 2 days of rest. In some embodiments, the fixed intermittent dosing regimen includes administering an effective amount of the composition comprising the first component and / or the second component for 4 consecutive days, followed by a repetitive cycle of 3 days of rest. In some embodiments, the fixed intermittent dosing regimen includes administering an effective amount of the composition comprising the first component and / or the second component for 3 consecutive days, followed by a repetitive cycle of 4 days of rest.

[0283] In some embodiments, the fixed intermittent dosing regimen includes administering an effective amount of the composition comprising the first component and / or the second component for 1 to 5 consecutive days (such as 2 to 5 consecutive days), followed by a repetitive cycle of resting for 6 to 2 days (such as resting for 5 to 2 days). In some embodiments, a placebo is administered on the rest days.

[0284] The pharmaceutical compositions may be processed according to conventional methods of pharmacy to produce medicaments for administration to patients in need thereof, including humans and other mammals.

[0285] Modification of the neoantigenic peptide can affect the solubility, bioavailability and metabolic rate of the peptide, thereby controlling the delivery of the active substance. The solubility can be assessed by preparing the neoantigenic peptide and testing according to methods well known to conventional practitioners skilled in the art.

[0286] It has been found that pharmaceutical compositions comprising succinic acid or a pharmaceutically acceptable salt thereof (succinate) can provide improved solubility for neoantigenic peptides. Therefore, in one aspect, the present disclosure provides a pharmaceutical composition comprising: at least one neoantigenic peptide or a pharmaceutically acceptable salt thereof; a pH adjuster (such as a base, such as a dicarboxylate or tricarboxylate, for example a pharmaceutically acceptable salt of succinic acid or citric acid); and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can be prepared by combining a solution comprising at least one neoantigenic peptide with a base (such as a dicarboxylate or tricarboxylate, such as a pharmaceutically acceptable salt of succinic acid or citric acid (such as sodium succinate)), or combining a solution comprising at least one neoantigenic peptide with a solution comprising a base (such as a dicarboxylate or tricarboxylate, such as a pharmaceutically acceptable salt of succinic acid or citric acid) (including, for example, a succinate buffer solution). In certain embodiments, the pharmaceutical composition comprises sodium succinate. In certain embodiments, the pH adjuster (such as citrate or succinate) is present in the composition at a concentration of about 1 mM to about 10 mM, and in certain embodiments, at a concentration of about 1.5 mM to about 7.5 mM, or about 2.0 to about 6.0 mM, or about 3.75 to about 5.0 mM.

[0287] In certain embodiments of the pharmaceutical composition, the pharmaceutically acceptable carrier includes water. In certain embodiments, the pharmaceutically acceptable carrier also includes glucose. In certain embodiments, the pharmaceutically acceptable carrier also includes dimethyl sulfoxide. In certain embodiments, the pharmaceutical composition also includes an immunomodulator or adjuvant. In certain embodiments, the immunomodulator or adjuvant is selected from polyICLC, 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, carrier systems, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, and QS21 stimulator from Aquila. In certain embodiments, the immunomodulator or adjuvant comprises polyICLC.

[0288] According to embodiments of the present disclosure, xanthone derivatives (e.g., Vadimezan or AsA404 (also known as 5,6-dimethylxanthone-4-acetic acid (DMXAA))) may also be used as adjuvants. Alternatively, such derivatives may also be administered concurrently with the vaccine or immunogenic composition of the present disclosure, e.g., via systemic or intratumoral delivery, to stimulate immunity at the tumor site. Without being bound by theory, it is believed that such xanthone derivatives act by stimulating the production of interferons (IFNs) via the stimulator of IFN genes (STING) receptor (see, e.g., Conlon et al. (2013) Mouse, but not Human STING, Binds and Signals in Response to the Vascular Disrupting Agent 5,6-Di-methylxanthenone-4-Acetic Acid, journal of Immunology, 190:5216-25 and Kim et al. (2013) Anticancer Flavonoids are Mouse-Selective STING Agonists, 8:1396-1401).

[0289] The vaccine or immune composition may also include an adjuvant compound selected from acrylic acid or methacrylic acid polymers and copolymers of maleic anhydride and alkenyl derivatives. In particular, acrylic acid or methacrylic acid is cross-linked with a polyalkenyl ether of a sugar or polyol (carbomer), in particular with allyl sucrose or allyl pentaerythritol. It may also be a copolymer of maleic anhydride and ethylene (e.g., cross-linked with divinyl ether) (see U.S. Patent No. 6,713,068, incorporated herein by reference in its entirety).

[0290] In certain embodiments, a pH modifier can stabilize an adjuvant or immunomodulator as described herein.

[0291] In certain embodiments, the pharmaceutical composition comprises: one to five peptides, dimethyl sulfoxide (DMSO), glucose, water, succinate, poly I: poly C, poly L-lysine, carboxymethyl cellulose and chloride. In certain embodiments, each of one to five peptides is present at a concentration of 200 μg / ml and 500 μg / ml, 300-400 μg / ml. In certain embodiments, the pharmaceutical composition comprises ≤3% DMSO, about 4%-5% DMSO by volume. In certain embodiments, the pharmaceutical composition comprises 3.5%-5.5% glucose, 4.9%-5.0% glucose in water. In certain embodiments, the pharmaceutical composition comprises ≤5.0mM succinate, 3.6-3.7mM succinate (e.g., sodium succinate). In certain embodiments, the pharmaceutical composition comprises ≥0.4mg / ml poly I: poly C, e.g., 1.0-2.2mg / ml, e.g., 1.7-1.9mg / ml. In certain embodiments, the pharmaceutical composition comprises ≥0.375 mg / ml poly-L-lysine, 0.5-2.0 mg / ml or 1.5 mg / ml. In certain embodiments, the pharmaceutical composition comprises ≥1.25 mg / ml sodium carboxymethylcellulose, 2-7 mg / ml, such as 4-5 mg / ml. In certain embodiments, the pharmaceutical composition comprises ≥0.225% sodium chloride, 0.5%-1.0% sodium chloride or 0.8%-2.0% sodium chloride.

[0292] The pharmaceutical composition comprises a therapeutically effective amount of the tumor-specific neoantigenic peptides described herein for treating the diseases and conditions (e.g., tumors / tumors) described herein, optionally in combination with pharmaceutically acceptable additives, carriers and / or excipients. One of ordinary skill in the art will recognize, based on this disclosure and the knowledge in the art, that the therapeutically effective amount of one or more compounds according to the present disclosure may vary depending on the condition to be treated, its severity, the treatment regimen to be adopted, the pharmacokinetics of the agent used, and the patient (animal or human) treated.

[0293] In order to prepare a pharmaceutical composition according to the present disclosure, one or more compounds according to the present disclosure of a therapeutically effective amount can be closely mixed with a pharmaceutically acceptable carrier according to conventional drug compounding techniques to produce a dosage. The carrier can take a variety of forms according to the formulation form required for administration (e.g., eye, oral, topical or parenteral), including gel, cream, ointment, lotion and implantable formulations of delayed release, etc. When preparing a pharmaceutical composition of an oral dosage form, any conventional drug medium can be used. Therefore, for liquid oral preparations (such as suspensions, elixirs and solutions), suitable carriers and additives can be used, including water, ethylene glycol, oils, alcohols, flavoring agents, preservatives, coloring agents, etc. For solid oral preparations (such as powders, tablets, capsules) and solid preparations (such as suppositories), suitable carriers and additives can be used, including starch, sugar carriers (such as glucose, mannitol, lactose and related carriers), diluents, granulators, lubricants, adhesives, disintegrants, etc. If necessary, tablets or capsules can be enteric coated or slowly released by standard techniques.

[0294] The active compound is included in a pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount for the desired indication without causing serious toxic effects in the treated patient.

[0295] Oral compositions generally include an inert diluent or an edible carrier. They may be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound or its prodrug derivative may be incorporated with excipients and used in the form of tablets, lozenges or capsules. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition.

[0296] Tablets, pills, capsules, lozenges, and the like may contain any of the following ingredients or compounds of similar nature: binders such as microcrystalline cellulose, tragacanth, or gelatin; excipients such as starch or lactose; dispersants such as alginic acid or corn starch; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweeteners such as sucrose or saccharin; or flavorings such as mint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule, it may contain a liquid carrier such as a fatty oil in addition to the materials discussed herein. In addition, the dosage unit form may contain various other materials that modify the physical form of the dosage unit, such as sugar coatings, shellac, or enteric solvents.

[0297] Formulations of the present disclosure suitable for oral administration may be presented in discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil emulsion, as boluses, and the like.

[0298] Tablets can be made by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets can be prepared in the following manner: in a suitable machine, the active ingredient in a free-flowing form (such as a powder or granules) is compressed, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersant. Molded tablets can be prepared by molding a mixture of a powdered compound moistened with an inert liquid diluent in a suitable machine. Tablets can be optionally coated or scored, and can be formulated to provide a slow release or controlled release of the active ingredient therein.

[0299] Methods of formulating such slow-release or controlled-release compositions of pharmaceutically active ingredients are known in the art and are described in several issued U.S. patents, some of which include, but are not limited to, U.S. Pat. Nos. 3,870,790, 4,226,859, 4,369,172, 4,842,866, and 5,705,190, the disclosures of which are incorporated herein by reference in their entirety. Coatings can be used to deliver compounds to the small intestine (see, e.g., U.S. Pat. Nos. 6,638,534, 5,541,171, 5,217,720, and 6,569,457 and references cited therein).

[0300] The active compound or its pharmaceutically acceptable salt may also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum, etc. A syrup may contain, in addition to the active compound, sucrose or fructose as a sweetening agent and certain preservatives, dyes, colorings, and flavorings.

[0301] Solutions or suspensions for ophthalmic, parenteral, intradermal, subcutaneous or topical application may contain the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates or phosphates; and agents for adjusting tonicity, such as sodium chloride or dextrose.

[0302] In certain embodiments, the pharmaceutically acceptable carrier is an aqueous solvent, i.e., a solvent comprising water, optionally with another cosolvent. Exemplary pharmaceutically acceptable carriers include water, buffer solutions in water (such as phosphate buffered saline (PBS)) and 5% glucose (D5W) in water. In certain embodiments, the aqueous solvent also includes, for example, dimethyl sulfoxide (DMSO) in an amount of about 1%-4% or 2%-3%. In certain embodiments, the pharmaceutically acceptable carrier is isotonic (i.e., having an osmotic pressure substantially the same as that of body fluids such as blood plasma).

[0303] In one embodiment, the active compound is prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic-co-glycolic acid (PLGA) can be used. Methods for preparing such formulations are within the scope of the skilled artisan according to the disclosure and knowledge in the art.

[0304] Skilled technicians will recognize from this disclosure and knowledge in the art that, in addition to tablets, other dosage forms may be formulated to provide slow or controlled release of active ingredients. Such dosage forms include, but are not limited to, capsules, granules, and gel capsules.

[0305] Liposomal suspensions can also be pharmaceutically acceptable carriers. They can be prepared according to methods known to those skilled in the art. For example, liposome formulations can be prepared in the following manner: one or more suitable lipids are dissolved in an inorganic solvent, and then the solvent is evaporated, leaving a thin film of dried lipids on the surface of the container. Then an aqueous solution of the active compound can be introduced into the container. The container is then rotated by hand to release the lipid material from the side of the container and disperse the lipid aggregates, thereby forming a liposome suspension. Other formulation methods well known to those of ordinary skill can also be used in this aspect of the present disclosure.

[0306] The formulation can be conveniently provided in unit dosage form and can be prepared by conventional pharmaceutical techniques. Such techniques include the step of associating the active ingredient with one or more pharmaceutical carriers or one or more excipients. Typically, the formulation is prepared by uniformly and intimately associating the active ingredient with a liquid carrier or a finely dispersed solid carrier or both and then, if necessary, shaping the product.

[0307] Formulations and compositions suitable for topical administration in the mouth include lozenges comprising the ingredient in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the ingredient to be administered in a suitable liquid carrier.

[0308] Formulations suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes comprising the ingredient to be administered in a pharmaceutically acceptable carrier. Topical delivery systems that may be used include transdermal patches containing the ingredient to be administered.

[0309] Formulations for rectal administration may be presented as a suppository with a suitable base comprising for example cocoa butter or a salicylate.

[0310] Formulations suitable for nasal administration (wherein the carrier is a solid) include coarse powders with a particle size, for example, in the range of 20 to 500 microns, which are administered in the manner of administering a filler, i.e., by rapid inhalation through the nasal cavity from a powder container held against the nose. Suitable formulations for administration (wherein the carrier is a liquid), such as nasal sprays or nose drops, include aqueous or oily solutions of the active ingredient.

[0311] Formulations suitable for vaginal administration may be presented as pessaries, tampons, creams, gels, pastes, foams or spray formulations containing in addition to the active ingredient such carriers as are known in the art to be appropriate.

[0312] The parenteral preparation may be enclosed in an ampoule, a disposable syringe or a multiple dose vial made of glass or plastic. If administered intravenously, the carrier may include, for example, physiological saline or phosphate buffered saline (PBS).

[0313] For parenteral formulations, the carrier typically includes sterile water or aqueous sodium chloride solution, but may also include other ingredients (including those that aid dispersion). Of course, where sterile water is to be used and maintained sterile, the composition and carrier must also be sterilized. Injectable suspensions can also be prepared, in which case appropriate liquid carriers, suspending agents, etc. can be used.

[0314] Preparations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. Preparations may be provided in unit doses or multi-dose containers (e.g., sealed ampoules and vials), and may be stored under freeze drying (lyophilization) conditions, requiring only the addition of sterile liquid carriers (e.g., water for injection) immediately prior to use. Ready-to-use injection solutions and suspensions may be prepared by previously described sterile powders, granules, and tablets.

[0315] Administration of the active compound may range from continuous (intravenous drip) administration to oral administration several times a day (e.g., QID), and may include oral, topical, ocular or ocular, parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may include penetration enhancers), buccal and suppository administration, as well as other routes of administration (including via the ocular or ocular route).

[0316] Tumor vaccine or immunogenic composition and at least one inhibitor (such as checkpoint inhibitor or chemotherapeutic agent) and any other medicament can be injected, orally, parenterally, inhaled spray, rectum, vaginal or topically applied in dosage unit formulations, and the dosage unit formulations contain conventional pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes entering lymph nodes or nodules, subcutaneously, intravenously, intramuscularly, intrasternally, infusion techniques, intraperitoneally, eyes or eyes, intravitreal, intrabuccal, transdermal, intranasal, entering the brain (including intracranial and intradural), entering joints (including ankles, knees, hips, shoulders, elbows, wrists), directly into tumors, etc. and in the form of suppositories.

[0317] Various techniques can be used to deliver the subject composition at the intended site, such as injection, use of a catheter, trocar, projectile, pluronic gel, stent, slow drug release polymer, or other device providing internal access. In the case where an organ or tissue is available due to removal from a patient, such organ or tissue can be soaked in a medium containing the subject composition, the subject composition can be painted on the organ or can be applied in any convenient manner.

[0318] Tumor-specific neoantigen peptides can be administered via controlled-release and slow-release devices suitable for compositions that are effective in achieving desired local or systemic physiological or pharmacological effects. The method includes placing a slow-release drug delivery system in an area where the agent needs to be released and allowing the agent to pass through the device to the desired treatment area.

[0319] Tumor-specific neoantigen peptides can be used in combination with at least one other known therapeutic agent or a pharmaceutically acceptable salt of the agent. Examples of known therapeutic agents that can be used for combination therapy include, but are not limited to, corticosteroids (e.g., cortisone, prednisone, dexamethasone); nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., ibuprofen, celecoxib, aspirin, indomethacin, naproxen); alkylating agents such as busulfan, cisplatin, mitomycin C, and carboplatin; antimitotic agents such as colchicine, vinblastine, paclitaxel, and docetaxel; topo I inhibitors such as camptothecin and topotecan; topo II inhibitors such as doxorubicin and etoposide; and / or RNA / DNA antimetabolites such as 5-azacytidine, 5-fluorouracil, and methotrexate; DNA antimetabolites such as 5-fluoro-2'-deoxy-uridine, ara-C, hydroxyurea, and thioguanine; antibodies such as BERCEPT1N and RITUXAN.

[0320] In certain embodiments, administration of the compositions described herein can be combined with administration of antagonists that block the release of histamine and anti-inflammatory drugs to prevent adverse allergic reactions.H1 and H2 antagonists can be administered to the patient prior to administration of the compositions described herein.

[0321] It should be understood that in addition to the ingredients particularly mentioned herein, the formulations of this disclosure may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

[0322] Pharmaceutically acceptable salt forms may be the chemical forms of the compounds according to the present disclosure for inclusion in the pharmaceutical compositions according to the present disclosure.

[0323] The compounds of the present invention or their derivatives (including the prodrug forms of these agents) can be provided in the form of pharmaceutically acceptable salts. As used herein, the term pharmaceutically acceptable salt or complex refers to a suitable salt or complex of an active compound according to the present disclosure, which retains the desired biological activity of the parent compound and exhibits limited toxicological effects on normal cells. Non-limiting examples of such salts are (a) acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.), and salts formed with organic acids (such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid and polyglutamic acid, etc.); (b) base addition salts formed with metal cations (such as zinc, calcium, sodium, potassium, etc.), etc.

[0324] The compounds herein are commercially available or can be synthesized. As will be appreciated by a skilled artisan, other methods for synthesizing compounds having the formula herein are apparent to those of ordinary skill in the art. In addition, the various synthesis steps can be performed in alternating sequences or orders to obtain the desired compounds. Synthetic chemical transformations and protecting group methods (protection and deprotection) for synthesizing the compounds described herein are known in the art, and include, for example, those described in the following documents: R. Larock, Comprehensive Organic Transformations, 2nd Edition, Wiley-VCH Publishers (1999); T. W. Greene and P. G. Wuts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1999); and L. Paquette, editor, Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent versions thereof.

[0325] VI. dose

[0326] When the agents described herein are administered to humans or animals as medicines, they may be administered per se or as a pharmaceutical composition containing an active ingredient in combination with a pharmaceutically acceptable carrier, excipient or diluent.

[0327] The actual dosage level and administration schedule of the active ingredients in the pharmaceutical compositions of the present disclosure can be varied to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without being toxic to the patient. Typically, the medicament or pharmaceutical composition of the present disclosure is administered in an amount sufficient to reduce or eliminate the symptoms associated with viral infection and / or autoimmune disease.

[0328] The dose of the agent may be the maximum dose that a patient can tolerate without experiencing severe or unacceptable side effects.

[0329] The determination of effective amount is completely within the capabilities of those skilled in the art, especially in light of the specific disclosures provided herein. Typically, the effective amount of a medicament is determined by first administering a low dose of one or more medicaments, then increasing the administered dose incrementally until the desired effect (e.g., reducing or eliminating symptoms associated with viral infection or autoimmune disease) is observed in the treated subject, and with minimal or acceptable toxic side effects. Suitable methods for determining the appropriate dose and dosing schedule for administering the pharmaceutical composition of the present disclosure are described in, for example, Goodman and Oilman's The Pharmacological Basis of Therapeutics, Goodman et al., ed., 11th ed., McGraw-Hill 2005, and Remington: The Science and Practice of Pharmacy, 20th and 21st ed., Gennaro, and University of the Sciences in Philadelphia, Lippencott Williams and Wilkins (2003 and 2005), each of which is incorporated herein by reference.

[0330] Unit dosage formulations may be those containing a daily dose or unit, daily sub-dose, as herein discussed, or an appropriate fraction thereof, of the administered ingredient.

[0331] The dosage regimen for treating a condition or disease with the tumor-specific neoantigenic peptides of the present disclosure and / or the compositions of the present disclosure is based on a variety of factors, including the type of disease, the age, weight, sex, medical condition of the patient, the severity of the condition, the route of administration, and the specific compound used. Thus, the dosage regimen can vary widely, but can be routinely determined using standard methods.

[0332] The amount and dosage regimen administered to a subject may depend on many factors, such as the mode of administration, the nature of the condition being treated, the weight of the subject being treated, and the judgment of the prescribing physician; all of these factors are within the scope of the skilled artisan based on the disclosure and knowledge in the art. In some embodiments, a first series of closely spaced immunizations may be administered to induce an immune response, followed by a rest period to allow memory T cells to be established, and booster immunizations to expand the response. Alternatively, priming doses may be administered over a longer period of time, and booster doses may be administered more frequently for a longer period of time. For example, a longer priming period may be followed by booster immunizations administered once every 2 months for 1 year.

[0333] The amount of compound contained in a therapeutically active formulation according to the present disclosure is an amount effective to treat the disease or condition.

[0334] Typically, the therapeutically effective amount of the compound in dosage form can be slightly less than about 0.025mg / kg / day to about 2.5g / kg / day, about 0.1mg / kg / day to about 100mg / kg / day or quite large for the patient, depending on the compound used, the condition or infection for treatment and the route of administration, although the disclosure may consider the exceptions to the dosage range. In some embodiments, the compound according to the disclosure is administered with an amount ranging from about 1mg / kg / day to about 100mg / kg / day. The dosage of the compound may depend on the condition treated, the specific compound and other clinical factors (such as the patient's weight and condition) and the route of administration of the compound. It should be understood that the disclosure is applicable to both human and veterinary use.

[0335] According to certain exemplary embodiments, the vaccine or immunogenic composition is administered at a dose of about 10 μg-1 mg / neoantigen peptide. According to certain exemplary embodiments, the NEO-PV-01 vaccine / adjuvant + pembrolizumab + chemotherapy regimen includes administering the vaccine or immunogenic composition at an average weekly dose level of about 10 μg-2000 μg / neoantigen peptide. In some cases, the concentration of a single dose of one or more neoantigen peptides is between 100 μg / ml and 1000 μg / ml, between 300-600 μg / ml, or between 400-500 μg / ml. According to some embodiments, the NEO-PV-01 vaccine / adjuvant + pembrolizumab + chemotherapy regimen includes administering pembrolizumab at a dose of 200 mg by intravenous infusion (IV) plus chemotherapy using carboplatin (AUC 5) + pemetrexed (500 mg / m^2), once every 3 weeks for 4 cycles. At Week 12, all patients, regardless of disease status, will receive NEO-PV-01 + adjuvant (one vial of pooled peptide per injection site) administered subcutaneously at up to four different sites (each limb or flank) while continuing pembrolizumab therapy.

[0336] In some embodiments, the neoantigenic peptide is administered to a subject at a dose of 10 μg to 2,000 μg / peptide. In some embodiments, the neoantigenic peptide is administered to a subject at a dose of at least 10 μg, 50 μg, 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 400 μg, 500 μg, 600 μg, 800 μg, 1000 μg or 1500 μg / peptide. In some embodiments, the neoantigenic peptide is administered to a subject at a dose of up to 2,000 μg, 1500 μg, 1000 μg, 800 μg, 700 μg, 600 μg, 500 μg, 400 μg, 300 μg, 250 μg, 200 μg, 100 μg, 75 μg / peptide.In some embodiments, the dosage form is 10 μg to 50 μg, 10 μg to 100 μg, 10 μg to 200 μg, 10 μg to 300 μg, 10 μg to 400 μg, 10 μg to 500 μg, 10 μg to 600 μg, 10 μg to 800 μg, 10 μg to 1,000 μg, 10 μg to 1,500 μg, 10 μg to 2,000 μg, 50 μg to 100 μg, 50 μg to 200 μg, 50 μg to 300 μg, 50 μg to 400 μg, 50 μg to 500 μg, 50 μg to 600 μg, 50 μg to 800 μg, 50 μg to 1,000μg, 50μg to 1,500μg, 50μg to 2,000μg, 100μg to 200μg, 100μg to 300μg, 100μg to 400μg, 100μg to 500μg, 100μg to 600μg, 100μg to 800μg, 100μg to 1,000μg, 100μg to 1,500μg, 100μg to 2,000μg, 200μg to 300μg, 200μg to 400μg, 200μg to 500μg, 200μg to 600μg, 200μg to 800μg, 200μg to 1,000μg, 200μg to 1,500μg, 200μg to 2,000μg, 300μg to 400μg, 300μg to 500μg, 300μg to 600μg, 300μg to 800μg, 300μg to 1,000μg, 300μg to 1,500μg, 300μg to 2,000μg, 400μg to 500μg, 400μg to 600μg, 400μg to 800μg, 400μg to 1,000μg, 400μg to 1,500μg, 400μg to 2,000μg, 500μg to 600μg The neoantigen peptides are administered to a subject at a dose of 100 μg, 500 μg to 800 μg, 500 μg to 1,000 μg, 500 μg to 1,500 μg, 500 μg to 2,000 μg, 600 μg to 800 μg, 600 μg to 1,000 μg, 600 μg to 1,500 μg, 600 μg to 2,000 μg, 800 μg to 1,000 μg, 800 μg to 1,500 μg, 800 μg to 2,000 μg, 1,000 μg to 1,500 μg, 1,000 μg to 2,000 μg, or 1,500 μg to 2,000 μg per peptide. In some embodiments, the neoantigenic peptide is administered to a subject at a dose of 10 μg, 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 800 μg, 1,000 μg, 1500 μg, or 2000 μg per peptide.

[0337] In some embodiments, pembrolizumab is administered to a subject at a dose of 50 mg to 400 mg. In some embodiments, pembrolizumab is administered to a subject at a dose of at least 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 220 mg, 240 mg, 250 mg, 260 mg, 270 mg, 280 mg, 300 mg, 320 mg, or 350 mg. In some embodiments, pembrolizumab is administered to a subject at a dose of up to 400 mg, 350 mg, 300 mg, 260 mg, 240 mg, 200 mg, 150 mg, 100 mg, or 75 mg.In some embodiments, the dosage is 50 mg to 75 mg, 50 mg to 100 mg, 50 mg to 150 mg, 50 mg to 200 mg, 50 mg to 220 mg, 50 mg to 240 mg, 50 mg to 260 mg, 50 mg to 280 mg, 50 mg to 300 mg, 50 mg to 350 mg, 50 mg to 400 mg, 75 mg to 100 mg, 75 mg to 150 mg, 75 mg to 200 mg, 75 mg to 220 mg, 75 mg to 240 mg, 75 mg to 260 mg, 75 mg to 280mg, 75mg to 300mg, 75mg to 350mg, 75mg to 400mg, 100mg to 150mg, 100mg to 200mg, 100mg to 220mg, 100mg to 240mg, 100mg to 260mg, 100mg to 280mg, 100mg to 300mg, 100mg to 350mg, 100mg to 400mg, 150mg to 200mg, 150mg to 220mg, 150mg to 240mg, 150mg to 260mg, 150mg to 280mg, 150mg to 300mg, 150mg to 350mg, 150mg to 400mg, 200mg to 220mg, 200mg to 240mg, 200mg to 260mg, 200mg to 280mg, 200mg to 300mg, 200mg to 350mg, 200mg to 400mg, 220mg to 240mg, 220mg to 260mg, 220mg to 280mg, 220mg to 300mg, 220mg to 350mg, 220mg to In some embodiments, pembrolizumab is administered to a subject at a dose of 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 220 mg, 240 mg, 260 mg, 280 mg, 300 mg, 350 mg, or 400 mg.

[0338] The concentration of the active compound in the pharmaceutical composition will depend on the absorption, distribution, inactivation and excretion rate of the drug and other factors known to those skilled in the art. It should be noted that the dosage value will also vary with the severity of the condition to be alleviated. It should also be understood that for any particular object, the specific dosage regimen should be adjusted over time according to individual needs and the professional judgment of the personnel administering the composition or supervising the administration of the composition, and the concentration ranges described herein are only exemplary and are not intended to limit the scope or practice of the desired composition. The active ingredient can be administered once, or can be divided into multiple smaller doses to be administered at different time intervals.

[0339] The present disclosure provides a pharmaceutical composition containing at least one tumor-specific neoantigen described herein. In an embodiment, the pharmaceutical composition contains a pharmaceutically acceptable carrier, excipient or diluent, which includes any agent that does not itself induce the production of an immune response that is harmful to the subject receiving the composition and can be administered without abnormal toxicity. As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the federal or state government or listed in the U.S. Pharmacopoeia, the European Pharmacopoeia or other generally recognized pharmacopoeia for use in mammals and more particularly in humans. These compositions can be used to treat and / or prevent viral infections and / or autoimmune diseases.

[0340] An in-depth discussion of pharmaceutically acceptable carriers, diluents, and other excipients is provided in Remington's Pharmaceutical Sciences (17th edition, Mack Publishing Company) and Remington: The Science and Practice of Pharmacy (21st edition, Lippincott Williams & Wilkins), which are incorporated herein by reference. The formulation of the pharmaceutical composition should be suitable for the mode of administration. In an embodiment, the pharmaceutical composition is suitable for administration to humans and may be sterile, particulate-free, and / or pyrogen-free.

[0341] Pharmaceutically acceptable carriers, excipients or diluents include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer, and combinations thereof.

[0342] Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.

[0343] Examples of pharmaceutically acceptable antioxidants include, but are not limited to: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0344] In an embodiment, the pharmaceutical composition is provided in solid form (such as a lyophilized powder suitable for reconstitution), liquid solution, suspension, emulsion, tablet, pill, capsule, slow release formulation, or powder.

[0345] In an embodiment, the pharmaceutical composition is provided in liquid form, for example in a sealed container indicating the amount and concentration of the active ingredient in the pharmaceutical composition. In a related embodiment, the liquid form of the pharmaceutical composition is supplied in a sealed container.

[0346] Methods for formulating the pharmaceutical compositions of the present disclosure are routine and well known in the art (see Remington and Remington's).One skilled in the art can readily formulate pharmaceutical compositions having desired properties (eg, route of administration, biological safety, and release profile).

[0347] The method for preparing the pharmaceutical composition comprises the step of associating the active ingredient with a pharmaceutically acceptable carrier and optionally one or more auxiliary ingredients. The pharmaceutical composition can be prepared by uniformly and intimately associating the active ingredient with a liquid carrier or a finely dispersed solid carrier or both and then (if necessary) shaping the product. Additional methods for preparing pharmaceutical compositions (including formulations of multilayer dosage forms) are described in Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (9th Edition, Lippincott Williams & Wilkins), which is incorporated herein by reference.

[0348] Pharmaceutical compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, etc., each containing a predetermined amount of one or more compounds described herein, their derivatives, or pharmaceutically acceptable salts or prodrugs thereof as one or more active ingredients. The active ingredient may also be administered in the form of a bolus, electuary or paste.

[0349] In solid dosage forms for oral administration (e.g., capsules, tablets, pills, dragees, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, excipients or diluents, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) ) disintegrants such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarder such as paraffin; (6) absorption accelerator such as quaternary ammonium compounds; (7) wetting agents such as cetyl alcohol and glyceryl monostearate; (8) absorbents such as kaolin and bentonite; (9) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical composition may also contain a buffer. Similar types of solid compositions can also be prepared using fillers in soft and hard-filled gelatin capsules, as well as excipients such as lactose or milk sugar and high molecular weight polyethylene glycols and the like.

[0350] Tablets can be made by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets can be prepared using a binder (e.g., gelatin or hydroxypropyl methylcellulose), a lubricant, an inert diluent, a preservative, a disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), a surfactant and / or a dispersant. Molded tablets can be prepared by molding a mixture of powdered active ingredients moistened with an inert liquid diluent in a suitable machine.

[0351] Tablets and other solid dosage forms such as dragees, capsules, pills and granules may optionally be scored or prepared with coatings and shells such as enteric coatings and other coatings well known in the art.

[0352] In some embodiments, in order to prolong the effect of active ingredients, it is desirable to slow down the absorption of the compound by subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline material or amorphous material with poor water solubility. Then, the absorption rate of the active ingredient depends on its dissolution rate, and the dissolution rate can then depend on the crystal size and crystal form. Alternatively, the absorption of the active ingredient administered parenterally can be delayed by dissolving or suspending the compound in an oily vehicle. In addition, the absorption of injectable drug forms can be prolonged by comprising a medicament (such as aluminum monostearate and gelatin) that delays absorption.

[0353] Controlled release parenteral compositions can be in the form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, emulsions, or the active ingredient can be incorporated into one or more biocompatible carriers, liposomes, nanoparticles, implants or infusion devices.

[0354] Materials used to prepare microspheres and / or microcapsules include biodegradable / bioerodible polymers such as polyglactin, poly(isobutylcyanoacrylate), poly(2-hydroxyethyl-L-glutamine), and poly(lactic acid).

[0355] Biocompatible carriers that may be used when formulating controlled release parenteral formulations include carbohydrates (such as dextran), proteins (such as albumin, lipoproteins or antibodies).

[0356] The material used in the implant can be a non-biodegradable material (eg, polydimethylsiloxane) or a biodegradable material (eg, poly(caprolactone), poly(lactic acid), poly(glycolic acid), or poly(orthoesters)).

[0357] In embodiments, the active ingredient is administered by aerosol. This is accomplished by preparing an aqueous aerosol, a liposomal formulation, or solid particles containing the compound. Non-aqueous (e.g., fluorocarbon propellants) suspensions may be used. The pharmaceutical composition may also be administered using an ultrasonic nebulizer, which will minimize exposure of the agent to shearing (which may result in compound degradation).

[0358] Typically, aqueous aerosols are prepared by preparing an aqueous solution or suspension of one or more active ingredients with conventional pharmaceutically acceptable carriers and stabilizers. Carriers and stabilizers vary with the requirements of the specific compound, but typically include nonionic surfactants (Tweens, Pluronics or polyethylene glycols), harmless proteins (such as serum albumin), sorbitan esters, oleic acid, lecithin, amino acids (such as glycine), buffers, salts, sugars or sugar alcohols. Aerosols are usually prepared from isotonic solutions.

[0359] Dosage forms for topical or transdermal administration of one or more active ingredients include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches and inhalants. The one or more active ingredients may be mixed under sterile conditions with a pharmaceutically acceptable carrier and, if appropriate, any preservatives, buffers or propellants.

[0360] Transdermal patches suitable for use in the present disclosure are disclosed in Transdermal Drug Delivery: Developmental Issues and Research Initiatives (Marcel Dekker Inc., 1989) and U.S. Patent Nos. 4,743,249, 4,906,169, 5,198,223, 4,816,540, 5,422,119, 5,023,084, which are incorporated herein by reference. Transdermal patches can also be any transdermal patches well known in the art, including scrotal patches. The pharmaceutical composition in such transdermal patches can contain one or more absorption enhancers or skin penetration enhancers well known in the art (see, for example, U.S. Patent Nos. 4,379,454 and 4,973,468, which are incorporated herein by reference). The transdermal therapeutic system used in the present disclosure can be based on iontophoresis, diffusion or a combination of these two effects.

[0361] Transdermal patches have the additional advantage of providing controlled delivery of one or more active ingredients to the body. Such dosage forms can be prepared by dissolving or dispersing one or more active ingredients in a suitable medium. Absorption enhancers can also be used to increase the flux of active ingredients through the skin. The rate of circulation can be controlled by providing a rate-controlled membrane or by dispersing one or more active ingredients in a polymer matrix or a gel.

[0362] Such pharmaceutical compositions can be in the form of creams, ointments, lotions, liniments, gels, hydrogels, solutions, suspensions, sticks, sprays, pastes, plasters and other types of transdermal drug delivery systems. The composition can also include pharmaceutically acceptable carriers or excipients, such as emulsifiers, antioxidants, buffers, preservatives, moisturizers, penetration enhancers, chelating agents, gelling agents, ointment bases, fragrances and skin protectants.

[0363] Examples of emulsifiers include, but are not limited to, naturally occurring gums (eg, gum arabic or gum tragacanth), naturally occurring phospholipids (eg, soy lecithin and sorbitan monooleate derivatives).

[0364] Examples of antioxidants include, but are not limited to, butylated hydroxyanisole (BHA), ascorbic acid and its derivatives, tocopherol and its derivatives, and cysteine.

[0365] Examples of preservatives include, but are not limited to, trehalose, parabens (such as methyl or propyl paraben), and benzalkonium chloride.

[0366] Examples of humectants include, but are not limited to, glycerin, propylene glycol, sorbitol, and urea.

[0367] Examples of penetration enhancers include, but are not limited to, propylene glycol, DMSO, triethanolamine, N,N-dimethylacetamide, N,N-dimethylformamide, 2-pyrrolidone and its derivatives, tetrahydrofurfuryl alcohol, propylene glycol, diethylene glycol monoethyl ether or monomethyl ether and propylene glycol monolaurate or methyl laurate, eucalyptol, lecithin, TRANSCUTOL, and AZONE.

[0368] Examples of chelating agents include, but are not limited to, sodium EDTA, citric acid, and phosphoric acid.

[0369] Examples of gelling agents include, but are not limited to, carbomer, cellulose derivatives, bentonite, alginates, gelatin, and polyvinylpyrrolidone.

[0370] Ointments, pastes, creams and gels of the present disclosure may contain, in addition to one or more active ingredients, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicone, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0371] Powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate and polyamide powder, or mixtures of these substances. Sprays may additionally contain conventional propellants, such as chlorofluorocarbons and volatile unsubstituted hydrocarbons (such as butane and propane).

[0372] Injectable depot forms are prepared by forming a microencapsulation matrix of one or more compounds of the present disclosure in a biodegradable polymer such as polylactide-polyglycolide. Depending on the ratio of compound to polymer and the properties of the specific polymer employed, the compound release rate can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by embedding the drug in liposomes or microemulsions compatible with body tissues.

[0373] Subcutaneous implants are well known in the art, and are suitable for use in the present disclosure. Subcutaneous implantation methods are preferably non-irritating and have mechanical elasticity. Implants can be matrix type, reservoir type or their mixtures. In matrix type devices, the carrier material can be a porous or non-porous solid or semisolid, and permeable or impermeable one or more active compounds. The carrier material can be biodegradable, or can be slowly eroded after application. In some cases, the matrix is ​​non-degradable, but relies on the active compound to diffuse through the matrix of the carrier material to degrade. Alternative subcutaneous implantation methods use a reservoir device, wherein one or more active compounds are surrounded by a rate-controlled membrane (for example, a membrane (with zero-order kinetics) that is independent of component concentration). The device composed of a matrix surrounded by a rate-controlled membrane is also suitable for use.

[0374] Both reservoir and matrix devices may contain materials such as polydimethylsiloxane (such as SILASTIC) or other silicone rubbers. Matrix materials may be insoluble polypropylene, polyethylene, polyvinyl chloride, ethylvinyl acetate, polystyrene and polymethacrylate, and glyceryl palmitostearate, glyceryl stearate and glyceryl behenate types. Materials may be hydrophobic or hydrophilic polymers and may optionally contain a solubilizing agent.

[0375] The subcutaneous implant device may be a slow release capsule made of any suitable polymer, for example, as described in US Pat. Nos. 5,035,891 and 4,210,644, which are incorporated herein by reference.

[0376] Usually, in order to control the release and transdermal penetration of pharmaceutical compounds, at least four different methods can be used. These methods are: membrane buffer system, adhesive diffusion control system, matrix dispersion system and micro-reservoir system. It should be understood that the transdermal and / or local compositions of controlled release can be obtained by using a suitable mixture of these methods.

[0377] In the membrane buffer system, the active ingredient is present in a reservoir completely encapsulated in a shallow compartment, which is molded from a drug-impermeable laminate (such as a metal plastic laminate) and a rate-controlling polymer membrane (such as a multi-microporous or non-porous polymer membrane, such as ethylene-vinyl acetate copolymer). The active ingredient is released through the rate-controlling polymer membrane. In the drug reservoir, the active ingredient can be dispersed in a solid polymer matrix or suspended in a non-leaching viscous liquid medium (such as silicone oil). On the outer surface of the polymer membrane, a thin layer of viscous polymer is applied to achieve close contact between the transdermal system and the skin surface. The viscous polymer can be a polymer that is hypoallergenic and compatible with the active drug substance.

[0378] In an adhesive diffusion controlled system, a reservoir of active ingredient is formed by dispersing the active ingredient directly in an adhesive polymer and then spreading the active ingredient-containing adhesive onto a substantially drug-impermeable metal-plastic backed flat sheet by, for example, solvent casting to form a thin drug reservoir layer.

[0379] Matrix dispersion systems are characterized in that a reservoir of active ingredients is formed by substantially uniformly dispersing the active ingredient in a hydrophilic or lipophilic polymer matrix. The drug-containing polymer is then molded into a disk having a substantially well-defined surface area and a controlled thickness. An adhesive polymer is spread circumferentially to form an adhesive band around the disk.

[0380] Microreservoir systems can be viewed as a combination of reservoir and matrix dispersion type systems. In this case, a reservoir of active substance is formed by first suspending the drug solid in an aqueous solution of a water-soluble polymer and then dispersing the drug suspension in a lipophilic polymer to form a plurality of non-leachable tiny drug reservoir spheres.

[0381] Any of the controlled release, extended release, and slow release compositions described herein can be formulated to release the active ingredient within about 30 minutes to about 1 week, within about 30 minutes to about 72 hours, within about 30 minutes to 24 hours, within about 30 minutes to 12 hours, within about 30 minutes to 6 hours, within about 30 minutes to 4 hours, and within about 3 hours to 10 hours. In embodiments, after administering the pharmaceutical composition to a subject, an effective concentration of one or more active ingredients is maintained in the subject for 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours or longer.

[0382] VII. Vaccine or immunogenic composition

[0383] The present disclosure relates to methods of combination therapy. The combination therapy includes at least one immunogenic composition, for example, a tumor vaccine or immunogenic composition capable of eliciting a specific T cell response. The tumor vaccine or immunogenic composition comprises a neoantigenic peptide and / or neoantigenic polypeptide corresponding to a tumor-specific neoantigen identified by the methods described herein.

[0384] Suitable tumor vaccines or immunogenic compositions may contain a variety of tumor-specific neoantigenic peptides. In an embodiment, a vaccine or immunogenic composition may include peptides between 1 and 100 sets, such peptides between 1 and 50, peptides between 10 and 30 sets, or peptides between 15 and 25. According to another embodiment, a vaccine or immunogenic composition may include at least one peptide, such as 2, 3, 4 or 5 peptides. In certain embodiments, a vaccine or immunogenic composition may include 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 different peptides. Multiple doses of a vaccine or immunogenic composition may be administered to a subject. Each dose of a vaccine composition may include peptides of different sets. For example, one dose of the composition or a portion of one dose of the composition may contain 5 peptides. Another portion of the dose may contain a different set of 5 peptides.

[0385] The optimal amount and optimal dosing regimen of each peptide contained in the vaccine or immunogenic composition can be determined by those skilled in the art without excessive experimentation. For example, a peptide or variant thereof can be prepared for intravenous (iv) injection, subcutaneous (sc) injection, intradermal (id) injection, intraperitoneal (ip) injection, intramuscular (im) injection. Peptide injection methods include sc, id, ip, im and iv. DNA injection methods include id, im, sc, ip and iv. For example, a dose of a peptide or DNA between 1 and 500 mg, between 50 μg and 1.5 mg, or between 10 μg and 500 μg can be given, and it may depend on the corresponding peptide or DNA. This range of doses has been successfully used in previous experiments (Brunsvig PF et al., Cancer Immunol Immunother. 2006; 55 (12): 1553-1564; M. Staehler et al., ASCO meeting 2007; Abstract No. 3017). Other methods of administering a vaccine or immunogenic composition are known to those skilled in the art.

[0386] Vaccines or immunogenic compositions can be administered to a subject in the form of one or more subcutaneous injections. In one embodiment, a single dose of a composition can be divided into one or more subcutaneous injections for administration. For example, a single dose of a composition can be divided into 1, 2, 3, 4, 5, 6, 7 or 8 different subcutaneous injections. Each injection of the composition can include one or more peptides. The peptides in each injection of a single dose of a composition can include peptides of different sets. In some examples, each injection of a single dose of a composition includes 1, 2, 3, 4, 5 or 6 different peptides. Alternatively, each subcutaneous injection of a single dose of a composition can include peptides of the same set. In some cases, multiple injections as a part of a single dose of a vaccine or immunogenic composition can include peptides of different sets. For example, a single dose of a composition can be divided into 4 injections, each injection including 5 different sets of peptides.

[0387] Vaccine or immunogenic composition can be applied to a single part of the patient with subcutaneous injection.For example, a single dose of vaccine or immunogenic composition can be applied to the patient's extremities with a single injection.In the case where the composition of a single dose is divided into one or more injections, the dosage can be applied to multiple parts of the object.For example, in the case where the dosage is divided into 4 different injections, 4 different injections can be applied to different extremities.In some cases, multiple injections as a part of the composition of a single dose can be applied at the same site in different time periods.For example, a time period of 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 50 minutes or 60 minutes can be provided between different injections of a single dose of vaccine or immunogenic composition.

[0388] In one embodiment of the present disclosure, different tumor-specific neoantigenic peptides and / or polypeptides are selected for use in tumor vaccines or immunogenic compositions to maximize the possibility of generating an immune attack against a patient's tumor / tumor. Without wishing to be bound by theory, it is believed that the inclusion of a variety of tumor-specific neoantigenic peptides can generate a large-scale immune attack against a tumor / tumor. In one embodiment, the selected tumor-specific neoantigenic peptide / polypeptide is encoded by a missense mutation. In a second embodiment, the selected tumor-specific neoantigenic peptide polypeptide is encoded by a combination of a missense mutation and a neoORF mutation. In a third embodiment, the selected tumor-specific neoantigenic peptide / polypeptide is encoded by a neoORF mutation.

[0389] In one embodiment where the selected tumor-specific neoantigenic peptides / polypeptides are encoded by missense mutations, the peptides and / or polypeptides are selected based on their ability to associate with the patient's specific MHC molecules. Peptides / polypeptides derived from neoORF mutations can also be selected based on their ability to associate with the patient's specific MHC molecules, but can be selected even if association with the patient's specific MHC molecules is not predicted.

[0390] The vaccine or immunogenic composition is capable of eliciting a specific cytotoxic T cell response and / or a specific helper T cell response.

[0391] Vaccines or immunogenic compositions may also include adjuvants and / or carriers. Examples of useful adjuvants and carriers are given herein. The peptides and / or polypeptides in the composition may be associated with a carrier (e.g., a protein or an antigen presenting cell, such as a dendritic cell (DC) capable of presenting the peptide to a T cell).

[0392] An adjuvant is any substance that is mixed into a vaccine or immunogenic composition to increase or otherwise alter the immune response to the mutant peptide. A carrier is a scaffold structure, such as a polypeptide or polysaccharide, to which the neoantigenic peptide can associate. Optionally, the adjuvant is covalently or non-covalently conjugated to the peptide or polypeptide of the present disclosure.

[0393] The ability of an adjuvant to increase the immune response to an antigen is generally manifested as a significant increase in an immune-mediated reaction or a decrease in disease symptoms. For example, an increase in humoral immunity is generally manifested as a significant increase in the titer of antibodies produced against the antigen, and an increase in T cell activity is generally manifested as increased cell proliferation or cytotoxicity or cytokine secretion. An adjuvant can also alter the immune response, for example by changing a predominantly humoral or Th2 response to a predominantly cellular or Th1 response.

[0394] Suitable adjuvants include, but are not limited to, 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS1312, Montanide ISA206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL vector system, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, Aquila's QS21 stimulator (Aquila Biotech, Worcester, Mass., USA, which is derived from saponins, mycobacterial extracts and synthetic bacterial cell wall mimics), and other proprietary adjuvants such as Ribi's Detox, Quil or Superfos. Several immunoadjuvants (e.g., F59) specific for dendritic cells and their preparation have been described previously (Dupuis M et al., Cell Immunol. 1998; 186 (1): 18-27; Allison AC; Dev Biol Stand. 1998; 92: 3-11). Cytokines can also be used. Several cytokines have been directly implicated in influencing the migration of dendritic cells to lymphoid tissues (e.g., TNF-α), accelerating the maturation of dendritic cells into potent antigen-presenting cells of T lymphocytes (e.g., GM-CSF, IL-1, and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich DI et al., J Immunother Emphasis Tumor Immunol. 1996(6):414-418).

[0395] Toll-like receptors (TLRs) can also be used as adjuvants and are important members of the pattern recognition receptor (PRR) family, which recognize conserved motifs (called "pathogen-associated molecular patterns" (PAMPs)) shared by many microorganisms. Recognition of these "danger signals" activates multiple elements of the innate and adaptive immune systems. TLRs are expressed by cells of the innate and adaptive immune systems (such as dendritic cells (DCs), macrophages, T and B cells, mast cells, and granulocytes) and are located in different cell compartments (such as plasma membranes, lysosomes, endosomes, and endolysosomes). Different TLRs recognize different PAMPs. For example, TLR4 is activated by LPS contained in bacterial cell walls, TLR9 is activated by unmethylated bacterial or viral CpG DNA, and TLRS is activated by double-stranded RNA. TLR ligand binding leads to the activation of one or more intracellular signal transduction pathways, ultimately leading to the production of many key molecules associated with inflammation and immunity (particularly transcription factors NF-κB and type I interferons). TLR-mediated DC activation leads to enhanced DC activation, phagocytosis, upregulation of activation and co-stimulatory markers (such as CD80, CD83 and CD86), CCR7 expression (allowing DC to migrate to draining lymph nodes and promote antigen presentation to T cells), and increased secretion of cytokines (such as type I interferons, IL-12 and IL-6). All of these downstream events are critical for the induction of adaptive immune responses.

[0396] The most promising cancer vaccine or immunogenic composition adjuvants currently in clinical development include the TLR9 agonist CpG and the synthetic double-stranded RNA (dsRNA) TLR3 ligand polyICLC. In preclinical studies, polyICLC appears to be the most effective TLR adjuvant compared to LPS and CpG because it induces proinflammatory cytokines, lacks stimulation of IL-IO, and maintains high levels of co-stimulatory molecules in IXsi. Furthermore, polyICLC was recently directly compared to CpG (composed of human papillomavirus (HPV) 16 capsids) as adjuvants for protein vaccines or immunogenic compositions in non-human primates (rhesus macaques) (Stahl-Hennig C, Eisenblatter M, Jasny E et al. Synthetic double-stranded R As are adjuvants for the induction of T helper I and humoral immune responses to human papillomavirus in rhesus macaques. PLoS pathogens. 2009 Apr;5(4)).

[0397] It has also been reported that CpG immunostimulatory oligonucleotides enhance the effect of adjuvants in vaccines or immunogenic composition environments. Without being bound by theory, CpG oligonucleotides play a role by activating the innate (non-adaptive) immune system via Toll-like receptors (TLR, mainly TLR9). The TLR9 activation triggered by CpG enhances the antigen-specific humoral and cellular responses to a variety of antigens (including peptide or protein antigens, live viruses or inactivated viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in preventive and therapeutic vaccines). More importantly, it enhances the maturation and differentiation of dendritic cells, thereby enhancing the activation of Th1 cells and the generation of strong cytotoxic T lymphocytes (CTL), even in the absence of CD4 T cells. Even in the presence of vaccine adjuvants (such as alum or incomplete Freund's adjuvant (IFA)) that usually promote Th2 bias, the Th1 bias induced by TLR9 stimulation is still maintained. When CpG oligonucleotides are formulated or co-administered with other adjuvants or in formulations (such as microparticles, nanoparticles, lipids, emulsions or similar formulations), CpG oligonucleotides show even greater adjuvant activity, which is particularly necessary for inducing strong responses when antigens are relatively weak. They also accelerate immune responses and reduce antigen doses by about two orders of magnitude, wherein in some experiments, there is an antibody response (Arthur M. Krieg, Nature Reviews, Drug Discovery, June 5, 2006, 471-484) comparable to a full dose vaccine without CpG. U.S. Patent No. 6,406,705 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants and antigens to induce antigen-specific immune responses. Commercially available CpG TLR9 antagonists are dSLIM (double stem loop immunomodulators) of Mologen (Berlin, GERMANY), which can be a component of a pharmaceutical composition disclosed herein. Other TLR binding molecules, such as TLR 7, TLR 8 and / or TLR 9 that bind RNA may also be used.

[0398] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), poly(I:C) (e.g., poly I:CI2U), non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies (such as cyclophosphamide, sunitinib, bevacizumab, Celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which can act therapeutically and / or as adjuvants). The skilled artisan can readily determine the amounts and concentrations of adjuvants and additives useful in the context of the present disclosure without undue experimentation. Additional adjuvants include colony stimulating factors, such as granulocyte macrophage colony stimulating factor (GM-CSF, sargramostim).

[0399] PolyICLC is a synthetically prepared double-stranded RNA, which consists of poly-I and poly-C chains with an average length of about 5000 nucleotides, and is stabilized to heat denaturation and hydrolysis by serum nucleases by adding polylysine and carboxymethyl cellulose. The compound activates the RNA helicase domains of TLR3 and MDA5 (both members of the PAMP family), leading to DC and natural killer (NK) cell activation and production of a "natural mixture" of type I interferons, cytokines and chemokines. In addition, polyICLC exerts a more direct, broad host-targeted anti-infective effect and possible anti-tumor effect through two IFN-inducible ribozyme systems (2'5'-OAS and Pl / eIF2a kinase (also known as PKR (4-6)) and RIG-I helicase and MDA5).

[0400] In rodents and nonhuman primates, polyICLC has been shown to enhance T cell responses to viral antigens, cross-prime, and induce tumor-specific, virus-specific, and self-antigen-specific CD8+ T cells. In recent studies in nonhuman primates, polyICLC was found to be essential for the generation of antibody responses and T cell immunity against either DC-targeted or non-targeted HIV Gag p24 protein, underscoring its usefulness as a vaccine adjuvant.

[0401] In human subjects, transcriptional analysis of serial whole blood samples revealed similar gene expression profiles in eight healthy human volunteers who received a single subcutaneous (sc) administration of polyICLC, with up to 212 genes differentially expressed between these eight subjects and four subjects who received placebo. Strikingly, comparison of the polyICLC gene expression data with previous data from volunteers immunized with the highly effective yellow fever vaccine YF17D revealed that a large number of transcriptional pathways and canonical pathways of signal transduction, including those of the innate immune system, were similarly upregulated at peak time points.

[0402] Recently, an immunological analysis was reported of patients with ovarian, fallopian tube, and primary peritoneal cancer who were in second or third complete clinical remission and were treated with synthetic overlapping long peptides (OLP) from the cancer-testis antigen NY-ESO-1 alone, or Montanide-ISA-51, or 1.4 mg polyICLC and Montanide in a phase 1 study of subcutaneous vaccination. The addition of polyICLC and Montanide significantly enhanced the generation of NY-ESO-1-specific CD4+ and CD8+ T cells and antibody responses compared with OLP alone or OLP and Montanide.

[0403] Vaccines or immunogenic compositions according to the present disclosure may contain more than one different adjuvant. In addition, the present disclosure encompasses therapeutic compositions comprising any adjuvant substance, including any adjuvant substance discussed herein. Peptides or polypeptides are also contemplated, and the adjuvants may be administered separately in any appropriate order.

[0404] The carrier may be independent of the presence of an adjuvant. The carrier may be covalently linked to the antigen. The carrier may also be added to the antigen by inserting the DNA encoding the carrier into the frame with the DNA encoding the antigen. The function of the carrier may be, for example, to impart stability, increase biological activity, or increase serum half-life. Extending the half-life may help reduce the number of applications and reduce the dosage, which is therefore beneficial for treatment and also for economic reasons. In addition, the carrier may help the peptide to be presented to T cells. The carrier may be any suitable carrier known to those skilled in the art, such as a protein or an antigen presenting cell. The carrier protein may be, but is not limited to, keyhole limpet hemocyanin, serum proteins (such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin), immunoglobulins, or hormones (such as insulin or palmitic acid). For human immunity, the carrier may be a physiologically acceptable carrier that is safe and acceptable to humans. However, in one embodiment of the present disclosure, tetanus toxoid and / or diphtheria toxoid are suitable carriers. Alternatively, the carrier may be a dextran, such as agarose.

[0405] Cytotoxic T cells (CTLs) recognize antigens in the form of peptides bound to MHC molecules, without binding to the complete exogenous antigen itself. The MHC molecules themselves are located on the cell surface of antigen presenting cells. Therefore, CTLs can only be activated when there is a trimeric complex of peptide antigens, MHC molecules and APCs. Accordingly, it can enhance the immune response if not only the peptides are used for the activation of CTLs, but also if APCs with corresponding MHC molecules are added in addition. Therefore, in some embodiments, the vaccine or immunogenic composition according to the present disclosure additionally contains at least one antigen presenting cell.

[0406] Antigen presenting cells (or stimulator cells) typically have MHC class I or class II molecules on their surface, and in one embodiment, are themselves substantially incapable of loading the MHC class I or class II molecules with selected antigens. As described in more detail herein, MHC class I or class II molecules can be readily loaded with selected antigens in vitro.

[0407] CD8+ cell activity can be enhanced by using CD4+ cells. The identification of CD4T+ cell epitopes of tumor antigens has attracted people's attention, because if both CD8+ and CD4+ T lymphocytes are used to target the patient's tumor, many immune-based anticancer therapies can be more effective, and CD4+ cells can enhance CD8 T cell responses. Many studies conducted in animal models clearly show that when both CD4+ and CD8+ T cells are involved in anti-tumor responses, the results are better (see, for example, Nishimura et al. (1999) Distinct role of antigen-specific T helper type I (Th1) and Th2 cells in tumor eradication in vivo. J Ex Med 190: 617-27). Universal CD4+ T cell epitopes suitable for developing therapies for different types of cancer have been identified (see, for example, Kobayashi et al. (2008) Current Opinion in Immunology 20: 221-27). For example, HLA-DR restricted adjuvant peptides from tetanus toxoid are used in melanoma vaccines to non-specifically activate CD4+ T cells (see, e.g., Slingluff et al. (2007) Immunologic and Clinical Outcomes of a Randomized Phase II Trial of Two Multipeptide Vaccines for Melanoma in the Adjuvant Setting, Clinical Cancer Research 13(21):6386-95). It is contemplated within the scope of the present disclosure that such CD4+ cells can be applied at three levels that differ in tumor specificity: 1) a broad level, where universal CD4+ epitopes (e.g., tetanus toxoid) can be used to enhance CD8+ cells; 2) an intermediate level, where natural tumor-associated CD4+ epitopes can be used to enhance CD8+ cells; and 3) a patient-specific level, where neoantigen CD4+ epitopes can be used to enhance CD8+ cells in a patient-specific manner.

[0408] CD8+ cellular immunity can also be generated with dendritic cell (DC) vaccines loaded with neoantigens. DC is a potent antigen presenting cell that initiates T cell immunity, and when it is loaded with one or more target peptides (e.g., by direct peptide injection), it can be used as a cancer vaccine. For example, newly diagnosed patients with metastatic melanoma show that, via the production of IL-12p70 patient DC vaccines, mature DC activated with autologous peptide pulsed CD40 L / IFN-g produce immunity to 3 HLA-A*0201 restricted gp100 melanoma antigen derived peptides (see, e.g., Carreno et al. (2013) L-12p70-producing patient DC vaccine elicits Tel-polarized immunity, Journal of Clinical Investigation, 123 (8): 3383-94, and Ali et al. (2009) In situ regulation of DC subsets and T cells mediates tumor regression in mice, Cancer Immunotherapy, 1 (8): 1-10). It is expected within the scope of the present disclosure that DC loaded with neoantigens can be prepared by stimulating DC with the synthetic TLR 3 agonist polyinosinic acid-polycytidylic acid-poly-L-lysine carboxymethylcellulose (polyICLC). PolyICLC is a potent individual maturation stimulator of human DC as assessed by upregulating CD83 and CD86, inducing interleukin-12 (IL-I2), tumor necrosis factor (TNF), interferon gamma-induced protein 10 (IP-10), interleukin 1 (IL-1) and type I interferon (IFN), and very little interleukin 10 (IL-10) production. DC can be distinguished from frozen peripheral blood mononuclear cells (PBMC) obtained by leukocyte apheresis, and PBMC can be separated by Ficoll gradient centrifugation and frozen in equal parts.

[0409] Illustratively, the following 7-day activation scheme can be used. On the 1st day, PBMCs were thawed and inoculated in tissue culture bottles to select monocytes that adhere to plastic surfaces after incubation at 37°C for 1-2 hours in a tissue culture incubator. After incubation, lymphocytes were washed away, and adhered monocytes were cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte macrophage colony stimulating factor (GM-CSF) to differentiate them into immature DCs. On the 6th day, immature DCs were pulsed with keyhole limpet hemocyanin (KLH), which served as a control of vaccine quality and could strengthen the immunogenicity of the vaccine. DCs were stimulated to maturity, loaded with peptide antigens, and incubated overnight. On the 7th day, cells were washed and frozen in 1 ml aliquots containing 4-20x10(6) individual cells using a controlled rate refrigerator. Batches of DCs can be batch-release tested to meet minimum specifications before injection into patients (see, e.g., Sabado et al. (2013) Preparation of tumor antigen-loaded mature dendritic cells for immunotherapy, J. Vis Exp. Aug 1;(78). doi:10.3791 / 50085).

[0410] DC vaccine can be incorporated into the support system, to promote delivery to the patient. Therapeutic treatment of patient tumor with DC vaccine can utilize biomaterial system, the biomaterial system releases the factor that host dendritic cells are recruited into the device, while releasing antigen, distinguishes the immature DC that resides by local presentation adjuvant (for example, danger signal), and promotes activation, DC loaded with antigen to be released into lymph node (or desired site of action), wherein DC can interact with T cell to generate the strong cytotoxic T lymphocyte response for cancer neoantigen. Implantable biomaterial can be used to generate the strong cytotoxic T lymphocyte response for tumor in a patient-specific manner. Then, the dendritic cell activation can be made by exposing the dendritic cell that biomaterial resides to the danger signal of simulated infection, cooperating with releasing antigen from biomaterial. Then, the activated dendritic cell migrates to lymph node from biomaterial to respond to induce cytotoxic T effector cells. This approach has previously been demonstrated to result in regression of established melanomas in preclinical studies using lysates prepared from tumor biopsies (see, e.g., Ali et al. (2009) In situ regulation of DC subsets and T cells mediates tumor regression in mice, Cancer Immunotherapy 1(8):1-10; Ali et al. (2009)).

[0411] In some embodiments, the antigen presenting cell is a dendritic cell. Suitably, the dendritic cell is an autologous dendritic cell pulsed with a neoantigen peptide. The peptide can be any suitable peptide that produces an appropriate T cell response. T cell therapy using autologous dendritic cells pulsed with peptides from tumor-associated antigens is disclosed in Murphy et al. (1996) The Prostate 29, 371-380 and Tjua et al. (1997) The Prostate 32, 272-278.

[0412] Therefore, in one embodiment of the present disclosure, a vaccine or immunogenic composition containing at least one antigen presenting cell is pulsed with one or more peptides of the present disclosure or is loaded with one or more peptides of the present disclosure. Alternatively, peripheral blood mononuclear cells (PBMCs) isolated from patients can be loaded with peptides in vivo and injected back into the patient. Alternatively, antigen presenting cells comprise expression constructs encoding peptides of the present disclosure. The polynucleotides can be any suitable polynucleotides and can transduce dendritic cells, thereby producing peptide presentation and immune induction.

[0413] The pharmaceutical compositions of the present invention can be edited so that the selection, quantity and / or amount of peptides present in the composition is tissue-specific, cancer-specific and / or patient-specific. For example, the precise selection of peptides can be guided by the expression pattern of the parent protein in a given tissue to avoid side effects. The selection can depend on the specific type of cancer, the state of the disease, the early treatment regimen, the patient's immune status, and of course the patient's HLA haplotype. In addition, vaccines or immunogenic compositions according to the present disclosure can contain personalized components according to the personalized needs of a particular patient. Examples include varying the amount of peptides according to the expression of relevant neoantigens in a particular patient, adverse side effects due to personalized allergies or other treatments, and adjustments to secondary treatments after a first round of treatment or a first treatment regimen.

[0414] Pharmaceutical compositions comprising peptides of the present disclosure can be administered to individuals who already have cancer. In therapeutic applications, the composition is administered to a patient in an amount sufficient to induce an effective CTL response to a tumor antigen and to cure or at least partially inhibit symptoms and / or complications. An amount sufficient to achieve this purpose is defined as a "therapeutically effective dose". The amount effective for this purpose may depend on, for example, the peptide composition, the mode of administration, the stage and severity of the disease being treated, the patient's weight and general health, and the judgment of the prescribing physician, but the amount is generally about 1.0 μg to about 50,000 μg of peptide for a 70 kg patient for the first immunization (i.e., for therapeutic or prophylactic administration), followed by a booster dose or about 1.0 μg to about 10,000 μg of peptide according to a booster regimen of several weeks to several months, depending on the patient's response and condition, and may be determined by measuring specific CTL activity in the patient's blood. It should be remembered that the peptides and compositions of the present disclosure can generally be used in severe disease states, i.e., life-threatening or life-threatening situations, especially when the cancer has metastasized. For therapeutic use, administration should be initiated as soon as possible after detection or surgical removal of a tumor, followed by booster doses until symptoms are at least substantially alleviated, and for a period thereafter.

[0415] Pharmaceutical compositions (e.g., vaccine compositions) for therapeutic treatment are intended for parenteral, topical, nasal, oral or local administration. In some embodiments, pharmaceutical compositions are administered parenterally (e.g., intravenously, subcutaneously, intradermally or intramuscularly). Compositions can be administered at the site of surgical resection to induce a local immune response to a tumor. The present disclosure provides compositions for parenteral administration, comprising a solution of a peptide and a vaccine, or dissolving or suspending an immunogenic composition in an acceptable carrier (e.g., an aqueous carrier). Various aqueous carriers can be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, etc. These compositions can be sterilized by conventional, well-known sterilization techniques, or can be aseptically filtered. The resulting aqueous solution can be packaged and used as is, or lyophilized, and the lyophilized preparation is merged with a sterile solution before administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusters and buffers, toxicity adjusters, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.

[0416] Liposomal suspensions containing peptides can be administered intravenously, locally, topically, etc., with dosages varying depending, inter alia, on the mode of administration, the peptide being delivered, and the stage of the disease being treated. To target immune cells, ligands (e.g., antibodies or fragments thereof specific for cell surface determinants of desired immune system cells) can be incorporated into the liposomes.

[0417] For solid compositions, conventional carriers or nanoparticulate non-toxic solid carriers can be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. For oral administration, a pharmaceutically acceptable non-toxic composition is formed by incorporating any common excipients (such as those carriers listed previously) and typically 10%-95% of the active ingredient (i.e., one or more peptides of the present disclosure), for example, at a concentration of 25%-75%.

[0418] For aerosol administration, the immunogenic peptide can be provided in finely dispersed form with a surfactant and a propellant. Typical percentages of peptides are 0.01%-20% by weight, for example 1%-10%. Of course, the surfactant can be non-toxic and soluble in the propellant. Representatives of such agents are esters or partial esters of fatty acids containing 6 to 22 carbon atoms (such as caproic acid, caprylic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric acid and oleic acid) with aliphatic polyols or their cyclic anhydrides. Mixed esters such as mixed or natural glycerides can be used. The surfactant can account for 0.1%-20% of the weight of the composition, for example 0.25%-5%. The balance of the composition is usually a propellant. A carrier can also be included as needed, for example, lecithin for intranasal delivery.

[0419] The peptides and polypeptides disclosed herein can be readily chemically synthesized using reagents that do not contain contaminating bacteria or animal matter (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of atetrapeptide. J. Am. Chem, Soc. 85:2149-54, 1963).

[0420] The peptides and polypeptides of the present disclosure may also be expressed by vectors, such as nucleic acid molecules (e.g., RNA or DNA plasmids), viral vectors (such as poxviruses (e.g., orthopoxviruses, fowlpoxviruses) or adenoviruses, AAV or lentiviruses) as discussed herein. The method involves the use of vectors to express nucleotide sequences encoding the peptides of the present disclosure. Upon introduction into an acutely or chronically infected host or an uninfected host, the vector expresses the immunogenic peptide, thereby eliciting a host CTL response.

[0421] For therapeutic or immune purposes, nucleic acids encoding the disclosed peptides and optionally one or more peptides described herein can also be administered to patients. Many methods are conveniently used to deliver nucleic acids to patients. For example, nucleic acids can be delivered directly as "naked DNA". This method is described, for example, in Wolff et al., Science 247:1465-1468 (1990) and U.S. Patent Nos. 5,580,859 and 5,589,466. Nucleic acids can also be administered using ballistic delivery as described in, for example, U.S. Patent No. 5,204,253. Particles containing only DNA can be administered. Alternatively, DNA can be adhered to particles (such as gold particles). Typically, a plasmid for a vaccine or immune composition may include DNA encoding an antigen (e.g., one or more neoantigens) operably linked to a regulatory sequence that controls expression or expression and secretion of the antigen from a host cell (e.g., a mammalian cell); for example, from upstream to downstream, DNA comprising a promoter such as a mammalian viral promoter (e.g., a CMV promoter, such as an hCMV or mCMV promoter, e.g., an immediate early promoter or an SV40 promoter, for useful promoters, see references cited or incorporated herein), DNA of a secreted eukaryotic leader peptide (e.g., tissue plasminogen activator), DNA of one or more neoantigens, and DNA encoding a terminator (e.g., a 3'UTR transcriptional terminator from a gene encoding bovine growth hormone or bGH polyA). The composition may contain more than one plasmid or vector, whereby each vector contains and expresses a different neoantigen. Wasmoen's U.S. Pat. No. 5,849,303 and Dale's U.S. Pat. No. 5,811,104 make reference, and its text can be, useful DNA or DNA plasmid preparation can be prepared with cationic lipid or in cationic lipid; And about cationic lipid and adjuvant, Loosmore's U.S. Patent Application 2003 / 0104008 makes reference. In addition, Audonnet's U.S. Pat. Nos. 6,228,846 and 6,159,477 teaching content can rely on DNA plasmid teaching content, and described teaching content can be used for constructing and using DNA plasmid that contains and expresses in vivo.

[0422] Nucleic acids can also be delivered in combination with cationic compounds such as cationic lipids. Lipid-mediated gene delivery methods are described in, for example, WO 1996 / 18372; WO 1993 / 24640; Mannino and Gould-Fogerite, BioTechniques 6(7):682-691 (1988); U.S. Patent No. 5,279,833; WO 1991 / 06309; and Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7414 (1987).

[0423] RNA (e.g., mRNA) encoding a peptide of interest can also be used for delivery (see, e.g., Kiken et al., 2011; Su et al., 2011; see also US 8278036; Halabi et al. J Clin Oncol (2003) 21: 1232-1237; Petsch et al., Nature Biotechnology 2012 Dec 7; 30(12): 1210-6).

[0424] With regard to poxviruses that can be used to practice the present disclosure, such as Chordopoxvirinae poxviruses (poxviruses of vertebrates), e.g., orthopoxviruses and avipoxviruses, e.g., vaccinia virus (e.g., Wyeth strain, WR strain (e.g., VR-1354), Copenhagen strain, NYVAC, NYVAC.1, NYVAC.2, MVA, VA-BN), canarypox virus (e.g., Wheatley C93 strain, ALVAC), fowlpox virus (e.g., FP9 strain, Webster strain, TROVAC), dovepox virus, pigeonpox virus, quailpox virus, and raccoonpox virus, and information on synthetic or non-naturally occurring recombinants thereof, their uses, and methods for making and using such recombinants can be found in the following scientific and patent literature, such as: U.S. Pat. Nos. 4,603,112, 4,769,330, 5 ,110,587、5,174,993、5,364,773、5,762,938、5,494,807、5,766,597、7,767,449、6,780,407、6,537,594、6,265,189、6,214,353、6,130,066、6,004,777、5,990,091、5,942,235、5,833,975、5,766,597、5,7 56,101、7,045,313、6,780,417、8,470,598、8,372,622、8,268,329、8,268,325、8,236,560、8,163,293、7,964,398、7,964,396、7,964,395、7,939,086、7,923,017、7,897,156、7,892,533、7,628,980、7,459 ,270, 7,445,924, 7,384,644, 7,335,364, 7,189,536, 7,097,842, 6,913,752, 6,761,893, 6,682,743, 5,770,212, 5,766,882, and 5,989,562, and Panicali, D. Proc. Natl. Acad. Sci. 1982; 79; 4927-493; Panicali. D. Proc. Natl. Acad. Sci. 1983; 80 (17): 5364-8; Mackett, M. Proc. Natl. Acad. Sci. 1982; 79: 7415-7419; Smith GL. 1983;302:490-5; SullivanVJ.Gen.Vir.1987;68:2587-98;Perkus M Journal of Leukocyte Biology 1995;58:1-13;Yilma TD.Vaccine 1989;7:484-485;Brochier B.Nature 1991;354:520-22;Wiktor,TJ.Proc.Natl Acad,Sci.1984;81:7194-8;Rupprecht,CE.Proc,Natl Acd.Sci.1986;83:7947-50;Pocciulet,H. 2007:25(Jul):5606-12;Weyer J.Vaccine 2009;27(Nov):7198-201;Buller,RM Nature1985;317(6040):813-5;Boiler RM.J.Virol.1988;62(3):866-74;Flexner,C.Nature 1987;330(6145):259-62;Shida,HJVirol.1988;62(12):4474-80;Kotwal; GJ.J.Virol.1989;63(2):600-6;Child,SJ.Virology1990;174(2):625-9;Mayr A.Zentralbl Bacteriol 1978;167(5,6):375-9;Antoine G.Virology.1998;244(2):365-96;Wyatt,LS.Virology 1998;251(2):334-42;Sancho,MC.J.Virol.2002;76(16);8313-34;Gallego-Gomez,JC.J.Virol.2003;77(19);106-22Gobel; SJ.Virology1990;(a,b)179:247-66;Tartaglia,J.Virol.1992;188(1):217-32;Najera JL.J.Virol.2006;80(12):6033-47;Najera,JL.J.Virol.2006;80:6033-6047;Gomez,CE.J.Gen.Virol.2007;88:2473-Jooij,P.Of. Virol.2008;82:2975-2988;Gomez,CE.Curr.Gene Ther.2011;11:189-217;Cox,W.Virology 1993;195:845-50;Perkus,M.Jour.OfLeukocyteBiology 1995;58:1-13;Blanchard TJ.J Gen Virology 1998;79(5):1159-67;Amara R.Science 2001;292:69-74;Hel,Z.,J.Immunol.2001;167:7180-9;Gherardi MM.J.Virol.2003;77:7048-57;Didierlaurent,A.Vaccine 2004;22:3395-3403;Bissht H.Proc.Nat.Aca.Sci.2004;101:6641-46;McCurdy LH.Clin.Inf.Dis 2004;38:1749-53;Earl PL.Nature2004;428:182-85;Chen Z.J.Virol.2005;79:2678-2688;Najera JL.J.Virol.2006;80(12):6033-47;Nam JH.Acta.Virol.2007;51:125-30;Antonis AF.Vaccine 2007;25:4818~4827;B Weyer J.Vaccine 2007;25:4213-22;Ferrier-Rembert A.Vaccine 2008;26(14):1794-804;CorbettM.Proc.Natl.Acad.Sci.2008:105(6):2046-51;Kaufman HL.,J.Clin.Oncol.2004;22:2122-32;Amato,RJ.Clin.Cancer Res.2008;14(22):7504-10;Dreicer R.Invest New Drugs 2009;27(4):379-86;KantoffPW.J.Clin.Oncol.2010,28,1099-1105;Amato RJ.J.Clin.Can.Res.2010;16(22):5539-47;Kim,DW.Hum.Vaccine.2010:6:784-791;Oudard,S.Cancer Immunol.]mmanother.2011;60:261-71;Wyatt,LS.Aids Res.Hum.Retroviruses.2004;20:645-53;Gomez,CE.Virus Research 2004;105:11-22;Webster,DP.Proc.Natl Acad.Sci.2005;102;4836-4;Huang,X.Vaccine 2007;25:8874-84;Gomez,CE.Vaccine 2007a;25:2863-85;Esteban M.Hum.Vaccine 2009;5:867-871;Gomez,CE.Curr.Genetherapy 2008;8(2):97-120;Whelan,KT.PLoS One 2009;4(6):5934; Scriba, TJ. Eur, Jour. Immuno. 2010;40(1):279-90; Corbett, M. Proc. Natl, Acad. Sci. 2008;105:2046-2051; Midgley, CM. J. Gen. Virol. 2008;89:2992-97; Von Krempelhuber, A. Vaccine 2010;28:1209-16; Perreau, MJ Of Virol. 2011;Oct:9854-62; Pantaleo, G. Curr Opin HIV-AIDS. 2010;5:391-396, each of which is incorporated herein by reference.

[0425] As for adenoviral vectors for practicing the present disclosure, U.S. Pat. No. 6,955,808 makes reference. The adenoviral vector used can be selected from Ad5, Ad35, Ad11, C6 and C7 vectors. The sequence of the adenovirus 5 ("Ad5") genome has been disclosed. (Chroboczek, J., Bieber, F., and Jacrot, B. (1992) The Sequence of the Genome of Adenovirus Type 5 and Its Comparison with the Genome of Adenovirus Type 2, Virology 186, 280-285; the contents of which are incorporated herein by reference). Ad35 vectors are described in U.S. Pat. Nos. 6,974,695, 6,913,922 and 6,869,794. Ad11 vectors are described in U.S. Pat. No. 6,913,922. C6 adenovirus vectors are described in U.S. Pat. Nos. 6,780,407, 6,537,594, 6,309,647, 6,265,189, 6,156,567, 6,090,393, 5,942,235 and 5,833,975. C7 vectors are described in U.S. Pat. No. 6,277,558. Adenovirus vectors with E1 defect or deletion, E3-d defect or deletion, and / or E4 defect or deletion may also be used. Certain adenoviruses with E1 region mutations have an increased safety margin because E1-deficient adenovirus mutants are replication-defective or at least highly attenuated in non-permissive cells. Adenoviruses with E3 region mutations may enhance immunogenicity by disrupting the mechanism by which adenoviruses downregulate MHC class I molecules. Adenoviruses with E4 mutations may have reduced immunogenicity of adenovirus vectors due to inhibition of late gene expression. Such vectors are particularly useful when repeated revaccination with the same vector is required. According to the present disclosure, adenoviral vectors in which E1, E3, E4, E1 and E3, and E1 and E4 are deleted or mutated can be used. In addition, according to the present disclosure, "gutless" adenoviral vectors in which all viral genes are deleted can also be used. Such vectors require a helper virus for replication and require a special human 293 cell line that expresses both E1a and Cre (a situation that does not exist in the natural environment). This "gutless" vector is non-immunogenic, so the vector can be inoculated multiple times for re-vaccination. "Gutless" adenoviral vectors can be used to insert heterologous inserts / genes (such as the transgenes of the present disclosure), and can even be used to co-deliver large numbers of heterologous inserts / genes.

[0426] As to lentiviral vector systems useful in practicing the present disclosure, reference is made to US Patent Nos. 6428953, 6165782, 6013516, 5994136, 6312682, and 7,198,784, and references cited therein.

[0427] As to AAV vectors useful in practicing the present disclosure, reference is made to U.S. Patent Nos. 5658785, 7115391, 7172893, 6953690, 6936466, 6924128, 6893865, 6793926, 6537540, 6475769, and 6258595, and references cited therein.

[0428] Another carrier is BCG (Bacille Calmette Guerin). BCG carriers are described in Stover et al. (Nature 351: 456-460 (1991)). Based on the description herein, a variety of other carriers for therapeutic administration or immunization of the peptides of the present disclosure (e.g., Salmonella typhi carriers, etc.) are apparent to those skilled in the art.

[0429] The vector may be administered so as to have an in vivo expression and response similar to the dose and / or response elicited by administration of the antigen.

[0430] In some embodiments, the method of administering a nucleic acid encoding a peptide of the present disclosure uses a minigene construct encoding multiple epitopes. In order to generate a DNA sequence encoding a selected CTL epitope (minigene) expressed in human cells, the amino acid sequence of the epitope is reverse translated. The codon selection of each amino acid is guided by the human codon usage table. These DNA sequences encoding epitopes are directly connected to produce a continuous polypeptide sequence. In order to optimize expression and / or immunogenicity, additional elements can be incorporated into the minigene design. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include: helper T lymphocytes, epitopes, leader (signal) sequences, and endoplasmic reticulum retention signals. In addition, the MHC presentation of CTL epitopes can be improved by including synthetic (e.g., polyalanine) or naturally occurring flanking sequences adjacent to the CTL epitope.

[0431] The minigene sequence is converted to DNA by assembling oligonucleotides encoding the plus and minus strands of the minigene. Using well-known techniques, overlapping oligonucleotides (30-100 bases long) are synthesized, phosphorylated, purified, and annealed under appropriate conditions. The ends of the oligonucleotides are ligated using T4 DNA ligase. This synthetic minigene encoding the CTL epitope polypeptide can then be cloned into the desired expression vector.

[0432] Standard regulatory sequences well known to those skilled in the art are included in the vector to ensure expression in the target cell. Several vector elements are required: a promoter with a downstream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g., ampicillin or kanamycin resistance). Many promoters can be used for this purpose, for example, the human cytomegalovirus (hCMV) promoter. For other suitable promoter sequences, see U.S. Patent Nos. 5,580,859 and 5,589,466.

[0433] Additional vector modifications may be required to optimize minigene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally occurring introns can be incorporated into the transcribed region of the minigene. It is also possible to consider including mRNA stabilizing sequences for increasing minigene expression. Recently, it has been suggested that immunostimulatory sequences (ISS or CpG) play a role in the immunogenicity of DNA vaccines. If these sequences are found to enhance immunogenicity, these sequences can be included in the vector outside the minigene coding sequence.

[0434] In some embodiments, a bicistronic expression vector can be used to allow the production of epitopes encoded by minigenes and a second protein included for enhancing or reducing immunogenicity. If co-expressed, examples of proteins or polypeptides that can beneficially enhance immune responses include cytokines (e.g., IL2, IL11, IL12, GM-CSF), cytokine inducing molecules (e.g., LeIF), or co-stimulatory molecules. Auxiliary (HTL) epitopes can be connected to intracellular targeting signals and expressed separately from CTL epitopes. This will allow HTL epitopes to be directed to cell compartments that are different from CTL epitopes. If necessary, this can promote HTL epitopes to enter the MHC II class pathway more efficiently, thereby improving CTL induction. In contrast to CTL induction, reducing immune responses, particularly by co-expressing immunosuppressive molecules (e.g., TGF-β), can be beneficial in certain diseases.

[0435] Once the expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. The plasmid is transformed into an appropriate strain of E. coli and DNA is prepared using standard techniques. Restriction mapping and DNA sequence analysis are used to confirm the orientation and DNA sequence of the minigene and all other elements contained in the vector. Bacterial cells carrying the correct plasmid can be stored as master cell banks and working cell banks.

[0436] Purified plasmid DNA for injection can be prepared using a variety of formulations. The simplest is to reconstitute lyophilized DNA in sterile phosphate buffered saline (PBS). A variety of methods have been described, and new technologies can be used. As described herein, nucleic acids are conveniently prepared with cationic lipids. In addition, glycolipids, fusogenic liposomes, peptides and compounds (collectively referred to as protective, interactive, non-condensing (P1NC)) can also be compounded with purified plasmid DNA to affect variables such as stability, intramuscular dispersion or transportation to specific organs or cell types.

[0437] Target cell sensitization can be used as a functional assay for the expression of CTL epitopes encoded by minigenes and MHC class I presentation. Plasmid DNA is introduced into a mammalian cell line suitable as a target for standard CTL chromium release assay. The transfection method used depends on the final formulation. Electroporation can be used for "naked" DNA, while cationic lipids allow direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be co-transfected to allow the use of fluorescence activated cell sorting (FACS) to enrich transfected cells. These cells are then labeled with chromium-51 and used as target cells for epitope-specific CTL lines. Cell lysis detected by 51Cr release indicates that MHC presentation of CTL epitopes encoded by minigenes has been produced.

[0438] In vivo immunogenicity is a second method for functional testing of minigene DNA formulations. Transgenic mice expressing appropriate human MHC molecules are immunized with DNA products. The dosage and route of administration are formulation dependent (e.g., IM for DNA in PBS, IP for lipid-complexed DNA). Twenty-one days after immunization, spleen cells are harvested and restimulated for 1 week in the presence of peptides encoding each test epitope. Standard techniques are used to measure the cytolysis of chromium-51 labeled target cells loaded with peptides in these effector cells (CTL). The lysis of target cells sensitized by MHC loading with peptides corresponding to epitopes encoded by minigenes demonstrates the function of DNA vaccines in inducing CTLs in vivo.

[0439] Peptides can also be used to trigger CTL in vitro. The resulting CTL can be used to treat chronic tumors in patients in need, and the patient is unresponsive to other conventional forms of therapy or unresponsive to peptide vaccine treatment methods. Induce in vitro CTL responses to specific tumor antigens by incubating the patient's CTL precursor cells (CTLp) with the source of antigen presenting cells (APC) and appropriate peptides in tissue culture. After an appropriate incubation time (usually 1-4 weeks, wherein CTLp is activated and mature and amplified as effector CTL), the cells are transferred back into the patient's body, where the cells destroy their specific target cells (i.e., tumor cells). In order to optimize the in vitro conditions for generating specific cytotoxic T cells, the stimulator cell culture is maintained in an appropriate serum-free medium.

[0440] Before the stimulator cells are incubated with the cells to be activated (e.g., precursor CD8+ cells), a sufficient amount of antigenic peptide is added to the stimulator cell culture to load it onto the human class I molecules to be expressed on the surface of the stimulator cells. In the present disclosure, a sufficient amount of peptide is an amount that allows about 200, or 200 or more human class I MHC molecules loaded with peptide to be expressed on the surface of each stimulator cell. In some embodiments, the stimulator cells are incubated with>2 μg / ml of peptide. For example, the stimulator cells are incubated with>3μg / ml, 4μg / ml, 5μg / ml, 10μg / ml, 15μg / ml or higher μg / ml of peptide.

[0441] The resting or precursor CD8+ cells are then incubated in culture with appropriate stimulator cells for a period of time sufficient to activate the CD8+ cells. In some embodiments, the CD8+ cells are activated in an antigen-specific manner. The ratio of resting or precursor CD8+ (effector) cells to stimulator cells can vary from individual to individual, and can further depend on variables such as the adaptability of individual lymphocytes to culture conditions and the nature and severity of the disease condition or other conditions for which the treatment is used. The ratio of lymphocytes: stimulator cells can be in the range of about 30:1 to 300:1. The effector / stimulator culture can be maintained for as long as the time required to stimulate a therapeutically available number or effective number of CD8+ cells.

[0442] In vitro induction of CTL requires specific recognition of peptides bound to allele-specific MHC class I molecules on APCs. The number of specific MHC / peptide complexes on each APC is crucial for stimulating CTLs (particularly in primary immune responses). Although a small amount of peptide / MHC complexes on each cell is sufficient to sensitize cells to lysis caused by CTLs, or to stimulate secondary CTL responses, significantly more MHC / peptide complexes are required for successful activation of CTL precursors (pCTLs) during primary responses. Peptide loading of empty major histocompatibility complex molecules on cells allows induction of primary cytotoxic T lymphocyte responses.

[0443] Since not every human MHC allele has mutant cell lines, it is advantageous to use a technique to remove endogenous MHC-associated peptides from the surface of APCs and then load the resulting empty MHC molecules with the immunogenic peptide of interest. The use of non-transformed (non-tumorigenic) non-infected cells and autologous cells of patients as APCs is ideal for designing CTL induction protocols for developing in vitro CTL therapies. The present application discloses a method for stripping endogenous MHC-associated peptides from the surface of APCs and then loading the desired peptides.

[0444] Stable MHC class I molecules are trimeric complexes formed by the following elements: 1) a peptide usually having 8-10 residues, 2) a transmembrane polymorphic heavy protein chain with peptide binding sites in its a1 and a2 domains, and 3) a non-covalently associated non-polymorphic light chain, i.e., p2 microglobulin. Removing the bound peptide and / or dissociating the p2 microglobulin from the complex renders the MHC class I molecules nonfunctional and unstable, resulting in rapid degradation. All MHC class I molecules isolated from PBMC have endogenous peptides bound to them. Therefore, the first step is to remove all endogenous peptides bound to the MHC class I molecules on APC without causing the molecules to degrade, after which exogenous peptides can be added thereto.

[0445] Two possible methods for releasing bound peptides from MHC class I molecules include lowering the culture temperature from 37°C to 26°C overnight to destabilize p2 microglobulin, and stripping endogenous peptides from cells using mild acid treatment. The method releases previously bound peptides into the extracellular environment, allowing new exogenous peptides to bind to empty class I molecules. The low-temperature incubation method allows exogenous peptides to bind to MHC complexes efficiently, but requires overnight incubation at 26°C, which slows down the metabolic rate of the cells. Similarly, cells that do not actively synthesize MHC molecules (e.g., resting PBMCs) may not produce a large number of empty surface MHC molecules through the low-temperature process.

[0446] Strict acid stripping involves extracting peptides with trifluoroacetic acid (pH 2), or acid denaturing immunoaffinity purified class I-peptide complexes. These methods are not feasible for CTL induction because it is important to remove endogenous peptides while retaining APC viability and the optimal metabolic state that is critical for antigen presentation. Weak acid solutions at pH 3 (such as glycine or citrate-phosphate buffer) have been used to identify endogenous peptides and tumor-associated T cell epitopes. The treatment is particularly effective because only MHC class I molecules are unstable (and release associated peptides), while other surface antigens (including MHC class II molecules) remain intact. Most importantly, treating cells with weak acid solutions does not affect the viability or metabolic state of the cells. Weak acid treatment is rapid because the stripping of endogenous peptides occurs within two minutes at 4°C, and the APC is ready to perform its function after being loaded with the appropriate peptide. The technology is used herein to prepare peptide-specific APCs for generating primary antigen-specific CTLs. The resulting APCs can effectively induce peptide-specific CD8+CTLs.

[0447] One of a variety of known methods can be used to effectively separate activated CD8+ cells from stimulator cells. For example, monoclonal antibodies specific to stimulator cells, peptides loaded onto stimulator cells, or CD8+ cells (or segments thereof) can be used to bind their appropriate complementary ligands. Antibody-labeled molecules can then be extracted from the stimulator cell-effector cell mixture via an appropriate method, such as via well-known immunoprecipitation or immunoassay methods.

[0448] The effective cytotoxic amount of activated CD8+ cells may vary between in vitro and in vivo use and with the number and type of cells that are the ultimate target of these killer cells. The amount may also vary depending on the patient's condition and should be determined by the practitioner considering all appropriate factors. Approximately 1x10 6 About 1x10 12 , about 1x10 8 About 1x10 11 , or about 1x10 9 About 1x 10 10 activated CD8+ cells can be used in adults, compared to approximately 5x10 6 -5x 10 7 The cells can be used in mice.

[0449] As discussed herein, activated CD8+ cells are harvested from cell culture and thereafter administered to the individual being treated. However, it is important to note that, unlike other existing and proposed treatment modalities, the methods of the present invention utilize a non-tumorigenic cell culture system. Thus, if complete separation of stimulatory cells and activated CD8+ cells is not achieved, there are no inherent risks known to be associated with administration of small amounts of stimulatory cells, whereas administration of mammalian tumor-promoting cells can be extremely dangerous.

[0450] Methods of reintroducing cellular components are known in the art and include methods such as those exemplified in U.S. Patent No. 4,844,893 to Honsik et al. and U.S. Patent No. 4,690,915 to Rosenberg. For example, administration of activated CD8+ cells via intravenous infusion is appropriate.

[0451] Unless otherwise indicated, the practice of the present disclosure employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the knowledge of the skilled artisan. Such techniques are fully explained in the following literature, such as: "Molecular Cloning; A Laboratory Manual", 2nd Edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Wei, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PGR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of polynucleotides and polypeptides of the present disclosure, and thus can be considered for use in making and practicing the present disclosure.Techniques that are particularly useful for specific embodiments are discussed in the following sections.

[0452] VIII. Treatment

[0453] The present disclosure provides methods for inducing a tumor / tumor-specific immune response in a subject, vaccinating against a tumor / tumor, treating and or alleviating cancer symptoms in a subject by administering to the subject a tumor vaccine or neoantigenic peptide or composition of the present disclosure and at least one inhibitor (such as a checkpoint inhibitor or a chemotherapeutic agent).

[0454] In particular, the present disclosure relates to methods of treating or preventing tumors, comprising the steps of administering to a subject (a) a tumor vaccine or immunogenic composition, and (b) at least one inhibitor, such as a checkpoint inhibitor or a chemotherapeutic agent.

[0455] In one aspect, provided herein is a method for treating or preventing cancer in a human subject in need thereof, the method comprising administering to a human subject in need thereof: a first component comprising: (i) a polypeptide comprising a cancer-specific neo-epitope of a protein expressed by a cancer cell of the cancer, (ii) a polynucleotide encoding the polypeptide of (i), (iii) one or more APCs comprising the polypeptide of (i) or the polynucleotide of (ii), (iv) a T cell receptor (TCR) specific for a complex comprising an HLA protein expressed by the human subject and a T cell receptor having the cancer neo-epitope; The invention relates to a human subject comprising: (i) a human subject that has not previously received systemic treatment for metastatic disease, (ii) a human subject that has not previously received immunotherapy with an anti-PD-1 antibody, and (iii) a human subject that has not previously received immunotherapy with an anti-PD-L1 antibody.

[0456] According to the present disclosure, the tumor vaccines or immunogenic compositions described herein can be used in patients who have been diagnosed with cancer or are at risk of developing cancer.

[0457] The combination of the present disclosure is administered in an amount sufficient to induce a CTL response.

[0458] A vaccine or immunogenic composition comprising a neoantigenic peptide can be administered to a subject to treat a condition. In addition to the neoantigenic peptide, one or more inhibitors, such as a checkpoint inhibitor or a chemotherapeutic drug, can also be administered to the subject. One or more inhibitors, such as a checkpoint inhibitor or a chemotherapeutic drug, can be administered before the neoantigenic peptide is administered. In some embodiments, more than one inhibitor, such as a checkpoint inhibitor or a chemotherapeutic drug, is being administered to the patient. In such cases, one inhibitor, such as a checkpoint inhibitor or a chemotherapeutic drug, can be administered before the neoantigenic peptide is administered. For example, in the case of a combination of neoantigenic peptides.

[0459] Patients can be screened before and after the administration of the compositions described herein. Patients can undergo a screening assessment that records their past health status and their current and future general health status and health status associated with underlying diseases. Screening assessments can include examinations such as: vital signs (including diastolic and systolic blood pressure, heart rate, temperature, weight and height), electrocardiogram, symptom-oriented physical examination, hematology (including hematocrit, hemoglobin, RBC count, WBC count and differential count, and platelet count), chemistry (including glucose, urea nitrogen, creatinine, sodium, potassium, calcium, total and direct bilirubin, AST, ALT, alkaline phosphatase, lactate dehydrogenase (LDH) and adrenocorticotropic hormone), liver function tests (such as testing AST, ALT, total and direct bilirubin levels), pregnancy testing, CT or MRI, surgery or core needle biopsy of primary or metastatic tumor sites for DNA and RNA sequencing, and immunological analysis. Biopsies can be used to evaluate the presence of T cell infiltration in the tumor and its localization relative to the tumor margin by tests such as immunohistochemistry, Western blot analysis, RNA and DNA analysis. The presence of tumor-associated macrophages and DCs in the tumor microenvironment can also be evaluated. The list of markers used for analysis can include, but is not limited to, the following: CD3, CD4, CD8, CD45RO, PD-L1, PD-1, FoxP3, granzyme B, perforin, CD68, CD163, MHC class I, MHC class II, CD83, and CD11b.

[0460] The changes of immune response parameters over time from the baseline level before the administration of treatment can be analyzed, and it can include a summary of the characterization of nucleic acids in tissues obtained before treatment, before vaccination treatment and vaccination treatment period and during preliminary assessment (e.g., DNA mutations, transcript abundance), histopathology and immune cell analysis. Reporting test results can include a table describing the changes from an earlier time point to compare the changes in immune parameters. Descriptive statistics and frequency distribution can be used as appropriate. Immunological analysis can include a summary of CD8+ and CD4+ T cell responses measured by ex vivo IFN-γ ELISpot and evaluated by spot counting. Analysis can be used to evaluate changes from before treatment to before vaccination treatment, to vaccination treatment, to preliminary assessment. The report can include medians and interquartile ranges, as well as a table describing the changes of each patient from an earlier time point. In addition, nonparametric tests (e.g., Wilcoxon signed rank test) can be used to determine the differences in ELISpot data between time points (if applicable). Between the treatment groups in each cohort, the Wilcoxon rank sum test can be used to summarize and compare the changes in biomarkers measured on a continuous scale between each response category. For multi-gene assays, genes can be grouped into analysis panels to characterize the biological function of the cell, as applicable.

[0461] Primary objectives may include: the incidence of adverse events and serious adverse events leading to treatment discontinuation [time frame: baseline to 90 days after the last dose of pembrolizumab], those leading to treatment discontinuation and those detected during symptom-driven physical examinations (safety laboratory evaluations, physical examination results, changes in vital signs and ECOGPs).

[0462] Secondary outcome measures may include:

[0463] 1. The date of first documented PD based on objective response rate (ORR), which was defined as the proportion of patients achieving complete response (CR) or partial response (PR) based on Response Criteria in Solid Tumors (RECIST) v1.1.

[0464] 2. Clinical benefit rate (CBR), defined as the proportion of patients achieving CR, PR, or stable disease (SD) based on RECIST v1.1.

[0465] 3. Duration of response, DOR, defined as the date of first documented confirmed response to the date of first documented PD based on RECIST v1.1.

[0466] 4. Response conversion rate (RCR), defined as the proportion of patients with improvement in RECIST v1.1 category (e.g., PD to SD / PR / CR, SD to PR / CR, PR / CR) after vaccination.

[0467] 5. Progression-free survival (PFS), defined as the time from the first dose to the first

[0468] The time between the date of PD or death was recorded.

[0469] 6. Overall survival (OS), defined as the period from the date of enrollment to death from any cause.

[0470] Detection and results can include detailed characterization of the phenotype and abundance of antigen-specific T cells in the periphery and tumor microenvironment. The abundance of regulatory cells (such as regulatory T cells or myeloid-derived suppressor cells) and T cell recognition, activation and cytotoxicity can also be evaluated with PBMC and tumor cells. In addition, the ex vivo induction of neoantigen T cell responses can also be performed on peripheral blood and leukocyte apheresis samples. After treatment with the compositions described herein, the presence of cycle tumor DNA (ctDNA) and vaccine-specific antibody responses can be evaluated.

[0471] In one aspect, provided herein is a method for treating or preventing cancer in a human subject in need thereof, the method comprising administering to the human subject in need thereof: (a) a first component comprising: (i) a polypeptide comprising a cancer-specific neo-epitope of a protein expressed by a cancer cell of the cancer, (ii) a polynucleotide encoding the polypeptide of (i), (iii) one or more APCs comprising the polypeptide of (i) or the polynucleotide of (ii), (iv) a T cell receptor (TCR) specific for a complex comprising an HLA protein expressed by the human subject and a cancer-specific neo-epitope of a protein expressed by a cancer cell having the cancer neo-epitope, or (v) a T cell containing the TCR of (iv); and (b) a second component comprising an anticancer agent, which is an antibody or an antigen-binding portion thereof that specifically binds to a programmed death protein-1 (PD-1) receptor and inhibits PD-1 activity; and (c) a third component comprising a platinum-based chemotherapy; wherein: (i) the method promotes epitope expansion of epitopes that are different from any of the cancer-specific neoepitopes; and (ii) the median progression-free survival (PFS) of a first population of human subjects suffering from the cancer treated with the first component, the second component, and the third component is longer than that of a second population of human subjects suffering from the cancer treated with the second component and / or the third component but not the first component. (iii) the overall response rate (ORR) of a first population of human subjects with the cancer treated with the first, second and third components is higher than the ORR of a second population of subjects with the cancer treated with the second component and / or the third component but not the first component; (iv) the percentage of subjects with at least 12 months progression-free survival (PFS) in the first population of human subjects with the cancer treated with the first, second and third components is higher than the percentage of subjects with at least 12 months PFS in the second population of subjects with the cancer treated with the second component and / or the third component but not the first component. (v) the median overall survival (OS) of a first population of human subjects with the cancer treated with the first, second, and third components is longer than the median OS of a second population of subjects with the cancer treated with the second component and / or the third component but not the first component; (vi) the percentage of subjects achieving complete remission, partial remission, prolonged disease stabilization, or disease stabilization for 6 months or longer (CBR) in the first population of human subjects with the cancer treated with the first, second, and third components is higher than the CBR of a second population of subjects with the cancer treated with the second component and / or the third component but not the first component;(vii) a reduction in tumor size in a first population of human subjects with the cancer treated with the first, second, and third components is greater than a reduction in tumor size in a second population of subjects with the cancer treated with the second and / or third components but not the first component; (viii) a level of CD4+ T cells infiltrating the tumor in the first population of human subjects treated with the first, second, and third components is greater than a level of CD4+ T cells infiltrating the tumor in the second population of subjects treated with the second and / or third components but not the first component; (ix) a level of effector cells and cytotoxic CD4+ T cells produced in the first population of human subjects treated with the first, second, and third components is greater than a level of CD4+ T cells infiltrating the tumor in the second population of subjects treated with the second and / or third components but not the first component; The method increases the level of effector cells and cytotoxic CD4+T cells produced in a second subject population treated with the first component but not the first component; (x) the method increases the level of CD4+T cells specific for a cancer-specific neoepitope that upregulates the expression of ZEB2, PDCD1, TOX, TIGT, CXCR3, ITGB1, GZMA and / or ICOS; (xi) the method increases the level of CD4+ / CD62Lhi / CD69+ / CD27+ / CCR7+T cells specific for a cancer-specific neoepitope; and / or (xii) the method increases the level of CD4+ / NKG7+ / CCL4+ / CCL5+ / GNLY+ / LAG3+T cells specific for a cancer-specific neoepitope. ;

[0472] For inclusion in clinical trials, the following criteria were followed: Study Eligible Age: 18 years and older (Adults, Elderly); Study Eligible Gender: All. Adverse Events (NCI CTCAE Version 4.03), Grade 0 or 1, except for toxicities not considered a safety risk (e.g., alopecia or vitiligo). Screening laboratory values ​​must meet the following criteria and should be obtained within 30 days (or 45 days if repeat biopsy) prior to study treatment:

[0473] 1. White blood cell (WBC) count ≥ 3 × 10e3 / μL

[0474] 2. Absolute neutrophil count (ANC) ≥ 1.5 × 10e3 / μL

[0475] 3. Platelet count ≥100×10e3 / μL

[0476] 4. Hemoglobin > 9 g / dL

[0477] 5. Serum creatinine ≤1.5×upper limit of normal (ULN) or creatinine clearance (CrCl) ≥40mL / min / 1.73m^2

[0478] 6. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) ≤ 2.5 × ULN, or ≤ 5 × ULN (for patients with liver metastases)

[0479] 7. Total bilirubin ≤1.5×ULN (except for patients with Gilbert's syndrome, who may have total bilirubin <3.0 mg / dL). Direct bilirubin ≤ULN, for patients with total bilirubin levels >1.5×ULN

[0480] 8. International normalized ratio (INR) or prothrombin time (PT) ≤ 1.5 × ULN, unless the patient is receiving anticoagulant therapy, as long as the PT or PTT is within the therapeutic range for the intended use of the anticoagulant

[0481] 9. Activated partial thromboplastin time (aPTT) ≤ 1.5 × ULN, unless the patient is receiving anticoagulant therapy, as long as the PT or PTT is within the therapeutic range for the intended use of the anticoagulant. Female patients of reproductive potential must have a negative urine or serum pregnancy test with...

Claims

1. A method of treating or preventing cancer in a human subject in need thereof, the method comprising administering to the human subject in need thereof: (a) a first component comprising: (i) a polypeptide comprising a cancer-specific neo-epitope of a protein expressed by cancer cells of the cancer, (ii) a polynucleotide encoding the polypeptide of (i), (iii) one or more APCs containing the polypeptide of (i) or the polynucleotide of (ii), (iv) a T cell receptor (TCR) specific for a complex comprising an HLA protein expressed by the human subject and a cancer-specific neo-epitope of a protein expressed by a cancer cell having the cancer neo-epitope, or (v) a T cell comprising the TCR of (iv); and (b) a second component comprising an anticancer agent, which is an antibody or an antigen-binding portion thereof that specifically binds to a programmed death protein-1 (PD-1) receptor and inhibits PD-1 activity; and (c) a third component comprising a platinum-based chemotherapy; Wherein the human subject: (i) No previous systemic therapy for metastatic disease, (ii) not previously treated with anti-PD-1 antibody immunotherapy, and (iii) No prior immunotherapy with anti-PD-L1 antibodies.

2. The method of claim 1, wherein the method comprises administering to the human subject a combination of the second component and the third component prior to administering the first component.

3. The method of claim 2, wherein the method comprises administering to the human subject a combination of the second component and the third component for a period of 12 weeks prior to administering the first component.

4. The method of claim 3, wherein production of the first component occurs during a 12 week period of administering the combination of the second component and the third component.

5. The method of claim 3 or 4, wherein the method comprises administering the first component for a period of 12 weeks followed by a period of 12 weeks of administering the combination of the second component and the third component.

6. The method of claim 5, wherein the administering of the first component for a period of 12 weeks after the administering of the combination of the second component and the third component for a period of 12 weeks comprises administering the first component at four separate anatomical sites on the human subject.

7. The method of claim 5 or 6, wherein the period of 12 weeks of administering the first component after the period of 12 weeks of administering the combination of the second component and the third component comprises administering five priming doses of the first component and two booster doses of the first component.

8. The method of claim 7, wherein the period of 12 weeks of administering the first component after the period of 12 weeks of administering the combination of the second component and the third component comprises administering a priming dose of the first component on days 1 and 4 and then once a week in weeks 13, 14, and 15; and administering a booster dose in weeks 19 and 23.

9. The method of any one of claims 5-8, wherein the second component is administered to the human subject during the 12-week period of administration of the first component.

10. The method of claim 9, wherein the second component is administered to the human subject after the 12 week period of administration of the first and second components.

11. The method of claim 10, wherein the second component is administered to the human subject for a period of at least 28 weeks after the 12 week period of administration of the first and second components.

12. The method of claim 11, wherein the second component is administered to the human subject for a period of 80 weeks after the 12-week period of administration of the first and second components.

13. The method of any one of claims 2-12, wherein the second component is administered to the human subject for a total period of at least 52 weeks, or about 103 weeks, or about 104 weeks.

14. The method of any one of claims 2-13, wherein the third component is not administered to the human subject during or after administration of the first component.

15. The method of any one of claims 3-14, wherein after the 12-week period of administering the combination of the second component and the third component prior to administering the first component, the third component is not administered to the human subject.

16. The method of any one of claims 1-15, wherein the human subject has a KRAS mutation, a TP53 mutation, and / or a KEAP1 mutation. 17 . The method of claim 16 , wherein the cancer-specific neo-epitopes of the first component do not include KRAS neo-epitopes, TP53 neo-epitopes, and / or KEAP1 neo-epitopes.

18. The method of claim 1, wherein the cancer is lung cancer.

19. The method of claim 18, wherein the lung cancer is non-small cell lung cancer (NSCLC).

20. The method of claim 19, wherein the NSCLC has squamous histology.

21. The method of claim 19, wherein the NSCLC has non-squamous histology.

22. The method of claim 19, wherein the NSCLC is metastatic NSCLC.

23. The method of any one of claims 1-22, wherein the first component comprises a polypeptide comprising a cancer-specific neo-epitope of a protein expressed by cancer cells of the cancer.

24. The method of claim 23, wherein the first component comprises an adjuvant.

25. The method of claim 24, wherein the adjuvant comprises poly I:poly C.

26. The method of claims 1-25, wherein the cancer-specific neo-epitopes comprise at least two different cancer-specific neo-epitopes of proteins expressed by cancer cells of the cancer.

27. The method of claims 1-25, wherein the cancer-specific neo-epitopes comprise up to twenty different cancer-specific neo-epitopes of proteins expressed by cancer cells of the cancer.

28. The method of any one of claims 1-27, wherein the method comprises comparing: (i) a nucleic acid sequence obtained by whole genome sequencing or whole exome sequencing of a cancer cell from a single subject, with (ii) a nucleic acid sequence obtained by whole genome sequencing or whole exome sequencing of a non-cancerous cell from a single subject.

29. The method of claim 28, wherein the method comprises identifying a plurality of cancer-specific nucleic acid sequences unique to cancer cells of the human subject based on the comparison.

30. The method of claim 29, wherein the method comprises predicting or calculating the binding affinity of the cancer-specific neo-epitope sequences encoded by the identified plurality of cancer-specific nucleic acid sequences to the proteins encoded by the HLA alleles of the human subject by performing an HLA peptide binding analysis using a program executed on a computer system.

31. The method of claim 30, wherein the method comprises selecting an IC for a protein predicted or calculated to be encoded by an HLA allele of the human subject. 50 At least two cancer-specific neo-epitope sequences at less than 500 nM or 150 nM or less.

32. The method of any one of claims 1-31, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, cross-competes with nivolumab for binding to human PD-1.

33. The method of any one of claims 1-31, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, comprises a heavy chain constant region of human IgG1 or IgG4 isotype.

34. The method of any one of claims 1-31, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is a chimeric antibody, a humanized antibody, or a human monoclonal antibody, or a portion thereof.

35. The method of any one of claims 1-31, wherein the anti-PD-1 antibody is pembrolizumab.

36. The method of claim 35, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered once every 2 weeks, every 3 weeks, or every 4 weeks at a dose ranging from 0.1 to 10.0 mg / kg body weight.

37. The method of claim 36, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered once every 3 weeks at a dose of 5 or 10 mg / kg body weight.

38. The method of claim 36, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered once every 2 weeks at a dose of 3 mg / kg body weight.

39. The method of claim 35, wherein the anti-PD-1 antibody, or antigen-binding portion thereof, is administered via intravenous infusion at a dose of 200 mg on day 1 of every 3-week cycle.

40. The method of any one of claims 1-39, wherein the platinum-based chemotherapy is platinum-based dual chemotherapy (PT-DC).

41. The method of claim 40, wherein the PT-DC is a combination of pemetrexed and carboplatin.

42. The method of claim 41, wherein the carboplatin is administered at a dose that achieves an area under the free carboplatin plasma concentration versus time curve (AUC) of 5.

43. The method of claim 41 or 42, wherein the pemetrexed is administered at a dose of 500 mg / m^2.

44. The method of any one of claims 40-43, wherein the PT-DCs are administered simultaneously with the anti-PD-1 antibody, or an antigen-binding portion thereof, for 4 doses of the anti-PD-1 antibody, or an antigen-binding portion thereof, followed by repeated administration of the anti-PD-1 antibody, or an antigen-binding portion thereof, alone.

45. The method of any one of claims 1-44, wherein the method promotes epitope spreading.

46. ​​The method of any one of claims 1-44, wherein the method promotes epitope spreading of epitopes that are distinct from any of the cancer-specific neo-epitopes.

47. The method of claim 46, wherein the epitope different from any of the cancer-specific neo-epitopes comprises a KRAS neo-epitope, a TP53 neo-epitope, and / or a KEAP1 neo-epitope.

48. The method of claim 47, wherein the KRAS neo-epitope comprises a G12C or G12V mutation.

49. The method of any one of claims 1-44, wherein a first population of human subjects having the cancer treated with the first, second, and third components has a median progression-free survival (PFS) that is longer than the median PFS of a second population of subjects having the cancer treated with the second and / or third components but not the first component.

50. The method of any one of claims 1-44, wherein the overall response rate (ORR) of a first population of human subjects suffering from the cancer treated with the first, second, and third components is greater than the ORR of a second population of subjects suffering from the cancer treated with the second and / or third components but not the first component.

51. The method of any one of claims 1-44, wherein the percentage of subjects in a first population of human subjects having the cancer treated with the first, second, and third components who have a progression-free survival (PFS) of at least 9 or 12 months is greater than the percentage of subjects in a second population of subjects having the cancer treated with the second and / or third components but not the first component who have a PFS of at least 9 or 12 months.

52. The method of any one of claims 1-44, wherein a first population of human subjects suffering from the cancer treated with the first, second, and third components has a median overall survival (OS) that is longer than the median OS of a second population of subjects suffering from the cancer treated with the second and / or third components but not the first component.

53. The method of any one of claims 1-44, wherein the percentage of subjects achieving complete remission, partial remission, prolonged disease stabilization, or disease stabilization for 6 months or longer (CBR) in a first population of human subjects suffering from the cancer treated with the first, second, and third components is higher than the CBR in a second population of subjects suffering from the cancer treated with the second and / or third components but not the first component.

54. The method of any one of claims 1-44, wherein the reduction in tumor size in a first population of human subjects suffering from the cancer treated with the first, second, and third components is greater than the reduction in tumor size in a second population of subjects suffering from the cancer treated with the second and / or third components but not the first component.

55. The method of any one of claims 1-44, wherein the level of tumor-infiltrating CD4+ T cells in a first population of human subjects treated with the first, second, and third components is higher than the level of tumor-infiltrating CD4+ T cells in a second population of subjects treated with the second and / or third components but not the first component.

56. The method of any one of claims 1-44, wherein the levels of effector cells and cytotoxic CD4+ T cells generated in a first population of human subjects treated with the first, second, and third components are higher than the levels of effector cells and cytotoxic CD4+ T cells generated in a second population of subjects treated with the second and / or third components but not the first component.

57. The method of any one of claims 1-44, wherein the method increases the level of CD4+ T cells specific for a cancer-specific neo-epitope that upregulates the expression of ZEB2, PDCD1, TOX, TIGT, CXCR3, ITGB1, GZMA and / or ICOS.

58. The method of any one of claims 1-44, wherein the method increases CD4+ / CD62L specific for cancer-specific neoepitopes. hi / Levels of CD69+ / CD27+ / CCR7+T cells.

59. The method of any one of claims 1-44, wherein the method increases the level of CD4+ / NKG7+ / CCL4+ / CCL5+ / GNLY+ / LAG3+ T cells specific for cancer-specific neoepitopes.

60. The method of any one of claims 1-59, wherein prior to administering the first component, the second component, and / or the third component, the human subject is identified as having a PD-L1 positive cancer.

61. A method of treating or preventing cancer in a human subject in need thereof, the method comprising administering to the human subject in need thereof: (a) a first component comprising: (i) a polypeptide comprising a cancer-specific neo-epitope of a protein expressed by cancer cells of the cancer, (ii) a polynucleotide encoding the polypeptide of (i), (iii) one or more APCs containing the polypeptide of (i) or the polynucleotide of (ii), (iv) a T cell receptor (TCR) specific for a complex comprising an HLA protein expressed by the human subject and a cancer-specific neo-epitope of a protein expressed by a cancer cell having the cancer neo-epitope, or (v) a T cell comprising the TCR of (iv); and (b) a second component comprising an anticancer agent, which is an antibody or an antigen-binding portion thereof that specifically binds to a programmed death protein-1 (PD-1) receptor and inhibits PD-1 activity; and (c) a third component comprising a platinum-based chemotherapy; wherein: (i) the method promotes epitope spreading of epitopes that are different from any of the cancer-specific neo-epitopes; (ii) a first population of human subjects having the cancer treated with the first, second, and third components has a median progression-free survival (PFS) that is longer than a second population of subjects having the cancer treated with the second and / or third components but not the first component; (iii) an overall response rate (ORR) in a first population of human subjects suffering from the cancer treated with the first, second, and third components is greater than an ORR in a second population of subjects suffering from the cancer treated with the second and / or third components but not the first component; (iv) the percentage of subjects in a first population of human subjects having the cancer treated with the first, second, and third components who have a progression-free survival (PFS) of at least 9 or 12 months is greater than the percentage of subjects in a second population of human subjects having the cancer treated with the second and / or third components but not the first component who have a PFS of at least 9 or 12 months; (v) a first population of human subjects suffering from the cancer treated with the first, second, and third components has a median overall survival (OS) that is longer than a second population of subjects suffering from the cancer treated with the second and / or third components but not the first component; (vi) a percentage of subjects achieving complete remission, partial remission, prolonged disease stabilization, or disease stabilization for 6 months or longer (CBR) in a first population of human subjects suffering from the cancer treated with the first, second, and third components is higher than the CBR in a second population of subjects suffering from the cancer treated with the second and / or third components but not the first component; (vii) a reduction in tumor size in a first population of human subjects suffering from said cancer treated with said first, second and third components is greater than a reduction in tumor size in a second population of subjects suffering from said cancer treated with said second and / or third components but not with said first component; (viii) the level of tumor-infiltrating CD4+ T cells in a first population of human subjects treated with the first, second, and third components is higher than the level of tumor-infiltrating CD4+ T cells in a second population of subjects treated with the second and / or third components but not the first component; (ix) the levels of effector cells and cytotoxic CD4+ T cells generated in a first population of human subjects treated with the first, second and third components are higher than the levels of effector cells and cytotoxic CD4+ T cells generated in a second population of subjects treated with the second and / or third components but not the first component; (x) the method increases the level of CD4+ T cells specific for cancer-specific neoepitopes that upregulate the expression of ZEB2, PDCD1, TOX, TIGT, CXCR3, ITGB1, GZMA and / or ICOS; (xi) The method increases CD4+ / CD62L specific for cancer-specific neoepitopes hi / levels of CD69+ / CD27+ / CCR7+ T cells; and / or (xii) The method increases the levels of CD4+ / NKG7+ / CCL4+ / CCL5+ / GNLY+ / LAG3+ T cells specific for cancer-specific neoepitopes.

62. The method of claim 61, wherein prior to administering the first component, the second component, and / or the third component, the human subject is identified as having a PD-L1 positive cancer.

63. The method of claim 61, wherein the human subject has not previously received systemic treatment for metastatic disease, has not previously received immunotherapy with an anti-PD-1 antibody, and / or has not previously received immunotherapy with an anti-PD-L1 antibody.

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