Methods and compositions for the use of tumor autoantigens in adoptive immunotherapy

Abstract

To provide methods and compositions for immunotherapy employing a modified T cell or NK cell comprising a receptor that binds to newly identified tumor antigens that can be administered to patients for disease (e.g., cancer) treatment.SOLUTION: The present invention provides a chimeric antigen receptor (CAR) protein, which binds to a specific antigen. For example, a CAR protein binding to FLDETLRSLA (SEQ ID NO: 2) and a CAR protein binding to YNPIRTTF (SEQ ID NO: 3) are illustrated.SELECTED DRAWING: Figure 6B

Description

[Technical Field]

[0001] Priority Information This application claims priority to U.S. Provisional Patent Application No. 62 / 641,541, filed on 12 March 2018, the entire contents of which are incorporated herein by reference.

[0002] 1. Technical Fields This disclosure generally pertains to the fields of medicine, cancer, oncology, immunology, immunotherapy, cell biology, and molecular biology. In certain aspects, the field of this disclosure pertains to adoptive immunotherapy. More specifically, it pertains to tumor antigens, vaccines, chimeric antigen receptor (CAR) engineered T cells, T cells having engineered T cell receptors (TCRs), engineered antibodies, and therapeutic methods using such means. [Background technology]

[0003] 2. Background The immune system is thought to play a dual role in carcinogenesis (Ichim, 2005). Firstly, once a proper immune response is initiated, the immune system can eliminate newly generated cells arising from early tumor-initiating events (immunoediting). Conversely, the immune system can initiate signaling of wound healing pathways that can help cultivate an environment that promotes tumorigenesis. Human leukocyte antigen (HLA) proteins present a snapshot of the proteome of all nucleated cells on the cell surface for T cell surveillance. Individuals possess six distinct HLA class I alleles (A, B, and C), but to date, a total of 13,145 unique class I alleles have been characterized at these highly polymorphic loci (Robinson et al., 2018). Presentation of processed pathogen-derived peptides by at least one of these HLA alleles is a major bottleneck in the initiation of an adaptive immune response. Each HLA allele possesses the ability to present a different set of peptides to the immune system based on biophysical properties within the peptide binding groove, which limit specificity to a limited set of available peptides. Peptide bonds are primarily determined by two HLA-facing anchor residues restricted to a few amino acids at these positions (Fritsch et al., 2014). Recently, algorithms such as NetMHC and SYFPEITHI have enabled the prediction of peptide sequence binding affinity to specific HLA alleles, yielding accurate predictions of over 75% binding with positive prediction values ​​ranging from 90–95% (Andreatta et al., 2016; He et al., 2010; Nielsen et al., 2007).

[0004] The novel antigens presented can be divided into two distinct classes: Group 1, arising from mutations in TCR-facing residues and less likely to alter peptide / HLA complex binding affinity accordingly; and Group 2, arising from peptide anchor residues and therefore presenting longer novel polypeptide sequences to the immune system compared to single-residue modifications of Group 1 antigens (Fritsch et al., 2014). Appropriately mediated interactions between HLA proteins, presented peptides, and T cells help maintain the integrity of the organism's genome by eliminating cells carrying foreign genetic material from both external pathogens and somatic mutations. Tumor immunoediting theory predicts that early pathogenic events leading to precancerous cell proliferation can be eliminated by the adaptive immune system unless cancer cells evolve the ability to evade this selective pressure (Dunn et al., 2004).

[0005] While the adaptive immune system is increasingly recognized as potentially playing a crucial role in the elimination of pre-existing tumors, its role in the clearance of cancer cells during initial triggering events remains challenging to study. Although immunosuppression in humans has been well-documented as being associated with increased cancer incidence (Grulich et al., 2007; Gallagher et al., 2010; Penn et al., 1973), quantifying initial immunoediting events and attributing the clearance of precancerous lesions in immunocompetent individuals to the clearance of neo-tumor antigens remains difficult, in contrast to other mechanisms such as the elimination of cells carrying oncogenic viruses. [Overview of the Initiative]

[0006] overview This disclosure provides chimeric antigen receptor (CAR) proteins that bind to the antigens listed in Table 1 or 2 or Figure 6B. The CAR proteins are: TIFF0007853938000001.tif can be combined with 31150. A polynucleotide molecule encoding the above CAR protein is also provided. The polynucleotide molecule may further include a promoter active in eukaryotic cells and may be further defined as an expression vector.

[0007] In another aspect, a T cell receptor protein that binds to an antigen described in Table 1 or 2 or FIG. 6B is provided. The T cell receptor protein TIFF0007853938000002.tif can be combined with 32159. A polynucleotide molecule encoding the above T cell receptor protein is also provided. The polynucleotide molecule may further include a promoter active in eukaryotic cells and may be further defined as an expression vector.

[0008] In yet another aspect, An engineered T cell containing a polynucleotide molecule encoding a T cell receptor that binds to an antigen of Table 1 or 2 or FIG. 6B, such as TIFF0007853938000003.tif 32159, is provided. The cell can be a T cell or a NK cell. The cell may further include a transposase.

[0009] In a further aspect, a method of treating cancer in a human subject in need thereof is provided, comprising administering to the subject an effective amount of a cell therapy comprising one or more of the above cells. The method may further comprise administering to the human subject a second cancer therapy, such as chemotherapy, immunotherapy, radiation therapy, hormone therapy, or surgery. The second cancer therapy can be administered concurrently with, before, or after the cell therapy. The method may similarly further comprise administering to the human subject a second administration of an effective amount of one or more of the cells as defined above.

[0010] Cancer can be metastatic, recurrent, or drug-resistant. Cell therapy can be administered locally to the cancer site, to a region of the cancer site, or systemically. Cancer can be neuroblastoma, lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancers, and melanoma.

[0011] In a further embodiment, a method for identifying non-standard tumor antigens is provided, comprising the steps of: (a) obtaining tumor RNA and / or DNA from a tumor sample; (b) sequencing the RNA and / or DNA; (c) comparing tumor gene expression data with tumor-specific difference analysis of TARGET / TCGA vs. GTEx to identify highly expressed genes that are specific to tumors and are known not to be present in normal tissues; (d) obtaining MHC molecules from the tumor sample; (e) characterizing MHC-binding ligands by elution and LC / MS / MS proteomics; (f) filtering the MHC ligands identified based on differential expression in the tumor; (g) filtering the remaining ligands from step (f) based on their absence in a database of normal ligands; and (h) identifying non-standard tumor antigens by matching empirically characterized ligands with RNA sequencing reads that do not map to standard proteins.

[0012] This method may further include a step of applying neural network machine learning to prioritize tumor antigens based on peptide / MHC binding affinity, quantitative abundance of tumor ligands on the MHC surface determined by LC / MS / MS, biological relevance to tumor type, recurrence across other tumors, population frequency of presented HLA alleles, and / or antigen recurrence across multiple tumors. This method may similarly further include a step of (i) preparing tumor antigen / MHC dextramers and selecting antigen-specific CD8 cells from healthy HLA-matched donors; and (j) validating the identified antigens by performing single-cell sequencing on the selected antigen-specific T cells to obtain paired α / β TCR sequences. This method may similarly further include a step of pairing the α / β TCR sequences with mouse constant regions, followed by codon optimization, and expressing the codon-optimized construct in mammalian cells. This method may similarly further include a step of co-culturing mammalian cells with tumor cells.

[0013] In a further form, (i) A fusion protein is provided comprising (ii) a first single-chain antibody that selectively binds to an antigen in Table 1 or 2 or Figure 6B, such as TIFF0007853938000004.tif32159; and (ii) a second single-chain antibody that binds to T or B cells. The second single-chain antibody may bind to CD3, T cells, or B cells. The fusion protein may further comprise a labeled or therapeutic moiety.

[0014] In a further manner, A vaccine composition is provided comprising one or more antigens as described in Tables 1 and 2 or Figure 6B, such as TIFF0007853938000005.tif25150. The vaccine composition may further comprise an adjuvant, a biological response modifier, and / or a chemokine. One or more antigens may be delivered by intact dendritic cells. A method is also provided for inducing an anti-cancer immune response in a subject, comprising the step of administering the vaccine composition defined above to the subject.

[0015] [Invention 1001] A chimeric antigen receptor (CAR) protein that binds to the antigens listed in Table 1 or 2 or Figure 6B. [Invention 1002] The CAR protein of the present invention 1001, which binds to TIFF0007853938000006.tif11141. [Invention 1003] The CAR protein 1001 of the present invention, which binds to FLDETLRSLA (SEQ ID NO: 2). [Invention 1004] The CAR protein 1001 of the present invention, which binds to QYNPIRTTF (SEQ ID NO: 3). [Invention 1005] The CAR protein 1001 of the present invention, which binds to SYQKVIELF (SEQ ID NO: 4). [Invention 1006] The CAR protein 1001 of the present invention, which binds to IYPDITYSL (SEQ ID NO: 5). [Invention 1007] The CAR protein 1001 of the present invention, which binds to FLIENLLAA (SEQ ID NO: 6). [Invention 1008] The CAR protein 1001 of the present invention binds to ALLSGVRQV (SEQ ID NO: 7). [Invention 1009] The CAR protein 1001 of the present invention binds to VLFENTDSVHL (SEQ ID NO: 8). [Invention 1010] The CAR protein 1001 of the present invention, which binds to SAAMVFSAL (SEQ ID NO: 9). [Invention 1011] A polynucleotide molecule encoding any of the CAR proteins described in invention 1001 to 1010. [Invention 1012] A polynucleotide molecule of the present invention 1011, further comprising an active promoter in eukaryotic cells. [Invention 1013] A polynucleotide molecule of the present invention 1011, further defined as an expression vector. [Invention 1014] A T cell receptor protein that binds to the antigens listed in Table 1 or 2 or Figure 6B. [Invention 1015] The T cell receptor protein of the present invention 1014, which binds to TIFF0007853938000007.tif11146. [Invention 1016] The T cell receptor protein of the present invention 1014, which binds to FLDETLRSLA (SEQ ID NO: 2). [Invention 1017] The T cell receptor protein of the present invention 1014, which binds to QYNPIRTTF (SEQ ID NO: 3). [Invention 1018] The T cell receptor protein of the present invention 1014, which binds to SYQKVIELF (SEQ ID NO: 4). [Invention 1019] The T cell receptor protein of the present invention 1014, which binds to IYPDITYSL (SEQ ID NO: 5). [Invention 1020] The T cell receptor protein of the present invention 1014, which binds to FLIENLLAA (SEQ ID NO: 6). [Invention 1021] The T cell receptor protein of the present invention 1014, which binds to ALLSGVRQV (SEQ ID NO: 7). [Invention 1022] The T cell receptor protein of the present invention 1014, which binds to VLFENTDSVHL (SEQ ID NO: 8). [Invention 1023] The T cell receptor protein of the present invention 1014, which binds to SAAMVFSAL (SEQ ID NO: 9). [Invention 1024] A polynucleotide molecule encoding any of the T cell receptor proteins described in invention 1014 to 1023. [Invention 1025] A polynucleotide molecule of the present invention 1024, further comprising an active promoter in eukaryotic cells. [Invention 1026] A polynucleotide molecule of the present invention 1024, further defined as an expression vector. [Invention 1027] Engineered T cells containing a polynucleotide molecule encoding a T cell receptor that binds to the antigen shown in Table 1 or 2 or Figure 6B. [Invention 1028] A cell according to the present invention 1027, wherein a polynucleotide molecule encodes one of the T cell receptor proteins of the present invention 1015 to 1023. [Invention 1029] A T cell, according to the cell of invention 1027. [Invention 1030] NK cells, the cells of the present invention 1027. [Invention 1031] Cells of the present invention 1027, further comprising a transposase. [Invention 1032] A method for treating cancer in a human subject in need, comprising the step of administering an effective amount of a cell therapy comprising one or more cells according to any of the present invention 1014-1018 or 1027-1031 to the target. [Invention 1033] The method of the present invention 1032, further comprising the step of administering a second cancer therapy to a human subject. [Invention 1034] The method of the present invention 1033, wherein the second cancer therapy is chemotherapy, immunotherapy, radiation therapy, hormone therapy, or surgery. [Invention 1035] The method of the present invention 1033, wherein a second cancer therapy is administered simultaneously with cell therapy. [Invention 1036] The method of the present invention 1033, wherein a second cancer therapy is administered before or after cell therapy. [Invention 1037] The method of the present invention 1032, further comprising the step of administering a second effective dose of one or more cells of any of the present invention 1014-1018 or 1027-1031 to a human subject. [Invention 1038] The method of the present invention 1032, wherein the cancer is metastatic, recurrent, or drug-resistant. [Invention 1039] The method of the present invention 1032, wherein cell therapy is administered locally to the cancer site, to a region of the cancer site, or systemically. [Invention 1040] The method of the present invention 1032, wherein the cancer is neuroblastoma. [Invention 1041] The method of the present invention 1032, wherein the cancer is lung cancer (including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), peritoneal cancer, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, various types of head and neck cancer, and melanoma. [Invention 1042] (a) The step of obtaining tumor RNA and / or DNA from a tumor sample; (b) The step of sequencing the RNA and / or DNA; (c) A step in which tumor gene expression data is compared with tumor-specific difference analysis of TARGET / TCGA vs. GTEx to identify highly expressed genes that are specific to tumors and are known not to be present in normal tissues; (d) The step of obtaining MHC molecules from the tumor sample; (e) Characterizing MHC-binding ligands by elution and LC / MS / MS proteomics; (f) A step of filtering MHC ligands identified based on differential expression in the tumor; (g) A step of filtering out the remaining ligands from step (f) based on the fact that they do not exist in the database of normal ligands; and (h) Identifying non-standard tumor antigens by matching empirically characterized ligands with RNA sequencing reads that do not map to standard proteins. A method for identifying non-standard tumor antigens, including [specific antigens]. [Invention 1043] The method of the present invention 1042 further comprises the step of applying neural network machine learning to prioritize tumor antigens based on peptide / MHC binding affinity, quantitative abundance of tumor ligands on the MHC surface determined by LC / MS / MS, biological relevance to tumor type, recurrence extending to other tumors, population frequency of presented HLA alleles, and / or antigen recurrence extending to multiple tumors. [Invention 1044] (i) Prepare tumor antigen / MHC dextramers and select antigen-specific CD8 cells from healthy HLA-matched donors; (j) Single-cell sequencing is performed on the selected antigen-specific T cells to obtain paired α / β TCR sequences. The method of the present invention 1042 further comprises the step of verifying the identified antigen. [Invention 1045] The method of the present invention 1042 further comprises the steps of pairing an α / β TCR sequence with a mouse constant region, followed by codon optimization, and expressing the codon-optimized construct in mammalian cells. [Invention 1046] The method of the present invention 1045, further comprising the step of co-culturing mammalian cells with tumor cells. [Invention 1047] (i) A first single-chain antibody that selectively binds to the antigen in Table 1; and (ii) A second single-chain antibody that binds to T or B cells A fusion protein containing the above. [Invention 1048] A fusion protein according to the present invention 1047, in which a second single-chain antibody binds to CD3. [Invention 1049] A fusion protein according to the present invention 1047, in which a second single-chain antibody binds to T cells. [Invention 1050] A fusion protein according to Invention 1047, in which a second single-chain antibody binds to B cells. [Invention 1051] A fusion protein according to the present invention 1047, wherein the fusion protein further comprises a labeled or therapeutic moiety. [Invention 1052] A vaccine composition comprising one or more antigens listed in Table 1 or Figure 6B. [Invention 1053] A vaccine composition according to the present invention 1052, further comprising an adjuvant. [Invention 1054] The vaccine composition of the present invention 1052, further comprising a biological response modifier. [Invention 1055] A vaccine composition according to the present invention 1052, further comprising a chemokine. [Invention 1056] A vaccine composition according to the present invention 1042, wherein one or more antigens are delivered by intact dendritic cells. [Invention 1057] A method for inducing an anti-cancer immune response in a subject, comprising the step of administering the vaccine composition of the present invention 1052 to the subject. Other purposes, features, and advantages of this disclosure will become apparent from the following detailed description. However, while the detailed description and specific examples illustrate certain aspects of the disclosure, they are for illustrative purposes only, and various changes and modifications within the spirit and scope of this disclosure will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawing]

[0016] The following drawings form part of this specification and are included to further illustrate certain aspects of this disclosure. This disclosure may be better understood by referring to one or more of these drawings in conjunction with the detailed descriptions of the particular embodiments presented herein.

[0017] (Figure 1) Workflow of the strategy used to identify tumor-specific antigens. MHC class I molecules were purified from xenografts from eight patients and eight primary patient tumors, and peptides were acid-eluted for LC / MS / MS characterization to identify the MHC antigens found. Using HLA typing predicted from exome data with the PHLAT algorithm, the binding affinity of the peptides was predicted using the machine learning algorithms NetMCH and SYFPEITHI to determine which peptides best bound to MHC and which were most likely to induce a T cell response. Next, the inventors filtered these peptides again against a differential expression database created by comparing 1641 healthy tissues against 153 neuroblastoma tumors. The inventors searched for peptides derived from the obtained genes against a database of 190 healthy tissues and 273 tumor samples characterized by MHC proteomics. Furthermore, the inventors processed non-standard readouts to create a proteomics search library and identified antigens generated from alternative splice sites, insertions / deletions, frameshifts, and intron misreadings. (Figure 2) Identification of MHC class I tumor-specific antigens. Schematic diagram of tumor-specific antigen filtering and identification using a combination of proteomics, RNA-seq, and ligand-mix databases. (Figure 3) Examples of recurrent tumor-specific antigen (PHOX2B) observed in 4 / 4 tested tumors. FPKM values ​​of tumor vs. normal tissue in PDX tumors, and structure of the target peptide PHOX2B. (Figure 4)(Figure 4A) Schematic diagram of peptide-MHC multimers. (Figure 4B) Dextramer staining of a rare population of tumor antigen-specific CD8 T cells identified from HLA-matched donors. (Figure 5)(Figure 5A) Representative surface MHC expression in NB measured by W6 / 32 pan-MHC class I antibody (red: no antibody; blue: baseline; orange: IFN-γ stimulated cells). (Figure 5B) Antigen presentation experiment using influenza CEF1 matrix protein antigen. HLA-A2 neuroblastoma cells were treated with peptide and influenza virus, and IL-2 release was assayed by ELISA. (Figure 5C) IL-2 release after co-incubation with influenza antigen-specific T cell hybridoma. (Figure 6A) Figures 6A and B. Nonprotective domains and protein regions that are widely applicable as cancer vaccine candidates are revealed from HLA presentation scores by protein regions. (Figure 6A) 29 peptides detected by ligandmix in 16 neuroblastoma tumors were mapped to HLA population presentation scores. Empirically detected peptides were highly enriched in high-scoring regions of the protein (p=0.000011). (Figure 6B) Analysis of MYCN HLA presentation across protein spans. Analysis of individual peptides (above) reveals that the most highly presented peptide derived from MYCN, TVRPKNAAL (SEQ ID NO: 25), is presented on 9 HLA alleles, representing 58.1% of the population. The KATEYVHSL (SEQ ID NO: 26) peptide detected by ligandmix is ​​predicted to be presented on a total of 10 HLA alleles (31.9% of the population). From 17mer region (central) analysis, a peptide is produced that is predicted to bind to 19 HLA alleles (73.1% of the population). TIFF0007853938000008.tif4128 is identified. From a 33mer region analysis, it is identified as the highest scoring peptide presented in 18 HLA alleles in 85.4% of the population. TIFF0007853938000009.tif4128 has been identified, suggesting that these represent promising areas of MYCN proteins for widely applicable vaccines. (Figure 6B) See the explanation for Figure 6A. (Figure 7) NPY is highly differentially expressed in neuroblastoma and is a promising target for vaccination. RNA sequencing data from 153 neuroblastoma tumors in the target (first column) were compared with 1643 normal tissues compiled from GTEx by organ (second column), revealing high NPY expression in neuroblastoma compared to normal tissue. See Table 2. [Modes for carrying out the invention]

[0018] Exemplary Description While immunotherapies, including adoptive T-cell therapy, have shown noteworthy recent results, most of these cases were within highly mutated tumors such as melanoma and lung cancer (Figure 1; line extending to the right from the HLA typing box). Furthermore, as discussed above, the generation of antigen-specific T cells remains a specialized, labor-intensive process, entrusted to a small number of laboratories capable of performing these techniques. There remains a great need for methods to identify tumor antigens in tumors that do not present standard novel antigens (antigens derived from proteins with SNVs).

[0019] Neuroblastoma is a tumor characterized by low MHC expression and low mutational burden (Figure 1), and therefore a good model for more "challenging" targets for adoptive immunotherapy. We have developed a scalable method for identifying patient-specific tumor antigens that we expect to be specific and potent targets for adoptive T-cell immunotherapy. Furthermore, we have created a streamlined workflow for identifying T-cell receptors (TCRs) that bind to these tumor antigens for use in research and clinical settings.

[0020] In summary, the inventors have developed novel methods for scalable tumor antigen identification and the development of TCRs specific to these antigens. In addition, the inventors have identified neuroblastoma-specific antigens and antigens in other “challenging” tumors that are promising targets for adoptive cancer immunotherapy. These and other aspects of the disclosure are described in detail below.

[0021] I. Definition In this disclosure, the use of the singular form includes the plural form, the words "a" or "an" mean "at least one," and the use of "or" means "and / or" unless otherwise specified. Furthermore, the use of the term "including," as well as other forms such as "includes" and "included," is not limiting. Also, terms such as "element" or "component" include both elements and components containing one unit and elements or components containing two or more units, unless otherwise specified.

[0022] As used herein, the term "about" means plus or minus 10% when used in conjunction with a percentage or other numerical quantity. For example, the term "about 80%" includes 80% plus or minus 8%.

[0023] Section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described herein. All documents or parts of documents cited herein, including but not limited to patents, patent applications, articles, books, and treaties, are expressly incorporated herein by reference in their entirety for all purposes. If one or more incorporated documents or similar materials define terms in a manner that conflicts with their definitions herein, this application shall prevail.

[0024] As used herein, and unless otherwise specified, the terms “disease,” “disorder,” or “condition” mean a condition or health state of a patient or subject that can be treated with the compounds, pharmaceutical compositions, or methods provided herein. In some embodiments, disease is cancer (e.g., pancreatic cancer, colon cancer, stomach cancer, lung cancer, ovarian cancer, osteosarcoma, bladder cancer, cervical cancer, liver cancer, kidney cancer, skin cancer (e.g., Merkel cell carcinoma), testicular cancer, leukemia, lymphoma, head and neck cancer, colorectal cancer, prostate cancer, pancreatic cancer, melanoma, breast cancer, neuroblastoma, stomach cancer).

[0025] As used herein, and unless otherwise specified, the terms “to treat” or “treatment” mean any indication of success in treating or improving an injury, disease, disease, disease, or condition, including any objective or subjective parameters such as: mitigation; remission; reduction of symptoms, or making an injury, disease, or condition more tolerable to a patient; slowing the rate of degeneration or decay; making the final stage of degeneration less debilitating; or improving the patient’s physical or mental health. Treatment or improvement of symptoms may be based on objective or subjective parameters, including the results of a physical examination, neuropsychiatric examination, and / or psychiatric evaluation. The terms “to treat” and their inflections include the prevention of injury, disease, condition, or disease. In some embodiments, “to treat” means treatment of cancer.

[0026] As used herein, and unless otherwise specified, the terms “prevent,” “prevent,” and “prevention” refer to actions taken before a patient begins to suffer from cancer-related disorders, which would delay the onset of cancer and / or inhibit or reduce the severity of cancer.

[0027] As used herein, and unless otherwise specified, the terms “manage,” “control,” and “administer” include preventing, delaying, or reducing the severity of recurrence of a cancer-like disorder in a patient already suffering from such a disease, injury, or condition. The terms also include modulating the threshold, development, and / or duration of a cancer-related disorder, or altering how a patient responds to a cancer-related disorder.

[0028] As used herein, and unless otherwise specified, “therapeutic effective dose” of a compound means an amount sufficient to provide any therapeutic utility in the treatment or management of an electrically active cell disorder, such as neuronal dysfunction, neuron-mediated disorders, ocular disorders, or cardiac disorders, or, without limitation, to delay or minimize one or more symptoms associated with an electrically active cell disorder, such as neuronal dysfunction, neuron-mediated disorders, ocular disorders, or cardiac disorders. The therapeutic effective dose of a compound means the amount of the compound, either alone or in combination with one or more other treatments and / or therapeutic agents, that provides therapeutic utility in the treatment or management of an electrically active cell disorder, such as neuronal dysfunction, neuron-mediated disorders, ocular disorders, or cardiac disorders.

[0029] As used herein, and unless otherwise specified, “effective dose” is an amount sufficient to achieve the specified purpose of the compound compared to the absence of the compound (e.g., to achieve the effect it is administered, to treat a disease, to reduce enzyme activity, to increase enzyme activity, to reduce a signaling pathway, or to reduce one or more symptoms of a disease or condition). An example of a “therapeutic effective dose” is an amount sufficient to contribute to the treatment, prevention, or reduction of symptoms of a disease, which may also be called a “therapeutic effective dose.” “Reduction” of symptoms (and grammatical synonyms thereof) means a reduction in the severity or frequency of symptoms, or the elimination of symptoms. The exact amount depends on the purpose of the treatment and can be determined by those skilled in the art using known techniques (see, for example, Lieberman, Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins).

[0030] As used herein, and unless otherwise specified, “prophylactic effective dose” of a compound means an amount sufficient to prevent or delay the onset of one or more cancer-related symptoms, or to prevent or delay their recurrence. The prophylactic effective dose of a compound means the amount of the compound alone or in combination with one or more other treatments and / or prophylactic agents that provide prophylactic utility in the prevention of a cancer-like disorder. The term “prophylactic effective dose” can encompass an amount that prevents a cancer-like disorder, improves overall prevention, or enhances the prophylactic effect of another prophylactic agent. A “prophylactic effective dose” may, for example, be prescribed before the onset of a cancer-like disorder.

[0031] As used herein, “patient” or “subject requiring it” means a living organism that is suffering from or susceptible to a disease or condition that can be treated by administration of the compositions or pharmaceutical compositions provided herein. Non-limiting examples include humans, primates, companion animals (dogs, cats, etc.), other mammals such as cattle, rats, mice, monkeys, goats, sheep, deer, and other non-mammalian animals, but not limited to these. In some embodiments, the patient is human.

[0032] As used herein, the term “conservative substitution” generally refers to an amino acid substitution that preserves the structural and functional properties of a protein or polypeptide. Such functionally equivalent (conservative substitution) peptide amino acid sequences include, but are not limited to, the addition or substitution of amino acid residues in an amino acid sequence encoded by a nucleotide sequence that results in a silent change and thus produces a functionally equivalent gene product. Conservative amino acid substitutions can be made based on the similarity of the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0033] Abbreviations used herein have their conventional meanings within the fields of chemistry and biology. Chemical structures and formulas described herein are constructed in accordance with the standard rules of chemical valence known in the field of chemistry.

[0034] Unless otherwise defined, technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. See, for example, Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2nd ed., J. Wiley & Sons (New York, NY 1994); Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, Cold Springs Harbor Press (Cold Springs Harbor, NY 1989). Any methods, apparatus, and materials similar or equivalent to those described herein may be used in the practice of this disclosure. The following definitions are provided to facilitate the understanding of certain terms frequently used herein and are not intended to limit the scope of this disclosure.

[0035] A “biological sample” or “sample” refers to material obtained from or originating from a subject or patient. Biological samples include tissue sections such as biopsy and autopsy samples, as well as frozen sections taken for histological purposes. Such samples include body fluids, such as blood and blood fractions or preparations (e.g., serum, plasma, platelets, red blood cells), sputum, tissues, cultured cells (e.g., primary cultures, explants, and transformed cells), feces, urine, synovial fluid, joint tissue, synovial tissue, synovial cells, fibroblast-like synovial cells, macrophage-like synovial cells, immune cells, hematopoietic cells, fibroblasts, macrophages, T cells, etc. Biological samples are typically obtained from eukaryotes such as mammals, e.g., primates, e.g., chimpanzees or humans; cattle; dogs; cats; rodents, e.g., guinea pigs, rats, mice; rabbits; or birds; reptiles; or fish.

[0036] As used herein, “cell” means a cell that performs metabolic or other functions sufficient to preserve or replicate its genomic DNA. Cells can be identified by methods well known in the art, including, for example, the presence of an intact membrane, staining with a specific dye, the ability to produce offspring, or, in the case of gametes, the ability to combine with a second gamete to produce viable offspring. Cells may include prokaryotic and eukaryotic cells. Prokaryotic cells include, but are not limited to, bacteria. Eukaryotic cells include, but are not limited to, yeast cells, as well as cells derived from plants and animals, such as mammals, insects (e.g., Spodoptera), and human cells. Cells may be useful if they are naturally non-adherent or treated, for example, by trypsin treatment, so that they do not adhere to surfaces.

[0037] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues, where the polymer may optionally have non-amino acid portions attached. This term applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding natural amino acids, as well as to natural amino acid polymers and non-natural amino acid polymers. A “fusion protein” is a chimeric protein that encodes two or more distinct protein sequences expressed by recombination as a single portion.

[0038] "Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and polymers thereof, in either single-stranded or double-stranded form, as well as their complements. The term "polynucleotide" refers to a linear sequence of nucleotides. The term "nucleotide" typically refers to a single unit, i.e., monomer, of a polynucleotide. A nucleotide can be a ribonucleotide, a deoxyribonucleotide, or a variant thereof. Examples of polynucleotides as intended herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA (including siRNA), and hybrid molecules having mixtures of single-stranded and double-stranded DNA and RNA. As used herein, nucleic acid also refers to nucleic acid that has the same basic chemical structure as naturally occurring nucleic acids. Such analogs have modified sugars and / or modified ring substituents but retain the same basic chemical structure as naturally occurring nucleic acids. Nucleic acid mimes are compounds that have a different structure from the general chemical structure of nucleic acids but function similarly to naturally occurring nucleic acids. Examples of such analogues include, but are not limited to, phosphorothioates, phosphoramidites, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, and peptide nucleic acids (PNAs).

[0039] The "sequence identity ratio" is determined by comparing two optimally aligned sequences across a comparison window, where portions of the polynucleotide or polypeptide sequence within the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. This ratio is calculated by determining the number of positions where identical nucleic acid bases or amino acid residues exist in both sequences, finding the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity ratio.

[0040] In the context of two or more nucleic acid or polypeptide sequences, the term “identical” or “identical” percentage means two or more sequences or subsequences that are identical or have a specific percentage of amino acid residues or nucleotides that are identical (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identity, over a specific region of the entire polypeptide sequence of the disclosed or individual domains of the polypeptide of the disclosed) when compared and aligned to obtain the greatest possible correspondence across a comparison window or over a specified region measured by manual alignment and visual inspection. Such sequences are said to be “substantially identical.” This definition also refers to the complement of the test sequence. Optionally, identity exists over a region of at least approximately 50 nucleotides in length, or more specifically, over a region of 100–500 or 1000 or more nucleotides in length. This disclosure includes polypeptides that are substantially identical to any of those identified herein.

[0041] As used herein with respect to genes, the terms “expressed” or “expressed” refer to the transcript and / or translation product of that gene. The expression level of a DNA molecule in a cell can be determined based on either the amount of corresponding mRNA present in the cell or the amount of protein encoded by that DNA produced by the cell. The expression level of non-coding nucleic acid molecules (e.g., siRNA) can be detected by standard PCR or Northern blotting methods well known in the art. See Sambrook et al., 1989 MOLECULAR CLONING: A LABORATORY MANUAL, 18.1–18.88. The expression of a transfected gene can occur transiently or stably within the cell. During “transient expression,” the transfected gene is not transferred to daughter cells during cell division. Because its expression is limited to the transfected cell, gene expression is lost over time. In contrast, stably expressed gene can occur if the gene is co-transfected with another gene that gives the transfected cell a selective advantage. Such selective advantages may include resistance to certain toxins presented to the cell. Expression of transfected genes can be further achieved by transposon-mediated insertion into the host genome. During transposon-mediated insertion, the gene is positioned in a predictable manner between two transposon linker sequences that enable insertion into the host genome and subsequent excision. Stable expression of transfected genes can be further achieved by infecting cells with lentiviral vectors that result in stable gene expression by forming (integrating) a part of the cell genome after infection.

[0042] The terms "plasmid," "vector," or "expression vector" refer to nucleic acid molecules that encode a gene and / or regulatory elements necessary for gene expression. Gene expression from a plasmid can occur in cis or trans configuration. When a gene is expressed in cis configuration, the gene and its regulatory elements are encoded by the same plasmid. Trans expression refers to the case where the gene and its regulatory elements are encoded by separate plasmids.

[0043] The terms “transfection,” “transfer,” “transfect,” or “transfer” are interchangeable and are defined as the process of introducing nucleic acid molecules or proteins into cells. Nucleic acids are introduced into cells using non-viral or virus-based methods. Nucleic acid molecules can be gene sequences encoding complete proteins or functional portions thereof. Non-viral transfection methods include any suitable transfection method that does not use viral DNA or viral particles as a delivery system for introducing nucleic acid molecules into cells. Exemplary non-viral transfection methods include calcium phosphate transfection, liposome transfection, nucleofection, sonoporation, heat shock transfection, magnetization, and electroporation. In some embodiments, nucleic acid molecules are introduced into cells using electroporation according to standard procedures well known in the art. In the case of virus-based transfection methods, any useful viral vector may be used in the methods described herein. Examples of viral vectors include, but are not limited to, retroviral vectors, adenovirus vectors, lentiviral vectors, and adeno-associated virus vectors. In some embodiments, nucleic acid molecules are introduced into cells using retroviral vectors according to standard procedures well known in the art. The term “transfection” also refers to the introduction of proteins into cells from the external environment. Typically, protein transfection or introduction relies on the attachment of peptides or proteins that can cross the cell membrane and migrate to the protein of interest. See, for example, Ford et al. (2001) and Prochiantz (2007).

[0044] An "antibody" is a polypeptide containing a framework region from an immunoglobulin gene or fragment that specifically binds to and recognizes an antigen. Recognized immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. The light chain is classified as either κ or λ. The heavy chain is classified as γ, μ, α, δ, or ε, which define the immunoglobulin classes, IgG, IgM, IgA, IgD, and IgE, respectively. Typically, the antigen-binding region of an antibody plays a crucial role in determining the specificity and affinity of binding. In some embodiments, antibodies or fragments of antibodies may originate from different organisms, including humans, mice, rats, hamsters, and camels. Antibodies may include antibodies that have been modified or mutated at one or more amino acid positions to improve or modulate the desired function of the antibody (e.g., glycosylation, expression, antigen recognition, effector function, antigen binding, specificity, etc.).

[0045] When referring to proteins or peptides, the phrases "specifically (or selectively) bind" or "specifically (or selectively) immunize with" an antibody often refer to a binding reaction that determines the presence of a protein in a heterogeneous population of proteins and other biologics. Therefore, under specified immunoassay conditions, a specified antibody will bind to a particular protein at least twice the background level, more typically 10 to 100 times or more. Specific binding to an antibody under such conditions typically requires an antibody selected for its specificity to a particular protein. For example, by selecting polyclonal antibodies, one can obtain only a subset of antibodies that immunize specifically with a selected antigen but not with other proteins. This selection can be achieved by subtracting antibodies that cross-react with other molecules. Various immunoassay formats can be used to select antibodies that immunize specifically with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies that specifically react with proteins (see, for example, Harlow & Lane, Using Antibodies, A Laboratory Manual (1998) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity).

[0046] The term "isolated," when applied to nucleic acids or proteins, indicates that the nucleic acid or protein essentially contains no other cellular components to which it is bound in its native state. It can be, for example, homogeneous and may be either dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high-performance liquid chromatography. The protein, which is the dominant species present in the preparation, is substantially purified.

[0047] A "control" sample or value refers to a sample that serves as a reference for comparison with a test sample, usually as a known reference. For example, a test sample may be taken from test conditions, for example, in the presence of the test compound, and can be compared to a sample from known conditions, for example, in the absence of the test compound (negative control) or in the presence of a known compound (positive control). A control may also represent the mean value collected from several tests or results. Those skilled in the art will recognize that controls can be designed for the evaluation of any number of parameters. For example, controls can be devised to compare therapeutic benefits based on pharmacological data (e.g., half-life) or therapeutic means (e.g., comparison of side effects). Those skilled in the art will understand which controls are useful in a given situation and will be able to analyze data based on comparison with control values. Controls are also useful for determining the significance of data. For example, if the values ​​of a given parameter differ significantly in the controls, the variability in the test samples will not be considered significant.

[0048] As used herein, the terms “metastasis,” “metastatic,” and “metastatic cancer” are interchangeable and refer to the spread of a proliferative disease or disorder, such as cancer, from one organ or another non-adjacent organ or part of the body. Cancer originates in a primary tumor, such as a primary breast cancer, in a site of origin such as the breast. Some cancer cells of the primary tumor or site of origin acquire the ability to penetrate and invade the surrounding normal tissue in the local area, and / or penetrate the walls of the lymphatic or vascular system and circulate through that system to reach other parts and tissues in the body. A clinically detectable second tumor formed from cancer cells of the primary tumor is called a metastatic or secondary tumor. When cancer cells metastasize, the metastatic tumor and its cells are presumed to be similar to those of the original tumor. Thus, if lung cancer metastasizes to the breast, the secondary tumor in the breast site consists of abnormal lung cells, not abnormal mammary cells. The secondary tumor in the breast is called metastatic lung cancer. Therefore, the term "metastatic cancer" refers to a disease in which the subject has or has had a primary tumor and has one or more secondary tumors. The term "non-metastatic cancer" or "subject with non-metastatic cancer" refers to a disease in which the subject has a primary tumor but does not have one or more secondary tumors. For example, metastatic lung cancer refers to a disease in a subject who has or has a history of a primary lung tumor and has one or more secondary tumors in a second or more locations, such as the breast.

[0049] "Anticancer" is used in its simple, ordinary sense and refers to a composition (e.g., compound, drug, antagonist, inhibitor, regulator) having antineoplastic properties or the ability to inhibit cell growth or proliferation. In some embodiments, an anticancer agent is a chemotherapeutic agent. In some embodiments, an anticancer agent is a drug identified herein that is useful in a method of treating cancer. In some embodiments, an anticancer agent is a drug approved by the FDA or a similar regulatory authority in a country other than the United States for the treatment of cancer.

[0050] II. Neuroblastoma Neuroblastoma (NB) is a type of cancer that develops in certain types of nerve tissue. Most frequently, it begins in one of the adrenal glands, but it can also occur in the neck, chest, abdomen, or spine. Symptoms may include bone pain, lumps in the abdomen, neck, or chest, or painless, bluish lumps under the skin.

[0051] Neuroblastoma can sometimes be caused by mutations inherited from one's parents. Environmental factors have not been found to be involved. Diagnosis is based on tissue biopsy. It can sometimes be detected in babies by ultrasound during pregnancy. At the time of diagnosis, the cancer has usually already spread. Cancers are classified into low-risk, intermediate-risk, and high-risk groups based on the child's age, the stage of the cancer, and how the cancer looks.

[0052] Treatment and outcomes depend on the individual's risk group. Treatment may include observation, surgery, radiotherapy, chemotherapy, or stem cell transplantation. Low-risk diseases in infants usually have a good outcome with surgery or simply observation. However, in high-risk diseases, the likelihood of long-term survival is less than 40%, even with aggressive treatment.

[0053] Neuroblastoma is the most common cancer in infants and the third most common cancer in children, after leukemia and brain tumors. At one point in time, about 1 in 7,000 children is affected. About 90% of cases occur in children under 5 years of age, and it is rare in adults. About 15% of childhood cancer deaths are due to neuroblastoma.

[0054] A. Signs and Symptoms The initial symptoms of neuroblastoma are often vague, making diagnosis difficult. Common symptoms include fatigue, loss of appetite, fever, and joint pain. Symptoms depend on the location of the primary tumor and, if present, metastatic cancer. In the abdomen, tumors can cause abdominal distension and constipation. Tumors in the chest can cause respiratory problems. • If a tumor compresses the spinal cord, it can cause weakness, making it impossible to stand, crawl, or walk. Bone lesions in the legs and buttocks can cause pain and limping. Tumors of the orbit or bone around the eye can cause noticeable bruising and swelling. Bone marrow infiltration can cause pallor due to anemia. Neuroblastoma often spreads to other parts of the body before symptoms appear, and 50-60% of all neuroblastoma cases show metastasis.

[0055] The most common site where neuroblastoma develops (i.e., the primary tumor) is in the adrenal gland. This occurs in 40% of localized tumors and 60% of cases of widespread disease. Neuroblastoma can also develop anywhere along the sympathetic nervous system chain from the neck to the pelvis. The frequency of different locations includes the neck (1%), chest (19%), abdomen (30% non-adrenal), or pelvis (1%). Rarely, the primary tumor may not be identifiable.

[0056] Rare but characteristic findings include transverse myelopathy (tumor compression of the spinal cord, 5% of cases), treatment-resistant diarrhea (tumor vasoactive intestinal peptide secretion, 4% of cases), Horner's syndrome (cervical tumor, 2.4% of cases), opsoclonus-myoclonus syndrome, ataxia (suspected paraneoplastic cause, 1.3% of cases), and hypertension (catecholamine secretion or renal artery compression, 1.3% of cases).

[0057] B. Cause The cause of neuroblastoma is not well understood. The majority of cases are sporadic and non-familial. Approximately 1-2% of cases occur within families and are associated with specific gene mutations. Familial neuroblastoma is sometimes caused by rare germline mutations in the anaplastic lymphoma kinase (ALK) gene. Germline mutations in the PHOX2A or KIF1B genes are also associated with familial neuroblastoma. Neuroblastoma is also a characteristic feature of neurofibromatosis type 1 and Beckwith-Wiedemann syndrome.

[0058] Amplification of the MYCN oncogene within tumors is a common finding in neuroblastoma. The degree of amplification shows a bimodal distribution, either 3-10 times or 100-300 times. The presence of this mutation is highly correlated with the stage of disease progression.

[0059] Duplicate segments of the LMO1 gene in neuroblastoma tumor cells have been shown to increase the risk of developing aggressive cancer morphologies.

[0060] Neuroblastoma is associated with copy number variations in the NBPF10 gene, which causes 1q21.1 deletion syndrome or 1q21.1 duplication syndrome.

[0061] Several risk factors have been proposed and are the subject of ongoing research. Due to the characteristic early onset, many studies have focused on parental factors before, during, and after conception. Factors investigated included occupation (i.e., exposure to chemicals in specific industries), smoking, alcohol consumption, medication use during pregnancy, and birth factors; however, the results have not been conclusive.

[0062] Other studies have investigated possible associations between atopic dermatitis and early life exposure to infections, the use of hormones and ovulation-inducing drugs, and maternal use of hair dyes.

[0063] C. Diagnosis The diagnosis is usually confirmed by a surgical pathologist, taking into account clinical findings, microscopic findings, and other clinical trials. It can arise from any neural crest element of the sympathetic nervous system (SNS).

[0064] Nerve neuroblastoma, also known as olfactory neuroblastoma, is thought to originate from the olfactory epithelium, and its classification is still debatable. However, because it is not a malignant tumor of the sympathetic nervous system, nerve neuroblastoma is a distinct clinical entity and should not be confused with neuroblastoma.

[0065] D. Biochemistry In approximately 90% of neuroblastoma cases, elevated levels of catecholamines or their metabolites are found in the urine or blood. Catecholamines and their metabolites include dopamine, homovanillic acid (HVA), and / or vanillylmandelic acid (VMA).

[0066] E. Image creation Another method for detecting neuroblastoma is the mIBG scan (metaiodobenzylguanidine), often called "mIBG-avid," which is used and practiced in 90-95% of all neuroblastomas. The mechanism involves mIBG being taken up by sympathetic nerve cells, as it is a functional analogue of the neurotransmitter norepinephrine. 131 or I 123 When radioactively ionized with radioactive iodine isotopes, it is an excellent radiopharmaceutical for diagnosing and monitoring the response to treatment for this disease. (Half-life 13 hours) 123 I is a special isotope for imaging sensitivity and quality. 131 It has a half-life of 8 days and, at high doses, is an effective targeted radiation therapy for recurrent and refractory neuroblastoma.

[0067] F. Histology Microscopic examination typically describes tumor cells as small, round, and blue, and may exhibit a rosette pattern (Homerright rosette). Homerright rosettes are tumor cells around the neural network and should not be confused with pseudorosettes, which are tumor cells around blood vessels. They also differ from pseudorosettes of ependymoma (and thus a combination of the two), which consist of tumor cells with a glial fibrillary acidic protein (GFAP)-positive process that tapers towards blood vessels. Pathologists use various immunohistochemical stains to distinguish neuroblastoma from histological mimetic tumors such as rhabdomyosarcoma, Ewing's sarcoma, lymphoma, and Wilms' tumor.

[0068] Neuroblastoma is a type of peripheral neuroblastoma (pNT) that shares a common origin and exhibits a wide differentiation pattern, ranging from benign ganglioneuromas to stroma-rich ganglioblastomas with mixed neuroblasts or nodules, and highly malignant neuroblastomas. This distinction in tumor pathology before treatment, along with age and mitotic-nuclear decay index (MKI), is an important prognostic factor. This pathological classification system (Shimada System) describes “favorable” and “unfavorable” tumors by the International Neuroblastoma Pathology Committee (INPC), established in 1999 and revised in 2003.

[0069] G. Disease Staging The International Neuroblastoma Staging System (INSS), established in 1986 and revised in 1988, stratifies neuroblastomas based on their anatomical features at the time of diagnosis: • Stage 1: A localized tumor confined to the area of ​​origin. Stage 2A: Unilateral tumor with incomplete macroscopic resection; tumor-negative ipsilateral and contralateral lymph nodes. Stage 2B: Unilateral tumor with complete or incomplete macroscopic resection; ipsilateral lymph node positive for tumor; identifiable contralateral lymph node negative for tumor. Stage 3: Tumors that invade beyond the midline, with or without regional lymph node metastasis; or unilateral tumors with contralateral lymph node metastasis; or midline tumors with bilateral lymph node metastasis. • Stage 4: Except as defined in Stage 4S, the tumor has spread to distant lymph nodes, bone marrow, bone, liver, or other organs. • Stage 4S: A patient under 1 year of age has a localized primary tumor as defined in Stage 1 or 2, with transmission limited to the liver, skin, or bone marrow (less than 10% of nucleated bone marrow cells are tumorous).

[0070] While the International Staging Standard (INSS) is used, the need for an international consensus on risk allocation is recognized in order to compare similar cohorts in research results. Since 2005, representatives from major pediatric oncology collaboration groups have met to review data from 8,800 neuroblastoma patients treated in Europe, Japan, the United States, Canada, and Australia between 1990 and 2002. This working group proposed the International Neuroblastoma Risk Group (INRG) classification system. Retrospective studies have revealed high survival rates in the 12-18 month age group, which was previously classified as high-risk, prompting the decision to reclassify 12-18 month age children without N-myc (commonly known as MYCN) amplification into the intermediate-risk category.

[0071] The new INRG risk assignment classifies neuroblastomas at diagnosis based on a new International Neuroblastoma Risk Grouping System (INRGSS): • Stage L1: A localized disease without risk factors defined by imaging. • Stage L2: A localized disease with risk factors defined by imaging. • Stage M: Metastatic disease. • Stage MS: A "special" metastatic disease in which MS is equivalent to stage 4S.

[0072] The new risk stratification is based on a new INRGSS staging system, age (divided at 18 months), tumor grade, N-myc amplification, disproportionate 11q abnormalities, and ploidy, resulting in four pre-treatment risk groups: very low risk, low risk, intermediate risk, and high risk.

[0073] H. Screening Urinary catecholamine levels can be elevated in preclinical neuroblastoma. Since the 1980s, screening of asymptomatic infants at 3 weeks, 6 months, and 1 year has been conducted in Japan, Canada, Austria, and Germany. In Japan, screening for neuroblastoma at 6 months of age began in 1984 through analysis of homovanillic acid and vanylmandelic acid levels. Studies in Canada and Germany did not show a reduction in neuroblastoma mortality, but rather resulted in an increase in diagnoses that would have resolved on their own, leading to unnecessary surgery and chemotherapy for these infants, after which screening was discontinued in 2004.

[0074] I. Treatment If the lesions are localized, it is generally curable. However, despite aggressive and intensive treatment (intensive chemotherapy, surgery, radiotherapy, stem cell transplantation, the differentiation agent isotretinoin, also known as 13-cis-retinoic acid, and frequently immunotherapy with anti-GD2 monoclonal antibody therapy), long-term survival is poor in children over 18 months of age with progressive disease.

[0075] Biological and genetic characteristics have been identified, which, when added to the classical clinical staging classification, allow for the assignment of patients to risk groups for planning treatment intensity. These criteria include the patient's age, the extent of disease spread, microscopic appearance, and genetic features such as DNA ploidy and N-myc oncogene amplification (N-myc regulates microRNAs) to low-risk, intermediate-risk, and high-risk diseases. A recent biological study (COG ANBL00B1) analyzed 2687 neuroblastoma patients to determine the range of risk assignments: 37% of neuroblastoma cases were low-risk, 18% intermediate-risk, and 45% high-risk. There is some evidence that high-risk and low-risk types are caused by different mechanisms, not simply two different levels of expression of the same mechanism.

[0076] Treatment for these different risk categories varies considerably. Low-risk diseases are often managed with observation alone or cured by surgery alone. Intermediate-risk diseases are treated with surgery and chemotherapy. High-risk neuroblastomas are treated with intensive chemotherapy, surgery, radiotherapy, bone marrow / hematopoietic stem cell transplantation, biology-based therapy with 13-cis-retinoic acid (isotretinoin or Accutane), and antibody therapy, usually administered with cytokines GM-CSF and IL-2.

[0077] With current treatments, patients with low- and intermediate-risk neuroblastoma have an excellent prognosis, with cure rates exceeding 90% for low-risk patients and 70-90% for intermediate-risk patients. In contrast, treatment of high-risk neuroblastoma over the past 20 years has resulted in a cure rate of only about 30%. The addition of antibody therapy has significantly increased survival rates for high-risk patients. In March 2009, an initial analysis of the Children's Oncology Group (COG) study involving 226 high-risk patients showed that 66% of the group randomized to receive ch14.18 antibodies along with GM-CSF and IL-2 two years after stem cell transplantation were alive and disease-free compared to only 46% in the group that did not receive antibodies. Randomization was stopped so that all patients enrolled in the trial would receive antibody therapy.

[0078] Chemotherapy agents used in combination have been found to be effective against neuroblastoma. Commonly used agents for conditioning induction and stem cell transplantation include platinum compounds (cisplatin, carboplatin), alkylating agents (cyclophosphamide, ifosfamide, melphalan), topoisomerase II inhibitors (etoposide), anthracycline antibiotics (doxorubicin), and vinca alkaloids (vincristine). Some newer regimens include topoisomerase I inhibitors (topotecan and irinotecan) in induction, which have been shown to be effective against relapsing disease.

[0079] J. Prognosis Twenty to fifty percent of high-risk cases do not respond adequately to the initiation of high-dose chemotherapy and are progressive or refractory. Relapse after completion of frontline treatment is also common. While Phase I and Phase II clinical trials testing new drugs and drug combinations for neuroblastoma offer further treatment options, outcomes for relapsed high-risk disease remain very poor.

[0080] Most long-term survivors alive today had low- or intermediate-risk diseases and had a milder treatment course compared to those with high-risk diseases. The majority of survivors have long-term effects from treatment. Survivors of intermediate- and high-risk treatments often experience hearing loss. Growth retardation, thyroid dysfunction, learning disabilities, and an increased risk of secondary cancers affect survivors of high-risk diseases. An estimated two out of three childhood cancer survivors eventually develop at least one chronic, sometimes life-threatening, health problem within 20–30 years of cancer diagnosis.

[0081] K. Cytogenetic Profile Based on a series of 493 neuroblastoma samples, it has been reported that the overall genomic pattern, tested by array-based karyotype analysis, is a predictor of outcomes in neuroblastoma: Tumors showing only changes in the total copy number of chromosomes were associated with superior survival. Tumors exhibiting any type of partial chromosomal copy number alteration were associated with a higher risk of recurrence. Within tumors exhibiting partial changes, independent further predictors of decreased overall survival were N-myc amplification, 1p and 11q deletions, and 1q acquisition.

[0082] In previous publications, neuroblastoma was classified into three main subtypes based on its cytogenetic profile: • Subtype 1: A favorable neuroblastoma that is nearly triploid, predominantly characterized by numerical fluctuations, and mainly representing non-metastatic NB stages 1, 2, and 4S. Subtypes 2A and 2B: Undesirable widespread neuroblastoma, seen in stages 3 and 4, with 11q loss and 17q gain without N-myc amplification (subtype 2A), or with N-myc amplification, often accompanied by 1p deletion and 17q gain (subtype 2B).

[0083] Virtual karyotype analysis can be performed on fresh tumors or paraffin-embedded tumors to assess copy numbers at these loci. SNP array virtual karyotype analysis can be used on tumor samples, including neuroblastoma, because it can detect copy neutral loss of heterozygosity (acquired uniparental disomy). Copy neutral LOH may be biologically equivalent to deletions and has been detected at major loci in neuroblastoma. ArrayCGH, FISH, or conventional cytogenetics cannot detect copy neutral LOH.

[0084] L. Epidemiology Neuroblastoma accounts for 6–10% of all childhood cancers and 15% of childhood cancer deaths. The annual mortality rate is 10 per million children in the 0–4 year group and 4 per million children in the 4–9 year group.

[0085] The highest incidence occurs in the first year of life, and it can be congenital. While the age range is broad, including older children and adults, only about 10% of cases occur in individuals over 5 years of age. A large European study reported that less than 2% of over 4,000 neuroblastoma cases occurred in individuals over 18 years of age.

[0086] M. Treatment Guidelines The recent focus has been on reducing treatment for low- and intermediate-risk neuroblastoma while maintaining a 90% survival rate. A study of 467 intermediate-risk patients enrolled in A3961 from 1997 to 2005 confirmed the hypothesis that treatment in this risk group could be successfully reduced. Patients with favorable features (tumor grade and response) received four cycles of chemotherapy, while those with unfavorable features received eight cycles, with 3-year event-free survival and overall survival stable in 90% of the cohort. Future plans include intensifying treatment for patients with 1p36 or 11q23 chromosomal abnormalities and those with no initial response to treatment.

[0087] In contrast, the focus over the past 20 years has been on enhancing the management of high-risk neuroblastoma. Differences in chemotherapy initiation, timing of surgery, stem cell transplantation regimens, various radiation delivery schemes, and the use of monoclonal antibodies and retinoids to manage minimal residual disease continue to be investigated. In response to these issues, recent randomized Phase III clinical trials are being conducted to improve survival in high-risk patients.

[0088] III. Methods for identifying tumor-specific autoantigens In one aspect, this disclosure describes novel methods for identifying tumor antigens that can act as targets for immune cells, particularly those specifically genetically engineered to express receptors capable of targeting tumor antigens. These methods represent a significant improvement over current techniques and enable the identification of antigens from cancers with relatively low mutation rates, as well as the identification of recurrent tumor-specific antigens in tumors with high mutational burdens.

[0089] To identify antigens, tumor RNA and DNA are obtained from tumor samples and sequenced in parallel with MHC ligands empirically characterized by tumor cell elution and LC / MS / MS proteomics (ligandmics). Tumor gene expression data are compared with tumor-specific differential analysis of TARGET / TCGA vs. GTEx to identify highly expressed genes known to be tumor-specific and absent in normal tissues. Ligands identified by MHC ligandmics are filtered based on differential expression in tumors, and remaining ligands are further filtered based on their absence in the normal ligandome database. Further classes of non-standard tumor antigens are identified using algorithms that match empirically characterized ligands with RNA-seq reads that do not map to standard proteins. Tumor antigens are prioritized based on peptide / MHC binding affinity determined by the neural network machine learning algorithm NetMHC, quantitative presence of tumor ligands on the MHC surface determined by LC / MS / MS, biological relevance to tumor type, recurrence across other tumors, population frequency presenting HLA alleles, and antigen recurrence across multiple tumors.

[0090] To validate the selected antigen, peptide / MHC dextramers are specifically synthesized for tumor ligands and used to select antigen-specific CD8 cells from healthy HLA-matched donors. Next, single-cell sequencing is performed on a subset of antigen-specific T cells to obtain paired α / β TCR sequences. Variable regions of the TCR sequences are estimated from the data, paired with mouse constant regions, and codon-optimized for improved expression. Expression cassettes of the TCR β chain and subsequent α chain, linked by a P2A peptide for equimolar expression, are cloned into a pMP71 T cell-specific expression vector. The retroviral TCR vector is transduced into Jurkat / MA reporter cell lines manipulated by NFAT-driven luciferase expression. To validate TCR expression, transduced Jurkat / MA cells are co-cultured with tumor cells, and luciferase expression is assayed.

[0091] Using this approach, the inventors identified several different peptide antigens in polypeptides expressed by tumors. These proteins include IGFBPL1 (NP_001007564), GFRA2 (NM_001158510), PHOX2B (NP_003915), PBK (NP_001265874), CHRNA3 (NP_000743), HMX1 (NP_001293071.1), tyrosine hydroxylase (AAI43612.1), RBM34 (NP_001155005), and ATP6V0C (NP_001185498). Specific peptide sequences are shown in Table 1 below.

[0092] IV. Immunotherapy involving vaccines, engineered receptor molecules, and cells A. Bites Bispecific T-cell engagers (BiTEs) are a class of artificially created bispecific monoclonal antibodies being investigated for use as anticancer agents. They instruct the host immune system against cancer cells, more specifically, the cytotoxic activity of T cells. BiTE is a registered trademark of Micromet AG.

[0093] BiTE is a fusion protein consisting of two single-chain variable fragments (scFv) of different antibodies, or amino acid sequences derived from four different genes, on a single peptide chain of approximately 55 kilodaltons. One scFv binds to T cells via the CD3 receptor, while the other binds to tumor cells via tumor-specific molecules, in this case the antigens listed in Table 1.

[0094] Like other bispecific antibodies, and unlike typical monoclonal antibodies, BiTE forms a link between T cells and tumor cells. This allows T cells to exert cytotoxic activity against tumor cells by producing proteins such as perforin and granzymes, independently of the presence of MHC I or costimulatory molecules. These proteins enter tumor cells and initiate apoptosis. This action mimics the physiological processes observed during T cell attacks on tumor cells.

[0095] As of July 2010, BiTEs in clinical trials included blinatumomab (MT103), which was developed against the surface molecule CD19 expressed on B cells for the treatment of non-Hodgkin lymphoma and acute lymphoblastic leukemia; and MT110, which was developed against the EpCAM antigen for the treatment of gastrointestinal cancer and lung cancer.

[0096] The same technology can be used to target melanoma (by MCSP-specific BiTE) and acute myeloid leukemia (by CD33-specific BiTE). Research in this area is currently underway. Another approach to novel anticancer therapy involves reworking some of the conventional antibodies currently in use, such as trastuzumab (targeting HER2 / neu), cetuximab, and panitumumab (both targeting the EGF receptor), using the BiTE approach. BiTEs targeting CD66e and EphA2 are also being developed.

[0097] Another example of pf BiTE technology is HLA-A * This paper concerns a BiTE antibody derived from ESK1, a T cell receptor (TCR)-mimicking monoclonal antibody (mAb) that binds to a peptide derived from the intracellular oncoprotein WT1, presented at 02:01. Despite the very low density of the complex on the cell surface, ESK1-BiTE selectively activated and induced the proliferation of cytolytic human T cells, which killed cells from multiple leukemias and solid tumors in vitro and in mice. In autologous in vitro settings, ESK1-BiTE induced a potent secondary CD8 T cell response specific to tumor-associated antigens other than WT1 (Dao et al., Nature Biotechnol. 33: 1079-1086, 2015).

[0098] B. Other forms of polyvalent antibodies Currently, various multivalent antibody constructs are being designed. Some of these have specificity to a single epitope, while others have specificity to multiple different binding targets. Bispecific antibodies include both full-length and Fab2 constructs, as well as molecules called diabodies. Diabodies are heavy-chain variable (V) connected by small peptide linkers. H ) region and light chain variable (V L Diabody is a non-covalent dimer of a single-stranded Fv (scFv) fragment consisting of a region. Another form of diabody is single-stranded (Fv)2, in which two scFv fragments are covalently bonded to each other. Triple-specific constructs include Fab3 and triabody, the latter being three scFv types of diabody. scFv-Fc consists of two linked single-stranded variable fragments fused to an intact Fc region. Minibody is similar to scFv-Fc but contains only a CH1 domain instead of a complete Fc region. Other multivalent constructs include IgNAR and hcIgG.

[0099] C. Manipulated T cells with peptide antigen specificity In another aspect of the present invention, engineered target cells expressing a TCR having specificity for the peptide antigens listed in Table 1 are disclosed. Aspects of this disclosure include compositions encoding the α and β subunits of the TCR, as well as instructions for use of the compositions. The compositions may be, for example, recombinant viruses or viral vectors. In some embodiments, the compositions comprise one or more sequences encoding a TCR subunit substantially comprising at least one of the variable regions described herein.

[0100] In some embodiments, a vector can be used to introduce a polynucleotide sequence encoding all or part of a functional TCR into a packaging cell line for the preparation of recombinant viruses. In addition to the elements described herein, the vector may include polynucleotide sequences encoding various components of recombinant viruses and at least one variable region described herein, as well as any components necessary for the production of viruses not provided by the packaging cell line. In other embodiments, in addition to the elements described herein, the vector may include polynucleotide sequences encoding various components of recombinant viruses and at least one variable region described herein, as well as any components necessary for the production of viruses not provided by the packaging cell line. Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources.

[0101] In some embodiments, one or more polycistronic expression vectors are used that contain two or more elements necessary for the production of the desired recombinant virus in packaging cells (e.g., viral genes, at least one of the following: m1-α sequence and m1-β sequence, suicide gene). The use of polycistronic vectors reduces the total number of vectors required and thus avoids the potentially difficult problems associated with coordinating expression from multiple vectors. In polycistronic vectors, the various elements to be expressed are functionally linked to one or more promoters (and other expression regulatory elements as needed). In some embodiments, polycistronic vectors are used that contain a suicide gene and / or reporter gene, viral elements, and nucleotide sequences encoding all or part of the α or β subunit of the TCR, where the nucleotide sequences are substantially as described herein.

[0102] Each component expressed in a polycistronic expression vector can be isolated, for example, from an IRES element or a viral 2A element, enabling the separate expression of various proteins from the same promoter. IRES and 2A elements are known in the art (U.S. Patent No. 4,937,190; de Felipe et al., 2004. Traffic 5: 616-626; each of these is incorporated herein by reference in whole). In one embodiment, genetic elements in a polycistronic vector are isolated using oligonucleotides encoding furin cleavage site sequences (RAKRs) linked to 2A-like sequences derived from foot-and-mouth disease virus (FMDV), equine rhinitis virus (ERAV), and thosea asigna virus (TaV) (Fang et al., 2005. Nat. Biotech 23: 584-590, which is incorporated herein by reference in its entirety) (Szymczak et al., 2004. Nat. Biotechnol. 22: 589-594, which is incorporated herein by reference in its entirety). The effectiveness of a particular polycistronic vector for use in the synthesis of a desired recombinant virus can be easily tested by detecting the expression of each gene using standard protocols. Exemplary protocols well known in the art include, but are not limited to, antibody-specific immunoassays such as Western blotting.

[0103] Vectors typically contain promoters that are recognized by packaging cells and functionally linked to polynucleotides encoding targeting molecules, viral components, etc. Promoters are expression regulators formed by nucleic acid sequences that enable RNA polymerase binding and transcription. Promoters are untranslated sequences located upstream (5') of the start codon of a structural gene (generally within approximately 100–1000 bp) and control the transcription and translation of the antigen-specific polynucleotide sequences to which they are functionally linked. Promoters can be inductive or constitutive. The activity of inductive promoters is induced by the presence or absence of biological or abiotic factors. Inductive promoters can be useful tools in genetic engineering because they can turn the expression of the genes to which they are functionally linked on or off at certain stages of biological development or in specific tissues. Inductive promoters can be grouped into chemically regulated promoters and physically regulated promoters. Typical chemically regulated promoters include, but are not limited to, alcohol-regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter), tetracycline-regulated promoters (e.g., tetracycline-responsive promoter), steroid-regulated promoters (e.g., rat glucocorticoid receptor (GR)-based promoter, human estrogen receptor (ER)-based promoter, mosecdysone receptor-based promoter, and steroid / retinoid / thyroid receptor superfamily-based promoters), metal-regulated promoters (e.g., metallothionein gene-based promoter), and pathogen-related promoters (e.g., Arabidopsis (Arabidopsis) and maize pathogen-associated (PR) protein-based promoters). Typical physically regulated promoters include, but are not limited to, temperature-regulated promoters (e.g., heat shock promoter) and light-regulated promoters (e.g., soybean SSU promoter).Other exemplary promoters are listed elsewhere on the World Wide Web, for example, in the Hypertext Transfer Protocol section at patentlens.net / daisy / promoters / 768 / 271.html.

[0104] Those skilled in the art will be able to select an appropriate promoter based on specific circumstances. Many different promoters are well known in the art, as are methods for functionally linking promoters to the genes to be expressed. Both natural promoter sequences and many heterologous promoters can be used to direct expression in packaging and target cells. However, heterologous promoters are preferred because they generally allow for greater transcription and higher yields of the desired protein compared to natural promoters.

[0105] Promoter may be obtained from the genomes of viruses such as polyomavirus, fowlpox virus, adenovirus, bovine papillomavirus, aerosarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and Simian virus 40 (SV40). The promoter may be, for example, a heterozoan promoter, such as an actin promoter or immunoglobulin promoter, a heat shock promoter, or a promoter that is usually linked to a native sequence, provided that such a promoter is compatible with the target cell. In one embodiment, the promoter is a naturally occurring viral promoter in the viral expression system.

[0106] Transcription can be increased by inserting enhancer sequences into a vector. Enhancers are typically cis-acting elements of DNA, usually about 10–300 bp in length, that act on the promoter to increase its transcription. Many enhancer sequences are currently known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). The use of enhancers from eukaryotic viruses is particularly intended. Examples include the SV40 enhancer (bp 100–270) posterior to the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer posterior to the origin of replication, and the adenovirus enhancer. Enhancers can be spliced ​​into the vector at the 5' or 3' end of the antigen-specific polynucleotide sequence and positioned 5' from the promoter.

[0107] Other vectors and methods suitable for the expression of viral polypeptides are well known in the art and can be easily adapted to specific situations.

[0108] Using the teachings provided herein, those skilled in the art will recognize that the effectiveness of a particular expression system can be tested, for example, by measuring fluorescence from a green fluorescent protein conjugate, by transforming packaging cells with a vector containing a gene encoding a reporter protein and measuring its expression using an appropriate technique. Suitable reporter genes are well known in the art.

[0109] Vectors encoding core viruses are also known as “viral vectors.” There are many available viral vectors suitable for use in the present invention, including those identified for human gene therapy applications, such as those described by Pfeifer and Verma (2001, the entire work is incorporated herein by reference). Suitable viral vectors include retrovirus-derived vectors, such as RNA virus-based vectors like those derived from Moloney's mouse leukemia virus (MLV), and more complex retrovirus-derived vectors, such as those derived from lentiviruses. Vectors derived from human immunodeficiency virus (HIV-1) belong to this category. Other examples include lentiviral vectors derived from HIV-2, feline immunodeficiency virus (Hy), equine infectious anemia virus, simian immunodeficiency virus (SIV), and maedi / visna virus.

[0110] In particular, viral vectors may contain one or more genes encoding components of a recombinant virus, as well as nucleic acids encoding all or part of a functional MART-1 TCR. In some embodiments, the viral vector encodes components of a recombinant virus and at least one of the m1-α, m1-β, and m2-β variable regions, optionally a suicide gene or a reporter gene. In other embodiments, the viral vector encodes components of a recombinant virus and at least one of the m1-α subunit, m1-β subunit, and m2-β subunit, optionally a suicide gene or a reporter gene. Viral vectors may also contain genetic elements that promote the expression of corresponding α and β polynucleotide sequences in target cells, such as promoter and enhancer sequences. To prevent replication in target cells, endogenous viral genes necessary for replication may be removed and provided separately in a packaging cell line.

[0111] In certain embodiments, the viral vector comprises intact retrovirus 5' LTR and self-inactivated 3' LTR.

[0112] Infectious retroviruses and / or lentiviral particles can be produced using any method known in the art, the genome of which contains an RNA copy of a viral vector. For this purpose, a viral vector (at least one of the m1-α subunit and m1-β subunit of the TCR that recognizes the peptide antigens of Table 1, along with optionally another vector encoding a suicide gene) can be introduced into a packaging cell line that packages viral genomic RNA based on the viral vector into viral particles.

[0113] Packaging cell lines provide the viral proteins required in trans for packaging viral genomic RNA into viral particles. A packaging cell line can be any cell line capable of expressing retroviral proteins. Specific packaging cell lines include 293 (ATCC CCL X), Platinum A, HeLa (ATCC CCL 2), D17 (ATCC CCL 183), MDCK (ATCC CCL 34), BHK (ATCC CCL-10), and Cf2Th (ATCC CRL 1430). Packaging cell lines can stably express the required viral proteins. Such packaging cell lines are described, for example, in U.S. Patent No. 6,218,181, which is incorporated herein by reference in its entirety. Alternatively, the packaging cell line may be transiently transfected with a plasmid containing nucleic acids encoding one or more required viral proteins, including but not limited to gag, pol, rev, and any envelope proteins that facilitate the transduction of target cells, along with a viral vector encoding at least one of the m1-α and m1-β subunits of the TCR that recognizes the peptide antigens of Table 1.

[0114] Typically, viral particles containing polynucleotides comprising a gene of interest, at least one of the m1-α variable region nucleotide sequence, the m1-β variable region nucleotide sequence, and the m2-β variable region nucleotide sequence, and optionally a suicide gene or reporter gene, are collected and used to infect target cells. In some embodiments, the gene of interest comprises at least one of the m1-α subunit nucleotide sequence, the m1-β subunit nucleotide sequence, and the m2-β subunit nucleotide sequence. In some embodiments, the virus is pseudotyped to achieve target cell specificity. Methods of pseudotyped viruses are well known in the art and are described herein.

[0115] In one embodiment, the recombinant virus used to deliver the gene of interest is a modified lentivirus, and the viral vector is lentivirus-based. Since lentiviruses can infect both dividing and non-dividing cells, in this embodiment, the target cells do not need to be dividing (or stimulated to divide).

[0116] In another embodiment, the recombinant virus used to deliver the gene of interest is a modified gamma retrovirus, and the viral vector is based on a gamma retrovirus.

[0117] In another embodiment, the vector is based on mouse stem cell virus (MSCV; (Hawley, RG, et al. (1996) Proc. Natl. Acad. Sci. USA 93:10297-10302; Keller, G., et al. (1998) Blood 92:877-887; Hawley, RG, et al. (1994) Gene Ther. 1:136-138, each of the above is incorporated herein by reference in whole). The MSCV vector provides long-term stable expression in target cells, particularly hematopoietic progenitor cells and their differentiated offspring.

[0118] In another embodiment, the vector is based on a modified Moloney virus, such as Moloney mouse leukemia virus. Viral vectors can similarly be based on hybrid viruses, such as those described by Choi, JK et al. (2001. Stem Cells 19, No. 3, 236-246, which is incorporated herein by reference in its entirety).

[0119] DNA viral vectors can be used, including, for example, vectors based on adenoviruses and vectors based on adeno-associated viruses (AAVs). Similarly, retrovirus-adenovirus vectors can also be used in the method of the present invention.

[0120] Other vectors, including those derived from herpes simplex virus (HSV), such as amplicon vectors, replication-deficient HSV, and attenuated HSV, can also be used for polynucleotide delivery (Krisky et al. 1998. Gene Ther. 5: 1517-30, which is incorporated herein by reference in its entirety).

[0121] Other vectors recently developed for use in gene therapy can also be used in the method of the present invention. Such vectors include those derived from baculoviruses and alphaviruses. Jolly, DJ (1999). Emerging viral vectors. pp 209-40 in Friedmann T, ed. (1999). The development of human gene therapy. New York: Cold Spring Harbor Lab. This is incorporated herein by reference in its entirety.

[0122] In some specific embodiments, the viral construct includes sequences from a lentiviral genome, such as an HIV genome or an SIV genome. The viral construct may include sequences from the 5' and 3' LTRs of a lentivirus. More specifically, the viral construct includes R and U5 sequences from the 5' LTR of a lentivirus, as well as an inactivated or self-inactivated 3' LTR from a lentivirus. The LTR sequences can be LTR sequences from any lentivirus of any species. For example, they can be LTR sequences from HIV, SIV, FIV, or BIV. More specifically, the LTR sequences are HIV LTR sequences.

[0123] The viral construct may include an inactivated or self-inactivated 3' LTR. The 3' LTR may be self-inactivated by any method known in the art. In certain embodiments, the U3 element of the 3' LTR includes a deletion of its enhancer sequence, such as a TATA box, Spl, and NFκB site. As a result of the self-inactivated 3' LTR, the provirus incorporated into the host cell genome includes an inactivated 5' LTR.

[0124] Optionally, the U3 sequence from the lentivirus 5' LTR may be replaced with a promoter sequence in the viral construct. This may increase the titer of the virus recovered from the packaging cell line. Enhancer sequences may also be included. Any enhancer / promoter combination that increases the expression of the viral RNA genome in the packaging cell line may be used. In certain embodiments, CMV enhancer / promoter sequences are used.

[0125] In some embodiments, the viral construct may include an inactivated or self-inactivated 3' LTR. The 3' LTR may be self-inactivated by any method known in the art. In certain embodiments, the U3 element of the 3' LTR includes a deletion of its enhancer sequence, such as a TATA box, Spl, and NFκB site. As a result of the self-inactivated 3' LTR, the provirus incorporated into the host cell genome includes an inactivated 5' LTR.

[0126] A viral construct generally includes a gene of interest, typically comprising at least one of the following: an m1-α variable region nucleotide sequence, an m1-β variable region nucleotide sequence, an m2-β variable region nucleotide sequence, an m1-α subunit nucleotide sequence, an m1-β subunit nucleotide sequence, and an m2-β subunit nucleotide sequence, and optionally, a suicide gene or reporter gene that is desirablely expressed in one or more target cells. The gene of interest may be located between the 5' LTR sequence and the 3' LTR sequence. Furthermore, the gene of interest may have a functional relationship with other genetic elements, such as transcriptional regulatory sequences like promoters and / or enhancers, which, once incorporated into target cells, can regulate the expression of the gene of interest in a specific way. In certain embodiments, useful transcriptional regulatory sequences are highly regulated in terms of activity, both temporally and spatially.

[0127] In some embodiments, the gene of interest is functionally related to an internal promoter / enhancer regulatory sequence. The “internal” promoter / enhancer is located between the 5' LTR and 3' LTR sequences in the viral construct and is functionally linked to the gene to be expressed.

[0128] An internal promoter / enhancer can be any promoter, enhancer, or promoter / enhancer combination known to increase the expression of a gene with which it has a functional relationship. "Functional relationship" and "functionally linked" mean, non-limitingly, that the gene is in the correct position and orientation with respect to the promoter and / or enhancer, so that the gene's expression is affected when the promoter and / or enhancer is in contact with the appropriate molecule.

[0129] Internal promoters / enhancers can be selected based on the desired expression pattern of the gene of interest and specific characteristics of known promoters / enhancers. Therefore, internal promoters can be constitutive promoters. Non-limiting examples of constitutive promoters that may be used include the ubiquitin promoter, CMV (Karasuyama et al., 1989. J. Exp. Med. 169:13, which is incorporated herein by reference in its entirety), β-actin (Gunning et al., 1989. Proc. Natl. Acad. Sci. USA 84:4831-4835, which is incorporated herein by reference in its entirety), and pgk (see, for example, Adra et al., 1987. Gene 60:65-74; Singer-Sam et al., 1984. Gene 32:409-417; and Dobson et al., 1982. Nucleic Acids Res. 10:2635-2637, each of which is incorporated herein by reference in its entirety).

[0130] Furthermore, promoters can be selected to enable inducible gene expression. Several systems for inducible expression are known in the art, including tetracycline response systems and LAC operator-repressor systems. It is also intended that combinations of promoters can be used to obtain the desired expression of a gene of interest. Those skilled in the art will be able to select promoters based on the desired expression pattern of the gene in the organism of interest and / or target cells.

[0131] D. Chimeric antigen receptor As used herein, “chimeric antigen receptor” (CAR) refers to an engineered receptor that can transfer desired specificity to an antigen to immune effector cells, such as T cells and NK cells. Typically, a CAR protein comprises an extracellular domain that introduces the desired specificity, a transmembrane domain, and an intracellular domain that signals to immune effector cells when they bind to the antigen. In certain embodiments, the extracellular domain comprises a leader peptide, an antigen recognition region, and a spacer region. In certain embodiments, the antigen recognition region is derived from an antibody that specifically binds to the antigen. In certain embodiments, the antigen recognition region is a single-stranded variable fragment (scFv) derived from the antibody. In certain embodiments, the single-stranded variable fragment comprises a heavy-stranded variable region fused to a light-stranded variable region via a flexible linker.

[0132] As used herein, the term “leader peptide” is used in accordance with its common meaning in the art and refers to a peptide having a length of approximately 5 to 30 amino acids. Leader peptides are located at the N-terminus of newly synthesized proteins that form part of a secretory pathway. Proteins in the secretory pathway include, but are not limited to, proteins located in certain organelles (endoplasmic reticulum, Golgi apparatus, or endosomes), proteins secreted from cells, or proteins inserted into the cell membrane. In some embodiments, leader peptides form part of the transmembrane domain of a protein.

[0133] In one aspect, this disclosure provides CAR proteins that bind to antigens described herein. In some embodiments, the CAR protein comprises, in the direction from N-terminus to C-terminus, a leader peptide, an anti-antigen heavy chain variable domain, a linker domain, an anti-antigen light chain variable domain, a CD8α hinge region, a CD8α transmembrane domain (or CD28 transmembrane domain), a 4-1BB intracellular costimulatory signaling domain (or a CD28 intracellular costimulatory signaling domain, or a CD28 intracellular costimulatory signaling domain followed by a 4-1BB intracellular costimulatory signaling domain), and a CD3-ζ intracellular T cell signaling domain.

[0134] In some embodiments, the protein comprises, from N-terminus to C-terminus, a CD8a leader peptide, the HuCAR scFV antigen, a human CD8a hinge domain, a CD28 transmembrane domain, and a zeta (ζ) chain of the human CD3 complex T cell signaling domain.

[0135] In another embodiment, the protein comprises, from N-terminus to C-terminus, a CD8a leader peptide, the antigen HuCAR scFV, a human CD8a hinge domain, a 4-1BB intracellular costimulatory signaling domain, and a zeta (ζ) chain of a human CD3 complex T cell signaling domain.

[0136] In an alternative embodiment, the protein comprises, from N-terminus to C-terminus, a CD8a leader peptide, the antigen HuCAR scFV, a human CD8a hinge domain, a 4-1BB intracellular costimulatory signaling domain, a CD28 transmembrane domain, and a zeta (ζ) chain of a human CD3 complex T cell signaling domain.

[0137] In another embodiment, the protein comprises, from N-terminus to C-terminus, a leader peptide, an antigen heavy chain variable domain, a linker domain, an antigen light chain variable domain, a human IgG1-CH2-CH3 domain, a spacer region, a CD28 transmembrane domain, a 4-1BB intracellular costimulatory signaling domain, and a zeta (ζ) chain of a human CD3 complex T cell signaling domain.

[0138] In some embodiments, nucleic acids encode antibody heavy chain variable domains and antibody light chain variable domains from antibodies that bind to antigens.

[0139] In another aspect, an expression vector comprising nucleic acids provided herein, including that aspect, is provided. In another aspect, T lymphocytes comprising an expression vector provided herein, including that aspect, are provided. In another aspect, mammalian cells comprising an expression vector provided herein, including that aspect, are provided. In another aspect, a recombinant protein is provided. The recombinant protein comprises (i) an antibody region comprising a central cavity formed by a heavy chain variable (VH) region and a light chain variable (VL) region, the central cavity comprising a peptide bonding site comprising framework region amino acid residues; and (ii) a transmembrane domain.

[0140] In another context, a recombinant protein is provided. The recombinant protein comprises a first portion containing an antibody heavy chain variable domain and a second portion containing an antibody light chain variable domain and an antibody light chain constant domain, wherein the first portion further comprises a transmembrane domain, where the antibody heavy chain variable domain, the antibody light chain variable domain and the antibody light chain constant domain together form an antibody region.

[0141] In another context, a recombinant protein is provided. The recombinant protein comprises a first portion containing an antibody heavy chain variable domain and a second portion containing an antibody light chain variable domain, wherein the first portion further comprises a transmembrane domain, and thereafter the antibody heavy chain variable domain and the antibody light chain variable domain together form an antibody region.

[0142] In another context, a recombinant protein is provided. The recombinant protein comprises a first portion containing an antibody heavy chain variable domain and an antibody heavy chain constant domain, and a second portion containing an antibody light chain variable domain, wherein the first portion further comprises a transmembrane domain, where the antibody heavy chain variable domain, the antibody heavy chain constant domain, and the antibody light chain variable domain together form an antibody region.

[0143] In another context, a recombinant protein is provided. The recombinant protein comprises a first portion containing an antibody heavy chain variable domain and a second portion containing an antibody light chain variable domain and an antibody light chain constant domain, the second portion further comprising a transmembrane domain, wherein the antibody heavy chain variable domain, the antibody light chain variable domain and the antibody light chain constant domain together form an antibody region.

[0144] In another context, a recombinant protein is provided. The recombinant protein comprises a first portion containing an antibody heavy chain variable domain and a second portion containing an antibody light chain variable domain, the second portion further comprising a transmembrane domain, where the antibody heavy chain variable domain and the antibody light chain variable domain together form an antibody region.

[0145] In another aspect, a mammalian cell containing a recombinant protein provided herein, including its embodiments, is provided, where the transmembrane domain is located within the cell membrane of the mammalian cell.

[0146] In some embodiments, the transmembrane domain is a CD8α transmembrane domain. The term “CD8α transmembrane domain” as provided herein includes either recombinant or native forms of the CD8α transmembrane domain. In some aspects, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole or a portion of the sequence compared to a naturally occurring CD8α transmembrane domain polypeptide. In some embodiments, the CD8α transmembrane domain is It has the polypeptide sequence TIFF0007853938000010.tif4128. In some embodiments, the CD8α transmembrane domain is It is a protein encoded by the nucleic acid sequence TIFF0007853938000011.tif11149.

[0147] In some embodiments, the transmembrane domain is the CD28 transmembrane domain. The term “CD28 transmembrane domain” as provided herein includes either a recombinant or native form of the CD28 transmembrane domain, or a variant or homolog thereof that maintains CD28 transmembrane domain activity. In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the whole or a portion of the sequence compared to the naturally occurring CD28 transmembrane domain polypeptide. In some embodiments, the CD28 transmembrane domain is It has the polypeptide sequence TIFF0007853938000012.tif4128. In some embodiments, the CD28 transmembrane domain is It is a protein encoded by the nucleic acid sequence TIFF0007853938000013.tif11148.

[0148] In some embodiments, the intracellular T cell signaling domain is the CD3-ζ intracellular T cell signaling domain. In some embodiments, the intracellular T cell signaling domain includes the signaling domain of the zeta (ζ) chain of the human CD3 complex. In some embodiments, the intracellular T cell signaling domain is the CD3-ζ intracellular T cell signaling domain. In some embodiments, the intracellular T cell signaling domain is It is a protein encoded by the nucleic acid sequence TIFF0007853938000014.tif45150.

[0149] In some embodiments, the isolated nucleic acids provided herein include an intracellular costimulatory signaling sequence encoding an intracellular costimulatory signaling domain. The “intracellular costimulatory signaling domain” provided herein includes an amino acid sequence capable of providing costimulatory signaling in response to antigen binding to an antibody region provided herein, including its embodiments. In some embodiments, signaling of the costimulatory signaling domain results in cytokine production and proliferation of T cells expressing it. In some embodiments, the intracellular costimulatory signaling domain is the CD28 intracellular costimulatory signaling domain, the 4-1BB intracellular costimulatory signaling domain. In some embodiments, the intracellular costimulatory signaling domain includes the CD28 intracellular costimulatory signaling domain, the 4-1BB intracellular costimulatory signaling domain, the ICOS intracellular costimulatory signaling domain, the OX-40 intracellular costimulatory signaling domain, or any combination thereof. In some embodiments, the CD28 costimulatory domain is It has the polypeptide sequence TIFF0007853938000015.tif4143. In some embodiments, the CD28 intracellular costimulatory signaling domain is It is a protein encoded by the nucleic acid sequence TIFF0007853938000016.tif18150. In some embodiments, the 4-1BB intracellular costimulatory signaling domain is It has the polypeptide sequence TIFF0007853938000017.tif4143. In some embodiments, the 4-1BB intracellular costimulatory signaling domain is It is a protein encoded by the nucleic acid sequence TIFF0007853938000018.tif18149.

[0150] In some embodiments, the isolated nucleic acids provided herein include a spacer sequence encoding a spacer region. The “spacer region” provided herein is a polypeptide that connects an antibody region to a transmembrane domain or to various components of an antibody region. In some embodiments, the spacer region is located between the antibody region and the transmembrane domain. In some embodiments, the spacer region connects the heavy chain variable region to the transmembrane domain. In some embodiments, the spacer region connects the heavy chain constant region to the transmembrane domain. In some embodiments, the spacer region connects the light chain variable region to the transmembrane domain. In some embodiments, the spacer region connects the light chain constant region to the transmembrane domain. In some embodiments, the binding affinity of the antibody region to the antigen is increased compared to the case where the spacer region is absent. In some embodiments, steric hindrance between the antibody region and the antigen is reduced in the presence of the spacer region.

[0151] In some embodiments, the spacer region includes a hinge region. In some embodiments, the hinge region is the CD8α hinge region. In some embodiments, the hinge region is the CD28 hinge region.

[0152] In some embodiments, the spacer region includes an Fc region. Examples of spacer regions intended for the compositions and methods provided herein include, but are not limited to, immunoglobulin molecules or fragments thereof (e.g., IgG1, IgG2, IgG3, IgG4) and immunoglobulin molecules or fragments thereof (e.g., IgG1, IgG2, IgG3, IgG4) containing mutations that affect Fc receptor binding. In some embodiments, the spacer region is a fragment of IgG (e.g., IgG4), where the fragment includes a deletion of the CH2 domain. The spacer region may be a peptide linker. In some embodiments, the nucleic acid does not contain a spacer sequence encoding the spacer region.

[0153] In some embodiments, the spacer region connects various components of the antibody region. In some embodiments, the spacer region connects the heavy chain variable region to the light chain variable region.

[0154] In some embodiments, the isolated nucleic acids provided herein include a linker sequence encoding a linker domain. In some embodiments, the linker domain is inserted between the VH and VL of scFv. In some embodiments, the linker domain is located between the transmembrane domain and the intracellular T cell signaling domain. In some embodiments, the linker domain is located between the intracellular T cell signaling domain and the intracellular costimulatory signaling domain. In some embodiments, the linker domain is a sequence Includes TIFF0007853938000019.tif4128.

[0155] In some embodiments, the isolated nucleic acids provided herein do not contain a linker sequence encoding a linker domain.

[0156] In some embodiments, the nucleic acid comprises (i) a heavy chain sequence encoding the heavy chain domain of a protein, wherein the heavy chain domain includes a variable heavy chain domain and a transmembrane domain; and (ii) a light chain sequence encoding the light chain domain of a protein, wherein the light chain domain includes a variable light chain domain, where together the variable heavy chain domain and the variable light chain domain form at least a portion of the antibody region.

[0157] In some embodiments, the nucleic acid comprises (i) a heavy chain sequence encoding a heavy chain domain of a protein, wherein the heavy chain domain includes a variable heavy chain domain; and (ii) a light chain sequence encoding a light chain domain of a protein, wherein the light chain domain includes a variable light chain domain and a transmembrane domain, wherein the variable heavy chain domain and the variable light chain domain together form at least a portion of the antibody region.

[0158] The “heavy chain sequence” provided herein refers to a nucleic acid sequence encoding a heavy chain domain provided herein. The heavy chain domain provided herein may include a heavy chain variable (VH) region and / or a heavy chain constant region (CH). The “light chain sequence” provided herein refers to a nucleic acid sequence encoding a light chain domain provided herein. The light chain domain provided herein may include a light chain variable (VL) region and / or a light chain constant region (CL). The term “heavy chain domain” as used herein is used in accordance with its ordinary meaning in the art and refers to a polypeptide containing a heavy chain variable (VH) region and a heavy chain constant region (CH). The term “light chain domain” as used herein is used in accordance with its ordinary meaning in the art and refers to a polypeptide containing a light chain variable (VL) region and a light chain constant region (CL). In some embodiments, antibody heavy chain variable domains and antibody light chain variable domains are humanized.

[0159] In some embodiments, the protein or antibody region provided herein, including such embodiments, competes for antigen binding with one, more, or all CDRs (or CDRs having at least or about 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% identity with the CDR) of an antibody that binds to an antigen, including, for example, heavy chain CDRs 1, 2, and / or 3, and / or light chain CDRs 1, 2, and / or 3, and specifically binds to and / or that antigen or epitope.

[0160] In some embodiments, nucleic acids encode antibody heavy chain variable domains and antibody light chain variable domains from antibodies that bind to antigens.

[0161] In some embodiments, the protein comprises an intracellular costimulatory signaling domain and a CD3-ζ intracellular T cell signaling domain. In some embodiments, the protein comprises a heavy chain variable domain, a light chain variable domain, a transmembrane domain, an intracellular costimulatory signaling domain, and a CD3-ζ intracellular T cell signaling domain, arranged from the amino terminus to the carboxyl terminus.

[0162] In some embodiments, the protein comprises an intracellular costimulatory signaling domain and a CD3-ζ intracellular T cell signaling domain. In some embodiments, the protein comprises, from the amino terminus to the carboxyl terminus, a light chain variable domain, a heavy chain variable domain, a transmembrane domain, an intracellular costimulatory signaling domain, and a CD3-ζ intracellular T cell signaling domain.

[0163] In some embodiments, the recombinant protein comprises a first portion containing an antibody heavy chain variable domain and a second portion containing an antibody light chain variable domain. In some embodiments, the first portion comprises an intracellular costimulatory signaling domain and a CD3-ζ intracellular T cell signaling domain. In some embodiments, the first portion comprises, from the amino terminus to the carboxyl terminus, a heavy chain variable domain, a transmembrane domain, an intracellular costimulatory signaling domain, and a CD3-ζ intracellular T cell signaling domain.

[0164] In some embodiments, the recombinant protein comprises a first portion containing an antibody heavy chain variable domain and a heavy chain constant domain, and a second portion containing an antibody light chain variable domain. In some embodiments, the first portion contains an intracellular costimulatory signaling domain and a CD3-ζ intracellular T cell signaling domain. In some embodiments, the first portion comprises, from the amino terminus to the carboxyl terminus, a heavy chain variable domain, a heavy chain constant domain, a transmembrane domain, an intracellular costimulatory signaling domain, and a CD3-ζ intracellular T cell signaling domain.

[0165] In some embodiments, the protein comprises a CD3-ζ intracellular T cell signaling domain and an intracellular co-stimulatory signaling domain. In some embodiments, the protein comprises, from the amino terminus to the carboxyl terminus, a heavy chain variable domain, a light chain variable domain, a transmembrane domain, a CD3-ζ intracellular T cell signaling domain, and an intracellular co-stimulatory signaling domain.

[0166] In some embodiments, the recombinant protein comprises a first portion containing an antibody heavy chain variable domain and a second portion containing an antibody light chain variable domain. In some embodiments, the first portion comprises a CD3-ζ intracellular T cell signaling domain and an intracellular costimulatory signaling domain. In some embodiments, the first portion comprises, from the amino terminus to the carboxyl terminus, a heavy chain variable domain, a transmembrane domain, a CD3-ζ intracellular T cell signaling domain, and an intracellular costimulatory signaling domain.

[0167] In some embodiments, the recombinant protein comprises a first portion containing an antibody heavy chain variable domain and a heavy chain constant domain, and a second portion containing an antibody light chain variable domain. In some embodiments, the first portion contains a CD3-ζ intracellular T cell signaling domain and an intracellular co-stimulatory signaling domain. In some embodiments, the first portion comprises, from the amino terminus to the carboxyl terminus, a heavy chain variable domain, a heavy chain constant domain, a transmembrane domain, a CD3-ζ intracellular T cell signaling domain, and an intracellular co-stimulatory signaling domain.

[0168] In some embodiments, isolated nucleic acids encode proteins in the N-terminus to C-terminus direction: leader peptide, anti-antigen heavy chain variable domain, linker domain, anti-antigen light chain variable domain, human IgG1-CH2-CH3 domain, spacer region, CD28 domain, 4-1BB intracellular costimulatory signaling domain, and CD3-ζ intracellular T cell signaling domain.

[0169] In some embodiments, isolated nucleic acids encode proteins in the N-terminus-C-terminus direction: leader peptide, anti-antigen heavy chain variable domain, linker domain, anti-antigen light chain variable domain, spacer region, CD28 domain, 4-1BB intracellular costimulatory signaling domain, and CD3-ζ intracellular T cell signaling domain.

[0170] In some embodiments, isolated nucleic acids encode proteins in the N-terminus-C-terminus direction: leader peptide, anti-antigen heavy chain variable domain, linker domain, anti-antigen light chain variable domain, spacer region, CD28 transmembrane, and costimulatory domain, as well as CD3-ζ intracellular T cell signaling domain.

[0171] In some embodiments, the isolated nucleic acid encodes a protein in the N-terminal to C-terminal direction: leader peptide, antigen-binding heavy chain variable domain, linker domain, antigen-binding light chain variable domain, spacer region, CD8α transmembrane domain (or CD28 transmembrane domain), 4-1BB intracellular co-stimulatory signaling domain, and CD3-ζ intracellular T cell signaling domain.

[0172] In some embodiments, the protein is, in the direction from the N-terminus to the C-terminus, a leader peptide encoded by the nucleic acid of TIFF0007853938000020.tif11148, an antigen-binding heavy chain variable domain coding region, a linker domain encoded by the nucleic acid of TIFF0007853938000021.tif11150, an antigen-binding light chain variable domain coding region, a hinge region encoded by the nucleic acid of TIFF0007853938000022.tif18150, a CD28 domain encoded by the nucleic acid of SEQ ID NO: 16; a 4-1BB intracellular co-stimulatory signaling domain encoded by the nucleic acid of SEQ ID NO: 18, and a CD3-ζ intracellular T cell signaling domain encoded by the nucleic acid of SEQ ID NO: 14.

[0173] In certain embodiments, the antigen CAR proteins provided herein demonstrate high affinity for the antigen. In certain embodiments, the CAR proteins provided herein have a binding affinity (EC measured by ELISA) for the antigen of less than 1 nM, 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, 0.1 nM, 0.09 nM, 0.08 nM, 0.07 nM, 0.06 nM, or 0.05 nM. 50 ) For the purposes of this application, ELISA EC 50The values ​​can be determined as follows: Antigen-4 extracellular domain protein (with 6 HIS tag at the C-terminus) was recombinantly produced in HEK293 cells and coated high-binding 96-well clear plates (Corning-Costar, Fisher Scientific) at a concentration of 1 μg / ml (100 μl / well) at 4°C for 14-16 hours. The coated plates were briefly washed with PBS, pH 7.4 and blocked at 37°C for 2 hours with 5% skim milk in 200 μl / well PBS. Serial dilutions of the test monoclonal antibody (IgG or scFv fragment) were added to the 96-well plates by covering the assay plate and incubating at 37°C for 45 minutes, starting from 10 μg / ml and decreasing by 3-fold in 12 steps. The plates were then washed three times with PBS containing Tween 20 (0.05% concentration) and once with PBS. Secondary antibodies (Jackson ImmunoResearch) containing HRP conjugates, anti-human, anti-rabbit, or other IgG-specific antibodies, were added for incubation at room temperature for 1 hour according to the manufacturer's recommended dilution ratio. Detection was performed by adding HRP substrate TMB (ThermoFisher) for 10 minutes, followed by the addition of 50 μl / well of 2N H2SO4 to stop the reaction. The absorbance at 450 nm was read from the plate using a plate reader (SpectraMax M4, Molecular Devices). The data were recorded in EC. 50 For calculations, data was collected and graphed using a 4-parameter fitted curve with GrapPad Prism 7 software.

[0174] In another aspect, T lymphocytes comprising recombinant proteins provided herein, including in their respective embodiments, are provided, where the transmembrane domain is located within the cell membrane of the T lymphocyte.

[0175] E. Vaccine Cancer vaccines are a form of active immunotherapy in which antigenic peptides, polypeptides, or proteins, or autologous or allogeneic tumor cell compositions or "vaccines," such as the antigens disclosed in Table 1, are administered to the target. Vaccines may be administered systemically, for example, intravenously or intradermally. Vaccines may also be administered multiple times to enhance the immune response to the administered antigen.

[0176] 1. Adjuvant As is also well known in the art, the immunogenicity of certain immunogenic compositions can be enhanced by the use of nonspecific immune response stimulants known as adjuvants. Adjuvants have been experimentally used to promote an overall increase in immunity to immunogenic antigens (e.g., U.S. Patent No. 4,877,611). Immunotherapy protocols have long used adjuvants to stimulate responses, and thus, adjuvants are well known to those skilled in the art. Some adjuvants affect the way antigens are presented. For example, the immune response increases when protein antigens are adsorbed onto an alum. Emulsification of antigens also extends the duration of antigen presentation and initiates an innate immune response. Suitable molecular adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins, or synthetic compositions.

[0177] In some respects, the compositions described herein may further include other adjuvants. Although alum is an approved adjuvant for human use, adjuvants in experimental animals include Freund's complete adjuvant (a nonspecific stimulant of the immune response containing dead Mycobacterium tuberculosis bacteria), Freund's incomplete adjuvant, and aluminum hydroxide adjuvant. Other adjuvants that may also be used in animals and sometimes in humans include interleukin (IL)-1, IL-2, IL-4, IL-7, IL-12, interferon, Bacillus Calmette-Guerin (BCG), aluminum hydroxide, muramyl dipeptide (MDP) (N-acetylmuramyl-L-alanyl-D-isoglutamine MDP) compounds, such as thur-MDP and nor-MDP, lipid A, and monophosphoryl lipid A (MPL). A RIBI containing three bacterial extracts—MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS)—in a 2% squalene / Tween 80 emulsion is also being considered. Even MHC antigens may be used.

[0178] In one approved application for human use, the adjuvant effect is achieved by using drugs such as alum, which are used as a solution of approximately 0.05 to 0.1% in phosphate-buffered saline. Alternatively, in experimental animals, the antigen is prepared as a mixture with a synthetic polymer of sugar (Carbopol®) used as a solution of approximately 0.25%. The adjuvant effect can also be achieved by agglutinating the antigen in the vaccine by heat treatment at temperatures ranging from approximately 70°C to 101°C for 30 seconds to 2 minutes each. Agglutination by reactivation with pepsin-treated (Fab) antibodies against albumin, mixing with bacterial cells such as C. parvum, endotoxins or lipopolysaccharide components of Gram-negative bacteria, emulsification in a physiologically acceptable oily vehicle such as mannidomonoleate (Aracel A), or emulsification with a 20% solution of perfluorocarbon (Fluosol-DA®) used as a block substitute may also be employed.

[0179] Some adjuvants, such as certain organic molecules derived from bacteria, act on the host rather than the antigen. An example is bacterial peptidoglycan (MDP). Although the effects of MDP, like most adjuvants, are not fully understood, it is now beginning to be understood that MDP activates cells of the innate immune system, such as dendritic cells, macrophages, neutrophils, NKT cells, and NK cells. MDP stimulates macrophages, but it also appears to directly stimulate B cells. Therefore, the effects of adjuvants are non-specific to the antigen. However, if adjuvants are administered together with purified antigens, they can be used to selectively enhance the response to the antigen.

[0180] In certain embodiments, hemocyanins and hemoerythrins may also be used in the compositions of this disclosure. While the use of keyhole limpet-derived hemocyanins (KLH) is used in certain embodiments, hemocyanins and hemoerythrins from other mollusks and arthropods may also be employed.

[0181] Various polysaccharide adjuvants may also be used. For example, the use of various pneumococcal polysaccharide adjuvants for antibody responses in mice has been documented (Yin et al., 1989). The dose that produces the optimal response, or a dose that does not cause suppression, should be adopted as indicated (Yin et al., 1989). Polysaccharide polyamine heterogenes, such as chitin and chitosan, including deacetylated chitin, are particularly noteworthy.

[0182] Another group of adjuvants is bacterial peptidoglycans of the muramyl dipeptide (MDP, N-acetylmuramyl-L-alanyl-D-isoglutamine) group. Muramyl dipeptide derivatives such as the amino acid derivative threonyl-MDP, and the fatty acid derivative muramyl tripeptide phosphatidylethanolamine (MTPPE) are also being considered.

[0183] U.S. Patent No. 4,950,645 describes a lipophilic disaccharide-tripeptide derivative of muramyl dipeptide, described for use in artificial liposomes formed from phosphatidylcholine and phosphatidylglycerol. This is effective in activating human monocytes and destroying tumor cells, but is generally non-toxic at high doses. The compounds described in U.S. Patent No. 4,950,645 and the PCT patent application, International Publication No. 91 / 16347, are intended for use in cell carriers and other embodiments of this disclosure.

[0184] BCG and BCG-cell wall skeleton (CWS), with or without trehalose dimicolate, can also be used as adjuvants. Trehalose dimicolate itself can be used. Administration of trehalose dimicolate has been shown to correlate with enhanced resistance to influenza virus infection in mice (Azuma et al., 1988). Trehalose dimicolate can be prepared as described in U.S. Patent No. 4,579,945. BCG is an important clinical tool thanks to its immunostimulatory properties. BCG acts to stimulate the reticuloendothelial system (RES), activate natural killer (NK) cells, and increase the proliferation of hematopoietic stem cells. Cell wall extracts of BCG have been shown to have excellent immunoadjuvant activity. Molecular genetic tools and methods for mycobacteria have provided means to introduce exogenous genes into BCG (Jacobs et al., 1987; Snapper et al., 1988; Husson et al., 1990; Martin et al., 1990). Live BCG is an effective and safe vaccine used worldwide to prevent tuberculosis. BCG and other mycobacteria are highly effective adjuvants, and the immune response to mycobacteria has been extensively studied. With nearly 2 billion immunizations performed, BCG has a long-standing record of safe use in humans (Luelmo, 1982; Lotte et al., 1984). BCG is one of the few vaccines that can be given at birth, producing a long-lasting immune response with just one dose, and has a global distribution network with experience in BCG vaccination. An exemplary BCG vaccine is marketed as TICE BCG (Organon Inc., West Orange, NJ).

[0185] Amphiphilic agents and surfactants, such as saponins and derivatives including QS21 (Cambridge Biotech), form another group of adjuvants for use in immunogens of this disclosure. Nonionic block copolymer surfactants (Rabinovich et al., 1994) may also be employed. Oligonucleotides are another useful group of adjuvants (Yamamoto et al., 1988). Quil A and lentinen are other adjuvants that may be used in certain embodiments of this disclosure.

[0186] Another group of adjuvants are detoxified endotoxins, such as purified and detoxified endotoxins described in U.S. Patent No. 4,866,034. These purified and detoxified endotoxins are effective in producing adjuvant responses in mammals. Of course, detoxified endotoxins can be combined with other adjuvants to prepare cells incorporating multiple adjuvants. For example, the combination of detoxified endotoxin and trehalose dimycolate is particularly intended, as described in U.S. Patent No. 4,435,386. Combinations of detoxified endotoxin with trehalose dimycolate and endotoxinous glycolipids are also intended (U.S. Patent No. 4,505,899), as are combinations of detoxified endotoxin with cCWS or CWS and trehalose dimycolate, as described in U.S. Patents No. 4,436,727, 4,436,728, and 4,505,900. As described in U.S. Patent No. 4,520,019, a combination of CWS without detoxified endotoxins and trehalose dimicholate alone is also expected to be useful.

[0187] Those skilled in the art are aware of different types of adjuvants that can be conjugated to vaccines pursuant to this disclosure and are approved for human use compared to experimental use. These include, among others, alkyllysophospholipids (ALP); BCG; and biotin (including biotinylated derivatives). Certain adjuvants particularly intended for use are teicoic acids from Gram-negative bacterial cells. These include lipoteichoic acid (LTA), ribitol teicoic acid (RTA), and glycerol teicoic acid (GTA). Active forms of their synthetic counterparts may also be employed in conjunction with the compositions of this disclosure (Takada et al., 1995).

[0188] Various adjuvants, even those not typically used in humans, may still be employed in animals. Adjuvants can be encoded by nucleic acids (e.g., DNA or RNA). Such adjuvants may also be encoded by nucleic acids encoding antigens (e.g., expression vectors) or by different vectors or other constructs. The nucleic acids encoding the adjuvant can be delivered directly, for example, using lipids or liposomes.

[0189] 2. Biological response modifiers (BRMs) In addition to adjuvants, it may be desirable to co-administer a BRM that has been shown to upregulate T-cell immunity or downregulate suppressor cell activity. Such BRMs include cimetidine (CIM; 1200 mg / d) (Smith / Kline, PA); and low-dose cyclophosphamide (CYP; 300 mg / m²). 2 This includes, but is not limited to, genes encoding proteins involved in immune helper functions, such as cytokines, interferon, IL-2, or IL-12, or B-7. Further biological response moduli include those described in Gupta and Kanodia, 2002 and Bisht, et al., 2010, both of which are incorporated herein by reference.

[0190] 3. Chemokines Chemokines, nucleic acids encoding chemokines, and / or cells expressing them can also be used as vaccine components. Chemokines generally act as chemotaxis to recruit immune effector cells to the chemokine expression site. It may be advantageous to express specific chemokine-coding sequences together, for example, with cytokine-coding sequences, to enhance the recruitment of other immune system components to the treatment site. Such chemokines include, for example, RANTES, MCAF, MIP1-α, MIP1-β, IP-10, and combinations thereof. Those skilled in the art will recognize that certain cytokines are also known to have chemotaxis effects and can be classified under the term chemokine.

[0191] 4. Immunogenic carrier proteins In some embodiments, the vaccine antigens described herein may be chemically conjugated to a carrier or recombinantly expressed together with an immunogenic carrier peptide or polypeptide (e.g., an antigen-carrier fusion peptide or polypeptide) to enhance the immune response. Exemplary immunogenic carrier amino acid sequences include hepatitis B surface antigen (HBSA), tetanus toxoid (TT), keyhole limpet hemocyanin (KLH), and BSA. In humans, TT is advantageous because it is an already approved protein vaccine. For experimental animals, other albumins such as OVA, mouse serum albumin, or rabbit serum albumin can also be used as immunogenic carrier proteins. Means for conjugating polypeptides or peptides with immunogenic carrier proteins are well known in the art and include, for example, glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide, and bis-biazotized benzidine.

[0192] 5. Manipulated dendritic cells In some embodiments, this disclosure relates to dendritic cell (DC) vaccines. The DC vaccines contain antigen-presenting cells capable of inducing specific T-cell immunization, which are collected from a patient or donor. The DCs can then be exposed in vitro to the peptide antigens listed in Table 1, which will produce T cells in the patient. The antigen-loaded dendritic cells are then injected back into the patient. If necessary, the immunization can be repeated multiple times. Methods for collecting, expanding, and administering dendritic cells are well known in the art, for example, as described in Fong et al. (2001). DC vaccines are further described elsewhere, for example, in U.S. Patent Application No. 11 / 517,814, filed on 8 September 2006, titled "METHOD FOR THE GENERATION OF ANTIGEN-SPECIFIC LYMPHOCYTES"; U.S. Patent Application No. 11 / 071,785, filed on 2 March 2005, titled "ANTIGEN SPECIFIC T CELL THERAPY"; and U.S. Patent Application No. 11 / 446,353, filed on 1 June 2006, titled "METHOD OF TARGETED GENE DELIVERY USING VIRAL VECTORS," each of which is incorporated herein by reference in its entirety. A typical dose of DC administered to a patient contains at least approximately 10 million cells.

[0193] 6. MHC Class I Antigen For MHC class I peptides to induce a cellular immune response, they must also bind to MHC molecules. This process depends on specific polymorphisms in the MHC molecule's alleles and the peptide's amino acid sequence. Therefore, when considering this type of vaccine, it is crucial to match the MHC antigen profile to the patient's MHC profile.

[0194] MHC class I binding peptides are typically 8-12 amino acid residues long and usually contain two conserved residues ("anchors") in their sequence that interact with the corresponding binding groove of the MHC molecule. Thus, each MHC allele has a "binding motif" that determines which peptides can specifically bind to the binding groove. In MHC class I-dependent immune responses, peptides not only need to be able to bind to certain MHC class I molecules expressed by tumor cells, but also need to be recognized by T cells carrying a specific T cell receptor (TCR).

[0195] V. Host cells Certain aspects of this disclosure relate to immune cells expressing chimeric antigen receptors (CARs). These immune cells may be T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or γ-δ T cells), natural killer (NK) cells, invariant NK cells, or NKT cells. Methods for producing and manipulating immune cells, as well as methods for using and administering cells for adoptive cell therapy, are also provided herein, in which case the cells may be autologous or allogeneic. Thus, immune cells can be used as immunotherapies, such as targeting cancer cells.

[0196] Immune cells can be isolated from subjects, particularly human subjects. Immune cells can be obtained from subjects of interest, such as those suspected of having a specific disease or condition, those suspected of being predisposed to a specific disease or condition, those receiving treatment for a specific disease or condition, healthy volunteers or healthy donors, or from blood banks. Immune cells can be collected from any location present in the subject, including, but not limited to, blood, umbilical cord blood, spleen, thymus, lymph nodes, and bone marrow. Isolated immune cells may be used immediately or stored for a predetermined period, for example, by freezing.

[0197] Immune cells may be concentrated / purified from any tissue in which immune cells are present, including, but not limited to, blood (including blood collected from blood banks or umbilical cord blood banks), spleen, bone marrow, tissues removed and / or exposed during surgery, and tissues obtained by biopsy procedures. The tissues / organs from which immune cells are concentrated, isolated, and / or purified may be isolated from both abiotic and abiotic subjects, where abiotic subjects are organ donors. In certain embodiments, immune cells are isolated from blood, such as peripheral blood or umbilical cord blood. In some aspects, immune cells isolated from umbilical cord blood have enhanced immunomodulatory capacity, as measured by CD4-positive or CD8-positive T cell suppression. In specific aspects, immune cells are isolated from pooled blood, particularly from pooled umbilical cord blood, for enhanced immunomodulatory capacity. Pooled blood may originate from two or more sources, such as three, four, five, six, seven, eight, nine, ten, or more sources (e.g., donor subjects).

[0198] A population of immune cells can be obtained from a subject requiring treatment or from a subject suffering from a disease associated with reduced immune cell activity. Therefore, the cells would be autologous to the subject requiring treatment. Alternatively, a population of immune cells can be obtained from a donor, such as a histocompatibility-matched donor. The immune cell population can be collected from peripheral blood, umbilical cord blood, bone marrow, spleen, or any other organ / tissue where immune cells are present in the subject or donor. Immune cells can be isolated from a pool of subjects and / or donors, for example, from pooled umbilical cord blood.

[0199] When a population of immune cells is obtained from a donor different from the target, the donor may be allogeneic, but the obtained cells are compatible with the target in that they can be introduced into the target. Allogeneic donor cells may or may not be human leukocyte antigen (HLA) compatible. To make them compatible with the target, allogeneic cells can be treated to reduce their immunogenicity.

[0200] A. T cells In some embodiments, immune cells are T cells. Several fundamental approaches for inducing, activating, and expanding functional antitumor effector cells have been described over the past two decades. These include autologous cells such as tumor-infiltrating lymphocytes (TILs); T cells activated ex vivo using autologous dendritic cells, lymphocytes, artificial antigen-presenting cells (APCs) or beads coated with T cell ligands and activating antibodies, or cells isolated by capturing target cell membranes; allogeneic cells that naturally express anti-host tumor T cell receptors (TCRs); and non-tumor-specific autologous or allogeneic cells that have been genetically reprogrammed or "redirected" to express tumor-reactive TCRs or chimeric TCR molecules that exhibit antibody-like tumor recognition ability known as "T bodies." These approaches have given rise to numerous protocols for the preparation and immunization of T cells that can be used in the manner described herein.

[0201] In some aspects, T cells originate from blood, bone marrow, lymph, umbilical cord, or lymphoid organs. In some aspects, the cells are human cells. The cells are typically primary cells, such as those isolated directly from the subject and / or those isolated from the subject and frozen. In some aspects, the cells include one or more subsets of T cells or other cell types, e.g., the whole T cell population, CD4 + cells, CD8 +This includes cells and their subpopulations, defined, for example, by function, activation state, maturity, differentiation, expansion, recirculation, potential for localization and / or persistence, antigen specificity, antigen receptor type, presence in a specific organ or compartment, marker or cytokine secretion profile, and / or degree of differentiation. With respect to the subject being treated, the cells may be allogeneic and / or autologous. In some aspects, for example in the case of ready-made techniques, the cells are pluripotent and / or multipotent, and are stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, this method includes steps of isolating cells from a subject, preparing them, processing them, culturing them, and / or manipulating them, and reintroducing them to the same patient before or after cryopreservation, as described herein.

[0202] T cells (e.g., CD4 + and / or CD8 + Among the subtypes and subpopulations of T cells, there are naive T cells (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes, for example, stem cell memory T (TSC) M ), central memory T (TC M ), effector memory T (T EM These include terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TILs), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T (MAIT) cells, spontaneously generated and adaptively regulatory T (Treg) cells, helper T cells such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells.

[0203] In some embodiments, one or more T cell populations are enriched or depleted of cells that are positive for or negative for a particular marker, such as a surface marker. In some cases, such markers are absent or expressed at relatively low levels in certain populations of T cells (e.g., non-memory cells), but present or expressed at relatively high levels in certain other populations of T cells (e.g., memory cells).

[0204] In some embodiments, T cells are isolated from PBMC samples by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other leukocytes, such as CD4. In some aspects, CD4 + or CD8 + The selection stage is CD4 + Helper and CD8 + It is used to isolate cytotoxic T cells. Such CD4 + and CD8 + The population can be further subdivided into subpopulations by positive or negative selection of one or more markers expressed or relatively highly expressed in naive, memory, and / or effector T cell subpopulations.

[0205] In some embodiments, CD8 + T cells are further enriched or depleted of naive, central memory, effector memory, and / or central memory stem cells by positive or negative selection based, for example, on surface antigens associated with each subpopulation. In some embodiments, central memory T (T CM Cell enrichment is performed to enhance efficacy, such as improving long-term survival, expansion, and / or engraftment after administration, which is particularly evident in such subpopulations in some aspects. See Terakura et al. (2012); Wang et al. (2012).

[0206] In some embodiments, the T cells are autologous T cells. In this method, a tumor sample is obtained from a patient and a single cell suspension is obtained. The single cell suspension can be obtained by any suitable method, for example, mechanically (e.g., by using a gentleMACS™ Dissociator, Miltenyi Biotec, Auburn, Calif. to break up the tumor) or enzymatically (e.g., with collagenase or DNase). The single cell suspension of the tumor enzymatic digest is cultured in interleukin-2 (IL-2). The cells are cultured until confluent (e.g., about 2×10 6 lymphocytes), for example, for about 5 to about 21 days, for example about 10 days to about 14 days. For example, the cells can be cultured for 5 days, 5.5 days or 5.8 days to 21 days, 21.5 days or 21.8 days, for example 10 days, 10.5 days or 10.8 days to 14 days, 14.5 days or 14.8 days.

[0207] The cultured T cells can be pooled and rapidly expanded. The rapid expansion provides an increase in the number of antigen-specific T cells of at least about 50-fold (e.g., 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more) over a period of about 10 days to about 14 days. The rapid expansion can provide an increase of at least about 200-fold (e.g., 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or more) over a period of about 10 days to about 14 days.

[0208] Expansion can be achieved by any of several methods, as is known in the art. For example, T cells can be rapidly expanded using non-specific T cell receptor stimulation in the presence of feeder lymphocytes and either interleukin-2 (IL-2) or interleukin-15 (IL-15), with IL-2 being particularly contemplated. Non-specific T cell receptor stimulation can include OKT3 at around 30 ng / ml, i.e., a murine monoclonal anti-CD3 antibody (available from Ortho-McNeil®, Raritan, N.J.). Alternatively, T cells can be rapidly expanded by in vitro stimulation of peripheral blood mononuclear cells (PBMC) with one or more antigens of cancer (such as its antigenic portion, like an epitope, or including cells), which may optionally be expressed from a vector in the presence of a T cell growth factor such as 300 IU / ml IL-2 or IL-15, such as a human leukocyte antigen A2 (HLA-A2) binding peptide, with IL-2 being contemplated. T cells induced in vitro are rapidly expanded by restimulation with the same antigen of cancer pulsed onto antigen presenting cells expressing HLA-A2. Alternatively, T cells can be restimulated with, for example, irradiated autologous lymphocytes, or irradiated HLA-A2+ allogeneic lymphocytes and IL-2.

[0209] Autologous T cells can be modified to express a T cell growth factor that promotes the growth and activation of autologous T cells. Suitable T cell growth factors include, for example, interleukin (IL)-2, IL-7, IL-15, and IL-12. Suitable methods of modification are known in the art. For example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 3 rdSee ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001; and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and John Wiley & Sons, NY, 1994. In certain contexts, modified autologous T cells express high levels of T cell growth factor. T cell growth factor coding sequences, such as those for IL-12, are readily available in the art, as are their promoters, and functional linking of these promoters to T cell growth factor coding sequences promotes high levels of expression.

[0210] B. NK cells In some aspects, immune cells are natural killer (NK) cells. Natural killer (NK) cells are a subpopulation of lymphocytes that exhibit spontaneous cytotoxicity against various tumor cells, virus-infected cells, and some normal cells in the bone marrow and thymus. NK cells are important effectors in the initial innate immune response to transformed and virus-infected cells. NK cells make up about 10% of lymphocytes in human peripheral blood. When lymphocytes are cultured in the presence of interleukin-2 (IL-2), they exhibit a strong cytotoxic reactivity. NK cells are effector cells known as macrogranular lymphocytes because of their large size and the presence of characteristic azurophilic granules in their cytoplasm. NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus. NK cells can be detected in humans by certain surface markers such as CD16, CD56, and CD8. NK cells do not express T cell antigen receptors, the pan T marker CD3, or surface immunoglobulin B cell receptors.

[0211] Stimulation of NK cells is achieved through crosstalk of signals originating from activating and inhibitory receptors on the cell surface. The activation state of NK cells is regulated by the balance of intracellular signals received from a range of germline-encoded activating and inhibitory receptors. When NK cells encounter abnormal cells (e.g., tumor or virus-infected cells) and activation signals are dominant, NK cells can rapidly induce apoptosis of target cells through the direct secretion of cytolytic granules containing perforin and granzymes or through the involvement of receptors containing death domains. Activated NK cells can also secrete type I cytokines such as interferon-γ, tumor necrosis factor-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF), thereby activating both innate and adaptive immune cells, as well as other cytokines. The production of these soluble factors by NK cells in the initial innate immune response significantly impacts the recruitment and function of other hematopoietic cells. Furthermore, NK cells are also central players in the regulatory crosstalk network with dendritic cells and neutrophils, which promote or suppress the immune response through physical contact and cytokine production.

[0212] In certain embodiments, NK cells are derived from human peripheral blood mononuclear cells (PBMCs), unstimulated leukocyte apheresis products (PBSCs), human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs), bone marrow, or umbilical cord blood by methods well known in the art. In particular, umbilical cord CBs are used to derive NK cells. In certain aspects, NK cells are isolated and expanded by the previously described method of ex vivo expansion of NK cells (Spanholtz et al., 2011; Shah et al., 2013). In this method, CB mononuclear cells are isolated by Ficol density gradient centrifugation and cultured in a bioreactor containing IL-2 and artificial antigen-presenting cells (aAPCs). After 7 days, the cell culture is depleted of CD3-expressing cells and cultured for another 7 days. The cells are again depleted of CD3 and CD56 + / CD3 -It is characterized to determine the proportion of cells or NK cells. Alternatively, umbilical cord CB is used to characterize CD34 + CD56 by isolating cells and culturing them in a medium containing SCF, IL-7, IL-15, and IL-2. + / CD3 - NK cells are derived through cell differentiation.

[0213] C. Manipulation of host cells Immune cells (e.g., autologous or allogeneic T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or γ-δ T cells)), NK cells, invariant NK cells, or NKT cells) can be genetically engineered to express antigen receptors such as engineered TCRs and / or chimeric antigen receptors (CARs). For example, host cells (e.g., autologous or allogeneic T cells) can be modified to express T cell receptors (TCRs) that have antigen specificity for cancer antigens. In certain embodiments, NK cells can be engineered to express TCRs. NK cells can be further engineered to express CARs. Multiple CARs and / or TCRs for different antigens, etc., can be added to a single cell type, such as T cells or NK cells.

[0214] Suitable modification methods are known in the art. For example, see Sambrook and Ausubel, mentioned above. For example, cells can be transduced to express T cell receptors (TCRs) that have antigen specificity for cancer antigens using transduction techniques described in Heemskerk et al. (2008) and Johnson et al. (2009).

[0215] In some embodiments, the cell comprises one or more nucleic acids / expression constructs / vectors introduced via genetic engineering encoding one or more antigen receptors, as well as genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterogeneous, i.e., not typically found in cells or samples obtained from cells, e.g., those obtained from another organism or cell that are not typically found in the cell being engineered and / or the organism from which such cells originate. In some embodiments, the nucleic acids are not naturally occurring, such as nucleic acids not found in nature (e.g., chimeras).

[0216] The delivery of vectors to T cells has been discussed above, and that discussion is incorporated herein by reference.

[0217] VI. How to use A. Treatment In some embodiments, the present disclosure provides methods for immunotherapy, comprising the step of administering an effective amount of the immune cells of the present disclosure. In one embodiment, a medical disease or disorder is treated by the transfer of an immune cell population that elicits an immune response. In certain embodiments of the present disclosure, cancer is treated by the transfer of an immune cell population that elicits an immune response. Provided herein are methods for treating or delaying the progression of cancer in an individual, comprising the step of administering an effective amount of antigen-specific cell therapy to the individual. The methods of the present invention may be applied to the treatment of immunodisorders, solid tumors, or hematological malignancies.

[0218] In certain aspects of this disclosure, immune cells are delivered to an individual in need, such as an individual with cancer. These cells then enhance the individual's immune system to attack the respective cancer cells. In some cases, the individual is provided with one or more doses of immune cells. If the individual is provided with two or more doses of immune cells, the period between doses must be sufficient to give the individual time for proliferation, and in specific embodiments, the period between doses is one, two, three, four, five, six, seven, or more days.

[0219] In certain embodiments, growth factors that promote the growth and activation of immune cells are administered to the target simultaneously with or after the immune cells. Immune cell growth factors can be any suitable growth factors that promote the growth and activation of immune cells. Examples of suitable immune cell growth factors include interleukin (IL)-2, IL-7, IL-15, and IL-12, which can be used alone or in various combinations, for example, IL-2 and IL-7, IL-2 and IL-15, IL-7 and IL-15, IL-2, IL-7 and IL-15, IL-12 and IL-7, IL-12 and IL-15, or IL-12 and IL-2.

[0220] A therapeutically effective dose of immune cells can be administered by parenteral administration via several routes, including intravenous, intraperitoneal, intramuscular, intrasternal, or intra-articular injection or infusion.

[0221] The immune cell population can be administered in a disease-appropriate treatment regimen, such as a single or multiple doses over one to several days to improve the disease state, or in regular doses over a long period to inhibit disease progression and prevent disease recurrence. The exact dose adopted in the prescription will depend on the route of administration and the severity of the disease or disorder, and should be determined according to the practitioner's judgment and the circumstances of each patient. The number of therapeutically effective immune cells will depend on the subject being treated, the severity and type of distress, and the method of administration. In some embodiments, the dose that can be used in the treatment of human subjects is at least 3.8 × 10⁶ 4 , at least 3.8 × 10 5 , at least 3.8 × 10 6 , at least 3.8 × 10 7 , at least 3.8 × 10 8 , at least 3.8 × 10 9 , or at least 3.8 × 10 10 individual immune cells / m 2 It extends to approximately 3.8 × 10⁻⁶. In certain embodiments, the dose used in treatment of human subjects is approximately 3.8 × 10⁻⁶. 9 From approximately 3.8 x 1010 individual immune cells / m 2 In a further embodiment, the number of therapeutically effective immune cells is approximately 5 × 10⁶ per kg of body weight. 6 From individual cells, approximately 7.5 × 10⁶ cells are produced per kg of body weight. 8 For example, approximately 2 x 10⁶ cells per kg of body weight. 7 Approximately 5 x 10 from a single cell 8 Each cell, or approximately 5 x 10⁶ cells per kg of body weight. 7 Approximately 2 × 10⁶ cells 8 Changes can occur down to the individual cell level. The exact number of immune cells can be easily determined by those skilled in the art based on the subject's age, weight, sex, and physiological state. The effective dose can be estimated from dose-response curves obtained from in vitro or animal model test systems.

[0222] B. Pharmaceutical Compositions Pharmaceutical compositions and formulations comprising immune cells (e.g., T cells, CAR-T cells, dendritic cells, or NK cells) and pharmaceutically acceptable carriers are also provided herein.

[0223] The pharmaceutical compositions and formulations described herein contain an active ingredient (e.g., an antibody or polypeptide) having a desired degree of purity, carried on one or more pharmaceutically acceptable carriers in the form of lyophilized formulations or aqueous solutions (Remington's Pharmaceutical Sciences 22). ndIt can be prepared by mixing it with (edition, 2012). Pharmacochemically acceptable carriers are generally non-toxic to the recipient at the dose and concentration used and include: buffers, e.g., phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives, e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; low molecular weight (less than approximately 10 residues) polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; These include, but are not limited to, chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include intervening drug dispersants such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publications 2005 / 0260186 and 2006 / 0104968. In one phase, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

[0224] C. Combination therapy In certain embodiments, compositions and methods of the present invention involve a population of immune cells combined with at least one further therapeutic agent. The further therapeutic agent may be radiotherapy, surgery (e.g., mammary tumor removal and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination thereof. The further therapeutic agent may be in the form of adjuvant or neoadjuvant therapy.

[0225] In some embodiments, further treatment is the administration of small molecule enzyme inhibitors or metastasis inhibitors. In some embodiments, further treatment is the administration of side-effect-reducing agents (e.g., agents intended to reduce the occurrence and / or severity of side effects of the procedure, such as antiemetics). In some embodiments, further treatment is radiotherapy. In some embodiments, further treatment is surgery. In some embodiments, further treatment is a combination of radiotherapy and surgery. In some embodiments, further treatment is gamma irradiation. In some embodiments, further treatment is therapies targeting the PBK / AKT / mTOR pathway, HSP90 inhibitors, tubulin inhibitors, apoptosis inhibitors, and / or chemopreventive agents. Further treatment may be one or more chemotherapeutic agents known in the art.

[0226] Immunotherapy can be administered before, during, after, or in various combinations with additional cancer therapies, such as immune checkpoint therapy. Administration can be made at intervals ranging from simultaneous to several minutes, days, or weeks apart. In some aspects where immunotherapy is provided to a patient separately from additional therapeutic agents, it will generally be ensured that no significant period elapses between the times of each delivery so that two compounds can exert an advantageously combined effect in the patient. In such situations, it is contemplated that antibody therapy and anti-cancer therapy can be provided to the patient within about 12 to 24 or within 72 hours of each other, more specifically, within about 6 to 12 hours of each other. In some situations, it may be desirable to significantly extend the treatment period, with several days (2 days, 3 days, 4 days, 5 days, 6 days, or 7 days) to several weeks (1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, or 8 weeks) elapsing between each administration.

[0227] Various combinations can be utilized. For the following examples, immunotherapy is "A" and anti-cancer therapy is "B". TIFF0007853938000023.tif17128

[0228] The administration of any compound or therapy of an aspect of the present invention to a patient, if any, will follow the general protocol for the administration of such a compound, taking into account the toxicity of the agent. Thus, in some aspects, there is a stage of monitoring the toxicity resulting from the combination therapy.

[0229] 1. Chemotherapy A variety of chemotherapeutic agents may be used according to embodiments of the present invention. The term "chemotherapy" refers to the use of drugs to treat cancer. "Chemotherapeutic agent" is used to imply a compound or composition administered in the treatment of cancer. These agents or drugs are classified according to their mode of activity within cells, for example, whether or not they affect the cell cycle and at what stage they affect it. Alternatively, agents may be characterized based on their ability to directly crosslink DNA, intercalate into DNA, or induce chromosomal and cephalic abnormalities by affecting nucleic acid synthesis.

[0230] Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carbocone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolomelamine; acetogenins (specifically, bratacin and bratacinone); camptothecin (including its synthetic analog topotecan); bryostatin; calistatin; CC-1065 (including its synthetic analogs adzeresin, karzeresin, and bizeresin); cryptophycin (specifically, cryptophycin 1 and cryptophycin 8); drastatin; Duocalmycin (including synthetic analogs KW-2189 and CB1-TM1); eryuterobin; pancratistatin; sarcodictiin; spongistatin; chlorambucil, chlornafadin, colophosphamide, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, nobuenvicin, fenesterine, prednimustine, trophosphamide, and nitrogen mustards such as uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as engine antibiotics (e.g., calitiamycin, specifically calitiamycin γ1I and calitiamycin ωI1); dinemisins, including dinemisin A; bisphosphonates such as clodronate; esperamicin;Furthermore, neocardinostatin chromophore and related pigment protein enediin antibiotics chromophore, acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, cactinomycin, carabicin, carminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (morpholino-doxorubicin, cyanomorpholino -Doxorubicin (including 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin such as mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin, queramycin, rodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, and zolubicin; Antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folate analogs such as denopterin, pteropterin, and trimethotrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and phloxuridine; androgens such as carsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; antiadrenal drugs such as mitotane and trilostane; folate supplements such as folinic acid; acegraton; aldofsphamide glycosides; aminolevulinic acid; enyluracil; amsacrine; bestrabusil; bisantren; Edatrexate; Defofamine; Demecoltin; Diadiquan; Elformithine; Elliptinium acetate; Epotilon; Etoglucid; Gallium nitrate; Hydroxyurea; Lentinan; Ronidamine;Mytansinoids such as mytansine and anthamitosine; mitogwazone; mitoxantrone; mopidanmol; nitracrine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex; razoxane; lyzoxin; schizophyllan; spirogermanium; tenuazonic acid; triadicone; 2,2',2"-trichlorotriethylamine; trichothecin (specifically T-2 toxin, verracurin A, loridine A, and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitractol; pipobromane; gacytosine; arabinoside ("Ara-C"); Cyclophosphamide; Taxoids, e.g., paclitaxel, docetaxel, gemcitabine; 6-thioguanine; mercaptopurine; platinum-coordinated compounds such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; capecitabine; Examples include carboplatin, procarbazine, plicomycin, gemcitabine, navelbine, farnesyl protein transferase inhibitors, transplatinum, and any pharmaceutically acceptable salts, acids, or derivatives thereof.

[0231] 2. Radiation therapy Other widely used factors that cause DNA damage include gamma rays, commonly known as X-rays, and / or directional delivery of radioisotopes to tumor cells. Other types of DNA damaging factors such as microwaves, proton beam irradiation (US Patents 5,760,395 and 4,870,287), and UV irradiation are also considered. All of these factors will cause widespread damage to DNA, DNA precursors, DNA replication and repair, and chromosome assembly and maintenance. The dose range for X-rays ranges from a daily dose of 50–200 roentgens over a long period (3–4 weeks) to a single dose of 2,000–6,000 roentgens. The dose range for radioisotopes varies widely, depending on the half-life of the isotope, the intensity and type of radiation emitted, and uptake by neoplastic cells.

[0232] 3. Immunotherapy Those skilled in the art will understand that further immunotherapies may be used in combination with or in conjunction with the methods of this embodiment. In the context of cancer treatment, immunotherapeutic agents generally rely on the use of immune effector cells and immune effector molecules to target and destroy cancer cells. Rituximab (RITUXAN®) is one such example. Immune effectors can be antibodies specific to certain markers on the surface of tumor cells, for example. Antibodies can function as therapeutic effectors on their own, or they can mobilize other cells that actually have an effect on cell death. Antibodies can also be conjugated with drugs or toxins (chemotherapeutic agents, radionuclides, lysine A chain, cholera toxin, pertussis toxin, etc.) and function as targeting agents. Alternatively, effectors can be lymphocytes that hold surface molecules that directly or indirectly interact with tumor cell targets. Various effector cells include cytotoxic T cells and NK cells.

[0233] Antibody-drug conjugates have emerged as a groundbreaking approach to the development of cancer treatments. Cancer is one of the leading causes of death worldwide. Antibody-drug conjugates (ADCs) contain monoclonal antibodies (MAbs) covalently bound to a drug that kills cells. This approach combines the high specificity of MAbs to their antigenic targets with highly potent cytotoxic drugs, resulting in "armed" MAbs that deliver the payload (drug) to tumor cells along with abundant levels of antigen. Targeted drug delivery also minimizes drug exposure into normal tissues, leading to reduced toxicity and improved therapeutic metrics. The approval of two ADC drugs, ADCETRIS® (brentuximab vedotin), approved by the FDA in 2011, and KADCYLA® (trastuzumab emtansine or T-DM1), approved in 2013, confirms this approach. Currently, more than 30 ADC drug candidates are in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization become increasingly mature, the discovery and development of new ADCs largely depend on the identification and validation of novel targets suitable for this approach and the creation of targeted MAbs. Two criteria for ADC targets are upregulated / high levels of expression and robust internal translocation in tumor cells.

[0234] In one aspect of immunotherapy, tumor cells must possess some targetable marker, i.e., one that is not present in the majority of other cells. Many tumor markers exist, and any of these may be suitable for targeting in the context of the embodiments of this invention. Common tumor markers include CD20, carcinoembryonic antigen, tyrosinase (p97), gp68, TAG-72, HMFG, sialyl Lewis antigen, MucA, MucB, PLAP, laminin receptor, erb B, and p155. Another aspect of immunotherapy is combining anticancer effects with immunostimulatory effects. Immunostimulatory molecules also exist, including: cytokines such as IL-2, IL-4, IL-12, GM-CSF, and γIFN; chemokines such as MIP-1, MCP-1, and IL-8; and growth factors such as FLT3 ligand.

[0235] Examples of immunotherapies currently under investigation or in use include: immunoadjuvants, e.g., Mycobacterium bovis, Plasmodium falciparum, dinitrochlorobenzene, and aromatic compounds (U.S. Patent Nos. 5,801,005 and 5,739,169; Hui and Hashimoto, 1998; Christodoulides et al., 1998); cytokine therapies, e.g., interferon α, β, and γ, IL-1, GM-CSF, and TNF (Bukowski et al., 1998; Davidson et al., 1998; Hellstrand et al., 1998); gene therapies, e.g., TNF, IL-1, IL-2, and p53 (Qin et al., 1998; Austin-Ward and Villaseca, 1998; U.S. Patents No. 5,830,880 and 5,846,945; as well as monoclonal antibodies, e.g., anti-CD20, anti-ganglioside GM2, and anti-p185 (Hollander, 2012; Hanibuchi et al., 1998; U.S. Patent No. 5,824,311). One or more anticancer therapies are intended to be used in conjunction with the antibody therapies described herein.

[0236] In some embodiments, immunotherapy can be immune checkpoint inhibitors. Immune checkpoints either increase or decrease signals (e.g., co-stimulatory molecules). Inhibitory immune checkpoint molecules that can be targeted by immune checkpoint blockade include adenosine A2A receptor (A2AR), B7-H3 (also known as CD276), B and T lymphocyte attenuators (BTLA), cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152), indoleamine 2,3-dioxygenase (IDO), killer cell immunoglobulin (KIR), lymphocyte activator gene-3 (LAG3), programmed cell death 1 (PD-1), T cell immunoglobulin domain and mucin domain 3 (TIM-3), and V-domain Ig inhibitor of T cell activation (VISTA). Specifically, immune checkpoint inhibitors target the PD-1 axis and / or CTLA-4.

[0237] Immune checkpoint inhibitors may be drugs such as small molecules, recombinant forms of ligands or receptors, or antibodies such as human antibodies (see, for example, International Patent Publication WO2015016718; Pardoll, Nat Rev Cancer, 12(4): 252-64, 2012, both of which are incorporated herein by reference). Known inhibitors of immune checkpoint proteins or their analogues may be used, in particular chimeric, humanized, or human forms of antibodies. Those skilled in the art will understand that alternative names and / or equivalent names may be used for certain antibodies mentioned herein. Such alternative names and / or equivalent names are interchangeable in the context of the present invention. For example, lambrolizumab is known by the alternative and equivalent names MK-3475 and pembrolizumab.

[0238] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. In certain contexts, the PD-1 ligand-binding partner is PDL1 and / or PDL2. In another embodiment, a PDL1 binding antagonist is a molecule that inhibits the binding of PDL1 to its binding partner. In certain contexts, the PDL1 binding partner is PD-1 and / or B7-1. In another embodiment, a PDL2 binding antagonist is a molecule that inhibits the binding of PDL2 to its binding partner. In certain contexts, the PDL2 binding partner is PD-1. The antagonist may be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide. Exemplary antibodies are described in U.S. Patents 8,735,553, 8,354,509, and 8,008,449, all of which are incorporated herein by reference. Other PD-1 axis antagonists for use in the manner provided herein are known in the art, including those described in U.S. Patent Applications No. 20140294898, No. 2014022021, and No. 20110008369, all of which are incorporated herein by reference.

[0239] In some embodiments, the PD-1 conjugated antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of nivolumab, pembrolizumab, and CT-011. In some embodiments, the PD-1 conjugated antagonist is an immunoadhesin (e.g., an immunoadhesin containing the extracellular portion or PD-1 binding portion of PDL1 or PDL2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 conjugated antagonist is AMP-224. Nivolumab is the anti-PD-1 antibody described in WO2006 / 121168, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OPDIVO®. Pembrolizumab is an anti-PD-1 antibody described in WO2009 / 114335, also known as MK-3475, Merck 3475, lambrolizumab, KEYTRUDA®, and SCH-900475. CT-011 is an anti-PD-1 antibody described in WO2009 / 101611, also known as hBAT or hBAT-1. AMP-224 is a PDL2-Fc fusion soluble receptor described in WO2010 / 027827 and WO2011 / 066342, also known as B7-DCIg.

[0240] Another immune checkpoint that can be targeted in the methods provided herein is cytotoxic T lymphocyte-associated protein 4 (CTLA-4), also known as CD152. The complete cDNA sequence of human CTLA-4 has Genbank accession number L15006. CTLA-4 is found on the surface of T cells and functions as an "off" switch when it binds to CD80 or CD86 on the surface of antigen-presenting cells. CTLA4 is a member of the immunoglobulin superfamily that is expressed on the surface of helper T cells and transmits inhibitory signals to T cells. CTLA4 is analogous to the T cell costimulatory protein CD28, both of which bind to CD80 and CD86, also known as B7-1 and B7-2, on antigen-presenting cells, respectively. CTLA4 transmits inhibitory signals to T cells, while CD28 transmits stimulatory signals. Intracellular CTLA4 is also found in regulatory T cells and may be important for their function. T cell activation by T cell receptors and CD28 leads to increased expression of CTLA-4, an inhibitory receptor for the B7 molecule.

[0241] In some embodiments, immune checkpoint inhibitors are anti-CTLA-4 antibodies (e.g., human antibodies, humanized antibodies, or chimeric antibodies), their antigen-binding fragments, immunoadhesins, fusion proteins, or oligopeptides.

[0242] An anti-human CTLA-4 antibody (or a VH and / or VL domain derived therefrom) suitable for use in the method of the present invention can be prepared using methods well known in the art. Alternatively, an anti-CTLA-4 antibody recognized in the art may be used. For example, the anti-CTLA-4 antibodies disclosed in U.S. Patent No. 8,119,129, WO 01 / 14424, WO 98 / 42752; WO 00 / 37504 (CP675,206, also known as tremelimumab; formerly tisilimmab), U.S. Patent No. 6,207,156; Hurwitz et al. (1998) Proc Natl Acad Sci USA 95(17): 10067-10071; Camacho et al. (2004) J Clin Oncology 22(145): Abstract No. 2505 (antibody CP-675206); and Mokyr et al. (1998) Cancer Res 58:5301-5304 can be used in the methods disclosed herein. The teachings of each of the aforementioned publications are incorporated herein by reference. Antibodies that compete with any of these antibodies recognized in the art for binding to CTLA-4 can also be used. For example, humanized CTLA-4 antibodies are described in International Patent Application No. WO2001014424, International Patent Application No. WO2000037504, and U.S. Patent No. 8,017,114, all of which are incorporated herein by reference.

[0243] Exemplary anti-CTLA-4 antibodies are ipilimumab (also known as 10D1, MDX-010, MDX-101, and Yervoy®) or antigen-binding fragments and their variants (see, e.g., WO 01 / 14424). In other embodiments, the antibody comprises the CDR or VR of the heavy and light chains of ipilimumab. Thus, in one embodiment, the antibody comprises the CDR1, CDR2, and CDR3 domains of the VH region of ipilimumab, as well as the CDR1, CDR2, and CDR3 domains of the VL region of ipilimumab. In another embodiment, the antibody competes for and / or binds to the same epitope on CTLA-4 as the antibody described above. In yet another embodiment, the antibody has at least about 90% variable region amino acid sequence identity with the antibody described above (e.g., at least about 90%, 95%, or 99% variable region identity with ipilimumab).

[0244] Other molecules for modulating CTLA-4 include CTLA-4 ligands and receptors, such as those described in U.S. Patent No. 5,844,905, U.S. Patent No. 5,885,796, and International Patent Applications WO1995,001,994 and WO1998,042,752, all of which are incorporated herein by reference, as well as immunoadhesins, such as those described in U.S. Patent No. 8,329,867, also incorporated herein by reference.

[0245] 4. Surgery Approximately 60% of people with cancer will undergo some form of surgery, including surgery for prevention, diagnosis or staging, curative treatment, and palliative care. Curative surgery includes excision, in which all or part of the cancerous tissue is physically removed, cut out, and / or destroyed, and may be used in conjunction with other treatments such as the treatments of the embodiments of the invention, chemotherapy, radiation therapy, hormone therapy, gene therapy, immunotherapy, and / or alternative therapies. Tumor excision means the material removal of at least a portion of the tumor. In addition to tumor excision, surgical treatments include laser surgery, cryosurgery, electrosurgery, and microsurgery (Mohs procedure).

[0246] A cavity may be formed in the body by excision of cancerous cells, tissue, or part or all of a tumor. Treatment may be achieved by perfusion, direct injection, or local application of additional anticancer treatment to that area. For example, such treatment may be repeated every day, every two days, every three days, every four days, every five days, every six days, or every seven days, or every week, every two weeks, every three weeks, every four weeks, and every five weeks, or every month, every two months, every three months, every four months, every five months, every six months, every seven months, every eight months, every nine months, every ten months, every eleven months, or every twelve months. These treatments may be performed with varying dosages.

[0247] 5. Other medications To improve the therapeutic efficacy of the treatment, other agents may be used in combination with certain aspects of the embodiments of the present invention. These additional agents include agents that affect the upregulation of cell surface receptors and gap junctions, cell division inhibitors and differentiation agents, cell adhesion inhibitors, agents that increase the sensitivity of hyperproliferating cells to apoptosis inducers, or other biological agents. Increasing intercellular signaling by increasing the number of gap junctions will increase the anti-hyperproliferative effect on neighboring hyperproliferating cell populations. In other embodiments, cell division inhibitors or differentiation agents may be used in combination with certain aspects of the embodiments of the present invention to improve the anti-hyperproliferative efficacy of the treatment. Cell adhesion inhibitors are intended to improve the efficacy of the embodiments of the present invention. Examples of cell adhesion inhibitors are adhesion plaque kinase (FAK) inhibitors and lovastatin. To further improve the efficacy of the treatment, other agents that increase the sensitivity of hyperproliferating cells to apoptosis, such as antibody c225, may be used in combination with certain aspects of the embodiments of the present invention.

[0248] VII. Manufactured goods or kits Products or kits containing immune cells are also provided herein. The products or kits may further include a package insert containing instructions for using the immune cells to treat or delay the progression of cancer in an individual, or to enhance the immune function of an individual with cancer. Any of the antigen-specific immune cells described herein may be included in the products or kits. Suitable containers include, for example, bottles, vials, bags, and syringes. Containers may be formed from a variety of materials such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloys (e.g., stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and labels on or accompanying the container may indicate instructions for use. The products or kits may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, syringes, and a package insert with instructions for use. In some embodiments, the products further include one or more other agents (e.g., chemotherapeutic agents and antitumor agents). Suitable containers for one or more agents include, for example, bottles, vials, bags, and syringes. [Examples]

[0249] VIII. Examples The following section of examples provides further details regarding examples of various embodiments. Those skilled in the art will understand that the techniques disclosed in the following examples represent techniques and / or compositions that the inventors have found to work well. However, those skilled in the art will understand that many modifications may be made in light of the disclosed particular embodiments, and that similar or comparable results can still be obtained without departing from the spirit and scope of the disclosure. These examples are illustrative of the methods and systems described herein and are not intended to limit the scope of the disclosure. Such non-limiting examples include, but are not limited to, those presented below.

[0250] Example 1 The inventors collected purified CD8+ cells from HLA-matched donors using negative selection. Peptide / MHC multimers (dextramers) were synthesized for each antigen of interest and used to stain CD8 cells with antibodies against CCR7, CD14, CD19, CD4, CD8, CD45RO, CD27, CD3, perforin, granzyme B, T-bet, and Eomes, and / or for viable / dead staining. Cells were fluid-sorted from a dextramer-positive population expressing CD3 and CD8 and with contaminating lymphocytes excluded by nonspecific dextramer staining. Antigen-specific CD8+ cells were loaded onto a 10× Genomics V(D)J single-cell TCR sequencing platform, and the amplified paired alpha / beta TCR DNAs were sequenced using a MiSeq next-generation sequencing instrument. To prioritize TCR sequences encoding homologous CDR3 hypervariable loop domains within the TCR, single-cell barcodes were computationally deconvoluted and analyzed using the TCRdist pipeline (Nature, 2017 doi:10.1038 / nature22383). DNA encoding a bicistronic expression cassette, encoding both alpha and beta constant domains and the variable domain, was synthesized using the P2A ribosome skipping peptide and cloned into the pMP71 TCR expression vector. The TCR construct is a transducer for the Jurkat / MA cell line and contains an NFAT-driven luciferase reporter, allowing the construct to be screened for tumor antigen specificity using a luciferase assay after co-culture with tumor cells known to express congeneral tumor antigens or T2 cells lacking antigen presentation pulsed with synthetic tumor antigen peptides. TCR constructs found to induce antigen-specific responses are further screened using cytokine release assays, which measure IL-2 and IFN-γ by ELISA after co-culturing with antigen-expressing cells.

[0251] (Table 1) List of antigens TIFF0007853938000024.tif172164

[0252] (Table 2) NPY peptides detected by ligand mix in PDX in HLA classes I and II and across primary tumors TIFF0007853938000025.tif214120TIFF0007853938000026.tif229115TIFF0007853938000027.tif229109TIFF0007853938000028.tif30128

[0253] Example 2 A. Materials and Methods Isolation of HLA ligands by immunoaffinity purification Eight patient-derived xenograft tumors and eight primary patient tumors were lysed in 10 mM CHAPS / PBS (AppliChem / Lonza) containing a 1× protease inhibitor (Complete; Roche, Basel, Switzerland). Mouse MHC molecules were reduced by 1 hour of immunoaffinity purification with H-2K-specific mAb 20-8-4S covalently bound to CNBr-activated Sepharose (GE Healthcare, Little Chalfont, UK). The remaining HLA molecules were purified overnight using a mixture of pan-HLA class I-specific mAb W6 / 32 or pan-HLA class II-specific mAb Tu39 and HLA-DR-specific mAb L243 covalently bound to CNBr-activated material. MHC-peptide complexes were eluted by repeated addition of 0.2% trifluoroacetic acid (Merck). Eluted fractions E1-E4 were pooled, and free MHC ligands were isolated by ultrafiltration using a centrifugal filter unit (Amicon; Merck Millipore). MHC ligand, ZipTip C 18 The filtrate was extracted and desalted using a pipette tip (Merck Millipore). The extracted peptide was eluted in 35 μl of acetonitrile (Merck) / 0.1% trifluoroacetic acid, centrifuged to dry completely, and resuspended in 25 μl of 1% acetonitrile / 0.05% trifluoroacetic acid. The sample was stored at -20°C until analysis by LC-MS / MS.

[0254] Analysis of HLA ligands by LC-MS / MS Peptide samples were separated by reversed-phase liquid chromatography (nanoUHPLC, UltiMate 3000 RSLCnano, Dionex) and then analyzed using online-coupled Orbitrap Fusion Lumos (Thermo Fisher Scientific). Samples were analyzed using three technical replicas. A sample volume of 5 μl (20% sample share) was injected at a rate of 4 μl / min for 5.75 minutes into a 75 μm × 2 cm trapping column (Acclaim PepMap RSLC, Dionex). Peptide separation was then performed on a 50 μm × 25 cm separation column (Acclaim PepMap RSLC, Dionex) at 50°C and a flow rate of 300 nl / min for 90 minutes, applying an acetonitrile gradient ranging from 2.4% to 32.0%. Eluted peptides were ionized by nanospray ionization and analyzed using a mass spectrometer with the TopSpeed ​​method. Survey scans were created with Orbitrap at a resolution of 120,000. Precursor ions were separated using a quadrupole and fragmented by either collision-induced dissociation (CID) of purified MHC class I peptides in a double-pressure linear ion trap, or high-energy collision dissociation (HCD) of purified MHC class II peptides in an ion-routing multipole. Finally, the fragmented ions were recorded in Orbitrap. For fragmentation, the mass range was restricted to 400–650 m / z with charge states of 2+ and 3+ for MHC class I, and to 300–1500 m / z with charge states of 2+–5+ for MHC class II, respectively.

[0255] Database search and spectral annotation Data were processed against the human proteome contained in the Swiss-Prot database (world-wide-web at uniport.org, release: September 27, 2013; containing 20,279 re-examined protein sequences) using the SequestHT algorithm of the Proteome Discoverer (v1.3, ThermoFisher) software. For non-standard peptide searches, data were processed against sample-specific fasta files created from RNA-Seq reads. The mass tolerance for precursors was set to 5 ppm, and the mass tolerance for fragments was set to 0.02 Da. The search was not limited to enzyme specificity. Oxidized methionine was recognized as a dynamic modification. The false discovery rate (FDR) was determined by the Percolator algorithm based on processing a decoy database consisting of shuffled sequences. The FDR was set to a target value of q ≤ 0.05 (5% FDR). Peptide-spectral matches (PSM) with q ≤ 0.05 were filtered by further orthogonal parameters to ensure spectral quality and validity. Peptide lengths were limited to 8–12 amino acids for MHC class I and 8–25 amino acids for MHC class II. HLA annotation was performed using SYFPEITHI and NetMHC-4.0 for HLA class I, or NetMHCIIpan for HLA class II.

[0256] B. Results In addition to understanding the immune evasion of proteins arising from unprotected protein domains, the inventors attempted to apply local HLA presentation to identify co-occurring tumor epitopes derived from clinically relevant oncogenes that can be broadly therapeutically applied across the broadest patient population. The inventors performed mass spectrometry on 16 neuroblastoma tumors to characterize the ligandme and test the predictive ability of local HLA scoring across the entire protein span. The inventors mapped the local presentation scores of NPY (29 MHC class I peptides detected in the 16 neuroblastomas), the protein most highly expressed in the neuroblastoma ligandme (Figure 6A; p=0.000011), and found a very significant agreement between empirically detected peptides and regions of proteins expected to be highly presented, and did not find any peptides in the ligandme derived from the signal peptide region (aa 1-28) cleaved from the full-length pro-NPY protein. Based on the high level of presentation of the entire NPY protein across 68 / 84 HLA alleles, its high level of differential expression (Figure 7), and its role in promoting tumor growth (Tilan and Kitlinska, 2016), we hypothesize that NPY is a promising candidate for vaccination strategies. Surprisingly, despite the increased population presentation scores in the highly presented regions, none of the peptides presented in these regions were HLA-A * It was found that binding at 02:01 was not expected, and HLA-A * This highlights the usefulness of population-scale analysis of HLA presentation in identifying widely presented epitopes that may be missed due to the lack of presentation by 02:01. Next, we searched a neuroblastoma immunopeptide mix dataset created for peptides derived from the MYCN oncogene, a major cancer driver in neuroblastoma, and identified relatively rare HLA-C *Only a single peptide (KATEYVHSL; (SEQ ID NO: 26)) presented at 16:01 was identified (Figure 6B). Applying an HLA protein scoring map, the inventors found that this peptide was predicted to bind strongly to 10 / 84 HLA alleles, representing 31.9% of the population (ranked 15th out of 456 peptides in population binding score), suggesting that this peptide has broad applications as a therapeutic target in this pediatric cancer population. This peptide is related to previously reported immunogenic HLA-A * 02:01 Peptide VILKKATEYV (SEQ ID NO: 163) (Himoudi et al., 2008) overlaps, and immunization using this region of MYCN is HLA-A * 02:01 This suggests that it may have a greater impact beyond the patient population. Using this analysis, the inventors found that the highest-scoring MYCN peptide (TVRPKNAAL; (SEQ ID NO: 25)) is predicted to bind to nine HLA alleles, representing 58.1% of the population, and they expect this prediction to be validated by analysis of more neuroblastoma tumor specimens. The inventors further analyzed region scores across 17-mer and 33-mer and found that these regions are predicted to produce peptides that bind to 73.1% and 85.4% of the population, respectively (Figure 6BF). The inventors suggest that these tools can be used to design and prioritize more broadly applicable cancer therapeutic targets and vaccines, particularly when combined with ligand mix data. Analysis of population-scale presentations of individual proteins and specific novel antigens along their time spans is available through the Shiny-NAP web application (reslnmaris01.research.chop.edu:3838 / shinyNAP / ).

[0257] C. Discussion Here, we describe a model for quantifying immunoediting during early tumorigenesis, which provides insights into the immunological contributions to recurrent somatic mutation hotspots observed in human cancers, as well as to the immunological contributions to cancer susceptibility. The model described herein employs methods orthogonal to recent studies demonstrating evidence for immunoediting in the TCGA cohort (Rech et al., 2018; Marty et al., 2017 and Rooney et al., 2015). Using an HLA-based hypothesis, we converge on the conclusion that common driver mutations evade the immune system, providing a population-scale HLA-centric basis for their overpresentation in human cancers. Each of these studies has demonstrated that the immunoediting process is incomplete, highlighting the need to understand this discrepancy in the immune response. Here, we provide methods that can be employed to elucidate immunoediting across HLA alleles, patients, individual variants, and other genomic or clinical features. The inventors believe that an HLA-centric population-scale model provides a comparative baseline for estimating the degree of immunoediting, and that some examples of differences between these features are highlighted herein.

[0258] This is the first report, with the inventors consciously mapping known driver novel antigens across common HLA alleles, that, remarkably, the most recurrent hotspot mutations in human cancer are not predicted to bind to common HLA alleles with sufficiently high affinity to be involved in the adaptive immune system, highlighting the immune mechanisms underlying the evolutionary advantages of common mutations, in addition to their oncogenic potential. This is also the first report, with the inventors consciously quantifying the contribution of individual HLA alleles to the cancer immunoediting process, revealing significant differences in the immunoprotective properties of HLA alleles across cancer. These data suggest that the ability of individual HLA alleles to bind to cancer novel antigens with high affinity is strongly correlated with their ability to contribute to cancer immunoediting. While these data support the cancer immunoediting theory, the inventors and others have shown that the immunoediting process leads to incomplete elimination of novel antigens arising from early driver mutations. While the inventors demonstrate that a considerable degree of immune evasion can be attributable to immunogenic silent mutations, the absence of complete immunoediting in patients without immunogenic silent mutations may be attributable to factors including tumor-endogenous immune evasion, MHC downregulation, lack of adequate T cell response size and quality, poor TCR repertoire, T cell elimination from tissues, or peripheral tolerance. The inventors believe that these models, combined with genomic surrogates of these features, can be used to investigate these variations in future studies using tumor genomic data.

[0259] Here, we show that not all HLA alleles have been found to be significantly protective against the novel antigens they present. We hypothesize that alleles not found to be significantly involved in immunoediting may possess biophysical and / or geometric properties that induce a lethal T-cell response or provide suboptimal interaction with the germline-encoded binding region of the TCR. The finding that certain regions of cancer driver proteins are not protected by HLA presentation, combined with differences in binding across the HLA alleles, raises the question of whether HLA alleles have evolved to provide protection against specific viral domains that coincide with motifs found in oncoproteins, and whether the unpresented regions remain unprotected due to a lack of evolutionary pressure on these motifs. These results also raise the question of whether HLA presentation of Group 2 novel antigens is associated with mutational signatures resulting from specific groups of DNA damage that produce variants with more favorable binding properties at anchor residues (Alexandrov et al., 2013). The inventors have made available tools for other researchers to test these and other hypotheses (reslnmaris01.research.chop.edu:3838 / shinyNAP / ).

[0260] The inventors also present a tool for mapping presentation scores across the entire span of any given protein within a population. The inventors found a highly significant agreement between peptides empirically detected in a complex ligandme of 16 neuroblastoma tumors with diverse HLA alleles and regions of the NPY protein predicted to be most highly presented by HLA across the population. Based on these results, paired with high levels of differential expression and its role in promoting tumor growth, the inventors suggest that NPY is a promising candidate for vaccine development for neuroblastoma patients. Using this tool, the inventors also suggest that current vaccination strategies used against MYCN may have broader applicability across the population.

[0261] As access to genomic data from cancer patients continues to expand and peptide / MHC binding and T-cell epitope prediction tools improve, this model will benefit from greater statistical power in stratifying subsets of patient populations based on molecular features occurring in smaller subpopulations. Despite the fact that this model did not predict HLA alleles binding to novel antigens derived from the KRAS G12D mutation, the KRAS G12D novel antigen GADGVGKSA (SEQ ID NO: 164) is HLA-C *It was recently reported by Tran and colleagues that 08:02 can mediate T cell responses. This antigen is predicted to be a weak HLA-binding factor (15,390 nM), and it is noteworthy that this algorithm does not necessarily predict T cell epitopes, especially for rare alleles with limited training data, and that a new method can help identify novel antigens with non-standard motifs (Abelin et al., 2017). Here, we limited our analysis of immunoediting by CD8 T cells via MHC class I presentation of the 9-mer antigen only, and did not consider immunoediting that may be triggered by other class I antigens of various lengths, class II antigens, or innate immune system activity from NK cells or macrophages. This is because we focused on maintaining statistical power by using common HLA alleles and the most common class I peptide lengths. In the future, we plan to extend the analysis to further peptide lengths and release further features for Shiny-NAP applications.

[0262] The inventors found that statistically significant immunoediting occurs in glioblastoma, while overall, sarcomas, pancreatic tumors, ovarian and adrenocortical tumors, and lymphomas do not show significant evidence for immunoediting. Given that immunogenically silent KRAS G12D mutations are characteristic of pancreatic cancer, these findings may help explain the lack of efficacy of checkpoint inhibitor-like treatments in pancreatic cancer (Winograd et al., 2015) and the lack of immunoediting observed in the inventors' analysis, as these tumors are primarily driven by immunogenically silent mutations. The inventors suggest that these methods can ultimately be used to signal the stratification of patient groups most likely to respond to immunotherapies such as checkpoint inhibitors, and to prioritize peptide vaccines based on patient HLA and antigenic immunogenicity. Using this model, it is also possible to predict how an individual's HLA profile can determine the type of mutation most likely to develop or be protected from.

[0263] Using the immunogenicity models described herein, it may be possible to infer the physical properties of novel antigens that induce high levels of immunoediting compared to other novel antigens presented but not eliminated by the immune system, and to study the contributions of various molecular pathways as a basis for exploring the mechanisms by which specific HLA alleles, as well as tumor types and individual patients, may contribute to cancer protection and predisposition, contributing to varying degrees of immunoediting. *Because alleles like 68:01 have emerged as having disproportionately high immunoediting scores, we are interested in determining whether the contribution of HLA alleles to early immunoediting leads to an improved ability to induce a patient's T-cell response to novel tumor antigens, and whether such alleles are associated with improved outcomes in patients treated with modern immunotherapy. We suggest that the immunogenicity maps, HLA typing data, and immunoediting models contained herein facilitate the investigation of novel antigen immunogenicity at the HLA allele, mutation, patient, and histological levels, supporting the prioritization of shared tumor epitopes for therapeutic development, and further supporting our mechanistic understanding of immune evasion in tumor evolution.

[0264] (Table S1) Driver oncogenes and tumor suppressor genes TIFF0007853938000029.tif117134

[0265] A list of 125 cancer driver genes involved in carcinogenesis, including oncogenes and tumor suppressor genes that regulate cell fate, cell survival, and genome maintenance (Vogelstein et al., 2013).

[0266] (Table S3) Verification of HLA genotyping TIFF0007853938000030.tif20695

[0267] A comparison of HLA predictions across 15 HLA alleles inferred by the PHLAT algorithm from exome sequencing data of three neuroblastoma cell lines, compared with clinical genotyping performed using next-generation sequencing of amplified HLA loci.

[0268] (Table S5) Immunoedited variants from TCGA TIFF0007853938000031.tif23262

[0269] List of the most under-presented variants (p ≤ 0.05) when measured within a population of patients with HLA alleles predicted to bind to novel antigens derived from the variant. The proportion of the population with binding factors is the probability of a TCGA subject having an HLA allele capable of binding to a novel epitope derived from a specific variant. Observed variants are frequencies calculated from the number of patients with at least one HLA allele from the set of patients capable of binding to the variant.

[0270] (Table S6) Immunoedited subjects from TCGA TIFF0007853938000032.tif23295

[0271] List of subjects with the highest degree of immunoediting in TCGA (p ≤ 0.05). Expected binding factors are calculated by summing the probabilities of all individual variants binding to HLA alleles in TCGA in each patient. Observed binding factors are the total number of variant / HLA pairs that create at least one epitope across all variants. Observed / expected represents the degree of under-presentation of the presented novel antigen in each patient (0 is complete immunoediting). Despite being ranked as having the lowest importance of immunoediting, uterine cancer accounts for 5 of the top 10 patients with the highest degree of immunoediting. The most significantly immunoedited subjects also rank 3rd out of 7300 among the number of immunogenically silent variants.

[0272] All methods disclosed and asserted herein can be constructed and carried out in light of this disclosure without undue experimentation. While the compositions and methods of this disclosure have been described in relation to preferred embodiments, it will be apparent to those skilled in the art that modifications may be made to the methods and steps, or the order of the steps of the methods, described herein without departing from the concepts, spirit, and scope of this disclosure. More specifically, it will be apparent that certain chemically and physiologically relevant agents may be used in place of the agents described herein, and that they may achieve the same or similar results. All such similar substitutions and modifications, which would be apparent to those skilled in the art, are deemed to be within the spirit, scope, and concepts of this disclosure as defined by the appended claims.

[0273] IX. References The following references provide exemplary procedural or other details that supplement the references set forth herein and are incorporated herein by reference. TIFF0007853938000033.tif190149TIFF0007853938000034.tif231146TIFF0007853938000035.tif23088 TIFF0007853938000036.tif223150TIFF0007853938000037.tif230150TIFF0007853938000038.tif168151

[0274] Sequence information SEQUENCE LISTING <110> THE CHILDREN'S HOSPITAL OF PHILADELPHIA <120> METHODS AND COMPOSITIONS FOR USE OF TUMOR SELF-ANTIGENS IN ADOPTIVE IMMUNOTHERAPY <150> US 62 / 641,541 <151> 2018-03-12 <160> 164 <170> PatentIn version 3.5 <210> 1 <211> 10 <212> PRT <213> Homo sapiens <400> 1 Leu Leu Pro Leu Leu Pro Pro Leu Ser Pro 1 5 10 <210> 2 <211> 10 <212> PRT <213> Zea mays <400> 2 Phe Leu Asp Glu Thr Leu Arg Ser Leu Ala 1 5 10 <210> 3 <211> 9 <212> PRT <213> Homo sapiens <400> 3 Gln Tyr Asn Pro Ile Arg Thr Thr Phe 1 5 <210> 4 <211> 9 <212> PRT <213> Homo sapiens <400> 4 Ser Tyr Gln Lys Val Ile Glu Leu Phe 1 5 <210> 5 <211> 9 <212> PRT <213> Homo sapiens <400> 5 Ile Tyr Pro Asp Ile Thr Tyr Ser Leu 1 5 <210> 6 <211> 9 <212> PRT <213> Homo sapiens <400> 6 Phe Leu Ile Glu Asn Leu Leu Ala Ala 1 5 <210> 7 <211> 9 <212> PRT <213> Homo sapiens <400> 7 Ala Leu Leu Ser Gly Val Arg Gln Val 1 5 <210> 8 <211> 11 <212> PRT <213> Homo sapiens <400> 8 Val Leu Phe Glu Asn Thr Asp Ser Val His Leu 1 5 10 <210> 9 <211> 9 <212> PRT <213> Homo sapiens <400> 9 Ser Ala Ala Met Val Phe Ser Ala Leu 1 5 <210> 10 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 10 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Ser Leu Val Ile Thr 20 <210> 11 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 11 atctacatct gggctccact ggcaggaacc tgtggcgtgc tgctgctgtc cctggtcatc 60 and 63 <210> 12 <211> 27 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 12 Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu 1 5 10 15 Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val 20 25 <210> 13 <211> 81 <212> DNA <213> Artificial Sequence <220> <223> Synthetic peptide <400> 13 ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60 gccttatta ttttctgggt g 81 <210> 14 <211> 339 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 14 agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60 tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120 cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180 gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240 cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300 tacgacgccc ttcacatgca ggccctgccc cctcgctaa 339 <210> 15 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 15 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr 1 5 10 15 Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro 20 25 30 Pro Arg Asp Phe Ala Ala Tyr Arg Ser 35 40 <210> 16 <211> 123 <212> DNA <213> Artificial Sequence <220> <223> Synthetic peptide <400> 16 aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60 gggcccaccc gcaagcatta gccccaccac gcgactcgc agcctaccgc 120 tcc 123 <210> 17 <211> 42 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 17 Lys Arg Gly Arg Lys Leu Leu Tyr Ile Phe Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 40 <210> 18 <211> 126 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 18 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60 actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120 gaactg 126 <210> 19 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 19 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 20 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 20 atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60 ccg 63 <210> 21 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 21 ggtggaggcg gttcaggtgg cggcggttcg ggcggtggcg gctct 45 <210> 22 <211> 135 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 22 accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60 tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120 gacttcgcct gtgat 135 <210> 23 <211> 9 <212> PRT <213> Homo sapiens <400> 23 Leu Leu Leu Pro Leu Leu Pro Pro Leu 1 5 <210> 24 <211> 12 <212> PRT <213> Homo sapiens <400> 24 Leu Leu Leu Pro Leu Leu Pro Pro Leu Ser Pro Ser 1 5 10 <210> 25 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 25 Thr Val Arg Pro Lys Asn Ala Ala Leu 1 5 <210> 26 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 26 Lys Ala Thr Glu Tyr Val His Ser Leu 1 5 <210> 27 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 27 Leu Glu Arg Gln Arg Arg Asn Asp Leu Arg Ser Ser Phe Leu Thr Leu 1 5 10 15 Arg <210> 28 <211> 33 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 28 Thr Val Arg Pro Lys Asn Ala Ala Leu Gly Pro Gly Arg Ala Gln Ser 1 5 10 15 Ser Glu Leu Ile Leu Lys Arg Cys Leu Pro Ile His Gln Gln His Asn 20 25 30 Tyr <210> 29 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 29 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp 1 5 10 <210> 30 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 30 Arg Tyr Tyr Ser Ala Leu Arg His Tyr 1 5 <210> 31 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 31 Ser Pro Glu Thr Leu Ile Ser Asp Leu Leu Met 1 5 10 <210> 32 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 32 Thr Leu Ile Ser Asp Leu Leu Met 1 5 <210> 33 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 33 Ser Ser Pro Glu Thr Leu Ile Ser Asp Leu Leu 1 5 10 <210> 34 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 34 Ser Ala Leu Arg His Tyr Ile Asn Leu 1 5 <210> 35 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 35 Ser Ser Pro Glu Thr Leu Ile Ser Asp Leu 1 5 10 <210> 36 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 36 Ser Pro Glu Thr Leu Ile Ser Asp Leu Leu 1 5 10 <210> 37 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 37 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu 1 5 10 <210> 38 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 38 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala 1 5 10 <210> 39 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 39 Ser Thr Glu Asn Val Pro Arg Thr 1 5 <210> 40 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 40 Thr Glu Asn Val Pro Arg Thr Arg 1 5 <210> 41 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 41 Arg Glu Ser Thr Glu Asn Val Pro Arg Thr 1 5 10 <210> 42 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 42 Tyr Pro Ser Lys Pro Asp Asn Pro Gly 1 5 <210> 43 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 43 Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro 1 5 10 <210> 44 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 44 Arg Glu Ser Thr Glu Asn Val Pro Arg 1 5 <210> 45 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 45 Glu Ser Thr Glu Asn Val Pro Arg Thr 1 5 <210> 46 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 46 Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg 1 5 10 <210> 47 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 47 Arg Tyr Tyr Ser Ala Leu Arg His Tyr 1 5 <210> 48 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 48 Ser Ala Leu Arg His Tyr Ile Asn Leu 1 5 <210> 49 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 49 His Tyr Ile Asn Leu Ile Thr Arg 1 5 <210> 50 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 50 Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu 1 5 10 <210> 51 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 51 Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu 1 5 10 <210> 52 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 52 Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu 1 5 10 <210> 53 <211> 22 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 53 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 Met Ala Arg Tyr Tyr Ser 20 <210> 54 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 54 Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg 1 5 10 <210> 55 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 55 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp 1 5 10 <210> 56 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 56 Arg Glu Ser Thr Glu Asn Val Pro Arg Thr Arg Leu Glu Asp Pro Ala 1 5 10 15 <210> 57 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 57 Glu Ser Thr Glu Asn Val Pro Arg Thr Arg Leu Glu Asp Pro Ala Met 1 5 10 15 Trp <210> 58 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 58 Met Arg Glu Ser Thr Glu Asn Val Pro Arg Thr Arg Leu Glu Asp Pro 1 5 10 15 Ala <210> 59 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 59 Arg Glu Ser Thr Glu Asn Val Pro Arg Thr Arg Leu Glu 1 5 10 <210> 60 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 60 Ser Thr Glu Asn Val Pro Arg Thr Arg Leu Glu Asp Pro Ala Met Trp 1 5 10 15 <210> 61 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 61 Tyr Pro Ser Lys Pro Asp Asn Pro Gly 1 5 <210> 62 <211> 21 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 62 Ser Ser Pro Glu Thr Leu Ile Ser Asp Leu Leu Met Arg Glu Ser Thr 1 5 10 15 Glu Asn Val Pro Arg 20 <210> 63 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 63 Met Arg Glu Ser Thr Glu Asn Val Pro Arg Thr Arg Leu Glu Asp Pro 1 5 10 15 Ala Met Trp <210> 64 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 64 Glu Ser Thr Glu Asn Val Pro Arg Thr Arg Leu Glu 1 5 10 <210> 65 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 65 Leu Arg His Tyr Ile Asn Leu Ile Thr Arg 1 5 10 <210> 66 <211> 21 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 66 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 Met Ala Arg Tyr Tyr 20 <210> 67 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 67 Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr 1 5 10 15 Ile Asn Leu <210> 68 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 68 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 Met Ala Arg Tyr 20 <210> 69 <211> 24 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 69 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 Met Ala Arg Tyr Tyr Ser Ala Leu 20 <210> 70 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 70 Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln 1 5 10 <210> 71 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 71 Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr 1 5 10 15 Ile Asn <210> 72 <211> 23 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 72 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 Met Ala Arg Tyr Tyr Ser Ala 20 <210> 73 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 73 Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala 1 5 10 15 Arg Tyr Tyr Ser Ala Leu Arg His Tyr 20 25 <210> 74 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 74 Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr Gly Lys Arg 1 5 10 15 <210> 75 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 75 Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn 1 5 10 <210> 76 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 76 Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu 1 5 10 15 <210> 77 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 77 Only Glu Asp Met Only Arg Tyr Tyr Ser Only Leu Arg His Tyr Ile Asn 1 5 10 15 Leu <210> 78 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 78 Only Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu 1 5 10 <210> 79 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 79 Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile 1 5 10 15 Thr pure Arg <210> 80 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 80 Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His 1 5 10 15 Tyr Ile Asn Leu 20 <210> 81 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 81 Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn 1 5 10 15 <210> 82 <211> 23 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 82 Ser Ser Pro Glu Thr Leu Ile Ser Asp Leu Leu Met Arg Glu Ser Thr 1 5 10 15 Glu Asn Val Pro Arg Thr Arg 20 <210> 83 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 83 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 Met Ala <210> 84 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 84 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 Met Ala Arg <210> 85 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 85 Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu 1 5 10 <210> 86 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 86 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu 1 5 10 15 <210> 87 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 87 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 <210> 88 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 88 Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile 1 5 10 15 Asn Leu <210> 89 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 89 Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu 1 5 10 15 <210> 90 <211> 23 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 90 Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala 1 5 10 15 Leu Arg His Tyr Ile Asn Leu 20 <210> 91 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 91 Ser Ser Pro Glu Thr Leu Ile Ser Asp Leu Leu Met Arg Glu Ser Thr 1 5 10 15 Glu <210> 92 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 92 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp 1 5 10 15 Met <210> 93 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 93 Ser Ser Pro Glu Thr Leu Ile Ser Asp Leu Leu 1 5 10 <210> 94 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 94 Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu 1 5 10 <210> 95 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 95 Ser Pro Glu Thr Leu Ile Ser Asp Leu Leu 1 5 10 <210> 96 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 96 Ser Ser Pro Glu Thr Leu Ile Ser Asp Leu Leu Met 1 5 10 <210> 97 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 97 Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg 1 5 10 15 Gln <210> 98 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 98 Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln 1 5 10 <210> 99 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 99 Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr Gly 1 5 10 <210> 100 <211> 22 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 100 Ser Ser Pro Glu Thr Leu Ile Ser Asp Leu Leu Met Arg Glu Ser Thr 1 5 10 15 Glu Asn Val Pro Arg Thr 20 <210> 101 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 101 Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg 1 5 10 <210> 102 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 102 Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr Gly 1 5 10 15 <210> 103 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 103 Ser Ser Pro Glu Thr Leu Ile Ser Asp Leu Leu Met Arg Glu Ser Thr 1 5 10 15 Glu Asn Val Pro Arg Thr Arg Leu Glu 20 25 <210> 104 <211> 22 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 104 Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu 1 5 10 15 Arg His Tyr Ile Asn Leu 20 <210> 105 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 105 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala 1 5 10 <210> 106 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 106 Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr 1 5 10 15 <210> 107 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 107 Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr 1 5 10 <210> 108 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 108 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu 1 5 10 <210> 109 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 109 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro 1 5 10 <210> 110 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 110 Glu Ser Thr Glu Asn Val Pro Arg Thr 1 5 <210> 111 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 111 Tyr Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala 1 5 10 <210> 112 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 112 Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn 1 5 10 <210> 113 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 113 Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu 1 5 10 <210> 114 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 114 Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr 1 5 10 15 <210> 115 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 115 Ser Ser Pro Glu Thr Leu Ile Ser Asp Leu 1 5 10 <210> 116 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 116 Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg 1 5 10 15 <210> 117 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 117 Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg 1 5 10 <210> 118 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 118 Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr 1 5 10 15 Ile Asn Leu <210> 119 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 119 Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln 1 5 10 15 Arg Tyr Gly <210> 120 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 120 Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile 1 5 10 15 Thr Arg <210> 121 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 121 Only Arg Tyr Tyr Ser Only Leu Arg His Tyr With Asn Leu With Thr Arg 1 5 10 15 Clean Arg Tire <210> 122 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 122 Tyr Ser Only Leo Arg His Tyr With Asn Leo With Thr Arg 1 5 10 <210> 123 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 123 Serves Only One Arg His Tyr and Only One Only Thr Arg Gln Arg Tyr Gly 1 5 10 15 <210> 124 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 124 Arg Tyr Tyr Ser Ala Leu Arg His Tyr With Asn Leu With Thr Arg Gln 1 5 10 15 Arg Tyr <210> 125 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 125 Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg 1 5 10 <210> 126 <211> 23 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 126 Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr 1 5 10 15 Ile Asn Leu Ile Thr Arg Gln 20 <210> 127 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 127 Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr 1 5 10 15 <210> 128 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 128 Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg 1 5 10 <210> 129 <211> 22 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 129 Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile 1 5 10 15 Thr Arg Gln Arg Tyr Gly 20 <210> 130 <211> 19 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 130 Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile 1 5 10 15 Thr Arg Gln <210> 131 <211> 21 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 131 Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn 1 5 10 15 Leu Ile Thr Arg Gln 20 <210> 132 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 132 Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg 1 5 10 15 Tyr Gly <210> 133 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 133 Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr Gly 1 5 10 15 <210> 134 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 134 Ser Ser Pro Glu Thr Leu Ile Ser Asp Leu Leu Met 1 5 10 <210> 135 <211> 21 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 135 Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr 1 5 10 15 Ile Asn Leu Ile Thr 20 <210> 136 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 136 Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg 1 5 10 15 <210> 137 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 137 Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile 1 5 10 15 Thr pure copper 20 <210> 138 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 138 Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr 1 5 10 15 I Have Asn I Have Thr Arg 20 <210> 139 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 139 Arg Tyr Tyr Ser Ala Leu Arg His Tyr With Asn Leu With Thr Arg Gln 1 5 10 15 Arg <210> 140 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 140 Only Glu Asp Met Only Arg Tyr Tyr Ser Only Leu Arg His Tyr Ile Asn 1 5 10 15 Leu With Three Arg Gln Arg Tyr Gly 20 <210> 141 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 141 Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu 1 5 10 15 <210> 142 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 142 Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr 1 5 10 15 House Asn Leu House 20 <210> 143 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic peptide <400> 143 Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn 1 5 10 15 Leu Ile Thr Arg 20 <210> 144 <211> 24 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 144 Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr 1 5 10 15 Ile Asn Leu Ile Thr Arg Gln Arg 20 <210> 145 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 145 Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr 1 5 10 <210> 146 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 146 Ala Pro Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr 1 5 10 15 Ile Asn Leu Ile Thr Arg Gln Arg Tyr 20 25 <210> 147 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 147 Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg 1 5 10 15 Gln Arg Tyr Gly 20 <210> 148 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 148 Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr 1 5 10 <210> 149 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 149 Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg Tyr 1 5 10 15 <210> 150 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 150 Ser Pro Glu Thr Leu Ile Ser Asp Leu Leu Met 1 5 10 <210> 151 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 151 Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu 1 5 10 <210> 152 <211> 16 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 152 Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg 1 5 10 15 <210> 153 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 153 Ser Ser Pro Glu Thr Leu Ile Ser Asp Leu Leu 1 5 10 <210> 154 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 154 Ser Pro Glu Thr Leu Ile Ser Asp Leu Leu 1 5 10 <210> 155 <211> 21 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 155 Ser Ser Pro Glu Thr Leu Ile Ser Asp Leu Leu Met Arg Glu Ser Thr 1 5 10 15 Glu Asn Val Pro Arg 20 <210> 156 <211> 13 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 156 Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln 1 5 10 <210> 157 <211> 24 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 157 Ile Ser Asp Leu Leu Met Arg Glu Ser Thr Glu Asn Val Pro Arg Thr 1 5 10 15 Arg Leu Glu Asp Pro Ala Met Trp 20 <210> 158 <211> 17 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 158 Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg Gln Arg 1 5 10 15 Tyr <210> 159 <211> 25 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 159 Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile 1 5 10 15 Thr Arg Gln Arg Tyr Gly Lys Arg Ser 20 25 <210> 160 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 160 Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr 1 5 10 <210> 161 <211> 23 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 161 Ala Glu Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn 1 5 10 15 Leu Ile Thr Arg Gln Arg Tyr 20 <210> 162 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 162 Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp 1 5 10 <210> 163 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 163 Val Ile Leu Lys Lys Ala Thr Glu Tyr Val 1 5 10 <210> 164 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptide <400> 164 Gly Ala Asp Gly Val Gly Lys Ser Ala 1 5

Claims

1. A chimeric antigen receptor (CAR) protein that binds to an antigen consisting of the amino acid sequence described in SEQ ID NO:

3.

2. A T cell receptor protein that binds to an antigen, consisting of the amino acid sequence described in SEQ ID NO:

3.

3. A polynucleotide molecule encoding the CAR protein according to claim 1 or the T cell receptor protein according to claim 2, further comprising a promoter active in eukaryotic cells, or further defined as an expression vector.

4. A modified T cell comprising a polynucleotide molecule encoding an antigen-binding T cell receptor consisting of the amino acid sequence described in SEQ ID NO: 3, further comprising a transposase.

5. (i) A first single-chain antibody that selectively binds to an antigen consisting of the amino acid sequence described in SEQ ID NO: 3; and (ii) A second single-chain antibody that binds to T or B cells A fusion protein comprising a labeled or therapeutic moiety.

6. The fusion protein according to claim 5, wherein the second single-chain antibody binds to CD3, T cells, or B cells.

7. A vaccine composition for use in a method of treating cancer in a subject, comprising an antigen comprising the amino acid sequence described in SEQ ID NO: 3, wherein the antigen is delivered by intact dendritic cells, and the vaccine composition further comprises an adjuvant, a biological response modifier, or a chemokine, wherein the method comprises the step of administering the vaccine composition to the subject.

Images