Combination therapy with neoantigen vaccines

Abstract

The present disclosure relates to neoplasia vaccines or immunogenic compositions administered in combination with other agents, such as checkpoint inhibitors, for the treatment or prevention of a neoplasia in a subject.

Description

[Technical Field]

[0001] cross reference

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 368,963, filed July 20, 2022, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Cancer immunotherapy is the use of the immune system to treat cancer. Immunotherapy exploits the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor antigens, often proteins or other macromolecules (e.g., carbohydrates). Active immunotherapy directs the immune system to attack tumor cells by targeting tumor antigens. Passive immunotherapy enhances existing antitumor responses and involves 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 critical obstacles in developing curative and tumor-specific immunotherapy is the identification and selection of highly specific, defined antigens to circumvent autoimmunity.

[0003]

[0003] Tumor antigens, which arise as a result of genetic alterations 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 unique to tumor cells because the mutations and their corresponding proteins are present only in tumors. They also evade central tolerance and are therefore more likely to be immunogenic. Therefore, tumor neoantigens provide excellent targets for immune recognition, including immune recognition by both humoral and cellular immunity. Therefore, the development of additional cancer therapeutics remains necessary. Summary of the Invention [Means for solving the problem]

[0004]

[0004] A method for treating or preventing cancer in a human subject in need thereof, comprising administering to the human subject in need thereof (a) (i) a polypeptide comprising a cancer-specific neoepitope 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 complex comprising an HLA protein expressed by the human subject and a cancer-specific neoepitope of a protein expressed by a cancer cell of the cancer neoepitope. (b) a second component comprising an anti-cancer agent that is an antibody or antigen-binding portion thereof that specifically binds to the programmed death-1 (PD-1) receptor and inhibits PD-1 activity; and (c) a third component comprising 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.

[0005]

[0005] In some embodiments, the method includes administering a combination of the second and third components to the human subject prior to the step of administering the first component.

[0006] In some embodiments, the method includes administering a combination of a second component and a third component to a human subject for a 12-week period prior to administering the first component. In some embodiments, manufacturing the first component occurs during the 12-week period during which the combination of the second component and the third component is administered. In some embodiments, the method includes administering the first component for a 12-week period after administering the combination of the second component and the third component for a 12-week period. In some embodiments, administering the first component for a 12-week period after administering the combination of the second component and the third component for a 12-week period includes administering the first component to four separate anatomical locations in the human subject. In some embodiments, administering the first component for a 12-week period after administering the combination of the second component and the third component for a 12-week period includes administering five priming doses of the first component and two booster doses of the first component. In some embodiments, administering the first component for a 12-week period after administering the combination of the second and third components for a 12-week period includes administering a priming dose of the first component on days 1 and 4, then once a week at weeks 13, 14, and 15, and administering a boosting dose at weeks 19 and 23. In some embodiments, the second component is administered to the human subject during the 12-week period in which the first component is administered. In some embodiments, the second component is administered to the human subject after the 12-week period in which the first and second components are administered. In some embodiments, the second component is administered to the human subject for a period of at least 28 weeks after the 12-week period in which the first and second components are administered. In some embodiments, the second component is administered to the human subject for a period of 80 weeks after the 12-week period in which the first and second components are administered. In some embodiments, the second component is administered to the human subject for a total period of at least 52 weeks, or about 103 or about 104 weeks.In some embodiments, the third component is not administered to the human subject during or after the administration of the first component. In some embodiments, the third component is not administered to the human subject after the administration of the combination of the second and third components for a 12-week period prior to administering the first component.

[0006]

[0007] In some embodiments, the human subject has a KRAS mutation, a TP53 mutation, and / or a KEAP1 mutation. In some embodiments, the cancer-specific neoepitope of the first component does not include a KRAS neoepitope, a TP53 neoepitope, and / or a KEAP1 neoepitope.

[0007]

[0008] In some embodiments, the cancer is lung cancer.

[0009] In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC).

[0010] In some embodiments, the NSCLC has squamous histology.

[0008]

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

[0012] In some embodiments, the NSCLC is metastatic NSCLC.

[0013] In some embodiments, the first component comprises a polypeptide comprising a cancer-specific neoepitope of a protein expressed by a cancer cell of the cancer.

[0009]

[0014] In some embodiments, the first component comprises an adjuvant.

[0015] In some embodiments, the adjuvant comprises poly I:poly C.

[0016] In some embodiments, the cancer-specific neoepitope comprises at least two different cancer-specific neoepitopes of proteins expressed by cancer cells of the cancer.

[0010]

[0017] In some embodiments, the cancer-specific neoepitopes comprise at most 20 different cancer-specific neoepitopes of proteins expressed by cancer cells of the cancer.

[0018] In some embodiments, the method includes comparing (i) nucleic acid sequences obtained by whole genome or whole exome sequencing of cancer cells from a single subject with (ii) nucleic acid sequences obtained by whole genome or whole exome sequencing of non-cancerous cells from the single subject, hi some embodiments, the method includes identifying a plurality of cancer-specific nucleic acid sequences that are unique to cancer cells of the human subject based on the comparing step.

[0011]

[0019] In some embodiments, the method includes predicting or calculating the binding affinity of cancer-specific neoepitope sequences encoded by the identified plurality of cancer-specific nucleic acid sequences to proteins encoded by HLA alleles of the human subject by HLA peptide binding analysis using a program implemented in a computer system.

[0012]

[0020] In some embodiments, the method comprises administering to a subject an IC50 antibody against a protein encoded by an HLA allele of a human subject that is less than 500 nM or 150 nM or less. 50 The method includes selecting at least two cancer-specific neoepitopes predicted or calculated to have:

[0013]

[0021] In some embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, cross-competes with nivolumab for binding to human PD-1.

[0022] In some embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, comprises a heavy chain constant region that is of the human IgG1 or IgG4 isotype.

[0014]

[0023] In some embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, is a chimeric, humanized, or human monoclonal antibody, or portion thereof.

[0024] In some embodiments, the anti-PD-1 antibody is pembrolizumab.

[0015]

[0025] 36. The method of claim 35, wherein the anti-PD-1 antibody or antigen-binding portion thereof is administered at a dose ranging from 0.1 to 10.0 mg / kg body weight once every 2, 3, or 4 weeks.

[0026] In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a dose of 5 or 10 mg / kg body weight once every three weeks.

[0016]

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

[0028] In some embodiments, the anti-PD-1 antibody, or antigen-binding portion thereof, is administered by intravenous infusion at a dose of 200 mg on day 1 of a 3-week cycle.

[0017]

[0029] In some embodiments, the platinum-based chemotherapy is platinum-doublet chemotherapy (PT-DC).

[0030] In some embodiments, the PT-DC is a combination of pemetrexed and carboplatin.

[0018]

[0031] In some embodiments, carboplatin is administered at a dose that achieves an area under the free carboplatin plasma concentration versus time curve (AUC) of 5.

[0032] In some embodiments, pemetrexed is administered at 500 mg / m 2 is administered at a dose of

[0019]

[0033] In some embodiments, the PT-DCs are administered in conjunction with the anti-PD-1 antibody, or antigen-binding portion thereof, for four doses of the anti-PD-1 antibody, or antigen-binding portion thereof, followed by repeated administration of the anti-PD-1 antibody, or antigen-binding portion thereof, alone.

[0020]

[0034] In some embodiments, the method promotes epitope spreading.

[0035] In some embodiments, the method promotes epitope spreading of an epitope that is distinct from any of the cancer-specific neoepitopes.

[0021]

[0036] In some embodiments, the epitope that is distinct from any of the cancer-specific neoepitopes comprises a KRAS neoepitope, a TP53 neoepitope, and / or a KEAP1 neoepitope.

[0022]

[0037] In some embodiments, the KRAS neoepitope comprises a G12C or G12V mutation.

[0038] In some embodiments, the median progression-free survival (PFS) of a first population of human subjects treated for cancer with the first, second, and third components is longer than the median PFS of a second population of subjects treated for cancer with the second and / or third components but not the first component.

[0023]

[0039] In some embodiments, the overall response rate (ORR) of a first population of human subjects treated for cancer with the first, second, and third components is higher than the ORR of a second population of subjects treated for cancer with the second and / or third components but not the first component.

[0024]

[0040] In some embodiments, the percentage of subjects in a first population of human subjects treated for cancer with the first, second, and third components who have at least a progression-free survival (PFS) of at least 9 months is higher than the percentage of subjects in a second population of subjects treated for cancer with the second and / or third components but not the first component who have at least a PFS of at least 9 months.

[0025]

[0041] In some embodiments, the percentage of subjects in a first population of human subjects treated for cancer with the first, second, and third components who have at least a 12-month progression-free survival (PFS) is higher than the percentage of subjects in a second population of subjects treated for cancer with the second and / or third components but not the first component who have at least a 12-month PFS.

[0026]

[0042] In some embodiments, the median overall survival (OS) of a first population of human subjects treated for cancer with the first, second, and third components is longer than the median OS of a second population of subjects treated for cancer with the second and / or third components but not the first component.

[0027]

[0043] In some embodiments, the percentage of subjects achieving a complete response, partial response, long-term stable disease, or stable disease for 6 months or more (CBR) of a first population of human subjects treated for cancer with the first, second, and third components is higher than the CBR of a second population of subjects treated for cancer with the second and / or third components but not the first component.

[0028]

[0044] In some embodiments, the reduction in tumor size in a first population of human subjects treated for cancer with the first, second, and third components is greater than the reduction in tumor size in a second population of subjects treated for cancer with the second and / or third components but not the first component.

[0029]

[0045] In some embodiments, the level of tumor-infiltrating CD4+ T cells in a first population of human subjects treated for cancer 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 for cancer with the second and / or third components but not the first component.

[0030]

[0046] In some embodiments, the levels of effector and cytotoxic CD4+ T cells generated in a first population of human subjects treated for cancer with the first, second, and third components are higher than the levels of effector and cytotoxic CD4+ T cells generated in a second population of subjects treated for cancer with the second and / or third components but not the first component. In some embodiments, the method increases the levels of CD4+ T cells specific for cancer-specific neoepitopes that upregulate expression of ZEB2, PDCD1, TOX, TIGT, CXCR3, ITGB1, GZMA, and / or ICOS.

[0031]

[0047] In some embodiments, the method comprises detecting CD4+ / CD62L specific for a cancer-specific neoepitope. hi / CD69+ / CD27+ / CCR7+ T cell levels.

[0048] In some embodiments, the method increases the level of CD4+ / NKG7+ / CCL4+ / CCL5+ / GNLY+ / LAG3+ T cells specific for a cancer-specific neoepitope.

[0032]

[0049] In some embodiments, the human subject is identified as having a PD-L1 positive cancer prior to administration of the first, second and / or third components.

[0050] In one aspect, a method of treating or preventing cancer in a human subject in need thereof comprises administering to the human subject in need thereof (a) a first component comprising (i) a polypeptide comprising a cancer-specific neoepitope 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 neoepitope of a protein expressed by a cancer cell of the cancer neoepitope, or (v) a T cell comprising the TCR of (iv); (a) a second component comprising an anti-cancer agent that is an antibody or antigen-binding portion thereof that specifically binds to Programmed Death-1 (PD-1) receptor and inhibits PD-1 activity; and (b) a third component comprising platinum-based chemotherapy; wherein (i) the method comprises administering to the human subject an antibody or antigen-binding portion thereof that specifically binds to the cancer-specific neoepitope of a protein expressed by a cancer cell of the cancer. (ii) the median progression-free survival (PFS) of a first population of human subjects whose cancer has been treated with the first, second, and third components is longer than the median PFS of a second population of subjects whose cancer has been 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 whose cancer has been treated with the first, second, and third components is longer than the median PFS of a second population of subjects whose cancer has been treated with the second and / or third components but not the first component; (iv) the percentage of subjects having at least a 9 or 12 month progression-free survival (PFS) in a first population of human subjects whose cancer is treated with the first, second, and third components is higher than the percentage of subjects having at least a 9 or 12 month PFS in a second population of subjects whose cancer is treated with the second and / or third components but not the first component;(v) the median overall survival (OS) of a first population of human subjects whose cancer is treated with the first, second, and third components is longer than the median OS of a second population of subjects whose cancer is treated with the second and / or third components but not the first component; (vi) the percentage of subjects achieving a complete response, partial response, long-term stable disease, or stable disease for 6 months or more (CBR) of a first population of human subjects whose cancer is treated with the first, second, and third components is higher than the CBR of a second population of subjects whose cancer is treated with the second and / or third components but not the first component; (vii) the reduction in tumor size in a first population of human subjects whose cancer is treated with the first, second, and third components is greater than the reduction in tumor size in a second population of subjects whose cancer is treated with the second and / or third components but not the first component; (viii) the percentage of subjects whose cancer is treated with the first, second, and third components is higher than the percentage of subjects whose cancer is treated with the first, second, and third components. (ix) the level of effector and cytotoxic CD4+ T cells generated in the first population of human subjects treated with the first, second, and third components is higher than the level of tumor-infiltrating CD4+ T cells generated in the 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 a cancer-specific neoepitope that upregulates expression of ZEB2, PDCD1, TOX, TIGT, CXCR3, ITGB1, GZMA, and / or ICOS; (xi) the method increases the level of CD4+ / CD62L specific for a cancer-specific neoepitope; hi and / or (xii) the method increases the level of CD4+ / NKG7+ / CCL4+ / CCL5+ / GNLY+ / LAG3+ T cells specific for a cancer-specific neoepitope.

[0033]

[0051] In some embodiments, the human subject is identified as having a PD-L1 positive cancer prior to administration of the first, second and / or third components.

[0052] In some embodiments, the human subject has had no prior systemic treatment for metastatic disease, has had no prior immunotherapy with an anti-PD-1 antibody, and / or has had no prior immunotherapy with an anti-PD-L1 antibody.

[0034]

[0053] In some embodiments, provided herein are methods of treating or preventing a neoplasm in a human subject in need thereof, the method comprising administering to the human subject in need thereof a first component comprising: (i) a peptide comprising a neoepitope of a protein, (ii) a polynucleotide encoding the peptide, (iii) one or more APCs comprising a polynucleotide that comprises or encodes the peptide, or (iv) a T cell receptor (TCR) specific for the neoepitope in complex 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.

[0035]

[0054] In some embodiments, the first component comprises a neoplastic vaccine or immunogenic composition.

[0055] In some embodiments, the first component further comprises an adjuvant. In some embodiments, the adjuvant is poly-ICLC.

[0036]

[0056] In some embodiments, the first component comprises a neoplasia vaccine or immunogenic composition comprising neo-antigenic peptides, wherein the peptides comprise at least two, at least three, at least four, or at least five peptides. In some embodiments, the peptides comprise at most 15, at most 20, at most 25, or at most 30 peptides. In some embodiments, the peptides are about 5-50 amino acids in length. In some embodiments, the peptides are about 14-35 amino acids in length. In some embodiments, the neo-epitope of each peptide is unique.

[0037]

[0057] In some embodiments, the first component further comprises a pH adjuster. In some embodiments, the first component further comprises a pharmaceutically acceptable carrier.

[0058] In some embodiments, the subject is suffering from a neoplasm selected from the group consisting of non-Hodgkin's lymphoma (NHL), clear cell renal cell carcinoma (ccRCC), melanoma, sarcoma, leukemia, or cancer of the bladder, colon, brain, breast, head and neck, endometrium, lung, ovary, pancreas, or prostate. In some embodiments, the neoplasm is metastatic melanoma. In some embodiments, the subject does not have a detectable neoplasm but is at high risk of disease recurrence. In embodiments, the cancer is selected from the group consisting of adrenal gland cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, glioblastoma, head and neck cancer, chromophobe renal cell carcinoma, clear cell renal carcinoma, papillary renal carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, ovarian cancer, pancreatic cancer, melanoma, gastric cancer, endometrial cancer, and uterine carcinosarcoma. In some embodiments, the cancer is selected from the group consisting of prostate cancer, bladder cancer, lung squamous cell carcinoma, 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 some embodiments, the cancer is selected from the group consisting of CRC, head and neck cancer, gastric cancer, lung squamous cell carcinoma, lung adenocarcinoma, prostate cancer, bladder cancer, gastric cancer, renal cell carcinoma, and uterine cancer. In some embodiments, the cancer is selected from the group consisting of melanoma, lung squamous cell carcinoma, DLBCL, uterine cancer, head and neck cancer, uterine cancer, liver cancer, and CRC. In embodiments, the cancer is selected from the group consisting of lymphatic cancer, Burkitt's lymphoma, neuroblastoma, prostate cancer, colorectal adenocarcinoma, uterine / endometrial adenocarcinoma, MSI+, endometrial serous carcinoma, endometrial carcinosarcoma-malignant mixed mesodermal tumor, glioma, astrocytoma, GBM, acute myeloid leukemia associated with MDS, chronic lymphocytic leukemia-small lymphocytic lymphoma, myelodysplastic syndrome, acute myeloid leukemia, luminal NS carcinoma of breast, chronic myeloid leukemia, pancreatic ductal carcinoma, chronic myelomonocytic leukemia, myelofibrosis, myelodysplastic syndrome, prostate cancer, essential thrombocythemia, and medullomyoblastoma. 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 some embodiments, the cancer is selected from the group consisting of bladder cancer, colon cancer, and gastric cancer. In some embodiments, the cancer is selected from the group consisting of lung cancer, CRC, melanoma, breast cancer, NSCLC, and CLL. In some embodiments, the subject is a partial or non-responder to checkpoint inhibitor therapy. In some 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 adenocarcinoma (CESC), chronic lymphocytic leukemia (CLL), colorectal cancer (CRC), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney papillary renal cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), prostate cancer, skin cutaneous melanoma (SKCM), gastric adenocarcinoma (STAD), thyroid cancer (THCA), and uterine endometrioid carcinoma (UCEC). In some embodiments, the cancer is selected from the group consisting of colorectal cancer, uterine cancer, endometrial cancer, gastric cancer, and Lynch syndrome. In some embodiments, the cancer is MSI+ cancer.

[0038]

[0059] 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 is administered on the same day as the second component. In some embodiments, the second component is administered before the first component. In some embodiments, administration of pembrolizumab is initiated before the start of administration of the first component. In some embodiments, administration of pembrolizumab is initiated before the start of administration of carboplatin or pemetrexed. In some embodiments, administration of pembrolizumab is initiated before the start of administration of carboplatin or pemetrexed. In some embodiments, administration of pembrolizumab is on the same day as administration of the first component. In some embodiments, administration of carboplatin is initiated on the same day as the first administration of the first component. In some embodiments, administration of nivolumab continues every 12 to 36 weeks or more after the first administration of pembrolizumab. In some embodiments, administration of nivolumab continues every 2, 3, 4, 6, or 8 weeks after the initial administration of pembrolizumab. In some embodiments, administration of the inhibitor, e.g., a checkpoint inhibitor or chemotherapeutic agent, is initiated after tumor resection. In some embodiments, administration of the first component is in a prime-boost regimen.

[0039]

[0060] In some embodiments, administration of the first component is at week 1, 2, 3, or 4 for the prime. In some embodiments, administration of the first component is at month 2, 3, 4, or 5 for the boost. In some embodiments, administration of the first component is at week 19, 20, 21, 22, 23, or 24 for the boost.

[0040]

[0061] In some embodiments, the peptide is 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.

[0041]

[0062] In some embodiments, the first component and / or the second component are administered intravenously or subcutaneously.

[0063] In some embodiments, the dose of peptide is divided into at least 2, at least 3, at least 4, or at least 5 sub-doses, hi some embodiments, each sub-dose of peptide comprises at least 4 or at least 5 peptides.

[0042]

[0064] In some embodiments, each peptide is administered in a dose of 200-400 μg, hi some embodiments, each divided dose is administered to a different location in the subject.

[0065] In some embodiments, the method further comprises administering one or more additional agents, hi some embodiments, the additional agents are selected from the group consisting of chemotherapeutic agents, anti-angiogenic agents, and agents that reduce immunosuppression.

[0043]

[0066] In some aspects, provided herein are methods 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 that is 1-95% of the dosage of the chemotherapeutic agent typically administered in a monotherapy regimen.

[0044]

[0067] In some aspects, provided herein are methods of treating or preventing cancer in a human subject in need thereof who has previously been treated with pembrolizumab at a dose of 1-95% of the dosage of pembrolizumab typically administered in a monotherapy regimen, the method comprising administering to the subject one or more chemotherapeutic agents, e.g., at a dose that is lower than their usual dose for monotherapy.

[0045]

[0068] In some aspects, provided herein are methods 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 that is 1-95% of the dose typically administered in monotherapy regimens, and pemetrexed at a dose that is less than the dose typically administered in monotherapy regimens.

[0046]

[0069] In some embodiments, the method further comprises administering to the subject at least five peptides, each comprising a unique neoepitope of the protein, at a dose of 100-500 μg of each peptide.

[0047]

[0070] In some aspects, provided herein are compositions comprising: a first component comprising: (i) a peptide comprising a neoepitope of a protein; (ii) a polynucleotide encoding the peptide; (iii) one or more APCs comprising the peptide or comprising a polynucleotide encoding the peptide; or (iv) a T cell receptor (TCR) specific for the neoepitope in complex with an HLA protein; and a second component comprising at least two inhibitors, wherein the at least two inhibitors comprise pembrolizumab and platinum-based chemotherapy; pembrolizumab and pemetrexed; or pembrolizumab, platinum-based chemotherapy, and pemetrexed.

[0048]

[0071]

[0013] Further aspects and advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, in which merely illustrative embodiments of the present disclosure are shown and described. As will be understood, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious aspects, all without departing from the present disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive. Incorporation by Reference

[0072] All publications, patents, and patent applications mentioned herein are incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that the publications and patents or patent applications incorporated by reference conflict with the present disclosure contained herein, the present specification is intended to supersede and / or take precedence over any such conflicting material.

[0049]

[0073] The novel features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments in which the principles of the invention are utilized, and the accompanying drawings (also referred to herein as "FIG" and "FIG"). [Brief explanation of the drawings]

[0050] [Figure 1A]

[0074] Figures 1A-1C: NEO-PV-01 vaccine generation, clinical study design, and patient breakdown.

[0075] Schematic diagram of patient tumor sequencing, prediction of neoantigens restricted to class I MHC molecules, and generation of synthetic long peptides for inclusion in personalized neoantigen vaccines. [Figure 1B]

[0076] Treatment with pembrolizumab and pemetrexed plus carboplatin began at week 0, then NEO-PV-01 was administered between weeks 12 and 24, with pembrolizumab continued for up to 2 years. [Figure 1C]

[0077] Breakdown of study patients: Seventeen of 38 patients (45%) in the ITT set were not vaccinated due to reasons including inability to produce vaccine due to inappropriate tumor and / or insufficient neoplasm count (10 patients), adverse events (2 patients), patient consent withdrawal (2 patients), disease progression, investigator's discretion, or early study termination due to administration of concomitant medications prohibited by the study (1 patient each). [Figure 2A]

[0078] Figures 2A-2E: Response rate and durability after treatment with NEO-PV-01 plus chemotherapy and anti-PD-1.

[0079] Best radiographic change (%) of the sum of target lesions for each patient (VAX set) who received at least one dose of vaccine. Darker, thinner bars represent best change before NEO-PV-01, and lighter, wider bars represent best overall change on study for patients who received at least one dose of NEO-PV-01 + anti-PD-1. Red indicates progressive disease, gray indicates stable disease, and blue indicates partial response. [Figure 2B]

[0080] Percent radiographic change in target lesions after initiation of pembrolizumab treatment for each patient (colors are the same as in a), including 30 of 38 ITT patients who had at least one post-baseline RECIST assessment. [Figure 2C]

[0081] Swimmer plot summarizing all patients during the study period. Each bar represents one subject in the study, and the length of the bar represents the duration of study participation. [Figure 2D]

[0082] Kaplan-Meier estimates of PFS (top) and OS (bottom) for both the ITT patient set (left) and the vaccinated patient set (right). [Figure 2E]

[0083] Measurement of ctDNA in peripheral blood of a subset of patients measured by 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 patients without PFS-9 (blue) and in patients with PFS-9 (green). [Figure 3A]

[0084] Figures 3A-3F: Correlates of clinical response, including T cell infiltration, MHC class II expression, and TCR diversity, are observed in the tumor microenvironment (TME) pre-treatment.

[0085] Correlation of PFS in months with CD4+ (top) and CD8+ (bottom) T cells per mm2 either extratumoral (left) or intratumoral (right) in patient tumor biopsies by multiplex IHC at pretreatment time points. Pearson correlation coefficients (R) and associated p-values are shown. [Figure 3B]

[0086] Representative images of IHC analysis of CD4+ and CD8+ T cells in patient tumor biopsies from one patient without PFS-9 (2L3) and one patient with PFS-9 (2L5) stained with DAPI (blue), CD3 (red), CD4 (green), CD8 (white), and PanCK (cyan). White arrows indicate CD8+ T cells surrounding the tumor area, and yellow arrows indicate CD4+ T cells infiltrating the TME. (Scale bar, 50 μM). [Figure 3C]

[0087] Correlation of HLA class II gene expression in pre-treatment tumor biopsies with PFS in months. Pearson correlation coefficients (R) and associated p-values are shown. [Figure 3D]

[0088] Representative images of MHC class II expression by multiplex IHC in patient tumor biopsies from one patient (2L3) without PFS-9 and one patient (2L5) with PFS-9 stained with HLA-DR / DP / DQ (red), PanCK (green), DAPI (blue), CD11c (white), and CD14 (yellow). Individual channel images for patient 2L5 are shown below the multiplex images for HLA-DR / DP / DQ, CD14, CD11c, and PanCK. 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). [Figure 3E]

[0089] Analysis of Shannon entropy (left) and unique amino acid (UniAA) counts (right) in tumor biopsies at pretreatment time point and correlation with PFS in months. Pearson correlation coefficients (R) and associated p-values are shown. [Figure 3F]

[0090] Analysis of Shannon entropy (left) and unique amino acid counts (right) in tumor biopsies at pre-treatment (week 0), pre-vaccine (week 12), and post-vaccine (week 24) time points, with PFS-9 patients represented in green and non-PFS-9 patients represented in blue. [Figure 4A]

[0091] Figures 4A-4E: NEO-PV-01 plus chemotherapy and anti-PD-1 induce neoantigen-reactive T cell responses that are neoepitope-specific, durable, and exhibit cytotoxic potential.

[0092] Percentage of all NEO-PV-01 vaccination peptides that elicited an IFNγ response in a series of PBMCs at the indicated time points. [Figure 4B]

[0093] Table summarizing the overall immune response detected for the 13 patients analyzed, also characterized as CD4+ or CD8+ response percentage. [Figure 4C]

[0094] Specificity measured by IFNγ ELISpot assay for mutant peptide (green) compared to wild-type peptide (red) across a range of peptide concentrations. Representative responses from patients 2L7 (IM13 and IM04) and 2L3 (IM03 and IM15). [Figure 4D]

[0095] 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 columns 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. [Figure 4E]

[0096] Cytotoxicity of the immune response generated against NEO-PV-01, as measured by surface expression of the marker CD107a in conjunction with intracellular IFNγ expression at post-vaccine time points. Representative flow plots for patient 2L7 are shown on the left, comparing peptide recall (bottom) and DMSO recall (top) in both the ex vivo assay design (left) and the 5-day stimulation assay design (right). Data are summarized on the right and presented as stacked columns for individual patients, with positive (cytotoxic) responses shown in dark green and negative (no cytotoxicity) responses shown in light green. The table below summarizes the responses detected for all patients analyzed and categorizes responses as either CD4+, CD8+, or both. Aggregate data are presented as mean + / - SEM. [Figure 5A]

[0097] Figures 5A-5E: NEO-PV-01 plus chemotherapy and anti-PD-1 induces epitope spreading responses in the majority of analyzed patients in whom mutKRAS responses were observed.

[0098] Epitope spreading was measured in 13 patients. Postvaccine PBMC reactivity to a range of 10-25 predicted neoantigenic peptides not included in the vaccine was tested by IFNγ ELISpot assay. Responses characterized as epitope spreading were detected only at the postvaccine time point (week 20) but not at the prevaccine time point (week 10). [Figure 5B]

[0099] T cell responses to individual non-immunizing (NIM) peptides across nine patients at post-vaccination time points are shown. NIM peptides that did not elicit reactivity after vaccination are not shown. Each NIM peptide was tested for the generation of an immune response using overlapping assay peptides. The assay peptide that produced the maximal response in either the ex vivo assay or the 5-day assay is shown. Responses marked with an * indicate ex vivo responses. [Figure 5C]

[0100] Four patients mounted mutKRAS-specific epitope-spreading responses as determined by IFNγ ELISpot assay. Each patient's PFS-9 status is indicated below the graph, and the specific G12 mutation is indicated above the graph. Responses marked with an * indicate ex vivo responses. [Figure 5D]

[0101] Specificity of the immune response measured by IFNγ ELISpot assay to the mutant peptide (solid line) compared to the wild-type peptide (dotted line) over a range of peptide concentrations for each of the four patients in whom an epitope-spreading response to mutKRAS was observed. [Figure 5E]

[0102] Surface expression of the cytotoxic marker CD107a (x-axis) and intracellular expression of IFNγ (y-axis) are shown by FACS analysis of the mutKRAS-specific epitope spreading response observed for patient 2L15. Individual plots depict the control (DMSO) on the left and the NIM peptide on the right. Positivity in this assay was defined as >1.5-fold stimulation over DMSO in the double-positive gate. The parent gate is shown below the paired FACS plot. [Figure 6A]

[0103] Figures 6A-6H: Neoantigen-specific CD4+ T cell responses share an activated effector phenotype in the periphery after vaccination.

[0104] Table summarizing five patients analyzed using multimer-based sorting of neoantigen-specific CD4+ T cells (representative flow panel on the right) and a combination of CITE-Seq and TCRSeq with gene expression analysis. [Figure 6B]

[0105] Unsupervised clustering of tetramer+ and tetramer- CD4+ T cell samples from five patients (left) and heatmap of normalized gene expression used for clustering analysis (right). [Figure 6C]

[0106] Unsupervised clustering plots separated to visualize tetramer- cells (top) and tetramer+ cells (bottom) from five patients. [Figure 6D]

[0107] Proportions of each UMAP cluster comparing tetramer+ population to tetramer- population. [Figure 6E]

[0108] Measurement of the Gini coefficient (a measure of clonality of the TCR repertoire) for both the tetramer+ and tetramer- populations. Each point represents an individual patient. [Figure 6F]

[0109] Measurement of clonotypes covering the top 30% of the TCR repertoire of tetramer+ CD4+ T cells detected by single-cell TCR sequencing utilizing bulk TCR-seq data across pre-treatment, pre-vaccine, and post-vaccine time points. [Figure 6G]

[0110] Measurement of the number of CD3+CD4+ T cells per mm2 of tumor biopsy tissue using multiplex IHC at pre-treatment, pre-vaccine, and post-vaccine time points when available. Aggregate data are expressed as mean + / - SEM. [Figure 6H]

[0111] Representative IHC images for patient 2L7 (PFS-9) at pre-treatment (left), pre-vaccine (center), and post-vaccine (right) time points stained with DAPI (blue), CD3 (red), CD4 (yellow), and PanCK (green). (Scale bar, 50 μm.) Serial biopsies were from the same lung lesion in this patient. [Figure 7A]

[0112] Figures 7A-7C: Radiographic response by pre-treatment tumor PD-L1 levels in vaccinated patients, as well as tracking of individual ctDNA molecules over time for each patient measuring the abundance of predicted neoantigen genes and genes included in the personalized Signatera pool.

[0113] Radiographic response by pretreatment PD-L1 levels in tumors of patients who received at least one dose of NEO-PV-01. PD-L1 levels are shown below the bars in the waterfall graph. Scoring was based on PD-L1 on tumor cells as follows: <1% is indicated as -, 1 to <50% is indicated as +, and ≥50% is indicated as ++. [Figure 7B]

[0114] ctDNA measurements over time across 17 patients for 16 variants predicted to be high-quality neoepitopes based on an internal bioinformatics algorithm. [Figure 7C]

[0115] 16 selected somatic targeted mutations selected by Natera variant calling method. [Figure 8A]

[0116] Figures 8A-8B: NEO-PV-01 plus chemotherapy and anti-PD-1 induces sustained T cell reactivity against multiple vaccine neoepitopes.

[0117] T cell responses to individual immunizing (IM) peptides across nine patients are shown. Immunizing peptides that did not elicit reactivity are not shown. Each IM peptide was tested for the generation of an immune response using overlapping assay peptides. The assay peptide that produced the maximal response for each IM peptide is shown across pre-vaccine and post-vaccine time points. Each bar corresponds to the IM peptide and corresponding assay peptide that produced the maximal response in either the ex vivo or 5-day assay. Immunizing peptides shown in red on the x-axis elicited a pre-vaccine response. [Figure 8B]

[0118] Additional analyses were performed on four patients 52 weeks after initiation of chemotherapy plus pembrolizumab, and T cell responses were quantified as in panel A. The inset for patient 2L11 is enlarged to allow visualization of responses to IM17 and IM07. Immunizing peptides shown in red on the x-axis elicited pre-vaccine responses, while those shown in green elicited responses that were only detected at week 52. Aggregate data are presented as mean + / - SEM. [Figure 9A-1]

[0119] Figures 9A-9B show that NEO-PV-01 plus chemotherapy and anti-PD-1 induce cytotoxic CD4+ and CD8+ T cell responses. Surface expression of the cytotoxic marker CD107a (x-axis) and intracellular expression of IFNγ (y-axis) are shown by FACS analysis for 56 IM peptides across 12 patients. Individual plots depict the control (DMSO) on the left and the IM peptide on the right. Only IM peptides that were positive in this assay (>1.5-fold stimulation over the DMSO control in the double-positive gate) are shown. The parent gate is shown below each pair of FACS plots, containing responses categorized as either CD4+ T cell responses (Figure 9A) or CD8+ T cell responses (Figure 9B). [Figure 9A-2] Figures 9A-9B show that NEO-PV-01 plus chemotherapy and anti-PD-1@0034 induce cytotoxic CD4+ and CD8+ T cell responses. Surface expression of the cytotoxic marker CD107a (x-axis) and intracellular expression of IFNγ (y-axis) are shown by FACS analysis for 56 IM peptides across 12 patients. Individual plots depict the control (DMSO) on the left and the IM peptide on the right. Only IM peptides that were positive in this assay (>1.5-fold stimulation over the DMSO control in the double-positive gate) are shown. The parent gate is shown below each pair of FACS plots, including responses categorized as either CD4+ T cell responses (Figure 9A) or CD8+ T cell responses (Figure 9B). [Figure 9B]Figures 9A-9B show that NEO-PV-01 plus chemotherapy and anti-PD-1 induce cytotoxic CD4+ and CD8+ T cell responses. Surface expression of the cytotoxic marker CD107a (x-axis) and intracellular expression of IFNγ (y-axis) are shown by FACS analysis for 56 IM peptides across 12 patients. Individual plots depict the control (DMSO) on the left and the IM peptide on the right. Only IM peptides that were positive in this assay (>1.5-fold stimulation over the DMSO control in the double-positive gate) are shown. The parent gate is shown below each pair of FACS plots, containing responses categorized as either CD4+ T cell responses (Figure 9A) or CD8+ T cell responses (Figure 9B). [Figure 10A]

[0120] Figures 10A-10E: NEO-PV-01 plus chemotherapy and anti-PD-1 induces an epitope-spreading response that is cytotoxic and durable.

[0121] Cytotoxicity of epitope spreading responses measured by surface expression of the marker CD107a in conjunction with intracellular IFNγ expression at post-vaccine time points. Data are presented as stacked columns for individual patients, with positive (cytotoxic) responses shown in dark green and negative (no cytotoxicity) responses shown in light green. The table below summarizes the responses detected for all patients analyzed and categorizes responses as either CD4+, CD8+, or both. [Figure 10B]

[0122] Individual plots depict the control (DMSO) on the left and the non-immunizing (NIM) peptide on the right. Only NIM peptides that were positive in this assay (>1.5-fold stimulation over DMSO control in the double positive gate) are shown. The parent gate is shown below each pair of FACS plots. [Figure 10C]

[0123] Distribution of PFS in months for patients (ITT set) who either did or did not have KRAS mutations in their pretreatment tumor biopsies. Vaccinated patients are shown as "+" and unvaccinated patients as "·". Four patients in whom an epitope spreading response to mutKRAS was observed are shown in green. All other patients are shown in black. Box plots show the 25th, 50th, and 75th percentiles, with whiskers extending to the 95% confidence interval. p-values are derived from a two-tailed Student's t-test. [Figure 10D]

[0124] T cell responses to individual non-immunization (NIM) peptides are shown across three patients at pre-vaccine (week 10), post-vaccine (week 20), and week 52 time points. NIM peptides that did not elicit reactivity are not shown. Each NIM peptide was tested for the generation of an immune response using overlapping assay peptides. The assay peptide that produced the maximal response for each NIM peptide is shown across the three time points. Each bar corresponds to the NIM peptide and corresponding assay peptide that produced the maximal response in either the ex vivo or 5-day assay. NIM peptides shown in red on the x-axis elicited a pre-vaccine response, while those shown in green elicited a response that was only detected at week 52. The inset for patient 2L16 has been enlarged to allow visualization of responses to NIM101 and NIM12. [Figure 10E]

[0125] Durability of the immune response induced by the NIM peptide 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 columns 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. [Figure 11A]

[0126] 11A-11D: The neoantigen-reactive CD4+ effector cell phenotype is distinct from other effector cells.

[0127] Table summarizing the five patients analyzed using combination multimeric selection of neoantigen-specific CD4+ T cells and the corresponding mutant and wild-type peptide sequences shown for the responses analyzed. NA = not applicable. [Figure 11B]

[0128] Comparison of antibody (lines 1, 3 and 5) and RNA (lines 2, 4 and 6) expression levels of genes included in the CITE antibody panel. [Figure 11C]

[0129] Unsupervised clustering of tetramer- and tetramer+ samples using normalized expression of selected RNA markers for clustering. [Figure 11D]

[0130] Effector cells (designated CD45RO+CD45RA-CD62LloCCR7lo) identified in blue in unsupervised clustering of tetramer+ and tetramer- CD4+ T cell samples. All other cells are shown in pink. [Figure 11E]

[0131] (Top) Unsupervised clustering of effector cells only (both tetramer+ and tetramer-) results in five unique clusters. (Bottom) Unsupervised clustering plot separated to visualize tetramer- and tetramer+ cells among all effector cells. [Figure 11F]

[0132] Unsupervised clustering of effector cells partitioned for each individual patient. [Figure 11G]

[0133] Heatmap of normalized gene expression for each of the five unique clusters identified based on clustering of all effector cells. [Figure 12-1]

[0134] Figure 12 shows that neoantigen-specific CD4+ T cell responses were detected by MHC class II tetramer staining and demonstrated effector and central memory phenotypes by flow cytometry. MHC class II tetramer analysis was performed across 27 peptide-MHC combinations using CD4+ T cells from eight patients at post-vaccination time points. The leftmost plot for each patient depicts the tetramer+ population (quantified by both total tetramer+ cell count and percent of the bulk CD4+ population) with a green circle. For a subset of patients, additional phenotyping was performed by flow cytometry to characterize cells as naive, effector memory, or central memory based on CD45RA and CD62L expression (rightmost plot). [Figure 12-2] Figure 12 shows that neoantigen-specific CD4+ T cell responses were detected by MHC class II tetramer staining and demonstrated effector and central memory phenotypes by flow cytometry. MHC class II tetramer analysis was performed across 27 peptide-MHC combinations using CD4+ T cells from eight patients at post-vaccination time points. The leftmost plot for each patient depicts the tetramer+ population (quantified by both total tetramer+ cell count and percent of the bulk CD4+ population) with a green circle. For a subset of patients, additional phenotyping was performed by flow cytometry to characterize cells as naive, effector memory, or central memory based on CD45RA and CD62L expression (rightmost plot). [Figure 13A]

[0135] Figures 13A-13I: Neoantigen-specific CD4+ T cells display distinct phenotypes across patients but consistent phenotypes when compared across clones targeting individual epitopes, neoantigen-reactive TCRs are functional after vaccination, and tumors display peripherally expanded TCR accumulation.

[0136] Table outlining TCR clones covering the top 30% of the repertoire per patient from single-cell TCRseq data at post-vaccination time points and the corresponding frequency of tetramer+ CD4+ T cells for that patient in peripheral blood. [Figure 13B]

[0137] Unsupervised clustering of tetramer+ samples only using corresponding gene and protein expression markers specific for each cluster. [Figure 13C]

[0138] Unsupervised clustering of tetramer+ samples depicting the normalized expression of selected genes used in clustering. [Figure 13D]

[0139] Heatmap of normalized gene expression based on clustering of tetramer+ samples only. [Figure 13E]

[0140] Unsupervised clustering of reclustered tetramer+ samples, partitioned by individual patient. [Figure 13F]

[0141] Heatmap showing the abundance of cells of each clonotype across clusters of tetramer+ CD4+ T cells. Top expanded clones are defined as those covering the top 30% of the TCR repertoire for each patient as detected by single-cell TCR-Seq. [Figure 13G]

[0142] CD4+ TCR clones identified by single-cell TCR sequencing as specifically recognizing the corresponding immunizing peptide epitope in the context of MHC class II allele matching were cloned into Jurkat cell lines (separately for each patient indicated). Each of the four clonal Jurkat cell lines was cocultured with matched patient APCs containing the corresponding IM peptide at various peptide concentrations, and IL-2 secretion into the supernatant was measured as a readout for TCR recognition of the peptide:MHC complex. Reactivity to the corresponding wild-type sequence was also tested, except for patient 2L11 / IM18, for whom the wild-type peptide was unavailable and the mutation was a frameshift. [Figure 13H]

[0143] Measurement of the number of CD3+CD8+ T cells per mm2 of tumor biopsy tissue using multiplex IHC at pre-treatment, pre-vaccine, and post-vaccine time points when available. Aggregate data are expressed as mean + / - SEM. [Figure 13I]

[0144] TCR sequencing was performed on selected post-vaccination tumor biopsies, for which corresponding pre-treatment or post-vaccination biopsies were also available.TCRs found exclusively in post-vaccination biopsies were then cross-referenced with TCRs found in peripheral blood.Only TCRs found to increase in peripheral blood at post-vaccination time points were visualized.TCRs that expanded in the periphery at the time of vaccination are shown in red, and TCRs that were only detected in the periphery at post-vaccination time points are shown in blue. DETAILED DESCRIPTION OF THE INVENTION

[0051]

[0145] Described herein are new immunotherapeutic agents and their uses based on the discovery of neoantigens that arise from mutational events unique to individual tumors. Thus, the disclosure described herein provides peptides, polynucleotides encoding the peptides, and peptide-binding agents that can be used to stimulate an immune response against tumor-associated antigens or neoepitopes, for example, to create immunogenic compositions or cancer vaccines for use in treating disease.

[0052]

[0146] The following description and examples are provided to illustrate embodiments of the present disclosure in detail. It is understood that the present disclosure is not limited to the specific embodiments described herein, and as such may vary. Those skilled in the art will recognize that numerous variations and modifications of the present disclosure exist and are encompassed within the scope of the present disclosure.

[0053]

[0147] All terms are intended to be understood as they are understood by one of ordinary skill in the art. Unless otherwise defined, 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 pertains.

[0054]

[0148] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0149] While various features of the present disclosure may be described in the context of a single embodiment, those features may also be provided separately or in any suitable combination. Conversely, while the present disclosure may be described herein for clarity in the context of separate embodiments, the present disclosure may also be implemented in a single embodiment.

[0055]

[0150] The following definitions supplement those in the art and relate to the present application and are not to be construed as being related or unrelated to, for example, any co-owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in carrying out the tests of the present disclosure, the preferred materials and methods are described herein. Therefore, the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting. I. Definition

[0151] The terminology used herein is for the purpose of describing particular instances only and is not intended to be limiting. In this application, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the singular forms "a," "an," and "the" are intended to include the plural as well, unless the context clearly dictates otherwise.

[0056]

[0152] In some embodiments of the present application, the use of "or" means "and / or" unless otherwise stated. The terms "and / or" and "any combination thereof," and their grammatical equivalents, may be used interchangeably when used herein. In some embodiments, these terms may convey that any combination is specifically contemplated. For purposes of explanation only, the following phrases "A, B, and / or C," or "A, B, C, or any combination thereof" may mean "A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C." The term "or" may be used conjunctively or disjunctively, unless the context specifically dictates disjunctive use.

[0057]

[0153] In some embodiments, the term "about" or "approximately" means within an acceptable error range for a particular value, as determined by one of ordinary skill in the art, which will depend in part on the limitations of the method by which the value is measured or determined, i.e., the measurement system. For example, in some embodiments, "about" can mean within 1 or more standard deviations, according to practice in the art. Alternatively, "about" can mean a range of 20% or less, 10% or less, 5% or less, or 1% or less of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, 5-fold, or 2-fold of a value. When a particular value is described in this application or claims, unless otherwise stated, the term "about" should be construed to mean within an acceptable error range of the particular value.

[0058]

[0154] As used in the specification and claims, the words "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has"), "including" (and any form of including, e.g., "includes" and "include"), or "containing (and any form of containing, e.g., "contains" and "contain") are inclusive or open-ended and do not exclude additional, unstated elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and conversely, any method or composition of the disclosure can also be implemented with respect to any embodiment discussed herein. Furthermore, the compositions of the disclosure can be used to achieve the methods of the disclosure.

[0059]

[0155] References herein to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least some embodiments of the present disclosure, but not necessarily in all embodiments. To facilitate understanding of this disclosure, a number of terms and phrases are defined below.

[0060]

[0156] A "major histocompatibility complex" or "MHC" may, in some embodiments, be a cluster of genes involved in regulating cellular interactions that result in a physiological immune response. In humans, the MHC complex is also known as the human leukocyte antigen (HLA) complex. For a detailed description of MHC and HLA complexes, see Paul, Fundamental Immunology, 3rd ed., Raven Press, New York (1993). A "major histocompatibility complex (MHC) protein or molecule," "MHC molecule," "MHC protein," or "HLA protein" may be taken to mean a protein that results from proteolytic cleavage of a protein antigen and is capable of binding peptides corresponding to potential lymphocyte epitopes (e.g., T cell epitopes and B cell epitopes) that transport them to the cell surface, where they are presented to specific cells, particularly cytotoxic T lymphocytes, helper T cells, or B cells. The major histocompatibility complex in the genome contains genetic regions whose gene products, which can be expressed on the cell surface, are important for binding and presentation of endogenous and / or foreign antigens and, therefore, for regulating immunological processes. The major histocompatibility complex can be divided into two groups of genes that encode different proteins: MHC class I molecules and MHC class II molecules. The cell biology and expression patterns of the two MHC classes are thought to be adapted to these different roles.

[0061]

[0157] "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)).

[0062]

[0158] As used herein, "polypeptide," "peptide," and their grammatical equivalents can refer to a polymer of amino acid residues in some embodiments. A "mature protein" can be a full-length protein, optionally including glycosylation or other modifications typical of that protein in a given cellular environment. The polypeptides and proteins disclosed herein (including functional portions and functional variants thereof) can contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids may be known in the art and 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-phenylalanine. The polypeptides may include β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indole-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 further contemplates that expression of the polypeptides described herein in engineered cells may be associated with post-translational modification of one or more amino acids of the polypeptide construct.Non-limiting examples of post-translational modifications include phosphorylation; acylation, including acetylation and formylation; glycosylation (including N-linked and O-linked); amidation; hydroxylation; alkylation, including methylation and ethylation; ubiquitination; addition of pyrrolidine carboxylic acid; formation of disulfide bridges; sulfation; myristoylation; palmitoylation; isoprenylation; farnesylation; geranylation; GIPylation; lipoylation; and iodination. The term "polypeptide" or "peptide" can also refer to a polypeptide separated from components naturally associated with the polypeptide. Typically, a polypeptide can be isolated, in which case the polypeptide is at least 60% by weight free from the proteins and naturally occurring organic molecules with which it is naturally associated. In some embodiments, the preparation can be at least 75%, at least 90%, or at least 99% by weight polypeptide. An isolated polypeptide can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, eg, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0063]

[0159] In some embodiments, an "immunogenic" peptide or "immunogenic" epitope or "peptide epitope" can be a peptide that contains an allele-specific motif, such that the peptide binds to an HLA molecule and elicits a cell-mediated or humoral response, such as the activation of cytotoxic T lymphocytes (CTLs (e.g., CD8 + )), helper T lymphocytes (Th (e.g., CD4 + )) and / or B lymphocyte responses. Thus, the immunogenic peptides described herein may be capable of binding to the appropriate HLA molecule and subsequently inducing a CTL (cytotoxic) response, or an HTL (and humoral) response against the peptide.

[0064]

[0160] In some embodiments, the term "neoantigen" or "neoantigenicity" can refer to a class of tumor antigens that arise from tumor-specific mutations that alter the amino acid sequence of a genomically encoded protein. Neoantigens include, but are not limited to, tumor antigens that arise from, for example, protein sequence substitutions, frameshift mutations, fusion polypeptides, in-frame deletions, insertions, expression of endogenous retroviral polypeptides, and tumor-specific overexpression of polypeptides.

[0065]

[0161] In some embodiments, the terms "neo-antigenic peptide" and "neo-antigenic peptide," which may be used interchangeably herein with "peptide," can refer to a series of residues, typically L-amino acids, connected to one another by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. Similarly, in some embodiments, the term "polypeptide," which may be used interchangeably herein with "variant polypeptide," "neo-antigenic polypeptide," and "neo-antigenic polypeptide," can refer to a series of residues, typically L-amino acids, connected to one another by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. In some embodiments, polypeptides or peptides can be of various lengths in their neutral (uncharged) form, or alternatively as salts, either free of or containing modifications such as glycosylation, side chain oxidation, or phosphorylation, provided that the modifications do not destroy the biological activity of the polypeptides described herein. In some embodiments, a peptide or polypeptide, as used herein, can include at least one flanking sequence. In some embodiments, the term "flanking sequence," as used herein, can refer to a fragment or region of a neo-antigenic peptide that is not part of a neoepitope. In some embodiments, the term "residue" can refer to an amino acid residue or amino acid mimetic residue incorporated into a peptide or protein by an amide bond or amide bond mimetic, or a nucleic acid (DNA or RNA) that can encode an amino acid or amino acid mimetic.

[0066]

[0162] In some embodiments, "neoplasia" can refer to any disease caused by or resulting in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. For example, cancer is an example of a neoplasia. Examples of cancer include leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's hypergammaglobulinemia, heavy chain disease, and solid tumors, such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovium, mesothelioma, These include, but are not limited to, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, 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 to be proliferative diseases.

[0067]

[0163] In some embodiments, the term "neoplasm vaccine" can refer to a pooled sample of neoplasm / 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" is understood to mean a composition for generating immunity for the prevention and / or treatment of disease (e.g., neoplasm / tumor). Thus, a vaccine can be a drug containing an antigen and intended for use in humans or animals to generate specific defense and protection by vaccination. In some embodiments, a "vaccine composition" or "neoplasm vaccine composition" can include a pharmaceutically acceptable excipient, carrier, or diluent.

[0068]

[0164] In some embodiments, immune checkpoints can affect inhibitory pathways that slow or halt immune responses, preventing excessive tissue damage due to uncontrolled immune cell activity. A "checkpoint inhibitor" can refer to any small molecule chemical, antibody, nucleic acid molecule, or polypeptide, or fragment thereof, that can inhibit an inhibitory pathway, thereby allowing broader immune activity. In certain embodiments, the checkpoint inhibitor is an inhibitor of the programmed death-1 (PD-1) pathway, such as an anti-PD1 antibody, including, but not limited to, nivolumab. In other embodiments, the checkpoint inhibitor is an anti-cytotoxic T-lymphocyte-associated antigen (CTLA-4) antibody. In additional embodiments, the checkpoint inhibitor targets another member of the CD28 CTLA4 (g superfamily, e.g., BTLA, LAG3, ICOS, PDL1, or KIR; Page et al., Annual Review of Medicine 65:27 (2014)). In some cases, targeting of the checkpoint inhibitor is accomplished by an inhibitory antibody or similar molecule. In other cases, it is accomplished by agonists of the target.

[0069]

[0165] In yet further 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 by an inhibitory antibody or similar molecule. In other cases, it can be accomplished by an agonist of the target, examples of this class include the stimulatory targets CD40, OX40, and GITR.

[0070]

[0166] The term "combination," in some embodiments, can encompass the administration of a vaccine or vaccine composition (e.g., a pooled sample of neoplasm / tumor-specific neoantigens) and one or more inhibitors, e.g., checkpoint inhibitors or chemotherapeutic agents, as part of a treatment regimen intended to provide a beneficial (additive or synergistic) effect from the synergistic action of one or more of these therapeutic agents. The combination can also include one or more additional agents, such as, but not limited to, chemotherapeutic agents, antiangiogenic agents, and agents that reduce immunosuppression. The beneficial effect of the combination includes, but is not limited to, pharmacokinetic or pharmacodynamic synergy resulting from the combination of therapeutic agents. The administration of these therapeutic agents in combination can occur over a defined period of time (e.g., minutes, hours, days, or weeks, depending on the combination selected).

[0071]

[0167] "Combination therapy" may be intended to encompass the administration of these therapeutic agents in a sequential manner, i.e., where each therapeutic agent can be administered at different times, as well as the administration of these therapeutic agents, or at least two of the therapeutic agents, in a simultaneous manner. Substantially simultaneous administration can be accomplished, for example, by administering to a subject a single capsule having a fixed ratio of each therapeutic agent, or by administering a single capsule for each of the therapeutic agents multiple times. For example, a combination of the present disclosure can include a pooled sample of tumor-specific neoantigens and an inhibitor, e.g., a checkpoint inhibitor or a chemotherapeutic agent, administered at the same or different times, or the composition can be formulated as a single co-formulated pharmaceutical composition containing the two compounds. As another example, a combination of the present disclosure (e.g., a pooled sample of tumor-specific neoantigens and an inhibitor, e.g., a checkpoint inhibitor (e.g., an anti-PD-L1 antibody), and / or a chemotherapeutic agent) can be formulated as separate pharmaceutical compositions that can be administered at the same or different times. As used herein, the term "concurrently" can refer to the administration of one or more agents at the same time. For example, in certain embodiments, the vaccine or immunogenic composition and the inhibitor, e.g., a checkpoint inhibitor or chemotherapeutic agent, are administered simultaneously. Simultaneous includes administration contemporaneously, i.e., during the same period of time. In certain embodiments, one or more agents can be administered simultaneously at the same time or on the same day. Sequential or substantially simultaneous administration of each therapeutic agent can be accomplished by any suitable route, including, but not limited to, oral, intravenous, subcutaneous, intramuscular, direct absorption through mucosal tissues (e.g., nasal, oral, vaginal, and rectal), and ocular (e.g., intravitreal, intraocular, etc.). The therapeutic agents can be administered by the same route or by different routes. For example, one component of a particular combination can be administered by intravenous injection, while other components of the combination can be administered orally. The components can be administered in any therapeutically effective amount. The term "combination" encompasses a group of compounds or non-drug therapies useful as part of a combined therapy.

[0072]

[0168] The term "pharmaceutically acceptable" refers to approved or approvable by a regulatory agency of a federal or state government, or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia, for use in animals, including humans. Examples of cancer include leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphomas (e.g., Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's hypergammaglobulinemia, heavy chain disease, and solid tumors, such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovium, mesothelioma, euthyroidism, thyroid cancer ... Cancers that may be present include, but are not limited to, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, 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 to be proliferative diseases.

[0073]

[0169] A "pharmaceutically acceptable excipient, carrier, or diluent" can refer to an excipient, carrier, or diluent that can be administered to a subject together with a drug, which does not destroy the pharmacological activity of the drug when administered in an amount sufficient to deliver a therapeutic amount of the drug, and which is non-toxic.

[0074]

[0170] A "pharmaceutically acceptable salt" of a pooled tumor-specific neo-antigen, as referred to herein, can in some embodiments be an acid or base salt generally regarded in the art as suitable for use in contact with human or animal tissues without undue toxicity, irritation, allergic response, or other problems or complications. Such salts include inorganic and organic acid salts of basic residues such as amines, and alkali or organic salts of acidic residues such as carboxylic acids. Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, sulfanilic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethylsulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, pamoic acid, succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodic acid, phenylacetic acid, alkanoic acids such as acetic acid, HOOC-(CH)-COOH (where n=0=4), and the like. Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those skilled in the art will recognize, from this disclosure and knowledge in the art, additional pharmaceutically acceptable salts of the pooled tumor-specific neoantigens provided herein, including those listed in 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 the parent compound containing a basic or acidic moiety by conventional chemical methods. Briefly, such salts can be prepared by reacting the free acid or base form of these compounds with a stoichiometric amount of the appropriate base or acid in a suitable solvent.

[0075]

[0171] As used herein, in some embodiments, the terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like can refer to reducing the chance of developing a disease or condition in a subject who does not have the disease or condition but is at risk of or susceptible to developing it.

[0076]

[0172] The term "prime / boost" or "prime / boost administration regimen" can refer to sequential administration of a vaccine or immunogenic or immunological composition. A priming administration (priming) can be the administration of a first vaccine or immunogenic or immunological composition type and can include one, two, or more administrations. A boosting administration can be the second administration of a vaccine or immunogenic or immunological composition type and can include one, two, or more administrations, for example, can include or consist essentially of annual administrations. In certain embodiments, the administration of a neoplastic vaccine or immunogenic composition can be in a prime / boost administration regimen.

[0077]

[0173] Ranges provided herein may be understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50, as well as all intervening decimal values between the aforementioned integers, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. With respect to subranges, "nested subranges" extending from both endpoints of the range are specifically contemplated. For example, nested subranges of the exemplary range of 1 to 50 could 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 opposite direction.

[0078]

[0174] "Receptor" may be understood to mean a biological molecule or molecular configuration capable of binding to a ligand. Receptors may serve to transmit information about cells, cell formation, or organisms. Receptors contain at least one receptor unit, often two or more receptor units, and each receptor unit may consist of a protein molecule, particularly a glycoprotein molecule. Receptors may have a structure complementary to that of a ligand and may complex with the ligand as a binding partner. Signaling information may be transmitted by a conformational change of the receptor after binding to the ligand on the surface of a cell. According to the present disclosure, receptors may refer to specific proteins of MHC class I and II that can form receptor / ligand complexes with ligands, particularly peptides or peptide fragments of a suitable length.

[0079]

[0175] The term "subject" refers to an animal that may be the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, which includes, but is not limited to, a human or non-human primate, such as a human primate, a bovine, an equine, a canine, an ovine, or a feline.

[0080]

[0176] The terms "treat," "treated," "treating," "treatment," and the like may be intended to refer to alleviating or ameliorating a disorder (e.g., a neoplasm or tumor) and / or its associated symptoms. "Treating" may refer to the administration of a combination therapy to a subject after the onset of, or suspected of the onset of, cancer. "Treating" may include the concept of "alleviating," which refers to reducing the frequency of occurrence or recurrence, or the severity, of any symptoms or other pathological effects related to cancer and / or side effects associated with cancer therapy. The term "treating" may also encompass the concept of "managing," which refers to reducing the severity of a particular disease or disorder in a patient or delaying its recurrence, e.g., extending the period of remission in a patient afflicted with a disease. It may be understood that treating a disorder or condition does not require, but does not exclude, the elimination of the disorder, condition, or its associated symptoms.

[0081]

[0177] The term "therapeutic effect" can refer to some degree of alleviation of one or more symptoms of a disorder (e.g., a neoplasm or tumor) or its related pathology. A "therapeutically effective amount," as used herein, can refer to an amount of an agent that, upon single or multiple dose administration to a cell or subject, is effective in prolonging survival of a patient with such a disorder, reducing, preventing, or delaying one or more signs or symptoms of the disorder beyond that expected in the absence of such treatment. A therapeutically effective amount may be intended to limit the amount necessary to achieve a therapeutic effect. A physician or veterinarian of ordinary skill in the art can readily determine and prescribe the required "therapeutically effective amount" (e.g., ED50) of the pharmaceutical composition. For example, a physician or veterinarian could start doses of the disclosed compounds used in the pharmaceutical composition at levels lower than those required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved.

[0082]

[0178] "Adverse reaction" or AE can generally refer to any untoward medical occurrence in a patient administered a pharmaceutical product that may not necessarily be causally related to treatment. An AE can be any untoward, unintended sign (including, for example, abnormal laboratory findings), symptom, or disease that is temporally related to the use of an investigational drug, whether or not it is considered to be study treatment-related. This includes any new occurrence or pre-existing condition that has increased in severity or frequency since administration of the study treatment. Abnormal laboratory values or test results can constitute AEs only if they induce clinical signs or symptoms, are considered clinically significant, or require treatment.

[0083]

[0179] A progression of the cancer under investigation is not considered an AE unless it is deemed drug-related by the patient care team. An "adverse drug reaction (ADR)" may be defined as any non-toxic, unintended response to a drug, at any dose. A causal relationship between the drug and the AE is at least reasonably possible—i.e., the relationship cannot be excluded. An expected AE may be one that is listed or characterized in the applicable product information, e.g., the current IB. An unexpected AE may not be identified by the nature, severity, or frequency described in the applicable product information, e.g., the current IB. An unexpected ADR is an ADR whose nature or severity is inconsistent with the applicable product information. An ADR that is more specific or severe than those described in the IB may also be considered an unexpected ADR. A "serious adverse event" or SAE is a fatal, life-threatening event (life-threatening means that the patient is at imminent risk of dying from the reaction if it occurs; i.e., it does not include reactions that, hypothetically, might have caused death if they had occurred in a more severe form) that requires in-patient hospitalization or an extension of an existing hospitalization (hospitalizations that are scheduled to occur during the study period but that were planned prior to study enrollment). An illness or disease, including but not limited to: a medical condition (such as a breast cancer, breast cancer, or breast cancer), which may be present before the patient enrolled in the study, may be a birth defect / anomaly that results in persistent or significant disability / incapacity (disability may be defined as a significant disruption of a person's ability to perform normal life functions), or is a significant medical event, which is defined as an event that does not result in death or is life-threatening or requires hospitalization. An AE may occur at any dose, regardless of causation, but may be considered an SAE if, based on sound medical judgment, it may endanger the patient or patients and may require medical or surgical intervention to prevent one of the outcomes listed in the definition of an SAE.Such medical events include allergic bronchospasm requiring intensive care in an emergency room or at home, blood disorders or seizures that do not result in patient hospitalization, or the development of drug addiction or drug abuse.

[0084]

[0180] Each patient will be closely monitored for the development of any AEs from the time the consent form is signed until 30 days after treatment discontinuation. This information can come from signs and symptoms detected during each examination, investigator observations, and spontaneous patient reports in the form of non-leading questions (e.g., "How are you feeling?"). All AEs (serious and non-serious) spontaneously reported by the patient and / or investigator in response to open-ended questions, or revealed by observations, physician examinations, or other diagnostic procedures, can be recorded on the appropriate page of the eCRF. When possible, signs and symptoms indicative of a common underlying condition can be described as a single umbrella event. The investigator must report all SAEs occurring between the time the ICF is signed and 90 days after the last dose of nivolumab, or 30 days after the last dose of nivolumab if the patient has started a new anticancer treatment, to the study sponsor (see below) within one working day of the investigator's awareness of the SAE. If the sponsor is unavailable for any reason, an alternate physician may be contacted. All SAEs may be reported, whether or not they are considered causally related to the study treatment. Information collected on the SAE form may include patient number, a textual description of the event, and the investigator's assessment of the event's severity and relationship to study treatment. Samples of follow-up information about SAEs may be requested by the sponsor or CRO.

[0085]

[0181] The present disclosure relates to methods for the treatment of neoplasms, more particularly tumors, by administering to a subject a neoplasm vaccine or immunogenic composition comprising multiple neoplasm / tumor-specific neoantigens and at least one inhibitor, e.g., a checkpoint inhibitor or a chemotherapeutic agent.

[0086]

[0182] Human tumors can contain numerous unique deoxyribonucleic acid (DNA) mutations that result in alterations in the amino acid sequences of encoded proteins. These novel protein sequences, often known as neoantigens, range from single amino acid changes (caused by missense mutations) to the addition of long stretches of novel amino acid sequences due to frameshifts, stop codon readthrough, or translation of intronic regions (novel open reading frames [neoORFs]). Tumor neoantigens arise primarily due to mutations in tumors. Therefore, they are highly tumor-specific and not subject to the immune-compromising effects of self-tolerance.

[0087]

[0183] Immune responses to neoantigens can depend largely on the ability of major histocompatibility complex (MHC) molecules to effectively bind and present small peptides (epitopes) containing altered amino acid sequences to T cells. Such epitopes can be synthetically produced and used in vaccines to mount antigen-specific T cell responses that target tumor cells expressing the mutant protein.

[0088]

[0184] Peptide binding to MHC can be used as a surrogate for the immunogenicity of a given peptide sequence. Advanced algorithms that predict peptide binding to MHC have been built using binding data from a large number of peptides to different MHC molecules (Lundegaard, 2011). These algorithms can be used to predict with high accuracy whether a particular peptide sequence will bind to an MHC molecule with what affinity. These algorithms can be used to evaluate protein sequences containing tumor-encoded mutations (both missense and neoORF) in silico for binding to specific MHC molecules.

[0089]

[0185] In some embodiments, the subject may have a mutant epitope that contains an altered amino acid sequence, for example, when the subject has cancer. In one embodiment, the mutant epitope is determined by sequencing the genome and / or exome of tumor tissue and healthy tissue from cancer patients using next-generation sequencing technology. In another embodiment, the gene selected based on the frequency of mutation and its ability to act as a neoantigen is sequenced using next-generation sequencing technology. Next-generation sequencing is applied to genome sequencing, genome resequencing, transcriptome profiling (RNA-Seq), DNA-protein interactions (ChiP sequencing), and epigenome 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 Thing in Genetic Medicine", The American Journal of Human Genetics 85(2):142-154).

[0090]

[0186] Next-generation sequencing now rapidly reveals the presence of distinct mutations, such as coding mutations in individual tumors; most commonly single amino acid changes (e.g., missense mutations) and, less commonly, novel stretches of amino acids resulting from frameshift insertions, deletions, or gene fusions; stop codon readthrough mutations; and improperly spliced introns (e.g., neoORFs). NeoORFs are particularly useful as immunogens because their entire sequences resemble viral or bacterial foreign antigens. Therefore, neoORFs are (1) highly tumor-specific (i.e., not expressed in any normal cells); and (2) can increase the precursor frequency of neoantigen-specific CTLs by bypassing central tolerance. For example, the power of utilizing similar foreign sequences in therapeutic anticancer vaccines or immunogenic compositions was recently demonstrated using peptides derived from human papillomavirus (HPV). Of 19 patients with preneoplastic, virus-induced disease who received three to four vaccinations with a mix of HPV peptides derived from the viral oncogenes E6 and E7, 50% maintained a complete response for >24 months (Kenter et al., Vaccination against HPV-16 Oncoproteins for Vulvar Intraepithelial Neoplasia, NEJM 361:1838 (2009)).

[0091]

[0187] Sequencing technologies have revealed that each tumor contains multiple, patient-specific mutations that alter the protein-coding content of genes. Such mutations result in protein modifications ranging from single amino acid changes (caused by missense mutations) to the addition of long stretches of novel amino acid sequence due to frameshifts, stop codon readthrough, or translation of intronic regions (novel open reading frame mutations; neoORFs). These mutant proteins are useful targets for the host's immune response against tumors because, unlike native proteins, they are not subject to the immune-compromising effects of self-tolerance. Therefore, mutant proteins are more likely to be immunogenic and also more specific to tumor cells compared with the patient's normal cells.

[0092]

[0188] 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 the neoantigens of the present disclosure. Such proteomic approaches allow for rapid, highly automated analysis (see, for example, 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, for example, Gutaliás et al. (2012) Shotgun Protein Sequencing with Meta-contig Assembly, Molecular and Cellular Proteomics 11(30):3084-96).

[0093]

[0189] MHC multimers can also be used to identify tumor-specific neoantigens to identify neoantigen-specific T cell responses. For example, high-throughput analysis of neoantigen-specific T cell responses in patient samples can be performed using MHC tetramer-based screening approaches (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 approaches can be used to initially identify tumor-specific neoantigens, or alternatively, to assess what antigens a patient may have already been exposed to, thereby facilitating the selection of candidate neoantigens of the present disclosure.

[0094]

[0190] In one embodiment, sequencing data obtained from determining the presence of mutations in cancer patients is analyzed to predict personalized variant peptides that can bind to the individual's HLA molecules. In one embodiment, the data is analyzed using a computer. 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. Efficient selection of specific variants to utilize as immunogens requires identification of the patient's HLA type and the ability to predict which variant peptides will efficiently bind to the patient's HLA alleles. Recently, neural network-based learning approaches involving validation of binding and non-binding peptides have improved the accuracy of prediction algorithms for the major HLA-A and HLA-B alleles. Recently improved algorithms for predicting which missense mutations will result in strongly binding peptides to a patient's cognate MHC molecules can be used to identify and prioritize a set of peptides representing the optimal mutant epitopes (both neoORF and missense) for each patient (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)).

[0095]

[0191] Targeting as many mutant epitopes as practically possible harnesses the immune system's enormous capabilities, thwarts opportunities for immune escape through downregulation of specific immune-targeted gene products, and compensates for the known inaccuracies of epitope prediction approaches. Synthetic peptides provide a particularly useful means for efficiently preparing multiple immunogens and rapidly translating the identification of mutant epitopes into effective vaccines or immunogenic compositions. Peptides can be easily chemically synthesized and purified using reagents free of bacterial or animal contaminants. Their small size allows for a clear focus on the mutant region of a protein and also reduces inappropriate antigen competition from other components (non-mutated proteins or viral vector antigens).

[0096]

[0192] In one embodiment, the drug formulation is a long peptide multi-epitope vaccine or immunogenic composition. Such "long" peptides have been shown to be efficiently internalized, processed, and cross-presented by professional antigen-presenting cells, such as dendritic cells, and to induce CTLs in humans (Melief and van der Burg, Immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines Nature Rev Cancer 8:351 (2008)). In 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 neo-antigenic peptide ranges from about 5 to about 50 amino acids in length. In another embodiment, peptides from about 15 to about 35 amino acids in length are synthesized. In some embodiments, the neo-antigenic peptide ranges from about 20 to about 35 amino acids in length.

[0097]

[0193] In some embodiments, personalized cancer vaccines are provided that consist of up to 20 synthetic peptides, approximately 14-35 amino acids in length, derived from an individual patient's mutant tumor DNA (neoantigens). Because these mutations are not expressed in the patient's normal cells, they are specific targets expressed only on tumor cells.

[0098]

[0194] Unlike most previously used cancer vaccines, this neo-antigen peptide vaccine is based on the production of a novel and specific product for each individual patient or cancer phenotype. Due to the degree of possible tumor mutations and the wide range of patient human leukocyte antigen (HLA) haplotypes, the chances that any two patients will receive the same vaccine are extremely low.

[0099]

[0195] The generation of neoantigens can begin with whole-exome DNA and ribonucleic acid (RNA) sequencing of tumor and normal tissue samples from a subject, as well as HLA-A, HLA-B, and HLA-C genotypes. These data can then be used to identify coding sequence mutations that have occurred in the subject's tumor. These mutations may, in some cases, include single amino acid missense mutations, fusion proteins, and neoORFs, which can vary in length from one amino acid to several hundred amino acids. Long peptides, 14 to 35 residues in length, can then be specifically designed from the specific mutations identified in the individual's tumor. Vaccines can then be composed of a mixture of peptides predicted to induce responses in CD4+ and / or CD8+ T cells. To predict which are most likely to induce such an immune response, multiple filters can be applied to the entire set of long peptides covering the subject's tumor mutagenesis. The primary 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 predicted to bind to MHC class I molecules in each subject (Lundegaard, 2011). Other key criteria include RNA expression, the type of mutation (e.g., missense neoORF), the likelihood that the mutation is an oncogenic driver, and the physical location of the mutant residue on the peptide. Up to 35 peptides can be selected for synthesis and prioritized. Up to 20 synthesized peptides can then be combined into up to four pools of up to five peptides per injection. Each of the four pools can be injected into the subject. II. Production of tumor-specific neoantigens

[0196] The present disclosure is based, at least in part, on the ability to present a patient's immune system with a pool of tumor-specific neoantigens. From this disclosure and (ad) knowledge in the art, those skilled in the art will understand that there are various methods for producing such tumor-specific neoantigens. Generally, such tumor-specific neoantigens can be produced either in vitro or in vivo. Tumor-specific neoantigens can be produced in vitro as peptides or polypeptides, which can then be formulated into personalized neoplasia vaccines or immunogenic compositions and administered to a subject. As described in further detail herein, such in vitro production can be achieved by various methods known to those skilled in the art, such as peptide synthesis from DNA or RNA molecules or expression of peptides / polypeptides 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 a molecule (e.g., DNA, RNA, viral expression system, etc.) encoding the tumor-specific neoantigen into a subject, resulting in expression of the encoded tumor-specific neoantigen. Methods for in vitro and (ad) in vivo production of neoantigens are also further described herein as they relate to pharmaceutical compositions and methods of deliver of combination therapies. A. In vitro peptide / polypeptide synthesis

[0197] Proteins or peptides can be produced by any method known to those skilled in the art, including expressing proteins, polypeptides, or peptides using standard molecular biology techniques, isolating proteins or peptides from natural sources, in vitro translation, or chemically synthesizing proteins or peptides. 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 skilled in the art. Exemplary databases can be found in the National Center for Biotechnology Information, Genbank, and GenPept databases at the National Institutes of Health website. The coding regions of known genes can be amplified and / or expressed using the methods disclosed herein or known to those skilled in the art. Alternatively, various commercially available preparations of proteins, polypeptides, and peptides are known to those skilled in the art.

[0100]

[0198] Peptides can be readily chemically synthesized using reagents free of bacterial or animal contamination (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. 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 homogeneous synthesis and cleavage conditions; (2) P-HPLC column purification and column stripping; and re-cleaning, rather than replacement, between peptides; followed by (3) analysis with a limited set of the most informative assays. Because a Good Manufacturing Practice (GMP) footprint can be defined for the set of peptides for an individual patient, only suite change procedures are required between the synthesis of peptides for different patients.

[0101]

[0199] Alternatively, a nucleic acid (e.g., polynucleotide) encoding the neo-antigenic peptide of the present disclosure can be used to produce the neo-antigenic peptide. The polynucleotide can be, for example, single-stranded and / or double-stranded DNA, cDNA, PNA, CNA, RNA, or a native or stabilized form of a polynucleotide, such as a polynucleotide with a phosphorothioate backbone, or a combination thereof, which may or may not contain introns, provided that it encodes the peptide. In one embodiment, in vitro translation is used to produce the peptide. Many exemplary systems are available to those skilled in the art (e.g., Retic Lysate IVT Kit, Life Technologies, Waltham, MA).

[0102]

[0200] Expression vectors capable of expressing polypeptides can also be prepared.Expression vectors for different cell types are well known in the art, and they can be selected without undue experimentation.Generally, DNA is inserted into an expression vector such as a plasmid for expression in appropriate direction and correct reading frame, and if necessary, DNA can be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host (e.g., bacteria), and such control elements are generally available in the expression vector.The vector is then introduced into host cells for cloning using standard methods (see, for example, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0103]

[0201] Expression vectors comprising the isolated polynucleotides and host cells containing the expression vectors are also contemplated. The neo-antigenic peptides may be provided in the form of RNA or cDNA molecules encoding the desired neo-antigenic peptide. One or more neo-antigenic peptides of the present disclosure may be encoded by a single expression vector.

[0104]

[0202] The term "polynucleotide encoding a polypeptide" encompasses polynucleotides that contain only the coding sequence for a polypeptide, as well as polynucleotides that contain additional coding and / or non-coding sequences. The polynucleotide may be in the form of RNA or in the form of DNA. The DNA includes cDNA, genomic DNA, and synthetic DNA, and may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding (antisense) strand.

[0105]

[0203] In embodiments, the polynucleotide can include, in the same reading frame, a coding sequence for a tumor-specific neo-antigenic peptide fused to the polynucleotide, e.g., useful for expression and / or secretion of the polypeptide from the host (e.g., a leader sequence that functions as a secretory sequence to control transport of the polypeptide out of the cell). Polypeptides with leader sequences can be preproteins, with the leader sequence being cleaved by the host cell to form the mature form of the polypeptide.

[0106]

[0204] In embodiments, the polynucleotide can include, in the same reading frame, a coding sequence for a tumor-specific neo-antigenic peptide fused to a marker sequence, e.g., that allows for purification of the encoded polypeptide, thus allowing the polynucleotide to be incorporated into a personalized neoplasia vaccine or immunogenic composition. For example, the marker sequence can be a hexahistidine tag provided by the pQE-9 vector, which provides for purification of the polypeptide fused to the marker in the case of a bacterial host, or the marker sequence can be a hemagglutinin (HA) tag, derived from the influenza hemagglutinin protein, when a mammalian host (e.g., COS-7 cells) is used. Additional tags include, but are not limited to, calmodulin tag, FLAG tag, Myc tag, S tag, SBP tag, Softag 1, Softag 3, V5 tag, Xpress tag, Isopeptag, SpyTag, biotin carboxyl carrier protein (BCCP) tag, GST tag, fluorescent protein tag (e.g., green fluorescent protein tag), maltose binding protein tag, Nus tag, Strep tag, thioredoxin tag, TC tag, Ty tag, and the like.

[0205] In embodiments, a polynucleotide may contain coding sequences for one or more tumor-specific neo-antigenic peptides fused in the same reading frame to create a single concatameric neo-antigenic peptide construct capable of producing multiple neo-antigenic peptides.

[0107]

[0206] In certain embodiments, isolated nucleic acid molecules may be provided having a nucleotide sequence 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 neo-antigenic peptide of the present disclosure.

[0108]

[0207] A polynucleotide sequence having a nucleotide sequence at least, for example, 95%, "identical" to a reference nucleotide sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence, except that the polynucleotide sequence may contain no more than 5 point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference polynucleotide sequence, no more than 5% of the nucleotides in the reference sequence may be deleted or replaced with other nucleotides, or a number of nucleotides equal to or less than 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may be present at the amino- or carboxy-terminal positions of the reference nucleotide sequence, or may be present somewhere between these terminal positions, interspersed between nucleotides in the reference sequence, either individually or in one or more consecutive groups within the reference sequence.

[0109]

[0208] In practice, whether any particular nucleic acid molecule is at least 80% identical, at least 85% identical, at least 90% identical, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identical to a reference sequence can be conventionally 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 most homologous segment between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present disclosure, parameters are set so that the percentage of identity is calculated over the entire length of the reference nucleotide sequence and so that homology gaps of no more than 5% of the total number of nucleotides in the reference sequence are allowed.

[0110]

[0209] The isolated tumor-specific neo-antigenic 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 to constructing DNA sequences encoding the isolated polypeptide sequences and expressing those sequences in a suitable transformed host. In some embodiments, the DNA sequences are constructed by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest using recombinant techniques. If necessary, the sequence can be mutated by site-directed mutagenesis to obtain functional analogs thereof. See, for example, Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

[0111]

[0210] In embodiments, a DNA sequence encoding a polypeptide of interest is constructed by chemical synthesis using an oligonucleotide synthesizer. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and by selecting the codons preferred by the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize an isolated polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a reverse-translated gene. Furthermore, a DNA oligomer containing a nucleotide sequence encoding a specific isolated polypeptide can be synthesized. For example, several small oligonucleotides encoding portions of the desired polypeptide can be synthesized and then ligated. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary assembly.

[0112]

[0211] Once assembled (for example, by synthesis, site-directed mutagenesis, or another method), the polynucleotide sequence encoding the specific isolated polypeptide of interest is inserted into a vector, and, if necessary, is operably linked to an expression control sequence suitable for protein expression in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction mapping, and the expression of a biologically active polypeptide in a suitable host. As is well known in the art, to obtain high expression levels of transfected genes in a host, the gene can be operably linked to transcriptional and translational expression control sequences that are functional in the selected expression host.

[0113]

[0212] Recombinant expression vectors can be used to amplify and express DNA encoding tumor-specific neo-antigenic peptides. A recombinant expression vector is a replicable DNA construct containing a synthetic or cDNA-derived DNA fragment encoding a tumor-specific neo-antigenic peptide or a biologically equivalent analog operably linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. A transcription unit, as described in detail herein, generally comprises an assembly of (1) a genetic element or elements that play a regulatory role in gene expression, such as a transcriptional promoter or enhancer; (2) a structural or coding sequence that is transcribed into mRNA and translated into protein; and (3) appropriate transcriptional and translational initiation and termination sequences. Such regulatory elements may include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication and a selection gene to facilitate recognition of transformants, can also be incorporated. DNA regions are operably linked when they are functionally related to each other. For example, 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 the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. Generally, operably linked means contiguous, and in the case of a secretory leader, operably linked means contiguous and in-frame. Structural elements intended for use in yeast expression systems include a leader sequence that enables extracellular secretion of translated protein by a host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, it may contain an N-terminal methionine residue, which can then be cleaved from the expressed recombinant protein, as needed, to provide the final product.

[0114]

[0213] Expression vectors useful for eukaryotic hosts, particularly mammalian or human, include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Expression vectors useful for bacterial hosts include known bacterial plasmids, such as those from Escherichia coli, including pCR1, pBR322, pMB9, and their derivatives; broader host range plasmids, such as M13; and filamentous single-stranded DNA phages.

[0115]

[0214] Suitable host cells for expressing polypeptides include prokaryotes, yeast, insect, or higher eukaryotic cells under the control of an appropriate promoter. Prokaryotes include gram-negative or gram-positive organisms, such as E. coli or bacilli. Higher eukaryotic cells include established cell lines of mammalian origin. Cell-free translation systems may also be utilized. Suitable cloning and expression vectors for use with 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).

[0116]

[0215] Various mammalian or insect cell culture systems can also be advantageously used to express recombinant proteins. Expression of recombinant proteins in mammals is feasible because such proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cells include the COS-7 system of monkey kidney cells described by Gluzman (Cell 23:175, 1981); as well as other cell lines capable of expressing suitable vectors, including L cells, C127, 3T3, Chinese hamster ovary (CHO), 293, HeLa, and BHK cell lines. Mammalian expression vectors may include nontranscribed sequences, such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5'- or 3'-flanking nontranscribed sequences, and 5'- or 3'-untranslated sequences, such as necessary ribosome binding sites, polyadenylation sites, splitter donors and acceptors, and transcription termination sequences. Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio / Technology 6:47 (1988).

[0117]

[0216] Proteins produced by transformed host cells can be purified according to any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Affinity tags, such as hexahistidine, maltose-binding domain, influenza coat protein sequence, glutathione-S-transferase, etc., can be attached to the jl protein to enable easy purification by passage through an appropriate affinity column. Isolated proteins can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0118]

[0217] For example, supernatant from a system secreting recombinant ammonium into the culture medium can be first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. After the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin, such as a matrix or substrate with pendant diethylaminoethyl (DEAE) groups, can be utilized. The matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Alternatively, a cation exchange step can be utilized. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, one or more reverse-phase high-performance liquid chromatography (RP-HPLC) steps utilizing hydrophobic RP-HPLC media, such as silica gel with pendant methyl or other aliphatic groups, can be utilized to further purify the cancer stem cell protein-Fc composition. Some or all of the above purification steps can also be utilized in various combinations to obtain a homogeneous recombinant protein.

[0119]

[0218] Recombinant proteins produced in bacterial culture 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 to express recombinant proteins can be disrupted by any conventional method, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents. B. In vivo peptide / polypeptide synthesis

[0219] The present disclosure also contemplates the use of nucleic acid molecules in vivo, e.g., in the form of DNA / RNA vaccines, as vehicles for delivering neo-antigenic peptides / polypeptides to a subject in need thereof (see, e.g., WO2012 / 159643 and WO2012 / 159754, which are hereby incorporated by reference in their entireties).

[0120]

[0220] In one embodiment, neoantigens can be administered to patients in need thereof by the use of plasmids. These typically consist of a strong viral promoter to drive in vivo transcription and translation of the gene of interest (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 thus increase protein expression (Leitner et al., (1997). The Journal of Immunology 159(12):6112-6119). The plasmid also contains a strong polyadenylation / transcription termination signal, such as the bovine growth hormone or rabbit beta-globulin polyadenylation sequence (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 may also be 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).

[0121]

[0221] Because the plasmid is the "vehicle" in which the immunogen is expressed, optimizing vector design for maximum protein expression is essential (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 eukaryotic pathogenic mRNA. Another consideration is the choice of promoter. Such promoters can be the SV40 promoter or the Rous Sarcoma Virus (Vims) (RSV).

[0122]

[0222] Plasmids can be introduced into animal tissues by a number of different methods. The two most prominent approaches are injection of DNA in saline using a standard hypodermic needle and gene gun delivery; the construction of DNA vaccine plasmids and their subsequent delivery to the host by these two methods is described in Scientific American (Weiner et al., (1999) Scientific American 281(I):34-41). Saline injection is usually performed intramuscularly (IM) or intradermally (ID) in skeletal muscle, and the DNA is delivered to the extracellular space. This can be assisted by electroporation by transiently damaging muscle fibers with myotoxins such as bupivacaine (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42:343-410). The immune response to this delivery method can be affected by many factors, including needle type, needle arrangement, injection rate, injection volume, muscle type, age, sex, and physiological condition of the injected animal (Alarcon et al. (1999). Adv. Parasitol. Advances in Parasitology 42:343-410).

[0123]

[0223] Another commonly used delivery method, gene gun delivery, uses compressed helium as an accelerator 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).

[0124]

[0224] Alternative delivery methods include aerosol injection of naked DNA into mucosal surfaces such as the nasal and pulmonary membranes (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54:129-88), and topical administration of pDNA to the ocular and vaginal mucosa (Lewis et al., (1999) Advances in Virus Research (Academic Press) 54:129-88). Mucosal surface delivery has also been achieved using cationic liposome-DNA preparations, biodegradable microspheres, attenuated Shigella or Listeria vectors for oral administration to the intestinal mucosa, and recombinant adenovirus vectors.

[0125]

[0225] The delivery method determines the dose of DNA required to generate an effective immune response. Saline injections require variable amounts of DNA, ranging from 10 μg to 1 mg, whereas gene gun delivery requires 100-1000 times less DNA than intramuscular saline injections to generate an effective immune response. Generally, 0.2 μg to 20 μg is required, although amounts as low as 16 ng have been reported. These amounts vary by species; for example, mice require roughly 10 times less DNA than primates. Saline injection requires more DNA because the DNA is delivered to the extracellular space of the target tissue (usually muscle) where it must overcome physical barriers (e.g., the basal lamina and abundant connective tissue, to name a few) before it can be taken up by the cell, whereas gene gun delivery shoots the DNA directly into the cell, resulting in less loss (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).

[0126]

[0226] In one embodiment, a neoplasia vaccine or immunogenic composition may comprise separate DNA plasmids encoding one or more neoantigenic peptides / polypeptides, for example, as identified in accordance with the present disclosure. As discussed herein, the exact choice of expression vector may depend on the peptide / polypeptide to be expressed and is well within the skill of one of ordinary skill in the art. The expected persistence of the DNA constructs (e.g., in an episomal, non-replicating, unintegrated form in muscle cells) is expected to provide an extended period of protection.

[0127]

[0227] One or more neo-antigenic peptides of the present disclosure can be encoded and expressed in vivo using a viral-based system (e.g., an adenovirus system, an adeno-associated virus (AAV) vector, a poxvirus, or a lentivirus). In one embodiment, a neoplasia vaccine or immunogenic composition can comprise a viral-based vector, such as an adenovirus, for use in a human patient in need of the neoplasia vaccine or immunogenic composition (see, e.g., Baden et al., First-in-human evaluation of the safety and immunogenicity of a recombinant adenovirus serotype 26 HIV-1 Env vaccine (1PCAVD 001). J Infect Dis. 2013 Jan. 15;207(2):240-7, hereby incorporated 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. Pat. Nos. 6,955,808 and 6,943,019 and U.S. Patent Application Publication No. 20080254008, which are hereby incorporated by reference).

[0128]

[0228] Among the vectors that can be used in practicing the present disclosure, retroviral gene transfer methods allow integration into the cellular host genome, often resulting in long-term expression of the inserted transgene. In some embodiments, the retrovirus is a lentivirus. Therefore, high transduction efficiency has been observed in many different cell types and target tissues. The tropism of retroviruses can be altered by incorporating foreign envelope proteins, thereby expanding the potential target population of target cells. Retroviruses can also be engineered to allow conditional expression of the inserted transgene, such that the retrovirus infects only certain cells. Cell-type-specific promoters can be used to target expression in specific cell types. Lentiviral vectors are retroviral vectors (and therefore both lentiviral and retroviral vectors can be used in practicing the present disclosure). Furthermore, lentiviral vectors can transduce or infect non-dividing cells and typically produce high viral titers. Therefore, the choice of retroviral gene transfer system depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats capable of packaging up to 6-10 kb of foreign sequence. The minimal cis-acting LTRs are sufficient for vector replication and packaging, and are therefore used to integrate the desired nucleic acid into target cells for permanent expression.Widely used retroviral vectors that can be used in the practice of 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. 9 Approximately 10 μl of recombinant lentivirus with a titer of transducing units (TU) / ml was administered via an intrathecal catheter. These types of dosages can be adapted or extrapolated for use with retroviral or lentiviral vectors in this disclosure.

[0129]

[0229] Minimal non-primate lentiviral vectors, such as those based on equine infectious anemia virus (vims) (EIAV), are also useful in practicing the present disclosure (see, e.g., Balagaan, (2006) J Gene Med;8:275-285, published online 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. Accordingly, the present disclosure contemplates viral vectors, including retroviral and lentiviral vectors, among other vectors useful in practicing the present disclosure.

[0130]

[0230] Adenovirus vectors are also useful in implementing the present disclosure. One advantage is the ability of recombinant adenovirus to efficiently introduce and express recombinant genes in various mammalian cells and tissues in vitro and in vivo, thereby resulting in high expression of transferred nucleic acids. Furthermore, the ability to productively infect quiescent cells expands the usefulness of recombinant adenovirus vectors. In addition, high expression levels ensure that the nucleic acid product is expressed at a level sufficient to generate an immune response (see, for example, U.S. Patent No. 7,029,848, which is hereby incorporated by reference).

[0131]

[0231] In certain embodiments herein, the delivery is by adenovirus, which is at least 1×10 5 The adenoviral vector may be delivered in a single booster dose containing particles (also called particle units, pu). In certain embodiments herein, the dose is at least about 1 x 10 6 particles (e.g., about 1 × 10 6 ~1×10 2 particles), at least about 1 × 10 7 particles, at least about 1 x 10 8 particles (e.g., about 1 × 10 8 ~1×10 11 particles or approximately 1 x 10 8 ~1×10 12 particles), or at least about 1 × 10 9 particles (e.g., about 1 × 10 9 ~1×10 10 particles or approximately 1 x 10 9 ~1×10 12 particles), or even at least about 1 × 10 10 particles (e.g., about 1 × 10 10 ~1×10 12 Alternatively, the dose may be up to about 1 x 10 14 Grains, maximum approximately 1×10 13 Particles, up to about 1×10 12 Particles, up to about 1×10 11 particles, or up to about 1 × 1010 particles (e.g., up to about 1 × 10 9 Thus, the dose may be, for example, about 1 x 10 6 Particle unit (pu), approximately 2 x 10 6 pu, approx. 4×10 6 pu, about 1×10 7 pu, approx. 2×10 7 pu, approx. 4×10 7 pu, about 1×10 8 pu, approx. 2×10 8 pu, approx. 4×10 8 pu, about 1×10 9 pu, approx. 2×10 9 pu, approx. 4×10 9 pu, about 1×10 10 pu, approx. 2×10 10 pu, approx. 4×10 10 pu, about 1×10 11 pu, approx. 2×10 11 pu, approx. 4×10 11 pu, about 1×10 12 pu, about 2×10 12 pu, or approximately 4 × 10 12 The adenovirus vector may contain a single dose of an adenovirus vector, with the adenovirus vector having pu. See, for example, the adenovirus vector in U.S. Patent No. 8,454,972 (B2) to Nabel et al., issued June 4, 2013, and the dosage amounts at column 29, lines 36-58, which are incorporated herein by reference. In some embodiments herein, the adenovirus is delivered in multiple doses.

[0132]

[0232] For in vivo delivery, AAV has advantages over other viral vectors due to its low toxicity and the low likelihood of causing insertional mutagenesis because it does not integrate into the host genome. AAV has a packaging limit of 4.5 or 4.75 kb. Constructs larger than 4.5 or 4.75 kb result in significantly reduced virus production. Many promoters can be used to drive nucleic acid molecule expression. AAV ITRs can serve as promoters, advantageously eliminating the need for additional promoter elements. For ubiquitous expression, the following promoters can be used: CMV, CAG, CBh, PGK, SV40, ferritin heavy or light chain, etc. For expression in the brain, the following promoters can be used: synapsin I for all neurons, CaMKII alpha for excitatory neurons, and GAD67, GAD65, or VGAT for GABAergic neurons. Promoters used to drive RNA synthesis can include Pol II promoters, such as U6 or HI, and the use of Pol II promoters and intron cassettes can be used to express guide RNAs (gRNAs).

[0133]

[0233] As for AAV, AAV can be AAV1, AAV2, AAV5 or any combination thereof.AAV can be selected according to the cell to be targeted, for example, when targeting brain or nerve cells, AAV serotype 1, 2, 5 or hybrid capsid AAV1, AAV2, AAV5 or any combination thereof can be selected, and when targeting heart tissue, AAV4 can be selected.AAV8 is useful for delivery to liver.The promoter and vector mentioned above can be used individually.

[0134]

[0234] In some embodiments herein, the delivery is by AAV. The therapeutically effective dosage for in vivo delivery of AAV to humans is about 1 x 10 per ml of solution. 10 ~Approx. 1×10 50The dosage is expected to be in the range of about 20 to about 50 ml of saline solution containing functional AAV. Dosage can be adjusted to balance the therapeutic benefit against any side effects. In certain embodiments herein, the AAV dose is generally about 1 x 10 5 ~Approx. 1×10 50 Genomic AAV, approximately 1 × 10 8 ~Approx. 1×10 20 Genomic AAV, approximately 1 × 10 10 ~Approx. 1×10 16 genome, or approximately 1 × 10 11 ~Approx. 1×10 16 The concentration of genomic AAV can range from about 1 x 10 13 The AAV may be genomic AAV. Such concentrations may be delivered in the form of a carrier solution of about 0.001 ml to about 100 ml, about 0.05 ml to about 50 ml, or about 10 ml to about 25 ml. In some embodiments, the concentration may be about 2×10 13 AAV with a titer of 10 ...

[0135]

[0235] In another embodiment, effective activation of a cellular immune response to a neoplastic vaccine or immunogenic composition can be achieved by expressing the relevant neoantigen 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, e.g., U.S. Patent No. 6,991,797, hereby incorporated by reference in its entirety).

[0136]

[0236] In another embodiment, poxviruses are used in neoplastic vaccines or immunogenic compositions. These include orthopoxviruses, avian pox, vaccinia, MVA, NYVAC, canarypox, TROVAC, and the like (see, e.g., Verardiet 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 quickly became widely used in research in numerous fields as well as vaccine development. Advantages of these vectors include easy construction, the ability to accommodate large amounts of foreign DNA, and high expression levels.

[0137]

[0237] In another embodiment, vaccinia virus is used in neoplasia 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 can replicate within the cytoplasm of infected host cells, thus allowing the polypeptide of interest to induce an immune response. Furthermore, poxviruses are widely used as vaccine or immunogenic composition vectors due to their ability to target encoded antigens for processing by the major histocompatibility complex class I pathway by directly infecting immune cells at specific antigen-presenting cells, but also due to their self-adjuvanting ability.

[0138]

[0238] In another embodiment, ALVAC is used as a vector in a neoplastic vaccine or immunogenic composition. ALVAC is a canarypox virus (avim) that can be modified to express heterologous transgenes and has been used as a method for vaccination against both prokaryotic and eukaryotic antigens (Horig H, Lee DS, Conkright W, et al., Phase I clinical trial of a recombinant 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 gene recombinant 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: an update.Proc Natl Acad Sci USA 1996;93:11349-53;US 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, but no objective clinical responses were observed (Marshall JL, Hawkins MJ, Tsang KY, et al. Phase I study in cancer patients of a replication-defective avipox recombinant, vaccine that expresses human carcinoembryonic antigen. J Clin Oncol 1999;17:332-7).

[0139]

[0239] In another embodiment, modified vaccinia Ankara (MVA) virus can be used as a viral vector in neoantigen vaccines or immunogenic compositions. MVA is a member of the orthopoxvirus family and was generated by approximately 570 serial passages of the Ankara strain of vaccinia virus (CVA) in chicken embryo fibroblasts (for a review, see Mayr, A. et al., Infection 3, 6-14, 1975). As a result of these passages, the resulting MVA virus (VV) contains 3.1 kilobytes less genomic information compared to 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, yet retains excellent immunogenicity. When tested in various animal models, MVA has proven avirulent, even in immunosuppressed individuals. Additionally, MVA-BN®-HER2 is a candidate immunotherapy designed for HER-2-positive breast cancer and is currently in clinical trials. (Mandl et al. Cancer Immunol Immunother. 2012 January;61(1):19-29). Methods for making and using recombinant MVA have been described (see, e.g., U.S. Patent Nos. 8,309,098 and 5,185,146, which are hereby incorporated in their entireties).

[0140]

[0240] In another embodiment, the modified Copenhagen strain of vaccinia virus, NYVAC and NYVAC variations, are used as vectors (see, e.g., U.S. Patent No. 7,255,862; PCT WO95 / 30018; U.S. Patent Nos. 5,364,773 and 5,494,807, which are hereby incorporated by reference in their entireties).

[0141]

[0241] In one embodiment, recombinant viral particles of a vaccine or immunogenic composition are administered to a patient in need of such recombinant viral particles. The dosage of expressed neoantigen can range from a few micrograms to several hundred micrograms, e.g., 7 to 500 μg. The vaccine or immunogenic composition can be administered in any suitable amount to achieve expression at these dosage levels. 3.5 pfu of viral particles can be administered to a patient in need thereof or transfected into cells; thus, at least about 10 4 pfu~about 10 6 pfu of viral particles can be administered to a patient in need of the viral particles or to infect or transfect cells; however, a patient in need of viral particles should not be administered more than about 10 pfu of viral particles. 8 pfu can be administered, and therefore the amount for administration is at least about 10 7 pfu~about 10 9 The doses for NYVAC are applicable to ALVAC, MVA, MVA-BN, and avipox, such as canarypox and fowlpox. III. Vaccine and Immunogenic Composition Adjuvants

[0242] Toll-like receptors (TLRs) are important members of the family of pattern recognition receptors (PRRs) expressed by cells of the innate and adaptive immune systems. TLRs recognize conserved motifs shared by many microorganisms, termed pathogen-associated molecular patterns (PAMPs). Different TLRs recognize distinct PAMPs, and TLR ligand binding leads to the activation of inflammatory signaling cascades, including the nuclear factor kappa-light chain of activated B cells (NF-κB) transcription factor and type I interferon (IFN). Toll-like receptor-mediated activation of APCs, such as dendritic cells (DCs), leads to increased expression of MHC and T cell costimulatory molecules, which can help initiate peptide-specific T cell responses.

[0142]

[0243] Non-limiting examples of cancer vaccine adjuvants include the TLR9 agonist 5'-C-phosphate-G-3' (CpG) and the synthetic double-stranded ribonucleic acid (dsRNA) TLR3 ligand polyinosinic-polycytidylic acid carboxymethylcellulose (adjuvant) (poly-ICLC) [Hiltonol®] (polyinosinic acid:polycytidylic acid). CpG is a synthetic dinucleotide, and pICLC is a synthetic dsRNA stabilized by polylysine and carboxymethylcellulose.

[0143]

[0244] Poly-ICLC is a synthetic dsRNA "host-targeted" therapeutic viral mimic and PAMP with broad innate and adaptive immune adjuvant functions. Poly-ICLC exerts its functions 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).

[0144]

[0245] Stimulation with poly-ICLC leads to DC and natural killer (NK) cell activation and the production of a natural mix 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, poly-ICLC appears to be a potent TLR adjuvant, based on its induction of proinflammatory cytokines, lack of stimulation of interleukin-10 (IL-10), and maintenance of high levels of costimulatory molecules in DCs (Bogunovic, 2011). Furthermore, poly-ICLC has been directly compared with CpG in nonhuman primates as an adjuvant for a protein vaccine consisting of human papillomavirus (HPV) 16 capsomeres, resulting in the upregulation of HPV-specific T cells. H It has been found to be much more effective in inducing T-helper 1 (T-cell 1) immune responses (Stahl-Hennig, 2009).

[0145]

[0246] Poly-ICLC can induce sustained CD4+ and CD8+ responses in humans. Remarkable similarities in the upregulation of transcriptional and signaling pathways were observed between patients vaccinated with poly-ICLC and in volunteers who received a highly effective replication-deficient yellow fever vaccine (Okada, 2011). In a recent phase 1 study, >90% of ovarian cancer patients immunized with poly-ICLC in combination with the NY-ESO-1 peptide vaccine demonstrated induction of CD4+ and CD8+ T cells and antibody responses to the peptide (Sabbatini, 2012). Without being bound by theory, these neoantigens are expected to bypass central thymic tolerance (thus enabling stronger antitumor T cell responses) while reducing the potential for autoimmunity (e.g., by avoiding targeting of normal self-antigens). Effective immune responses benefit from the inclusion of strong adjuvants to activate the immune system (Speiser and Romero, Molecularly defined vaccines for cancer immunotherapy, and protective T cell immunity Seminars in Immunol 22:144 (2010)). For example, Toll-like receptors (TLRs) have emerged as powerful sensors of microbial and viral pathogen “danger signals” that effectively induce the innate immune system, and consequently the adaptive immune system (Bhardwaj and Gnjatic, TLR AGONISTS: Are They Good Adjuvants? Cancer J. 16:382-391 (2010)). Among TLR agonists, poly-ICLC (a synthetic double-stranded RNA mimic) is one of the most potent activators of myeloid-derived dendritic cells.In human volunteer studies, poly-ICLC has been shown to be safe and to induce a gene expression profile in peripheral blood cells comparable to that induced by the yellow fever vaccine YF-17D, one of the most potent live attenuated viral vaccines (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, HiitonoI®, a GMP preparation of poly-ICLC prepared by Oncovir, Inc., is utilized as an adjuvant. In other embodiments, other adjuvants described herein are contemplated, such as oil-in-water, water-in-oil, or multiphase W / O / W; see, e.g., U.S. Pat. No. 7,608,279 and Aucouturier et al., Vaccine 19 (2001), 2666-2672, and references cited therein. IV. Immune Checkpoint Modulators

[0247] Immune checkpoints are crucial signaling pathways in the immune system that maintain self-tolerance and modulate the duration and amplitude of physiological immune responses. Under normal conditions, these pathways prevent excessive effector activity by T cells. Two key examples of these pathways are the cell surface receptors CTLA-4 and PD-1 (Teft, 2006; Keir, 2008). In some cases, tumors express or overexpress inhibitory immune checkpoints as a primary mechanism of immune evasion. Because many immune checkpoints are initiated by ligand-receptor interactions, these signals can be easily blocked by antibodies or modulated by recombination of the ligand or receptor (Pardoll, 2012).

[0146]

[0248] Immune checkpoints are important targets for pharmacological blockade (Teft, 2006; Keir, 2008), and dramatic 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). Accordingly, the present disclosure, in exemplary embodiments, features novel combinations of neoplastic vaccines or immunogenic compositions with anti-PD-1 antibodies.

[0147]

[0249] The PD-1 receptor refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 is expressed on numerous cell types, including Tregs, activated B cells, and natural killer (NK) cells, and is primarily expressed in vivo on activated T cells. It binds to two ligands, PD-L1 and PD-L2. The endogenous ligands of PD1, PD-L1 and PD-L2, are expressed on activated immune cells as well as non-hematopoietic cells, including tumor cells. As used herein, PD-1 is intended to include human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs that share at least one epitope with hPD-1. The complete hPD-1 sequence can be found in GENBANK Accession No. U64863. Programmed death-ligand-1 (PD-L1) is one of two cell surface glycoprotein ligands for PD-1 (the other is PD-L2), which downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, PD-L3 includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, as well as analogs that share at least one epitope with hPD-L1. The complete hPD-L1 sequence can be found under GENBAN accession number Q9NZQ7. It has been demonstrated that tumors evade immune surveillance by expressing PD-L1 / L2, thereby suppressing tumor-infiltrating lymphocytes through PD-1 / PD-L1,2 interactions (Dong et al., Nat. Med. 8:793-800, 2002).

[0148]

[0250] In some embodiments, the anti-PD-1 antibody is nivolumab. Nivolumab (Opdivo®, Bristol-Myers Squibb Company, NY) is a human immunoglobulin G4 (IgG4) mAb that binds to the programmed death 1 (PD-1) receptor and blocks its interaction with PD-L1 and programmed death-ligand 2 (PD-L2), thereby reversing 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. Upregulation of PD-1 ligand occurs in some tumors, and signaling through this pathway may contribute to the inhibition of active T cell immunosurveillance of tumors. Antibodies of the present disclosure include, but are not limited to, all of the 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 are described, for example, in U.S. Patent Nos. 7,488,802 and 8,168,757, and anti-PD-L1 mAbs are described, for example, in U.S. Patent Nos. 7,635,757 and 8,217,149, and U.S. Patent Application Publication No. 2009 / 0317368. U.S. Patent No. 8,008,449 lists seven illustrative anti-PD-1 HuMAbs: 1708.2D3, 4M, 5C4 (also referred to herein as nivolumab or BMS-936558), 4A11, 7D3, and 5F4.

[0149]

[0251] In addition to CTLA-4 and PD-1 / PD-L1, numerous other immunomodulatory targets have been preliminarily identified, and many are under investigation in clinical trials with corresponding therapeutic antibodies. Page et al. (Annu.Rev.Med.2014.65) detail targets of antibody immunomodulators in Figure 1, which is incorporated herein by reference.

[0150]

[0252] In exemplary aspects, the present disclosure features novel combinations of neoplastic vaccines or immunogenic compositions with one or more inhibitors of the anti-PD-L1 pathway. In some embodiments, the PD-1 pathway inhibitor is a PD-L1 antibody, e.g., pembrolizumab.

[0151]

[0253] In another aspect, the present disclosure also features a novel combination of a neoplastic vaccine or immunogenic composition in combination with the anti-PD-L1 antibody pembrolizumab and one or more chemotherapeutic agents. In one embodiment, the one or more chemotherapeutic agents include a platinum-based anticancer therapeutic agent. The platinum-based anticancer agent may be selected from carboplatin, dicycloplatin, oxaliplatin, satraplatin, and nedaplatin, or pharmaceutically acceptable salts or solvates thereof, for use in combination with the neoplastic vaccine or immunogenic composition and pembrolizumab in treating cancer. In certain preferred embodiments, the platinum-based anticancer agent is carboplatin. In some embodiments, the 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 novel combination further includes an adjuvant.

[0152]

[0254] In some embodiments, a PD-L1 inhibitor such as pembrolizumab may be administered once or more than once with neo-antigen administration. In some dosing regimens, a PD-L1 inhibitor such as __ may be administered once as a priming dose at the beginning of the vaccination period, followed by one, two, three, four, five or more boost doses during and / or after the neo-antigen vaccine dose. In some embodiments, a pembrolizumab dose is administered once before administering the neo-antigen vaccine. In some embodiments, a pembrolizumab dose is administered more than once before administering the neo-antigen vaccine. In some embodiments, a pembrolizumab dose is administered once after administering the neo-antigen vaccine. In some embodiments, a pembrolizumab dose is administered more than once after administering the neo-antigen vaccine. In some embodiments, a pembrolizumab dose is administered two, three, four, five or more times after administering the neo-antigen vaccine. In some embodiments, the nivolumab dose is administered both before and after the neo-antigen vaccine is administered.

[0153]

[0255] In some embodiments, the adjuvant is Hiltolol. In some embodiments, the adjuvant is Poly-ICLC. In some embodiments, Hiltolol and Poly-ICLC are used.

[0154]

[0256] There is growing recognition that somatic mutations in tumor cells can lead to the presentation of neoantigens, which 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). Numerous clinical trials have attempted to exploit this mechanism to induce tumor control by utilizing personalized neoantigen-based vaccinations, particularly in the context of solid tumors using high neoantigen loads, with some promising success in adjuvant therapy (Ott et al., 2017; Keskin et al., 2019; Sahin et al., 2017; Hilf et al., 2019). The inventors previously reported on a similar personalized neoantigen-based vaccine trial in combination with anti-PD1 in patients with melanoma, non-small cell lung cancer (NSCLC), and urothelial carcinoma of the bladder (TCC), which demonstrated safety and feasibility, as well as the ability to deeply characterize the immune responses generated against the vaccine peptide ( Ott et al., 2020 ).

[0155]

[0257] The treatment of patients with NSCLC using immunotherapy modalities is a relatively recent trend and has had encouraging effects on the prognosis of patients diagnosed with this disease. Several pivotal studies have led to the approval of immune checkpoint inhibitors (ICIs), including PD-1 inhibitors or pembrolizumab, either 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 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, there remains a clear and urgent need for additional treatment options to further extend 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 combining them with neoantigen-based vaccination strategies to further boost antitumor immune responses in a targeted manner.

[0156]

[0258] The addition of neoantigen vaccine to chemotherapy and pembrolizumab regimens enhances tumor-specific CD4 + and CD8 + This provides an opportunity to induce and expand T cell responses. The use of neoantigen vaccines in combination with anti-PD-1 as a treatment for advanced NSCLC has been reported by the present inventors (Ott et al., 2020), with CD4 + Response and CD8 + Both responses were robust. In addition, neoantigen vaccines were studied in combination with tyrosine kinase inhibitors in a recent small study of 16 patients with late-stage NSCLC harboring EGFR mutations (Li et al., 2021). Both studies demonstrated that such an approach of adding neoantigen vaccines is feasible, safe, and provides additional tumor-specific immunity.

[0157]

[0259] In some embodiments, the subject is suffering from a neoplasm selected from the group consisting of non-Hodgkin's lymphoma (NHL), clear cell renal cell carcinoma (ccRCC), melanoma, sarcoma, leukemia, or cancer of the bladder, colon, brain, breast, head and neck, endometrium, lung, ovary, pancreas, or prostate. In some embodiments, the neoplasm is metastatic melanoma. In some embodiments, the subject does not have a detectable neoplasm but is at high risk of disease recurrence. In embodiments, the cancer is selected from the group consisting of adrenal gland cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, glioblastoma, head and neck cancer, chromophobe renal cell carcinoma, clear cell renal carcinoma, papillary renal carcinoma, liver cancer, lung adenocarcinoma, lung squamous cell carcinoma, ovarian cancer, pancreatic cancer, melanoma, gastric cancer, endometrial cancer, and uterine carcinosarcoma. In some embodiments, the cancer is selected from the group consisting of prostate cancer, bladder cancer, lung squamous cell carcinoma, 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 some embodiments, the cancer is selected from the group consisting of CRC, head and neck cancer, gastric cancer, lung squamous cell carcinoma, lung adenocarcinoma, prostate cancer, bladder cancer, gastric cancer, renal cell carcinoma, and uterine cancer. In some embodiments, the cancer is selected from the group consisting of melanoma, lung squamous cell carcinoma, DLBCL, uterine cancer, head and neck cancer, uterine cancer, liver cancer, and CRC. In some embodiments, the cancer is selected from the group consisting of lymphatic cancer, Burkitt's lymphoma, neuroblastoma, prostate cancer, colorectal adenocarcinoma, uterine / endometrial adenocarcinoma, MSI+, endometrial serous carcinoma, endometrial carcinosarcoma-malignant mixed mesodermal tumor, glioma, astrocytoma, GBM, acute myeloid leukemia associated with MDS, chronic lymphocytic leukemia-small lymphocytic lymphoma, myelodysplastic syndrome, acute myeloid leukemia, luminal NS breast carcinoma, chronic myeloid leukemia, pancreatic ductal carcinoma, chronic myelomonocytic leukemia, myelofibrosis, myelodysplastic syndrome, prostate cancer, essential thrombocythemia, and medullary myoblastoma. In some embodiments, the cancer is selected from the group consisting of colorectal cancer, uterine cancer, endometrial cancer, and gastric cancer. In some 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, colon cancer, and gastric cancer.In some embodiments, the cancer is selected from the group consisting of lung cancer, CRC, melanoma, breast cancer, NSCLC, and CLL. In some embodiments, the subject is a partial or non-responder to checkpoint inhibitor therapy. In some embodiments, the subject is a partial or non-responder to CD40 agonist therapy. In some 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 adenocarcinoma (CESC), chronic lymphocytic leukemia (CLL), colorectal cancer (CRC), glioblastoma multiforme (GBM), head and neck squamous cell carcinoma (HNSC), kidney papillary renal cell carcinoma (KIRP), liver hepatocellular carcinoma (LIHC), lung adenocarcinoma (LUAD), lung squamous cell carcinoma (LUSC), pancreatic adenocarcinoma (PAAD), prostate cancer, skin cutaneous melanoma (SKCM), gastric adenocarcinoma (STAD), thyroid cancer (THCA), and uterine endometrioid carcinoma (UCEC). In some embodiments, the cancer is selected from the group consisting of colorectal cancer, uterine cancer, endometrial cancer, gastric cancer, and Lynch syndrome. In some embodiments, the cancer is MSI+ cancer. V. Pharmaceutical Compositions / Delivery Methods

[0260] 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, diluent, or excipient.

[0158]

[0261] When administered as a combination, the therapeutic agents (i.e., the neoplastic vaccine or immunogenic composition and the one or more inhibitors, e.g., one or more checkpoint inhibitors or immunotherapeutic agents) can be formulated as separate compositions given at the same or different times, or the therapeutic agents can be given as a single composition.

[0159]

[0262] The composition can be administered once a day, twice a day, every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 2 weeks, every 3 weeks, every 4 weeks, every 2 months, every 6 months, or once a year. The administration interval can be adjusted according to the needs of individual patients. For longer administration intervals, sustained release or depot formulations can be used.

[0160]

[0263] The compositions of the present disclosure can be used to treat diseases and conditions that are acute, as well as chronic conditions. In particular, the compositions of the present disclosure are used in methods of treating or preventing neoplasms.

[0161]

[0264] In certain embodiments, the compounds of the present disclosure are administered for a period of 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, or more than 5 years, 10 years, or 15 years, or for any period of time ranging from days, months, or years, with the lower end of the range being any period between 14 days and 15 years and the upper end of the range being between 15 days and 20 years (e.g., 4 weeks = 15 years, 6 months = 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 monitor the progression of the disease or disorder, and the dosage is adjusted accordingly. In some embodiments, treatment according to the present disclosure is effective for at least 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 1 year, 2 years, 3 years, 4 years, or 5 years, 10 years, 15 years, 20 years, or for the rest of the subject's life.

[0162]

[0265] As described herein, in certain embodiments, administration of the inhibitor is initiated prior to the initiation of administration of the neoplastic vaccine or immunogenic composition. In other embodiments, administration of the inhibitor is initiated after the initiation of administration of the neoplastic vaccine or immunogenic composition. In yet other embodiments, administration of the inhibitor is initiated simultaneously with the initiation of administration of the neoplastic vaccine or immunogenic composition.

[0163]

[0266] Administration of the inhibitor, e.g., checkpoint inhibitor or chemotherapeutic agent, can continue every 2, 3, 4, 5, 6, 7, 8 or more weeks after the initial administration of the inhibitor, e.g., checkpoint inhibitor or chemotherapeutic agent. It is understood that week 1 is intended to include days 1-7, week 2 is intended to include days 8-14, week 3 is intended to include days 15-21, and week 4 is intended to include days 22-28. When administration is described as being at weekly intervals, this means approximately 7 days apart, but within any given week, the days may be one or more days earlier or later than the scheduled day.

[0164]

[0267] In certain embodiments, administration of the inhibitor, e.g., a checkpoint inhibitor or chemotherapeutic agent, is withheld for the week prior to administration of the neoplastic vaccine or immunogenic composition, hi other embodiments, administration of the inhibitor, e.g., a checkpoint inhibitor, is withheld during administration of the neoplastic vaccine or immunogenic composition.

[0165]

[0268] Surgical resection uses surgery to remove abnormal tissue in cancer, such as mediastinal, neurogenic or germ cell tumors, or thymoma. In other embodiments, administration of the neoplasia vaccine or immunogenic composition begins 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 weeks or more after tumor resection. In some embodiments, administration of the neoplasia vaccine or immunogenic composition begins 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after tumor resection.

[0166]

[0269] A prime / boost regimen refers to sequential administration of a vaccine or immunogenic or immunological composition. In certain embodiments, the neoplastic vaccine or immunogenic composition is administered in a prime / boost regimen, e.g., a prime administration of the neoplastic vaccine or immunogenic composition occurs at week 1, 2, 3, or 4, followed by a boost administration of the neoplastic vaccine or immunogenic composition at month 2, 3, or 4. In another embodiment, a heterologous prime-boost strategy is used to elicit a larger cytotoxic T cell response (see Schneider et al., "Induction of CD8+ T cells using heterologous prime-boost immunization strategies," Immunological Reviews, Vol. 170, No. 1, pp. 29-38, August 1999). In another embodiment, DNA encoding a neoantigen is used to prime, followed by a protein boost. In another embodiment, a protein is used to prime, followed by boosting with a virus encoding the neoantigen. In another embodiment, a virus encoding a neoantigen is used to prime and another virus is used to boost. In another embodiment, a protein is used to prime and DNA is used to boost. In some embodiments, a DNA vaccine or immunogenic composition is used to prime T cells and a recombinant viral vaccine or immunogenic composition is used to boost the response. In some embodiments, a viral vaccine or immunogenic composition is co-administered with a protein or DNA vaccine or immunogenic composition to act as an adjuvant for the protein or DNA vaccine or immunogenic composition.The patient can then be boosted with either a viral vaccine or immunogenic composition, a protein, or a DNA vaccine or 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, 2007 December;75(12):5S19-26. Epub 2007 October 1).

[0167]

[0270] As used herein, the term "fixed intermittent dosing regimen" refers to a pre-planned repeated cycle of drug administration in which a drug is administered on one or multiple consecutive days ("on days"), followed by one or multiple consecutive days of rest ("off days") during which the drug is not administered.

[0168]

[0271] In some embodiments, the cycles are regular because the pattern of on and off days is the same for each cycle. In some embodiments, the cycles are irregular because the pattern of on and off days varies from cycle to cycle. However, in some embodiments, each repeated cycle is pre-planned because it is not determined solely in response to the occurrence of one or more adverse events. In some embodiments, the administration of the composition comprising the first component and / or the second component is repeated for 1 to 10 cycles, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cycles.

[0169]

[0272] In some embodiments, a cycle comprises 3 to 60 days. In some embodiments, a cycle comprises 7 to 50 days, e.g., 7 to 30 days, 7 to 21 days, or 7 to 14 days. In some embodiments, a cycle consists of 7 days.

[0170]

[0273] In some embodiments, the fixed intermittent dosing regimen comprises repeated cycles of administering an effective amount of the composition comprising the first component and / or the second component for 1 to 5 consecutive days, e.g., 2 to 5 consecutive days, followed by 6 to 2 days of rest, e.g., 5 to 2 days of rest. In some embodiments, the fixed intermittent dosing regimen comprises repeated cycles of administering an effective amount of the composition comprising the first component and / or the second component for 5 consecutive days, followed by 2 days of rest. In some embodiments, the fixed intermittent dosing regimen comprises repeated cycles of administering an effective amount of the composition comprising the first component and / or the second component for 4 consecutive days, followed by 3 days of rest. In some embodiments, the fixed intermittent dosing regimen comprises repeated cycles of administering an effective amount of the composition comprising the first component and / or the second component for 3 consecutive days, followed by 4 days of rest.

[0171]

[0274] In some embodiments, the fixed intermittent dosing regimen comprises repeated cycles of administering an effective amount of the composition comprising the first component and / or the second component for 1 to 5 consecutive days, e.g., 2 to 5 consecutive days, followed by 6 to 2 days of rest, e.g., 5 to 2 days of rest, in some embodiments, where a placebo is administered on the rest days.

[0172]

[0275] The pharmaceutical compositions can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to subjects in need thereof, including humans and other mammals.

[0276] Modifications of the neo-antigenic peptides can affect the solubility, bioavailability, and metabolic rate of the peptide, thus controlling the delivery of the active species. Solubility can be assessed by preparing and testing the neo-antigenic peptides according to known methods well within the capabilities of a routine practitioner in the art.

[0173]

[0277] It has been found that pharmaceutical compositions comprising succinic acid or a pharmaceutically acceptable salt (succinate) can improve the solubility of neo-antigenic peptides. Accordingly, in one aspect, the present disclosure provides pharmaceutical compositions comprising at least one neo-antigenic peptide or a pharmaceutically acceptable salt thereof, a pH adjuster (e.g., a base, e.g., a pharmaceutically acceptable salt of a di- or tricarboxylate, e.g., a succinic acid salt), and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can be prepared by combining a solution comprising at least one neo-antigenic peptide with a base, e.g., a pharmaceutically acceptable salt of a di- or tricarboxylate, e.g., succinic acid or citric acid (e.g., sodium citrate), or by combining a solution comprising at least one neo-antigenic peptide with a solution comprising a base, e.g., a pharmaceutically acceptable salt of a di- or tricarboxylate, e.g., succinic acid or citric acid (e.g., a succinic acid buffer solution). In certain embodiments, the pharmaceutical composition comprises sodium succinate. In certain embodiments, the pH adjuster (e.g., citrate or succinate) is present in the composition at a concentration of about 1 mM to 10 mM, and in certain embodiments, 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.

[0174]

[0278] In certain embodiments of the pharmaceutical composition, the pharmaceutically acceptable carrier comprises water. In certain embodiments, the pharmaceutically acceptable carrier further comprises dextrose. In certain embodiments, the pharmaceutically acceptable carrier further comprises dimethyl sulfoxide. In certain embodiments, the pharmaceutical composition comprises an immune modulator. In certain embodiments, the immune modulator or adjuvant is selected from the group consisting of poly ICLC, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, JuvImmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide In certain embodiments, the immune modulator or adjuvant is selected from the group consisting of ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, vector systems, PLGA microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, and Aquila's QS21 stimulon. In certain embodiments, the immune modulator or adjuvant is poly-ICLC.

[0175]

[0279] Xanthenone derivatives, such as vadimezan or AsA404 (also known as 5,6-dimethylaxanthenone-4-acetic acid (DMXAA)), can also be used as adjuvants in accordance with embodiments of the present disclosure. Alternatively, such derivatives can be administered in parallel with the vaccine or immunogenic compositions of the present disclosure, for example, by systemic or intratumoral delivery, to stimulate immunity at the tumor site. Without being bound by theory, such xanthenone derivatives are thought to act by stimulating interferon (IFN) production through 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).

[0176]

[0280] The vaccine or immunogenic composition may also contain an adjuvant compound selected from acrylic or methacrylic acid polymers and copolymers of maleic anhydride and alkenyl derivatives, particularly polymers of acrylic or methacrylic acid cross-linked with polyalkenyl ethers of sugars or polyalcohols (carbomers), especially those cross-linked with allyl sucrose or allyl pentaerythritol. It may also be a copolymer of maleic anhydride and ethylene cross-linked, for example, with a divinyl ether (see U.S. Pat. No. 6,713,068, hereby incorporated by reference in its entirety).

[0177]

[0281] In certain embodiments, the pH adjusting agent can stabilize the adjuvants or immune modulators described herein.

[0282] In certain embodiments, the pharmaceutical composition comprises one to five peptides, dimethyl sulfoxide (DMSO), dextrose, water, succinate, poly I:poly C, poly-L-lysine, carboxymethylcellulose, and chloride. In certain embodiments, each of the one to five peptides is present at a concentration of between 200 μg / ml and 500 μg / ml, such as 300-400 μg / ml. In certain embodiments, the pharmaceutical composition comprises ≦3% DMSO by volume, about 4-5% DMSO. In certain embodiments, the pharmaceutical composition comprises 3.5-5.5% dextrose, 4.9-5.0% dextrose in water. In certain embodiments, the pharmaceutical composition comprises ≦5.0 mM succinate, 3.6-3.7 mM succinate (e.g., as sodium succinate). In certain embodiments, the pharmaceutical composition comprises ≥0.4 mg / ml poly I:poly C, e.g., 1.0-2.2 mg / ml, e.g., 1.7-1.9 mg / ml. In certain embodiments, the pharmaceutical composition comprises ≥0.375 mg / ml poly-L-lysine, e.g., 0.5-2.0 mg / ml, or 1.5 mg / ml. In certain embodiments, the pharmaceutical composition comprises ≥1.25 mg / ml sodium carboxymethylcellulose, e.g., 2-7 mg / ml, e.g., 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.

[0178]

[0283] The pharmaceutical compositions comprise the tumor-specific neo-antigenic peptides described herein in a therapeutically effective amount to treat a disease or condition (e.g., neoplasm / tumor) as described herein, in combination with pharmaceutically acceptable additives, carriers and / or excipients as needed. From this disclosure and knowledge in the art, one skilled in the art will recognize that the therapeutically effective amount of one of the many compounds according to the present disclosure may vary depending on the condition to be treated, its severity, the treatment regimen to be utilized, the pharmacokinetics of the agent used, as well as the patient (animal or human) to be treated.

[0179]

[0284] To prepare pharmaceutical compositions according to the present disclosure, a therapeutically effective amount of one or more compounds according to the present disclosure can be intimately mixed with a pharmaceutically acceptable carrier according to conventional pharmaceutical compounding techniques to produce a dosage. Carriers can take a wide variety of forms, depending on the form of preparation desired for administration, e.g., ophthalmic, oral, topical, or parenteral administration, including gels, creams, ointments, lotions, and sustained release implant preparations, among others. When preparing pharmaceutical compositions in oral dosage form, any of the usual pharmaceutical media can be used. Thus, for liquid oral preparations, e.g., suspensions, elixirs, and solutions, suitable carriers and additives can be used, including water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, etc. For solid oral preparations, e.g., powders, tablets, and capsules, and for solid preparations, e.g., suppositories, suitable carriers and additives can be used, including starches, sugar carriers, e.g., dextrose, mannitol, lactose, and related carriers, diluents, granulating agents, lubricants, binders, disintegrating agents, etc. If desired, tablets or capsules may be enteric-coated or sustained release by standard techniques.

[0180]

[0285] The active compound is included in a pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to the patient a therapeutically effective amount for the desired indication without causing serious toxic effects in the patient being treated.

[0181]

[0286] Oral compositions generally contain an inert diluent or an edible carrier.They can be sealed in gelatin capsules or compressed into tablets.For oral therapeutic administration, the active compound or its prodrug derivative can be mixed with excipients and used in the form of tablets, troches or capsules.Pharmaceutically compatible binders and / or adjuvants can be included as part of the composition.

[0182]

[0287] Tablets, pills, capsules, troches, etc. may contain any of the following ingredients or compounds of similar nature: binders, such as microcrystalline cellulose, tragacanth gum or gelatin; excipients, such as starch or lactose, dispersing agents, such as alginic acid or corn starch; lubricants, such as magnesium stearate; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavoring agents, such as peppermint, methyl salicylate, or orange flavoring. When the dosage unit form is a capsule, it can contain a liquid carrier such as fatty oil in addition to the materials discussed herein. In addition, dosage unit form can contain various other materials that modify the physical form of the dosage unit, such as sugar, shellac, or enteric coating.

[0183]

[0288] Formulations of the present disclosure suitable for oral administration may be presented as discrete units each containing a predetermined amount of the active ingredient, such as capsules, cachets, or tablets; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or water-in-oil emulsion, and as a bolus, etc.

[0184]

[0289] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared by compressing the active ingredient in a free-flowing form, such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant or dispersant, in a suitable machine. Molded tablets can be made by molding a mixture of powdered compounds moistened with an inert liquid diluent in a suitable machine. If necessary, tablets can be coated or scored, and can be formulated to provide slow or controlled release of the active ingredient therein.

[0185]

[0290] Methods for formulating such slow-release or controlled-release compositions of pharmaceutical active ingredients are known in the art and are described in several issued U.S. patents, including but not limited to U.S. Patent Nos. 3,870,790, 4,226,859, 4,369,172, 4,842,866 and 5,705,190, the disclosures of which are incorporated herein by reference in their entirety.Coatings can be used to deliver compounds to the intestine (see, for example, U.S. Patent Nos. 6,638,534, 5,541,171, 5,217,720 and 6,569,457, and the references cited therein).

[0186]

[0291] The active compound or its pharmaceutically acceptable salts can 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, certain preservatives, dyes and colorings, and flavors.

[0187]

[0292] Solutions or suspensions used 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 glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methylparaben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetates, citrates, or phosphates; and an agent for adjusting isotonicity, such as sodium chloride or dextrose.

[0188]

[0293] In certain embodiments, the pharmaceutically acceptable carrier is an aqueous solvent, i.e., a solvent containing water, optionally with additional cosolvents. Exemplary pharmaceutically acceptable carriers include water, buffer solutions in water, such as phosphate-buffered saline (PBS), and 5% dextrose in water (D5W). In certain embodiments, the aqueous solvent further contains dimethyl sulfoxide (DMSO), for example, in an amount of about 1-4% or 2-3%. In certain embodiments, the pharmaceutically acceptable carrier is isotonic (i.e., has substantially the same osmotic pressure as a body fluid such as plasma).

[0189]

[0294] In one embodiment, the active compounds are prepared using carriers that protect the compounds from rapid elimination from the body, such as controlled-release formulations, 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 skill of those in the art, given the present disclosure and knowledge in the art.

[0190]

[0295] From this disclosure and knowledge in the art, one skilled in the art will recognize that in addition to tablets, other dosage forms can be formulated to provide slow or controlled release of the active ingredient, including, but not limited to, capsules, granules, and gelcaps.

[0191]

[0296] Liposomal suspension can also be a pharmaceutically acceptable carrier.It can be prepared according to the method known to those skilled in the art.For example, liposomal preparation can be prepared by dissolving suitable lipid in inorganic solvent, then evaporating it, so that a thin film of dried lipid remains on the surface of the container.Then, an aqueous solution of active compound can be introduced into the container.Then, the container is manually rotated to release lipid material from the side of the container and disperse lipid aggregates, thereby forming a liposomal suspension.Other preparation methods known to those skilled in the art can also be used in this aspect of the present disclosure.

[0192]

[0297] Preparations can be conveniently provided in unit dosage form and can be prepared by conventional pharmaceutical methods.Such methods include the step of combining active ingredient with pharmaceutically acceptable carrier or excipient.Generally, preparations are prepared by uniformly and intimately combining active ingredient with liquid carrier or finely divided solid carrier or both, and then, if necessary, shaping the product.

[0193]

[0298] Formulations and compositions suitable for topical administration in the mouth include lozenges which comprise the ingredient in a flavored base, usually sucrose and gum arabic or tragacanth; pastilles which comprise the active ingredient in an inert base, such as gelatin and glycerin, or sucrose and gum arabic; and mouthwashes which comprise the ingredient to be administered in a suitable liquid carrier.

[0194]

[0299] Formulations suitable for topical administration to the skin can be provided as ointments, creams, gels, and pastes containing the ingredient to be administered in a pharmaceutically acceptable carrier. A topical delivery system that can be used is a transdermal patch containing the ingredient to be administered.

[0195]

[0300] Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

[0301] Formulations suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size, for example, in the range 20 to 500 micrometers, and administered in the manner in which substances are administered, i.e., by rapid inhalation through the nasal passage from a closely held container of the powder into the nose. Formulations suitable for administration wherein the carrier is a liquid, for example, as a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.

[0196]

[0302] 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.

[0197]

[0303] The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.If administered intravenously, the carrier can include, for example, saline or phosphate-buffered saline.

[0198]

[0304] For parenteral preparations, carrier usually comprises sterilized water or aqueous sodium chloride solution, but may also comprise other components, including those that aid dispersion.Of course, when sterilized water is used and must be maintained as sterile, the composition and carrier are also sterilized.Injectable suspension can also be prepared, and in this case, suitable liquid carrier, suspending agent, etc. can be used.

[0199]

[0305] Preparations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions, which may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may contain suspending agents and thickening agents.Preparations can be provided in unit-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) state, which only requires the addition of sterile liquid carriers, such as distilled water for injection, immediately before use.Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the type described above.

[0200]

[0306] Administration of the active compound may vary from continuous administration (infusion) to oral administration several times daily (e.g., QID) and may include oral, topical, ocular or ophthalmic, parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may include penetration enhancers), buccal, and suppository routes of administration, among others, including by the ocular or ocular route.

[0201]

[0307] The neoplastic vaccine or immunogenic composition, and at least one inhibitor, such as a checkpoint inhibitor or chemotherapeutic agent, and any additional agents, can be administered by injection, orally, parenterally, by inhalation spray, rectally, vaginally, or topically in a dosage unit formulation containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral, as used herein, includes intralymph node(s) or nodes, subcutaneously, intravenously, intramuscularly, intrasternally, by injection, intraperitoneally, intraocularly, intravitreously, buccally, transdermally, intranasally, intracranially and intradurally, intraarticularly, including ankle, knee, hip, shoulder, elbow, wrist, directly intratumorally, and the like, as well as in suppository form.

[0202]

[0308] A variety of techniques can be used to provide the subject compositions to the desired site, such as the use of catheters, trocars, projectiles, Pluronic® gels, stents, drug-releasing polymers, or other devices that provide internal access. If an organ or tissue is available by removal from a patient, such organ or tissue can be immersed in a medium containing the subject composition, the subject composition can be painted onto the organ, or can be applied in any convenient manner.

[0203]

[0309] The tumor-specific neoantigenic peptides can be administered by a device suitable for controlled or sustained release of the composition effective to achieve the desired local or systemic physiological or pharmacological effect. The method involves placing a sustained release drug delivery system in the area where drug release is desired and directing the drug through the device to the desired treatment area.

[0204]

[0310] The tumor-specific neoantigenic peptides can be used in combination with at least one other known therapeutic agent or a pharmaceutically acceptable salt thereof. Examples of known therapeutic agents that can be used in combination therapy include corticosteroids (e.g., cortisone, prednisone, dexamethasone), nonsteroidal anti-inflammatory drugs (NSAIDS) (e.g., ibuprofen, celecoxib, aspirin, indomethacin, naproxen), alkylating agents (e.g., busulfan, cisplatin, mitomycin C, and carboplatin); antimitotic agents (e.g., colchicine, vinblastine, paclitaxel, and docetaxel); topoisomerase I inhibitors, For example, 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, and thioguanine; antibiotics, such as berceptin and rituxan, but are not limited to these.

[0205]

[0311] In certain embodiments, administration of the compositions described herein can be combined with antagonists that block the release of histamine and anti-inflammatory drugs to prevent adverse allergic reactions. H1 and H2 antagonists can be administered to patients prior to administration of the compositions described herein.

[0206]

[0312] It should be understood that in addition to the ingredients specifically mentioned herein, the formulations of the present 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.

[0207]

[0313] A pharmaceutically acceptable salt form can be the chemical form of a compound according to the present disclosure for inclusion in a pharmaceutical composition according to the present disclosure.

[0314] The present compounds or their derivatives, including the prodrug forms of these drugs, can be provided in the form of pharmaceutically acceptable salts.As used herein, the term pharmaceutically acceptable salts or complexes refers to suitable salts or complexes of the active compounds of the present disclosure, which retain the desired biological activity of the parent compound and show limited toxicological effects on normal cells.Non-limiting examples of such salts include: (a) acid addition salts formed with inorganic acids (for example, 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, among others; (b) base addition salts formed with metal cations, such as zinc, calcium, sodium, potassium, among others.

[0208]

[0315] The compounds herein can be commercially available or synthesized. As can be understood by those skilled in the art, additional methods for synthesizing the compounds described herein will be apparent to those skilled in the art. In addition, various synthetic steps can be performed in a different order or sequence to obtain the desired compound. Synthetic chemical transformations and protecting group methodologies (protection and deprotection) useful for synthesizing the compounds described herein are known in the art, and include, for example, those described in R. Larock, Comprehensive Organic Transformations, 2nd Edition, Wiley-VCH Publishers (1999); T.W. Greene and P.G.M. Buts, 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, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof. VI. Dosage

[0316] When the agents described herein are administered as pharmaceuticals to humans or animals, they can be given per se, or they can be given as pharmaceutical compositions containing the active ingredient in combination with a pharmaceutically acceptable carrier, excipient, or diluent.

[0209]

[0317] The actual dosage levels and time course of administration of the active ingredients in the pharmaceutical compositions of the present disclosure can be varied to provide an amount of the active ingredient that is effective to achieve the desired therapeutic response without being toxic to the patient for a particular patient, composition, and method of administration. Generally, the medicaments or pharmaceutical compositions of the present disclosure are administered in an amount sufficient to reduce or eliminate symptoms associated with viral infections and / or autoimmune diseases.

[0210]

[0318] The dose of the drug is the maximum that the patient can tolerate and not develop serious or intolerable side effects.

[0319] Determining an effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, the effective amount or effective dose of a drug is determined by first administering a low dose of the drug, and then gradually increasing the administered dose or dosage until the desired effect (e.g., reducing or eliminating symptoms associated with viral infections or autoimmune diseases) is achieved with minimal or tolerable toxic side effects. Applicable methods for determining the appropriate dose and administration schedule for the administration of the pharmaceutical compositions of the present disclosure are described, for example, in Goodman and Oilman's The Pharmacological Basis of Therapeutics, edited by Goodman et al., 11th Edition, McGraw-Hill 2005, and Remington: The Science and Practice of Pharmacy, 20th and 21st Editions, edited by Gennaro and University of the Sciences in Philadelphia, Lippencott Williams and Wilkins (2003 and 2005), each of which is hereby incorporated by reference herein.

[0211]

[0320] Unit dosage formulations are those containing a daily dose or unit, or daily sub-dose, as discussed herein, of the administered ingredient, or an appropriate fraction thereof.

[0212]

[0321] Dosage regimens for treating disorders or diseases with the tumor-specific neo-antigenic peptides of the present disclosure and / or compositions of the present disclosure are based on a variety of factors, including the type of disease, the patient's age, weight, sex, medical condition, severity of the condition, route of administration, and the particular compound utilized. Thus, dosage regimens vary widely but can be routinely determined using standard methods.

[0213]

[0322] The amount and dosage regimen administered to a subject can depend on numerous factors, such as the method of administration, the nature of the condition being treated, the weight of the subject being treated, and the judgment of the prescribing physician, all of which are within the skill of those in the art from this disclosure and knowledge in the art. In some embodiments, an initial series of closely spaced immunizations can be administered to induce an immune response, followed by a resting period to establish memory T cells and then boosts to expand the response. Alternatively, priming doses can be administered over a longer period, with boosts administered more frequently and for a longer period. For example, a long priming period can be followed by boosts every two months for a year.

[0214]

[0323] The amount of compound contained in a therapeutically active formulation according to the present disclosure is an amount effective to treat a disease or condition.

[0324] Generally, a therapeutically effective amount of a compound in a dosage form may range from just under about 0.025 mg / kg / day to about 2.5 g / kg / day, from about 0.1 mg / kg / day to about 100 mg / kg / day of a patient, or significantly more, depending on the compound used, the condition or infection being treated, and the route of administration, although exceptions to this dosage range may be contemplated by the present disclosure. In some embodiments, a compound according to the present disclosure is administered in an amount ranging from about 1 mg / kg / day to about 100 mg / kg / day. The dosage of the compound may depend on the condition being treated, the particular compound, other clinical factors such as the patient's weight and condition, and the route of administration of the compound. It is understood that the present disclosure applies to both human and veterinary use.

[0215]

[0325] According to certain exemplary embodiments, the vaccine or immunogenic composition is administered at a dose of about 10 μg to 1 mg per neo-antigenic peptide. According to certain exemplary embodiments, the NEO-PV-01 vaccine / adjuvant + pembrolizumab + chemotherapy regimen comprises administering the vaccine or immunogenic composition at an average weekly dose level of about 10 μg to 2000 μg per neo-antigenic peptide. In some cases, a single dose of one or more neo-antigenic peptides has a concentration of between 100 μg / ml and 1000 μg / ml, between 300 and 600 μg / ml, or between 400 and 500 μg / ml. According to some embodiments, the NEO-PV-01 vaccine / adjuvant + pembrolizumab + chemotherapy regimen comprises pembrolizumab at a dose of 200 mg by intravenous infusion (IV), plus carboplatin (AUC5) + pemetrexed (500 mg / m 2 The study involves chemotherapy with NEO-PV-01 administered every three weeks for four cycles. At week 12, all patients, regardless of their disease status, will receive NEO-PV-01 plus adjuvant (one vial of pooled peptide per injection site) administered subcutaneously at up to four different sites (each limb or flank) while continuing treatment with pembrolizumab.

[0216]

[0326] In some embodiments, a subject is administered a neo-antigenic peptide at a dosage of 10 μg to 2,000 μg per peptide. In some embodiments, a subject is administered a neo-antigenic peptide at a dosage 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 per peptide. In some embodiments, a subject is administered a neo-antigenic peptide at a dosage of at most 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, or 75 μg per peptide.In some embodiments, the subject receives between 10 μg and 50 μg, 10 μg and 100 μg, 10 μg and 200 μg, 10 μg and 300 μg, 10 μg and 400 μg, 10 μg and 500 μg, 10 μg and 600 μg, 10 μg and 800 μg, 10 μg and 1,000 μg, 10 μg and 1,500 μg, 10 μg and 2,000 μg, 50 μg and 100 μg, 50 μg and 200 μg, 50 μg and 300 μg, 50 μg and 400 μg, 50 μg and 500 μg, 50 μg and 600 μg of neo-antigenic peptide per peptide. μg, 50μg~800μg, 50μg~1,000μg, 50μg~1,500μg, 50μg~2,000μg, 100μg~200μg, 100μg~300μg, 100μg~400μg, 100μg~500μg, 100μg~600 μg, 100μg~800μg, 100μg~1,000μg, 100μg~1,500μg, 100μg~2,000μg, 200μg~300μg, 200μg~400μg, 200μg~500μg, 200μg~600μg, 200μg ~800μg, 200μg~1,000μg, 200μg~1,500μg, 200μg~2,000μg, 300μg~400μg, 300μg~500μg, 300μg~600μg, 300μg~800μg, 300μg~1,000μg , 300μg~1,500μg, 300μg~2,000μg, 400μg~500μg, 400μg~600μg, 400μg~800μg, 400μg~1,000μg, 400μg~1,500μg, 400μg~2,000μg, 500 It is administered in dosages of 600μg, 500μg to 800μg, 500μg to 1,000μg, 500μg to 1,500μg, 500μg to 2,000μg, 600μg to 800μg, 600μg to 1,000μg, 600μg to 1,500μg, 600μg to 2,000μg, 800μg to 1,000μg, 800μg to 1,500μg, 800μg to 2,000μg, 1,000μg to 1,500μg, 1,000μg to 2,000μg, or 1,500μg to 2,000μg. In some embodiments, the subject is administered a neo-antigenic peptide at a dosage of 10 μg, 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg, 600 μg, 800 μg, 1,000 μg, 1,500 μg, or 2,000 μg per peptide.

[0217]

[0327] In some embodiments, the subject is administered pembrolizumab at a dosage of 50 mg to 400 mg. In some embodiments, the subject is administered pembrolizumab at a dosage 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, the subject is administered pembrolizumab at a dosage of at most 400 mg, 350 mg, 300 mg, 260 mg, 240 mg, 200 mg, 150 mg, 100 mg, or 75 mg.In some embodiments, the subject is administered pembrolizumab at a dose of between 50 mg and 75 mg, 50 mg and 100 mg, 50 mg and 150 mg, 50 mg and 200 mg, 50 mg and 220 mg, 50 mg and 240 mg, 50 mg and 260 mg, 50 mg and 280 mg, 50 mg and 300 mg, 50 mg and 350 mg, 50 mg and 400 mg, 75 mg and 100 mg, 75 mg and 150 mg, 75 mg and 200 mg, 75 mg and 220 mg, 75 mg and 240 mg, 75 ... ~260mg, 75mg~280mg, 75mg~300mg, 75mg~350mg, 75mg~400mg, 100mg~150mg, 100mg~200mg, 100mg~220mg, 100mg~240mg, 1 00mg~260mg, 100mg~280mg, 100mg~300mg, 100mg~350mg, 100mg~400mg, 150mg~200mg, 150mg~220mg, 150mg~240mg, 150mg~ 260mg, 150mg~280mg, 150mg~300mg, 150mg~350mg, 150mg~400mg, 200mg~220mg, 200mg~240mg, 200mg~260mg, 200mg~280m g, 200mg~300mg, 200mg~350mg, 200mg~400mg, 220mg~240mg, 220mg~260mg, 220mg~280mg, 220mg~300mg, 220mg~350mg, 220 In some embodiments, the subject is administered pembrolizumab at a dosage 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.

[0218]

[0328] The concentration of active compound in drug composition will depend on the absorption, distribution, inactivation and excretion rate of drug and other factors known to those skilled in the art.It should be noted that dosage value will also vary according to the severity of the condition to be alleviated.It should be further understood that for any specific subject, specific dosage regimen should be adjusted over time according to individual need and the professional judgment of the person who administers or supervises the administration of composition, and the concentration ranges shown herein are merely illustrative and are not intended to limit the scope or implementation of the compositions described in claims.Active ingredient can be administered once, or can be divided into multiple smaller doses and administered at various time intervals.

[0219]

[0329] The present disclosure provides pharmaceutical compositions containing at least one tumor-specific neoantigen described herein. In embodiments, the pharmaceutical compositions contain a pharmaceutically acceptable carrier, excipient, or diluent, including any pharmaceutical agent that does not itself induce the production of an immune response harmful to the subject receiving the composition and can be administered without undue toxicity. As used herein, the term "pharmaceutically acceptable" means approved by a federal or state government regulatory agency or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias for use in animals, and more particularly in humans. These compositions may be useful for the treatment and / or prevention of viral infections and / or autoimmune diseases.

[0220]

[0330] A thorough discussion of pharmaceutically acceptable carriers, diluents, and other excipients is provided in Remington's Pharmaceutical Sciences (17th ed., Mack Publishing Company) and Remington: The Science and Practice of Pharmacy (21st ed., Lippincott Williams & Wilkins), which are hereby incorporated by reference. The formulation of a pharmaceutical composition should be suitable for the method of administration. In embodiments, the pharmaceutical composition is suitable for human administration and may be sterile, non-particulate, and / or non-pyrogenic.

[0221]

[0331] 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.

[0222]

[0332] Wetting agents, emulsifiers, and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0223]

[0333] 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, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0224]

[0334] In embodiments, the pharmaceutical composition is provided in a solid form, for example, a lyophilized powder, solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder suitable for reconstitution.

[0225]

[0335] In embodiments, the pharmaceutical composition is supplied in liquid form, e.g., in a sealed container indicating the quantity and concentration of the active ingredient in the pharmaceutical composition, hi a related embodiment, the liquid form of the pharmaceutical composition is supplied in a sealed container.

[0226]

[0336] Methods for formulating pharmaceutical compositions of the present disclosure are conventional and well known in the art (see Remington and Remington's). One of ordinary skill in the art can readily formulate pharmaceutical compositions having desired characteristics (e.g., route of administration, biosafety, and release profile).

[0227]

[0337] Methods for preparing pharmaceutical compositions include bringing the active ingredient into association with a pharmaceutically acceptable carrier and, optionally, one or more accessory ingredients. Pharmaceutical compositions can be prepared by uniformly and intimately bringing the active ingredient into association with a liquid carrier or a finely divided solid carrier, or both, and then, if necessary, shaping the product. Additional methodologies for preparing pharmaceutical compositions, including the preparation of multilayer dosage forms, are described in Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (9th Edition) (9th cd.), Lippincott Williams & Wilkins), which is hereby incorporated by reference herein.

[0228]

[0338] Pharmaceutical compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and gum arabic or tragacanth), powders, granules, or in such forms as a solution or suspension in an aqueous or non-aqueous liquid, or in such forms as an oil-in-water or water-in-oil liquid emulsion, or in such forms as an elixir or syrup, or in such forms as a troche (using an inert base, e.g., gelatin and glycerin, or sucrose and gum arabic), and / or as a mouthwash, each containing, as an active ingredient, a predetermined amount of a compound described herein, its derivative, or a pharmaceutically acceptable salt or prodrug thereof. The active ingredient may also be administered as a bolus, electuary, or paste.

[0229]

[0339] In solid dosage forms for oral administration (e.g., capsules, tablets, pills, dragees, powders, granules, and the like), the active ingredient may be admixed with one or more pharmaceutically acceptable carriers, excipients, or diluents, such as sodium citrate or calcium hydrogen phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or gum acacia; (3) humectants, such as glycerol; and (4) disintegrating agents, such as agar-agar, calcium carbonate. , potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarders, such as paraffin; (6) absorption promoters, such as quaternary ammonium compounds; (7) wetting agents, such as acetyl alcohol and glycerol monostearate; (8) adsorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets, and pills, the pharmaceutical compositions may also contain buffering agents. Solid compositions of a similar type can also be prepared using fillers in soft and hard gelatin capsules, and excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols.

[0230]

[0340] Tablets can be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants, and / or dispersants. Molded tablets can be made by molding a mixture of the powdered active ingredient moistened with an inert liquid diluent in a suitable machine.

[0231]

[0341] If desired, tablets or other solid dosage forms, such as dragees, capsules, pills, and granules, can be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the art.

[0232]

[0342] In some embodiments, in order to prolong the effect of active ingredient, it is desirable to slow down the absorption of compound from subcutaneous or intramuscular injection.This can be achieved by using a liquid suspension of crystalline or amorphous material with poor water solubility.Therefore, the absorption rate of active ingredient depends on its dissolution rate, and the dissolution rate can also depend on crystal size and crystalline form.Alternatively, the delayed absorption of parenterally administered active ingredient can be achieved by dissolving or suspending the compound in an oil vehicle.In addition, the inclusion of an agent that delays absorption, such as aluminum monostearate and gelatin, can result in the sustained absorption of injectable pharmaceutical forms.

[0233]

[0343] Controlled release parenteral compositions may be in the form of aqueous suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, emulsions, or the active ingredient may be incorporated into biocompatible carriers, liposomes, nanoparticles, implants, or infusion devices.

[0234]

[0344] Materials used in the preparation of microspheres and / or microcapsules include biodegradable / bioerodible polymers such as polyglactin, poly-(isobutyl cyanoacrylate), poly(2-hydroxy-L-glutamine) and poly(lactic acid).

[0235]

[0345] Biocompatible carriers that can be used in formulating controlled release parenteral formulations include carbohydrates, such as dextrans, proteins, such as albumin, lipoproteins, or antibodies.

[0236]

[0346] The material used in the implant can be non-biodegradable, for example, polydimethylsiloxane, or biodegradable, for example, poly(carbohydrate lactone), poly(lactic acid), poly(glycolic acid), or (polyorthoester).

[0237]

[0347] In embodiments, the active ingredient is administered by aerosol.This can be achieved by preparing aqueous aerosol, liposome preparation or solid particles containing the compound.Non-aqueous (e.g., fluorocarbon propellant) suspension can be used.The pharmaceutical composition can also be administered using ultrasonic nebulizer, which minimizes the exposure of the drug to shear, which can cause the compound to decompose.

[0238]

[0348] Usually, aqueous aerosol is prepared by preparing the aqueous solution or suspension of active ingredient together with conventional pharmaceutically acceptable carrier and stabilizer.Carrier and stabilizer vary according to the requirements of specific compound, but typically comprise nonionic surfactant (Tween, Pluronic or polyethylene glycol), harmless protein such as serum albumin, sorbitan ester, oleic acid, lecithin, such as glycine, buffer, salt, sugar or sugar alcohol.Aerosol is generally prepared from isotonic solution.

[0239]

[0349] Dosage forms for topical or transdermal administration of the active ingredient include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants as required.

[0240]

[0350] Suitable transdermal patches 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, and 5,023,084, which are incorporated herein by reference. The transdermal patch may also be any transdermal patch known in the art, including transscrotal patches. The pharmaceutical composition in such a transdermal patch may contain one or more absorption enhancers or skin permeation enhancers 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 systems used in the present disclosure may be based on iontophoresis, diffusion, or a combination of these two effects.

[0241]

[0351] Transdermal patches have the additional advantage of providing controlled delivery of active ingredients to the body.Such dosage forms can be prepared by dissolving or dispersing the active ingredients in a suitable medium.Absorption enhancers can also be used to increase the flux of the active ingredients across the skin.The rate of such flux can be controlled by either providing a rate-limiting membrane or dispersing the active ingredients in a polymer matrix or gel.

[0242]

[0352] Such pharmaceutical compositions may be in the form of creams, ointments, lotions, liniments, gels, hydrogels, solutions, suspensions, sticks, sprays, pastes, plasters, and other types of transdermal drug delivery systems. The compositions may also include pharmaceutically acceptable carriers or excipients, such as emulsifiers, antioxidants, buffers, preservatives, humectants, penetration enhancers, chelating agents, gel-forming agents, ointment bases, fragrances, and skin protectants.

[0243]

[0353] Examples of emulsifying agents include, but are not limited to, naturally occurring gums such as gum acacia or gum tragacanth, naturally occurring phosphatides such as soybean lecithin, and sorbitan monooleate derivatives.

[0244]

[0354] Examples of antioxidants include, but are not limited to, butylhydroxyanisole (BHA), ascorbic acid and derivatives, tocopherol and its derivatives, and cysteine.

[0245]

[0355] Examples of preservatives include, but are not limited to, trehalose, parabens such as methyl and propyl p-hydroxybenzoate, and benzalkonium chloride.

[0246]

[0356] Examples of humectants include, but are not limited to, glycerin, propylene glycol, sorbitol, and urea.

[0357] 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 or monomethyl ether with propylene glycol monolaurate or methyl laurate, eucalyptol, lecithin, TRANSCUTOL, and AZONE.

[0247]

[0358] Examples of chelating agents include, but are not limited to, sodium EDTA, citric acid, and phosphoric acid.

[0359] Examples of gel-forming agents include, but are not limited to, Carbopol, cellulose derivatives, bentonite, alginates, gelatin, and polyvinylpyrrolidone.

[0248]

[0360] In addition to the active ingredient, the ointments, pastes, creams and gels of the present disclosure may contain excipients such as animal and vegetable fats, oils, waxes, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc and zinc oxide, or mixtures thereof.

[0249]

[0361] Powders and sprays can contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.

[0250]

[0362] Injectable depot forms are prepared by forming microencapsulated matrices of the compounds of the present disclosure in biodegradable polymers such as polylactic acid-glycolic acid. Depending on the ratio of compound to polymer and the properties of the specific polymer used, the compound release rate can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations can also be prepared by entrapping drugs in liposomes or microemulsions that are compatible with body tissues.

[0251]

[0363] Subcutaneous implants are well known in the art and are suitable for use in the present disclosure. Subcutaneous implants are preferably non-irritating and mechanically resilient. The implants can be matrix-type, reservoir-type, or a hybrid of these. In matrix-type devices, the carrier material can be porous or non-porous, solid or semi-solid, and permeable or impermeable to the active compound(s) or compounds. The carrier material can be biodegradable or slowly erode after administration. In some cases, the matrix is non-degradable, but instead undergoes protein degradation depending on the diffusion of the active compound from the matrix. An alternative subcutaneous implant utilizes a reservoir device in which the active compound(s) or compounds are surrounded by a rate-limiting membrane, e.g., a membrane that is independent of component concentration (having zero-order kinetics). Also suitable for use are devices consisting of a matrix surrounded by a rate-limiting membrane.

[0252]

[0364] Both reservoir-type and matrix-type devices contain materials such as polydimethylsiloxane, e.g., SILASTIC, or other silicone rubbers. The matrix materials can be insoluble polypropylene, polyethylene, polyvinyl chloride, vinyl acetate, polystyrene, and polymethacrylate, as well as glycerol esters of the glycerol palmitate-stearate, glycerol stearate, and glycerol behenate types. The materials can be hydrophobic or hydrophilic polymers and, if necessary, can contain solubilizers.

[0253]

[0365] The subcutaneously implanted device may be a slow release capsule made of any suitable polymer, for example, as described in U.S. Pat. Nos. 5,035,891 and 4,210,644, which references are hereby incorporated by reference herein.

[0254]

[0366] Generally, at least four different approaches are applicable to rate-controlling the release and transdermal permeation of drug compounds: membrane-moderated systems, adhesive diffusion-controlled systems, matrix dispersion systems, and microreservoir systems. It is understood that controlled-release transdermal and / or topical compositions can be achieved by using a suitable mixture of these approaches.

[0255]

[0367] In membrane-controlled systems, the active ingredient resides in a reservoir completely enclosed in a shallow compartment molded from a drug-impermeable laminate, such as a metal-plastic laminate, and a rate-controlling polymer membrane, such as a microporous or nonporous polymer membrane, such as an ethylene-vinyl acetate copolymer. The active ingredient is released through the rate-controlling polymer membrane. Within the drug reservoir, the active ingredient may be dispersed in a solid polymer matrix or suspended in a non-leaching viscous liquid medium, such as a silicone solution. A thin layer of adhesive polymer is applied to the outer surface of the polymer membrane to achieve intimate contact between the transdermal system and the skin surface. The adhesive polymer may be a polymer that is hypoallergenic and compatible with the active drug substance.

[0256]

[0368] The active ingredient reservoir in the adhesive diffusion-controlled system is formed by dispersing the active ingredient directly into an adhesive polymer and then spreading the active ingredient-containing adhesive onto a flat sheet of substantially drug-impermeable metal-plastic backing, e.g., by solvent casting, to form a thin drug reservoir layer.

[0257]

[0369] Matrix dispersion systems are characterized in that a reservoir of active ingredient is formed by dispersing the active ingredient substantially homogeneously in a hydrophilic or lipophilic polymer matrix. The drug-containing polymer is then molded into a disk with a well-defined surface area and controlled thickness. An adhesive polymer is spread around the periphery to form an adhesive strip around the disk.

[0258]

[0370] Microreservoir systems can be considered a combination of reservoir and matrix dispersion systems, where the active agent reservoir is formed by first suspending drug solids in an aqueous solution of a water-soluble polymer, and then dispersing the drug suspension in a lipophilic polymer to form non-leaching microspheres of the drug reservoir.

[0259]

[0371] Any of the controlled-release, sustained-release, and extended-release compositions described herein can be formulated to release the active ingredient in about 30 minutes to about 1 week, about 30 minutes to about 72 hours, about 30 minutes to 24 hours, about 30 minutes to 12 hours, about 30 minutes to 6 hours, about 30 minutes to 4 hours, and about 3 hours to 10 hours. In embodiments, an effective concentration of the active ingredient 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 more after administration of the pharmaceutical composition to the subject. VII. Vaccines or Immunogenic Compositions

[0372] The present disclosure relates to methods of combination treatments, which include at least an immunogenic composition, e.g., a neoplastic vaccine or immunogenic composition, capable of generating a specific T cell response. The neoplastic vaccine or immunogenic composition includes neoantigenic peptides and / or neoantigenic polypeptides corresponding to tumor-specific neoantigens identified by the methods described herein.

[0260]

[0373] A suitable neoplasia vaccine or immunogenic composition may contain multiple tumor-specific neoantigenic peptides. In certain embodiments, the vaccine or immunogenic composition may contain between 1 and 100 sets of peptides, between 1 and 50 such peptides, between 10 and 30 sets of peptides, or between 15 and 25 peptides. According to other embodiments, the vaccine or immunogenic composition may contain at least one peptide, e.g., 2, 3, 4, or 5 peptides. In certain embodiments, the vaccine or immunogenic composition may contain 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 the vaccine or immunogenic composition can be administered to a subject. Each dose of the vaccine composition may contain a different set of peptides. For example, one dose, or a portion of one dose, of the composition may contain five peptides. Another portion of the dose may contain a different set of 5 peptides.

[0261]

[0374] Those skilled in the art can determine the optimal amount of each peptide to be included in a vaccine or immunogenic composition and the optimal administration regimen without undue experimentation. For example, the peptide or its variant can be prepared for intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. Methods of peptide injection include sc, id, ip, im, and iv. Methods of DNA injection include id, im, sc, ip, and iv. For example, a dose of between 1 and 500 mg, between 50 μg and 1.5 mg, or between 10 μg and 500 μg of peptide or DNA can be given, and the dose may depend on the respective peptide or DNA. Doses in this range have been used successfully in previous studies (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 vaccines or immunogenic compositions are known to those skilled in the art.

[0262]

[0375] The vaccine or immunogenic composition can be administered to a subject via one or more subcutaneous injections. In one embodiment, a single dose of the composition can be divided into one or more subcutaneous injections for administration. For example, a single dose of the composition can be divided into 1, 2, 3, 4, 5, 6, 7, or 8 different subcutaneous injections. Each injection of the composition can contain one or more peptides. The peptides in each injection of the single dose of the composition can contain different sets of peptides. In some examples, each injection of the single dose of the composition contains 1, 2, 3, 4, 5, or 6 different peptides. Alternatively, each subcutaneous injection of the single dose of the composition can contain the same set of peptides. In some cases, multiple injections as part of a single dose of the vaccine or immunogenic composition can contain different sets of peptides. For example, a single dose of the composition can be divided into four injections, each containing five different sets of peptides.

[0263]

[0376] Vaccine or immunogenic composition can be administered to a single location of a patient as a subcutaneous injection.For example, a single dose of vaccine or immunogenic composition can be administered to a patient's limb as a single injection.If a single dose of composition is divided into one or more injections, the dose can be administered to multiple locations of the subject.For example, if the dose is divided into four different injections, the four different injections can be administered to different limbs.In some cases, multiple injections as part of a single dose of composition can be administered to the same location at different time periods.For example, a period of 5, 10, 15, 20, 30, 50 or 60 minutes can be provided between different injections of one single dose of vaccine or immunogenic composition.

[0264]

[0377] In one embodiment of the present disclosure, different tumor-specific neo-antigenic peptides and / or polypeptides are selected for use in a neoplasia vaccine or immunogenic composition to maximize the likelihood of generating an immune attack against a patient's neoplasm / tumor. Without wishing to be bound by theory, it is believed that including a variety of tumor-specific neo-antigenic peptides can generate a broad range of immune antigens against neoplasms / tumors. In one embodiment, the selected tumor-specific neo-antigenic peptide / polypeptide is encoded by a missense mutation. In a second embodiment, the selected tumor-specific neo-antigenic peptide / polypeptide is encoded by a combination of a missense mutation and a neoORF mutation. In a third embodiment, the selected tumor-specific neo-antigenic peptide / polypeptide is encoded by a neoORF mutation.

[0265]

[0378] In one embodiment, where the selected tumor-specific neo-antigenic peptides / polypeptides are encoded by missense mutations, the peptides and / or polypeptides are selected based on their ability to associate with specific MHC molecules of the patient. Peptides / polypeptides derived from neoORF mutations can also be selected based on their ability to associate with specific MHC molecules of the patient, but may also be selected even if they are not predicted to associate with specific MHC molecules of the patient.

[0266]

[0379] The vaccine or immunogenic composition may generate a specific cytotoxic T cell response and / or a specific helper T cell response.

[0380] The vaccine or immunogenic composition may further comprise an adjuvant and / or a carrier. Examples of useful adjuvants and carriers are provided herein. The peptides and / or polypeptides in the composition may be associated with a carrier, such as, for example, a protein or antigen-presenting cell, such as a dendritic cell (DC), that can present the peptide to T cells.

[0267]

[0381] An adjuvant is any substance whose incorporation into a vaccine or immunogenic composition increases or otherwise modifies the immune response to the mutant peptide. A carrier is a scaffold, such as a polypeptide or polysaccharide, to which the neo-antigenic peptide can be associated. Optionally, the adjuvant is covalently or non-covalently conjugated to the peptide or polypeptide of the present disclosure.

[0268]

[0382] The ability of adjuvants to increase the immune response to antigens is typically manifested by a significant increase in immune-mediated reactions or the alleviation of disease symptoms.For example, the increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies raised against antigens, and the increase in T cell activity is typically manifested by an increase in cell proliferation or cytotoxicity or cytokine secretion.Adjuvants can also modify immune responses, for example, by changing primary humoral or Th2 responses into primary cellular responses or Th1 responses.

[0269]

[0383] Suitable adjuvants include 1018 ISS, aluminum salts, Amplivax, AS 15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Julvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL vector systems, PLG microparticles, resiquimod, SRL172, virosomes and other virus-like particles, YF-17D, VEGF These include, but are not limited to, Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA), derived from saponin; mycobacterial extracts and synthetic bacterial cell wall mimics, as well as other proprietary adjuvants such as Ribi's Detox, Quil, or Superfos. Several immunological adjuvants specific for dendritic cells (e.g., F59) and their preparation have been previously described (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 dendritic cell migration to lymphoid tissues (e.g., TNF-alpha), accelerating dendritic cell maturation into efficient antigen-presenting cells for 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).

[0270]

[0384] Toll-like receptors (TLRs) can also be used as adjuvants. TLRs are important members of the family of pattern recognition receptors (PRRs) that recognize conserved motifs shared by many microorganisms, called "pathogen-associated molecular patterns" (PAMPS). Recognition of these "danger signals" activates multiple components of the innate and adaptive immune systems. TLRs are expressed by cells of the innate and adaptive immune systems, such as dendritic cells (DCs), macrophages, T and B cells, mast cells, and granulocytes, and are localized in different intracellular compartments, such as the plasma membrane, lysosomes, endosomes, and endolysosomes. Different TLRs recognize distinct 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 are activated by double-stranded RNA. TLR ligand binding leads to the activation of one or more intracellular signaling pathways, ultimately resulting in the production of many key molecules associated with inflammation and immunity (particularly the transcription factor NF-κB and type I interferon). TLR-mediated DC activation leads to enhanced DC activation; phagocytosis; upregulation of activation and costimulatory markers, such as CD80, CD83, and CD86; expression of CCR7, which enables DC migration to draining lymph nodes and facilitates antigen presentation to T cells; and increased secretion of cytokines, such as type I interferon, IL-12, and IL-6. All of these downstream events are crucial for the induction of adaptive immune responses.

[0271]

[0385] Among the most promising cancer vaccine or immunogenic composition adjuvants currently in clinical development are the TLR9 agonist CpG and the synthetic double-stranded RNA (dsRNA) TLR3 ligand poly-ICLC. In preclinical studies, poly-ICLC appears to be the most potent TLR adjuvant compared to LPS and CpG due to its induction of proinflammatory cytokines, lack of stimulation of IL-10, and maintenance of high levels of costimulatory molecules in IX si. Furthermore, poly-ICLC was recently directly compared with CpG in non-human primates (rhesus macaques) as an adjuvant for a protein vaccine or immunogenic composition consisting of human papillomavirus (HPV) 16 capsomers (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 April;5(4)).

[0272]

[0386] CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in vaccine or immunogenic composition settings. Without being bound by theory, CpG oligonucleotides act by activating the innate (non-adaptive) immune system through Toll-like receptors (TLRs), primarily TLR9. CpG-induced TLR9 activation enhances antigen-specific humoral and cellular responses to a wide variety of antigens, including peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates in both prophylactic and therapeutic vaccines. More importantly, it promotes dendritic cell maturation and differentiation, resulting in enhanced Th1 cell activation and the generation of potent cytotoxic T lymphocytes (CTLs), even in the absence of CD4 T cell help. The Th1 bias induced by TLR9 stimulation is maintained even in the presence of vaccine adjuvants that normally promote Th2 bias, such as alum or incomplete Freund's adjuvant (IFA). CpG oligonucleotides exhibit even greater adjuvant activity when formulated or co-administered with other adjuvants, or in formulations such as microparticles, nanoparticles, lipids, emulsions, or similar formulations, which are particularly needed to induce strong responses when the antigen is relatively weak. They also accelerate immune responses, allowing the antigen dose to be reduced by approximately two orders of magnitude in some experiments with antibody responses equivalent to those to a full-dose vaccine without CpG (Arthur M. Krieg, Nature Reviews, Drug Discovery, 5, June 2006, pp. 471-484). U.S. Patent No. 6,406,705 describes the use of CpG oligonucleotides in combination with non-nucleic acid adjuvants and antigens to induce antigen-specific immune responses. A commercially available CpG TLR9 antagonist is dSLIM (double stem-loop immunomodulator) from Mologen (Berlin, Germany), which can be a component of the pharmaceutical composition of the present disclosure. Other TLR binding molecules, for example, RNA binding TLR 7, TLR 8 and / or TLR 9, can also be used.

[0273]

[0387] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), Poly(I:C) (e.g., polyI: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 may act therapeutically and / or as an adjuvant. The amounts and concentrations of adjuvants and additives useful in the present disclosure can be determined by those skilled in the art without undue experimentation. Additional adjuvants include colony-stimulating factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim).

[0274]

[0388] Poly-ICLC is a synthetically prepared double-stranded RNA consisting of polyI and polyC strands with an average length of approximately 5000 nucleotides, stabilized against heat denaturation and hydrolysis by serum nucleases by the addition of polylysine and carboxymethylcellulose. This compound activates the RNA helicase domains of TLR3 and MDA5, both of which are members of the PAMP family. This activation leads to DC and natural killer (NK) cell activation and the production of a "natural mix" of type I interferons, cytokines, and chemokines. Furthermore, poly-ICLC exerts more direct, broad-spectrum anti-infective and potentially anti-tumor effects, mediated by two IFN-inducible nuclear enzyme systems, 2'5'-OAS and P1 / eIF2a kinase (also known as PKR(4-6)), as well as RIG-I helicase and MDA5.

[0275]

[0389] In rodents and non-human primates, poly-ICLC has been shown to enhance T cell responses to viral antigens, cross-priming, and the induction of tumor-, virus-, and autoantigen-specific CD8+ T cells. In a recent study in non-human primates, poly-ICLC was found to be essential for the generation of antibody responses and T cell immunity against HIV Gag p24 protein, either targeted or untargeted to DCs. This highlights its effectiveness as a vaccine adjuvant.

[0276]

[0390] In human subjects, transcriptional analysis of serial whole blood samples revealed similar gene expression profiles among eight healthy human volunteers who received a single subcutaneous (sc) dose of poly-ICLC, and differential expression of ∼212 genes between these eight subjects and four subjects who received placebo. Unexpectedly, comparison of the poly-ICLC gene expression data with previous data from volunteers immunized with the highly effective yellow fever vaccine YF17D showed that numerous transcriptional and signaling canonical pathways, including those of the innate immune system, were similarly upregulated at their peak.

[0277]

[0391] More recently, immunological analyses were reported for patients with ovarian, fallopian tube, and primary peritoneal cancer in second or third complete clinical remission who were treated with synthetic overlapping long peptides (OLPs) from the cancer-testis antigen NY-ESO-1 alone, with Montanide-ISA-51, or with 1.4 mg of poly-ICLC and Montanide in a phase 1 subcutaneous vaccination study. The generation of NY-ESO-1-specific CD4+ and CD8+ T cell and antibody responses was significantly enhanced by the addition of poly-ICLC and Montanide compared with OLP alone or OLP and Montanide.

[0278]

[0392] A vaccine or immunogenic composition according to the present disclosure may contain more than one different adjuvant. Furthermore, the present disclosure encompasses therapeutic compositions containing any adjuvant substance, including any of those discussed herein. It is also contemplated that the peptide or polypeptide and the adjuvant can be administered separately in any suitable order.

[0279]

[0393] The carrier can be present independently of the adjuvant. The carrier can be covalently linked to the antigen. The carrier can also be added to the antigen by inserting DNA encoding the carrier in frame with the DNA encoding the antigen. The function of the carrier can be, for example, to confer stability, increase biological activity, or extend serum half-life. Extending half-life can help reduce the number of applications and lower the dose, which is therefore beneficial for therapeutic reasons but also for economic reasons. Furthermore, the carrier can assist in presenting the peptide to T cells. The carrier can be any suitable carrier known to those skilled in the art, such as a protein or antigen-presenting cell. The carrier protein can be, but is not limited to, keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones such as insulin or palmitic acid. For human immunization, the carrier can be a physiologically acceptable carrier that is acceptable and safe for humans. However, tetanus toxoid and / or diphtheria toxoid are suitable carriers in one embodiment of the present disclosure. Alternatively, the carrier can be a dextran, such as sepharose.

[0280]

[0394] Cytotoxic T cells (CTLs) recognize antigens in the form of peptides bound to MHC molecules, rather than intact foreign antigens themselves. MHC molecules themselves are located on the cell surface of antigen-presenting cells. Therefore, CTL activation is possible only in the presence of a trimeric complex of peptide antigen, MHC molecule, and APC. Correspondingly, CTLs can enhance immune responses not only when peptides are used to activate CTLs, but also when APCs bearing the respective MHC molecules are added. Thus, in some embodiments, a vaccine or immunogenic composition according to the present disclosure further contains at least one antigen-presenting cell.

[0281]

[0395] Antigen-presenting cells (or stimulator cells) typically have MHC class I or II molecules on their surface and, in one embodiment, are substantially incapable of loading MHC class I or II molecules with a selected antigen themselves. As described in more detail herein, MHC class I or II molecules can be readily loaded with a selected antigen in vitro.

[0282]

[0396] CD8+ cell activity can be augmented by the use of CD4+ cells. Identification of CD4 T+ cell epitopes of tumor antigens has attracted attention because many immune-based treatments for cancer may be more effective if both CD8+ and CD4+ T lymphocytes are used to target a patient's tumor, and CD4+ cells can enhance CD8 T cell responses. Many studies in animal models have clearly demonstrated better results when both CD4+ and CD8+ T cells are involved in the antitumor response (see, e.g., 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 have been identified that are applicable for developing treatments for different types of cancer (see, e.g., Kobayashi et al. (2008) Current Opinion in Immunology 20:221-27). For example, HLA-DR-restricted helper peptides from tetanus toxoid were used in melanoma vaccines to nonspecifically 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 may be applicable at three levels that differ in their tumor specificity: 1) a broad level, where universal CD4+ epitopes (e.g., tetanus toxoid) can be used to enrich for CD8+ cells; 2) an intermediate level, where native tumor-associated CD4+ epitopes can be used to enrich for CD8+ cells; and 3) a patient-specific level, where neoantigen CD4+ epitopes can be used to enrich for CD8+ cells in a patient-specific manner.

[0283]

[0397] CD8+ cell immunity can also be generated by neoantigen-loaded dendritic cell (DC) vaccines. DCs are potent antigen-presenting cells that initiate T cell immunity and can be used as cancer vaccines when loaded with one or more peptides of interest, e.g., by direct peptide injection. For example, patients newly diagnosed with metastatic melanoma were immunized with IL-12p70-producing patient DC vaccines using CD40 L / IFN-g-activated mature DCs pulsed with self-peptides against three 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 contemplated within the scope of the present disclosure that neoantigen-loaded DCs can be prepared using the synthetic TLR 3 agonist polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC) to stimulate DCs. Poly-ICLC is a potent, individualized stimulator of human DC maturation, as assessed by upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon gamma-inducible protein 10 (IP-10), interleukin-1 (IL-1), and type I interferon (IFN), and minimal interleukin-10 (IL-10) production. DCs can be differentiated from frozen peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis, or PBMCs can be isolated by Ficoll gradient centrifugation and frozen in aliquots.

[0284]

[0398] As an example, the following 7-day activation protocol can be used: Day 1 - PBMCs are thawed and seeded onto tissue culture flasks to select for monocytes that adhere to the plastic surface after 1-2 hours of incubation at 37°C in a tissue culture incubator. After incubation, lymphocytes are washed out and cultured for 5 days in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) to differentiate into immature DCs. On day 6, immature DCs are pulsed with keyhole limpet hemocyanin (KLH) protein, which serves as a control for vaccine quality and can boost vaccine immunogenicity. DCs are stimulated to mature, loaded with peptide antigen, and incubated overnight. On day 7, cells are washed and frozen in 1 ml aliquots containing 4-20 x 10(6) cells using a controlled-rate freezer. Before DCs are injected into patients, batches of DCs are lot-release tested to ensure they meet minimum specifications (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).

[0285]

[0399] DC vaccines can be incorporated into scaffold systems to facilitate delivery to patients. Therapeutic treatment of patient neoplasms with DC vaccines can utilize biomaterial systems that release factors that recruit host dendritic cells to the device; differentiate resident immature DCs by locally presenting adjuvants (e.g., danger signals) while releasing antigens; and promote the release of activated, antigen-loaded DCs to lymph nodes (or desired sites of action), where they can interact with T cells to generate potent cytotoxic T lymphocyte responses against cancer neoantigens. Implantable biomaterials can be used to generate potent cytotoxic T lymphocyte responses against neoplasms in a patient-specific manner. In this case, biomaterial-resident dendritic cells can be activated by exposure to danger signals that mimic infection, in concert with the release of antigens from the biomaterial. Activated dendritic cells then migrate from the biomaterial to lymph nodes to induce cytotoxic T effector responses. This approach has previously been demonstrated to lead to regression of established melanoma in preclinical studies using lysates prepared from tumor biopsies (see, e.g., Ali et al., (2209) In situ regulation of DC subsets and T cells mediates tumor regression in mice, Cancer Immunotherapy 1(8):1-10; Ali et al. (2009)).

[0286]

[0400] In some embodiments, the antigen-presenting cells are dendritic cells. Preferably, the dendritic cells are autologous dendritic cells pulsed with neoantigenic peptides. The peptides can be any suitable peptide that generates 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.

[0287]

[0401] Thus, in one embodiment of the present disclosure, a vaccine or immunogenic composition containing at least one antigen-presenting cell is pulsed or loaded with one or more peptides of the present disclosure. Alternatively, peripheral blood mononuclear cells (PBMCs) isolated from a patient can be loaded with peptides in vivo and then injected back into the patient. Alternatively, the antigen-presenting cell comprises an expression construct encoding a peptide of the present disclosure. The polynucleotide can be any suitable polynucleotide and can be transduced into dendritic cells, resulting in peptide presentation and immune induction.

[0288]

[0402] The pharmaceutical compositions of the present invention can be edited so that the selection, number, and / or amount of peptides present in the composition are tissue-, cancer-, 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. Selection can depend on the specific type of cancer, the status of the disease, previous treatment regimens, the patient's immune status, and, of course, the patient's HLA-halotype. Furthermore, vaccine or immunogenic compositions according to the present disclosure can contain components tailored to the individual needs of a particular patient. Examples include varying the amount of peptide according to the expression of relevant neoantigens in a particular patient, unwanted side effects due to an individual's allergies or other treatments, and adjustments to secondary treatments after a first treatment round or scheme.

[0289]

[0403] Pharmaceutical compositions containing the peptides of the present disclosure can be administered to individuals already suffering from cancer. In therapeutic applications, the compositions are administered to patients in an amount sufficient to elicit an effective CTL response against tumor antigens and to cure or at least partially arrest symptoms and / or complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use can depend, for example, on the peptide composition, the method of administration, the stage and severity of the disease being treated, the patient's weight and general health, and the judgment of the prescribing physician, but generally can range from about 1.0 μg to about 50,000 μg of peptide for an initial immunization (whether for therapeutic or prophylactic administration) for a 70 kg patient, followed by boosting dosages or about 1.0 μg to about 10,000 μg of peptide according to a boosting regimen over several weeks to months, depending on the patient's response and condition, and possibly by measuring specific CTL activity in the patient's blood. It should be noted that the peptides and compositions of the present disclosure can generally be utilized in serious conditions, i.e., potentially life-threatening or life-threatening situations, especially when cancer metastasizes.For therapeutic use, administration should be initiated as soon as possible after tumor detection or surgical removal.This is followed by boosting doses until at least significant symptom relief and for a period thereafter.

[0290]

[0404] Pharmaceutical compositions for therapeutic treatment (e.g., vaccine compositions) 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. The compositions can be administered at a surgical excision site to induce local immunity against tumors. The present disclosure provides compositions for parenteral administration comprising a solution of peptides, where the vaccine or immunogenic composition is dissolved or suspended in an acceptable carrier, e.g., an aqueous carrier. Various aqueous carriers can be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions can be sterilized by conventional, well-known sterilization techniques or sterile filtered. The resulting aqueous solutions can be packaged for use as is or lyophilized, with the lyophilized preparation being combined with a sterile solution prior to use. The compositions may contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.

[0291]

[0405] Liposomal suspensions containing peptides can be administered intravenously, topically, locally, etc., at doses that vary depending on, inter alia, the method of administration, the peptide being delivered, and the stage of disease being treated. To target immune cells, a ligand, such as, for example, an antibody or fragment thereof specific for a cell surface determinant of the desired immune system cell, can be incorporated into the liposome.

[0292]

[0406] For solid compositions, conventional or nanoparticulate non-toxic solid carriers can be used, including, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. Pharmaceutically acceptable non-toxic compositions for oral administration are formed by mixing any of the commonly employed excipients, such as those previously listed, with the active ingredient, generally 10-95%, i.e., one or more peptides of the present disclosure, in a concentration of, for example, 25%-75%.

[0293]

[0407] For aerosol administration, the immunogenic peptide can be supplied in finely divided form along with a surfactant and propellant. Typical percentages of peptide are 0.01% to 20% by weight, e.g., 1% to 10%. The surfactant, of course, is nontoxic and can be soluble in the propellant. Representative of such agents are esters or partial esters of fatty acids containing 6 to 22 carbon atoms, such as caproic acid, octanoic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric acid, and oleic acid, with aliphatic polyhydric alcohols or their cyclic anhydrides. Mixed esters, e.g., mixed or natural glycerides, can also be utilized. The surfactant can comprise 0.1% to 20% by weight, e.g., 0.25 to 5%, of the composition. The remainder of the composition is usually the propellant. A carrier can also be included as desired, e.g., lecithin for intranasal delivery.

[0294]

[0408] The peptides and polypeptides of the present disclosure can be readily chemically synthesized utilizing reagents that are free of bacterial or animal contaminants (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149-54, 1963).

[0295]

[0409] The peptides and polypeptides of the present disclosure can also be expressed by vectors, such as nucleic acid molecules as discussed herein, for example, RNA or DNA plasmids, viral vectors, such as poxviruses, for example, orthopoxviruses, avian poxviruses, or adenoviruses, AAVs, or lentiviruses.This approach involves the use of vectors to express the nucleotide sequences encoding the peptides of the present disclosure.When introduced into acutely or chronically infected hosts or into non-infected hosts, the vectors express immunogenic peptides, thereby eliciting host CTL responses.

[0296]

[0410] For treatment or immunization, nucleic acids encoding the peptides of the present disclosure and, if necessary, one or more of the 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 directly delivered as "naked DNA".This approach 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 delivered using ballistic delivery, for example, as described in U.S. Patent No. 5,204,253.Particles composed entirely of DNA can be administered.Alternatively, DNA can be attached to particles, such as gold particles. Generally, a plasmid for a vaccine or immunogenic composition may include DNA encoding an antigen (e.g., one or more neo-antigens) operably linked to regulatory sequences that control the expression or expression and secretion of the antigen from a host cell, e.g., a mammalian cell, e.g., from upstream to downstream: DNA for a promoter such as a mammalian promoter (e.g., a CMV promoter, e.g., an hCMV or mCMV promoter, e.g., an early-middle promoter, or an SV40 promoter—see the references cited or incorporated herein for useful promoters), DNA for a eukaryotic leader peptide for secretion (e.g., tissue plasminogen activator), DNA for the neo-antigen, and DNA encoding a terminator (e.g., a 3' UTR transcription terminator from a gene encoding bovine growth hormone or bGH polyA). A composition can contain more than one plasmid or vector, whereby each vector contains and expresses a different neo-antigen. Reference is made to Wasmoen U.S. Pat. No. 5,849,303 and Dale U.S. Pat. No. 5,811,104, which may be useful DNA or DNA plasmid formulations that can be formulated with or in cationic lipids; and to Loosmore U.S. Patent Application Publication No. 2003 / 0104008, which may be useful DNA or DNA plasmid formulations that can be formulated with or in cationic lipids.Additionally, the teachings in U.S. Patent Nos. 6,228,846 and 6,159,477 may be relied upon for teaching DNA plasmids that may be utilized in constructing and using DNA plasmids that contain and express in vivo.

[0297]

[0411] Nucleic acid can also be complexed with cationic compounds such as cationic lipids to deliver nucleic acid.Lipid-mediated gene delivery method is described in, for example, WO1996 / 18372; WO1993 / 24640; Mannino and Gould-Fogerite, BioTechniques 6(7):682-691(1988); U.S. Patent No. 5,279,833; WO1991 / 06309; and Feigner et al., Proc.Natl.Acad.Sci.USA 84:7413-7414(1987).

[0298]

[0412] RNA (e.g., mRNA) encoding the peptide of interest can also be used for delivery (see, e.g., Kiken et al., 2011; Su et al., 2011; see also U.S. Patent No. 8,278,036; Halabi et al., J Clin Oncol (2003) 21:1232-1237; Petsch et al., Nature Biotechnology 2012 Dec. 7;30(12):1210-6).

[0299]

[0413] Poxviruses that can be used in the practice of the present disclosure include, among others, poxviruses of the subfamily Chordopoxvirinae (vertebrate poxviruses), such as orthopoxviruses and avian poxviruses, such as vaccinia viruses (e.g., Wyeth strain, WR strain (e.g., ATCC® 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, pigeonpox, quailpox, and raccoonpox), synthetic or naturally occurring recombinant forms thereof, their uses, and methods for making and using such recombinants can be found in the scientific and patent literature, e.g., U.S. Pat. Nos. 4,603,112, 4,769,330, 5,110,587, 5, No. 174,993, No. 5,364,773, No. 5,762,938, No. 5,494,807, No. 5,766,597, No. 7,767,449, No. 6,780,407, No. 6,537,594, No. 6,265 ,189, No. 6,214,353, No. 6,130,066, No. 6,004,777, No. 5,990,091, No. 5,942,235, No. 5,833,975, No. 5,766,597, No. 5,756,10 No. 1, No. 7,045,313, No. 6,780,417, No. 8,470,598, No. 8,372,622, No. 8,268,329, No. 8,268,325, No. 8,236,560, No. 8,163,293, No. 7,964,398, No. 7,964,396, No. 7,964,395, No. 7,939,086, No. 7,923,017, No. 7,897,156, No. 7,892,533, No. 7,628,980, No. 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.Proc, Natl. Acad. Sci. 1983; 80(23): 7155 - 9, Smith GL. Nature 1983; 302: 490 - 5, Sullivan VJ. 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, Poulet, H Vaccine 2007; 25(July): 5606 - 12, Weyer J. Vaccine 2009; 27(November): 7198 - 201, Buller, RM Nature 1985; 317(6040): 813 - 5, Boiler RM. J. Virol. 1988; 62(3): 866 - 74, Flexner, C. Nature 1987; 330(6145): 259 - 62, Shida, H. J. Virol. 1988; 62(12): 4474 - 80, Kotwal GJ. J. Virol. 1989; 63(2): 600 - 6, Child, SJ. Virology 1990; 174(2): 625 - 9, Mayr A. Zentralbl Bakteriol 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); 10606 - 22), Goebel SJ. Virology 1990; (a, b)179: 247 - 66, Tartaglia, J. Virol. 1992; 188(1): 217 - 32, Najera J L. J. Virol. 2006; 80(12): 6033 - 47, Najera, JL. J. Virol. 2006; 80: 6033 - 6047, Gomez, CE. J. Gen. Virol.2007;88:2473-78、Mooij,P.Jour.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.Of Leukocyte Biology 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.Nature 2004;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、Corbett M.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、Kantoff PW.J.Clin.Oncol.2010、28、1099-1 105、Amato RJ.J.Clin.Can.Res.2010;16(22):5539-47、Kim,DW.Hum.Vaccine.2010:6:784-791, Oudard, S. Cancer Immunol.]mm another.201 1;60:261-71, Wyatt, LS. Aids Res. Hum. Retroviruses.2004;20:645-53, Gomez, CE. Virus Research 2004;105:1 1-22, 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. Gene therapy 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, MJOf Virol.2011;Oct:9854-62, Pantaleo, G. Curr Opin HIV-AIDS. 2010;5:391-396, each of which is incorporated herein by reference.

[0300]

[0414] For adenovirus vectors useful in the practice of the present disclosure, reference is made to U.S. Patent No. 6,955,808. Adenovirus vectors that can be used can be selected from the group consisting of Ad5, Ad35, Ad11, C6, and C7 vectors. The sequence of the adenovirus 5 ("Ad5") genome has been published. (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 this reference are hereby incorporated by reference.) Ad35 vectors are described in U.S. Patent Nos. 6,974,695, 6,913,922, and 6,869,794. Ad11 vectors are described in U.S. Patent No. 6,913,922. C6 adenoviral vectors are described in U.S. Patent 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. Patent No. 6,277,558. E1-deleted, E3-deleted, and / or E4-deleted adenoviral vectors can also be used. E1-deleted adenoviral mutants are replication-deficient in nonpermissive cells, or at least highly attenuated, so certain adenoviruses with mutations in the E1 region have an improved safety margin. Adenoviruses with mutations in the E3 region may enhance immunogenicity by disrupting the mechanism by which adenovirus downregulates MHC class I molecules.Adenoviruses with mutations in E4 may have low immunogenicity due to the suppression of late gene expression.Such vectors may be particularly useful when repeated vaccination using the same vector is desired.Adenoviral vectors in which E1, E3, E4, E1 and E3, and E1 and E4 are deleted or mutated can be used in accordance with the present disclosure. Additionally, "gutless" adenoviral vectors in which all viral genes are deleted can also be used in accordance with the present disclosure. Such vectors require a helper virus for their replication and specialized human 293 cells that express both Ela and Cre, conditions not present in the natural environment. Such "gutless" vectors are non-immunogenic, and therefore, the vectors can be administered multiple times for revaccination. "Gutless" adenoviral vectors can be used to insert heterologous inserts / genes, such as the transgenes of the present disclosure, and can also be used to simultaneously deliver multiple heterologous inserts / genes.

[0301]

[0415] For lentiviral vectors useful in the practice of this disclosure, reference is made to U.S. Patent Nos. 6,428,953, 6,165,782, 6,013,516, 5,994,136, 6,312,682, and 7,198,784, and the references cited therein.

[0302]

[0416] With regard to AAV vectors useful in practicing the present disclosure, reference is made to U.S. Patent Nos. 5,658,785, 7,115,391, 7,172,893, 6,953,690, 6,936,466, 6,924,128, 6,893,865, 6,793,926, 6,537,540, 6,475,769, and 6,258,595, and the references cited therein.

[0303]

[0417] Another vector is Bacille Calmette-Guerin (BCG). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vectors useful for therapeutic administration or immunization of the peptides of the present disclosure, such as Salmonella typhi vectors, will be apparent to those skilled in the art from the description herein.

[0304]

[0418] The vectors can be administered to have in vivo expression and responses that resemble the dose and / or response elicited by antigen challenge.

[0419] In some embodiments, a means of administering nucleic acids encoding peptides of the present disclosure uses a minigene construct encoding multiple epitopes. To create a DNA sequence encoding a selected CTL epitope (minigene) for expression in human cells, the amino acid sequence of the epitope is reverse-translated. A human codon usage table is used to guide codon selection for each amino acid sequence. These epitope-encoding DNA sequences are directly spliced, thereby creating a continuous polypeptide sequence. Additional elements can be incorporated into the minigene design to optimize expression and / or immunogenicity. Examples of amino acid sequences that can be reverse-translated and included in the minigene sequence include helper T lymphocytes, epitopes, leader (signal) sequences, and endoplasmic reticulum retention signals. Additionally, MHC presentation of CTL epitopes can be improved by including synthetic (e.g., polyalanine) or naturally occurring flanking sequences adjacent to the CTL epitope.

[0305]

[0420] The minigene sequence is converted to DNA by assembling oligonucleotides encoding the plus and minus strands of the minigene. Overlapping oligonucleotides (30-100 bases long) are synthesized under appropriate conditions using well-known techniques, phosphorylated, purified, and annealed. The ends of the oligonucleotides are joined using T4 DNA ligase. This synthetic minigene sequence, encoding the CTL epitope polypeptide, can then be cloned into the desired expression vector.

[0306]

[0421] Standard regulatory sequences well known to those skilled in the art are included in the vector to ensure expression in target cells.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).A large number of promoters can be used for this purpose, such as the human cytomegalovirus (hCMV) promoter.For other suitable promoter sequences, see U.S. Patent Nos. 5,580,859 and 5,589,466.

[0307]

[0422] Additional vector modifications may be desired 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 may be incorporated into the transcribed region of the minigene. To increase the minigene sequence, it may also be considered to include mRNA stabilization sequences. It has recently been reported that immune stimulatory sequences (ISS or CpG) contribute to the immunogenicity of DNA vaccines. If found to enhance immunogenicity, these sequences could be included outside the minigene coding sequence in the vector.

[0308]

[0423] In some embodiments, bicistronic vectors can be used to allow production of the minigene-encoded epitope and a second protein included to enhance or increase immunogenicity. Examples of proteins or polypeptides that can beneficially enhance immune responses when coexpressed include cytokines (e.g., IL2, IL11, IL12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), or costimulatory molecules. Helper (HTL) epitopes could be linked to intracellular targeting signals and expressed separately from CTL epitopes. This would allow for the HTL epitopes to be directed to different cellular compartments than the CTL epitopes. If necessary, this could facilitate more efficient entry of the HTL epitopes into the MHC class II pathway, thereby improving CTL induction. Specifically reducing the immune response by coexpressing immunosuppressive molecules (e.g., TGF-β) as opposed to CTL induction may be beneficial in certain diseases.

[0309]

[0424] Once an expression vector is selected, the minigene is cloned into the polylinker region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and DNA is prepared using standard techniques. The orientation and DNA sequence of the minigene, as well as all other elements contained in the vector, are confirmed using restriction mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as master and working cell banks.

[0310]

[0425] Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate-buffered saline (PBS). Various methods have been described, and new approaches may become available. As described herein, nucleic acids are conveniently formulated with cationic lipids. In addition, glycolipids, fusogenic liposomes, peptides, and compounds collectively referred to as protective, interactive, and non-aggregating (PINC) compounds may be complexed with purified plasmid DNA to affect variables such as stability, intramuscular distribution, or transport to specific organs or cell types.

[0311]

[0426] Target cell sensitization can be used as a functional assay for expression and MHC class I presentation of the minigene-encoded CTL epitope. Plasmid DNA is introduced into a mammalian cell line suitable as a target for a 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 for direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) can be cotransfected, and transfected cells can be enriched using fluorescence-activated cell sorting (FACS). These cells are then labeled with chromium-51 and used as target cells for epitope-specific CTL systems. Cell lysis, detected by 51Cr release, indicates the generation of MHC presentation of the minigene-encoded CTL epitope.

[0312]

[0427] In vivo immunogenicity is a second approach for functional testing of minigene-DNA formulations. Transgenic mice expressing the appropriate MHC molecules are immunized with the DNA product. The dose and route of administration are formulation-dependent (e.g., IM for DNA in PBS, IP for lipid-complexed DNA). 21 days after immunization, splenocytes are harvested and restimulated for one week in the presence of peptides encoding each epitope being tested. These effector cells (CTLs) are assayed for lysis of peptide-loaded, chromium-51-labeled target cells using standard techniques. Lysis of target cells sensitized by MHC loading of peptides corresponding to minigene-encoded epitopes demonstrates that the DNA vaccine is functional for in vivo induction of CTLs.

[0313]

[0428] Peptides can also be used to elicit CTLs ex vivo. The resulting CTLs can be used to treat chronic tumors in patients in need of treatment who have not responded to other conventional forms of therapy or to peptide vaccine therapeutic approaches. Ex vivo CTL responses against specific tumor antigens are induced by incubating the patient's CTL precursors (CTLp) with a source of antigen-presenting cells (APCs) and the appropriate peptide in tissue culture. After an appropriate incubation period (typically 1-4 weeks), during which the CTLp are activated, mature, and expand to become effector CTLs, the cells are returned to the patient, where they destroy their specific target cells (i.e., tumor cells). To optimize in vitro conditions for the generation of specific cytotoxic T cells, stimulator cell cultures are maintained in an appropriate serum-free medium.

[0314]

[0429] Prior to incubation of the stimulator cells 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 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 approximately 200 or more human class I MHC molecules 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 >3 μg / ml, 4 μg / ml, 5 μg / ml, 10 μg / ml, 15 μg / ml, or more of peptide.

[0315]

[0430] 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 CD8+ (effector) cells to stimulator cells may vary from individual to individual and may depend, in addition, on the suitability of the individual's lymphocytes for the culture conditions and the nature and severity of the disease state or other condition for which the described treatment modality is being used. The lymphocyte:stimulator cell ratio may be within the range of about 30:1 to 300:1. The effector / stimulator culture may be maintained for as long as necessary to stimulate a therapeutically usable or effective number of CD8+ cells.

[0316]

[0431] In vitro CTL induction requires specific recognition of peptides bound to allele-specific MHC class I molecules on APCs. The number of specific MHC / peptide complexes per APC is crucial for CTL stimulation, especially in primary immune responses. Although a small number of peptide / MHC complexes per cell is sufficient to render cells susceptible to CTL lysis or to stimulate secondary CTL responses, a significantly higher number of MHC / peptide complexes is required for successful activation of CTL precursors (pCTLs) during primary responses. Peptide loading of empty major histocompatibility complex molecules on cells allows for the induction of primary cytotoxic T lymphocyte responses.

[0317]

[0432] Because mutant cell lines do not necessarily represent all human MHC alleles, it is advantageous to use a method for removing endogenous MHC-associated peptides from the surface of APCs and then loading the resulting empty MHC molecules with the immunogenic peptide of interest. The use of non-transformed (non-tumorigenic), non-infected cells, and the patient's autologous cells as APCs is desirable for designing CTL induction protocols for the development of ex vivo CTL therapy. This application discloses a method for stripping endogenous MHC-associated peptides from the surface of APCs and then loading the desired peptide.

[0318]

[0433] Stable MHC class I molecules are trimeric complexes formed from the following elements: 1) a peptide, usually 8–10 residues long; 2) a transmembrane heavy chain polymorphic protein chain with peptide-binding sites in its a1 and a2 domains; and 3) a noncovalently associated nonpolymorphic light chain, p2 microglobulin. Removal of the bound peptide from the complex and / or dissociation of p2 microglobulin renders the MHC class I molecule nonfunctional and unstable, resulting in rapid degradation. All MHC class I molecules isolated from PBMCs have endogenous peptides bound to them. Therefore, the first step is to remove all endogenous peptides bound to MHC class I molecules on APCs without causing their degradation before exogenous peptides can be added to them.

[0319]

[0434] Two potential methods for completely liberating MHC class I molecules from bound peptides include destabilizing p2 microglobulin by lowering the culture temperature from 37°C to 26°C overnight, and stripping endogenous peptides from cells using mild acid treatment. These methods release previously bound peptides into the extracellular environment, thereby allowing new exogenous peptides to bind to empty class I molecules. The low-temperature incubation method allows exogenous peptides to efficiently bind to MHC complexes, but requires overnight incubation at 26°C, which may slow the metabolic rate of cells. Furthermore, cells that do not actively synthesize MHC molecules (e.g., resting PBMCs) are unlikely to produce large amounts of empty surface MHC molecules by the low-temperature procedure.

[0320]

[0435] Strong acid stripping involves peptide extraction with trifluoroacetic acid, pH 2, or acid denaturation of immunoaffinity-purified class I-peptide complexes. These methods are not feasible for CTL induction because it is important to remove endogenous peptides while maintaining APC viability and an optimal metabolic state, which is crucial for antigen presentation. Weakly acidic solutions at pH 3, such as glycine or citrate-phosphate buffer, have been used to identify endogenous peptides and tumor-associated T cell epitopes. This treatment is particularly effective because only MHC class I molecules are destabilized (and associated peptides are released), while other surface antigens, including MHC class II molecules, remain intact. Most importantly, treatment of cells with weakly acidic solutions does not affect cell viability or metabolic state. Weak acid treatment is rapid, as stripping of endogenous peptides is performed for 2 minutes at 4°C, allowing APCs to perform their functions whenever they are loaded with the appropriate peptide. This approach is utilized herein to generate peptide-specific APCs for the generation of primary antigen-specific CTLs. The resulting APCs are efficient in inducing peptide-specific CD8+ CTLs.

[0321]

[0436] Activated CD8+ cells can be effectively separated from stimulator cells by using one of various known methods.For example, monoclonal antibodies specific to stimulator cells, specific to the peptide loaded on stimulator cells, or specific to CD8+ cells (or their segments) can be used to bind to their appropriate complementary ligands.Then, antibody-tagged molecules can be extracted from the mixture of stimulator cells and effector cells by suitable means, for example, by well-known immunoprecipitation or immunoassay methods.

[0322]

[0437] The effective cytotoxic dose of activated CD8+ cells may differ between in vitro and in vivo use and may also vary depending on the amount and type of cells that are the ultimate target of these killer cells. This amount may also vary depending on the condition of the patient and should be determined by the practitioner, taking all appropriate factors into consideration. Approximately 5 × 10 6 ~5×10 7 cells, compared with approximately 1 × 10 6 ~Approx. 1×10 12 , about 1×10 8 ~Approx. 1×10 11 , or approximately 1 × 10 9 ~Approx. 1×10 10 of activated CD8+ cells can be utilized.

[0323]

[0438] As discussed herein, activated CD8+ cells are recovered from cell culture prior to administration of the CD8+ cells to the individual being treated. However, it is important to note that, unlike other current and proposed treatment modalities, the present method uses a cell culture system that is not non-tumorigenic. Therefore, if complete separation of stimulator cells and activated CD8+ cells is not achieved, there is no inherent risk known to be associated with administering a small number of stimulator cells, whereas administration of mammalian tumor-promoting cells can be extremely dangerous.

[0324]

[0439] Methods for reintroducing cellular components are known in the art and include procedures 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 by intravenous infusion is suitable.

[0325]

[0440] The practice of the present disclosure will utilize, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are well within the skill of those in the art. Such techniques are fully explained in such publications as "Molecular Cloning: A Laboratory Manual," 2nd Edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" and "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); and "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the present disclosure and therefore may be considered in making and practicing the present disclosure. Techniques particularly useful for certain embodiments are discussed in subsequent sections. VIII. Treatment method

[0441] The present disclosure provides methods of inducing a neoplasm / tumor-specific immune response in a subject, vaccinating against a neoplasm / tumor, treating and / or alleviating symptoms of cancer in a subject by administering to the subject a neoplasm vaccine or neo-antigenic peptide or composition of the present disclosure and at least one inhibitor, e.g., a checkpoint inhibitor or a chemotherapeutic agent.

[0326]

[0442] In particular, the present disclosure relates to methods of treating or preventing a neoplasm, comprising administering to a subject (a) a neoplasm vaccine or immunogenic composition, and (b) at least one inhibitor, e.g., a checkpoint inhibitor or chemotherapeutic agent.

[0327]

[0443] In one embodiment, a method of treating or preventing cancer in a human subject in need thereof includes administering to the human subject in need thereof (i) a polypeptide comprising a cancer-specific neoepitope 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) an HLA protein specific for a complex comprising an HLA protein expressed by the human subject and the cancer-specific neoepitope of a protein expressed by a cancer cell of the cancer. and (v) a T cell comprising a TCR of (iv); (a) a second component comprising an anti-cancer agent that is an antibody or antigen-binding portion thereof that specifically binds to a programmed death-1 (PD-1) receptor and inhibits PD-1 activity; and (b) a third component comprising 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.

[0328]

[0444] In accordance with the present disclosure, the neoplastic vaccines or immunogenic compositions described herein can be used in patients who have cancer or are at risk of developing cancer.

[0445] The disclosed combinations described are administered in amounts sufficient to induce a CTL response.

[0329]

[0446] A vaccine or immunogenic composition comprising a neo-antigenic peptide can be administered to a subject for the treatment of a condition. In addition to the neo-antigenic peptide, one or more inhibitors, such as checkpoint inhibitors or chemotherapeutic drugs, can be administered to the subject. The administration of one or more inhibitors, such as checkpoint inhibitors or chemotherapeutic drugs, can be performed before the administration of the neo-antigenic peptide. In some embodiments, one or more inhibitors, such as checkpoint inhibitors or chemotherapeutic drugs, are currently being administered to the patient. In such cases, one inhibitor, such as a checkpoint inhibitor or chemotherapeutic drug, can be administered before the administration of the neo-antigenic peptide. For example, in the case of a combination of neo-antigenic peptides.

[0330]

[0447] Patients can be screened before and after administration as described herein.Patients can undergo screening assessments to document their past health status and their current and future health status in general and in relation to their underlying diseases.Screening assessments can include tests such as vital signs (including diastolic and systolic blood pressure, heart rate, body temperature, weight and extension), electrocardiogram, symptom-based physical examination, hematology (including hematocrit, hemoglobin, RBC count, WBC count with differential, and platelet count), chemistry (including tests for glucose, urinary nitrogen, creatinine, sodium, potassium, calcium, total and direct bilirubin, AST, ALT, alkaline phosphatase, lactate dehydrogenase (LDH), and adrenocorticotropic hormone), liver function tests (for example, detecting the levels of AST, ALT, total and direct bilirubin), pregnancy test, CT or MRI; surgical or core needle biopsy of primary or metastatic tumor site for DNA and RNA sequencing; immunological analysis. Biopsy can be used to evaluate the presence of T cell infiltration into tumors and their location relative to the tumor border through 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 for analysis can include, but is not limited to, for example, CD3, CD4, CD8, CD45RO, PD-L1, PD-1, FoxP3, granzyme B, perforin, CD68, CD163, MHC class I, MHC class II, CD83 and CD11b.

[0331]

[0448] Immune response parameters can be analyzed for changes over time from basal levels before administration of treatment, and can include summaries of nucleic acid (e.g., DNA mutations, transcript abundance) characterization, histopathology, and immune cell analysis in tissues obtained during the pretreatment, prevaccination, and vaccination phases, as well as during the preliminary evaluation. Reports of test results can include tables depicting shifts from earlier time points to compare changes in immune parameters. Descriptive statistics and frequency distributions can be used as needed. Immunological analysis can include summaries of CD8+ and CD4+ T cell responses measured by ex vivo IFN-γ ELISpot and assessed by spot counting. Analysis can be used to assess changes from pretreatment to prevaccination, then to vaccination, and then to the preliminary evaluation. Reports can include medians and interquartile ranges, as well as tables depicting shifts from earlier time points for each patient. Additionally, nonparametric tests (e.g., Wilcoxon signed-rank tests) can be used to determine differences in ELISpot data between time points, if needed. Fold changes in biomarkers measured on a continuous scale can be summarized and compared across response categories using Wilcoxon rank sum tests between treatment arms for every cohort. For multigene assays, genes can be grouped into analysis sets to characterize cellular biological functions, as needed.

[0332]

[0449] Primary objectives may include: rates of adverse events and serious adverse events leading to treatment discontinuation [time frame: baseline to 90 days after the last dose of pembrolizumab]; rates of adverse events and serious adverse events leading to treatment discontinuation; and adverse events and serious adverse events detected during symptom-based physical examination (safety laboratory assessments, physical examination findings, vital signs, and changes in ECOG PS).

[0333]

[0450] Secondary endpoints may include: 1. Date of first documented PD based on objective response rate (ORR), defined as the proportion of patients achieving a complete response (CR) or partial response (PR) per Response Criteria in Solid Tumors (RECIST) v1.1. 2. Clinical benefit rate (CBR), defined as the proportion of patients achieving CR, PR, or stable disease (SD) per RECIST v1.1. 3. Duration of response, DOR, defined as the date of first documentation of confirmed response per RECIST v1.1 to the date of first documented PD. 4. Response conversion rate (RCR), defined as the proportion of patients experiencing an improvement in RECIST v1.1 category (e.g., PD to SD / PR / CR, SD to PR / CR, PR / CR) after vaccination. 5. Progression-free survival (PFS), defined as the time from the date of first dose to the date of first documented PD or death. 6. Overall survival, defined as the time from the date of enrollment to death from any cause.

[0334]

[0451] The tests and results may include detailed characterization of the phenotype and abundance of antigen-specific T cells in both the periphery and the tumor microenvironment. The abundance of regulatory cells, such as regulatory T cells or myeloid-derived suppressor cells, as well as T cell recognition, activation, and cytotoxicity, can also be evaluated using PBMCs and tumor cells. In addition, ex vivo induction of neoantigen T cell responses can also be performed in peripheral blood and leukapheresis samples. The presence of circulating tumor DNA (ctDNA) and vaccine-specific antibody responses can be evaluated after treatment with the compositions described herein.

[0335]

[0452] In one aspect, a method of treating or preventing cancer in a human subject in need thereof comprises administering to the human subject in need thereof (a) a first component comprising (i) a polypeptide comprising a cancer-specific neoepitope 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 neoepitope of a protein expressed by a cancer cell of the cancer neoepitope, or (v) a T cell comprising the TCR of (iv); (b) a second component comprising an anti-cancer agent that is an antibody or antigen-binding portion thereof that specifically binds to Programmed Death-1 (PD-1) receptor and inhibits PD-1 activity; and (c) a third component comprising platinum-based chemotherapy; (i) the method is characterized in that the cancer-specific neoepitope of a protein expressed by a cancer cell of the cancer is administered to the human subject. promotes epitope spreading of an epitope distinct from any of the neoepitopes; (ii) the median progression-free survival (PFS) of a first population of human subjects whose cancer has been treated with the first, second, and third components is longer than the median PFS of a second population of subjects whose cancer has been 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 whose cancer has been treated with the first, second, and third components is longer than the median PFS of a second population of subjects whose cancer has been treated with the second and / or third components but not the first component; or the ORR of a second population of subjects treated with the third component but not the first component; (iv) the percentage of subjects having at least a 12-month progression-free survival (PFS) in a first population of human subjects whose cancer is treated with the first, second, and third components is higher than the percentage of subjects having at least a 12-month PFS in a second population of subjects whose cancer is treated with the second and / or third components but not the first component;(v) the median overall survival (OS) of a first population of human subjects whose cancer is treated with the first, second, and third components is longer than the median OS of a second population of subjects whose cancer is treated with the second and / or third components but not the first component; (vi) the percentage of subjects who achieve a complete response, partial response, prolonged stable disease, or stable disease for 6 months or more (CBR) of a first population of human subjects whose cancer is treated with the first, second, and third components is longer than the median OS of a second population of subjects whose cancer is treated with the second and / or third components but not the first component. (vii) the reduction in tumor size in a first population of human subjects whose cancer is treated with the first, second, and third components is greater than the reduction in tumor size in a second population of subjects whose cancer is treated with the second and / or third components but not the first component; (viii) the level of tumor-infiltrating CD4+ T cells in a first population of human subjects whose cancer is treated with the first, second, and third components i...

Claims

1. 1. A method of treating or preventing cancer in a human subject in need thereof, comprising administering to said human subject in need thereof: (a)(i) a polypeptide comprising a cancer-specific neoepitope of a protein expressed by a cancer cell of said 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 neoepitope of a protein expressed by a cancer cell of the cancer neoepitope; or (v) T cells comprising the TCR of (iv). a first component comprising: (b) a second component comprising an anti-cancer agent that is an antibody or antigen-binding portion thereof that specifically binds to the Programmed Death-1 (PD-1) receptor and inhibits PD-1 activity; (c) a third component comprising platinum-based chemotherapy; administering the human subject (i) have not received previous systemic treatment for metastatic disease; (ii) have not previously received immunotherapy with an anti-PD-1 antibody, and (iii) have not previously received immunotherapy with an anti-PD-L1 antibody; method.

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

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

4. 4. The method of claim 3, wherein the manufacturing of the first component occurs during the 12 week period during which the combination of the second component and the third component is administered.

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

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

7. 7. The method of claim 5 or 6, wherein the step of administering the first component for a period of 12 weeks after the step of administering the combination of the second component and the third component for a period of 12 weeks comprises administering 5 priming doses of the first component, and 2 booster doses of the first component.

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

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

10. 10. The method of claim 9, wherein the second component is administered to the human subject after the period of 12 weeks during which the first and second components are administered.

11. 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 period of 12 weeks during which the first and second components are administered.

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

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

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

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

16. 16. The method of any one of claims 1 to 15, wherein the human subject has a KRAS mutation, a TP53 mutation, and / or a KEAP1 mutation.

17. 17. The method of claim 16, wherein the cancer-specific neoepitope of the first component does not include a KRAS neoepitope, a TP53 neoepitope, and / or a KEAP1 neoepitope.

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

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

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

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

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

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

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

25. 25. The method of claim 24, wherein the adjuvant comprises polyI:polyC.

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

27. 26. The method of claims 1 to 25, wherein the cancer-specific neoepitopes comprise at most 20 different cancer-specific neoepitopes of proteins expressed by cancer cells of the cancer.

28. 28. The method of any one of claims 1 to 27, comprising comparing (i) nucleic acid sequences obtained by whole genome or whole exome sequencing of cancer cells from a single subject with (ii) nucleic acid sequences obtained by whole genome or whole exome sequencing of non-cancerous cells from said single subject.

29. 30. The method of claim 28, comprising identifying a plurality of cancer-specific nucleic acid sequences that are unique to cancer cells of the human subject based on said comparing step.

30. 30. The method of claim 29, comprising predicting or calculating the binding affinity of cancer-specific neoepitope sequences encoded by the identified plurality of cancer-specific nucleic acid sequences to proteins encoded by HLA alleles of the human subject by HLA peptide binding analysis using a program implemented in a computer system.

31. an IC for a protein encoded by an HLA allele of said human subject that is less than 500 nM or 150 nM or less; ５０ 31. The method of claim 30, comprising selecting at least two cancer-specific neoepitopes predicted or calculated to have:

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

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

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

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

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

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

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

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

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

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

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

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

45. 45. The method of any one of claims 1 to 44, which promotes epitope spreading.

46. 45. The method of any one of claims 1 to 44, wherein the method promotes epitope spreading of an epitope that is distinct from any of the cancer-specific neoepitopes.

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

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

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

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

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

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

53. 45. The method of any one of claims 1-44, wherein the percentage of subjects achieving a complete response, partial response, long-term stable disease, or stable disease for 6 months or more (CBR) in a first population of human subjects treated for the cancer with the first, second, and third components is higher than the CBR in a second population of subjects treated for the cancer with the second and / or third components but not the first component.

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

55. 45. 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 whose cancer has been 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 whose cancer has been treated with the second and / or third components but not the first component.

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

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

58. CD4+ / CD62L specific for cancer-specific neoepitopes ｈｉ 45. The method of any one of claims 1 to 44, wherein the level of CD69+ / CD27+ / CCR7+ T cells is increased.

59. 45. The method of any one of claims 1 to 44, wherein the level of CD4+ / NKG7+ / CCL4+ / CCL5+ / GNLY+ / LAG3+ T cells specific for a cancer-specific neoepitope is increased.

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

61. 1. A method of treating or preventing cancer in a human subject in need thereof, comprising administering to said human subject in need thereof: (a)(i) a polypeptide comprising a cancer-specific neoepitope of a protein expressed by a cancer cell of said 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 neoepitope of a protein expressed by a cancer cell of the cancer neoepitope; or (v) T cells comprising the TCR of (iv). a first component comprising: (b) a second component comprising an anti-cancer agent that is an antibody or antigen-binding portion thereof that specifically binds to the Programmed Death-1 (PD-1) receptor and inhibits PD-1 activity; (c) a third component comprising platinum-based chemotherapy; administering (i) the method promotes epitope spreading of an epitope distinct from any of the cancer-specific neoepitopes; (ii) the median progression-free survival (PFS) of a first population of human subjects whose cancer is treated with the first, second, and third components is longer than the median PFS of a second population of subjects whose cancer is 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 whose cancer is treated with the first, second, and third components is higher than the ORR of a second population of subjects whose cancer is 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 treated for said cancer with said first, second, and third components having at least a 9 or 12 month progression-free survival (PFS) is higher than the percentage of subjects in a second population of subjects treated for said cancer with said second and / or third components but not with said first component having at least a 9 or 12 month PFS; (v) the median overall survival (OS) of a first population of human subjects treated for said cancer with said first, second, and third components is longer than the median OS of a second population of subjects treated for said cancer with said second and / or third components but not with said first component; (vi) the percentage of subjects achieving a complete response, partial response, long-term stable disease, or stable disease for 6 months or more (CBR) in a first population of human subjects treated for said cancer with said first, second, and third components is higher than the CBR in a second population of subjects treated for said cancer with said second and / or third components but not with said first component; (vii) the reduction in tumor size in a first population of human subjects whose cancer is treated with the first, second, and third components is greater than the reduction in tumor size in a second population of subjects whose cancer is treated with the second and / or third components but not the first component; (viii) the level of tumor-infiltrating CD4+ T cells in a first population of human subjects treated for said cancer with said first, second, and third components is higher than the level of tumor-infiltrating CD4+ T cells in a second population of subjects treated for said cancer with said second and / or third components but not with said first component; (ix) the level of effector and cytotoxic CD4+ T cells generated in a first population of human subjects treated for the cancer with the first, second, and third components is higher than the level of effector and cytotoxic CD4+ T cells generated in a second population of subjects treated for the cancer with the second and / or third components but not the first component; (x) the method increases the level of CD4+ T cells specific for a cancer-specific neoepitope that upregulates expression of ZEB2, PDCD1, TOX, TIGT, CXCR3, ITGB1, GZMA, and / or ICOS; (xi) the method comprises: ｈｉ / increase the level of CD69+ / CD27+ / CCR7+ T cells; and / or (xii) the method increases the level of CD4+ / NKG7+ / CCL4+ / CCL5+ / GNLY+ / LAG3+ T cells specific for the cancer-specific neoepitope. The method.

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

63. 62. 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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