Combination therapy by neo-antigen vaccine

Abstract

To provide use of a cell therapy composition in manufacturing a medicine for personalized treatment of patient-specific cancer which includes administering Nivolumab or Pembrolizumab.SOLUTION: Provided is (A) an antigen-presenting cell (APC) including: (i) one or more polypeptides having the length of 5 to 50 amino acid which include at least two peptide sequences which are calculated to have binding affinity of less being IC50 of than 500nM to protein encoded by HLA class I allele of the patient by HLA peptide bond analysis using a program executed on a computer system; or (ii) one or more polynucleotide encoding at least two of the peptide sequences, or (B) use of a cell therapy composition including T-cell stimulated by APC including the (i) or (ii).SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] Related Applications and Incorporation by Reference This application claims the benefit of and priority to U.S. Provisional Patent Application No. 61 / 919,576, filed December 20, 2013, and U.S. Provisional Patent Application No. 61 / 976,274, filed April 7, 2014.

[0002] The aforementioned applications, and all citations in such applications or in the prosecution thereof ("Application Citations") and all documents cited or referenced in the Application Citations, and all documents cited or referenced herein ("Prescription Citations"), and all documents cited or referenced in the Prescription Citations, together with any manufacturer's instructions, manuals, product specifications, and product sheets for any products mentioned in this specification or in any document incorporated herein by reference, are hereby incorporated by reference and may be used in the practice of this invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document were specifically and individually indicated to be incorporated by reference.

[0003] The present invention relates to a method for treating neoplasms, and more particularly tumors, by administering to a subject a neoplasm vaccine comprising multiple neoplasm / tumor-specific neoantigens and at least one checkpoint inhibitor.

[0004] Federal Grant Statement This invention was made with federal support under R01 CA155010-03 awarded by the NIH / NCI. The federal government has certain rights in this invention. [Background technology]

[0005] Approximately 1.6 million Americans are diagnosed with neoplasms each year, and approximately 580,000 people are expected to die from the disease in the United States in 2013. Significant improvements in neoplasm detection, diagnosis, and treatment have been made in recent decades, resulting in significant increases in survival rates for many types of neoplasms. However, only about 60% of people diagnosed with a neoplasm are still alive five years after initiating treatment, making neoplasms the second leading cause of death in the United States.

[0006] Currently, there are a variety of existing cancer therapies, including ablative techniques (e.g., surgical procedures, cryogenic / thermal treatments, ultrasound, radiofrequency, and radiation) and chemical techniques (e.g., pharmaceuticals, cytotoxic / chemotherapeutic agents, monoclonal antibodies, and various combinations thereof). Unfortunately, such treatments often involve serious risks, toxic side effects, and prohibitive costs, as well as uncertain efficacy.

[0007] Cancer therapies that attempt to target cancerous cells using a patient's own immune system (e.g., cancer vaccines) have gained increasing interest because such therapies may mitigate / eliminate some of the drawbacks described herein. Cancer vaccines typically consist of tumor antigens and immune stimulatory molecules (e.g., cytokines or TLR ligands), which work together to induce antigen-specific cytotoxic T cells, which then target and destroy tumor cells. Current cancer vaccines typically contain common tumor antigens, which are naturally occurring proteins (i.e., proteins encoded by the DNA of all normal cells in an individual) that are selectively expressed or overexpressed in tumors found in many individuals. While such common tumor antigens are useful for identifying specific types of tumors, they are not suitable as immunogens for targeting T cell responses to specific tumor types. However, vaccines containing tumor-specific and patient-specific neoantigens may overcome some of the drawbacks of vaccines containing common tumor antigens, as they are subject to the immunodepressant effects of self-tolerance. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] US Patent Application Publication No. 20110293637 Summary of the Invention [Problem to be solved by the invention]

[0009] This application describes combination therapies to achieve desirable therapeutic results, particularly in the treatment of cancer. Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention. [Means for solving the problem]

[0010] The present invention relates to neoplastic vaccine or immunogenic compositions administered in combination with one or more other agents, such as one or more checkpoint blockade inhibitors, for the treatment or prevention of a neoplasm in a subject.

[0011] In one aspect, the invention features a method of treating or preventing a neoplasm in a subject in need thereof, which can include administering to a subject in need thereof: (a) a neoplasm vaccine or immunogenic composition; and (b) at least one checkpoint inhibitor. The administration can be sequential, sequential, at substantially the same time, or substantially simultaneous. For example, the administration of the neoplasm vaccine or immunogenic composition and the administration of the at least one checkpoint inhibitor can be at about the same time or substantially simultaneous. Alternatively, the administration of the neoplastic vaccine or immunogenic composition may be on a one time schedule, e.g., weekly, every other week, every three weeks, monthly, every other month, every quarter of a year (every three months), every third of a year (every four months), every five months, twice a year (every six months), every seven months, eight months, nine months, ten months, eleven months, yearly, etc., and the administration of the at least one checkpoint blockade inhibitor may be on a different schedule typical for that checkpoint blockade inhibitor, and the administration of the neoplastic vaccine or immunogenic composition and the administration of the at least one checkpoint inhibitor may be sequential or consecutive, such that the subject or patient has two different treatment schedules progressing in parallel. The neoplastic vaccine or immunogenic composition advantageously comprises at least four different neo-antigens (and by different antigens it is intended that each antigen has a different neo-epitope), for example there may be at least 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 or 31 or 32 or 33 or 34 or 35 or 36 or 37 or 38 or 39 or 40 or more different neo-antigens in the neoplastic vaccine or immunogenic composition.A neoplastic vaccine or immunogenic composition can be administered in subcompositions, each containing a portion of a neo-antigen, and the subcompositions can be administered to different locations in a subject or patient; for example, a composition containing 20 different neo-antigens can be administered in four subcompositions, each containing five of the 20 different neo-antigens, and the four subcompositions can be administered in an attempt to deliver each subcomposition to or near the patient's draining lymph nodes, such as to each of the patient's upper and lower extremities (e.g., the thigh or upper thigh, or near the buttocks or lower back on each side of the patient's body). Of course, the number of locations, and therefore the number of subcompositions, can vary; for example, one skilled in the art might contemplate administration with a fifth administration point at or near the spleen, and one skilled in the art would recognize the need for only one, two, or more subcompositions. The positions can be varied so that three are used (e.g., each upper limb and one lower limb, each lower limb and one upper limb, each lower limb and no upper limbs, or both upper limbs only). The vaccine or immunogenic compositions administered at the various intervals described above may be different formulations, and sub-compositions administered to different locations in a subject or patient during a single administration may be of different compositions. For example, the first administration may be a whole antigen vaccine or immunogenic composition, and the next or subsequent administration may be a vector (e.g., a viral vector or a plasmid) capable of expressing one or more antigens in vivo. Similarly, when different sub-compositions are administered to different locations in a patient or subject, some sub-compositions may comprise the whole antigen, and some sub-compositions may comprise a vector (e.g., a viral vector or a plasmid) capable of expressing one or more antigens in vivo. Also, some compositions and sub-compositions may comprise both a vector (e.g., a viral vector or a plasmid) capable of expressing one or more antigens in vivo and the whole antigen. Some vectors (e.g., poxviruses) that express one or more antigens in vivo can have an immunostimulatory or adjuvant effect, and thus compositions or subcompositions containing such vectors can be self-adjuvanting. Furthermore, administration can "prime" and then "boost" the immune system by altering the nature of how antigens are presented to the immune system. Also, in this document, when a "vaccine" is mentioned, it is intended that the invention encompasses immunogenic compositions, and when a patient or subject is mentioned, it is intended that such individual is a patient or subject in need of the treatments, administrations, compositions, and generally the invention disclosed herein.

[0012] In one embodiment, the neoplasm vaccine or immunogenic composition comprises at least two, at least three, at least four, or at least five neo-antigenic peptides. In another embodiment, the neo-antigenic peptides range from about 5 to about 50 amino acids in length. In another related embodiment, the neo-antigenic peptides range from about 15 to about 35 amino acids in length. Typically, the length is greater than about 15 or 20 amino acids, e.g., 15 to 50 or about 75 amino acids.

[0013] In one embodiment, the neoplastic vaccine or immunogenic composition further comprises a pH adjusting agent and a pharmaceutically acceptable carrier.

[0014] In one embodiment, the method further comprises administration of an immunomodulatory agent or adjuvant (and thus the vaccine or immunogenic composition may comprise an immunomodulatory agent or adjuvant). In another related embodiment, the immunomodulatory agent 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 another further embodiment, the immunomodulatory agent 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.

[0015] Dissolving these polymers in water produces an acidic solution, which, upon neutralization, preferably to physiological pH, provides an adjuvant solution for formulation with a vaccine or immunogenic composition or its antigen or vector. In this case, the carboxyl groups of the polymer are partially COO - is.

[0016] Preferably, a solution of the adjuvant according to the invention, particularly a carbomer solution, is prepared in distilled water, preferably in the presence of sodium chloride, resulting in an acidic pH. This stock solution is diluted all at once or in several portions with the required volume (to reach the desired final concentration) or a substantial portion thereof of water containing a salt such as NaCl, preferably physiological saline (9 g / L NaCl), followed by simultaneous or subsequent neutralization (pH 7.3-7.4), preferably with a base such as NaOH. This solution at physiological pH is used for the reconstitution of vaccines, particularly those stored in freeze-dried or lyophilized form.

[0017] The polymer concentration in the final vaccine composition is 0.01% to 2% w / v, more particularly 0.06 to 1% w / v, and preferably 0.1 to 0.6% w / v.

[0018] Furthermore, the present invention applies to the use of any type of expression vector, for example, a viral expression vector, such as a poxvirus (e.g., an orthopoxvirus or an avipoxvirus, such as a vaccinia virus, e.g., modified vaccinia Ankara or MVA, MVA-BN, NYVAC according to WO-A-92 / 15672, fowlpox, e.g., TROVAX, canarypox, e.g., ALVAC (WO-A-95 / 27780 and WO-A-92 / 15672), pigeonpox, swinepox, etc.), adenovirus, AAV, herpesvirus, and lentivirus; or a plasmid or DNA or nucleic acid molecule vector. Some vectors that are cytoplasmic, such as poxvirus vectors, may be advantageous. However, adenovirus, AAV, and lentivirus may also be advantageous for use in practicing the present invention.

[0019] In a ready-to-use, particularly reconstituted, vaccine or immunogenic composition, the vector, e.g., viral vector, is present in an amount that is within the purview of one skilled in the art from this disclosure and knowledge in the art (such as that in the patents and scientific literature cited herein).

[0020] Whole antigens or vectors, such as live recombinant vaccines, are generally present in a freeze-dried form to allow their storage and are reconstituted immediately prior to use in a solvent or excipient which may include an adjuvant as discussed herein.

[0021] A subject of the present invention is therefore also a vaccination or immunization set or kit comprising a separately packaged freeze-dried vaccine and a solution for reconstituting the freeze-dried vaccine, advantageously containing an adjuvant compound as discussed herein.

[0022] A subject of the present invention is also a vaccination or immunization method comprising, consisting essentially of or consisting of administering a vaccine or immunogenic composition according to the invention at one or more dosage rates, for example by a parenteral route, preferably by the subcutaneous, intramuscular or intradermal route, or by a mucosal route. Optionally, this method comprises a preliminary step of reconstituting a freeze-dried vaccine or immunogenic composition (for example in the case of a lyophilized whole antigen or vector) in a solution advantageously also containing an adjuvant.

[0023] In one embodiment, the checkpoint inhibitor is an inhibitor of the programmed death 1 (PD-1) pathway. In another embodiment, the PD-1 pathway inhibitor is an anti-PD1 antibody. In a related embodiment, the PD-1 pathway inhibitor is nivolumab.

[0024] In one embodiment, the checkpoint inhibitor is an anti-cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) antibody. In a related embodiment, the anti-CTLA4 antibody is ipilimumab or tremelimumab.

[0025] In one embodiment, the subject is afflicted with a neoplasm selected from the group consisting of non-Hodgkin's lymphoma (NHL), clear cell renal cell carcinoma (ccRCC), melanoma, sarcoma, leukemia, or bladder, colon, brain, breast, head and neck, endometrial, lung, ovarian, pancreatic, or prostate cancer. In another embodiment, the neoplasm is metastatic. In a further embodiment, the subject does not have a detectable neoplasm but is at high risk of disease recurrence. In a further related embodiment, the subject has previously undergone autologous hematopoietic stem cell transplantation (AHSCT).

[0026] In one embodiment, administration of the checkpoint inhibitor is initiated prior to initiation of administration of the neoplastic vaccine or immunogenic composition. In one embodiment, administration of the checkpoint inhibitor is initiated after initiation of administration of the neoplastic vaccine or immunogenic composition. In one embodiment, administration of the checkpoint inhibitor is initiated simultaneously with initiation of administration of the neoplastic vaccine or immunogenic composition.

[0027] In another embodiment, administration of the checkpoint inhibitor continues every 2-8 weeks or more after the initial administration of the checkpoint inhibitor. In a further embodiment, administration of the checkpoint inhibitor continues every 2, 3, or 4, 6, or 8 weeks after the initial administration of the checkpoint inhibitor. In another further embodiment, administration of the checkpoint inhibitor is withheld for one week prior to administration of the neoplastic vaccine or immunogenic composition. In yet another further embodiment, administration of the checkpoint inhibitor is withheld during administration of the neoplastic vaccine or immunogenic composition.

[0028] In one embodiment, administration of the checkpoint inhibitor is initiated after tumor resection. In another embodiment, administration of the neoplastic vaccine or immunogenic composition is initiated 1 to 15 weeks after tumor resection. In another further embodiment, administration of the neoplastic vaccine or immunogenic composition is initiated 4 to 12 weeks after tumor resection.

[0029] In one embodiment, administration of the neoplastic vaccine or immunogenic composition is a prime / boost regimen. In another embodiment, administration of the neoplastic vaccine or immunogenic composition is at week 1, 2, 3, or 4 as a priming. In another further embodiment, administration of the neoplastic vaccine or immunogenic composition is at month 2, 3, 4, or 5 as a boost.

[0030] In one embodiment, the vaccine or immunogenic composition is administered at a dose of about 10 μg to 1 mg per 70 kg individual for each neo-antigenic peptide. In another embodiment, the vaccine or immunogenic composition is administered at an average weekly dose level of about 10 μg to 2000 μg per 70 kg individual for each neo-antigenic peptide. In another further embodiment, the checkpoint inhibitor is administered at a dose of about 0.1 to 10 mg / kg. In another related embodiment, administration is intravenous.

[0031] In one embodiment, the anti-CTLA4 antibody is administered at a dose of about 1 mg / kg to 3 mg / kg.

[0032] In one embodiment, the vaccine or immunogenic composition is administered intravenously or subcutaneously.

[0033] In another embodiment, the checkpoint inhibitor is administered intravenously or subcutaneously.

[0034] In another embodiment, the checkpoint inhibitor is administered subcutaneously within about 2 cm of the site of administration of the neoplastic vaccine or immunogenic composition.

[0035] In one embodiment, the checkpoint inhibitor is administered as a neoplasia vaccine per 70 kg individual. The vaccine or immunogenic composition is administered at a dose of about 0.1 to 1 mg per administration site.

[0036] In one embodiment, the method further comprises administering one or more additional agents. In another embodiment, the additional agents are selected from the group consisting of chemotherapeutic agents, anti-angiogenic agents, and agents that reduce immunosuppression. In a further embodiment, the one or more additional agents are one or more anti-glucocorticoid-inducible tumor necrosis factor family receptor (GITR) agonist antibodies.

[0037] In one embodiment, the method may include administering a combination therapy within the standard of care for a particular cancer, hi another embodiment, the combination therapy is administered within the standard of care when the addition of the combination therapy is synergistic with the standard of care step.

[0038] The present invention relates to a method as in U.S. Patent Application Publication No. 20110293637 (incorporated herein by reference), e.g., a method of identifying a plurality of at least four subject-specific peptides and preparing a subject-specific immunogenic composition that, upon administration, presents the plurality of at least four subject-specific peptides to a subject's immune system, wherein the subject has a tumor and the subject-specific peptides are specific to the subject and the subject's tumor, the method comprising: (i) a plurality of at least four tumor-specific non-silent mutations that are not present in non-tumor samples; Nucleic acid sequencing of a sample of the subject's tumor; and Nucleic acid sequencing of a non-tumor sample of a subject identifying, including by; (ii) selecting from the identified plurality of tumor-specific mutations a plurality of at least four subject-specific peptides, each peptide having a different tumor neoepitope that is an epitope specific for the tumor of interest, from the identified plurality of tumor-specific mutations; each neoepitope is the expression product of a tumor-specific non-silent mutation not present in non-tumor samples, each neoepitope binds to an HLA protein of interest, Determining binding of target-specific peptides to HLA proteins a selecting step, (iii) formulating the subject-specific immunogenic composition for administration to a subject such that upon administration, a plurality of at least four subject-specific peptides are presented to the subject's immune system; Including, The selecting or formulating step comprises: including in the subject-specific immunogenic composition a subject-specific peptide comprising the expression product of the identified neo-ORF, wherein the neo-ORF is a tumor-specific non-silent mutation not present in the non-tumor sample that creates a novel open reading frame; and comprising in the subject-specific immunogenic composition a subject-specific peptide that comprises the expression product of the identified point mutation and whose binding to the subject's HLA protein is determined to be at an IC50 of less than 500 nM. and whereby a plurality of at least four subject-specific peptides are identified and a subject-specific immunogenic composition is prepared which, upon administration, presents the plurality of at least four subject-specific peptides to the subject's immune system, the subject-specific peptides being specific to the subject and the subject's tumor; or a method for identifying neoantigens, comprising: a. identifying tumor-specific mutations in expressed genes in subjects with cancer; b. If the mutation identified in step (a) is a point mutation: i. identifying a mutant peptide having the mutation identified in step (a), wherein the mutant peptide binds to a Class I HLA protein with a higher affinity than the wild-type peptide; and has an IC50 of less than 500 nM; c. If the mutation identified in step (a) is a splice site, frameshift, readthrough, or gene fusion mutation: i. identifying a mutant polypeptide encoded by the mutation identified in step (a), wherein said mutant polypeptide binds to a class I HLA protein; or a method of inducing a tumor-specific immune response in a subject, comprising administering one or more of the identified peptides or polypeptides and an adjuvant; or a method of vaccinating or treating a subject for cancer, comprising the steps of: a. Identifying a plurality of tumor-specific mutations in expressed genes of a subject, wherein the identified mutations are: i. if it is a point mutation, further identifying a mutant peptide having the point mutation; and / or ii. further identifying the mutant polypeptide encoded by the mutation if it is a splice site, frameshift, readthrough, or gene fusion mutation; b. selecting one or more mutant peptides or polypeptides identified in step (a) that bind to a class I HLA protein; c. selecting one or more mutant peptides or polypeptides identified in step (b) that have the ability to activate anti-tumor CD8 T cells; and d. administering to a subject one or more peptides or polypeptides selected in step (c), autologous dendritic cells or antigen-presenting cells pulsed with the one or more peptides or polypeptides; or performing a method comprising preparing a pharmaceutical composition comprising one or more identified peptides, and performing one or more methods as discussed herein. Thus, the neoplastic vaccine or immunogenic composition herein may be as described in U.S. Patent Application Publication No. 20110293637.

[0039] Accordingly, it is an object of the present invention not to include within its scope any previously known product, process for making a product, or method for using a product, as applicants hereby reserve the right, and hereby disclose any disclaimer of any previously known product, process, or method. It is further noted that the present invention is not intended to include within its scope any product, process for making a product, or method for using a product that does not meet the description and enablement requirements of the United States Patent and Trademark Office (USPTO) (35 U.S.C. § 112, first paragraph) or the European Patent Office (EPO) (Article 83 EPC), as applicants hereby disclose any disclaimer of any previously described product, process for making a product, or method for using a product.

[0040] It is noted that in this disclosure, and particularly in the claims and / or paragraphs, terms such as "comprises," "comprised," "comprising," and the like may have the meaning ascribed to them in U.S. patent law; e.g., they may mean "includes," "included," "including," and the like; and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. patent law, e.g., they may permit elements not expressly recited, but exclude elements found in the prior art or that affect a basic or novel characteristic of the invention.

[0041] These and other embodiments are disclosed or are apparent from and encompassed by the following detailed description.

[0042] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication, including any drawings, will be provided by the Office upon request and payment of the necessary fee.

[0043] The following detailed description is not intended to limit the invention to the specific embodiments described but is provided by way of example and may be best understood in conjunction with the accompanying drawings, which are incorporated herein by reference. [Brief explanation of the drawings]

[0044] [Figure 1] 1 shows a flow process for the production of a personalized cancer vaccine or immunogenic composition. [Figure 2] 1 shows a flow process of pre-treatment steps for creating a cancer vaccine or immunogenic composition for melanoma patients. [Figure 3] 1 shows a prime-boost immunization schedule according to an exemplary embodiment of the present invention. [Figure 4] 1 shows a timeline indicating primary immunological endpoints, according to an exemplary embodiment of the present invention. [Figure 5] 1 shows a timeline for administering co-therapy with a checkpoint blockade antibody to evaluate the combination of alleviating local immunosuppression combined with stimulating de novo immunity, according to an exemplary embodiment of the present invention. As shown in this scheme, patients who enroll as suitable candidates for checkpoint blockade therapy, e.g., anti-PDL1 as shown herein, are treated with the antibody immediately upon enrollment, during which time a vaccine or immunogenic composition may be prepared. The patient may then be vaccinated. Administration of the checkpoint blockade antibody may be continued or potentially delayed while the priming phase of vaccination is taking place. [Figure 6] 1 shows a schematic diagram illustrating the formulation process of individual neo-antigenic peptides into pools of four subgroups according to an exemplary embodiment of the present invention. [Figure 7] 1 shows the results of quantitative PCR evaluating the level of induction of several key immune markers after stimulation of mouse dendritic cells with neoantigen preparations. [Figure 8] Study 1: Schematic overview of nivolumab versus nivolumab and NeoVax after AHSCT in non-Hodgkin lymphoma. [Figure 9] Study 2: Schematic overview of nivolumab and NeoVax in metastatic melanoma and metastatic RCC. [Figure 10A] Figure 10A shows an overview of the four studies. (A) Study 3a shows dose escalation of reduced-intensity intravenous ipilimumab in metastatic melanoma. (B) Study 3b shows dose escalation of subcutaneous ipilimumab (topical) in metastatic melanoma. (C) Study 3c shows dose escalation of reduced-intensity intravenous ipilimumab in high-risk melanoma. (D) Study 3d shows dose escalation of subcutaneous ipilimumab (topical) in high-risk melanoma. [Figure 10B] Figure 10B shows an overview of the four studies. (A) Study 3a shows dose escalation of reduced-intensity intravenous ipilimumab in metastatic melanoma. (B) Study 3b shows dose escalation of subcutaneous ipilimumab (topical) in metastatic melanoma. (C) Study 3c shows dose escalation of reduced-intensity intravenous ipilimumab in high-risk melanoma. (D) Study 3d shows dose escalation of subcutaneous ipilimumab (topical) in high-risk melanoma. [Figure 10C] Figure 10C shows an overview of the four studies. (A) Study 3a shows dose escalation of reduced-intensity intravenous ipilimumab in metastatic melanoma. (B) Study 3b shows dose escalation of subcutaneous ipilimumab (topical) in metastatic melanoma. (C) Study 3c shows dose escalation of reduced-intensity intravenous ipilimumab in high-risk melanoma. (D) Study 3d shows dose escalation of subcutaneous ipilimumab (topical) in high-risk melanoma. [Figure 10D] Figure 10D shows an overview of the four studies. (A) Study 3a shows dose escalation of reduced-intensity intravenous ipilimumab in metastatic melanoma. (B) Study 3b shows dose escalation of subcutaneous ipilimumab (topical) in metastatic melanoma. (C) Study 3c shows dose escalation of reduced-intensity intravenous ipilimumab in high-risk melanoma. (D) Study 3d shows dose escalation of subcutaneous ipilimumab (topical) in high-risk melanoma. [Figure 11]1 shows a trial scheme for the combination of NeoVax and ipilimumab to treat high-risk renal cell carcinoma. [Figure 12] 1 shows the treatment scheme for a trial using NeoVax in combination with ipilimumab to treat high-risk renal cell carcinoma. DETAILED DESCRIPTION OF THE INVENTION

[0045] definition To facilitate the understanding of the invention, certain terms and phrases are defined herein.

[0046] Unless specifically stated or clear from the context, the term "about" as used herein is understood to mean within the normal tolerance range in the art, for example, within 2 standard deviations of the mean. About can be understood to mean within 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from the context, all numerical values ​​provided herein are modified by the term about.

[0047] Unless specifically stated or clear from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or clear from context, as used herein, the terms "a," "an," and "the" are understood to be singular or plural.

[0048] By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof.

[0049] Immune checkpoints are inhibitory pathways that stall or halt immune responses and prevent excessive tissue damage due to uncontrolled immune cell activity. The term "checkpoint inhibitor" is intended to refer to any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragment thereof, that inhibits an inhibitory pathway, 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 further embodiments, the checkpoint inhibitor targets another member of the CD28CTLA4 Ig superfamily, such as BTLA, LAG3, ICOS, PDL1, or KIR (Page et al., Annual Review of Medicine 65:27 (2014)). In yet another embodiment, the checkpoint inhibitor is targeted to a member of the TNFR superfamily, such as CD40, OX40, CD137, GITR, CD27, or TIM-3. In some cases, targeting of the checkpoint inhibitor is achieved by an inhibitory antibody or similar molecule. In other cases, it is achieved by an agonist to the target; examples of this class include the stimulatory targets OX40 and GITR.

[0050] The term "combination" encompasses the administration of a neoplastic vaccine or immunogenic composition (e.g., a pooled sample of neoplasm / tumor-specific neoantigens) with one or more checkpoint inhibitors as part of a treatment regimen intended to produce a beneficial (additive or synergistic) effect from the co-action of one or more of the therapeutic agents. The combination may also include one or more additional agents, such as, but not limited to, chemotherapeutic agents, anti-angiogenic agents, and agents that reduce immunosuppression. Beneficial effects of the combination include, but are not limited to, pharmacokinetic or pharmacodynamic co-actions resulting from the combination of therapeutic agents. The combined administration of these therapeutic agents is typically for a defined period of time (e.g., a selected period of time). The treatment may take place over a period of minutes, hours, days, or weeks, depending on the combination of treatment options.

[0051] "Combination therapy" is intended to encompass administration of these therapeutic agents in a sequential manner, i.e., administration in which each therapeutic agent is administered at a different time, as well as administration in which the therapeutic agents, or at least two of the therapeutic agents, are administered substantially simultaneously. Substantially simultaneous administration can be achieved, for example, by administering to the subject a single capsule having a fixed ratio of each therapeutic agent, or multiple single capsules of each of the therapeutic agents. For example, a combination of the present invention may include a pooled sample of tumor-specific neoantigens and a checkpoint inhibitor administered at the same or different times, or they can be formulated as a single co-formulated pharmaceutical composition containing the two compounds. As another example, a combination of the present invention (e.g., a pooled sample of tumor-specific neoantigens and a checkpoint inhibitor and / or an anti-CTLA4 antibody) may be formulated as separate pharmaceutical compositions that can be administered at the same or different times. As used herein, the term "concurrently" is intended to refer to administration of one or more agents at the same time. For example, in certain embodiments, a neoplastic vaccine or immunogenic composition and a checkpoint inhibitor are administered simultaneously. Concurrently includes administration contemporaneously, i.e., within the same time period. In certain embodiments, one or more agents are administered simultaneously at the same time or on the same day. Sequential or substantially simultaneous administration of each therapeutic agent can be achieved by any suitable route, including, but not limited to, oral, intravenous, subcutaneous, intramuscular, direct absorption through mucosal tissues (e.g., nose, mouth, vagina, and rectum), and intraocular (e.g., intravitreal, intraocular, etc.). The therapeutic agents may be administered by the same route or by different routes. For example, one component of a particular combination may be administered by intravenous injection, while one or more other components of the combination may be administered orally. The components may be administered in any therapeutically effective order. The phrase "combination" encompasses compounds or non-drug therapies useful as part of a combination therapy.

[0052] The term "neoantigen" or "neoantigenic" refers to a class of tumor antigens that arise from one or more tumor-specific mutations that alter the amino acid sequence of a genome-encoded protein.

[0053] By "neoplasm" is meant 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 neoplasm. Examples of cancer include, without limitation, leukemia (e.g., acute leukemia, acute lymphocytic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors. Tumors, such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial tumor, lymphangiosarcoma, lymphangioendothelial tumor, synovioma, mesothelioma, 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, hepatocellular carcinoma, bile duct (bile duct) carcinoma, duct cancer, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). Lymphoproliferative disorders are also considered proliferative diseases.

[0054] The term "neoplasm vaccine" is intended to refer to a pooled preparation of neoplasm / tumor specific neoantigens, e.g., at least two, at least three, at least four, at least five, or more neoantigenic peptides. A "vaccine" is a vaccine that is used to prevent or treat a disease (e.g., a neoplasm / tumor). "Neoplastic vaccine compositions" should be understood to mean compositions that generate protective and / or therapeutic immunity. A vaccine is thus an agent containing an antigen and intended to be used to generate specific defense and protection in humans or animals by vaccination. A "neoplastic vaccine composition" may include a pharmaceutically acceptable excipient, carrier, or diluent.

[0055] The term "pharmaceutically acceptable" refers to being approved or expected to be approved by a federal or state regulatory agency for use in animals, including humans, or being listed in the United States Pharmacopoeia or other generally recognized pharmacopeia.

[0056] A "pharmaceutically acceptable excipient, carrier, or diluent" refers to an excipient, carrier, or diluent that can be administered to a subject together with a drug and that does not destroy the pharmacological activity of the drug and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the drug.

[0057] A "pharmaceutically acceptable salt" of the pooled tumor-specific neo-antigens as described herein may be an acidic or basic salt generally considered in the art to be suitable for use in contact with human or animal tissues without undue toxicity, irritation, allergic response, or other problem or complication. 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, 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 acid, such as acetic acid, HOOC-(CH2) nIncluded are salts of acids such as -COOH (where n is 0-4). Similarly, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium. Those skilled in the art will recognize additional pharmaceutically acceptable salts of the pooled tumor-specific neoantigens provided herein, including those listed in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, p. 1418 (1985), from this disclosure and knowledge in the art. In general, pharmaceutically acceptable acid or base salts can be synthesized from a parent compound containing a basic or acidic moiety by any conventional chemical method. Briefly, such salts can be prepared by reacting the free acid or free base form of these compounds with a stoichiometric amount of the appropriate base or acid in a suitable solvent.

[0058] By "polypeptide" or "peptide" is meant a polypeptide that has been separated from components that naturally accompany it. Typically, a polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, the polypeptide. Isolated polypeptides can be obtained, for example, by extraction from a natural source, by expressing a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0059] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like refer to a condition that affects a person who does not have a disease or condition, but is at risk of developing or is suffering from a disease or condition. "Ablation" refers to reducing the likelihood of a disease or condition developing in a subject who is susceptible to developing the disease or condition.

[0060] The terms "prime / boost" or "prime / boost dosing regimen" are intended to refer to sequential administration of a vaccine or immunogenic or immunological composition. A priming administration (priming) is the administration of a first vaccine or immunogenic or immunological composition type and may include one, two or more administrations. A boosting administration is the administration of a second vaccine or immunogenic or immunological composition type and may include one, two or more administrations, and may include or consist essentially of, for example, annual administration. In certain embodiments, the administration of a neoplastic vaccine or immunogenic composition is a prime / boost dosing regimen.

[0061] Ranges provided herein are understood to be shorthand for all values ​​within that 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 either endpoint 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 other direction.

[0062] "Receptor" should be understood to mean a biological molecule or molecular class capable of binding to a ligand. Receptors can function to transmit information in cells, cell formations, or organisms. A receptor contains at least one receptor unit, and often contains two or more receptor units, where each receptor unit can consist of a protein molecule, particularly a glycoprotein molecule. A receptor has a structure complementary to that of a ligand and can form a complex with the ligand as a binding partner. After binding to a ligand on the surface of a cell, the receptor undergoes a conformational change, which can transmit signal information. According to the present invention, receptors can refer to specific proteins of MHC class I and II that have the ability to form a receptor / ligand complex with a ligand, particularly a peptide or peptide fragment of a suitable length.

[0063] The term "subject" refers to an animal that has been the object of treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, such as, but not limited to, a human or a non-human mammal, such as, but not limited to, a non-human primate, cow, horse, dog, sheep, or cat.

[0064] The terms "treat," "treated," "treating," "treatment," and the like are intended to refer to the reduction or amelioration of a disorder and / or its associated symptoms (e.g., a neoplasm or tumor). "Treating" can refer to the administration of a combination therapy to a subject after the onset or suspected onset of cancer. "Treating" encompasses the concept of "alleviating," which refers to reducing the frequency of occurrence or recurrence, or severity, of any symptoms or other pathological effects associated with cancer and / or side effects associated with cancer therapy. The term "treating" also encompasses the concept of "managing," which refers to reducing the severity or delaying the recurrence of a particular disease or disorder in a patient, for example, extending the period of remission in a patient who has previously suffered from the disease. It is understood, although not excluded, that treating a disorder or condition does not require complete disappearance of the disorder, condition, or its associated symptoms.

[0065] The term "therapeutic effect" refers to the reduction or improvement of one or more of the symptoms of a disorder (e.g., a neoplasm or tumor) or related thereto. "Therapeutically effective amount" refers to any degree of alleviation of the associated pathology. As used herein, "therapeutically effective amount" refers to an amount of an agent that, upon single or multiple dose administration to a cell or subject, is effective in prolonging the 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. "Therapeutically effective amount" is intended to qualify the amount necessary to achieve a therapeutic effect. A physician or veterinarian having 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 may start doses of a compound of the present invention used in a 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.

[0066] The recitation of a list of chemical groups in any definition of a variable herein includes a definition of that variable as any single group or combination of the listed groups. The recitation of an embodiment of a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0067] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0068] The combination therapies disclosed herein represent novel methods for treating various cancers. The combination therapies described herein also provide a therapeutic method for achieving clinical benefit without unacceptable levels of side effects.

[0069] The present invention relates to a method of treating 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 checkpoint inhibitor.

[0070] As described in further detail herein, whole genome / exome sequencing can be used to identify all or nearly all mutant neoantigens uniquely present in an individual patient's neoplasm / tumor, and analyzing this collection of mutant neoantigens can identify specific, optimized subsets of neoantigens to be used as personalized cancer vaccines or immunogenic compositions to treat the patient's neoplasm / tumor. For example, by sequencing each patient's neoplasm / tumor and normal DNA to identify tumor-specific mutations, a population of neoplasm / tumor-specific neoantigens can be identified, and the patient's HLA allotype can be identified. The population of neoplasm / tumor-specific neoantigens and their cognate natural antigens can then be subjected to bioinformatics analysis using validated algorithms to predict which tumor-specific mutations create epitopes that can bind to the patient's HLA allotype. Based on this analysis, multiple peptides corresponding to a subset of these mutations for each patient can be designed and synthesized and pooled together for use as cancer vaccines or immunogenic compositions in immunizing the patient.

[0071] The immune system can be categorized into two functional subsystems: the innate immune system and the adaptive immune system. The innate immune system is the first line of defense against infection; most potential pathogens are quickly neutralized by this system before they can cause, for example, a recognizable infection. The adaptive immune system responds to the molecular structures of invading organisms, called antigens. There are two types of adaptive immune responses, including humoral and cell-mediated immune responses. In the humoral immune response, antibodies secreted into bodily fluids by B cells bind to pathogen-derived antigens, leading to their elimination through various mechanisms, such as complement-mediated lysis. In the cell-mediated immune response, T cells, which have the ability to destroy other cells, are activated. For example, when disease-related proteins are present in cells, they are fragmented into peptides by proteolysis within the cell. Specific cellular proteins then attach to the antigens or peptides thus formed and destroy them. They transport these antigens or peptides to the surface of cells, where they are presented to the body's molecular defense mechanisms, specifically T cells. Cytotoxic T cells recognize these antigens and kill cells that carry them.

[0072] Molecules that transport and present peptides to the cell surface are called major histocompatibility complex (MHC) proteins. MHC proteins are classified into two types, MHC class I and MHC class II. The structures of these two MHC class proteins are very similar; however, they have very different functions. MHC class I proteins are present on the surface of almost all cells in the body, including many tumor cells. MHC class I proteins are typically loaded with antigens derived from endogenous proteins or intracellular pathogens, which are then presented to naive or cytotoxic T lymphocytes (CTLs). MHC class II proteins are present on dendritic cells, B lymphocytes, macrophages, and other antigen-presenting cells. MHC class II proteins primarily present peptides from external antigen sources, i.e., processed outside the cell, to T helper (Th) cells. Most peptides bound by MHC class I proteins are derived from cytoplasmic proteins occurring in the organism's own healthy host cells and do not typically stimulate an immune response. Therefore, cytotoxic T lymphocytes that recognize such self-peptide-presenting MHC molecules of class I are either eliminated in the thymus (central tolerance) or eliminated or inactivated after release from the thymus, i.e., tolerized (peripheral tolerance). MHC molecules have the ability to stimulate an immune response when they present peptides to non-tolerized T lymphocytes. Cytotoxic T lymphocytes have both T cell receptors (TCRs) and CD8 molecules on their surface. T cell receptors have the ability to recognize and bind peptides complexed with MHC class I molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor capable of binding to a specific MHC / peptide complex.

[0073] Before being presented on the cell surface, peptide antigens attach to MHC class I molecules in the endoplasmic reticulum by competitive affinity binding, where the affinity of an individual peptide antigen is directly related to its amino acid sequence and the presence of specific binding motifs at defined positions within the amino acid sequence. If the sequences of such peptides are known, it is possible to manipulate the immune system against diseased cells, for example, using peptide vaccines.

[0074] One of the major obstacles hindering the development of curative and tumor-specific immunotherapy is the identification and selection of highly specific and restrictive tumor antigens to avoid autoimmunity. Tumor neoantigens, 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 antigen class. Neoantigens have rarely been used in cancer vaccines or immunogenic compositions due to the technical challenges of their identification, optimized neoantigen selection, and engineering of neoantigens for use in vaccines or immunogenic compositions. These problems can be addressed by: Identifying all or nearly all mutations in the neoplasm / tumor at the DNA level using whole-genome, whole-exome (e.g., captured exons only), or RNA sequencing of the tumor on each patient's corresponding germline sample; Analyzing the identified mutations with one or more peptide-MHC binding prediction algorithms to generate multiple candidate neo-antigen T cell epitopes that are expressed within the neoplasm / tumor and that can bind to the patient's HLA alleles; and ·Synthesizing multiple candidate neo-antigenic peptides selected from a set of all neo-ORF peptides and predicted binding peptides for use in cancer vaccines or immunogenic compositions.

[0075] As described herein, mutant epitopes are effective in inducing immune responses in both animals and humans, and cases of spontaneous tumor regression or long-term survival correlate with CD8+ T cell responses to mutant epitopes (Buckwalter and Srivastava PK. "'It's the antigen, silly' and human cancer vaccines"). "It is the antigen(s), stupid" and other lessons from over a decade of vaccitherapy of human cancer." Seminars in immunology 20:296-300 (2008); Karanikas et al., "High frequency of cytolytic T lymphocytes directed against a tumor-specific mutated antigen detectable with HLA tetramers in the blood of a lung carcinoma patient with long-term survival." long survival" Cancer Res. 61:3718-3724 (2001); Lennerz et al., "The response of autologous T cells to a human melanoma is dominated by mutated neo-antigens" Proc Natl Acad Sci U S A. 102:16013 (2005)) and that "immunoediting" can be traced to altered expression of dominant mutant antigens in mice and humans (Matsushita et al., "Cancer exome analysis reveals a T cell-dependent mechanism of cancer immunoediting" a T-cell-dependent mechanism of cancer immunoediting,” Nature 482:400 (2012); DuPage et al., “Expression of tumor-specific antigens underlies cancer immunoediting,” Nature 482:405 (2012); and Sampson et al., “Immunologic escape after prolonged progression-free survival with epidermal growth factor receptor variant III peptide vaccination in newly diagnosed glioblastoma patients.” There is a large body of evidence for vaccination in patients with newly diagnosed glioblastoma (J Clin Oncol. 28:4722-4729 (2010)). In one embodiment, mutant epitopes in cancer patients are determined.

[0076] In one embodiment, mutant epitopes are determined by sequencing the genomes and / or exomes of tumor and healthy tissue from cancer patients using next-generation sequencing techniques. In another embodiment, genes selected based on their mutation frequency and ability to act as neoantigens are sequenced using next-generation sequencing techniques. Next-generation sequencing applies to genome sequencing, genome resequencing, transcriptome profiling (RNA-Seq), DNA-protein interactions (ChIP-sequencing), and epigenomic characterization (de Magalhaes JP, Finch CE, Janssens G (2010). "Next-generation sequencing in aging research: emerging applications, problems, pitfalls and possible solutions". Ageing Research Reviews 9(3):315-323; Hall N (May 2007). "Advanced sequencing technologies and their wider impact in microbiology". J. Exp. Biol.209(Pt 9):1518-1525; Church GM(January 2006). "Genomes for all". Sci.Am.294(1):46-54; Bosch JR, Grody WW(2008). "Keeping Up with the Next Next-generation sequencing can now rapidly reveal the presence of coding mutations in individual tumors, most commonly single amino acid changes (e.g., missense mutations) and, less frequently, characteristic mutations such as frameshift insertions / deletions / gene fusions, read-through mutations at stop codons, and novel stretches of amino acids generated by translation of improperly spliced ​​introns (e.g., neo-ORFs). Neo-ORFs are particularly useful as immunogens because their entire sequence is completely novel to the immune system and therefore resembles viral or bacterial foreign antigens. Thus, neo-ORFs are: (1) highly specific for tumors (i.e., not expressed in any normal cells); and (2) capable of circumventing central tolerance, thereby increasing the precursor frequency of neoantigen-specific CTLs. For example, peptides derived from human papillomavirus (HPV) have recently demonstrated the power of utilizing analogous foreign sequences in therapeutic anti-cancer vaccines or immunogenic compositions. Approximately 50% of 19 patients with pre-neoplastic virus-induced disease who received 3-4 vaccinations with a mixture of HPV peptides derived from the viral oncogenes E6 and E7 maintained complete remission for more than 24 months (Kenter et al., "Vaccination against HPV-16 Oncoproteins for Vulvar Intraepithelial Neoplasia"). NEJM 361:1838(2009)).

[0077] Sequencing technologies have revealed that each tumor contains multiple patient-specific mutations that alter the protein-coding content of genes. Such mutations create altered proteins ranging from single amino acid changes (caused by missense mutations) to the addition of long stretches of novel amino acid sequence due to frameshifts, readthrough of stop codons, or translation of intronic regions (novel open reading frame mutations; neo-ORFs). These mutant proteins are useful targets for the host's immune response against tumors because, unlike native proteins, they are not subject to the immunodepressant effects of self-tolerance. Thus, mutant proteins are more likely to be immunogenic and more specific for tumor cells compared to the patient's normal cells.

[0078] An alternative method for identifying tumor-specific neoantigens is direct protein sequencing. Protein sequencing of enzymatic digests using multidimensional MS techniques (MSn), including tandem mass spectrometry (MS / MS), can also be used to identify neoantigens of the present invention. Such proteomic approaches allow for rapid and highly automated analysis (see, e.g., K. Gevaert and J. Vandekerckhove, Electrophoresis 21:1145-1154 (2000)). Furthermore, it is contemplated within the scope of the present invention that high-throughput de novo sequencing methods for unknown proteins can be used to analyze the proteome of a patient's tumor and identify expressed neoantigens. For example, meta-shotgun protein sequencing can be used to identify expressed neoantigens (see, e.g., Guthals et al. (2012) "Shotgun Protein Sequencing by Metacontig Assembly"). ncing with Meta-contig Assembly), Molecular and Cellular Proteomics 11(10):1084-96).

[0079] Tumor-specific neoantigens may also be identified by using MHC multimers to identify neoantigen-specific T cell responses. For example, high-throughput analysis of neoantigen-specific T cell responses in patient samples may be performed using MHC tetramer-based screening techniques (see, e.g., Hombrink et al. (2011) "High-Throughput Identification of Potential Minor Histocompatibility Antigens by MHC Tetramer-Based Screening: Feasibility and Limitations"). (2009) "Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers," Nature Methods, 6(7):520-26; van Rooij et al. (2013) "Tumor exome analysis reveals neoantigen-specific T-cell reactivity in an ipilimumab-responsive melanoma," Journal of Clinical Oncology, 31:1-4; and Heemskerk et al. (2013) "The cancer antigenome," EMBO Journal, 32(2):194-203). Such tetramer-based screening techniques can be used for the initial identification of tumor-specific neoantigens or as a secondary screening protocol to assess which neoantigens a patient has already been exposed to, thereby facilitating the selection of candidate neoantigens of the present invention.

[0080] In one embodiment, sequencing data obtained from determining the presence of mutations in cancer patients is analyzed to predict personalized mutant 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 which specific mutations to utilize as immunogens requires identification of the patient's HLA type and the ability to predict which mutant peptides can efficiently bind to the patient's HLA alleles. Recently, neural network-based training methods using validated binding and non-binding peptides have improved the accuracy of prediction algorithms for major HLA-A and -B alleles. Recently improved algorithms that predict which missense mutations will result in strong binding peptides for a patient's cognate MHC molecule can be used to identify and prioritize a set of peptides that represent the best mutant epitopes (both neo-ORF 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)).

[0081] Targeting as many mutant epitopes as practically possible harnesses the tremendous power of the immune system, thwarts opportunities for immune escape through downregulation of specific immune-targeted gene products, and compensates for the known inaccuracies of epitope prediction methods. 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 are easily chemically synthesized using reagents that are free of contaminating bacterial or animal substances and can be easily purified. Their small size allows for a clear focus on the mutated region of the protein and also reduces irrelevant antigenic competition from other components (non-mutated proteins or viral vector antigens).

[0082] In one embodiment, the pharmaceutical formulation is a long peptide multi-epitope vaccine or immunogenic composition. Such "long" peptides have been shown to undergo efficient internalization, processing, and cross-presentation in 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"). (Vaccines)" Nature Rev Cancer 8:351 (2008)). In one embodiment, at least one peptide is prepared for immunization. In a preferred embodiment, 20 or more peptides are prepared for immunization. In one embodiment, the neo-antigenic peptides range from about 5 to about 50 amino acids in length. In another embodiment, peptides ranging from about 15 to about 35 amino acids in length are synthesized. In a preferred embodiment, the neo-antigenic peptides range from about 20 to about 35 amino acids in length.

[0083] Generation of tumor-specific neoantigens The present invention is based, at least in part, on the ability to present a pool of tumor-specific neoantigens to a patient's immune system. Those skilled in the art will recognize, from this disclosure and knowledge in the art, that there are various methods for generating such tumor-specific neoantigens. Generally, such tumor-specific neoantigens can be generated either in vitro or in vivo. Tumor-specific neoantigens may be generated in vitro as peptides or polypeptides, which may then be formulated into personalized neoplasia vaccines or immunogenic compositions and administered to a subject. As described in further detail herein, such in vitro generation can be accomplished by various methods known to those skilled in the art, such as peptide synthesis or expression of the peptide / polypeptide from DNA or RNA molecules in any of a variety of bacterial, eukaryotic, or viral recombinant expression systems, followed by purification of the expressed peptide / polypeptide. Alternatively, tumor-specific neoantigens may be generated in vivo by introducing into a subject molecules (e.g., DNA, RNA, viral expression systems, etc.) encoding the tumor-specific neoantigen, followed by expression of the encoded tumor-specific neoantigen. Methods for in vitro and in vivo production of neoantigens are also further described herein, as are methods for delivery of pharmaceutical compositions and combination therapies.

[0084] In vitro peptide / polypeptide synthesis Proteins or peptides can be produced by any technique known to those of skill in the art, including expression of the protein, polypeptide, or peptide by standard molecular biology techniques, isolation of the protein, polypeptide, or peptide from a natural source, in vitro translation, or chemical synthesis of the protein or peptide. Nucleotide and protein, polypeptide, and peptide sequences corresponding to various genes have been disclosed and can be found in computerized databases known to those of skill in the art. One such database is maintained by the National Institutes of Health (NIH). and the Genbank and GenPept databases of the National Center for Biotechnology Information at the National Institutes of Health website. The coding regions of known genes can be amplified and / or expressed using the techniques disclosed herein or as would be known to one of skill in the art. Alternatively, a variety of commercially available protein, polypeptide and peptide preparations are known to those of skill in the art.

[0085] Peptides can be readily chemically synthesized using reagents that are free of contaminating bacterial or animal substances (Merrifield RB: "Solid phase peptide synthesis. I. The synthesis of tetrapeptides"). "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) purification by column stripping on an RP-HPLC column; and re-cleaning between peptides but without exchange; followed by (3) analysis by a limited set of the most informative assays. A Good Manufacturing Practice (GMP) footprint can be defined for each patient's set of peptides, so that suite switching procedures are only required between different patient peptide syntheses.

[0086] Alternatively, neo-antigenic peptides can be produced in vitro using nucleic acids (e.g., polynucleotides) encoding the neo-antigenic peptides of the present invention. The polynucleotides can be, for example, DNA, cDNA, PNA, CNA, RNA, single-stranded and / or double-stranded polynucleotides, or natural or stabilized forms, such as polynucleotides with phosphorothioate backbones, or combinations thereof, and may or may not contain introns, so long as they encode the peptide. In one embodiment, the peptides are produced using in vitro translation. Many exemplary systems are available to those skilled in the art (e.g., Retic Lysate IVT Kit, Life Technologies, Waltham, MA).

[0087] An expression vector capable of expressing a polypeptide can also be prepared. Expression vectors for various cell types are well known in the art and can be selected without undue experimentation. Generally, DNA is inserted into an expression vector such as a plasmid in the appropriate orientation and correct reading frame for expression. If necessary, the DNA may be linked to appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host (e.g., bacteria), although such controls are generally available in the expression vector. The vector is then introduced into a host bacterium for cloning using standard techniques (see, for example, Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0088] Expression vectors containing the isolated polynucleotides, as well as 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 peptides. One or more neo-antigenic peptides of the present invention may be provided by a single expression vector.

[0089] The term "polynucleotide encoding a polypeptide" encompasses polynucleotides containing only the coding sequence for the polypeptide as well as polynucleotides containing additional coding and / or non-coding sequences. Polynucleotides may be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; it may be double-stranded or single-stranded, and if single-stranded, may be the coding strand or the non-coding strand (antisense strand). Good too.

[0090] In embodiments, the polynucleotide may include a coding sequence for a tumor-specific neo-antigenic peptide fused in the same reading frame as a polynucleotide that aids in the expression and / or secretion of the polypeptide from a host cell (e.g., a leader sequence that functions as a secretory sequence to control transport of the polypeptide from the cell). Polypeptides with leader sequences are preproteins and may have a leader sequence that is cleaved by the host cell to form the mature form of the polypeptide.

[0091] In embodiments, the polynucleotide may comprise a coding sequence for a tumor-specific neo-antigenic peptide fused in the same reading frame to a marker sequence that allows for, for example, purification of the encoded polypeptide so that it can then be incorporated into a personalized neoplasia vaccine or immunogenic composition. For example, the marker sequence may be a hexahistidine tag provided by the pQE-9 vector, which in the case of a bacterial host provides for purification of the mature polypeptide fused to the marker, or the marker sequence may 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.

[0092] In embodiments, the polynucleotide may comprise one or more coding sequences for tumor-specific neo-antigenic peptides fused in the same reading frame to create a single concatemerized neo-antigenic peptide construct capable of producing multiple neo-antigenic peptides.

[0093] 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 invention.

[0094] A polynucleotide 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 up to 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 nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with other nucleotides, or up to 5% of the total number of nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the amino- or carboxy-terminal positions of the reference nucleotide sequence, or anywhere between these terminal positions, either individually interspersed among nucleotides in the reference sequence, or as one or more contiguous groups within the reference sequence.

[0095] As a practical matter, 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 determined using the Bestfit program (Wisconsin Sequence Analysis Package). This can be determined conventionally using known computer programs such as the lysis Package, version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best 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 of the present invention, parameters are set such that the percentage of identity is calculated over the entire length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.

[0096] The isolated tumor-specific neo-antigenic peptides described herein can be produced in vitro (e.g., in the laboratory) by any suitable method known in the art. Such methods range from direct protein synthesis methods 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 using recombinant techniques to isolate or synthesize a DNA sequence encoding a wild-type protein of interest. Optionally, the sequence can be mutated by site-directed mutagenesis to provide a functional analog thereof. See, e.g., Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81:5662-5066 (1984) and U.S. Pat. No. 4,588,585.

[0097] In embodiments, DNA sequences encoding a polypeptide of interest can be 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 codons preferred by the host cell in which the recombinant polypeptide of interest will be produced. Standard methods are used to synthesize isolated polynucleotide sequences encoding isolated polypeptides of interest. For example, a complete amino acid sequence can be used to construct a reverse-translated gene. Additionally, DNA oligomers containing nucleotide sequences encoding specific isolated polypeptides 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.

[0098] After assembly (e.g., by synthesis, site-directed mutagenesis, or otherwise), the polynucleotide sequence encoding the specific isolated polypeptide of interest is inserted into an expression vector and, optionally, operably linked to expression control sequences appropriate for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and expression of a biologically active polypeptide in a suitable host. As is well known in the art, to achieve high levels of expression of a transfected gene in a host, the gene can be operably linked to transcriptional and translational expression control sequences that function in the selected expression host.

[0099] Recombinant expression vectors may be used to amplify and express DNA encoding tumor-specific neo-antigenic peptides. A recombinant expression vector is a replicable DNA construct that contains 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 generally contains (1) one or more genetic elements that have a regulatory role in gene expression, such as a transcriptional promoter or enhancer, and (2) a transcriptional regulator, as described in more detail herein. The assembly of a structural or coding sequence to be transcribed into RNA and translated into protein includes (3) appropriate transcription and translation 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 may also be incorporated. DNA regions are operably linked when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operably linked to DNA for 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, this means contiguous and in reading frame. Structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by the host cell. Alternatively, if the recombinant protein is expressed without a leader or transport sequence, it may include an N-terminal methionine residue. This residue may optionally be subsequently cleaved from the expressed recombinant protein to yield the final product.

[0100] Useful expression vectors for eukaryotic hosts, particularly mammalian or human, include, for example, vectors containing expression control sequences from SV40, bovine papillomavirus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, e.g., Escherichia coli-derived plasmids, including pCR1, pBR322, pMB9, and their derivatives, as well as broader host range plasmids such as M13 and filamentous single-stranded DNA phages.

[0101] Suitable host cells for expressing polypeptides include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of a suitable 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 can also be used. Suitable cloning and expression vectors for use in bacterial, fungal, yeast, and mammalian cell hosts are well known in the art (Pouwels, (See et al., Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985).

[0102] Various mammalian or insect cell culture systems are also advantageously used to express recombinant proteins. Expression of recombinant proteins in mammalian cells is feasible because such proteins are generally correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cell lines include the COS-7 system of monkey kidney cells described by Gluzman (Cell 23:175, 1981), as well as other cell lines capable of expressing appropriate vectors, such as L cells, C127, 3T3, Chinese hamster ovary (CHO), 293, HeLa, and BHK cell lines. Mammalian expression vectors may contain nontranscribed elements, 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 essential ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. Baculovirus systems for producing heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio / Technology 6:47 (1988).

[0103] Proteins produced by transformed hosts can be purified by any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity, and size exclusion column chromatography), centrifugation, differential solubility, or any other standard method. These include conventional protein purification techniques. Attaching affinity tags, such as hexahistidine, maltose-binding domain, influenza coat sequence, glutathione-S-transferase, etc., to the protein can allow for easy purification by passage over an appropriate affinity column. Isolated proteins can also be physically characterized using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0104] For example, supernatants from systems secreting recombinant proteins into culture media can be first concentrated using commercially available protein concentration filters, such as Amicon or Millipore Pellicon ultrafiltration units. Following the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, an anion exchange resin, such as a matrix or substrate bearing pendant diethylaminoethyl (DEAE) groups, can be used. The matrix can be acrylamide, agarose, dextran, cellulose, or other types commonly used in protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Finally, the cancer stem cell protein-Fc composition can be further purified using one or more reverse-phase high-performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium, such as silica gel bearing pendant methyl or other aliphatic groups. Some or all of the aforementioned purification steps, in various combinations, can also be used to provide a homogeneous recombinant protein.

[0105] Recombinant proteins produced in bacterial cultures can be isolated, for example, by initial extraction from cell pellets, followed by one or more concentration, salting out, aqueous ion exchange, or size-exclusion chromatography steps. High-performance liquid chromatography (HPLC) may be used for final purification steps. Microbial cells used for recombinant protein expression can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents.

[0106] In vivo peptide / polypeptide synthesis The present invention also contemplates the use of nucleic acid molecules as vehicles for in vivo delivery of neo-antigenic peptides / polypeptides to a subject in need thereof, e.g., in the form of a DNA / RNA vaccine (see, e.g., WO 2012 / 159643 and WO 2012 / 159754, which are hereby incorporated by reference in their entireties).

[0107] In one embodiment, neoantigens can be administered to patients in need thereof using plasmids. These are plasmids that usually consist of a strong viral promoter to drive in vivo transcription and translation of the gene (or complementary DNA) of interest (Mor, et al., (1995). The Journal of Immunology 155(4):2039-2046). Sometimes, intron A can be included to improve mRNA stability and thus increase protein expression (Leitner et al. (1997). The Journal of Immunology 159(12):6112-6119). Plasmids also contain strong polyadenylation / transcription termination signals, such as the bovine growth hormone or rabbit β-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; Boehm et al., (1996). Journal of Immunological Methods 193(1):29-40). Sometimes, to express two or more immunogens, or to express an immunogen and an immunostimulatory protein, A multicistronic vector is constructed to express the vector (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54:129-88).

[0108] Because the plasmid is the "vehicle" from which the immunogen is expressed, it is essential to optimize the vector design to maximize protein expression (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54:129-88). One way to enhance protein expression is by optimizing the codon usage of the pathogenic mRNA for eukaryotic cells. Another consideration is the choice of promoter. Such a promoter can be the SV40 promoter or the Rous Sarcoma Virus (RSV).

[0109] Plasmids can be introduced into animal tissues by several different methods. The two most common techniques are injection of DNA in saline using a standard hypodermic needle and gene gun delivery. An overview of the construction of DNA vaccine plasmids and their subsequent delivery to the host by these two methods is provided in Scientific American (Weiner et al., (1999) Scientific American 281(1):34-41). Injections in saline are usually performed intramuscularly (IM) or intradermally (ID) in skeletal muscle, with the DNA being delivered to the extracellular space. This can be assisted by electroporation, by transiently damaging muscle fibers with myotoxins such as bupivacaine, or by using hypertonic solutions of saline or sucrose (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 alignment, injection speed, injection volume, muscle type, and the age, sex, and physiological condition of the animal receiving the injection (Alarcon et al., (1999). Adv. Parasitol. Advances in Parasitology 42:343-410).

[0110] 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) 54:129-88).

[0111] Alternative delivery methods include aerosol instillation of naked DNA onto mucosal surfaces such as the nasal and pulmonary mucosa (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.

[0112] The dose of DNA required to generate an effective immune response depends on the delivery method. Saline injections require varying amounts of DNA, 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 as little as 16 ng may be needed. Smaller amounts have also been reported. These amounts vary between species; for example, mice require about one-tenth the amount of DNA compared to primates. While saline injection requires more DNA because it must overcome physical barriers (such as the basement membrane and abundant connective tissue, to name a few) before being delivered to the extracellular space of the target tissue (usually muscle) and taken up by the cell, gene gun delivery shoots the DNA directly into the cell, thus resulting in less "waste" (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. al., (1993) Proc. Natl. Acad. Sci. USA 90(24):11478-82).

[0113] In one embodiment, a neoplasia vaccine or immunogenic composition may comprise a separate DNA plasmid encoding one or more neo-antigenic peptides / polypeptides, e.g., as identified in the present invention. 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 in the art. The expected persistence of the DNA construct (e.g., in an episomal, non-replicating, non-integrated form in muscle cells) is expected to result in an increased duration of protection.

[0114] One or more neo-antigenic peptides of the present invention can be encoded and expressed in vivo using a viral-based system (e.g., an adenoviral system, an adeno-associated viral (AAV) vector, a poxvirus, or a lentivirus). In one embodiment, the neoplasia vaccine or immunogenic composition can comprise a viral-based vector, such as an adenovirus, for use in a human patient in need thereof (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 (IPCAVD 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 viral, adenoviral, and lentiviral 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).

[0115] Among the vectors that can be used in the practice of the present invention, retroviral gene transfer methods allow integration into the cellular host genome, often resulting in long-term expression of the inserted transgene. In a preferred embodiment, the retrovirus is a lentivirus. In addition, high transduction efficiency has been observed in many different cell types and target tissues. The tropism of retroviruses can be altered by introducing exogenous envelope proteins, expanding the potential target population of target cells. Retroviruses can also be engineered to allow conditional expression of the inserted transgene, thus allowing only specific cell types to be infected by the lentivirus. Cell-type specific promoters can be used to target expression in specific cell types. Lentiviral vectors are retroviral vectors (and thus, in the practice of the present invention, lentiviral vectors and retroviral vectors are used). (Both retroviral and lentiviral vectors may be used.) Furthermore, lentiviral vectors are preferred because they are capable of transducing or infecting non-dividing cells and typically produce high viral titers. Therefore, the choice of retroviral gene transfer system may depend on the target tissue. Retroviral vectors contain cis-acting long terminal repeats capable of packaging up to 6-10 kb of foreign sequence. A minimal set of cis-acting LTRs is sufficient for vector replication and packaging, which is then used to integrate the desired nucleic acid into target cells, resulting in persistent expression. Widely used retroviral vectors that can be used in the practice of the present invention 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). Zou et al. 9 Approximately 10 μl of recombinant lentivirus was administered, with a titer of transducing units (TU) / ml. Such dosages can be adapted or extrapolated for use with retroviral or lentiviral vectors in the present invention.

[0116] Also useful in the practice of the present invention are minimal non-primate lentiviral vectors, such as those based on equine infectious anemia virus (EIAV) (see, e.g., Balagaan, (2006) J Gene Med;8:275-285, published online 21 November 2005 in Wiley InterScience (www.interscience.wiley.com). DOI:10.1002 / jgm.845). The vector may have a cytomegalovirus (CMV) promoter driving expression of the target gene. Accordingly, the present invention contemplates viral vectors, including, among other vectors, useful in the practice of the present invention: retroviral vectors and lentiviral vectors.

[0117] Adenoviral vectors are also useful in practicing the present invention. One advantage is that recombinant adenoviruses can efficiently transfer and express recombinant genes in various mammalian cells and tissues in vitro and in vivo, resulting in high expression of the transferred nucleic acid. Furthermore, the ability to productively infect quiescent cells broadens the usefulness of recombinant adenoviral vectors. In addition, high expression levels ensure expression of nucleic acid products at levels sufficient to generate an immune response (see, e.g., U.S. Pat. No. 7,029,848, incorporated herein by reference).

[0118] In certain embodiments herein, delivery is via adenovirus, and the adenovirus is at least 1 x 10 5 In certain embodiments herein, the dose is preferably at least about 1 x 10 6 particles (e.g., about 1 × 10 6 ~1×10 12 particles), more preferably at least about 1×10 7 particles, more preferably at least about 1×10 8 particles (e.g., about 1 × 10 8 ~1×10 11 particles or approximately 1 x 108 ~1×10 12 particles), and most preferably 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 particles) of adenoviral vector. Alternatively, the dose is about 1 x 10 14 particles or less, preferably about 1 x 10 13 particles or less, and even more preferably about 1×10 12 particles or less, and even more preferably about 1×10 11 particles or less, and most preferably about 1×10 10 particles or less (e.g., about 1 × 10 9 particle s) or less). 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, approx. 2×10 12 pu, or approximately 4 x 10 12The adenovirus vector may comprise a single dose of the adenovirus vector having pu of the adenovirus vector. See, for example, U.S. Patent No. 8,454,972 B2 to Nabel, et al., granted June 4, 2013 (incorporated herein by reference), adenovirus vectors, and dosages at column 29, lines 36-58 thereof. In some embodiments herein, the adenovirus is delivered in multiple doses.

[0119] From the perspective of in vivo delivery, AAV is advantageous over other viral vectors because it has low toxicity and is less likely to cause 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 significantly reduce viral production. Numerous promoters are available for driving nucleic acid molecule expression. AAV ITRs can serve as promoters, 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, CaMKIIα for excitatory neurons, and GAD67, GAD65, or VGAT for GABAergic neurons. Promoters used to drive RNA synthesis include Pol III promoters such as U6 or H1. For guide RNA (gRNA) expression, Pol II promoters and intron cassettes can be used.

[0120] Regarding AAV, AAV may be AAV1, AAV2, AAV5, or any combination thereof. AAV can be selected based on the target cells; for example, AAV serotype 1, 2, 5, or hybrid capsid AAV1, AAV2, AAV5, or any combination thereof can be selected for targeting brain or neural cells; and AAV4 can be selected for targeting cardiac tissue. AAV8 is useful for delivery to the liver. The above promoters and vectors are individually preferred.

[0121] In certain embodiments herein, delivery is via AAV. The therapeutically effective dosage for in vivo delivery of AAV to humans is about 1×10 10 ~Approx. 1×10 50 The dosage is considered to be in the range of about 20 to about 50 ml of saline containing 1 x 10 functional AAV / ml solution. Dosage can be adjusted to balance the therapeutic benefit with any side effects. In certain embodiments herein, the AAV dose is generally about 1 x 10 5 ~1×10 50 Genomic AAV, approximately 1 × 10 8 ~1×10 20 Genomic AAV, approximately 1 × 10 10 ~Approx. 1×10 16 genome, or approximately 1 x 10 11 ~Approx. 1×10 16 The concentration of genomic AAV is in the range of 1 × 10 13 The concentration can be about 0.001 ml to about 100 ml, about 0.05 ml to about 50 ml, or about 10 ml to about 25 ml of carrier solution. In a preferred embodiment, the concentration can be about 2 × 10 13 AAV is used at a titer of 1000 viral genomes per milliliter, with each mouse striatal hemisphere receiving a single 500 nanoliter injection. Other effective dosages can be readily established by those skilled in the art through routine testing to generate dose-response curves. See, for example, U.S. Patent No. 8,404,658 B2 to Hajjar, et al., issued March 26, 2013, column 27, lines 45-60.

[0122] In another embodiment, the cellular immune response to the neoplastic vaccine or immunogenic composition is Effective activation can be achieved by expressing the relevant neoantigen in a vaccine or immunogenic composition in a non-pathogenic microorganism, well-known examples of which are Mycobacterium bovis BCG, Salmonella, and Pseudomonas (see U.S. Pat. No. 6,991,797, hereby incorporated by reference in its entirety).

[0123] In another embodiment, a poxvirus is used in the neoplasia vaccine or immunogenic composition. Poxviruses include orthopoxvirus, avipox, vaccinia, MVA, NYVAC, canarypox, ALVAC, fowlpox, TROVAC, etc. (See, e.g., Verardiet al., Hum Vaccin Immunother. 2012). (See, e.g., J. J. Vaccine. 2013;31(39):4220-4222.) Poxvirus expression vectors were described in 1982 and quickly became widely used in vaccine development and in numerous areas of research. Advantages of these vectors include simple construction, the ability to accommodate large amounts of foreign DNA, and high expression levels.

[0124] In another embodiment, neoplasia vaccines or immunogenic compositions use vaccinia virus to express neoantigens (Rolph et al., "Recombinant viruses as vaccines and immunological tools." Curr Opin Immunol 9:517-524, 1997). Recombinant vaccinia virus is capable of replicating in the cytoplasm of infected host cells, thus allowing the polypeptide of interest to induce an immune response. Furthermore, poxviruses have been 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, particularly antigen-presenting cells, and their self-adjuvanting capabilities.

[0125] In another embodiment, ALVAC is used as a vector in a neoplastic vaccine or immunogenic composition. ALVAC is a canarypox virus that can be modified to express foreign transgenes and has been used as a vaccination strategy 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 adenocarcinoma." 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. "Intramuscularly administered HIV-1 vaccination can induce both systemic and mucosal T-cell immunity in HIV-1-naive individuals (HIV-1 vaccination administered intramuscularly can induce both systemic and mucosal T "Poxvirus vectors 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; U.S. Patent No. 7,255,862. In a phase I clinical trial, ALVAC virus expressing the tumor antigen CEA demonstrated a favorable safety profile in selected patients and induced increased CEA-specific T cell responses; however, 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).

[0126] In another embodiment, modified vaccinia Ankara (MVA) virus can be used as a viral vector for neoantigen vaccines or immunogenic compositions. MVA is a member of the orthopoxvirus family and was generated by serial passage of the Ankara strain of vaccinia virus (CVA) in chicken embryo fibroblasts for approximately 570 generations (for a review, see Mayr, A., et al., Infection 3, 6-14, 1975). As a result of such passage, the resulting MVA virus contains 31 kilobases less genomic information than CVA and is highly host-cell restricted (Meyer, H. et al., J. Gen. Virol. 72, 1031-1038, 1991). MVA is characterized by its severe attenuation, i.e., reduced virulence or infectivity, while still retaining excellent immunogenicity. When tested in various animal models, MVA has proven avirulent, even in immunosuppressed individuals. Additionally, MVA-BN®-HER2 is an immunotherapy candidate designed for the treatment of HER-2-positive breast cancer and is currently in clinical trials (Mandl et al., Cancer Immunol Immunother. Jan 2012;61(1):19-29). Methods for making and using recombinant MVA have been described (see, e.g., U.S. Pat. Nos. 8,309,098 and 5,185,146, which are hereby incorporated by reference in their entireties).

[0127] In another embodiment, modified vaccinia virus Copenhagen strain, NYVAC and NYVAC variants are used as vectors (see U.S. Pat. No. 7,255,862; WO 95 / 30018; U.S. Pat. Nos. 5,364,773 and 5,494,807, which are hereby incorporated by reference in their entireties).

[0128] In one embodiment, recombinant viral particles of a vaccine or immunogenic composition are administered to a patient in need thereof. The dosage of the neoantigen to be expressed can range from a few micrograms to hundreds of micrograms, e.g., 5-500 μg. The vaccine or immunogenic composition can be administered in any suitable amount that achieves expression at such dosage levels. The viral particles are at least about 10 3.5 The virus particles can be administered to a patient in need thereof or transfected into cells in amounts of at least about 10 pfu; therefore, the virus particles can be administered in amounts of at least about 10 pfu. 4 pfu~about 10 6 pfu to a patient in need thereof, or to infect or transfect cells; however, a patient in need thereof may be administered at least about 10 8 pfu, and more preferably at least about 10 7 pfu~about 10 9 The dose for NYVAC may be expressed as pfu. C, MVA, MVA-BN, and avipox, such as canarypox and fowlpox.

[0129] Vaccine or Immunogenic Composition Adjuvants An effective vaccine or immunogenic composition advantageously includes a potent adjuvant to elicit an immune response. As described herein, poly-ICLC, an agonist of the RNA helicase domains of TLR3 and MDA5 and RIG3, exhibits several desirable properties for a vaccine or immunogenic composition adjuvant. These properties include inducing local and systemic activation of immune cells in vivo, producing stimulatory chemokines and cytokines, and stimulating antigen presentation by DCs. Furthermore, poly-ICLC can induce sustained CD4+ and CD8+ responses in humans. Importantly, striking similarities were observed in the upregulation of transcriptional and signaling pathways between subjects vaccinated with poly-ICLC and volunteers previously administered a highly efficacious, replication-competent yellow fever vaccine. Furthermore, in a recent phase 1 trial, over 90% of ovarian cancer patients immunized with poly-ICLC in combination with the NY-ESO-1 peptide vaccine (in addition to Montanide) demonstrated induction of CD4+ and CD8+ T cells and antibody responses to the peptide. At the same time, poly-ICLC has been extensively tested in over 25 clinical trials to date and exhibits a relatively benign toxicity profile. In addition to being potent and specific immunogens, neoantigen peptides can be combined with adjuvants (e.g., poly-ICLC) or other anti-neoplastic agents. Without being bound by theory, these neoantigens are expected to circumvent central thymic tolerance (thus enabling a stronger anti-tumor T cell response) while reducing the likelihood of autoimmunity (e.g., by avoiding targeting of normal self-antigens). Effective immune responses advantageously include a potent adjuvant 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 and, in turn, adaptive immune systems (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 bone marrow-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 a preferred embodiment, Hiltonol®, a GMP formulation of poly-ICLC prepared by Oncovir, Inc., is utilized as the adjuvant. In other embodiments, other adjuvants described herein are contemplated. For example, oil-in-water, water-in-oil or multiphase W / O / W; see, for example, US Pat. No. 7,608,279 and Aucouturier et al, Vaccine 19 (2001), 2666-2672, and references cited therein.

[0130] Checkpoint inhibitors The present invention relates to a neoplastic vaccine or immunogenic composition as described herein and at least one The present invention features a method for treating or preventing a neoplasm, comprising administering to a subject a combination of a medicament for treating or preventing a neoplasm and at least one checkpoint inhibitor. Thus, one, two, three, four, five, or more checkpoint inhibitors may be administered. In certain exemplary embodiments, one checkpoint inhibitor is administered. In other exemplary embodiments, two checkpoint inhibitors are administered.

[0131] Page et al. (Annu. Rev. Med. 2014.65) summarizes published studies examining checkpoint regulators in solid tumors. Mullard, A. (Nature Reviews, Drug Discovery. Vol. 12, July 2013) provides a review of checkpoint inhibitors. A summary table of exemplary checkpoint inhibitors is provided herein.

[0132] [Table 1]

[0133] Anti-CTLA4 antibody Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), also known as CD152, is a co-inhibitory molecule that acts to regulate T cell activation.

[0134] CTLA4 was originally identified as a negative regulator on the surface of T cells that is upregulated immediately after the elicitation of a de novo immune response or stimulation of a pre-existing response to dampen subsequent immune T cell responses and prevent autoimmunity or uncontrolled inflammation. Thus, the magnitude of the immune response is closely linked to CTLA4 activity. In a specific embodiment, the anti-CTLA4 antibody is ipilimumab or tremelimumab.

[0135] Checkpoint inhibitors work by modulating the immune system's endogenous T cell regulatory mechanisms. Ipilimumab (YERVOY, Bristol-Meyers Squibb, New York, NY), a monoclonal antibody and the first such checkpoint inhibitor approved by the U.S. Food and Drug Administration (FDA), has become the standard treatment for metastatic melanoma (Hodi et al., N. Engl. J. Med. 363:711-23, 2010; Robert et al., N. Engl. J. Med. 364:2517-26, 2011). Ipilimumab binds to the T cell surface co-inhibitory molecule cytotoxic T-lymphocyte antigen 4 (CTLA-4) and blocks the inhibitory signaling mediated by it. Because its mechanism of action is not specific to one tumor type, and abundant preclinical data support a role for tumor immune surveillance across multiple malignancies (Andre et al., Clin. Cancer Res. 19:28-33. 2013; May et al. Clin. Cancer Res. 17:5233-38. 2011), ipilimumab is a promising treatment for prostate cancer, lung cancer, among other tumor types. It is being studied as a treatment for patients with breast cancer, renal cancer, and ovarian cancer. Ipilimumab works by targeting CTLA-4, activating the immune system.

[0136] Another CTLA-4-blocking antibody, tremelimumab, continues to be investigated in clinical trials and has also demonstrated durable responses in melanoma patients (Kirkwood et al., Clin. Cancer Res. 16:1042-48. 2010; Ribas et al. al.J.Clin.Oncol.31:616-22,2013).

[0137] Thus, in exemplary embodiments, the present invention features novel combinations of a neoplastic vaccine or immunogenic composition and one or more anti-CTLA4 antibodies. The present invention also features, in other exemplary embodiments, novel combinations of a neoplastic vaccine or immunogenic composition, ipilimumab and / or nivolumab, and one or more anti-CTLA4 antibodies.

[0138] Inhibitors of the programmed cell death-1 pathway CTLA-4 functions to regulate early T cell activation, while programmed death 1 (PD-1) signaling functions, in part, to regulate T cell activation in peripheral tissues. The PD-1 receptor refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed on several cell types, including Tregs, activated B cells, and natural killer (NK) cells, primarily on previously activated T cells in vivo, and binds to two ligands, PD-L1 and PD-L2. The endogenous ligands for PD1, PD-L1 and PD-L2, are expressed on activated immune cells and 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, and analogs that share at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under 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) that downregulates T-cell activation and cytokine secretion upon binding to PD-1. PD-L1, as used herein, includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, and analogs that share at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found under GENBANK Accession No. Q9NZQ7. Tumors have been demonstrated to 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). Blockade of these interactions with therapeutic antibodies has been shown to enhance T-cell responses and stimulate anti-tumor activity (Freeman et al. al.J.Exp.Med.192:1027-34.2000).

[0139] Abs of the present invention 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 exemplifies seven anti-PD-1 HuMAbs: 17D8, 2D3, 4H1, 5C4 (also referred to herein as nivolumab or BMS-936558), 4A11, 7D3, and 5F4.

[0140] In some embodiments, the anti-PD-1 antibody is nivolumab. Other names for nivolumab include MDX-1 106, MDX-1 106-04, ONO-4538, and BMS-936558. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946 414-94-4).

[0141] Nivolumab is a fully human IgG4-blocking monoclonal antibody against PD-1 (Topaliam et al., N. Engl. J. Med. 366:2443-54. 2012). Nivolumab specifically blocks PD-1 and can overcome immune resistance. Ligands for PD-1 have been identified as PD-L1 (B7-H1), which is expressed on all hematopoietic cells and many non-hematopoietic tissues, and PD-L2 (B7-DC), whose expression is primarily restricted to dendritic cells and macrophages (Dong, H. et al. 1999. Nat. Med. 5:1365; Freeman, GJ et al. 2000. J. Exp. Med. 192:1027; Latchman, Y. et al. 2001. Nat. Immunol. 2:261; Tseng, S. Y. et al. 2001. J. Exp. Med. 193:839). PD-L1 is overexpressed in many cancers and is often associated with poor prognosis (Okazaki T et al., Intern. Immun. 2007 19(7):813) (Thompson RH et al., Cancer Res 2006,66(7):3381). Interestingly, the majority of tumor-infiltrating T lymphocytes, in contrast to T lymphocytes in normal tissues and peripheral blood, predominantly express PD-1, suggesting that upregulation of PD-1 in tumor-reactive T cells may contribute to the attenuation of antitumor immune responses (Blood 2009 1 14(8):1537). Specifically, tumor cells express the immunosuppressive PD-1 ligand PD-L1, and inhibition of the interaction between PD-1 and PD-L1 can enhance T cell responses in vitro and mediate preclinical antitumor activity.

[0142] Several clinical trials (phase I, II, and III) involving nivolumab have been conducted or are ongoing (see clinicaltrials.gov / ct2 / results?term=nivolumab&pg=1, accessed December 20, 2013). For example, in a phase I dose-escalation study, nivolumab was safe, with objective responses of 16–31% across all tumor types, with most responses lasting more than 1 year (Topaliam et al., Presented at Annu. Meet. Am. Soc. Clin. Oncol., Chicago, May 31–June 4, 2013). Another study investigated the safety and clinical activity of nivolumab (anti-PD-1, BMS-936558, ONO-4538) in combination with ipilimumab in patients with advanced melanoma (Wolchok, J Clin Oncol 31, 2013 (suppl; abstr 9012 2013 ASCO Annual Meeting).

[0143] Two anti-PD-L1 inhibitor antibodies, MPDL3280A (Genentech, South Clinical trials are underway for PD-1 / PD-L1 antibodies (BMS-936559, San Francisco, CA) and BMS-936559 (Bristol Meyers Squibb, New York, NY). Similar to nivolumab and MK-3475, these antibodies are thought to function primarily by blocking PD-1 / PD-L1 signaling. Unlike PD-1 antibodies, PD-L1 antibodies allow the potential interaction between PD-L2 and PD-1, but also block the interaction between PD-L1 and CD80 (Park et al., 2010. Blood 116:1291-98). MPDL3280A has been evaluated in multiple tumor types, with safety and preliminary efficacy identified in melanoma; renal cell carcinoma; non-small cell lung cancer (NSCLC); and colorectal, gastric, and head and neck squamous cell carcinoma (Herbst et al. resented at Annu. Meet. Am. Soc. Clin. Oncol., Chicago, May 31-June 4, 2013). Similarly, BMS-936559 was shown to be safe and clinically active across multiple tumor types in a Phase I trial. MEDI-4736 is another PD-L1-blocking antibody currently in clinical development (NCT01693562).

[0144] In addition to CTLA-4 and PD-1 / PD-L1, numerous other immunomodulatory targets have been identified preclinically, many with corresponding therapeutic antibodies that are being investigated in clinical trials. Page et al. (Annu.Rev.Med.2014.65) detail targets of antibody immunomodulatory drugs in Figure 1 (incorporated herein by reference).

[0145] In an exemplary embodiment, the present invention features a novel combination of a neoplastic vaccine or immunogenic composition and one or more PD-1 pathway inhibitors. In a preferred embodiment, the PD-1 pathway inhibitor is an anti-PD1 antibody, such as nivolumab.

[0146] The present invention also features, in other exemplary embodiments, novel combinations of neoplastic vaccines or immunogenic compositions with nivolumab and / or one or more anti-CTLA4 antibodies.

[0147] Indications Examples of cancers and cancer conditions that may be treated by the combination therapy herein include, but are not limited to, patients in need thereof who have been diagnosed with cancer or are at risk of developing cancer. Subjects include solid tumors, e.g., breast, ovarian, prostate, lung, kidney, stomach, colon, testicular, head and neck, pancreatic, brain, melanoma, and other tissue and organ tumors, and hematological tumors, e.g., lymphomas and leukemias, including acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma; tumors of the brain and central nervous system (e.g., tumors of the meninges, brain, spinal cord, cranial nerves, and other CNS sites, e.g., glioblastoma or medulloblastoma); head and / or neck cancer; breast tumors; tumors of the circulatory system (e.g., tumors of the heart, mediastinum, and pleura, and other intrathoracic organs, vascular tumors, and tumor-associated vascular tissue); tumors of the blood and lymphatic system (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt's lymphoma, AIDS-related lymphoma, malignant immunoproliferative disorders, and cancers of the brain and central nervous system, e.g., tumors of the brain, spinal cord, cranial nerves, and other CNS sites, e.g., glioblastoma or medulloblastoma); lymphoid leukemia, multiple myeloma, and malignant plasma cell neoplasms, lymphocytic leukemia, myeloid leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, other leukemias of a specific cell type, leukemias of an unspecified cell type, unspecified malignant neoplasms of lymphoid tissue, hematopoietic tissue, and related tissues, e.g., diffuse large cell lymphoma, T-cell lymphoma, or cutaneous T-cell lymphoma; excretory system (e.g., kidney, renal pelvis, urinary tumors of the gastrointestinal tract (e.g., esophagus, stomach, small intestine, colon, colorectum, rectosigmoid junction, rectum, anus, and anal canal); tumors involving the liver and intrahepatic bile ducts, gallbladder, and other biliary tract sites, pancreas, and other digestive tract; tumors of the oral cavity (e.g., lips, tongue, gums, floor of mouth, palate, parotid glands, salivary glands, tonsils, oropharynx, nasopharynx, piriform sinus, tumors of the reproductive system (e.g., vulva, vagina, cervix, uterus, ovaries, and other female reproductive organs, placenta, penis, prostate, testes, and other male reproductive organs); tumors of the respiratory tract (e.g., nasal cavity, middle ear, paranasal sinuses, larynx, trachea, bronchi, and lungs, including small cell lung cancer and non-small cell lung cancer); tumors of the skeletal system (e.g., extremities, bones, and articular cartilage of other bones and joints); tumors of the skin (e.g., skin malignant melanoma, non-melanoma skin cancer, basal cell carcinoma of the skin, squamous cell carcinoma of the skin, mesothelioma, Kaposi's sarcoma); and tumors involving other tissues including the peripheral and autonomic nervous system, connective and soft tissue, retroperitoneum and peritoneum, eyes, thyroid, adrenal glands, and other endocrine glands and associated structures, secondary and unspecified malignant neoplasms of lymph nodes, secondary malignant neoplasms of the respiratory and digestive systems, and secondary malignant neoplasms of other sites.

[0148] Of particular interest is the treatment of non-Hodgkin's lymphoma (NHL), clear cell renal cell carcinoma (ccRCC), metastatic melanoma, sarcoma, leukemia, or bladder, colon, brain, breast, head and neck, endometrial, lung, ovarian, pancreatic, or prostate cancer. In certain embodiments, the melanoma is a high-risk melanoma.

[0149] Cancers that can be treated using the present combination therapy can include, among others, cases that are refractory to treatment with other chemotherapy. The term "refractory," as used herein, refers to cancers that show no or only a weak antiproliferative response after treatment with another chemotherapy agent (e.g., tumor growth is slowed). Refractory cancers refer to cancers (and / or their metastases) that do not or only weakly inhibit the growth factor receptor agonist (GFAP) receptor agonist (GFAP). These are cancers that cannot be adequately treated with other chemotherapies. Refractory cancers include not only (i) cancers that have already failed one or more chemotherapies in treating patients, but also (ii) cancers that can be shown to be refractory by other means, such as biopsy and culture in the presence of chemotherapy.

[0150] The combination therapy described herein is also applicable to the treatment of previously untreated patients in need thereof.

[0151] The combination therapy described herein is also applicable when a subject has no detectable neoplasia but is at high risk of disease recurrence.

[0152] Also of particular interest is the treatment of patients in need who have undergone autologous hematopoietic stem cell transplantation (AHSCT), particularly those who exhibit residual disease after AHSCT. The post-AHSCT setting is characterized by a small amount of residual disease, infusion of immune cells for a homeostatic proliferation state, and the absence of any standard therapy to delay relapse. These characteristics provide a unique opportunity to delay disease relapse using the claimed neoplastic vaccine or immunogenic compositions and checkpoint inhibitor compositions.

[0153] Pharmaceutical Compositions / Delivery Methods The present invention also relates to pharmaceutical compositions comprising an effective amount of one or more compounds according to the invention (including pharmaceutically acceptable salts thereof), optionally in combination with a pharmaceutically acceptable carrier, diluent or excipient.

[0154] When administered as a combination, the therapeutic agents (i.e., the neoplastic vaccine or immunogenic composition and one or more checkpoint inhibitors) may be formulated as separate compositions that are administered at the same or different times, or the therapeutic agents may be administered as a single composition.

[0155] 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 long administration intervals, sustained release or depot formulations can be used.

[0156] The compositions of the present invention can be used to treat acute diseases and disease conditions, and can also be used to treat chronic conditions. In particular, the compositions of the present invention are used in methods for treating or preventing neoplasms.

[0157] In certain embodiments, the compounds of the invention 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 5 years, 10 years, or more than 15 years; or for any range of days, months, or years (e.g., 4 weeks to 15 years, 6 months to 20 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 any period between 15 days and 20 years. In some cases, it may be advantageous to administer the compounds of the invention for the patient's lifetime. In preferred embodiments, the patient is monitored to identify progression of the disease or disorder, and the dosage is adjusted accordingly. In preferred embodiments, the treatment of the invention 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 subject's lifetime.

[0158] As described herein, in certain embodiments, administration of the checkpoint inhibitor is initiated prior to initiation of administration of the neoplastic vaccine or immunogenic composition. In other embodiments, administration of the checkpoint inhibitor is initiated after initiation of administration of the neoplastic vaccine or immunogenic composition. In yet other embodiments, administration of the checkpoint inhibitor is initiated simultaneously with initiation of administration of the neoplastic vaccine or immunogenic composition.

[0159] Administration of the checkpoint inhibitor can continue every 2, 3, 4, 5, 6, 7, 8 or more weeks after the initial administration of the checkpoint inhibitor. It is understood that week 1 is meant to include days 1 through 7, week 2 is meant to include days 8 through 14, week 3 is meant to include days 15 through 21, and week 4 is meant to include days 22 through 28. When administration is described as being weekly, it means that there are about 7 days between administrations, but in any given week, the days can be one or more days before or one or more days after the expected date.

[0160] In certain embodiments, administration of the checkpoint inhibitor is withheld for one week prior to administration of the neoplastic vaccine or immunogenic composition, hi other embodiments, administration of the checkpoint inhibitor is withheld during administration of the neoplastic vaccine or immunogenic composition.

[0161] Surgical resection involves the use of surgery to remove abnormal cancerous tissue, such as a mediastinal tumor, neurogenic tumor, germ cell tumor, or thymoma. In certain embodiments, administration of a checkpoint inhibitor is initiated after tumor resection. In other embodiments, administration of a neoplastic vaccine or immunogenic composition is initiated 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more weeks after tumor resection. Preferably, administration of a neoplastic vaccine or immunogenic composition is initiated 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after tumor resection.

[0162] A prime / boost regimen refers to the sequential administration of a vaccine or immunogenic or immunological composition. In certain embodiments, the administration of a neoplastic vaccine or immunogenic composition is a prime / boost dosing regimen, e.g., administration of a neoplastic vaccine or immunogenic composition at 1, 2, 3, or 4 weeks as a priming and administration of a neoplastic vaccine or immunogenic composition at 2, 3, or 4 months as a boost. In another embodiment, a heterologous prime-boost method is used to elicit a higher cytotoxic T cell response (Schneider et al., "Induction of CD8+ T cells using heterologous prime-boost immunization strategies," Immunological Reviews Volume 170, Issue 1). 1, pages 29-38, August 1999). In another embodiment, priming is performed with DNA encoding the neoantigen, followed by a protein boost. In another embodiment, priming is performed with a protein, followed by a boost with a virus encoding the neoantigen. In another embodiment, priming is performed with a virus encoding the neoantigen and boosting with another virus. In another embodiment, priming is performed with a protein and boosting with DNA. In a preferred embodiment, a DNA vaccine or immunogenic composition is used to prime a T cell response, and the response is boosted with a recombinant viral vaccine or immunogenic composition. In another preferred embodiment, the viral vaccine or immunogenic composition is co-administered with a protein or DNA vaccine or immunogenic composition, and serves 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 (Hutchings et al., "Combination of Protein and Viral Vaccines Induces Strong Cellular and Humoral Immune Responses and Protection Against Murine Malaria Challenge Infection"). "Combination of protein and viral vaccines induces potent cellular and humoral immune responses and enhanced protection from murine malaria challenge." Infect Immun. 2007 Dec;75(12):5819-26. Epub 2007 Oct 1).

[0163] Pharmaceutical compositions can be processed in accordance with conventional methods of pharmacy to produce medicinal agents for administration to patients in need thereof, including humans and other mammals.

[0164] Modifications of the neo-antigenic peptide can affect the solubility, bioavailability, and metabolic rate of the peptide, thus resulting in controlled delivery of the active species. Solubility can be assessed by preparing the neo-antigenic peptide and testing it by known methods well within the routine skill of one of ordinary skill in the art.

[0165] It has been found that pharmaceutical compositions containing succinic acid or a pharmaceutically acceptable salt thereof (succinate) can provide improved solubility for neo-antigenic peptides. Accordingly, in one aspect, the present invention provides pharmaceutical compositions comprising at least one neo-antigenic peptide or a pharmaceutically acceptable salt thereof; a pH adjuster (a base, e.g., a pharmaceutically acceptable salt of a di- or tri-carboxylate, e.g., succinic acid or citric acid); and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can be prepared by combining a solution containing at least one neo-antigenic peptide with a base, e.g., a pharmaceutically acceptable salt of a di- or tri-carboxylate, e.g., succinic acid or citric acid (e.g., sodium succinate), or by combining a solution containing at least one neo-antigenic peptide with a solution containing a base, e.g., a pharmaceutically acceptable salt of a di- or tri-carboxylate, e.g., succinic acid or citric acid (e.g., a succinate buffer solution). In certain embodiments, the pharmaceutical composition comprises sodium succinate. In certain embodiments, the pH adjuster (such as citrate or succinate) is present in the composition at a concentration of about 1 mM to about 10 mM, and in certain embodiments, at a concentration of about 1.5 mM to about 7.5 mM, or about 2.0 to about 6.0 mM, or about 3.75 to about 5.0 mM.

[0166] 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 further comprises an immunomodulator or adjuvant. In certain embodiments, the immunomodulator 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, and Montanide. In certain embodiments, the immunomodulatory agent 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 immunomodulatory agent or adjuvant comprises poly-ICLC.

[0167] Xanthenone derivatives, such as vadimezan or AsA404 (also known as 5,6-dimethylaxanthenone-4-acetic acid (DMXAA)) Alternatively, such derivatives may also be administered in conjunction with the vaccine or immunogenic composition of the invention, e.g., via 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-Dimethylxanthenone-4-Acetic Acid," Journal of Immunology, 190:5216-25 and Kim et al. (2013) "Anticancer Flavonoids are Mouse-Selective STING Agonists," 8:1396-1401).

[0168] The vaccine or immunological composition may also contain an adjuvant compound selected from acrylic or methacrylic polymers and maleic anhydride-alkenyl derivative copolymers. Specifically, it is a polymer of acrylic or methacrylic acid crosslinked with a polyalkenyl ether of a sugar or polyhydric alcohol (carbomer), specifically a polymer crosslinked with allyl sucrose or allyl pentaerythritol. It may also be a copolymer of maleic anhydride and ethylene crosslinked, for example, with a divinyl ether (see U.S. Pat. No. 6,713,068, hereby incorporated by reference in its entirety).

[0169] In certain embodiments, the pH adjusting agent can stabilize an adjuvant or immunomodulatory agent as described herein.

[0170] 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 300 μg / ml. In certain embodiments, the pharmaceutical composition comprises ≦3% by volume of DMSO. In certain embodiments, the pharmaceutical composition comprises 3.6-3.7% dextrose in water. In certain embodiments, the pharmaceutical composition comprises 3.6-3.7 mM succinate (e.g., sodium succinate). In certain embodiments, the pharmaceutical composition comprises 0.5 mg / ml of poly I:poly C. In certain embodiments, the pharmaceutical composition comprises 0.375 mg / ml of poly-L-lysine. In certain embodiments, the pharmaceutical composition comprises 1.25 mg / ml of sodium carboxymethylcellulose. In certain embodiments, the pharmaceutical composition comprises 0.225% sodium chloride.

[0171] The pharmaceutical compositions comprise the tumor-specific neo-antigenic peptides described herein in an amount therapeutically effective for treating the diseases and conditions (e.g., neoplasms / tumors) described herein, optionally in combination with pharmaceutically acceptable additives, carriers, and / or excipients. One skilled in the art will recognize from this disclosure and knowledge in the art that the therapeutically effective amount of one or more compounds of the present invention may vary depending on the condition to be treated, its severity, the treatment regimen employed, the pharmacokinetics of the drugs used, and the patient (animal or human) being treated.

[0172] To prepare pharmaceutical compositions according to the present invention, a therapeutically effective amount of one or more of the compounds according to the present invention is preferably formulated into a pharmaceutically acceptable carrier in accordance with conventional pharmaceutical compounding techniques to produce a dosage. The carrier is thoroughly mixed with the carrier. The carrier may take a variety of forms depending on the preparation desired for administration, for example, ophthalmic, oral, topical, or parenteral, such as gels, creams, ointments, lotions, and time-release implantable formulations, among others. When preparing pharmaceutical compositions in oral dosage form, any conventional pharmaceutical medium may be used. Thus, for liquid oral preparations such as suspensions, elixirs, and solutions, suitable carriers and additives, including water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, etc., may be used. For solid oral preparations such as powders, tablets, capsules, and for solid preparations such as suppositories, suitable carriers and additives, including starches, sugar carriers, e.g., dextrose, mannitol, lactose, and related carriers, diluents, granulating agents, lubricants, binders, disintegrating agents, etc., may be used. If necessary, tablets or capsules may be enterically coated or sustained-release by standard techniques.

[0173] 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 significant toxic effects to the patient being treated.

[0174] Oral compositions generally include an inert diluent or an edible carrier. Oral compositions can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound or its prodrug derivative can be incorporated with excipients and used in the form of tablets, troches, or capsules. Pharmaceutically compatible binding agents and / or adjuvant materials can also be included as part of the composition.

[0175] Tablets, pills, capsules, troches, etc. may contain any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, gum tragacanth, 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 may contain a liquid carrier such as fatty oil in addition to the materials discussed herein. In addition, dosage unit forms may contain various other materials that modify the physical form of the dosage unit, for example, sugar, shellac, or enteric coatings.

[0176] Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil emulsion, and as a bolus, etc.

[0177] Tablets may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing the active ingredient in a free-flowing form, such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface active agent or dispersant, in a suitable machine. Molded tablets may be made by molding a mixture of powdered compound moistened with an inert liquid diluent in a suitable machine. Tablets may optionally be coated or scored, and may be formulated to provide sustained or controlled release of the active ingredient therein.

[0178] Methods for formulating such sustained- or controlled-release compositions of pharmaceutically active ingredients are known in the art and are described in several issued U.S. patents, including, but not limited to, U.S. Pat. Nos. 3,870,790; 4,226,859; 4,369,172; 4,842,866; and 5,705,190, the disclosures of which are incorporated herein by reference in their entireties. Coatings can be used to deliver compounds to the intestine (see, e.g., U.S. Pat. Nos. 6,638,534, 5,541,171, 5,217,720, and 6,569,457, and references cited therein).

[0179] The active compound or a pharmaceutically acceptable salt thereof may also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum, etc. A syrup may contain, in addition to the active compound, sucrose or fructose as a sweetening agent and certain preservatives, dyes and colorings and flavors.

[0180] Solutions or suspensions used for ocular, parenteral, intradermal, subcutaneous, or topical application may include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates, and agents for the adjustment of tonicity, such as sodium chloride or dextrose.

[0181] In certain embodiments, the pharmaceutically acceptable carrier is an aqueous solvent, i.e., a solvent comprising water, optionally with an additional co-solvent. Exemplary pharmaceutically acceptable carriers include water, aqueous buffer solutions (such as phosphate buffered saline (PBS)), and 5% dextrose in water (D5W). In certain embodiments, the aqueous solvent further comprises dimethyl sulfoxide (DMSO), e.g., in an amount of about 1-4%, or 1-3%. In certain embodiments, the pharmaceutically acceptable carrier is isotonic (i.e., has substantially the same osmotic pressure as a body fluid, such as plasma).

[0182] In one embodiment, the active compound is prepared with a carrier that protects the compound from rapid elimination from the body, such as a controlled-release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic-co-glycolic acid (PLGA) may also be used. Methods for preparing such formulations are within the skill of those in the art in light of this disclosure and knowledge in the art.

[0183] Those skilled in the art will recognize from this disclosure and knowledge in the art that, in addition to tablets, other dosage forms can be formulated to provide sustained or controlled release of the active ingredient, including, but not limited to, capsules, granules, and gelcaps.

[0184] Liposomal suspensions can also be pharmaceutically acceptable carriers.They can be prepared by methods known to those skilled in the art.For example, liposomal preparations can be prepared by dissolving one or more suitable lipids in an inorganic solvent, and then evaporating the solvent, leaving a thin film of dried lipid on the surface of the container.Then, an aqueous solution of active compound is introduced into the container.Then, the container is swirled by hand 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 invention.

[0185] The formulations may conveniently be provided in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient with one or more pharmaceutical carriers or one or more excipients. Generally, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with a liquid carrier, or by finely pulverizing a solid carrier, or both, and then, if necessary, shaping the product.

[0186] Formulations and compositions suitable for topical administration in the mouth include lozenges which comprise the ingredient in a flavored base, usually sucrose and acacia or tragacanth; pastilles which comprise the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes which comprise the ingredient to be administered in a suitable liquid carrier.

[0187] Formulations suitable for topical administration to the skin may be presented as ointments, creams, gels, and pastes comprising the ingredient to be administered in a pharmaceutically acceptable carrier. A preferred topical delivery system is a transdermal patch containing the ingredient to be administered.

[0188] Formulations for rectal administration may be presented as a suppository with a suitable base comprising, for example, cocoa butter or a salicylate.

[0189] Suitable formulations for nasal administration, where the carrier is a solid, include a coarse powder having a particle size in the range 20 to 500 microns, which may be administered in the manner in which snuff is administered, i.e. by rapid inhalation through the nasal passage from a container of the powder held against the nose. Suitable formulations where the carrier is a liquid are liquids for administration, for example, as a nasal spray or as nasal drops, and include aqueous or oily solutions of the active ingredient.

[0190] 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.

[0191] Parenteral preparations can be enclosed in glass or plastic ampoules, disposable syringes or multiple dose vials. If administered intravenously, preferred carriers include, for example, physiological saline or phosphate buffered saline (PBS).

[0192] For parenteral formulations, the carrier usually comprises sterile water or aqueous sodium chloride solution, but other ingredients, including those that aid dispersion, may be included. Of course, when sterile water is used and maintained as sterile, the composition and carrier are also sterilized. Injectable suspensions may also be prepared, in which case appropriate liquid carriers, suspending agents, and the like may be used.

[0193] Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may contain suspending agents and thickening agents. These formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.

[0194] Administration of the active compound may range from continuous administration (intravenous infusion) to several oral doses per day (e.g., QID) and may include oral, topical, intraocular or ocular, parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may include penetration enhancers), buccal, and suppository administration, among other routes of administration, including intraocular or ocular routes.

[0195] The neoplastic vaccine or immunogenic composition and at least one checkpoint inhibitor, and any additional agents, may be administered by injection, orally, parenterally, by inhalation spray, rectally, intravaginally, or topically in dosage unit formulations containing conventional pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral, as used herein, includes intralymph node(s), subcutaneously, intravenously, intramuscularly, intrasternally, by infusion, intraperitoneally, intraocularly, intravitreally, intrabuccally, transdermally, intranasally, intracranially and intradurally, intracerebrally, including intracranially and intradurally, intrathecal ... , intra-articularly including the hip, shoulder, elbow and wrist, directly into a tumor, etc., and in suppository form.

[0196] In certain embodiments, the vaccine or immunogenic composition or one or more checkpoint inhibitors are administered intravenously or subcutaneously.

[0197] The application of the subject therapy may be localized, such that it is administered to the desired site. In certain embodiments, the checkpoint inhibitor is administered subcutaneously near the site of administration of the neoplastic vaccine or immunogenic composition, e.g., within about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 cm of the site of administration of the vaccine or immunogenic composition, and preferably within 5 cm of the site of administration of the neoplastic vaccine or immunogenic composition. Those skilled in the art of administering compositions should understand that the concentration of the checkpoint inhibitor administered to a subject may vary based on the location of administration. For example, the concentration of the checkpoint inhibitor may be reduced if the checkpoint inhibitor is administered near the site of administration of the neoplastic vaccine or immunogenic composition.

[0198] A variety of techniques can be used to provide the subject compositions to the desired site, including injection, use of a catheter, a trocar, a projectile, a pluronic gel, a stent, a sustained drug release polymer, or other devices that provide internal access. If the organ or tissue is accessible because it has been removed from the patient, the organ or tissue may be placed in a bath of a medium containing the subject composition, the subject composition may be painted onto the organ, or it may be applied in any convenient manner.

[0199] The tumor-specific neo-antigenic peptides may be administered by a device suitable for the controlled and sustained release of compositions effective in achieving a desired local or systemic physiological or pharmacological effect. The method includes positioning a sustained release drug delivery system in the area where drug release is desired and allowing the drug to travel from the device to the desired treatment area.

[0200] The tumor-specific neo-antigenic peptides may 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, but are not limited to, corticosteroids (e.g., cortisone, prednisone, dexamethasone), nonsteroidal anti-inflammatory drugs (NSAIDs) (e.g., ibuprofen, celecoxib, aspirin, indomethacin, naproxen), alkylating agents such as busulfan, cisplatin, mitomycin C, and carboplatin; antimitotic agents such as colchicine, vinblastine, and the like. topo I inhibitors such as camptothecin and topotecan; topo II inhibitors such as doxorubicin and etoposide; and / or RNA / DNA antimetabolites such as 5-azacytidine, 5-fluorouracil and methotrexate; DNA antimetabolites such as 5-fluoro-2'-deoxy-uridine, ara-C, hydroxyurea and thioguanine; antibodies such as HERCEPTIN and RITUXAN.

[0201] It should be understood that in addition to the ingredients particularly listed herein, the formulations of the present invention 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.

[0202] Pharmaceutically acceptable salt forms may be the preferred chemical form of the compounds of the present invention for inclusion in pharmaceutical compositions of the present invention.

[0203] The compounds or derivatives thereof may be provided in the form of pharmaceutically acceptable salts, including prodrug forms of these agents. As used herein, the term pharmaceutically acceptable "Suitable salts or complexes" refer to suitable salts or complexes of the active compounds of the present invention that retain the desired biological activity of the parent compound and exhibit limited toxic effects on normal cells. Non-limiting examples of such salts include, among others, (a) acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.) and salts formed with organic acids such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pamoic acid, alginic acid, and polyglutamic acid; and (b) base addition salts formed with metal cations such as zinc, calcium, sodium, potassium, etc.

[0204] The compounds herein are commercially available or can be synthesized. As one of ordinary skill in the art will appreciate, additional methods for synthesizing the compounds of the formulae herein will be apparent to one of ordinary skill in the art. In addition, various synthetic steps may be performed in an alternate order or sequence to obtain the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those described in R. Larock, Comprehensive Organic Transformations, 2nd Ed., Wiley-VCH Publishers (1999); T.W. Greene and P.G.M. Buts, Protective Groups in Organic Synthesis, 3rd Ed., 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 successive editions thereof.

[0205] Dosage When the agents described herein are administered to humans or animals as pharmaceuticals, the agents can be administered per se or as a pharmaceutical composition containing the active ingredient in combination with a pharmaceutically acceptable carrier, excipient, or diluent.

[0206] The actual dosage levels and time course of administration of the active ingredients in the pharmaceutical compositions of the present invention can be varied for a particular patient, composition, and method of administration to achieve an amount of the active ingredient effective to achieve the desired therapeutic response without causing toxicity to the patient. Generally, the medicaments or pharmaceutical compositions of the present invention are administered in an amount sufficient to reduce or eliminate symptoms associated with viral infections and / or autoimmune diseases.

[0207] The preferred dose of a drug is the maximum that a patient can tolerate and that does not cause serious or intolerable side effects.

[0208] 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, an effective or effective amount of a drug is determined by initially administering a low dose of the drug and then gradually increasing the administered dose or dosage until the desired effect (e.g., reduction or elimination of symptoms associated with a viral infection or autoimmune disease) is observed in the treated subject with minimal or tolerable toxic side effects. Methods applicable to determining appropriate doses and dosing schedules for administering the pharmaceutical compositions of the present invention can be found, for example, in Goodman and Gilman's The Pharmacological Basis of Therapeutics, Goodman et al., eds., 11th Edition, McGraw-Hill 2005, and Remington: The Science and Practice of Pharmacy, 20th and 21st Editions, Gennaro and University of the Sciences in Philadelphia, Eds., Lippencott Williams & Wilkins (2003 and 2005), each of which is hereby incorporated by reference.

[0209] Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein discussed, of the administered ingredient, or an appropriate fraction thereof.

[0210] Dosage regimens for treating disorders or diseases with the tumor-specific neo-antigenic peptides of the invention and / or compositions of the invention 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 used. Thus, dosage regimens can vary widely but can be routinely determined using standard methods.

[0211] The amount and dosage regimen administered to a subject will depend on many factors, including 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 such factors are within the purview of one of ordinary skill in the art from this disclosure and knowledge in the art.

[0212] The amount of compound contained in the therapeutically active formulations of the present invention is an amount effective to treat a disease or condition.

[0213] In general, the therapeutically effective amount of the preferred compounds in dosage form will typically range from just under about 0.025 mg / kg / day to about 2.5 g / kg / day of a patient, preferably from about 0.1 mg / kg / day to about 100 mg / kg / day 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 are contemplated by the present invention. In its most preferred form, the compounds of the present invention are administered in amounts ranging from about 1 mg / kg / day to about 100 mg / kg / day. The dosage of the compound will depend on the condition being treated, the particular compound, and other clinical factors, such as the patient's weight and condition, and the route of administration of the compound. It should be understood that the present invention has application for both human and veterinary use.

[0214] 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 vaccine or immunogenic composition is administered at an average weekly dose level of about 10 μg to 2000 μg per neo-antigenic peptide. According to certain exemplary embodiments, the checkpoint inhibitor is administered at a dose of about 0.1 to 10 mg / kg. According to certain exemplary embodiments, the anti-CTLA4 antibody is administered at a dose of about 1 mg / kg to 3 mg / kg. For example, in certain exemplary embodiments, nivolumab is administered at a standard single-agent dose level of 3 mg / kg. When one or more checkpoint inhibitors are administered at the site of administration of the vaccine or immunogenic composition, the inhibitors are preferably administered at a dose of about 0.1 to 1 mg per site of administration of the neoplastic vaccine or immunogenic composition.

[0215] In a preferred embodiment, concentrations and timing used in clinical trials of checkpoint inhibitors, either alone or in combination with neoantigen vaccines or immunogenic compositions, are used. Topalian, et al., N Engl J Med 2012;366:2443-2454, describes a phase 1 study evaluating the safety, antitumor activity, and pharmacokinetics of BMS-936558, a fully human IgG4-blocking monoclonal antibody against PD-1, in patients with select advanced solid tumors in need thereof. The antibody was administered as an intravenous infusion every 2 weeks for each 8-week treatment cycle. Response was assessed after each treatment cycle. Patients received treatment for up to 2 years (12 cycles). Patients with advanced melanoma, non-small cell lung cancer, renal cell carcinoma, castration-resistant prostate cancer, or colorectal cancer were enrolled. Cohorts of 3 to 6 patients per dose level were sequentially enrolled at doses of 1.0, 3.0, or 10.0 mg per kilogram of body weight. Initially, five expansion sessions of approximately 16 patients each were conducted. Cohorts were enrolled at a dose of 10.0 mg per kilogram for melanoma, non-small cell lung cancer, renal cell carcinoma, castration-resistant prostate cancer, and colorectal cancer. Based on initial activity signals, additional expansion cohorts of approximately 16 patients each were enrolled for melanoma (at a dose of 1.0 or 3.0 mg per kilogram, followed by randomization of cohorts to 0.1, 0.3, or 1.0 mg per kilogram), lung cancer (patients with squamous or non-squamous subtypes randomized to doses of 1.0, 3.0, or 10.0 mg per kilogram), and renal cell carcinoma (at a dose of 1.0 mg per kilogram).

[0216] Wolchok, et al., N Engl J Med 2013;369:122-133, described a clinical trial using nivolumab plus ipilimumab in advanced melanoma. In this study, patients received intravenous doses of nivolumab and ipilimumab every three weeks for four doses, followed by nivolumab alone every three weeks for four doses. The combination treatment was then administered every 12 weeks for up to eight doses. In the sequential regimen, patients previously treated with ipilimumab received nivolumab every two weeks for up to 48 doses. The maximum doses associated with acceptable levels of adverse events were nivolumab at a dose of 1 mg per kilogram of body weight and ipilimumab at a dose of 3 mg per kilogram.

[0217] Wolchok et al., Clin. Cancer Res. 15, 7412; 2009, described a phase II clinical trial program with ipilimumab. Patients were treated with induction therapy (ipilimumab 10 mg / kg every 3 weeks × 4), followed by maintenance therapy in eligible patients (ipilimumab 10 mg / kg every 12 weeks, starting at week 24).

[0218] Hamid et al., N Engl J Med 2013;369:134-144, described the safety and tumor response of lambrolizumab (anti-PD-1) in melanoma. Patients with advanced melanoma received intravenous lambrolizumab at a dose of 10 mg per kilogram of body weight every 2 or 3 weeks or 2 mg per kilogram every 3 weeks. Patients included both those who had received prior treatment with the immune checkpoint inhibitor ipilimumab and those who had not.

[0219] Spigel et al., J Clin Oncol 31, 2013 (suppl; abstr 8008) describe a phase I trial of MPDL3280A, a human monoclonal Ab containing an engineered Fc-domain targeting PD-L1, designed to optimize efficacy and safety. Patients with squamous or non-squamous NSCLC received MPDL3280A IV at doses of 1 to 20 mg / kg for up to one year.

[0220] The concentration of the active compound in the drug composition may depend on the absorption, distribution, inactivation, and excretion rate of the drug, as well as other factors known to those skilled in the art. It should be noted that the dosage value may also vary depending on the severity of the condition to be alleviated. Furthermore, it should be understood that for any particular subject, the specific dosage regimen must be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the composition, and that the concentration ranges set forth herein are merely exemplary and are not intended to limit the scope or practice of the claimed compositions. The active ingredient may be administered at once, or may be divided into several smaller doses to be administered at various time intervals.

[0221] The present invention provides pharmaceutical compositions containing at least one tumor-specific neo-antigen 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 development of an immune response harmful to the subject receiving the composition and that may be administered without undue toxicity. As used herein, the term "pharmaceutically acceptable" means approved by a federal or state regulatory agency for use in mammals, more particularly humans, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias. These compositions may be useful for the treatment and / or prevention of viral infections and / or autoimmune diseases.

[0222] 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, particulate-free, and / or non-pyrogenic.

[0223] Pharmaceutically acceptable carriers, excipients, or diluents include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, sterile isotonic aqueous buffer solution, and combinations thereof.

[0224] Wetting agents, emulsifying agents, and lubricating agents, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring, and perfuming agents, preservatives, and antioxidants can also be present in the composition.

[0225] 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, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0226] In embodiments, the pharmaceutical composition is provided in a solid form such as a lyophilized powder suitable for reconstitution, a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder.

[0227] In embodiments, the pharmaceutical composition is provided in liquid form, e.g., in a hermetically sealed container indicating the quantity and concentration of the active ingredient in the pharmaceutical composition. In a related embodiment, the pharmaceutical composition in liquid form is provided in a hermetically sealed container.

[0228] Methods for formulating pharmaceutical compositions of the present invention are conventional and well known in the art (see Remington and Remington's). One of skill in the art can readily formulate pharmaceutical compositions having desired characteristics (e.g., route of administration, biosafety, and release profile).

[0229] Methods for preparing pharmaceutical compositions include the step of bringing into association the active ingredient with a pharmaceutically acceptable carrier, and optionally one or more accessory ingredients. Pharmaceutical compositions may be prepared by uniformly and intimately bringing into association the active ingredient with a liquid carrier, or by finely pulverizing a 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 Deliverer. System Systems (9th ed., Lippincott Williams & Wilkins), hereby incorporated by reference.

[0230] Pharmaceutical compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as a troche (using an inert base such as gelatin and glycerin, or sucrose and acacia), and / or as a mouthwash, each containing a predetermined amount of one or more compounds described herein, their derivatives, or pharmaceutically acceptable salts or prodrugs thereof as one or more active ingredients. The active ingredient may also be administered as a bolus, electuary, or paste.

[0231] In solid dosage forms for oral administration (e.g., capsules, tablets, pills, dragees, powders, granules, and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, excipients, or diluents, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants, such as glycerol; (4) disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) dissolution inhibitors, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as acetyl alcohol and glycerol monostearate; (8) absorbents, 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 and excipients, such as lactose or milk sugar, and high molecular weight polyethylene glycols, in soft- and hard-filled gelatin capsules.

[0232] Tablets may 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), surface active agents, and / or dispersing agents. Molded tablets may be made by molding in a suitable machine a mixture of the powdered active ingredient moistened with an inert liquid diluent.

[0233] Tablets and other solid dosage forms such as dragees, capsules, pills, and granules may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the art.

[0234] In some embodiments, in order to prolong the effect of an active ingredient, it is desirable to delay the absorption of the compound from subcutaneous or intramuscular injection. This can be achieved by using a liquid suspension of a crystalline or amorphous material that is poorly water-soluble. In this case, the absorption rate of the active ingredient depends on its dissolution rate, which in turn depends on the size and crystalline form of the crystals. Alternatively, delayed absorption of a parenterally administered active ingredient can be achieved by dissolving or suspending the compound in an oil vehicle. In addition, prolonged absorption of an injectable pharmaceutical dosage form can be achieved by incorporating an agent that delays absorption, such as aluminum monostearate and gelatin.

[0235] 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 one or more biocompatible carriers, liposomes, nanoparticles, implants, or infusion devices.

[0236] Materials used in preparing microspheres and / or microcapsules include biodegradable / bioerodible polymers such as polyglactin, poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutamine), and poly(lactic acid).

[0237] Biocompatible carriers that can be used in formulating controlled release parenteral formulations include carbohydrates such as dextrans, proteins such as albumin, lipoproteins, or antibodies.

[0238] The materials used in the implants can be non-biodegradable, such as polydimethylsiloxane, or biodegradable, such as poly(caprolactone), poly(lactic acid), poly(glycolic acid), or poly(orthoesters).

[0239] In embodiments, one or more active ingredients are administered by aerosol.This can be achieved by preparing an aqueous aerosol, liposomal formulation, or solid particles containing the compound.Non-aqueous (e.g., fluorocarbon propellant) suspension can also be used.The pharmaceutical composition can also be administered using a sonic nebulizer, which can minimize the exposure of the drug to shear, which can cause the compound to decompose.

[0240] Usually, aqueous aerosol is prepared by mixing aqueous solution or aqueous suspension of one or more active ingredients with conventional pharmaceutically acceptable carriers and stabilizers.Carriers and stabilizers vary according to the requirements of specific compounds, but typically include nonionic surfactants (Tween, Pluronic, or polyethylene glycol), harmless proteins, such as serum albumin, sorbitan esters, oleic acid, lecithin, amino acids such as glycine, buffers, salts, sugars or sugar alcohols.Aerosols are generally prepared from isotonic solutions.

[0241] Dosage forms for topical or transdermal administration of the active ingredient(s) include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active ingredient(s) may be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants as appropriate.

[0242] Transdermal patches suitable for use in the present invention are disclosed in "Transdermal Drug Delivery: Developmental Issues and Research Initiatives" (Marcel Dekker Inc., 1989) and U.S. Pat. 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, e.g., U.S. Pat. Nos. 4,379,454 and 4,973,468, which are incorporated herein by reference). The transdermal therapeutic drug systems used in the present invention may be based on iontophoresis, diffusion, or a combination of these two effects.

[0243] Transdermal patches have the added advantage of providing controlled delivery of one or more active ingredients to the body. Such dosage forms comprise one or more active ingredients dissolved or dispersed in a suitable medium. Absorption enhancers can also be used to increase the flux of the active ingredient across the skin. The rate of such flux can be controlled by either providing a rate-controlling membrane or dispersing the active ingredient(s) in a polymer matrix or gel.

[0244] 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.

[0245] 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.

[0246] Examples of antioxidants include, but are not limited to, butylated hydroxyanisole (BHA), ascorbic acid and its derivatives, tocopherol and its derivatives, and cysteine.

[0247] Examples of preservatives include, but are not limited to, parabens, such as methyl or propyl p-hydroxybenzoate, and benzalkonium chloride.

[0248] Examples of humectants include, but are not limited to, glycerin, propylene glycol, sorbitol, and urea.

[0249] 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.

[0250] Examples of chelating agents include, but are not limited to, sodium EDTA, citric acid, and phosphoric acid.

[0251] Examples of gel-forming agents include, but are not limited to, carbopol, cellulose derivatives, bentonite, alginates, gelatin, and polyvinylpyrrolidone.

[0252] In addition to the active ingredient(s), the ointments, pastes, creams, and gels of the present invention may contain excipients such as animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonite, silicic acid, talc, and zinc oxide, or mixtures thereof.

[0253] 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.

[0254] Injectable depot forms are made by forming microencapsule matrices of one or more compounds of the invention in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

[0255] Subcutaneous implants are well known in the art and are suitable for use in the present invention. Subcutaneous implantation methods are preferably non-irritating and mechanically resilient. The implants may be matrix-type, reservoir-type, or a hybrid thereof. In matrix-type devices, the carrier material may be porous or non-porous, solid or semi-solid, and permeable or impermeable to one or more active compounds. The carrier material may be biodegradable or slowly erode after administration. In some cases, the matrix is ​​non-degradable, but instead relies on the diffusion of the active compound through the matrix as the carrier material degrades. An alternative subcutaneous implant method utilizes a reservoir device, in which one or more active compounds are surrounded by a rate-limiting membrane, e.g., a membrane that is independent of component concentration (having zero-order kinetics). Devices consisting of a matrix surrounded by a rate-limiting membrane are also suitable for use.

[0256] Both reservoir-type and matrix-type devices may contain polydimethylsiloxanes, such as SILASTIC, or other silicone rubbers. Matrix materials may be insoluble polypropylene, polyethylene, polyvinyl chloride, ethyl vinyl acetate, polystyrene, and polymethacrylates, as well as glycerol esters of the glycerol palmitostearate, glycerol stearate, and glycerol behenate types. Materials may be hydrophobic or hydrophilic polymers, and may optionally contain solubilizers.

[0257] The subcutaneous implant device may be a delayed release capsule made of any suitable polymer, for example, as described in U.S. Pat. Nos. 5,035,891 and 4,210,644, which are hereby incorporated by reference.

[0258] Generally, at least four different approaches can be applied to provide rate-limiting release and skin permeation of drug compounds. These approaches are: membrane-restricted systems, adhesive diffusion-controlled systems, matrix-dispersed systems, and microreservoir systems. It will be appreciated that controlled-release transdermal and / or topical compositions can be achieved by any suitable combination of these approaches.

[0259] In membrane-based systems, the active ingredient resides in a reservoir completely encapsulated between 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, e.g., ethylene-vinyl acetate copolymer. The active ingredient is released through the rate-controlling polymer membrane. In the drug reservoir, the active ingredient can be either dispersed in a solid polymer matrix or suspended in a non-leachable viscous liquid medium, such as silicone fluid. A thin layer of adhesive polymer is applied to the outer surface of the polymer membrane, ensuring intimate contact between the transdermal system and the skin surface. The adhesive polymer is preferably a polymer that is hypoallergenic and compatible with the active drug substance.

[0260] In adhesive diffusion-controlled systems, a reservoir of the active ingredient is formed by dispersing the active ingredient directly into an adhesive polymer and then applying, for example, by solvent casting, the adhesive containing the active ingredient onto a flat sheet of substantially drug-impermeable metal-plastic backing to form a thin drug reservoir layer.

[0261] Matrix dispersion systems are characterized by the formation of a reservoir of active ingredient by substantially uniformly dispersing the active ingredient in a hydrophilic or lipophilic polymer matrix. The drug-containing polymer is then molded into a disk with a substantially well-defined surface area and controlled thickness. An adhesive polymer is applied along the periphery to form an adhesive strip around the disk.

[0262] Microreservoir systems can be thought of as 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 this drug suspension in a lipophilic polymer to form a multiplicity of non-leachable microspheres of drug reservoirs.

[0263] Any of the controlled-release, extended-release, and sustained-release compositions herein can be formulated to release the active ingredient(s) 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 one or more active ingredients is sustained 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.

[0264] Vaccine or Immunogenic Composition The present invention relates to a combination therapy method. The combination therapy includes 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. The combination therapy also includes at least one checkpoint inhibitor. In particular, the present invention relates to a method for treating or preventing a neoplasm, comprising administering to a subject (a) a neoplastic vaccine or immunogenic composition, and (b) at least one checkpoint inhibitor.

[0265] A suitable neoplasia vaccine or immunogenic composition may preferably comprise multiple tumor-specific neoantigenic peptides. In certain embodiments, the vaccine or immunogenic composition may comprise 1 to 100 pairs of peptides, more preferably 1 to 50 such peptides, even more preferably 10 to 30 pairs of peptides, and even more preferably 15 to 25 peptides. According to another preferred embodiment, the vaccine or immunogenic composition may comprise at least one peptide, more preferably two, three, four, or five peptides. In certain embodiments, the vaccine or immunogenic composition may comprise 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.

[0266] The optimal amount and optimal administration regimen of each peptide to be included in a vaccine or immunogenic composition can be determined by one of ordinary skill in the art 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. Preferred peptide injection methods include sc, id, ip, im, and iv. Preferred DNA injection methods include id, im, sc, ip, and iv. For example, a dose of 1 to 500 mg, 50 μg to 1.5 mg, preferably 10 μg to 500 μg of peptide or DNA may be administered, depending on the respective peptide or DNA. This dose range has 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 the immunogenic composition are known to those skilled in the art.

[0267] In one embodiment of the present invention, 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 the patient's neoplasm / tumor. Without being bound by theory, it is believed that including a variety of tumor-specific neo-antigenic peptides may generate a broad-scale immune attack against the neoplasm / tumor. 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 neo-ORF mutation. In a third embodiment, the selected tumor-specific neo-antigenic peptide / polypeptide is encoded by a neo-ORF mutation.

[0268] In one embodiment where the selected tumor-specific neo-antigenic peptide / polypeptide is encoded by a missense mutation, the peptide and / or polypeptide is selected based on its ability to associate with a particular MHC molecule of the patient. Peptides / polypeptides derived from neo-ORF mutations can also be selected based on their ability to associate with a particular MHC molecule of the patient, but can also be selected even if they are not predicted to associate with a particular MHC molecule of the patient.

[0269] A vaccine or immunogenic composition is capable of generating a specific cytotoxic T cell response and / or a specific helper T cell response.

[0270] 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, e.g., a protein capable of presenting the peptide to T cells or antigen-presenting cells, e.g., dendritic cells (DCs).

[0271] An adjuvant is any substance that, when incorporated 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 invention.

[0272] The ability of an adjuvant to increase the immune response to an antigen is typically manifested as a significant increase in immune-mediated responses or a reduction in disease symptoms. For example, an increase in humoral immunity is typically manifested as a significant increase in the titer of antibodies raised against the antigen, and an increase in T cell activity is typically manifested as an increase in cell proliferation, cytotoxicity, or cytokine secretion. Adjuvants can also alter the immune response, for example, by shifting a primary humoral or Th2 response to a primary cellular or Th1 response.

[0273] Suitable adjuvants include, but are not limited to, 1018 ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS patch, ISS, ISCOMATRIX, Julmune, 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 trap. These include Aquila's QS21 stimulon (Aquila Biotech, Worcester, Mass., USA), derived from stimulants such as R848, beta-glucan, Pam3Cys, and saponins; mycobacterial extracts and synthetic bacterial cell wall mimics; and other proprietary adjuvants, such as Ribi's Detox, Quil, or Superfos. Several immunological adjuvants specific for dendritic cells (e.g., MF59) and their formulations 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 may also be used. Several cytokines have been directly implicated in influencing dendritic cell migration to lymphoid tissues (e.g., TNF-α), accelerating dendritic cell maturation into efficient antigen-presenting 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).

[0274] Toll-like receptors (TLRs) can also be used as adjuvants. They are important members of the pattern recognition receptor (PRR) family, which 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 various intracellular compartments, such as the plasma membrane, lysosomes, endosomes, and endolysosomes. Different TLRs recognize distinct PAMPs. For example, TLR4 is activated by LPS, which is found in bacterial cell walls; TLR9 is activated by unmethylated bacterial or viral CpG DNA; and TLR3 is activated by double-stranded RNA. TLR ligand binding triggers the activation of one or more intracellular signaling pathways, ultimately resulting in the production of many key molecules related to inflammation and immunity, particularly the transcription factor NF-κB and type I interferons. 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 allows DC migration to draining lymph nodes and promotes 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 critical for the induction of adaptive immune responses.

[0275] 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 and lack of stimulation of IL-10, as well as the maintenance of high levels of costimulatory molecules in DC1. Furthermore, poly-ICLC was recently directly compared with CpG in non-human primates (rhesus macaques) as an adjuvant for protein vaccines or immunogenic compositions consisting of human papillomavirus (HPV) 16 capsomers (Stahl-Hennig C, Eisenblatter M, Jasny E, et al. "Synthetic double-stranded RNAs are adjuvants for the induction of T helper 1 and humoral immune responses to human papillomavirus in rhesus macaques." PLoS pathogens. Apr 2009;5(4)).

[0276] 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 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 enhances dendritic cell maturation and differentiation, leading to enhanced Th1 cell activation and potent cytotoxic T lymphocyte (CTL) generation, 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 such as alum or incomplete Freund's adjuvant (IFA), which normally promote a Th2 bias. CpG oligonucleotides exhibit even greater adjuvant activity when formulated or co-administered with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations, which are particularly necessary to induce strong responses when the antigen is relatively weak. CpG oligonucleotides also accelerate immune responses, allowing antigen doses to be reduced by approximately two orders of magnitude with antibody responses equivalent to those to full-dose vaccines without CpG in some experiments (Arthur M. Krieg, Nature Reviews, Drug Discovery, 5, Jun. 2006, 471-484). U.S. Patent No. 6,406,705 B1 describes the induction of antigen-specific immune responses by the combined use of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens. A commercially available CpG TLR9 antagonist is dSLIM (double stem loop immunomodulator) by Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical composition of the present invention. Other TLR-binding molecules, such as RNA-binding TLR7, TLR8, and / or TLR9, can also be used.

[0277] 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, sorafinib, 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 context of the present invention can be readily determined by one of ordinary skill in the art without undue experimentation. Further adjuvants include colony-stimulating factors such as granulocyte-macrophage colony-stimulating factor (GM-CSF, sargramostim).

[0278] Poly-ICLC is a synthetically prepared double-stranded RNA consisting of poly-I and poly-C strands with an average length of approximately 5000 nucleotides. It is 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 members of the PAMP family, leading to the activation of DCs and natural killer (NK) cells and the production of a "natural mixture" 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.

[0279] In rodents and non-human primates, poly-ICLC inhibits T cell responses to viral antigens. Response, cross-priming, and tumor-, virus-, and autoantigen-specific CD8 + It has been shown to enhance the induction of T cells. A recent study in non-human primates found that poly-ICLC was essential for the generation of antibody responses and T cell immunity against DC-targeted or non-targeted HIV Gag p24 protein, highlighting its effectiveness as a vaccine adjuvant.

[0280] In human subjects, transcriptional analysis of serial whole blood samples revealed similar gene expression profiles among eight healthy human volunteers who received one single subcutaneous dose of poly-ICLC, with up to 212 genes differentially expressed between these eight subjects versus four subjects receiving placebo. Notably, comparison of poly-ICLC gene expression data with previous data from volunteers immunized with the highly efficacious yellow fever vaccine YF17D showed that many canonical transcriptional and signaling pathways, including those of the innate immune system, were similarly upregulated at their peak time points.

[0281] More recently, immunological analyses were reported on patients with ovarian, fallopian tube, and primary peritoneal cancer in second or third complete clinical remission who were treated in a phase 1 study of subcutaneous vaccination with synthetic overlapping long peptides (OLPs) derived from the cancer-testis antigen NY-ESO-1, alone or in combination with Montanide-ISA-51, or with 1.4 mg of poly-ICLC and Montanide. The addition of poly-ICLC and Montanide significantly increased NY-ESO-1-specific CD4 expression compared with OLP alone or OLP and Montanide. + and CD8 + T cell generation and antibody responses were significantly enhanced.

[0282] A vaccine or immunogenic composition according to the invention may comprise two or more different adjuvants. Furthermore, the invention encompasses therapeutic compositions comprising any adjuvant material, including any of those discussed herein. It is also contemplated that the peptide or polypeptide and the adjuvant may be administered separately in any suitable order.

[0283] A carrier may be present independent of the adjuvant. The carrier may be covalently linked to the antigen. A carrier can also be added to an antigen by inserting DNA encoding the carrier in frame with DNA encoding the antigen. The function of the carrier may be, for example, to confer stability, increase biological activity, or increase serum half-life. Prolonged half-life can help reduce the frequency of applications and doses, which is therefore beneficial for therapeutic and economic reasons. Furthermore, the carrier may aid in the presentation of peptides to T cells. The carrier may be any suitable carrier known to those skilled in the art, such as a protein or antigen-presenting cells. Carrier proteins may include, but are 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 may be a physiologically acceptable carrier that is acceptable and safe for humans. However, tetanus toxoid and / or diptheria toxoid are preferred carriers in one embodiment of the present invention. Alternatively, the carrier may be a dextran, such as sepharose.

[0284] 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 molecules, and APCs. Correspondingly, immune responses of CTLs can be enhanced not only when peptides alone are used to activate CTLs, but also when APCs bearing the respective MHC molecules are added. Thus, in some embodiments, the vaccine or immunogenic composition of the present invention further comprises at least one antigen-presenting cell. .

[0285] 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 an antigen of choice themselves. As described in more detail herein, MHC class I or II molecules can be readily loaded with an antigen of choice in vitro.

[0286] CD8+ cell activity can be enhanced using CD4+ cells. Identification of CD4 T+ cell epitopes of tumor antigens has attracted interest because many immune-based anticancer therapies may be more effective when targeting a patient's tumor using both CD8+ and CD4+ T lymphocytes. CD4+ cells have the ability to enhance CD8 T cell responses. Many studies in animal models have clearly demonstrated that better outcomes are achieved when both CD4+ and CD8+ T cells participate in the antitumor response (see, e.g., Nishimura et al. (1999) "Distinct role of antigen-specific T helper type 1 (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 to the development of therapies for different types of cancer (e.g., Kobayashi et al. (2008) Current Opinion in Immunology 20:221-27). For example, HLA-DR-restricted helper peptides derived from tetanus toxoid have been 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 an Adjuvant Setting"). (See "The Adjuvant Setting," Clinical Cancer Research 13(21):6386-95.) Within the scope of the present invention, it is contemplated that such CD4+ cells may be applicable at three levels that differ in their tumor specificity: 1) a broad level, where a universal CD4+ epitope (e.g., tetanus toxoid) may be used to enhance CD8+ cells; 2) an intermediate level, where a natural tumor-associated CD4+ epitope may be used to enhance CD8+ cells; and 3) a patient-specific level, where a neoantigen CD4+ epitope may be used to enhance CD8+ cells in a patient-specific manner.

[0287] CD8+ cell immunity can also be generated by neoantigen-loaded dendritic cell (DC) vaccines. DCs are potent antigen-presenting cells that elicit 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, it has been shown that patients with newly diagnosed metastatic melanoma can be immunized with autologous peptide-pulsed CD40L / IFN-γ-activated mature DCs against three HLA-A*0201-restricted gp100 melanoma antigen-derived peptides using an IL-12p70-producing patient DC vaccine (e.g., Carreno et al. (2013) "L-12p70-producing patient DC vaccine elicits Tc1-polarized immunity"). (2009) "In situ regulation of DC subsets and T cells mediates tumor regression in mice," Cancer Immunotherapy, 1(8):1 (See references 1-10.) Within the scope of the present invention, it is contemplated that neoantigen-loaded DCs can be prepared by stimulating DCs with the synthetic TLR 3 agonist polyinosinic-polycytidylic acid-poly-L-lysine carboxymethylcellulose (poly-ICLC). Poly-ICLC is a potent, individualized maturation stimulus for human DCs, as assessed by upregulation of CD83 and CD86, induction of interleukin-12 (IL-12), tumor necrosis factor (TNF), interferon-γ-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, while PBMCs can be isolated by Ficoll gradient centrifugation and frozen in aliquots.

[0288] As an example, the following 7-day activation protocol can be used: Day 1 - PBMCs are thawed and plated into tissue culture flasks and incubated at 37°C in a tissue culture incubator for 1-2 hours, followed by selection of monocytes that adhere to the plastic surface. After incubation, lymphocytes are washed away, and adherent monocytes are 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 may 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 may undergo lot release testing 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).

[0289] DC vaccines can be incorporated into scaffold systems to facilitate delivery to patients. Therapeutic treatment of neoplasms in patients with DC vaccines can utilize biomaterial systems that release factors that recruit host dendritic cells within the device, differentiate resident immature DCs by locally presenting adjuvants (e.g., danger signals) during antigen release, 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. Potent cytotoxic T lymphocyte responses against neoplasms can also be generated in a patient-specific manner using implantable biomaterials. These biomaterial-resident dendritic cells can then be activated by exposure to danger signals that mimic infection, in conjunction with the release of antigens from the biomaterial. Activated dendritic cells then migrate from the biomaterial to lymph nodes and induce cytotoxic T effector responses. This technique has previously been demonstrated to cause regression of established melanoma in preclinical studies using lysates prepared from tumor biopsies (e.g., Ali et al. (2009) "In situ regulation of DC subsets and T cells mediates tumor regression in mice"). (2009) "Tumor regression in mice," Cancer Immunotherapy 1(8):1-10; Ali et al. (2009) "Infection-mimicking materials to program dendritic cells in situ," Nat Mater 8:151-8; such a vaccine is currently being tested in a recently initiated phase I clinical trial at Dana-Farber Cancer Institute. This approach is also Furthermore, the current proposal, using a C6 rat glioma model, has also been shown to result in glioblastoma regression and the induction of a strong memory response that prevents relapse. The ability of such implantable biomatrix vaccine delivery scaffolds to amplify and sustain tumor-specific dendritic cell activation may result in more robust antitumor immunity than can be achieved by conventional subcutaneous or intranodal vaccination.

[0290] Preferably, the antigen-presenting cells are dendritic cells. Suitably, the dendritic cells are autologous dendritic cells pulsed with a neoantigenic peptide. The peptide may be any suitable peptide that generates an appropriate T cell response. T cell therapy using autologous dendritic cells pulsed with peptides derived 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.

[0291] Thus, in one embodiment of the present invention, a vaccine or immunogenic composition containing at least one antigen-presenting cell is pulsed or loaded with one or more peptides of the present invention. Alternatively, peripheral blood mononuclear cells (PBMCs) isolated from a patient may be loaded with peptides ex vivo and infused back into the patient. Alternatively, the antigen-presenting cell comprises an expression construct encoding a peptide of the present invention. The polynucleotide may be any suitable polynucleotide, preferably one capable of transducing dendritic cells, thus resulting in presentation of the peptide and induction of immunity.

[0292] The pharmaceutical compositions of the present invention can be tailored such that the selection, number, and / or amount of peptides present in the composition are tissue-, cancer-, and / or patient-specific. For example, the exact selection of peptides can be guided by the expression pattern of the parent protein in a given tissue to avoid side effects. The selection can depend on the specific type of cancer, the state of the disease, previous treatment regimens, the patient's immune status, and, of course, the patient's HLA-haplotype. Furthermore, the vaccine or immunogenic composition of the present invention can contain personalized components according 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 individual allergies or other treatments, and adjustments to secondary treatments after an initial treatment round or scheme.

[0293] Pharmaceutical compositions containing the peptides of the present invention 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 cure or at least partially arrest symptoms and / or complications. An amount sufficient to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will 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 range from about 1.0 μg to about 50,000 μg of peptide for a 70 kg patient for an initial immunization (for therapeutic or prophylactic administration), followed by boosting dosages, or from about 1.0 μg to about 10,000 μg of peptide, followed by a boosting regimen over several weeks to months, depending on the patient's response and condition, and optionally by measuring specific CTL activity in the patient's blood. It should be noted that the peptides and compositions of the present invention may generally be used in severe disease states, i.e., life-threatening or potentially life-threatening situations, particularly when the cancer has metastasized. For therapeutic use, administration should begin as soon as possible after tumor detection or surgical removal. This should be followed by booster doses until at least substantial remission of symptoms and for a period thereafter.

[0294] Pharmaceutical compositions for therapeutic treatment (e.g., vaccine compositions) are intended for parenteral, topical, nasal, oral, or local administration. Preferably, the pharmaceutical compositions are administered parenterally, for example, intravenously, subcutaneously, intradermally, or intramuscularly. The compositions stimulate a local immune response against the tumor. The present invention provides compositions for parenteral administration comprising a solution of the peptide, wherein the vaccine or immunogenic composition is dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, and the like. These compositions may be sterilized by conventional, well-known sterilization techniques or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as needed to approximate physiological conditions, such as pH adjusting and buffering agents, isotonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.

[0295] Liposomal suspensions containing peptides can be administered intravenously, locally, topically, etc., at doses that vary depending, inter alia, on the method of administration, the peptide being delivered, and the stage of disease being treated. To target immune cells, a ligand, such as an antibody or fragment thereof specific for a cell surface determinant on the desired immune system cell, can be incorporated into the liposome.

[0296] 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. For oral administration, a pharmaceutically acceptable non-toxic composition can be formed by incorporating a commonly used excipient, such as any of the carriers previously listed, and approximately 10-95% of the active ingredient, i.e., one or more peptides of the invention, more preferably in a concentration of 25%-75%.

[0297] For aerosol administration, the immunogenic peptide is preferably provided in finely divided form together with a surfactant and propellant. Typical proportions of peptide are 0.01% to 20% by weight, preferably 1% to 10% by weight. The surfactant is, of course, nontoxic and preferably soluble in the propellant. Representative examples 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, oleic acid, and the like, with aliphatic polyhydric alcohols or their cyclic anhydrides. Mixed esters, such as mixed or natural glycerides, may also be used. The surfactant may comprise 0.1% to 20% by weight, preferably 0.25 to 5% by weight, of the composition. The remainder of the composition is a conventional propellant. A carrier may also be included as needed, such as lecithin for intranasal delivery.

[0298] The peptides and polypeptides of the present invention can be readily chemically synthesized using reagents that are free of bacterial or animal substances (Merrifield RB: "Solid phase peptide synthesis. I. The synthesis of a tetrapeptide". J. Am. Chem. Soc. 85:2149-54, 1963).

[0299] The peptides and polypeptides of the invention may also be expressed by vectors, such as nucleic acid molecules as discussed herein, e.g., RNA or DNA plasmids, viral vectors, such as poxviruses, e.g., orthopoxviruses, avipoxviruses, or adenoviruses, AAV, or lentiviruses. This approach involves the use of vectors to express nucleotide sequences encoding the peptides of the invention. When introduced into an acutely or chronically infected or uninfected host, the vectors confer immunity. They express an immunogenic peptide and thus induce a host CTL response.

[0300] Nucleic acids encoding the peptides of the present invention and optionally one or more of the peptides described herein can also be administered to patients for treatment or immunization. Numerous methods are conveniently used to deliver nucleic acids to patients. For example, nucleic acids can be delivered directly as "naked DNA." This technique is described, for example, in Wolff et al., Science 247:1465-1468 (1990) and U.S. Pat. Nos. 5,580,859 and 5,589,466. Nucleic acids can also be administered using ballistic delivery, for example, as described in U.S. Pat. No. 5,204,253. Particles composed solely of DNA can also be administered. Alternatively, DNA can be attached to particles, such as gold particles. Generally, a plasmid for a vaccine or immunological composition can contain DNA encoding an antigen (e.g., one or more neoantigens) 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; for example, from upstream to downstream, DNA for a promoter, e.g., a mammalian viral promoter (e.g., a CMV promoter such as the hCMV or mCMV promoter, e.g., an early intermediate promoter, or an SV40 promoter—see documents cited or incorporated herein for useful promoters), DNA for a eukaryotic leader peptide for secretion (e.g., tissue plasminogen activator), DNA for one or more neoantigens, and DNA encoding a terminator (e.g., the 3' UTR transcription terminator from the gene encoding bovine growth hormone, i.e., bGH polyA). The composition can contain two or more plasmids or vectors, so that each vector contains and expresses a different neoantigen. Reference is also made to U.S. Pat. No. 5,849,303 to Wasmoen and U.S. Pat. No. 5,811,104 to Dale, the texts of which may be useful. The DNA or DNA plasmid formulations can be formulated with or in cationic lipids; and reference is also made to Loosmore, U.S. Patent Application Publication No. 2003 / 0104008, regarding cationic lipids and adjuvants.Reliance may also be placed on the teachings of Audonnet, US Pat. Nos. 6,228,846 and 6,159,477 for teachings of DNA plasmids that can be used in the construction and use of DNA plasmids that contain and express in vivo.

[0301] Nucleic acids can also be delivered complexed to cationic compounds, such as cationic lipids. Lipid-mediated gene delivery methods are described, for example, in WO 1996 / 18372; WO 1993 / 24640; Mannino & Gould-Fogerite, BioTechniques 6(7):682-691 (1988); U.S. Pat. No. 5,279,833; WO 1991 / 06309; and Feigner et al., Proc. Natl. Acad. Sci. USA 84:7413-7414 (1987).

[0302] 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. Pat. 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).

[0303] Poxviruses that can be used in the practice of the present invention include, inter alia, poxviruses of the subfamily Chordopoxvirinae (vertebrate poxviruses), such as orthopoxviruses and avipoxviruses, including vaccinia viruses (e.g., Wyeth strain, WR strain (e.g., ATCC® VR 16166), and poxviruses of the subfamily Chordopoxvirinae (vertebrate poxviruses), such as orthopoxviruses and avipoxviruses, including vaccinia viruses (e.g., Wyeth strain, WR strain (e.g., ATCC® VR 16166). Information regarding viruses such as variola, flu, flu-like viruses (e.g., -1354), Copenhagen strain, NYVAC, NYVAC.1, NYVAC.2, MVA, MVA-BN), canarypox (e.g., Wheatley C93 strain, ALVAC), fowlpox (e.g., FP9 strain, Webster strain, TROVAC), dovepox, pigeonpox, quailpox, and raccoonpox, synthetic or non-naturally occurring recombinant forms thereof, their uses, and methods of making and using such recombinants may be found in the scientific and patent literature, such as: U.S. Patent Nos. 4,603,112, 4,769,330, 5,110,587, 5,174,993, 5,364,773, 5,762,938, 5,494,807, 5,766,597, 7,767,449, 6,780,407, 6,537,594, and 6,265,189 6,214,353, 6,130,066, 6,004,777, 5,990,091, 5,942,235, 5,833,975 , Specification No. 5,766,597, Specification No. 5,756,101, Specification No. 7,045,313, Specification No. 6,780,417, Specification No. 8,470,598, Specification No. 8,372,622, Specification No. 8,268,329, Specification No. 8,268,325, Specification No. 8,236,560, Specification No. 8,163,293, Specification No. 7,964,398, Specification No. 7,964,396, Specification No. 7,964,395, Specification No. 7,939,086, Specification No. 7,923,017, Specification No. 7,897,156, Specification No. 7,892,533, Specification No. 7,628,980, Specification No. Nos. 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 Acd.Sci.1984;81:7194-8,Rupprecht,CE.Proc.Natl Acd.Sci.1986;83:7947-50,Poulet,H Vaccine 2007;25(Jul):5606-12,Weyer J.Vaccine 2009;27(Nov):7198-201,Buller,RM Nature 1985;317(6040):813-5,Buller 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);106 06-22),Goebel SJ.Virology 1990;(a,b)179:247-66,Tartaglia,J.Virol.1992;188(1):217-32,Najera JL.J.Virol.2006;80(12):6033-47,Najera,JL.J.Virol.2006;80:6033-6047,Gomez,CE.J.Gen.Virol.2007;88:2473-78,Mooij.Of,P.Jour. 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.1ukocyte-15 TJ3;5 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,Didierlaucci-70ne 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:18,Chen-4 2004 ZJVirol.2005;79:2678-2688,Najera JL.J.Virol.2006;80(12):6033-47,Nam JH.Acta.Virol.2007;51:125-30,Antonis AF.B.B71;8-40487 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.2010,28,1099-1105,Amato RJ.J.Clin.Can.Res.2010;16(22):5539-47,Kim,DW.Hum.Vaccine.2010;6:784-791,Oudard,S.Cancer Immunol.Immunother.2011;60:261-71,Wyatt,LS.Aids Res.Hum.Retroviruses.2004;20:645-53,Gomez,CE.Virus Research 2004;105:11-22,Webster,DP.Proc.Natl.Acad.Sci.2005;102:4836-4,Huang,X.Vaccine 2007;25:8874-84,Gomez,CE.Vaccine 2007a;25:2863-85,Esteban M.Hum.Vaccine 2009;5:867-871,Gomez,CE.Curr.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).

[0304] With regard to adenoviral vectors useful in the practice of the present invention, reference is made to U.S. Patent No. 6,955,808. Adenoviral vectors used include Ad5, Ad3, and Ad6. The vector can be selected from the group consisting of Ad5, 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 Adenovirus Type 5 Genome and Its Comparison with the Adenovirus Type 2 Genome"). "Comparison of the Genome of Adenovirus Type 5 and Its Comparison with the Genome of Adenovirus Type 2," Virology 186, 280-285, the contents of which are 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 or -deleted, E3-deleted or -deleted, and / or E4-deleted or -deleted adenoviral vectors can also be used. Certain adenoviruses with mutations in the E1 region offer improved safety margins because E1-deleted adenoviral mutants are replication-deficient or, at the very least, highly attenuated in nonpermissive cells. Adenoviruses with mutations in the E3 region have enhanced immunogenicity due to disruption of the mechanism by which adenovirus downregulates MHC class I molecules. Adenoviruses with E4 mutations may reduce the immunogenicity of adenoviral vectors due to suppression of late gene expression. Such vectors may be particularly useful when repeated revaccination using the same vector is desired. Adenoviral vectors with deletions or mutations in E1, E3, E4, E1 and E3, and E1 and E4 can be used in the present invention. Furthermore, "gutless" adenoviral vectors in which all viral genes are deleted can also be used in the present invention. Such vectors require a helper virus for replication and require a specific human 293 cell line that expresses both E1a and Cre, a condition not present in the natural environment.Such "gutless" vectors are non-immunogenic and therefore the vectors can be administered multiple times for re-vaccination. "Gutless" adenoviral vectors can be used to insert heterologous inserts / genes, such as the transgenes of the present invention, and can even be used to co-deliver multiple heterologous inserts / genes.

[0305] With regard to lentiviral vector systems useful in the practice of the present invention, 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 references cited therein.

[0306] With regard to AAV vectors useful in the practice of the present invention, 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 references therein.

[0307] Another vector is BCG (bacille Calmette-Guerin). BCG vectors are described by 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 invention, such as Salmonella typhi vectors, are described herein. This will be clear to those skilled in the art from the description in the document.

[0308] The vectors can be co-administered to have in vivo expression and responses similar to the dose and / or response elicited by antigen challenge.

[0309] A preferred means of administering nucleic acids encoding the peptides of the invention uses minigene constructs encoding multiple epitopes. To create DNA sequences (minigenes) encoding selected CTL epitopes for expression in human cells, the amino acid sequences of the epitopes are reverse-translated. A human codon usage table is used to guide the codon selection for each amino acid. These DNA sequences encoding the epitopes are directly adjacent, creating a continuous polypeptide sequence. Additional elements may 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 may be improved by including synthetic (e.g., polyalanine) or naturally occurring flanking sequences adjacent to the CTL epitopes.

[0310] 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, phosphorylated, purified, and annealed under appropriate conditions using well-known techniques. The ends of the oligonucleotides are joined using T4 DNA ligase. This synthetic minigene, encoding the CTL epitope polypeptide, can then be cloned into the desired expression vector.

[0311] Standard regulatory sequences well known to those skilled in the art are included in the vector to ensure expression in the target cells. Several vector elements are necessary: ​​a promoter with a downstream cloning site for minigene insertion; a polyadenylation signal for efficient transcription termination; an E. coli origin of replication; and an E. coli selectable marker (e.g., ampicillin or kanamycin resistance). Many promoters can be used for this purpose, such as the human cytomegalovirus (hCMV) promoter. See U.S. Patent Nos. 5,580,859 and 5,589,466 for other suitable promoter sequences.

[0312] Further vector modifications may be desirable to optimize minigene expression and immunogenicity. In some cases, introns are necessary for efficient gene expression, and one or more synthetic or naturally occurring introns can be incorporated into the transcribed region of the minigene. The inclusion of mRNA stabilization sequences to increase minigene expression may also be considered. Recently, immune stimulatory sequences (ISS or CpG) have been proposed to play a role in the immunogenicity of DNA vaccines. These sequences can be included in the vector outside the minigene coding sequence if found to enhance immunogenicity.

[0313] In some embodiments, bicistronic expression vectors can be used that allow for the production of a minigene-encoded epitope and a second protein that can be included to enhance or reduce immunogenicity. Examples of proteins or polypeptides that can advantageously enhance an immune response when coexpressed include cytokines (e.g., IL2, IL12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), or costimulatory molecules. Helper (HTL) epitopes can be coupled to intracellular targeting signals and expressed separately from CTL epitopes. This can allow for the targeting of HTL epitopes to different cellular compartments than CTL epitopes. If necessary, this can facilitate more efficient entry of HTL epitopes into the MHC class II pathway, thus improving CTL induction. CTL In contrast to induction, specifically dampening the immune response by co-expression of immunosuppressive molecules (eg, TGF-β) may be beneficial in certain diseases.

[0314] After 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 and all other elements contained in the vector are confirmed using restriction enzyme mapping and DNA sequence analysis. Bacterial cells harboring the correct plasmid can be stored as master and working cell banks.

[0315] Purified plasmid DNA can be prepared for injection using a variety of formulations, the simplest of which is reconstitution of lyophilized DNA in sterile phosphate-buffered saline (PBS). Various methods have been described, and new technologies may become available. As described herein, nucleic acids are advantageously formulated with cationic lipids. Additionally, glycolipids, fusogenic liposomes, peptides, and compounds collectively referred to as protective, interactive, non-condensing (PINC) compounds can also be complexed to purify plasmid DNA and affect variables such as stability, intramuscular distribution, or transport to specific organs or cell types.

[0316] 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 standard CTL chromium release assays. The transfection method used depends on the final formulation. Electroporation may be used for "naked" DNA, while cationic lipids allow for direct in vitro transfection. A plasmid expressing green fluorescent protein (GFP) may be cotransfected to allow enrichment of transfected cells using fluorescence-activated cell sorting (FACS). These cells are then labeled with chromium-51 and used as target cells for epitope-specific CTL lines. Cell lysis, detected by 51Cr release, indicates MHC presentation of the minigene-encoded CTL epitope.

[0317] In vivo immunogenicity is a second approach to testing the function of minigene-DNA formulations. Transgenic mice expressing the appropriate human MHC molecules are immunized with the DNA formulation. The dose and route of administration depend on the formulation (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 tested. These effector cells (CTLs) are assayed for cytolysis of peptide-loaded, chromium-51-labeled target cells using standard techniques. Lysis of target cells primed by MHC loading of peptides corresponding to minigene-encoded epitopes demonstrates DNA vaccine function for in vivo induction of CTLs.

[0318] Peptides can also be used to induce CTLs ex vivo. The resulting CTLs can be used to treat chronic tumors in patients in need of treatment that are unresponsive to other conventional therapies or to peptide vaccine 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), the CTLp are activated, mature, and expand into effector CTLs, and the cells are infused back into 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.

[0319] 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 expressed on the surface of the stimulator cells. In the present invention, a sufficient amount of peptide is an amount sufficient to allow approximately 200, and preferably more than 200, human class I MHC molecules to be loaded with the peptide and to be expressed on the surface of each stimulator cell. Preferably, the stimulator cells are incubated with >2 μg / ml of peptide. For example, the stimulator cells may be incubated with >3, 4, 5, 10, 15 μg / ml or more of peptide.

[0320] The resting or precursor CD8+ cells are then incubated in culture with appropriate stimulator cells for a period of time sufficient to activate the CD8+ cells. Preferably, the CD8+ cells are activated in an antigen-specific manner. The ratio of resting or precursor CD8+ (effector) cells to stimulator cells may vary from individual to individual and may further depend on variables such as the amenability of the individual's lymphocytes to the culture conditions and the nature and severity of the disease state or other condition for which the described therapeutic modality is used. Preferably, however, the lymphocyte:stimulator cell ratio ranges from about 30:1 to 300:1. The effector / stimulator culture may be maintained for the time necessary to stimulate a therapeutically usable or effective number of CD8+ cells.

[0321] 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 a cell susceptible to CTL lysis or to stimulate a secondary CTL response, a significantly higher number of MHC / peptide complexes is required for successful activation of CTL precursors (pCTLs) during primary responses. Loading empty major histocompatibility complex molecules on cells with peptides allows for the induction of primary cytotoxic T lymphocyte responses.

[0322] Because mutant cell lines do not exist for every human MHC allele, it is advantageous to use techniques to remove endogenous MHC-associated peptides from the surface of APCs and then load the resulting empty MHC molecules with the immunogenic peptide of interest. The use of non-transformed (non-tumorigenic), non-infected cells, preferably 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.

[0323] Stable MHC class I molecules are trimeric complexes formed by the following elements: 1) a peptide, usually 8–10 residues long; 2) a transmembrane heavy 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 and / or dissociation of p2 microglobulin from the complex renders the MHC class I molecule nonfunctional, destabilizing it and leading to its 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, after which exogenous peptides can be added to them.

[0324] Two possible methods for removing bound peptides from MHC class I molecules include destabilizing p2 microglobulin by lowering the incubation 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, allowing new exogenous peptides to bind to the empty class I molecules. The low-temperature incubation method Although exogenous peptides can be efficiently bound to MHC complexes, overnight incubation at 26°C is required, which may slow the metabolic rate of the cells. Also, cells that do not actively synthesize MHC molecules (e.g., resting PBMCs) may not generate large amounts of empty surface MHC molecules by the low-temperature procedure.

[0325] Harsh 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 the optimal metabolic state of APCs, which is crucial for their viability and 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 it destabilizes only MHC class I molecules (and releases associated peptides), while other surface antigens, including MHC class II molecules, remain intact. Most importantly, treatment of cells with weakly acidic solutions does not affect their viability or metabolic state. Endogenous peptide stripping occurs in 2 minutes at 4°C, and mild acid treatment is rapid, as APCs are immediately ready to perform their functions after loading with the appropriate peptide. This technique is utilized herein to generate peptide-specific APCs that give rise to primary antigen-specific CTLs. The resulting APCs are efficient in inducing peptide-specific CD8+ CTLs.

[0326] Activated CD8+ cells can be effectively separated from stimulator cells using one of a variety of known methods. For example, monoclonal antibodies specific for stimulator cells, peptides loaded onto stimulator cells, or CD8+ cells (or segments thereof) can be used to bind to their appropriate complementary ligand. The antibody-tagged molecules can then be extracted from the stimulated-effector cell mixture by appropriate means, such as well-known immunoprecipitation or immunoassay methods.

[0327] The effective cytotoxic dose of activated CD8+ cells may vary between in vitro and in vivo use, as well as depending on the amount and type of cells that are the ultimate target of these killer cells. The amount may also vary depending on the patient's condition and must be determined by a specialist, taking into account all appropriate factors. However, preferably, the dose is about 5×10 for use in mice. 6 ~5×10 7 cells, compared to approximately 1 × 10 for an adult human. 6 ~Approx. 1×10 12 , more preferably about 1×10 8 ~Approx. 1×10 11 , and even more preferably about 1×10 9 ~Approx. 1×10 10 Activated CD8+ cells are used.

[0328] Preferably, as discussed herein, activated CD8+ cells are recovered from cell culture prior to administering the CD8+ cells to the individual to be treated. However, it is important to note that, unlike other currently proposed treatment modalities, the present method utilizes a non-tumorigenic cell culture system. Thus, when complete separation of stimulator cells and activated CD8+ cells is achieved, there are no inherent risks known to be associated with administering small numbers of stimulator cells, whereas administering mammalian tumor-promoting cells can be quite dangerous.

[0329] 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.

[0330] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the skill of the art. Such techniques are described in "Molecular Cloning: A Laboratory Manual," second edition (Sambrook, 1989). These techniques are described in detail in such publications as "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); "PCR: 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 invention and, therefore, may be considered in making and practicing the invention. Techniques particularly useful for specific embodiments are discussed in the following sections.

[0331] treatment method The present invention provides methods for inducing a neoplasm / tumor-specific immune response in a subject, methods for vaccinating against a neoplasm / tumor, and methods for 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 invention and at least one checkpoint inhibitor.

[0332] In particular, the present invention relates to methods for treating or preventing a neoplasm, comprising administering to a subject (a) a neoplasm vaccine or immunogenic composition, and (b) at least one checkpoint inhibitor.

[0333] In accordance with the present invention, the neoplastic vaccines or immunogenic compositions described herein may be used in patients diagnosed with or at risk of developing cancer.

[0334] The combinations of the invention described are administered in amounts sufficient to induce a CTL response.

[0335] Additional treatments The tumor-specific neo-antigenic peptides and pharmaceutical compositions described herein may also be administered in combination with another agent, such as a therapeutic agent. In certain embodiments, the additional agent may be, but is not limited to, a chemotherapeutic agent, an anti-angiogenic agent, and an agent that reduces immunosuppression.

[0336] The neoplastic vaccine or immunogenic composition and one or more checkpoint inhibitors may be administered before, during, or after the administration of the additional agent. In embodiments, the neoplastic vaccine or immunogenic composition and / or one or more checkpoint inhibitors are administered before the first administration of the additional agent. In other embodiments, the neoplastic vaccine or immunogenic composition and / or one or more checkpoint inhibitors are administered after the first administration of the additional therapeutic agent (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days). In embodiments, the neoplastic vaccine or immunogenic composition and one or more checkpoint inhibitors are administered simultaneously with the first administration of the additional therapeutic agent.

[0337] Therapeutic agents may be, for example, chemotherapeutic or biological agents, radiation, or immunotherapy. Any suitable therapeutic treatment for the particular cancer may be administered. Examples of chemotherapeutic and biological agents include, but are not limited to, angiogenesis inhibitors, such as hydroxyangiostatin K1-3, DL-α-difluoromethyl-ornithine, endostatin, fumagillin, genistein, minocycline, staurosporine, and thalidomide; DNA intercalators / crosslinkers, such as bleomycin, carboplatin, carmustine, clopidogrel, and clopidogrel;lambucil, cyclophosphamide, cis-diammineplatinum(II) dichloride (cisplatin), melphalan, mitoxantrone, and oxaliplatin; DNA synthesis inhibitors, such as (±)-amethopterin (methotrexate), 3-amino-1,2,4-benzotriazine 1,4-dioxide, aminopterin, cytosine β-D-arabinofuranoside, 5-fluoro-5'-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea, and mitomycin DNA-RNA transcription factors such as actinomycin D, daunorubicin, doxorubicin, homoharringtonine, and idarubicin; enzyme inhibitors such as S(+)-camptothecin, curcumin, (-)-deguelin, 5,6-dichlorobenzimidazole 1-β-D-ribofuranoside, etoposide, formestane, fostriecin, hispidin, 2-imino-1-imidazolidinic acid (cyclocreatine), mevinolin, trichostatin A, tyrphostin AG 34, and tyrphostin AG 879; Gene regulators, e.g., 5-aza-2'-deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3), 4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, all-trans retinal (vitamin A aldehyde), retinoic acid all-trans (vitamin A acid), 9-cis retinoic acid, 13-cis retinoic acid, retinol (vitamin A), tamoxifen, and troglitazone; Microtubule inhibitors, e.g., colchicine, docetaxel, dolastatin 15, nocodazole, paclitaxel, podophyllotoxin and unclassified therapeutic agents such as 17-(allylamino)-17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide, apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid, leuprolide (leuprorelin), luteinizing hormone-releasing hormone, pifithrin-α, rapamycin, sex hormone-binding globulin, thapsigargin, and urinary trypsin inhibitor fragment (bikunin).The therapeutic agent may be altretamine, amifostine, asparaginase, capecitabine, cladribine, cisapride, cytarabine, dacarbazine (DTIC), dactinomycin, dronabinol, epoetin alfa, filgrastim, fludarabine, gemcitabine, granisetron, ifosfamide, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, metoclopramide, mitotane, omeprazole, ondansetron, pilocarpine, prochloroperazine, or topotecan hydrochloride.Therapeutic agents include monoclonal antibodies, such as rituximab (Rituxan®), alemtuzumab (Campath®), bevacizumab (Avastin®), cetuximab (Erbitux®), panitumumab (Vectibix®), and trastuzumab (Herceptin®), vemurafenib (Zelboraf®), imatinib mesylate (Gleevec®), and cerebrospinal fluid (CFL) therapy. trademark), erlotinib (Tarceva®), gefitinib (Iressa®), vismodegib (Erivedge™), 90Y-ibritumomab tiuxetan, 131I-tositumomab, ado-trastuzumab emtansine, lapatinib (Tykerb®), pertuzumab (Perjeta™), ado-trastuzumab emtansine (Kadcyla™), regorafenib (Stivarga®), sunitinib (Sutent®), denosumab (Xgeva®), sorafenib (Nexavar®), pazopanib (Votrient®), axitinib (Inlyta®), dasatinib (Sprycel®), nilotinib (Tasigna®), bosutinib (Bosulif®), ofatumumab (Arzeworx®), rra®), obinutuzumab (Gazyva™), ibrutinib (Imbruvica™), idelalisib (Zydelig®), crizotinib (Xalkori®), erlotinib (Tarceva®), afatinib dimaleate (Gilotrif®), ceritinib (LDK378 / Zykadia), tositumomab and 131I-tositumomab (Bexxa). r®), ibritumomab tiuxetan (Zevalin®), brentuximab vedotin (Adcetris®), bortezomib (Velcade®), siltuximab (Sylvant™), trametinib (Mekinist®), dabrafenib (Tafinlar®), pembrolizumab (Keytruda®), carfilzomib (Kyprolis®), ramucirumab (Cyramza™), cabozantinib (Cometriq™), vandetanib (Caprelsa®), and optionally, the therapeutic agent is a neoantigen. The therapeutic agent may be a cytokine, e.g., an interferon (INF), an interleukin (IL), or a hematopoietic growth factor. The therapeutic agent may be INF-α, IL-2, aldesleukin, IL-2, erythropoietin, granulocyte macrophage colony-stimulating factor (GM-CSF), or granulocyte colony-stimulating factor.Therapeutic agents include targeted therapies, such as toremifene (Fareston®), fulvestrant (Faslodex®), anastrozole (Arimidex®), exemestane (Aromasin®), letrozole (Femara®), ziv-aflibercept (Zaltrap®), alitretinoin (Panretin®), temsirolimus (Torisel®), tretinoin (Vesanoid®), denileukin diftitox (Ontak®), vorinostat (Zolinza®), romidepsin (Istodax®), and rituximab (Tetraglio®). The therapeutic agent may be an epigenetic targeted drug, such as an HDAC inhibitor, a kinase inhibitor, a DNA methyltransferase inhibitor, a histone demethylase inhibitor, or a histone methylation inhibitor. The epigenetic drug may be azacitidine (Vidaza), decitabine (Dacogen), vorinostat (Zolinza), romidepsin (Istodax), or ruxolitinib (Jakafi). For prostate cancer treatment, a preferred chemotherapeutic agent that may be used in combination with anti-CTLA-4 is paclitaxel (TAXOL).

[0338] In certain embodiments, the one or more additional agents are one or more anti-glucocorticoid-inducible tumor necrosis factor family receptor (GITR) agonist antibodies. GITR is a costimulatory molecule for T lymphocytes, regulates the innate and adaptive immune systems, and has been shown to be involved in various immune responses and inflammatory processes. GITR was originally reported by Nocentini et al. after cloning from a dexamethasone-treated murine T cell hybridoma (Nocentini et al. Proc Natl Acad Sci USA 94:6216-6221, 1997). Unlike CD28 and CTLA-4, GITR has very low basal expression in natural CD4+ and CD8+ T cells (Ronchetti et al. Eur J Immunol 34:613-622, 2004). The observation that GITR stimulation had an immunostimulatory effect in vitro and induced autoimmunity in vivo prompted research into the antitumor efficacy of triggering this pathway. A review of Modulation of Ctla 4 and Gitr for Cancer Immunotherapy can be found in Cancer Immunology and Immunotherapy (Avogadri et al. Current Topics in Microbiology and Immunology 344.2011). Other agents that may contribute to reducing immunosuppression include the CD28 / CTLA4 Ig superfamily. Examples of checkpoint inhibitors include checkpoint inhibitors targeted to other members of the TNFR superfamily, such as BTLA, LAG3, ICOS, PDL1, or KIR (Page et al., Annual Review of Medicine 65:27 (2014)). In yet another embodiment, the checkpoint inhibitor is targeted to a member of the TNFR superfamily, such as CD40, OX40, CD137, GITR, CD27, or TIM-3. In some cases, targeting of the checkpoint inhibitor is achieved by an inhibitory antibody or similar molecule. In other cases, it is achieved by an agonist against the target; examples of this class include the stimulatory targets OX40 and GITR.

[0339] In certain embodiments, the one or more additional agents are synergistic in that they increase immunogenicity after treatment. In one embodiment, the additional agent allows for a lower dose of the additional therapeutic agent or any component of the combination therapy described herein, thereby allowing for reduced toxicity and / or discomfort. In another embodiment, the additional agent increases the efficacy of the combination therapy described herein, thereby resulting in a longer lifespan. Chemotherapeutic treatments that enhance a patient's immune response have been reviewed (Zitvogel et al., "Immunological aspects of cancer chemotherapy." Nat Rev Immunol. 2008 Jan;8(1):59-73). In addition, chemotherapeutic agents can be safely administered with immunotherapy without inhibiting vaccine-specific T cell responses (Perez et al., "A new era in anticancer peptide vaccines." Cancer May 2010). In one embodiment, administration of the additional agent increases the efficacy of the combination therapy described herein. In one embodiment, the additional agent is a chemotherapy treatment. In one embodiment, low-dose chemotherapy enhances delayed-type hypersensitivity (DTH) responses. In one embodiment, the chemotherapy agent targets regulatory T cells. In one embodiment, cyclophosphamide is the therapeutic agent. In one embodiment, cyclophosphamide is administered prior to vaccination. In one embodiment, cyclophosphamide is administered as a single dose prior to vaccination (Walter et al., "Multipeptide immune response to cancer vaccine IMA901 after single-dose cyclophosphamide associates with longer patient survival." Nature Medicine; 18:8 2012).In another embodiment, cyclophosphamide is administered according to a metronomic program, in which daily doses are administered for one month (Ghiringhelli et al., "Metronomic cyclophosphamide regimen selectively depletes CD4+CD25+ regulatory T cells and restores T and NK effector functions in end stage cancer patients." Cancer Immunol Immunother 2007 56:641-648). In another embodiment, taxanes are administered prior to vaccination to enhance T cell and NK cell function (Zitvogel et al., 2008). In another embodiment, a low dose of a chemotherapeutic agent is administered with the combination therapy described herein. In one embodiment, the chemotherapeutic agent is estramustine. In one embodiment, the cancer is hormone-refractory prostate cancer. While personalized vaccination alone resulted in a ≥50% reduction in serum prostate-specific antigen (PSA) in 8.7% of patients with advanced hormone-refractory prostate cancer, such a reduction occurred in 54% of patients when personalized vaccination was combined with low-dose estramustine (Itoh et al., "Personalized Peptide Vaccines: A Novel Cancer Treatment Modality"). "Alized peptide vaccines: A new therapeutic modality for cancer." Cancer Sci 2006;97:970-976). In another embodiment, glucocorticoids are not administered with or before the combination therapy described herein (Zitvogel et al., 2008). In another embodiment, glucocorticoids are administered after the combination therapy described herein. In another embodiment, gemcitabine is administered before, simultaneously with, or after the combination therapy described herein to enhance the frequency of tumor-specific CTL precursors (Zitvogel et al., 2008). In another embodiment, 5-fluorouracil is administered with the combination therapy described herein because of the synergistic effects observed with peptide-based vaccines (Zitvogel et al., 2008). In another embodiment, a Braf inhibitor, e.g., vemurafenib, is used as an additional agent. Braf inhibition has been shown to be associated with increased melanoma antigen expression and T cell infiltration in tumors of treated patients, as well as a decrease in immunosuppressive cytokines (Frederick et al., "BRAF inhibition is associated with enhanced melanoma antigen expression and a more favorable tumor microenvironment in patients with metastatic melanoma." Clin Cancer Res. 2013;19:1225-1231). In another embodiment, a tyrosine kinase inhibitor is used as the additional agent. In one embodiment, a tyrosine kinase inhibitor is used prior to vaccination with a combination therapy described herein. In one embodiment, a tyrosine kinase inhibitor is used concurrently with a combination therapy described herein. In another embodiment, a tyrosine kinase inhibitor is used to create a more immunopermissive environment. In another embodiment, the tyrosine kinase inhibitor is sunitinib or imatinib mesylate.It has previously been shown that sequential administration of daily dosing of sunitinib and a recombinant vaccine can achieve favorable outcomes (Farsaci et al., "Consequence of dose scheduling of sunitinib on host immune response elements and vaccine combination therapy." Int J Cancer; 130:1948-1959). Sunitinib has also been shown to reverse type 1 immunosuppression using a daily dose of 50 mg / day (Finke et al., "Sunitinib Reverses Type-1 Immune Suppression and Decreases T-Regulatory Cells in Renal Cell Carcinoma Patients." Clin Cancer Res 2008; 14(20)). In another embodiment, a targeted therapy is administered in conjunction with the combination therapy described herein. Doses of targeted therapy have been previously described (Alvarez, "Present and future evolution of advanced breast cancer therapy." Breast Cancer Research 2010, 12(Suppl 2):S1). In another embodiment, temozolomide is administered with a combination therapy described herein. In one embodiment, temozolomide is administered at 200 mg / day for 5 days every 4 weeks of combination therapy with a combination therapy described herein.Results of a similar strategy have shown low toxicity (Kyte et al., "Telomerase Peptide Vaccination Combined with Temozolomide: A Clinical Trial in Stage IV Melanoma Patients." Clin Cancer Res;17(13)). 2011). In another embodiment, the combination therapy is administered with an additional therapeutic agent that produces lymphopenia. In one embodiment, the additional agent is temozolomide. Under these conditions, an immune response can still be induced (Sampson et al., "Greater chemotherapy-induced lymphopenia enhances tumor-specific immune responses that eliminate EGFRvIII-expressing tumor cells in patients with glioblastoma." Neuro-Oncology 13(3):324-333, 2011).

[0340] The compositions and methods described herein can be used on patients with any cancer in need thereof, following the general flow process shown in Figure 2. Patients in need thereof can receive a series of priming vaccinations with a mixture of personalized tumor-specific peptides. In addition, priming over a four-week period can be followed by two boosts during a maintenance phase. All vaccinations are delivered subcutaneously. The vaccine or immunogenic composition can be evaluated for safety, tolerability, immune response, and clinical efficacy in patients, as well as the feasibility of producing the vaccine or immunogenic composition and successfully initiating vaccination within an appropriate timeframe. The first cohort may consist of five patients, and after safety is fully demonstrated, additional cohorts of 10 patients can be enrolled. Peripheral blood is extensively monitored for peptide-specific T cell responses, and patients are followed for up to two years to assess disease recurrence.

[0341] Administration of combination therapy aligned with standard of care In another aspect, the combination therapy described herein provides for selecting an appropriate time point for administration of the combination therapy relative to and within the standard of care for the cancer under treatment for a patient in need thereof. The studies described herein demonstrate that the combination therapy can be effectively administered even within the standard of care, including surgery, radiation, or chemotherapy. Standard of care for the most common cancers can be found on the National Cancer Institute's website (http: / / www.cancer.gov / cancertopics). Standard of care is the current treatment recognized by medical professionals as appropriate for a particular type of disease and widely used by medical professionals. Standard of care is also referred to as best practice, standard medical care, and standard therapy. Standard of care for cancer generally includes surgery, lymph node removal, radiation, chemotherapy, targeted therapy, tumor-targeting antibodies, and immunotherapy. Immunotherapies may include checkpoint blockers (CBPs), chimeric antigen receptors (CARs), and adoptive T-cell therapy. The combination therapy described herein can be incorporated into standard of care. The combination therapies described herein may also be administered when the standard of care changes due to medical advances.

[0342] The incorporation of the combination therapies described herein may rely on therapeutic steps within the standard of care that may result in activation of the immune system. Therapeutic steps that may activate and function synergistically with the combination therapy are described herein. This therapy may advantageously be administered simultaneously with or after the treatment that activates the immune system.

[0343] The incorporation of the combination therapy described herein may depend on treatment steps during standard therapy that result in suppression of the immune system. Such treatment steps may include irradiation, high doses of alkylating agents and / or methotrexate, steroids, e.g., glucosteroids, surgery, e.g., lymph node removal, imatinib mesylate, high doses of TNF, and taxanes (Zitvogel et al., 2008). The combination therapy may be administered prior to such steps. It may be administered before or after.

[0344] In one embodiment, the combination therapy may be administered after bone marrow transplantation and peripheral blood stem cell transplantation. Bone marrow transplantation and peripheral blood stem cell transplantation are procedures that restore stem cells destroyed by high-dose chemotherapy and / or high-dose radiation therapy. After treatment with high-dose anti-cancer drugs and / or high-dose radiation, patients are transplanted with harvested stem cells, which migrate to the bone marrow and begin producing new blood cells. A "mini-transplant" uses less toxic, low-dose chemotherapy and / or low-dose radiation to prepare the patient for transplantation. A "tandem transplant" involves two sequential courses of high-dose chemotherapy and stem cell transplantation. In an autologous transplant, a patient is transplanted with their own stem cells. In a syngeneic transplant, a patient is transplanted with stem cells from their identical twin. In an allogeneic transplant, a patient is transplanted with stem cells from their brother, sister, or parent. An unrelated donor may also be used. In certain types of leukemia, the graft-versus-tumor (GVT) effect that occurs after allogeneic BMT and PBSCT is critical to the effectiveness of treatment. GVT occurs when donor-derived white blood cells (the graft) identify and attack cancer cells (the tumor) remaining in the patient's body after chemotherapy and / or radiation therapy. Immunotherapy using the combination therapy described herein can take advantage of this by vaccinating after transplantation. In addition, the transplanted cells can be presented with neoantigens from the combination therapy described herein before transplantation.

[0345] In one embodiment, the combination therapy is administered to a patient in need of such treatment whose cancer requires surgery. In one embodiment, the combination therapy described herein is administered to a patient in need of such treatment whose cancer, e.g., breast cancer, is where the standard of care is primary surgery followed by treatment to remove possible micrometastases. Breast cancer is typically treated with various combinations of surgery, radiation therapy, chemotherapy, and hormonal therapy depending on the stage and grade of the cancer. Adjuvant therapy for breast cancer is any treatment given after primary therapy to increase the chances of long-term survival. Neoadjuvant therapy is treatment given before primary therapy. Adjuvant therapy for breast cancer is any treatment given after primary therapy to increase the chances of long-term disease-free survival. Primary therapy is the main treatment used to reduce or eliminate cancer. Primary therapy for breast cancer typically includes surgery, mastectomy (removal of the breast) or lumpectomy (surgery to remove the tumor and a small amount of surrounding normal tissue; a type of breast-conserving surgery). During either type of surgery, one or more nearby lymph nodes are also removed to see if cancer cells have spread to the lymphatic system. When women undergo breast-conserving surgery, primary therapy almost always includes radiation therapy. Even in early-stage breast cancer, cells can break away from the primary tumor and spread (metastasize) to other parts of the body. Therefore, doctors use adjuvant therapy to kill any cancer cells that may have spread, even if they cannot be detected by imaging or clinical tests.

[0346] In one embodiment, the combination therapy is administered in conjunction with standard of care for ductal carcinoma in situ (DCIS). The standard of care for this type of breast cancer is: 1. Breast-conserving surgery and radiation therapy with or without tamoxifen. 2. Total mastectomy with or without tamoxifen. 3. Breast-conserving surgery without radiation therapy.

[0347] The combination therapy may be administered prior to breast-conserving surgery or mastectomy to shrink the tumor before surgery, hi another embodiment, the combination therapy may be administered as adjuvant therapy to eliminate any remaining cancer cells.

[0348] In another embodiment, patients diagnosed with stage I, II, IIIA, and operable IIIC breast cancer are treated with a combination therapy as described herein. The standard of care for this type of breast cancer is: 1. Local-topical treatment: · Breast-conserving therapy (lumpectomy, breast radiation, and axillary surgical staging). · Modified radical mastectomy (removal of the entire breast with level I-II axillary incision) with or without breast reconstruction. Sentinel lymph node biopsy. 2. Post-mastectomy adjuvant radiation therapy for axillary node-positive tumors: To 1-3 lymph nodes: Role of local radiation (subclavian / supraclavicular nodes, internal mammary nodes, axillary nodes, and chest wall) is unclear. For 4 or more lymph nodes or extranodal disease: Local radiation therapy is recommended. 3. Adjuvant systemic therapy

[0349] In one embodiment, the combination therapy is administered as neoadjuvant therapy to shrink tumors, hi another embodiment, the combination is administered as adjuvant systemic therapy.

[0350] In another embodiment, patients diagnosed with inoperable stage IIIB or IIIC or inflammatory breast cancer are treated with a combination therapy as described herein. The standard of care for this type of breast cancer is: 1. Multimodality therapy administered with curative intent is the standard of care for patients with clinical stage IIIB disease. 2. Initial surgery is generally limited to biopsies that allow for determination of histology, estrogen receptor (ER) and progesterone receptor (PR) levels, and human epidermal growth factor receptor 2 (HER2 / neu) overexpression. Initial treatment with anthracycline-based chemotherapy and / or taxane-based therapy is standard. For patients who respond to neoadjuvant chemotherapy, local therapy may consist of total mastectomy with axillary lymph node dissection, followed by postoperative radiation therapy to the chest wall and regional lymphatics. Breast-conserving therapy can be considered in patients who achieve a good partial or complete response to neoadjuvant chemotherapy. Subsequent systemic therapy may consist of additional chemotherapy. Patients with ER-positive or unknown tumors should receive hormonal therapy. All patients should be considered candidates for clinical trials to evaluate the optimal mode of administering the various components of a multimodality regimen.

[0351] In one embodiment, the combination therapy is administered as part of various components of a multimodality regimen. In another embodiment, the combination therapy is administered before, simultaneously with, or after a multimodality regimen. In another embodiment, the combination therapy is administered based on synergy between the modalities. In another embodiment, the combination therapy is administered after treatment with anthracycline-based chemotherapy and / or taxane-based therapy (Zitvogel et al., 2008). Treatment after administration of the combination therapy can negatively affect the division of effector T cells. The combination therapy may also be administered after radiation.

[0352] In another embodiment, the combination therapy described herein is used in the treatment of cancers where the standard of care is primarily based on systemic therapy, not primarily surgery, such as chronic lymphocytic leukemia (CLL). In another embodiment, patients diagnosed with stage I, II, III, and IV chronic lymphocytic leukemia are treated with a combination therapy as described herein. The standard of care for this cancer type is: 1. Observation in asymptomatic or minimally affected patients 2. Rituximab 3. Ofatumumab 4. Oral alkylating agents with or without corticosteroids 5. Fludarabine, 2-chlorodeoxyadenosine, or pentostatin 6. Bendamustine 7. Lenalidomide 8. Combination chemotherapy Combination chemotherapy regimens include: Fludarabine + cyclophosphamide + rituximab Fludarabine plus rituximab as seen in the CLB-9712 ​​and CLB-9011 trials Fludarabine + cyclophosphamide vs. fludarabine + cyclophosphamide + rituximab For example, pentostatin + cyclophosphamide + rituximab, as seen in the MAYO-MC0183 trial Ofatumumab + fludarabine + cyclophosphamide ○CVP: Cyclophosphamide + Vincristine + Prednisone CHOP: Cyclophosphamide + Doxorubicin + Vincristine + Prednisone Fludarabine plus cyclophosphamide versus fludarabine, as seen in the E2997 trial [NCT00003764] and the LRF-CLL4 trial For example, fludarabine plus chlorambucil, as seen in the CLB-9011 trial 9. Involved field radiation therapy 10. Alemtuzumab 11. Bone marrow and peripheral blood stem cell transplants are under clinical evaluation 12. Ibrutinib

[0353] In one embodiment, the combination therapy is administered before, concurrently with, or after treatment with rituximab or ofatumumab. Because these are monoclonal antibodies that target B cells, treatment with the combination therapy can be synergistic. In another embodiment, the combination therapy is administered after treatment with oral alkylating agents, with or without corticosteroids, and fludarabine, 2-chlorodeoxyadenosine, or pentostatin, because these treatments can negatively affect the immune system if administered beforehand. In one embodiment, bendamustine is administered with the combination therapy at a low dose based on the prostate cancer results described herein. In one embodiment, the combination therapy is administered after treatment with bendamustine.

[0354] Vaccine or immunogenic composition kits and co-packaging In certain aspects, the present invention provides kits containing any one or more of the components discussed herein to enable administration of a combination therapy. Components may be provided individually or in combination and in any suitable container, e.g., vials, bottles, or tubes. In some embodiments, the kits include instructions in one or more languages, e.g., two or more languages. In some embodiments, the kits include one or more reagents for use in a process utilizing one or more of the components described herein. Reagents may be provided in any suitable container. For example, the kit may provide one or more delivery or storage buffers. Reagents may be provided in a form ready for use in a particular process or may be provided in a form requiring the addition of one or more other components prior to use (e.g., concentrated or lyophilized). The buffer may be any buffer, including, but not limited to, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH of about 7 to about 10. In some embodiments, a kit comprises one or more of the vectors, proteins, and / or one or more polynucleotides described herein. The kit may advantageously allow for the provision of all elements of the system of the present invention. The kit may include one or more vectors and / or one or more particles and / or one or more nanoparticles containing or encoding one or more RNAs of 1 to 50 or more neo-antigen mutations to be administered to an animal, mammal, primate, rodent, etc., and such kits may be used for administration to such eukaryotic organisms. and such kits may optionally include any of the anti-cancer agents described herein. The kits may include any of the above components (e.g., one or more vectors and / or one or more particles and / or one or more nanoparticles containing or encoding RNA for one or more of 1-50 or more neo-antigen mutations, neo-antigen proteins or peptides, checkpoint inhibitors) and instructions for use in any of the methods of the invention.

[0355] In one embodiment, the kit includes at least one vial containing an immunogenic composition or vaccine and at least one vial containing an anti-cancer agent. In one embodiment, the kit may contain ready-to-use components that are mixed together and ready for administration. In one aspect, the kit includes a ready-to-use immunogenic or vaccine composition and a ready-to-use anti-cancer agent. The ready-to-use immunogenic or vaccine composition may include separate vials containing different pools of immunogenic compositions. The immunogenic composition may include one vial containing a viral vector or DNA plasmid, and another vial containing an immunogenic protein. The ready-to-use anti-cancer agent may include a cocktail of anti-cancer agents or a single anti-cancer agent. Separate vials may contain different anti-cancer agents. In another embodiment, the kit may include a ready-to-use anti-cancer agent and an immunogenic composition or vaccine in a ready-to-reconstitute form. The immunogenic or vaccine composition may be freeze-dried or lyophilized. The kit may include a separate vial containing a reconstitution buffer that can be added to the lyophilized composition to enable it to be administered immediately. The buffer may advantageously include an adjuvant or emulsion according to the present invention. In another embodiment, the kit may include a ready-to-reconstitute anti-cancer agent and a ready-to-reconstitute immunogenic composition or vaccine. In this aspect, both may be lyophilized. In this aspect, the kit may include separate reconstitution buffers for each. The buffers may advantageously include an adjuvant or emulsion according to the present invention. In another embodiment, the kit may include a single vial containing a dose of the immunogenic composition and the anti-cancer agent to be administered together. In another aspect, multiple vials are included, such that one vial is administered according to a treatment timeline. One vial may contain only one therapeutic dose of the anti-cancer agent, another may contain both another therapeutic dose of the anti-cancer agent and the immunogenic composition, and yet another dose of the immunogenic composition alone. In a further aspect, the vials are labeled with labeling regarding their appropriate administration to a patient in need thereof.The immunogen or anti-cancer agent of any embodiment may be in lyophilized form, dry form, or in aqueous solution as described herein. The immunogen may be a live attenuated virus, protein, or nucleic acid as described herein.

[0356] In one embodiment, the anti-cancer agent enhances the immune system to enhance the efficacy of the immunogenic composition or vaccine. In a preferred embodiment, the anti-cancer agent is a checkpoint inhibitor. In another embodiment, the kit includes multiple vials of an immunogenic composition and an anti-cancer agent to be administered at different time intervals along a treatment regimen. In another embodiment, the kit may include separate vials of an immunogenic composition used in priming the immune response and another immunogenic composition used for boosting. In one aspect, the priming immunogenic composition may be a DNA or viral vector, and the boosting immunogenic composition may be a protein. Either composition may be lyophilized or ready for immediate administration. In another embodiment, different anti-cancer agent cocktails containing at least one anti-cancer agent are contained in different vials for administration along a treatment regimen.

[0357] Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions, and alterations could be made therein without departing from the spirit and scope of the invention as defined in the appended claims.

[0358] The present invention is further illustrated in the following examples, which are provided for illustrative purposes only and are not intended to limit the invention in any way. [Example]

[0359] Example 1 Cancer Vaccine Trial Protocol The compositions and methods described herein may be tested in 15 patients with high-risk melanoma (stage IIIB, IIIC, and IVM1a, b after complete resection) following the general flow process shown in Figure 2. Patients may receive a series of priming vaccinations with a mixture of personalized tumor-specific peptides and poly-ICLC over a 4-week period, followed by two boosts during the maintenance phase. All vaccinations are delivered subcutaneously. The vaccine or immunogenic composition may be evaluated for safety, tolerability, immune response, and clinical efficacy in patients, as well as the feasibility of producing the vaccine or immunogenic composition and successfully initiating vaccination within an appropriate timeframe. The first cohort may consist of five patients, and after safety is fully demonstrated, additional cohorts of 10 patients may be enrolled. Peripheral blood will be extensively monitored for peptide-specific T cell responses, and patients will be followed for up to two years to assess disease recurrence.

[0360] As described herein, the effectiveness of mutant epitopes in inducing immune responses and cases of spontaneous tumor regression or long-term survival in both animals and humans have been demonstrated by CD8 expression of mutant epitopes. + There is a great deal of evidence that it correlates with T cell responses (Buckwalter and Srivastava PK. "It is the antigen, silly" and other lessons from a decade of human cancer vaccine therapy ("It is the "antigen(s), stupid" and other lessons from over a decade of vaccitherapy of human cancer" Seminars in immunology 20:296-300 (2008); Karanikas et al., "High frequency of cytolytic T lymphocytes against tumor-specific mutant antigens detectable by HLA tetramers in the blood of long-term surviving lung cancer patients" Cancer Res. 61:3718-3724 (2001); Lennerz et al., "The response of autologous T cells to a human melanoma is dominated by mutated neo-antigens" Proc Natl Acad Sci U S A. 102:16013 (2005)) and that "immunoediting" can be traced to altered expression of dominant mutant antigens in mice and humans (Matsushita et al., "Cancer exome analysis reveals a T cell-dependent mechanism of cancer immunoediting" a T-cell-dependent mechanism of cancer immunoediting,” Nature 482:400 (2012); DuPage et al., “Expression of tumor-specific antigens underlies cancer immunoediting,” Nature 482:405 (2012); and Sampson et al., “Immunologic escape following long-term progression-free survival following epidermal growth factor receptor variant III peptide vaccination in newly diagnosed glioblastoma patients.” escape after prolonged progression-free survival with epidermal growth factor receptor variant III peptide vaccination in patients with newly diagnosed glioblastoma)” J Clin Oncol. 28:4722-4729 (2010)).

[0361] Next-generation sequencing can now rapidly reveal the presence of discrete mutations in individual tumors, most commonly single amino acid changes (e.g., missense mutations) and, less commonly, frameshift insertions / deletions / gene fusions, read-through mutations at stop codons, and novel stretches of amino acids generated by translation of improperly spliced ​​introns (e.g., neo-ORFs). Neo-ORFs are particularly valuable as immunogens because their entire sequences are completely novel to the immune system and thus resemble viral or bacterial foreign antigens. Thus, neo-ORFs are: (1) highly specific for tumors (i.e., not expressed in any normal cells); and (2) capable of bypassing central tolerance, thereby increasing the precursor frequency of neo-antigen-specific CTLs. For example, peptides derived from human papillomavirus (HPV) recently demonstrated the power of utilizing similar foreign sequences in therapeutic anti-cancer vaccines. Approximately 50% of 19 patients with preneoplastic virus-induced disease who received three to four vaccinations with a mixture of HPV peptides derived from the viral oncogenes E6 and E7 maintained complete remission for more than 24 months (Kenter et al., "Vaccination against HPV-16 oncoproteins for vulvar intraepithelial neoplasia"). HPV-16 Oncoproteins for Vulvar Intraepithelial Neoplasia)” NEJM 361:1838 (2009)).

[0362] Sequencing technologies have revealed that each tumor contains multiple patient-specific mutations that alter the protein-coding content of genes. Such mutations create modified proteins ranging from single amino acid changes (caused by missense mutations) to the addition of long stretches of novel amino acid sequence due to frameshifts, readthrough of stop codons, or translation of intronic regions (novel open reading frame mutations; neo-ORFs). These mutant proteins are useful targets for the host's immune response against tumors because, unlike native proteins, they are not subject to the immunosuppressive effects of self-tolerance. Thus, mutant proteins are more likely to be immunogenic and more specific for tumor cells compared to normal cells in the patient.

[0363] Using recently improved algorithms that predict which missense mutations will result in peptides that bind strongly to a patient's cognate MHC molecule, a set of peptides representing the optimal mutant epitopes (both neo-ORF and missense) for each patient is identified and prioritized, and up to 20 or more peptides are prepared for immunization (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)). Peptides of approximately 20-35 amino acids in length were synthesized because such "long" peptides have been shown to undergo efficient internalization, processing, and cross-presentation in 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).

[0364] In addition to a potent and specific immunogen, an effective immune response advantageously includes a powerful adjuvant to activate the immune system (Speiser and Romero, "Cancer Immunology"). "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 and, in turn, adaptive immune systems (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 bone marrow-derived dendritic cells. In human volunteer studies, poly-ICLC was 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"). Exp Med 208:2357 (2011)). Hiltonol®, a GMP formulation of poly-ICLC prepared by Oncovir, Inc., is utilized as the adjuvant.

[0365] Example 2 Target Patient Population Patients with stage IIIB, IIIC, and IVM1a,b melanoma are at significantly increased risk of disease recurrence and death, even with complete surgical resection of the disease (Balch et al., "Final Version of 2009 AJCC Melanoma Staging and Classification," J Clin Oncol 27:6199-6206 (2009)). The only systemic adjuvant therapy available for this patient population is interferon-alpha (IFNα), which provides measurable, but marginal, benefit and is associated with significant, often dose-limiting, toxicity (Kirkwood et al., "Interferon alfa-2b Adjuvant Therapy of High-Risk Resected Cutaneous Melanoma: The Eastern Cooperative Oncology Group Trial EST 1684"). 1684),” J Clin Oncol 14:7-17 (1996); Kirkwood et al., “High- and Low-dose Interferon Alpha-2b in High-Risk Melanoma: First Analysis of Intergroup Trial E1690 / S9111 / C9190,” J Clin Oncol 18:2444-2458 (2000)). These patients are not immunocompromised by previous cancer-targeted therapy or active cancer and therefore represent an excellent patient population for evaluating the safety and immunological impact of vaccines. Finally, the current standard of care for these patients does not mandate any treatment after surgery, allowing an 8- to 10-week window for vaccine formulation.

[0366] The target population is completely resected, disease-free cutaneous melanoma patients with clinically detectable, histologically confirmed lymph node (regional or distant) or in-transit metastases. These patients may be: many with stage IIIB (excluding patients with ulcerated primary tumors but micrometastatic lymph nodes (T1-4b, N1a, or N2a) due to the need to have sufficient tumor tissue for sequencing and cell line development), all with stage IIIC, and stage IVM1a,b. These may be patients with a first diagnosis of early-stage melanoma or patients with recurrent disease after a previous diagnosis.

[0367] Tumor Resection: Patients may undergo complete resection of their primary melanoma (if not already removed) and all locally metastatic disease with the goal of rendering them melanoma-free. After sufficient tumor for pathological evaluation has been removed, the remaining tumor tissue is placed in sterile media in a sterile container and prepared for disaggregation. A portion of the tumor tissue is used for whole exome and transcriptome sequencing and cell line generation, and the remaining tumor is frozen.

[0368] Normal tissue collection: Collect a normal tissue sample (blood or sputum sample) for whole exome sequencing.

[0369] Patients with clinically evident locoregional metastatic disease or completely resectable distant lymph node, skin, or lung metastatic disease (but no unresectable distant or visceral metastatic disease) will be identified and enrolled in the study. Pre-operative patient enrollment is required to obtain fresh tumor tissue for melanoma cell line establishment (thereby generating target cells for in vitro cytotoxicity assays as part of the immune monitoring program).

[0370] Example 3 Dosage and Schedule For patients who meet all pretreatment criteria, vaccination may begin as soon as possible after the study drug arrives and meets acceptance specifications. There will be four separate study drugs for each patient, each containing five of the 20 patient-specific peptides. Immunizations may generally proceed according to the schedule shown in Figure 3.

[0371] Patients are treated in the outpatient department. Immunization on each treatment day consists of four 1 ml subcutaneous injections, and each injection may be given in a different limb to target different regions of the lymphatic system and reduce antigen competition. If the patient has had a complete axillary or inguinal lymph node dissection, the vaccine is alternatively administered into the right or left diaphragm. Each injection consists of one of the four study drugs for that patient, with the same study drug injected into the same limb for each cycle. The composition of each 1 ml injection is as follows: 0.75ml test drug containing 300µg each of five patient-specific peptides 0.25 ml (0.5 mg) of 2 mg / ml Poly ICLC (Hiltonol®)

[0372] During the induction / priming phase, patients are immunized on days 1, 4, 8, 15, and 22. During the maintenance phase, patients receive booster doses at 12 and 24 weeks.

[0373] Blood samples may be collected at multiple time points: before (baseline; two samples on different days); on day 15 during the priming vaccination; 4 weeks after the induction / priming vaccination (week 8); before (week 12) and after (week 16) the first boost; and before (week 24) and after (week 28) the second boost, with 50-150 ml of blood collected for each sample (except week 16). The primary immunological endpoint is week 16, and therefore patients will undergo leukapheresis (unless otherwise indicated based on patient and physician assessment).

[0374] Example 4 immune monitoring The immunization strategy is a "prime-boost" approach, involving an initial series of closely spaced immunizations to induce an immune response, followed by a rest period to establish memory T cells. This is followed by a booster immunization, with the T cell response 4 weeks after the boost expected to produce the strongest response and representing the primary immunological endpoint. Global immune responses are initially monitored using peripheral blood mononuclear cells stimulated with a pool of overlapping 15-mer peptides (11 aa overlap) encompassing all immune epitopes in an ex vivo ELISPOT assay 18 hours from this point. Pre-vaccination samples are evaluated to establish a baseline response to this peptide pool. Additional PBMC samples are evaluated as needed to examine the kinetics of the immune response to the total peptide mixture. For patients who demonstrate a response significantly above baseline, the entire 15-mer pool is deconvolved to determine which specific immunizing peptides were immunogenic. Additionally, appropriate samples are subjected to several additional assays, depending on the individual case: Use the entire 15mer pool or subpools as stimulatory peptides in intracellular cytokine staining assays to identify and quantify antigen-specific CD4+, CD8+, central memory, and effector memory populations Similarly, these pools can be used to assess the patterns of cytokines secreted by these cells and to identify T H 1 vs T H 2. Determine the Phenotype · Quantify Tregs and myeloid-derived suppressor cells (MDSCs) using extracellular cytokine staining of unstimulated cells and flow cytometry. If melanoma cell lines from responding patients are successfully established and activating epitopes are identified, T cell cytotoxicity assays will be performed using the mutant peptides and the corresponding wild-type peptides. The primary immunological endpoint, PBMCs, will be assessed for "epitope spreading" by using known melanoma tumor-associated antigens as stimuli and several additional identified mutant epitopes that were not selected among the immunogens, as shown in Figure 4.

[0375] Immunohistochemistry of tumor samples will be performed to quantify CD4+, CD8+, MDSC, and Treg infiltrating populations.

[0376] Example 5 Clinical efficacy in patients with metastatic disease Vaccine therapy for patients with metastatic disease is complicated by the need for effective treatment of the active cancer and the resulting lack of a treatment-free window for vaccine preparation. Furthermore, these cancer treatments can impair the patient's immune system, potentially preventing the induction of an immune response. With these considerations in mind, settings can be selected in which the timing of vaccine preparation is temporally compatible with other standard treatment approaches for a particular patient population and / or in which such standard treatments can be reliably compatible with immunotherapeutic approaches. There are two types of settings that can be explored: 1. Combination with checkpoint blockade: Checkpoint blockade antibodies have emerged as an effective immunotherapy for metastatic melanoma (Hodi et al., "Improved Survival with Ipilimumab in Patients with Metastatic Melanoma"). Topalian et al., “Safety, Activity, and Immune Correlates of Anti-PD-1 Antibody in Cancer” NEJM 366:2443-2454 (2012); Brahmer et al., “Safety and Activity of Anti-PD-L1 Antibody in Patients with Advanced Cancer.” It is being actively explored in other disease settings, including "In Patients with Advanced Cancer" NEJM 366:2455-2465 (2012)). The mechanism of action is unclear, but both reversal of local immunosuppression and enhancement of the immune response are possible explanations. Integrating potent vaccines to elicit immune responses with checkpoint-blocking antibodies can result in synergistic effects, as observed in several animal studies (van Elsas et al., "Combined immunotherapy of B16 melanoma with an anti-cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and granulocyte / macrophage colony-stimulating factor (GM-CSF)-producing vaccine induces rejection of subcutaneous and metastatic tumors with autoimmune depigmentation," J Exp Med 190:35-366 (1999); Li et al., "Anti-programmed death-1 synergizes with granulocyte macrophage colony-stimulating factor-secreting tumor cell immunotherapy providing therapeutic benefit to mice with established tumors," Clin Cancer Res. 15:1623-1634(2009); Pardoll, DM "Immune checkpoint blockade in cancer immunotherapy" of immune checkpoints in cancer immunotherapy,” Nature Reviews Cancer 12:252-264 (2012); Curran et al. “PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors.” melanoma tumors.” Proc Natl Acad Sci U S A. 2010 Mar 2;107(9):4275-80; Curran et al. “Tumor vaccines expressing flt3 ligand synergize with ctla-4 blockade to reject preimplanted tumors.” Cancer Res. 2009 Oct 1;69(19):7747-55. As shown in Figure 5, patients can immediately begin checkpoint blockade therapy while the vaccine is being prepared, and then vaccine administration can be integrated with antibody therapy; and 2. Combination with standard treatment regimens that exhibit beneficial immune properties a) Patients with renal cell carcinoma (RCC) who present with metastatic disease typically undergo surgical debulking, followed by systemic treatment, commonly with one of the approved tyrosine kinase inhibitors (TKIs), such as sunitinib, pazopanib, and sorafenib. HSunitinib has been shown to increase type 1 immune suppression and decrease Treg and myeloid-derived suppressor cells (Finke et al., "Sunitinib reverses Type-1 immune suppression and decreases T-regulatory cells in renal cell carcinoma patients," Clin Can Res 14:6674-6682 (2008); Terme et al., "VEGFA-VEGFR pathway blockade inhibits tumor-induced regulatory T cell proliferation in colorectal cancer," Cancer Research Author Manuscript published Online (2012)). Approval that does not impair the immune system The ability to immediately treat patients with a pre-existing therapeutic provides a necessary window for vaccine formulation and potential synergistic effects with the vaccine therapeutic. Additionally, multiple animal and human studies have shown that cyclophosphamide (CTX) has an inhibitory effect on Treg cells, and a single dose of CTX prior to vaccination has recently been shown to improve survival in RCC patients who responded to the vaccine (Walter et al., "Multiple-peptide immune responses to the cancer vaccine IMA901 following a single dose of cyclophosphamide are associated with longer patient survival," Nature Medicine 18:1254-1260 (2012)). Both of these immune synergy approaches have been utilized in a recently completed phase 3 trial of a natural peptide vaccine in RCC (ClinicalTrials.gov, NCT01265901 IMA901 in patients receiving sunitinib for advanced / metastatic renal cell carcinoma (NCT01265901 IMA901 in Patients Receiving Sunitinib for Advanced / Metastatic Renal Cell Carcinoma)); b) Alternatively, standard treatment for glioblastoma (GBM) involves surgery, recovery, and follow-up radiation and low-dose temozolomide (TMZ), followed by a 4-week rest period followed by initiation of standard-dose TMZ. This standard treatment provides a window for vaccine preparation, followed by initiation of vaccination and then initiation of standard-dos...

Claims

1. 1. A method of treating or preventing a neoplasm in a subject in need thereof, comprising administering to a subject in need thereof: (a) a neoplastic vaccine or immunogenic composition; and (b) at least one checkpoint inhibitor The method of claim 1, further comprising administering

2. 2. The method of claim 1, wherein the neoplasm vaccine comprises at least two, at least three, at least four, or at least five neoantigenic peptides.

3. 3. The method of claim 1 or 2, wherein the neo-antigenic peptide ranges from about 5 to about 50 amino acids in length.

4. The method of any one of claims 1 to 3, wherein the neo-antigenic peptide ranges from about 15 to about 35 amino acids in length.

5. 10. The method of claim 1, wherein the neoplasm vaccine composition further comprises a pH adjusting agent and a pharma- ceutically acceptable carrier.

6. 10. The method of claim 1, further comprising administration of an immunomodulatory agent or adjuvant.

7. The immunomodulatory agent 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 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, acrylic or methacrylic polymers, copolymers of maleic anhydride and Aquila's QS21 stimulon.

8. 8. The method of claim 7, wherein the immunomodulatory agent or adjuvant is poly-ICLC.

9. 2. The method of claim 1, wherein the checkpoint inhibitor is an inhibitor of the programmed death 1 (PD-1) pathway.

10. The method of claim 9, wherein the PD-1 pathway inhibitor is an anti-PD1 antibody.

11. The method of claim 10, wherein the PD-1 pathway inhibitor is nivolumab.

12. 2. The method of claim 1, wherein the checkpoint inhibitor is an anti-cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) antibody.

13. 13. The method of claim 12, wherein the anti-CTLA4 antibody is ipilimumab or tremelimumab.

14. 2. The method of claim 1, wherein the subject is afflicted with a neoplasm selected from the group consisting of non-Hodgkin's lymphoma (NHL), clear cell renal cell carcinoma (ccRCC), melanoma, sarcoma, leukemia, or bladder, colon, brain, breast, head and neck, endometrial, lung, ovarian, pancreatic, or prostate cancer.

15. The method of claim 14, wherein the neoplasm is metastatic.

16. 10. The method of claim 1, wherein the subject does not have a detectable neoplasm but is at high risk of disease recurrence.

17. 10. The method of claim 1, wherein the subject has previously undergone an autologous hematopoietic stem cell transplant (AHSCT).

18. 13. The method of claim 1, wherein administration of the checkpoint inhibitor is initiated prior to initiation of administration of the neoplastic vaccine.

19. 10. The method of claim 1, wherein administration of the checkpoint inhibitor is initiated after administration of the neoplasm vaccine is initiated.

20. 10. The method of claim 1, wherein administration of the checkpoint inhibitor is initiated simultaneously with initiation of administration of the neoplastic vaccine.

21. 21. The method of any one of claims 18-20, wherein administration of the checkpoint inhibitor continues every 2-8 weeks or more after an initial administration of the checkpoint inhibitor.

22. 22. The method of claim 21, wherein administration of the checkpoint inhibitor is continued every 2, 3, or 4, 6, or 8 weeks following an initial administration of the checkpoint inhibitor.

23. 20. The method of claim 18, wherein administration of the checkpoint inhibitor is withheld for one week prior to administration of the neoplasm vaccine.

24. 20. The method of claim 18, wherein administration of the checkpoint inhibitor is withheld during administration of the neoplasm vaccine.

25. The method of any one of claims 18 to 20, wherein administration of the checkpoint inhibitor is initiated after tumor resection.

26. The method of any one of claims 18 to 20, wherein administration of the neoplasm vaccine is initiated 1 to 15 weeks after tumor resection.

27. The method of any one of claims 18 to 20, wherein administration of the neoplasm vaccine is initiated 4 to 12 weeks after tumor resection.

28. 10. The method of claim 1, wherein the administration of the neoplastic vaccine is a prime / boost dosing regimen.

29. 30. The method of claim 28, wherein administration of the neoplastic vaccine is at week 1, 2, 3 or 4 as a prime.

30. 29. The method of claim 28, wherein administration of the neoplastic vaccine is at 2, 3, 4 or 5 months as a boost.

31. The method of any one of claims 2 to 30, wherein the vaccine is administered at a dose of about 10 μg to 1 mg per neo-antigenic peptide.

32. 31. The method of any one of claims 2 to 30, wherein the vaccine is administered at an average weekly dosage level of about 10 μg to 2000 μg per neo-antigenic peptide.

33. 31. The method of any one of claims 1-30, wherein the checkpoint inhibitor is administered at a dose of about 0.1-10 mg / kg.

34. 34. The method of claim 33, wherein the administration is intravenous.

35. The method of claim 12, wherein the anti-CTLA4 antibody is administered at a dose of about 1 mg / kg to 3 mg / kg.

36. The method of any one of claims 1 to 32, wherein the vaccine is administered intravenously or subcutaneously.

37. 36. The method of any one of claims 1 to 35, wherein the checkpoint inhibitor is administered intravenously or subcutaneously.

38. 36. The method of any one of claims 1-35, wherein the checkpoint inhibitor is administered subcutaneously within about 2 cm of the site of administration of the neoplasm vaccine.

39. 40. The method of claim 38, wherein the checkpoint inhibitor is administered at a dosage of about 0.1-1 mg per administration site of the neoplasm vaccine.

40. 10. The method of claim 1, further comprising administration of one or more additional agents.

41. 41. The method of claim 40, wherein the additional agent is selected from the group consisting of a chemotherapeutic agent, an anti-angiogenic agent, and an agent that reduces immunosuppression.

42. 41. The method of claim 40, wherein the one or more additional agents are one or more anti-glucocorticoid-induced tumor necrosis factor family receptor (GITR) agonist antibodies.

43. (a) a separately packaged freeze-dried immunogenic composition configured to elicit an immune response against at least one neo-antigen; and (b) Solution for reconstitution of freeze-dried vaccines 20. A vaccination or immunization kit comprising:

44. A vaccination or immunization kit according to claim 43, wherein the solution contains an adjuvant.

45. 44. A vaccination or immunization kit according to claim 43, wherein said immunogenic composition is an antigen.

46. 44. The vaccination or immunization kit of claim 43, wherein the immunogenic composition is a viral vector.

47. 44. The kit of claim 43, further comprising a checkpoint inhibitor.

Images