Personalized longitudinal analysis of circulating substances to monitor and modulate neoantigen cancer vaccines

Abstract

Disclosed herein is a method comprising administering to a subject in need thereof an initial immunogenic composition comprising a plurality of tumor-specific neoantigens, each tumor-specific neoantigen corresponding to a member of a first group of tumor-associated mutations in the subject, and no member corresponding to a second set of tumor-associated mutations in the subject; and quantifying each member of the first set of tumor-associated mutations and each member of the second set of tumor-associated mutations in a circulating substance comprising tumor-associated mutations isolated from the subject at each of a plurality of time points.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Nos. 63 / 389,384 and 63 / 380,286, filed on July 15, 2022 and October 20, 2022, respectively, the entire contents of which are incorporated herein by reference. Technical Field

[0003] It will be apparent to those skilled in the art that other suitable modifications and adaptations of the methods of the invention described herein will be apparent and may be made using suitable equivalents without departing from the scope of the disclosure or embodiments. Background Art

[0004] Cancer is the leading cause of death worldwide, accounting for a 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 many existing standard of care cancer therapies, including ablative techniques (such as surgical procedures and radiation) and chemical techniques (such as chemotherapeutic agents). Unfortunately, such therapies are frequently associated with serious risks, toxic side effects, extremely high costs, and uncertain efficacy.

[0005] Cancer immunotherapy (e.g., cancer vaccines) has emerged as a promising cancer treatment modality. The goal of cancer immunotherapy is to harness the immune system to selectively destroy cancer while leaving normal tissue unharmed. Traditional cancer vaccines typically target tumor-associated antigens. Tumor-associated antigens are often present in normal tissues but are overexpressed in cancer. However, because these antigens are often present in normal tissues, immune tolerance prevents immune activation. Several clinical trials targeting tumor-associated antigens have failed to demonstrate lasting beneficial effects compared to standard of care treatment. Li et al., Ann Oncol., 28(Suppl 12): xii11–xii17(2017).

[0006] Neoantigens represent attractive targets for cancer immunotherapy. Neoantigens are non-self proteins with individual specificity. Neoantigens are derived from random somatic mutations in the genome of tumor cells and are not expressed on the surface of normal cells. Same as above. Since neoantigens are only expressed on tumor cells and therefore do not induce central immune tolerance, cancer vaccines targeting cancer neoantigens have potential advantages, including reducing central immune tolerance and improving safety characteristics. Same as above. The mutation situation of cancer is complex, and the tumor mutations of each individual subject are usually unique. Most somatic mutations detected by sequencing do not produce effective neoantigens. Only a small part of the mutations in tumor DNA or tumor cells are transcribed, translated and processed into tumor-specific neoantigens, and their accuracy is sufficient to design a potentially effective vaccine. In addition, not all neoantigens are immunogenic. In fact, the proportion of T cells that spontaneously recognize endogenous neoantigens is about 1% to 2%. See Karpanen et al., Front Immunol., 8: 1718 (2017). In addition, the cost and time associated with the manufacture of neoantigen vaccines are huge.

[0007] Compounding these challenges is that predictions of neoantigen immunogenicity are not perfect, which can result in overall neoantigen vaccine efficacy being lower than expected.

[0008] Therefore, there is a need to identify neoantigens that are ineffective when included in a neoantigen vaccine. There is also a need to replace such ineffective neoantigens with effective neoantigens during the course of a subject's therapeutic vaccination regimen. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 A hypothetical model of various aspects of the methods disclosed herein is shown. DETAILED DESCRIPTION

[0010] The present disclosure relates to methods for identifying neoantigens that are ineffective when included in a neoantigen vaccine. The present disclosure also relates to methods for replacing ineffective neoantigens with effective neoantigens during a subject's treatment regimen.

[0011] In some embodiments, the present disclosure relates to a method comprising administering to a subject in need thereof an initial immunogenic composition comprising a plurality of tumor-specific neoantigens, wherein each tumor-specific neoantigen corresponds to a member of a first group of tumor-associated mutations in the subject, and no tumor-specific neoantigen corresponds to a member of a second group of tumor-associated mutations in the subject. The method further comprises quantifying each member of the first group of tumor-associated mutations and each member of the second group of tumor-associated mutations in circulating material isolated from the subject at each of a plurality of time points. The circulating material may be circulating tumor DNA (ctDNA), circulating free DNA (cfDNA), circulating tumor cells (CTCs), circulating tumor proteins, extracellular vesicles, or two or more thereof.

[0012] The method may further include replacing the tumor-specific neo-antigens corresponding to members of the first group of tumor-associated mutations in the initial immunogenic composition with replacement tumor-specific neo-antigens corresponding to members of the second group of tumor-associated mutations in response to an increase in the number of members of the first group of tumor-associated mutations from an earlier time point to a later time point to produce a reformulated immunogenic composition.

[0013] The method may further comprise administering the reconstituted immunogenic composition to the subject.

[0014] The subject may have any type of cancer in which the cancer cells have a genetic mutation, including but not limited to melanoma, breast cancer, sarcoma, 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, colon cancer, urothelial carcinoma, or lung cancer.

[0015] The tumor-associated mutation may include at least one subject-specific mutation.

[0016] The tumor-associated mutation may include at least one tumor hotspot mutation.

[0017] The tumor may be ER+ / HER2- breast cancer, and at least one tumor hotspot mutation may be located in a gene selected from the group consisting of: AKT1, APC, ARID1A, ATM, BRAF, BRCA1, BRCA2, CDH1, CDKN2A, ESR1, GATA3, GNAS, HER2, KRAS, NF1, PIK3CA, PTEN, RB1, SMAD4 and TP53.

[0018] The tumor may be a melanoma.

[0019] In some embodiments, each tumor-specific neo-antigen corresponding to a member of a first set of tumor-associated mutations may have a higher immunogenicity score than any tumor-specific neo-antigen corresponding to a member of a second set of tumor-associated mutations.

[0020] At least one of the plurality of time points may be prior to administration of the initial immunogenic composition.

[0021] The initial immunogenic composition may be administered multiple times before the tumor-specific neoantigen is replaced.

[0022] The reformulated immunogenic composition can be administered multiple times after replacing the tumor-specific neoantigen.

[0023] In some embodiments, the number of members of the second set of tumor-associated mutations corresponding to replacement tumor-specific neoantigens may remain the same or increase (i.e., not decrease) from an earlier time point to a later time point.

[0024] Quantifying tumor-associated mutations may include a variety of methods, depending on the circulating material being quantified for tumor-associated mutations. Quantifying tumor-associated mutations may include sequencing ctDNA using whole exome sequencing (WES), whole genome sequencing (WGS), targeted sequencing, polymerase chain reaction (PCR), or hybridization methods; sequencing cfDNA using quantitative polymerase chain reaction (qPCR) or next-generation sequencing; measuring the methylation or chromatin content of ctDNA, cfDNA, or DNA from CTCs; performing mass spectrometry or elution assays for circulating tumor proteins, proteins from CTCs, or proteins from extracellular vesicles; performing fluorescence activated cell sorting (FACS) on CTCs; or sequencing nucleic acids from CTCs or extracellular vesicles using WES, WGS, targeted sequencing, PCR, qPCR, next-generation sequencing, single-cell RNA sequencing, or hybridization methods.

[0025] One of the multiple time points can be at least 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, or about 12 weeks after administration of the initial immunogenic composition or the reconstituted immunogenic composition.

[0026] One of the multiple time points can be at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months after administration of the initial immunogenic composition or the reformulated immunogenic composition.

[0027] One of the multiple time points can be at least about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years after administration of the initial immunogenic composition or the reformulated immunogenic composition.

[0028] The circulating material can be isolated from a blood sample, a serum sample, a plasma sample, a urine sample, or a cerebrospinal fluid sample.

[0029] The circulating material can be isolated from at least about 10 ml of whole blood from a subject.

[0030] The circulating material can be isolated from at least about 20 ml of whole blood from a subject.

[0031] The method may further include detecting the presence of at least one tumor-associated mutation that is not included in the first set of tumor-associated mutations or the second set of tumor-associated mutations at the first time point.

[0032] The method may further include adding tumor-associated mutations that occur at a time point after the first time point to the second set of tumor-associated mutations.

[0033] All publications and patents cited in this disclosure are incorporated by reference in their entirety. Insofar as the materials incorporated herein by reference are contradictory or inconsistent with this specification, this specification will replace any such materials. The citation of any reference herein is not an admission that such reference is the prior art of the present disclosure. When stating the range of values, it includes embodiments using any specific value within the range. In addition, reference to the numerical values ​​stated in the range includes each and all values ​​within the range. All ranges include their endpoints and can be combined. When a value is expressed as an approximation by using the antecedent "about", it should be understood that the specific value forms another embodiment. Unless the context clearly indicates otherwise, mentioning a specific numerical value includes at least that specific value. Unless the specific context of its use provides otherwise, the use of "or" will mean "and / or".

[0034] Various terms related to various aspects of this specification are used throughout the specification and claims. Unless otherwise stated, such terms should have their ordinary meaning in the art. Other specifically defined terms will be interpreted in a manner consistent with the definitions provided herein. The techniques and procedures described or quoted herein are generally known to those skilled in the art and are generally adopted using conventional methodology, such as the widely used molecular cloning methods described in: Sambrook et al., Molecular Cloning: A Laboratory Manual 4th Edition (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Where appropriate, unless otherwise stated, procedures involving the use of commercially available kits and / or reagents are generally performed according to the protocols and conditions defined by the manufacturer.

[0035] As used herein, the singular forms "a / an," "the," and "the" include the plural forms unless the context clearly dictates otherwise. Unless clearly stated otherwise, the terms "including," "such as," and the like are intended to convey inclusion without limitation.

[0036] Unless otherwise indicated, the terms "at least," "less than," and "about," or similar terms preceding a series of elements or ranges should be understood to refer to each element in the series or range. Those skilled in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the invention described herein using only routine experimentation. Such equivalents are intended to be covered by the following claims.

[0037] The term "cancer" refers to a physiological condition in a subject in which a cell population is characterized by uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and / or certain morphological characteristics. Typically, cancer may be in the form of a tumor or mass, but may be present alone in a subject, or may circulate in the bloodstream as an independent cell such as a leukemia cell or lymphoma cell. The term cancer includes all types of cancer and metastasis, 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, lung adenocarcinoma, lung squamous carcinoma, peritoneal cancer, hepatocellular carcinoma, gastrointestinal 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) cancer or uterine cancer, salivary gland cancer, kidney cancer, liver cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatoma, and various types of head and neck cancer.

[0038] The term "subject" as used herein refers to any animal, such as any mammal, including but not limited to humans, non-human primates, rodents, mammals commonly kept as pets (e.g., dogs and cats, etc.), livestock (e.g., cattle, sheep, goats, pigs, horses, camels, etc.), etc. In some embodiments, the mammal is a mouse. In some embodiments, the mammal is a human.

[0039] As used herein, the term "tumor cell" refers to any cell that is a cancer cell or is derived from a cancer cell. The term "tumor cell" may also refer to a cell that exhibits cancer-like properties, such as uncontrolled proliferation, resistance to anti-growth signals, the ability to metastasize, and the loss of the ability to undergo programmed cell death.

[0040] This article provides additional description of the method and guidance for practicing the method.

[0041] A. Tumor-associated mutations and corresponding tumor-specific neoantigens

[0042] Relative to the DNA of normal or healthy tissue, the DNA from the tumor usually shows one or more mutations. Some of these tumor-related mutations are relatively common in patient populations. This relatively common tumor-related mutation can be referred to as a tumor hotspot mutation. The gene in which a tumor hotspot mutation occurs depends on a specific cancer. For example, in ER+ / HER2- breast cancer, a tumor hotspot mutation may be located in a gene selected from the group consisting of: AKT1, APC, ARID1A, ATM, BRAF, BRCA1, BRCA2, CDH1, CDKN2A, ESR1, GATA3, GNAS, HER2, KRAS, NF1, PIK3CA, PTEN, RB1, SMAD4 and TP53. Other tumor hotspot mutations and genes thereof are known in other cancers (such as melanoma).

[0043] In some embodiments, the tumor-associated mutation includes at least one tumor hotspot mutation.

[0044] Other tumor-associated mutations may be subject-specific. These specific tumor-associated mutations cannot be predicted in advance and can only be determined through a workflow that sequences healthy and tumor nucleic acid sequences, such as whole exome sequencing (WES) or whole genome sequencing (WGS) of DNA from healthy and tumor tissues, single-cell RNA (scRNA) sequencing of healthy or tumor cells, or other technologies. The normal and tumor sequences can then be aligned and compared, and the differences between the two types of aligned sequences can be identified as tumor-associated mutations.

[0045] In WES and scRNA sequencing, sequencing data comes from nucleic acid sequences that are transcribed and translated into peptides. The data of WGS includes nucleic acid sequences that are transcribed and translated into peptides. Therefore, any tumor-associated mutations discovered by WES or scRNA sequencing of tumor nucleic acids will produce peptides that are expected to be present in tumors and are not expected to be present in healthy tissues after translation. Such peptides can be considered as tumor-specific neoantigens. Tumor-specific neoantigens can be considered to correspond to tumor-associated mutations. Similarly, at least some tumor-associated mutations discovered by WGS sequencing of tumor DNA will have tumor-specific neoantigens corresponding thereto.

[0046] Figure 1 A hypothetical model is presented to help understand. In the upper left, WES, WGS, or scRNA sequencing (RNA-seq) is performed on normal and healthy tissues. The sequencing data has been aligned and shows three tumor-associated mutations: CATTGG→CCTTGG, CGATTT→CGATGT, and ACAGAG→ACAGCG. Each of these three tumor-associated mutations has a tumor-specific neoantigen corresponding to it: peptide 1, containing ProTrp; peptide 2, containing ArgCys; peptide 3, containing ThrAla. In reality, more tumor-associated mutations and longer tumor-specific neoantigen sequences will be found.

[0047] Two factors should be kept in mind when identifying subject-specific tumor-associated mutations. First, after initial identification, subclonal mutations may emerge that can render the tumor resistant to immunogenic compositions that do not target tumor-specific neoantigens corresponding to such subclonal mutations. Second, clonal hematopoiesis of indeterminate potential (CHIP) is the accumulation of mutations in leukocytes with age. The latter factor can be at least partially mitigated by additional sequence analysis of leukocytes to exclude CHIP-associated variants.

[0048] In some embodiments, the tumor-associated mutation includes at least one subject-specific mutation.

[0049] B. Immunogenic Compositions

[0050] Once the polypeptide sequence data for one or more tumor-specific neoantigens is obtained, a numerical probability score can be generated to predict whether the one or more tumor-specific neoantigens are immunogenic (e.g., whether they will elicit an immune response in a subject). For example, the polypeptide sequence data and MHC molecules can be input into a machine learning platform (i.e., a model). The machine learning platform can generate a numerical probability score.

[0051] MHC molecules transport and present peptides on the cell surface. MHC molecules are divided into class I and class II MHC molecules. MHC class I molecules are present on the surface of almost all body cells, including most tumor cells. MHC class I proteins are loaded with antigens usually from endogenous proteins or pathogens present in cells, and then presented to cytotoxic T lymphocytes (i.e. CD8+). MHC class I molecules may include HLA-A, HLA-B or HLA-C. Class II MHC molecules are only present on dendritic cells, B lymphocytes, macrophages and other antigen presenting cells. It mainly presents peptides processed from external antigen sources (i.e. outside the cell) to T helper (Th) cells (i.e. CD4+). MHC class II molecules may include HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA and HLA-DRB1. In some cases, MHC class II molecules may also be expressed on cancer cells.

[0052] MHC class I molecules and / or MHC class II molecules may be input into a machine learning platform. Typically, MHC class I molecules or MHC class II molecules are input into a machine learning platform. In some embodiments, MHC class I molecules are input into a machine learning platform. In other embodiments, MHC class II molecules are input into a machine learning platform. In some embodiments, an MHC class I machine learning platform may be trained based on MHC class I training data. In some embodiments, an MHC class II machine learning platform may be trained based on MHC class II training data. In some embodiments, the same machine learning platform may be trained based on both MHC class I and class II training data. In some embodiments, a machine learning platform may include an MHC class I model and an MHC class II model.

[0053] MHC class I molecules bind to short peptides. MHC class I molecules can accommodate peptides that are typically about 8 amino acids in length to about 10 amino acids in length. In embodiments, the sequence data encoding one or more tumor-specific neoantigens is a short peptide of about 8 amino acids to about 10 amino acids in length. MHC class II molecules bind to longer peptides. MHC class II can accommodate peptides that are typically about 13 amino acids in length to about 25 amino acids in length. In embodiments, the sequence data encoding one or more tumor-specific neoantigens is a long peptide of about 13 to 25 amino acids in length.

[0054] The sequence data encoding one or more tumor specific neoantigens 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, about 15 amino acids in length, about 16 amino acids in length, about 17 amino acids in length, about 18 amino acids in length, about 19 amino acids in length, about 20 amino acids in length, about 21 amino acids in length, about 22 amino acids in length, about 23 amino acids in length, about 24 amino acids in length, about 25 amino acids in length, about 26 amino acids in length, about 27 amino acids in length, about 28 amino acids in length, about 29 amino acids in length, or about 30 amino acids in length.

[0055] Immunogenic tumor-specific neoantigens are not expressed in normal tissues. They can be presented to CD4+ and CD8+ T cells by antigen presenting cells to produce an immune response. In the embodiments, the immune response of the subject triggered by one or more tumor-specific neoantigens includes presenting one or more tumor-specific neoantigens to the surface of tumor cells. More specifically, the immune response of the subject triggered by one or more tumor-specific neoantigens includes presenting one or more tumor-specific neoantigens by one or more MHC molecules on tumor cells. It is expected that the immune response triggered by one or more tumor-specific neoantigens is a T cell-mediated response. The immune response triggered by one or more tumor-specific neoantigens in the subject may involve one or more tumor-specific neoantigens that can be presented to T cells by antigen presenting cells (e.g., dendritic cells). In one aspect, one or more tumor-specific neoantigens can activate CD8+T cells and / or CD4+T cells.

[0056] In embodiments, the machine learning platform may predict the likelihood that one or more tumor-specific neoantigens will activate CD8+T cells. In embodiments, the machine learning platform may predict the likelihood that one or more tumor-specific neoantigens will activate CD4+T cells. In some cases, the machine learning platform may predict the antibody titer that one or more tumor-specific neoantigens may elicit. In other cases, the machine learning platform may predict the frequency with which one or more tumor-specific neoantigens activate CD8+.

[0057] The machine learning platform may include a model trained according to training data. The training data may be obtained from a series of different subjects. The training data may include data from healthy subjects and subjects with cancer. The training data may include various data that can be used to generate probability scores, and the probability scores indicate whether one or more tumor-specific neoantigens will trigger an immune response in a subject. Exemplary training data may include data representing nucleotide or polypeptide sequences from normal tissues and / or cells, data representing nucleotide or polypeptide sequences from tumor tissues, data representing MHC peptide group sequences from normal and tumor tissues, peptide-MHC binding affinity measurements, or a combination thereof. The training data may also include mass spectrometry data, DNA sequencing data, RNA sequencing data, clinical data from healthy subjects and subjects with cancer, cytokine analysis data, T cell cytotoxicity assay data, peptide-MHC monomer or polymer data and proteomics data, for being engineered to express a predetermined MHC allele mono-allelic cell line, the cell line is subsequently exposed to synthetic proteins, normal and tumor human cell lines, fresh and frozen original samples, and T cell assays.

[0058] The machine learning platform can be a supervised learning platform, an unsupervised learning platform, or a semi-supervised learning platform. The machine learning platform can use a sequence-based method to generate a numerical probability that one or more tumor-specific neoantigens can trigger an immune response (e.g., will induce a high or low antibody response or CD8+ response). Sequence-based predictions may include supervised machine learning modules, including artificial neural networks (e.g., deep neural networks or other), support vector machines, K-nearest neighbors, logistic multiple network constrained regression (LogMiNeR), regression trees, random forests, adaBoost, XGBoost, or hidden Markov models. These platforms require training data sets containing known MHC-binding peptides.

[0059] Many prediction programs have been used to predict whether tumor-specific neoantigens can be presented on MHC molecules and trigger an immune response. Exemplary prediction programs include, for example, HLAminer (Warren et al., Genome Med., 4: 95 (2012); HLA type predicted by orienting the assembly of shotgun sequence data and comparing it with the reference allele sequence database), Ensembl Variant Effect Predictor (McLaren et al., Genome Biol., 17: 122 (2016)), NetMHCpan (Andreatta et al., Bioinformatics, 32: 511–517 (2016); sequence comparison method based on artificial neural network, and predict the affinity of peptide-MHC-I typemolecular), UCSC browser (Kent et al., Genome Res., 12:996–1006 (2002)), CloudNeo pipeline (Bais et al., Bioinformatics, 33:3110–2 (2017)), OptiType (Szolek et al., Bioinformatics, 30:3310–3316 (2014)), ATHLATES (Liu C et al., Nucleic Acids Res. 41:e142 (2013)), pVAC-Seq (Hundal et al., Genome Med. 8:11 (2016)), MuPeXI (Bjerregaard et al., Cancer Immunol. Immunother., 66:1123–30 (2017)), Strelka (Saunders et al., Bioinformatics, 28:1811–7 (2012)), Strelka2 (Kim et al., Nat Biol. Methods.15:591–4(2018)), VarScan2(Koboldt et al., Genome Res.,22:568–76(2012)), SomaticSeq(Fang L et al., Genome Biol., 16:197 (2015)), SMMPMBEC (Kim et al., BMC Bioinformatics, 10:394 (2009)), NeoPredPipe (Schenck RO, BMC Bioinformatics, 20:264 (2019)), Weka (Witten et al., Data mining: practical machine-learning tools and techniques. 4th edition Elsevier, ISBN:97801280435578 (e-book) (2017)) or Orange (Demsar et al., Orange: Data Mining Toolbox in Python., J. Mach. Learn. Res., 14:2349-2353 (2013)). Any known prediction program can be used as a machine learning platform to generate a numerical probability score indicating whether a new antigen will elicit an immune response. .

[0060] Depending on the machine learning platform employed, additional filters may be applied to prioritize tumor-specific neoantigen candidates, including elimination of hypothetical (Riken) proteins; use of antigen processing algorithms to eliminate epitopes that are unlikely to be generated by constitutive or immunoproteasomal proteolysis; and prioritization of neoantigens with higher predicted binding affinity than the corresponding wild-type sequence.

[0061] The numerical probability score may be a number between 0 and 1. In embodiments, the numerical probability score may be a number of 0, 0.0001, 0.0002, 0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.20, 0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, or 1. Tumor-specific neoantigens with higher numerical probability scores relative to lower numerical probability scores indicate that the tumor-specific neoantigen will elicit a stronger immune response in a subject and, therefore, may be a suitable candidate for an immunogenic composition. For example, a tumor-specific neoantigen with a numerical probability score of 1 will likely elicit a greater immune response in a subject than a tumor-specific neoantigen with a numerical probability score of 0.05. Similarly, a tumor-specific neoantigen with a numerical probability score of 0.5 will likely elicit a greater immune response in a subject than a tumor-specific neoantigen with a numerical probability score of 0.1.

[0062] Higher numerical probability scores are preferred relative to lower numerical probability scores. Preferably, tumor-specific neoantigens having a numerical probability score of at least 0.80, 0.81, 0.82, 0.83, 0.84, 0.85, 0.86, 0.87, 0.88, 0.89, 0.90, 0.95, 0.96, 0.97, 0.98, 0.99, or 1 indicate that they will likely elicit an immune response in a subject.

[0063] Although higher numerical probability scores are preferred, lower numerical probability scores may still indicate that the tumor-specific neoantigen is able to elicit a sufficient immune response and therefore the tumor-specific neoantigen may be a suitable candidate.

[0064] For example, the machine learning platform can also predict the likelihood that one or more tumor-specific neoantigens will be presented by MHC molecules on tumor cells. The machine learning platform can predict the likelihood that one or more tumor-specific neoantigens will be presented by MHC class I molecules or MHC class II molecules.

[0065] The method for selecting one or more tumor-specific neoantigens may also include a step of measuring the affinity of one or more tumor-specific neoantigens to MHC molecules in the subject by a computer. Tumor-specific neoantigens with a binding affinity to MHC molecules less than about 1000nM indicate that one or more tumor-specific neoantigens may be suitable for immunogenic compositions. Tumor-specific neoantigens with a binding affinity to MHC molecules less than about 500nM, less than about 400nM, less than about 300nM, less than about 200nM, less than about 100nM, or less than about 50nM may indicate that one or more tumor-specific neoantigens may be suitable for immunogenic compositions. The affinity of one or more tumor-specific neoantigens to MHC molecules in the subject can predict the immunogenicity of tumor-specific neoantigens. Alternatively, median affinity can be an effective method for predicting the immunogenicity of tumor-specific neoantigens. Epitope prediction algorithms can be used to calculate median affinity, such as NetMHCpan, artificial neural networks (ANN), stable matrix methods (SMM), and SMMPMBEC.

[0066] The RNA expression of one or more tumor-specific neoantigens can also be quantified. The RNA expression of one or more tumor-specific neoantigens can be quantified to identify one or more neoantigens that will trigger an immune response in a subject. There are multiple methods for measuring RNA expression. Known techniques for measuring RNA expression include RNA-seq, in situ hybridization (e.g., FISH), Northern blots, DNA microarrays, Tiling arrays, and quantitative polymerase chain reaction (qPCR). Other known techniques in the art can be used to quantify RNA expression. RNA can be messenger RNA (mRNA), short interfering RNA (siRNA), microRNA (miRNA), circular RNA (circRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nucleolar RNA (snRNA), Piwi interacting RNA (piRNA), long non-coding RNA (long ncRNA), subgenomic RNA (sgRNA), RNA from integrated or non-integrated viruses, or any other RNA. In one aspect, mRNA expression is measured.

[0067] The present technology can further reduce the possibility that the selected tumor-specific neoantigens can induce an autoimmune response in normal tissues. Tumor-specific neoantigens with sequences similar to normal antigens are expected to induce autoimmune responses in normal tissues. For example, tumor-specific neoantigens that are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% similar to normal antigens can induce autoimmune responses. Tumor-specific neoantigens that are predicted to induce autoimmune responses are not preferentially used in immunogenic compositions. Tumor-specific neoantigens that are predicted to induce autoimmune responses are generally not selected for use in immunogenic compositions. The method may also include measuring the ability of one or more tumor-specific neoantigens to induce immune tolerance. Tumor-specific neoantigens that are predicted to induce immune tolerance are not preferentially used in immunogenic compositions.

[0068] Tumor subclone profiles can be used to inform the selection of tumor-specific neoantigens for immunogenic compositions. For example, for any tumor subclones identified prior to formulating the initial immunogenic composition, at least one tumor-specific neoantigen associated with each subclone can be considered for inclusion in the initial immunogenic composition.

[0069] The immunogenicity score for each tumor-specific neoantigen can be determined based on one or more of the following: a numerical probability score, likelihood of CD8+ T cell activation, predicted antibody titer, one or more additional filters, likelihood of presentation by MHC class I or MHC class II molecules, affinity for binding to MHC molecules, RNA quantification, or likelihood of autoimmune response.

[0070] Given the immunogenicity score of each tumor-specific neoantigen, one or more tumor-specific neoantigens can be selected to formulate a subject-specific initial immunogenic composition. The selection can be directed to any tumor-specific neoantigen with an immunogenicity score equal to or higher than the desired value, or to the n highest tumor-specific neoantigens ranked by immunogenicity score. In some embodiments, each tumor-specific neoantigen corresponding to a member of the first set of tumor-associated mutations has a higher immunogenicity score than any tumor-specific neoantigen corresponding to a member of the second set of tumor-associated mutations.

[0071] In embodiments, at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 25, at least about 30, at least about 35, at least about 40, at least about 50 or more tumor-specific neoantigens are selected for the initial immunogenic composition. Typically, at least about 10 tumor-specific neoantigens are selected. In other cases, at least about 20 tumor-specific neoantigens are selected.

[0072] The immunogenic composition may also include natural or synthetic antigens. Natural or synthetic antigens may increase the immune response. Exemplary natural or synthetic antigens include, but are not limited to, pan-DR epitopes (PADRE) and tetanus toxin antigens.

[0073] The immunogenic composition may be in any form, such as a synthetic long peptide, RNA, DNA, a cell, a dendritic cell, a nucleotide sequence, a polypeptide sequence, a plasmid or a vector.

[0074] Tumor-specific neoantigens may also be included in a viral vector-based vaccine platform, such as vaccinia, fowlpox, self-replicating alphavirus, Maraba virus, adenovirus (see, e.g., Tatsis et al., Molecular Therapy, 10:616-629 (2004)) or lentivirus, including but not limited to second generation, third generation or hybrid second / third generation lentivirus and any generation of recombinant lentivirus designed to target a specific cell type or receptor (see, e.g., Hu et al., Immunol Rev., 239(1):45-61 (2011); Sakma et al., Biochem J., 443(3):603-18 (2012)). Depending on the packaging capacity of the viral vector-based vaccine platform described above, the method may deliver one or more nucleotide sequences encoding one or more tumor-specific neoantigenic peptides. These sequences may be flanked by non-mutated sequences, may be separated by linkers, or may be preceded by one or more sequences that target subcellular compartments (see, e.g., Gros et al., Nat Med., 22(4):433-8 (2016); et al., Science., 352(6291):1337-1341(2016); Lu et al., Clin Cancer Res., 20(13):3401-3410(2014)). After introduction into the host, the infected cells express the one or more tumor-specific neoantigens, and thereby induce a host immune (e.g., CD8+ or CD4+) response against the one or more tumor-specific neoantigens. Vaccinia vectors and methods that can be used for immunization protocols are described, for example, in U.S. Pat. No. 4,722,848. Another vector is BCG (bacillus Calmette-Guérin). BCG vectors are described in Stover et al. (Nature 351:456-460(1991)). A variety of other vaccine vectors that can be used for therapeutic administration or immunization of neoantigens that will be apparent to those skilled in the art from the description herein may also be used.

[0075] The immunogenic composition may contain individualized components according to the individual needs of a particular subject.

[0076] The immunogenic compositions described herein may also include an adjuvant. An adjuvant is any substance that, when incorporated into an immunogenic composition, increases or otherwise enhances and / or strengthens an immune response to a tumor-specific neoantigen, but does not produce an immune response to a tumor-specific neoantigen when the substance is administered alone. The adjuvant preferably produces an immune response to the neoantigen and does not produce an allergic or other adverse reaction. It is contemplated herein that the immunogenic composition may be administered before, together with, simultaneously with, or after the administration of the immunogenic composition.

[0077] Adjuvants can enhance immune responses by several mechanisms, including, for example, lymphocyte recruitment, stimulation of B and / or T cells, and stimulation of macrophages. When the immunogenic compositions of the present invention comprise an adjuvant or are administered with one or more adjuvants, adjuvants that can be used include, but are not limited to, mineral salt adjuvants or mineral salt gel adjuvants, granular adjuvants, microparticle adjuvants, mucosal adjuvants, and immunostimulatory adjuvants. Examples of adjuvants include, but are not limited to, aluminum salts (alum) (e.g., aluminum hydroxide, aluminum phosphate, and aluminum sulfate), 3 de-O-acylated monophosphoryl lipid A (MPL) (see GB 2220211), MF59 (Novartis), AS03 (GlaxoSmithKline), AS04 (Glaxo SmithKline), polysorbate 80 (Tween 80; ICL Americas, Inc.), imidazopyridine compounds (see International Application No. PCT / US2007 / 064857, published as International Publication No. WO2007 / 109812), imidazoquinoxaline compounds (see International Application No. PCT / US2007 / 064858, published as International Publication No. WO2007 / 109813), and saponins such as QS21 (see Kensil et al., Vaccine Design: The Subunit and Adjuvant Approach (Powell and Newman, eds., 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 emulsions (such as squalene or peanut oil), optionally combined with an immunostimulant such as monophosphoryl lipid A (see Stoute et al., N. Engl. J. Med. 336, 86-91 (1997)).

[0078] It has been reported that CpG immunostimulatory oligonucleotides can enhance the effect of adjuvants in a vaccine setting.Other TLR binding molecules, such as RNA binding TLR 7, TLR 8 and / or TLR 9, may also be used.

[0079] Other examples of useful adjuvants include, but are not limited to, chemically modified CpG (e.g., CpR, Idera), poly(I:C) (e.g., poly i:CI2U), polyICLC, non-CpG bacterial DNA or RNA, and immunologically active small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, Celebrex (celecoxib), NCX-4016, sildenafil, tadalafil, vardenafil, sorafenib, XL-999, CP-547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which may act therapeutically and / or as an adjuvant. In embodiments, polyICLC is an adjuvant.

[0080] The immunogenic composition may comprise one or more tumor-specific neoantigens described herein, alone or together with a pharmaceutically acceptable carrier. A suspension or dispersion of one or more tumor-specific neoantigens may be used, in particular an isotonic aqueous suspension, dispersion or amphiphilic solvent. The immunogenic composition may be sterilized and / or may comprise excipients, such as preservatives, stabilizers, wetting agents and / or emulsifiers, solubilizers, salts for regulating osmotic pressure and / or buffers, and may be prepared in a manner known per se, for example by conventional dispersion and suspension methods. In certain embodiments, such dispersions or suspensions may comprise a viscosity modifier. The suspension or dispersion is stored at a temperature of about 2°C to 8°C, or may be frozen for long-term storage and then thawed shortly before use. For injection, the vaccine or immunogenic formulation may be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hanks's solution, Ringer's solution or saline buffer. The solution may contain formulatory agents, such as suspending, stabilizing and / or dispersing agents.

[0081] In certain embodiments, the compositions described herein further comprise a preservative, such as the mercury derivative thimerosal. In a specific embodiment, the pharmaceutical compositions described herein comprise 0.001% to 0.01% thimerosal. In other embodiments, the pharmaceutical compositions described herein do not comprise a preservative.

[0082] Excipients may exist independently of adjuvants. The function of an excipient may be, for example, to increase the molecular weight of an immunogenic composition, to increase activity or immunogenicity, to confer stability, to increase biological activity, or to increase serum half-life. Excipients may also be used to help present one or more tumor-specific neoantigens to T cells (e.g., CD4+ or CD8+ T cells). Excipients may be carrier proteins, such as, but not limited to, keyhole limpet hemocyanin, serum proteins (e.g., transferrin, bovine serum albumin, human serum albumin, thyroglobulin, or ovalbumin), immunoglobulins, or hormones (e.g., insulin or palmitic acid). For immunization of humans, carriers are generally physiologically acceptable and safe for humans. Alternatively, the carrier may be a dextran, such as Sepharose.

[0083] Cytotoxic T cells recognize antigens in the form of peptides bound to MHC molecules, rather than the complete exogenous antigens themselves. The MHC molecules themselves are located on the cell surface of antigen presenting cells. Therefore, if there is a trimeric complex of peptide antigens, MHC molecules and antigen presenting cells (APCs), activation of cytotoxic T cells is possible. If additional APCs with corresponding MHC molecules are added, rather than using only one or more tumor-specific antigens to activate cytotoxic T cells, the immune response can be enhanced. Therefore, in some embodiments, the immunogenic composition additionally contains at least one APC.

[0084] The immunogenic composition may include an acceptable carrier (e.g., an aqueous carrier). A variety of aqueous carriers may be used, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, etc. These compositions may be sterilized by conventional well-known sterilization techniques, or may be aseptically filtered. The resulting aqueous solution may be packaged for use as is or lyophilized, and the lyophilized preparation may contain pharmaceutically acceptable auxiliary substances such as pH regulators and buffers, tension regulators, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc., before administration.

[0085] Neoantigens can also be administered via liposomes, which target specific cell tissues, such as lymphoid tissue. Liposomes can also be used to extend half-life. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. In these preparations, the neoantigens to be delivered are incorporated as part of the liposomes, either alone or in combination with molecules that bind to receptors that are ubiquitous in lymphocytes, such as monoclonal antibodies that bind to CD45 antigens, or in combination with other therapeutic or immunogenic compositions. Therefore, liposomes filled with the desired neoantigens can be directed to the site of lymphoid cells, where the liposomes then deliver the selected immunogenic composition. Liposomes can be formed by standard vesicle-forming lipids, which typically include neutral or negatively charged phospholipids and sterols, such as cholesterol. The choice of lipids is typically guided by considerations such as liposome size, acid instability, and the stability of liposomes in the bloodstream. A variety of methods are available for preparing liposomes as described, for example, in Szoka et al., Annu. 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] For targeting immune cells, the ligand incorporated into the liposomes may include, for example, antibodies or fragments thereof that are specific for cell surface determinants of the desired immune system cells. The liposomal suspension may be administered intravenously, locally, etc., at doses that vary, inter alia, depending on the mode of administration, the peptide being delivered, and the stage of the disease being treated.

[0087] As an alternative approach to 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-co-glycolic acid) microspheres. Poly(lactic-co-glycolic acid) microspheres can serve as endosomal delivery devices to entrap components of the immunogenic composition.

[0088] For treatment or immunization purposes, nucleic acids encoding tumor-specific neoantigens described herein may also be administered to patients. Many methods are conveniently used to deliver these nucleic acids to patients. For example, nucleic acids can be delivered directly as "naked DNA". For example, this method is described in Wolff et al., Science 247: 1465-1468 (1990) and U.S. Patent Nos. 5,580,859 and 5,589,466. Nucleic acids can also be administered using ballistic delivery, such as described in U.S. Patent No. 5,204,253. Particles consisting only of DNA may be administered. Alternatively, DNA may be adhered to particles (such as gold particles). Methods for delivering nucleic acid sequences may include viral vectors, mRNA vectors, and DNA vectors, with or without electroporation. Nucleic acids may also be delivered by compounding with cationic compounds (such as cationic lipids).

[0089] The immunogenic compositions provided herein can be administered to a subject by a variety of routes, including, but not limited to, oral, intradermal, intratumoral, intramuscular, intraperitoneal, intravenous, topical, subcutaneous, transdermal, intranasal, and inhalation routes, as well as via scarification (e.g., using a bifurcated needle to scrape the top layer of the skin). The immunogenic compositions can be administered at the tumor site to induce a local immune response against the tumor.

[0090] The dosage of the one or more tumor-specific neoantigens may depend on the type of composition and the subject's age, weight, body surface area, individual condition, individual pharmacokinetic data and mode of administration.

[0091] Also disclosed herein is a method for making an immunogenic composition comprising one or more tumor-specific neoantigens selected by performing the steps of the method disclosed herein. The immunogenic composition described herein can be made using methods known in the art. For example, a method for producing a tumor-specific neoantigen or vector disclosed herein (e.g., a vector comprising at least one sequence encoding one or more tumor-specific neoantigens) may include culturing a host cell under conditions suitable for expressing the neoantigen or vector, wherein the host cell comprises at least one polynucleotide encoding the neoantigen or vector, and purifying the neoantigen or vector. Standard purification methods include chromatography, electrophoresis, immunology, precipitation, dialysis, filtration, concentration, and chromatofocusing techniques.

[0092] The host cell may include a Chinese hamster ovary (CHO) cell, a NS0 cell, a yeast, or a HEK293 cell. The host cell may be transformed with one or more polynucleotides comprising at least one nucleic acid sequence encoding one or more tumor-specific neoantigens or vectors disclosed herein. In certain embodiments, the isolated polynucleotide may be a cDNA.

[0093] After selecting tumor-specific neoantigens for the initial immunogenic composition, tumor-associated mutations can be divided into multiple groups, such as two groups. The first group includes tumor-associated mutations corresponding to the selected tumor-specific neoantigens. The second group includes tumor-associated mutations that do not correspond to any of the selected tumor-specific neoantigens.

[0094] Figure 1 Further assistance is provided for visualization. In the upper right corner, the immunogenicity of three peptides of the hypothetical model are scored and ranked: peptide 1, containing ProTrp, with an immunogenicity score of 0.8; peptide 2, containing ArgCys, with an immunogenicity score of 0.6; and peptide 3, containing ThrAla, with an immunogenicity score of 0.5. As shown in the lower left, the initial immunogenic composition was selected to include the two highest ranked tumor-specific neoantigens, peptide 1 and peptide 2. Peptide 3 was excluded from the initial immunogenic composition.

[0095] C. Administering the Immunogenic Composition to a Subject in Need thereof

[0096] The above-described immunogenic compositions comprising tumor-specific neoantigens corresponding to a first set of tumor-associated mutations and not corresponding to a second set of tumor-associated mutations can be administered to a subject in need thereof, such as a subject having cancer or at risk of having cancer.

[0097] Cancer can be any solid tumor or any blood tumor.Tumor can be a primary tumor or a metastatic tumor.Solid tumors can 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.Hematological tumors can include but are not limited to tumors from lymphomas (e.g., B cell lymphomas) and leukemias (e.g., acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia and T cell lymphocytic leukemia).

[0098] The methods disclosed herein can be used with 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 lymphocytic leukemia (ALL), acute myeloid leukemia (AML), adrenocortical carcinoma, anal cancer, adnexal cancer, astrocytoma, basal cell carcinoma, brain tumor, biliary tract cancer, bladder cancer, bone cancer, breast cancer, bronchial tumor, cancer of unknown primary cause, cardiac tumor, cervical cancer, chordoma, colon cancer, colorectal cancer, craniopharyngioma, ductal carcinoma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, nasal glioma, fibrous histiocytoma, Ewing sarcoma, eye cancer, germ cell tumor, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, glioma, head and neck cancer, hepatocellular carcinoma, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, Kaposi sarcoma, 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, occult primary metastatic squamous neck cancer, midline airway cancer involving the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndrome, myelodysplasia / bone marrow Hyperplastic tumors, cancer of the nasal cavity and paranasal sinuses, nasopharyngeal carcinoma, neuroblastoma, non-small cell lung cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, cancer of the renal pelvis and ureter, retinoblastoma, rhabdoid tumor, salivary gland cancer, Sezary syndrome, skin cancer, small cell lung cancer, small intestine cancer, soft tissue sarcoma, spinal cord tumor, gastric cancer, T-cell lymphoma, teratoma, testicular cancer, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, urethral cancer, uterine cancer, vaginal cancer, vulvar cancer and Wilms tumor. In one aspect, the cancer is 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. Melanoma is of particular concern. Breast cancer, lung cancer and bladder cancer are also of particular concern.

[0099] The immunogenic composition stimulates the subject's immune system, especially the response of specific CD8+T cells or CD4+T cells. Interferon gamma produced by CD8+ and T helper CD4+ cells regulates the expression of PD-L1. When attacked by T cells, PD-L1 expression in tumor cells is upregulated. Therefore, tumor vaccines can induce the production of specific T cells and simultaneously upregulate the expression of PD-L1, which may limit the efficacy of the immunogenic composition. In addition, when the immune system is activated, the expression of the T cell surface reporter gene CTLA-4 also increases accordingly, which binds to the ligand B7-1 / B7-2 on the antigen presenting cell and exerts an immunosuppressive effect. Therefore, in some cases, anti-immunosuppressive drugs or immunostimulatory drugs, such as checkpoint inhibitors, may be further administered to the subject. Checkpoint inhibitors may include, but are not limited to, anti-CTL4-A antibodies, anti-PD-1 antibodies, and anti-PD-L1 antibodies. These checkpoint inhibitors bind to the immune checkpoint proteins of T cells to remove the inhibition of T cell function by tumor cells. Blocking CTLA-4 or PD-L1 by antibodies can enhance the patient's immune response to cancerous cells. CTLA-4 has been shown to be effective when following a vaccination regimen.

[0100] Immunogenic compositions comprising one or more tumor-specific neoantigens can be administered to subjects who have been diagnosed with cancer, have cancer, have recurrent cancer (i.e., relapse), or are at risk of developing cancer. Immunogenic compositions comprising one or more tumor-specific neoantigens can be administered to subjects who are resistant to other forms of cancer treatment (e.g., chemotherapy, immunotherapy, or radiation). Immunogenic compositions comprising one or more tumor-specific neoantigens can be administered to subjects before other standard of care cancer therapies (e.g., chemotherapy, immunotherapy, or radiation). Immunogenic compositions comprising one or more tumor-specific neoantigens can be administered to subjects simultaneously with, after, or in combination with other standard of care cancer therapies (e.g., chemotherapy, immunotherapy, or radiation).

[0101] The subject can be a human, dog, cat, horse, or any animal in which a tumor-specific response is desired.

[0102] The immunogenic composition is administered to the subject in an amount sufficient to induce an immune response to the tumor-specific neoantigen and to destroy or at least partially prevent symptoms and / or complications. In embodiments, the immunogenic composition can provide a lasting immune response. A lasting immune response can be established by administering a booster dose of the immunogenic composition to the subject. The immune response to the immunogenic composition can be prolonged by administering a booster dose to the subject. In embodiments, at least one, at least two, at least three or more booster doses can be administered to alleviate cancer. The first booster dose can 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% relative to the initial immune response. The second booster dose can 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% relative to the initial immune response. The third booster 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% relative to the initial immune response.

[0103] An amount sufficient to elicit an immune response is defined as a "therapeutically effective dose". An amount 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 patient's weight and general health, and the judgment of the prescribing physician. It should be remembered that immunogenic compositions are often used in serious disease states, i.e., life-threatening or potentially life-threatening conditions, especially when the cancer has metastasized. In such cases, it is possible and desirable for the treating physician to administer a significant excess of these immunogenic compositions in view of the minimization of foreign material and the relatively non-toxic nature of the neoantigens.

[0104] The immunogenic composition comprising one or more tumor-specific neoantigens can be administered to a subject alone or in combination with other therapeutic agents. The therapeutic agent can be, for example, a chemotherapeutic agent, radiation, or immunotherapy. Any suitable therapeutic treatment for a particular cancer can be administered. Exemplary chemotherapeutic agents include, but are not limited to, 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, ifosfamide, interferon alpha, irinotecan, lansoprazole, levamisole, folinic acid, megestrol acetate, mesna, methotrexate, metoclopramide, mitomycin, mitotane, mitoxantrone, omeprazole, ondansetron, paclitaxel Pilocarpine, prochlorperazine, rituximab, tamoxifen, topotecan hydrochloride, trastuzumab, vinblastine, vincristine, and vinorelbine tartrate. Small molecules or targeted therapies (e.g., kinase inhibitors) may be administered to the subject. Anti-CTLA antibodies, anti-PD-1 antibodies, or anti-PD-L1 antibodies may be further administered to the subject. Blocking CTLA-4 or PD-L1 by antibodies can enhance the patient's immune response to cancerous cells.

[0105] D. Quantification of Circulating Substances and Tumor-Associated Mutations

[0106] Although it is expected that every tumor-specific neoantigen in the initial immunogenic composition will be effective against the tumor, in reality, at least some of the tumor-specific neoantigens may be ineffective targets for the initial immunogenic composition. Even if all tumor-specific neoantigens are effective, this cannot be predicted in advance.

[0107] The present disclosure also relates to monitoring the level of each tumor-associated mutation in the subject's tumor at multiple time points. If a tumor-associated mutation in the first group of tumor-associated mutations increases over time, the tumor-specific neoantigen corresponding to the tumor-associated mutation can be determined to be an ineffective target.

[0108] The level of each tumor-associated mutation can be determined by quantifying each member of the first set of tumor-associated mutations and each member of the second set of tumor-associated mutations in circulating material isolated from the subject.

[0109] Circulating substances can be any substance produced by a subject's tumor or healthy cells and can be found in the subject's blood. Examples of circulating substances may include circulating tumor DNA (ctDNA), circulating free DNA (cfDNA), circulating tumor cells (CTCs), circulating tumor proteins, extracellular vesicles, or two or more thereof.

[0110] Circulating tumor DNA is DNA from tumor cells and is therefore expected to contain tumor-associated mutations. Even if the tumor itself is growing, individual tumor cells may die and release ctDNA directly or through the normal action of macrophages.

[0111] The abundance of ctDNA is a function of the total area of ​​the tumor-vascular interface. This includes all metastases. Thus, ctDNA summarizes information from all tumors in a subject, whereas quantification of tumor-associated mutations by WES, WGS, or scRNA sequencing of tissue (rather than blood) is limited to the specific tumor from which tissue was extracted for sequencing. In addition, drawing blood is a simpler and less invasive procedure than extracting tissue from most internal tumors.

[0112] An exemplary amount of ctDNA in a biological sample (e.g., plasma or serum) may be in the range of about 1 femtogram (fg) to about 1 picogram (pg), about 1 pg to about 200 nanograms (ng), about 1 ng to about 100 ng, or about 10 ng to about 1000 ng. For example, the amount may be at most about 600 ng, at most about 500 ng, at most about 400 ng, at most about 300 ng, at most about 200 ng, at most about 100 ng, at most about 50 ng, or at most about 20 ng of ctDNA. The amount may be at least 1 fg, at least 10 fg, at least 100 fg, at least 1 pg, at least 10 pg, at least 100 pg, at least 1 ng, at least 10 ng, at least 100 ng, at least 150 ng, or at least 200 ng of ctDNA. The amount may be at most 1 fg, 10 fg, 100 fg, 1 pg, 10 pg, 100 pg, 1 ng, 10 ng, 100 ng, 150 ng or 200 ng of ctDNA molecules. The method may comprise obtaining 1 fg to 200 ng of ctDNA.

[0113] The ctDNA can have an exemplary size distribution of about 100-500 nucleotides. The ctDNA can be about 100, about 105, about 110, about 115, about 120, about 125, about 130, about 135, about 140, about 145, about 150, about 155, about 160, about 165, about 170, about 175, about 180, about 185, about 190, about 195, about 200, about 210, about 215, about 220, about 225, about 230, about 235, about 240, about 245, about 250, about 255, about 260, about 265, about 270, about 275, about 280, about 285, about 290, about 295, about 30 430, about 435, about 440, about 445, about 450, about 455, about 460, about 465, about 470, about 475, about 480, about 485, about 490, about 495, or about 500 nucleotides.

[0114] From the broadest definition, circulating free DNA is any DNA present in the blood. Herein, cfDNA refers to DNA released into the blood from non-tumor cells. In other words, ctDNA and cfDNA are different aspects of the present disclosure.

[0115] In addition to ctDNA, the blood also contains circulating free DNA (cfDNA) from non-tumor sources, namely normal cells. In general, 1 ml of blood contains an average of 6 ng of circulating free DNA, or approximately 1,800 cell genomes. The concentrations of cfDNA and ctDNA fluctuate greatly between individuals and depend on many factors, including age, lifestyle, physical condition, and in the context of cancer treatment, the type and stage of cancer. Of these cell genomes, most will come from healthy cells. For example, a breast cancer tumor with a diameter of 10 mm is expected to contribute approximately 0.03 cancer genomes per milliliter of blood. For another example, a melanoma with a diameter of 10 mm is expected to contribute approximately 1-2 cancer genomes per milliliter of blood.

[0116] Although cfDNA is DNA from non-tumor cells and therefore at first glance seems unlikely to contain tumor-associated mutations, DNA from non-tumor cells present in the tumor microenvironment may undergo epigenetic changes, such as changes in methylation or changes in chromatin content, that reflect tumor-associated mutations.

[0117] Circulating tumor proteins are proteins from tumor cells and are therefore expected to include tumor-associated mutations. Circulating tumor proteins may be released from dead or dying tumor cells, may be secreted by living tumor cells, or introduced into the blood of a subject by any other possible known mechanism.

[0118] CTCs are tumor cells, whether alive, dying or dead, that are present in the bloodstream. As cells, they contain tumor DNA and tumor proteins and are therefore expected to contain tumor-associated mutations.

[0119] Extracellular vesicles are membrane-bound capsules that can be released from the surface of tumor or normal cells. Extracellular vesicles usually contain RNA and proteins from the parent cell and can provide information about tumor-related mutations. For example, extracellular vesicles from tumor cells can be a source of tumor proteins, and any extracellular vesicle can be a source of nucleic acids indicating epigenetic changes in tumor cells or normal cells, which result in changes in the DNA transcription of one or more genes (and the expected changes in the RNA levels resulting therefrom).

[0120] To quantify tumor-associated mutations in circulating material, a sample containing circulating material can be collected from a subject, and the circulating material can be isolated therefrom.

[0121] The sample can be obtained from a human or non-human subject. In one aspect, the sample is obtained from a human. The sample can be obtained from a variety of biological sources.

[0122] Various assays (e.g., sequencing assays) can be used to detect circulating substances. The methods provided herein can include separating and analyzing circulating substances from the blood (e.g., plasma or serum) of a subject of interest (i.e., a subject with cancer, a subject in remission of cancer, or a subject suspected of having cancer). The methods can include separating plasma and circulating substances from blood depleted of intact cells. The methods can include centrifugation to produce plasma and extracting nucleic acids from the plasma.

[0123] Circulating material can be from the body fluid (for example, blood sample) of the subject of interest. Circulating material can be obtained from the plasma fraction, serum fraction or both of the blood sample. In some embodiments, body sample is whole blood, serum, plasma, cerebrospinal fluid, synovial fluid, lymph fluid, ascites, interstitial fluid or fluid in extracellular fluid, intercellular space, including gingival crevice fluid, bone marrow, pleural effusion, cerebrospinal fluid, saliva, mucus, sputum, semen, sweat, urine or any combination thereof. In some embodiments, circulating DNA is obtained from blood and its fraction. Sample can be in the form of initial separation from subject, or can be further processed to remove or add components, such as cells, or to enrich one component relative to another component. Sample can be separated or obtained from subject and transported to sample analysis site. Sample can be stored and transported at desired temperature such as room temperature, 4 ° C, -20 ° C and / or -80 ° C. Sample can be separated or obtained from subject at sample analysis site. Subject can be human, mammal, animal, companion animal, service animal or pet. Subject may not suffer from cancer or have detectable cancer symptoms. The subject may have been treated with one or more cancer therapies, such as any one or more of chemotherapy, antibodies, vaccines, or biologics. The subject may be in remission. The subject may be suspected of having cancer or having any cancer-related genetic mutation.

[0124] Biological samples can be obtained from subjects by any means, including but not limited to tumor biopsy, needle aspiration, scraping, surgical resection, surgical incision, venipuncture or other means known in the art. Those skilled in the art will recognize other suitable techniques for obtaining biological samples.

[0125] The biological sample can be obtained from the subject in a single procedure. The biological sample can be obtained from the subject repeatedly over a period of time. For example, the biological sample can be obtained once a day, once a week, once a month, once every two years, or once a year. Obtaining a large number of samples over a period of time can be used to identify and select new tumor-specific neoantigens.

[0126] Circulating material can be separated from body fluid (for example, blood plasma) by fractionation or separation step, in which the circulating material found in the solution is separated from other insoluble components of intact cells and body fluid. Separation may include techniques such as centrifugation or filtration. Alternatively, cells in body fluids may be lysed and cell-free nucleic acid and cell nucleic acid are processed together. Typically, after adding buffer and washing steps, nucleic acid can be precipitated with alcohol. Further removal steps, such as silica-based columns, can be used to remove pollutants or salts. After such treatment, the sample may include various forms of nucleic acid, including double-stranded DNA and single-stranded DNA. In some embodiments, single-stranded DNA can be converted into double-stranded form, so it is included in subsequent processing and analysis steps.

[0127] In some embodiments, the circulating material is isolated from a blood sample, a serum sample, a plasma sample, a urine sample, or a cerebrospinal fluid sample. In one aspect, the sample is a blood sample, a serum sample, or a plasma sample.

[0128] In embodiments where the sample is a blood sample, a serum sample, or a plasma sample, the circulating material can be isolated from at least about 10 ml of whole blood of the subject, such as at least about 20 ml of whole blood of the subject. These blood volumes may be particularly useful when the circulating material comprises ctDNA, given the relatively low number of cancer genomes per milliliter of blood.

[0129] In some embodiments, for ctDNA, a tumor-associated mutation can be found whose allele frequency in the cfDNA pool is less than 10.0%, such as less than 9.9%, less than 9.8%, less than 9.7%, less than 9.6%, less than 9.5%, less than 9.4%, less than 9.3%, less than 9.2%, less than 9.1%, less than 9.0%, less than 8.9%, less than 8.8%, less than 8.7%, less than 8.6%, less than 8.5%, less than 8.4%, less than 8.3%, less than 8.2%, less than 8.1%, Less than 8.0%, less than 7.9%, less than 7.8%, less than 7.7%, less than 7.6%, less than 7.5%, less than 7.4%, less than 7.3%, less than 7.2%, less than 7.1%, less than 7.0%, less than 6.9%, less than 6.8%, less than 6.7%, less than 6.6%, less than 6.5%, less than 6.4%, less than 6.3%, less than 6.2%, less than 6.1%, less than 6.0%, less than 5.9%, less than 5.8%, less than 5.7%, less than 5.6%, less than 5.5%, less than 5.4 %, less than 5.3%, less than 5.2%, less than 5.1%, less than 5.0%, less than 4.9%, less than 4.8%, less than 4.7%, less than 4.6%, less than 4.5%, less than 4.4%, less than 4.3%, less than 4.2%, less than 4.1%, less than 4.0%, less than 3.9%, less than 3.8%, less than 3.7%, less than 3.6%, less than 3.5%, less than 3.4%, less than 3.3%, less than 3.2%, less than 3.1%, less than 3.0%, less than 2.9%, less than 2.8%, less than 2.7%, less than 2.6%, less than 2.5%, less than 2.4%, less than 2.3%, less than 2.2%, less than 2.1%, less than 2.0%, less than 1.9%, less than 1.8%, less than 1.7%, less than 1.6%, less than 1.5%, less than 1.4%, less than 1.3%, less than 1.2%, less than 1.1%, less than 1.0%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2% or less than 0.1%.

[0130] In some embodiments, blood samples may be collected at least once prior to treatment and then serially as guided by the frequency of monitoring of immunogenic composition efficacy and disease progression.

[0131] Prior to treatment, 10-20 ml of blood may be collected from the subject for paired genomic (including but not limited to WGS / WES) tumor-normal sequencing within 5 days of biopsy collection, and within 2-4 weeks prior to administration of the immunogenic composition.

[0132] Following treatment, serial collection of blood samples may be performed as follows: at regularly scheduled intervals based on medical information indicating disease progression, clinically observed responses to the immunogenic composition, or when the patient is scheduled to receive subsequent doses of the initial immunogenic composition or a reformulated immunogenic composition, and at least once at the time of administration of the initial immunogenic composition and once within 10 weeks after administration of the initial immunogenic composition.

[0133] Quantifying tumor-associated mutations may involve a variety of approaches, depending on the circulating material under consideration. Quantification may include sequencing of ctDNA using WES, WGS, targeted sequencing, polymerase chain reaction (PCR), or hybridization methods.

[0134] For cfDNA, quantification of tumor-associated mutations may include quantitative polymerase chain reaction (qPCR) or next-generation sequencing.

[0135] Tumor-associated mutations in nucleic acids from CTCs or extracellular vesicles can be quantified by any of the techniques described herein, keeping in mind that a reverse transcription process (e.g., using a reverse transcriptase) can be used to prepare DNA from RNA present in CTCs or extracellular vesicles. In addition, RNA can be directly subjected to single-cell RNA sequencing (scRNAseq) to identify and quantify tumor-associated mutations from RNA.

[0136] For any genomic DNA-based method, such as ctDNA, cfDNA, or DNA from CTCs, quantifying tumor-associated mutations can include determining the methylation or chromatin content of one or more epigenetic markers. Chromatin content can be determined by high-throughput sequencing transposase accessible chromatin assay (ATAC-Seq) and other techniques known to those of ordinary skill in the art.

[0137] There are various methods for obtaining genome sequence data as described herein. Sequencing methods are well known in the art, including but not limited to WGS, WES, targeted sequencing, PCR-based methods (including real-time PCR (RT-PCR)), deep sequencing, high-throughput sequencing, or combinations thereof. In some cases, the aforementioned techniques and procedures can be carried out according to the method described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 4th edition (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. See also Austell et al., Current Protocols in Molecular Biology, ed., Greene Publishing and Wiley-Interscience New York (1992) (with regular updates).

[0138] The genomic sequence data can be obtained from WGS, WES, targeted sequencing, DNA hybridization methods or a combination thereof. The genomic sequencing data can be sequence data derived from high-depth WGS data.

[0139] In some embodiments, the DNA can be sequenced to obtain sequence reads. The sequence data includes sequence reads of multiple polynucleotides from a subject. The sequence reads can include about 2 to about 5000 nucleotides. For example, about 2, about 3, about 4, about 5, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500 80, about 290, about 300, about 310, about 320, about 330, about 340, about 350, about 360, about 370, about 380, about 390, about 400, about 410, about 420, about 430, about 440, about 450, about 460, about 470, about 480, about 490, about 500, about 550, about 600, about 700, about 800, about 900, about 1,000 0, about 1,100, about 1,200, about 1,300, about 1,400, about 1,500, about 1,600, about 1,700, about 1,800, about 1,900, about 2,000, about 2,100, about 2,200, about 2,300, about 2,400, about 2,500, about 2,600, about 2,700, about 2,800, about 2,900, about 3,000, about about 3,100, about 3,200, about 3,300, about 3,400, about 3,500, about 3,600, about 3,700, about 3,800, about 3,900, about 4,000, about 4,100, about 4,200, about 4,300, about 4,400, about 4,500, about 4,600, about 4,700, about 4,800, about 4,900, or about 5,000 nucleotides.

[0140] DNA sequence data can be analyzed using integration of variant reads (INVAR) as described in International Application No. PCT / EP2019 / 055610 for circulating tumor DNA sequence data. DNA sequence data can be analyzed using the objective function outlined in Equation 1:

[0141]

[0142] where ξi is the estimated cellular prevalence of the ith subclone. P~iα is the probability that peptide α is in the ith subclone, and sα is the individual score of the αth peptide from machine learning modeling. All of these prevalence and subclonality estimates are derived from a combination of biopsies and DNA sequencing. The “big-OR” is taken over the best L peptides, where L=20, and the sum is taken over all C subclones, where C is the parameter returned by the subclonality estimation algorithm. Typically, 1 <C<15。

[0143] DNA sequence data can be analyzed using the objective function outlined in Equation 2:

[0144]

[0145] where ξα is the cellular prevalence of the αth mutation and sα is the individual score of the αth peptide from machine learning modeling. This function is optimized by direct ranking of all peptides by the product ξαsα.

[0146] A machine learning platform can be used to analyze DNA sequence data. Applicable exemplary machine learning models include, but are not limited to, neural networks, Bayesian classifiers, logistic regression, decision trees, gradient-enhanced decision trees, random forests, support vector machines, gradient-enhanced trees, multilayer perceptrons, one-vs-rest, or Gaussian Naive Bayes.

[0147] In some embodiments, blood samples can be processed within 4 hours and can be obtained using standard methods or kits (e.g., DNeasy TM Kit (Qiagen NV, Venlo, Netherlands), QIAmp TM Kit (described above) or Quick-cfDNA TM Kit (Zymo Research, Irvine, CA)) is used to separate circulating free DNA from plasma. In the pre-treatment sample processing, separate from the circulating free DNA isolated from plasma, the peripheral blood lymphocytes centrifuged in the first step of circulating free DNA isolation can also be used to extract germline genomic DNA, which can be used as a matched normal value for genomic analysis. The DNA yield of each blood aliquot is expected to be at least 20ng.

[0148] As described herein, circulating material may include circulating tumor proteins, proteins from CTCs, or proteins from extracellular vesicles, any of which may contain tumor-associated mutations. Quantifying tumor-associated mutations in proteins may include performing mass spectrometry or elution assays on circulating tumor proteins, proteins from CTCs, or proteins from extracellular vesicles.

[0149] As described herein, CTCs can be a source of tumor proteins or tumor nucleic acids. In addition, quantifying tumor-associated mutations in CTC material can include performing fluorescence activated cell sorting (FACS) on CTCs. This can be part of the preliminary classification or separation of CTCs from other cells in the circulation. In some embodiments, particularly when tumor-associated mutations are located in proteins exposed to the surface of tumor cells, FACS can be used to sort CTCs based at least in part on tumor-associated mutations, and thus direct quantification of tumor-associated mutations can be provided.

[0150] The unit of quantification can be molecules / ml, or another measure of the number of copies of a tumor-associated mutation per unit weight or volume of the sample. If desired, the quantification can be normalized, for example, by normalizing the number of copies of a tumor-associated mutation to the total amount of the source material, the number of copies of a healthy tissue marker gene that is expected to have a constant copy number in the source material, etc.

[0151] Quantification of tumor-associated mutations can be performed at each of multiple time points. By doing so, the number of each tumor-associated mutation can be tracked over time.

[0152] The same circulating material can be used at each time point to quantify tumor-associated mutations. As a hypothetical example, if a first tumor-associated mutation is quantified from ctDNA at a first time point, the mutation can be quantified from ctDNA at a second time point. This may be desirable to provide greater consistency and more accurate measurements at multiple time points.

[0153] It is envisioned that a first tumor-associated mutation is quantified from a first circulating material at a first time point, and from a second circulating material at a second time point. As another hypothetical example, a first tumor-associated mutation may be quantified from ctDNA at a first time point, and from CTCs at a second time point. By appropriately normalizing the quantified values ​​determined by each technique to a reference value, which is expected to remain substantially constant within each circulating material at each time point and substantially proportional between the two circulating materials, the accuracy of the measurements at multiple time points can be maintained at an acceptably high level.

[0154] Multiple circulating species can be used to quantify multiple tumor-associated mutations. As another hypothetical example, a first tumor-associated mutation can be quantified from ctDNA at a first time point and a second time point, and a second tumor-associated mutation can be quantified from circulating tumor proteins, for example, at the first time point and the second time point.

[0155] In some embodiments, at least one of the multiple time points may be prior to administration of the initial immunogenic composition.

[0156] Figure 1The hypothetical model shown in shows the quantification of three tumor-specific neoantigens (peptides 1-3) from ctDNA. As described above, peptides 1 and 2 are included in the immunogenic composition and correspond to members of the first group of tumor-associated mutations. Peptide 3 is not included in the immunogenic composition and corresponds to a member of the second group of tumor-associated mutations. The three tumor-associated mutations corresponding to peptides 1-3 in ctDNA are quantified at four time points (week E, week 0, week 4, and week 8). Week E is the time point for identifying tumor-associated mutations in subjects. Figure 1 The immunogenic composition referred to as a vaccine in the present invention is manufactured prior to week 0. The immunogenic composition is administered to the subject at week 0, week 4, and week 8, as indicated by the symbol "VAC". Thus, at least one quantitative time point, week E, is prior to administration of the initial immunogenic composition.

[0157] continue Figure 1 The number of associated mutations in each tumor is plotted over time based on the hypothetical model. After week 8, the following is observed:

[0158] The number of tumor-associated mutations corresponding to peptide 1 (including ProTrp) decreased over time. This suggests that the tumor-specific neoantigens of peptide 1 in the initial immunogenic composition inhibited tumor cells containing this tumor-associated mutation;

[0159] The number of tumor-associated mutations corresponding to peptide 2 (including ArgCys) increased over time. This suggests that the tumor-specific neoantigen of peptide 2 in the initial immunogenic composition failed to inhibit tumor cells containing this tumor-associated mutation;

[0160] The number of tumor-associated mutations corresponding to peptide 3 (including ThrAla) increased slightly over time. However, it should be remembered that the tumor-specific neoantigen of peptide 3 was not included in the initial immunogenic composition.

[0161] The multiple time points for quantifying tumor-associated mutations can occur at various intervals and / or at different times relative to other acts according to the method, as will be discussed in greater detail herein.

[0162] E. Reconstitution of Immunogenic Compositions

[0163] By quantifying tumor-associated mutations in circulating material at multiple time points, it can be found that the number of members of the first group of tumor-associated mutations (i.e., tumor-associated mutations with corresponding tumor-specific neoantigens included in the initial immunogenic composition) increases from earlier time points to later time points. As described above, such a finding indicates that the corresponding tumor-specific neoantigens are ineffective components of the initial immunogenic composition.

[0164] The method may further include replacing the tumor-specific neo-antigens corresponding to members of the first group of tumor-associated mutations in the initial immunogenic composition with replacement tumor-specific neo-antigens corresponding to members of the second group of tumor-associated mutations in response to an increase in the number of members of the first group of tumor-associated mutations from an earlier time point to a later time point to produce a reformulated immunogenic composition.

[0165] It is apparent that if the condition is met that the number of members of the first group of tumor-associated mutations corresponding to the tumor-specific neo-antigens increases from an earlier time point to a later time point, a plurality of tumor-specific neo-antigens, each tumor-specific neo-antigen corresponding to a member of the first group of tumor-associated mutations, may be replaced.

[0166] In some embodiments, the number of members of the second set of tumor-associated mutations to which the replacement tumor-specific neoantigen corresponds does not decrease from an earlier time point to a later time point. This may be desirable, given that if the number of the tumor-associated mutation in question decreases, even if its corresponding tumor-specific neoantigen is not targeted by the immunogenic composition, then there will be no further benefit in targeting it with the replacement tumor-specific neoantigen.

[0167] If the method is repeated multiple times, the membership of the second set of tumor-associated mutations may change. This may involve periodic monitoring of the subject's circulating material for the emergence of new neoantigens. When new tumor neoantigens emerge and are detected, their coding sequences may be added to the second set of tumor-associated mutations.

[0168] In some embodiments, the method may further include detecting the appearance of at least one tumor-associated mutation that is not included in the first set of tumor-associated mutations or the second set of tumor-associated mutations at a first time point; and adding the appearing tumor-associated mutation to the second set of tumor-associated mutations at a time point after the first time point. After adding the appearing tumor-associated mutation to the second set, at other time points, the immunogenic composition may be reformulated to include the appearing tumor-associated mutation.

[0169] As a specific hypothetical example, an initial immunogenic composition may be prepared based in part on the discovery that peptide p is not encoded by the subject's ctDNA. In other words, peptide p is not from a tumor-associated mutation and is therefore not suitable for inclusion in the initial immunogenic composition. At a first time point (e.g., after at least one round of quantifying members of a second set of tumor-associated mutations and monitoring the emergence of new neoantigens), new neoantigens are detected. New neoantigens may be added to the second set of tumor-associated mutations, and the method will continue to a later time point. If the number of new neoantigens does not decrease at two or more time points, it may be a very ideal candidate for reformulation, because the new neoantigens may reflect the dynamic response of the tumor to treatment (e.g., a new subclone carrying a new neoantigen may have developed resistance to all components in the initial immunogenic composition). Reformulating the composition to include new neoantigens can minimize the ability of subclones to escape treatment.

[0170] The replacement tumor-specific neoantigens can be selected based on one or more criteria that will be apparent to a person skilled in the art. In some embodiments, the tumor-specific neoantigens corresponding to the second set of tumor-associated mutations can be ranked by immunogenicity score, and the highest-ranked tumor-specific neoantigens can be selected as replacement tumor-specific neoantigens. In some embodiments, the change in the number of the second set of tumor-associated mutations at multiple time points can be ranked, and the tumor-specific neoantigens corresponding to the tumor-associated mutations with the greatest increase can be selected as replacement tumor-specific neoantigens.

[0171] The replacement may be performed at any time after administration of the initial immunogenic composition. In some embodiments, the initial immunogenic composition may be administered once prior to replacement of the tumor-specific neoantigen.

[0172] In some embodiments, the initial immunogenic composition may be administered multiple times before the tumor-specific neoantigen is replaced.

[0173] Back to Figure 1In the hypothetical model of the invention, the initial immunogenic composition is administered three times (week 0, week 4, and week 8). Thereafter, the tumor-associated mutation in the first group of tumor-associated mutations (mutations of the corresponding tumor-specific neoantigens contained in the initial immunogenic composition) that increases in number from week E to week 8, i.e., peptide 2 containing ArgCys, is replaced by peptide 3 containing ThrAla in the immunogenic composition. Although not required in the present method, the number of tumor-associated mutations corresponding to peptide 3 does not decrease from week E to week 8. The resulting reformulated immunogenic composition comprises peptide 1 (comprising ProTrp) and peptide 3 (comprising ThrAla). The reformulated immunogenic composition can be administered at weeks 12, 16, and 20. In other words, the immunogenic composition administration regimen can remain unchanged, except that after replacing the previous tumor-specific neoantigen with the replacement tumor-specific neoantigen, the reformulated immunogenic composition instead of the initial immunogenic composition is administered at the predetermined administration time.

[0174] The replacement immunogenic composition may be administered at any time after said replacement. In some embodiments, the reconstituted immunogenic composition is administered once after replacement of the tumor-specific neoantigen.

[0175] The reformulated immunogenic composition can be administered multiple times after replacing the tumor-specific neoantigens.

[0176] F. Selection of Quantification Time Points

[0177] As described above, in some implementations, at least one of the multiple time points used for quantification may be selected according to the timing of the method relative to other actions.

[0178] In some embodiments, one of the multiple time points is at least 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, or about 12 weeks after administration of the initial immunogenic composition or the reconstituted immunogenic composition.

[0179] In some embodiments, one of the multiple time points is at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months after administration of the initial immunogenic composition or the reconstituted immunogenic composition.

[0180] In some embodiments, one of the multiple time points is at least about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years after administration of the initial immunogenic composition or the reformulated immunogenic composition.

[0181] G. Treatment regimens comprising more than two reconstituted immunogenic compositions

[0182] The foregoing discussion of initial immunogenic compositions and reconstituted immunogenic compositions is not limited to, for example, Figure 1 The case of the hypothetical model shown in , where the initial immunogenic composition is the first immunogenic composition administered to the subject and the reconstituted immunogenic composition is the second and last immunogenic composition administered to the subject. More generally, the immunogenic composition referred to herein as the initial immunogenic composition can be the xth immunogenic composition among n immunogenic compositions administered to the subject during the treatment regimen, where x is 1 to n-1 (inclusive), and the reconstituted immunogenic composition is the x+1th immunogenic composition among n immunogenic compositions administered to the subject, where x+1 is 2 to n (inclusive). This definition covers Figure 1 A hypothetical model of where n=2, as well as implementations where n=3, 4, 5, 6 or greater.

[0183] Thus, in some embodiments, the methods disclosed herein can be performed multiple times during the treatment regimen. For example, if n=3, the methods disclosed herein can be performed twice. In the first execution, the first immunogenic composition administered during the treatment regimen is the first initial immunogenic composition, and the second immunogenic composition administered during the treatment regimen is the first re-formulated immunogenic composition. In the second execution, the first re-formulated immunogenic composition is also the second initial immunogenic composition, and the third immunogenic composition administered during the treatment regimen is the second re-formulated immunogenic composition.

Claims

1. A method, comprising: administering to a subject in need thereof an initial immunogenic composition comprising a plurality of tumor-specific neo-antigens, wherein each tumor-specific neo-antigen corresponds to a member of a first set of tumor-associated mutations in the subject, and no tumor-specific neo-antigen corresponds to a member of a second set of tumor-associated mutations in the subject; and Each member of the first set of tumor-associated mutations and each member of the second set of tumor-associated mutations are quantified in circulating material comprising tumor-associated mutations isolated from the subject at each time point in a plurality of time points.

2. The method of claim 1, wherein the circulating material comprises circulating tumor DNA (ctDNA).

3. The method of claim 1 or 2, wherein the circulating material comprises circulating free DNA (cfDNA).

4. The method of any one of claims 1 to 3, wherein the circulating material comprises circulating tumor cells (CTCs).

5. The method of any one of claims 1 to 4, wherein the circulating substance comprises a circulating tumor protein.

6. The method of any one of claims 1 to 5, wherein the circulating material comprises extracellular vesicles.

7. The method of any of the preceding claims, wherein the circulating material comprises circulating tumor DNA (ctDNA), circulating free DNA (cfDNA), circulating tumor cells (CTCs), circulating tumor proteins, extracellular vesicles, or a combination thereof.

8. The method according to any one of the preceding claims, further comprising: responsive to an increase in the number of members of the first set of tumor-associated mutations from an earlier time point to a later time point, replacing the tumor-specific neo-antigens corresponding to members of the first set of tumor-associated mutations in the initial immunogenic composition with replacement tumor-specific neo-antigens corresponding to members of the second set of tumor-associated mutations to produce a reformulated immunogenic composition; as well as The reconstituted immunogenic composition is administered to the subject.

9. The method of any of the preceding claims, wherein the subject has melanoma, breast cancer, sarcoma, ovarian cancer, prostate cancer, kidney cancer, stomach cancer, colon cancer, testicular cancer, head and neck cancer, pancreatic cancer, brain cancer, bone cancer, B-cell lymphoma, acute myeloid leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, T-cell lymphocytic leukemia, colon cancer, urothelial carcinoma, or lung cancer.

10. The method of any of the preceding claims, wherein the tumor-associated mutations include at least one mutation that is specific to the subject.

11. The method of any of the preceding claims, wherein the tumor-associated mutation comprises at least one tumor hotspot mutation.

12. The method of claim 11, wherein the tumor is ER+ / HER2- breast cancer and the at least one tumor hotspot mutation is located in a gene selected from the group consisting of: AKT1, APC, ARID1A, ATM, BRAF, BRCA1, BRCA2, CDH1, CDKN2A, ESR1, GATA3, GNAS, HER2, KRAS, NF1, PIK3CA, PTEN, RB1, SMAD4, and TP53.

13. The method of claim 11, wherein the tumor is melanoma.

14. The method of any of the preceding claims, wherein each tumor-specific neo-antigen corresponding to a member of the first set of tumor-associated mutations has a higher immunogenicity score than any tumor-specific neo-antigen corresponding to a member of the second set of tumor-associated mutations.

15. The method of any of the preceding claims, wherein at least one of the plurality of time points is prior to administration of the initial immunogenic composition.

16. The method of any one of claims 8 to 15, wherein the initial immunogenic composition is administered multiple times prior to the step of replacing the tumor-specific neoantigens.

17. The method of any one of claims 8 to 16, wherein the reformulated immunogenic composition is administered multiple times after the step of replacing the tumor-specific neoantigens.

18. The method of any one of claims 8 to 17, wherein the number of members of the second set of tumor-associated mutations corresponding to the replacement tumor-specific neoantigen does not decrease from the earlier time point to the later time point.

19. The method of any one of claims 7 to 18, wherein quantifying the tumor-associated mutation comprises: Sequencing of ctDNA using whole exome sequencing (WES), whole genome sequencing (WGS), targeted sequencing, polymerase chain reaction (PCR), or hybridization methods; Sequencing of cfDNA using quantitative polymerase chain reaction (qPCR) or next-generation sequencing; Determine the methylation or chromatin content of ctDNA, cfDNA, or DNA from CTCs; Mass spectrometry or elution assays of circulating tumor proteins, CTC proteins, or proteins from extracellular vesicles; Fluorescence activated cell sorting (FACS) of CTCs; or Sequence nucleic acids from CTCs or extracellular vesicles using WES, WGS, targeted sequencing, PCR, qPCR, next-generation sequencing, single-cell RNA sequencing, or hybridization methods.

20. The method of any of the preceding claims, wherein one of the multiple time points is at least 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, or about 12 weeks after administration of the initial immunogenic composition or the reformulated immunogenic composition.

21. The method of any one of claims 1 to 20, wherein one of the multiple time points is at least about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, or about 12 months after administration of the initial immunogenic composition or the reformulated immunogenic composition.

22. The method of any one of claims 1 to 20, wherein one of the multiple time points is at least about 1 year, about 2 years, about 3 years, about 4 years, about 5 years, about 6 years, about 7 years, about 8 years, about 9 years, or about 10 years after administration of the initial immunogenic composition or the reformulated immunogenic composition.

23. The method of any one of the preceding claims, wherein the circulating material is isolated from a blood sample, a serum sample, a plasma sample, a urine sample, or a cerebrospinal fluid sample.

24. The method of any of the preceding claims, wherein the circulating material is isolated from at least about 10 ml of whole blood of the subject.

25. The method of claim 24, wherein the circulating material is isolated from at least about 20 ml of whole blood of the subject.

26. The method of any one of the preceding claims, further comprising: detecting the presence of at least one tumor-associated mutation not included in the first set of tumor-associated mutations or the second set of tumor-associated mutations at a first time point; as well as Tumor-associated mutations that occur at a time point after the first time point are added to the second set of tumor-associated mutations.

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