Immunotherapy targeting tumor neoantigen peptides

Abstract

The present disclosure provides: - identifying a fusion transcript sequence from mRNA sequences derived from the cancer cells of the subject, the fusion transcript sequence including a transposable element (TE) sequence and an exon sequence, and including an open reading frame (ORF); - selecting a tumor neoantigenic peptide of at least 8 amino acids encoded by a portion of the ORF of the fusion transcript sequence, the ORF overlaps the junction between the TE and the exon sequence, is a pure TE, and / or is non-canonical; The tumor neoantigen peptide binds to at least one major histocompatibility complex (MHC) molecule of the subject. The present disclosure also relates to tumor neoantigen peptides, vaccines or immunogenic compositions obtained according to the present methods, antibodies and immune cells derived therefrom, and their use in the treatment of cancer.

Description

Technical Field

[0001] The present disclosure provides transposable element (TE) exon fusion transcripts, nucleic acids, vaccines, antibodies, and neoantigen peptides encoded by immune cells that can be used for cancer treatment.

Background Art

[0002] Generating an effective response against tumors by harnessing the immune system is a central goal of cancer immunotherapy. Part of an effective immune response involves T lymphocytes specific for tumor antigens. Activation of T cells requires interaction with antigen-presenting cells (APCs), typically dendritic cells (DCs), which express TCR-cognate peptides presented in the context of major histocompatibility molecules (MHC) and co-stimulatory signals. Subsequently, activated T cells can recognize peptide-MHC complexes presented by all cell types, including malignant cells. Neoplasms often contain infiltrating T lymphocytes that react with tumor cells.

[0003] However, the efficiency of the immune response against tumors is significantly blunted by various immunosuppressive strategies developed by tumors. For example, tumor cells express receptors that provide inhibitory signals to infiltrating T cells or secrete inhibitory cytokines. The development of checkpoint blockade therapy provides a means to bypass some of these mechanisms and results in more efficient killing of cancer cells. The promising results obtained by this approach have opened new avenues for the development of T cell-based immunotherapies. However, checkpoint inhibitors are effective in only a small number of patients and are effective only for limited types of cancer.

[0004] The main goal in immunotherapy is to increase the proportion of responding patients and expand the indications for cancer. Vaccination, administration of anti-tumor antibodies, or administration of immune cells specific to tumor antigens have all been proposed to increase the anti-tumor immune response and can be administered alone, together with other therapies such as chemotherapy or radiation, or as combination therapy with checkpoint blockers. The selection of antigens that can trigger anti-tumor immunity without targeting healthy tissues has been a challenge over the years.

[0005] The search for tumor neoantigens has mainly focused on mutated sequences as found in cancer cells. These antigens are unique to each patient. However, tumor antigens (those preferentially expressed in tumor cells) are self-antigens that represent poor targets for vaccination (presumably due to central tolerance). The identification of shared true neoantigens (not present in normal tissues) is a major challenge for the field.

[0006] Some past reports on transposable elements (TEs) in tumors include the following: (Helman, E. et al. (2014). Genome Res.), (Schiavetti, F. et al. (2002). Cancer Res., Takahashi, Y. et al. (2008). J. Clin. Invest.), (Chiappinelli, K. B. et al. (2015). Cell, Roulois, D. et al. (2015). Cell). However, the relationship between the antigen landscape presented by tumor cells and TEs has not been studied in detail.

[0007] New tumor neoantigens are of interest, especially in the case of vaccination and adoptive cell therapy, as they may improve or reduce the cost of cancer therapy. SUMMARY OF THE INVENTION

[0008] The present disclosure provides a tumor neoantigen peptide comprising at least 8 amino acids, wherein the neoantigen peptide is encoded by a part of an open reading frame (ORF) from a fusion transcript sequence comprising a transposable element (TE) sequence and an exon sequence.

[0009] Typically, the ORF - overlaps with the junction between the TE and the exon sequence, - is a pure TE, and / or - can be non-standard.

[0010] The present disclosure also provides - a step of identifying a fusion transcript sequence comprising a transposable element (TE) sequence and an exon sequence, which contains an open reading frame (ORF), from among the mRNA sequences of the cancer cells of a subject; and - a step of selecting a tumor neoantigen peptide of at least 8 amino acids encoded by a part of the ORF of the fusion transcript sequence, and provides a method for selecting a tumor neoantigen peptide, wherein the ORF overlaps with the junction between the TE and the exon sequence, is a pure TE, and / or is non-standard, and the tumor neoantigen peptide binds to at least one major histocompatibility complex (MHC) molecule of the subject.

[0011] In one embodiment, the tumor neoantigen peptide is 8 or 9 amino acids in length, particularly 8 - 11 amino acids in length, and binds to at least one MHC class I molecule.

[0012] In another embodiment, the tumor neoantigen peptide is 13 - 25 amino acids in length and binds to at least one MHC class II molecule of the subject.

[0013] According to the present disclosure, "neoantigen peptide characteristics" include the following: - The TE sequence may be located at the 5' end of the fusion transcript sequence, the exon sequence may be located at the 3' end of the fusion transcript sequence, and the portion of the ORF of the fusion transcript sequence encoding the neoantigen peptide may overlap with the junction; - The TE sequence may be located at the 5' end of the fusion transcript sequence, the exon sequence may be located at the 3' end of the fusion transcript sequence, and the portion of the ORF encoding the tumor neoantigen peptide may be present downstream of the junction such that the open reading frame is non-standard; - The TE sequence may be located at the 3' end of the fusion transcript sequence, the exon sequence may be located at the 5' end of the fusion transcript sequence, and the portion of the ORF of the fusion transcript sequence encoding the tumor neoantigen peptide may overlap with the junction; or - The TE sequence is located at the 3' end of the fusion transcript sequence, the exon sequence is located at the 5' end of the chimeric transcript sequence, the portion of the ORF encoding the tumor neoantigen peptide is present downstream of the junction between the exon sequence and the TE sequence, and optionally, the peptide sequence encoded by the pure TE sequence is non-standard.

[0014] The TE sequence can be selected from TE class I: endogenous retroviruses (ERVs), long interspersed nuclear elements (LINEs), and short interspersed nuclear elements (SINEs), and MaLR sequences, or class II DNA transposons.

[0015] The present disclosure also encompasses peptides obtainable by the methods disclosed herein.

[0016] The present disclosure also provides a neoantigen peptide comprising at least 8 amino acids and encoded by any one of the open reading frames (ORFs) of any one of the fusion transcripts of SEQ ID NOs: 118 - 17492, preferably a peptide having one or more of the neoantigen peptide characteristics described above. More specifically, neoantigen peptides comprising at least 8 amino acids of any one of SEQ ID NOs: 1 - 117 are provided herein.

[0017] The neoantigen peptide is typically expressed at a higher level or frequency in tumor samples compared to normal, and optionally, the neoantigen peptide is not expressed or not detectably expressed in normal tissue samples (i.e., normal and healthy cells).

[0018] In some embodiments, the neoantigen peptide is expressed in at least 1%, 5%, 10%, 15%, 20%, 25%, or at least 30% of a population of subjects suffering from cancer, particularly lung cancer, more specifically non-small cell lung cancer (NSCLC), and even more specifically lung adenocarcinoma (LUAD).

[0019] Typically, the neoantigen peptide binds to MHC class I or class II with a binding affinity Kd of less than about 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 , or 10 -9 M (where lower numbers indicate higher binding affinity).

[0020] Typically, the neoantigen peptide binds to MHC class I with a binding affinity at a percentile rank score of less than 2% predicted by NetMHCpan 4.0.

[0021] Typically, the neoantigen peptide binds to MHC class II with a binding affinity at a percentile rank score of less than 10% predicted by NetMHCpanII 3.2.

[0022] The present disclosure also includes: - A population of autologous dendritic cells or antigen-presenting cells pulsed with one or more peptides as defined herein or transfected with a polynucleotide encoding one or more peptides described herein, - A vaccine or immunogenic composition, particularly a sterilized vaccine or immunogenic composition, comprising: a. One or more neoantigen peptides as defined herein; b. One or more polynucleotides encoding a neoantigen peptide as defined herein, optionally wherein one or more polynucleotides are linked to a heterologous regulatory nucleotide sequence; or c. A specific T cell response comprising a population of autologous dendritic cells or antigen-presenting cells (particularly artificial APCs) pulsed or loaded with one or more peptides as defined herein, Optionally, a vaccine or immunogenic composition that can be enhanced in combination with a physiologically or pharmacologically acceptable buffer, carrier, excipient, immunostimulant, and / or adjuvant; - An antibody selected for its binding affinity to a neoantigen peptide as defined herein, or an antigen-binding fragment thereof, a T cell receptor (TCR), or a chimeric antigen receptor (CAR), or a composition comprising such an antibody, antigen-binding fragment, TCR, or CAR; - A polynucleotide encoding a neoantigen peptide, antibody, CAR, or TCR as defined herein, typically operably linked to a heterologous regulatory nucleotide sequence, and a vector encoding such a polynucleotide, or a vaccine or immunogenic composition comprising such a polynucleotide or vector; - An immune cell or population of immune cells targeting one or more neoantigen peptides as defined herein, wherein the population of immune cells preferably targets a plurality of different tumor neoantigen peptides disclosed herein, or optionally a composition comprising such immune cells or population of immune cells in combination with a physiologically or pharmacologically acceptable buffer, carrier, excipient, immunostimulant, and / or adjuvant.

[0023] Typically, an antibody or an antigen-binding fragment thereof, a TCR, or a CAR binds to a neoantigen peptide with a Kd affinity of about 10 -6 M or less, optionally in association with an MHC molecule.

[0024] In some embodiments, a T cell receptor can be made soluble and fused to an antibody fragment directed against a T cell antigen, and optionally, the target antigen is CD3 or CD16.

[0025] In some embodiments, the antibody may be a multispecific antibody that further targets at least an immune cell antigen, and optionally, the immune cell is a T cell, an NK cell, or a dendritic cell, and optionally, the target antigen is CD3, CD16, CD30, or a TCR. In any of the embodiments regarding the antibody, the antibody may be chimeric, humanized, or human, and may be IgG, for example, IgG1, IgG2, IgG3, IgG4.

[0026] The immune cells can typically be T cells or NK cells, CD4+ and / or CD8+ cells, TIL / tumor-derived CD8 T cells, central memory CD8+ T cells, Tregs, MAIT, or γδ T cells. The cells can also be autologous or allogeneic.

[0027] The T cells can comprise a recombinant antigen receptor selected from a T cell receptor and a chimeric antigen receptor as defined herein, and the antigen is a tumor neoantigen receptor as disclosed herein.

[0028] The present disclosure also produces an antibody, TCR, or CAR that specifically binds to a neoantigen peptide as defined herein, optionally in association with an MHC or HLA molecule, and optionally, about 10 -6A method is included that comprises the step of selecting an antibody, TCR, or CAR that binds to a tumor neoantigen peptide of the present disclosure with a Kd binding affinity of less than M. The antibodies, TCRs, and CARs selected by the method are also part of the present application, and thus any reference herein to an antibody, TCR, or CAR also means an antibody, TCR, or CAR selected by the method.

[0029] Optionally, a polynucleotide encoding a neoantigen peptide as defined herein, or a polynucleotide encoding an antibody, CAR, or TCR as defined herein, linked to a heterologous regulatory control sequence, is also part of the present application.

[0030] According to the present disclosure, a neoantigen peptide, dendritic cell population, vaccine or immunogenic composition, polynucleotide or vector encoding a peptide can be used in a cancer vaccination therapy of a subject or to treat cancer in a subject suffering from or at risk of cancer, or to inhibit the growth of cancer cells. Typically, the peptide binds to at least one MHC molecule of the subject.

[0031] According to the present disclosure, an antibody as defined herein or an antigen-binding fragment thereof, a bispecific antibody, a TCR, a CAR, a polynucleotide or vector encoding such an antibody, TCR or CAR can be used to treat cancer in a subject in need thereof, a subject suffering from or at risk of cancer, or to inhibit the growth of cancer cells. Further according to the present disclosure, an immune cell population as defined herein can be used in cell therapy of a subject suffering from or at risk of cancer, or to inhibit the growth of cancer cells.

[0032] In particular, neoantigen peptides, dendritic cell populations, vaccines or immunogenic compositions, polynucleotides or vectors encoding peptides, antibodies or antigen-binding fragments thereof, bispecific antibodies, TCRs, CARs, polynucleotides or vectors encoding such antibodies, TCRs or CARs, or immune cell populations (collectively referred to herein as "cancer therapeutics") are used for the treatment of subjects suffering from NSCLC or at risk of developing NSCLC and / or for the treatment of NSCLC.

[0033] Pharmaceutical compositions comprising any of the foregoing and optionally comprising a sterile pharmaceutically acceptable excipient, carrier, and / or buffer are also contemplated, as are methods of using them.

[0034] In any of the embodiments described herein, the cancer therapeutics as defined above can be administered in combination with at least one additional therapeutic agent. Such additional therapeutic agents can typically be chemotherapeutic agents or immunotherapeutic agents.

[0035] For example, according to the present disclosure, any of the cancer therapeutics can be administered in combination with an anti-immunosuppressive / immunostimulatory agent. For example, the subject is typically further administered one or more checkpoint inhibitors selected from a PD-1 inhibitor, a PD-L1 inhibitor, a Lag-3 inhibitor, a Tim-3 inhibitor, a TIGIT inhibitor, a BTLA inhibitor, an inhibitor of the V domain Ig suppressor of T cell activation (VISTA), and a CTLA-4 inhibitor, or an IDO inhibitor.

[0036] Various embodiments of the methods, neoantigen peptides, and cancer therapeutics are detailed below. Combinations of such embodiments are included in this application, except where specific alternatives are explicitly mentioned.

DETAILED DESCRIPTION OF THE INVENTION

[0037] In normal tissues, the expression of transposable elements (TEs) is silenced by DNA methylation established in the early stage of embryonic development. Histone modifications provide an additional inhibitory layer. TEs can be reactivated within tumor cells.

[0038] The inventors have developed a method for selecting tumor neoantigen peptides encoded by fusion transcript sequences that include a portion of a TE sequence and a portion of an exon sequence.

[0039] Neoantigen tumor-specific peptides identified by the methods according to the present disclosure are highly immunogenic. Indeed, since they are derived from fusion transcripts (consisting of transposable elements, TEs, and exon sequences) that do not exist in normal cells, the peptides of the present disclosure are expected to exhibit very low immune tolerance.

[0040] The present disclosure also enables the selection of peptides having shared tumor neoepitopes among patient populations. Such shared tumor peptides are of high therapeutic interest as they can be used for immunotherapy of a large number of patients.

[0041] Definitions According to the present disclosure, the term "disease" refers to any pathological condition, including cancerous diseases, particularly the forms of cancerous diseases described herein.

[0042] The term "normal" refers to a healthy state or a state in a healthy subject or tissue, i.e., a non-pathological state, where "healthy" preferably means non-cancerous.

[0043] Cancer (medical term: malignant neoplasm) is a type of disease in which a group of cells shows uncontrolled growth (division beyond the normal range), invasion (penetration and destruction of adjacent tissues), and sometimes metastasis (spread to other parts of the body via lymph or blood). These three malignant characteristics of cancer distinguish it from benign tumors, which are self-limiting and do not invade or metastasize. Most cancers form tumors, although some cancers, such as leukemia, do not.

[0044] A malignant tumor is essentially synonymous with cancer. Malignancy, malignant neoplasm, and malignant tumor are essentially synonymous with cancer.

[0045] As used herein, the terms "tumor" or "tumor disease" refer to the abnormal growth of cells (referred to as neoplastic cells, carcinogenic cells, or tumor cells), preferably forming a swelling or lesion. "Tumor cells" mean abnormal cells that grow by rapid and uncontrollable cell proliferation and continue to grow even after the stimulus that initiated the new growth has ceased. A tumor shows a partial or complete lack of structural constitution and functional coordination with normal tissue and usually forms a distinct mass of tissue that can be either benign, pre-malignant, or malignant.

[0046] A benign tumor is a tumor that lacks all three of the malignant characteristics of cancer. Thus, by definition, a benign tumor does not grow invasively without limit, does not infiltrate surrounding tissues, and does not spread to non-adjacent tissues (metastasize).

[0047] A neoplasm is a mass of abnormal tissue as a result of neoplasia. Neoplasia (from the Greek for new growth) is abnormal cell proliferation. The cell proliferation is excessive and not coordinated with the growth of the normal tissue around it. The excessive proliferation persists in the same way even after the stimulus has ceased. It usually causes a lump or tumor. A neoplasm can be benign, pre-malignant, or malignant.

[0048] "Tumor growth" or "tumor growth" according to the present disclosure relates to the tendency of a tumor to increase in size and / or the tendency of tumor cells to proliferate.

[0049] For the purposes of the present disclosure, the terms "cancer" and "cancer disease" are used interchangeably with the terms "tumor" and "tumor disease".

[0050] Cancer is classified by the type of cells that resemble tumors and thus the tissue from which the tumor is presumed to originate. These are the histotype and site, respectively.

[0051] According to the present application, cancer can affect any one of the following tissues or organs: breast; liver; kidney; heart, mediastinum, pleura; floor of the mouth; lips; salivary gland; tongue; gingiva; oral cavity; palate; tonsil; larynx; trachea; bronchi, lungs; pharynx, hypopharynx, mid-pharynx, nasopharynx; esophagus; digestive organs such as stomach, intrahepatic bile duct, biliary tract, pancreas, small intestine, colon, etc.; rectum; urinary organs such as bladder, gallbladder, ureter, etc.; sigmoid colon-rectum junction; anus, anal canal; skin; bone; joints, articular cartilage of hands and feet; eyes and appendages; brain; peripheral nerves, autonomic nervous system; spinal cord, cranial nerves, meninges; and various parts of the central nervous system; connective tissue, subcutaneous tissue and other soft tissues; retroperitoneum, peritoneum; adrenal gland; thyroid gland; endocrine glands and related structures; female genital organs such as ovaries, uterus, cervix, etc.; corpus uteri, vagina, vulva; male genital organs such as penis, testis and prostate; hematopoietic system and reticuloendothelial system; blood; lymph nodes; thymus.

[0052] Thus, the term "cancer" according to the present disclosure includes leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, renal cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, gastric cancer, bowel cancer, head and neck cancer, digestive tract cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer and lung cancer, as well as metastases thereof. Examples thereof are lung cancer, breast cancer, prostate cancer, colon cancer, renal cell cancer, cervical cancer, or metastases of the above-mentioned cancer types or tumors. The term cancer according to the present disclosure also includes cancer metastasis and cancer recurrence.

[0053] The main types of lung cancer are small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). Non-small cell lung cancer has three main subtypes: squamous cell lung cancer, lung adenocarcinoma (LUAD), and large cell lung cancer. Adenocarcinoma accounts for about 10% of lung cancer. This cancer is usually found in the periphery of the lung, in contrast to the tendency of both small cell lung cancer and squamous cell lung cancer to be centrally located.

[0054] "Metastasis" means the spread of cancer cells from the original site in the body to another site. The formation of metastasis is a very complex process, which depends on the detachment of malignant cells from the primary tumor, the invasion of the extracellular matrix, the penetration of the endothelial basement membrane into the body cavity and blood vessels, then the transportation by the blood, and finally the invasion of the target organ. Finally, the growth of a new tumor at the target site, i.e., the secondary tumor or metastatic tumor, depends on angiogenesis. Tumor metastasis often occurs because tumor cells or components may remain and develop the ability to metastasize even after the removal of the primary tumor. In one embodiment, the term "metastasis" according to the present disclosure relates to "distant metastasis" regarding metastasis away from the primary tumor and the associated lymph node system.

[0055] The cells of a secondary or metastatic tumor resemble the cells of the original tumor. This means that, for example, when ovarian cancer metastasizes to the liver, the secondary tumor is composed of abnormal ovarian cells rather than abnormal liver cells. Then, the tumor in the liver is called metastatic ovarian cancer rather than liver cancer.

[0056] Recurrence or relapse occurs when a patient develops the condition they had in the past again. For example, if a patient has a tumor disease, successfully treats the disease, and then develops the disease again, the newly developed disease may be regarded as recurrence or relapse. However, according to the present disclosure, recurrence or relapse of a tumor disease may occur at the site of the original tumor disease, but not necessarily. Therefore, for example, if a patient has an ovarian tumor and the treatment is successful, recurrence or relapse may be the occurrence of an ovarian tumor or the occurrence of a tumor at a site different from the ovary. Recurrence or relapse of a tumor also includes the situation where the tumor occurs not only at the site of the original tumor but also at a site different from the site of the original tumor. Preferably, the original tumor from which the patient is receiving treatment is the primary tumor, and the tumor at a site different from the site of the original tumor is a secondary or metastatic tumor.

[0057] "Treatment" means administering to a subject a compound or composition described herein for preventing or removing a disease, including reducing the size or number of tumors in the subject; arresting or delaying the disease in the subject; inhibiting or delaying the onset of a new disease in the subject; reducing the frequency or severity of symptoms and / or relapses in a subject having or having had the disease; and / or prolonging, i.e., extending the lifespan of the subject. In particular, the term "treatment of a disease" includes cure of the disease or its symptoms, shortening of the duration, improvement, prevention, delay or inhibition of progression or worsening, or prevention or delay of onset.

[0058] "At risk" means a subject, i.e., a patient, who is identified as having a higher than normal likelihood of developing a disease, particularly cancer, compared to the general population. Further, a subject who has had or currently has a disease, particularly cancer, is a subject at high risk of developing the disease and such a subject may continue to develop the disease. A subject having or having had cancer is at high risk of cancer metastasis.

[0059] The therapeutic agents, vaccines, and compositions described herein can be administered via any conventional route including injection or infusion.

[0060] The agents described herein are administered in an effective amount. "Effective amount" refers to the amount that achieves the desired response or desired effect, alone, or in combination with additional dosages, or in combination with additional therapeutic agents. For the treatment of a particular disease or particular condition, the desired response preferably relates to inhibition of the course of the disease. This includes delaying the progression of the disease, particularly interrupting or reversing the progression of the disease. The desired response in the treatment of a disease or condition may also be delay of onset or prevention of onset of the disease or condition.

[0061] The effective amount of the agent described herein depends on the individual parameters of the patient, including the condition being treated, the severity of the disease, age, physiological state, height and weight, treatment duration, the type of concomitant therapy (if any), the particular route of administration, and similar factors. Thus, the dosage of the agent described herein may depend on such various parameters. If the patient's response is inadequate at the initial dosage, higher dosages (or substantially higher dosages achieved by different, more local routes of administration) may be used.

[0062] The pharmaceutical compositions described herein are preferably sterilized and contain an effective amount of a therapeutic active substance to produce a desired response or desired effect.

[0063] The pharmaceutical compositions described herein are generally administered in a pharmaceutically compatible amount and in a pharmaceutically compatible preparation. The term "pharmaceutically compatible" refers to non-toxic substances that do not interact with the action of the active ingredient of the pharmaceutical composition. This type of preparation usually supplements salts, buffering substances, preservatives, carriers, immunostimulatory substances such as adjuvants, e.g., CpG oligonucleotides, cytokines, chemokines, saponins, GM-CSF and / or RNA, and, where appropriate, other therapeutically active compounds. When used in medicine, the salts should be pharmaceutically compatible.

[0064] In this application, the terms "fusion transcript" or "chimeric transcript" are used interchangeably as synonyms. A "fusion or chimeric" "transcript or sequence" according to the present disclosure aligns partially with an exon sequence and partially with a transposable element (TE) sequence and is defined as a transcript with a normalized read count greater than 2.10 -6 The normalized read count is defined as the number of reads covering the fusion divided by the library size of the sample.

[0065] As used herein, unless specifically recited or clear from the context, the term "about" should be understood to be within the normal tolerance range in the art, e.g., within two standard deviations of the average. About can be understood to be within 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the recited value. Unless clear from the context, all numerical values provided herein are modified by the term about.

[0066] "Transposable elements" should be understood to mean that both Class I (retrotransposons including those containing LTRs, LINEs, and SINEs) and Class II (DNA transposons) are inherently part of the genome (i.e., not due to infection). This includes both autonomous and non-autonomous elements of both classes. According to the present disclosure, TE sequences can be selected from, for example, Class I TEs such as endogenous retroviruses (ERVs), long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs), and retrotransposons including mammalian-wide interspersed repeats (MaLRs), and Class II TEs such as DNA transposons that are inherently part of the genome.

[0067] A reading frame is a way of dividing the sequence of nucleotides in a nucleic acid (DNA or RNA) molecule into successive, non-overlapping triplets.

[0068] An open reading frame (ORF) is a part of a reading frame that has the ability to be translated into a peptide. An ORF is a continuous sequence of codons that contains a start codon (e.g., AUG) at the transcription start site (TSS) and a stop codon (e.g., UAA, UAG, or UGA). The ATG codon within the ORF (not necessarily the first one) can indicate where translation begins. The transcription termination site is located after the ORF, beyond the translation stop codon. In eukaryotic genes with multiple exons, the ORF spans the intron / exon regions and can be spliced together after transcription to produce the final mRNA for protein translation.

[0069] As intended herein, a "standard ORF" is a protein-coding sequence having a specific reading frame within an mRNA sequence that is described or annotated in a database such as, for example, the Ensembl genome / transcriptome / proteome database collection (typically HG19). Typically, a standard ORF is the same as one of the exons in a normal and healthy cell.

[0070] As intended herein, a "non-standard ORF" is a protein-coding sequence having a specific reading frame within an mRNA sequence that is not described (i.e., not annotated) in a genomic database such as, for example, the Ensembl genomic database. Typically, a non-standard ORF thus means that the reading frame is shifted compared to the normal reading frame of the exons in a normal and healthy cell. However, in some embodiments, a non-standard can be described in a genomic database (e.g., the Ensembl database), but the mRNA sequence represents a minor species in normal cells. Minor species typically intend species that are less than 5%, particularly less than 2%, or preferably less than 1% in normal cells.

[0071] An exon is any part of a gene that codes for a portion of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term exon refers to both the DNA sequence within a gene and the corresponding sequence of the RNA transcript. In RNA splicing, introns are removed and exons are covalently joined to each other as part of generating mature messenger RNA. The exon sequences according to the present applicant include at least a part of one or more exons. Typically, an exon sequence includes at least a part of one or two exons.

[0072] Thus, the untranslated sequences of the 3’ and 5’ ends (3’UTR and 5’UTR) present in the mature RNA after splicing are exon sequences, but these sequences are non-coding sequences because they are located upstream of the start codon for translation (5’UTR) or downstream of the stop codon that ends translation (3’UTR).

[0073] The term “peptide or polypeptide” is used interchangeably herein with “neoantigen peptide or polypeptide” and typically refers to a series of residues, typically L-amino acids, linked to each other by peptide bonds between the α-amino and carboxyl groups of adjacent amino acids. A polypeptide or peptide may be in its neutral (uncharged) form or in the form of a salt, and may or may not contain modifications such as glycosylation, side-chain oxidation, or phosphorylation, and may be of various lengths, provided that the modifications do not destroy the biological activity of the polypeptides described herein.

[0074] A “reference genome,” or “representative genome,” is a digital nucleic acid sequence database assembled by scientists as a representative example of a set of genes of a species. Since a reference genome is often assembled from the sequencing of DNA from multiple donors, it does not precisely represent the set of genes of any single individual (animal or human). Instead, the reference provides a haploid mosaic of different DNA sequences from each donor.

[0075] Method for selecting tumor neoantigen peptides The method for selecting tumor neoantigen peptides according to the present disclosure is as follows - identifying a fusion transcript sequence that includes a translocation element (TE) sequence and an exon sequence, and includes an open reading frame (ORF), from among mRNA sequences derived from a cancer cell sample of a subject - selecting a tumor neoantigen peptide of at least 8 amino acids encoded by a part of the ORF of the fusion transcript sequence wherein the ORF overlaps with the junction between the TE and the exon sequence, is a pure TE, and / or is non-standard wherein the tumor neoantigen peptide binds to at least one major histocompatibility complex (MHC) molecule of the subject

[0076] Typically, a peptide translated from a part of a non-standard ORF of an exon sequence is recognized as non-self by the immune system

[0077] In some embodiments, the exon sequences are from oncogenes and / or tumor suppressor genes, and their mutant variants

[0078] Conceptually, cancer is the result of the accumulation of successive somatic mutations. Many studies have shown that both gain-of-function in oncogenes and loss-of-function in tumor suppressor genes are required for the development of cancer from normal cells. In the case of diploid organisms, gain-of-function mutations are often dominant or semi-dominant, whereas loss-of-function mutations are usually recessive. The two-hit hypothesis of carcinogenesis suggests that the development of cancer is initiated by the loss of both alleles of a tumor suppressor gene

[0079] An oncogene (also called a cancer gene) is a gene whose action actively promotes cell proliferation or growth. The normal non-mutated form is known as a proto-oncogene. The mutated form is over-activated or inappropriately activated, leading to tumor growth. Oncogenes can be identified in the Cancer Gene Marker Database (CGMD) (Pradeepkiran, J., Sainath, S., Kramthi Kumar, K. et al. CGMD:. Sci Rep 5, 12035 (2015) “An integrated database of cancer genes and marker”). Oncogenes (ONC) can also be downloaded from the Network of Cancer Genes Database (NCG 5.0) (An O, Dall’Olio GM, Mourikis TP, Ciccarelli FD, Nucleic Acids Res. 2016 Jan 4; 44(D1):D992-9; “NCG 5.0: updates of a manually curated repository of cancer genes and associated properties from cancer mutationalscreenings ”). Non-limiting examples of oncogenes include the following: L-MYC, LYL-1, LYT-10, LYT-10 / Cα1, MAS, MDM-2, MLL, MOS, MTG8 / AML1, MYB, MYH11 / CBFB, NEU, N-MYC, OST, PAX-5, PBX1 / E2A, PIM-1, PRAD-1, RAF, RAR / PML, RAS-H, RAS-K, RAS-N, REL / NRG, RET, RHOM1, RHOM2, ROS, SKI, SIS, SET / CAN, SRC, TAL1, TAL2, TAN-1, TIAM1, TSC2, and TRK.

[0080] Tumor suppressor genes (also called anti-oncogenes) represent the opposite of cell growth control and usually act to inhibit cell growth and tumorigenesis. Thus, tumor suppressor genes are generally genes that suppress cell division or growth. Loss of TSG function promotes uncontrolled cell division and tumor growth. Rb is a tumor suppressor gene identified by genetic analysis of retinoblastoma, which encodes a transcriptional regulatory protein, and has served as a prototype for the identification of additional tumor suppressor genes that contribute to the development of many different human cancers. Tumor suppressor genes are described in particular in "Cooper GM. The Cell: A Molecular Approach. 2nd edition. Sunderland (MA): Sinauer Associates; 2000. Tumor Suppressor Genes". Tumor suppressor genes (TSGs) can also be downloaded from the Tumor Suppressor Gene Database (TSGene 2.0) (see Zhao M, Kim P, Mitra R, Zhao J, Zhao Z; Nucleic Acids Res. 2016 Jan 4; 44(D1):D1023-31; "TSGene 2.0: an updated pature-based knowledgebase for tumor suppressor genes"). Non-limiting examples of tumor suppressor genes in this context include: APC, BRCA1, BRCA2, DPC4, INK4, MADR2, NF1, NF2, p53, PTC, PTEN, Rb, RB1, VHL, WT1, BUB1, BUBR1, TGF-βRII, Axin, DPC4, p300, PPARγ, p16, DPC4, PTEN, and hSNF5.

[0081] Oncogenes, tumor suppressor genes, or "dual-spy" genes (having both oncogenic and tumor suppressor functions) can be systematically identified via database searches and text mining. In fact, information on oncogenes or tumor suppressor genes can typically be found in the Ensembl database (but also see Shen L, Shi Q, Wang W. Double agents: genes with both oncogenic and tumor-suppressor functions. Oncogenesis. 2018;7(3):25. Published March 13, 2018). Dual-spy genes can be identified as overlapping genes between the two databases described above (see Shen et al., Oncogenesis 2018).

[0082] Without being bound by any theory, the inventors believe that the selection of fusions whose exon sequences are derived from oncogenes and / or tumor suppressor genes is highly relevant for the following reasons.

[0083] TE insertions into oncogenes can change their oncogenic activity. Thus, insertion of a TE sequence into the active domain of an oncogene can result in constitutive activation of the oncogene, similar to a driver mutation. Thus, these fusions that give rise to chimeric oncogenes can represent a new family of oncogenic proteins. In this case, targeting the activity of these new "fusion oncogenes" with small molecule antagonists can represent a potential therapeutic approach to cancers in which these chimeric oncogenes are expressed.

[0084] TE insertions in tumor suppressors inactivate their suppressor function and typically result in loss of function (e.g., through introduction of a stop codon, change in ORF, or disruptive amino acid elongation), thereby potentially contributing to the oncogenic process.

[0085] Fusions involving cancer driver genes would be excellent targets for adoptive cell therapy, antibodies, ADCs, T cell engagers, etc. When they are involved in carcinogenesis, the fusion oncogenes are more specific to cancer cells and are thus expected to reduce the occurrence of resistance (due to the oncogenic activity of the target).

[0086] In one embodiment, with respect to the junction, the TE sequence is located at the 5' end of the fusion transcript sequence (the TE sequence is also said to be the donor sequence), and the exon sequence is located at the 3' end of the fusion transcript sequence (the exon sequence is thus called the acceptor sequence). The expression "located at the 5' end of the fusion transcript sequence" means that the element is located upstream of the junction of the fusion transcript sequence. The expression "located at the 3' end of the fusion transcript sequence" means that the element is located downstream of the junction of the fusion transcript sequence.

[0087] In certain embodiments, the TE sequence is located at the 5' end of the fusion transcript sequence, the exon sequence is located at the 3' end of the fusion transcript sequence, and the portion of the ORF of the fusion transcript sequence encoding the neoantigen peptide overlaps with the junction. In this case, the ORF can be standard or non-standard. It is understood that the ORF may include the junction, but the neoantigen peptide does not have to include the junction. In some embodiments, when the neoantigen peptide includes the junction, the resulting peptide is encoded by both the TE sequence and the exon sequence.

[0088] The expression "the portion of the ORF overlaps or overlaps with the junction between the TE sequence and the exon sequence" means that the junction is contained in the portion of the ORF of the fusion transcript sequence encoding the neoantigen peptide.

[0089] (i) In an embodiment where the portion of the ORF encoding the neoantigen peptide overlaps with the junction between the TE sequence and the exon sequence, and (ii) the TE sequence and the exon sequence are located at the 5'-end and 3'-end of the fusion transcript sequence respectively, the portion of the ORF typically encodes a neoantigen peptide of at least 8 amino acids, including at least 1-6 amino acids, particularly 2-6 amino acids, from the TE sequence and at least 1-6 amino acids, particularly 2-6 amino acids, from the exon sequence.

[0090] In another embodiment where the TE sequence is located at the 5'-end of the fusion transcript sequence and the exon sequence is located at the 3'-end of the fusion transcript sequence, the portion of the ORF encoding the neoantigen peptide is downstream of the junction, and the ORF is thus non-standard.

[0091] The expression "the portion of the ORF is downstream of the junction" means that the portion of the ORF encoding the neoantigen peptide does not overlap with the junction, but it is contained in the 3'-end portion of the fusion transcript sequence with respect to the junction. In this embodiment, since the 3'-end portion with respect to the junction is the exon sequence, the portion of the ORF encoding the neoantigen peptide is thus contained in the exon sequence. Therefore, since the portion of the ORF is located only in the exon sequence, the resulting peptide is encoded by the exon sequence of the non-standard ORF. Thus, in a specific embodiment where the exon sequence is located at the 3'-end of the fusion transcript sequence with respect to the junction and the portion of the ORF encoding the neoantigen peptide is downstream of the junction with a non-standard reading frame, the portion of the ORF of the fusion transcript sequence encodes a neoantigen peptide containing 0 amino acids from the TE sequence and at least 8 amino acids from the exon sequence.

[0092] In another embodiment, with respect to the junction, the TE sequence is located at the 3'-end of the fusion transcript sequence and the exon sequence is located at the 5'-end of the fusion transcript sequence.

[0093] In some embodiments, the TE sequence is located at the 3' end of the fusion transcript sequence, the exon sequence is located at the 5' end of the fusion transcript sequence, and the portion of the ORF of the fusion transcript sequence encoding the neoantigen peptide overlaps with the junction between the TE sequence and the exon sequence. In this case, the ORF may also be standard or non-standard. The resulting peptide is encoded by both the TE sequence and the exon sequence.

[0094] In certain embodiments where the portion of the ORF encoding the neoantigen peptide overlaps with the junction between the exon sequence and the TE sequence, and the exon sequence and the TE sequence are at the 5' end and 3' end of the fusion transcript sequence respectively, the portion of the ORF encodes a neoantigen peptide of at least 8 amino acids, including at least 1-6 amino acids, particularly 2-6 amino acids, from the TE sequence and at least 1-6 amino acids, particularly 2-6 amino acids, from the exon sequence.

[0095] In yet another embodiment, the TE sequence is located at the 3' end of the fusion transcript sequence, the exon sequence is located at the 5' end of the fusion transcript sequence, and the portion of the ORF encoding the neoantigen peptide is downstream of the junction between the exon sequence and the TE sequence. Optionally, the peptide sequence thus encoded by the pure TE sequence is non-standard.

[0096] In this embodiment, since the 3' end portion relative to the junction is the TE sequence, the portion of the ORF encoding the neoantigen peptide is thus encoded by the TE sequence. Therefore, the portion of the ORF encodes a neoantigen peptide that does not contain amino acids from the exon sequence and contains at least 8 amino acids from the TE sequence. In certain embodiments where the TE sequence is located at the 3' end of the fusion transcript sequence relative to the junction and the portion of the ORF encoding the neoantigen peptide is downstream of the junction, the portion of the ORF of the fusion transcript sequence encodes a neoantigen peptide that contains 0 amino acids from the exon sequence and at least 8 amino acids from the TE sequence.

[0097] Tumor neoantigen peptides are peptides that result from somatic changes (classically DNA sequence mutations), are recognized as foreign to self, and are presented by antigen-presenting cells (APCs) such as dendritic cells (DCs) and the tumor cells themselves. APCs play an important role in cross-presentation because they can move exogenous antigens from the phagosome to the cytosol and perform proteolytic cleavage of the proteins into major histocompatibility complex I (MHC I) epitopes by the proteasome.

[0098] In the present disclosure, the changes correspond to the transcription of fusion mRNA sequences that include transposable element (TE) sequences and exon sequences. This can result from somatic (i.e., specifically within tumor clones) translocations. It can also result from tumor-specific transcriptional derepression such that the TE and nearby genes are co-transcribed, rather than from de novo translocations.

[0099] Neoantigen peptides according to the present disclosure are not present at all in normal healthy samples (i.e., not expressed in normal healthy samples) and can thus be specific to tumor samples. Alternatively, they may be expressed at low levels in normal cells and / or may be preferentially expressed on tumor samples as compared to normal (healthy) samples.

[0100] They may also be selectively expressed by the cell lineage in which cancer has occurred.

[0101] Cancer or tumor samples according to the present disclosure can be isolated from any solid or non-solid tumor of any previously defined tissue or organ, such as breast cancer, lung cancer, and / or melanoma. In some embodiments, the cancer sample is from acute myeloid leukemia, adrenocortical carcinoma, bladder urothelial carcinoma, ductal carcinoma of the breast, lobular carcinoma of the breast, cervical cancer, cholangiocarcinoma, colorectal adenocarcinoma, esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, hepatocellular carcinoma, renal chromophobe carcinoma, renal clear cell carcinoma, renal papillary cell carcinoma, low-grade glioma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglioma and pheochromocytoma, prostate adenocarcinoma, sarcoma, cutaneous melanoma, testicular germ cell carcinoma, thymoma, papillary thyroid carcinoma, uterine carcinosarcoma, endometrial carcinoma of the uterine body or uveal melanoma sample. In certain embodiments, the cancer sample is from a lung cancer sample, particularly a LUAD sample.

[0102] Typically, according to the present disclosure, the step of identifying the fusion transcript sequence is performed by mapping the mRNA sequence from the cancer sample to the reference genome and then distinguishing normal junctions from abnormal junctions.

[0103] According to the present disclosure, a normal junction corresponds to junction donors and acceptors that are on the same strand and not too far apart (e.g., not on different chromosomes).

[0104] According to the present disclosure, an abnormal junction corresponds to a junction between a donor sequence and an acceptor sequence that are on different chromosomes or, in cis, on different strands (regardless of order and 5'-3' sense).

[0105] In one embodiment, the mRNA sequence can be mapped to the corresponding reference genome with adapted software such as, for example, Spliced Transcripts Alignment to a Reference (i.e., STAR - see Dobin, Alexander et al. “STAR: ultrafast universal RNA-seq aligner.” Bioinformatics (Oxford, England) vol. 29, 1 (2013): 15-21), TopHat2 (Kim, Daehwan et al. “TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions.” Genome biology vol. 14, 4 R36.25 Apr. 2013, doi:10.1186 / gb-2013-14-4-r36) or HISAT (Kim, Daehwan et al. “HISAT: a fast spliced aligner with low memory requirements.” Nature methods vol. 12, 4 (2015): 357-60. doi:10.1038 / nmeth.3317). STAR is a stand-alone software that aligns RNA-seq reads using sequential maximal mappable seed search, followed by seed clustering and stitching. It can detect standard junctions, non-standard splices, and fusion / chimeric transcripts.

[0106] In certain embodiments, normal and abnormal junctions are determined in silico using dedicated databases such as, for example, the Ensembl and Repeatmasker databases, and fusion transcripts having a junction between a TE and an exon sequence are extracted in silico.

[0107] According to the present disclosure, the mRNA sequences can be derived from all types of cancer cells or tumor cell samples. The tumor may be a solid tumor or a non-solid tumor. In particular, the mRNA sequences are derived from any tissue or organ affected by a previously defined cancer or tumor, such as breast cancer, lung cancer, and / or melanoma. In certain embodiments, the mRNA sequences are derived from LUAD samples.

[0108] Typically, according to the present disclosure, the fusion transcript sequences are shared in more than 1%, particularly more than 5%, more than 10%, more than 15%, more than 20%, or even more than 25% of cancer samples. In other words, the fusion transcript sequences according to the present disclosure are shared in more than 1%, particularly more than 5%, more than 10%, more than 15%, more than 20%, or even more than 25% of cancer samples from subjects suffering from cancer. Thus, the fusion transcript sequences can be cancer-type specific and shared among several cancers.

[0109] According to the present disclosure, the fusion transcript sequences are expressed at high levels in tumor cells as compared to normal and healthy cells. In some embodiments, the fusion transcript sequences are expressed in cancer cells and not in healthy cells, particularly not in healthy cells of the thymus. Such fusion transcripts can be referred to as tumor-specific fusions according to the present disclosure. Fusion transcripts that are expressed at higher levels in tumor cells as compared to normal cells, typically those that are disproportionately expressed in cancer cells as compared to normal cells as defined above, can be referred to as tumor-associated fusion transcripts (TAFs) according to the present disclosure. Tumor-associated fusion transcripts can be selected according to the present application if they are present in more than 10% of tumor samples and less than 20% of normal samples.

[0110] In some embodiments, the method further comprises determining, optionally in silico or using in vitro techniques (particularly referring to the exemplary embodiments), the binding affinity of at least one MHC molecule of the subject suffering from cancer to a tumor neoantigen peptide.

[0111] MHC class I proteins form functional receptors on most nucleated cells of the body. There are three major MHC class I genes in HLA: HLA-A, HLA-B, HLA-C, and three minor genes HLA-E, HLA-F, and HLA-G. β2-microglobulin binds to both major and minor gene subunits to produce a heterodimer. Class I MHC molecules consist of a heavy chain and a light chain and can bind peptides of about 8 - 11 amino acids, usually 8 or 9 amino acids, and when this peptide has an appropriate binding motif, it can present it to cytotoxic T-lymphocytes. Peptide binding is stabilized at both ends by contacts between the atoms of the peptide backbone and invariant sites in the peptide-binding groove of all MHC class I molecules. There are invariant sites at both ends of the groove that bind to the amino and carboxy termini of the peptide. Changes in peptide length are often accommodated by kinking of the peptide backbone at proline or glycine residues that allow the required flexibility. Peptides bound by class I MHC molecules usually derive from endogenous protein antigens. As an example, the heavy chain of class I MHC molecules is typically an HLA-A, HLA-B, or HLA-C monomer in humans, and the light chain is β-2-microglobulin.

[0112] There are three major MHC class II proteins and two minor MHC class II proteins encoded by HLA. The class II genes combine to form a heterodimer (αβ) protein receptor that is typically expressed on the surface of antigen-presenting cells. The peptides bound by class II MHC molecules usually derive from extracellular or exogenous protein antigens. As an example, the α-chain and β-chain are, in humans, especially the HLA-DR, HLA-DQ, and HLA-DP monomers. MHC class II molecules can bind peptides of about 8-20 amino acids, especially 10-25, or 13-25 amino acids if this peptide has an appropriate binding motif, and present it to T-helper cells. These peptides are in an extended structure along the MHC II peptide-binding groove that is open at both ends (unlike the MHC class I peptide-binding groove). The peptide is held in place by the main-chain atoms contacting conserved residues that are mainly aligned in the peptide-binding groove.

[0113] When the method is carried out on a human sample, the method may include the step of determining the class I or class I major histocompatibility complex (MHC, also known as human leukocyte antigen (HLA) alleles) of the patient. Note that this step may not be necessary in certain situations since the MHC alleles of laboratory mice are generally known. In this application, "MHC molecule" refers to at least one MHC class I molecule or at least one MHC class II molecule.

[0114] The MHC allele database is implemented by analyzing known sequences of MHC I and MHC II and determining the allelic variability of each domain. This can typically be determined in silico using appropriate software algorithms well known in the art. Several tools have been developed to obtain HLA allele information from genome-wide sequencing data (whole exome, whole genome, and RNA sequencing data), such as OptiType, Polysolver, PHLAT, HLAreporter, HLAforest, HLAminer, and seq2HLA (see Kiyotani K et al., Immunopharmacogenomics to personized cancer waiming targeting neo antigens; Cancer Science 2018;109:542-549). For example, the seq2hla tool (see Boegel S, Lower M, Schafer M, et al. HLA typing from RNA-Seq sequence reads. Genome Med. 2012;4:102), which is well designed to implement the method as disclosed herein, is an in silico method written in python and R. It takes as input standard RNA-Seq sequence reads in fastq format and uses a Bowtie index (Langmead B, et al., Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 2009, 10:R25-10.1186 / gb-2009-10-3-r25) containing all HLA alleles to output the most likely HLA class I and class II genotypes (4-digit resolution), the p-value for each call, and the expression of each class.

[0115] Typically, sequences having a junction between the TE and the exon sequence are extracted in silico. The affinity of all possible peptides encoded by each sequence for each MHC allele from a patient (or mouse) can be determined in silico using, for example, computational methods for predicting peptide binding affinity to HLA molecules. Indeed, the exact prediction approach is the predicted IC 50Based on an artificial neural network. For example, the NetMHCpan software, which is modified from NetMHC, is well-suited to implement the methods disclosed herein for predicting peptides that bind to alleles for which no ligand has been reported (Lundegaard C et al., NetMHC-3.0: accurate web accessible predictions of human, mouse and monkey MHC class I affinities for peptides of length 8-11; Nucleic Acids Res. 2008;36:W509-W512; Nielsen M et al., NetMHCpan, a method for quantitative predictions of peptide binding to any HLA-A and -B locus protein of known sequence. PLoS One. 2007;2:e796, see also Kiyotani K et al., Immunopharmacogenomics towards personalized cancer immunotherapy targeting neoantigens; Cancer Science 2018; 109:542-549 and Yarchoan M et al., Nat rev. cancer 2017; 17(4):209-222). The NetMHCpan software uses an artificial neural network (ANN) to predict peptide binding to any MHC molecule of known sequence. This method is directed towards a combination of over 180,000 quantitative binding data and MS-derived MHC eluted ligands. The binding affinity data are for 172 MHC molecules from human (HLA-A, B, C, E), mouse (H-2), bovine (BoLA), primate (Patr, Mamu, GOGO) and pig (SLA). The MS eluted ligand data are for 55 HLA and mouse alleles.

[0116] In an exemplary embodiment, the neoantigen peptide encoded by the above-described fusion transcript, and the Kd affinity of the predicted peptide for the MHC allele, are 10 -4 , 10 -5 , 10 -6 , 10 -7 M or less, or less than 500 nM, particularly less than 50 nM, are selected as tumor neoantigen peptides.

[0117] As described above, the affinity of the selected peptide for the MHC allele can be determined in silico using suitable software such as netMHCpan. Thus, in some embodiments, the neoantigen peptide binds to MHC class I with a binding affinity at a percentile rank score of less than 2% predicted by NetMHCpan 4.0. In other embodiments, the neoantigen peptide binds to MHC class II with a binding affinity at a percentile rank score of less than 10% predicted by NetMHCpanII 3.2.

[0118] The affinity can also be (alternatively or additionally) estimated in vitro, for example, using an MHC tetramer formation assay as described in the results contained therein (see Example 2, points 2.1 and 2.2.2). For example, commercially available assays from ImmunAware® can typically be used by those skilled in the art (the EasYmers® kits are from ImmunAware®, and are specifically used according to their training guides). Typically, the binding affinity is determined as the ratio of binding to a positive control. Generally, peptides showing a binding ratio of at least 30%, particularly at least 40%, or even at least 50% of the positive control are selected. Typically, the neoantigen peptides according to the present disclosure, and typically obtainable by the present method, bind to at least one HLA / MHC molecule with an affinity sufficient for the peptide to be presented on the surface of the cell as an antigen. Generally, the neoantigen peptide binds to at least one HLA / MHC molecule, typically the molecule of the subject suffering from cancer, with a 10-4 less than, or 10 -5 less than, or 10 -6 less than, or 10 -7 less than, or less than 500 nM, at least less than 250 nM, at least less than 200 nM, at least less than 150 nM, at least less than 100 nM, at least less than 50 nM (the smaller the number, the greater the binding affinity), having an IC50 affinity.

[0119] Accordingly, any further steps according to this method are, independently, - a step of excluding fusion transcripts or predicted peptides that are expressed at a high level or frequency on healthy cells, wherein the alignment of the fusion transcript sequence to the RNAseq data of healthy cells typically enables determination of the relative amount of the fusion transcript sequence present in healthy cells, and in one embodiment, the fusion transcripts or predicted peptides expressed on healthy cells are discarded; - a step of confirming that the tumor neoantigen peptide is not expressed in the healthy cells of the subject, which step can typically be performed by aligning the sequence of the neoantigen peptide to the proteome of healthy cells using a basic local alignment search tool (BLAST), and preferably, peptides that align to the proteome of normal and healthy cells (e.g., using BLAST) are discarded; - a step of confirming that the fusion transcript or predicted peptide is expressed in the cancer cells of the subject, wherein the presence of the selected fusion transcript sequence in cancer cells can typically be checked by RT-PCR in the mRNA extracted from cancer cell samples.

[0120] Neoantigen Peptides, Polynucleotides, and Vectors The present disclosure also relates to an isolated tumor neoantigen peptide comprising at least 8, 9, 10, 11, or 12 amino acids, encoded by a portion of an open reading frame (ORF) derived from a fusion transcript that is a human mRNA sequence comprising a transposable element (TE) sequence and an exon sequence. The peptide may be 8-9, 8-10, 8-11, 12-25, 13-25, 12-20, or 13-20 amino acids in length. It is understood that the ORF overlaps with the junction between the TE sequence and the exon sequence, but the tumor neoantigen peptide itself may not include the junction.

[0121] The present disclosure also more specifically encompasses an isolated tumor neoantigen peptide encoded by a portion of a human fusion mRNA sequence derived from cancer cells, the fusion mRNA comprising a TE sequence and an exon sequence. In an exemplary embodiment, the neoantigen peptide comprising at least 8, 9, 10, 11, or 12 amino acids is encoded by a portion of an open reading frame (ORF) of any one of the fusion transcript sequences of SEQ ID NOs: 118 - 910, and is preferably a peptide having one or more of the neoantigen peptide characteristics described above.

[0122] The peptide may be 8-9, 8-10, 8-11, 12-25, 13-25, 12-20, or 13-20 amino acids in length and meets one or more of the neoantigen peptide characteristics described above. The N-terminus of the peptide of at least 8 amino acids may be encoded by a triplet codon starting at any of nucleotide positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, and more (it is understood that the present disclosure contemplates a starting position that is any integer between 1 and 8000 without enumerating all numbers between 1 and 8000).

[0123] The above-mentioned peptides are typically obtainable according to the methods of the present disclosure and thus encompass one or more of the aforementioned features. In particular, neoantigen peptides according to the present disclosure may exhibit one or a combination of the following further features: - It binds or specifically binds to the MHC class I of the subject and is 8 - 11 amino acids, particularly 8, 9, 10, or 11 amino acids. Typically, the neoantigen peptide is 8 or 9 amino acids in length, binds to at least one MHC class I molecule of the subject, or alternatively, binds to at least one MHC class II molecule of the subject and contains 12 - 25 amino acids, particularly 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. - It binds to at least one HLA / MHC molecule of the subject suffering from cancer and has sufficient affinity for the peptide presented on the cell surface as an antigen. Typically, the neoantigen peptide has an IC50 of less than 10 -4 or less than 10 -5 or less than 10 -6 or less than 10 -7 or less than 500 nM, at least less than 250 nM, at least less than 200 nM, at least less than 150 nM, at least less than 100 nM, at least less than 50 nM (the smaller the number, the greater the binding affinity). - When administered to the subject, it does not induce a significant autoimmune reaction and / or does not cause immune tolerance. - It is expressed at a high level in tumor samples compared to normal and healthy samples. Typically, according to the present disclosure, the fusion transcript may be selected when it is present in more than 10% of the tumor sample and less than 20% of the normal sample. In some embodiments, the neoantigen is more specifically a tumor-specific antigen (TSA), i.e., it is not expressed in normal samples, is expressed only in cancer samples, or is expressed at a relatively low level in normal samples (e.g., the expressed mRNA sequence represents a minor species in normal cells from normal samples). - This includes the junction between the TE sequence and the exon sequence. In other words, this is encoded by a part of the TE sequence and a part of the exon sequence, and the ORF can be either standard or non-standard, or - This is encoded by a non-standard ORF of the exon sequence, or - This is encoded by the TE sequence and optionally by a non-standard ORF. Tumor neoantigen peptides may first be verified by RT transcript analysis of the fusion transcript sequence in tumor cells from a subject. Typically, immunization with tumor neoantigen peptides according to the present disclosure also induces a T cell response.

[0124] In certain embodiments, the present disclosure encompasses NSCLC neoantigen peptides comprising at least 8 amino acids of any one of SEQ ID NOs: 1-117. Typically, the neoantigen peptides of SEQ ID NOs: 1-117 bind to HLA-A02, which has sufficient affinity for the peptides presented on the surface of cells as antigens. The affinity for MHC alleles can be determined by techniques known in the art, particularly in silico or in vitro as exemplified above.

[0125] In certain embodiments, the tumor neoantigen peptides according to the present disclosure bind to MHC molecules present in at least 1%, 5%, 10%, 15%, 20%, 25%, or more of a subject. In particular, the tumor neoantigen peptides disclosed herein are expressed in at least 1%, 5%, 10%, 15%, 20%, 25% of subjects from a population of subjects suffering from cancer.

[0126] More specifically, the tumor neoantigen peptides of the present disclosure can induce an immune response against tumors present in at least 1%, 5%, 10%, 15%, 20%, or 25% of subjects from a population of subjects suffering from cancer.

[0127] As previously defined, cancer can affect any one of the following tissues or organs: breast; liver; kidney; heart, mediastinum, pleura; floor of the mouth; lips; salivary glands; tongue; gums; oral cavity; palate; tonsils; larynx; trachea; bronchi, lungs; pharynx, hypopharynx, mid-pharynx, nasopharynx; esophagus; digestive organs such as the stomach, intrahepatic bile ducts, biliary tract, pancreas, small intestine, colon, etc.; rectum; urinary organs such as the bladder, gallbladder, ureters; rectosigmoid junction; anus, anal canal; skin; bones; joints, articular cartilage of hands and feet; eyes and appendages; brain; peripheral nerves, autonomic nervous system; spinal cord, cranial nerves, meninges; and various parts of the central nervous system; connective tissue, subcutaneous tissue and other soft tissues; retroperitoneum, peritoneum; adrenal glands; thyroid gland; endocrine glands and related structures; female genital organs such as ovaries, uterus, cervix; uterine body, vagina, vulva; male genital organs such as penis, testis and prostate; hematopoietic and reticuloendothelial systems; blood; lymph nodes; thymus. For example, tumors or cancers according to the present application include leukemia, seminoma, melanoma, teratoma, lymphoma, neuroblastoma, glioma, rectal cancer, endometrial cancer, kidney cancer, adrenal cancer, thyroid cancer, blood cancer, skin cancer, brain cancer, cervical cancer, intestinal cancer, liver cancer, colon cancer, gastric cancer, bowel cancer, head and neck cancer, digestive organ cancer, lymph node cancer, esophageal cancer, colorectal cancer, pancreatic cancer, ear, nose and throat (ENT) cancer, breast cancer, prostate cancer, uterine cancer, ovarian cancer and lung cancer, as well as metastases thereof. Examples thereof are lung cancer, breast cancer, prostate cancer, colon cancer, renal cell cancer, cervical cancer, or metastases of the above-mentioned cancer types or tumors. The term cancer according to the present disclosure also includes cancer metastasis and cancer recurrence.

[0128] Typically, the neoantigen peptides according to the present disclosure do not induce a significant autoimmune response and / or do not cause immune tolerance when administered to a subject. Mechanisms of tolerance include clonal deletion, ignorance, anergy, or suppression in the host, which is accompanied by a decrease in the number of high-affinity autoreactive T cells.

[0129] Neoantigen peptides can also be modified by extending or reducing the amino acid sequence of the compound, for example, by addition or deletion of amino acids. Peptides can also be modified by altering the order or composition of specific residues, and it is readily understood that certain amino acid residues essential for biological activity, such as critical contact sites or conserved residues, generally cannot be modified without adversely affecting biological activity. Non-essential amino acids need not be limited to those naturally occurring in proteins such as L-α-amino acids, or their D-isomers, and may include non-natural amino acids such as β-γ-δ-amino acids, and many derivatives of L-α-amino acids.

[0130] Typically, a series of peptides with single amino acid substitutions are used to determine the effect on binding, such as electrostatic charge, hydrophobicity. For example, a series of positively charged (e.g., Lys or Arg) or negatively charged (e.g., Glu) amino acid substitutions are made along the length of the peptide, revealing different patterns of sensitivity to various MHC molecules and T cell receptors. Additionally, multiple substitutions using small relatively neutral moieties such as Ala, Gly, Pro, or similar residues may be employed. The substitutions may be homo-oligomers or hetero-oligomers. The number and type of residues substituted or added depend on the required spacing between essential contact points and the specific functional properties desired (e.g., hydrophobicity vs. hydrophilicity). An increase in binding affinity for an MHC molecule or T cell receptor may also be achieved by such substitutions compared to the affinity of the parent peptide. In any case, such substitutions should utilize amino acid residues or other molecular fragments selected to avoid steric hindrance and charge interference that may disrupt binding.

[0131] Amino acid substitutions are typically single residue substitutions. Combinations of substitutions, deletions, insertions, or any combination thereof may be combined to arrive at the final peptide. Substitution variants are those in which at least one residue of the peptide is removed and a different residue is inserted in its place. Such substitutions are generally made according to Table 1 below when it is desired to finely adjust the properties of the peptide.

Table 1

[0132] Substantial changes in function (e.g., affinity for MHC molecules or T cell receptors) are made by selecting substitutions that are less conservative than those in the table above. That is, by selecting residues that have more significant differences in (a) the structure of the peptide backbone in the substitution region, such as a sheet or helical structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the effect on the maintenance of most of the side chain. Generally, substitutions that are expected to result in the greatest change in peptide properties are: (a) hydrophilic residues, such as seryl, are substituted with hydrophobic residues, such as leucyl, isoleucyl, phenylalanyl, valyl, or alanyl (or vice versa); (b) residues with electropositive side chains, such as lysyl, arginyl, or histidyl, are substituted with electronegative residues, such as glutamyl or aspartyl (or vice versa); or (c) residues with bulky side chains, such as phenylalanine, are substituted with residues having no side chain, such as glycine (or vice versa).

[0133] Peptides and polypeptides may also contain isosteres of two or more residues in the neoantigen peptide or polypeptide. An isostere, as defined herein, is a sequence of two or more residues that can be substituted for a second sequence because the conformation of the first sequence is compatible with the binding site specific for the second sequence. This term specifically includes peptide backbone modifications well known to those of skill in the art. Such modifications include modifications of the amide nitrogen, α-carbon, amide carbonyl, complete substitution, extension, deletion, or cross-linking of the backbone. Generally, see Spatola, Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. VII (Weinstein ed., 1983).

[0134] Furthermore, the neoantigen peptide may be conjugated to a carrier protein, ligand, or antibody. The half-life of the peptide can be improved by PEGylation, glycosylation, polysialylation, HESylation, recombinant PEG mimetics, Fc fusion, albumin fusion, nanoparticle attachment, nanoparticle encapsulation, cholesterol fusion, iron fusion, or acylation.

[0135] The modification of peptides and polypeptides with various amino acid mimics or unnatural amino acids is particularly useful for enhancing the stability of peptides and polypeptides in vivo. Stability can be assayed in several ways. For example, stability has been tested using various biological media such as peptidases and human plasma and serum. See, for example, Verhoef et al., Eur. J. Drug Metab Pharmacokin. 11:291-302 (1986). The half-life of the peptides of the present disclosure is conveniently determined using a 25% human serum (v / v) assay. The protocol is generally as follows. Pooled human serum (AB type, non-heat inactivated) is defatted by centrifugation prior to use. The serum is then diluted to 25% with RPMI tissue culture medium and used to test peptide stability. At predetermined time intervals, a small amount of the reaction solution is removed and added to either 6% aqueous trichloroacetic acid or ethanol. The turbid reaction sample is cooled for 15 minutes (4 °C) and then centrifuged to pellet the precipitated serum proteins. The presence of the peptide is then determined by reverse phase HPLC using stability-specific chromatography conditions.

[0136] Peptides and polypeptides may be modified to provide desired attributes other than an improved serum half-life. For example, the ability of a peptide to induce CTL activity can be enhanced by linking it to a sequence containing at least one epitope capable of inducing a T helper cell response. Particularly preferred immunogenic peptide / T helper conjugates are linked by a spacer molecule. The spacer typically consists of relatively small neutral molecules such as amino acids or amino acid mimics that are substantially uncharged under physiological conditions. The spacer is typically selected from, for example, Ala, Gly, or other neutral spacers of nonpolar or neutral polar amino acids. It will be understood that any optional spacer present need not consist of the same residues and may thus be a hetero-oligomer or a homo-oligomer. When present, the spacer is usually at least 1 or 2 residues, more commonly 3 to 6 residues. Alternatively, the peptide may be linked to the T helper peptide without a spacer.

[0137] Neoantigen peptides may be linked to T helper peptides either directly or via a spacer at either the amino or carboxy terminus of the peptide. The amino terminus of either the neoantigen peptide or the T helper peptide may be acylated. Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria sporozoite circumsporozoite 382-398 and 378-389.

[0138] Multiple neoantigen peptides described herein may also optionally be linked together by a spacer.

[0139] A protein or peptide can be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides, or peptides through standard molecular biology techniques, the isolation of proteins or peptides from natural sources, or the chemical synthesis of proteins or peptides. Nucleotide and protein, polypeptide, and peptide sequences corresponding to various genes have been previously disclosed and can be found in computerized databases known to those of skill in the art. One such database is the Genbank and GenPept databases of the National Center for Biotechnology Information at the website of the National Institutes of Health. The coding regions of known genes can be amplified and / or expressed using the techniques disclosed herein or as known to those of skill in the art. Alternatively, various commercially available preparations of proteins, polypeptides, and peptides are known to those of skill in the art.

[0140] In a further aspect, the disclosure provides a nucleic acid (e.g., a polynucleotide) encoding a neoantigen peptide disclosed herein. The polynucleotide may be selected from single-stranded and / or double-stranded DNA, cDNA, PNA, CNA, RNA, or polynucleotides in native or stabilized forms, e.g., polynucleotides having a phosphorothioate backbone, or combinations thereof, and may or may not contain introns so long as it encodes a peptide. Only peptides containing naturally occurring amino acid residues linked by naturally occurring peptide bonds can be encoded by a polynucleotide.

[0141] Yet another aspect of the disclosure provides an expression vector capable of expressing the neoantigen peptides disclosed herein. Expression vectors for different cell types are well known in the art and can be selected without undue experimentation. Generally, the DNA is inserted into an expression vector, such as a plasmid, in the appropriate orientation and correct reading frame for expression. The expression vector contains appropriate heterologous transcriptional and / or translational regulatory nucleotide sequences recognized by the desired host. The polynucleotide encoding the tumor neoantigen peptide may be operably linked to such heterologous regulatory nucleotide sequences, or may be operably linked without being adjacent to such heterologous regulatory nucleotide sequences. The vector is then introduced into the host through standard techniques. Guidance can be found, for example, in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.

[0142] antigen-presenting cell (APC) The present disclosure also encompasses a population of antigen-presenting cells pulsed with one or more peptides that are pre-defined and / or obtainable in the aforementioned method. The antigen-presenting cells are preferably dendritic cells (DCs) or artificial antigen-presenting cells (aAPCs) (see Neal, Lillian R et al. “The Basics of Artificial Antigen Presenting Cells in T Cell-Based Cancer Immunotherapies.” Journal of Immunology research and therapy vol.2, 1 (2017):68-79). Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) with the extraordinary ability to stimulate naive T cells and initiate a primary immune response against pathogens. Indeed, the main role of mature DCs is to sense antigens and produce mediators that activate other immune cells, particularly T cells. DCs are powerful stimulators for lymphocyte activation because they express MHC molecules that trigger the TCR (signal 1) and co-stimulatory molecules (signal 2) on T cells. Furthermore, DCs also secrete cytokines that support T cell proliferation. T cells require antigens presented in the form of processed peptides to recognize foreign pathogens or tumors. Presentation of peptide epitopes derived from pathogen / tumor proteins is achieved via MHC molecules. MHC class I (MHC-I) molecules and MHC class II (MHC-II) molecules present processed peptides to CD8+ T cells and CD4+ T cells, respectively. Importantly, DCs are present at the site of inflammation containing a rich population of T cells and promote the immune response. Thus, DCs can be an important element of any immunotherapy approach because they are closely involved in the activation of the adaptive immune response. In the context of vaccines, DC therapy can enhance the T cell immune response against a desired target in healthy volunteers or patients with infectious diseases or cancer. In one embodiment, the APCs are artificial APCs, which are genetically modified to express desired T cell co-stimulatory molecules, human HLA alleles, and / or cytokines.Such artificial antigen-presenting cells (aAPCs) can provide the requirements for proper T cell engagement, co-stimulation, and sustained release of cytokines that enable controlled T cell proliferation. These cells can be stored in small amounts for use in generating T cell lines from different donors without being subject to time constraints and availability limitations, thus serving as off-the-shelf reagents for immunotherapy applications. The expression of potent co-stimulatory signals on these aAPCs brings higher efficiency to this system for enhancing the effectiveness of adoptive immunotherapy. Furthermore, aAPCs can be engineered to express genes that direct the release of specific cytokines, for example, to promote the preferential expansion of desirable T cell subsets for adoptive transfer such as long-lived memory T cells (see Hasan AH et al., Artificial Antigen Presenting Cells: An Off the Shelf Approach for Generation of Desirable T-Cell Populations for Broad Application of Adoptive Immunotherapy; Adv Genet Eng. 2015; 4(3):130, Kim JV, Latouche JB, Riviere I, Sadelain M. The ABCs of artificial antigen presentation. Nat Biotechnol. 2004;22:403-410 or Wang C, Sun W, Ye Y, Bomba HN, Gu Z. Bioengineering of Artificial Antigen Presenting Cells and Lymphoid Organs. Theranostics 2017; 7(14):3504-3516).

[0143] Typically, the dendritic cells are autologous dendritic cells pulsed with the neoantigen peptides disclosed herein. The peptide may be any suitable peptide that elicits an appropriate T cell response. The antigen-presenting cell (or stimulatory cell) typically has MHC class I or II molecules on its surface, and in one embodiment, it is substantially impossible to load the selected antigen onto the MHC class I or II molecules. The MHC class I molecules or MHC class II molecules can be readily loaded with the selected antigen in vitro.

[0144] Alternatively, the antigen-presenting cell may comprise an expression construct encoding the tumor neoantigen peptides disclosed herein. The polynucleotide may be any suitable polynucleotide as previously defined, and preferably can transduce dendritic cells and thus result in the presentation of peptides and induction of immunity.

[0145] Accordingly, the present disclosure encompasses a population of APCs that can be pulsed or loaded with the neoantigen peptides disclosed herein, genetically modified (via DNA or RNA transfer) to express at least one of the neoantigen peptides disclosed herein, or that comprise an expression construct encoding the tumor neoantigen peptides of the present disclosure. Typically, the population of APCs is modified or comprises pulsed or loaded and expressing at least 1, at least 5, at least 10, at least 15, or at least 20 different neoantigen peptides or expression constructs encoding the same.

[0146] The present disclosure also encompasses compositions comprising the APCs disclosed herein. The APCs can be suspended in any known physiologically compatible pharmaceutical carrier such as cell culture medium, saline, phosphate buffered saline, cell culture medium, etc. to form a physiologically acceptable aqueous pharmaceutical composition. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, and lactated Ringer's. Other substances, such as antibacterial agents, can be added if desired. As used herein, "carrier" refers to any substance suitable as a vehicle for delivering the APCs to a suitable in vitro or in vivo site of action. Thus, the carrier can function as an excipient for formulating a therapeutic or experimental reagent containing the APCs. Preferred carriers are capable of maintaining the APCs in a form that can interact with T cells. Examples of such carriers include, but are not limited to, water, phosphate buffered saline, saline, Ringer's solution, dextrose solution, serum-containing solution, Hank's solution, and other physiologically balanced aqueous solutions or cell culture media. The aqueous carrier may also contain appropriate adjunct substances necessary to approximate the physiological conditions of the recipient, such as, for example, enhancement of chemical stability and isotonicity. Appropriate adjunct substances include, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and other substances used to generate phosphate buffers, Tris buffers, and bicarbonate buffers.

[0147] Vaccine composition The present disclosure further encompasses a vaccine or immunogenic composition capable of eliciting a specific T cell response, - one or more neoantigen peptides as defined herein, - one or more polynucleotides encoding the neoantigen peptides as defined herein, - a population of the above-described antigen-presenting cells (such as autologous dendritic cells or artificial APCs). Preferably, the neoantigen peptides encoded by previously defined tumor-specific fusions are used in the vaccine compositions according to the present disclosure. The neoantigen peptides may also be named tumor-specific peptides. Polynucleotides encoding tumor-specific peptides are also preferably used according to the present disclosure.

[0148] Suitable vaccines or immunogenic compositions preferably contain 1 to 20 neoantigen peptides, more preferably 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 different neoantigen peptides, even more preferably 6, 7, 8, 9, 10, 11, 12, 13, or 14 different neoantigen peptides, and most preferably 12, 13 or 14 different neoantigen peptides.

[0149] The neoantigen peptides may be linked to a carrier protein. When the composition contains two or more neoantigen peptides, two or more (e.g., 2 to 25) peptides may be linearly linked by a spacer molecule as described above, e.g., a spacer containing 2 to 6 non-polar or neutral amino acids.

[0150] In one embodiment of the present disclosure, different neoantigen peptides encoding a polynucleotide, vector, or APC are selected such that one vaccine or immunogenic composition comprises neoantigen peptides capable of associating with different MHC molecules, such as different MHC class I molecules. Preferably, such neoantigen peptides are capable of associating with the most frequently occurring MHC class I molecules, for example, different fragments capable of associating with at least two, more preferably at least three, even more preferably at least four preferred MHC class I molecules. In some embodiments, the composition comprises a peptide encoding a polynucleotide, vector, or APC capable of associating with one or more MHC class II molecules. The MHC is optionally HLA-A, -B, -C, -DP, -DQ, or -DR.

[0151] The vaccine or immunogenic composition can elicit a specific cytotoxic T cell response and / or a specific helper T cell response.

[0152] Thus, in certain embodiments, the present disclosure also relates to the neoantigen peptides described above, wherein the neoantigen peptides have tumor-specific neoepitopes and are included in the vaccine or immunogenic composition. The vaccine composition should be understood to mean a composition for generating immunity for the prevention and / or treatment of a disease. Thus, a vaccine is an agent that contains or generates an antigen and is intended for use in humans or animals to generate specific defensive and protective substances by vaccination. An "immunogenic composition" should be understood to mean a composition that contains or generates an antigen and is capable of inducing an antigen-specific humoral or cellular immune response, such as a T cell response.

[0153] In a preferred embodiment, the neoantigen peptide according to the present disclosure is 8 or 9 residues in length, or 13 to 25 residues in length. When the peptide is less than 20 residues in length, in order to have a peptide suitable for in vivo immunization, the neoantigen peptide is optionally adjacent to additional amino acids to obtain an immunogenic peptide of more, usually more than 20, amino acids.

[0154] A pharmaceutical composition (i.e., a vaccine or immunogenic composition) containing the peptides described herein may be administered to an individual already suffering from cancer. For therapeutic use, the composition is administered to the patient in an amount sufficient to induce an effective CTL response against the tumor antigen and to cure or at least partially prevent the symptoms and / or complications. The amount appropriate to achieve this is defined as the "therapeutically effective dose". The amount effective for this use depends, for example, on the peptide composition, the method of administration, the stage and severity of the disease being treated, the patient's weight and general health, and the judgment of the prescribing physician, but generally, the range for the initial immunization (i.e., therapeutic or prophylactic administration) is from about 1.0 μg to about 50,000 μg of the peptide for a patient weighing 70 kg, and then, depending on the patient's response and condition as measured by the specific CTL activity in the patient's blood, an additional dose of the peptide from about 1.0 μg to about 10,000 μg is administered according to an additional regimen over several weeks to several months. It should be noted that the peptides and compositions of the present invention can generally be employed in severe disease states, i.e., life-threatening or potentially life-threatening states, especially when cancer has metastasized. In such cases, considering the minimization of foreign substances and the relatively non-toxic nature of the peptides, substantial over-administration of these peptide compositions may be possible and may be desired by the treating physician.

[0155] For therapeutic use, administration should be initiated from the detection or surgical removal of the tumor. Thereafter, the dose is increased until at least the symptoms are substantially reduced and then for a certain period.

[0156] The vaccine or immunogenic composition for treatment is intended for parenteral, topical, nasal, oral, or local administration. Preferably, the pharmaceutical composition is administered parenterally, for example, by intravenous, subcutaneous, intradermal, or intramuscular injection. The composition may be administered at the site of surgical resection to induce a local immune response against the tumor.

[0157] The vaccine or immunogenic composition may be a pharmaceutical composition additionally comprising a pharmaceutically acceptable adjuvant, immunostimulant, stabilizer, carrier, diluent, excipient, and / or any other material well-known to those skilled in the art. Such materials should be non-toxic and should not interfere with the effectiveness of the active ingredient. The carrier is preferably an aqueous carrier, but the exact nature of the carrier or other materials depends on the route of administration. Various aqueous carriers, such as water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid, etc. may be used. These compositions may be sterilized by conventional well-known sterilization techniques or may be sterile filtered. The resulting aqueous solution may be packaged for use as is or may be lyophilized, but the lyophilized preparation is combined with a sterile solution before administration. The composition may further contain pharmaceutically acceptable auxiliary substances necessary to approximate physiological conditions, such as pH adjusters and buffers, tonicity adjusters, wetting agents, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc. For considerations regarding cancer vaccines, see, for example, Butterfield, BMJ. 2015 22;350.

[0158] Examples of adjuvants that increase or broaden the host's immune response to an antigenic compound include emulsifiers, muramyl dipeptide, abrin, aqueous adjuvants such as aluminum hydroxide, chitosan-based adjuvants, saponins, oils, amphigen, LPS, bacterial cell wall extracts, bacterial DNA, CpG sequences, synthetic oligonucleotides, cytokines, and combinations thereof. Examples of emulsifiers include potassium, sodium, and ammonium salts of lauric acid and oleic acid, calcium, magnesium, and aluminum salts of fatty acids, organic sulfonates such as sodium lauryl sulfate, cetyltrhethylammonlum bromide, glyceryl esters, polyoxyethylene glycol esters and ethers, and sorbitan fatty acid esters and their polyoxyethylene, acacia, gelatin, lecithin, and / or cholesterol. Adjuvants containing an oil component include mineral oil, vegetable oil, or animal oil. Other adjuvants include complete Freund's adjuvant (FCA) or incomplete Freund's adjuvant (FIA). Cytokines useful as additional immunostimulants include interferon alpha, interleukin-2 (IL-2), and granulocyte macrophage-colony stimulating factor (GM-CSF), or combinations thereof.

[0159] The concentration of the peptides described herein in a vaccine or immunogenic formulation can vary widely, i.e., as low as less than about 0.1% by weight, usually or at least about 2% by weight, to as high as 20% to 50% by weight or more, and is selected mainly by the fluid volume, viscosity, etc., according to the particular mode of administration selected.

[0160] The peptides described herein may also be administered via liposomes that target the peptide to specific tissues such as lymphoid tissue. Liposomes are also useful for increasing the half-life of the peptide. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers, and the like. In these preparations, the peptide to be delivered is incorporated as part of the liposome, alone, or together with a molecule that binds to a receptor commonly found on lymphoid cells, such as a monoclonal antibody that binds to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, liposomes filled with the desired peptide of the invention are directed to the site of lymphoid cells, where the liposomes can then deliver the selected therapeutic / immunogenic peptide composition. Liposomes for use in the present invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids, as well as sterols such as cholesterol. The choice of lipid is generally guided by, for example, considering the size of the liposome, acid lability, and stability of the liposome in the bloodstream. The various methods available for the preparation of liposomes are described, for example, in Szoka et al., Ann. Rev. Biophys. Bioeng. 9;467 (1980), U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0161] For targeting to immune cells, the ligand incorporated into the liposome may include, for example, an antibody or fragment thereof specific for a cell surface determinant of the desired immune system cell. The liposome suspension containing the peptide can be administered intravenously, locally, topically, etc., at a dose that varies particularly according to the method of administration, the peptide being delivered, and the stage of the disease being treated.

[0162] For solid compositions, for example, conventional or nanoparticle non-toxic solid carriers including pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, etc. can be used. For oral administration, pharmaceutically acceptable non-toxic compositions are formed by incorporating any of the commonly used excipients such as the carriers listed previously, and generally 10 to 95% of the active ingredient, i.e., one or more peptides of the present invention, more preferably at a concentration of 25% to 75%.

[0163] For aerosol administration, the immunogenic peptide is preferably supplied in a finely divided form together with a surfactant and a propellant. Typical proportions of the peptide are 0.01% to 20% by weight, preferably 1% to 10%. The surfactant must of course be non-toxic and preferably soluble in the propellant. Representatives of such agents include fatty acids containing 6 to 22 carbon atoms, such as caproic acid, octanoic acid, lauric acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, olesteric acid, and esters or partial esters of oleic acid with aliphatic polyhydric alcohols or their cyclic anhydrides. Mixed esters, such as mixed or natural glycerides, may be used. The surfactant may constitute 0.1% to 20% by weight of the composition, preferably 0.25 to 5% by weight. The remainder of the composition is usually the propellant. The carrier can optionally contain, for example, lecithin for nasal delivery.

[0164] Cytotoxic T lymphocytes (CTLs) recognize antigens not in the form of intact foreign antigens themselves, but rather in the form of peptides bound to MHC molecules. The MHC molecules themselves are located on the cell surface of antigen-presenting cells. Therefore, CTL activation is only possible when a trimeric complex of peptide antigen, MHC molecule, and antigen-presenting cell (APC) is present. Similarly, not only using peptides for CTL activation, but also additionally adding APCs having respective MHC molecules can enhance the immune response. Therefore, in some embodiments, the vaccines or immunogenic compositions according to the present disclosure alternatively or additionally contain at least one antigen-presenting cell, preferably a population of APCs.

[0165] Therefore, the vaccine or immunogenic composition may be delivered, for example, as a dendritic cell vaccine, in the form of cells such as antigen-presenting cells. Antigen-presenting cells such as dendritic cells may be pulsed or loaded with the neoantigen peptides disclosed herein, may contain an expression construct encoding the neoantigen peptides disclosed herein, or may be genetically modified to express one, two or more, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 of the neoantigen peptides disclosed herein (via DNA or RNA transfer).

[0166] Suitable vaccines or immunogenic compositions may also be in the form of DNA or RNA related to the neoantigen peptides described herein. For example, DNA or RNA encoding one or more neoantigen peptides or proteins derived therefrom may be used as a vaccine, for example, by directly injecting it into a subject. For example, DNA or RNA encoding at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 neoantigen peptides or proteins derived therefrom.

[0167] For delivering nucleic acids to a patient, many methods are conveniently used. For example, the nucleic acid may be delivered directly as "naked DNA". This approach is described, for example, in Wolff et al., Science 247:1465-1468 (1990), and U.S. Pat. Nos. 5,580,859 and 5,589,466. The nucleic acid can also be administered using, for example, ballistic delivery as described in U.S. Pat. No. 5,204,253. Particles consisting of only DNA can be administered. Alternatively, the DNA can be attached to particles such as gold particles.

[0168] The nucleic acid may also be complexed with a cationic compound such as a cationic lipid and delivered. Lipid-mediated gene delivery methods are described, for example, in 9618372WOAWO 96 / 18372; 9324640WOAWO 93 / 24640; Mannino & Gould-Fogerite, BioTechniques 6(7):682-691(1988), 5279833USARose U.S. Pat. No. 5,279,833, 9106309WOAWO 91 / 06309, and Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7414 (1987).

[0169] The delivery system may optionally include a cell-penetrating peptide, nanoparticle encapsulation, virus-like particles, liposomes, or any combination thereof. Cell-penetrating peptides include the TAT peptide, herpes simplex virus VP22, transportan, Antp. Liposomes can be used as a delivery system. Listeria vaccines or electroporation can also be used.

[0170] One or more neoantigen peptides may also be delivered via a bacterial or viral vector containing a DNA or RNA sequence encoding the one or more neoantigen peptides. The DNA or RNA may be delivered as the vector itself, or within an attenuated bacterial virus, or an attenuated live virus such as vaccinia or fowlpox. This approach involves the use of vaccinia virus as a vector for expressing the nucleotide sequence encoding the peptides of the invention. When introduced into an acute or chronic infected host or a non-infected host, the recombinant vaccinia virus expresses immunogenic peptides, thereby inducing a CTL response in the host. Vaccinia vectors and methods useful in immunization protocols are described, for example, in U.S. Patent No. 4,722,848. Another vector is BCG (Bacillus Calmette-Guerin). The BCG vector is described in Stover et al. (Nature 351:456-460 (1991)). A wide variety of other vectors useful for the therapeutic administration or immunization of the peptides of the invention, such as typhoid vectors, will be apparent to those skilled in the art from the description herein.

[0171] Suitable means for administering the nucleic acids encoding the peptides described herein involve the use of minigene constructs encoding multiple epitopes. To create a DNA sequence encoding a CTL epitope (minigene) selected for expression in human cells, the amino acid sequence of the epitope is reverse translated. Using a human codon usage table, the codon selection for each amino acid is derived. The DNA sequences encoding these epitopes are directly adjacent, creating a continuous polypeptide sequence. Additional elements can be incorporated into the minigene design to optimize expression and / or immunogenicity. Examples of amino acid sequences that can be reverse translated and included in the minigene sequence include helper T lymphocyte, epitope, leader (signal) sequence, and endoplasmic reticulum retention signal. Furthermore, MHC presentation of the CTL epitope can be improved by including a synthetic (e.g., polyalanine) or naturally occurring flanking sequence adjacent to the CTL epitope.

[0172] The mini gene sequence is converted into DNA by assembling oligonucleotides encoding the plus and minus strands of the mini gene. Overlapping oligonucleotides (30 - 100 bases in length) are synthesized, phosphorylated, purified, and annealed under appropriate conditions using well-known techniques. The ends of the oligonucleotides are ligated using T4 DNA ligase. This synthetic mini gene encoding the CTL epitope polypeptide can then be cloned into a desired expression vector.

[0173] Standard regulatory sequences well-known to those skilled in the art are included in the vector to ensure expression in target cells. Thus, DNA or RNA encoding a neoantigen peptide may typically be operably linked to one or more of the following: - A promoter that can be used to drive nucleic acid molecule expression. AAV ITR can function as a promoter and is advantageous for eliminating the need for additional promoter elements. For ubiquitous expression, the following promoters can be used: CMV (especially the human cytomegalovirus immediate early promoter (hCMV-IE)), CAG, CBh, PGK, SV40, RSV, ferritin heavy or light chain, etc. For brain expression, the following promoters can be used: synapsin for all neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc. Promoters used to drive RNA synthesis may include: Pol III promoters such as U6 or HI. A guide RNA (gRNA) can be expressed using a Pol II promoter and an intron cassette. Typically, the promoter includes a downstream cloning site for mini gene insertion. For examples of suitable promoter sequences, see U.S. Patent Nos. 5,580,859 and 5,589,466. - A transcriptional transactivator or other enhancer element capable of enhancing transcriptional activity, for example, the regulatory R region from the 5' terminal repeat sequence (LTR) of human T cell leukemia virus type 1 (HTLV-1), which has been shown to induce a higher cellular immune response when combined with the CMV promoter. - A translation optimization sequence, for example, the Kozak sequence adjacent to the AUG start codon (ACCAUGG) in mRNA, and codon optimization.

[0174] To optimize the expression and immunogenicity of the minigene, further vector modifications may be desirable. In some cases, introns are required for efficient gene expression, and one or more synthetic or naturally occurring introns can be incorporated into the transcriptional region of the minigene. To increase the expression of the minigene, it is also possible to consider including an mRNA stabilization sequence. Recently, it has been proposed that immunostimulatory sequences (ISS or CpG) are involved in the immunogenicity of DNA vaccines. These sequences can be included in the vector outside the minigene coding sequence if they are found to enhance immunogenicity.

[0175] In some embodiments, a bicistronic expression vector can be used to enable the production of a second protein included to enhance or decrease the epitope and immunogenicity encoded by the minigene.

[0176] The DNA vaccines or immunogenic compositions described herein can be enhanced by co-delivering cytokines that promote a cell-mediated immune response, such as IL-2, IL-12, IL-18, GM-CSF, and IFNγ. CXC chemokines such as IL-8, and CC chemokines such as macrophage inflammatory protein (MIP)-1α, MIP-3α, MIP-3β, and RANTES may increase the efficacy of the immune response. DNA vaccine immunogenicity can also be enhanced by co-delivering cytokine-inducing molecules (e.g., LeIF), costimulatory and adhesion molecules encoded by the plasmid, such as B7-1 (CD80) and / or B7-2 (CD86). Helper (HTL) epitopes can be linked to intracellular target signals and expressed separately from CTL epitopes. This allows for the targeting of HTL epitopes to different cellular compartments than CTL epitopes. Optionally, this can facilitate more efficient entry of HTL epitopes into the MHC class II pathway, thereby improving CTL induction. In contrast to CTL induction, specific downregulation of the immune response by co-expression of immunosuppressive molecules (e.g., TGF-β) can be beneficial in certain diseases.

[0177] Once the expression vector is selected, the mini-gene is cloned into the polylinker region downstream of the promoter. The plasmid is transformed into a suitable E. coli strain and the DNA is prepared using standard techniques. The orientation and DNA sequence of the mini-gene, as well as all other elements contained in the vector, are confirmed using restriction enzyme mapping and DNA sequence analysis. Bacterial cells carrying the correct plasmid can be stored as a master cell bank and a working cell bank.

[0178] The purified plasmid DNA can be prepared for injection using various formulations. The simplest of these is the reconstitution of lyophilized DNA in sterile phosphate-buffered saline (PBS). Various methods have been described and new technologies may become available. As noted above, nucleic acids are readily formulated with cationic lipids. In addition, glycolipids, fusogenic liposomes, peptides, and compounds collectively referred to as protective, interactive, non-condensing (PINC) can be complexed with the purified plasmid DNA to affect variables such as stability, intramuscular dispersion, or transport to specific organs or cell types.

[0179] A vaccine or immunogenic composition comprising a peptide may be administered in combination with a vaccine or immunogenic composition comprising a polynucleotide encoding the peptide. For example, the administration of a peptide vaccine and a DNA vaccine may be alternated in a prime-boost protocol. For example, priming with a peptide immunogenic composition and boosting with a DNA immunogenic composition, as well as priming with a DNA immunogenic composition and boosting with a peptide immunogenic composition, are contemplated.

[0180] The present disclosure also provides a) optionally, identifying at least one neoantigen peptide according to the methods described above; b) producing at least one neoantigen peptide, at least one polypeptide encoding the neoantigen peptide, or at least one vector comprising the polypeptide as described herein; c) optionally, adding a physiologically acceptable buffer, excipient, and / or adjuvant and producing a vaccine using the at least one neoantigen peptide, polypeptide, or vector. The present disclosure encompasses methods for producing a vaccine composition comprising these steps.

[0181] Another aspect of the disclosure is a method of producing a DC vaccine, wherein the DC presents at least one neoantigen peptide disclosed herein.

[0182] Antibody TCR, CAR and derivatives thereof The disclosure also relates to an antibody or antigen-binding fragment thereof that specifically binds to a neoantigen peptide as defined herein.

[0183] In some embodiments, the neoantigen peptide is associated with an MHC molecule or an HLA molecule.

[0184] Typically, the antibody or antigen-binding fragment thereof binds to the neoantigen peptide as defined herein, alone or optionally in association with an MHC molecule or an HLA molecule, with a Kd binding affinity of 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, or 10 -11 M or less.

[0185] To promote the infiltration and recognition of tumor cells by lymphocytes T (LTs), another strategy consists of using antibodies that can recognize multiple antigen targets simultaneously, and more specifically two antigen targets simultaneously. There are many formats for bispecific antibodies. BiTEs (bispecific T-cell engagers) were the first ones developed. These are fusion proteins consisting of two scFvs (heavy-chain VH variable domain and light-chain VL variable domain) from two antibodies linked by a binding peptide, one recognizing the LT marker (CD3+) and the other recognizing a tumor antigen. The goal is to promote the recruitment and activation of LTs in contact with the tumor, and thus to bring about cell-lytic tumors (see Patrick A. Baeuerle and Carsten Reinhardt; Bispecific T-Cell Engageing Antibodies for Cancer Therapy; Cancer Res 2009; 69:(12).June 15, 2009; and Galaine et al., Innovations & Therapeutiques en Oncologie, vol.3-n°3-7, mai-aout 2017).

[0186] In certain embodiments, the antibody is a bispecific T-cell engager that targets a tumor neoantigen peptide as defined herein, optionally in association with an MHC molecule or an HLA molecule, and further targets at least an immune cell antigen. Typically, the immune cell is a T cell, an NK cell, or a dendritic cell. In this context, the target immune cell antigen may be, for example, CD3, CD16, CD30, or a TCR.

[0187] As used herein, the term "antibody" is used in its broadest sense and includes polyclonal and monoclonal antibodies, intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, variable heavy chain (VH) regions capable of specifically binding to an antigen, single-chain antibody fragments, including single-chain variable fragments (scFv), and single-domain antibody (e.g., VHH antibody, sdAb, sdFv, nanobody) fragments. The term includes, for example, intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetra-bodies, tandem di-scFv, tandem tri-scFv, and other genetically engineered and / or otherwise variant-modified forms of immunoglobulins. Unless otherwise indicated, the term "antibody" should be understood to encompass functional antibodies and fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgG1, IgG2, IgG3, IgG4, IgM, IgE, IgA, and IgD. In some embodiments, the antibody comprises a light chain variable domain and a heavy chain variable domain and is, for example, in scFv format.

[0188] Antibodies include variant polypeptide species having one or more amino acid substitutions, insertions, or deletions within the native amino acid sequence, provided that the antibody retains or substantially retains its specific binding function. Conservative substitutions of amino acids are well known and have been described above.

[0189] The present disclosure further provides for about 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, or 10 -11A method of making an antibody or an antigen-binding fragment thereof, comprising the step of selecting an antibody having a Kd binding affinity of less than M that optionally associates with an MHC molecule or an HLA molecule and binds to a tumor neoantigen peptide as defined herein.

[0190] In some embodiments, the antibody is selected from a library of human antibody sequences. In some embodiments, the antibody is generated by immunizing an animal with a polypeptide comprising a neoantigen peptide optionally associated with an MHC molecule or an HLA molecule, followed by a selection step.

[0191] Antibodies, including chimeric antibodies, humanized antibodies, or human antibodies, can be further affinity matured and selected as described above. A humanized antibody contains CDR regions derived from rodent sequences. Typically, the rodent CDRs are grafted onto a human framework, and some of the human framework residues may be mutated to the original rodent framework residues to retain affinity, and / or one or some of the CDR residues may be mutated to enhance affinity. A fully human antibody has no mouse sequences and is typically produced by phage display technology of a human antibody library or immunization of a transgenic mouse in which the native immunoglobulin locus is replaced with segments of the human immunoglobulin locus.

[0192] Also encompassed by the present disclosure are antibodies produced by the method, as well as immune cells expressing such antibodies or fragments thereof.

[0193] The present disclosure also encompasses pharmaceutical compositions comprising one or more of the antibodies disclosed herein, alone or in combination with at least one other agent such as a stabilizing compound, which may be administered in any sterile biocompatible pharmaceutical carrier and may optionally be formulated by incorporating a sterile pharmaceutically acceptable buffer, diluent, and / or excipient. Pharmaceutically acceptable carriers can typically be used to enhance or stabilize the composition and / or to facilitate the preparation of the composition. Pharmaceutically acceptable carriers include solvents, dispersion media, coating agents, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., which are physiologically compatible and, in some embodiments, pharmaceutically inert.

[0194] Administration of the pharmaceutical compositions comprising the antibodies disclosed herein can be achieved orally or parenterally. Methods of parenteral delivery include topical, intraarterial (directly to the tumor), intramuscular, spinal, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.

[0195] Accordingly, in addition to the active ingredient, these pharmaceutical compositions may contain a suitable pharmaceutically acceptable carrier including excipients and adjuvants that facilitate processing the active compound into a preparation that can be pharmaceutically used. Further details regarding techniques of formulation and administration can be found in the latest edition of Remington’s Pharmaceutical Sciences (Ed. Maack Publishing Co, Easton, Pa.).

[0196] Depending on the route of administration, the active compounds, i.e., the antibodies, bispecific molecules, and multispecific molecules, can be coated with a material to protect the compounds from the action of acids and other natural conditions that can inactivate the compounds.

[0197] The composition is typically sterilized and is preferably fluid. Suitable fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintaining the particle size required in the case of dispersion, and by the use of surfactants. In many cases, it is preferred to include in the composition an isotonic agent, for example, a polyhydric alcohol such as sugar, mannitol or sorbitol, and sodium chloride. The long-term absorption of the injectable composition can be brought about, for example, by including in the composition an agent that delays absorption, such as aluminum monostearate or gelatin.

[0198] Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art at a dosage suitable for oral administration. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. for ingestion by the patient.

[0199] Pharmaceuticals for oral use can be obtained by combining the active compound with a solid excipient, optionally grinding the resulting mixture, adding suitable auxiliaries if desired, and then processing the mixture of granules to obtain tablets or dragee cores. Suitable excipients are carbohydrates or protein fillers such as sugars including lactose, sucrose, mannitol, or sorbitol; starches from corn, wheat, rice, potatoes, or other plants; celluloses such as methyl, cellulose, hydroxypropylmethylcellulose, or sodium carboxymethylcellulose; and gums including arabia and tragacanth; and proteins such as gelatin and collagen. Optionally, a disintegrant or solubilizing agent such as crosslinked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate, may be added.

[0200] The sugar-coated tablet core may also contain a suitable coating such as a concentrated sugar solution, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and / or titanium dioxide, a lacquer solution, and a suitable organic solvent or solvent mixture. Dyes or pigments may be added to the tablets or the sugar-coated tablet coating for product identification or to characterize the amount of the active compound, i.e., the dosage.

[0201] Pharmaceuticals that can be used orally include push-fit capsules made of gelatin and soft sealed capsules made of gelatin and coated with glycerol or sorbitol, etc. Push-fit capsules may contain a filler or binder such as lactose or starch, a lubricant such as talc or magnesium stearate, and an active ingredient optionally mixed with a stabilizer. In soft capsules, the active compound can be dissolved or suspended in a suitable liquid such as a fatty oil, liquid paraffin, or liquid polyethylene glycol, regardless of the presence or absence of a stabilizer.

[0202] Pharmaceutical formulations for parenteral administration include an aqueous solution of the active compound. In the case of injection, the pharmaceutical composition of the present invention can be formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiologically buffered saline. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, or dextran. Furthermore, a suspension of the active compound may be prepared as a suitable oily injection suspension. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, synthetic fatty acid esters such as ethyl oleate or triglycerides, or liposomes. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compound and enable the preparation of a highly concentrated solution.

[0203] For topical or nasal administration, penetrants suitable for the specific barriers to be penetrated are used in the formulation. Such penetrants are generally known in the art.

[0204] The pharmaceutical compositions of the present disclosure can be prepared according to methods well known and routinely practiced in the art. See, for example, Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. The pharmaceutical compositions are preferably manufactured under GMP conditions.

[0205] The present disclosure also encompasses T cell receptors (TCRs) that target neoantigen peptides as defined herein in relation to MHC or HLA molecules.

[0206] The present disclosure further includes a method of producing a TCR or an antigen-binding fragment thereof, which binds to a tumor neoantigen peptide as defined herein, optionally in association with an MHC or HLA molecule, with a Kd binding affinity of about 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, or 10-11M or less, and includes the step of selecting a TCR.

[0207] Nucleic acids encoding TCRs can be obtained from a variety of sources, such as polymerase chain reaction (PCR) amplification of naturally occurring TCR DNA sequences, followed by expression of antibody variable regions, followed by the selection process described above. In some embodiments, the TCR is obtained from T cells isolated from a patient or cultured T cell hybridomas. In some embodiments, TCR clones specific for a target antigen are generated in transgenic mice engineered with human immune system genes (e.g., the human leukocyte antigen system, or HLA). For example, tumor antigens (see, e.g., Parkhurst et al. (2009) Clin Cancer Res. 15:169-180 and Cohen et al. (2005) J Immunol. 175:5799-5808). In some embodiments, phage display is used to isolate TCRs specific for a target antigen (see, e.g., Varela-Rohena et al. (2008) Nat Med. 14:1390-1395 and Li (2005) Nat Biotechnol. 23:349-354).

[0208] "T cell receptor" or "TCR" refers to a molecule containing variable alpha and beta chains (also known as TCRα and TCRβ, respectively), or variable gamma and delta chains (also known as TCRγ and TCRδ, respectively), and having the ability to specifically bind to antigenic peptides bound to MHC receptors. In some embodiments, the TCR is in the αβ form. Typically, TCRs present in the αβ and γδ forms are generally structurally similar, but the T cells expressing them can have different anatomical locations or functions. TCRs can be found on the surface of cells or in soluble form. Generally, TCRs are found on the surface of T cells (or T lymphocytes) and are commonly involved in recognizing antigens bound to major histocompatibility complex (MHC) molecules. In some embodiments, the TCR may also contain constant domains, transmembrane domains, and / or a short cytoplasmic tail (see, for example, Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997). For example, in some aspects, each chain of the TCR can have, at its C-terminus, one N-terminal immunoglobulin variable domain, one immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail. In some embodiments, the TCR is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. Unless otherwise stated, the term "TCR" should be understood to encompass its functional TCR fragments. This term also encompasses intact or full-length TCRs, including TCRs in the αβ or γδ forms.

[0209] Accordingly, for the purposes of this specification, reference to a TCR includes any TCR or functional fragment thereof, such as the antigen-binding portion of a TCR that binds to a specific antigenic peptide bound to an MHC molecule, i.e., an MHC-peptide complex. The "antigen-binding portion" or "antigen-binding fragment" of a TCR can be used interchangeably and refers to a molecule that contains a part of the structural domain of the TCR but binds to the antigen (e.g., an MHC-peptide complex) to which the full TCR binds. In some instances, the antigen-binding portion contains the variable domains of the TCR, such as the variable alpha and variable beta chains, and generally forms a binding site that is sufficient to bind to a particular MHC-peptide complex, such as each chain containing three complementarity-determining regions.

[0210] In some embodiments, the variable domains of the TCR chains bind to form loops or complementarity-determining regions (CDRs) similar to immunoglobulins, which confer antigen recognition by forming the binding site of the TCR molecule and determine peptide specificity. Typically, as with immunoglobulins, the CDRs are separated by framework regions (FRs) (see, e.g., Jores et al., Proc. Nat'l Acad. Sci. U.S.A. 87:9138, 1990; Chothia et al., EMBO J. 7:3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27:55, 2003). In some embodiments, CDR3 is the major CDR for recognition of processed antigen, although it has also been shown that CDR1 of the alpha chain interacts with the N-terminal portion of the antigen peptide and CDR1 of the beta chain interacts with the C-terminal portion of the peptide. CDR2 is thought to recognize the MHC molecule. In some embodiments, the variable region of the beta chain may contain an additional hypervariable (HV4) region.

[0211] In some embodiments, the TCR chain contains a constant domain. For example, similar to immunoglobulins, the extracellular portion of the TCR chain (e.g., α-chain, β-chain) can include two immunoglobulin domains, an N-terminal variable domain (e.g., Va or Vp; typically, amino acids 1-116 based on Kabat numbering, Kabat et al., “Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.), and one constant domain adjacent to the cell membrane (e.g., α-chain constant domain or Ca, typically, amino acids 117-259 based on Kabat, β-chain constant domain or Cp, typically, amino acids 117-295 based on Kabat). For example, in some cases, the extracellular portion of the TCR formed by two chains contains two membrane-proximal constant domains and two membrane-distal variable domains containing CDRs. The constant domain of the TCR domain contains a short connecting sequence where cysteine residues form disulfide bonds, forming a link between the two chains. In some embodiments, the TCR may have additional cysteine residues in each of the α-chain and β-chain such that the TCR contains two disulfide bonds in the constant domain.

[0212] In some embodiments, the TCR chain may contain a transmembrane domain. In some embodiments, the transmembrane domain is positively charged. In some cases, the TCR chain contains a cytoplasmic tail. In some cases, this structure enables the TCR to associate with other molecules such as CD3. For example, a TCR containing a constant domain with a transmembrane region can anchor the protein within the cell membrane and associate with an invariant subunit of the CD3 signaling apparatus or complex.

[0213] Generally, CD3 is a multi - protein complex that can have three different chains (γ, δ, and ε) and a ζ - chain in mammals. For example, in mammals, the complex can contain a CD3γ chain, a CD3δ chain, two CD3ε chains, and a homodimer of the CD3ζ chain. The CD3γ chain, CD3δ chain, and CD3ε chain are highly related cell - surface proteins of the immunoglobulin superfamily that contain a single immunoglobulin domain. The transmembrane regions of the CD3γ, CD3δ, and CD3ε chains are negatively charged, which is a characteristic that allows these chains to associate with the positively charged T - cell receptor chains. The intracellular tails of the CD3γ chain, CD3δ chain, and CD3ε chain each contain a single conserved motif known as an immunoreceptor tyrosine - based activation motif or ITAM, and there are three in each CD3ζ chain. Generally, ITAMs are involved in the signaling ability of the TCR complex. These accessory molecules have negatively charged transmembrane regions and play a role in propagating signals from the TCR intracellularly. The CD3 chains and the ζ - chain, together with the TCR, form what is known as the T - cell receptor complex.

[0214] In some embodiments, the TCR may be a heterodimer of two chains α and β (or optionally, γ and δ), or it may be a single - chain TCR construct. In some embodiments, the TCR is a heterodimer containing two different chains (α - chain and β - chain or γ - chain and δ - chain) linked by one or more disulfide bonds, etc.

[0215] The T - cell receptor (TCR) is a transmembrane protein and does not naturally exist in a soluble form, while antibodies can be not only membrane - bound but also secreted. Importantly, the TCR has the advantage over antibodies in that, when presented in the context of MHC molecules, it can, in principle, recognize peptides generated from all degraded cellular proteins both intracellularly and extracellularly. Thus, the TCR has important therapeutic potential.

[0216] The present disclosure also relates to soluble T cell receptors (sTCRs) containing antigen recognition moieties directed against the tumor neoantigen peptides disclosed herein (see, in particular, Walseng E, Walchli S, Fallang L-E, Yang W, Vefferstad A, Areffard A, et al. (2015) Soluble T-Cell Receptors Produced in Human Cells for Targeted Delivery. PLoS ONE 10(4):e0119559). In certain embodiments, the soluble TCR may be fused to an antibody fragment directed against a T cell antigen, and optionally, the target antigen is CD3 or CD16 (see, for example, Boudousquie, Caroline et al. “Polyfunctional response by ImmTAC (IMCgp100) redirected CD8+ and CD4+ T cells.” Immunology vol. 152, 3 (2017):425-438. doi:10.1111 / imm.12779).

[0217] The present disclosure also encompasses chimeric antigen receptors (CARs) directed against the tumor neoantigen peptides disclosed herein. A CAR is a fusion protein that includes an antigen-binding domain, typically derived from an antibody, linked to a signaling domain of the TCR complex. CARs can be used to direct immune cells, such as T cells or NK cells, against tumor neoantigen peptides, as previously defined, together with a selected and appropriate antigen-binding domain.

[0218] The antigen-binding domain of a CAR is typically based on a scFv (single-chain variable fragment) derived from an antibody. In addition to the N-terminal extracellular antibody-binding domain, a CAR typically includes a hinge domain that functions as a spacer to extend the antigen-binding domain away from the cell membrane of the immune effector cell in which it is expressed, a transmembrane (TM) domain, an intracellular signaling domain (e.g., a signaling domain from the zeta chain of the CD3 molecule (CD3ζ) of the TCR complex, or an equivalent), and optionally, one or more co-stimulatory domains that may assist in the signaling or functionality of the cell expressing the CAR. Signaling domains from co-stimulatory molecules including CD28, OX-40 (CD134), and 4-1BB (CD137) can be added either alone (second generation) or in combination (third generation) to enhance the survival and increase the proliferation of CAR-modified T cells. Potential co-stimulatory domains include ICOS-1, CD27, GITR, and DAP10.

[0219] Thus, a CAR can include the following: (1) In its extracellular portion, one or more antigen-binding molecules such as one or more antigen-binding fragments, domains, or portions of an antibody, or one or more antibody variable domains, and / or antibody molecules. (2) In its transmembrane portion, a transmembrane domain derived from the human T cell receptor-alpha or -beta chain, CD3 zeta chain, CD28, CD3-epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, ICOS, CD154, or GITR. In some embodiments, the transmembrane domain is derived from CD28, CD8, or CD3-zeta. (3) One or more co-stimulatory domains such as co-stimulatory domains derived from human CD28, 4-1BB (CD137), ICOS-1, CD27, OX 40 (CD137), DAP10, and GITR (AITR). In some embodiments, the CAR includes co-stimulatory domains of both CD28 and 4-1BB. (4) In the intracellular signaling domain, an intracellular signaling domain containing one or more ITAMs, for example, the intracellular signaling domain may be CD3-zeta, or a variant thereof lacking one or two ITAMs (e.g., ITAM3 and ITAM2), or the intracellular signaling domain is derived from FcεRIγ.

[0220] The CAR can be designed to recognize a tumor neoantigen peptide alone or in association with an HLA or MHC molecule.

[0221] Exemplary antigen receptors including CARs and recombinant TCRs, as well as methods of manipulating the receptors and methods of introducing them into cells, are described, for example, in International Patent Application Publication Nos. WO200014257, WO2013126726, WO2012 / 129514, WO2014031687, WO2013 / 166321, WO2013 / 071154, WO2013 / 123061, U.S. Patent Application Publication Nos. US2002131960, US2013287748, US20130149337, U.S. Patent Nos.: 6,451,995, 7,446,190, 8,252,592, 8,339,645, 8,398,282, 7,446,179, 6,410,319, 7,070,995, 7,265,209, 7,354,762, 7,446,191, 8,324,353, and 8,479,118, European Patent Application No. EP2537416, and / or those described in Sadelain et al. Cancer Discov. 2013 April; 3(4):388 - 398; Davila et al. (2013) PLoS ONE 8(4):e61338; Turtle et al., Curr. Opin. Immunol., 2012 October; 24(5):633 - 39; Wu et al., Cancer, 2012 March 18(2):160 - 75. In some embodiments, the genetically engineered antigen receptor includes the CAR described in U.S. Patent No. 7,446,190 and those described in International Patent Application Publication No. WO / 2014055668 A1.

[0222] The present disclosure also encompasses polynucleotides encoding antibodies, antigen-binding fragments or derivatives thereof, the aforementioned TCRs and CARs, and vectors comprising the polynucleotides.

[0223] Immune cells The present disclosure further encompasses immune cells that target one or more of the aforementioned tumor neoantigen peptides.

[0224] As used herein, the term "immune cell" includes cells of hematopoietic origin that play a role in the immune response. Immune cells include lymphocytes such as B cells and T cells, natural killer cells, monocytes, macrophages, eosinophils, mast cells, basophils, and myeloid cells such as granulocytes.

[0225] As used herein, the term "T cell" includes cells having a T cell receptor (TCR), particularly a TCR directed against a tumor neoantigen peptide disclosed herein. T cells according to the present disclosure may be selected from the group consisting of helper T lymphocytes including inflammatory T lymphocytes, cytotoxic T lymphocytes, regulatory T lymphocytes, mucosal-associated invariant T cells (MAIT), Υδ T cells, tumor-infiltrating lymphocytes (TIL), or both type 1 and 2 helper T cells and Th17 helper T cells. In another embodiment, the cells can be derived from the group consisting of CD4+ T-lymphocytes and CD8+ T-lymphocytes. The immune cells may be derived from a healthy donor or a subject suffering from cancer.

[0226] The immune cells may be extracted from blood or may be derived from stem cells. The stem cells can be adult stem cells, embryonic stem cells, more specifically, non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells. A representative human cell is a CD34+ cell.

[0227] T cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood collected from a subject using any number of techniques known to those of skill in the art, such as FICOLL™ separation. In one embodiment, cells from the subject's circulating blood are obtained by apheresis. In certain embodiments, T cells are isolated from PBMCs. PBMCs can be isolated from the buffy coat obtained by density gradient centrifugation of whole blood, e.g., centrifugation on a LYMPHOPREP™ gradient, a PERCOLL™ gradient, or a FICOLL™ gradient. T cells can be isolated from PBMCs by depleting monocytes, e.g., using CD14 DYNABEADS®. In some embodiments, red blood cells may be lysed prior to density gradient centrifugation.

[0228] In another embodiment, the cells can be derived from a healthy donor, a subject diagnosed with cancer. The cells can be autologous or allogeneic.

[0229] In allogeneic immunotherapy, immune cells are collected from healthy donors rather than the patient. Typically, these are HLA-matched to reduce the likelihood of graft-versus-host disease. Alternatively, common "off-the-shelf" products that may not require HLA matching include modifications designed to reduce graft-versus-host disease, such as disruption or removal of the TCRαβ receptor. For a review, see Graham et al., Cells. 2018 Oct; 7(10):155. Since a single gene, rather than two genes encoding the beta chain, encodes the alpha chain (TRAC), the TRAC locus is a typical target for removing or disrupting TCRαβ receptor expression. Alternatively, an inhibitor of TCRαβ signaling may be expressed; for example, a truncated form of CD3ζ can act as a TCR inhibitory molecule. Disruption or removal of HLA class I molecules is also employed. For example, Torikai et al., Blood. 2013;122:1341-1349 used ZFNs to knockout the HLA-A locus, while Ren et al., Clin. Cancer Res. 2017;23:2255-2266 knocked out beta-2 microglobulin (B2M) required for HLA class I expression. Ren et al. knocked out TCRαβ, B2M, and the immune checkpoint PD1 simultaneously. Generally, immune cells are activated and expanded as used in adoptive cell therapy. The immune cells disclosed herein can be expanded in vivo or ex vivo. Immune cells, particularly T cells, can generally be activated and expanded using methods known in the art. Generally, T cells proliferate by contacting a surface to which an agent that stimulates CD3 / TCR complex-related signals and a ligand that stimulates co-stimulatory molecules on the surface of the T cells are attached.

[0230] In one embodiment of the present disclosure, immune cells can be modified to be directed against previously defined tumor neoantigen peptides. In certain embodiments, the immune cells may express a recombinant antigen receptor on their cell surface that is directed against the neoantigen peptide. "Recombinant" means an antigen receptor that is not encoded by the cell in its native state, i.e., is heterologous and non-endogenous. Thus, expression of the recombinant antigen receptor can be seen as introducing new antigen specificity into the immune cell, allowing the cell to recognize and bind the aforementioned peptide. The antigen receptor may be isolated from any useful source. In some embodiments, the cell contains one or more nucleic acids introduced via genetic engineering encoding one or more antigen receptors, and the antigen contains at least one tumor neoantigen peptide according to the present disclosure.

[0231] Among the antigen receptors according to the present disclosure are genetically engineered T cell receptors (TCRs) and their components, as well as functional non-TCR antigen receptors such as the chimeric antigen receptors (CARs) described above.

[0232] Methods by which immune cells can be genetically modified to express a recombinant antigen receptor are well known in the art. The nucleic acid molecule encoding the antigen receptor may be introduced into the cell, for example, in the form of a vector or any other suitable nucleic acid construct. Vectors, and their essential components, are well known in the art. The nucleic acid molecule encoding the antigen receptor can be produced using any method known in the art, such as molecular cloning using PCR. The antigen receptor sequence can be modified using commonly used methods such as site-directed mutagenesis.

[0233] The present disclosure also relates to a method of providing a population of T cells that target the tumor neoantigen peptides disclosed herein.

[0234] The population of T cells may include CD8+ T cells, CD4+ T cells, or both CD8+ and CD4+ T cells.

[0235] The T cell population produced according to the present disclosure may be enriched in T cells specific for, i.e., targeting, the tumor neoantigen peptides of the present disclosure. That is, the number of T cells targeting one or more tumor neoantigen peptides of the present disclosure will increase in the T cell population produced according to the present disclosure. For example, the T cell population of the present disclosure has an increased number of T cells targeting tumor neoantigen peptides as compared to the T cells in a sample isolated from a subject. That is, the composition of the T cell population differs from that of a "natural" T cell population (i.e., a population that has not undergone the identification and expansion processes discussed herein) in that the proportion or ratio of T cells targeting tumor neoantigen peptides is increased.

[0236] The T cell population produced according to the present disclosure may be enriched in T cells specific for, i.e., targeting, the tumor neoantigen peptides of the present disclosure. That is, the number of T cells targeting one or more tumor neoantigen peptides of the present disclosure will increase in the T cell population produced according to the present disclosure. For example, the T cell population of the present disclosure has an increased number of T cells targeting tumor neoantigen peptides as compared to the T cells in a sample isolated from a subject. That is, the composition of the T cell population differs from that of a "natural" T cell population (i.e., a population that has not undergone the identification and expansion processes discussed herein) in that the proportion or ratio of T cells targeting tumor neoantigen peptides is increased.

[0237] The T cell population according to the present disclosure may have at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% of T cells targeting the tumor neoantigen peptides disclosed herein. For example, the T cell population may have about 0.2% - 5%, 5% - 10%, 10 - 20%, 20 - 30%, 30 - 40%, 40 - 50%, 50 - 70%, or 70 - 100% of T cells targeting the tumor neoantigen peptides of the present disclosure.

[0238] A population of tumor neoantigen peptide-reactive T cells can have, for example, higher activity than a population of T cells that do not proliferate, using, for example, a tumor neoantigen peptide. The reference to "activity" can represent the response of a T cell population to restimulation with a tumor neoantigen peptide, for example, a peptide corresponding to the peptide used for proliferation, or a mixture of tumor neoantigen peptides. Suitable methods for assaying the response are known in the art. For example, cytokine production can be measured (e.g., IL2 or IFNγ production can be measured). The reference to "higher activity" can include, for example, an increase in activity of 1 to 5-fold, 5 to 10-fold, 10 to 20-fold, 20 to 50-fold, 50 to 100-fold, 100 to 500-fold, 500 to 1000-fold. In one embodiment, the activity can be 1000-fold higher.

[0239] In a preferred embodiment, the present disclosure provides a plurality or population, i.e., two or more T cells, and the plurality of T cells includes T cells that recognize a clonal tumor neoantigen peptide and T cells that recognize a different clonal tumor neoantigen peptide. Accordingly, the present disclosure provides a plurality of T cells that recognize different clonal tumor neoantigen peptides. The different T cells in the plurality or population can alternatively have different TCRs that recognize the same tumor neoantigen peptide.

[0240] In a preferred embodiment, the number of clonal tumor neoantigen peptides recognized by the plurality of T cells is from 2 to 1000. For example, the number of clonal neoantigens recognized can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000, preferably from 2 to 100. There may be a plurality of T cells that have different TCRs but recognize the same clonal neoantigen.

[0241] The T cell population may consist of all or mainly CD8+ T cells, or may consist of all or mainly a mixture of CD8+ T cells and CD4+ T cells, or may consist of all or mainly CD4+ T cells.

[0242] In certain embodiments, the T cell population is generated from T cells isolated from a subject having a tumor. For example, the T cell population may be generated from T cells in a sample isolated from a subject having a tumor. The sample may be a tumor sample, a peripheral blood sample, or a sample from other tissue of the subject.

[0243] In certain embodiments, the T cell population is generated from a sample from a tumor in which a tumor neoantigen peptide has been identified. In other words, the T cell population is isolated from a sample derived from the tumor of the patient to be treated. Such T cells are referred to herein as "tumor infiltrating lymphocytes" (TIL).

[0244] T cells may be isolated using methods well known in the art. For example, T cells may be purified from a single cell suspension generated from a sample based on the expression of CD3, CD4, or CD8. T cells can be concentrated from a sample by passing through a Ficoll-Paque gradient.

[0245] Cancer treatment method In any embodiment, the cancer therapeutics described herein may be used in a method for inhibiting the growth of cancer cells. The cancer therapeutics described herein may also be used for the treatment of cancer in a patient suffering from cancer, or for prophylactic treatment of cancer in a patient at risk of cancer. Cancers that can be treated using the therapies described herein include any solid or non-solid tumor as previously defined. According to the present disclosure, of particular interest are breast cancer, melanoma, and lung cancer. In certain embodiments of the present disclosure, the cancer is non-small cell lung cancer (NSCLC).

[0246] Cancer also includes cancers that are resistant to treatment with other chemotherapeutic agents. As used herein, the term "refractory" refers to cancers (and / or metastases thereof) that do not exhibit an antiproliferative response, or exhibit only a weak response (e.g., no or only weak inhibition of tumor growth) after treatment with another chemotherapeutic agent. These are cancers that cannot be satisfactorily treated with other chemotherapeutic agents. Refractory cancers include (i) not only cancers in which one or more chemotherapeutic agents have already failed during the treatment of the patient, but also (ii) cancers that can be shown to be refractory by other means such as biopsy and culture in the presence of chemotherapeutic agents.

[0247] The therapies described herein are also applicable to the treatment of patients in need thereof who have not been previously treated.

[0248] Subjects according to the present disclosure are typically patients in need thereof who have been diagnosed with cancer or are at risk of developing cancer. Subjects are typically humans, dogs, cats, horses, or any animal in which a tumor-specific immune response is desired.

[0249] The present disclosure also relates to neoantigen peptides, populations of APCs, vaccines or immunogenic compositions, polynucleotides encoding neoantigen peptides or vectors previously defined for use in cancer vaccination therapy of a subject, or polynucleotides for treating cancer of a subject, wherein the peptide binds to at least one MHC molecule of the subject.

[0250] The present disclosure also provides a method for treating cancer in a subject, comprising administering to the subject a vaccine or immunogenic composition described herein in a therapeutically effective amount for treating the subject. The method may additionally include the step of identifying a subject having cancer.

[0251] The present disclosure also relates to a method of treating cancer, which comprises producing an antibody or an antigen-binding fragment thereof by the methods described herein, and administering to a subject having cancer or having immune cells expressing the antibody or the antigen-binding fragment thereof, a therapeutically effective amount thereof for treating the subject.

[0252] The present disclosure also relates to antibodies (including variants and derivatives thereof), T cell receptors (TCRs) (including variants and derivatives thereof), or chimeric antigen receptors (CARs) (including variants and derivatives thereof) directed against the tumor neoantigen peptides described herein, optionally in association with MHC or HLA molecules, for use in the treatment of cancer in a subject, wherein the tumor neoantigen peptides bind to at least one MHC molecule of the subject.

[0253] The present disclosure also relates to antibodies (including variants and derivatives thereof), T cell receptors (TCRs) (including variants and derivatives thereof), or chimeric antigen receptors (CARs) (including variants and derivatives thereof) directed against the tumor neoantigen peptides described herein, optionally in association with MHC or HLA molecules, or immune cells targeting previously defined neoantigen peptides, for use in adoptive cell or CAR-T cell therapy of a subject, wherein the tumor neoantigen peptides bind to at least one MHC molecule of the subject.

[0254] Typically, one of ordinary skill in the art can select an appropriate antigen receptor that binds and recognizes a tumor neoantigen peptide as previously defined to redirect immune cells for use in cancer cell therapy. In certain embodiments, the immune cells for use in the methods of the present disclosure are redirected T cells, such as redirected CD8+ and / or CD4+ T cells.

[0255] In some embodiments, the cancer treatment, vaccination therapy, and / or adoptive cell cancer therapy described above are administered in combination with an additional cancer therapy. In particular, the T cell compositions according to the present disclosure may be administered in combination with checkpoint blockade therapy, co-stimulatory antibodies, chemotherapy and / or radiation therapy, targeted therapy, or monoclonal antibody therapy.

[0256] Checkpoint inhibitors include, but are not limited to, PD-1 inhibitors, PD-L1 inhibitors, Lag-3 inhibitors, Tim-3 inhibitors, TIGIT inhibitors, BTLA inhibitors, V domain Ig suppressor of T cell activation (VISTA) inhibitors, and CTLA-4 inhibitors, IDO inhibitors, and the like. Co-stimulatory antibodies deliver a positive signal via immunomodulatory receptors including, but not limited to, ICOS, CD137, CD27, OX-40, and GITR. In a preferred embodiment, the checkpoint inhibitor is a CTLA-4 inhibitor.

[0257] As used herein, a chemotherapy entity refers to an entity that is destructive to cells, i.e., an entity that reduces the viability of cells. The chemotherapy entity may be a cytotoxic drug. Intended chemotherapeutic agents include, but are not limited to, alkylating agents, anthracyclines, epothilones, nitrosoureas, ethyleneimines / methylmelamines, alkyl sulfonates, alkylating agents, antimetabolites, pyrimidine analogs, epipodophyllotoxins, enzymes such as L-asparaginase; biological response modifiers such as IFNα, IL-2, G-CSF and GM-CSF; cisplatin, oxaliplatin and carboplatin, anthracenediones, substituted ureas such as hydroxyurea, methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine, adrenal cortex suppressants such as mitotane (ο,ρ’-DDD) and aminoglutethimide; hormones and antagonists including adrenal cortex steroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogen agents such as flutamide and the like.

[0258] "In combination" may refer to the administration of additional therapy before, at the time of, or after administration of the T cell composition according to the present disclosure.

[0259] In addition to, or as an alternative to, combination with checkpoint blockade, the T cell compositions of the present disclosure may be genetically modified to be resistant to immune checkpoints using gene editing techniques including but not limited to TALEN and Crispr / Cas. Such methods are known in the art, see, for example, US20140120622. Gene editing techniques can be used to prevent the expression of immune checkpoints expressed by T cells, including but not limited to PD-1, Lag-3, Tim-3, TIGIT, BTLA CTLA-4, and combinations thereof. The T cells discussed herein may be modified by any of these methods.

[0260] The T cells according to the present disclosure may also be genetically modified to express molecules that increase homing to tumors and / or to deliver inflammatory mediators into the tumor microenvironment, including but not limited to cytokines, soluble immune regulatory receptors and / or ligands.

[0261] In certain embodiments, the tumor neoantigen peptides are used in cancer vaccination therapies in combination with another immunotherapy, such as immune checkpoint therapy, and more specifically in combination with anti-PD1 antibody, anti-PDL1 antibody, anti-CTLA-4 antibody, anti-TIM-3 antibody, anti-LAG3 antibody, anti-GITR antibody.

Brief Description of the Drawings

[0262]

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Example

[0263] 1. Example 1: Identification of fusion transcript sequences encoding tumor neoantigen peptides 1.1 Proof of concept in mice To detect individual shared tumor neoantigen peptides emitted from the fusion transcript array, a bioinformatics pipeline has been developed. This pipeline is designed to identify tumor-specific mRNA sequences composed of a part of the TE sequence and a part of the exon sequence. This pipeline means determining the MHC alleles. For each human sample, the class I and class II MHC alleles can be determined using the seq2hla (v2.2) tool (bitbucket.org / sebastian_boegel / seq2hla). In the case of the mouse model, the mouse H-2 alleles are generally known. The bioinformatics method includes mapping of transcripts from RNA sequencing to the reference genome. For the proof-of-concept analysis described here, mm10 was used for mice and hg19 was used for humans. Different versions of the assembled genome can be used, for example, hg19, hg38, mm9, or mm10. This mapping is performed using STAR (v2.5.3a) (github.com / alexdobin / STAR) with the following settings: - To enable multi-hit mapping of the parameter outFilterMultimapNmax that sets the maximum number of loci, make the reads mappable and set it to 1000, - To detect abnormal junctions (fusions), set the parameter chimSegmentMin that sets the minimum length of the fusion segment to 10, and set the parameter chimJunctionOverhangMin that sets the minimum overhang of the fusion junction to 10.

[0264] Junctions from normal (from SJ.out.tab output file) and abnormal (from Chimeric.out.junction output file) are annotated using Ensembl and repeatmasker databases. Normal junctions define all junctions that match the parameters used for mapping (maximum intron length <= 1,000,000 bp (set by --alignIntronMax), same chromosome and well orientation), and abnormal junctions are those that do not match at least one of the previous criteria. This means that TE / exon junctions can be of both junction types, but exon / exon junctions must be in the standard file (SJ.out.tab). Transcript sequences containing junctions between TE sequences and exon sequences are extracted in silico. For regions of the transcript sequence that overlap with the junction, or downstream of the junction if out of frame (non-standard reading frame), the software predicts all possible peptides of 8 or 9 mers in all reading frames. Then, the binding affinity of all these possible peptides to previously defined MHC alleles for the matched samples was determined by netMHCpan (v3.4) (cbs.dtu.dk / services / NetMHCpan / ). Currently, there are more than a dozen different prediction algorithms for predicting peptide binding affinity, and NetMHC is the most widely used and validated algorithm in the neoantigen prediction pipeline.

[0265] Peptides with less than 500 nM or percentile rank less than 2% are considered potential neoantigens. Each splice site (donor or acceptor) is uniquely annotated as either TE or exon. A part of the 5' end is recognized as "donor", and a part of the 3' end is recognized as "acceptor".

[0266] Predicted HLA-binding peptides shared between cancer and normal tissues are excluded from further analysis.

[0267] This method has been applied to RNAseq data obtained from seven well-characterized mouse tumor cell lines (B16F10, B16F10-OVA, MCA101, MCA101-OVA, MC38, MC38-GFP, MC38-GFP-OVA). Cell lines with -OVA extensions correspond to the same model but further express ovalbumin. In this study, this lineage is considered a similar model. For example, assays performed on cell lines from B16F10-OVA are considered a repeat of assays performed on cell lines from B16F10.

[0268] Using these parameters (Figures 1A, 1B, and 1C), a list of candidate peptides was obtained, some of which were specific to a particular cell line (Figures 1A and 1B), and some of which were shared between two tumor cell lines (Figure 1C).

[0269] For validation, we selected a range of peptides expressed in either B16F10-OVA or MCA101-OVA with a predicted affinity of less than 500 nM. Peptides were selected to optimize the ratio between read counts and predicted affinity for MHC-I.

[0270] Four predicted tumor neoantigen peptides were selected and characterized by identifying the TE and exon sequences (Table 3).

Table 2

[0271] 1.2 Verification by RT-PCR of Fusion Transcript Sequences First, verification by conventional RT-PCR has been performed using a primer pair of one primer for the TE sequence and the other primer for the exon sequence.

[0272] For RNA extraction and reverse transcription, 3 - 5.10 6The cells were lysed in 500 μL of TRIzol, and 100 μL of phenol-chloroform was added to the solubilized solution before centrifugation. The aqueous phase was collected, mixed with 100% EtOH in a 1:1 ratio, and transferred to an RNAeasy minikit column. Subsequently, RNA was recovered according to the manufacturer's instructions (including on-column DNase treatment). After RNA elution, DNA contaminants were further removed by treatment with Turbo DNase (Fisher scientific) according to the manufacturer's instructions. RNA concentration was measured using a nanodrop, and 1 μg of RNA was used for reverse transcription. First-strand synthesis was performed with Superscript III (Life technologies) using oligo dT(15) as a primer according to the manufacturer's instructions. Primers were ordered from Eurogentec. PCR reactions were carried out using Taq polymerase. After identifying the optimal conditions for each reaction, the PCR products were extracted from the agarose gel and sequenced using GATC lightrun. Sequence alignment was confirmed with APE software.

[0273] Using this approach, bands corresponding to the predicted sizes of N25, N26, N90, and N94 were each detected in the cell lines identified in Table 1 (see Figure 2A for N25). Interestingly, N26 was detected only in silico in MCA and MC38 cells by RNAseq as described above in the pipeline, but using RT-PCR, a band corresponding to N26 in B16F10-OVA cells was detected (Figure 2B), indicating that this sequence is shared among three independent tumor cell lines (MCA, MC38, and B16F10). By re-analyzing the RNAseq data, it was discovered that the N26 junction is present in B16F10-OVA cells but below the detection threshold of the algorithm. Furthermore, sequencing of the RT-PCR product showed a perfect match with the sequence predicted by the algorithm.

[0274] 1.3 In vivo immunization of mice To verify these candidates in vivo, short (9-mer) peptides corresponding to neoantigen peptides that bind to the MHC class I sequence were synthesized. For in vivo assays, long (27-mer) peptides containing the adjacent region of the short peptides with predicted MHC binding of 9mers were synthesized. This is because this length is more suitable for in vivo immunity. B16F10 OVA and MCA101-OVA were maintained in RPMI, Glutamax, 10% FCS, 1% penicillin-streptomycin and passaged using TrypLE. Cells were kept in culture for up to one month and new vials were thawed for each in vivo experiment. C57BL6J recipient mice were immunized by subcutaneous injection into the flank with 100 μg of long peptide (N25L or N26L), SIINFEKL peptide (short OVA peptide), OVA (Sigma) or DMSO, each with 50 μg of poly I:C. Seven days after the primary immunization, the immunization was repeated. Three days later (10 days after the primary immunization), the animals were sacrificed and the number of peptide-specific IFNg-secreting CD8 T cells in the inguinal lymph nodes was detected by ELISPOT (Figure 3A). Short peptides (N25, N26, or SIINFEKL) or DMSO were used at 10 μg.mL -1 to restimulate the T cells. Alternatively, seven days after the secondary immunization, the animals were injected subcutaneously with 2.5.10 5 of B16F10-OVA or 5.10 5 of MCA-OVA cells in PBS. We found that N25, and to a lesser extent N26, were able to induce an immune response (Figure 3B).

[0275] 1.4 In Vivo Treatment of Mice with Tumors To verify whether these peptides are protective against tumor cells, C57BL6 mice were immunized on d0 and d7 with 100 mg of peptide N25L or N26L, or OVA (control peptide), and 50 μg of poly I:C in PBS, and on d14, 2.5.10 5B16F10-OVA cells were injected. B16F10 OVA and MCA101-OVA were maintained in RPMI, Glutamax, 10% FCS, 1% penicillin-streptomycin and passaged using TrypLE. Cells were kept in culture for up to one month and new vials were thawed for each in vivo experiment. C57BL6J recipient mice were immunized by subcutaneous injection into the flank with 100 μg of the long peptides (N25L or N26L), OVA (Sigma) or DMSO, each with 50 μg of poly I:C. The immunization was repeated 7 days after the primary immunization.

[0276] Short peptides (N25, N26, or SIINFEKL) or DMSO at 10 μg.mL -1 were used to restimulate the T cells. Alternatively, 7 days after the secondary immunization, the animals were injected subcutaneously with 2.5×10 5 B16F10-OVA or 5×10 5 MCA-OVA cells in PBS. Tumor size was measured twice a week using manual calipers and the health status of the animals was monitored throughout the experimental period (Figures 4A and 4B). Animals were sacrificed when the tumor volume reached 1 mm 3 . Surprisingly, it was observed that N25L significantly delayed the formation of B16OVA tumors in a more efficient manner than OVA. Furthermore, similar results were obtained with N26L immunization.

[0277] Example 2: Identification of human lung adenocarcinoma (LUAD) neoantigen peptides derived from fusion transcripts composed of TE elements and exon sequences 2.1 Materials and methods RNA extraction. Tumor samples and adjacent tumor samples were #1 mm 3Cut into fragments and resuspended in 700 μl of RTL lysis buffer (Quiagen) supplemented with 1% β-mercaptoethanol, and homogenized using a Perecellys 24 Tissue Homogenizer (Bertin Technologies). Total RNA isolation was performed using the RNeasy Micro Kit (Qiagen) according to the manufacturer's instructions. Total RNA from the tumor cell line was extracted from the 5.10 6 tumor cell line using the same procedure.

[0278] PCR and sequencing. Primers were designed using APE software. For each sample, 1 μg of RNA was reverse transcribed into cDNA using SuperScript III reverse transcriptase (ThermoFisher) as per the provider's instructions. The PCR reaction was performed using GoTaq G2 Hot Start Polymarase (Promega). All primers were used at a concentration of 0.5 μM. The reaction was carried out in a Veriti™ 96-well thermal cycler (ThermoFisher). The PCR products were loaded onto a LabChip GX (Caliper LifeSciences) and analyzed using LabChip GX software (v4.2).

[0279] For these samples containing amplification products of the expected size, the PCR reaction was repeated. The PCR products were then electrophoresed on a 2% agarose gel with SYBR Free Dye (1 / 10000) (Invitrogen). Specific bands were excised and the DNA products were purified using the QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer's instructions. Finally, these products were sequenced by EuroFins Scientific. The obtained sequences were compared with those predicted using Serial Cloner software.

[0280] Tetramer formation. HLA-A2 monomers were purchased from ImmunAware®. Tetramer formation was evaluated according to the manufacturer's instructions using synthetic ER-derived peptides. Briefly, synthetic HLA-A2 monomers were incubated with synthetic peptides for 48 hours at 18°C. Tetramer formation was performed by further incubating the monomers with biotinylated sepharose. Finally, tetramer formation was measured by flow cytometry using a PE-conjugated anti-β2 microglobulin antibody. A CMV-derived peptide provided by the manufacturer was used as a positive control.

[0281] In experiments to evaluate the presence of specific CD8+ T cells, the tetramerization step was performed by incubating the monomers with different combinations of fluorescent streptavidin (PE, APC, PE-Cy5, PE-CF594, BV421, BV711, and FITC).

[0282] Naïve CTL priming. PBMCs were obtained by Ficoll gradient separation from HLA-A2+ healthy blood donors. CD14+, CD4+, and CD8+ cells were purified by positive selection using magnetic beads (Miltenyi Biotec). CD4+ T cells and CD8+ T cells were cryopreserved until the day of the experiment, while the CD14+ fraction was cultured at 106 cells / mL for 5 days in the presence of IL-4 (50 ng / mL) and GM-CSF (10 ng / mL) to obtain moDCs. After this period, moDCs were matured with LPS and incubated with synthetic ER-derived peptides at a final concentration of 1 μg / mL for 2 hours. Finally, peptide-loaded moDCs were co-cultured with autologous CD4+ and CD8+ T cells in culture medium supplemented with IL-2 (10 U / ml) and IL-7 (100 ng / ml). ER-derived peptide stimulation of specific CD8+ CTL populations was evaluated by MHC-I tetramer staining by flow cytometry using a combination of two-color tetramers for each peptide.

[0283] Tetramer staining. Cells were resuspended in PBS, stained with Live / Dead Aqua-405nm (ThermoFisher) for 20 minutes at 4°C, and washed once. Then, the cells were resuspended in PBS-1% BSA containing a mixture of SA-conjugated tetramers and incubated for 20 minutes at room temperature in the dark. Without further washing, surface antibodies were added to PBS-1% BSA and the cells were incubated for 20 minutes at 4°C in the dark. The surface antibodies were, as needed, a combination of anti-CD3-BV650 + anti-CD8-PECy7 and anti-CCR7-AF700 + anti-CD45RA-BUV395. Finally, the cells were washed twice and resuspended in FACS buffer for flow cytometry analysis.

[0284] CTL-clone generation. Tetramer-positive cells were single-cell FACS sorted (ARIA sorter, BD) in a U-bottom 96-well plate. The sorted cells were collected in 100 μL of RPMI 10% human serum AB (Sigma-Aldrich) containing 150,000 feeder cells. Finally, 100 μL of AIM medium containing IL-2 (3000 IU / ml) and anti-CD3 (100 μg / ml, Miltenyi's OKT3 clone) was added and the cells were cultured for up to 15 - 20 days. If obvious cell growth was observed in the wells, a second round of growth was performed for up to 15 days using fresh feeder cells. The cells were fed and split during this period as needed with the same culture medium (AIM-RPMI 50 / 50 + 5% human serum) but containing IL-2 at 500 IU / ml. Finally, the specificity of the expanded clones was checked by FAC-tetramer staining and only clones with more than 85% of tetramer-positive clones were used for further analysis.

[0285] Cytotoxicity assay. To perform the cytotoxicity assay, an xCELLigence RTCA S16 Real Time Cell Analyzer was used. The H1650 cell line was seeded at 0.5×10 6They were seeded at cells / ml. One day later, the cells were either incubated for 1 hour with the corresponding synthetic peptides at different concentrations or not. Then, the cells were washed twice with the medium and either incubated for an additional 30 minutes with the anti-MHC-I antibody (clone W6 / 32, 50 μg / well) at the same concentration or an isotype control or not. Without additional washing, CTL-clones were added at the corresponding ratios. The complete assay was performed at 37 °C in 200 μl of serum-free medium connected to the xCELLigence system. Impedance variations (cell index) were measured in real time for 40 hours. Each condition was performed in duplicate.

[0286] Cytokine secretion and activation of Jurkat cells. 50,000 H1650 cells were seeded in a 96-well plate with medium supplemented with 5% fetal bovine serum. The next day, the cells were cultured for 1 - 2 hours with synthetic peptides at different final concentrations. Then, the cells were washed twice, CTL clones were added at a 1:1 ratio, and co-cultured with peptide-loaded target cells for 18 hours. The culture supernatant was collected and cytokine concentrations were analyzed by cytokine bead array (CBA, BD Biosciences) according to the manufacturer's instructions.

[0287] The same experiment was performed using Jurkat cells transduced instead of CTL-clones and two different types of target cells: H1650 and H1395 cell lines. In this assay, after co-culture with peptide-loaded target cells, Jurkat cells were evaluated by flow cytometry analyzing the expression of a reporter marker. PMA / ionomycin was used as a positive control to activate Jurkat cells.

[0288] Tissue and blood samples. Lung tumor, adjacent tumor, and lymph node samples were minced and digested in a final volume of 2 ml of medium (CO2 Independent Medium + 5) with a mixture of collagenase-I (2 mg / ml), hyaluronidase (2 mg / ml), and DNase (25 μg / ml) for 40 minutes at 37°C. After digestion, single cell suspensions were collected via a cell strainer and washed. Tumor and adjacent tumor suspensions were enriched in the lymphocyte fraction by Ficoll gradient. Subsequently, cells were stained for tetramer analysis by FAC as described above.

[0289] Blood samples were seeded onto Ficoll gradients and PBMCs were isolated. Subsequently, PBMCs were enriched for CD8+ T cells using the EasyStep Human CD8+ T Cell Enrichment Kit (STEMCELL Technologies). Finally, the enriched cells were stained for tetramer analysis as described above.

[0290] Tumor-infiltrating lymphocyte (TIL) culture. Tumor tissues were minced (1 - 3 mm 3 in size, up to 6 - 12 pieces). Each tumor fragment was transferred to an individual well from a 24-well plate and cultured in a final volume of 2 ml of RPMI 10% human serum + IL-2 6000 IU / ml. Cells were fed / split as needed over 15 - 20 days and cryopreserved or analyzed for tetramer staining.

[0291] TCR cloning. Total RNA was extracted from CTL-clones and reverse transcribed into cDNA using SuperScript III (ThermoFisher). TCRα and β were amplified by PCR as described by Li et al 2019. DNA products were electrophoresed on a 2% agarose gel and sequenced after gel band extraction (Qiagen). TCR V regions (α and β) were ligated to mouse TCR constant chains, cloned into the PEW-pEF1A-inactEGFP vector, and amplified in transformed bacteria.

[0292] Jacket transduction. Lentiviral particles were produced by HEK-293FT cell lines transfected with a TCR expression plasmid together with an envelope (pVSVG) plasmid and a packaging (psPAX2) plasmid. After 64 hours, the supernatant was collected and the lentiviral particles were concentrated using a 100 kDa centrifugal filter (Sigma-Aldrich). The lentiviral suspension was transferred by spinoculation to TCR-negative jacket cells expressing reporter genes (NFAT-GPF, NF-KB-CFP, and AP-1-mCherry). Five days later, the gene transfer efficiency was evaluated by FACS using an anti-mouse TCR-β antibody (clone H57-597). This jacket cell is described in Rosskopf S. et al. 2018.

[0293] Mass spectrometry data analysis. Raw data of the public immune peptide mix derived from MHC eluted peptides were analyzed using ProteomeDiscoverer 1.4 (ThermoFisher) with the following parameters: no enzyme, peptide length 8 - 15 aa, precursor mass tolerance 20 ppm, and fragment mass tolerance 0.02 Da. Methionine was enabled as a variable modification and a 1% false discovery rate (FDR) was applied. MS / MS spectra were searched against the human proteome from Uniprot / SwissProt (updated 06.03.2020) linked to a list of all fusion transcript-derived proteins from the lung TCGA project. Finally, peptides matching the Uniprot database or peptides matching the translated fusion transcripts present in lung normal samples were discarded.

[0294] 2.2 Results: Identification of fusion transcript sequences encoding tumor neoantigen peptides in human subjects 2.2.1 Characterization of neoantigens First, the TE-exon fusion transcript landscape was characterized in normal samples from the TCGA public database. A total of 8,876 unique fusions were identified in 679 normal samples from 19 different tissues (bile duct, bladder, brain, breast, cervix, colon, head and neck, kidney, liver, pancreas, PCPG, prostate, rectum, sarcoma, skin, thymus, thyroid, uterus). Specific fusions for each tissue type were found in only a small fraction of pan-tissue fusion transcripts. These results suggest that dedicated tissue-specific regulatory mechanisms are associated with these fusion transcripts.

[0295] Next, the number of fusions identified in 514 LUAD samples from TCGA was compared with 59 normal related lung samples present in TCGA. An average of 235 fusions were identified in NSCLC samples compared to 200 in healthy lung samples (Wilcoxon pvalue = 9×10. -10 )). A total of 8,269 unique fusions were identified in NSCLC tumors.

[0296] The first category, called TSF (tumor-specific fusion), was obtained as fusions seen in at least 1% of tumor samples and not at all in normal samples. Thus, 210 fusions were defined as TSF.

[0297] Fusion transcripts that are frequent in tumors and low-frequency in normal cells may also be good candidates for neoantigens. Thus, a second category, called TAF (tumor-associated fusion), was specifically defined as fusions present in less than 4%, particularly less than 2%, and more than 10% of tumors in normal tissue and overexpressed in tumors compared to normal tissue samples.

[0298] Tables 3 and 4 (see below) describe the fusions according to whether the exon or TE is the donor. The first column shows the frequency of the fusions in the NSCLC cohort. The donor and acceptor columns introduce the type of each element. All columns starting with "donor" (each "acceptor") are information about the donor (each acceptor). The sequences of the fusions can be retrieved as follows: - Donor sequence: on chromosome "Donor_chromosome_X" from "Donor_start_X" to "Donor_breakpoint_X" of the strand "Donor_strand_X" - Acceptor sequence: on chromosome "Acceptor_chromosome_X" from "Acceptor_end_X" to "Acceptor_breakpoint_X" of the strand "Donor_strand_X"

[0299] Note that if the fusion is on the minus strand, the reverse complement of the sequence should be taken.

[0300] Fusion sequence: - To reconstruct the fusion nucleotide sequence, the donor sequence on chromosome "Donor_chromosome_X" from "Donor_start_X" to "Donor_breakpoint_X" of the strand "Donor_strand_X" and the acceptor sequence on chromosome "Acceptor_chromosome_X" from "Acceptor_breakpoint_X" to "Acceptor_end_X" of the strand "Acceptor_strand_X" were extracted from the Ensembl HG19 human assembly database. Note that the use of the Ensembl HG19 human database is not restrictive, and other compatible databases such as the NCBI reference sequence database (RefSeq) can also be used. - Note that if the fusion is on the minus strand, the reverse complement of the sequence should be taken. - The "fusion sequence" consists of the acceptor sequence following the donor sequence.

[0301] Nucleotide sequence of the fusion transcript: All "fusion transcripts" were reconstructed based on known standard transcripts involving exons. When the donor is an exon (see Figure 9A) · It starts from the start of the standard transcript to the donor exon and replaces the complete standard exon sequence with the fusion sequence. In this case, the fusion transcript stops after the TE sequence of the acceptor. When the donor is a TE (Figure 9B) · The array starts at the standard position of the acceptor exon in the transcript, forgetting all upstream exons. The standard array of the acceptor exon was replaced with the fusion array, and the transcript was reconstituted to the end.

[0302] Next, each nucleotide sequence of size k of the fusion transcript (i.e., 24 - 75 nucleotides) (the translation of the first k-mer starts at the first nucleotide of the fusion transcript, the translation of the second k-mer starts at the second nucleotide of the fusion transcript, etc.) was translated into a peptide sequence.

[0303] Next, the resulting peptides were further analyzed using NetMHCpan for MHC binding prediction. Thus, the affinity to bind to at least one of the known human alleles was predicted for each k-mer present in the sequence (see also Example 1 for further explanation).

[0304] Table 3. Coordinates of the fusion sequences where the donor is an exon. The column names are as follows. 1. Frequency in the LUAD cohort 2. Donor chromosomal exon 3. Donor start exon 4. Donor breakpoint exon 5. Donor strand exon 6. Donor transcript (i.e., donor_tx_name_exon) 7. Acceptor chromosomal TE 8. Acceptor breakpoint TE 9. Acceptor end TE 10. Acceptor strand TE 11. Type of fusion

[0305] The fusion transcript arrays in Table 3 correspond to SEQ ID NOs: 118 to 431 in the same order (typically, the first row corresponds to SEQ ID NO: 118, the second row corresponds to SEQ ID NO: 119, and the third row corresponds to SEQ ID NOs: 120 and 121. This is due to, for example, two donor transcripts (ENST00000296474 and ENST00000344206, respectively, as shown in the sixth column)).

Table 3-1

Table 3-2

Table 3-3

Table 3-4

Table 3-5

Table 3-6

Table 3-7

Table 3-8

Table 4-1

Table 4-2

Table 4-3

Table 4-4

Table 4-5

Table 4-6

Table 4-7

Table 4-8

Table 4-9

Table 4-10

Table 4-11

Table 4-12

Table 4-13

Table 4-14

Table 4-15

[0306] Next, the peptides were further screened against the reference proteome, typically against all the sequences present in Uniprot (representing all the sequences encoded by the human exome) in the case of a human subject. Peptides were considered equivalent to Uniprot peptides if they had the same amino acid sequence or differed only in amino acids at the first or last position. Then, all these equal sequences were discarded from the candidate list. Thus, 117 peptide sequences derived from these 230 fusion transcripts predicted to bind to HLA-A2:01 (see Table 5 below).

Table 5

[0307] 2.2.2 Validation of HLA-A2-related peptides Considering that the HLA-A2 allele is expressed in approximately 50% of the white population, along with the existence of different technical tools, the validation focused on HLA-A2-related peptides.

[0308] In the following paragraphs, TE-exon-derived transcripts are used interchangeably with "fusion transcripts", and the term "TE-derived peptide" is used interchangeably with "fusion-transcript-derived peptide".

[0309] Expression of TE-exon-derived transcripts in lung adenocarcinoma samples To experimentally validate the predicted TE-exon transcripts, expression by PCR in LUAD tumor samples and tumor cell lines was first verified. Thus, specific primers for each chimeric fusion were designed such that one of them binds to the TE part of the fusion and the other binds to the exon part of the fusion. The results were further confirmed by sequencing of the PCR products.

[0310] Specifically, specific primers were designed such that the forward primer binds to the "donor" sequence of the rearranged fusion array and the reverse primer binds to the "acceptor" sequence of the rearranged fusion array. The PCR reaction was carried out on RNA derived from lung tumor samples and human tumor cell lines. The amplification products were seeded on an agarose gel, and the bands found at the expected sizes were excised and sequenced. Finally, the sequenced PCR products were compared with the rearranged fusion sequences.

[0311] Using this approach, the presence of predicted fusion transcripts could be confirmed in both LUAD tumor samples and tumor cell lines. Table 6 below summarizes the results obtained for the 8 most frequent chimeric fusions having peptides predicted to bind with high affinity to the HLA-A2 allele.

[0312] Table 6: Validation of the most frequent fusion transcripts. The most frequent fusion peptides were verified by PCR in 15 LUAD tumor samples and 6 LUAD tumor cell lines. The status "present" or "absent" in the following table indicates the presence or absence of the PCR product at the expected size. When the PCR product was further verified by sequencing, it is indicated as "present".

Table 6

[0313] Binding of ER-derived peptides to HLA-A2 molecules Once the expression of the chimeric transcripts was confirmed, the induction peptides were synthesized and their binding to HLA-A2 was confirmed. Since monomer stabilization and tetramer formation are only possible in the presence of high-affinity binding peptides, the formation of HLA-A2 tetramers was estimated by flow cytometry in the presence of the synthetic peptides. All of the predicted peptides showed a fluorescence percentage exceeding 50% compared to the positive control and were able to stabilize tetramer formation. As a positive control, a known high-affinity binding peptide to HLA-A2 derived from cytomegalovirus (CMV) was used. This result confirmed the predicted high-affinity binding to the HLA-A2 allele. Figure 10 shows the results of 10 peptides derived from the most frequent fusion peptides.

[0314] Immunogenicity of ER-derived peptides The next step after verifying the binding to the HLA-A2 allele was to test the immunogenicity of the predicted peptides. Therefore, a priming assay was performed to verify the ability of the identified peptides to expand specific cytotoxic T cells. Monocyte-derived DCs (moDCs) were generated using PBMCs from healthy HLA-A2+ donors. After loading a mixture of synthetic peptides onto the moDCs, autologous co-culture was performed with CD4+ and CD8+ T cells. Finally, the expansion of specific CD8+ T cells was analyzed by flow cytometry using two-color tetramer staining. As a control for specific expansion, co-culture was performed in the absence of peptides. By using this approach for one donor, it was possible to identify and expand specific CD8+ T cells that recognize six of the most frequent chimeric fusion-derived peptides (RLLHLESFL, LLGETKVYV, AILPKANTV, RLADHLSFC, FLIVAEILI, YLWTFFPL). This result was shown by at least an order of magnitude increase in the percentage of tetramer-positive cells compared to the control test of total CD8+ T cells.

[0315] To evaluate the responses in an additional five donors, the same experiment was performed. Figure 11A summarizes the results obtained for a total of six donors analyzed, and CD8+ T proliferation specific for 21 of the most frequent fusion transcript-derived peptides was observed (YLWTTFFPL, FLGTRVTRV, RLADHLSFC, LLGETKVYV, MLVTWELAL, MLMKTVWQA, SLMQSGSPV, AILPKANTV, AMDGKELSL, LLDRFGYHV, GLLNISHTA, ILTASITSI, ILSGYGPCV, RQAPGFHHA, GLPSHVELA, ILHSLVTGV, LLHLESFLV, VLLTNTIWL, LLTSWHLYL). These experiments demonstrate that these peptides can induce an immune response and confirm the immunogenicity of ER-derived peptides.

[0316] Generation of cytotoxic T lymphocyte clones recognizing ER-derived peptides Proliferating CD8+ tetramer-positive T cells from the immunogenicity assay (Figure 11A) were single cell FACS sorted to generate cytotoxic T lymphocyte (CTL) clones. Ten clones were generated that recognize five different ER-derived peptides: YLWTTFFPL, LLGETKVYV, MLVTWELAL, MLMKTVWQA, RLADHLSF. These peptides are listed in Table 5 as peptide 9, 86, 53, 80 and 64 respectively. Referring to these numbers indicates the specificity of each CTL-clone generated. As an example, CTL-clone 9 recognizes ER-derived peptide 9.

[0317] To evaluate the cytotoxic ability of the generated CTL-clones, two different functional assays were performed using the H1650 cell line as the target cell. This is a LUAD-derived tumor cell line that expresses the HLA-A2 allele.

[0318] First, the ability of CTL-clones to secrete cytokines after exposure to ER-derived peptides was measured. CTL-clones were co-cultured with target cells loaded with a specific ER-derived peptide for 18 hours, and then the secretion of INF-γ, TNF, and granzyme-B (Gr-B) in the culture supernatant was measured. All CTL-clones were activated after exposure to a specific ER-derived peptide and secreted cytokines in a dose-dependent manner (Figure 11B).

[0319] In the second set of experiments, the killing ability of CTL clones was evaluated. CTL-clones were co-cultured with target cells loaded or not loaded with ER-derived peptides under different conditions. The xCELLigence system was used to measure real-time impedance fluctuations in the monolayer of target cells. In these assays, a decrease in the cell index is associated with a decrease in the number of cells in the monolayer, which reflects cell viability.

[0320] When CTL-clone 9 was co-cultured with target cells loaded with peptide 9 derived from ER at a ratio of 1:1, a decrease in the cell index was observed over time compared to control cells (target cells only). When co-culture was performed in the presence of a blocking anti-MHC-I antibody (+ anti-MHC-I), this decrease in the cell index was suppressed. When co-culture was performed using an isotype control at the same concentration (+ isotype), the decrease in the cell index was not suppressed. Furthermore, when target cells were loaded with a high concentration of peptide (1 pM compared to 1 uM), these decreases increased (Figure 11C, left panel). This result indicates that cytotoxic T cells that recognize the peptide identified by CTL-clone 9 in the method disclosed herein are killing target tumor cells.

[0321] When ER-derived peptides are naturally expressed and presented by target cells, it was inferred that they should be able to kill them by co-culturing with CTL-clones without adding the peptides externally. For this purpose, co-cultures of CTL-clone 9 and H1650 target cells were performed at different ratios to find a ratio sufficient for the effector to kill the target cells. In the right panel of Figure 11C, it was found that CTL-9 could kill target cells at an effector-to-target ratio of 4:1 compared to control cells (target cells only). Furthermore, the killing power increases when the ratio is increased (8:1). Target cell death was not observed at lower ratios (2:1).

[0322] Finally, similar experiments were performed using CTL-clone 9, CTL-clone 64, and CTL-clone 80, and it was found that specific killing of target cells was observed, but this could also be suppressed when co-culture was performed in the presence of anti-MHC-I antibody (Figure 11D).

[0323] Collectively, these results confirm that cytotoxic T cells that recognize several different peptides identified by the methods disclosed herein can recognize and kill tumor cells expressing peptides derived from specific fusion transcripts, and that this effect is due to specific recognition of the peptides in the context of MHC-I molecules. Furthermore, the fact that CTL-clones can kill target cells without adding external peptides indicates that fusion transcript-derived peptides 9, 64, and 80 are naturally expressed and presented by the H1650 LUAD tumor cell line.

[0324] Generation of Engineered T Cells Recognizing Fusion-Derived Peptides Jurkat cells transduced with a lentiviral vector encoding the CTL-9 TCR sequence were co-cultured with two different target cells, H1650 and H1395. Both are LUAD-derived cell lines that express the HLA-A2 allele. Activation of Jurkat cells via the TCR was evaluated by flow cytometry analyzing the increase in fluorescence of reporter genes (NFAT-GFP, NF-κB-CFP, and AP-1-mCherry). Preliminary results indicate that Jurkat cells are activated when co-cultured with both target cells compared to the negative control (non-transduced Jurkat cells). Furthermore, this activation increased in a dose-dependent manner when co-culture was performed with target cells loaded with specific peptides. PMA / ionomycin was used as a positive control (Figure 12). These results are consistent with those shown in Figures 11C and D, suggesting that LUAD-derived tumor cells express TE-derived peptides. Furthermore, the potential use of CTL-clone TCR sequences in the generation of engineered T cells was demonstrated.

[0325] Presence of CD8+ cells recognizing fusion-derived peptides in LUAD patients The aim was to identify the presence of CTL cells recognizing fusion-derived peptides in LUAD tumor samples.

[0326] In a first series of experiments, tumor-infiltrating lymphocytes (TILs) grown with a mixture of TE-derived peptides and Il-2, or Il-2 alone, were analyzed by tetramer staining. As shown in Figures 13A and B, CD8+ T cells recognizing fusion-derived peptides were found in TILs derived from LUAD patients.

[0327] Next, we analyzed whether tetramer-positive cells and their phenotypes were detected in non-proliferating CD8+ T cells derived from fresh tumor samples. Using this strategy, we analyzed CD8+ T cells present in tumors, adjacent-tumor, infiltrating lymph nodes, and blood derived from LUAD patient samples. Phenotypes were determined as naive (CCR7+CD45+), central memory (CM, CCR7+CD45RA-), effector memory (EM, CCR7-CD45-), and terminal effector (TE, CCR7-CD45+), considering the expression of the surface markers CCR7 and CD45RA. Interestingly, tetramer-positive cells present in tumor tissue preferentially shared a memory phenotype, and naive cells (CCR7+CD45+) were mainly found in cells derived from lymph nodes (Figures 14A and B). Patients 2 and 3 are the same in Figures 13 and 14.

[0328] All samples examined were derived from HLA-A2+ patients.

[0329] The presence of tetramer-positive cells with a memory phenotype in tumor tissue, together with the presence of tetramer-positive cells in TIL, is consistent with the immune response generated against TE-derived peptides in these patients. Furthermore, the presence of naive tetramer-positive cells in lymph nodes suggests the potential of these to generate an immune response, particularly against TE-derived peptides.

[0330] Peptide identification by mass spectrometry in LUAD biopsies. To be recognized by cytotoxic T cells, ER-derived peptides need to be presented by MHC class I molecules on the surface of tumor cells. To confirm that the predicted peptides are expressed on MHC class I molecules, public data from the MHC I immunopeptidomes derived from three LUAD biopsies (Laumont CM et al., “Noncoding regions are the main source of targetable tumor-specific antigens” Sci Transl Med. 2018 10(470)) were used. Raw data uploaded to the PRIDE database from MHC-I immunopurification of three LUAD tumors (PXD009752, PXD009754, and PXD009755) were analyzed using OpenMS software. It should be noted that data-dependent acquisition in proteomics only enables the identification of those sequences included in the target database (generally the entire human proteome). Since the peptides according to the present application are derived from non-coding sequences, they have not been previously identified. MS / MS identifications incorporating the sequences of the peptides predicted herein into the target database were reanalyzed. Five peptides (peptide IDs: 3304, 269, 757, 1810, 3953) were found in three of the sample biopsies. To perform this analysis, all predicted peptides derived from chimeric fusions present in at least five samples in TCGA that bind to any MHC I allele were considered. This result confirms the expression of chimeric fusion-derived peptides on MHC class I molecules in LUAD tumors.

[0331] Subsequently, the analysis was extended to a dataset of new lung immune peptide mixes (Bulik-Sullivan et al. Nat. Biotec 2018, Chong et al. Nat. Comm. 2020 and Javitt et al. Front Immunol 2019). Notably, all databases were generated from fresh lung tumor samples, except for Javitt et al. Front Immunol 2019 which contained LUAD tumor cell lines. In this second analysis, ProteomeDiscoverer 1.4 Software was used to identify ER-derived peptides. Considering four datasets, 23 unique ER-derived peptides were present in at least one of a total of 19 immunopeptidomic samples. In Figure 15, the ER-derived peptides (rows) identified in each MHC sample (columns) are shown as gray squares. On the right, the found peptide sequences are shown. Interestingly, some of them were observed in multiple MHC samples, indicating that they are shared among samples. From these results, it was confirmed that peptides derived from fusion transcripts are processed and presented by HLA-I molecules on the tumor cell surface.

[0332] The peptide RLADHLSFC (fusion ID: chr22:29117506:->chr22:29115473:- / involved gene: CHEK2) derived from a fusion transcript where the gene part of the fusion is a tumor suppressor gene, and the peptide GLPSHVELA (fusion ID: chr6:117763597:->chr6:117739669:- / involved gene: ROS1) derived from a fusion transcript where the gene part of the fusion is an oncogene. Interestingly, both peptides were found to be immunogenic (Figure 11A), and in particular, in the case of the peptide RLADHLSFC, the results shown in Figure 11D indicate that it can be expressed by the H1650 cell line. Furthermore, TILs recognizing the peptide GLPSHVELA were found (Figure 12A), indicating that this fusion transcript-derived peptide can be expressed in LUAD tumor samples.

[0333] Example 3: Identification of neoantigen peptides derived from fusion transcripts composed of TE elements and exon sequences from various cancer samples. 9184 samples from 32 different cancer types (acute myeloid leukemia, adrenocortical carcinoma, bladder urothelial carcinoma, ductal breast carcinoma, lobular breast carcinoma, cervical cancer, cholangiocarcinoma, colorectal adenocarcinoma, esophageal cancer, gastric adenocarcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, hepatocellular carcinoma, kidney chromophobe carcinoma, kidney clear cell carcinoma, kidney papillary cell carcinoma, low-grade glioma, lung adenocarcinoma, lung squamous cell carcinoma, mesothelioma, ovarian serous adenocarcinoma, pancreatic ductal adenocarcinoma, paraganglioma and pheochromocytoma, prostate adenocarcinoma, sarcoma, cutaneous melanoma, testicular germ cell carcinoma, thymoma, papillary thyroid carcinoma, uterine carcinosarcoma, endometrial carcinoma of the uterine body, and uveal melanoma) were analyzed according to the method described above.

[0334] Fusion transcripts with SEQ ID NOs: 911 - 17492 were identified.

[0335] In the following table, the columns are referred to as follows: 1. Frequency in cohort 2. Donor chromosome exon / 2’ donor chromosome exon 3. Donor start exon / 3’ donor start TE 4. Donor breakpoint exon / 4’ donor breakpoint TE 5. Donor strand exon / 5’ donor strand TE 6. Donor transcript (i.e., donor_tx_name_Exon) / 6’ acceptor chromosome exon 7. Acceptor chromosome TE / 7’ acceptor breakpoint exon 8. Acceptor breakpoint TE / 8’ acceptor end exon 9. Acceptor end TE / 9’ acceptor strand exon 10. Acceptor strand TE / 10’ acceptor transcript (i.e., acceptor_tx_name_exon) 11. Type of fusion / 11’ type of fusion [Table 7-1] [Table 7-2]

Table 7-3

Table 7-4

Table 7-5

Table 8-1

Table 8-2

Table 8-3

Table 8-4

Table 8-5

Table 8-6

Table 8-7

Table 8-8

Table 8-9

Table 9-1

Table 9-2

Table 9-3

Table 9-4

Table 9-5

Table 9-6

Table 9-7

Table 9-8

Table 9-9

Table 9-10

Table 9-11

Table 9-12

Table 9-13

Table 9-14

Table 9-15

Table 9-16

Table 9-17

Table 9-18

Table 9-19

Table 9-20

Table 9-21

Table 10-1

Table 10-2

Table 10-3

Table 10-4

Table 10-5

Table 10-6

Table 10-7

Table 10-8

Table 10-9

Table 10-10

Table 10-11

Table 10-12

Table 10-13

Table 10-14

Table 10-15

Table 10-16

Table 10-17

Table 10-18

Table 10-19

Table 10-20

Table 10-21

Table 10-22

Table 10-23

Table 10-24

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Table 10-27

Table 11-1

Table 11-2

Table 11-3

Table 11-4

Table 11-5

Table 11-6

Table 11-7

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Table 11-10

Table 11-11

Table 11-12

Table 11-13

Table 11-14

Table 11-15

Table 12-1

Table 12-2

Table 12-3

Table 12-4

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Table 12-6

Table 12-7

Table 12-8

Table 12-9

Table 12-10

Table 12-11

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Table 12-13

Table 12-14

Table 12-15

Table 12-16

Table 12-17

Table 12-18

Table 12-19

Table 12-20

Table 12-21

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Table 12-23

Table 12-24

Table 12-25

Table 12-26

Table 12-27

Table 13-1

Table 13-2

Table 13-3

Table 13-4

Table 13-5

Table 13-6

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Table 13-8

Table 13-9

Table 13-10

Table 13-11

Table 13-12

Table 13-13

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Table 14-2

Table 14-3

Table 14-4

Table 14-5

Table 14-6

Table 14-7

Table 14-8

Table 14-9

Table 14-10

Table 14-11

Table 14-12

Table 14-13

Table 14-14

Table 14-15

Table 14-16

Table 15-1

Table 15-2

Table 15-3

Table 15-4

Table 16-1

Table 16-2

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Table 16-4

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Table 16-7

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Table 17-2

Table 17-3

Table 17-4

Table 18-1

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Table 18-4

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Table 18-6

Table 19-1

Table 19-2

Table 19-3

Table 19-4

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Table 19-6

Table 19-7

Table 19-8

Table 19-9

Table 19-10

Table 19-11

Table 19-12

Table 19-13

Table 20-1

Table 20-2

Table 20-3

Table 20-4

Table 20-5

Table 20-6

Table 21-1

Table 21-2

Table 21-3

Table 21-4

Table 21-5

Table 21-6

Table 21-7

Table 21-8

Table 21-9

Table 21-10

Table 21-11

Table 21-12

Table 21-13

Table 21-14

Table 21-15

Table 21-16

Table 22-1

Table 22-2

Table 22-3

Table 22-4

Table 22-5

Table 22-6

Table 22-7

Table 22-8

Table 22-9

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Table 22-14

Table 22-15

Table 23-1

Table 23-2

Table 23-3

Table 23-4

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Table 23-6

Table 23-7

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Table 23-10

Table 23-11

Table 23-12

Table 23-13

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Table 23-16

Table 23-17

Table 23-18

Table 23-19

Table 23-20

Table 23-21

Table 23-22

Table 23-23

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Table 23-27

Table 23-28

Table 23-29

Table 23-30

Table 23-31

Table 23-32

Table 23-33

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Table 23-36

Table 23-37

Table 23-38

Table 23-39

Table 23-40

Table 23-41

Table 23-42

Table 23-43

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Table 23-45

Table 23-46

Table 24-1

Table 24-2

Table 24-3

Table 24-4

Table 24-5

Table 24-6

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Table 24-9

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Table 24-14

Table 24-15

Table 24-16

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Table 25-2

Table 25-3

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Table 25-7

Table 25-8

Table 25-9

Table 25-10

Table 25-11

Table 25-12

Table 25-13

Table 25-14

Table 25-15

Table 25-16

Table 25-17

Table 25-18

Table 25-19

Table 25-20

Table 25-21

Table 25-22

Table 25-23

Table 25-24

Table 25-25

Table 25-26

Table 25-27

Table 25-28

Table 25-29

Table 26-1

Table 26-2

Table 26-3

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Table 26-6

Table 26-7

Table 26-8

Table 26-9

Table 26-10

Table 26-11

Table 26-12

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Table 26-14

Table 26-15

Table 27-1

Table 27-2

Table 27-3

Table 27-4

Table 27-5

Table 27-6

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Table 27-9

Table 27-10

Table 27-11

Table 27-12

Table 27-13

Table 27-14

Table 27-15

Table 27-16

Table 27-17

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Table 27-19

Table 27-20

Table 27-21

Table 27-22

Table 27-23

Table 27-24

Table 27-25

Table 27-26

Table 27-27

Table 27-28

Table 27-29

Table 27-30

Table 27-31

Table 27-32

Table 27-33

Table 27-34

Table 27-35

Table 27-36

Table 28-1

Table 28-2

Table 28-3

Table 28-4

Table 28-5

Table 28-6

Table 29-1

Table 29-2

Table 29-3

Table 29-4

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Table 29-6

Table 29-7

Table 29-8

Table 29-9

Table 29-10

Table 29-11

Table 29-12

Table 29-13

Table 29-14

Table 30-1

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Table 30-3

Table 30-4

Table 30-5

Table 30-6

Table 30-7

Table 30-8

Table 30-9

Table 30-10

Table 30-11

Table 31-1

Table 31-2

Table 31-3

Table 31-4

Table 31-5

Table 31-6

Table 31-7

Table 31-8

Table 31-9

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Table 31-12

Table 31-13

Table 31-14

Table 31-15

Table 31-16

Table 31-17

Table 31-18

Table 31-19

Table 31-20

Table 31-21

Table 31-22

Table 31-23

Table 31-24

Table 31-25

Table 31-26

Table 31-27

Table 32-1

Table 32-2

Table 32-3

Table 32-4

Table 32-5

Table 33-1

Table 33-2

Table 33-3

Table 33-4

Table 33-5

Table 33-6

Table 33-7

Table 33-8

Table 33-9

Table 33-10

Table 33-11

Table 33-12

Table 33-13

Table 34-1

Table 34-2

Table 34-3

Table 34-4

Table 34-5

Table 34-6

Table 34-7

Table 34-8

Table 34-9

Table 34-10

Table 34-11

Table 34-12

Table 34-13

Table 34-14

Table 34-15

Table 34-16

Table 34-17

Table 34-18

Table 35-1

Table 35-2

Table 35-3

Table 35-4

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Table 35-6

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Table 35-8

Table 35-9

Table 35-10

Table 35-11

Table 35-12

Table 35-13

Table 35-14

Table 35-15

Table 35-16

Table 35-17

Table 35-18

Table 35-19

Table 35-20

Table 35-21

Table 35-22

Table 35-23

Table 35-24

Table 35-25

Table 35-26

Table 35-27

Table 35-28

Table 35-29

Table 35-30

Table 35-31

Table 35-32

Table 35-33

Table 35-34

Table 35-35

Table 35-36

Table 35-37

Table 35-38

Table 35-39

Table 35-40

Table 35-41

Table 35-42

Table 36-1

Table 36-2

Table 36-3

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Table 36-5

Table 36-6

Table 36-7

Table 36-8

Table 37-1

Table 37-2

Table 37-3

Table 37-4

Table 37-5

Table 37-6

Table 37-7

Table 37-8

Table 37-9

Table 37-10

Table 37-11

Table 37-12

Table 37-13

Table 37-14

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Table 37-16

Table 37-17

Table 37-18

Table 37-19

Table 37-20

Table 37-21

Table 37-22

Table 37-23

Table 37-24

Table 37-25

Table 37-26

Table 37-27

Table 37-28

Table 37-29

Table 37-30

Table 38-1

Table 38-2

Table 38-3

Table 38-4

Table 38-5

Table 38-6

Table 38-7

Table 38-8

Table 38-9

Table 38-10

Table 38-11

Table 38-12

Table 39-1

Table 39-2

Table 39-3

Table 39-4

Table 39-5

Table 39-6

Table 39-7

Table 39-8

Table 39-9

Table 39-10

Table 39-11

Table 39-12

Table 39-13

Table 39-14

Table 39-15

Table 39-16

Table 39-17

Table 39-18

Table 39-19

Table 39-20

Table 39-21

Table 40-1

Table 40-2

Table 40-3

Table 40-4

Table 40-5

Table 40-6

Table 40-7

Table 40-8

Table 40-9

Table 40-10

Table 40-11

Table 40-12

Table 40-13

Table 40-14

Table 40-15

Table 40-16

Table 40-17

Table 40-18

Table 40-19

Table 40-20

Table 41-1

Table 41-2

Table 41-3

Table 41-4

Table 41-5

Table 41-6

Table 41-7

Table 41-8

Table 41-9

Table 41-10

Table 41-11

Table 41-12

Table 41-13

Table 41-14

Table 41-15

Table 41-16

Table 41-17

Table 41-18

Table 41-19

Table 41-20

Table 41-21

Table 41-22

Table 41-23

Table 41-24

Table 41-25

Table 41-26

Table 41-27

Table 41-28

Table 41-29

Table 41-30

Table 41-31

Table 41-32

Table 41-33

Table 41-34

Table 41-35

Table 41-36

Table 41-37

Table 41-38

Table 41-39

Table 41-40

Table 41-41

Table 41-42

Table 41-43

Table 41-44

Table 41-45

Table 41-46

Table 41-47

Table 41-48

Table 42-1

Table 42-2

Table 42-3

Table 42-4

Table 43-1

Table 43-2

Table 43-3

Table 43-4

Table 43-5

Table 43-6

Table 43-7

Table 44-1

Table 44-2

Table 44-3

Table 44-4

Table 44-5

Table 44-6

Table 44-7

Table 44-8

Table 44-9

Table 44-10

Table 44-11

Table 44-12

Table 44-13

Table 44-14

Table 44-15

Table 44-16

Table 44-17

Table 44-18

Table 44-19

Table 44-20

Table 44-21

Table 44-22

Table 44-23

Table 44-24

Table 44-25

Table 44-26

Table 44-27

Table 44-28

Table 44-29

Table 44-30

Table 44-31

Table 44-32

Table 44-33

Table 44-34

Table 44-35

Table 44-36

Table 44-37

Table 45-1

Table 45-2

Table 45-3

Table 45-4

Table 45-5

Table 45-6

Table 45-7

Table 45-8

Table 45-9

Table 45-10

Table 45-11

Table 45-12

Table 45-13

Table 45-14

Table 45-15

Table 45-16

Table 45-17

Table 45-18

Table 45-19

Table 45-20

Table 45-21

Table 45-22

Table 45-23

Table 45-24

Table 45-25

Table 45-26

Table 45-27

Table 45-28

Table 45-29

Table 45-30

Table 45-31

Table 45-32

Table 45-33

Table 45-34

Table 45-35

Table 45-36

Table 45-37

Table 45-38

Table 45-39

Table 45-40

Table 45-41

Table 45-42

Table 45-43

Table 45-44

Table 45-45

Table 45-46

Table 45-47

Table 45-48

Table 45-49

Table 45-50

Table 45-51

Table 45-52

Table 45-53

Table 45-54

Table 45-55

Table 45-56

Table 45-57

Table 45-58

Table 45-59

Table 45-60

Table 45-61

Table 45-62

Table 45-63

Table 45-64

Table 45-65

Table 45-66

Table 45-67

Table 45-68

Table 45-69

Table 46-1

Table 46-2

Table 46-3

Table 46-4

Table 47-1

Table 47-2

Table 47-3

Table 47-4

Table 48-1

Table 48-2

Table 48-3

Table 48-4

Table 48-5

Table 48-6

Table 48-7

Table 48-8

Table 48-9

Table 48-10

Table 49-1

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Table 49-6

Table 49-7

Table 49-8

Table 49-9

Table 49-10

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Table 49-13

Table 50-1

Table 50-2

Table 50-3

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Table 50-5

Table 50-6

Table 50-7

Table 50-8

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Table 50-10

Table 51-1

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Table 51-3

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Table 51-5

Table 51-6

Table 51-7

Table 51-8

Table 51-9

Table 51-10

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Table 51-14

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Table 51-16

Table 51-17

Table 51-18

Table 51-19

Table 51-20

Table 51-21

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Table 51-23

Table 51-24

Table 51-25

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Table 51-27

Table 52-1

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Table 52-3

Table 52-4

Table 53-1

Table 53-2

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Table 53-6

Table 53-7

Table 54-1

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Table 54-7

Table 55-1

Table 55-2

Table 55-3

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Table 55-6

Table 55-7

Table 55-8

Table 55-9

Table 55-10

Table 55-11

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Table 55-16

Table 56-1

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Table 56-10

Table 57-1

Table 57-2

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Table 57-6

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Table 57-18

Table 58-1

Table 58-2

Table 58-3

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Table 58-6

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Table 58-8

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Table 58-10

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Table 58-16

Table 58-17

Table 58-18

Table 58-19

Table 59-1

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Table 59-3

Table 59-4

Table 59-5

Table 59-6

Table 59-7

Table 59-8

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Table 59-10

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Table 59-16

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Table 59-22

Table 59-23

Table 59-24

Table 59-25

Table 59-26

Table 59-27

Table 59-28

Table 59-29

Table 59-30

Table 59-31

Table 59-32

Table 59-33

Table 59-34

Table 59-35

Table 59-36

Table 59-37

Table 59-38

Table 59-39

Table 59-40

Table 59-41

Table 59-42

Table 59-43

Table 59-44

Table 59-45

Table 59-46

Table 59-47

Table 59-48

Table 59-49

Table 59-50

Table 59-51

Table 59-52

Table 59-53

Table 59-54

Table 59-55

Table 59-56

Table 59-57

Table 60-1

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Table 60-3

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Table 60-5

Table 60-6

Table 60-7

Table 60-8

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Table 60-10

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Table 60-14

Table 61-1

Table 61-2

Table 61-3

Table 61-4

Table 61-5

Table 61-6

Table 61-7

Table 61-8

Table 61-9

Table 61-10

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Table 61-16

Table 61-17

Table 61-18

Table 61-19

Table 61-20

Table 61-21

Table 61-22

Table 61-23

Table 61-24

Table 61-25

Table 61-26

Table 61-27

Table 61-28

Table 61-29

Table 61-30

Table 61-31

Table 61-32

Table 61-33

Table 61-34

Table 61-35

Table 61-36

Table 61-37

Table 61-38

Table 61-39

Table 61-40

Table 61-41

Table 61-42

Table 62-1

Table 62-2

Table 62-3

Table 62-4

Table 62-5

Table 62-6

Table 63-1

Table 63-2

Table 63-3

Table 63-4

Table 63-5

Table 63-6

Table 63-7

Table 63-8

Table 63-9

Table 63-10

Table 63-11

Table 64-1

Table 64-2

Table 64-3

Table 64-4

Table 64-5

Table 64-6

Table 64-7

Table 64-8

Table 65-1

Table 65-2

Table 65-3

Table 65-4

Table 65-5

Table 65-6

Table 65-7

Table 65-8

Table 65-9

Table 65-10

Table 65-11

Table 65-12

Table 65-13

Table 65-14

Table 65-15

Table 65-16

Table 65-17

Table 65-18

Table 65-19

Table 66-1

Table 66-2

Table 66-3

Table 66-4

Table 67-1

Table 67-2

Table 67-3

Table 67-4

Table 67-5

Table 67-6

Table 67-7

Table 67-8

Table 67-9

Table 68-1

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Table 68-3

Table 69-1

Table 69-2

Table 69-3

Table 69-4

Table 69-5

Table 69-6

Table 69-7

Table 69-8

Table 69-9

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Table 69-11

Table 70-1

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Table 70-4

Table 71-1

Table 71-2

Table 71-3

Table 71-4

Table 71-5

Table 71-6

Table 71-7

Table 71-8

Table 71-9

Table 71-10

Table 71-11

Table 71-12

Table 71-13

Table 71-14

Table 71-15

Table 72-1

Table 72-2

Claims

**Claim 1** A method for selecting a tumor neoantigen peptide, comprising: - identifying a fusion transcript sequence comprising a translocation element (TE) sequence and an exon sequence and containing an open reading frame (ORF) from among mRNA sequences derived from cancer cells of a subject; - selecting a tumor neoantigen peptide of at least 8 amino acids encoded by a part of the ORF of the fusion transcript sequence; wherein the ORF overlaps a junction between the TE and the exon sequence, is a pure TE, and / or is non-standard; and wherein the tumor neoantigen peptide binds to at least one major histocompatibility complex (MHC) molecule of the subject. **Claim 2** The method according to claim 1, wherein the tumor neoantigen peptide is 8 to 11 amino acids in length and binds to at least one MHC class I molecule of the subject, or the tumor neoantigen peptide is 13 to 25 amino acids in length and binds to at least one MHC class II molecule of the subject. **Claim 3** The neoantigen peptide is - expressed at a higher level in tumor cells compared to normal and healthy cells; - expressed in at least 1% of subjects from a population of subjects with cancer; and / or - binds to MHC class I or class II with a Kd binding affinity of less than about 10−5 M. The method according to claim 1 or 2. **Claim 4** The method according to any one of claims 1 to 3, wherein the cancer is NSCLC.

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