Formulations for neoplasia vaccines

Abstract

The present invention relates to neoplasia vaccine or immunogenic composition formulations for use in the treatment or prevention of neoplasia in a subject.

Description

[0001] Related applications and references This application is a divisional application of Chinese Patent Application No. 201480074770.9, filed on December 5, 2014, entitled "Preparation for Tumor Formation Vaccine". The original application was a Chinese national phase application of PCT Patent Application No. PCT / US2014 / 068893, which claimed the benefit and priority of U.S. Provisional Patent Application Serial No. 61 / 913,172, filed on December 6, 2013.

[0002] All documents cited in or during the examination of the foregoing applications (“Application References”) and all documents referenced or cited in those Application References, and all documents referenced or cited herein (“Documents Referenced herein”), together with any manufacturer’s specifications, descriptions, product specifications, and product tables for any product mentioned herein or incorporated herein by reference, are hereby incorporated by reference and may be used in the practice of the invention. More specifically, all referenced documents are incorporated herein by reference to the extent that each individual document is explicitly and individually indicated to be incorporated herein by reference. Invention Field This invention relates to formulations for treating tumor formation. More specifically, this invention relates to formulations for tumor vaccines used to treat tumor formation in a subject.

[0003] Federal Funding Explanation This invention is supported by the U.S. National Institutes of Health (NIH) under license numbers NIH / NCI-1R01CA155010-02 and NHLBI-5R01HL103532-03. The U.S. government holds certain rights to this invention. Background of the Invention Approximately 1.6 million Americans are diagnosed with neoplasia each year, and about 580,000 people were expected to die from the disease in the United States in 2013. Significant improvements have been made in the detection, diagnosis, and treatment of neoplasia over the past few decades, which have significantly improved survival rates for many types of neoplasia. However, only about 60% of those diagnosed with neoplasia survive five years after treatment begins, making neoplasia the second leading cause of death in the United States.

[0004] Currently, there are many different existing cancer treatments, including resection techniques (e.g., surgery, cryo / thermal therapy, ultrasound, radiofrequency, and radiation) and chemotherapeutic techniques (e.g., drugs, cytotoxic agents / chemotherapeutic agents, monoclonal antibodies, and different combinations thereof). Unfortunately, such therapies are frequently associated with serious risks, toxic side effects, extremely high costs, and uncertain efficacy.

[0005] There is growing interest in cancer therapies that target cancerous cells using the patient's own immune system (e.g., cancer vaccines) because such therapies can mitigate / eliminate some of the drawbacks described herein. Cancer vaccines typically consist of tumor antigens and immunostimulatory molecules (e.g., cytokines or TLR ligands) that work together to induce antigen-specific cytotoxic T cells that 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) selectively expressed or overexpressed in tumors found in many individuals. While such common tumor antigens are useful in identifying specific types of tumors, they are undesirable as immunogens for targeting T-cell responses specific to a particular tumor type because they are susceptible to self-suppressive immunosuppression. Vaccines containing both tumor-specific and patient-specific neoantigens can overcome some of the drawbacks of vaccines containing common tumor antigens.

[0006] In general, any vaccine should have a sufficiently long shelf life to ensure it does not degrade or spoil before use. Storage stability also requires that vaccine components do not precipitate from the solution during storage. However, achieving adequate storage stability can be challenging. Consequently, new formulations are needed for vaccines.

[0007] The citation or designation of any reference in this application does not imply an admission that such reference can be obtained as prior art to this invention. Invention Overview This invention relates to a tumorigenesis vaccine or immunogenic composition for treating tumor formation, and more particularly to a vaccine formulation comprising a pool of tumor-specific and patient-specific neoantigens for treating tumors in a subject.

[0008] In one aspect, the present invention provides a pharmaceutical composition comprising: at least one neoantigen peptide or a pharmaceutically acceptable salt thereof; a pH modifier; and a pharmaceutically acceptable carrier.

[0009] In some embodiments, the pharmaceutical composition is a vaccine composition.

[0010] In some embodiments, the pharmaceutical composition comprises at least two neoantigen peptides. In some embodiments, the pharmaceutical composition comprises at least three neoantigen peptides. In some embodiments, the pharmaceutical composition comprises at least four neoantigen peptides. In some embodiments, the pharmaceutical composition comprises at least five neoantigen peptides. The tumorigenesis vaccine or immunogenic composition advantageously comprises at least four different neoantigens (and different antigens are intended to refer to each antigen having a different neoepithelial site), for example, 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 neoantigens may be present in the tumorigenesis vaccine or immunogenic composition.

[0011] In some embodiments, the length of the neoantigen peptide ranges from about 5 to about 50 amino acids. In another related embodiment, the length of the neoantigen peptide ranges from about 15 to about 35 amino acids. Typically, the length is greater than about 15 or 20 amino acids, for example from 15 to 50 or about 75 amino acids.

[0012] In one embodiment, the tumor-forming vaccine or immunogenic composition further comprises a pH-modifying agent and a pharmaceutically acceptable carrier.

[0013] In some embodiments, the pH changer is a base. In some embodiments, the pH changer is a dicarboxylate or tricarboxylate. In some embodiments, the pH changer is a succinate. In some embodiments, the pH changer is a citrate.

[0014] In some embodiments, succinic acid or a pharmaceutically acceptable salt thereof includes disodium succinate.

[0015] In some embodiments, succinate is present in the formulation at a concentration from about 1 mM to about 10 mM. In some embodiments, succinate is present in the formulation at a concentration from about 2 mM to about 5 mM.

[0016] In some embodiments, the pharmaceutically acceptable carrier includes water.

[0017] In some embodiments, the pharmaceutically acceptable carrier further comprises dextrose.

[0018] In some embodiments, the pharmaceutically acceptable carrier further comprises trehalose.

[0019] In some embodiments, the pharmaceutically acceptable carrier further comprises sucrose.

[0020] In some embodiments, the pharmaceutically acceptable carrier further comprises dimethyl sulfoxide.

[0021] In some embodiments, the pharmaceutical composition further comprises an immunomodulator or adjuvant. In one embodiment, the method further comprises administering an immunomodulator or adjuvant. In another related embodiment, 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, ISCMATRIX, Juvlmmune, LipoVac, MF59, monophospholipase A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanid ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, vector system, PLGA microparticles, Remiquimod, SRL172, virions and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, and Aquila's QS21 stimulon. In another further embodiment, the immunomodulator or adjuvant is poly-ICLC.

[0022] The dissolution of these polymers in water produces an acidic solution preferably neutralized to a physiological pH, providing an adjuvant solution in which a vaccine or immunogenic composition or one or more of its antigens or carriers is incorporated. The carboxyl groups of the polymer are then partially located at COO-.

[0023] Preferably, the solution of the adjuvant according to the invention, especially the carbomer solution, is prepared in distilled water, preferably in the presence of sodium chloride, and the resulting solution is at an acidic pH. This stock solution is diluted by adding the required amount (for obtaining the desired final concentration) or a substantial portion thereof of water containing salt (e.g., NaCl), preferably physiological saline (NaCl 9 g / L), either together or in portions, with or subsequently neutralizing (pH 7.3 to 7.4), preferably with an alkali, such as NaOH. This solution at a physiological pH is used to rehydrate vaccines, especially those stored in lyophilized or freeze-dried form.

[0024] The final polymer concentration in the vaccine composition is 0.01% to 2% w / v, more specifically 0.06% to 1% w / v, and preferably 0.1% to 0.6% w / v.

[0025] In another aspect, the present invention provides a pharmaceutical composition for a tumorigenesis vaccine, comprising one to five neoantigenic peptides or pharmaceutically acceptable salts thereof; 1%-3% dimethyl sulfoxide; 3.6%-3.7% dextran aqueous solution; 3.6-3.7 mM succinic acid or a salt thereof; 0.5 mg / ml poly(I:polyC); 0.375 mg / ml poly(L-lysine); 1.25 mg / ml sodium carboxymethyl cellulose; and 0.225% sodium chloride. In some embodiments, each of the one to five neoantigenic peptides or pharmaceutically acceptable salts thereof is present at a concentration of about 300 μg / ml.

[0026] In another aspect, the present invention provides a method for preparing a neoantigen peptide solution for a tumorigenesis vaccine, the method comprising: providing a solution containing at least one neoantigen peptide or a pharmaceutically acceptable salt thereof; and combining the solution containing at least one neoantigen peptide or a pharmaceutically acceptable salt thereof with a solution containing succinic acid or a pharmaceutically acceptable salt thereof, thereby preparing a peptide solution for a tumorigenesis vaccine.

[0027] In some embodiments, the solution containing at least one neoantigen peptide or a pharmaceutically acceptable salt thereof comprises at least two (or three, four, or five) neoantigen peptides. In some embodiments, the peptide solution for the tumorigenesis vaccine comprises water, dextran or trehalose or sucrose, succinate, and dimethyl sulfoxide. In some embodiments, the method further comprises filtering the peptide solution for the tumorigenesis vaccine after the combination step.

[0028] In another aspect, the present invention provides a method for preparing a tumor-forming vaccine, the method comprising: providing a peptide solution for the tumor-forming vaccine; and combining the peptide solution with a solution of an immunomodulator or adjuvant, thereby preparing the tumor-forming vaccine.

[0029] In another aspect, the present invention provides tumorigenesis vaccines manufactured by any of the methods described herein (such as those described above).

[0030] In another aspect, the present invention provides a neoantigen peptide solution for a tumor-forming vaccine, the solution comprising: at least one neoantigen peptide or a pharmaceutically acceptable salt thereof; and succinic acid or a pharmaceutically acceptable salt thereof.

[0031] In another aspect, the present invention provides a method for treating a subject diagnosed with tumor formation, the method comprising administering a pharmaceutical composition of the present invention (e.g., the pharmaceutical composition described herein) to the subject, thereby treating the tumor formation.

[0032] In some embodiments, the method further includes administering a second pharmaceutical composition of the present invention (e.g., the pharmaceutical composition described herein) to the subject.

[0033] In some embodiments, the method further includes administering a third pharmaceutical composition of the invention (e.g., the pharmaceutical composition described herein) to the subject.

[0034] In some embodiments, the method further includes administering a fourth pharmaceutical composition of the invention (e.g., the pharmaceutical composition described herein) to the subject.

[0035] The tumor-forming vaccine or immunogenic composition can be administered on a schedule, such as weekly, bi-weekly, bi-weekly, monthly, bi-monthly, quarterly (every three months), every third of a year (every four months), every five months, twice a year (every six months), every seven months, every eight months, every nine months, every ten months, every eleven months, annually, etc.

[0036] The tumor-forming vaccine or immunogenic composition can be administered via sub-compositions, each containing a portion of a neoantigen, and these sub-compositions can be administered to different locations on the subject or patient; for example, a composition containing 20 different neoantigens can be divided into four (4) sub-compositions for administration, each sub-composition containing 5 of the 20 different neoantigens, and these four (4) sub-compositions can be administered to deliver each sub-composition at or near the patient's draining lymph nodes, for example, to each arm and each leg (e.g., the thigh or upper thigh on each side of the patient or near the buttock or lower back), to deliver each sub-composition at or near the patient's or subject's draining lymph nodes. Of course, the number of locations and thus the number of sub-compositions can vary; for example, a skilled practitioner can consider administration at or near the spleen with a fifth point, and a skilled practitioner can change the locations so that only one, two, or three of the limbs are used (e.g., each arm and one leg, each leg and one arm, each leg and no arm, or only two arms).

[0037] The vaccines or immunogenic compositions administered at the aforementioned intervals may be different formulations, and the sub-compositions administered at different sites on the subject or patient during a single administration may be different compositions. For example, a whole-antigen vaccine or immunogenic composition may be administered first, followed or later by an in vivo vector (e.g., a viral vector or plasmid) having one or more antigens expressed in vivo. Similarly, in the administration of different sub-compositions at different sites on a patient or subject, some sub-compositions may include whole antigens and some sub-compositions may include vectors (e.g., viral vectors or plasmids) having one or more antigens expressed in vivo. Furthermore, some compositions and sub-compositions may include both one or more vectors (e.g., viral vectors or plasmids) having one or more antigens expressed in vivo and whole antigens. Vectors having one or more antigens expressed in vivo (e.g., poxviruses) may have an immunostimulatory or adjuvant effect, and therefore compositions or sub-compositions containing such vectors may be adjuvants themselves. Moreover, by demonstrating the nature of how antigens are presented to the immune system, these administrations may be "priming" and then "boosting" the immune system. Furthermore, when “vaccine” is mentioned in this document, it is intended to refer to the invention comprising an immunogenic composition, and when “patient” or “subject” is mentioned, it is intended to refer to an individual who is a patient or subject who needs the treatment, administration, composition and general subject matter invention disclosed herein.

[0038] Furthermore, this invention is applicable to the use of any type of expression vector, such as viral expression vectors, such as poxviruses (e.g., orthopoxvirus or avipoxvirus, such as cowpoxvirus, including modified Ankara vaccinia vaccine or MVA, MVA-BN, NYVAC (according to WO-A-92 / 15672), fowlpox, such as TROVAX, canarypox, such as ALVAC (WO-A-95 / 27780 and WO-A-92 / 15672), pigeonpox virus, swinepox, and the like), adenoviruses, AAV herpesviruses, and lentiviruses; or plasmid or DNA or nucleic acid molecular vectors. Some vectors that are cytoplasmic, such as poxvirus vectors, will be advantageous. However, in the practice of this invention, the use of adenoviruses, AAVs, and lentiviruses will also be advantageous.

[0039] In ready-to-use, especially rehydrated, vaccines or immunogenic compositions, the vector, such as a viral vector, is present in an amount that is within the skill of a person skilled in the art, as disclosed herein and known in the art (e.g., in the patents and scientific literature cited herein).

[0040] Whole antigens or vectors, such as recombinant live vaccines, are generally in a freeze-dried form that allows them to be stored and rehydrated immediately in a solvent or excipient before use, which may include adjuvants as discussed herein.

[0041] Therefore, the subject of this invention is still a vaccination or immunization device or kit comprising separately packaged, freeze-dried vaccines and solutions, advantageously including adjuvant compounds for rehydration freeze-dried vaccines as discussed herein.

[0042] The subject of this invention is also a method of vaccination or immunization, which comprises or consists essentially of: administering, for example, via a parenteral route, preferably subcutaneous, intramuscular, or intradermal route, or via a mucosal route, at one or more administration ratios, a vaccine or immunogenic composition according to the invention. Optionally, this method includes a preparation step of rehydrating a freeze-dried vaccine or immunogenic composition (e.g., if a freeze-dried whole antigen or carrier) in a solution (advantageously also including an adjuvant).

[0043] In one embodiment, the subject has a tumor selected from the group consisting of: non-Hodgkin's lymphoma (NHL), clear cell renal cell carcinoma (ccRCC), melanoma, sarcoma, leukemia, or bladder cancer, colon cancer, brain cancer, breast cancer, head and neck cancer, endometrial cancer, lung cancer, ovarian cancer, pancreatic cancer, or prostate cancer. In another embodiment, the tumor is metastatic. In yet another embodiment, the subject does not have a detectable tumor but is at high risk of disease recurrence. In a further related embodiment, the subject has previously undergone autologous hematopoietic stem cell transplantation (AHSCT).

[0044] In one embodiment, the tumorigenesis vaccine or immunogenic composition is administered according to a primary / booster dose regimen. In another embodiment, the tumorigenesis vaccine or immunogenic composition is administered at week 1, 2, 3, or 4 as a primary immunization. In yet another embodiment, the tumorigenesis vaccine or immunogenic composition is administered at month 2, 3, 4, or 5 as a booster.

[0045] 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 neoantigenic 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 neoantigenic peptide.

[0046] In one embodiment, the vaccine or immunogenic composition is administered intravenously or subcutaneously.

[0047] In another aspect, the present invention provides a neoantigen peptide solution for a tumor-forming vaccine, the solution comprising: at least one neoantigen peptide or a pharmaceutically acceptable salt thereof; and succinic acid or a pharmaceutically acceptable salt thereof.

[0048] The present invention includes methods as described in U.S. Patent Application No. 20110293637, which is incorporated herein by reference, such methods as: identifying at least four subject-specific peptides of a plurality of species, and preparing a subject-specific immunogenic composition, which, when administered, presents the at least four subject-specific peptides of the plurality of species to the immune system of a subject, wherein the subject has a tumor, and the subject-specific peptides are specifically targeted at the subject and the subject's tumor, the methods comprising: (i) Identification is carried out through the following means, including Nucleic acid sequencing of tumor samples from the subjects and Nucleic acid sequencing of non-tumor samples from the subjects. At least four tumor-specific non-silent mutations that are not present in non-tumor samples; and (ii) Select at least four subject-specific peptides from identified non-silent mutations, each peptide having a different novel tumor epitope specifically targeting the subject's tumor, from multiple identified tumor-specific mutations. Each novel epitope is the expression product of a tumor-specific, non-silent mutation not present in non-tumor samples. Each novel epitope binds to the subject's HLA protein and is selected from those including... To determine the binding of subject-specific peptides to HLA proteins, and (iii) Formulating a subject-specific immunogenic composition for administration to a subject, such that, upon administration, at least four of the plurality of subject-specific peptides are presented to the subject's immune system. The selection or preparation includes at least one of the following: The subject-specific immunogenic composition includes a subject-specific peptide comprising the expression product of an identified novel ORF, wherein the novel ORF is a tumor-specific non-silenced mutation not present in non-tumor samples that produce the new open reading frame. The subject-specific immunogenic composition includes a subject-specific peptide comprising the expression product of an identified point mutation and having a defined binding affinity to the subject's HLA protein with an IC50 of less than 500 nM. Therefore, at least four subject-specific peptides of the plurality of peptides were identified, and a subject-specific immunogenic composition was prepared, which, when administered, presents the at least four subject-specific peptides of the plurality of peptides to the subject's immune system, wherein the subject-specific peptides specifically target the subject and the subject's tumor, or a method for identifying neoantigens, the method comprising: a. Identify tumor-specific mutations in genes expressed by subjects with cancer; b. Where the mutation identified in step (a) is a point mutation: i. Identify mutant peptides having the mutation identified in step (a), wherein the mutant peptides bind class I HLA proteins with a greater affinity than wild-type peptides; and have an IC50 of less than 500 nm; c. Wherein the mutation identified in step (a) is a splicing site, frameshift, read-through, or gene fusion mutation: i. Identifying a mutant polypeptide encoded by the mutation identified in step (a), wherein the mutant polypeptide binds to a class I HLA protein; or a method for inducing a tumor-specific immune response in a subject, the method comprising administering one or more identified peptides or polypeptides and an adjuvant; or a method for inoculating or treating a subject against cancer, the method comprising: a. Identifying multiple tumor-specific mutations in genes expressed by the subject, wherein the identified mutations are: i. In the case of a point mutation, further identification of the mutant peptide possessing that point mutation; and / or ii. When the mutation is a splice site, frameshift, read linking, or gene fusion mutation, further identify the mutant polypeptide encoded by the mutation; b. Select one or more mutant peptides or polypeptides identified in step (a) that bind to class I HLA proteins; c. Select one or more mutant peptides or polypeptides identified in step (b) that can activate anti-tumor CD8 T cells; and d. Administering the subject the one or more peptides or polypeptides, pulse-treating autologous dendritic cells or antigen-presenting cells with one or more peptides or polypeptides selected in step (c); or preparing a pharmaceutical composition comprising one or more identified peptides and performing one or more methods as discussed herein. Therefore, the tumorigenesis vaccine or immunogenic composition herein may be as described in U.S. Patent Application No. 20110293637.

[0049] Therefore, the object of this invention is to exclude any previously known products, processes for manufacturing such products, or methods of using such products, thereby reserving and hereby publicly waiving any rights to any previously known products, processes, or methods. Furthermore, within the scope of this invention, it is not intended to cover any product, process, or method of manufacturing or using such product that does not meet the written description and implementability requirements of the USPTO (35 USC §112, paragraph 1) or EPO (Section 83 of the EPC), thereby reserving and hereby publicly waiving any rights to any previously described products, processes for manufacturing such products, or methods of using such products.

[0050] It should be noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as “comprise,” “comprised,” “comprising,” etc., may have the meanings that fall under the jurisdiction of U.S. patent law; for example, they may mean “includes,” “included,” “including,” etc.; and these terms such as “consisting essentially of” and “consists essentially of” have the meanings that fall under the jurisdiction of U.S. patent law, for example, they allow elements not explicitly stated but exclude elements found in the prior art or affecting the essential or novel features of the invention.

[0051] These and other embodiments are disclosed in the following detailed description, or are obvious from thereto and are covered therein. Brief description of the attached figures This patent or application contains at least one color drawing. A copy of this patent or patent application publication with one or more color drawings shall be provided by the Office upon request and payment of the necessary fees.

[0052] The following detailed description, given by way of example, can be best understood in conjunction with the accompanying drawings, which are incorporated herein by reference, but the invention is not intended to be limited to the specific embodiments described, wherein: Figure 1 The process for manufacturing personalized cancer vaccines or immunogenic compositions is described.

[0053] Figure 2 A process flow is shown for pretreatment steps used to generate cancer vaccines or immunogenic compositions for cancer patients.

[0054] Figure 3 An exemplary embodiment of the present invention is illustrated, based on an immunization procedure using a primary-boost strategy. Multiple immunizations can occur within approximately 3 weeks prior to the primary immunization phase to maintain early, high antigen exposure during the initial phase of the immune response. The patient can then rest for eight weeks to allow memory T cells to develop, and these T cells are subsequently boosted to maintain a strong, sustained response.

[0055] Figure 4 A timeline is shown according to an exemplary aspect of the invention, indicating a primary immune endpoint.

[0056] Figure 5 A schematic diagram is shown, depicting a pool of pharmaceutically processed individual neoantigen peptides into four subgroups according to an exemplary embodiment of the present invention.

[0057] Figure 6 The results of quantitative PCR used to assess the induction levels of several key immune markers after stimulating mouse dendritic cells with a neoantigen formulation are shown.

[0058] Figure 7 MDSC analysis of 5% dextrose and 0.8% DMSO is shown.

[0059] Figure 8 MDSC analysis of 10% trehalose and 0.8% DMSO is shown.

[0060] Figure 9 MDSC analysis of 10% sucrose and 0.8% DMSO is shown.

[0061] Figure 10 An exemplary pressure curve for freeze-drying is shown.

[0062] Figure 11 An exemplary temperature profile for freeze-drying is shown.

[0063] Figure 12 The physical appearance of a freeze-dried cake using an exemplary formulation of the present invention is shown.

[0064] Detailed Description of the Invention definition To aid in understanding this invention, several terms and phrases are defined herein: Unless explicitly stated or obvious from the context, as used herein, the term "about" is understood to mean within the normal tolerance range in this field, for example, within 2 standard deviations of the mean. "About" can be understood as 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 specified value. All numerical values ​​provided herein are modified by the term "about" unless otherwise clearly apparent from the context.

[0065] Unless expressly stated or obvious from the context, the term “or” as used herein is to be understood as inclusive. Unless expressly stated or obvious from the context, the terms “a,” “an,” and “the” as used herein are to be understood as singular or plural.

[0066] The term "agent" refers to any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide or fragment thereof.

[0067] The term "ameliorate" refers to reducing, inhibiting, weakening, diminishing, blocking, or stabilizing the development or progression of a disease (e.g., tumor formation, tumors, etc.).

[0068] The term "change" means a change (increase or decrease) in the expression level or activity of a gene or polypeptide, as detected by methods known by standard techniques (such as those described herein). As used herein, a change includes a 10% change in expression level, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change.

[0069] The term "analog" refers to molecules that are not identical but have similar functions or structural features. For example, tumor-specific neoantigen peptide analogs retain the biological activity of the corresponding naturally occurring tumor-specific neoantigen peptide while possessing certain biochemical modifications that enhance the function of the analog compared to the naturally occurring peptide. Such biochemical modifications can increase its protease resistance, membrane permeability, or half-life without altering, for example, ligand binding. Analogs may include non-natural amino acids.

[0070] The term “neo-antigen” or “neo-antigenic” refers to a class of tumor antigens that arise from one or more tumor-specific mutations that alter the amino acid sequence of proteins encoded by the genome.

[0071] The term "neoplasia" refers to any disease caused by an inappropriately high level of cell division, an inappropriately low level of cell apoptosis, or both. Cancer, for example, is an instance of neoplasm. Examples of cancer include, but are not limited to, leukemia (e.g., acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (e.g., Hodgkin's disease, non-Hodgkin's disease), and Waldenstrom's disease. s) Macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, chordoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovoma, 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, etc.). Cystic adenocarcinoma, medullary carcinoma, bronchial carcinoma, renal cell carcinoma, liver cancer, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, renal blastoma, cervical cancer, uterine cancer, testicular cancer, lung cancer, small cell lung cancer, bladder cancer, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pineal tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma. Lymphoproliferative disorders are also considered proliferative diseases.

[0072] The term "tumor formation vaccine" refers to a pooled sample of neoantigens specific to tumor formation, such as at least two, three, four, five, or more neoantigen peptides. "Vaccine" should be understood to mean a composition used to generate immunity for the prevention and / or treatment of a disease (e.g., tumor formation / tumor). Accordingly, a vaccine is a pharmaceutical agent containing antigens and intended to be used in humans or animals to generate specific defensive and protective substances through vaccination. A "tumor formation vaccine composition" may include a pharmaceutically acceptable excipient, carrier, or diluent.

[0073] The term "pharmaceutical acceptable" means that a substance approved or permitted for use in animals (including humans) by a federal or state regulatory agency or listed in the United States Pharmacopeia or other recognized pharmacopoeia.

[0074] "Pharmaceutical acceptable excipients, carriers or diluents" means excipients, carriers or diluents that can be administered to a subject together with a reagent and that do not impair the pharmacological activity of the reagent and are non-toxic when administered at a dose that effectively delivers a therapeutic amount of the reagent.

[0075] As listed herein, a “pharmaceutically acceptable salt” of a pooled tumor-specific neoantigen can be an acid salt or base salt that is generally considered in the art to be suitable for contact with human or animal tissues without excessive toxicity, irritation, allergic reactions or other problems or complications. Such salts include mineral salts and organic acid salts of basic residues (such as amines) and base or organic salts of acidic residues (such as carboxylic acids). Specific pharmaceutical salts include, but are not limited to, salts of acids, such as hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, glycolic acid, fumaric acid, sulfuric acid, sulfamic acid, sulfanilic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethylsulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, pyric acid, succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodic acid, phenylacetic acid, alkanonic acids such as acetic acid, and HOOC-(CH2)n-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 from this disclosure and the knowledge of the art that other pharmaceutically acceptable salts of the pooled tumor-specific neoantigens provided herein include those listed in Remington's Pharmaceutical Sciences, 17th edition, Mack Publishing Company, Easton, PA, p. 1418 (1985). Generally, pharmaceutically acceptable acid or base salts can be synthesized by any conventional chemical method from a parent compound containing a basic or acidic moiety. In short, such salts can be prepared by reacting the free acidic or basic form of these compounds with a stoichiometric amount of an appropriate base or acid in a suitable solvent.

[0076] The term "polypeptide" or "peptide" refers to a polypeptide that has been isolated from its naturally occurring components. Typically, when the polypeptide comprises at least 60% by weight, it is isolated from its naturally associated proteins and naturally occurring organic molecules. Preferably, the formulation comprises at least 75% by weight, more preferably at least 90%, and most preferably at least 99% polypeptide by weight. The isolated polypeptide can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide, or by chemical synthesis of the protein. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or analysis by HPLC.

[0077] As used herein, the terms “prevent,” “preventing,” “prevention,” “prophylactic treatment,” etc., refer to reducing the likelihood that a subject who does not have a disease or condition but is at risk of developing or is susceptible to a disease or condition will develop one.

[0078] The terms "primer / booster" or "primer / booster dosage regimen" refer to the continuous administration of a vaccine or immunogenic or immunological composition. A primary immunization (primer) is the administration of a first type of vaccine or immunogenic or immunological composition and may include one, two, or more administrations. A booster immunization is a second administration of a type of vaccine or immunogenic or immunological composition and may include one, two, or more administrations, and may, for example, include or consist substantially of an annual administration. In some embodiments, the administration of a tumorigenesis vaccine or immunogenic composition is performed according to a primary / booster dosage regimen.

[0079] The ranges provided herein are to be understood as abbreviations of all values ​​within that range. For example, the range 1 to 50 is to be understood as including any number, combination of numbers, or subrange from the following group, which consists 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, and all intermediate decimal values ​​between the integers mentioned above, such as 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, and 1.9. Regarding subranges, “nested subranges” extending from any endpoint of the range are specifically considered. For example, nested subranges of the exemplary range of 1 to 50 may include 1 to 10, 1 to 20, 1 to 30, and 1 to 40 in one direction, or 50 to 40, 50 to 30, 50 to 20, and 50 to 10 in another direction.

[0080] "Receptor" should be understood to mean a biomolecule or group of molecules capable of binding a ligand. Receptors can be used to transmit information within cells, cell formation, or organisms. A receptor comprises at least one receptor unit and often contains two or more receptor units, each of which may be composed of a protein molecule, particularly a glycoprotein molecule. Receptors have a structure complementary to that of a ligand and can complex the ligand into a binding partner. Signal transduction information can be transmitted through conformational changes in the receptor following binding to a ligand on the cell surface. According to the invention, a receptor can refer to specific MHC class I and II proteins capable of forming receptor / ligand complexes with a ligand, particularly peptides or peptide fragments of suitable length.

[0081] "Receptor / ligand complex" should also be understood to mean "receptor / peptide complex" or "receptor / peptide fragment complex", especially class I or class II MHC molecules that present peptides or peptide fragments.

[0082] The term "reduce" refers to a negative change of at least 10%, 25%, 50%, 75%, or 100%.

[0083] The term "reference" refers to a standard or benchmark.

[0084] A “reference sequence” is a defined sequence used as the basis for sequence comparison. The reference sequence can be a subset or the whole of a specified sequence; for example, a segment of a full-length cDNA or genome sequence, or a complete cDNA or genome sequence. For peptides, the reference peptide sequence is typically at least about 10-2,000, 10-1,500, 10-1,000, 10-500, or 10-100 amino acids in length. Preferably, the reference peptide sequence is at least about 10-50 amino acids in length, more preferably at least about 10-40 amino acids, and even more preferably about 10-30 amino acids, about 10-20 amino acids, about 15-25 amino acids, or about 20 amino acids. For nucleic acids, the reference nucleic acid sequence is typically at least about 50 nucleotides in length, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides, or any integer between or near these.

[0085] The term "specific binding" refers to a compound or antibody recognizing and binding to a polypeptide, but not necessarily recognizing and binding to other molecules in a sample (e.g., a biological sample).

[0086] The nucleic acid molecules useful in the methods of this invention include any nucleic acid molecule encoding the polypeptide or a fragment thereof of the invention. Such nucleic acid molecules do not need to be 100% identical to an endogenous nucleic acid sequence, but will typically exhibit substantially similarity. Polynucleotides having “substantially similarity” to an endogenous sequence are typically capable of hybridizing with at least one strand of a double-stranded nucleic acid molecule. “Hybridization” means pairing under different stringent conditions to form a double-stranded molecule between complementary polynucleotide sequences (e.g., the gene described herein) or portions thereof. (See, for example, Wahl, GM and SL Berger (1987), Methods Enzymol. 152:399; Kimmel, AR (1987), Methods Enzymol. 152:507).

[0087] For example, the stringent salt concentration will typically be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringent hybridization can be obtained in the absence of organic solvents (e.g., formamide), while high stringent hybridization can be obtained in the presence of at least about 35% formamide, and more preferably at least about 50% formamide. The stringent temperature conditions will typically include at least about 30°C, more preferably at least about 37°C, and most preferably at least about 42°C. Different additional parameters, such as hybridization time, the concentration of the cleaning agent (e.g., sodium dodecyl sulfate (SDS)), and the inclusion or exclusion of the vector DNA, are well known to those skilled in the art. Different levels of stringency can be achieved by combining these different conditions as needed. In a preferred embodiment, hybridization will occur at 30°C in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur at 37°C in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA). In a most preferred embodiment, hybridization will occur at 42°C in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA. Useful variations of these conditions should readily be apparent to those skilled in the art.

[0088] For most applications, the washing steps following hybridization will also vary in terms of stringency. The stringency of the washing can be defined by salt concentration and temperature. As mentioned above, the stringency of the washing can be increased by decreasing the salt concentration or by increasing the temperature. For example, the stringent salt concentration for the washing step will preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. The stringent temperature conditions for the washing step will generally include at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In a preferred embodiment, the washing step will occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step will occur at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the washing step will occur at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Further variations of these conditions will be readily apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and described, for example, by Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proceedings of the National Academy of Sciences of the United States of America 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York).

[0089] The term "subject" refers to an animal used as a subject of treatment, observation, or experimentation. By way of example only, subjects include, but are not limited to, mammals, including, but not limited to, humans or non-human mammals such as non-human primates, cattle, horses, dogs, sheep, or cats.

[0090] The term "substantially identical" means that a polypeptide or nucleic acid molecule exhibits at least 50% similarity to a reference amino acid sequence (e.g., any amino acid sequence described herein) or nucleic acid sequence (e.g., any nucleic acid sequence described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and even more preferably 90%, 95%, or even 99% similar to the sequence used for comparison at the amino acid level or at the nucleic acid level.

[0091] Sequence identity is typically measured using sequence analysis software, such as the sequence analysis package from the Genetic Computation Group at the University of Wisconsin-Madison Biotechnology Center (1710 University Avenue, Madison, Wisconsin 53705), including programs like BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX. This software matches identical or similar sequences by assigning values ​​to the degree of homology of different substitutions, deletions, and / or other modifications. Conserved substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In one exemplary method for determining the degree of identity, the BLAST program can be used, where probability scores between e-3 and e-100 indicate closely related sequences.

[0092] "T cell epitope" should be understood as meaning a peptide sequence that can be bound by class I or II MHC molecules in the form of a peptide-presenting MHC molecule or MHC complex, and subsequently recognized and bound in this form by naive T cells, cytotoxic T lymphocytes, or T helper cells.

[0093] The term "treatment" (treat, treated, treating, etc.) refers to reducing or improving a disorder and / or symptoms associated with it (e.g., tumor formation or tumor). "Treatment" includes the concept of "relief," which refers to reducing the frequency or severity of the occurrence or recurrence of any symptoms or other adverse effects related to cancer and / or cancer treatment. The term "treatment" also encompasses the concept of "management," which refers to reducing the severity of a specific disease or disorder in a patient, or delaying its recurrence, such as prolonging remission periods for patients with the disease. It should be understood that, although not excluded, treating a disorder or condition does not require the complete elimination of the disorder, condition, or associated symptoms.

[0094] The term "therapeutic effect" refers to the reduction to a certain extent of one or more symptoms of an impairment (e.g., tumor formation or tumor) or its associated lesions. As used herein, "therapeutic effective amount" refers to the amount of agent that, when administered to cells or a subject in a single or multiple doses, is effective in prolonging the survival of a patient with such an impairment, reducing one or more signs or symptoms of the impairment, preventing or delaying, and exceeding the expected outcome without such treatment. "Therapeutic effective amount" is intended to define the amount required to achieve a therapeutic effect. A physician or veterinarian with ordinary skill in the art can readily determine and prescribe the desired "therapeutic effective amount" (e.g., ED50) of a pharmaceutical composition. For example, a physician or veterinarian may begin by administering the composition of the present invention used in a pharmaceutical composition at a level below that required to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved.

[0095] These pharmaceutical compositions should typically be provided at a dose ranging from about 0.0001 mg to about 200 mg of compound per kilogram of body weight per day. For example, the systemic dosage range for human patients can be 0.01-10 µg / kg, 20-80 µg / kg, 5-50 µg / kg, 75-150 µg / kg, 100-500 µg / kg, 250-750 µg / kg, 500-1000 µg / kg, 1-10 mg / kg, 5-50 mg / kg, 25-75 mg / kg, 50-100 mg / kg, 100-250 mg / kg, 50-100 mg / kg, 250-500 mg / kg, 500-750 mg / kg, 750-1000 mg / kg, 1000-1500 mg / kg, 1500-2000 mg / kg, 5 mg / kg, 20 mg / kg, 50 mg / kg, 100 mg / kg, or 200 mg / kg. Prepare pharmaceutical unit dosage forms to provide combinations of compounds or essential ingredients in quantities ranging from about 0.001 mg to about 5000 mg (e.g., from about 100 mg to about 2500 mg) per unit dosage form.

[0096] "Vaccine" should be understood to mean a composition used to generate immunity for the prevention and / or treatment of a disease (e.g., tumor formation / tumor). Accordingly, a vaccine is a pharmaceutical agent containing an antigen and intended to be used in humans or animals to generate specific defensive and protective substances through inoculation.

[0097] In any definition of the variables herein, the statement of the list of chemical groups includes the definition of the variable as any single group or a combination of the listed groups. Statements of embodiments of the variables or aspects herein include the embodiment as any single embodiment or a combination with any other embodiment or part thereof.

[0098] Any composition or method provided herein may be combined with one or more other compositions and methods provided herein.

[0099] This invention relates to vaccines and methods for treating tumor formation by administering a therapeutically effective amount of a pharmaceutical composition (e.g., a cancer vaccine) to a subject (e.g., a mammal, such as a human), and more specifically, vaccines and methods for treating tumors, the pharmaceutical composition comprising a plurality of tumor-forming / tumor-specific neoantigens. As described in more detail herein, whole-genome / exome sequencing can be used to identify all or almost all mutated neoantigens uniquely present in the tumor formation / tumor of an individual patient, and the collection of these mutated neoantigens can be analyzed to identify optimized neoantigen subpopulations specific to be used as personalized cancer vaccines or immunogenic compositions for treating the tumor formation / tumor of that patient. For example, tumor-specific mutations can be identified by sequencing both the tumor formation / tumor and normal DNA of each patient, thereby identifying the tumor formation / tumor-specific neoantigen population, and the patient's HLA allotype can be identified. The tumor formation / tumor-specific neoantigen population and its homologous natural antigens can then be subjected to bioinformatics analysis using validated algorithms to predict which tumor-specific mutation produces an epitope that can bind to the patient's HLA allotype. Based on this analysis, multiple peptides corresponding to these mutation subsets can be designed and synthesized for each patient and pooled together for use as cancer vaccines or immunogenic compositions in immunization of that patient. These neoantigen peptides can be combined with adjuvants (e.g., poly-ICLC) or another antitumor agent. Unbound by theory, these neoantigens are expected to circumvent central thymic tolerance (thus allowing for a stronger antitumor T-cell response) while reducing the likelihood of autoimmunity (e.g., by avoiding targeting normal autoantigens).

[0100] The immune system can be divided into two functional subsystems: the innate and the adaptive immune system. The innate immune system is the first line of defense against infection, and most potential pathogens are rapidly neutralized by this system before they can cause, for example, significant infection. The adaptive immune system reacts with molecular structures called antigens that invade an organism. There are two types of adaptive immune responses: humoral immune responses and cell-mediated immune responses. In humoral immune responses, antibodies secreted into the body fluids by B cells bind to pathogen-derived antigens, leading to the elimination of the pathogen through various mechanisms, such as complement-mediated cleavage. In cell-mediated immune responses, T cells capable of destroying other cells are activated. For example, if disease-associated proteins are present in a cell, they are proteasically cleaved into peptides within that cell. Specific cellular proteins then attach themselves to the antigens or peptides formed in this way and transport them to the cell surface, where they are presented to the body's molecular defense mechanisms, particularly T cells. Cytotoxic T cells recognize these antigens and kill cells containing them.

[0101] The molecules that transport and present peptides on the cell surface are called proteins of the major histocompatibility complex (MHC). MHC proteins are divided into two types, called MHC class I and MHC class II. The proteins of the two MHC classes are structurally 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 most tumor cells. MHC class I proteins load antigens, which are usually derived from endogenous proteins or from pathogens present within the cell, and are subsequently presented to naïve or cytotoxic T lymphocytes (CTLs). MHC class II proteins are present on dendritic cells, B lymphocytes, macrophages, and other antigen-presenting cells. They primarily present peptides processed from in vitro antigen sources (i.e., extracellularly) to T helper (Th) cells. Most peptides bound by MHC class I proteins originate from cytoplasmic proteins produced in the organism's own healthy host and generally do not stimulate an immune response. Accordingly, cytotoxic T lymphocytes that recognize class I MHC molecules that present their own peptides are absent in the thymus (central tolerance) or, after release from the thymus, are absent or inactivated, i.e., tolerated (peripheral tolerance). MHC molecules can stimulate an immune response when they present peptides to non-tolerant T lymphocytes. Cytotoxic T lymphocytes possess both T cell receptors (TCRs) and CD8 molecules on their surface. T cell receptors can recognize and bind peptides complexed with class I MHC molecules. Each cytotoxic T lymphocyte expresses a unique T cell receptor that can bind to a specific MHC / peptide complex.

[0102] Before being presented on the cell surface, peptide antigens attach themselves to MHC class I molecules via competitive affinity binding within the endoplasmic reticulum. Here, the affinity of a single peptide antigen is directly related to its amino acid sequence and the presence of a specific binding motif at a defined position within that amino acid sequence. If the sequence of such a peptide is known, the immune system can be manipulated to fight diseased cells, for example, using peptide vaccines.

[0103] One of the key obstacles to developing curative and tumor-specific immunotherapies is the identification and selection of highly specific and restrictive tumor antigens to avoid autoimmunity. Tumor neoantigens, which arise as a result of genetic changes within malignant cells (e.g., inversions, translocations, deletions, missense mutations, splice site mutations, etc.), represent the most tumor-specific classes of antigens. Neoantigens are rarely used in cancer vaccines or immunogenic compositions due to technical challenges in identifying them, selecting optimized neoantigens, and generating neoantigens for use in vaccines or immunogenic compositions. These challenges can be addressed through the following: • Identify all or almost all mutations in tumor formation / tumor at the DNA level using whole-genome, whole-exome (e.g., captured exons only) or RNA sequencing of tumors compared to matched germline samples from each patient; • These identified mutations were analyzed using one or more peptide-MHC binding prediction algorithms to generate multiple candidate neoantigen T-cell epitopes that are expressed within the tumor and can bind to the patient's HLA alleles; and • Synthesize these multiple candidate neoantigen peptides, selected from all novel ORF peptides in these collections and predicted binding peptides for use in cancer vaccines or immunogenic compositions.

[0104] For example, converting sequencing information into therapeutic vaccines can include: (1) Predicting personalized mutant peptides that can bind to an individual's HLA molecules. Effective selection of which specific mutations to use as immunogens requires identifying a patient's HLA type and predicting which mutant peptides will effectively bind to that patient's HLA alleles. Recently, neural network-based learning methods using validated binding and non-binding peptides have improved the accuracy of prediction algorithms targeting major HLA-A and -B alleles.

[0105] (2) Formulating drugs as multi-epitope vaccines using long peptides. Practically targeting as many mutant epitopes as possible leverages the immense power of the immune system, preventing immune escape through downregulation of specific immune-targeting gene products and compensating for known inaccuracies in epitope prediction methods. Synthetic peptides provide a particularly useful means for the efficient preparation of multiple immunogens and the rapid conversion of mutant epitope identification into effective vaccines. Peptides can be readily synthesized chemically and easily purified using reagents free from contaminating bacteria or animal matter. Small size allows for precise targeting of mutant regions of the protein and also reduces competition from irrelevant antigens from other components (unmutated proteins or viral vector antigens).

[0106] (3) Combination with strong vaccine adjuvants. Effective vaccines require strong adjuvants to initiate an immune response. As described below, poly-ICLCs have shown several promising properties as vaccine adjuvants, acting as agonists of the TLR3 and RNA helicase-MDA5 and RIG3 domains. These properties include inducing local and systemic activation of immune cells in vivo, producing stimulating chemokines and cytokines, and stimulating antigen presentation via dendritic cells. Furthermore, poly-ICLCs can induce durable CD4+ and CD8+ responses in humans. Importantly, striking similarities were observed in the upregulation of transcriptional and signal transduction pathways in subjects vaccinated with poly-ICLCs and in volunteers who had received a highly effective, replicating yellow fever vaccine. In addition, in a recent phase 1 study, >90% of ovarian cancer patients immunized with poly-ICLCs in combination with NY-ESO-1 peptide vaccines (excluding Montaned) showed induction of CD4+ and CD8+ T cells, as well as antibody responses to the peptide. Furthermore, poly-ICLC has been extensively tested in over 25 clinical trials to date and has demonstrated relatively benign toxicity characteristics. The advantages of this invention are further described herein.

[0107] As described here, there is substantial evidence in both animals and humans that mutated epitopes are effective in inducing immune responses, and cases of spontaneous tumor regression or long-term survival are associated with CD8+ T cell responses against mutated epitopes (Buckwalter and Srivastava PK, “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. 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. Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci US A).)102:16013(2005)) and "immunoediting" can be traced back to changes in the expression of dominant mutant antigens in mice and humans (Matsushita et al. Cancer exome analysis reveals 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 patients with newly diagnosed glioblastoma Journal of Clinical Oncology (J Clin Oncol. 28:4722-4729 (2010). In one embodiment, mutated epitopes in cancer patients are identified.

[0108] In one embodiment, mutated epitopes are identified by sequencing the genome and / or exons of tumor tissue and healthy tissue from cancer patients using next-generation sequencing technology. In another embodiment, next-generation sequencing technology is used to sequence genes selected based on mutation frequency and their ability to act as neoantigens.Next-generation sequencing is applicable to genome sequencing, genome resequencing, transcriptome profiling (RNA-Seq), DNA-protein interaction sequencing (ChIP-sequencing), and genome characterization (de Magalhães 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." Journal of Experimental Biology 209 (Pt 9): 1518-1525; Church Gm (January 2006). "Targeting all genomes (Genomes) “For all”, Scientific American (Sci. Am.) 294 (1): 46-54; Ten Bosch Jr, Grody WW (2008) “Keeping Up with the Next Generation”. The Journal of Molecular Diagnostics 10 (6): 484-492; Tucker T, Marra M, Friedman JM (2009) “Massively Parallel Sequencing: The Next Big Thing in Genetic Medicine”. The American Journal of Human Genetics 85 (2): 142-154.Next-generation sequencing can now rapidly reveal the presence of discrete mutations, such as coding mutations in a single tumor, most commonly single-amino acid alterations (e.g., missense mutations) and infrequently occurring novel amino acid segments (e.g., novel ORFs) resulting from frameshift insertions / deletions / gene fusions in stop codons, read linking mutations, and translation of inappropriately spliced ​​introns. Novel ORFs are particularly valuable as immunogens because their sequences as a whole are completely novel to the immune system and thus resemble viral or bacterial exogenous antigens. Therefore, novel ORFs: (1) are highly specific to tumors (i.e., not expressed in any normal cells); and (2) can bypass central tolerance, thereby increasing the precursor frequency of neoantigen-specific CTLs. For example, the efficacy of utilizing similar exogenous sequences in therapeutic anticancer vaccines or immunogenic compositions has recently been demonstrated with peptides derived from human papillomavirus (HPV). Approximately 50% of 19 patients with pretumoral viral-induced disease who received 3–4 doses of a mixture of HPV peptides derived from viral oncogenes E6 and E7 maintained a complete response for ≥ 24 months (Kenter et al., Vaccination against HPV-16 Oncoproteins for Vulvar Intraepithelial Neoplasia NEJM 361:1838 (2009)).

[0109] Sequencing technology has revealed that each tumor contains multiple patient-specific mutations that alter the protein-coding content of genes. These mutations produce altered proteins ranging from single-amino acid changes (caused by missense mutations) to novel amino acid sequences that add long regions (novel open reading frame mutations; new ORFs) attributable to frameshifts, read linking, or translation of intron regions of stop codons. These mutant proteins are valuable targets for the host's immune response to the tumor because, unlike natural proteins, they are not affected by the host's own immunosuppressive effects. Therefore, mutant proteins are more likely to be immunogenic and more specific to tumor cells than to the patient's normal cells.

[0110] An alternative method for identifying tumor-specific neoantigens is direct protein sequencing. Protein sequencing of enzyme digests using multidimensional MS technologies (MSn) (including tandem mass spectrometry (MS / MS)) can also be used to identify the neoantigens of this invention. Such proteomics methods allow for rapid, highly automated analysis (see, for example, K. Gevaert and J. Vandekerckhove, Electrophoresis 21: 1145-1154 (2000)). Further within the scope of this invention, high-throughput methods for re-sequencing unknown proteins can be used to analyze the proteome of a patient's tumor to identify expressed neoantigens. For example, meta-shotgun protein sequencing can be used to identify expressed neoantigens (see, for example, Guthals et al. (2012) Shotgun Protein Sequencing with Meta-contig Assembly, Molecular and Cellular Proteomics 11(10): 1084-96).

[0111] MHC multimers can also be used to identify tumor-specific neoantigens in order to identify neoantigen-specific T cell responses. For example, high-throughput analysis of neoantigen-specific T-cell responses in patient samples can be performed using MHC tetramer-based screening techniques (see, for example, Hombrink et al. (2011), High-Throughput Identification of Potential Minor Histocompatibility Antigens by MHC Tetramer-Based Screening: Feasibility and Limitations, 6(8): 1-11; Hadrup et al. (2009), Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers, Nature Methods, 6(7): 520-26; van Rooij et al. (2013), Tumor exome analysis reveals neoantigen-specific T-cell reactivity in iplimma-reactive melanoma). Analysis reveals neoantigen-specific T-cell reactivity in anIpilimumab-responsive melanoma (Journal of Clinical Oncology, 31: 1-4); and Heemskerk et al. (2013), The cancer antigenome (EMBO), 32(2): 194-203). Such tetramer-based screening techniques can be used for initial identification of tumor-specific neoantigens, or alternatively as a secondary screening protocol to assess which neoantigens a patient may have been exposed to, thereby aiding in the selection of candidate neoantigens for this invention.

[0112] In one embodiment, sequencing data derived from determining the presence of mutations in cancer patients is analyzed to predict personalized mutant peptides that bind to an individual's HLA molecules. In one embodiment, computer analysis is used to analyze the data. In another embodiment, sequence data is analyzed for the presence of neoantigens. In one embodiment, neoantigens are identified by their affinity for MHC molecules. Effective selection of which specific mutations to utilize as immunogens requires the ability to identify a patient's HLA type and predict which mutant peptides will effectively bind to that patient's HLA alleles. Recently, neural network-based learning methods using validated binding and non-binding peptides have improved the accuracy of prediction algorithms for major HLA-A and -B alleles. Using recently improved algorithms for predicting which missense mutations result in strong-binding peptides of homologous MHC molecules in patients, a set of peptides representing the best mutant epitopes (both novel ORFs and mistranslations) for each patient can be identified and prioritized (Zhang et al. Machine learning competition in immunology - Prediction of HLA class I binding peptides, J Immunol Methods 374: 1 (2011); Lundegaard et al. Prediction of epitopes using neural network based methods, J Immunol Methods 374: 26 (2011)).

[0113] Practically targeting as many mutant epitopes as possible leverages the immense power of the immune system, preventing immune escape through the downregulation of specific immune-targeting gene products and compensating for known inaccuracies in epitope prediction methods. Synthetic peptides offer a particularly useful means of efficiently preparing multiple immunogens and rapidly converting the identification of mutant epitopes into effective vaccines or immunogenic compositions. Peptides can be readily synthesized chemically and easily purified using reagents free from contaminating bacteria or animal matter. Their small size allows for precise targeting of mutant regions of the protein and also reduces competition from irrelevant antigens from other components (unmutated proteins or viral vector antigens).

[0114] In one embodiment, the pharmaceutical formulation is a long peptide multi-epitope vaccine or immunogenic composition. Such “long” peptides undergo efficient internalization, processing, and cross-presentation in professional antigen-presenting cells (such as dendritic cells) and have been shown to induce CTLs in humans (Melief and van der Burg, Immunotherapy of established (pre)malignant disease by synthetic long peptide vaccines, Nature RevCancer 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 length of the neoantigen peptide ranges from about 5 to about 50 amino acids. In another embodiment, a peptide with a length of about 15 to about 35 amino acids is synthesized. In a preferred embodiment, the length of the neoantigen peptide ranges from about 20 to about 35 amino acids.

[0115] Production of tumor-specific neoantigens This invention is based, at least in part, on the ability to provide a pool of tumor-specific neoantigens to a patient's immune system. Those skilled in the art will recognize from this disclosure and the knowledge of the art that there are various ways to generate such tumor-specific neoantigens. Typically, such tumor-specific neoantigens can be generated in vitro or in vivo. Tumor-specific neoantigens can be generated in vitro as peptides or polypeptides, which can then be formulated into personalized tumor-forming vaccines or immunogenic compositions and administered to a subject. As further detailed herein, such in vitro generation can occur by a variety of methods known to those skilled in the art, such as, for example, synthesizing peptides or expressing peptides / polypeptides from DNA or RNA molecules in any of a variety of bacterial, eukaryotic, or viral recombinant expression systems, followed by purification of the expressed peptides / polypeptides. Alternatively, tumor-specific neoantigens can be generated in vivo by introducing a molecule encoding a tumor-specific neoantigen (e.g., DNA, RNA, viral expression systems, etc.) into a subject, expressing the encoded tumor-specific neoantigen in that subject's body. The methods for producing neoantigens in vitro and in vivo are further described herein as relating to pharmaceutical compositions and delivery methods.

[0116] In vitro peptide / peptide synthesis Proteins or peptides can be prepared using any technique known to those skilled in the art, including expressing proteins, polypeptides, or peptides using standard molecular biotechnology, isolating proteins or peptides from natural sources, through in vitro translation, or through chemical synthesis. Nucleotide and protein, polypeptide, and peptide sequences corresponding to various genes have previously been disclosed and can be found in computerized databases known to those skilled in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information, located on the website of the National Institutes of Health. The coding regions of known genes can be amplified and / or expressed using techniques disclosed herein or that should be known to those skilled in the art. Alternatively, various commercial formulations of proteins, polypeptides, and peptides are known to those skilled in the art.

[0117] Peptides can be readily synthesized chemically using reagents free from contaminating bacteria or animal matter (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of the American Chemical Society (J. Am. Chem. Soc.) 85: 2149-54, 1963). In some embodiments, neoantigen peptides are prepared by the following steps: (1) parallel solid-phase synthesis on a multichannel instrument using uniform synthesis and cleavage conditions; (2) purification on an RP-HPLC column using column stripping; and rewashing, but without substitution between peptides; followed by (3) analysis using a limited set of assays with the highest information content. A standard production specification (GMP) footprint can be defined around a set of peptides for individual patients, thus requiring only a set of conversion procedures between the synthesis of peptides for different patients.

[0118] Alternatively, a nucleic acid (e.g., a polynucleotide) encoding the neoantigenic peptide of the present invention can be used to produce the neoantigenic peptide in vitro. The polynucleotide can be, for example, single-stranded and / or double-stranded DNA, cDNA, PNA, CNA, RNA, or a natural or stable form of a polynucleotide (such as a polynucleotide having a phosphate-thiocyanate backbone) or a combination thereof, and it may or may not contain introns, as long as it encodes the peptide. In one embodiment, in vitro translation is used to produce the peptide. Many exemplary systems exist that can be utilized by those skilled in the art (e.g., the Retic Lysis IVT Kit, Life Technologies, Waltham, MA).

[0119] Expression vectors capable of expressing peptides have also been prepared. Expression vectors for different cell types are well known in the art and can be selected without excessive experimentation. Typically, DNA is inserted into an expression vector (such as a plasmid) with appropriate orientation and within the correct reading frame for expression. If necessary, the DNA can be ligated to appropriate transcriptional and translational regulatory nucleotide sequences that are recognized by the desired host (e.g., bacteria), although such controls are usually available in the expression vector. The vector is then introduced into the host bacteria for cloning using standard techniques (see, for example, Sambrook et al. (1989), Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, New York).

[0120] Expression vectors containing isolated polynucleotides, along with host cells containing the expression vectors, are also considered. Neoantigen peptides can be provided in the form of RNA or cDNA molecules encoding the desired neoantigen peptide. One or more neoantigen peptides of the present invention can be encoded by a single expression vector.

[0121] The term "polynucleotide encoding a polypeptide" encompasses polynucleotides that include only the coding sequence for the polypeptide, as well as polynucleotides that include additional coding and / or non-coding sequences. Polynucleotides can be in RNA or DNA form. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single-stranded, can be a coding strand or a non-coding (antisense) strand.

[0122] In embodiments, these polynucleotides may include coding sequences for tumor-specific neoantigen peptides fused to a polynucleotide within the same reading frame, such as an aid to the expression and / or secretion of a polypeptide by a host cell (e.g., acting as a leader sequence for controlling the transport of the polypeptide from the cell). The polypeptide having the leader sequence is a pre-protein and the leader sequence can be cleaved by the host cell to form the mature form of the polypeptide.

[0123] In embodiments, these polynucleotides may include a coding sequence for a tumor-specific neoantigen peptide fused to a marker sequence within the same reading frame. This marker sequence, for example, allows for the purification of the encoded polypeptide, which can then be incorporated into a personalized tumorigenesis vaccine or immunogenic composition. For instance, in the case of a bacterial host, the marker sequence may be a hexahistine tag provided by the pQE-9 vector to prepare for the purification of the mature polypeptide fused to the marker, or when using a mammalian host (e.g., COS-7 cells), the marker sequence may be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein. Other 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 tags (e.g., green fluorescent protein tag), maltose-binding protein tag, Nus tag, Strep- tag, thioredoxin tag, TC tag, Ty tag, etc.

[0124] In embodiments, these polynucleotides may include coding sequences for one or more of these tumor-specific neoantigen peptides, the coding sequences being concatenated in the same reading frame to produce a single concatamerized neoantigen peptide construct capable of generating multiple neoantigen peptides.

[0125] In some embodiments, isolated nucleic acid molecules may be provided having a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 96%, 97%, 98%, or 99% identical to the polynucleotide encoding the tumor-specific neoantigen peptide of the present invention.

[0126] A polynucleotide having at least, for example, 95% "identical" nucleotide sequence to a reference nucleotide sequence means that the polynucleotide's nucleotide sequence is identical to the reference sequence, except that the polynucleotide sequence may include up to five point mutations per 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having at least 95% identical nucleotide sequence to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. These mutations in the reference sequence may occur at the amino-terminal or carboxyl-terminal position of the reference nucleotide sequence, or at any position between those positions, and may be either scattered individually among the nucleotides in the reference sequence or scattered in one or more consecutive groups within the reference sequence.

[0127] As a practical problem, known computer programs can be used to routinely determine whether any specific nucleic acid molecule is at least 80%, at least 85%, at least 90%, and in some embodiments, at least 95%, 96%, 97%, 98%, or 99% identical to a reference sequence. These computer programs are such as the Bestfit program (Wisconsin Sequence Analysis Package, Unix-based version 8, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wisconsin 53711). Bestfit uses a local homology algorithm (Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981)) to find the optimal homology region between two sequences. When using Bestfit or any other sequence alignment program to determine whether a particular sequence is 95% identical to a reference sequence according to the invention, these parameters are set such that the percentage of similarity is calculated over the full length of the reference nucleotide sequence and allows for homology vacancies of up to 5% of the total number of nucleotides in the reference sequence.

[0128] The isolated tumor-specific neoantigen peptides described herein can be generated in vitro (e.g., in a laboratory) by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing DNA sequences encoding isolated polypeptide sequences and expressing those sequences in a suitable transforming host. In some embodiments, the DNA sequence is constructed by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest using recombinant techniques. Optionally, the sequence can be mutagenized by site-specific mutagenesis to provide its functional analogues. See, for example, Zoeller et al., Proc. Nat'l. Acad. Sci. USA 81: 5662-5066 (1984) and U.S. Patent No. 4,588,585.

[0129] In this embodiment, an oligonucleotide synthesizer is used to construct DNA sequences encoding polypeptides of interest through chemical synthesis. Such oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and by selecting codons preferred in the host cell that produces the recombinant polypeptide of interest. Standard methods can be used to synthesize isolated polynucleotide sequences encoding isolated polypeptides of interest. For example, complete amino acid sequences can be used to construct back-translated genes. Furthermore, 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 together. Individual oligonucleotides typically contain 5' or 3' overhangs for complementary components.

[0130] Once assembled (e.g., by synthesis, site-directed mutagenesis, or another method), the polynucleotide sequence encoding the specific isolated polypeptide of interest is inserted into the expression vector and optionally operably ligated to an expression control sequence adapted to express the protein in a desired host. Appropriate assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and expression of the bioactive polypeptide in a suitable host. As is well known in the art, to obtain high expression levels of the transfected gene in a host, the gene can be operably ligated to transcriptional and translational expression control sequences that are functional in the selected expression host.

[0131] Recombinant expression vectors can be used to amplify and express DNA encoding tumor-specific neoantigen peptides. Recombinant expression vectors are reproducible DNA constructs containing synthetic or cDNA-derived DNA fragments encoding tumor-specific neoantigen peptides or bioequivalent analogs, operatively linked to suitable transcriptional or translational regulatory elements derived from mammalian, microbial, viral, or insect genes. Transcriptional units typically comprise a set of: (1) one or more genetic elements that play a regulatory role in gene expression, such as transcription promoters or enhancers; (2) a structural or coding sequence transcribed into mRNA and translated into protein; and (3) appropriate transcriptional and translational initiation and termination sequences, as detailed herein. Such regulatory elements may include operon sequences for controlling transcription. An origin of replication, which typically confers the ability to replicate in the host, and a selection gene that helps identify the transformant may also be incorporated. DNA regions are operatively linked when they are functionally related to each other. For example, the DNA of a signal peptide (secretory precursor) can be operatively linked to the DNA of a polypeptide if it is expressed as a precursor involved in the secretion of the polypeptide; a promoter can be operatively linked to a coding sequence if it controls the transcription of that sequence; or a ribosome-binding site can be operatively linked to a coding sequence if it is positionally permitted for translation. Generally, operatively linked means sequential, and in the case of a secretory precursor, means sequential and within the reading frame. Structural elements intended for use in yeast expression systems include a precursor sequence that allows the host cell to secrete the translated protein extracellularly. Alternatively, in the absence of a precursor or transport sequence for expressing recombinant proteins, it may include an N-terminal methionine residue. This residue may then optionally be cleaved from the expressed recombinant protein to provide the final product.

[0132] Useful expression vectors for eukaryotic hosts, especially mammals or humans, 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, such as plasmids from Escherichia coli (including pCR1, pBR322, pMB9, and their derivatives), broader host-range plasmids such as M13, and filamentous single-stranded DNA bacteriophages.

[0133] Suitable host cells for expressing peptides include prokaryotes, yeast, insects, or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include Gram-negative or Gram-positive organisms such as *Escherichia coli* or *Bacillus*. Higher eukaryotic cells include established mammalian-derived cell lines. Cell-free translation systems can also be used. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are well known in the art (see Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985).

[0134] Recombinant proteins can also be expressed using a variety of mammalian or insect cell culture systems. Recombinant proteins can be expressed in mammalian cells because such proteins are typically correctly folded, appropriately modified, and fully functional. Examples of suitable mammalian host cells include the COS-7 monkey kidney cell line described by Gluzman (Cell 23: 175, 1981), as well as other cell lines capable of expressing appropriate vectors, including, for example, L cells, C127, 3T3, Chinese hamster ovary (CHO), 293, HeLa, and BHK cell lines. Mammalian expression vectors may include non-transcriptional elements (such as origin of replication), appropriate promoters and enhancers linked to the gene to be expressed, and other 5' or 3' flanking non-transcriptional and 5' or 3' untranslated sequences, such as necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, and transcription termination sequences. The baculovirus system for producing heterologous proteins in insect cells was outlined by Luckow and Summers, Bio / Technology 6: 47 (1988).

[0135] Proteins produced by a transformed host can be purified using any suitable method. Such standard methods include chromatography (e.g., ion exchange, affinity and size fractionation column chromatography, etc.), centrifugation, differential solubility, or any other standard technique used for protein purification. Affinity tags (such as hexahistidine, maltose-binding domains, influenza coating sequences, glutathione S-transferases, etc.) can be attached to proteins to allow for easy purification by passing them through a suitable affinity column. Separated proteins can also be characterized physically using techniques such as proteolysis, nuclear magnetic resonance, and X-ray crystallography.

[0136] For example, the supernatant from the system that secretes the recombinant protein into the culture medium can be first concentrated using a commercially available protein concentrator filter (e.g., an Amicon or Millipore Pellicon ultrafiltration unit). Following the concentration step, the concentrate can be applied to a suitable purification matrix. Alternatively, anion exchange resins, such as matrices or substrates with 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, one or more reversed-phase high-performance liquid chromatography (RP-HPLC) steps using a hydrophobic RP-HPLC medium (e.g., silica gel with pendant methyl or other aliphatic groups) can be used for further purification of the cancer stem cell protein-Fc composition. Some or all of the foregoing purification steps, in different combinations, can also be used to provide homogeneous recombinant protein.

[0137] Recombinant proteins produced in bacterial cultures can be separated, 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) can be used for the final purification step. The microbial cells used in expressing the recombinant protein can be destroyed by any conventional method, including freeze-thaw cycles, sonication, mechanical destruction, or the use of cell lysis agents.

[0138] In vivo peptide / polypeptide synthesis The present invention also contemplates the use of nucleic acid molecules as a carrier for delivering neoantigen peptides / polypeptides, in the form of, for example, DNA / RNA vaccines, to subjects in need (see, for example, WO 2012 / 159643 and WO 2012 / 159754, which are incorporated herein by reference in their entirety).

[0139] In one embodiment, a neoantigen can be administered to a patient in need by using plasmids. These are plasmids that typically consist of strong viral promoters to drive in vivo transcription and translation of the gene of interest (or complementary DNA) (Mor et al., (1995). The Journal of Immunology 155(4): 2039-204). Introns A may sometimes 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 include strong polyadenylation / transcription termination signals, such as bovine growth hormone or rabbit β-globulin polyadenylation sequences (Alarcon et al., (1999). Advances in Parasitology 42: 343-410; Robinson et al. (2000). Advances in Virus Research 55: 1-74; Böhmet et al., (1996). Journal of Immunological Methods 193 (1): 29-40). Sometimes polycistronic vectors are constructed to express more than one immunogen, or to express both immunogens and immunostimulatory proteins (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88).

[0140] Because plasmids are the “vectors” from which they express immunogens, optimized vector design for maximizing protein expression is crucial (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 pathogenic mRNAs targeting eukaryotic cells. Another consideration is promoter selection. Such promoters could be the SV40 promoter or Rous sarcoma virus (RSV).

[0141] Plasmids can be introduced into animal tissues using a variety of different methods. The two most common routes are injection of DNA in saline using a standard hypodermic needle and delivery using a gene gun. A schematic diagram of the construction of DNA vaccine plasmids and their subsequent delivery into the host via these two methods is illustrated in Scientific American (Weiner et al., (1999) Scientific American 281(1): 34-41). Injection in saline is typically performed in the intramuscular (IM) or intradermal (ID) space of skeletal muscle, where DNA is delivered into the extracellular space. This can be done by using myotoxic agents such as bupivacaine; or by temporarily damaging muscle fibers with a hypertonic solution of saline or sucrose, assisted by electroporation (Alarcon et al., (1999) Advances in Parasitology 42: 343-410). The immune response to this delivery method can be influenced by many factors, including the type of needle, needle alignment, injection speed, injection volume, muscle type, and the age, sex, and physiological condition of the animal being injected (Alarcon et al., (1999). Advances in Parasitology 42: 343-410).

[0142] Gene gun delivery (another commonly used delivery method) uses compressed helium as a accelerator to ballistically accelerate plasmid DNA (pDNA) already adsorbed onto gold or tungsten microparticles into target cells (Alarcon et al., (1999). Advances in Parasitology 42: 343-410; Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88).

[0143] Alternative delivery methods may include aerosol instillation of naked DNA onto mucosal surfaces, such as the nasal and lung mucosa (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88), and topical administration of pDNA to the eye and vaginal mucosa (Lewis et al., (1999). Advances in Virus Research (Academic Press) 54: 129-88). Mucosal surface delivery has been achieved using positively ionized liposome-DNA formulations, biodegradable microspheres, attenuated Shigella or Listeria vectors, and recombinant adenovirus vectors for oral administration to the intestinal mucosa.

[0144] The delivery method determines the dosage of DNA required to elicit an effective immune response. Saline injection requires a variable amount of DNA, ranging from 10 μg to 1 mg, while gene gun delivery requires 100 to 1000 times less DNA compared to intramuscular saline injection to elicit an effective immune response. Typically, 0.2 μg to 20 μg is required, although amounts as low as 16 ng have been reported. These amounts vary between species; for example, mice require approximately 10 times less DNA than primates. Saline injection requires more DNA because the DNA is delivered to the extracellular space of the target tissue (usually muscle), where it must overcome physical barriers (such as the substrate and large amounts of connective tissue, to name a few) before it can be taken up by the cell. In contrast, gene gun delivery bombards DNA directly into the cell, resulting in less “loss” (see, for example, Sedegah et al. (1994). Proceedings of the National Academy of Sciences of the United States of America 91 (21): 9866-9870; Daheshiaet et al. (1997). The Journal of Immunology 159 (4): 1945-1952; Chen et al. (1998). The Journal of Immunology 160 (5): 2425-2432; Sizemore (1995) Science 270 (5234): 299-302; Fynan et al. (1993) Proceedings of the National Academy of Sciences of the United States of America 90 (24): 11478-82.

[0145] In one embodiment, the tumor-forming vaccine or immunogenic composition may comprise a separate DNA plasmid encoding, for example, one or more neoantigen peptides / peptides as identified according to the invention. As discussed herein, the exact choice of expression vector will depend on the peptide / peptide to be expressed and is entirely within the skill of a person of ordinary skill. The anticipated persistence of the DNA construct (e.g., in an appendage-type, non-replicating, non-integrating form in muscle cells) is expected to provide an increased duration of protection.

[0146] One or more of the novel antigenic peptides of the present invention can be encoded and expressed in vivo using virus-based systems (e.g., adenovirus systems, adeno-associated virus (AAV) vectors, poxviruses, or lentiviruses). In one embodiment, the tumorigenesis vaccine or immunogenic composition may include a virus-based vector for use in human patients in need of it, such as adenovirus (see, for example, 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, which is incorporated herein by reference in its entirety). Plasmids that can be used for delivery of adeno-associated viruses, adenoviruses, and lentiviruses have been previously described (see, for example, U.S. Patent Nos. 6,955,808 and 6,943,019, and U.S. Patent Application No. 20080254008, which are incorporated herein by reference).

[0147] In vectors suitable for use in the practice of this invention, integration into the host genome of a cell using retroviral gene transfer methods is possible, which typically leads to long-term expression of the inserted transgene. In a preferred embodiment, the retrovirus is a lentivirus. Furthermore, high transduction efficiency has been observed in various cell types and target tissues. The tropism of the retrovirus can be altered by incorporating exogenous envelope proteins to expand the potential target population of the target cells. Retroviruses can also be engineered to allow conditional expression of the inserted transgene, thus enabling certain cell types to be infected by the lentivirus. Cell type-specific promoters can be used for targeted expression in specific cell types. Lentiviral vectors are retroviral vectors (and therefore both lentiviral and retroviral vectors can be used in the practice of this invention). 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 depends on the target tissue. Retroviral vectors consist of cis-acting long terminal repeats that have the ability to package up to 6-10 kb of exogenous sequences. The minimum amount of cis-acting LTR is sufficient for vector replication and packaging, and then these vectors are used to integrate the desired nucleic acid into target cells to provide permanent expression. Widely used retroviral vectors that can be used in the practice of this invention include those based on murine leukemia virus (MuLV), gibberish leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, for example, Buchscher et al., (1992) Journal of Virology 66: 2731-2739; Johann et al., (1992) Journal of Virology 66: 1635-1640; Sommnerfelt et al., (1990) Virology 176: 58-59; Wilson et al., (1998) Journal of Virology 63: 2374-2378; Miller et al., (1991) Journal of Virology 65: 2220-2224; PCT / US 94 / 05700). Zou et al. administered approximately 10 µl of a 1 x 10-element solution via an intrathecal catheter. 9 Recombinant lentivirus at titers of transduction units (TU) / ml. These types of dosages can be adapted or extrapolated to allow the use of retroviral or lentiviral vectors in this invention.

[0148] Also useful in the practice of this invention are minimal nonprimate lentiviral vectors, such as equine infectious anemia virus-based lentiviral vectors (EIAV) (see, for example, Balagaan. (2006) J GeneMed; 8: 275-285, published online on 21 November 2005 in the Wiley InterScience database (www.interscience.wiley.com) DOI: 10.1002 / jgm.845). These vectors may have cytomegalovirus (CMV) promoters that drive the expression of target genes. Accordingly, the invention contemplates one or more vectors useful in the practice of this invention: viral vectors, including retroviral vectors and lentiviral vectors.

[0149] Adenoviral vectors are also useful in the practice of this invention. One advantage is the ability of recombinant adenoviruses to efficiently transfer and express recombinant genes, resulting in high expression of the transferred nucleic acids, both in vitro and in vivo in a variety of mammalian cells and tissues. Additionally, the ability to efficiently infect dormant cells extends the utility of the recombinant adenoviral vector. Furthermore, the high expression level ensures that the nucleic acid product will be expressed at a sufficient level to generate an immune response (see, for example, U.S. Patent No. 7,029,848, which is incorporated herein by reference).

[0150] In one embodiment of this document, delivery is carried out via adenovirus, which may contain at least 1 x 10 5 A single booster dose of one adenovirus vector particle (also known as a particle unit, pu). In one embodiment herein, this dose is preferably at least about 1 x 10⁻⁶ of the adenovirus vector. 6 1 x 103 particles (e.g., approximately 1 x 103) 6 -1 x 10 12 (1 x 10^12 particles), more preferably at least about 1 x 10^12 particles. 7 One particle, more preferably at least about 1 x 10 8 1 x 103 particles (e.g., approximately 1 x 103) 8 -1 x 10 11 One particle or approximately 1 x 10 8 -1 x 10 12 (number of particles), and preferably at least about 1 x 10^6 particles. 9 Particles (e.g., approximately 1 x 10⁻⁶) 9 -1 x 10 10 One particle or approximately 1 x 10 9 -1 x 10 12 (a few particles), or even at least about 1 x 10^6 particles. 101 x 103 particles (e.g., approximately 1 x 103) 10 -1 x10 12 (particles). Alternatively, the dose contains no more than about 1 x 10⁻⁶ particles. 14 Particles, preferably no more than about 1 x 103 13 One particle, or even more preferably no more than about 1 x 10⁻⁶. 12 One particle, or even more preferably no more than about 1 x 10⁻⁶. 11 The number of particles, and preferably no more than about 1 x 103 10 1 x 103 particles (e.g., no more than about 1 x 103) 9 (particles). Therefore, this dose may contain a single dose of adenovirus vector, having, for example, about 1 x 10⁻⁶ particles. 6 Particle unit (pu), approximately 2 x 10 6 PU, approximately 4 x 10 6 PU, approximately 1 x 10 7 PU, approximately 2 x 10 7 PU, approximately 4 x 10 7 PU, approximately 1 x 10 8 PU, approximately 2 x 10 8 PU, approximately 4 x 10 8 PU, approximately 1 x 10 9 PU, approximately 2 x 10 9 PU, approximately 4 x 10 9 PU, approximately 1 x 10 10 PU, approximately 2 x 10 10 PU, approximately 4 x 10 10 PU, approximately 1 x 10 11 PU, approximately 2 x 10 11 PU, approximately 4 x 10 11 PU, approximately 1 x 10 12 PU, approximately 2 x 10 12 pu, or approximately 4 x 10 12 The adenovirus vector of pu. See, for example, the adenovirus vector (incorporated herein by reference) in U.S. Patent No. 8,454,972 B2 to Nabel et al., issued June 4, 2013, and its dosage form in column 29, lines 36-58. In one embodiment herein, the adenovirus is delivered via multiple doses.

[0151] In terms of in vivo delivery, AAV is superior to other viral vectors due to its low toxicity and low likelihood of inducing insertional mutagenesis, as it does not integrate into the host genome. AAV has a packaging limit of 4.5 or 4.75 kb. Constructs larger than 4.5 or 4.75 kb result in a significant reduction in viral production. Many promoters exist that can be used to drive nucleic acid molecule expression. AAVITR can be used as one promoter and is advantageous for eliminating the need for additional promoter elements. For pervasive expression, the following promoters can be used: CMV, CAG, CBh, PGK, SV40, ferritin heavy or light chains, etc. For brain expression, the following promoters can be used: synaptic I for all neurons, CaMKIIα for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. Promoters for driving RNA synthesis can include: Pol III promoters, such as U6 or H1. Pol II promoters and intron cassettes can be used to express guide RNA (gRNA).

[0152] Regarding AAV, the AAV can be AAV1, AAV2, AAV5, or any combination thereof. The AAV can be selected relative to the cells to be targeted; for example, AAV serotypes 1, 2, 5, or hybrid capsids AAV1, AAV2, AAV5, or any combination thereof can be selected for targeting brain or neuronal cells; and AAV4 can be selected for targeting cardiac tissue. AAV8 can be used for delivery to the liver. The above promoters and vectors are individually preferred.

[0153] In one embodiment of this document, the delivery is performed via AAV. Therapeuticly effective doses for in vivo delivery of AAV to humans are considered to be those containing approximately 1 x 10⁻⁶. 10 To approximately 1 x 10 50 The dosage of a functional AAV / ml solution ranges from about 20 to about 50 ml of saline solution. This dosage can be adjusted to balance the therapeutic benefit with any side effects. In one embodiment described herein, the AAV dosage is approximately from about 1 x 10⁻⁶. 5 To 1 x 10 50 One genome AAV, from approximately 1 x 10 8 To 1 x 10 20 One genome of AAV, from approximately 1 x 10 10 To approximately 1 x 10 16 One genome, or approximately 1 x 10 11 To approximately 1 x 10 16 The concentration range of AAV in each genome. The human dose can be approximately 1 x 10⁻⁶. 13One genomic AAV. Such concentrations can be delivered in vector solutions ranging from about 0.001 ml to about 100 ml, about 0.05 ml to about 50 ml, or about 10 ml to about 25 ml. In a preferred embodiment, about 2 x 10 13 The viral genome / mL titer was measured using AAV, and each striatal hemisphere of the mouse received a single 500 nanoliter injection. Other effective doses can be readily determined by those skilled in the art through routine experiments that establish 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.

[0154] In another embodiment, effective activation of a cellular immune response to a neoantigen vaccine or immunogenic composition can be achieved by expressing the relevant neoantigen in a vaccine or immunogenic composition in a non-pathogenic microorganism. Well-known examples of such microorganisms are Mycobacterium bovis (BCG), Salmonella spp., and Pseudomonas spp. (see U.S. Patent No. 6,991,797, which is incorporated herein by reference in its entirety).

[0155] In another embodiment, poxviruses are used in tumorigenesis vaccines or immunogenic compositions. These include orthopoxvirus, fowlpox, cowpox, MVA, NYVAC, canarypox, ALVAC, fowlpox, TROVAC, etc. (see, for example, Verardiet et al., Human Vaccines and Immunother. 2012 July; 8(7):961-70; and Moss, Vaccine. 2013; 31(39): 4220-4222). Poxvirus expression vectors were described in 1982 and quickly became widely used in vaccine development along with research in multiple fields. The advantages of the vectors include simple construction, the ability to adapt to large amounts of exogenous DNA, and high expression levels.

[0156] In another embodiment, vaccinia virus is used for tumorigenesis vaccines or immunogenic compositions to express neoantigens. (Rolph et al., Recombinant viruses as vaccines and immunological tools. Curr Opin Immunol 9: 517-524, 1997). Recombinant vaccinia virus is able to replicate within the cytoplasm of infected host cells, and therefore the peptides of interest can induce an immune response. Furthermore, poxviruses have been widely used as carriers for vaccines or immunogenic compositions due to their ability to target antigens encoded by the major histocompatibility complex class I pathway through direct infection of immune cells, specifically antigen-presenting cells, but also due to their adjuvant capabilities.

[0157] In another embodiment, ALVAC is used as a carrier in tumorigenesis vaccines or immunogenic compositions. ALVAC is a canarypox virus that can be modified to express exogenous transgenes and has been used as a method for vaccination against both prokaryotic and eukaryotic antigens. (Horig H, Lee DS, Conkright W, et al., Phase I clinical trial of a recombinant canarypoxvirus (ALVAC) vaccine expressing human carcinoembryonic antigen and the B7.1 co-stimulatory molecule. Cancer Immunol Immunother 2000;49:504-14; von Mehren M, Arlen P, Tsang KY, et al., Pilot study of a dual gene recombinant fowlpox virus containing both carcinoembryonic antigen (CEA) and B7.1 transgenes in patients with recurrent CEA-expressing adenocarcinoma.) Avipox vaccine containing both carcinoembryonic antigen (CEA) and B7.1 transgenes in patients with recurrent CEA-expressing adenocarcinoma. Clin Cancer Res 2000; 6: 2219-28; Musey L, Ding Y, Elizaga M et al. HIV-1 vaccination administered intramuscularly can induce both systemic and mucosal T cell immunity in HIV-1-uninfected individuals. J Immunol 2003; 171: 1094-101; Paoletti E.Applications of pox virus vectors to vaccination: an update. Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci USA) 1996; 93:11349-53; US Patent No. 7,255,862). In a phase I clinical trial, ALVAC virus expressing the tumor antigen CEA showed excellent safety and resulted in increased CEA-specific T-cell responses in selected patients; however, the target clinical response was not 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). Journal of Clinical Oncology (J Clin Oncol) 1999; 17: 332-7).

[0158] In another embodiment, the modified Ankara vaccinia virus (MVA) can be used as a viral vector for neoantigen vaccines or immunogenic compositions. MVA is a member of the orthopoxvirus family and has been propagated in chicken embryo fibroblasts of the Ankara strain of vaccinia virus for approximately 570 consecutive passages (for review, see Mayr A. et al., Infection 3, 6-14, 1975). As a result of these passages, 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 extreme attenuation, i.e., reduced virulence or infectivity, but still retains excellent immunogenicity. When tested in various animal models, MVA has demonstrated to be non-virulent, even in immunosuppressed individuals. In addition, MVA-BN®-HER2 is a candidate immunotherapy designed for the treatment of HER-2 positive breast cancer and is currently in clinical trials. (Mandl et al., Cancer Immunol Immunother. 2012 Jan; 61(1): 19-29). Methods for preparing and using recombinant MVA have been described (see, for example, U.S. Patent Nos. 8,309,098 and 5,185,146, which are incorporated herein by reference in their entirety).

[0159] In another embodiment, modified Copenhagen strains of vaccinia virus, NYVAC and NYVAC variants are used as vectors (see U.S. Patent Nos. 7,255,862; PCT WO 95 / 30018; U.S. Patent Nos. 5,364,773 and 5,494,807, which are incorporated herein by reference in their entirety).

[0160] In one embodiment, recombinant viral particles of a vaccine or immunogenic composition are administered to a patient in need. The dose range of the expressed neoantigen can be from a few micrograms to hundreds of micrograms, for example, 5 to 500 μg. The vaccine or immunogenic composition can be administered in any suitable amount to achieve expression at these dose levels. It can be administered at about 10 μg. 3.5 The amount of PFU is used to administer viral particles to patients who need them or to transfect cells; therefore, preferably, at least about 10 4 pfu to approximately 10 6 PFU (Prophylactic Fuel) delivers viral particles to patients who need them or infects or transfects cells; however, at least approximately 10 PFU can be given to patients who need them. 8 pfu, thus allowing for a more preferred amount to be at least 107 pfu to approximately 10 9 PFU. The dosage for NYVAC is applicable to ALVAC, MVA, MVA-BN, and fowlpox, such as canarypox and chickenpox.

[0161] Vaccine or immunogenic composition adjuvant Effective vaccines or immunogenic compositions advantageously include strong adjuvants to initiate immune responses. As described herein, poly-ICLCs have demonstrated several promising properties for use as adjuvants in vaccines or immunogenic compositions; poly-ICLCs are agonists of the TLR3 and RNA helicase-MDA5 and RIG3 domains. These properties include inducing local and systemic activation of immune cells in vivo, producing stimulating chemokines and cytokines, and stimulating antigen presentation via dendritic cells (DCs). Furthermore, poly-ICLCs can induce durable CD4+ and CD8+ responses in humans. Importantly, striking similarities were observed in the upregulation of transcriptional and signal transduction pathways in subjects vaccinated with poly-ICLCs and in volunteers who had received a highly effective, replicating yellow fever vaccine. Moreover, in a recent phase 1 study, >90% of ovarian cancer patients immunized with poly-ICLCs in combination with NY-ESO-1 peptide vaccines (excluding Montaned) showed induction of CD4+ and CD8+ T cells, as well as antibody responses to the peptide. Meanwhile, poly-ICLCs have been extensively tested in more than 25 clinical trials to date and have demonstrated relatively benign toxicity profiles. In addition to being potent and specific immunogens, these neoantigen peptides can be combined with adjuvants (e.g., poly-ICLCs) or other antitumor agents. Unbound by theory, these neoantigens are expected to circumvent central thymic tolerance (thus allowing for stronger antitumor T-cell responses) while reducing the likelihood of autoimmunity (e.g., by avoiding targeting normal autoantigens). Effective immune responses favorably include strong adjuvants to activate the immune system (Speiser and Romero, Molecularly Defined Vaccines for Cancer Immunotherapy, and Seminars in Immunol 22: 144 (2010)). For example, Toll-like receptors (TLRs) have been shown to be powerful sensors of “danger signals” from microbial and viral pathogens, effectively inducing the innate immune system and consequently, the adaptive immune system (Bhardwaj and Gnjatic, TLR agonists: Are They Good Adjuvants?, Cancer J. 16: 382-391 (2010)). Among TLR agonists, poly-ICLC (a synthetic double-stranded RNA mimic) is one of the most potent activators of bone marrow-derived dendritic cells.In a human volunteer study, poly-ICLCs have been shown to be safe and to induce a gene expression profile in peripheral blood cells comparable to that induced by yellow fever vaccine YF-17D, one of the most effective live attenuated viral vaccines (Caskey et al., Synthetic double-stranded RNA induces innate immune responses similar to a live viral vaccine in humans. Journal of Experimental Medicine (J Exp Med) 208: 2357 (2011)). In a preferred embodiment, Hiltonol® (a GMP formulation of poly-ICLCs prepared by Oncovir) is used as an adjuvant. In other embodiments, other adjuvants described herein are contemplated. For example, oil-in-water, water-in-oil, or multiphase W / O / W; see, for example, US 7,608,279 and Aucouturier et al., Vaccine 19 (2001), 2666-2672, and the documents cited herein.

[0162] Indications Examples of cancers and cancerous conditions that can be treated with the immunogenic compositions or vaccines described in this document include, but are not limited to, patients who have been diagnosed with cancer or are at risk of developing cancer and who require treatment. Subjects may have solid tumors such as breast cancer, ovarian cancer, prostate cancer, lung cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, melanoma, and other tumors of tissues and organs, as well as hematologic malignancies such as lymphoma and leukemia, including acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic lymphoma, 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 parts of the CNS, such as malignant gliomas or medulloblastomas); head and / or neck cancer; breast tumors; circulatory system tumors (e.g., heart, mediastinum and pleura, and other intrathoracic organs, vascular tumors); and tumors of blood vessels. Tumors of the blood and lymphatic system (e.g., Hodgkin's disease, non-Hodgkin's lymphoma, Burkitt lymphoma, AIDS-related lymphoma, malignant immunoproliferative disorders, multiple myeloma, and malignant plasma cell tumors, lymphoid leukemia, myeloid leukemia, acute or chronic lymphocytic leukemia, monocytic leukemia, other leukemias of specific cell types, leukemias of unspecified cell types, unspecified malignancies of lymphoid, hematopoietic, and related tissues, such as diffuse large cell lymphoma, T-cell lymphoma, or T-cell lymphoma of the skin); tumors of the excretory system (e.g., kidneys, renal pelvis, ureters, bladder, and other urinary organs). Tumors; tumors of the gastrointestinal tract (e.g., esophagus, stomach, small intestine, colon, colorectal, rectosigmoid junction, rectum, anus, and anal canal); tumors involving the liver and intrahepatic bile ducts, gallbladder, and other parts of the bile ducts, pancreas, and other digestive organs; tumors of the oral cavity (e.g., lips, tongue, gums, floor of mouth, palate, parotid glands, salivary glands, tonsils, oropharynx, nasopharynx, piriform sinus, hypopharynx, and other parts of the oral cavity); tumors of the reproductive system (e.g., vulva, vagina, cervix, uterus, ovaries, and other parts associated with the female reproductive organs, placenta, penis, prostate, testes, and other parts associated with the male reproductive organs); tumors of the respiratory tract (e.g., nasal cavity, middle ear, paranasal sinuses, larynx, trachea, bronchi). Tumors of the trachea and lungs (e.g., small cell lung cancer and non-small cell lung cancer); tumors of the skeletal system (e.g., bones and articular cartilage of the limbs, joint cartilage, and other sites); tumors of the skin (e.g., malignant melanoma of the skin, 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 peripheral nerves and the autonomic nervous system, connective tissue and soft tissue, retroperitoneum and peritoneum, eyes, thyroid gland, adrenal glands and other endocrine glands and related structures, secondary and unspecified malignancies of lymph nodes, secondary malignancies of the respiratory and digestive systems, and secondary malignancies of other sites.

[0163] Of particular significance is the treatment of non-Hodgkin's lymphoma (NHL), clear cell renal cell carcinoma (ccRCC), metastatic melanoma, sarcoma, leukemia, or bladder cancer, colon cancer, brain cancer, breast cancer, head and neck cancer, endometrial cancer, lung cancer, ovarian cancer, pancreatic cancer, or prostate cancer. In some embodiments, the melanoma is high-risk melanoma.

[0164] Among other things, cancers that can be treated with this immunogenic composition or vaccine may include cases that are refractory to treatment with other chemotherapeutic agents. As used herein, the term "refractory" means cancer (and / or its metastases) that, after treatment with another chemotherapeutic agent, shows no or only a weak antiproliferative response (e.g., no or only a weak inhibition of tumor growth). These are cancers that cannot be satisfactorily treated with other chemotherapeutic agents. Refractory cancers encompass not only (i) cancers in which one or more chemotherapeutic agents have failed during the patient's treatment, but also (ii) cancers that may be refractory by other means, such as biopsy cultures in the presence of a chemotherapeutic agent.

[0165] The immunogenic compositions or vaccines described herein are also suitable for treating previously untreated patients in need of treatment.

[0166] The immunogenic compositions or vaccines described herein are also suitable for subjects who do not have detectable tumor formation but are at high risk of disease recurrence.

[0167] Of particular interest is the treatment of patients in need who have undergone autologous hematopoietic stem cell transplantation (AHSCT), and specifically, patients who have demonstrated residual disease after undergoing AHSCT. The post-AHSCT background is characterized by low levels of residual disease, immune cell infusion leading to self-balancing expansion, and the absence of any standard relapse-delaying treatments. These characteristics offer a rare opportunity to delay disease relapse using the described tumorigenesis vaccines or immunogenic compositions.

[0168] Pharmaceutical Composition / Delivery Method The present invention also relates to pharmaceutical compositions comprising an effective amount of one or more compounds (including pharmaceutically acceptable salts thereof) according to the invention, optionally in combination with a pharmaceutically acceptable carrier, excipient or additive.

[0169] While tumor-specific neoantigen peptides can be administered as the sole active agent, they can also be used in combination with one or more other agents and / or adjuvants. When administered in combination, the therapeutic agents can be formulated as separate compositions that are administered simultaneously or at different times, or these therapeutic agents can be administered as a single composition.

[0170] These compositions can be administered once daily, twice daily, every two days, every three days, every four days, every five days, every six days, every seven days, every two weeks, every three weeks, every four weeks, every two months, every six months, or once a year. The dosing interval can be adjusted according to the individual patient's needs. For longer dosing intervals, extended-release or long-acting formulations can be used.

[0171] The compositions of the present invention can be used to treat acute diseases and symptoms, and also to treat chronic conditions. Specifically, the compositions of the present invention can be used in methods for treating or preventing tumor formation. In some embodiments, the compounds of the present invention are administered for a period of time exceeding two weeks, three weeks, one month, two months, three months, four months, five months, six months, one year, two years, three years, four years, or five years, ten years, or fifteen years; or, for example, any range of time periods in days, months, or years, wherein the lower limit of the range is any period between 14 days and 15 years and the upper limit of the range is between 15 days and 20 years (e.g., between 4 weeks and 15 years, between 6 months and 20 years). In some cases, it may be advantageous to administer the compounds of the present invention to the patient for the remainder of their life. In a preferred embodiment, the patient is monitored to examine the progression of the disease or disorder and the dosage is adjusted accordingly. In a preferred embodiment, treatment according to the present invention is effectively sustained for at least two weeks, three weeks, one month, two months, three months, four months, five months, six months, one year, two years, three years, four years, or five years, ten years, fifteen years, twenty years, or for the remainder of the subject's life.

[0172] Tumor-specific neoantigen peptides can be administered via injection, oral administration, parenteral administration, inhalation spray, rectal administration, vaginal administration, or topical administration in unit dosage forms containing conventional pharmaceutically acceptable carriers, adjuvants, and delivery vehicles. As used herein, parenteral administration includes delivery to one or more lymph nodes, subcutaneously, intravenously, intramuscularly, intrasternally, via infusion techniques, intraperitoneally, through the eye or via the eye, intravitreal, buccally, percutaneously, intranasally, into the brain (including intracranial and intradural), into joints (including ankle, knee, hip, shoulder, elbow, and wrist), directly into the tumor, and in suppository form.

[0173] Surgical resection uses surgical procedures to remove abnormal tissue from cancerous tissue, such as mediastinal, neurogenic, or germ cell tumors, or thymomas. In some embodiments, administration of a tumor-forming 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 tumor-forming vaccine or immunogenic composition is initiated 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks after tumor resection.

[0174] A prime / booster regimen refers to the continuous administration of a vaccine or immunogenic or immunological composition. In some embodiments, the tumorigenesis vaccine or immunogenic composition is administered in a prime / booster dosing regimen, for example, at weeks 1, 2, 3, or 4 as a prime and at months 2, 3, or 4 as a booster. In another embodiment, a heterologous prime strategy is used to elicit a greater cytotoxic T cell response (see Schneider et al., Induction of CD8+ T cells using heterologous prime-boost immunization strategies, Immunological Reviews, Vol. 170, No. 1, pp. 29-38, August 1999). In another embodiment, DNA encoding a neoantigen is used for the prime, followed by a protein booster. In yet another embodiment, a protein is used for the prime, followed by a booster with a virus encoding a neoantigen. In another embodiment, a virus encoding a neoantigen is used for primary immunization, and another virus is used for booster. In another embodiment, a protein is used for primary immunization, and DNA is used for booster. In a preferred embodiment, a DNA vaccine or immunogenic composition is used for the primary T-cell response, and a recombinant viral vaccine or immunogenic composition is used to boost that response. In another preferred embodiment, the viral vaccine or immunogenic composition is administered co-administered with a protein or DNA vaccine or immunogenic composition used as an adjuvant for the protein or DNA vaccine or immunogenic composition. The patient can then be enhanced with a viral vaccine or immunogenic composition, a protein, or a DNA vaccine or immunogenic composition (see Hutchings et al., Combination of protein and viral vaccines induces potent cellular and humoral immune responses and enhanced protection from murine malaria challenge. Infect Immun. 2007 Dec; 75 (12): 5819-26. Published electronically on 1 October 2007).

[0175] Pharmaceutical compositions can be processed using conventional pharmaceutical methods to produce a medicament for administration to patients (including humans and other mammals) in need of it.

[0176] Modification of neoantigen peptides can affect their solubility, bioavailability, and metabolic rate, thereby providing control over the delivery of these active species. Solubility can be assessed by preparing and testing the neoantigen peptides using known methods entirely within the art of those skilled in the art.

[0177] It has been unexpectedly discovered that pharmaceutical compositions comprising succinic acid or a pharmaceutically acceptable salt thereof (succinate) can provide improved solubility for neoantigenic peptides. Therefore, in one aspect, the present invention provides pharmaceutical compositions comprising: at least one neoantigenic peptide or a pharmaceutically acceptable salt thereof; a pH modifier (e.g., a base, such as a dicarboxylic acid or tricarboxylic acid salt, such as a pharmaceutically acceptable salt of succinic acid or citric acid); and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can be prepared by combining a solution containing at least one neoantigenic peptide with a base, such as a dicarboxylic acid or tricarboxylic acid salt, such as a pharmaceutically acceptable salt of succinic acid or citric acid (e.g., sodium succinate), or by combining a solution containing at least one neoantigenic peptide with a solution containing a base, such as a dicarboxylic acid or tricarboxylic acid salt, such as a pharmaceutically acceptable salt of succinic acid or citric acid (including, for example, a succinate buffer solution). In some embodiments, the pharmaceutical composition comprises sodium succinate. In some embodiments, the pH-changing agent (e.g., citrate or succinate) is present in the composition at a concentration from about 1 mM to about 10 mM, and in some embodiments at a concentration from 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.

[0178] In some embodiments of the pharmaceutical composition, the pharmaceutically acceptable carrier comprises water. In some embodiments, the pharmaceutically acceptable carrier further comprises dextran. In some embodiments, the pharmaceutically acceptable carrier further comprises dimethyl sulfoxide. In some embodiments, the pharmaceutical composition further comprises an immunomodulator or adjuvant. In some 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, ISCMATRIX, Juvlmmune, LipoVac, MF59, monophospholipase A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montaned ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, vector system, PLGA microparticles, Remiquimod, SRL172, virions and other virus-like particles, YF-17D, VEGF trap, R848, β-glucan, Pam3Cys, and Aquila's QS21 stimulon. In some embodiments, the immunomodulator or adjuvant includes poly-ICLC.

[0179] Cortexyl derivatives (such as, for example, Vadimezan or AsA404 (also known as 5,6-dimethylcortexyl-4-acetic acid (DMXAA))) can also be used as adjuvants according to embodiments of the invention. Alternatively, such derivatives can also be administered, for example, in parallel with the vaccine or immunogenic composition of the invention via systemic or intratumoral delivery to stimulate immunity at the tumor site. Unbound by theory, these glucanone derivatives are believed to act by stimulating interferon (IFN) via IFN gene-stimulating factor (ISTING) receptors (see, for example, 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).

[0180] The vaccine or immunological composition may also include adjuvant compounds selected from polymers of acrylic acid or methacrylic acid and copolymers of maleic anhydride and alkenyl derivatives. Specifically, it is a polymer (carbomer) crosslinked with a polyalkenyl ether of acrylic acid or methacrylic acid and a sugar or polyol, specifically crosslinked with allyl sucrose or with allyl pentaerythritol. For example, it may also be a copolymer of maleic anhydride and ethylene crosslinked with divinyl ether (see U.S. Patent No. 6,713,068, which is incorporated herein by reference in its entirety).

[0181] In some embodiments, the pH modifier can stabilize adjuvants or immunomodulators as described herein.

[0182] In some embodiments, the pharmaceutical composition comprises: one to five peptides, dimethyl sulfoxide (DMSO), dextran (or trehalose or sucrose), water, succinate, poly(I:polyC), poly(L-lysine), carboxymethyl cellulose, and chloride. In some embodiments, each of the one to five peptides is present at a concentration of 300 μg / ml. In some embodiments, the pharmaceutical composition comprises ≤ 3% DMSO by volume. In some embodiments, the pharmaceutical composition comprises a 3.6%-3.7% aqueous solution of dextran. In some embodiments, the pharmaceutical composition comprises 3.6-3.7 mM succinate (e.g., disodium succinate) or a salt thereof. In some embodiments, the pharmaceutical composition comprises 0.5 mg / ml poly(I:polyC). In some embodiments, the pharmaceutical composition comprises 0.375 mg / ml poly(L-lysine). In some embodiments, the pharmaceutical composition comprises 1.25 mg / ml sodium carboxymethyl cellulose. In some embodiments, the pharmaceutical composition comprises 0.225% sodium chloride.

[0183] The pharmaceutical composition comprises, optionally in combination with pharmaceutically acceptable additives, carriers, and / or excipients, a therapeutically effective amount of the tumor-specific neoantigen peptide described herein for the treatment of diseases and conditions already described herein (e.g., tumor formation / tumor). Those skilled in the art will recognize from this disclosure and the knowledge of the art that the therapeutically effective amount of one or more compounds according to the invention can vary depending on the condition to be treated, its severity, the treatment regimen to be utilized, the pharmacokinetics of the reagents used, and the patient (animal or human) being treated.

[0184] To prepare the pharmaceutical composition according to the invention, preferably, a therapeutically effective amount of one or more compounds according to the invention is closely mixed with a pharmaceutically acceptable carrier to produce a dose, according to conventional pharmaceutical compounding techniques. The carrier can take a variety of forms depending on the desired formulation to be administered (e.g., ocular, oral, topical, or parenteral), including gels, creams, ointments, lotions, and delayed-release implantable formulations. In preparing the pharmaceutical composition for oral dosage forms, any of the common pharmaceutical media can be used. Thus, for liquid oral formulations (e.g., suspensions, elixirs, and solutions), suitable carriers and additives can be used, including water, glycerin, oils, alcohols, flavoring agents, preservatives, coloring agents, etc. For solid oral formulations (e.g., powders, tablets, capsules) and for solid formulations (e.g., suppositories), suitable carriers and additives can be used, including starch, sugar carriers (e.g., glucose, mannitol, lactose) and related carriers, diluents, granulating agents, lubricants, binders, disintegrants, etc. If desired, tablets or capsules can be enteric-coated or sustained-released using standard techniques.

[0185] The active compound is included in a pharmaceutically acceptable carrier or diluent in an amount sufficient to deliver to a patient a therapeutically effective amount for the desired indication without causing serious toxicity in the patient being treated.

[0186] Oral compositions typically include an inert diluent or an edible carrier. They may be encapsulated in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound or its prodrug derivative may be incorporated with excipients and administered in tablet, lozenge, or capsule form. Pharmaceutically compatible binders and / or adjuvant materials may be included as part of the composition.

[0187] Tablets, pills, capsules, lozenges, etc., may contain any of the following ingredients or compounds with similar properties: binders, such as microcrystalline cellulose, astragalus gum, or gelatin; excipients, such as starch or lactose; dispersants, such as alginate or corn starch; lubricants, such as magnesium stearate; glidants, such as colloidal silica; sweeteners, such as sucrose or saccharin; or flavorings, such as peppermint, methyl salicylate, or orange flavoring. When the unit dosage form is a capsule, it may also contain a liquid carrier, such as fatty oil, in addition to the materials discussed herein. Furthermore, the unit dosage form may contain a variety of other materials in physical form that modify the dosage unit, such as coatings of sugar, shellac, or enteric solvents.

[0188] The formulations of the present invention suitable for oral administration can be presented in discrete units, such as capsules, flat capsules or tablets each containing a predetermined amount of active ingredient; in powder or granule form; in solution or suspension in aqueous or non-aqueous liquid; or in oil-in-water or water-in-oil liquid emulsions and in pellet form, etc.

[0189] Tablets can be prepared by compression or molding, optionally with one or more excipients. Compressed tablets can be prepared by compressing the active ingredient in a free-flowing form (such as powder or granules) in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, preservative, surfactant, or dispersant. Molded tablets can be prepared by molding a mixture of powdered compounds wetted with an inert liquid diluent in a suitable machine. Tablets can optionally be coated or scored and can be formulated to provide a slow or controlled release of the active ingredient therein.

[0190] Methods for formulating such slow- or controlled-release compositions of pharmaceutical active ingredients are known in the art and described in several published U.S. patents, some of which include, but are not limited to, U.S. Patent Nos. 3,870,790; 4,226,859; 4,369,172; 4,842,866 and 5,705,190, the disclosures of which are incorporated herein by reference in their entirety. Coating can be used for delivery of compounds to the intestine (see, for example, U.S. Patent Nos. 6,638,534, 5,541,171, 5,217,720 and 6,569,457, and the references cited therein).

[0191] Active compounds or their pharmaceutically acceptable salts may also be administered as components of elixirs, suspensions, syrups, wafers, chewing gum, etc. In addition to active compounds, syrups may also contain sucrose or fructose as sweeteners, as well as certain preservatives, dyes and colorants, and flavorings.

[0192] Solutions or suspensions intended for ocular, parenteral, intradermal, subcutaneous, or topical application may include the following components: sterile diluents, such as water for injection, saline solution, non-volatile oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; antimicrobial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetates, citrates, or phosphates; and surfactants for regulating tension, such as sodium chloride or glucose.

[0193] In some embodiments, the pharmaceutically acceptable carrier is an aqueous solvent, i.e., a solvent containing water, optionally with an additional co-solvent. Exemplary pharmaceutically acceptable carriers include water, a buffered solution in water (e.g., phosphate-buffered saline (PBS), and a 5% dextran aqueous solution (D5W) or 10% trehalose or 10% sucrose). In some embodiments, the aqueous solvent further includes dimethyl sulfoxide (DMSO), for example in an amount of about 1%-4%, or 1%-3%. In some embodiments, the pharmaceutically acceptable carrier is isotonic (i.e., having substantially the same osmotic pressure as body fluids, such as plasma).

[0194] In one embodiment, these active compounds are prepared together with a carrier that protects these compounds from rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and polylactic-co-glycolic acid (PLGA) can be used. In view of this disclosure and knowledge of the art, methods for preparing such formulations are within the scope of those skilled in the art.

[0195] Those skilled in the art will recognize from this disclosure and their knowledge that, in addition to tablets, other dosage forms can be formulated to provide a slow- or controlled release of the active ingredient. Such dosage forms include, but are not limited to, capsules, granules, and gel-capsule forms.

[0196] Liposome suspensions can also serve as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art. For example, a liposome formulation can be prepared by dissolving one or more suitable lipids in an inorganic solvent and then evaporating the solvent, thereby leaving a thin film of dried lipids on the surface of a container. An aqueous solution of the active compound can then be introduced into the container. The container is then manually swirled to release the lipid material from the side of the container and to disperse the lipid aggregates, thereby forming a liposome suspension. Other preparation methods well known to those skilled in the art can also be used in this aspect of the invention.

[0197] These formulations can be conveniently presented in unit dosage forms and can be prepared using conventional pharmaceutical techniques. Such techniques involve the step of binding the active ingredient with one or more drug carriers or excipients. Typically, formulations are prepared by uniformly and tightly binding the active ingredient with a liquid carrier or a finely dispersed solid carrier, or both, and then, if necessary, shaping the product.

[0198] Formulations and compositions suitable for topical administration in the mouth include sugar tablets containing these ingredients in a flavoring matrix (typically sucrose and gum arabic or astragalus gum); soft tablets containing the active ingredients in an inert matrix (such as gelatin and glycerin, or sucrose and gum arabic); and mouthwashes containing the ingredients to be administered in a suitable liquid carrier.

[0199] Formulations suitable for topical administration to the skin can be presented as ointments, creams, gels, and pastes containing 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.

[0200] Preparations for rectal administration can be presented as suppositories with a suitable base, including, for example, cocoa butter or salicylates.

[0201] Formulations suitable for nasal administration (where the carrier is a solid) include coarse powder having a particle size, for example, in the range of 20 to 500 micrometers, which is administered through the nasal passage from a powder container held close to the nose in a manner that delivers the odorant (i.e., by rapid inhalation). Suitable formulations suitable for administration, for example, as nasal sprays or as nasal drops (where the carrier is a liquid) include aqueous or oily solutions of the active ingredient.

[0202] Preparations suitable for vaginal administration may be presented as vaginal suppositories, tampons, creams, gels, pastes, foams or sprays, which, in addition to the active ingredient, contain, for example, suitable carriers known in the art.

[0203] Parenteral preparations can be packaged in ampoules made of glass or plastic, disposable syringes, or multi-dose vials. If administered intravenously, preferred carriers include, for example, normal saline or phosphate-buffered saline (PBS).

[0204] For parenteral preparations, the carrier typically comprises sterile water or an aqueous solution of sodium chloride, but may also include other components, including those that assist in dispersion. Of course, when sterile water is to be used and sterility must be maintained, these compositions and the carrier are also sterilized. Injectable suspensions can also be prepared, in which case appropriate liquid carriers, suspending agents, etc., can be used.

[0205] Preparations suitable for parenteral administration include aqueous and non-aqueous sterile injectable solutions, which may contain antioxidants, buffers, antibacterial agents, and solutes that make the preparation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickeners. Preparations can be presented in single-dose or multi-dose containers (e.g., sealed ampoules and vials) and can be stored under lyophilized (freeze-dried) conditions, requiring only the addition of a sterile liquid carrier (e.g., water for injection) immediately before use. Temporary injectable solutions and suspensions can be prepared from the aforementioned types of sterile powders, granules, and tablets.

[0206] The administration of the active compound can range from continuous (intravenous infusion) to several oral doses per day (e.g., QID), and among other routes of administration can be oral, topical, ocular or transocular, parenteral, intramuscular, intravenous, subcutaneous, transdermal (which may include penetration enhancers), buccal, and suppository administration, including via ocular or transocular routes.

[0207] Tumor-forming vaccines or immunogenic compositions may be administered by injection, oral administration, parenteral administration, inhalation spray, rectal administration, vaginal administration, or topical administration in unit dosage forms containing conventional pharmaceutically acceptable carriers, adjuvants, and delivery vehicles. As used herein, parenteral administration includes delivery to one or more lymph nodes, subcutaneously, intravenously, intramuscularly, intrasternally, via infusion techniques, intraperitoneally, through the eye or via the eye, intravitreal, buccally, percutaneously, intranasally, into the brain (including intracranial and intradural), into joints (including ankles, knees, hips, shoulders, elbows, and wrists), directly into tumors, and in suppository form.

[0208] Various techniques can be used to deliver the subject composition to the site of interest, such as injection, using catheters, cannulas, projectiles, pluronic gels, stents, sustained-release drug-releasing polymers, or other devices for internal access. When an organ or tissue is available due to removal from a patient, it can be immersed in a culture medium containing the subject composition, the subject composition can be applied to the organ, or it can be applied in any convenient manner.

[0209] Tumor-specific neoantigen peptides can be delivered via a device adapted to control and sustain the release of the composition in a manner that effectively achieves the desired local or systemic physiological or pharmacological effect. The method involves placing a sustained-release drug delivery system in the area where the drug is desired to be released and allowing the drug to pass through the device to reach the desired treatment area.

[0210] These tumor-specific neoantigen peptides can be used in combination with at least one other known therapeutic agent or a pharmaceutically acceptable salt of the said agent. Examples of known therapeutic agents that may be used 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 (e.g., busulfan, cisplatin, mitomycin C, and carboplatin); antimitotic agents such as colchicine, vincristine, paclitaxel, and docetaxel; topoisomerase I inhibitors such as camptothecin and topotecan; topoisomerase 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′-deoxyuridine, cytarabine, hydroxyurea, and thioguanine; and antibodies such as Herceptin and Rituxan.

[0211] It should be understood that, regarding the types of formulations under discussion, in addition to the ingredients specifically mentioned herein, the formulations of the present invention may also include other reagents conventional in the art, for example, those suitable for oral administration may also include flavoring agents.

[0212] Pharmaceutically acceptable salt forms may be preferred chemical forms of compounds according to the invention used in pharmaceutical compositions according to the invention.

[0213] The compounds of the present invention or their derivatives (including prodrug forms of these agents) may be provided in pharmaceutically acceptable salt forms. As used herein, the term pharmaceutically acceptable salt or complex refers to a suitable salt or complex of the active compound according to the invention that retains the desired biological activity of the compound and exhibits limited toxicological activity against normal cells. Non-limiting examples of such salts are (a) acid addition salts formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, etc.) and salts formed with organic acids, particularly such as acetic acid, oxalic acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic acid, tannic acid, pyruvic acid, alginic acid, and polyglutamic acid; and (b) base addition salts formed with metal cations, among a number of other cations such as zinc, calcium, sodium, potassium, etc.

[0214] The compounds described herein are commercially available or synthetically synthesizable. As will be understood by those skilled in the art, other methods for synthesizing compounds having the chemical formulas herein will be apparent to those of ordinary skill in the art. Furthermore, various synthetic steps may be performed in an alternating sequence or order to obtain the desired compound. Synthetic chemical transformations and protecting group methodologies (protection and deprotection) useful in the synthesis of the compounds described herein are known in the art and include, for example, those described in the following literature: R. Larock, Comprehensive Organic Transformations, 2nd ed., Wiley-VCH Publishers (1999); TW Greene and PGM Wuts, 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 Willie & Sons (1995), and subsequent editions.

[0215] Additional reagents that may be included with the tumor-specific neoantigen peptides of the present invention may contain one or more asymmetric centers and thus appear as racemic mixtures and mixtures of racemic compounds, single enantiomers, single diastereomers, and mixtures of diastereomers. All such isomeric forms of these compounds are explicitly included in this invention. The compounds of the present invention may also be represented in multiple tautomeric forms; in such cases, the present invention explicitly includes all tautomeric forms of the compounds described herein (e.g., alkylation of a ring system can lead to alkylation at multiple sites, and the present invention explicitly includes all such reaction products). All such isomeric forms of such compounds are explicitly included in this invention. All crystalline forms of the compounds described herein are explicitly included in this invention.

[0216] dose When these pharmaceutical agents described herein are given to humans or animals as medicines, they may be given on their own or as a composition comprising an active ingredient in combination with a pharmaceutically acceptable carrier, excipient or diluent.

[0217] The actual dose level and timing of administration of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of active ingredient that is effective in achieving the desired therapeutic response for a particular patient, composition, and route of administration, without toxicity to that patient. Typically, the agents or pharmaceutical compositions of the present invention are administered in amounts sufficient to reduce or eliminate symptoms associated with viral infections and / or autoimmune diseases.

[0218] The preferred dose of the drug is the maximum amount that is tolerable to the patient and does not produce serious or unacceptable side effects. Exemplary dose ranges include 0.01 mg to 250 mg / day, 0.01 mg to 100 mg / day, 1 mg to 100 mg / day, 10 mg to 100 mg / day, 1 mg to 10 mg / day, and 0.01 mg to 10 mg / day. The preferred dose of the drug is the maximum amount that is tolerable to the patient and does not produce serious or unacceptable side effects. In examples, the drug is administered at concentrations of about 10 micrograms to about 100 mg / kg body weight / day, about 0.1 to about 10 mg / kg / day, or about 1.0 mg to about 10 mg / kg body weight / day.

[0219] In the embodiments, the pharmaceutical composition comprises an amount ranging from 1 to 10 mg (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg).

[0220] In the examples, the therapeutically effective dose produces a serum concentration of the drug from about 0.1 ng / ml to about 50-100 mg / ml. These pharmaceutical compositions 5 should typically provide a dose from about 0.001 mg to about 2000 mg of compound per kilogram of body weight per day. For example, the dosage range for systemic administration to human patients can be 1-10 mg / kg, 20-80 mg / kg, 5-50 mg / kg, 75-150 mg / kg, 100-500 mg / kg, 250-750 mg / kg, 500-1000 mg / kg, 1-10 mg / kg, 5-50 mg / kg, 25-75 mg / kg, 50-100 mg / kg, 100-250 mg / kg, 50-100 mg / kg, 250-500 mg / kg, 500-750 mg / kg, 750-1000 mg / kg, 1000-1500 mg / kg, 1000-2000 mg / kg, 5 mg / kg, 20 mg / kg, 50 mg / kg, 100 mg / kg, 500 mg / kg, 1000 mg / kg, 15 ... mg / kg or 2000 mg / kg. Prepare pharmaceutical unit dosage forms to provide combinations of compounds or essential ingredients in quantities ranging from about 1 mg to about 5000 mg (e.g., from about 100 mg to about 2500 mg) per unit dosage form.

[0221] In one embodiment, the subject was given a dose of about 50 nM to about 1 μM of the drug. In related embodiments, the subject was given a dose of about 50-100 nM, 50-250 nM, 100-500 nM, 250-500 nM, 250-750 nM, 500-750 nM, 500 nM to 1 μM, or 750 nM to 1 μM of the drug.

[0222] The determination of an effective amount is entirely within the capabilities of a person skilled in the art, especially based on the specific disclosure provided herein. Typically, an effective or efficient amount of a drug is determined by first administering a low dose of this or these drugs and then incrementally increasing the administered dose or dose value until the desired effect (e.g., reduction or elimination of symptoms associated with viral infection or autoimmune disease) is observed in the treated subject with minimal or acceptable toxic side effects. The applicable methods for determining the appropriate dosage and dosing regimen for administering the pharmaceutical compositions of the present invention are described, for example, in Goodman and Gilman's The Pharmacological Basis of Therapeutics, edited by Goodman et al., 11th edition, McGraw-Hill 2005, and Remington: The Science and Practice of Pharmacy, 20th and 21st editions, edited by Gennaro and University of the Sciences in Philadelphia, Lippincott Williams & Wilkins (2003 and 2005), each of which is incorporated herein by reference.

[0223] Preferred unit-dose formulations are those containing the administered ingredient as discussed herein, such as daily doses or units, daily sub-dose, or appropriate portions thereof.

[0224] Dosing regimens for treating disorders or diseases using the tumor-specific neoantigen peptides and / or compositions of the present 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 specific compound used. Therefore, dosing regimens can vary widely, but can be routinely determined using standard methods.

[0225] The amount and regimen of administration given to a subject may depend on a variety of factors, such as the route 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 scope of a person skilled in the art, to the best of this disclosure and knowledge of the art.

[0226] The amount of compound included in a therapeutically active formulation according to the invention is an effective amount for treating a disease or condition. Generally, for a patient, the range of therapeutically effective amounts of the preferred compounds of the invention in a dosage form is typically from slightly less than about 0.025 mg / kg / day to about 2.5 g / kg / day, preferably about 0.1 mg / kg / day to about 100 mg / kg / day or much more, depending on the compound used, the condition or infection being treated, and the route of administration; however, exceptions to this dosage range are also considered in the invention. The compounds according to the invention are administered in their most preferred form at amounts ranging from about 1 mg / kg / day to about 100 mg / kg / day. The dosage of the compound can depend on the condition being treated, the specific compound, and other clinical factors such as the patient's weight and condition and the route of administration. It should be understood that the invention is applicable to both human and veterinary use.

[0227] For oral administration to humans, a dose between approximately 0.1 and 100 mg / kg / day, preferably between approximately 1 and 100 mg / kg / day, is generally sufficient.

[0228] In cases where drug delivery is systemic rather than local, this dosage range typically produces effective blood concentrations of the active compound ranging from less than about 0.04 to about 400 micrograms / cc of blood or more in the patient. The compound is conveniently administered in any suitable unit dosage form, including but not limited to unit dosage forms containing 0.001 to 3000 mg, preferably 0.05 to 500 mg of the active ingredient per unit. Oral doses of 10-250 mg are generally convenient.

[0229] According to certain exemplary embodiments, the vaccine or immunogenic composition is administered at a dose of about 10 μg to 1 mg for each neoantigenic 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 for each neoantigenic peptide.

[0230] The concentration of the active compound in the pharmaceutical composition will depend on the rate of absorption, distribution, inactivation, and excretion of the drug, as well as other factors known to those skilled in the art. It should be noted that dosage values ​​also vary depending on the severity of the condition to be alleviated. It should be further understood that, for any specific subject, the particular dosing regimen should be adjusted over time based on individual needs and the professional judgment of the person administering the composition or supervising its administration, and the concentration ranges listed herein are merely exemplary and not intended to limit the range or practice of the required compositions. The active ingredient may be administered once or divided into many smaller doses to be administered at different time intervals.

[0231] This invention provides pharmaceutical compositions comprising at least one tumor-specific neoantigen described herein. In embodiments, these pharmaceutical compositions comprise a pharmaceutically acceptable carrier, excipient, or diluent, which includes any pharmaceutical agent that, on its own, does not induce an adverse immune response in a subject receiving the composition and can be administered without adverse toxicity. As used herein, the term "pharmaceuticalally acceptable" means approved by a federal or state regulatory agency or listed in the United States Pharmacopeia, the European Pharmacopeia, or other recognized pharmacopoeia for use in mammals, and more specifically in humans. These compositions can be used to treat and / or prevent viral infections and / or autoimmune diseases.

[0232] A full discussion of pharmaceutically acceptable carriers, diluents, and other excipients can be found in Remington's Pharmaceutical Sciences (17th edition, Mack Publishing) and Remington: The Science and Practice of Pharmacy (21st edition, Lippincott Williams & Wilkins), which are incorporated herein by reference. The formulation of this pharmaceutical composition should be suitable for the route of administration. In the examples, the pharmaceutical composition is suitable for human administration and may be sterile, non-particulate, and / or pyrogen-free.

[0233] Pharmaceutically acceptable carriers, excipients, or diluents include, but are not limited to, physiological saline, buffered physiological saline, glucose, water, glycerol, ethanol, sterile isotonic buffer solutions, and combinations thereof.

[0234] These compositions may also contain wetting agents, emulsifiers and lubricants (such as sodium lauryl sulfate and magnesium stearate), as well as colorants, release agents, coating agents, sweeteners, flavoring agents, and aroma agents, preservatives and antioxidants.

[0235] Examples of pharmaceutically acceptable antioxidants include, but are not limited to: (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, etc.; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, etc.; and (3) metal chelating agents, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0236] In the embodiments, the pharmaceutical composition is provided in solid form (such as lyophilized powder suitable for rehydration), liquid solution, suspension, emulsion, tablet, pill, capsule, sustained-release formulation or powder.

[0237] In one embodiment, the pharmaceutical composition is provided in liquid form, for example, in a sealed container indicating the amount and concentration of the active ingredient in the pharmaceutical composition. In a related embodiment, the liquid form of the pharmaceutical composition is provided in a melt-sealed container.

[0238] The methods used to formulate the pharmaceutical compositions of the present invention are conventional and well known in the art (see Remington and Remington). Those skilled in the art can readily formulate pharmaceutical compositions having desired characteristics (e.g., route of administration, biocompatibility, and release profile).

[0239] Methods for preparing these pharmaceutical compositions include the step of combining an active ingredient with a pharmaceutically acceptable carrier and optionally one or more auxiliary ingredients. A pharmaceutical composition can be prepared by uniformly and tightly binding the active ingredient with a liquid carrier or a finely dispersed solid carrier, or both, and then, if necessary, shaping the product. Further methods for preparing pharmaceutical compositions (including the preparation of multilayers) are described in Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems (9th edition, Lippincott Williams & Wilkins), which is incorporated herein by reference.

[0240] Pharmaceutical compositions suitable for oral administration may be in the form of capsules, flat capsules, pills, tablets, sugar lozenges (using a flavoring matrix, typically sucrose and gum arabic or astragalus gum), powders, granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as soft lozenges (using an inert matrix, such as gelatin and glycerin, or sucrose and gum arabic), and / or as mouthwashes, each comprising a predetermined amount of one or more compounds described herein, their derivatives, pharmaceutically acceptable salts, or prodrugs as one or more active ingredients. The active ingredient may also be administered as a large pill, medicated sugar, or paste.

[0241] In solid dosage forms intended for oral administration (e.g., capsules, tablets, pills, sugar-coated pills, powders, granules, etc.), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, excipients, or diluents (e.g., sodium citrate or dicalcium phosphate) and / or any of the following: (1) fillers or extenders, such as starch, lactose, sucrose, glucose, mannitol, and / or silicate; (2) binders, such as carboxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or gum arabic; (3) humectants, such as glycerin; (4) disintegrants, such as agar, calcium carbonate, potato or cassava starch, alginate, certain silicates, and sodium carbonate; (5) dissolution inhibitors, such as paraffin; (6) absorption enhancers, such as quaternary ammonium compounds; (7) humectants, such as acetyl alcohol and glyceryl monostearate; (8) adsorbents, such as kaolin and bentonite; (9) Lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) colorants. In the case of capsules, tablets, and pills, these pharmaceutical compositions may also include buffers. Similar types of solid compositions may also be prepared in soft-filled and hard-filled gelatin capsules using fillers and excipients such as lactose or milk sugar and high molecular weight polyethylene glycol.

[0242] Tablets can be prepared by compression or molding, optionally with one or more excipients. Compressed tablets can be prepared using binders (e.g., gelatin or hydroxypropyl methylcellulose), lubricants, inert diluents, preservatives, disintegrants (e.g., sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surfactants, and / or dispersants. Molded tablets can be prepared by molding a mixture of powdered active ingredients wetted with an inert liquid diluent in a suitable machine.

[0243] These tablets and other solid dosage forms (such as sugar-coated pills, capsules, pellets and granules) can optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings known in the art.

[0244] In some embodiments, to prolong the effect of the active ingredient, it is desirable to slow the absorption of the subcutaneously or intramuscularly injected compound. This can be achieved by using a liquid suspension of crystals or amorphous materials with poor water solubility. Thus, the absorption rate of the active ingredient depends on its dissolution rate, which in turn can depend on the crystal size and crystal form. Alternatively, delayed absorption of the compound can be achieved by dissolving or suspending the parenterally administered active ingredient in an oil carrier. Furthermore, prolonged absorption of injectable drug forms can be achieved by using reagents that contain delayed absorption agents, such as aluminum monostearate and gelatin.

[0245] The controlled-release parenteral composition may be in the form of an aqueous suspension, microspheres, microcapsules, magnetic microspheres, oil solution, oil suspension, or emulsion, or the active ingredient may be incorporated into one or more biocompatible carriers, liposomes, nanoparticles, implants, or infusion devices.

[0246] Materials used in the preparation of microspheres and / or microcapsules include biodegradable / biodegradable polymers such as hydroxyacetic acid lactate polyester, poly(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutamine), and poly(lactic acid).

[0247] When preparing controlled-release parenteral preparations, biocompatible carriers that can be used include carbohydrates (such as dextran), proteins (such as albumin), lipoproteins, or antibodies.

[0248] Materials used in implants can be non-biodegradable (e.g., polydimethylsiloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid), or poly(orthoester)).

[0249] In the embodiments, this or these active ingredients are delivered via aerosol. This is achieved by preparing a wet aerosol, a liposome formulation, or solid particles containing the compound. A non-aqueous suspension, such as a fluorocarbon propellant, can be used. The pharmaceutical composition can also be delivered using a sonic nebulizer, which minimizes the agent's exposure to shear, which can lead to the degradation of the compound.

[0250] Typically, wet aerosols are prepared by formulating this or these active ingredients into an aqueous solution or suspension with conventional, pharmaceutically acceptable carriers and stabilizers. These carriers and stabilizers vary depending on the specific compound requirements, but typically include nonionic surfactants (Tweens, Pluronics, or polyethylene glycol), nontoxic proteins (like serum albumin), sorbitol esters, oleic acid, lecithin, amino acids (such as glycine), buffers, salts, sugars, or sugar alcohols. Aerosols are usually prepared from isotonic solutions.

[0251] Dosage forms for topical or transdermal administration of one or more active ingredients include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalers. These active ingredients may be mixed under sterile conditions with pharmaceutically acceptable carriers and with any suitable preservatives, buffers, or propellants.

[0252] Suitable transdermal patches for use in this invention are disclosed in Transdermal Drug Delivery: Developmental Issues and Research Initiatives (Marcel Dekker Inc., 1989) and U.S. Patent Nos. 4,743,249, 4,906,169, 5,198,223, 4,816,540, 5,422,119, and 5,023,084, which are incorporated herein by reference. The transdermal patch may also be any transdermal patch well known in the art, including transscrotal patches. Pharmaceutical compositions in such transdermal patch form may contain one or more absorption enhancers or skin penetration enhancers well known in the art (see, for example, U.S. Patent Nos. 4,379,454 and 4,973,468, which are incorporated herein by reference). Transdermal therapeutic systems for use in this invention may be based on iontophoresis, diffusion, or a combination of both.

[0253] Transdermal patches offer the additional advantage of providing controlled delivery of one or more active ingredients into the body. Such dosage forms can be prepared by dissolving or dispersing the active ingredient or ingredients in a suitable medium. Absorption enhancers can also be used to increase the flow rate of the active ingredient across the skin. This flow rate can be controlled by providing a rate-controlled membrane or by dispersing the active ingredient or ingredients in a polymer matrix or gel.

[0254] Such pharmaceutical compositions can be in the form of creams, ointments, lotions, liniments, gels, hydrogels, solutions, suspensions, adhesives, sprays, pastes, plasters, and other types of transdermal drug delivery systems. These compositions may also include pharmaceutically acceptable carriers or excipients, such as emulsifiers, antioxidants, buffers, preservatives, humectants, penetration enhancers, chelating agents, gelling agents, ointment bases, fragrances, and skin protectants.

[0255] Examples of emulsifiers include, but are not limited to, naturally occurring gums (such as gum arabic or astragalus gum), naturally occurring phospholipids (such as soybean lecithin), and sorbitol monooleate derivatives.

[0256] Examples of antioxidants include, but are not limited to, butylated hydroxyanisole (BHA), ascorbic acid and its derivatives, tocopherol and its derivatives, and cysteine.

[0257] Examples of preservatives include, but are not limited to, parabens (such as methylparaben or propylparaben) and benzyl alkyl chloride.

[0258] Examples of humectants include, but are not limited to, glycerol, propylene glycol, sorbitol, and urea.

[0259] 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 esters together with propylene glycol monolaurate or methyl laurate, eucalyptol, lecithin, TRANSCUTOL, and AZONE.

[0260] Examples of chelating agents include, but are not limited to, sodium EDTA, citric acid, and phosphoric acid.

[0261] Examples of gelling agents include, but are not limited to, carbomer, cellulose derivatives, bentonite, alginate, gelatin, and polyvinylpyrrolidone.

[0262] In addition to these active ingredients, the ointments, pastes, creams and gels of the present invention may also contain excipients (such as animal and vegetable fats), oils, waxes, paraffin, starch, astragalus gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silicic acid, talc and zinc oxide or mixtures thereof.

[0263] Powders and aerosols may contain excipients such as lactose, talc, silica, aluminum hydroxide, calcium silicate, and polyamide powders or mixtures thereof. Aerosols may also contain conventional propellants such as chlorofluorocarbons, and volatile unsubstituted hydrocarbons such as butane and propane.

[0264] Injectable long-acting formulations are prepared by forming microcapsule matrices of one or more compounds within a biodegradable polymer, such as polylactide-polyglycolic acid. The release rate of the compound can be controlled depending on the ratio of the compound to the polymer and the properties of the specific polymer used. Other examples of biodegradable polymers include poly(orthoesters) and poly(anhydrides). Long-acting injectable formulations are also prepared by encapsulating the drug in tissue-compatible liposomes or microemulsions.

[0265] Subcutaneous implants are well known in the art and are suitable for use in this invention. Subcutaneous implantation methods are preferably non-irritating and mechanoelastic. These implants can be matrix-type, reservoir-type, or hybrids thereof. In matrix-type devices, the carrier material can be porous or non-porous, solid or semi-solid, and permeable or impermeable to this or these active compounds. The carrier material can be biodegradable or slowly corroded after administration. In some cases, the matrix is ​​non-degradable, and the degradation of the carrier material depends on the diffusion of the active compound through the matrix. Alternative subcutaneous implantation methods utilize reservoir devices in which this or these active compounds are surrounded by a rate-controlled membrane, such as a membrane independent of component concentration (with zero-order kinetics). Devices consisting of a matrix surrounded by a rate-controlled membrane are also suitable for use.

[0266] Both storage and matrix-type devices can contain a variety of materials, such as polydimethylsiloxane (e.g., SILASTIC) or other silicone rubbers. The matrix material can be insoluble polypropylene, polyethylene, polyvinyl chloride, ethyl vinyl acetate, polystyrene and polymethacrylate, as well as glycerides of the types of glyceryl palmitate, glyceryl stearate, and glyceryl betaine. The material can be a hydrophobic or hydrophilic polymer and optionally contains a solubilizer.

[0267] The subcutaneous implantable device can be a sustained-release capsule made of any suitable polymer, such as those described in U.S. Patent Nos. 5,035,891 and 4,210,644, which are incorporated herein by reference.

[0268] Typically, at least four different methods are suitable for providing control over the release and transdermal permeation rate of drug compounds. These methods include: membrane-moderated systems, adhesive diffusion-controlled systems, matrix dispersion-type systems, and microreservoir systems. It should be understood that controlled release of transdermal and / or topical compositions can be obtained by using appropriate combinations of these methods.

[0269] In a modulated membrane system, the active ingredient is contained within a reservoir completely encapsulated in a shallow compartment molded from a drug-impermeable laminate (such as a metal-plastic laminate) and a rate-controlled polymeric membrane (such as a microporous or non-porous polymeric membrane, for example, ethylene-vinyl acetate copolymer). The active ingredient is released through this rate-controlled polymeric membrane. Within the drug reservoir, the active ingredient can be dispersed in a solid polymer matrix or suspended in a non-leaching viscous liquid matrix (such as silicone solution). A thin layer of adhesive polymer is applied to the outer surface of the polymeric membrane to achieve close contact between the transdermal system and the skin surface. The adhesive polymer is preferably a hypoallergenic polymer compatible with the active pharmaceutical ingredient.

[0270] In a controlled adhesive diffusion system, the storage chamber of the active ingredient is formed by directly dispersing the active ingredient in an adhesive polymer and then, for example, by solvent casting, dispersing the adhesive polymer containing the active ingredient onto a substantially impermeable metal-plastic backing plate to form a thin drug storage chamber layer.

[0271] The matrix dispersion type system is characterized in that the storage chamber of the active ingredient is formed by dispersing the active ingredient substantially homogeneously in a hydrophilic or lipophilic polymer matrix. The drug-containing polymer is then molded into a disc having a substantially well-defined surface area and a controlled thickness. A binding polymer extends circumferentially to form an adhesive strip around the disc.

[0272] Micro-reservoir systems can be considered a combination of reservoir and matrix dispersion systems. In this case, the reservoir of the active substance is formed by first suspending the drug solid in an aqueous solution of a water-soluble polymer and then dispersing the drug suspension in a lipophilic polymer to form a large number of non-leaching drug reservoir microspheres.

[0273] Any controlled-release, extended-release, and sustained-release compositions described herein can be formulated to release the active ingredient over approximately 30 minutes to approximately 1 week, approximately 30 minutes to approximately 72 hours, approximately 30 minutes to approximately 24 hours, approximately 30 minutes to approximately 12 hours, approximately 30 minutes to approximately 6 hours, approximately 30 minutes to approximately 4 hours, and approximately 3 hours to approximately 10 hours. In embodiments, the effective concentration of this or these active ingredients persists in the subject for 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, or longer after administration of these pharmaceutical compositions to the subject.

[0274] Vaccines or immunogenic compositions This invention relates to an immunogenic composition, such as a tumor-forming vaccine or immunogenic composition capable of evoking a specific T-cell response. The tumor-forming vaccine or immunogenic composition comprises neoantigen peptides and / or neoantigen polypeptides corresponding to tumor-specific neoantigens identified by the methods described herein.

[0275] Suitable tumor-forming vaccines or immunogenic compositions may preferably comprise a variety of tumor-specific neoantigen peptides. In one embodiment, the vaccine or immunogenic composition may comprise between 1 and 100 groups of peptides, more preferably between 1 and 50 such peptides, even more preferably between 10 and 30 groups of peptides, and even more preferably between 15 and 25 peptides. According to another preferred embodiment, the vaccine or immunogenic composition may comprise at least one peptide, more preferably 2, 3, 4, or 5 peptides. In some 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.

[0276] Those skilled in the art can determine the optimal amount and administration regimen for each peptide to be included in a vaccine or immunogenic composition without excessive experimentation. For example, the peptide or a variant thereof can be prepared for intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), and intramuscular (im) injection. Preferred methods of injecting peptides include sc, id, ip, im, and iv. Preferred methods of injecting DNA include id, im, sc, ip, and iv. For example, doses of peptide or DNA between 1 and 500 mg, between 50 μg and 1.5 mg, preferably between 10 μg and 500 μg, can be administered, depending on the specific peptide or DNA. This range of dosages has been successfully used in previous trials (Brunsvig PF et al., Cancer Immunol Immunother. 2006; 55(12): 1553-1564; M. Staehler et al., ASCO meeting 2007; Abstract No. 3017). Other methods of administering vaccines or immunogenic compositions are known to those skilled in the art.

[0277] In one embodiment of the invention, these different tumor-specific neoantigen peptides and / or polypeptides are selected for use in tumorigenesis vaccines or immunogenic compositions to maximize the potential for generating an immune attack against tumorigenesis / tumor in a patient. Not bound by theory, it is believed that a diverse range of tumor-specific neoantigen peptides can generate a large-scale immune attack against tumorigenesis / tumor. In one embodiment, these selected tumor-specific neoantigen peptides / polypeptides are encoded by missense mutations. In a second embodiment, these selected tumor-specific neoantigen peptides / polypeptides are encoded by a combination of missense mutations and novel ORF mutations. In a third embodiment, these selected tumor-specific neoantigen peptides / polypeptides are encoded by novel ORF mutations.

[0278] In one embodiment where these selected tumor-specific neoantigen peptides / peptides are encoded by missense mutations, these peptides and / or peptides are selected based on their ability to associate with a patient's specific MHC molecule. Peptides / peptides derived from novel ORF mutations may also be selected based on their ability to associate with a patient's specific MHC molecule, but may be selected even if association with a patient's specific MHC molecule is not predicted.

[0279] The vaccine or immunogenic composition can induce specific cytotoxic T-cell responses and / or specific helper T-cell responses.

[0280] The vaccine or immunogenic composition may further include adjuvants and / or carriers. Examples of useful adjuvants and carriers are given herein. The peptides and / or polypeptides in the composition may associate with a carrier (e.g., a protein) or antigen-presenting cells (e.g., dendritic cells (DCs) capable of presenting peptides to T cells).

[0281] Adjuvants are any substances incorporated into the vaccine or immunogenic composition to enhance or otherwise alter the immune response against the mutant peptide. A carrier is a scaffold structure, such as a polypeptide or polysaccharide, to which these neoantigenic peptides can associate. Optionally, the adjuvant is covalently or non-covalently conjugated to the peptide or polypeptide of the invention.

[0282] The ability of adjuvants to increase the immune response against an antigen is typically manifested through a significant increase in immune-mediated responses or a reduction in disease symptoms. For example, an increase in humoral immunity is typically manifested through a significant increase in the titer of antibodies produced against the antigen, and an increase in T cell activity is typically manifested through increases in cell proliferation, cytotoxicity, or cytokine secretion. Adjuvants can also alter the immune response, for example, by converting the primary humoral or Th2 response into a primary cellular or Th1 response.

[0283] 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, ISCMATRIX, Juvlmmune, LipoVac, MF59, monophospholipase A, Montaned IMS 1312, Montaned ISA 206, Montaned ISA 50V, Montanid ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PEPTEL, vector systems, PLG microparticles, Requimod, SRL172, virions and other virus-like particles, YF-17D, VEGF traps, R848, β-glucan, Pam3Cys, Aquila QS21 stimulator derived from saponins (Aquila Biotech, Worcester, MA, USA), mycobacterial extracts and synthetic bacterial cell wall mimics, and other patented adjuvants such as Ribi's Detox. Quil or Superfos. Several specific adjuvants against dendritic cells (e.g., MF59) and their preparation have been previously described (Dupuis M et al., Cell Immunol. 1998; 186(1): 18-27; Allison AC; Progress in Biostandardization 1998; 92: 3-11). Cytokines may also be used. Several cytokines have been directly associated with influencing dendritic cell migration to lymphoid tissues (e.g., TNF-α), accelerating dendritic cell maturation into T lymphocytes as effective antigen-presenting cells (e.g., GM-CSF, IL-1, and IL-4) (US Patent No. 5,849,589, which is specifically incorporated herein by reference in its entirety), and acting as immune adjuvants (e.g., IL-12) (Gabrilovich DI et al., Journal of Immunother Emphasis Tumor Immunol. 1996(6): 414-418).

[0284] Toll-like receptors (TLRs) can also be used as adjuvants and are important members of the pattern recognition receptor (PRR) family, which recognizes conserved motifs shared by many microorganisms, known as pathogen-associated molecular patterns (PAMPs). Recognition of these "danger signals" activates multiple elements of the innate and adaptive immune systems. TLRs are expressed by cells of the innate and adaptive immune systems, such as dendritic cells (DCs), macrophages, T cells and B cells, mast cells, and granulocytes, and are located in different cellular compartments, such as the plasma membrane, lysosomes, endosomes, and endolysinic lysosomes. Different TLRs recognize different PAMPs. For example, TLR4 is activated by LPS contained in the bacterial cell wall, TLR9 is activated by unmethylated bacterial or viral CpG DNA, and TLR3 is activated by double-stranded RNA. TLR ligand binding leads to the activation of one or more intracellular signal transduction pathways, ultimately resulting in the production of many key molecules related to inflammation and immunity, particularly the transcription factors NF-κB and type I interferons. TLR-mediated dendritic cell (DC) activation leads to enhanced DC activation, phagocytosis, upregulation of activation and co-stimulatory markers (such as CD80, CD83, and CD86), expression of CCR7, thereby allowing DCs to migrate to draining lymph nodes and facilitating antigen presentation to T cells, and increased secretion of cytokines (such as type I interferon, IL-12, and IL-6). All of these downstream events are crucial for inducing an adaptive immune response.

[0285] Currently, the most promising adjuvants for cancer vaccines or immunogenic compositions 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 because it induces pro-inflammatory cytokines, does not require IL-10 stimulation, and maintains high levels of co-stimulatory molecules in DCs1. Furthermore, as an adjuvant for a protein vaccine or immunogenic composition, poly-ICLC has recently been directly compared with CpG in non-human primates (rhesus macaques) consisting of a human papillomavirus (HPV) 16 capsid (Stahl-Hennig C, Eisenblatter M, Jasny E et al. synthesized double-stranded RNAs in rhesus macaques as adjuvants for the induction of T helper 1 and humoral immune responses to human papillomavirus). PLoS pathogens. April 2009; 5(4).

[0286] It has also been reported that CpG immunostimulatory oligonucleotides can enhance the effects of adjuvants in the context of vaccines or immunogenic compositions. Unbound by theory, CpG oligonucleotides act by activating the innate (adaptive) immune system via Toll-like receptors (TLRs), primarily TLR9. CpG-triggered TLR9 activation enhances antigen-specific humoral and cellular responses against a wide variety of antigens, including peptide or protein antigens in both prophylactic and therapeutic vaccines, live or dead viruses, dendritic cell vaccines, autologous cell vaccines, and polysaccharide conjugates. More importantly, it enhances dendritic cell maturation and differentiation, leading to enhanced Th1 cell activation and the production of more potent cytotoxic T lymphocytes (CTLs), even in the absence of CD4 T cells. The TLR9-induced Th1 bias is maintained even in the presence of vaccine adjuvants such as alum or incomplete Freund's adjuvant (IFA), which typically promote 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 for inducing stronger responses when the antigen is relatively weak. They also promote immune responses and allow for antigen dose reductions of approximately two orders of magnitude, while producing comparable antibody responses to full-dose vaccines in some experiments without CpG (Arthur M. Krieg, Nature Reviews, Drug Discovery, 5, June 2006, 471-484). U.S. Patent No. 6,406,705 B1 describes the combined use of CpG oligonucleotides, non-nucleic acid adjuvants, and antigens to induce antigen-specific immune responses. One commercially available CpG TLR9 antagonist is dSLIM (double stem-ring immunomodulator) from Mologen (Berlin, Germany), which is a preferred component of the pharmaceutical compositions of the present invention. Other TLR-binding molecules, such as TLR 7, TLR 8 and / or TLR 9 which bind RNA, can also be used.

[0287] Other examples of useful adjuvants include, but are not limited to, chemically modified CpGs (e.g., CpR, Idera), poly(I:C) molecules (e.g., polyi:CI2U), non-CpG bacterial DNA or RNA, and immunologically active small molecules, as well as antibodies that can act therapeutically and / or as adjuvants, such as cyclophosphamide, sunitinib, bevacizumab, celecoxib, NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175. Those skilled in the art can readily determine the amount and concentration of adjuvants and additives useful in the context of this invention without excessive experimentation. Additional adjuvants include colony-stimulating factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF, saxaglastine).

[0288] Poly-ICLC is a synthetically prepared double-stranded RNA composed of polyI and polyC chains with an average length of approximately 5000 nucleotides. It has been stabilized against heat denaturation and hydrolysis by serum nucleases through the addition of polylysine and carboxymethyl cellulose. This compound activates the RNA helicase-domains of TLR3 and MDA5 (two members of the PAMP family), leading to the activation of dendritic cells (DCs) and natural killer (NK) cells, and the production of a “natural mix” of type I interferons, cytokines, and chemokines. Furthermore, poly-ICLC exerts more direct, broad-targeted anti-infective and potentially antitumor effects mediated by two IFN-inducible ribozyme systems: 2'5'-OAS and P1 / eIF2a kinases (also known as PKR(4-6)) and RIG-I helicase and MDA5.

[0289] In rodents and non-human primates, poly-ICLCs have been shown to enhance T-cell responses to viral antigens, cross-sensitize, and induce tumor-, virus-, and autoantigen-specific CD8+ T cells. A recent study in non-human primates found that poly-ICLCs are essential for generating antibody responses and T-cell immunity against both DC-targeted and non-targeted HIV Gag p24 proteins, highlighting their effectiveness as a vaccine adjuvant.

[0290] In human subjects, transcriptional analysis of a series of whole blood samples revealed similar gene expression profiles among eight healthy human volunteers who received a single subcutaneous administration of poly-ICLC and differential expression of up to 212 genes between these eight subjects and four subjects who received placebo. Notably, comparisons of poly-ICLC gene expression data with previous data from volunteers immunized with the highly effective yellow fever vaccine YF17D showed that a large number of classical transcriptional and signal transduction pathways (including those of the innate immune system) were similarly upregulated at peak time points.

[0291] Recently, an immunological analysis was reported on patients with ovarian, fallopian tube, and primary peritoneal cancer who achieved complete clinical remission in phase II or III. These patients were treated in a phase I subcutaneous vaccination study with a synthetic overlapping long peptide (OLP) derived from the testicular cancer antigen NY-ESO-1, alone or in combination with Montanede-ISA-51, or in combination with 1.4 mg of poly-ICLC and Montanede. Compared with OLP alone or OLP and Montanede alone, the addition of poly-ICLC and Montanede significantly enhanced the generation of NY-ESO-1-specific CD4+ and CD8+ T cell and antibody responses.

[0292] The vaccine or immunogenic composition according to the invention may include more than one different adjuvant. Furthermore, the invention covers a therapeutic composition comprising any adjuvant substance, including any of those discussed herein. It is also contemplated that the peptide or polypeptide and the adjuvant may be administered in any suitable order.

[0293] The vector can exist independently of the adjuvant. The vector can be covalently linked to the antigen. The vector can also be added to the antigen by inserting the DNA encoding the vector into a frame containing DNA encoding the antigen. The function of the vector can be, for example, to confer stability, increase biological activity, or increase serum half-life. A prolonged half-life can help reduce the number of applications and thus the dosage, which is beneficial for treatment and also for economic reasons. Furthermore, the vector can assist in the presentation of peptides to T cells. The vector can be any suitable vector known to those skilled in the art, such as a protein or antigen-presenting cell. The carrier protein can be, but is not limited to, key-foraminifera 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 use in human immunization, the vector can be physiologically acceptable and safe for humans. However, tetanus toxoid and / or diphtheria toxoid are suitable vectors in one embodiment of the invention. Alternatively, the vector can be a dextran, such as agarose.

[0294] Cytotoxic T cells (CTLs) recognize antigens in the form of peptides bound to MHC molecules, rather than the intact exogenous antigens themselves. The MHC molecules themselves are located on the cell surface of antigen-presenting cells. Therefore, CTLs can only be activated in the presence of a trimerized complex of the peptide antigen, the MHC molecule, and the APC. Accordingly, this can enhance the immune response not only if the peptide is used for CTL activation, but also if an APC having the corresponding MHC molecule is additionally added. Therefore, in some embodiments, the vaccine or immunogenic composition according to the invention further comprises at least one antigen-presenting cell.

[0295] The antigen-presenting cell (or stimulating cell) typically has MHC class I or II molecules on its surface and, in one embodiment, is substantially unable to load MHC class I or II molecules with selected antigens on its own. As described in more detail herein, the MHC class I or II molecules can be readily loaded with selected antigens in vitro.

[0296] CD8+ cell activity can be increased by using CD4+ cells. Identification of CD4+ T+ cell epitopes against tumor antigens has attracted interest because many immunotherapy-based anticancer therapies can be more effective if both CD8+ and CD4+ T lymphocytes are used to target a patient's tumor. CD4+ cells can enhance CD8 T cell responses. Numerous animal model studies have clearly demonstrated that better results are achieved when both CD4+ and CD8+ T cells are involved in the antitumor response (see, for example, Nishimura et al. (1999), Distinct role of antigen-specific T helper type 1 (TH1) and Th2 cells intumor eradication in vivo. Journal of Experimental Medicine (J Ex Med) 190: 617-27). Universal CD4+ T cell epitopes have been identified that can be used to develop therapies against different types of cancer (see, for example, Kobayashi et al. (2008), Current Opinion in Immunology, 20: 221-27). For example, HLA-DR-restricted helper peptides from tetanus toxoid have been used in melanoma vaccines to nonspecifically activate CD4+ T cells (see, for example, Slingluff et al. (2007), Immunologic and Clinical Outcomes of a Randomized Phase II Trial of Two Multipeptide Vaccines for Melanoma in the Adjuvant Setting, Clinical Cancer Research, 13(21): 6386-95). Within the scope of this invention, it is contemplated that such CD4+ cells can be applied at three levels, depending on their tumor specificity: 1) a broad level, in which a universal CD4+ epitope (e.g., tetanus toxoid) can be used to increase CD8+ cells; 2) an intermediate level, in which a natural tumor-associated CD4+ epitope can be used to increase CD8+ cells; and 3) a patient-specific level, in which a neoantigen CD4+ epitope can be used to increase CD8+ cells in a patient-specific manner.

[0297] CD8+ cell immunity can also be generated using neoantigen-loaded dendritic cell (DC) vaccines. DCs are potent antigen-presenting cells that initiate T-cell immunity and can be used as cancer vaccines when loaded with one or more peptides of interest, for example, via direct peptide injection. For example, it has been shown that newly diagnosed patients with metastatic melanoma can be immunized with mature CD40L / IFN-g-activated DCs treated with autologous peptide pulses using a patient DC vaccine that produces IL-12p70 against three HLA-A*0201-restricted gp100 melanoma antigen-derived peptides (see, for example, L-12p70-producing patient DC vaccineelicits Tc1-polarized immunity, Journal of Clinical Investigation, 123(8): 3383-94 and Ali et al. (2009) In situ regulation of DC subsets and T cells mediates tumor regression in mice, Cancer Immunotherapy, 1(8): 1-10). Within the scope of this invention, it is contemplated that neoantigen-loaded DCs can be prepared using polyinosinic acid-polycytidylic acid-poly-L-lysine carboxymethyl cellulose (poly-ICLC), a synthetic TLR 3 agonist that stimulates DCs. Poly-ICLC is a potent individual maturation stimulant for human DCs, as assessed by upregulating CD83 and CD86, inducing 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 producing minimal interleukin-10 (IL-10). DCs can differentiate from frozen peripheral blood mononuclear cells (PBMCs) obtained through leukocyte ablation, which can be separated by polysucrose gradient centrifugation and frozen in aliquots.

[0298] Illustratively, the following 7-day activation protocol can be used. Day 1—Thaw PBMCs and plate them on tissue culture flasks to select monocytes that adhere to the plastic surface after incubation at 37°C for 1–2 hours in a tissue culture incubator. After incubation, lymphocytes are washed away and the adhered 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 key-foraminifera hemocyanin (KLH), which serves as a control for vaccine quality and can enhance the immunogenicity of the vaccine. These DCs are stimulated to maturity, loaded with peptide antigens, and incubated overnight. On Day 7, the cells are washed and frozen in 1 ml aliquots containing 4–20 x 10(6) cells using a rate-controlled freezer. Batch release testing can be performed on these DCs to meet minimum specifications before injecting them into patients (see, for example, Preparation of tumorantigen-loaded mature dendritic cells for immunotherapy by Sabado et al. (2013), Journal of Visual Experimentation, August 1; (78). doi: 10.3791 / 50085).

[0299] DC vaccines can be incorporated into scaffold systems to facilitate delivery to patients. Therapeutic treatment of tumor formation in patients with DC vaccines can utilize a biomaterial system that releases factors that recruit host dendritic cells into the device, simultaneously releases antigens via local adjuvant presentation (e.g., a danger signal), differentiates resident immature DCs, and promotes the release of activated antigen-loaded DCs to lymph nodes (or desired sites of action), where these DCs can interact with T cells to generate a potent cytotoxic T-lymphocyte response against cancer neoantigens. Implantable biomaterials can be used to generate a potent cytotoxic T-lymphocyte response against tumor formation in a patient-specific manner. These biomaterial-resident dendritic cells can then be activated by exposing them to a danger signal-mimicking infection, consistent with the release of antigens from the biomaterial. These activated dendritic cells then migrate from the biomaterial to lymph nodes to induce a cytotoxic T-effective response. This approach has previously been shown to induce regression of established melanomas in preclinical studies using lysates prepared from tumor biopsies (see, for example, Ali et al. (2209), In situ regulation of DC subsets and T cells mediates 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), and such a vaccine is currently being tested in a recently initiated Phase I clinical trial at the Dana-Farber Cancer Institute. It has also been shown that, using the C6 rat glioma model of the present proposal24, this approach induces regression of glioblastomas and induces a potent memory response to prevent recurrence. Such an implantable bio-based vaccine delivery scaffold can amplify and maintain tumor-specific dendritic cell activation, producing more robust anti-tumor immunosensitization than that achievable through conventional subcutaneous or intranodal vaccine administration.

[0300] Preferably, these antigen-presenting cells are dendritic cells. Suitablely, these dendritic cells are autologous dendritic cells treated with a pulse of the neoantigen peptide. The peptide can be any suitable peptide that elicits an appropriate T-cell response. T-cell therapy using autologous dendritic cells treated with a pulse of a peptide derived from a tumor-associated antigen is disclosed in Murphy et al. (1996), The Prostate, 29, 371-380, and Tjua et al. (1997), The Prostate, 32, 272-278.

[0301] Therefore, in one embodiment of the invention, a vaccine or immunogenic composition comprising at least one antigen-presenting cell is pulsed or loaded with one or more peptides of the invention. Alternatively, peripheral blood mononuclear cells (PBMCs) isolated from a patient can be loaded with peptides ex vivo and injected back into the patient. As an alternative, the antigen-presenting cell comprises an expression construct encoding the peptide of the invention. The polynucleotide can be any suitable polynucleotide and preferably it is capable of transducing dendritic cells to present the peptide and induce immunity.

[0302] The pharmaceutical compositions of the present invention can be formulated such that the selection, number, and / or amount of peptides present in the composition are tissue-, cancer-, and / or patient-specific. For example, the precise selection of peptides can be guided by the expression profile 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-monomer type. Furthermore, the vaccine or immunogenic compositions according to the present invention can contain personalized components according to the individual needs of a specific patient. Examples include altering the amount of peptides based on the expression of the relevant neoantigen in a specific patient, unwanted side effects due to individual allergies or other treatments, and adjusting secondary treatment after a first round of treatment or a first regimen of treatment.

[0303] Pharmaceutical compositions comprising the peptides of the present invention can be administered to individuals already diagnosed with cancer. In therapeutic use, the composition is administered to the patient in an amount sufficient to elicit an effective CTL response against the tumor antigen and sufficient to cure or at least partially prevent symptoms and / or complications. An amount sufficient to achieve this purpose is defined as a “therapeuticly effective dose.” The effective dose for this purpose can depend on, for example, the peptide composition, the route of administration, the stage and severity of the disease being treated, the patient’s weight and general health condition, and the prescribing physician’s judgment; however, the initial immunization range typically used for patients weighing 70 kg (for therapeutic or prophylactic administration) is from about 1.0 μg to about 50,000 μg of peptide, followed by increasing the dose, or according to a booster regimen, from about 1.0 μg to about 10,000 μg of peptide for several weeks to several months, depending on the patient’s response and condition and possibly by measuring the specific CTL activity in the patient’s blood. It should be remembered that the peptides and compositions of the present invention can generally be used for severe disease states, i.e., life-threatening or potentially life-threatening conditions, especially when cancer has metastasized. For therapeutic use, administration should be initiated as soon as possible after detection or surgical removal of the tumor. After that, increase the dosage until at least the symptoms are substantially relieved and then continue for some time.

[0304] Pharmaceutical compositions for therapeutic treatment (e.g., vaccine compositions) are intended for parenteral, topical, nasal, oral, or local administration. Preferably, these pharmaceutical compositions are administered parenterally (e.g., intravenously, subcutaneously, intradermally, or intramuscularly). These compositions can be administered at the surgical site to induce a local immune response against the tumor. The present invention provides compositions for parenteral administration comprising a solution of a peptide and a vaccine or immunogenic composition dissolved or suspended in an acceptable carrier (preferably an aqueous carrier). Various aqueous carriers can be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, etc. These compositions can be sterilized using conventional, well-known sterilization techniques, or can be sterile filtered. The resulting aqueous solution can be packaged for use as is, or lyophilized and combined with a sterile solution prior to administration. These compositions may contain pharmaceutically acceptable adjuvants that approximate physiological conditions, such as pH adjusters and buffers, tension modifiers, humectants, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitol monolaurate, triethanolamine oleate, etc.

[0305] Liposome suspensions containing peptides can be administered intravenously, locally, topically, etc., at doses that vary, particularly depending on the route of administration, the peptide being delivered, and the stage of the disease being treated. For targeting immune cells, ligands (such as, for example, antibodies or fragments thereof that specifically target cell surface determinants of desired immune system cells) can be incorporated into the liposomes.

[0306] For solid compositions, conventional or nanoparticle-based non-toxic solid carriers can be used, including, for example, pharmaceutical-grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. For oral administration, a pharmaceutically acceptable non-toxic composition is formed by incorporating any commonly used excipients (such as those carriers previously listed) and typically 10%-95% of the active ingredient (i.e., one or more peptides of the present invention), and more preferably at a concentration of 25%-75%.

[0307] For aerosol drug delivery, the immunogenic peptide is preferably provided in a finely dispersed form along with a surfactant and propellant. The typical percentage of the peptide is 0.01%-20% by weight, preferably 1%-10%. Of course, the surfactant can be non-toxic and is preferably soluble in the propellant. Representative of such agents are esters or metaesters of fatty acids containing 6 to 22 carbon atoms and aliphatic polyols or their cyclic anhydrides, such as hexanoic acid, caprylic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, oleostearic acid, and oleic acid. Mixed esters, such as mixed or natural glycerides, can be used. The surfactant can be 0.1%-20% by weight of the composition, preferably 0.25%-5%. The balance of the composition is typically the propellant. A carrier may also be included, as desired, such as lecithin for intranasal delivery.

[0308] The peptides and polypeptides of the present invention can be readily synthesized chemically using reagents free from contaminating bacteria or animal matter (Merrifield RB: Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. Journal of the American Chemical Society (J. Am. Chem. Soc.) 85: 2149-54, 1963).

[0309] The peptides and polypeptides of the present invention can also be expressed via vectors, such as nucleic acid molecules as discussed herein, such as RNA or DNA plasmids, viral vectors such as poxviruses, such as orthopoxvirus, fowlpoxvirus, or adenovirus, AAV, or lentivirus. This method involves using a vector to express the nucleotide sequence encoding the peptide of the present invention. Upon introduction into an acutely or chronically infected host or into an uninfected host, the vector expresses the immunogenic peptide and thereby elicits a host CTL response.

[0310] For therapeutic or immunization purposes, nucleic acids encoding the peptides of the present invention, and optionally one or more peptides described herein, may also be administered to patients. Many methods are conveniently used to deliver these nucleic acids to patients. For example, nucleic acids can be delivered directly as “naked DNA.” This approach is described, for example, in Wolff et al., Science 247: 1465-1468 (1990) and U.S. Patent Nos. 5,580,859 and 5,589,466. Nucleic acids can also be administered using ballistic delivery, as described, for example, in U.S. Patent No. 5,204,253. Particles consisting solely of DNA can be administered. Alternatively, DNA can be attached to particles such as gold particles. Typically, plasmids used in vaccines or immunological compositions may comprise DNA encoding an antigen (one or more neoantigens) that is operatively linked to a regulatory sequence controlling the expression or expression and secretion of an antigen from a host cell (e.g., a mammalian cell); for example, from upstream to downstream, DNA for a promoter, such as a mammalian viral promoter (e.g., a CMV promoter, such as an hCMV or mCMV promoter, such as an early intermediate promoter, or an SV40 promoter—see, for useful promoters, the documents cited or incorporated herein), DNA for a secreted eukaryotic leader peptide (tissue plasminogen activator), DNA for one or more neoantigens, and DNA encoding a terminator (e.g., a 3' UTR transcription terminator from a gene encoding bovine growth hormone or bGH polyA). The composition may contain more than one plasmid or vector, whereby each plasmid contains and expresses a different neoantigen. References also include U.S. Patent No. 5,849,303 of Wasmoen and U.S. Patent No. 5,811,104 of Dale, the contents of which may be useful. DNA or DNA plasmid formulations can be prepared using cationic lipids or within cationic lipids; and regarding cationic lipids, along with adjuvants, Loosmore's U.S. Patent Application 2003 / 0104008 is also mentioned. Furthermore, the teachings in Audonnet's U.S. Patents 6,228,846 and 6,159,477 can depend on the DNA plasmid teachings that can be employed when constructing and using DNA plasmids contained and expressed in vivo.

[0311] Nucleic acids can also be delivered by complexing with 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. Patent No. 5,279,833; WO 1991 / 06309; and Feigner et al., Proceedings of the National Academy of Sciences 84: 7413-7414 (1987).

[0312] RNA encoding the peptide of interest (e.g., mRNA) can also be used for delivery (see, for example, Kiken et al., 2011; Su et al., 2011; see also US 8278036; Halabi et al., J Clin Oncol (2003) 21: 1232-1237; Petsch et al., Nature Biotechnology 2012 Dec 7; 30(12):1210-6).

[0313] Information regarding poxviruses that can be used in the practice of this invention, such as vertebrate poxviruses (vertebrate poxviruses), such as orthopoxviruses and fowlpoxviruses, such as cowpoxviruses (e.g., Wyeth Strain, WR strains (e.g., ATCC® VR-1354), Copenhagen strain, NYVAC, NYVAC.1, NYVAC.2, MVA, MVA-BN), canarypoxviruses (e.g., Wheatley C93 strain, ALVAC), fowlpoxviruses (e.g., FP9 strain, Webster strain, TROVAC), pigeonpox, pigeonpox virus, quailpox, and raccoon pox, especially their synthetic or non-naturally occurring recombinants, their uses, and methods for manufacturing and using such recombinants, can be found in scientific and patent literature, for example: 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, 6,265,189, 6,214,353, 6,130,066, 6,004,777, 5,990,091, 5,942,235, 5,833,975, 5,766,597, 5,756,101, 7,045,313, 6,780,417, 8,470,598, 8,372,62 2. 8,268,329, 8,268,325, 8,236,560, 8,163,293, 7,964,398, 7,964,396 ,7,964,395, 7,939,086, 7,923,017, 7,897,156, 7,892,533, 7,628,980, 7,459,270, 7,445,924, 7,384,644, 7,335,364, 7,189,536, 7,097,842, 6 ,913,752, 6,761,893, 6,682,743, 5,770,212, 5,766,882, and 5,989,562, and Panicali, D. Proc. Natl. Acad. Sci. 1982; 79; 4927-493; Panicali, D. Proc. Natl. Acad. Sci. 1983; 80(17): 5364-8; Makett, 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. *Proceedings of the National Academy of Sciences* 1984; 81: 7194-8, Rupprecht, CE. *Proceedings of the National Academy of Sciences* 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. Journal of Virology (J. Virol.) 1988; 62(3): 866-74, Flexner, C. Nature 1987; 330(6145): 259-62, Shida, H. Journal of Virology (J. Virol.) 1988; 62(12): 4474-80, Kotwal, GJ. Journal of Virology (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. Journal of Virology 2002; 76(16); 8313-34, Gallego-Gomez, JC. Journal of Virology (J. Virol.) 2003; 77(19); 10606-22), Goebel SJ. Virology 1990; (a,b) 179: 247-66, Tartaglia, Journal of Virology 1992; 188(1): 217-32, Najera JL. Journal of Virology 2006; 80(12): 6033-47, Najera JL. Journal of Virology 2006; 80: 6033-6047, Gomez CE. Journal of Genetic Virology 2007; 88: 2473-78, Mooij, P. Journal of Virology 2008; 82:2975-2988, Gomez, CE. Curr. Gene Ther. 2011; 11: 189-217, Cox, W. Virology 1993; 195: 845-50, Perkus, M. Journal of Leukocyte Biology 1995; 58: 1-13, Blanchard, TJ. Journal of Genetic Virology 1998; 79(5): 1159-67, Amara, R. Science 2001; 292: 69-74, Hel, Z. Journal of Immunology (J. Immunol. 2001; 167: 7180-9, Gherardi MM.Journal of Virology (J. Virol.) 2003; 77: 7048-57, Didierlaurent, A. Vaccine 2004; 22: 3395-3403, Bissht, H. Proc. Nat. Aca. Sci. 2004; 101: 6641-46, McCurdy, LH. Clin. Inf. Dis 2004; 38: 1749-53, Earl, PL. Nature 2004; 428: 182-85, Chen, Z. Journal of Virology (J. Virol.) 2005; 79: 2678-2688, Najera, JL. Journal of Virology (J.Virol.) 2006; 80(12): 6033-47, Nam JH. Acta Virologica Sinica (Acta. Virol.) 2007; 51: 125-30, Antonis AF. Vaccine 2007; 25: 4818-4827, B Weyer J. Vaccine 2007; 25: 4213-22, Ferrier-Rembert A. Vaccine 2008; 26(14): 1794-804, Corbett M. Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci.) 2008; 105(6): 2046-51, Kaufman HL., Journal of Clinical Oncology (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. Journal of Clinical Oncology (J. Clin. Oncol.) 2010, 28, 1099-1105, Amato RJ. Journal of Clinical Cancer Res. 2010; 16(22): 5539-47, Kim, DW. Hum. Vaccine. 2010; 6: 784-791, Oudard, S. Cancer Immunology and Immunotherapy.Immunother. 2011; 60: 261-71, Wyatt, LS. AIDS research and human retroviruses (Aids Res. Hum. Retroviruses.) 2004; 20: 645-53, Gomez, CE. Virus Research 2004; 105: 11-22, Webster, DP. Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci.) 2005; 102: 4836-4, Huang, X. Vaccine 2007; 25: 8874-84, Gomez, CE. Vaccine 2007a; 25: 2863-85, Esteban, M. Hum. Vaccine 2009; 5: 867-871, Gomez, CE. Curr. Genetherapy 2008; 8(2): 97-120, Whelan, KT. PLOS ONE 2009; 4(6): 5934, Scriba, TJ. Eur. Jour. Immuno. 2010; 40(1): 279-90, Corbett, M. Proc. Natl. Acad. Sci. 2008; 105: 2046-2051, Midgley, CM. J. Gen. Virol. 2008; 89: 2992-97, Von Klenper Huber Krempelhuber, A. Vaccine 2010; 28: 1209-16; Perreau, M. Journal of Virology 2011; Oct: 9854-62; Pantaleo, G. Curr Opin HIV-AIDS 2010; 5: 391-396. Each reference is cited here.

[0314] Regarding adenovirus vectors useful in practicing this invention, U.S. Patent No. 6,955,808 is mentioned. The adenovirus vectors used may be selected from the group consisting of Ad5, Ad35, Ad11, C6, and C7 vectors. The sequence of the genome of adenovirus type 5 (“Ad5”) has been disclosed. (Chroboczek J., Bieber F., and Jacrot B. (1992) The Sequence of the Genome of Adenovirus Type 5 and Its Comparison with the Genome of Adenovirus Type 2, Virology 186, 280-285; the contents of which are incorporated herein by reference. The Ad35 vector is described in U.S. Patent Nos. 6,974,695, 6,913,922, and 6,869,794. The Ad11 vector is described in U.S. Patent No. 6,913,922. The C6 adenovirus vector is described in U.S. Patent Nos. 6,780,407; 6,537,594; 6,309,647; 6,265,189; 6,156,567; 6,090,393; 5,9 In 42,235 and 5,833,975, the C7 vector is described in U.S. Patent No. 6,277,558. Also usable are E1-deficient or deleted, E3-deficient or deleted, and / or E4-deficient or deleted adenovirus vectors. Certain adenoviruses with mutations in the E1 region have improved safety limits because, in non-permissive cells, E1-deficient adenovirus mutants are replication-deficient, or minimally attenuated, and highly attenuated. By disrupting this mechanism, the adenovirus thereby downregulates MHC. Class I molecules, particularly adenoviruses with mutations in the E3 region, may have enhanced immunogenicity. Adenoviruses with E4 mutations may exhibit reduced immunogenicity compared to adenovirus vectors due to suppression of late gene expression. Such vectors are particularly useful when repeated reinoculations using the same vector are desired. According to the invention, adenovirus vectors with deletions or mutations in E1, E3, E4, E1 and E3, and E1 and E4 can be used. Furthermore, according to the invention, "gutless" adenovirus vectors (where all viral genes are deleted) can also be used. For replication, such vectors require helper viruses and specific human 293 cell lines expressing both E1a and Cre, conditions not present in the natural environment. Such "gutless" vectors are non-immunogenic, and therefore can be reinoculated multiple times for reinoculation."Entericless" adenovirus vectors can be used to insert heterologous inserts / genes (such as the transgenes of this invention), and can even be used for the co-delivery of large quantities of heterologous inserts / genes.

[0315] Regarding lentivirus vector systems useful in the practice of this invention, references are made to U.S. Patent Nos. 6,428,953, 6,165,782, 6,013,516, 5,994,136, 6,312,682, and 7,198,784, and the documents referenced therein.

[0316] Regarding AAV carriers useful in the practice of this invention, references are made to U.S. Patent Nos. 6,58785, 7,115,391, 7,172,893, 6,953,690, 6,936,466, 6,924,128, 6,893,865, 6,793,926, 6,537,540, 6,475,769, and 6,258,595, and the documents referenced therein.

[0317] Another vector is BCG (Bacillus Calmette-Guérin). The BCG vector is described in Stover et al. (Nature 351:456-460 (1991)). There are a wide variety of other vectors (e.g., Salmonella typhi vectors, etc.) for the therapeutic administration or immunization of the peptides of the present invention, which will be apparent to those skilled in the art from the description herein.

[0318] A carrier can be administered to elicit an in vivo expression and a response similar to that induced by drug administration and / or by antigen administration.

[0319] A preferred method for providing nucleic acids encoding the peptides of the present invention is to use a microgene construct encoding multiple epitopes. To generate DNA sequences encoding selected CTL epitopes (microgenes) for expression in human cells, the amino acid sequences of these epitopes are reverse-translated. A human codon selection table is used to guide codon selection for each amino acid. These DNA sequences encoding epitopes are directly adjacent, resulting in a continuous polypeptide sequence. Additional elements can be incorporated into the microgene design to optimize expression and / or immunogenicity. Examples of amino acid sequences that can be reverse-translated and included in the microgene sequence include: helper T lymphocytes, epitopes, precursor (signal) sequences, and endoplasmic reticulum retention signals. Additionally, the presentation of these CTL epitopes by MHC can be improved by including synthetic (e.g., polyalanine) or naturally occurring flanking sequences adjacent to the CTL epitopes.

[0320] The microgene sequence is converted into DNA by assembling positive and negative strand oligonucleotides encoding the microgene. Overlapping oligonucleotides (30-100 bases long) are synthesized under appropriate conditions using well-known techniques, phosphorylated, purified, and annealed. The ends of these oligonucleotides are ligated using T4 DNA ligase. The synthesized microgene encoding a CTL epitope polypeptide can then be cloned into the desired expression vector.

[0321] The vector includes standard regulatory sequences well known to those skilled in the art to ensure expression in target cells. Several vector elements are required: a promoter with a downstream cloning site for inserting the microgene; a polyadenylation signal for efficient termination of transcription; an *E. coli* origin of replication; and an *E. coli* selectivity marker (e.g., ampicillin or kanamycin resistance). Numerous promoters can be used for this purpose, such as the human herpesvirus (hCMV) promoter. For other suitable promoter sequences, see U.S. Patent Nos. 5,580,859 and 5,589,466.

[0322] Further vector modifications may be needed to optimize microgene expression and immunogenicity. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally occurring introns can be incorporated into the transcriptional region of the microgene. Including mRNA stable sequences can also be considered for increasing microgene expression. Recently, it has been proposed that immunostimulatory sequences (ISS or CpG) play a role in the immunogenicity of DNA vaccines. If enhanced immunogenicity is found, these sequences can be included in the vector, located outside the microgene coding sequence.

[0323] In some embodiments, bicistronic expression vectors may be used to allow the generation of microgene-encoded epitopes and second proteins, which are included to enhance or reduce immunogenicity. Examples of proteins or peptides that, if co-expressed, can beneficially enhance the immune response include cytokines (e.g., IL2, IL12, GM-CSF), cytokine-inducing molecules (e.g., LeIF), or co-stimulatory molecules. Helper (HTL) epitopes may be linked to intracellular targeting signals and expressed independently of CTL epitopes. This allows HTL epitopes to be directed to cellular compartments different from those of CTL epitopes. If desired, this can facilitate more efficient entry of HTL epitopes into the MHC class II pathway, thereby improving CTL induction. In contrast to CTL induction, specific reduction of the immune response through co-expression of immunosuppressive molecules (e.g., TGF-β) may be beneficial in certain diseases.

[0324] After selecting the expression vector, the microgene is cloned into the multi-connector region downstream of the promoter. This plasmid is transformed into an appropriate E. coli strain, and the DNA is prepared using standard techniques. Restriction enzyme mapping and DNA sequence analysis are used to confirm the orientation and DNA sequence of the microgene, as well as all other elements included in the vector. Bacterial cells carrying the correct plasmid can be stored as master and working cell banks.

[0325] Purified plasmid DNA for injection can be prepared using a variety of formulations. The simplest of these is rehydration of lyophilized DNA in sterile phosphate-buffered saline (PBS). Several methods have been described, and newer techniques are becoming available. Nucleic acids can be conveniently formulated using cationic lipids, as noted herein. Additionally, glycolipids, fusion-promoting liposomes, peptides, and compounds collectively referred to as protective, interacting, non-condensing (PINC) compounds can be complexed with purified plasmid DNA to influence variables such as stability, intramuscular dispersibility, or delivery to specific organs or cell types.

[0326] Target cell sensitization can be used for functional assays of microgene-encoded CTL epitopes expression and MHC class I presentation. The plasmid DNA is introduced into mammalian cell lines suitable as targets for standard CTL chromium release assays. The transfection method used depends on the final formulation. Electroporation can be used for “naked” DNA, while cationic lipids allow for direct in vitro transfection. Plasmids expressing green fluorescent protein (GFP) can be co-transfected 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. Detection of cell lysis via 51Cr release indicates the production of MHC-encoded CTL epitopes.

[0327] In vivo immunogenicity is a second approach for functional testing of microgene DNA formulations. Transgenic mice expressing appropriate human MHC molecules are immunized with the DNA product. Dosage and route of administration are formulation-dependent (e.g., IM for DNA in PBS, IP for DNA in lipid complexes). Twenty-one days post-immunization, spleen cells are harvested and restimulated for one week in the presence of peptides encoding the various epitopes to be tested. These effector cells (CTLs) are measured using standard techniques to induce CTLs in vivo through cell lysis of target cells sensitized by MHC loading of peptides corresponding to the epitopes encoded by microgene. The function of the DNA vaccine in in vivo inducing CTLs is demonstrated by the lysis of target cells sensitized by MHC loading of peptides corresponding to epitopes encoded by microgene.

[0328] Peptides can also be used to induce CTLs in vitro. The resulting CTLs can be used to treat chronic tumors in patients who do not respond to other conventional forms of therapy or peptide vaccine therapy pathways. In vitro CTL responses against specific tumor antigens are induced by incubating the patient's CTL precursor cells (CTLp) in tissue culture along with antigen-presenting cell (APC) sources and appropriate peptides. After an appropriate incubation period (typically 1–4 weeks), during which the CTLp are activated, mature, and develop into effector CTLs, these cells are reinfused into the patient, where they destroy their specific target cells (i.e., tumor cells). To optimize the in vitro conditions for generating specific cytotoxic T cells, the culture of stimulating cells is maintained in an appropriate serum-free medium.

[0329] Before incubating these stimulating cells with cells to be activated (e.g., precursor CD8+ cells), a sufficient amount of antigenic peptide is added to the stimulating cell culture to be loaded onto human class I molecules to be expressed on the surface of these stimulating cells. In this invention, a sufficient amount of peptide is to allow about 200, preferably 200 or more, peptide-loaded human class I MHC molecules to be expressed on the surface of each stimulating cell. Preferably, these stimulating cells are incubated with >2 μg / ml of peptide. For example, these stimulating cells are incubated with >3, 4, 5, 10, 15 or more μg / ml of peptide.

[0330] The quiescent or precursor CD8+ cells are then incubated in a culture with appropriate stimulating cells for a period sufficient to activate these CD8+ cells. Preferably, these CD8+ cells are activated in an antigen-specific manner. The ratio of quiescent or precursor CD8+ (effective) cells to stimulating cells may vary from person to person and may further depend on variables such as the individual's lymphocyte compliance with culture conditions and the nature and severity of the disease or other conditions using the treatment methods described. However, preferably, the lymphocyte:stimulating cell ratio is in the range of about 30:1 to 300:1. The effector / stimulating culture can be maintained for as long as possible to stimulate a therapeutically available or effective number of CD8+ cells.

[0331] In vitro induction of CTLs requires the specific recognition of peptides that bind to allele-specific MHC class I molecules on APCs. The number of specific MHC / peptide complexes per APC is crucial for stimulating CTLs, particularly in the primary immune response. While a small number of peptide / MHC complexes per cell is sufficient to make the cell readily lysed by CTLs 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 the primary response. Peptide loading of empty major histocompatibility complex molecules on cells allows for the induction of a primary cytotoxic T lymphocyte response.

[0332] Since not every human MHC allele has a mutant cell line, it is advantageous to use a technique to remove endogenous MHC-related peptides from the surface of an APC, followed by loading the resulting empty MHC molecule with an immunogenic peptide of interest. Using untransformed (non-tumorigenic), uninfected cells from a patient, preferably autologous cells, as APCs is desirable for designing CTL induction protocols for the development of ex vivo CTL therapies. This application discloses a method for stripping endogenous MHC-related peptides from the surface of an APC and subsequently loading the desired peptide.

[0333] A stable MHC class I molecule is a trimeric complex formed by the following elements: 1) a peptide typically having 8-10 residues, 2) a transmembrane polymorphic protein heavy chain with peptide-binding sites in its a1 and a2 domains, and 3) a non-covalently associated non-polymorphic light chain p2 microglobulin. Removal of the bound peptides and / or isolation of the p2 microglobulin from this complex renders the MHC class I molecule nonfunctional and unstable, leading to rapid degradation. All MHC class I molecules isolated from PBMCs possess endogenous peptides bound to them. Therefore, before introducing exogenous peptides, the first step is to remove all endogenous peptides bound to MHC class I molecules from the APC without inducing their degradation.

[0334] Two possible methods to remove MHC class I molecules from binding peptides include lowering the culture temperature from 37°C to 26°C overnight to destabilize p2 microglobulins, and using mild acid treatment to strip endogenous peptides from the cells. These methods release previously bound peptides into the extracellular environment, allowing new exogenous peptides to bind to empty class I molecules. This cold incubation method allows exogenous peptides to bind efficiently to the MHC complex, but requires overnight incubation at 26°C, which slows down the cell's metabolic rate. It is also possible that cells that do not actively synthesize MHC molecules (e.g., quiescent PBMCs) will not produce large amounts of empty surface MHC molecules through this cold incubation procedure.

[0335] Rough acid stripping involves extracting peptides with trifluoroacetic acid (pH 2) or acid denaturing immunoaffinity-purified class I peptide complexes. These methods are not feasible for CTL induction because it is important to remove endogenous peptides while maintaining APC viability and optimal metabolic state, which is crucial for antigen presentation. Mild acid solutions at pH 3 (such as glycine or citrate-phosphate buffer) have been used to identify endogenous peptides and tumor-associated T cell epitopes. 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. Crucially, treatment with mild acid solutions does not affect cell viability or metabolic state. Mild acid treatment is rapid, as the stripping of endogenous peptides occurs within two minutes at 4°C, and APCs can immediately perform their functions after loading the appropriate peptides. This technique was used here to prepare peptide-specific APCs for generating primary antigen-specific CTLs. The resulting APCs were effective in inducing peptide-specific CD8+ CTLs.

[0336] Activated CD8+ cells can be efficiently separated from these stimulating cells using one of a number of known methods. For example, monoclonal antibodies specifically targeting stimulating cells, peptides loaded onto stimulating cells, or CD8+ cells (or segments thereof) can be used to bind appropriate complementary ligands. The antibody-labeled molecules can then be extracted from the stimulant-effect cell mixture by appropriate means, such as well-known immunoprecipitation or immunoassay methods.

[0337] The effective, cytotoxic dose of activated CD8+ cells can vary between in vitro and in vivo applications, and also varies with the quantity and type of cells that serve as the ultimate target of these killer cells. This dose can also vary depending on the patient's condition and should be determined by the practitioner by considering all appropriate factors. However, preferably, it is approximately 5 x 10⁻⁶ cells compared to that used in mice. 6 -5 x 10 7 Compared to cells, approximately 1 x 10 6 To approximately 1 x 10 12 More preferably about 1 x 10 8 To approximately 1 x 10 11 And even more preferably about 1 x 10 9 To approximately 1 x 10 10 One activated CD8+ cell for adults.

[0338] Preferably, as discussed herein, activated CD8+ cells are harvested from the cell culture before being administered to the individual being treated. However, it is important to note that, unlike other existing and proposed forms of treatment, this method uses a non-tumorigenic cell culture system. Therefore, there is no known inherent danger associated with administering small amounts of stimulating cells, whereas administering tumor-promoting cells to mammals can be extremely dangerous, unless complete separation of the stimulating cells from the activated CD8+ cells is achieved.

[0339] 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 via intravenous infusion is appropriate.

[0340] Unless otherwise specified, the present invention is carried out using conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, which are entirely within the knowledge of those skilled in the art. These techniques are well explained in the following literature: "Molecular Cloning: A Laboratory Manual," 2nd edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology"; "Handbook of Experimental Immunology" (Wei, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction." "Chain Reaction" (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of polynucleotides and polypeptides of the present invention, and can thus be considered for the preparation and implementation of the invention. Techniques particularly useful in specific embodiments are discussed in the following sections.

[0341] Treatment The present invention provides methods for inducing tumor-specific immune responses in subjects, immunizing against tumor formation / tumors, treating cancer in subjects, and / or alleviating cancer symptoms in subjects, by administering the tumor-forming vaccine or neoantigen peptide or composition of the present invention to the subject.

[0342] According to the present invention, the tumor-forming vaccine or immunogenic composition described herein can be used in patients who have been diagnosed with cancer or are at risk of developing cancer. In one embodiment, the patient may have solid tumors such as breast cancer, ovarian cancer, prostate cancer, lung cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, melanoma, and other tumors of tissues and organs, as well as hematologic malignancies such as lymphoma and leukemia, including acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, and B-cell lymphoma.

[0343] The peptide or composition of the present invention shall be administered in an amount sufficient to induce a CTL response.

[0344] According to the following Figure 2 The general workflow involves using the compositions and methods described herein on patients with any type of cancer who require it. Patients in need can receive a series of primary immunizations using a personalized mixture of tumor-specific peptides. Additionally, two booster immunizations can be administered during a maintenance phase after the primary immunization, following a 4-week period. All vaccinations are delivered subcutaneously. The safety, tolerability, immune response, and clinical efficacy of the vaccine or immunogenic composition in patients, as well as the feasibility of producing the vaccine or immunogenic composition and successfully initiating vaccination within an appropriate timeframe, will be evaluated. The first concurrent cohort may consist of 5 patients, and additional concurrent cohorts of 10 patients may be recruited after sufficient safety has been demonstrated. Peripheral blood peptide-specific T-cell responses will be extensively monitored, and patients will be followed up for up to two years to assess disease recurrence.

[0345] Vaccine or immunogenic composition kits and co-packaging In one aspect, the present invention provides kits containing any one or more of the elements discussed herein to allow administration of immunogenic compositions or vaccines. Elements may be provided individually or in combination and may be provided in any suitable container, such as vials, bottles, or tubes. In some embodiments, the kit includes instructions in one or more languages, such as instructions in more than one language. In some embodiments, the kit includes one or more reagents for use in methods utilizing one or more of the elements 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 available in a specific process or in a form requiring the addition of one or more other components prior to use (e.g., in concentrated or lyophilized form). Buffers 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 from about 7 to about 10. In some embodiments, the kit includes one or more of the carriers described herein, the proteins described herein, and / or one or more of the polynucleotides described herein. The kit may advantageously allow the provision of all elements of the system of the present invention. The kit may involve one or more vectors and / or one or more particles and / or one or more nanoparticles containing or encoding one or more RNAs for 1-50 or more neoantigen mutations in an animal, mammal, primate, rodent, etc., wherein such a kit includes instructions for administration to such a eukaryote, together with instructions for use with any of the methods of the present invention.

[0346] In one embodiment, the kit comprises at least one vial containing an immunogenic composition or vaccine. In one embodiment, the kit may comprise readily available components that are mixed and readily available. The readily available immunogenic or vaccine composition may comprise separate vials containing different pools of immunogenic composition. The immunogenic composition may comprise one vial containing a viral vector or DNA plasmid and other vials may comprise immunogenic proteins. In another embodiment, the kit may comprise an immunogenic composition or vaccine in a rehydrated form. The immunogenic or vaccine composition may be lyophilized or freeze-dried. The kit may comprise separate vials with a rehydration buffer that may be added to the lyophilized composition, thus making it ready for administration. The buffer may advantageously comprise an adjuvant or emulsion according to the invention. In another embodiment, the kit may comprise a single vial containing a dose of the immunogenic composition. In another aspect, multiple vials are included, such that a single vial is administered according to a treatment timeline. In a further embodiment, the vials are labeled for proper administration to patients in need. As described herein, the immunogen may be in lyophilized form, dried form, or in an aqueous solution. As described herein, the immunogen can be an attenuated live virus, a protein, or a nucleic acid.

[0347] In another embodiment, the kit may include separate vials for two immunogenic compositions, one for primary immunization of an immune response and the other for boosting. In one embodiment, the primary 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 administration.

[0348] This application provides the following non-limiting embodiments 1-44.

[0349] Implementation Scheme 1. A pharmaceutical composition comprising: (a) at least one neoantigenic peptide or a pharmaceutically acceptable salt thereof; (b) pH modifiers; and (c) Pharmaceutically acceptable carriers.

[0350] Implementation Scheme 2. The pharmaceutical composition as described in Implementation Scheme 1, wherein the pharmaceutical composition is a vaccine composition.

[0351] Implementation Scheme 3. The pharmaceutical composition as described in Implementation Scheme 1 or Implementation Scheme 2, wherein the pharmaceutical composition comprises at least two neoantigen peptides.

[0352] Implementation Scheme 4. The pharmaceutical composition as described in any one of Implementation Schemes 1-3, wherein the claimed pharmaceutical composition comprises at least three neoantigen peptides.

[0353] Implementation Scheme 5. The pharmaceutical composition as described in any one of Implementation Schemes 1-4, wherein the pharmaceutical composition comprises at least four neoantigen peptides.

[0354] Implementation Scheme 6. The pharmaceutical composition as described in any one of Implementation Schemes 1-5, wherein the pharmaceutical composition comprises at least five neoantigen peptides.

[0355] Implementation Scheme 7. The pharmaceutical composition of any one of Implementation Schemes 1-6, wherein the length of the at least one neoantigen peptide ranges from about 5 to about 50 amino acids.

[0356] Implementation Scheme 8. The pharmaceutical composition of any one of Implementation Schemes 1-7, wherein the length of the at least one neoantigen peptide ranges from about 15 to about 35 amino acids.

[0357] Implementation Scheme 9. The pharmaceutical composition as described in any one of Implementation Schemes 1-8, wherein the pH changer is a base.

[0358] Implementation Scheme 10. The pharmaceutical composition of any one of Implementation Schemes 1-9, wherein the pH changer is a dicarboxylate or a tricarboxylate.

[0359] Implementation Scheme 11. The pharmaceutical composition as described in any one of Implementation Schemes 1-10, wherein the pH changer is a succinate.

[0360] Implementation Scheme 12. The pharmaceutical composition as described in any one of Implementation Schemes 1-10, wherein the pH changer is a citrate.

[0361] Implementation Scheme 13. The pharmaceutical composition of any one of Implementation Schemes 1-11, wherein succinic acid or a pharmaceutically acceptable salt thereof includes sodium succinate.

[0362] Implementation Scheme 14. The pharmaceutical composition as described in any one of Implementation Schemes 1-11 or 13, wherein the succinate is present in the preparation at a concentration from about 1 mM to about 10 mM.

[0363] Implementation Scheme 15. The pharmaceutical composition as described in any one of Implementation Schemes 1-11, 13, or 14, wherein the succinate is present in the preparation at a concentration of about 2 mM to about 5 mM.

[0364] Implementation Scheme 16. The pharmaceutical composition of any one of Implementation Schemes 1-15, wherein the pharmaceutically acceptable carrier comprises water.

[0365] Implementation Scheme 17. The pharmaceutical composition of any one of Implementation Schemes 1-16, wherein the pharmaceutically acceptable carrier further comprises dextran.

[0366] Implementation Scheme 18. The pharmaceutical composition of any one of Implementation Schemes 1-16, wherein the pharmaceutically acceptable carrier further comprises trehalose.

[0367] Implementation Scheme 19. The pharmaceutical composition of any one of Implementation Schemes 1-16, wherein the pharmaceutically acceptable carrier further comprises sucrose.

[0368] Implementation Scheme 20. The pharmaceutical composition of any one of Implementation Schemes 1-19, wherein the pharmaceutically acceptable carrier further comprises dimethyl sulfoxide.

[0369] Implementation Scheme 21. The pharmaceutical composition as described in any one of Implementation Schemes 15-19, wherein the pharmaceutical composition is lyophilizable.

[0370] Implementation Scheme 22. The pharmaceutical composition as described in any one of Implementation Schemes 1-21, wherein the pharmaceutical composition further comprises an immunomodulator or adjuvant.

[0371] Implementation Scheme 23. The pharmaceutical composition as described in Implementation Scheme 22, wherein the immunomodulator or adjuvant is selected from the group consisting of: poly-ICLC, 1018 ISS, aluminum salt, Amplivax, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCMATRIX, Juvlmmune, LipoVac, MF59, monophospholipase A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montaneid ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel®, vector systems, PLGA microparticles, Requimod, SRL172, virions and other virus-like particles, YF-17D, VEGF traps, R848, β-glucan, Pam3Cys and Aquila's QS21 stimulon.

[0372] Implementation Scheme 24. The pharmaceutical composition as described in Implementation Scheme 23, wherein the immunomodulator or adjuvant comprises poly-ICLC.

[0373] Implementation Scheme 25. A pharmaceutical composition for a tumorigenesis vaccine, the pharmaceutical composition comprising: One to five neoantigenic peptides or their pharmaceutically acceptable salts; 1%-3% dimethyl sulfoxide; 3.6%-3.7% dextrose aqueous solution; 3.6 mM-3.7 mM succinic acid or its salt; 0.5 mg / ml PolyI:PolyC; 0.375 mg / ml poly-L-lysine; 1.25 mg / ml sodium carboxymethyl cellulose; and 0.225% sodium chloride.

[0374] Implementation Scheme 26. The pharmaceutical composition as described in Implementation Scheme 25, wherein each of the one to five neoantigenic peptides or pharmaceutically acceptable salts thereof is present at a concentration of about 300 μg / ml.

[0375] Implementation Scheme 27. A method for preparing a neoantigen peptide solution for a tumor formation vaccine, the method comprising: (a) Prepare a solution containing at least one neoantigen peptide or a pharmaceutically acceptable salt thereof; and (b) Combining the solution containing at least one neoantigen peptide or a pharmaceutically acceptable salt thereof with a solution containing succinic acid or a pharmaceutically acceptable salt thereof, thereby preparing a peptide solution for tumorigenesis vaccines.

[0376] Implementation Scheme 28. The method of Implementation Scheme 27, wherein the solution containing at least one neoantigen peptide or a pharmaceutically acceptable salt thereof contains at least two (or at least three, four, or five) neoantigen peptides.

[0377] Implementation Scheme 29. The method of Implementation Scheme 27, wherein the peptide solution for the tumor formation vaccine comprises water, dextran, succinate, and dimethyl sulfoxide.

[0378] Implementation Scheme 30. The method of Implementation Scheme 27, further comprising filtering the peptide solution for the tumor formation vaccine after the combination step.

[0379] Implementation Scheme 31. The method as described in Implementation Scheme 30, wherein the peptide solution for the tumor formation vaccine is lyophilizable.

[0380] Implementation Scheme 32. A method for preparing a tumor-forming vaccine, the method comprising: (a) Prepare a peptide solution; and (b) Combining the peptide solution with a solution of an immunomodulator or adjuvant to prepare a tumor-forming vaccine.

[0381] Implementation Scheme 33. The method as described in Implementation Scheme 32, wherein the immunomodulator or adjuvant is selected from the group consisting of: poly-ICLC, 1018 ISS, aluminum salt, Amplivax, AS15, BCG, CP-870, 893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, imiquimod, ImuFact IMP321, IS Patch, ISS, ISCMATRIX, Juvlmmune, LipoVac, MF59, monophospholipase A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montaneid ISA-51, OK-432, OM-174, OM-197-MPEC, ONTAK, PepTel®, vector systems, PLGA microparticles, Requimod, SRL172, virions and other virus-like particles, YF-17D, VEGF traps, R848, β-glucan, Pam3Cys and Aquila QS21 stimulators.

[0382] Implementation Scheme 34. The method as described in Implementation Scheme 33, wherein the immunomodulator or adjuvant is poly-ICLC.

[0383] Implementation Scheme 35. A method of treating a subject diagnosed with tumor formation, the method comprising administering a pharmaceutical composition as described in any one of Implementation Schemes 1-26 to the subject, thereby treating the tumor formation.

[0384] Implementation Scheme 36. The method of Implementation Scheme 35, further comprising administering the second pharmaceutical composition of any one of Implementation Schemes 1-26 to the subject.

[0385] Implementation Scheme 37. The method of Implementation Scheme 36, further comprising administering the third pharmaceutical composition of any one of Implementation Schemes 1-26 to the subject.

[0386] 38. The method of embodiment 33, further comprising administering the fourth pharmaceutical composition of any one of embodiments 1-26 to the subject.

[0387] Implementation Scheme 39. A tumor formation vaccine, manufactured by any one of Implementation Schemes 27-34.

[0388] Implementation Scheme 40. A neoantigen peptide for use in tumorigenesis vaccines, comprising: (a) at least one neoantigenic peptide or a pharmaceutically acceptable salt thereof; and (b) Succinic acid or a pharmaceutically acceptable salt thereof.

[0389] Implementation Scheme 41. A vaccine inoculation or immunization kit, comprising: (a) Separately packaged, freeze-dried immunogenic compositions configured to elicit an immune response to at least one novel antigen; and (b) A solution for rehydrating this freeze-dried vaccine.

[0390] Implementation Scheme 42. A vaccination or immunization kit as described in Implementation Scheme 41, wherein the solution contains an adjuvant.

[0391] Implementation Scheme 43. A vaccination or immunization kit as described in Implementation Scheme 41, wherein the immunogenic composition is an antigen.

[0392] Implementation Scheme 44. A vaccination or immunization kit as described in Implementation Scheme 41, wherein the immunogenic composition is a viral vector.

[0393] Although the invention and its advantages have been described in detail, it should be understood that various changes, substitutions and modifications may be made without departing from the spirit and scope of the invention as defined by the appended claims.

[0394] The invention is further illustrated in the following examples, which are given for illustrative purposes only and are not intended to limit the invention in any way.

[0395] Example Example 1 Cancer vaccine testing protocol According to the following Figure 2 The general workflow was used to test the compositions and methods described herein in 15 patients with high-risk melanoma (completely resected stage IIIB, IIIC, and IVM1a,b). Patients could receive a series of primary vaccinations with a mixture of personalized tumor-specific peptides and poly-ICLCs over a 4-week period, followed by two booster doses in the maintenance phase. All vaccinations were delivered subcutaneously. The safety, tolerability, immune response, and clinical efficacy of the vaccine or immunogenic composition in patients, as well as the feasibility of producing the vaccine or immunogenic composition and successfully initiating vaccination within an appropriate timeframe, will be evaluated. The first concurrent cohort may consist of 5 patients, and additional concurrent cohorts of 10 patients may be recruited after adequate safety is demonstrated. Peripheral blood peptide-specific T-cell responses will be extensively monitored, and patients will be followed up for up to two years to assess disease recurrence.

[0396] As described here, there is substantial evidence in both animals and humans that mutated epitopes are effective in inducing immune responses, and cases of spontaneous tumor regression or long-term survival are associated with CD8+ T cell responses against mutated epitopes (Buckwalter and Srivastava PK, "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 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. Proceedings of the National Academy of Sciences of the United States of America (Proc Natl Acad Sci US A).)102:16013(2005)) and "immunoediting" can be traced back to changes in the expression of dominant mutant antigens in mice and humans (Matsushita et al., Cancer exome analysis reveals 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 patients with newly diagnosed glioblastoma, Journal of Clinical Oncology (J Clin Oncol.) 28:4722-4729 (2010)). .

[0397] Next-generation sequencing can now rapidly reveal the presence of discrete mutations, such as coding mutations in a single tumor, most commonly single-amino acid alterations (e.g., missense mutations) and infrequently occurring novel amino acid segments (e.g., novel ORFs) resulting from frameshift insertions / deletions / gene fusions in stop codons, read linking mutations, and translation of inappropriately spliced ​​introns. Novel ORFs are particularly valuable as immunogens because their sequences as a whole are completely novel to the immune system and thus resemble viral or bacterial exogenous antigens. Therefore, novel ORFs: (1) are highly specific to tumors (i.e., not expressed in any normal cells); and (2) can bypass central tolerance, thereby increasing the precursor frequency of neoantigen-specific CTLs. For example, the efficacy of utilizing similar exogenous sequences in therapeutic anticancer vaccines has recently been demonstrated using peptides derived from human papillomavirus (HPV). Approximately 50% of 19 patients with pretumoral viral-induced disease who received 3–4 doses of a mixture of HPV peptides derived from viral oncogenes E6 and E7 maintained a complete response for ≥ 24 months (Kenter et al., Vaccination against HPV-16 Oncoproteins for Vulvar Intraepithelial Neoplasia, NEJM 361: 1838 (2009)).

[0398] Sequencing technology has revealed that each tumor contains multiple patient-specific mutations that alter the protein-coding content of genes. These mutations produce altered proteins ranging from single-amino acid changes (caused by missense mutations) to novel amino acid sequences that add long regions (novel open reading frame mutations; new ORFs) attributable to frameshifts, read linking, or translation of intron regions of stop codons. These mutant proteins are valuable targets for the host's immune response to the tumor because, unlike natural proteins, they are not affected by the host's own immunosuppressive effects. Therefore, mutant proteins are more likely to be immunogenic and more specific to tumor cells than to the patient's normal cells.

[0399] Using a recently improved algorithm for predicting which missense mutations result in strong-binding peptides of homologous MHC molecules in patients, a set of peptides representing the best mutant epitopes (both novel ORFs and mistranslations) for each patient were identified and prioritized, and up to 20 or more peptides were prepared for immunization (Zhang et al., Machine learning competition in immunology - Prediction of HLA class Ibinding peptides, J Immunol Methods 374: 1 (2011); Lundegaard et al., Prediction of epitopes using neural network based methods, J Immunol Methods 374: 26 (2011)). Synthesize peptides with a length of approximately 20-35 amino acids, because such “long” peptides undergo efficient internalization, processing, and cross-presentation in professional antigen-presenting cells (such as dendritic cells), and have been shown 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)).

[0400] In addition to potent and specific immunogens, effective immune responses also advantageously include strong adjuvants to activate the immune system (Speiser and Romero, Molecularly defined vaccines for cancer immunotherapy, and protective T cell immunity, Seminars in Immunol 22: 144 (2010)). For example, Toll-like receptors (TLRs) have been shown to be powerful sensors of “danger signals” from microbial and viral pathogens, effectively inducing the innate immune system and consequently the adaptive immune system (Bhardwaj and Gnjatic, TLR agonists: Are They Good Adjuvants?, Cancer J. 16: 382-391 (2010)). Among TLR agonists, poly-ICLC (a synthetic double-stranded RNA mimic) is one of the most potent activators of bone marrow-derived dendritic cells. In a human volunteer study, poly-ICLC has been shown to be safe and induce a gene expression profile in peripheral blood comparable to that induced by 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. Journal of Experimental Medicine (J Exp Med) 208: 2357 (2011)). Hiltonol® (a GMP formulation of poly-ICLC prepared by Oncovir) was used as an adjuvant.

[0401] Example 2 Target patient group Even with complete surgical resection of the disease, patients with stage IIIB, IIIC, and IVM1a,b melanoma still have a significant risk of disease recurrence and death (Balch et al., Final Version of 2009 AJCC Melanoma Staging and Classification, J Clin Oncol 27: 6199-6206 (2009)). One available systemic adjuvant therapy for this patient population is interferon-alpha (IFNα), which provides measurable but marginal benefits and is associated with significant and frequent dose-limiting toxicities (Kirkwood et al., Interferon alpha-2b Adjuvant Therapy of High-Risk Resected Cutaneous Melanoma: The Eastern Cooperative Oncology Group Trial EST1684, 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). (E1690 / S9111 / C9190) Clinical Oncology Journal 18: 2444-2458 (2000)). These patients' immune systems were not compromised by prior cancer-targeted therapies or by active cancer, and therefore represent an excellent patient population for evaluating the safety and immune effects of the vaccine. Finally, these patients' current standard of care does not require any treatment after surgery, thus allowing an 8-10 week window for vaccine preparation.

[0402] This target group consists of patients with clinically detectable, histologically confirmed lymph node (local or distant) metastases or transitional metastases who have undergone complete resection and are disease-free (most of stage IIIB; patients with ulcerative primary tumors but with micrometastatic lymph nodes (T1-4b, N1a, or N2a) are excluded due to the need for sufficient tumor tissue for sequencing and cell line development; all of stage IIIC and IVM1a, b). These can be patients newly diagnosed or those experiencing disease recurrence after a previous diagnosis of pre-existing melanoma.

[0403] Tumor Harvesting: Patients can undergo complete resection of their primary melanoma (if not already removed) and all regional metastases, with the intention of freeing them from melanoma. After sufficient tumor tissue has been collected for pathological evaluation, the remaining tumor tissue is placed in sterile culture medium in sterile containers and prepared for depolymerization. A portion of the tumor tissue is used for whole-exome and transcriptome sequencing and cell line establishment, and the remaining tumor is frozen.

[0404] Normal tissue harvest: Whole exome sequencing was performed on normal tissue samples (blood or sputum samples).

[0405] Patients with clinically significant localized metastatic disease or completely resectable distant lymph node, cutaneous, or lung metastases (but without unresectable distant or visceral metastases) will be identified and recruited for this study. Patient admission prior to surgery is required to obtain fresh tumor tissue for the development of melanoma cell lines (to generate target cells for in vitro cytotoxicity assays as part of an immune surveillance program).

[0406] Example 3 Dosage and regimen For patients who have met all pretreatment criteria, vaccine administration can begin as soon as possible after the investigational drug has arrived and met the entry specifications. For each patient, there are four separate investigational drugs, each containing five of 20 patient-specific peptides. This can be roughly based on the [details omitted]. Figure 3 The immunization was carried out according to the protocol.

[0407] Patients are treated at the outpatient clinic. Immunization for each treatment day may consist of four 1 ml subcutaneous injections, each injected into a separate limb to target different areas of the lymphatic system and reduce antigen competition. If the patient has already undergone complete axillary or inguinal lymph node dissection, the vaccine is administered into the right or left diaphragm as an alternative. Each injection may consist of one of the four study drugs used for that patient, and for each cycle, the same study drug is injected into the same limb. The composition of each 1 ml injection is: 0.75 ml contains 300 μg of each of the five patient-specific peptides in the investigational drug. 0.25 ml (0.5 mg) of 2 mg / ml poly-ICLC (Hiltonol®) During the induction / primitive phase, patients are immunized on days 1, 4, 8, 15, and 22. During the maintenance phase, patients can receive booster doses at weeks 12 and 24.

[0408] Blood samples can be obtained at multiple time points: before primitivism (baseline; two samples obtained on different dates); day 15 during primitivism; four weeks after induction / primitivism (week 8); before (week 2) and after the first booster (week 16); before (week 24) and after (week 28), with each sample collecting 50–150 ml of blood (except at week 16). The primary immunization endpoint is at week 16, and therefore patients may undergo leukoablation (unless otherwise specified, based on patient and physician assessment).

[0409] Example 4 Immune monitoring This 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 allow for the generation of memory T cells. This is followed by a booster immunization, with the expectation that the T-cell response will be strongest 4 weeks after this booster and will be the primary immune endpoint. In the 18-hr ex vivo ELISPOT assay, the overall immune response was initially monitored using peripheral blood mononuclear cells from this time point, stimulated with a pool of overlapping 15-mer peptides (11 AA overlaps) containing all immune epitopes. Samples prior to vaccination were evaluated to establish a baseline response to this peptide pool. Additional PBMC samples were evaluated, as per guidelines, to examine the kinetics of the immune response to the total peptide mixture. For patients exhibiting a significantly higher-than-baseline response, the pools of all 15-mer peptides were unconvoluted to determine which specific immune peptides(s) were immunogenic. Additionally, several further assays were performed on appropriate samples, as needed. The entire 15mer pool or sub-pools were used as stimulating peptides for intracellular cytokine staining assays to identify and quantify antigen-specific CD4+, CD8+, central memory, and effector memory populations. Similarly, these pools were used to assess patterns of cytokines secreted by these cells to determine the TH1 versus TH2 phenotype. Extracellular cytokine staining and flow cytometry of unstimulated cells were used to quantify Tregs and myeloid-derived suppressor cells (MDSCs).

[0410] If a melanoma cell line is successfully established from a responding patient and activating epitopes can be identified, T-cell cytotoxicity assays are performed using mutant and corresponding wild-type peptides. "Epitope expansion" of PBMCs from primary immune endpoints was assessed using known melanoma tumor-associated antigens as stimulants and by using several additional identified mutant epitopes not selected among immunogens, such as... Figure 4 As shown.

[0411] Immunohistochemistry was performed on the tumor sample to quantify the CD4+, CD8+, MDSC, and Treg infiltrating populations.

[0412] Example 5 Neoantigen preparations Immediately after surgical resection of the tumor, a portion of tumor tissue and blood samples are transferred to a facility where a unique identifier is assigned for further tracking. The tumor tissue is depolymerized with collagenase, and the separated portions are frozen for nucleic acid (DNA and RNA) extraction. Blood samples are immediately transferred to a facility for nucleic acid extraction. The DNA and / or RNA extracted from the tumor tissue are used for whole-exome sequencing (e.g., using the Illumina HiSeq platform) and to determine HLA typing information. Within the scope of this invention, it is contemplated that missense or novel ORF neoantigen peptides can be directly identified using protein-based techniques (e.g., mass spectrometry).

[0413] Bioinformatics analysis was performed as follows. Sequence analysis of exomes and RNA-SEQ fast Q files utilized existing bioinformatics channels that have been widely used and validated in large-scale projects, such as TCGA for many patient samples (e.g., Chapman et al., 2011; Stransky et al., 2011; Berger et al., 2012). Two categories of sequence analysis existed: data processing and cancer genomic analysis.

[0414] Data Processing Channel: The Picard data processing channel (picard.sourceforge.net / ) is developed by Sequencing Platforms. Using different modules within the Picard channel, raw data extracted from each tumor and normal sample (e.g., from a sequencer) undergoes the following processes: (i) Data transformation: The raw Illumina data was transformed into standard BAM form, and basic QC metrics involving base distributions exceeding different quality thresholds were generated.

[0415] (ii) Alignment: The read pairs were aligned with the human genome (hg19) using the Burrows-Wheeler alignment tool (BWA).

[0416] (iii) Mark duplicates: Identify PCR and optical duplicates based on read pair plots and mark them in the final BAM file.

[0417] (iv) Indel re-alignment: Readings aligned to known insertion and deletion polymorphic sites in the genome were examined, and those sites that were corrected for improved log odds scores of at least 0.4 when re-aligned were corrected.

[0418] (v) Quality Recalibration: The original base quality scores reported by Yimingda Channel were recalibrated based on the read cycles, lanes, flow cell tiles, the bases in question, and the aforementioned bases. The recalibration assumes that all mismatches at non-dbSNP locations are attributed to errors that allow recalibrating the error probability in each class of interest to the portion of the total number of mismatches.

[0419] (vi) Quality Control: The final BAM file is processed to generate a large number of QC metrics, including the quality of readings in cycles, the distribution of quality scores, the summary of alignments, and the distribution of insert fragment sizes. Data that fails quality QC is blacklisted.

[0420] (vii) Consistency Verification: Genotypic data from samples collected orthogonally at approximately 100 known SNP locations are examined for consistency of the sequence data. A LOD score of ≥10 is used as a threshold for confirming consistency. Data that fails consistency QC is blacklisted.

[0421] (viii) Data clustering: All data from the same sample are merged, and the relabeling step is repeated. Novel target regions containing assumed short insertions and deletions are identified, and indel realignment steps are performed at these loci.

[0422] (ix) Local realignment of hypothetical indels around aggregated data: Identify novel target regions containing hypothetical short insertions and deletions, and perform local realignment steps at these loci (e.g., using the GATKRealignerTargetCreator and IndelRealigner modules) to ensure the consistency and correctness of indel calls.

[0423] (x) Quality control of aggregated data: QC metrics were recalculated, such as alignment summaries and insert fragment size distributions. Additionally, a set of metrics was developed to assess the rate of oxidative damage in the early steps of library construction caused by acoustic shearing of DNA in the presence of reactive contaminants from the extraction process.

[0424] Picard's output is a BAM file (Li et al., 2009) (see, for example, http: / / samtools.sourceforge.net / SAM1.pdf), which stores the base sequence, quality score, and alignment details of all readings for a given sample.

[0425] Cancer mutation detection pathway: As described here, analysis of tumor and matched normal BAM files from the Picard pathway is performed. 1. Quality Control (i) The Capseg procedure is applied to tumor and matched normal exome samples to obtain copy number curves. The resulting curves can then be manually examined and tumor / normal sample mixtures evaluated using a copy number QC tool. The mixtures are examined through a data generation and analysis workflow, which identifies and tracks normal samples with noise spectra along with cases where tumor samples have lower copy number variations than their corresponding normal samples.

[0426] (ii) Based on the copy number curve generated by Capseg, estimate tumor purity and ploidy using the ABSOLUTE tool 15. Strong noise spectra may originate from sequencing highly degraded samples. In such cases, it will be possible to estimate tumor purity and ploidy without labeling the corresponding samples.

[0427] (iii) ContEst (Cibulskis et al., 2011) was used to determine the level of cross-sample contamination in a sample. Samples with more than 4% contamination were discarded.

[0428] 2. Identification of somatic single nucleotide variants (SSNVs) Somatic base pair substitutions were identified by analyzing tumors and matched normal bam samples from patients using a Bayesian statistical framework called muTect (Cibulskis et al., 2013). In this preprocessing step, reads with dominant low-quality bases or mismatches with the genome were filtered out. MuTect then calculated two log-odds (LOD) scores, which summarized the confidence levels for the presence and absence of variants in tumor and normal samples, respectively. In the post-processing stage, candidate mutations were filtered using six filters to account for artifacts from capture, sequencing, and alignment.

[0429] (i) Proximal gap: Removes false positives due to the presence of misaligned indels near the event. Reject samples with ≥ 3 readings that have insertions or deletions within an 11-bp window surrounding the candidate mutation.

[0430] (ii) Poor mapping: Discard false positives resulting from fuzzy placement of reads in the genome. Candidates are rejected if reads in ≥ 50% of tumor and normal samples have a mapping quality of zero, or if no reads are available that accommodate mutant alleles with a mapping quality of ≥ 20.

[0431] (iii) Trial allele loci: Discard heterozygous loci that are normally discarded because they tend to produce many false positives.

[0432] (iv) Chain bias: Remove false positives caused by background-specific sequencing errors, where most reads accommodating the mutation have the same orientation. Reject candidates with chain-specific LOD < 2, where the sensitivity to this threshold is ≥ 90%.

[0433] (v) Clustering location: False positives are rejected due to alignment errors of reciprocal alleles that occur at a fixed distance to the start or end of the alignment. If the median distance to the start or end of these alignments is ≤ 10, it means the mutation is at the start or end of the alignment, or if the absolute median difference of these distances is ≤ 3, it means the mutation is clustered, and these are rejected.

[0434] (vi) Observations in controls: Discard false positives in tumors where there is evidence of cross-allele occurrence in normal samples that exceeds the expected random sequencing error. Reject if there are ≥ 2 cross-allele readings contained in normal samples, or if they are in ≥ 3% of readings, and if the sum of their quality scores is > 20.

[0435] In addition to these six filters, candidates are compared with a set of normal samples, and those found to be germ cell variants in two or more normal samples are rejected. The final set of mutations can then be annotated using the Oncotator tool with several fields, including genomic regions, codons, cDNA, and protein changes.

[0436] 3. Identification of small insertions and deletions in somatic cells The local realignment output described here (see “Local Realignment of Hypothetical Indels Around Aggregated Data”, ibid.) was used to predict candidate somatic and germline indels based on assessments of reads supporting the exclusionary presence of variants in tumors or in both tumor and normal bam cells. Further filtering was performed based on the number and distribution of mismatches and base quality scores (McKenna et al., 2010; DePristo et al., 2011). All indels were manually examined using the Integrated Genomics Viewer (Robinson et al., 2011) (www.broadinstitute.org / igv) to ensure high-fidelity calls.

[0437] 4. Gene fusion detection The first step in the gene fusion detection pipeline is to align tumor RNA-Seq reads with a library of known gene sequences, and then plot this alignment on genomic coordinates. Genome mapping helps to compress multiple read pairs mapped to different transcript variants that share exons with common genomic locations. DNA alignment bam files are queried for read pairs where the two co-pairs map to two different coding regions located on different chromosomes or, if on the same chromosome, at least 1 MB apart. It is also possible to require that the ends of the aligned pairs in their respective genes be aligned with the coding --> coding 5' -> 3' direction of the (presumed) fusion mRNA transcript. A list of gene pairs (where at least two such 'chimeric' read pairs exist) is enumerated as an initial list of presumed events undergoing further refinement. Next, all unaligned reads are extracted from the original bam file, with the additional constraint that their co-pairs were initially aligned and plotted into one of the genes in the gene pairs obtained as described herein. Then, all such initially unaligned reads can be compared to a custom “reference” construct of all possible exon-exon connections (full length, boundary-to-boundary, in the 5' -> 3' orientation) between the discovered gene pairs. If such an initially unaligned read (uniquely) maps to a connection between an exon of gene X and an exon of gene Y, and its mate is indeed mapped to either gene X or Y, then such a read is labeled a “fusion” read. A gene fusion event is defined as the presence of at least one fusion read in the correct relative orientation with its mate, without an excessive number of mismatches around the exon:exon connection, and covering at least 10 bp of either gene. Gene fusions between highly homologous genes (e.g., HLA family members) are likely to be false positives and are filtered out.

[0438] 5. Estimation of clonalness Bioinformatics analysis can be used to estimate the clonality of mutations. For example, the ABSOLUTE algorithm (Carter et al., 2012; Landau et al., 2013) can be used to estimate tumor purity, ploidy, absolute copy number, and the clonality of mutations. A probability density distribution of the allele fraction for each mutation is generated, which is then converted into the cancer cell fraction (CCF) of those mutations. Mutations are classified as clonal or subclonal based on whether their posterior probability of a CCF exceeding 0.95 is greater than or less than 0.5.

[0439] 6. Quantitative expression The TopHat suite (Langmead et al., 2009) was used to align RNA-Seq reads from tumors and matched normal bam cells with the hg19 genome. The quality of the RNA-Seq data was assessed using the RNA-SeQC (DeLuca et al., 2012) package. The RSEM tool (Li et al., 2011) was then used to estimate gene and isotype expression levels. The resulting reads / kilobases / millions and τ estimates were used to rank the neoantigens identified in each patient, as described elsewhere.

[0440] 7. Mutation Validation in RNA-Seq 8. The confirmation of somatic mutations identified through analysis of whole-exome data (including single nucleotide variants, small insertions and deletions, and gene fusions) as described herein is assessed by examining the patient's corresponding RNA-Seq tumor BAM file. For each variant locus, a probability calculation based on a β-binomial distribution is performed to ensure a minimum 95% probability of detection in the RNA-Seq data. A mutation is considered validated if at least two reads with captured identified mutations are present at a site with sufficient probability.

[0441] Selection of epitopes containing tumor-specific mutations: The presence of epitopes containing mutations in all missense mutations and novel ORFs was analyzed using a neural network-based algorithm, MHC, provided and maintained by the Center for Biological Sequence Analysis at the Technical University of Denmark. This algorithm family was rated as a top epitope prediction algorithm based on competition among a series of recently completed related methods. The algorithm was trained using an artificial neural network-based approach on 69 distinct human HLA A and B alleles, covering 99% of the HLA-A alleles and 87% of the HLA-B alleles found in the Caucasian population (the major ethnic group in the target patient population within the local region). The latest version (v2.4) was utilized.

[0442] The accuracy of these algorithms was evaluated by predicting mutations found in CLL patients with known HLA allotypes. The allotypes included were A0101, A0201, A0310, A1101, A2402, A6801, B0702, B0801, and B1501. In mid-2011, netMHCpan was used to predict all 9-mer and 10-mer peptides across each mutation. Based on these predictions, seventy-four (74) 9-mer peptides and sixty-three (63) 10-mer peptides were synthesized, most with predicted affinities below 500 nM, and binding affinity was measured using a competitive binding assay (Sette).

[0443] These peptides were repeatedly predicted in March 2013 using each of the latest versions of the netMHCC server (netMHCpan, netMHC, and netMHCcons). These three algorithms were among the most highly regarded in a set of 20 algorithms used in a competition in 2012 (Zhang et al.). The observed binding affinity was then evaluated relative to each of these new predictions. For each set of predicted and observed values, the percentage of correct predictions for each range and the number of samples are given. Each range is defined as follows: 0-150: Predicted affinity equal to or less than 150 nM and measured affinity equal to or less than 150 nM.

[0444] 0-150*: Predicted affinity equal to or less than 150 nM and measured affinity equal to or less than 500 nM.

[0445] 151-500 nM: Predicted affinity is greater than 150 nM but equal to or less than 500 nM and measured affinity is equal to or less than 500 nM.

[0446] FN (> 500 nM): False negative - predicted affinity is greater than 500 nM but measured affinity is equal to or less than 500 nM.

[0447] For 9mer peptides (Table 1), there was little difference between these algorithms, with the slightly higher values ​​of netMHC cons in the 151-500 nM range not being judged as significant due to the small number of samples.

[0448] Table 1 For 10-mer peptides (Table 2), there was again little difference between these algorithms, except that netMHC produced significantly more false positives than netMHCpan or netMMHCcons. However, compared to 9-mer, the accuracy of 10-mer predictions was slightly lower in the 0–150 nM and 0–150* nM ranges and significantly lower in the 151–500 nM range.

[0449] Table 2 For 10mer, only predictions in the 0–150 nM range are utilized, because the accuracy for the complex is less than 50% in the 151–500 nM range.

[0450] For any single HLA allele, the sample size is too small to draw any conclusions about the accuracy of the prediction algorithm for different alleles. Data from the largest available subgroup (0-150* nM; 9mer) are shown in Table 3 as an example.

[0451] Table 3 Only predictions of HLA A and B alleles were used because there is almost no data available to determine the accuracy of predictions for HLA C alleles (Zhang et al.).

[0452] Information from the TCGA database was used to evaluate melanoma sequence information and peptide binding predictions. Information from 220 melanoma samples from different patients revealed that, on average, each patient had approximately 450 missense sequences and 5 novel ORFs. Twenty patients were randomly selected, and predicted binding affinities were calculated using netMHC for mutations in all missense and novel ORFs (Lundegaard et al., Prediction of epitopes using neural network based methods, J Immunol Methods 374:26 (2011)). Because the HLA allotypes of these patients were unknown, the number of predicted binding peptides for each allotype was adjusted based on the frequency of that allotype (for the Bone Marrow Registry dataset [Caucasian melanoma], representing the dominant population expected to be affected in the geographic region) to generate the number of predicted mutant epitopes available for each patient. For each of these mutant epitopes (MUTs), the corresponding natural (NAT) epitope binding was also predicted.

[0453] Utilize the priority order described herein: It is predicted that 90% (18 out of 20) of the patients have at least 20 peptides suitable for vaccination; For nearly a quarter of patients, the new ORF peptide can make up half of all 20 peptides; For just over half of the patients, only peptides from categories 1 and 2 will be used; For 80% of patients, only peptides from categories 1, 2, and 3 will be used.

[0454] Therefore, a sufficient number of mutations exist in melanoma, so a high proportion of patients are expected to produce a sufficient number of immunogenic peptides.

[0455] Example 6 Peptide production and formulation GMP-compliant neoantigen peptides for immunological use were prepared according to FDA regulations via chemical synthesis (Merrifield RB: Solidphase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 85:2149-54, 1963). Three rounds of development were conducted, each with approximately 20-30 mer peptides. Each round was performed in the same facility and using the same equipment used for GMP operation, utilizing draft GMP batch records. Each round successfully produced >50 mg of each peptide, which was tested by all currently planned release tests (e.g., appearance by MS, purity by RP-HPLC, content by elemental nitrogen, and TFA content by RP-HPLC) and met specific specifications where appropriate. These products were also produced within the expected timeframe for this part of the process (approximately 4 weeks). Large-volume lyophilized peptides were placed in long-term stability studies and evaluated at different time points for up to 12 months.

[0456] Materials from these runs have been used to test the planned dissolution and mixing methods. In short, each peptide was dissolved at a high concentration (50 mg / ml) in 100% DMSO and diluted to 2 mg / ml in an aqueous solvent. Initially, PBS was expected to be used as the diluent; however, salting out of a small number of peptides produced visible turbidity. D5W (5% glucose aqueous solution) showed to be much more effective; 37 out of 40 peptides were successfully diluted to clear solutions. 10% sucrose or 10% trehalose in water were also effective. Unlike formulations containing 5% dextrose, formulations containing 10% sucrose or 10% trehalose are lyophilizable. Only the peptides in question were highly hydrophobic.

[0457] Table 4 shows the results of the solubility assessment of 60 potential neoantigenic peptides classified based on the calculated fractions of hydrophobic amino acids. As shown, almost all peptides with a hydrophobic fraction below 0.4 are soluble in DMSO / D5W, but many peptides with a hydrophobic fraction greater than or equal to 0.4 are insoluble in DMSO / D5W (indicated by red highlighting in the column labeled "Solubility in DMSO / D5W"). Many of these can be dissolved by adding succinate (indicated by green highlighting in the column labeled "Solubility in DMSO / D5W / Succinate"). Three of these four peptides have hydrophobic fractions between 0.4 and 0.43. When succinate is added, four peptides become less soluble; three of these four peptides have a hydrophobic fraction greater than or equal to 0.45.

[0458] Table 4 The predicted biochemical properties of the immunopeptides in the study are evaluated and the synthesis plan is modified accordingly (using shorter peptides, changing the region to be synthesized in the direction of the N-terminus or C-terminus around the predicted epitope, or potentially utilizing alternative peptides) in order to limit the number of peptides with highly hydrophobic moieties.

[0459] Ten separate peptides in DMSO / D5W were subjected to two freeze / thaw cycles and showed complete recovery. Two separate peptides were dissolved in DMSO / D5W and their stability was assessed at two temperatures (-20°C and -80°C). These peptides were evaluated (RP-HPLC, pH, and visual inspection) for up to 24 weeks. Both peptides remained stable for up to 24 weeks; for either peptide, the percentage of impurities detected by RP-HPLC did not change significantly when stored at -20°C or -80°C. Any minor variations appear to be attributable to assay variability, as no trend was observed to be evaluated.

[0460] like Figure 5 As shown, the dosage form design involved preparing four patient-specific peptide pools, each consisting of five peptides. The RP-HPL assay was prepared and approved to evaluate these peptide mixtures. This assay achieves good resolution of multiple peptides within a single mixture and can also be used to quantify individual peptides.

[0461] Membrane filtration (0.2 μm pore size) was used to reduce bioburden and perform final filtration sterilization. Four different filter types with appropriate sizes were initially evaluated, and a Pall PES filter (#4612) was selected. To date, four different mixtures (each containing five different peptides) have been prepared and sequentially filtered individually through two PES filters. The recovery of each individual peptide was evaluated using RP-HPLC. For 18 of the 20 peptides, the recovery after two filtrations was >90%. For the two highly hydrophobic peptides, the recovery was less than 60% when evaluated on a small scale, but almost complete (87% and 97%) was achieved on a large scale. As described herein, the method attempts to limit the hydrophobicity of the selected sequences.

[0462] A peptide pool (pool 4) consisting of five peptides was prepared by dissolving them in DMSO, diluting them to 2 mg / ml with D5W / succinate (5 mM), and collecting the solutions to a final peptide concentration of 400 μg / ml and a final DMSO concentration of 4%. After preparation, the peptides were filtered through a 25 mm Pall PES filter (Cat # 4612) and dispersed in 1 ml aliquots into Nunc Cryo vials (# 375418). Samples were analyzed at time zero and at weeks 2 and 4 until the cutoff time. Additional samples were analyzed at weeks 8 and 24. No significant changes in the HPLC or impurity characteristic curves of peptide pool 4 were observed at -80°C over the four-week time points. Visual observation and pH remained unchanged for the peptide pool throughout the four-week time points.

[0463] Example 7 peptide synthesis GMP peptides were synthesized using standard solid-phase peptide chemistry (e.g., using a CS 536 XT peptide synthesizer) and purified by RP-HPLC. Each individual peptide was analyzed by a variety of approved assays to evaluate appearance (visual), purity (RP-HPLC), identity (by mass spectrometry), quantity (elemental nitrogen), and trifluoroacetate counterion (RP-HPLC), and release.

[0464] These personalized neoantigen peptides can be composed of up to 20 different peptides unique to each patient. Each peptide can be a linear polymer of approximately 20-30 L-amino acids linked by standard peptide bonds. The amino terminus can be a primary amine (NH2-) and the carboxyl terminus is a carbonyl group (-COOH). Twenty standard amino acids commonly found in mammalian cells are used (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine). The molecular weight of each peptide varies based on its length and sequence, and the molecular weight of each peptide is calculated.

[0465] For all synthetic reactions, amino acids are protected at the Fmoc (9-fluorenylmethoxycarbonyl)-N-terminus. Where appropriate, the side chains of the amino acids are protected with 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl (Pbf), triphenylmethyl (Trt), tert-butoxycarbonyl (Boc), or tert-butyl ether (tBu) groups. All high-capacity amino acids are dissolved in dimethylformamide (DMF). In separate reactions, condensation utilizes a combination of two catalysts: Diisopropylcarbodiimide / 1-hydroxybenzotriazole (DIC / HOBT) Diisopropylethylamine / 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea hexafluorophosphate (DIEA / HBTU) To ensure high levels of incorporation, each amino acid is coupled twice. The first coupling uses DIC / HOBT for 2–6 hours, and the second coupling uses DIEA / HBTU for 1–2 hours. UV absorbance is monitored for each coupling cycle, and the resin is thoroughly washed with DMF between coupling cycles to improve efficiency. After two coupling cycles, the calculated coupling efficiency must be at least 95% to proceed to the next cycle. Further synthesis of any peptide that does not meet the minimum coupling efficiency is stopped.

[0466] After all amino acids have been coupled, the resin was washed twice with DMF and then three times with methanol. The resin was then briefly vacuum-dried while still in the reaction vessel and transferred to a new, tare-de-weighed vessel for vacuum drying (greater than 12 hours) until it was free-flowing. The mass of the synthesized crude peptide was determined by weighing the vessel containing the dry resin, subtracting the mass of the tare-de-weighed vessel, and adjusting the resin mass. Expected mass yields range from 60% to 90%. Any synthesis that failed to produce at least 200 mg of crude peptide was terminated. The dry resin can be stored at 4°C for up to 28 days before cleavage begins.

[0467] The cutting reaction is carried out in a single chamber. Before transferring the patient-specific dry resin from the synthesis chamber to the cutting chamber, the cutting chamber is QA-certified as fully compliant for the synthesis of the new GMP product. Certification of compliance includes line gap inspection, verification of GMP kit cleaning, grading of all required materials and glassware, verification of equipment suitability and labeling, and verification that all required personnel are properly trained, certified as competent to perform the work, appropriately dressed, and free of obvious disease.

[0468] With the verification of the equipment to be used (rotary evaporator, vacuum pump, balance) and the review of documents indicating that the equipment has been properly cleaned and calibrated, the laboratory is ready for operation (if appropriate). A complete list of all raw materials required by QA (TFA, triisopropylsilane (TIS), and 1,2-ethylenedithiol) is prepared, and the batch numbers to be used are identified, the expiration dates are retested, and the amount of material to be dispersed for the daily reaction is determined.

[0469] In the presence of 2% triisopropylsilane (TIS) and 1% 1,2-ethylenedithiol as scavengers of acid-generating free radicals, under acidic conditions (95% TFA) for 3 to 4 hours at room temperature, the cleavage of peptide chains and side-chain protecting groups from the resin is achieved.

[0470] The resin was separated from the free crude peptide by filtration. The final solution...

Claims

1. A pharmaceutical composition comprising: (a) at least one neoantigenic peptide or a pharmaceutically acceptable salt thereof; (b) pH altering agent; and (c) Pharmaceutically acceptable carriers.

2. The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is a vaccine composition.

3. The pharmaceutical composition of claim 1 or claim 2, wherein the pharmaceutical composition comprises at least two neoantigen peptides.

4. The pharmaceutical composition according to any one of claims 1-3, wherein the claimed pharmaceutical composition comprises at least three neoantigen peptides.

5. The pharmaceutical composition according to any one of claims 1-4, wherein the pharmaceutical composition comprises at least four neoantigen peptides.

6. The pharmaceutical composition according to any one of claims 1-5, wherein the pharmaceutical composition comprises at least five neoantigen peptides.

7. The pharmaceutical composition according to any one of claims 1-6, wherein the length of the at least one neoantigen peptide ranges from about 5 to about 50 amino acids.

8. The pharmaceutical composition according to any one of claims 1-7, wherein the length of the at least one neoantigen peptide ranges from about 15 to about 35 amino acids.

9. The pharmaceutical composition according to any one of claims 1-8, wherein the pH changer is a base.

10. The pharmaceutical composition according to any one of claims 1-9, wherein the pH-changing agent is a dicarboxylate or a tricarboxylate.

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