Multicomponent chemical compositions of peptide-based neo-antigen vaccines

Abstract

Provided herein are immunogenic compositions comprising tumor-specific neo-antigen long peptides, tumor-specific neo-antigen short peptides, and adjuvants, optionally helper peptides, and optionally tumor-specific peptides. The disclosure also provides methods of using these immunogenic compositions to treat cancer.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 194,041, filed May 27, 2021, which is incorporated by reference in its entirety.

[0002] Sequence Listing Reference This application contains a sequence listing in computer readable form. The computer readable form is incorporated herein by reference. The ASCII copy was created on May 3, 2022, is named 146401_091808_SL.txt, and is 1,108 bytes in size. [Background technology]

[0003] Cancer is the leading cause of death worldwide, accounting for one-quarter of all deaths. Siegel et al., CA: A Cancer Journal for Clinicians, 68:7-30(2018). In 2018, there were 18.1 million new cancer cases and 9.6 million cancer-related deaths. Bray et al., CA: A Cancer Journal for Clinicians, 68(6):394-424. There are several existing standard of care cancer therapies, including ablative techniques (e.g., surgical procedures and radiation) and chemical techniques (e.g., chemotherapy agents). Unfortunately, such therapies are often associated with significant risks, adverse side effects, and prohibitive costs, as well as uncertain efficacy.

[0004] Cancer immunotherapy (e.g., cancer vaccines) has emerged as a promising cancer treatment modality. The goal of cancer immunotherapy is to direct the immune system for selective destruction of cancer while leaving normal tissues unharmed. Traditional cancer vaccines typically target tumor-associated antigens, which are typically present in normal tissues but overexpressed in cancer. However, because these antigens are often present in normal tissues, immune tolerance can prevent immune activation. Several clinical trials targeting tumor-associated antigens have failed to demonstrate long-lasting beneficial effects compared to standard treatments. Li et al.,Ann Oncol.,28(Suppl 12):xii11-xii17(2017).

[0005] Neoantigens represent attractive targets for cancer immunotherapy. Neoantigens are non-autologous proteins with individual specificity. Neoantigens originate from random somatic mutations in the tumor cell genome and are not expressed on the surface of normal cells. Id. Because neoantigens are expressed exclusively on tumor cells and therefore do not induce central immune tolerance, cancer vaccines targeting cancer neoantigens have potential advantages including attenuated central immune tolerance and improved safety profile. Id.

[0006] The cancer mutation landscape is complex, and tumor mutations are usually unique to each individual subject. Mouse somatic mutations detected by sequencing do not result in effective neoantigens. Only a small percentage of mutations in tumor DNA or tumor cells are transcribed, translated, and processed into tumor-specific neoantigens with sufficient accuracy to design a vaccine that is likely to be effective. Moreover, not all neoantigens are immunogenic. In fact, the percentage of T cells that naturally recognize endogenous neoantigens is about 1%-2%. See Karpanen et al., Front Immunol., 8:1718 (2017). Moreover, the costs and time associated with producing neoantigen vaccines are significant.

[0007] Thus, significant challenges remain in developing personalized cancer vaccines that contain neoantigens.

[0008] Summary of the Invention The present invention relates to personalized (i.e., subject-specific) immunogenic compositions (e.g., cancer vaccines) that contain unique combinations of components. The immunogenic compositions described herein include multiple tumor-specific neo-antigen long peptides, multiple tumor-specific neo-antigen short peptides, and an adjuvant. The immunogenic compositions may optionally include helper peptides. The immunogenic compositions may optionally include tumor-specific frameshift peptides.

[0009] The immunogenic composition may contain up to about 50 tumor-specific neoantigen long and / or short peptides. The immunogenic composition may contain about 10 to about 20 tumor-specific neoantigen long and / or short peptides. The immunogenic composition preferably contains about 19 tumor-specific neoantigen long and / or short peptides.

[0010] The immunogenic composition may comprise at least about 2 or more types of tumor-specific neo-antigen long peptides. The immunogenic composition may comprise about 2 to about 18 types of tumor-specific neo-antigen long peptides. The immunogenic composition may typically comprise at least about 10 to about 15 types of tumor-specific neo-antigen long peptides. The immunogenic composition may comprise at least about 2 or more types of tumor-specific neo-antigen short peptides. The immunogenic composition may comprise at least about 2 to about 10 types of tumor-specific neo-antigen short peptides.

[0011] Typically, each of the tumor-specific neo-antigen long peptides in the immunogenic composition is different. Typically, each of the tumor-specific short peptides in the immunogenic composition is different.

[0012] The immunogenic composition may comprise two or more tumor-specific frameshift peptides. The tumor-specific neo-antigen long and / or short peptides may be divided into two or more peptide pools. Preferably, the tumor-specific neo-antigen long and / or short peptides are divided into about four peptide pools. Usually, each peptide pool may comprise about five or less tumor-specific neo-antigen long and / or short peptides. One or more peptide pools may optionally comprise a helper peptide. One or more peptide pools may comprise one or more tumor-specific frameshift peptides.

[0013] As an example, three peptide pools may contain about five tumor-specific neo-antigen long and / or short peptides, and one peptide pool may contain four tumor-specific neo-antigen long and / or short peptides and a helper peptide. Each peptide pool may contain different tumor-specific neo-antigen long and / or short peptides. The tumor-specific neo-antigen long peptides may be about 15 to about 30 amino acids in length. The tumor-specific neo-antigen short peptides may be about 5 to about 15 amino acids in length.

[0014] The adjuvant used in the immunogenic composition can be a Toll-like receptor agonist, a NOD-like receptor agonist, an Mda5 agonist, a RIG-I, a PKR agonist, a STING agonist or other innate immune sensing pathway agonist. Each of the peptide pools can include an adjuvant.

[0015] The helper peptide may be a pan-DR helper epitope (PADRE), a tetanus helper peptide, a Hepatitis B surface antigen helper T cell epitope, a pertussis toxin helper T cell epitope, a measles virus F protein helper T cell epitope, a Chlamydia trachomitis major outer membrane protein helper T cell epitope, a diphtheria toxin helper T cell epitope, a Plasmodium falciparum circumsporozoite protein helper T cell epitope, a Schistosoma mansoni triosephosphate isomerase helper T cell epitope, a keyhole limpet hemocyanin, a Plasmodium vivax B cell epitope (PVB), an Escherichia coli TraT helper T cell epitope, a synthetic helper T epitope, an immunopotentiating analogue or segment of any of the above helper peptides. Preferably, the helper peptide is a pan-DR helper epitope (PADRE).

[0016] The immunogenic compositions disclosed herein include those that bind to polyfunctional CD4 + and CD8 + A response can be induced.

[0017] The present disclosure also relates to pharmaceutical compositions comprising the immunogenic compositions disclosed herein.

[0018] The present disclosure also relates to a method of treating cancer in a subject in need thereof, comprising administering a personalized immunogenic composition as described herein. The methods disclosed herein may be suitable for any number of cancers. Majority may be from melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, bladder cancer, or lung cancer. Preferably, the cancer is melanoma, breast cancer, lung cancer, colon cancer, and urothelial cancer. Subjects suitable for the methods disclosed herein may be diagnosed with cancer, already suffer from cancer, have recurrent cancer, or are at risk of developing cancer.

[0019] A subject may be administered at least one or more doses of the immunogenic compositions disclosed herein. Typically, a subject is administered at least six doses of the immunogenic composition at different times.

[0020] The immunogenic composition may be administered as one or more peptide pools per dose. The subject may be administered six doses of two or more peptide pools at different times. The subject may be administered each peptide pool in one to four limbs of the subject. The peptide pools may be administered in different locations. The subject may be administered each peptide pool in a different limb of the subject. The subject may be administered each peptide pool in the same limb of the subject at each dose. Each dose of the immunogenic composition may be administered at least about one week to about four weeks after administration of the preceding dose of the immunogenic composition.

[0021] The adjuvant can be administered between each dose of the immunogenic composition.The adjuvant can be administered once a week between each dose of the immunogenic composition.The adjuvant can be a Toll-like receptor agonist, a NOD-like receptor agonist, an Mda5 agonist, a RIG-I, a PKR agonist, a STING agonist, or other innate immune sensing pathway agonist.

[0022] The method disclosed herein may further comprise administering at least one or one checkpoint inhibitor. The checkpoint inhibitor may be an inhibitor of programmed death-1 (PD-1) pathway, Lag3 pathway, Tim3 pathway, ICOS pathway, OX-40, GITR pathway or 4-1BB pathway. In particular, the inhibitor of PD-1 pathway may be an anti-PD-1 antibody, a small molecule, a peptide, or the pathway may be inhibited by genetic means (e.g., short interfering RNA or CRISPR-mediated gene editing). The checkpoint inhibitor of interest may be an anti-cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) antibody, a small molecule, a peptide, or the pathway may be inhibited by genetic means (e.g., short interfering RNA or CRISPR-mediated gene editing).

[0023] The immunogenic compositions may be administered by subcutaneous, intramuscular, transcutaneous, intradermal, transdermal, intravenous, intratumoral, intralymphatic or intraperitoneal administration. The immunogenic compositions disclosed herein are preferably administered by intramuscular administration. [Brief description of the drawings]

[0024] [Figure 1] FIG. 1 is a schematic diagram showing the composition of vaccine peptide pools. [Diagram 2] FIG. 1 is a schematic diagram showing a treatment regimen for an immunogenic composition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The present disclosure relates to potent personalized cancer immunogenic compositions (e.g., subject-specific immunogenic compositions) that comprise unique combinations of components. The immunogenic compositions described herein comprise a plurality of tumor-specific neo-antigen long peptides, a plurality of tumor-specific neo-antigen short peptides, and an adjuvant. The immunogenic compositions may optionally comprise a helper peptide. The immunogenic compositions may optionally comprise a tumor-specific frameshift peptide. The immunogenic compositions may comprise up to about 50 tumor-specific neo-antigen long peptides and / or short peptides. Typically, the immunogenic compositions comprise about 10 to about 20 tumor-specific neo-antigen long peptides and / or short peptides. Preferably, the immunogenic compositions comprise about 19 tumor-specific neo-antigen long peptides and / or short peptides. The immunogenic compositions may be divided into two or more peptide pools that comprise the tumor-specific neo-antigen long peptides and / or short peptides, and an adjuvant. One or more peptide pools may further comprise a helper peptide. One or more of the peptide pools may further comprise a tumor-specific frameshift peptide. The immunogenic composition is preferably divided into about four peptide pools, each of which contains about five or less peptides (i.e., tumor-specific neoantigen long or short peptides, helper peptides, or tumor-specific frameshift peptides). Each peptide pool may also include an adjuvant.

[0026] The present disclosure also relates to a method of treating cancer in a subject in need thereof by administering an immunogenic composition comprising a tumor-specific neo-antigenic peptide. The subject may be administered at least one or more doses of the immunogenic composition. Typically, the subject may be administered about six doses of the immunogenic composition at different times. The immunogenic composition may be administered as one or more peptides per dose. The peptide pool may be administered to different limbs (e.g., about 1 to about 4 limbs). The peptide pool may be administered to the same limb of the subject each time.

[0027] All publications and patents cited in this disclosure are incorporated by reference in their entirety. To the extent that material incorporated by reference contradicts or is inconsistent with this specification, the specification will take precedence over any such material. The citation of any reference herein is not an admission that such reference is prior art to this disclosure. When a range of values ​​is given, it includes embodiments using any specific value within the range. Furthermore, reference to values ​​given in ranges includes any and all values ​​falling within that range. All ranges include their endpoints and can be combined. When values ​​are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. Reference to a particular numerical value includes at least that particular value unless otherwise clear from the context. The use of "or" is intended to mean "and / or" unless otherwise dictated by the specific context of its use.

[0028] Various terms relating to the described embodiments are used throughout the specification and claims. Such terms should be given their ordinary meaning in the art unless otherwise indicated. Other specifically defined terms should be interpreted consistent with the definitions provided herein. The techniques and procedures described or referred to herein are generally well understood and typically employed by those of skill in the art using conventional methodologies, such as the widely used molecular cloning methodologies described in Sambrook et al., Molecular Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, procedures involving the use of commercially available kits and reagents are typically performed according to manufacturer-specified protocols and conditions unless otherwise noted.

[0029] As used herein, the singular forms "a," "an," and "the" include the plural forms unless otherwise clear from the context. The terms "including," "including," and similar terms are intended to convey an open-ended inclusion unless specifically indicated otherwise.

[0030] Unless otherwise indicated, the terms "at least," "less than," and "about," or similar terms preceding a group of elements or range, should be understood to refer to every element in the group or range. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0031] The term "cancer" refers to a physiological condition in a subject in which a population of cells is characterized by uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and / or certain morphological features. Cancers often take the form of a tumor or mass, but may also exist alone in a subject or circulate in the bloodstream as independent cells, such as leukemia or lymphoma cells. The term cancer includes all types of cancer and metastatic cancer, including hematological malignancies, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumors. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specific examples of such cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, digestive cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer (e.g., triple-negative breast cancer, hormone receptor positive breast cancer), osteosarcoma, melanoma, colon cancer, colorectal cancer, endometrial (e.g., serous) or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatocellular carcinoma, and various types of head, neck and brain cancer. Triple-negative breast cancer refers to breast cancer that is negative for the expression of estrogen receptor (ER), progesterone receptor (PR) and Her2 / neu genes. Hormone receptor positive breast cancer refers to breast cancer that is positive for at least one of the following: ER or PR, and negative for Her2 / neu (HER2).

[0032] The term "helper peptide" as used herein refers to a foreign peptide that functions as a non-specific vaccine helper epitope and induces an increased immune response by activated CD4 T cells.

[0033] As used herein, the term "frameshift mutation" refers to a change in a nucleic acid sequence within an open reading frame encoding a protein that results in an alteration in the reading frame downstream of the mutation, thereby producing a protein with an altered sequence compared to the wild-type protein. Typically, frameshift mutations arise from indels (i.e., insertions or deletions of one or more nucleotides) that are not a multiple of three.

[0034] As used herein, the term "tumor-specific frameshift peptide" refers to a tumor-specific neo-antigenic peptide that contains a frameshift mutation.

[0035] The term "neo-antigen" as used herein refers to an antigen that has at least one modification that makes it different from the corresponding parent antigen, for example, a modification due to a mutation in tumor cells or a modification due to a post-translational modification specific to tumor cells. Mutations may include frameshifts, indels, missense or nonsense substitutions, splice site changes, genomic rearrangements or gene fusions, or any genomic expression changes that give rise to neo-antigens. Mutations may include splice mutations. Post-translational modifications specific to tumor cells may include aberrant phosphorylation. Post-translational modifications specific to tumor cells may also include splice antigens generated by the proteosome. See Lipe et al., Science, 354(6310):354:358 (2016). Typically, point mutations account for about 95% of mutations in tumors, with indels and frameshift mutations accounting for the remainder. See Snyder et al., N Engl J Med., 371:2189-2199 (2014).

[0036] As used herein, the term "tumor-specific neoantigen" is a neoantigen that is present in tumor cells or tissues of a subject but not in normal cells or tissues of the subject.

[0037] As used herein, the term "subject" refers to any animal, e.g., any mammal, including but not limited to humans, non-human primates, rodents, etc. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0038] Further description of the methods, and guidance for practicing the methods, is provided herein.

[0039] A. Immunogenic composition A neo-antigenic peptide The immunogenic composition can be formulated such that the selection and number of tumor-specific neoantigens are tailored to the particular cancer of the subject. For example, the selection of tumor-specific neoantigens can depend on the specific type of cancer, the state of the cancer, the immune status of the subject, and the MHC type of the subject.

[0040] The immunogenic composition may comprise at least about 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 108, 109, 109, 108, 1109, 111, 112, 113, 114, 115, 116 The immunogenic composition may comprise about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, or more tumor specific neo-antigen peptides (e.g., tumor specific neo-antigen long and / or short peptides). The immunogenic composition may comprise up to about 100 tumor specific. The immunogenic composition may contain about 10-20 tumor-specific neo-antigens, about 10-30 tumor-specific neo-antigens, about 10-40 tumor-specific neo-antigens, about 10-50 tumor-specific neo-antigens, about 10-60 tumor-specific neo-antigens, about 10-70 tumor-specific neo-antigens, about 10-80 tumor-specific neo-antigens, about 10-90 tumor-specific neo-antigens, or about 10-100 tumor-specific neo-antigens. Typically, the immunogenic composition comprises at least about 10 tumor-specific neo-antigens. The immunogenic composition disclosed herein preferably comprises 10 to about 20 tumor-specific neo-antigens. For example, the immunogenic composition may comprise about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 tumor-specific neo-antigens. Preferably, the immunogenic composition may comprise about 19 tumor-specific neoantigens. Preferably, the immunogenic composition may comprise about 20 tumor-specific neoantigens. Each of the tumor-specific neoantigens in the immunogenic composition is preferably different.

[0041] The immunogenic compositions disclosed herein include a plurality of tumor-specific neo-antigen long peptides and a plurality of tumor-specific neo-antigen short peptides. The tumor-specific neo-antigen long peptides are internalized by antigen-presenting cells and processed for MCH presentation. MHC class II molecules typically bind peptides that are longer in length. MHC class II can generally tolerate peptides from about 13 amino acids to about 25 amino acids in length. In embodiments, the one or more tumor-specific neo-antigens are long peptides about 13-25 amino acids in length. MHC class I molecules typically bind short peptides. The tumor-specific neo-antigen short peptides bind directly to the MHC molecule. MHC class I molecules can bind short peptides. MHC class I molecules can generally tolerate peptides about 8 amino acids to about 10 amino acids in length.

[0042] The immunogenic composition may comprise at least about two or more tumor-specific neo-antigen long peptides. The immunogenic composition may comprise at least about 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 108, 109, 109, 110, 111, 121, 122, The immunogenic composition may comprise about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, or more tumor-specific neo-antigen long peptides. The immunogenic composition may contain about 2-20 tumor-specific neo-antigen long peptides, about 10-20 tumor-specific neo-antigen long peptides, about 10-30 tumor-specific neo-antigen long peptides, about 10-40 tumor-specific neo-antigen long peptides, or about 10-50 tumor-specific neo-antigen long peptides. Typically, the immunogenic composition comprises at least about 10 tumor-specific neo-antigen long peptides.

[0043] The immunogenic composition disclosed herein preferably comprises between 10 and about 15 tumor-specific neo-antigen long peptides. For example, the immunogenic composition may comprise about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, or about 18 tumor-specific neo-antigen long peptides. Typically, the immunogenic composition disclosed herein comprises more tumor-specific neo-antigen long peptides than tumor-specific neo-antigen short peptides. Each of the tumor-specific neo-antigen long peptides in the immunogenic composition is preferably different.

[0044] The immunogenic composition may comprise at least about two or more tumor-specific neoantigen short peptides. The immunogenic composition may comprise at least about 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 121, 122, 131, 132, 143, 144, 150, The immunogenic composition may comprise about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, or more tumor-specific neo-antigen short peptides. The immunogenic composition may contain about 2 to about 10 tumor-specific neo-antigen short peptides. For example, the immunogenic composition may comprise at least about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 tumor-specific neo-antigen short peptides.

[0045] The immunogenic composition may contain about 2-15 tumor-specific neo-antigen short peptides, about 2-10 tumor-specific neo-antigen short peptides, about 4-10 tumor-specific neo-antigen short peptides, about 5-10 tumor-specific neo-antigen short peptides, about 6-10 tumor-specific neo-antigen short peptides, about 7-10 tumor-specific neo-antigen short peptides, or about 8-10 tumor-specific neo-antigen short peptides. The immunogenic composition disclosed herein preferably contains 2 to about 10 tumor-specific neo-antigen short peptides. For example, the immunogenic composition may contain about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 tumor-specific neo-antigen short peptides. Typically, the immunogenic composition disclosed herein contains fewer tumor-specific neo-antigen short peptides compared to the tumor-specific neo-antigen long peptides. It is preferred that each of the tumor-specific short peptides in the immunogenic composition is different.

[0046] The tumor specific neo-antigen long peptide can be about 15 to about 30 amino acids in length. The tumor specific neo-antigen long peptide can be about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids in length.

[0047] The tumor specific neoantigen short peptides can be from about 5 amino acids in length to about 15 amino acids in length. The tumor specific neoantigen short peptides can be about 5 amino acids in length, about 6 amino acids in length, about 7 amino acids in length, about 8 amino acids in length, about 9 amino acids in length, about 10 amino acids in length, about 11 amino acids in length, about 12 amino acids in length, about 13 amino acids in length, about 14 amino acids in length, or about 15 amino acids in length.

[0048] Tumor-specific neo-antigenic peptides, whether long or short, can arise from any mechanism that results in tumor-specific transcription or translated peptides, including, but not limited to, insertions, deletions, and / or rearrangements of tumor DNA, transcription errors, altered and / or incomplete intron splicing of the primary transcript, or translation errors.

[0049] b. Peptide pool The tumor-specific neo-antigen long and / or short peptides of the immunogenic composition may be divided into two or more peptide pools. For example, the tumor-specific neo-antigen long and / or short peptides of the immunogenic composition may be divided into about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, or more peptide pools. The desired number of peptide pools may depend on the number of tumor-specific neo-antigen long and / or short peptides in the immunogenic composition. For example, an immunogenic composition containing about 40 tumor-specific neo-antigen long and / or short peptides may be divided into about 5 to about 10 peptide pools. For example, an immunogenic composition containing about 30 tumor-specific neo-antigen long and / or short peptides may be divided into about 5 to about 8 peptide pools. For example, an immunogenic composition containing about 20 tumor-specific neo-antigen long and / or short peptides may be divided into about 2 to about 5 peptide pools.

[0050] In the preferred immunogenic compositions described herein, the tumor specific neo-antigen long and / or short peptides may be split into about 2 to about 5 peptide pools. Without wishing to be bound by theory, it is believed that splitting the tumor specific neo-antigen long and / or short peptides may facilitate cosolubility of the immunogenic composition. Preferably, the tumor specific neo-antigen long and / or short peptides may be split into about 4 peptide pools, or about 5 peptide pools.

[0051] Each peptide pool disclosed herein may contain up to about 20 tumor-specific neo-antigen long and / or short peptides. For example, each peptide pool disclosed herein may contain about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 tumor-specific neo-antigen long and / or short peptides. Each peptide pool preferably contains about 5 or less peptide pools. For example, each peptide pool may contain about 1, about 2, about 3, about 4, or about 5 tumor-specific neo-antigen long and / or short peptides.

[0052] Each peptide pool may contain the same number of tumor-specific neo-antigen long and / or short peptides, for example, each peptide pool may contain about 1, about 2, about 3, about 4 or about 5 tumor-specific neo-antigen long and / or short peptides.

[0053] Each peptide pool may contain a different number of tumor-specific neo-antigen long and / or short peptides. For example, four peptide pools may contain five tumor-specific neo-antigen long and / or short peptides, and one peptide pool may contain four tumor-specific neo-antigen long and / or short peptides. For example, three peptide pools may contain five tumor-specific neo-antigen long and / or short peptides, one peptide pool may contain four tumor-specific neo-antigen long and / or short peptides, and one peptide pool may contain three tumor-specific neo-antigen long and / or short peptides. For example, two peptide pools may contain five tumor-specific neo-antigen long and / or short peptides, one peptide pool may contain four tumor-specific neo-antigen long and / or short peptides, and one peptide pool may contain three tumor-specific neo-antigen long and / or short peptides.

[0054] The immunogenic composition disclosed herein may also include at least one helper peptide. In some examples, one peptide pool of the immunogenic composition may include a helper peptide. In some examples, one or more peptide pools disclosed herein may include a helper peptide. A peptide pool may include about one, about two, about three, about four, about five, or more helper peptides. However, it is usually preferred that a peptide pool includes a single helper peptide. In one example, if an immunogenic composition is divided into five peptide pools, a single peptide pool may include a helper peptide.

[0055] The immunogenic composition further comprises an adjuvant. One or more peptide pools can comprise an adjuvant. In some examples, each peptide pool can comprise an adjuvant. In other examples, the adjuvant can be present in a portion of the peptide pool. For example, if the immunogenic composition is divided into 5 peptide pools, about 1, about 2, about 3, about 4 or about 5 peptide pools can comprise an adjuvant. Preferably, each peptide pool comprises an adjuvant.

[0056] The immunogenic composition disclosed herein may also comprise at least one or more tumor-specific frameshift peptides.The immunogenic composition may comprise about 1, about 2, about 3, about 4, about 5, about 6, about, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, or more tumor-specific frameshift peptides.In some examples, one or more peptide pools may comprise tumor-specific frameshift peptides.The peptide pool may comprise about 1, about 2, about 3, about 4, about 5, or more tumor-specific frameshift peptides.

[0057] The immune response in the subject can include the presentation of one or more tumor-specific neoantigens on the surface of a tumor cell, the presentation of one or more tumor-specific neoantigens by one or more MHC molecules on a tumor cell, or the presentation of one or more tumor-specific neoantigens by an antigen-presenting cell to a T cell.

[0058] The immune response in the subject can be a CD4+ mediated response, a CD8+ mediated response, or a polyfunctional CD4+ and mediated response.

[0059] The immunogenic compositions may contain components that are individualized according to the personal needs of a particular subject.

[0060] C. helper peptide The immunogenic composition disclosed herein may also comprise at least one helper peptide.Helper peptide may be any suitable peptide that stimulates CD4+ T cell response that is not specific to cancer antigen, but can stimulate recall response or other non-specific help.Activation of CD4 T cells can help CD8+ T cells.

[0061] Helper peptides are sequences of amino acids (natural or unnatural) that have T cell helper activity. Helper peptides are recognized by helper T lymphocytes, which play an important role in establishing and maximizing the capabilities of the immune system, and are involved in the activation and induction of other immune cells, such as cytotoxic T lymphocytes.

[0062] Helper peptides may contain continuous or discontinuous epitopes. Helper peptides, including analogs and segments of helper peptides, have the ability to enhance or stimulate an immune response. Helper peptides may be about 10 to about 150 amino acids in length, and in particular about 10 to about 50 amino acids in length. When multiple helper peptides are present, each helper peptide typically acts independently.

[0063] Helper peptides are not usually tumor-specific neoantigens.

[0064] Helper peptides that may be used in the immunogenic compositions disclosed herein include, for example, Hepatitis B surface antigen helper T-cell epitope, pertussis toxin helper T-cell epitope, measles virus F protein helper T-cell epitope, Chlamydia trachomitis major outer membrane protein helper T-cell epitope, diphtheria toxin helper T-cell epitope, Plasmodium falciparum circumsporozoite protein helper T-cell epitope, Schistosoma mansoni triosephosphate isomerase helper T-cell epitope, keyhole limpet hemocyanin, Plasmodium vivax B-cell epitope (PVB), Escherichia coli TraT helper T-cell epitope, and immunostimulatory analogs and segments of any of these helper peptides.

[0065] The helper peptide can be a pan-helper T epitope. As used herein, a pan-helper T epitope refers to a peptide or other immunogenic molecule, or fragment thereof, that binds to multiple class II molecules to activate T cell function in a class II (CD4+ T cell) restricted manner. One example of a pan-helper T epitope is PADRE (pan-DR epitope) that comprises the peptide sequence AKXVAAWTLKAAA (SEQ ID NO: 1). X can be cyclohexylalanyl. PADRE specifically has a CD4+ helper T epitope, i.e., it stimulates the induction of PADRE-specific CD4+ helper T response. Another example of a pan-helper T epitope is a non-natural pan-DR helper T cell epitope (PADRE). PADRE is a preferred helper peptide that is particularly suitable for the immunogenic composition disclosed herein.

[0066] Tetanus toxoid has other helper T epitopes that act similarly to PADRE. Tetanus and diphtheria toxins have pan-epitopes for human CD4+ cells (Diethelm-Okita 2000). The helper peptide used in the immunogenic compositions disclosed herein can be a tetanus toxoid peptide, for example, F21E, which contains the peptide sequence FNNFTVSFWLRVPKVSASHLE (amino acids 947-967; SEQ ID NO: 2).

[0067] The immunogenic compositions disclosed herein may comprise about one or more helper peptides. For example, the immunogenic compositions may comprise about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 helper peptides. Typically, an immunogenic composition comprising about 19 tumor-specific neo-antigen long and / or short peptides preferably comprises at least about one helper peptide.

[0068] d. Tumor-specific frameshift peptide The immunogenic compositions described herein may further comprise a tumor-specific frameshift peptide. Without wishing to be bound by theory, tumor-specific frameshift peptides are believed to be highly immunogenic and may result in an enhanced response to the immunogenic composition (i.e., vaccine).

[0069] The tumor-specific frameshift peptide may be of any length. For example, the tumor-specific frameshift peptide may correspond to a tumor-specific neo-antigen long peptide or a tumor-specific neo-antigen short peptide. The tumor-specific frameshift peptide may be from about 2 amino acids in length to about 100 amino acids in length. Typically, the tumor-specific frameshift peptide may be from about 2 amino acids in length to about 30 amino acids in length. The tumor-specific frameshift peptide may be about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 amino acids in length or more.

[0070] The tumor-specific frameshift peptide can be modified by the addition or deletion of amino acids compared to the tumor-specific frameshift peptide, making the particular peptide more suitable for incorporation into an immunogenic composition.

[0071] The immunogenic compositions disclosed herein may comprise about one or more tumor-specific frameshift peptides. For example, the immunogenic compositions may comprise about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20 tumor-specific peptides. Typically, an immunogenic composition comprising about 19 tumor-specific neo-antigen long and / or short peptides preferably comprises at least about 2 tumor-specific frameshift peptides.

[0072] The tumor-specific frameshift peptide may be included in one or more of the peptide pools.

[0073] e. Adjuvants The immunogenic compositions described herein further comprise an adjuvant. An adjuvant is any substance that increases or enhances and / or strengthens the immune response to tumor-specific neoantigens when mixed with the immunogenic composition, but does not generate an immune response to tumor-specific neoantigens when administered alone. Preferably, the adjuvant generates an immune response to neoantigens but does not produce an allergic or other adverse reaction. It is contemplated herein that the immunogenic composition may be administered prior to, together with, simultaneously with, concomitantly with, or subsequent to administration of the immunogenic composition.

[0074] Adjuvants can enhance immune responses by several mechanisms, including, for example, activation of antigen-presenting cell (APC)-like dendritic cells, lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages. When the immunogenic compositions of the invention comprise an adjuvant or are administered with one or more adjuvants, adjuvants that may be used include, but are not limited to, mineral salt or mineral salt gel adjuvants, particulate adjuvants, microparticle adjuvants, mucosal adjuvants, and immune stimulating adjuvants. Examples of adjuvants include aluminum salts (alum) (e.g., aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3De-O-deacylated monophosphoryl lipid A (MPL) (see GB 2220211), MF59 (Novartis), AS03 (Glaxo SmithKline), AS04 (Glaxo SmithKline), polysorbate 80 (Tween 80; ICL Americas, Inc.), imidazopyridine compounds (see International Application No. PCT / US2007 / 064857, published as International Publication No. WO 2007 / 109812), imidazoquinoxaline compounds (see International Application No. PCT / US2007 / 064858, published as International Publication No. WO 2007 / 109813), and saponins, such as QS21 (Kensil et al, in Vaccine Design: The Subunit Adjuvants include, but are not limited to, the Adjuvant and Adjuvant Approach (eds. Powell & Newman, Plenum Press, NY, 1995), U.S. Pat. No. 5,057,540. In some embodiments, the adjuvant is Freund's adjuvant (complete or incomplete). Other adjuvants are oil-in-water (e.g., squalene or peanut oil) emulsions, optionally combined with an immunostimulant such as monophosphoryl lipid A (see Stoute et al, N. Engl. J. Med. 336, 86-91 (1997)).

[0075] CpG immunostimulatory oligonucleotides have also been reported to enhance the effect of adjuvants in a vaccine environment. Other TLR binding molecules, such as RNA-binding TLR3, TLR7, TLR8, TLR13, and DNA-binding TLR9, can also be used.

[0076] Other examples of useful adjuvants include poly ICLC (polyinosinic and polycytidylic acid stabilized with poly l-lysine and carboxymethylcellulose), 1018ISS, aluminum salts, Amplivax, AS15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact IMP321, IS Patch, ISS, ISCOMATRIX, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide These include, but are not limited to, ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel.RTM, Vector Systems, PLGA microparticles, resiquimod, SRL172, Virosomes and other virus-like particles, VEGF trap, R848, beta glucan, Pam3Cys, Aquila's QS21 stimulon, badimesan, and AsA404 (DMXAA).

[0077] f. Other Components of the Immunogenic Composition The immunogenic compositions described herein may further comprise a pharma- ceutically acceptable carrier.

[0078] Suspensions or dispersions of one or more tumor-specific neoantigens may be used, in particular aqueous isotonic suspensions, dispersions or amphipathic solvents. The immunogenic compositions may be sterilized and / or may contain excipients, such as preservatives, stabilizing agents, wetting agents and / or emulsifying agents, solubilizing agents, salts for adjusting the osmotic pressure, and / or buffers, and are prepared in a manner known per se, for example by means of conventional dispersion and suspension processes. In certain embodiments, such dispersions or suspensions may contain viscosity modifiers. The suspensions or dispersions may be kept at a temperature of around 2° C. to 8° C., or, preferentially, may be frozen for longer storage and then thawed shortly before use. For injection, the vaccine or immunogenic preparation may be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hank's solution, Ringer's solution, glucose solution for injection, or buffered saline. The solutions may contain formulating agents, such as suspending agents, stabilizing agents and / or dispersing agents.

[0079] In certain embodiments, the compositions described herein additionally comprise a preservative, e.g., the mercury derivative thimerosal. In specific embodiments, the pharmaceutical compositions described herein comprise 0.001%-0.01% thimerosal. In other embodiments, the pharmaceutical compositions described herein do not comprise a preservative.

[0080] An excipient may exist independent of an adjuvant. The function of an excipient may be, for example, to increase the solubility of the vaccine peptide, to increase the molecular weight of the immunogenic composition, to increase activity or immunogenicity, to confer stability, to increase biological activity, or to increase serum half-life. An excipient may also be used to aid in the presentation of one or more tumor-specific neoantigens to T cells (e.g., CD4+ or CD8+ T cells). An excipient may be a carrier protein, such as, but not limited to, keyhole limpet hemocyanin, a serum protein, such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, an immunoglobulin, or a hormone, such as insulin or palmitic acid. For human immunization, the carrier is usually a physiologically acceptable carrier that is acceptable and safe for humans. Alternatively, the carrier may be a dextran, such as sepharose.

[0081] Cytotoxic T cells recognize antigens in the form of peptides bound to MHC molecules, not the foreign antigens themselves. The MHC molecules themselves are located on the cell surface of antigen-presenting cells. Therefore, activation of cytotoxic T cells is possible when a trimeric complex of peptide antigens, MHC molecules and antigen-presenting cells (APCs) is present. It can enhance immune response when not only one or more tumor-specific antigens are used for activation of cytotoxic T cells, but also additional APCs with the respective MHC molecules are added. Thus, in some embodiments, the immunogenic composition further contains at least one APC.

[0082] The immunogenic composition may include an acceptable carrier (e.g., an aqueous carrier). A variety of aqueous carriers, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, and the like, may be used. These compositions may be sterilized by conventional, well-known sterilization techniques or may be sterile filtered. The resulting aqueous solution may be packaged for use as is or may be lyophilized, and the lyophilized preparation may be administered after mixing with a sterile solution. The composition may contain pharma- ceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and the like.

[0083] The immunogenic compositions disclosed herein may further comprise one or more emulsifiers. The emulsifier may be a pure emulsifier or a mixture of emulsifiers. The emulsifier(s) must be pharma- ceutically and / or immunologically acceptable. The emulsifier may be used to help stabilize the amphiphile, the mixture of amphiphile and antigen, or the mixture of amphiphile, antigen, and other vaccine components (e.g., adjuvants, helper peptides, or tumor-specific frameshift peptides) when the amphiphile or mixture is resuspended in the hydrophobic carrier. The use of an emulsifier may, for example, promote a more uniform distribution of the amphiphile or mixture in the hydrophobic carrier.

[0084] The emulsifier may be amphiphilic, and therefore may include a wide range of compounds. The emulsifier may be a surfactant, such as a non-ionic surfactant. Examples of emulsifiers that may be used include polysorbates, which are the only liquids derived from polyethylene glycolated sorbitol, and sorbitan esters. Polysorbates may include, for example, sorbitan monooleate. Exemplary emulsifiers are well known in the art and include, but are not limited to, mannide oleate (Arlacel A), lecithin, Tween 80, Spans 20, 80, 83, and 85.

[0085] Neo-antigens can be administered by liposomes, which direct them to specific cellular tissues, such as lymphoid tissues. Liposomes are also useful in extending half-life. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. In these preparations, the neo-antigens to be delivered are incorporated as part of the liposome, either alone or together with, for example, a molecule that binds to a receptor that is widely recognized among lymphoid cells, such as a monoclonal antibody that binds to the CD45 antigen, or together with other therapeutic or immunogenic compositions. Thus, liposomes filled with the desired neo-antigens can be directed to the site of lymphoid cells, where they then deliver the selected immunogenic composition. Liposomes can be formed from standard vesicle-forming lipids, which typically include neutral and negatively charged phospholipids and sterols, such as cholesterol. The choice of lipid is usually made according to considerations, for example, of the size of the liposome, the acid lability and stability of the liposomes in the bloodstream. A variety of methods are available for preparing liposomes, as described, for example, in Szoka et al., An. Rev. Biophys. Bioeng. 9;467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369.

[0086] When directed to immune cells, the ligand to be incorporated into the liposome may include, for example, an antibody or fragment thereof specific for a cell surface determinant of the desired immune system cell. The liposome suspension may be administered intravenously, locally, topically, etc., in doses that vary depending, among other things, on the mode of administration, the peptide being delivered, and the stage of the disease being treated.

[0087] As an alternative method for targeting immune cells, components of the immunogenic composition, such as antigens (i.e., tumor-specific neoantigens), ligands or adjuvants (e.g., TLRs), can be incorporated into poly(lactic acid-co-glycol) microspheres, which can entrap components of the immunogenic composition as phagoendosomal delivery devices.

[0088] For therapeutic or immunization purposes, a patient may be administered a nucleic acid encoding a tumor-specific neoantigen as described herein. Several methods are conveniently used to deliver the nucleic acid to the patient. For example, the nucleic acid may be delivered directly as "naked DNA". This technique is described, for example, in Wolff et al., Science 247:1465-1468 (1990), and in U.S. Pat. Nos. 5,580,859 and 5,589,466. The nucleic acid may also be administered using ballistic delivery, as described, for example, in U.S. Pat. No. 5,204,253. Particles composed solely of DNA may be administered. Alternatively, the DNA may be attached to particles, such as gold particles. Techniques for delivering nucleic acid sequences may include viral vectors, mRNA and DNA vectors, linearized or circular DNA or RNA, with or without electroporation. The nucleic acid may also be delivered complexed with cationic compounds, such as cationic lipids.

[0089] Also disclosed herein are methods of producing an immunogenic composition comprising one or more selected tumor specific neo-antigens by carrying out the steps of the methods disclosed herein. The immunogenic compositions described herein can be produced using methods known in the art. For example, a method of producing a tumor specific neo-antigen or vector disclosed herein (e.g., a vector comprising at least one sequence encoding one or more tumor specific neo-antigens) can include culturing a host cell under conditions suitable for expressing the neo-antigen or vector, where the host cell comprises at least one polynucleotide encoding the neo-antigen or vector, and purifying the neo-antigen or vector. Standard purification methods include chromatographic, electrophoretic, immunological, precipitation, dialysis, filtration, concentration, and chromatofocusing techniques.

[0090] The host cell may include Chinese Hamster Ovary (CHO) cells, NS0 cells, yeast or HEK293 cells. The host cell may be transformed with one or more polynucleotides comprising at least one nucleic acid sequence encoding one or more tumor-specific neo-antigens or vectors disclosed herein. In certain embodiments, the isolated polynucleotide may be cDNA.

[0091] B. Treatment method The present disclosure also relates to a method of treating cancer in a subject in need thereof, comprising administering an individualized immunogenic composition described herein.

[0092] The cancer may be any solid tumor or any hematological tumor. The methods disclosed herein are preferably suitable for solid tumors. The tumor may be a primary tumor (e.g., a tumor at the original site where the tumor first developed). Solid tumors may include, but are not limited to, breast cancer tumors, ovarian cancer tumors, prostate cancer tumors, lung cancer tumors, kidney cancer tumors, stomach cancer tumors, testicular cancer tumors, head and neck cancer tumors, pancreatic cancer tumors, brain cancer tumors, and melanoma tumors. Hematological tumors may include, but are not limited to, lymphomas (e.g., B-cell lymphomas) and leukemias (e.g., acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, and T-cell lymphocytic leukemia).

[0093] The methods disclosed herein may be used for any suitable cancerous tumor, including hematological malignancies, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumors. Exemplary suitable cancers include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, breast ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, and the like. cancer), gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell neck cancer of unknown primary, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and paranasal sinuses (par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor. Preferably, the cancer is melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, bladder cancer, or lung cancer. Melanoma is of particular interest.Also of particular interest are breast, lung and bladder cancers.

[0094] The methods disclosed herein may be used for any suitable cancerous tumor, including hematological malignancies, solid tumors, sarcomas, carcinomas, and other solid and non-solid tumors. Exemplary suitable cancers include, for example, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, appendix cancer, astrocytoma, basal cell carcinoma, brain tumor, bile duct cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, carcinoma of unknown primary, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, breast ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, olfactory neuroblastoma, fibrous histiocytoma, Ewing's sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, and the like. cancer), gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin's lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell tumor, Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, lip and oral cancer, liver cancer, lobular carcinoma in situ, lung cancer, macroglobulinemia, malignant fibrous histiocytoma, melanoma, Merkel cell carcinoma, mesothelioma, metastatic squamous cell neck cancer of unknown primary, midline carcinoma involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, nasal cavity and paranasal sinuses (par nasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter cancer, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, stomach cancer, T-cell lymphoma, teratoid tumor, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer, and Wilms' tumor. Preferably, the cancer is melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, bladder cancer, or lung cancer. Melanoma is of particular interest.Also of particular interest are breast, lung and bladder cancers.

[0095] The immunogenic composition stimulates the immune system of the subject, particularly the response of specific CD8+ T cells or CD4+ T cells. Interferon gamma produced by CD8+ and helper CD4+ T cells regulates the expression of PD-L1. PD-L1 expression on tumor cells is upregulated when attacked by T cells. Therefore, tumor vaccines can induce the production of specific T cells while at the same time upregulating the expression of PD-L1, which can limit the effectiveness of the immunogenic composition. Moreover, although the immune system is activated, there is a corresponding increase in the expression of the T cell surface receptor CTLA-4, which binds to the ligand B7-1 / B7-2 on antigen-presenting cells and acts as an immunosuppressant. Therefore, in some instances, the subject may be further administered an anti-immunosuppressant or an immunostimulant, such as a checkpoint inhibitor. Checkpoint inhibitors may include, but are not limited to, anti-CTL4-A antibodies, anti-PD-1 antibodies and anti-PD-L1 antibodies, inhibitors of the Lag3 pathway, the Tim3 pathway, the ICOS pathway, the OX-40 pathway, the GITR pathway or the 4-1BB pathway. These checkpoint inhibitors bind to immune checkpoint proteins on T cells to remove the suppression of T cell function by tumor cells. Blockade of CTLA-4 or PD-L1 with antibodies can enhance the immune response against cancerous cells in patients. CTLA-4 has been shown to be effective when following vaccination protocols.

[0096] The immunogenic compositions described herein may be administered to subjects who have been diagnosed with cancer, already have cancer, have recurrent cancer (i.e., relapse), or are at risk of developing cancer. The immunogenic compositions described herein may be administered to subjects who are resistant to other forms of cancer treatment (e.g., chemotherapy, immunotherapy, or radiation). The immunogenic compositions described herein may be administered to a subject before, together with, or after other standard of care cancer therapies (e.g., surgery, chemotherapy, immunotherapy, or radiation). The immunogenic compositions described herein may be administered simultaneously with, after, or in combination with other standard of care cancer therapies (e.g., surgery, chemotherapy, immunotherapy, or radiation).

[0097] The subject may be a human, dog, cat, horse, or any animal in which a tumor-specific response is desired.

[0098] The immunogenic compositions described herein may be administered to a subject in an amount sufficient to induce an immune response against tumor-specific neoantigens and to terminate or at least partially arrest symptoms and / or complications. In embodiments, the immunogenic compositions may result in a long-lasting immune response. A long-lasting immune response may be established by administering an immune-enhancing dose of the immunogenic composition to the subject. An immune response to the immunogenic composition may be prolonged by administering an immune-enhancing dose to the subject. In embodiments, at least one, at least two, at least three, or more doses may be administered to attenuate the cancer. A first immune-enhancing dose may increase the immune response by at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%. A second immune-enhancing dose may increase the immune response by at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%. The third immune boosting dose may increase the immune response by at least 50%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, or at least 1000%.

[0099] An amount sufficient to induce an immune response is defined as a "therapeutically effective amount." Amounts effective for this use will depend, for example, on the composition, the mode of administration, the stage and severity of the disease being treated, the weight and general health of the patient, and the judgment of the prescribing physician. It should be kept in mind that immunogenic compositions are usually employed in severe disease states, i.e., situations that are life-threatening or potentially life-threatening, particularly when cancer has metastasized. In such cases, the treating physician may find it possible and desirable to administer significant excesses of these immunogenic compositions, taking into account the minimization of foreign material and the relatively non-toxic nature of neoantigens.

[0100] The immunogenic compositions provided herein can be administered to a subject, for example, but not limited to, by oral, intradermal, intrathecal, intratumoral, intramuscular, intraperitoneal, intravenous, topical, subcutaneous, transdermal, intranasal and inhalation routes, as well as by scarification (e.g., using a bifurcated needle to scratch the upper layer of the skin). The immunogenic compositions can be administered at the tumor site to induce a local immune response against the tumor. The preferred administration route is by intrathecal or intramuscular injection.

[0101] The dosage of the immunogenic composition may depend on the type of composition, as well as the subject's age, weight, body surface area, individual condition, individual pharmacokinetic data, and mode of administration.

[0102] The subject may be administered one or more doses of the immunogenic composition. In some examples, the immunogenic composition is administered as one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine doses, ten doses or more. It is preferred to administer six doses of the immunogenic composition to the subject. Each dose may be administered at the same time, but it is preferred to administer each dose at a different time. Each dose may be administered at any suitable interval. Each dose may be administered about 2 days, about 5 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks, about 11 weeks, about 12 weeks, or much later after administration of the preceding dose. The interval between each dose of the immunogenic composition may be the same or different time intervals. Each dose is preferably administered at least about 1 week to about 4 weeks after administration of the preceding dose of the immunogenic composition, typically about 4 weeks after the preceding dose of the immunogenic composition.

[0103] Usually, it is preferred to administer the immunogenic composition as two or more peptide pools at each dose.The immunogenic composition can be administered as about two, about three, about four or about five peptide pools at each dose.Preferably, four peptide pools are administered at each dose.In some examples, the immunogenic composition is administered as four peptide pools in six doses.

[0104] The subject may be administered each peptide pool to one or more limbs of the subject. The subject may be administered each peptide pool to about one to about four limbs. Each peptide pool may be administered to the same limb of the subject. Each peptide pool may be administered to a different limb of the subject. It is preferred that each peptide pool is administered to a different limb of the subject.

[0105] Each peptide pool can be administered to the same limb of a subject in one or more doses.For example, the peptide pool is administered to the left arm for each dose.Alternatively, each peptide pool can be administered to different limbs of a subject in one or more doses.For example, the peptide pool can be administered to the left arm for one dose, to the right arm for a second dose, and to the left and right for a third dose.Preferably, each peptide pool is administered to the same limb of a subject for each dose.

[0106] The method may further comprise administering an adjuvant between each dose of the immunogenic composition. The purpose of the adjuvant is to allow T cell infiltration into the tumor induced by the vaccine and to support the immune response induced by the vaccine. The adjuvant may be administered one or more times between each dose of the immunogenic composition. In some examples, the adjuvant may be administered about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more times between each dose of the immunogenic composition. Typically, it is preferred to administer the adjuvant once a week between each dose of the immunogenic composition.

[0107] The subject may be administered the adjuvant prior to the initiation of treatment with the immunogenic composition. The adjuvant may be administered about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, or more prior to the initiation of the immunogenic composition.

[0108] The immunogenic compositions may contain from about 10 to about 500 μg of each tumor-specific neo-antigen long and / or short peptide per immunogenic composition. The immunogenic compositions may contain from about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about 160 μg, about 170 μg, about 180 μg, about 190 μg, about 200 μg, about 210 μg, about 220 μg, about 230 μg, about 240 μg, about 250 μg, about 260 μg, about 270 μg, about 280 μg, about 290 μg, about 300 μg, about 310 μg, about 320 μg, about 330 μg, about 340 μg, about 350 μg, about 360 μg, about 370 μg, about 380 μg, about 390 μg, about 400 μg, about 410 μg, about 420 μg, about 430 μg, about 440 μg, about 450 μg, about 460 μg, about 470 μg, about 480 μg, about 490 μg, about 500 μg, about 510 μg, about 520 μg, about 530 μg, about 540 μg, about 550 μg, about 560 μg, about 570 μg, about 580 μg, about 590 μg, about The immunogenic compositions may contain about 70 μg, about 280 μg, about 290 μg, about 300 μg, about 310 μg, about 320 μg, about 330 μg, about 340 μg, about 350 μg, about 360 μg, about 370 μg, about 380 μg, about 390 μg, about 400 μg, about 410 μg, about 420 μg, about 430 μg, about 440 μg, about 450 μg, about 460 μg, about 470 μg, about 480 μg, about 490 μg, about 500 μg of each tumor-specific neo-antigen long and / or short peptide. Typically, the immunogenic compositions contain about 300 μg of tumor-specific neo-antigen long and / or short peptide per immunogenic composition.

[0109] Each peptide pool can be mixed with up to about 900 μg of adjuvant. For example, each peptide pool can be mixed with about 10 μg, about 20 μg, about 30 μg, about 40 μg, about 50 μg, about 60 μg, about 70 μg, about 80 μg, about 90 μg, about 100 μg, about 110 μg, about 120 μg, about 130 μg, about 140 μg, about 150 μg, about 160 μg, about 170 μg, about 180 μg, about 190 μg, about 200 μg, about 210 μg, about 220 μg, about 230 μg, about 240 μg, about 250 μg, about 260 μg, about 270 μg, about 280 μg, about 290 μg, about 300 μg , about 310 μg, about 320 μg, about 330 μg, about 340 μg, about 350 μg, about 360 μg, about 370 μg, about 380 μg, about 390 μg, about 400 μg, about 410 μg, about 420 μg, about 430 μg, about 440 μg, about 450 μg, about 460 μg, about 470 μg, about 480 μg, about 490 μg, about 500 μg, about 550 μg, about 600 μg, about 650 μg, about 700 μg, about 750 μg, about 800 μg, about 850 μg or about 900 μg of adjuvant.

[0110] The immunogenic compositions described herein may be administered to a subject alone or in combination with other therapeutic agents. The therapeutic agents may be, for example, chemotherapeutic agents, hormone-regulating agents, signal transduction cascade inhibitors, radiation, or immunotherapy. Any therapeutic treatment suitable for the particular cancer may be administered. Exemplary chemotherapeutic agents include aldesleukin, altretamine, amifostine, asparaginase, bleomycin, capecitabine, carboplatin, carmustine, cladribine, cisapride, cisplatin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, docetaxel, doxorubicin, dronabinol, epoetin alfa, etoposide, filgrastim, fludarabine, fluorouracil, gemcitabine, granisetron, hydroxyurea, idarubicin, ifosfamidis, and the like. The therapeutic agents include, but are not limited to, cyclosporine, interferon alpha, irinotecan, lansoprazole, levamisole, leucovorin, megestrol, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel (Taxol®), pilocarpine, prochlorperazine, rituximab, tamoxifen, taxol, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate. The subject may be administered a small molecule or targeted therapy (e.g., a kinase inhibitor). The subject may further be administered an anti-CTLA antibody, or an anti-PD-1 antibody, or an anti-PD-L1 antibody. Blocking CTLA-4 or PD-L1 with an antibody may enhance the immune response against cancerous cells in the patient. EXAMPLES

[0111] The following examples serve to illustrate the present disclosure and are not intended to limit it in any way.

[0112] Example 1 1. Personalized vaccines Each personalized vaccine product will consist of four patient-specific unconjugated peptide pools, each with up to five peptides, mixed with a ready-made adjuvant, poly-ICLC (Hiltonol® (Oncovir, Inc., Washington, DC)) at the time of administration. To determine the composition of the personalized peptide pools and the number of pools, tissue samples from each patient will be subjected to DNA and RNA sequencing, in addition to subjecting the patient samples to HLA typing. Similar data will be analyzed using multiple biotechnology algorithms to identify a patient-specific unique set of neo-antigenic peptides with the potential for directed activity against the patient's disease. Up to four patient-specific peptide pools will be produced, each containing up to five peptides with one pan-DR CD4-helper epitope (PADRE) (Figure 1).

[0113] The composition of each vaccine pool (which peptides will be incorporated into which pool) will be guided by information about peptide solubility and predicted immunogenicity / recipient immunological responsiveness. Specifically, peptide physicochemical properties such as the proportion of hydrophobic amino acids will determine which peptides are most co-soluble. Manufacturers will be provided with predicted immunogenicity from computational pathways and machine learning models. Manufacturers will attempt to create peptide pools that include all peptides with similar average immunogenicity.

[0114] Essentially, each patient-specific vaccine will consist of less than 19 peptides representing naturally presented MHC class I epitopes or their extended forms, such as at least one neo-antigenic tumor-specific variant. These peptides will be allowed for presentation on antigen-presenting cells, which is expected to induce T cell-mediated anti-tumor immune responses. To ensure cosolubility of vaccine peptides, they will be split into up to four peptide pools for individual administration. During formulation, a vaccine adjuvant (here, poly ICLC, Hiltonol®) will be added to each peptide pool to help generate the desired T cell response. Additionally, the pan-DR helper epitope, PADRE, will be included in one of these peptide pool vaccines to induce a CD4 T cell-mediated "helper" response, thereby supporting the priming and expansion of CD8 T cells.

[0115] Each peptide will have a length of 30 amino acids (AA) or less, all of which are composed of naturally occurring L-amino acids without modification. Each peptide sequence will incorporate at least one mutation (nonsynonymous or indel) found in the patient's tumor, and therefore will differ from the patient's normal protein sequence. The PADRE peptide has been previously described (AKFVAAWTLKAAA (SEQ ID NO: 3)) and is composed of naturally occurring L-amino acids. With the exception of the PADRE peptide, the peptide sequences contained in the product will vary from patient to patient.

[0116] Poly-ICLC (also known as Hiltonol®) will be used as an adjuvant component of the personalized vaccine. Furthermore, Poly-ICLC is planned to be injected between the six planned vaccination visits to support tumor homing of vaccine-induced neoantigen-specific T cells and to mitigate the immunosuppressive tumor microenvironment.

[0117] Polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose is a stabilized double-stranded RNA (dsRNA) that was used several years ago as an interferon (IFN) inducer at high doses (up to 300 mcg / kg IV) in short-term cancer trials. On the other hand, lower doses (10-50 mcg / kg) of polyICLC have been shown to induce broader host defense, potent adjuvant activity, and specific antitumor and antiviral activity. PolyICLC has also been found to preferentially reduce tumor protein synthesis and cell proliferation in vivo.

[0118] 2. Dosage Form and Administration In the proposed Phase I clinical trial, tumor-specific neoantigens will be administered to study participants together with poly-ICLC. Common to both indication groups, a single dose of vaccine and of poly-ICLC will be given as shown in Table 1 below. [Table 1]

[0119] Six doses of the four vaccine pools (peptide pools mixed with poly-ICLC adjuvant) will be administered once every four weeks. These vaccinations will be accompanied by IM injections of poly-ICLC once a week, except in the week in which the vaccine is given. The purpose of the IM poly-ICLC injections is to allow vaccine-induced T cell infiltration into the tumor and to support the vaccine-induced Th1 immune response. The treatment plan is shown in Figure 2.

[0120] 3. Vaccine Preparations The vaccine will undergo final "bedside" formulation at the clinical site to be uniquely tailored to each patient's tumor antigen profile and predicted immune responsiveness. The peptide pools will be diluted to the desired concentration of 300 μg / peptide with adjuvant solution (500 μg polyICLC) for a total injection volume of 1 mL per pool. The procedure will follow standard procedures for vaccine preparation and administration, with the added step of filtering the vaccine peptide pool product through a sterile particulate filter before mixing with the adjuvant.

[0121] The selection of vaccine peptides to be formulated into the vaccine pool will be determined by their predicted immunogenicity, as well as the physicochemical properties governing solubility and manufacturability. Individual peptides to be formulated will be identified collaboratively by the test group and peptide manufacturers with the goal of incorporating the largest number of immunogenic peptides (up to 20, optionally including PADRE).

[0122] DMSO will be used to dissolve as many peptides as possible that are ranked highly immunogenic by our vaccine peptide prediction algorithm. DMSO is used as an excipient at low concentrations (approximately 4% v / v) in various approved drugs and has been shown to be safe when used as a cryoprotectant in stem cell infusions.

[0123] Example 2: Phase I Clinical Trial for Treating Subjects with Stage IIIC-IV Melanoma or Hormone Receptor Positive, Her2 Negative Breast Cancer A phase I clinical trial will be used to investigate the safety of a personalized neo-antigen peptide vaccine in treating approximately 20 subjects with metastatic or refractory stage IIIC-IV melanoma or hormone receptor-positive, Her2-negative breast cancer. Administering the personalized neo-antigen peptide vaccine together with the Th1-polarizing adjuvant poly-ICLC can induce polyclonal, multi-epitope cytotoxic T cell immunity against patients' tumors.

[0124] Specific diseases tested are: Anatomical Stage IV Breast Cancer AJCC v8, Clinical Stage III Cutaneous Melanoma AJCC v8, Clinical Stage IV Cutaneous Melanoma AJCC v8, Hormone Receptor Positive Breast Cancer, Locally Advanced Cutaneous Melanoma, Metastatic Acral Lentiginous Melanoma, Metastatic Conjunctival Melanoma, Metastatic Cutaneous Melanoma, Metastatic Her2 Negative Breast Cancer, Metastatic Mucosal Melanoma, Pathological Stage IIIC Cutaneous Melanoma AJCC v8, Pathological Stage IIID Cutaneous Melanoma AJCC v8, Pathological Stage IV Cutaneous Melanoma AJCC v8, Prognostic Stage IV Breast Cancer AJCC v8, Recurrent Acral Lentiginous Melanoma, Recurrent Cutaneous Melanoma, Recurrent Mucosal Melanoma, Refractory Her2 Negative Breast Cancer, Unresectable Acral Lentiginous Melanoma, Unresectable Cutaneous Melanoma, and Unresectable Mucosal Melanoma.

[0125] 1. Overview Subjects receive the personalized neo-antigen peptide vaccine by intramuscular injection once every 4 weeks. During vaccine-free weeks, subjects receive Poly-ICLC (Hiltonol®, Poly I:Poly C with poly-L-lysine stabilizer, Polyinosinic-polycytidylic acid stabilized with polylysine and carboxymethylcellulose, Polyriboinosinic-polyribocytidylic acid-polylysine carboxymethylcellulose, stabilized polyriboinosinic / polyribocytidylic acid) intramuscularly once a week. The vaccine is first administered 2 weeks after starting Poly-ICLC, and then once every 4 weeks. Nivolumab (BMS-936558, CMAB819, MDX-1106, NIVO, Nivolumab biosimilar CMAB819, ONO-4538, Opdivo® (Bristol Myers Squibb, New York, NY)) is administered intravenously every 2 or 4 weeks, 2 weeks after starting poly-ICLC. The duration of vaccine treatment is 25 weeks, unless the subject's disease progresses or the treatment causes unacceptable toxicity.

[0126] Following completion of vaccine treatment, subjects will receive nivolumab every 2 or 4 weeks for up to 12 months in the absence of disease progression or acceptable toxicity.

[0127] After completion of study treatment, subjects will be followed up for up to 24, 36, and 48 weeks.

[0128] 2. Outcome evaluation criteria The primary outcome measure was the incidence of adverse events during the year following the first dose of the personalized neoantigen vaccine. Adverse events were assessed by the Common Terminology Criteria for Adverse Events, version 5.0.

[0129] Secondary outcome measures were the number of personalized neo-antigen vaccines formulated and administered after 48 weeks; the number of personalized neo-antigen vaccines formulated with at least five vaccine peptides after 48 weeks; the number of personalized neo-antigen vaccines formulated through 16 weeks after the screening visit biopsy; best overall response as assessed by Response Evaluation Criteria in Immune-Related Solid Tumors criteria during the one year period following the first dose of personalized neo-antigen vaccine; and progression-free survival during the one year period following the first dose of personalized neo-antigen vaccine.

[0130] 3. Eligibility Criteria The study is open to adults of any gender, aged 18 years or older. Healthy volunteers are not being accepted.

[0131] Equivalent Those skilled in the art will readily appreciate that other suitable modifications and adaptations of the methods of the invention described herein will be obvious and may be effected using suitable equivalents without departing from the scope or embodiments of the present disclosure. Now that certain compositions and methods have been described in detail, they will be more clearly understood by reference to the following examples, which are presented merely for purposes of illustration and are not intended to be limiting.

Claims

1. An immunogenic composition comprising: (a) a plurality of tumor-specific neoantigen long-chain peptides; (b) a plurality of tumor-specific neoantigen short-chain peptides; (c) an adjuvant; (d) optionally, a helper peptide; and (e) optionally, one or more tumor-specific frameshift peptides is provided.

2. The immunogenic composition according to claim 1, wherein the immunogenic composition comprises up to about 50 of said tumor-specific neoantigen long-chain peptides and / or short-chain peptides.

3. The immunogenic composition according to claim 1 or 2, wherein the immunogenic composition comprises from about 10 to about 20 of said tumor-specific neoantigen long-chain peptides and / or short-chain peptides.

4. The immunogenic composition according to claim 1 or 2, wherein the immunogenic composition comprises about 19 of said tumor-specific neoantigen long-chain peptides and / or short-chain peptides.

5. The immunogenic composition according to claim 1 or 2, wherein the immunogenic composition comprises at least about 2 or more of said tumor-specific neoantigen long-chain peptides.

6. The immunogenic composition according to claim 5, wherein the immunogenic composition comprises from about 2 to about 18 of said tumor-specific neoantigen long-chain peptides.

7. The immunogenic composition according to claim 6, wherein the immunogenic composition comprises at least about 10 to about 15 of said tumor-specific neoantigen long-chain peptides.

8. The immunogenic composition according to claim 1 or 2, wherein the immunogenic composition comprises at least about 2 or more of said tumor-specific neoantigen short-chain peptides.

9. The immunogenic composition according to claim 8, wherein the immunogenic composition comprises at least about 2 to about 10 of said tumor-specific neoantigen short-chain peptides.

10. The immunogenic composition according to claim 1 or 2, wherein each of the tumor-specific neoantigen long-chain peptides in the immunogenic composition is different.

11. The immunogenic composition according to claim 1 or 2, wherein each of the tumor-specific neoantigen short-chain peptides in the immunogenic composition is different.

12. The immunogenic composition according to claim 1 or 2, wherein the immunogenic composition comprises two or more of said tumor-specific frameshift peptides.

13. The immunogenic composition according to claim 1 or 2, wherein the tumor-specific neoantigen long-chain peptides and / or short-chain peptides are divided into two or more peptide pools.

14. The immunogenic composition according to claim 13, wherein the tumor-specific neoantigen long-chain peptide and / or short-chain peptide is divided into about four peptide pools.

15. The immunogenic composition according to claim 13, wherein each peptide pool contains about five or fewer of the tumor-specific neoantigen long-chain peptides and / or short-chain peptides.

16. The immunogenic composition according to claim 13, wherein one or more of the peptide pools optionally contain the helper peptide.

17. The immunogenic composition according to claim 13, wherein three of the peptide pools contain about five of the tumor-specific neoantigen long-chain peptides and / or short-chain peptides, and one of the peptide pools contains four of the tumor-specific neoantigen long-chain peptides and / or short-chain peptides, and the helper peptide.

18. The immunogenic composition according to claim 13, wherein one or more of the peptide pools contain one or more of the tumor-specific frameshift peptides.

19. The immunogenic composition according to claim 13, wherein each peptide pool contains different tumor-specific neoantigen long-chain peptides and / or short-chain peptides.

20. The immunogenic composition according to claim 1 or 2, wherein the tumor-specific neoantigen long-chain peptide is about 15 to about 30 amino acids in length.

21. The immunogenic composition according to claim 1 or 2, wherein the tumor-specific neoantigen short-chain peptide is about 5 to about 15 amino acids in length.

22. The immunogenic composition according to claim 1 or 2, wherein the adjuvant is a Toll-like receptor agonist, NOD-like receptor agonist, Mda5 agonist, RIG-I, PKR agonist, STING agonist or other innate immune sensing pathway agonist.

23. The immunogenic composition according to claim 1 or 2, wherein the helper peptide is a pan-DR helper epitope (PADRE), a tetanus helper peptide, a hepatitis B surface antigen helper T cell epitope, a pertussis toxin helper T cell epitope, a measles virus F protein helper T cell epitope, a Chlamydia trachomatis major outer membrane protein helper T cell epitope, a diphtheria toxin helper T cell epitope, a Plasmodium falciparum sporozoite circumsporozoite protein helper T cell epitope, a Schistosoma mansoni triosephosphate isomerase helper T cell epitope, keyhole limpet hemocyanin, a Plasmodium vivax B cell epitope (PVB), an Escherichia coli TraT helper T cell epitope, a synthetic T helper epitope, an immunopotentiating analog and segment of any of the above helper peptides.

24. The immunogenic composition according to claim 23, wherein the helper peptide is a pan-DR helper epitope (PADRE).

25. The immunogenic composition according to claim 13, wherein each of the peptide pools further comprises the adjuvant.

26. The immunogenic composition induces a multifunctional CD4 ＋ and CD8 ＋ response, and the immunogenic composition according to claim 1 or 2.

27. A pharmaceutical composition comprising the immunogenic composition according to claim 1 or 2 and a pharmaceutically acceptable carrier.

28. A method for treating cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of the immunogenic composition according to claim 1 or 2.

29. The method according to claim 28, wherein the subject is administered at least one dose of the immunogenic composition.

30. The method according to claim 29, wherein the subject is administered at least six doses of the immunogenic composition at different times.

31. The method according to claim 28, wherein the immunogenic composition is administered as one or more peptide pools per dose.

32. The method according to claim 31, wherein the subject is administered six doses of two or more of the peptide pools at different times.

33. The method according to claim 31, wherein the subject is administered each peptide pool to 1 to 4 limbs of the subject.

34. The method according to claim 31, wherein each peptide pool is administered at a different site.

35. The method according to claim 31, wherein the subject is administered each peptide pool to different limbs of the subject.

36. The method according to claim 31, wherein the subject is administered each peptide pool to the same limb of the subject each time of administration.

37. The method according to claim 31, wherein each dose of the immunogenic composition is administered at least about 1 week to about 4 weeks after the administration of the preceding dose of the immunogenic composition.

38. The method according to claim 28, further comprising administering the adjuvant between each dose of the immunogenic composition.

39. The method according to claim 38, wherein the adjuvant is administered once a week between each dose of the immunogenic composition.

40. The method according to claim 38, wherein the adjuvant is a Toll-like receptor agonist, NOD-like receptor agonist, Mda5 agonist, RIG-I, PKR agonist, STING agonist, or other innate immune sensing pathway agonist.

41. The method according to claim 28, further comprising administering at least one checkpoint inhibitor.

42. The method according to claim 41, wherein the checkpoint inhibitor is an inhibitor of the programmed death-1 (PD-1) pathway, Lag3 pathway, Tim3 pathway, ICOS pathway, OX-40, GITR pathway or 4-1BB pathway.

43. The method according to claim 42, wherein the inhibitor of the PD-1 pathway is an anti-PD-1 antibody.

44. The method according to claim 42, wherein the checkpoint inhibitor is an anti-cytotoxic T lymphocyte-associated antigen 4 (CTLA4) antibody.

45. The method according to claim 28, wherein the cancer is melanoma, breast cancer, ovarian cancer, prostate cancer, kidney cancer, gastric cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, colon cancer, urothelial cancer, or lung cancer.

46. The method according to claim 45, wherein the cancer is melanoma.

47. The method according to claim 45, wherein the cancer is breast cancer.

48. The method according to claim 45, wherein the cancer is lung cancer.

49. The method according to claim 28, wherein the subject is diagnosed with cancer, already has cancer, has recurrent cancer, or has a risk of developing cancer.

50. The method according to claim 28, wherein the immunogenic composition is administered by subcutaneous, intramuscular, transcutaneous, intradermal, transdermal, intratumoral, intranodal, intravenous or intraperitoneal administration.

51. The method according to claim 50, wherein the immunogenic composition is administered by intramuscular administration.

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