Methods of obtaining tumor-specific t cell receptors

Abstract

This application provides methods for obtaining multiple T-cell receptors that specifically recognize target tumor antigen peptides from an individual who has clinically benefited from immunotherapy, such as multi-antigen-specific cell therapy. It also provides tumor-specific TCRs, engineered immune cells expressing these TCRs, and methods for treating diseases using these engineered immune cells.

Description

Technical Field Submit the sequence list as an ASCII text file.

[0001] The following content, submitted in ASCII text format, is incorporated herein by reference in its entirety: Computer-readable format of sequence lists (CRF) (File name: 776902000342SEQLIST.TXT, Record date: April 11, 2019, Size: 426KB).

[0002] This invention relates to the field of cancer immunotherapy. Specifically, this invention provides a method for obtaining TCRs, tumor-specific TCRs, engineered immune cells, pharmaceutical compositions, and kits for treating cancer. Prior Technology

[0003] Unlike hematologic malignancies, solid tumors lack cell surface antigens that are recognized by chimeric antigen receptors (CARs). Intracellular antigens from solid tumors must be presented on the cell surface as HLA-antigen epitope complexes via HLA molecules. These HLA-antigen epitope complexes are specifically recognized by T cell receptors (TCRs) on the surface of tumor-specific T cells, thereby triggering antitumor cytotoxicity by tumor-specific T cells.

[0004] Prior to February 2018, there were 75 clinical trials involving adoptive immune cells, which are engineered to possess tumor-specific TCRs for cancer treatment. Targets include tumor-associated antigens, tumor-associated viral antigens, and neoantigens. Indications include many common types of solid tumors. Examples of TCR-T cells currently in clinical trials include TCR-T targeting the WT1 / HLA-A*0201 complex (Juno Therapeutics), used to treat acute myeloid leukemia (AML) and non-small cell lung cancer; TCR-T targeting the MAGE-A3 / A6 / HLA-DPB1*0401 complex (Kite), used to treat malignant solid tumors; and TCR-T targeting MAGE-A4 / HLA-A*0201 (Adaptimmune), used to treat malignant solid tumors.

[0005] All publications, patents, patent applications, and disclosures of published patent applications referenced herein are hereby incorporated herein by reference. Summary of the Invention

[0006] The present invention provides methods, compositions, and kits for obtaining one or more T-cell receptors (TCRs) that specifically recognize tumor antigen peptides from an individual who has clinically benefited from immunotherapy, such as multiple antigen specific cell therapy (MASCT).

[0007] One embodiment of this application provides a method for obtaining a plurality of T-cell receptors (TCRs) that specifically recognize target tumor antigen peptides, comprising: a) a first co-culture step, comprising co-culturing a first dendritic cell (DC) population carrying the target tumor antigen peptide with a T-cell population from an individual to obtain a first co-culture; b) an enrichment step, comprising subjecting the first co-culture to an enrichment procedure to obtain enriched activated T cells; c) a second co-culture step, comprising co-culturing the enriched activated T cells with a second DC population carrying the target tumor antigen peptide to obtain a tumor antigen-specific T-cell population. At least about 10% of these tumor antigen-specific T cells specifically respond to the target tumor antigen peptide; and d) a sequencing step comprising subjecting the tumor antigen-specific T cells to next-generation sequencing to recognize multiple pairs of genes encoding TCRα and TCRβ, thereby providing the multiple T cell receptors based on the paired genes encoding TCRα and TCRβ; wherein the individual has clinically benefited from multi-antigen-specific cell therapy (MASCT), which comprises administering an effective amount of activated T cells to the individual, the activated T cells being prepared by co-culturing a population of T cells with a population of dendritic cells carrying multiple tumor antigen peptides, including the target tumor antigen peptide. In some embodiments, the first co-culture step is performed for about 1 to about 3 days prior to the enrichment step. In some embodiments, the ratio between the T cell population and the first DC population carrying the target tumor antigen peptide is not greater than about 30:1 (such as about 10:1 to about 20:1, or about 15:1, or about 20:1). In some embodiments, the T cell population in the first co-culture step is present in PBMCs. In some embodiments, the first DC population carrying the target tumor antigen peptide and the T cell population are co-cultured in a first co-culture medium containing one or more cytokines (e.g., IL-2 or multiple cytokines) and immune checkpoint inhibitors. In some embodiments, the first co-culture medium contains IL-2, IL-7, IL-15, and IL-21, and an anti-PD-1 antibody. In some embodiments, the first co-culture medium contains IL-2 and an anti-PD-1 antibody.

[0008] In some embodiments according to any of the above methods, the enrichment step includes: contacting the first co-culture with antigen-presenting cells (APCs) carrying the target tumor antigen peptide to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand that specifically recognizes a cytokine. In some embodiments, the cytokine is IFNγ. In some embodiments, the ratio between the enriched activated T cell population and the second DC population carrying the target tumor antigen peptide is about 1:1 to about 20:1. In some embodiments, the enriched activated T cell population and the second DC population carrying the target tumor antigen peptide are co-cultured for about 12 to 25 days.

[0009] In some embodiments according to any of the above methods, the second co-culture step comprises: co-culturing the second DC population carrying the target tumor antigen peptide with the enriched activated T cell population in an initial second co-culture medium to provide a second co-culture, the initial second co-culture medium containing an immune checkpoint inhibitor and optionally one or more cytokines (e.g., IL-2 or multiple cytokines); and adding an anti-CD3 antibody (e.g., OKT3) and optionally one or more cytokines (e.g., IL-2 or multiple cytokines) to the second co-culture to obtain a tumor antigen-specific T cell population. In some embodiments, the anti-CD3 antibody is added to the second co-culture no more than about 3 days after the start of the second co-culture step. In some embodiments, the anti-CD3 antibody is OKT3. In some embodiments, the one or more cytokines are added to the second co-culture no more than about 3 days (e.g., about 2 days) after the start of the second co-culture step. In some embodiments, the one or more cytokines comprise IL-2. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody (e.g., SHR-1210). In some embodiments, the initial second co-culture medium contains IL-2, IL-7, IL-15, and IL-21, and an anti-PD-1 antibody.

[0010] In some embodiments of the methods described above, the method further includes a third co-culture step, which involves co-culturing a population of tumor antigen-specific T cells with a population of antigen-presenting cells (APCs) carrying a target tumor antigen peptide to obtain a second population of tumor antigen-specific T cells, wherein the second population of tumor antigen-specific T cells undergoes next-generation sequencing in the sequencing step. In some embodiments, the APCs are PBMCs, DCs, or cell line APCs. In some embodiments, the ratio between the population of tumor antigen-specific T cells and the population of APCs carrying the target tumor antigen peptide is about 1:1 to about 20:1. In some embodiments, the population of tumor antigen-specific T cells and the population of APCs carrying the target tumor antigen peptide are co-cultured for about 5 to 9 days. In some embodiments, the population of tumor antigen-specific T cells and the population of APCs carrying the target tumor antigen peptide are co-cultured in a third co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an anti-CD3 antibody. In some embodiments, the third co-culture medium contains IL-2, IL-7, IL-15, and OKT3. In some embodiments, the third co-culture medium contains IL-2 and OKT3. In some embodiments, the third co-culture step is repeated (e.g., once, twice, or three times).

[0011] In some embodiments according to any of the methods described above, the plurality of tumor antigen peptides are a plurality of synthetic tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides are not derived from cell samples.

[0012] In some embodiments according to any of the methods described above, the plurality of tumor antigen peptides comprises (multiple) general tumor antigen peptides, (multiple) cancer type-specific antigen peptides, and / or neoantigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises one or more neoantigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises neoantigen peptides (e.g., composed of neoantigen peptides). In some embodiments, the plurality of tumor antigen peptides comprises at least about 5 (e.g., at least about 10, 20, 30, 40, or more) different tumor antigen peptides.

[0013] In some embodiments according to any of the above methods, the target tumor antigen peptide is derived from a tumor antigen selected from the group consisting of: hTERT, p53, Survivin, NY-ESO-1, CEA, CCND1, RGS5, MMP7, VEGFR1, VEGFR2, MUC1, HER2, MAGE-A1, MAGE-A3, CDCA1, WT1, KRAS, PARP4, MLL3, MTHFR, HPV16-E6, HPV16-E7, HPV18-E6, HPV18-E7, HPV58-E6, HPV58-E7, HBcAg, HBV polymerase, GPC3, SSX, and AFP.

[0014] In some embodiments of any of the above methods, the method further includes: identifying a target tumor epitope from the target tumor antigen peptide, wherein the target tumor epitope elicits a specific response in the enriched activated T cell population; and contacting the APC population with the target tumor epitope to obtain the population of APCs carrying the target tumor antigen peptide.

[0015] In some embodiments of any of the methods described above, the next-generation sequencing is single-cell sequencing. In some embodiments, the next-generation sequencing is bulk sequencing. In some embodiments, the sequencing step includes sequencing a bulk sample of tumor antigen-specific T cells and single-cell sequencing of a plurality of tumor antigen-specific T cells. In some embodiments, the sequencing step includes bulk sequencing of a first portion of the tumor antigen-specific T cells to provide a plurality of genes encoding TCRα and TCRβ; and single-cell sequencing of a second portion of the tumor antigen-specific T cells, which provides homology pairing information of the plurality of genes encoding TCRα and TCRβ, thereby providing a plurality of TCRs based on paired genes encoding TCRα and TCRβ. In some embodiments, prior to the next-generation sequencing, the tumor antigen-specific T cells are stimulated with an APC carrying the target tumor antigen peptide.

[0016] In some embodiments according to any of the methods described above, after receiving the MASCT, the individual has a partial response (PR), a complete response (CR), or stable disease (SD). In some embodiments, the MASCT includes: co-culturing a population of dendritic cells (DCs) loaded with multiple tumor antigen peptides with a population of T cells to obtain an activated T cell population, wherein the multiple tumor antigen peptides include the target tumor antigen peptide. In some embodiments, the MASCT includes: (i) co-culturing a population of DCs loaded with multiple tumor antigen peptides with a population of T cells in an initial co-culture medium to provide a co-culture, wherein the multiple tumor antigen peptides include the target tumor antigen peptide, the initial co-culture medium containing one or more (e.g., multiple cytokines) and immune checkpoint inhibitors; and (ii) adding an anti-CD3 antibody to the co-culture approximately 3 to 7 days after the start of the co-culture to obtain the activated T cell population. In some embodiments, the MASCT includes: (i) contacting a population of dendritic cells (DCs) with a plurality of tumor antigen peptides to obtain DCs loaded with the plurality of tumor antigen peptides, the plurality of tumor antigen peptides including the target tumor antigen peptide; and (ii) culturing the DCs loaded with the plurality of tumor antigen peptides in a DC maturation medium containing MPLA. In some embodiments, the DC maturation medium contains INFγ, MPLA, and PGE2. In some embodiments, the MASCT includes administering an effective amount of DCs loaded with the plurality of tumor antigen peptides to the individual. In some embodiments, the individual has previously received the MASCT at least three times.

[0017] In some embodiments according to any of the above methods, TCRs that specifically identify multiple target tumor antigen peptides are obtained simultaneously.

[0018] In some embodiments according to any of the above methods, the method further comprises: expressing each pair of genes encoding TCRα and TCRβ in host immune cells to provide engineered immune cells expressing TCRs; and evaluating the response of the engineered immune cells to the target tumor antigen peptide. A further provided method is a method for obtaining a TCR that specifically recognizes the target tumor antigen peptide using the above methods, wherein the TCR is selected based on the response of the engineered immune cells expressing the TCR to the target tumor antigen peptide. In some embodiments, the method further comprises determining HLA restriction of the TCR. In some embodiments, the TCR has a predominantly Asian HLA haplotype restriction. In some embodiments, the method further comprises affinity maturation of the TCR. In some embodiments, the method further comprises enhancing the pairing of the TCRα and TCRβ chains in the TCR. In some embodiments, the method further comprises enhancing the expression of the TCR. In some embodiments, the target tumor antigen peptide is derived from CEA, RSG-5, or HPV18-E7.

[0019] Also provided is a tumor-specific TCR, which is obtained using any of the methods described above.

[0020] Another embodiment of this application provides a tumor-specific TCR comprising: (a) a TCRα chain containing a complementarity-determining region (CDR) 3, wherein the CDR 3 has at least about 90% sequence identity with any of the amino acid sequences of SEQ ID NO: 4, 10, and 16; and a TCRβ chain containing a CDR 3, wherein the CDR 3 contains an amino acid sequence having at least about 90% sequence identity with any of the amino acid sequences of SEQ ID NO: 7, 13, and 19; (b) a TCRα chain containing a complementarity-determining region (CDR) 3, wherein the CDR 3 has at least about 90% sequence identity with any of the amino acid sequences of SEQ ID NO: 22, 28, 34, 40, 46, and 52; and a TCRβ chain containing a CDR 3, wherein the CDR 3 contains an amino acid sequence having at least about 90% sequence identity with any of the amino acid sequences of SEQ ID NO: 4, 10, and 16; and a TCRβ chain containing a CDR 3, wherein the CDR 3 contains an amino acid sequence having at least about 90% sequence identity with any of the amino acid sequences of SEQ ID NO: 7, 13, and 19 ... (c) An amino acid sequence having at least about 90% sequence identity with any of the amino acid sequences of SEQ ID NO: 7, 13, 19, 25, 31, 37, 43, 49, and 55; or (c) a TCRα chain containing a complementarity-determining region (CDR) 3, wherein the CDR 3 has at least about 90% sequence identity with any of the amino acid sequences of SEQ ID NO: 58, 64, 70, 76, 87, and 93; and a TCRβ chain containing a CDR 3, wherein the CDR 3 contains an amino acid sequence having at least about 90% sequence identity with any of the amino acid sequences of SEQ ID NO: 61, 67, 73, 79, 90, and 96.

[0021] In some embodiments, the tumor-specific TCR comprises: (a) a TCRα chain comprising CDR3, wherein the CDR3 comprises the amino acid sequence of SEQ ID NO:4; and a TCRβ chain comprising CDR3, wherein the CDR3 comprises the amino acid sequence of SEQ ID NO:7; (b) a TCRα chain comprising CDR3, wherein the CDR3 comprises the amino acid sequence of SEQ ID NO:10; and a TCRβ chain comprising CDR3, wherein the CDR3 comprises the amino acid sequence of SEQ ID NO:13; (c) a TCRα chain comprising CDR3, wherein the CDR3 comprises the amino acid sequence of SEQ ID NO:16; and a TCRβ chain comprising CDR3, wherein the CDR3 comprises the amino acid sequence of SEQ ID NO:19; (d) a TCRα chain comprising CDR3, wherein the CDR3 comprises the amino acid sequence of SEQ ID NO:22; and a TCRβ chain comprising CDR3, wherein the CDR3 comprises the amino acid sequence of SEQ ID NO:4. (e) An amino acid sequence containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:25; and an amino acid sequence containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:31; (f) An amino acid sequence containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:34; and an amino acid sequence containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:37; (g) An amino acid sequence containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:40; and an amino acid sequence containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:43; (h) An amino acid sequence containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:46; and an amino acid sequence containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:28. The amino acid sequence NO:49; (i) a TCRα chain containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:52; and a TCRβ chain containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:55; (j) a TCRα chain containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:58; and a TCRβ chain containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:61; (k) a TCRα chain containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:64; and a TCRβ chain containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:67; (l) a TCRα chain containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:70;and a TCRβ chain containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:73; (m) a TCRα chain containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:76; and a TCRβ chain containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:79; (n) a TCRα chain containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:87; and a TCRβ chain containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:90; or (o) a TCRα chain containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:93; and a TCRβ chain containing CDR3, wherein the CDR3 contains the amino acid sequence of SEQ ID NO:96.

[0022] Further provided is a tumor-specific TCR comprising: (a) a TCRα chain comprising a CDR of any one of the amino acid sequences of SEQ ID NO: 5, 11, and 17; and a TCRβ chain comprising a CDR of any one of the amino acid sequences of SEQ ID NO: 8, 14, and 20; (b) a TCRα chain comprising a CDR of any one of the amino acid sequences of SEQ ID NO: 23, 29, 35, 41, 47, and 53; and a TCRβ chain comprising a CDR of any one of the amino acid sequences of SEQ ID NO: 26, 32, 38, 44, 50, and 56; or (c) a TCRα chain comprising a CDR of any one of the amino acid sequences of SEQ ID NO: 59, 65, 71, 77, 88, and 94; and a TCRβ chain comprising a CDR of any one of the amino acid sequences of SEQ ID NO: 8, 14, and 20. CDR of any of the amino acid sequences NO: 62, 68, 74, 80, 91, and 97.

[0023] In some embodiments of any of the tumor-specific TCRs described above, the tumor-specific TCR is a human TCR. In some embodiments, the tumor-specific TCR is a chimeric TCR, such as a murine TCR, for example a TCR comprising murine constant regions of the TCR α and β chains.

[0024] In some embodiments according to any of the above-described tumor-specific TCRs, the tumor-specific TCR comprises: (a) a TCRα chain comprising an amino acid sequence having at least about 80% identity with any of the amino acid sequences of SEQ ID NO: 5, 11, and 17; and a TCRβ chain comprising an amino acid sequence having at least about 80% identity with any of the amino acid sequences of SEQ ID NO: 8, 14, and 20; (b) a TCRα chain comprising an amino acid sequence having at least about 80% identity with any of the amino acid sequences of SEQ ID NO: 23, 29, 35, 41, 47, and 53; and a TCRβ chain comprising an amino acid sequence having at least about 80% identity with any of the amino acid sequences of SEQ ID NO: 26, 32, 38, 44, 50, and 56; or (c) a TCRα chain comprising an amino acid sequence having at least about 80% identity with any of the amino acid sequences of SEQ ID NO: 5, 11, and 17; or (c) a TCRβ chain comprising an amino acid sequence having at least about 80% identity with any of the amino acid sequences of SEQ ID NO: 5, 11, and 17; or (d ... The amino acid sequence having at least about 80% identity with any one of the amino acid sequences NO:59, 65, 71, 77, 88, and 94; and the TCRβ chain containing an amino acid sequence having at least about 80% identity with any one of the amino acid sequences SEQ ID NO:62, 68, 74, 80, 91, and 97.

[0025] Also provided is a single nucleic acid that encodes the TCRα chain and / or the TCRβ chain of any of the aforementioned tumor-specific TCRs; and a vector containing the single nucleic acid(e).

[0026] One embodiment of this application provides an engineered immune cell comprising a tumor-specific TCR, isolated nucleic acid, or vector according to any of the tumor-specific TCRs described above.

[0027] In some embodiments, the immune cell line is a T cell. In some embodiments, a pharmaceutical composition is provided comprising an engineered immune cell according to any of the above-described engineered immune cells, and a pharmaceutically acceptable carrier.

[0028] In some embodiments, a method of treating cancer in an individual is provided, comprising administering to the individual an effective amount of a pharmaceutical composition according to any of the above-described pharmaceutical compositions.

[0029] In some embodiments, a library of tumor-specific TCRs is provided, which are obtained using a method according to any of the above methods.

[0030] Further provided are kits, medicines, and products comprising any of the components described above (such as isolated nucleic acids, vectors, and engineered immune cells).

[0031] These and other aspects and advantages of the invention will become apparent from the following embodiments and the appended claims. It should be understood that one, some, or all of the various embodiments described herein can be combined to form other embodiments of the invention. Simple Explanation of the Diagram

[0032] Figure 1 shows an illustrative overview of methods for selecting multiple TCRs that specifically identify target tumor antigen peptides.

[0033] Figure 2 shows clinical data of patients with metastatic cervical cancer treated with MASCT. The lower figure shows the ECT results of patients in December 2013 (before any MASCT treatment), November 2016 (after achieving disease stability after MASCT), and November 2017. Arrows and circles indicate metastatic sites on the right sacroiliac joint bone, showing a reduction in metastatic tumors and no additional metastases in response to MASCT treatment.

[0034] Figures 3A and 3B show, as determined by ELISPOT, the specific immune responses of the patient's PBMCs to the cervical cancer antigen peptide pool (peptide pool) and individual tumor antigen peptides within that pool after customized MASCT treatment. W / O = response without stimulation by any antigen peptides. ENV refers to the test using unrelated peptides. Dashed lines indicate the threshold of no increase in immune response, such as the number of spots per 200,000 cells (reflecting IFNγ secretion levels). Figure 3B shows the patient's PBMCs exhibiting a consistent and strong immune response to HPV18-E7, RGS-5, and CEA peptides.

[0035] Figure 4 shows an exemplary method for preparing tumor antigen-specific T cells as described in Example 2 ("Method 2"). Figure 5 shows cell proliferation at various time points during the preparation of tumor antigen-specific T cells. Figures 6A and 6B show the percentage of IFNγ+CD3+ tumor antigen-specific T cells in various co-culture samples.

[0036] Figure 7 shows the optimization of the illustrative method of Figure 4 for preparing tumor antigen-specific T cells as described in Example 2 ("Method 2m"). Figure 8 shows cell proliferation at various time points during the preparation of tumor antigen-specific T cells. Figures 9A and 9B show the percentage of IFNγ+CD3+ tumor antigen-specific T cells in various co-culture samples. Figure 9C shows the percentage of IFNγ+TNFα+ tumor antigen-specific T cells in various co-culture samples.

[0037] Figure 10 shows the percentages of IFNγ+CD4+ and IFNγ+CD8+ cells in tumor antigen-specific cells stimulated by various tumor antigen peptides. Tumor antigen-specific cells were prepared using PBMCs from patients who had clinically benefited from MASCT, and pools of tumor antigen peptides derived from CEA, RGS5, and HPV18-E7. Figure 11 shows the frequency of unique TCR clonotypes in various T cell samples, determined by next-generation sequencing.

[0038] Figure 12 summarizes the number of progeny types from tumor antigen-specific T cell preparations (using different PBMC samples from patients who have clinically benefited from MASCT), which may be specific to CEA, RGS5, and HPV18-E7 peptide, respectively. Progeny types were selected based on the frequency patterns in various samples as shown in Figure 11. Results were obtained through next-generation sequencing of bulk T cell samples.

[0039] Figure 13 shows the sub-pools of HPV18-E7, RGS-5, and CEA peptide fragments, screened for specific immune responses against tumor antigen-specific T cells. The RGS5-OLP5 peptide elicits the strongest specific response in tumor antigen-specific T cells prepared using methods 2 and 2m. Figure 14A shows the percentage of IFNγ+CD3+ tumor antigen-specific T cells after stimulation with each of the antigen peptide sub-pools using method 2m. Figure 14B shows the percentage of IFNγ+TNFα+ tumor antigen-specific T cells after stimulation with each of the antigen peptide sub-pools using method 2.

[0040] Figures 15A and 15B show exemplary methods for preparing tumor antigen-specific T cells from cryopreservation stock of tumor antigen-specific T cells prepared by method 2 or method 2m as described in Example 3. Figure 16 shows cell proliferation at various time points during the preparation of tumor antigen-specific T cells. Figure 17A shows the percentage of IFNγ+CD3+ tumor antigen-specific T cells in various co-culture samples. Figure 17B shows the percentage of IFNγ+TNFα+ tumor antigen-specific T cells in various co-culture samples.

[0041] Figures 18A and 18B show the frequencies of unique TCRα and TCRβ progenitors in various T cell samples, determined by next-generation sequencing.

[0042] Figure 19 shows the TCRα and TCRβ pairing results determined by single-cell sequencing using the iPair analyzer. Wells in the 96-well disk that produced successfully paired TCRα and TCRβ sequences are shown in gray and labeled with well position numbers. Only wells that produced TCRβ sequences are shown in light gray, and only wells that produced TCRα sequences are shown in dark gray.

[0043] Figure 20 shows an exemplary method for preparing tumor antigen-specific T cells as described in Example 2.

[0044] Figure 21A shows cell proliferation at various time points during the preparation of tumor antigen-specific T cells. Figure 21B shows the percentage of IFNγ+CD3+ tumor antigen-specific T cells before and after the enrichment step.

[0045] Figures 22A and 22B show the cell proliferation and percentage of tumor antigen-specific T cell populations in various co-culture samples.

[0046] Figure 23 shows an exemplary validation procedure for tumor-specific TCR.

[0047] Figure 24 shows an exemplary TCR structure.

[0048] Figures 25A and 25B show the composition and sequence of an exemplary TCR structure.

[0049] Figures 26A to 26D show the verification results of 09B03 and related TCR structures.

[0050] Figures 27A to 27D show the verification results of P09E06 and related TCR structures.

[0051] Figures 28A to 28C show the verification results of 09B12 and related TCR structures.

[0052] Figures 29A to 29C show the verification results of 09E01 and related TCR structures.

[0053] Figures 30A to 30D show the verification results of 10F04 and related TCR structures.

[0054] Figures 31A to 31C show the verification results of 33D05 and related TCR structures.

[0055] Figures 32A to 32D show the verification results of P09B08 and related TCR structures.

[0056] Figures 33A to 33G show the verification results of 09H05 and related TCR structures.

[0057] Figures 34A to 34G show the verification results of 09D01 and related TCR structures.

[0058] Figures 35A to 35G show the verification results of 33A02 and related TCR structures.

[0059] Figure 36 shows the specific immune responses of the patient's PBMCs to the tumor antigen peptide pool and individual tumor antigen peptides within that pool, as determined by the ELISPOT assay, after MASCT treatment. Percentages indicate the reduction in peptide concentration. For example, 1 / 20 basic peptide indicates the general tumor antigen peptide pool at a 20-fold dilution.

[0060] Figure 37 shows the first round of an exemplary two-round method for preparing tumor antigen-specific T cells using PBMCs from patient SMZ.

[0061] Figure 38A shows cell proliferation at various time points during round 1. Figure 38B shows the percentage of IFNγ+CD3+ tumor antigen-specific T cell population after the enrichment step.

[0062] Figures 39A to 39E show the percentage of tumor antigen-specific T cell populations in the various co-culture samples during round 1. Figure 39F shows the effector T cell population among the IFNγ+CD4+ tumor antigen-specific T cells obtained at the end of round 1.

[0063] Figure 40 shows the second round of an exemplary two-round method for preparing tumor antigen-specific T cells using PBMCs from a patient's SMZ.

[0064] Figure 41A shows the number of T cells and tumor-specific T cells at various time points in round 2. Figures 41B and 41C show the percentage of tumor antigen-specific T cell populations in various co-culture samples in round 2.

[0065] Figure 42 shows the number of T cells and tumor-specific T cells at various time points in round 1 and round 2. Implementation

[0066] This application provides a platform for selecting multiple T-cell receptors (TCRs) that specifically recognize one or more tumor antigen peptides using PBMCs or T cells derived from individuals who have clinically benefited from immunotherapy, such as multi-antigen-specific cell therapy (“MASCT”). The method described herein involves enriching activated T cells from co-cultures of T cells and antigen-loaded dendritic cells (“DCs”), and subsequently co-culturing these enriched activated T cells with antigen-loaded DCs to provide tumor antigen-specific T cells for bulk and / or single-cell sequencing to obtain multiple pairs of TCRα and TCRβ genes. The source of T cells, and the enrichment and co-culture steps in the methods described herein, contribute to achieving a high percentage (e.g., at least about 1% or more for bulk sequencing of T cell samples, or at least 10% or more for single T cell sequencing) of tumor antigen-specific T cells that specifically respond to the target tumor antigen peptide. This is essential for obtaining homologous pairing information of the TCRα and TCRβ genes of the major TCR progenitors via next-generation sequencing. Tumor-specific TCRs, engineered immune cells expressing these TCRs, and methods for treating cancer using these engineered immune cells are also provided.

[0067] Currently, TCRs under development and in clinical trials in this field are generally selected from PBMCs of healthy human donors, which are stimulated with predetermined tumor antigen epitope peptides. Therefore, the clinical response of patients treated with T cells expressing such TCRs is unpredictable. In contrast, the TCR of this application is obtained from individuals who have clinically benefited from MASCT treatment, indicating the antitumor potential of TCRs targeting (multiple) tumor antigen epitopes contained in the target tumor antigen peptide.

[0068] Furthermore, it is known that TCRs selected from PBMCs of healthy human donors have low affinity for their target tumor antigen epitope-HLA complex. Optimization of the amino acid sequences of the TCRα and TCRβ chains is required to improve TCR affinity, but this increases the risk of TCR cross-reactivity, off-target side effects, and serious toxicity. In contrast, since the TCR of this application is selected from cancer patients who have demonstrated clinical responses, the TCR described herein may not require affinity optimization, thus ensuring improved clinical safety.

[0069] In some embodiments, the TCR is selected from individuals belonging to an ethnic group to provide an HLA-restricted TCR that reflects the dominant HLA haplotype of that ethnic group. Most TCRs currently used in clinical trials are haplotype-restricted, such as the dominant HLA-DPB1*0401 and HLA-A*0201 in Caucasian populations. The methods described herein can be used to obtain HLA-restricted TCRs for any ethnic group of interest, including, for example, HLA-A*1101 or HLA-A*2402-restricted TCRs that may be more effective in treating Asian patients.

[0070] Therefore, one embodiment of this application provides a method for obtaining a plurality of T-cell receptors (TCRs) that specifically recognize target tumor antigen peptides, comprising: a) a first co-culture step, comprising co-culturing a first dendritic cell (DC) population carrying the target tumor antigen peptide with a T-cell population from an individual to obtain a first co-culture; b) an enrichment step, comprising subjecting the first co-culture to an enrichment procedure to obtain enriched activated T cells; c) a second co-culture step, comprising co-culturing the enriched activated T cells with a second DC population carrying the target tumor antigen peptide to obtain a tumor antigen-specific T-cell population, wherein at least about 10% (e.g., at least about 20%, or at least about 50%) of the activated T cells are enriched. These tumor antigen-specific T cells specifically respond to the target tumor antigen peptide; and d) a sequencing step comprising subjecting the tumor antigen-specific T cells to next-generation sequencing (e.g., single-cell sequencing) to recognize multiple pairs of genes encoding TCRα and TCRβ, thereby providing the multiple T cell receptors based on the paired genes encoding TCRα and TCRβ; wherein the individual has clinically benefited from multi-antigen-specific cell therapy (“MASCT”), which involves administering an effective amount of activated T cells to the individual, the activated T cells being prepared by co-culturing a population of T cells with a population of dendritic cells carrying multiple tumor antigen peptides, the multiple tumor antigen peptides including the target tumor antigen peptide. In some embodiments, TCRs that specifically recognize multiple target tumor antigens are simultaneously obtained in this method.

[0071] This application claims priority to International Patent Application No. PCT / CN2018 / 082947, filed on April 13, 2018, the full disclosure of which is incorporated herein by reference. [I. Definition]

[0072] Unless otherwise defined below, the terms used herein are as they are commonly used in the relevant technical field.

[0073] As used in this article, "a plurality of tumor antigen peptides", "multiple tumor antigen peptides", "a pool of tumor antigen peptides", and "a tumor antigen peptides pool" are used interchangeably to refer to a combination of two or more tumor antigen peptides.

[0074] As used herein, "antigen presenting cells loaded with a plurality of tumor antigen peptides" and "antigen presenting cells loaded with one or more tumor antigen peptides" are also referred to as "antigen-loaded antigen presenting cells." An antigen presenting cell loaded with a plurality of tumor antigen peptides ("APC") is an APC that exhibits enhanced presentation of one or more tumor antigen peptides or fragments thereof among the plurality of tumor antigen peptides. In some embodiments, an antigen-loaded APC is an antigen-loaded DC. In some embodiments, an antigen-loaded APC is an antigen-loaded PBMC.

[0075] As used herein, "activated T cells" refers to a group of monoclonal (e.g., encoding the same TCR) or multiple (e.g., having strains encoding different TCRs) T cells that recognize at least one tumor antigen peptide T cell receptor. Activated T cells may contain one or more T cell subtypes, including but not limited to cytotoxic T cells, helper T cells, natural killer T cells, γδ T cells, regulatory T cells, and memory T cells.

[0076] The terms "tumor antigen-specific T cells" and "tumor specific T cells" can be used interchangeably in this article.

[0077] As used herein, "T cell receptor" or "TCR" refers to an endogenous or engineered T cell receptor containing an extracellular antigen-binding domain that binds to a specific antigenic epitope bound to an MHC molecule. A TCR may contain a TCRα polypeptide chain and a TCRβ polypeptide chain. "Tumor-specific TCR" refers to a TCR that specifically recognizes tumor antigens expressed by tumor cells. "TCR-T" refers to T cells expressing a recombinant TCR.

[0078] As used herein, "immune checkpoint inhibitor" refers to agents (including antibodies) that inhibit or block inhibitory immune checkpoint molecules on immune cells (such as T cells) or tumor cells. "Immune checkpoint molecules" include molecules that enhance immune signaling against tumor cells (i.e., "co-stimulatory molecules") or molecules that reduce immune signaling against tumor cells (i.e., "inhibitory immune checkpoint molecules").

[0079] As used herein, "treatment" refers to a manner of achieving a beneficial or desired outcome (including clinical outcomes). For the purposes of this invention, a beneficial or desired clinical outcome includes, but is not limited to, one or more of the following: reducing symptoms of another disease, reducing the severity of the disease, stabilizing the disease (e.g., preventing or delaying disease progression), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the onset or recurrence of the disease, delaying or slowing disease progression, improving disease status, providing (partial or complete) remission of the disease, reducing the dosage of one or more other medications required to treat the disease, delaying disease progression, increasing quality of life, and / or prolonging survival. "Treatment" also encompasses a reduction in cancer pathological outcomes. The methods of this invention are contemplated for any or more of these therapeutic outcomes.

[0080] As used herein, "delaying" cancer development means postponing, hindering, slowing, stabilizing, and / or delaying the progression of the disease. This delay can be of varying lengths, depending on the individual's medical history and / or current treatment. A sufficient or significant delay that is apparent to someone of ordinary knowledge in the relevant technical field can effectively cover prevention, as the individual will not develop the disease. Methods of "delaying" cancer development, when compared to not using such methods, are methods that reduce the likelihood of disease development and / or the severity of the disease within a given time period. Such comparisons are generally based on clinical studies and use statistically significant numbers of individuals. Standard methods can be used to detect cancer development, including but not limited to computed tomography (CAT), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Development can also refer to cancer progression that may initially be undetectable, and includes occurrence, recurrence, and onset.

[0081] The terms "individual," "subject," and "patient" are used interchangeably in this document to describe mammals, including humans. An individual includes, but is not limited to, humans, cattle, horses, cats, dogs, rodents, or primates. In some embodiments, an individual is a human. In some embodiments, an individual suffers from a disease such as cancer. In some embodiments, an individual requires treatment.

[0082] As understood in the art, an "effective amount" refers to the quantity of a component (e.g., antigen-loaded dendritic cells, activated T cells, or engineered immune cells expressing TCRs) sufficient to produce the desired therapeutic outcome (e.g., reducing the severity or duration of one or more cancer symptoms, stabilizing the severity of one or more cancer symptoms, or eliminating one or more cancer symptoms). For therapeutic use, beneficial or desired outcomes include, for example, reducing symptoms (biochemical, histological, and / or behavioral) of one or more diseases, including complications and intermediate pathological phenotypes present during disease development; increasing the quality of life of patients with the disease; reducing the dosage of other medications required to treat the disease; enhancing the effect of another medication; delaying disease progression; and / or prolonging patient survival.

[0083] "Adjuvant setting" refers to a clinical setting in which an individual has a history of cancer and is generally (but not necessarily) responsive to treatments, including but not limited to surgery (e.g., surgical resection), radiation therapy, and chemotherapy. However, due to their cancer history, such individuals are considered at risk of disease progression. Treatment or administration in "adjuvant setting" refers to the subsequent treatment modality. The level of risk (e.g., whether an individual in adjuvant setting is considered "high-risk" or "low-risk") depends on several factors, most commonly the severity of the disease at the time of initial treatment.

[0084] "Neoadjuvant setting" refers to a clinical setting in which the methods are performed prior to the primary / decisive therapy.

[0085] As used herein, "combination therapy" means that a first agent is administered in conjunction with another agent. "In conjunction with" means that a treatment modality is administered in addition to another treatment modality, such as the components described herein (e.g., antigen-loaded DCs, activated T cells, or engineered immune cells expressing TCRs) being administered to the same individual in addition to another agent (such as an immune checkpoint inhibitor). Therefore, "in conjunction with" means that a treatment modality is administered before, during, or after the delivery of another treatment modality to the individual. Such combinations are considered part of a single treatment regimen (regime).

[0086] As used herein, the term "simultaneous administration" means that the first and second therapies in a combination therapy are administered at intervals not exceeding about 15 minutes (such as not exceeding about 10, 5, or 1 minute). When the first and second therapies are administered simultaneously, they may be contained in the same composition (e.g., a composition containing both the first and second therapies) or in different compositions (e.g., the first therapy is in one composition and the second therapy is in another composition).

[0087] As used herein, the term "sequential administration" means that the first and second therapies in a combination therapy are administered at intervals of more than about 15 minutes (such as more than about 20, 30, 40, 50, 60, or more minutes). The first or second therapy may be administered first. The first and second therapies may be contained in different components, which may be contained in the same or different packages or kits.

[0088] As used in this article, the term "concurrent administration" refers to the overlapping administration of the first therapy and the second therapy in a combination therapy.

[0089] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means that the material is not biologically or otherwise undesirable, for example, that it can be incorporated into a pharmaceutical composition administered to an individual without causing any significant undesirable biological effects or interacting in a harmful manner with any other component in the composition containing it. Pharmaceutically acceptable carriers or excipients preferably meet the required toxicology and manufacturing testing standards and / or are included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0090] The following definitions can be used to assess response based on the target lesion: "Complete response" or "CR" means that all target lesions have disappeared; "Partial response" or "PR" means that the sum of the longest diameters (SLD) of the target lesions has decreased by at least 30%, with the baseline SLD as a reference; "Stable disease" or "SD" means that the shrinkage of the target lesions is insufficient to meet the criteria for PR, and its increase is insufficient to meet the criteria for PD, with the lowest SLD since the start of treatment as a reference; and "Progressive disease" or "PD" means that the SLD of the target lesions has increased by at least 20% (with the lowest SLD recorded since the start of treatment as a reference), or that one or more new lesions have appeared.

[0091] The following definitions of response assessment can be used to assess non-target lesions: "Complete response" or "CR" means that all non-target lesions have disappeared; "Stable disease" or "SD" means that one or more non-target lesions do not meet the criteria for CR or PD and persist; and "Progressive disease" or "PD" means "unequivocal progression" of existing (multiple) non-target lesions, or the appearance of one or more new lesions is considered as disease progression (if an individual's PD is assessed based entirely on the progression of (multiple) non-target lesions at a single point in time, additional criteria must be met).

[0092] As used herein, the terms "cell," "cell line," and "cell culture" are used interchangeably, and all such names include progeny. It is understood that due to human or accidental mutations, the DNA content of all progeny cells may not be entirely identical. Variant progeny lines with the same function or biological activity as the original cell are included.

[0093] The term "peptide" refers to an amino acid polymer (including fragments of proteins) containing no more than about 100 amino acids, which may be linear or branched, containing modified amino acids, and / or separated by non-amino acids. This term also covers amino acid polymers that have undergone natural or engineered modifications, including, for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification. Also included in this term are, for example, peptides containing one or more amino acid analogs (including, for example, non-natural amino acids), and other modifications known in the art. The peptides described herein may be naturally occurring, i.e., obtained from or derived from natural sources (e.g., blood), or synthesized (e.g., chemically synthesized or synthesized using recombinant DNA technology).

[0094] The term "antibody" as used in this article is used in the broadest sense and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, as long as they exhibit the desired biological activity.

[0095] An "antibody fragment" contains a portion of a complete antibody, preferably including its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; bivalent antibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0096] As used herein, the terms "specifically binds to," "recognizes," "specifically recognizes," "targets," or "specific for" refer to measurable and reproducible interactions, such as binding between a target and an antibody, or between a receptor and a ligand, or between a receptor and an epitope / MHC complex, which determine the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, a TCR that binds to or specifically binds to a target epitope is a TCR that binds to the target epitope / MHC complex with greater affinity, binding strength, ease, and / or a longer duration (compared to its binding to other epitope / MHC complexes). In one embodiment, as measured (e.g., by radioimmunoassay (RIA)), the degree of binding of a TCR to an unrelated epitope / MHC complex is less than about 10% of the binding of the TCR to the target epitope / MHC complex. In some embodiments, TCRs that specifically bind to the target epitope (i.e., the target epitope / MHC complex) have 1μM, 100nM 10nM 1nM, or The dissociation constant (Kd) is 0.1 nM. In some embodiments, the TCR specifically binds to epitopes on conserved proteins from different species. In another embodiment, specific binding may include, but does not require, exclusive binding.

[0097] As used herein, the term "isolated nucleic acid" is intended to refer to a nucleic acid of genomic origin, cDNA, or synthetic origin or combination thereof, which, due to its origin, (1) is not associated with all or part of the polynucleotides of "isolated nucleic acids" found in nature, (2) is operatively linked to polynucleotides that it would not be linked to in nature, or (3) does not exist in nature as a part of a larger sequence.

[0098] It is understood that the present invention described herein includes states and embodiments that are "consisting" and / or "consisting essentially of".

[0099] The term "about" as used in this document includes (and describes) variations of that value or parameter itself. For example, a description of "about X" includes a description of "X".

[0100] The term "about X to Y" used in this article has the same meaning as "about X to about Y".

[0101] As used in this article, the reference to "not" as a value or parameter generally means and describes a value or parameter "other than". For example, "This method is not used to treat type X cancer" means that the method is used to treat types of cancer other than X.

[0102] As used herein and in the claims of the accompanying patent application, the singular forms “a / an” and “the” include the plural reference, unless the context clearly indicates otherwise. [II. Methods for obtaining tumor-specific TCRs]

[0103] This application provides a method for obtaining multiple T-cell receptors (TCRs) that specifically recognize one or more target tumor antigen peptides from an individual's PBMCs or T cells who have clinically benefited from immunotherapy. In some embodiments, the immunotherapy is an adoptive T-cell therapy comprising administering to the individual an effective amount of activated T cells that specifically recognize a target tumor antigen peptide or a fragment thereof. In some embodiments, the immunotherapy is a multi-antigen-specific cell therapy (“MASCT”) comprising administering to the individual an effective amount of activated T cells prepared by co-culturing a population of T cells with a population of dendritic cells carrying multiple tumor antigen peptides, including one or more target tumor antigen peptides. In some embodiments, a TCR that specifically recognizes a single target tumor antigen peptide or a fragment thereof (e.g., a target tumor epitope) is obtained. In some embodiments, the method is used to simultaneously obtain TCRs that specifically recognize multiple target tumor antigen peptides.

[0104] In some embodiments, a method is provided for obtaining a plurality of TCRs that specifically recognize a target tumor antigen peptide, comprising subjecting a population of tumor antigen-specific T cells to next-generation sequencing (e.g., single-cell sequencing) to recognize a plurality of pairs of genes encoding TCRα and TCRβ, thereby providing the plurality of T cell receptors based on the paired genes encoding TCRα and TCRβ; wherein at least about 10% (e.g., at least about 20%, or at least about 50%) of the tumor antigen-specific T cells specifically respond to the target tumor antigen peptide; wherein the tumor antigen-specific T cell population is prepared by: i) a first co-culture step comprising combining a first population of DCs carrying the target tumor antigen peptide with a population of T cells from an individual. ii) an enrichment step comprising subjecting the first coculture to an enrichment process to obtain enriched activated T cells; and iii) a second coculture step comprising coculturing the enriched activated T cells with a second DC population carrying the target tumor antigen peptide to obtain a tumor antigen-specific T cell population; wherein the individual has clinically benefited from multiantigen-specific cell therapy (“MASCT”), which involves administering an effective amount of activated T cells to the individual, the activated T cells being prepared by coculturing the T cell population with a DC population carrying a plurality of tumor antigen peptides, the plurality of tumor antigen peptides including the target tumor antigen peptide.

[0105] In some embodiments, a method is provided for obtaining a plurality of TCRs that specifically recognize a target tumor antigen peptide, comprising: a) co-culturing an enriched population of activated T cells with a second population of dendritic cells (DCs) carrying the target tumor antigen peptide to obtain a population of tumor antigen-specific T cells, wherein at least about 10% (e.g., at least about 20%, or at least about 50%) of the tumor antigen-specific T cells specifically respond to the target tumor antigen peptide; and b) subjecting the tumor antigen-specific T cells to next-generation sequencing (e.g., single-cell sequencing) to recognize a plurality of pairs of genes encoding TCRα and TCRβ, thereby providing the plurality of T cell receptors based on the paired genes encoding TCRα and TCRβ. The enriched activated T cell population is prepared by: i) a first co-culture step comprising co-culturing a first DC population carrying the target tumor antigen peptide with a T cell population from an individual to obtain a first co-culture; and ii) an enrichment step comprising subjecting the first co-culture to an enrichment process to obtain enriched activated T cells; and wherein the individual has clinically benefited from multi-antigen-specific cell therapy (“MASCT”), which comprises administering an effective amount of activated T cells to the individual, the activated T cells being prepared by co-culturing a T cell population with a DC population carrying a plurality of tumor antigen peptides, the plurality of tumor antigen peptides including the target tumor antigen peptide.

[0106] In some embodiments, a method is provided for obtaining a plurality of TCRs that specifically recognize a target tumor antigen peptide, comprising: a) subjecting a first co-culture to an enrichment process to obtain enriched activated T cells, the first co-culture comprising a first dendritic cell (DC) population carrying the target tumor antigen peptide and a population of T cells from an individual; b) co-culturing the enriched activated T cells with a second DC population carrying the target tumor antigen peptide to obtain a tumor antigen-specific T cell population, wherein at least about 10% (e.g., at least about 20%, or at least about 50%) of the tumor antigen-specific T cells are specific to the target tumor antigen peptide. The individual has a positive response; and c) subjecting the tumor antigen-specific T cells to next-generation sequencing (e.g., single-cell sequencing) to recognize multiple pairs of genes encoding TCRα and TCRβ, thereby providing the multiple T cell receptors based on the paired genes encoding TCRα and TCRβ; and wherein the individual has clinically benefited from multi-antigen-specific cell therapy (“MASCT”), which involves delivering an effective amount of activated T cells to the individual, the activated T cells being prepared by co-culturing a population of T cells with a population of DCs carrying multiple tumor antigen peptides, the multiple tumor antigen peptides including the target tumor antigen peptide.

[0107] In some embodiments, a method is provided for obtaining a plurality of TCRs that specifically recognize a target tumor antigen peptide, comprising: a) a first co-culture step, comprising co-culturing a first population of dendritic cells (DCs) carrying the target tumor antigen peptide with a population of T cells from an individual to obtain a first co-culture; b) an enrichment step, comprising subjecting the first co-culture to an enrichment procedure to obtain enriched activated T cells; c) a second co-culture step, comprising co-culturing the enriched activated T cells with a second population of DCs carrying the target tumor antigen peptide to obtain a tumor antigen-specific T cell population, wherein at least about 10% (e.g., at least about 20%, or at least about 50%) of the tumor antigens are specific. The individual exhibits a specific response to the target tumor antigen peptide by T cells; and a) a sequencing step comprising subjecting the tumor antigen-specific T cells to next-generation sequencing (e.g., single-cell sequencing) to recognize multiple pairs of genes encoding TCRα and TCRβ, thereby providing the multiple T cell receptors based on the paired genes encoding TCRα and TCRβ; wherein the individual has clinically benefited from multi-antigen-specific cell therapy (“MASCT”), which involves administering an effective amount of activated T cells to the individual, the activated T cells being prepared by co-culturing a population of T cells with a population of DCs carrying multiple tumor antigen peptides, including the target tumor antigen peptide. In some embodiments, the first co-culture step is performed for about 1 to 3 days prior to the enrichment step. In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is no greater than about 30:1 (e.g., about 20:1, 15:1, or 10:1). In some embodiments, the T cell population in the first co-culture step is present in PBMCs. In some embodiments, the first population of dendritic cells (DCs) carrying the target tumor antigen peptide and the T cell population are co-cultured in a first co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines such as IL-2, IL-7, IL-15, and IL-21) and an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody, such as SHR-1210). In some embodiments, the first co-culture medium contains IL-2 and an anti-PD-1 antibody. In some embodiments, the ratio between the enriched activated T cell population and the second population of DCs carrying the target tumor antigen peptide is about 1:1 to about 20:1 (e.g., about 1:1, 2:1, or 4:1). In some embodiments, the enriched activated T cell population and the second population of DCs carrying the target tumor antigen peptide are co-cultured for about 12 to 25 days. In some embodiments, after receiving the MASCT, the individual has a response (PR), complete response (CR), or stable disease (SD) for at least about 6 months (e.g., at least about 1 year, 2 years, or more).

[0108] In some embodiments, a method is provided for obtaining a plurality of TCRs that specifically recognize a target tumor antigen peptide, comprising: a) a first co-culture step, comprising co-culturing a first population of dendritic cells (DCs) carrying the target tumor antigen peptide with a population of T cells from an individual to obtain a first co-culture; b) an enrichment step, comprising: contacting the first co-culture with APCs (e.g., DCs or PBMCs) carrying the target tumor antigen peptide to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand that specifically recognizes cytokines or cell surface molecules; c) a second co-culture step, comprising co-culturing the enriched activated T cells with a second population of DCs carrying the target tumor antigen peptide to obtain a tumor antigen-specific T cell population. At least about 10% (e.g., at least about 20%, or at least about 50%) of the tumor antigen-specific T cells specifically respond to the target tumor antigen peptide; and d) a sequencing step comprising subjecting the tumor antigen-specific T cells to next-generation sequencing (e.g., single-cell sequencing) to recognize multiple pairs of genes encoding TCRα and TCRβ, thereby providing the multiple T cell receptors based on the paired genes encoding TCRα and TCRβ; wherein the individual has clinically benefited from multi-antigen-specific cell therapy (“MASCT”), which comprises delivering an effective amount of activated T cells to the individual, the activated T cells being prepared by co-culturing a population of T cells with a population of DCs carrying multiple tumor antigen peptides, the multiple tumor antigen peptides including the target tumor antigen peptide. In some embodiments, the enrichment step includes: contacting the first co-culture with an APC (e.g., DC or PBMC) loaded with the target tumor antigen peptide to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand that specifically recognizes IFNγ. In some embodiments, the first co-culture step is performed for about 1 to 3 days prior to the enrichment step. In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is no greater than about 30:1 (e.g., about 20:1, 15:1, or 10:1). In some embodiments, the T cell population in the first co-culture step is present in PBMCs. In some embodiments, the first DC population loaded with the target tumor antigen peptide and the T cell population are co-cultured in a first co-culture medium containing multiple cytokines (e.g., IL-2, IL-7, IL-15, and IL-21) and immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, such as SHR-1210). In some embodiments, the ratio between the enriched activated T cell population and the second DC population carrying the target tumor antigen peptide is about 1:1 to about 20:1 (e.g., about 1:1, 2:1, or 4:1).In some embodiments, the enriched activated T cell population is co-cultured with the second DC population carrying the target tumor antigen peptide for approximately 12 to 25 days. In some embodiments, after receiving the MASCT, the individual exhibits a partial response (PR), a complete response (CR), or stable disease (SD).

[0109] In some embodiments, a method is provided for obtaining multiple TCRs that specifically recognize a target tumor antigen peptide, comprising: a) a first co-culture step, comprising co-culturing a first DC population carrying the target tumor antigen peptide with a T cell population from an individual to obtain a first co-culture; b) an enrichment step, comprising subjecting the first co-culture to an enrichment procedure to obtain enriched activated T cells; c) a second co-culture step, comprising co-culturing a second DC population carrying the target tumor antigen peptide with the enriched activated T cell population in an initial second co-culture medium to provide a second co-culture, the initial second co-culture medium containing an immune checkpoint inhibitor and optionally one or more cytokines (e.g., multiple cytokines); and adding an anti-CD3 (e.g., OKT-3) antibody and optionally one or more cytokines to the second co-culture. The process involves obtaining a population of tumor antigen-specific T cells, wherein at least about 10% (e.g., at least about 20%, or at least about 50%) of these tumor antigen-specific T cells specifically respond to the target tumor antigen peptide; and d) a sequencing step comprising subjecting the tumor antigen-specific T cells to next-generation sequencing (e.g., single-cell sequencing) to recognize multiple pairs of genes encoding TCRα and TCRβ, thereby providing the multiple T cell receptors based on the paired genes encoding TCRα and TCRβ; wherein the individual has clinically benefited from multi-antigen-specific cell therapy (“MASCT”), which involves delivering an effective amount of activated T cells to the individual, the activated T cells being prepared by co-culturing a population of T cells with a population of dendritic cells carrying multiple tumor antigen peptides, including the target tumor antigen peptide. In some embodiments, the first co-culture step is performed for about 1 to 3 days prior to the enrichment step. In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is no greater than about 30:1 (e.g., about 20:1, 15:1, or 10:1). In some embodiments, the T cell population in the first co-culture step is present in PBMCs. In some embodiments, the first DC population carrying the target tumor antigen peptide and the T cell population are co-cultured in a first co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15, and IL-21) and immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, such as SHR-1210). In some embodiments, the enrichment step includes: contacting the first co-culture with APCs (e.g., DCs or PBMCs) carrying the target tumor antigen peptide to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand that specifically recognizes cytokines (e.g., IFNγ) or cell surface molecules.In some embodiments, the ratio between the enriched activated T cell population and the second DC population carrying the target tumor antigen peptide is approximately 1:1 to approximately 20:1 (e.g., approximately 1:1, 2:1, or 4:1). In some embodiments, the enriched activated T cell population and the second DC population carrying the target tumor antigen peptide are co-cultured for approximately 12 to 25 days. In some embodiments, the anti-CD3 antibody is added to the second co-culture no more than approximately 3 days after the start of the second co-culture step (e.g., approximately 2 days). In some embodiments, the initial second co-culture medium contains IL-2, IL-7, IL-15, and IL-21, and an anti-PD-1 antibody. In some embodiments, after receiving the MASCT, the individual has a PR, CR, or stable disease (SD).

[0110] In some embodiments, a method is provided for obtaining a plurality of TCRs that specifically recognize a target tumor antigen peptide, comprising: a) a first co-culture step, comprising co-culturing a first population of dendritic cells (DCs) carrying the target tumor antigen peptide with a population of T cells from an individual to obtain a first co-culture; b) an enrichment step, comprising subjecting the first co-culture to an enrichment process to obtain enriched activated T cells; c) a second co-culture step, comprising co-culturing the enriched activated T cells with a second population of DCs carrying the target tumor antigen peptide to obtain a population of tumor antigen-specific T cells; d) a third co-culture step, comprising co-culturing the population of tumor antigen-specific T cells with a population of APCs (e.g., PBMCs, DCs, or cell line APCs) carrying the target tumor antigen peptide to obtain a second tumor antigen-specific T cell population. The individual possesses a heterologous T cell population, wherein at least about 10% (e.g., at least about 20%, or at least about 50%) of the tumor antigen-specific T cells specifically respond to the target tumor antigen peptide; and e) a sequencing step comprising subjecting the tumor antigen-specific T cells to next-generation sequencing (e.g., single-cell sequencing) to recognize multiple pairs of genes encoding TCRα and TCRβ, thereby providing the multiple T cell receptors based on the paired genes encoding TCRα and TCRβ; wherein the individual has clinically benefited from multi-antigen-specific cell therapy (“MASCT”), which comprises administering to the individual an effective amount of activated T cells prepared by co-culturing a T cell population with a DC population carrying multiple tumor antigen peptides, including the target tumor antigen peptide. In some embodiments, the first co-culture step is performed for about 1 to 3 days prior to the enrichment step. In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is no greater than about 30:1 (e.g., about 20:1, 15:1, or 10:1). In some embodiments, the T cell population in the first co-culture step is present in PBMCs. In some embodiments, the first DC population carrying the target tumor antigen peptide and the T cell population are co-cultured in a first co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15, and IL-21) and immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, such as SHR-1210). In some embodiments, the enrichment step includes: contacting the first co-culture with APCs (e.g., DCs or PBMCs) carrying the target tumor antigen peptide to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand that specifically recognizes cytokines (e.g., IFNγ) or cell surface molecules.In some embodiments, the ratio between the enriched activated T cell population and the second DC population carrying the target tumor antigen peptide is approximately 1:1 to approximately 20:1 (e.g., approximately 1:1, 2:1, or 4:1). In some embodiments, the enriched activated T cell population and the second DC population carrying the target tumor antigen peptide are co-cultured for approximately 12 to 25 days. In some embodiments, the second co-culture step includes: co-culturing the second DC population carrying the target tumor antigen peptide and the enriched activated T cell population in an initial second co-culture medium to provide a second co-culture, the initial second co-culture medium containing an immune checkpoint inhibitor and optionally one or more cytokines (e.g., multiple cytokines); and adding an anti-CD3 antibody (e.g., OKT-3) and optionally one or more cytokines to the second co-culture to obtain a tumor antigen-specific T cell population. In some embodiments, the anti-CD3 antibody is added to the second co-culture no more than approximately 3 days (e.g., approximately 2 days) after the start of the second co-culture step. In some embodiments, the initial second co-culture medium comprises IL-2, IL-7, IL-15, and IL-21, and an anti-PD-1 antibody. In some embodiments, the ratio of the tumor antigen-specific T cell population to the APC population carrying the target tumor antigen peptide is approximately 1:1 to approximately 20:1 (e.g., approximately 4:1). In some embodiments, the tumor antigen-specific T cell population and the APC population carrying the target tumor antigen peptide are co-cultured for approximately 5 to 9 days (e.g., approximately 7 days). In some embodiments, the tumor antigen-specific T cell population and the APC population carrying the target tumor antigen peptide are co-cultured in a third co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, and IL-15) and an anti-CD3 antibody (e.g., OKT3). In some embodiments, the third co-culture step is repeated (e.g., once or twice). In some embodiments, after receiving the MASCT, the individual has a response (PR), a complete response (CR), or stable disease (SD).

[0111] In some embodiments, a method is provided for obtaining a plurality of TCRs that specifically recognize a target tumor antigen peptide, comprising: a) a first co-culture step, comprising co-culturing a first population of DCs carrying the target tumor antigen peptide with a population of T cells from an individual to obtain a first co-culture; b) an enrichment step, comprising: contacting the first co-culture with APCs (e.g., PBMCs or DCs) carrying the target tumor antigen peptide to obtain a stimulated co-culture; and using a ligand to isolate the enriched activated T cell population from the stimulated co-culture, the ligand specifically recognizing a cytokine (e.g., IFNγ); c) a second co-culture step. This includes co-culturing a second population of DCs carrying the target tumor antigen peptide with an enriched population of activated T cells in an initial second co-culture medium to provide a second co-culture, the initial second co-culture medium containing an immune checkpoint inhibitor and optionally one or more cytokines (e.g., multiple cytokines); and adding an anti-CD3 antibody (e.g., OKT-3) and optionally one or more cytokines to the second co-culture to obtain a population of tumor antigen-specific T cells; d) a third co-culture step, which includes: co-culturing the population of these tumor antigen-specific T cells with a first population of APCs (e.g., PBMCs, DCs, or cells) carrying the target tumor antigen peptide. The co-cultured population of APCs (e.g., cell line APCs) was further cultured for approximately 5 to 9 days (e.g., 7 days); a second population of APCs (e.g., PBMCs, DCs, or cell line APCs) carrying the target tumor antigen peptide was added to the third co-culture and cultured for approximately 5 to 9 days (e.g., 7 days); and a third population of APCs (e.g., PBMCs, DCs, or cell line APCs) carrying the target tumor antigen peptide was added to the co-culture and cultured for approximately 5 to 9 days (e.g., 7 days) to obtain a second tumor antigen-specific T cell population, wherein at least approximately 10% (e.g., at least approximately 20%, or at least approximately 50%) of these tumor antigen-specific T cells are specific for the target tumor antigen peptide. The individual exhibits heterologous response; and e) a sequencing step comprising subjecting the tumor antigen-specific T cells to next-generation sequencing (e.g., single-cell sequencing) to recognize multiple pairs of genes encoding TCRα and TCRβ, thereby providing the multiple T cell receptors based on the paired genes encoding TCRα and TCRβ; wherein the individual has clinically benefited from multi-antigen-specific cell therapy (“MASCT”), which involves administering an effective amount of activated T cells to the individual, the activated T cells being prepared by co-culturing a population of T cells with a population of DCs carrying multiple tumor antigen peptides, the multiple tumor antigen peptides including the target tumor antigen peptide. In some embodiments, the first co-culture step is performed for about 1 to 3 days prior to the enrichment step. In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is no greater than about 30:1 (e.g., about 20:1, 15:1, or 10:1). In some embodiments, the T cell population in the first co-culture step is present in PBMCs.In some embodiments, the first population of dendritic cells (DCs) carrying the target tumor antigen peptide and the T cell population are co-cultured in a first co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines such as IL-2, IL-7, IL-15, and IL-21) and an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody, such as SHR-1210). In some embodiments, the ratio between the enriched activated T cell population and the second population of DCs carrying the target tumor antigen peptide is about 1:1 to about 20:1 (e.g., about 1:1, 2:1, or 4:1). In some embodiments, the enriched activated T cell population and the second population of DCs carrying the target tumor antigen peptide are co-cultured for about 12 to 25 days. In some embodiments, the anti-CD3 antibody is added to the second co-culture no more than about 3 days (e.g., about 2 days) after the start of the second co-culture step. In some embodiments, the initial second co-culture medium comprises IL-2, IL-7, IL-15, and IL-21, and an anti-PD-1 antibody. In some embodiments, the ratio between the tumor antigen-specific T cell population and the first APC population carrying the target tumor antigen peptide is approximately 1:1 to approximately 20:1 (e.g., approximately 4:1). In some embodiments, the tumor antigen-specific T cell population and the APCs carrying the target tumor antigen peptide are co-cultured in a third co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, and IL-15) and an anti-CD3 antibody (e.g., OKT3). In some embodiments, after receiving the MASCT, the individual has a PR, CR, or stable disease (SD).

[0112] In some embodiments, a method is provided for obtaining a plurality of TCRs that specifically recognize a target tumor antigen peptide, comprising: a) a first co-culture step, comprising co-culturing a first population of dendritic cells (DCs) carrying the target tumor antigen peptide with a population of T cells from an individual to obtain a first co-culture; b) an enrichment step, comprising subjecting the first co-culture to an enrichment process to obtain enriched activated T cells; c) a second co-culture step, comprising co-culturing the enriched activated T cells with a second population of DCs carrying the target tumor antigen peptide to obtain a population of tumor antigen-specific T cells; d) a screening step, comprising: identifying a target tumor epitope from the target tumor antigen peptide, wherein the target tumor epitope elicits a specific response in the enriched activated T cell population; and contacting an APC population (e.g., PBMCs, DCs, or cell line APCs) with the target tumor epitope to obtain an APC population carrying the target tumor antigen peptide; e) a third co-culture step, wherein The method includes: co-culturing a population of tumor antigen-specific T cells with a population of APCs carrying a target tumor antigen peptide to obtain a second population of tumor antigen-specific T cells, wherein at least about 10% (e.g., at least about 20%, or at least about 50%) of the tumor antigen-specific T cells specifically respond to the target tumor antigen peptide; and f) a sequencing step comprising subjecting the tumor antigen-specific T cells to next-generation sequencing (e.g., single-cell sequencing) to recognize multiple pairs of genes encoding TCRα and TCRβ, thereby providing the multiple T cell receptors based on the paired genes encoding TCRα and TCRβ; wherein the individual has clinically benefited from multi-antigen-specific cell therapy (“MASCT”), which involves administering an effective amount of activated T cells to the individual, the activated T cells being prepared by co-culturing a population of T cells with a population of DCs carrying multiple tumor antigen peptides, the multiple tumor antigen peptides including the target tumor antigen peptide. In some embodiments, the first co-culture step is performed for about 1 to 3 days prior to the enrichment step. In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is no greater than about 30:1 (e.g., about 20:1, 15:1, or 10:1). In some embodiments, the T cell population in the first co-culture step is present in PBMCs. In some embodiments, the first DC population carrying the target tumor antigen peptide and the T cell population are co-cultured in a first co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15, and IL-21) and immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, such as SHR-1210).In some embodiments, the enrichment step includes: contacting the first co-culture with APCs (e.g., DCs or PBMCs) carrying the target tumor antigen peptide to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand that specifically recognizes cytokines (e.g., IFNγ) or cell surface molecules. In some embodiments, the ratio between the enriched activated T cell population and the second DC population carrying the target tumor antigen peptide is about 1:1 to about 20:1 (e.g., about 1:1, 2:1, or 4:1). In some embodiments, the enriched activated T cell population is co-cultured with the second DC population carrying the target tumor antigen peptide for about 12 to 25 days. In some embodiments, the second co-culture step includes: co-culturing the second DC population carrying the target tumor antigen peptide with the enriched activated T cell population in an initial second co-culture medium to provide a second co-culture, the initial second co-culture medium containing an immune checkpoint inhibitor and optionally one or more cytokines (e.g., multiple cytokines); and adding an anti-CD3 antibody (e.g., OKT-3) and optionally one or more cytokines to the second co-culture to obtain a tumor antigen-specific T cell population. In some embodiments, the anti-CD3 antibody is added to the second co-culture no more than about 3 days (e.g., about 2 days) after the start of the second co-culture step. In some embodiments, the initial second co-culture medium contains IL-2, IL-7, IL-15, and IL-21, and an anti-PD-1 antibody. In some embodiments, the ratio between the tumor antigen-specific T cell population and the APC population carrying the target tumor antigen peptide is about 1:1 to about 20:1 (e.g., about 4:1). In some embodiments, the tumor antigen-specific T cell population is co-cultured with the APC population carrying the target tumor antigen peptide for approximately 5 to 9 days (e.g., approximately 7 days). In some embodiments, the tumor antigen-specific T cell population and the APC population carrying the target tumor antigen peptide are co-cultured in a third co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15) and an anti-CD3 antibody (e.g., OKT3). In some embodiments, the third co-culture step is repeated (e.g., once or twice). In some embodiments, after receiving the MASCT, the individual has a response (PR), a complete response (CR), or stable disease (SD).

[0113] Any of the methods described herein can be used to simultaneously obtain TCRs that specifically recognize multiple target tumor antigen peptides, by using APCs (e.g., PBMCs or DCs) loaded with multiple target tumor antigen peptides in a first co-culture step, an enrichment step, and a second co-culture step. In some embodiments, APCs loaded with individual target tumor antigen peptides (e.g., PBMCs, DCs, or cell line APCs) are used in a third co-culture step to prepare individual tumor antigen-specific T cell populations for sequencing. In some embodiments, APCs loaded with multiple target tumor antigen peptides (e.g., PBMCs, DCs, or cell line APCs) are used in a third co-culture step to prepare aggregated tumor antigen-specific T cell populations for sequencing. In some embodiments, the method is used to obtain TCRs that specifically recognize at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or more target tumor antigen peptides.

[0114] Therefore, in some embodiments, a method is provided for obtaining a plurality of TCRs that specifically recognize a plurality of target tumor antigen peptides, comprising: a) a first co-culture step, comprising co-culturing a first DC population carrying the plurality of target tumor antigen peptides with a population of T cells from an individual to obtain a first co-culture; b) an enrichment step, comprising subjecting the co-culture to an enrichment procedure to obtain enriched activated T cells; c) a second co-culture step, comprising co-culturing the enriched activated T cells with a second DC population carrying the plurality of target tumor antigen peptides to obtain a tumor antigen-specific T cell population, wherein at least about 10% (e.g., at least about 20%, or at least about 50%) of the tumor antigen-specific T cells are tumor antigen-specific T cells. The individual exhibits a specific response to one or more of the plurality of target tumor antigen peptides; and d) a sequencing step comprising subjecting the tumor antigen-specific T cells to next-generation sequencing (e.g., single-cell sequencing) to recognize multiple pairs of genes encoding TCRα and TCRβ, thereby providing the plurality of T cell receptors based on the paired genes encoding TCRα and TCRβ; wherein the individual has clinically benefited from multi-antigen-specific cell therapy (“MASCT”), which comprises administering an effective amount of activated T cells to the individual, the activated T cells being prepared by co-culturing a population of T cells with a population of DCs carrying multiple tumor antigen peptides, the multiple tumor antigen peptides including the target tumor antigen peptides. In some embodiments, the first co-culture step is performed for about 1 to 3 days prior to the enrichment step. In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is no greater than about 30:1 (e.g., about 20:1, 15:1, or 10:1). In some embodiments, the T cell population in the first co-culture step is present in a PBMC. In some embodiments, the first antigen-loaded DC population and the T cell population are co-cultured in a first co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15, and IL-21) and immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, such as SHR-1210). In some embodiments, the enrichment step includes: contacting the first co-culture with an APC (e.g., DC or PBMC) loaded with the multiple target tumor antigen peptides to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand that specifically recognizes cytokines (e.g., IFNγ) or cell surface molecules. In some embodiments, the ratio between the enriched activated T cell population and the second antigen-loaded DC population is about 1:1 to about 20:1 (e.g., about 1:1, 2:1, or 4:1). In some embodiments, the enriched activated T cell population is co-cultured with the second antigen-loaded DC population for approximately 12 to 25 days.In some embodiments, the second co-culture step includes: co-culturing the second antigen-loaded DC population with the enriched activated T cell population in an initial second co-culture medium to provide a second co-culture, the initial second co-culture medium containing an immune checkpoint inhibitor and optionally one or more cytokines (e.g., multiple cytokines); and adding an anti-CD3 antibody (e.g., OKT-3) and optionally one or more cytokines to the second co-culture to obtain a tumor antigen-specific T cell population. In some embodiments, the anti-CD3 antibody is added to the second co-culture no more than about 3 days (e.g., about 2 days) after the start of the second co-culture step. In some embodiments, the initial second co-culture medium contains IL-2, IL-7, IL-15, and IL-21, and an anti-PD-1 antibody. In some embodiments, the ratio between the tumor antigen-specific T cell population and the antigen-loaded APC population is about 1:1 to about 20:1 (e.g., about 4:1). In some embodiments, the method further includes a third co-culture step, which involves co-culturing a population of tumor antigen-specific T cells with a population of APCs (e.g., PBMCs, DCs, or cell line APCs) carrying one or more target tumor antigen peptides to obtain a second population of tumor antigen-specific T cells, wherein the one or more target tumor antigen peptides are derived from the plurality of tumor antigen peptides, wherein the second population of tumor antigen-specific T cells undergoes next-generation sequencing in the sequencing step. In some embodiments, the population of tumor antigen-specific T cells is co-cultured with the antigen-loaded APC population for about 5 to 9 days (e.g., about 7 days). In some embodiments, the population of tumor antigen-specific T cells and the antigen-loaded APC population are co-cultured in a third co-culture medium containing one or more cytokines (such as IL-2, or a plurality of cytokines, such as IL-2, IL-7, IL-15) and an anti-CD3 antibody (e.g., OKT3). In some embodiments, the third co-culture step is repeated (e.g., once or twice). In some embodiments, the method further includes: identifying a target tumor epitope from the target tumor antigen peptide, wherein the target tumor epitope elicits a specific response in the enriched activated T cell population; and contacting the APC population with the target tumor epitope to obtain the population of APCs carrying the target tumor antigen peptide. In some embodiments, after receiving the MASCT, the individual has a PR, CR, or stable disease (SD).

[0115] TCR can be selected from any individual who has clinically benefited from MASCT, including any or a combination of the MASCT methods described in the "MASCT" section. In some embodiments, after receiving the MASCT, the individual has a partial response (PR) for at least about 6 months (e.g., at least about 1 year, 2 years, or more). In some embodiments, after receiving the MASCT, the individual has a complete response (CR) for at least about 6 months (e.g., at least about 1 year, 2 years, or more). In some embodiments, after receiving the MASCT, the individual has stable disease (SD).

[0116] In some embodiments, a method is provided for obtaining a plurality of TCRs that specifically recognize a target tumor antigen peptide, comprising: a) optionally administering an effective amount of DCs loaded with a plurality of tumor antigen peptides, the plurality of tumor antigen peptides including the target tumor antigen peptide; b) co-culturing a population of DCs loaded with the plurality of tumor antigen peptides with a first population of T cells to obtain an activated T cell population; c) administering an effective amount of the activated T cells to the individual; d) obtaining a second population of T cells from the individual after achieving PR, CR, or SD following administration of the activated T cells; e) a first co-culture step comprising co-culturing a first population of DCs loaded with the target tumor antigen peptide with the second population of T cells to obtain a first co-culture; f) enrichment. The steps include: g) subjecting the co-culture to an enrichment process to obtain enriched activated T cells; g) a second co-culture step, comprising co-culturing the enriched activated T cells with a second DC population carrying the target tumor antigen peptide to obtain a tumor antigen-specific T cell population, wherein at least about 10% (e.g., at least about 20%, or at least about 50%) of the tumor antigen-specific T cells specifically respond to the target tumor antigen peptide; and h) a sequencing step, comprising subjecting the tumor antigen-specific T cells to next-generation sequencing (e.g., single-cell sequencing) to recognize multiple pairs of genes encoding TCRα and TCRβ, thereby providing the multiple T cell receptors based on the paired genes encoding TCRα and TCRβ. In some embodiments, the first co-culture step is performed for about 1 to 3 days prior to the enrichment step. In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is no greater than about 30:1 (e.g., about 20:1, 15:1, or 10:1). In some embodiments, the second T cell population in the first co-culture step is present in a PBMC. In some embodiments, the first antigen-loaded DC population and the second T cell population are co-cultured in a first co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15, and IL-21) and immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, such as SHR-1210). In some embodiments, the enrichment step includes: contacting the first co-culture with an APC (e.g., DC or PBMC) loaded with the multiple target tumor antigen peptides to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand that specifically recognizes cytokines (e.g., IFNγ) or cell surface molecules. In some embodiments, the ratio between the enriched activated T cell population and the second antigen-loaded DC population is about 1:1 to about 20:1 (e.g., about 1:1, 2:1, or 4:1). In some embodiments, the enriched activated T cell population is co-cultured with the second antigen-loaded DC population for approximately 12 to 25 days.In some embodiments, the second co-culture step includes: co-culturing the second antigen-loaded DC population with the enriched activated T cell population in an initial second co-culture medium to provide a second co-culture, the initial second co-culture medium containing an immune checkpoint inhibitor and optionally one or more cytokines (e.g., multiple cytokines); and adding an anti-CD3 antibody (e.g., OKT-3) and optionally one or more cytokines to the second co-culture to obtain a tumor antigen-specific T cell population. In some embodiments, the anti-CD3 antibody is added to the second co-culture no more than about 3 days (e.g., about 2 days) after the start of the second co-culture step. In some embodiments, the initial second co-culture medium contains IL-2, IL-7, IL-15, and IL-21, and an anti-PD-1 antibody. In some embodiments, the ratio between the tumor antigen-specific T cell population and the antigen-loaded APC population is about 1:1 to about 20:1 (e.g., about 4:1). In some embodiments, the method further includes a third co-culture step, which involves co-culturing a population of tumor antigen-specific T cells with a population of APCs (e.g., PBMCs, DCs, or cell line APCs) carrying one or more target tumor antigen peptides to obtain a second population of tumor antigen-specific T cells, wherein the one or more target tumor antigen peptides are derived from the plurality of tumor antigen peptides, wherein the second population of tumor antigen-specific T cells undergoes next-generation sequencing in the sequencing step. In some embodiments, the population of tumor antigen-specific T cells is co-cultured with the antigen-loaded APC population for about 5 to 9 days (e.g., about 7 days). In some embodiments, the population of tumor antigen-specific T cells and the antigen-loaded APC population are co-cultured in a third co-culture medium containing one or more cytokines (such as IL-2, or a plurality of cytokines, such as IL-2, IL-7, IL-15) and an anti-CD3 antibody (e.g., OKT3). In some embodiments, the third co-culture step is repeated (e.g., once or twice). In some embodiments, the method further includes: identifying a target tumor epitope from the target tumor antigen peptide, wherein the target tumor epitope elicits a specific response in the enriched activated T cell population; and contacting the APC population with the target tumor epitope to obtain the population of APCs carrying the target tumor antigen peptide.

[0117] In some embodiments, a method is provided for obtaining a plurality of TCRs that specifically recognize a target tumor antigen peptide, comprising: a) contacting a population of dendritic cells (DCs) with a plurality of tumor antigen peptides to obtain a population of DCs loaded with the plurality of tumor antigen peptides, the plurality of tumor antigen peptides including the target tumor antigen peptide; b) culturing the population of DCs loaded with the plurality of tumor antigen peptides in a DC maturation medium containing MPLA; c) optionally administering an effective amount of DCs loaded with the plurality of tumor antigen peptides to the individual; d) co-culturing the population of DCs loaded with the plurality of tumor antigen peptides with a first T cell population to obtain an activated T cell population; e) administering an effective amount of the activated T cells to the individual; f) after achieving PR, CR, or SD following the administration of the activated T cells, obtaining a second T cell population from the individual; g) the first co-culturing step, comprising: The process involves: h) a co-culture step, which includes co-culturing a first population of dendritic cells (DCs) carrying the target tumor antigen peptide with a second population of T cells to obtain a first co-culture; i) a second co-culture step, which includes co-culturing the enriched activated T cells with the second population of DCs carrying the target tumor antigen peptide to obtain a population of tumor antigen-specific T cells, wherein at least about 10% (e.g., at least about 20%, or at least about 50%) of the tumor antigen-specific T cells specifically respond to the target tumor antigen peptide; and j) a sequencing step, which includes subjecting the tumor antigen-specific T cells to next-generation sequencing (e.g., single-cell sequencing) to recognize multiple pairs of genes encoding TCRα and TCRβ, thereby providing the multiple T cell receptors based on the paired genes encoding TCRα and TCRβ. In some embodiments, the DC maturation medium contains INFγ, MPLA, and PGE2. In some embodiments, the first co-culture step is performed for about 1 to 3 days prior to the enrichment step. In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is no greater than about 30:1 (e.g., about 20:1, 15:1, or 10:1). In some embodiments, the second T cell population in the first co-culture step is present in PBMCs. In some embodiments, the first antigen-loaded DC population and the second T cell population are co-cultured in a first co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15, and IL-21) and immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, such as SHR-1210). In some embodiments, the enrichment step includes: contacting the first co-culture with APCs (e.g., DCs or PBMCs) loaded with the multiple target tumor antigen peptides to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand that specifically recognizes cytokines (e.g., IFNγ) or cell surface molecules.In some embodiments, the ratio between the enriched activated T cell population and the second antigen-loaded DC population is approximately 1:1 to approximately 20:1 (e.g., approximately 1:1, 2:1, or 4:1). In some embodiments, the enriched activated T cell population and the second antigen-loaded DC population are co-cultured for approximately 12 to 25 days. In some embodiments, the second co-culture step includes: co-culturing the second antigen-loaded DC population and the enriched activated T cell population in an initial second co-culture medium containing an immune checkpoint inhibitor and optionally one or more cytokines (e.g., multiple cytokines); and adding an anti-CD3 antibody (e.g., OKT-3) and optionally one or more cytokines to the second co-culture to obtain a tumor antigen-specific T cell population. In some embodiments, the anti-CD3 antibody is added to the second co-culture no more than approximately 3 days (e.g., approximately 2 days) after the start of the second co-culture step. In some embodiments, the initial second co-culture medium comprises IL-2, IL-7, IL-15, and IL-21, and an anti-PD-1 antibody. In some embodiments, the ratio between the tumor antigen-specific T cell population and the antigen-loaded APC population is approximately 1:1 to approximately 20:1 (e.g., approximately 4:1). In some embodiments, the method further comprises a third co-culture step, which involves co-culturing the population of tumor antigen-specific T cells with a population of APCs (e.g., PBMCs, DCs, or cell line APCs) carrying one or more target tumor antigen peptides to obtain a second tumor antigen-specific T cell population, wherein the one or more target tumor antigen peptides are derived from the plurality of tumor antigen peptides, wherein the second tumor antigen-specific T cell population undergoes next-generation sequencing in the sequencing step. In some embodiments, the tumor antigen-specific T cell population is co-cultured with the antigen-loaded APC population for approximately 5 to 9 days (e.g., approximately 7 days). In some embodiments, the tumor antigen-specific T cell population and the antigen-loaded APC population are co-cultured in a third co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15) and an anti-CD3 antibody (e.g., OKT3). In some embodiments, the third co-culture step is repeated (e.g., once or twice). In some embodiments, the method further includes: recognizing a target tumor epitope from the target tumor antigen peptide, wherein the target tumor epitope elicits a specific response in the enriched activated T cell population; and contacting the APC population with the target tumor epitope to obtain the population of APCs loaded with the target tumor antigen peptide.

[0118] In some embodiments, a method is provided for obtaining a plurality of TCRs that specifically recognize target tumor antigen peptides, comprising: a) optionally administering an effective amount of DCs loaded with the plurality of tumor antigen peptides to the individual; b) co-culturing the DC population loaded with the plurality of tumor antigen peptides with a first T cell population in an initial co-culture medium to provide a co-culture, the initial co-culture medium containing one or more cytokines (such as IL-2, or a plurality of cytokines, such as IL-2, IL-7, IL-15, and IL-21) and an immune checkpoint inhibitor; and adding an anti-CD3 antibody to the co-culture approximately 3 to 7 days (e.g., approximately 5 days) after the start of the co-culture to obtain the activated T cell population; c) administering an effective amount of the activated T cells to the individual; d) obtaining a second T cell population from the individual after achieving PR, CR, or SD following the administration of the activated T cells; e) the second T cell population... The co-culture step includes co-culturing a first population of dendritic cells (DCs) carrying the target tumor antigen peptide with a second population of T cells to obtain a first co-culture; f) an enrichment step includes subjecting the co-culture to an enrichment process to obtain enriched activated T cells; g) a second co-culture step includes co-culturing the enriched activated T cells with a second population of DCs carrying the target tumor antigen peptide to obtain a tumor antigen-specific T cell population, wherein at least about 10% (e.g., at least about 20%, or at least about 50%) of the tumor antigen-specific T cells specifically respond to the target tumor antigen peptide; and h) a sequencing step includes subjecting the tumor antigen-specific T cells to next-generation sequencing (e.g., single-cell sequencing) to recognize multiple pairs of genes encoding TCRα and TCRβ, thereby providing the multiple T cell receptors based on the paired genes encoding TCRα and TCRβ. In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is no greater than about 30:1 (e.g., about 20:1, 15:1, or 10:1). In some embodiments, the second T cell population in the first co-culture step is present in PBMCs. In some embodiments, the first antigen-loaded DC population and the second T cell population are co-cultured in a first co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15, and IL-21) and immune checkpoint inhibitors (e.g., anti-PD-1 antibodies, such as SHR-1210). In some embodiments, the enrichment step includes: contacting the first co-culture with APCs (e.g., DCs or PBMCs) loaded with the multiple target tumor antigen peptides to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand that specifically recognizes cytokines (e.g., IFNγ) or cell surface molecules.In some embodiments, the ratio between the enriched activated T cell population and the second antigen-loaded DC population is approximately 1:1 to approximately 20:1 (e.g., approximately 1:1, 2:1, or 4:1). In some embodiments, the enriched activated T cell population and the second antigen-loaded DC population are co-cultured for approximately 12 to 25 days. In some embodiments, the second co-culture step includes: co-culturing the second antigen-loaded DC population and the enriched activated T cell population in an initial second co-culture medium containing an immune checkpoint inhibitor and optionally one or more cytokines (e.g., multiple cytokines); and adding an anti-CD3 antibody (e.g., OKT-3) and optionally one or more cytokines to the second co-culture to obtain a tumor antigen-specific T cell population. In some embodiments, the anti-CD3 antibody is added to the second co-culture no more than approximately 3 days (e.g., approximately 2 days) after the start of the second co-culture step. In some embodiments, the initial second co-culture medium comprises IL-2, IL-7, IL-15, and IL-21, and an anti-PD-1 antibody. In some embodiments, the ratio between the tumor antigen-specific T cell population and the antigen-loaded APC population is approximately 1:1 to approximately 20:1 (e.g., approximately 4:1). In some embodiments, the method further comprises a third co-culture step, which involves co-culturing the population of tumor antigen-specific T cells with a population of APCs (e.g., PBMCs, DCs, or cell line APCs) carrying one or more target tumor antigen peptides to obtain a second tumor antigen-specific T cell population, wherein the one or more target tumor antigen peptides are derived from the plurality of tumor antigen peptides, wherein the second tumor antigen-specific T cell population undergoes next-generation sequencing in the sequencing step. In some embodiments, the tumor antigen-specific T cell population is co-cultured with the antigen-loaded APC population for approximately 5 to 9 days (e.g., approximately 7 days). In some embodiments, the tumor antigen-specific T cell population and the antigen-loaded APC population are co-cultured in a third co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15) and an anti-CD3 antibody (e.g., OKT3). In some embodiments, the third co-culture step is repeated (e.g., once or twice). In some embodiments, the method further includes: recognizing a target tumor epitope from the target tumor antigen peptide, wherein the target tumor epitope elicits a specific response in the enriched activated T cell population; and contacting the APC population with the target tumor epitope to obtain the population of APCs loaded with the target tumor antigen peptide.

[0119] In some embodiments, a method is provided for obtaining a plurality of TCRs that specifically recognize a target tumor antigen peptide, comprising: a) contacting a population of dendritic cells (DCs) with a plurality of tumor antigen peptides to obtain a population of DCs loaded with the plurality of tumor antigen peptides, the plurality of tumor antigen peptides including the target tumor antigen peptide; b) culturing the population of DCs loaded with the plurality of tumor antigen peptides in a DC maturation medium containing MPLA; c) optionally administering an effective amount of DCs loaded with the plurality of tumor antigen peptides to the individual; d) co-culturing the population of DCs loaded with the plurality of tumor antigen peptides with a first population of T cells in an initial co-culture medium to provide a co-culture, the initial co-culture medium containing one or more cytokines (such as IL-2 or a plurality of cytokines) and an immune checkpoint inhibitor; and adding an anti-CD3 antibody to the co-culture approximately 3 to 7 days (e.g., approximately 5 days) after the start of the co-culture to obtain the activated T cell population; e) administering an effective amount of the activated T cells to the individual; f) in addition to the DCs loaded with MPLA; After the activated T cells achieve PR, CR, or SD, a second T cell population is obtained from the individual; g) a first co-culture step, which includes co-culturing a first DC population carrying the target tumor antigen peptide with the second T cell population to obtain a first co-culture; h) an enrichment step, which includes subjecting the co-culture to an enrichment procedure to obtain enriched activated T cells; i) a second co-culture step, which includes co-culturing the enriched activated T cells with a second DC population carrying the target tumor antigen peptide to obtain a tumor antigen-specific T cell population, wherein at least about 10% (e.g., at least about 20%, or at least about 50%) of the tumor antigen-specific T cells specifically respond to the target tumor antigen peptide; and j) a sequencing step, which includes subjecting the tumor antigen-specific T cells to next-generation sequencing (e.g., single-cell sequencing) to recognize multiple pairs of genes encoding TCRα and TCRβ, thereby providing the multiple T cell receptors based on the paired genes encoding TCRα and TCRβ. In some embodiments, the DC maturation medium contains INFγ, MPLA, and PGE2. In some embodiments, the first co-culture step is performed for about 1 to 3 days prior to the enrichment step. In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is no greater than about 30:1 (e.g., about 20:1, 15:1, or 10:1). In some embodiments, the second T cell population in the first co-culture step is present in PBMCs. In some embodiments, the first antigen-loaded DC population and the second T cell population are co-cultured in a first co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15, and IL-21) and an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody, such as SHR-1210).In some embodiments, the enrichment step includes: contacting the first co-culture with APCs (e.g., DCs or PBMCs) loaded with the plurality of target tumor antigen peptides to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand that specifically recognizes cytokines (e.g., IFNγ) or cell surface molecules. In some embodiments, the ratio between the enriched activated T cell population and the second antigen-loaded DC population is about 1:1 to about 20:1 (e.g., about 1:1, 2:1, or 4:1). In some embodiments, the enriched activated T cell population is co-cultured with the second antigen-loaded DC population for about 12 to 25 days. In some embodiments, the second co-culture step includes: co-culturing the second antigen-loaded DC population with the enriched activated T cell population in an initial second co-culture medium to provide a second co-culture, the initial second co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor; and adding an anti-CD3 antibody (e.g., OKT-3) to the second co-culture to obtain a tumor antigen-specific T cell population. In some embodiments, the anti-CD3 antibody is added to the second co-culture no more than about 3 days (e.g., about 2 days) after the start of the second co-culture step. In some embodiments, the initial second co-culture medium contains IL-2, IL-7, IL-15, and IL-21, and an anti-PD-1 antibody. In some embodiments, the ratio between the tumor antigen-specific T cell population and the antigen-loaded APC population is about 1:1 to about 20:1 (e.g., about 4:1). In some embodiments, the method further includes a third co-culture step, which involves co-culturing a population of tumor antigen-specific T cells with a population of APCs (e.g., PBMCs, DCs, or cell line APCs) carrying one or more target tumor antigen peptides to obtain a second population of tumor antigen-specific T cells, wherein the one or more target tumor antigen peptides are derived from the plurality of tumor antigen peptides, wherein the second population of tumor antigen-specific T cells undergoes next-generation sequencing in the sequencing step. In some embodiments, the population of tumor antigen-specific T cells is co-cultured with the antigen-loaded APC population for about 5 to 9 days (e.g., about 7 days). In some embodiments, the population of tumor antigen-specific T cells and the antigen-loaded APC population are co-cultured in a third co-culture medium containing one or more cytokines (such as IL-2, or a plurality of cytokines, such as IL-2, IL-7, IL-15) and an anti-CD3 antibody (e.g., OKT3). In some embodiments, the third co-culture step is repeated (e.g., once or twice).In some embodiments, the method further includes: identifying a target tumor epitope from the target tumor antigen peptide, wherein the target tumor epitope elicits a specific response in the enriched activated T cell population; and contacting the APC population with the target tumor epitope to obtain the population of APCs carrying the target tumor antigen peptide.

[0120] In some embodiments, TCR is selected from individuals who respond to the MASCT method, such as individuals with a reduced or low number of CTCs after the MASCT; individuals with a clinical assessment of stable disease (SD), complete response (CR), or partial response (PR). In some embodiments, the individual maintains PR, CR, or SD for at least about 6 months (e.g., at least about 1 year, 2 years, or more). In some embodiments, the individual has a strong specific immune response to the target tumor antigen peptide. Any method known in the art can be used to determine the specific immune response to the target tumor antigen peptide, such as by measuring the levels of cytotoxic factors (e.g., perforin or granzyme B) or cytokines released (e.g., IFNγ or TNFα) from T cells (or PBMCs) after stimulation with the target tumor antigen peptide. Antibody-based assays (e.g., ELISPOT) can be used to quantify the levels of cytotoxic factors or cytokines (e.g., IFNγ). In some embodiments, the level of cytokines (such as IFNγ) released from T cells (or PBMCs) in response to a target tumor antigen peptide is normalized to a reference (such as baseline cytokines release level, or nonspecific cytokines released from T cells (or PBMCs) in response to an unrelated peptide) to provide a fold change value for cytokines (such as IFNγ). In some embodiments, in the ELISPOT assay, a fold change value for cytokines (such as IFNγ) greater than any one of about 1.2, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, or more indicates a strong specific immune response to the target tumor antigen peptide. In some embodiments, the method for selecting TCRs further includes determining the specific immune response to each of a plurality of tumor antigen peptides in an individual (such as in a PBMC sample of that individual).

[0121] T cells can be isolated from a biological sample derived from an individual who has undergone a MASCT. In some embodiments, the biological sample is obtained from an individual after one cycle of MASCT. In some embodiments, the biological sample is obtained from an individual after at least 2, 3, 4, 5, or more cycles of MASCT. In some embodiments, the biological sample is obtained from the individual at least about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 2 months, or 3 months after receiving MASCT. In some embodiments, the biological sample is obtained from the individual no more than about 6 months, 3 months, 2 months, 1 month, or less after receiving MASCT. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a PBMC sample. In some embodiments, the biological sample is a T cell sample. In some embodiments, the biological sample is a tumor sample containing CTLs. T cells can be isolated from the biological sample using any method known in the art, such as flow cytometry or centrifugation. In some embodiments, multiple T cells obtained from biological samples are screened for their specific immune responses to multiple tumor antigen peptides, for example by staining with multimers (such as pentamers or dextramers) or by determining the levels of cytotoxic factors (such as perforin or granzyme B) or cytokines released (such as IFNγ or TNFα).

[0122] The target tumor antigen peptide specifically recognized by T cells can be any tumor antigen peptide or fragment thereof from a pool(s) of tumor antigen peptides used in MASCT. In some embodiments, the target tumor antigen peptide contains an MHC-I restricted epitope. In some embodiments, the target tumor antigen peptide contains an MHC-II restricted epitope. In some embodiments, the target tumor antigen peptide is a general cancer tumor antigen peptide. In some embodiments, the target tumor antigen peptide is a cancer type-specific tumor antigen peptide. In some embodiments, the target tumor antigen peptide is a neoantigen peptide. In some embodiments, the target tumor antigen peptide is derived from a tumor antigen selected from the group consisting of: hTERT, p53, survivin, NY-ESO-1, CEA, CCND1, RGS5, MMP7, VEGFR1, VEGFR2, MUC1, HER2, MAGE-A1, MAGE-A3, CDCA1, WT1, KRAS, PARP4, MLL3, MTHFR, HPV16-E6, HPV16-E7, HPV18-E6, HPV18-E7, HPV58-E6, HPV58-E7, HBcAg, HBV polymerase, GPC3, SSX, and AFP. In some embodiments, the target tumor antigen peptide comprises an epitope derived from CEA, RGS5, or HPV18-E7.

[0123] Tumor antigen-specific T cells can be sequenced using any known next-generation sequencing method to provide multiple pairs of genes encoding TCRα and TCRβ. In some embodiments, next-generation sequencing is single-cell next-generation sequencing. In some embodiments, next-generation sequencing is batch next-generation sequencing. In some embodiments, the sequencing step includes next-generation sequencing of an immune repertoire of TCRs from tumor antigen-specific T cells. In some embodiments, the sequencing step includes: amplifying genes encoding TCRα and TCRβ from tumor antigen-specific T cells (e.g., single cells or cell populations) to provide a sample of amplified nucleic acids; and subjecting the amplified nucleic acids to next-generation sequencing. In some embodiments, genes encoding TCRα and TCRβ are amplified using a PCR method with primers specifically bound to known TCR variable domains. In some embodiments, genes encoding TCRα and TCRβ are amplified using amplicon rescued multiplex PCR (or arm-PCR). See, for example, U.S. Patent No. 7,999,092. In some embodiments, tumor antigen-specific T cells are subjected to bulk next-generation sequencing data and single-cell next-generation sequencing to identify multiple pairs of genes encoding TCRα and TCRβ. In some embodiments, the sequencing step includes bulk sequencing of a first portion of the tumor antigen-specific T cells to provide multiple genes encoding TCRα and TCRβ; and single-cell sequencing of a second portion of the tumor antigen-specific T cells, which provides homology pairing information of the multiple genes encoding TCRα and TCRβ, thereby providing multiple TCRs based on paired genes encoding TCRα and TCRβ.

[0124] Commercially available kits and services are available for single-cell next-generation sequencing of TCRs, including but not limited to IPAR® (iReportoire), IMMUNOSEQ™ (Adaptive Biotech), SMARTER® Human TCRa / b Profiling Kit (Clontech), and ION AMPLISEQ® Immune Repertoire and Assay Plus (Thermofisher). For example, the IPAR® method includes two PCR amplification steps followed by next-generation sequencing. In short, tumor antigen-specific T cell samples are seeded as single cells in one or more 96-well discs. In the first PCR step, RT-PCR is performed in each well of the 96-well disc using nested multiplex primers that cover both the α and β loci of the TCR and include communal forward and reverse binding sites at the 5' end of the inner primers. Included on the C-region gene primer is an in-line barcode, which serves as a disk identifier, allowing multiple 96-well disks to be multiplexed on a sequencing flow cell. After RT-PCR, the products are rescued. A second PCR is performed using double-indexed primers, which complete the adapters introduced during the first PCR and provide disk location information. In this step, each well (and therefore each single cell) is represented by a unique barcode. In some cases, batch next-generation sequencing of individual strands is also performed on RNA from other cells (those not inoculated with single cells). The sequencing data are analyzed using IPAR® Analyzer software, which also facilitates easy comparison between single-cell and batch sequencing data. As determined by IPAR® Analyzer, homology pairing information for genes encoding TCRα and TCRβδ is obtained based on the data from each well. Additionally, see, for example, the iPair Analyzer User's Guide, iRepertoire Inc. (docs.wixstatic.com / ugd / c9f231_3c322d131c084e908eea039c42304ff7.pdf), the contents of which are incorporated herein by reference.

[0125] In some embodiments, prior to the next-generation sequencing, these tumor antigen-specific T cells are stimulated by an APC carrying the target tumor antigen peptide (e.g., PBMCs, DCs, or cell line APCs) (i.e., stimulated tumor antigen-specific T cells). In some embodiments, the tumor antigen-specific T cells stimulated by the APC carrying the target tumor antigen peptide are sorted to isolate IFNγ+ T cells for next-generation sequencing. In some embodiments, a control T cell population is subjected to next-generation sequencing to provide baseline TCRα and TCRβ progenitor profiles. In some embodiments, the control T cell population is derived from unstimulated PBMCs of an individual. In some embodiments, the control T cell population is composed of tumor antigen-specific T cells stimulated by one or more unrelated peptides. In some embodiments, TCRα and TCRβ genes that are identified only in the sequencing data of stimulated tumor antigen-specific T cells (but not in the sequencing data of the control T cell population) are selected to provide tumor-specific TCRs. In some embodiments, TCRα and TCRβ genes are selected in the sequencing data of stimulated tumor antigen-specific T cells (compared to the sequencing data of control T cell populations) at a frequency of at least about 2x, 5x, 10x, 20x, 50x, 100x, 1000x, or more to provide tumor-specific TCRs.

[0126] In some embodiments, multiple T cell populations (e.g., PBMCs) from an individual are obtained at different time points following MASCT treatment. TCRα and TCRβ genes consistently recognized by tumor antigen-specific T cells prepared using each T cell population are selected to provide tumor-specific TCR. In some embodiments, if TCRα and TCRβ genes are found in the sequencing data of two or more tumor antigen-specific T cell populations (e.g., 2, 3, 4, 5, 6, or more) prepared using different T cell samples from the individual, and the TCRα and TCRβ genes are paired by single-cell sequencing, then that pair of TCRα and TCRβ genes is selected to provide tumor-specific TCR.

[0127] Any of the methods described herein may include one or more of the following steps: (i) expressing each pair of genes encoding TCRα and TCRβ in host immune cells to provide engineered immune cells expressing TCRs; and assessing the response of the engineered immune cells to the target tumor antigen peptide; (ii) determining the antigenic epitopes recognized by each TCR; (iii) determining the HLA restriction (including MHC I or MHC II restriction, and optionally HLA haplotype restriction) of each TCR; (iv) determining the binding and cross-reactivity of each TCR; (v) affinity maturation of each TCR; (vi) enhancing the pairing of TCRα and TCRβ chains in each TCR; and (vii) enhancing the expression of each TCR. An illustrative overview of methods for obtaining multiple TCRs that specifically recognize a target tumor antigen peptide from an individual who has clinically benefited from MASCT is shown in Figure 1.

[0128] In some embodiments, isolated nucleic acids comprising each pair of TCRα and TCRβ genes are synthesized. In some embodiments, for each pair of TCRα and TCRβ genes, a first isolated nucleic acid encoding the TCRα gene and a second isolated nucleic acid encoding the TCRβ gene are synthesized. In some embodiments, the TCRα and TCRβ genes are codon-optimized (e.g., for expression in human cells). In some embodiments, each of the TCRα and TCRβ genes is operatively linked to a promoter. In some embodiments, the TCRα and TCRβ genes are operatively linked to the same promoter. In some embodiments, the TCRα and TCRβ genes are operatively linked to different promoters. In some embodiments, the isolated nucleic acids are incorporated into a vector, such as a viral vector, for example a lentiviral vector.

[0129] In some embodiments, a single nucleic acid is transduced (e.g., by a viral vector, or by physical or chemical methods) into host immune cells (such as T cells) to express the TCR encoded by the TCRα and TCRβ genes. In some embodiments, the host immune cell line is a CD3+ cell line. In some embodiments, the host immune cell line is a T cell line. In some embodiments, the host immune cell line is selected from the group consisting of PBMCs, cytotoxic T cells, helper T cells, natural killer T cells, and regulatory T cells. In some embodiments, the specific immune response of host immune cells expressing the TCR to a target tumor antigen peptide is tested for validation. In some embodiments, the host immune cell line is derived from a cell line. In some embodiments, the host immune cell line is a primary cell line. In some embodiments, the host immune cell line is derived from a cancer patient. In some embodiments, the host immune cell line is derived from a healthy donor.

[0130] Furthermore, a method for obtaining a TCR that specifically identifies a target tumor antigen peptide is provided, which uses any of the methods described herein for obtaining a plurality of TCRs that specifically identify a target tumor antigen peptide, wherein the TCR is selected based on the response of an engineered immune cell expressing the TCR to the target tumor antigen peptide.

[0131] HLA restriction of a TCR can be determined using any method known in the art. See, for example, Larche M. Methods Mol. Med. (2008), 138:57-72. In some embodiments, the TCR is MHC class I restricted. In some embodiments, the TCR is MHC class II restricted. In some embodiments, in some examples, the tumor-specific TCR has a predominant HLA haplotype restriction in certain ethnic groups, including but not limited to Africans, African Americans, Asians, Caucasians, Europeans, Hispanics, Pacific Islanders, etc. In some embodiments, the TCR has a predominant HLA haplotype restriction in Asians, such as an HLA-A*1101 or HLA-A*2402 restricted TCR. In some embodiments, the TCR has a predominant HLA haplotype restriction in Caucasians, such as an HLA-DPB1*0401 or HLA-A*0201 restricted TCR.

[0132] The TCRs obtained herein can be further engineered to improve their physical / chemical properties and / or function. For example, engineered tumor-specific TCRs may have enhanced performance, improved stability, enhanced binding affinity to MHC target tumor-specific antigen peptide complexes, and / or enhanced communication. In some embodiments, the TCRs are engineered based on the MHC subtype of the individual receiving immunotherapy using the TCR. In some embodiments, the engineering comprises mutating one or more sites in the variable region of the TCR. In some embodiments, the engineering comprises providing a fusion protein comprising one or more domains or fragments of the TCR. In some embodiments, the TCRα and TCRβ chains of the TCR may be engineered to have enhanced pairing. Any method known in the art can be used for epitope determination, affinity maturation, binding and cross-reactivity determination, performance enhancement, and pairing enhancement.

[0133] It is intended that any steps and parameters described herein for preparing antigen-loaded APCs, the first, second, and third co-culture steps, the enrichment step, the sequencing step, MASCT, etc., can be combined with each other as if each combination were described individually. [Method for preparing tumor antigen-specific T cells]

[0134] This application provides a method for preparing a T-cell population for TCR selection, comprising: co-culturing an enriched population of activated T cells or a stockpiled population of tumor antigen-specific T cells with a population of antigen-presenting cells (APCs) carrying one or more target tumor antigen peptides (hereinafter referred to as "antigen-loaded APCs"), wherein the enriched population of activated T cells or the stockpiled population of tumor antigen-specific T cells is obtained from an individual who has clinically benefited from MASCT, and at least about 10% (e.g., at least 20% or 50%) of the T-cell population for TCR selection specifically responds to the target tumor antigen peptide. In some embodiments, the tumor antigen-specific T cell line is obtained using any of the methods described in International Patent Application No. PCT / CN2018 / 082945, the entire contents of which are incorporated herein by reference.

[0135] In some embodiments, a method for preparing a T-cell population for TCR selection is provided, comprising: co-culturing an enriched activated T-cell population with a DC population carrying a target tumor antigen peptide, wherein the enriched activated T-cell population is prepared by subjecting a first co-culture to an enrichment process, and wherein the first co-culture comprises a T-cell population and a first DC population carrying the tumor antigen peptide, wherein the T-cell population in the first co-culture is obtained from an individual who has clinically benefited from MASCT, and wherein at least about 10% (e.g., at least 20% or 50%) of the T-cell population for TCR selection specifically responds to the target tumor antigen peptide. In some embodiments, the ratio between the enriched activated T-cell population and the antigen-loaded DC population is about 1:1 to about 20:1 (e.g., about 1:1 or about 2:1). In some embodiments, the enriched activated T-cell population and the antigen-loaded DC population are co-cultured for about 12 to 25 days. In some embodiments, the method includes: co-culturing the antigen-loaded DC population with the enriched activated T cell population in an initial co-culture medium to provide a co-culture, the initial co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody); and adding an anti-CD3 antibody (e.g., OKT-3) and optionally one or more cytokines to the co-culture to obtain a tumor antigen-specific T cell population. In some embodiments, the anti-CD3 antibody is added to the co-culture no more than about 3 days (e.g., about 2 days) after the start of the co-culture step.

[0136] In some embodiments, a method for preparing a T cell population for TCR selection is provided, comprising: a) subjecting a first co-culture to an enrichment process to obtain an enriched activated T cell population, wherein the first co-culture comprises a first DC population carrying a target tumor antigen peptide and a T cell population; and b) co-culturing the enriched activated T cell population with a second DC population carrying the target tumor antigen peptide to obtain a T cell population for TCR selection, wherein the T cell population in step a) is obtained from an individual who has clinically benefited from MASCT, and wherein at least about 10% (e.g., at least 20% or 50%) of the T cell population for TCR selection specifically responds to the target tumor antigen peptide. In some embodiments, the enrichment process comprises: contacting the first co-culture with an APC (e.g., PBMC) carrying the target tumor antigen peptide to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand that specifically recognizes cytokines (such as IFNγ) or cell surface molecules. In some embodiments, the ratio between the enriched activated T cell population and the second antigen-loaded DC population is approximately 1:1 to approximately 20:1 (e.g., approximately 1:1 or approximately 2:1). In some embodiments, the enriched activated T cell population and the second antigen-loaded DC population are co-cultured for approximately 12 to 25 days. In some embodiments, the method includes: co-culturing the second antigen-loaded DC population and the T cell population in an initial second co-culture medium to provide a second co-culture, the initial second co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody); and adding an anti-CD3 antibody (e.g., OKT3) to the second co-culture to obtain a tumor antigen-specific T cell population. In some embodiments, the anti-CD3 antibody is added to the second co-culture no more than approximately 3 days (e.g., approximately 2 days) after the start of the second co-culture step.

[0137] In some embodiments, a method is provided for preparing a T cell population for TCR selection, comprising: a) a first co-culture step comprising co-culturing a first DC population carrying a target tumor antigen peptide with a T cell population to obtain a first co-culture containing activated T cells; b) an enrichment step comprising subjecting the first co-culture to an enrichment procedure to obtain an enriched activated T cell population; and c) a second co-culture step comprising co-culturing the enriched activated T cell population with a second DC population carrying the target tumor antigen peptide to obtain a T cell population for TCR selection, wherein the T cell population in the first co-culture step is obtained from an individual who has clinically benefited from MASCT, and at least about 10% (e.g., at least 20% or 50%) of the T cell population for TCR selection specifically responds to the target tumor antigen peptide. In some embodiments, the first co-culture step is performed no more than about 7 days prior to the enrichment step (e.g., about 1 to 3 days, for example, about 3 days). In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is no greater than about 30:1 (e.g., about 20:1, 15:1, or 10:1). In some embodiments, the first antigen-loaded DC population and the T cell population are co-cultured in a first co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15, and IL-21) and an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody). In some embodiments, the ratio between the enriched activated T cell population and the second antigen-loaded DC population is about 1:1 to about 20:1 (e.g., about 1:1 or about 2:1). In some embodiments, the enriched activated T cell population and the second antigen-loaded DC population are co-cultured for about 12 to 25 days. In some embodiments, the method includes: co-culturing the second antigen-loaded DC population with the T cell population in an initial second co-culture medium to provide a second co-culture, the initial second co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody); and adding an anti-CD3 antibody (e.g., OKT3) to the second co-culture to obtain a tumor antigen-specific T cell population. In some embodiments, the anti-CD3 antibody is added to the second co-culture no more than about 3 days (e.g., about 2 days) after the start of the second co-culture step.

[0138] In some embodiments, a method is provided for preparing a T cell population for TCR selection, comprising: a) a first co-culture step comprising co-culturing a first DC population carrying a target tumor antigen peptide with a T cell population to obtain a first co-culture containing activated T cells; b) an enrichment step comprising: contacting the first co-culture with an APC (e.g., PBMC) carrying the target tumor antigen peptide to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand that specifically recognizes cytokines (such as IFNγ) or cell surface molecules; and c) a second co-culture step comprising co-culturing the enriched activated T cell population with a second DC population carrying the target tumor antigen peptide to obtain a T cell population for TCR selection, wherein the T cell population in the first co-culture step is obtained from an individual who has clinically benefited from MASCT, and at least about 10% (e.g., at least 20% or 50%) of the T cell population for TCR selection specifically responds to the target tumor antigen peptide. In some embodiments, the first co-culture step is performed no more than about 7 days prior to the enrichment step (e.g., about 1 to 3 days, for example, about 3 days). In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is no greater than about 30:1 (e.g., about 20:1, 15:1, or 10:1). In some embodiments, the first antigen-loaded DC population and the T cell population are co-cultured in a first co-medium containing one or more cytokines (e.g., IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15, and IL-21) and an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody). In some embodiments, the ratio between the enriched activated T cell population and the second antigen-loaded DC population is about 1:1 to about 20:1 (e.g., about 1:1 or about 2:1). In some embodiments, the enriched activated T cell population and the second antigen-loaded DC population are co-cultured for about 12 to 25 days. In some embodiments, the method includes: co-culturing the second antigen-loaded DC population with the T cell population in an initial second co-culture medium to provide a second co-culture, the initial second co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody); and adding an anti-CD3 antibody (e.g., OKT3) to the second co-culture to obtain a tumor antigen-specific T cell population. In some embodiments, the anti-CD3 antibody is added to the second co-culture no more than about 3 days (e.g., about 2 days) after the start of the second co-culture step. In some embodiments, the T cell population in the first co-culture step is present in a PBMC population.

[0139] In some embodiments, a method for preparing a T cell population for TCR selection is provided, comprising: a) a first co-culture step, comprising co-culturing a first DC population carrying a target tumor antigen peptide with a T cell population to obtain a first co-culture containing activated T cells; b) an enrichment step, comprising subjecting the first co-culture to an enrichment process to obtain an enriched activated T cell population; and c) a second co-culture step, comprising: co-culturing the enriched activated T cell population with a second DC population carrying the target tumor antigen peptide in an initial second co-culture medium to obtain a second co-culture medium. The first co-culture medium contains one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody); and an anti-CD3 antibody (e.g., OKT3) is added to the second co-culture to obtain a T cell population for TCR selection, wherein the T cell population in the first co-culture step is obtained from individuals who have clinically benefited from MASCT, and at least about 10% (e.g., at least 20% or 50%) of the T cell population for TCR selection specifically responds to the target tumor antigen peptide. In some embodiments, the anti-CD3 antibody is added to the second co-culture no more than about 3 days (e.g., about 2 days) after the start of the second co-culture step. In some embodiments, the first co-culture step is performed no more than about 7 days (e.g., about 1 to 3 days, e.g., about 3 days) prior to the enrichment step. In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is no greater than about 30:1 (e.g., about 20:1, 15:1, or 10:1). In some embodiments, the first antigen-loaded DC population and the T cell population are co-cultured in a first co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15, and IL-21) and an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody). In some embodiments, the enrichment procedure includes: contacting the first co-culture with an APC (e.g., PBMC) carrying the target tumor antigen peptide to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand that specifically recognizes cytokines (such as IFNγ) or cell surface molecules. In some embodiments, the ratio between the enriched activated T cell population and the second antigen-loaded DC population is about 1:1 to about 20:1 (e.g., about 1:1 or about 2:1). In some embodiments, the enriched activated T cell population and the second antigen-loaded DC population are co-cultured for about 12 to 25 days. In some embodiments, the T cell population in the first co-culture step is present in the PBMC population.

[0140] In some embodiments, a method for preparing a T cell population for TCR selection is provided, comprising: a) contacting a first dendritic cell population with a target tumor antigen peptide to obtain a first dendritic cell population loaded with the target tumor antigen peptide; b) a first co-culture step comprising co-culturing the first dendritic cell population loaded with the target tumor antigen peptide with a T cell population to obtain a first co-culture containing activated T cells; c) an enrichment step comprising subjecting the first co-culture to an enrichment process to obtain an enriched activated T cell population; d) contacting a second dendritic cell population with the target tumor antigen peptide to obtain a second dendritic cell population loaded with the target tumor antigen peptide; and e) a second co-culture step comprising: in an initial second co-culture medium... The enriched activated T cell population is co-cultured with a second DC population carrying the target tumor antigen peptide to obtain a second co-culture. The initial second co-culture medium contains one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody). An anti-CD3 antibody (e.g., OKT3) is added to the second co-culture to obtain a T cell population for TCR selection. The T cell population in the first co-culture step is obtained from individuals who have clinically benefited from MASCT, and at least about 10% (e.g., at least 20% or 50%) of the T cell population for TCR selection specifically responds to the target tumor antigen peptide. In some embodiments, the anti-CD3 antibody is added to the second co-culture no more than about 3 days (e.g., about 2 days) after the start of the second co-culture step. In some embodiments, the first co-culture step is performed no more than about 7 days (e.g., about 1 to 3 days, for example, about 3 days) prior to the enrichment step. In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is no greater than about 30:1 (e.g., about 20:1, 15:1, or 10:1). In some embodiments, the first antigen-loaded DC population and the T cell population are co-cultured in a first co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15, and IL-21) and an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody). In some embodiments, the enrichment procedure includes: contacting the first co-culture with an APC (e.g., PBMC) carrying the target tumor antigen peptide to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand that specifically recognizes cytokines (such as IFNγ) or cell surface molecules. In some embodiments, the ratio between the enriched activated T cell population and the second antigen-loaded DC population is about 1:1 to about 20:1 (e.g., about 1:1 or about 2:1). In some embodiments, the enriched activated T cell population is co-cultured with the second antigen-loaded DC population for approximately 12 to 25 days.In some embodiments, the T cell population in the first co-culture step is present in the PBMC population.

[0141] In some embodiments, a method for preparing a T cell population for TCR selection is provided, comprising: a) contacting a first DC population with a target tumor antigen peptide to obtain a first DC population loaded with the target tumor antigen peptide; b) culturing the first DC population loaded with the target tumor antigen peptide in a DC maturation medium containing a toll-like receptor. c) A receptor (TLR) activator; d) A first co-culture step comprising: co-culturing the first DC population carrying the target tumor antigen peptide with the T cell population in a first co-culture medium to obtain a first co-culture containing activated T cells, the first co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15, and IL-21) and immune checkpoint inhibitors (e.g., anti-PD-1 antibody); e) An enrichment step comprising: contacting the first co-culture with PBMCs carrying the target tumor antigen peptide to obtain a stimulated co-culture; and using a ligand to isolate the enriched activated T cell population from the stimulated co-culture, the ligand specifically recognizing cytokines (such as IFNγ) or cell surface molecules; f) Contacting a second DC population with the target tumor antigen peptide to obtain a second antigen-loaded DC population; The second antigen-loaded DC population is cultured in a DC maturation medium containing a TLR receptor (TLR) agonist; and g) a second co-culture step comprising: co-culturing the enriched activated T cell population with the second DC population carrying the target tumor antigen peptide in a second initial co-culture medium to obtain a second co-culture, the second initial co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody); and adding an anti-CD3 antibody (e.g., OKT3) to the second co-culture to provide a T cell population for TCR selection, wherein the T cell population in the first co-culture step is obtained from individuals who have clinically benefited from MASCT, and at least about 10% (e.g., at least 20% or 50%) of the T cell population for TCR selection specifically responds to the target tumor antigen peptide. In some embodiments, the anti-CD3 antibody is added to the second co-culture no more than about 3 days (e.g., about 2 days) after the start of the second co-culture step. In some embodiments, the first co-culture step is performed no more than about 7 days prior to the enrichment step (e.g., about 1 to 3 days, for example, about 3 days). In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is no greater than about 30:1 (e.g., about 20:1, 15:1, or 10:1).In some embodiments, the first antigen-loaded DC population and the T cell population are co-cultured in a first co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15, and IL-21) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody). In some embodiments, the ratio between the enriched activated T cell population and the second antigen-loaded DC population is about 1:1 to about 20:1 (e.g., about 1:1 or about 2:1). In some embodiments, the enriched activated T cell population and the second antigen-loaded DC population are co-cultured for about 12 to 25 days. In some embodiments, the T cell population in the first co-culture step is present in a PBMC population. In some embodiments, the DC maturation medium contains INFγ, MPLA, and PGE2. In some embodiments, the first DC population and / or the second DC population are obtained by inducing differentiation of mononuclear spheroids from PBMCs.

[0142] In some embodiments, a method for preparing a T-cell population for TCR selection is provided, comprising: co-culturing a tumor antigen-specific T-cell population with an APC population (e.g., PBMCs, DCs, or cell line APCs) loaded with one or more target tumor antigen peptides. In some embodiments, the tumor antigen-specific T-cell population is obtained using any of the methods described above for preparing T-cells for TCR selection. In some embodiments, the tumor antigen-specific T-cell line is obtained from PBMCs of individuals who have clinically benefited from MASCT. In some embodiments, the ratio between the tumor antigen-specific T-cell population and the antigen-loaded APC population is from about 1:1 to about 20:1 (e.g., about 1:1, 1:2, or 1:4). In some embodiments, the tumor antigen-specific T-cell population and the antigen-loaded APC population are co-cultured for about 5 to 9 days (e.g., about 7 days). In some embodiments, the tumor antigen-specific T cell population and the antigen-loaded APC population are co-cultured in a third co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15) and an anti-CD3 antibody (e.g., OKT3). In some embodiments, the co-culture is repeated, for example, once or twice. In some embodiments, the tumor antigen-specific T cell population is obtained from a cryopreservation stock of tumor antigen-specific T cells.

[0143] In some embodiments, a method for preparing a T cell population for TCR selection is provided, comprising: a) a first co-culture step, comprising co-culturing a first DC population carrying a target tumor antigen peptide with a T cell population to obtain a first co-culture containing activated T cells; b) an enrichment step, comprising subjecting the first co-culture to an enrichment process to obtain an enriched activated T cell population; c) a second co-culture step, comprising co-culturing the enriched activated T cell population with a second DC population carrying the target tumor antigen peptide to obtain a first tumor-antigen-specific T cell population; d) The third co-culture step includes co-culturing a subset of tumor antigen-specific T cells from the first tumor antigen-specific T cell population with a third APC (e.g., DC, PBMC, or cell line APC, such as LCL) carrying the target tumor antigen peptide, thereby providing a T cell population for TCR selection, wherein the T cell population in the first co-culture step is obtained from individuals who have clinically benefited from MASCT, and at least about 10% (e.g., at least 20% or 50%) of the T cell population for TCR selection specifically responds to the target tumor antigen peptide. In some embodiments, the ratio between the tumor antigen-specific T cell subset and the third antigen-loaded DC population is about 1:1 to about 20:1 (e.g., about 1:1, 1:2, or 1:4). In some embodiments, the tumor antigen-specific T cell subset and the third antigen-loaded DC population are co-cultured for about 5 to 9 days (e.g., about 7 days). In some embodiments, the tumor antigen-specific T cell subset and the third antigen-loaded DC population are co-cultured in a third co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15) and an anti-CD3 antibody (e.g., OKT3). In some embodiments, the third co-culture step is repeated, for example, once, twice, or three times. In some embodiments, the tumor antigen-specific T cell subset is obtained from a cryopreservation stock of the first tumor antigen-specific T cell population. In some embodiments, the first co-culture step is performed no more than about 7 days prior to the enrichment step (e.g., about 1 to 3 days, for example, about 3 days). In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is no greater than about 30:1 (e.g., about 20:1, 15:1, or 10:1). In some embodiments, the first antigen-loaded DC population and the T cell population are co-cultured in a first co-culture medium containing one or more cytokines (such as IL-2, or multiple cytokines, such as IL-2, IL-7, IL-15, and IL-21) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody). In some embodiments, the ratio between the enriched activated T cell population and the second antigen-loaded DC population is about 1:1 to about 20:1 (e.g., about 1:1 or about 2:1).In some embodiments, the enriched activated T cell population is co-cultured with the second antigen-loaded DC population for approximately 12 to 25 days. In some embodiments, the method includes: co-culturing the second antigen-loaded DC population with the T cell population in an initial second co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody); and adding an anti-CD3 antibody (e.g., OKT3) to the second co-culture to obtain a tumor antigen-specific T cell population. In some embodiments, the anti-CD3 antibody is added to the second co-culture no more than approximately 3 days (e.g., approximately 2 days) after the start of the second co-culture step. In some embodiments, the T cell population from the first co-culture step is present in a PBMC population.

[0144] Exemplary methods for preparing T cells for TCR selection or for preparing tumor antigen-specific T cells are illustrated in Figures 4, 7, and 15A to 15B, and described in Examples 2 to 3.

[0145] In some embodiments, a method for preparing a T cell population for TCR selection is provided, comprising: (a) contacting a population of dendritic cells (DCs) derived from a PBMC population from an individual with a target tumor antigen peptide to obtain antigen-loaded DCs; (b) a first co-culture step comprising: co-culturing the T cell population (e.g., present in PBMCs) with a first antigen-loaded DC population in an initial first co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody); and (c) an enrichment step comprising: contacting the first co-culture with PBMCs carrying the target tumor antigen peptide to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand, the ligand specifically recognizing the target tumor antigen peptide. (d) A second co-culture step comprising: co-culturing the enriched activated T cell population with a second antigen-loaded DC population in an initial second co-culture medium to obtain a second co-culture, the initial second co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody); and adding an anti-CD3 antibody (e.g., OKT3) to the second co-culture from about 1 to about 3 days (e.g., about 2 days) after the start of the second co-culture to provide a T cell population for TCR selection, wherein the T cell population in the first co-culture step is obtained from an individual who has clinically benefited from MASCT, and at least about 10% (e.g., at least 20% or 50%) of the T cell population for TCR selection specifically responds to the target tumor antigen peptide. In some embodiments, the antigen-loaded DCs are cultured in a DC maturation medium containing a TLR receptor (TLR) agonist. In some embodiments, the DC maturation medium contains INFγ, MPLA, and PGE2. In some embodiments, the antigen-loaded DCs are cultured in the DC maturation medium for about 8 to about 12 days. In some embodiments, the ratio of the T cell population to the first antigen-loaded DC population is about 20:1. In some embodiments, the T cell population and the antigen-loaded DC population are co-cultured for about 2 to 3 days. In some embodiments, the ratio of the enriched activated T cell population to the second antigen-loaded DC population is about 1:1. In some embodiments, the enriched activated T cell population and the second antigen-loaded DC population are co-cultured for about 15 to 20 days (e.g., about 16 days). Illustrative methods are shown in Figures 4 and 7.

[0146] In some embodiments, a method is provided for preparing a T-cell population for TCR selection, comprising: (a) co-culturing a tumor antigen-specific T-cell population with a first APC (e.g., PBMC, DC, or cell line APC) carrying a target tumor antigen peptide in a co-culture medium for about 5 to 9 days (e.g., about 7 days) to obtain a first tumor antigen-specific T-cell population, the co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an anti-CD3 antibody; and (b) co-culturing the first tumor antigen-specific T-cell population with a second APC carrying the target tumor antigen peptide for about 5 to 9 days (e.g., about 7 days) to provide a second tumor antigen-specific T-cell population for TCR selection, wherein the tumor antigen-specific T-cell population is obtained from an individual who has clinically benefited from MASCT, and wherein at least about 10% (e.g., at least 20% or 50%) of the T-cell population for TCR selection specifically responds to the target tumor antigen peptide. In some embodiments, the stimulation step is repeated once or twice. In some embodiments, the method further comprises co-culturing the second tumor antigen-specific T cell population with a third APC population carrying the tumor antigen-specific T cells for about 5 to 9 days (e.g., about 7 days) to provide a third tumor antigen-specific T cell population. In some embodiments, the APCs are LCL cells, with or without feeder cells. In some embodiments, the APCs are DCs. In some embodiments, the co-culture medium contains IL-2, IL-7, IL-15, and OKT3. In some embodiments, the ratio between antigen-loaded APCs and the first, second, or third tumor antigen-specific T cell populations is about 1:1 to about 1:10 (e.g., about 1:4). Illustrative methods are shown in Figures 15A to 15B.

[0147] The tumor antigen-specific T cells used in the sequencing step have a high percentage of T cells that specifically respond to the target tumor antigen peptide or its epitope. For example, at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher of the tumor antigen-specific T cells used in the sequencing step specifically respond to the target tumor antigen peptide or its epitope. In some embodiments, about 20% to 90%, 20% to 50%, 50% to 95%, 20% to 80%, 50% to 70%, 40% to 60%, or 40% to 80% of the tumor antigen-specific T cells used in the sequencing step specifically respond to the target tumor antigen peptide or its epitope. Because T cells exhibit a large number of TCRα and TCRβ progenitors, a high percentage of T cells that specifically respond to the tumor antigen peptide is important for obtaining information on successful homologous pairing of the TCRα and TCRβ genes.

[0148] In some embodiments, tumor antigen-specific T cells in any embodiment of a single cell population are capable of in vivo or in vitro initiating a specific immune response to one or more tumor antigen peptides. In some embodiments, tumor antigen-specific T cells are capable of increasing cytotoxic T cell activity against more than one tumor antigen peptide in a human individual. In some embodiments, tumor antigen-specific T cells are characterized by high expression or secretion levels of pro-inflammatory signaling molecules after stimulation with one or more tumor antigen peptides. In some embodiments, expression or secretion levels are determined by comparing the expression or secretion levels of molecules (such as pro-inflammatory signaling molecules) in tumor antigen-specific T cells after stimulation with one or more tumor antigen peptides with the expression or secretion levels after stimulation with unrelated peptides. In some embodiments, the control expression or secretion level of molecules is the expression or secretion level of molecules in a control T cell population measured under the same assay conditions. In some embodiments, the control T cell population is a T cell population induced by one or more unrelated peptides (such as peptides not corresponding to T cell receptor antigens, or random peptides). In some embodiments, the control expression or secretion level of molecules is the mean or median expression or secretion level of molecules in a plurality of control T cell populations. In some embodiments, the high expression or secretion level of the molecule in tumor antigen-specific T cells is at least about 1.5, 2, 2.5, 3, 4, 5, 10, 20, 50, 100, 1000, or more times the control expression or secretion level.

[0149] In some embodiments, upon stimulation with the target tumor antigen peptide, tumor antigen-specific T cells exhibit multiple pro-inflammatory molecules, such as IFNγ, TNFα, granzyme B, perforin, or any combination thereof. In some embodiments, upon stimulation with the target tumor antigen peptide, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a higher percentage of tumor antigen-specific T cells secrete IFNγ. In some embodiments, upon stimulation with the target tumor antigen peptide, at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or a higher percentage of tumor antigen-specific T cells secrete TNF-α.

[0150] In some embodiments, a tumor antigen-specific T cell line is prepared by comprising the following steps: (a) contacting a population of dendritic cells (DCs) derived from an individual's PBMC population with a target tumor antigen peptide to obtain a first antigen-loaded DC population; (b) a first co-culture step comprising: co-culturing the T cell population (e.g., present in PBMCs) with the first antigen-loaded DC population in an initial first co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody); and adding an anti-CD3 antibody to the first co-culture no more than about 7 days (e.g., about 5 days) after the start of the first co-culture to obtain a first co-culture. (c) An enrichment step comprising: contacting the first co-culture with PBMCs carrying the target tumor antigen peptide to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand specifically recognizing a cytokine (such as IFNγ); (d) A second co-culture step comprising: co-culturing the enriched activated T cell population with a second DC population carrying the target tumor antigen peptide in a co-culture medium to provide tumor antigen-specific T cells, the co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines), immune checkpoint inhibitors (e.g., anti-PD-1 antibody), and anti-CD3 antibody. In some embodiments, the ratio of the T cell population to the first antigen-loaded DC population is approximately 20:1. In some embodiments, the T cell population is co-cultured with the antigen-loaded DC population for approximately 13 to 14 days. In some embodiments, the ratio of the enriched activated T cell population to the second antigen-loaded DC population is approximately 2:1. In some embodiments, the enriched activated T cell population is co-cultured with the second antigen-loaded DC population for approximately 9 to 13 days.

[0151] In some embodiments, a tumor antigen-specific T cell line is prepared by comprising the following steps: (a) contacting a population of dendritic cells (DCs) derived from an individual's PBMC population with a target tumor antigen peptide to obtain antigen-loaded DCs; (b) a first co-culture step comprising: co-culturing the T cell population (e.g., present in PBMCs) with a first antigen-loaded DC population in an initial first co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody); and (c) an enrichment step comprising: reacting the first co-culture with the target antigen-loaded DCs. The process involves contacting PBMCs labeled with tumor antigen peptides to obtain a stimulated co-culture; and using a ligand to isolate the enriched activated T cell population from the stimulated co-culture to obtain an enriched activated T cell population, the ligand specifically recognizing cytokines (such as IFNγ); (d) a second co-culture step comprising: co-culturing the enriched activated T cell population with a second antigen-loaded DC population in a co-culture medium to provide tumor antigen-specific T cells, the co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines), immune checkpoint inhibitors (e.g., anti-PD-1 antibody), and anti-CD3 antibody. In some embodiments, the antigen-loaded DCs are cultured in a DC maturation medium containing a TLR receptor (TLR) agonist. In some embodiments, the DC maturation medium contains INFγ, MPLA, and PGE2. In some embodiments, the antigen-loaded DCs are cultured in the DC maturation medium for about 8 to about 12 days. In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population is about 15:1. In some embodiments, the T cell population is co-cultured with the antigen-loaded DC population for approximately 3 to 4 days. In some embodiments, the ratio of the enriched activated T cell population to the second antigen-loaded DC population is approximately 2:1. In some embodiments, the enriched activated T cell population is co-cultured with the second antigen-loaded DC population for approximately 13 to 23 days.

[0152] In some embodiments, a tumor antigen-specific T cell line is prepared by comprising the following steps: (a) contacting a population of dendritic cells (DCs) derived from an individual's PBMC population with a target tumor antigen peptide to obtain antigen-loaded DCs; (b) a first co-culture step comprising: co-culturing the T cell population (e.g., present in PBMCs) with a first antigen-loaded dendritic cell population in an initial first co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody); (c) an enrichment step comprising: contacting the first co-culture with PBMCs carrying the target tumor antigen peptide to obtain a stimulated co-culture; and using ligand autologous... The stimulated co-culture isolates enriched activated T cell populations to obtain enriched activated T cell populations that specifically recognize cytokines (such as IFNγ); (d) a second co-culture step comprising: co-culturing the enriched activated T cell populations with a second antigen-loaded DC population in an initial second co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody); and adding an anti-CD3 antibody (e.g., OKT3) to the second co-culture approximately 1 to 3 days (e.g., approximately 2 days) after the start of the second co-culture to provide tumor antigen-specific T cells. In some embodiments, the antigen-loaded DCs are cultured in a DC maturation medium containing a TLR receptor (TLR) agonist. In some embodiments, the DC maturation medium contains INFγ, MPLA, and PGE2. In some embodiments, the antigen-loaded DCs are cultured in the DC maturation medium for approximately 8 to 12 days. In some embodiments, the ratio of the T cell population to the first antigen-loaded DC population is approximately 20:1. In some embodiments, the T cell population and the antigen-loaded DC population are co-cultured for approximately 2 to 3 days. In some embodiments, the ratio of the enriched activated T cell population to the second antigen-loaded DC population is approximately 1:1. In some embodiments, the enriched activated T cell population and the second antigen-loaded DC population are co-cultured for approximately 15 to 20 days (e.g., approximately 16 days).

[0153] In some embodiments, a tumor antigen-specific T cell line is prepared by comprising the following steps: (a) contacting a population of dendritic cells (DCs) derived from an individual's PBMC population with a target tumor antigen peptide to obtain antigen-loaded DCs; (b) a first co-culture step comprising: co-culturing the T cell population (e.g., present in PBMCs) with the first antigen-loaded DC population in an initial first co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody); (c) an enrichment step comprising: contacting the first co-culture with PBMCs carrying the target tumor antigen peptide to obtain a stimulated co-culture; and using a ligand from the PBMCs to obtain a stimulated co-culture. (d) A second co-culture step comprising: (a) culturing the enriched activated T cell population in an initial second co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody); and (b) culturing the antigen-loaded DCs in an initial second co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., anti-PD-1 antibody) for approximately 1 to approximately 3 days (e.g., approximately 2 days) after the start of the second co-culture to provide tumor antigen-specific T cells. In some embodiments, the antigen-loaded DCs are cultured in a DC maturation medium containing a TLR receptor agonist. In some embodiments, the DC maturation medium contains INFγ, MPLA, and PGE2. In some embodiments, the antigen-loaded DCs are cultured in the DC maturation medium for approximately 8 to approximately 12 days. In some embodiments, the ratio of the T cell population to the first antigen-loaded DC population is approximately 20:1. In some embodiments, the T cell population and the antigen-loaded DC population are co-cultured for approximately 2 to 3 days. In some embodiments, the ratio of the enriched activated T cell population to the second antigen-loaded DC population is approximately 1:1. In some embodiments, the enriched activated T cell population and the second antigen-loaded DC population are co-cultured for approximately 15 to 20 days (e.g., approximately 16 days).

[0154] In some embodiments, the method uses PBMCs obtained from individuals who have previously received immunotherapy (e.g., MASCT) to prepare tumor antigen-specific T cells for the sequencing step.

[0155] In some embodiments, the method includes: a) contacting a first PBMC population from the individual with the target tumor antigen peptide to provide a PBMC population loaded with the target tumor antigen peptide; b) subjecting the PBMC population loaded with the target tumor antigen peptide to an enrichment process to provide an enriched population of activated T cells; c) optionally contacting an APC (e.g., PBMC or DC) population with the target tumor antigen peptide to provide an antigen-loaded APC population; d) a co-culture step comprising co-culturing the enriched population of activated T cells with the antigen-loaded APC population to obtain a tumor antigen-specific T cell population. In some embodiments, prior to the enrichment process, the PBMCs are contacted with the target tumor antigen peptide for no more than about 5, 4, 3, 2, or 1 day. In some embodiments, the enrichment process includes: contacting the first co-culture with PBMCs loaded with the target tumor antigen peptide to obtain a stimulated co-culture; and isolating the enriched population of activated T cells from the stimulated co-culture using a ligand that specifically recognizes cytokines (such as IFNγ) or cell surface molecules. In some embodiments, the co-culture step includes: co-culturing the enriched activated T cell population with the antigen-loaded APC population in a co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines), an immune checkpoint inhibitor, and an anti-CD3 antibody. In some embodiments, the co-culture step includes: co-culturing the enriched activated T cell population with the antigen-loaded APC population in an initial co-culture medium to provide a co-culture, the initial co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor; and adding the anti-CD3 antibody to the co-culture. In some embodiments, the anti-CD3 antibody is added to the co-culture approximately 1 to 3 days after the start of the co-culture. In some embodiments, the enriched activated T cell population is co-cultured with the antigen-loaded APC population for a total of approximately 12 to 25 days.

[0156] In some embodiments, PBMCs are obtained fresh. In some embodiments, PBMCs are obtained by thawing a cryopreservation solution of PBMCs. In some embodiments, PBMCs are autologous, i.e., obtained from an individual receiving treatment. In some embodiments, PBMCs are contacted with cytokines (such as IL-2, GM-CSF, or similar) to induce differentiation, maturation, or proliferation of certain cells (such as DCs, T cells, or combinations thereof) in the PBMCs simultaneously or after the contact step.

[0157] In some embodiments, tumor antigen-specific T cell lines are prepared by comprising the following steps: (a) contacting a population of PBMCs with a target tumor antigen peptide to obtain a stimulated population of PBMCs; (b) isolating an enriched population of activated T cells from the stimulated PBMCs using a ligand that specifically recognizes a cytokine (such as IFNγ) to obtain an enriched population of activated T cells; and (c) a co-culture step comprising: co-culturing the enriched population of activated T cells with a population of DCs carrying the target tumor antigen peptide in an initial co-culture medium to obtain a first co-culture, the initial co-culture medium containing one or more cytokines (such as IL-2 or multiple cytokines) and an immune checkpoint inhibitor (e.g., an anti-PD-1 antibody); and adding an anti-CD3 antibody (e.g., OKT3) to the first co-culture from about 1 to about 3 days (e.g., about 1 or 2 days) after the start of the first co-culture to provide the tumor antigen-specific T cells. In some embodiments, the PBMCs are derived from cryopreservation stock. In some embodiments, the PBMCs are obtained fresh from an individual. In some embodiments, antigen-loaded dendritic cells (DCs) are prepared by contacting a population of PBMCs with a target tumor antigen peptide. In some embodiments, the ratio of the enriched activated T cell population to the antigen-loaded DC population is approximately 1:1. In some embodiments, the enriched activated T cell population and the antigen-loaded DC population are co-cultured for approximately 7 to approximately 21 days. [Co-training]

[0158] The methods described herein and the MASCT method include one or more co-culture steps (such as 1, 2, 3, or more). In some embodiments, the method includes a first co-culture step, which includes co-culturing a population of T cells with a population of dendritic cells (DCs) loaded with a target tumor antigen peptide. In some embodiments, in the first co-culture step, the population of T cells is co-cultured with a first antigen-loaded DC population for no more than about 7 days, such as about 1, 2, 3, 4, 5, 6, or 7 days. In some embodiments, the population of T cells is co-cultured with a first antigen-loaded DC population for about 1 to 3 days, such as about 2 to 3 days.

[0159] In some embodiments, the first co-culture step includes co-culturing a first antigen-loaded DC population with a T cell population in a first co-culture medium containing one or more cytokines (such as multiple cytokines) and an immune checkpoint inhibitor. In some embodiments, the first co-culture medium contains an anti-CD3 antibody. In some embodiments, the first co-culture medium does not contain an anti-CD3 antibody. In some embodiments, the first co-culture step includes: co-culturing a first antigen-loaded DC population with a T cell population in a first initial co-culture medium to provide a first co-culture, the first initial co-culture medium containing one or more cytokines (such as multiple cytokines) and an immune checkpoint inhibitor; and adding an anti-CD3 antibody to the first co-culture.

[0160] In some embodiments, the method includes a second co-culture step, which includes co-culturing an enriched population of activated T cells with a population of dendritic cells (DCs) loaded with a target tumor antigen peptide. In some embodiments, in the second co-culture step, the enriched population of activated T cells is co-cultured with the second antigen-loaded DCs for a total of at least about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 days. In some embodiments, the enriched population of activated T cells is co-cultured with the second antigen-loaded DCs for about 12 days to about 25 days, such as about 12 to 15, 15 to 18, 18 to 21, 15 to 20, 20 to 25, 15, 18, 19, 20, 21, or 22 days.

[0161] In some embodiments, enriched activated T cell populations and second antigen-loaded DC populations are co-cultured in the presence of anti-CD3 antibody for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, or more days. In some embodiments, enriched activated T cell populations and second antigen-loaded DC populations are co-cultured in the presence of anti-CD3 antibody for at least 8 to 18, 10 to 20, 1 to 25, or 12 to 25 days. In some embodiments, enriched activated T cell populations and second antigen-loaded DC populations are initially co-cultured in the absence of anti-CD3 antibody for at least 1 to 5 days, such as at least 1, 2, or 3 days.

[0162] In some embodiments, the second co-culture step includes co-culturing a second antigen-loaded DC population with the enriched activated T cell population in a second co-culture medium containing one or more cytokines (e.g., multiple cytokines) and an immune checkpoint inhibitor. In some embodiments, the second co-culture medium contains an anti-CD3 antibody. In some embodiments, the second co-culture medium does not contain an anti-CD3 antibody. In some embodiments, the second co-culture step includes: co-culturing a second antigen-loaded DC population with the enriched activated T cell population in a second initial co-culture medium to provide a second co-culture, the second initial co-culture medium containing one or more cytokines (e.g., multiple cytokines) and an immune checkpoint inhibitor; and adding an anti-CD3 antibody to the second co-culture.

[0163] In some embodiments, a method for preparing tumor antigen-specific T cells includes: (1) a first co-culture step, which includes co-culturing a population of T cells with a first population of DCs carrying a plurality of tumor antigen peptides; and (2) a second co-culture step, which includes co-culturing an enriched population of activated T cells with a second population of DCs carrying one or more of the plurality of tumor antigen peptides.

[0164] In some embodiments, the method includes a third co-culture step, which involves co-culturing a population of tumor antigen-specific T cells with a population of APCs (e.g., PBMCs (such as fixed PBMCs), DCs, or cell line APCs (such as LCLs)) carrying the target tumor antigen peptide (or its epitope). In some embodiments, the third co-culture step is repeated one or more times (e.g., 1, 2, 3, 4, 5, 6, or more times) to obtain a further population of tumor antigen-specific T cells. In some embodiments, repeating the third co-culture step includes co-culturing a portion of the tumor antigen-specific T cells obtained from the third co-culture step with a second population of antigen-loaded APCs (e.g., PBMCs (such as fixed PBMCs), DCs, or cell line APCs (such as LCLs)). In some embodiments, repeating the third co-culture step includes adding fresh antigen-loaded APCs (e.g., PBMCs (such as fixed PBMCs), DCs, or cell line APCs (such as LCLs)) to the third co-culture at intervals of about 5 to 9 days (e.g., about 7 days).

[0165] In some embodiments, in the third co-culture step, a population of tumor antigen-specific T cells is co-cultured with a population of antigen-loaded APCs (e.g., PBMCs (such as fixed PBMCs), DCs, or cell line APCs (such as LCLs)) for at least about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 14 days. In some embodiments, a population of tumor antigen-specific T cells is co-cultured with a population of antigen-loaded APCs (e.g., PBMCs (such as fixed PBMCs), DCs, or cell line APCs (such as LCLs)) for about 5 days to about 15 days, such as about 5 to 9, 7 to 10, 10 to 12, 12 to 15, 7, 8, 9, 10, 11, 12, 13, or 15 days.

[0166] In some embodiments, the third co-culture step includes co-culturing an antigen-loaded APC population (e.g., PBMCs (such as immobilized PBMCs), DCs, or cell line APCs (such as LCLs)) with a tumor antigen-specific T cell population in a third co-culture medium containing one or more cytokines (e.g., multiple cytokines) and an immune checkpoint inhibitor. In some embodiments, the third co-culture medium does not contain an anti-CD3 antibody. In some embodiments, the third co-culture medium contains an anti-CD3 antibody. In some embodiments, the third co-culture step includes: co-culturing an antigen-loaded APC population (e.g., PBMCs (such as immobilized PBMCs), DCs, or cell line APCs (such as LCLs)) with a tumor antigen-specific T cell population in a third initial co-culture medium to provide a third co-culture, the third initial co-culture medium containing one or more cytokines (e.g., multiple cytokines) and an immune checkpoint inhibitor; and adding an anti-CD3 antibody to the third co-culture.

[0167] The co-culture medium or initial co-culture medium used for each culture step may be the same or different. Unless otherwise indicated, "co-culture medium" as discussed in the "Co-culture" section includes first, second, and third co-culture media; "initial co-culture medium" as discussed in this section includes first, second, and third initial co-culture media. In some embodiments, the co-culture medium (including the initial co-culture medium) contains one or more (e.g., 1, 2, 3, 4, 5, or more) cytokines. In some embodiments, the co-culture medium (including the initial co-culture medium) contains a plurality of cytokines (also referred to herein as a "cytokine mixture"). Exemplary cytokines include, but are not limited to, IL-2, IL-7, IL-15, IL-21, and the like. In some embodiments, the co-culture medium (including the initial co-culture medium) contains IL-2. In some embodiments, the co-culture medium (including the initial co-culture medium) contains IL-2, IL-7, IL-15, and IL-21. In some embodiments, IL-2 is present in the co-culture medium (including the initial co-culture medium) at a concentration of at least about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 5000, 6000, or higher IU / mL. In some embodiments, IL-2 is present in the co-culture medium (including the initial co-culture medium) at a concentration of no more than about 1000, 500, 200, 100, 50, 20, or lower IU / mL. In some embodiments, the first co-culture medium contains IL-2 at a concentration of no more than about 200 IU / mL (such as about 150, 100, or 50 IU / mL). In some embodiments, the second co-culture medium contains IL-2 at a concentration of at least about 2000 IU / mL (such as about 3000, 5000, or 6000 IU / mL). In some embodiments, IL-7 is present in the co-culture medium (including the initial co-culture medium) at a concentration of at least about 1, 2, 5, 10, 20, 50, or 100 ng / mL. In some embodiments, IL-15 is present in the co-culture medium (including the initial co-culture medium) at a concentration of at least about 1, 2, 5, 10, 20, 50, or 100 ng / mL. Cytokines can promote the activation, maturation, and / or proliferation of T cells to prepare them for later differentiation into memory T cells, and / or inhibit the percentage of TREG in the co-culture.

[0168] In some embodiments, the co-culture medium (including the initial co-culture medium) contains one or more immune checkpoint inhibitors (such as any one of 1, 2, 3, or more). Any known immune checkpoint inhibitor may be used. In some embodiments, immune checkpoint inhibitors are natural or engineered ligands of inhibitory immune checkpoint molecules, including, for example, ligands of CTLA-4 (e.g., B7.1, B7.2), ligands of TIM-3 (e.g., galactolectin-9), ligands of A2a receptors (e.g., adenosine, regadenoson), ligands of LAG-3 (e.g., MHC class I or MHC class II molecules), ligands of BTLA (e.g., HVEM, B7-H4), ligands of KIR (e.g., MHC class I or MHC class II molecules), ligands of PD-1 (e.g., PD-L1, PD-L2), ligands of IDO (e.g., NKTR-218, indoximod, NLG919), and ligands of CD47 (e.g., SIRPα receptor). Immune checkpoint inhibitors can belong to any suitable molecular form, including but not limited to small molecules, nucleic acids (such as DNA, RNAi, or aptamers), peptides, or proteins (such as antibodies).

[0169] In some embodiments, immune checkpoint inhibitors are antibodies (such as antagonist antibodies) that target inhibitory immune checkpoint proteins, selected from the group consisting of: anti-CTLA-4 (e.g., ipilimumab, tremelimumab, KAHR-102), anti-TIM-3 (e.g., F38-2E2, ENUM005), and anti-LAG-3 (e.g., BMS-986016, IMP701, IMP321, C9B). 7W), anti-KIR (e.g., lirilumab and IPH2101), anti-PD-1 (e.g., nivolumab, pidilizumab, pembrolizumab, BMS-936559, atezolizumab, pembrolizumab, MK-3475, AMP-224, AMP-514, STI-A1110, TSR-042), SHR1210), anti-PD-L1 (e.g., KY-1003 (EP20120194977), MCLA-145, RG7446, BMS-936559, MEDI-4736, MSB0010718C, AUR-012, STI-A1010, PCT / US2001 / 020964, MPDL3280A, AMP-224, pegylated dapirolizumab pegol (CDP-7657), MEDI-4920), anti-CD73 (e.g., AR-42 (OSU-HDAC42, HDAC-42, AR42, AR 42, OSU-HDAC 42, OSU-HDAC-42, NSC D736012, HDAC-42, HDAC) 42, HDAC42, NSCD736012, NSC-D736012), MEDI-9447), anti-B7-H3 (e.g., MGA271, DS-5573a, 8H9), anti-CD47 (e.g., CC-90002, TTI-621, VLST-007), anti-BTLA, anti-VISTA, anti-A2aR, anti-B7-1, anti-B7-H4, anti-CD52 (such as alemtuzumab), anti-IL-10, anti-IL-35, and anti-TGF-β (such as fresolumimab). In some embodiments, anti-system monoclonal antibodies. In some embodiments, anti-system full-length antibodies. In some embodiments, the antibody system is selected from the group consisting of antigen-binding fragments such as Fab, Fab', F(ab')2, Fv, scFv, BiTE, nanobody, and other antigen-binding sequences of full-length antibodies or engineered combinations thereof.In some embodiments, the anti-system may contain human antibodies, humanized antibodies, or chimeric antibodies. In some embodiments, the anti-system may contain bispecific or multispecific antibodies.

[0170] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody. Exemplary anti-PD-1 antibodies include, but are not limited to, nivorumab, pembrolizumab, pilizumab, BMS-936559, and atezolizumab, pembrolizumab, MK-3475, AMP-224, AMP-514, STI-A1110, TSR-042, and SHR-1210. In some embodiments, the immune checkpoint inhibitor is nivorumab (e.g., OPDIVO®). In some embodiments, the immune checkpoint inhibitor is pembrolizumab (e.g., KEYTRUDA®). In some embodiments, the immune checkpoint inhibitor is SHR-1210. In some embodiments, the initial co-culture medium contains IL-2, IL-7, IL-15, and IL-21, and an anti-PD-1 antibody (e.g., SHR-1210).

[0171] Suitable concentrations of immune checkpoint inhibitors (e.g., anti-PD-1 antibodies) in the co-culture medium (including the initial co-culture medium) include, but are not limited to, any one of at least about 1, 2, 5, 10, 15, 20, 25, or greater μg / mL. In some embodiments, the immune checkpoint inhibitor (e.g., anti-PD-1 antibody) is present in the co-culture medium (including the initial co-culture medium) at any one of about 1 μg / mL to about 10 μg / mL, about 10 μg / mL to about 20 μg / mL, about 1 μg / mL to about 25 μg / mL, or about 5 μg / mL to about 20 μg / mL.

[0172] Anti-CD3 antibodies may be present in the co-culture at the start of co-culture, or added to the co-culture after the start of co-culture of antigen-loaded DCs with T cells, enriched activated T cells, or tumor antigen-specific T cell populations. In some embodiments, the anti-CD3 antibody system is included in the co-culture medium (including the initial co-culture medium). In some embodiments, the initial co-culture medium does not contain anti-CD3 antibodies.

[0173] In some embodiments, an anti-CD3 antibody is added to a second co-culture comprising an enriched population of activated T cells and a second antigen-loaded dendritic cells (DCs) no more than about 5, 4, 3, 2, or 1 day after the start of the second co-culture. In some embodiments, an anti-CD3 antibody is added to a second co-culture comprising an enriched population of activated T cells and a second antigen-loaded DCs no more than 1, 2, or 3 days after the start of the second co-culture. Any suitable anti-CD3 antibody may be used, including but not limited to OKT3.

[0174] Regarding cell number, T cells (e.g., T cells, enriched activated T cell populations, or tumor antigen-specific T cells) and antigen-loaded APCs (such as PBMCs, DCs, or cell line APCs) may be present in an appropriate ratio in the co-culture. In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population in the first co-culture step is no greater than any one of about 30:1, 25:1, 20:1, 15:1, 10:1, 8:1, or 5:1. In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population in the first co-culture step is at least any one of about 5:1, 8:1, 10:1, 15:1, 20:1, 25:1, or greater. In some embodiments, the ratio between the T cell population and the first antigen-loaded DC population in the first co-culture step is any one of about 5:1 to about 10:1, about 5:1 to about 20:1, about 10:1 to about 20:1, about 20:1 to about 30:1, or about 5:1 to about 30:1. In some embodiments, the ratio between the enriched T cell population and the second antigen-loaded DC population is at least about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the ratio between the enriched T cell population and the second antigen-loaded DC population is not greater than about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1. In some embodiments, the ratio between the enriched T cell population and the second antigen-loaded DC population is any one of about 1:1 to about 20:1, about 1:1 to about 10:1, about 1:1 to about 5:1, about 5:1 to about 10:1, about 10:1 to about 15:1, about 15:1 to about 20:1, about 10:1 to about 20:1, about 1:1 to about 1:3, about 1:1 to about 2:1, or about 2:1 to about 5:1. In some embodiments, the ratio between the tumor antigen-specific T cell population and the antigen-loaded APC (e.g., PBMC (such as fixed PBMC), DC, or cell line APC) population is at least any one of about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, the ratio between the tumor antigen-specific T cell population and the antigen-loaded APC population (e.g., PBMCs (such as fixed PBMCs), DCs, or cell line APCs) is no greater than any of about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.In some embodiments, the ratio between the tumor antigen-specific T cell population and the antigen-loaded APC population (e.g., PBMCs (such as fixed PBMCs), DCs, or cell line APCs) is any one of about 1:1 to about 20:1, about 1:1 to about 10:1, about 1:1 to about 5:1, about 5:1 to about 10:1, about 10:1 to about 15:1, about 15:1 to about 20:1, about 10:1 to about 20:1, about 1:3 to about 3:1, about 1:1 to about 3:1, about 1:1 to about 2:1, or about 2:1 to about 5:1.

[0175] In some embodiments, T cells and APCs (e.g., PBMCs, DCs, or cell line APCs) are derived from the same individual, such as an individual who has clinically benefited from MASCT. In some embodiments, APCs (e.g., PBMCs, DCs, or cell line APCs) are not derived from an individual who has clinically benefited from MASCT. In some embodiments, T cells, APCs (e.g., PBMCs, DCs, or cell line APCs), or both, are derived from an autologous source, such as an individual who received engineered immune cells that express TCR. In some embodiments, T cells, APCs (e.g., PBMCs, DCs, or cell line APCs), or both, are derived from an allogeneic source.

[0176] In some embodiments, T cells and / or APCs (e.g., PBMCs or DCs) are obtained from an individual who has previously received immunotherapy. In some embodiments, the individual has an immune response to the immunotherapy. "Immuneally responsive" to immunotherapy means that the individual has developed a specific immune response to one or more tumor antigens in response to the immunotherapy. In some embodiments, T cells and / or APCs (e.g., PBMCs or DCs) are obtained from an individual who has clinically benefited from the immunotherapy. "Clinically benefited" means that the individual has demonstrated a clinical response to the therapy (as assessed by a physician). Exemplary clinical responses include, but are not limited to, complete response ("CR"), partial response ("PR"), and stable disease ("SD"). Immunotherapy includes, but is not limited to, immune checkpoint inhibitors, adoptive immunotherapy (e.g., adoptive T-cell therapy, CIK, TIL, CAR-T, and TCR-T therapy), cancer vaccines, oncolytic viruses, and combinations thereof. In some embodiments, T cells and / or APCs (e.g., PBMCs or DCs) are obtained from an individual who has previously received MASCT. In some embodiments, the individual is able to develop a specific immune response to tumor antigen peptides during MASCT. A specific immune response to tumor antigen peptides can be determined using assays known in the art, such as the ELISPOT assay. In some embodiments, the individual has clinically benefited from MASCT. In some embodiments, the individual has a tumor antigen-specific immune response (multiple types). In some embodiments, the individual has a tumor antigen-specific immune response and has clinically benefited from MASCT.

[0177] The T cell population used in any embodiment of the methods described herein can be derived from various sources. A convenient source of T cells is peripheral blood mononuclear cells (PBMCs) from humans. The T cell population can be isolated from PBMCs, or alternatively, a T cell-rich PBMC population (e.g., by adding T cell-specific antibodies and cytokines) can be used in a co-culture. In some embodiments, the T cell population used in the first co-culture step is obtained from peripheral blood mononuclear cells (PBMCs). In some embodiments, PBMCs are obtained by density gradient centrifugation of a peripheral blood sample. In some embodiments, the T cell population used in the first co-culture step is present in PBMCs. [Enrichment of activated T cells]

[0178] The method described herein includes an enrichment step comprising enriching activated T cells from a co-culture comprising a first antigen-loaded DC population and a T cell population. In some embodiments, the method includes an enrichment step comprising enriching activated T cells derived from PBMCs stimulated with a target tumor antigen peptide or a fragment thereof.

[0179] In some embodiments, the enrichment process includes selecting activated T cells based on one or more T cell activation biomarkers (such as any one of 1, 2, 3, or more) in a co-culture that responds to stimulation by a target tumor antigen peptide or fragment thereof. In some embodiments, activated T cells are stimulated in the co-culture using an APC (such as PBMC) carrying the target tumor antigen peptide. In some embodiments, the enrichment process includes isolating activated T cells expressing one or more biomarkers (such as cell surface molecules or secreted molecules) from the co-culture.

[0180] In some embodiments, the enrichment process includes isolating activated T cells that express or secrete one or more cytokines from a co-culture that has been stimulated with a target tumor antigen peptide or a fragment thereof. In some embodiments, the enrichment step includes: contacting a first co-culture with antigen-loaded PBMCs to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand that specifically recognizes a cytokine. Exemplary cytokines include, but are not limited to, IFNγ and TNFα. A ligand that specifically recognizes a cytokine (such as an antibody or receptor for the cytokine) can be used to isolate the enriched activated T cell population. In some embodiments, the enrichment step includes: contacting a first co-culture with antigen-loaded PBMCs to obtain a stimulated co-culture; and isolating the enriched activated T cell population from the stimulated co-culture using a ligand that specifically recognizes cell surface molecules, such as 4-1BB (also known as CD137).

[0181] In some embodiments, the method includes: contacting a co-culture with PBMCs carrying a target tumor antigen peptide or a fragment thereof to obtain a stimulated co-culture; and isolating an enriched population of activated T cells from the stimulated co-culture using a ligand that specifically recognizes a cytokine or cell surface molecule. In some embodiments, the cytokine is IFNγ. In some embodiments, the cell surface molecule is 4-1BB.

[0182] In some embodiments, the enrichment procedure includes isolating activated T cells secreting IFNγδ from a co-culture after stimulation with a target tumor antigen peptide or a fragment thereof. In some embodiments, the enrichment procedure includes isolating CD3+IFNγ+ cells from a co-culture after stimulation with a target tumor antigen peptide or a fragment thereof. In some embodiments, the enrichment procedure includes: (1) contacting a co-culture containing a first population of DCs and a population of T cells carrying a target tumor antigen peptide or a fragment thereof with PBMCs carrying a target tumor antigen peptide or a fragment thereof for about 10 to 24 hours (e.g., about 1 day) to obtain a stimulated co-culture; and (2) isolating activated T cells from the stimulated co-culture using a ligand that specifically recognizes IFNγ. In some embodiments, the first antigen-loaded DC population and the T cell population have been co-cultured for about 1 to 7 days (e.g., about 2 to 3 days) before contact with the antigen-loaded PBMCs. In some embodiments, the co-culture and the antigen-loaded PBMCs are contacted for at least 2, 4, 6, 12, 18, 24, or more hours before isolation.

[0183] Activated T cells expressing cytokines (such as IFNγ) can be isolated or enriched from stimulated co-cultures using any method known in the art. For example, commercially available kits, such as the IFNγ Secretion Assay-Cell Enrichment and Detection Kit from Miltenyi Biotec, can be used to isolate IFNγ-secreting T cells. In some embodiments, activated T cells secreting IFNγ are isolated by: (1) contacting a co-culture with an IFNγ capture agent that specifically binds to cell surface antigens and IFNγ on T cells; (2) contacting the IFNγ capture agent-treated co-culture with an anti-IFNγ antibody (e.g., an anti-IFNγ antibody conjugated to R-phycoerthrin or PE); (3) contacting the anti-IFNγ antibody-treated co-culture with magnetic beads containing a secondary antibody (e.g., an anti-PE antibody) that recognizes the anti-IFNγ antibody; and (4) isolating the magnetic beads using a magnetic field (e.g., using a MACSTM separation column) to obtain an enriched population of activated T cells.

[0184] In some embodiments, activated T cells are expressed by means of the following: (1) contacting a co-culture with a fluorescent antibody targeting the cell surface biomarker; and (2) detaching the co-culture from the cells to the fluorescent antibody by flow cytometry. [APC antigen loading]

[0185] The methods and MASCT methods described herein use APCs (such as PBMCs, dendritic cells, or cell line APCs) loaded with one or more tumor antigen peptides. In some embodiments, antigen-loaded APCs (e.g., antigen-loaded DCs) are freshly prepared for use in one or more co-culture steps. In some embodiments, antigen-loaded APCs (e.g., antigen-loaded DCs) are freshly prepared for use in each co-culture step. In some embodiments, antigen-loaded APCs (e.g., antigen-loaded DCs) are prepared, cultured in DC maturation medium, and used in one or more co-culture or stimulation steps. Antigen-loaded DCs used in the first, second, and third co-culture steps can be obtained from single batches or separate batches of antigen-loaded DCs. Unless otherwise indicated, the characteristics described in this section for APCs (e.g., DCs) apply to all APCs (e.g., DCs) used in each co-culture step; and the methods and characteristics described in this section for antigen-loaded APCs (e.g., DCs) apply to antigen-loaded APCs of the first, second, and third groups and other types of APCs. APCs include, but are not limited to, PBMCs, DCs, B cells, or macrophages. The APCs described herein may be primary cells or derived from cell lines. In some embodiments, the APCs are PBMCs. In some embodiments, the APCs are immobilized PBMCs. Immobilization of PBMCs can disrupt their proliferative capacity while maintaining their antigen-presenting ability.

[0186] The antigen-loaded DCs used in each co-culture step may carry the same or different tumor antigen peptide pools. In some embodiments, the tumor antigen peptide pool carried by the first DC group in the first co-culture step is the same as the tumor antigen peptide pool used to load the second DC group in the second co-culture step. In some embodiments, the second DC group in the second co-culture step carries a subset of the tumor antigen peptide pool used to load the first DC group in the first co-culture step. In some embodiments, the third DC group in the third co-culture step carries a subset of the tumor antigen peptide pool used to load the first DC group in the first co-culture step and / or a subset of the tumor antigen peptide pool used to load the second DC group in the second co-culture step. In some embodiments, the subset of the tumor antigen peptide pool includes fragments and combinations of tumor antigen peptides. In some embodiments, a single tumor antigen peptide (i.e., the target tumor antigen peptide) or a fragment thereof is used to load APCs (such as DCs) for use in the second and third co-culture steps.

[0187] In some embodiments, a first antigen-loaded DC population for the first co-culture step is prepared using a plurality of tumor antigen peptides used by the individual in a previous MASCT. In some embodiments, a first antigen-loaded DC population for the first co-culture step is prepared using one or more tumor antigen peptides to which the individual has a specific immune response in a previous MASCT. In some embodiments, individual tumor antigen peptides, and combinations thereof, derived from one or more target tumor antigen peptides or fragments thereof, are screened (e.g., by ELISPOT) based on a specific immune response to PBMCs, activated T cells, or tumor antigen-specific T cells derived from the individual to identify one or more target tumor antigen peptides (including fragments thereof) for subsequent preparation of tumor antigen-specific T cells.

[0188] In some embodiments, prior to each co-culture step, the method includes one or more of the following steps: (1) obtaining PBMCs from an individual; (2) obtaining mononuclear spheroids from PBMCs; (3) inducing the mononuclear spheroids to differentiate into immature DCs; (4) contacting the immature DCs with one or more tumor antigen peptides to obtain antigen-loaded DCs; and (5) culturing the antigen-loaded DCs in a DC maturation medium containing a TLR activator (such as MPLA).

[0189] In some embodiments, antigen-loaded DCs are prepared by: (a) contacting a group of DCs with one or more tumor antigen peptides to obtain antigen-loaded DCs; and (b) culturing the antigen-loaded DCs in a DC maturation medium containing a TLR receptor (TLR) agonist. Exemplary TLR agonists include, but are not limited to, MPLA (monophospholipid A), Poly I:C, resquimod, gardiquimod, and CL075. Cytokines and other suitable molecules, such as INFγ and PGE2 (prostaglandin E2), may be further included in the culture medium during the maturation step.

[0190] In some embodiments, antigen-loaded DCs are prepared by: (a) contacting a group of DCs with one or more tumor antigen peptides to obtain antigen-loaded DCs; and (b) culturing the antigen-loaded DCs in a DC maturation medium containing MPLA, INFγ, and PGE2.

[0191] In some embodiments, antigen-loaded DCs are prepared by: (a) inducing mononuclear spheroids to differentiate into immature DCs; (b) contacting the immature DCs with one or more tumor antigen peptides to obtain antigen-loaded DCs; and (c) culturing the antigen-loaded DCs in a DC maturation medium containing MPLA, INFγ, and PGE2. In some embodiments, the mononuclear spheroids are obtained from PBMCs.

[0192] In some embodiments, antigen-loaded PBMCs are prepared by contacting a population of PBMCs with one or more tumor antigen peptides. In some embodiments, antigen-loaded cell lines APCs (e.g., LCLs) are prepared by contacting a population of APCs with one or more tumor antigen peptides.

[0193] DC maturation medium may contain suitable concentrations of MPLA, INFγ, and / or PGE2. In some embodiments, the DC maturation medium contains MPLA at a concentration of at least about 0.5 μg / mL, such as at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or greater μg / mL. In some embodiments, the DC maturation medium contains MPLA at a concentration of about 0.5 to 10, 1 to 5, 5 to 10, or 2.5 to 7.5 μg / mL. In some embodiments, the DC maturation medium contains INFγ at a concentration of at least about 100 IU / mL, such as at least about 150, 200, 250, 300, 400, 500, 600, 800, 1000, or greater IU / mL. In some embodiments, the DC maturation medium contains INFγ at a concentration of about 100 to 1000, 100 to 250, 250 to 500, 500 to 1000, or 250 to 750 IU / mL. In some embodiments, the DC maturation medium contains PGE2 at a concentration of at least about 0.1 μg / mL, such as at least about 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, or greater μg / mL. In some embodiments, the DC maturation medium contains PGE2 at a concentration of about 0.1 to 0.5, 0.1 to 0.3, 0.25 to 0.5, or 0.2 to 0.4 μg / mL.

[0194] Immature dendritic cells (DCs) carrying one or more tumor antigen peptides can be induced to mature for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 days using a TLR activator. In some embodiments, DCs carrying one or more tumor antigen peptides are induced to mature for about 8, 9, 10, 11, or 12 days.

[0195] In some embodiments, the antigen-loaded DC is a mature DC presenting one or more tumor antigen peptides. Mature DCs prepared by any of the methods described herein may present at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, or more tumor antigen peptides. Compared to naïve DCs, or DCs not yet loaded with multiple tumor antigen peptides, multi-antigen-loaded DCs may have enhanced presentation levels of at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 50, or more tumor antigen peptides. In some embodiments, mature DCs have enhanced presentation levels of more than 10 tumor antigen peptides. In some embodiments, mature DCs have enhanced presentation levels of tumor antigen peptides from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more, which are derived from proteins selected from the group consisting of: hTERT, p53, survivin, NY-ESO-1, CEA, CCND1, RGS5, MMP7, VEGFR1, VEGFR2, MUC1, HER2, MAGE-A1, MAGE-A3, CDCA1, WT1, KRAS, PARP4, MLL3, MTHFR, HPV16-E6, HPV16-E7, HPV18-E6, HPV18-E7, HPV58-E6, HPV58-E7, HBcAg, HBV polymerase, GPC3, SSX, and AFP.

[0196] In some embodiments, antigen-loaded APCs (e.g., DCs, PBMCs, or cell line APCs) are prepared by pulsed one or more tumor antigen peptides into a group of APCs. In some embodiments, antigen-loaded DCs, such as immature DCs or DCs contained in or derived from PBMCs, are prepared by pulsed one or more tumor antigen peptides into a group of DCs. As known in the art, pulsed refers to a procedure of mixing cells (such as APCs (e.g., PBMCs or DCs, or cell line APCs)) with a solution containing antigen peptides and optionally subsequently removing the antigen peptides from the mixture. The DC group can be contacted with one or more tumor antigen peptides for seconds, minutes, or hours, such as at least any of the following: 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 5 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, or longer. The concentration of each tumor antigen peptide used in the contacting step can be at least any of about 0.1, 0.5, 1, 2, 3, 5, or 10 μg / mL. In some embodiments, the concentration of the tumor antigen peptide is about 0.1 to 200 μg / mL, including, for example, any of about 0.1 to 0.5, 0.5 to 1, 1 to 10, 10 to 50, 50 to 100, 100 to 150, or 150 to 200 μg / mL.

[0197] In some embodiments, a group of APCs (e.g., DCs or PBMCs, or cell line APCs) is contacted with one or more tumor antigen peptides in the presence of a composition that promotes the uptake of the one or more tumor antigen peptides by the APCs (e.g., DCs or PBMCs, or cell line APCs). In some embodiments, compounds, materials, or compositions may be included in a solution of one or more tumor antigen peptides to promote the uptake of the peptides by the APCs (e.g., DCs or PBMCs, or cell line APCs). Compounds, materials, or compositions that promote the uptake of one or more tumor antigen peptides by APCs (e.g., DCs or PBMCs, or cell line APCs) include, but are not limited to, lipid molecules and peptides having a plurality of positively charged amino acids. In some embodiments, a group of APCs (e.g., DCs or PBMCs, or cell line APCs) takes up more than 50%, 60%, 70%, 80%, 90%, or 95% of the tumor antigen peptides. In some embodiments, more than 50%, 60%, 70%, 80%, 90%, or 95% of the APCs (e.g., DCs or PBMCs, or cell line APCs) in the population take up at least one tumor antigen peptide.

[0198] Dendritic cells (such as immature DCs) can be obtained from various sources, including autologous sources, i.e., from individuals receiving TCR therapy. A convenient source of DC cells is peripheral blood mononuclear cells (PBMCs). For example, monocytes (a type of white blood cell) are abundant in PBMCs, constituting approximately 5 to 30% of the total PBMCs. Cytokines can be used to induce monocytes to differentiate into DCs, such as immature DCs. In some embodiments, immature DCs are prepared by: obtaining a PBMC population; obtaining a monocyte population from the PBMC population; and contacting the monocyte population with one or more cytokines (e.g., multiple cytokines) to obtain an immature DC population. Exemplary cytokines that can be used to induce monocyte differentiation include, but are not limited to, GM-CSF and IL-4, wherein the induction is performed under conditions known in the art (such as concentration, temperature, CO2 level, etc.).

[0199] The adhering portion of PBMCs contains most of the mononuclear globules in the PBMCs. In some embodiments, mononuclear globules from the adhering portion of PBMCs are contacted with cytokines to obtain immature DC populations. PBMCs can be conveniently obtained from individuals by centrifuging peripheral blood samples or by collecting them using apheresis methods. In some embodiments, PBMC populations are obtained by density gradient centrifugation of human peripheral blood samples. In some embodiments, the samples are from individuals receiving multiantigen-loaded DCs, activated T cells, engineered immune cells expressing TCR, or other immunotherapeutic components prepared using multiantigen-loaded DCs. [Tumor antigen peptide]

[0200] The methods described herein and the MASCT method utilize one or more tumor antigen peptides (including target tumor antigen peptides) to prepare antigen-loaded APCs (such as antigen-loaded DCs), activated T cells, and tumor antigen-specific T cells (which can trigger specific immune responses both in vitro and in vivo). In some embodiments, the plurality of tumor antigen peptides are a plurality of synthetic tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides are not derived from cell samples (such as lysed cell components). As used herein, "one or more tumor antigen peptides from a plurality of tumor antigen peptides" refers to a subset or all of the plurality of tumor antigen peptides, including fragments and combinations thereof. The features and parameters described in this subsection may be applied to (multiple) target tumor antigen peptides.

[0201] In some embodiments, each tumor antigen peptide comprises at least one, two, three, four, five, or ten epitopes derived from a single protein antigen (including neoantigens). In some embodiments, each tumor antigen peptide in a plurality of tumor antigen peptides comprises at least one epitope recognizable by a T-cell receptor. In some embodiments, a plurality of tumor antigen peptides comprises at least one tumor antigen peptide comprising at least two epitopes derived from a single protein antigen. Tumor antigen peptides may be naturally derived peptide fragments from protein antigens containing one or more epitopes, or artificially designed peptides having one or more natural epitope sequences, wherein linker peptides may optionally be positioned between adjacent epitope sequences. In some preferred embodiments, epitopes contained in the same tumor antigen peptide are derived from the same protein antigen.

[0202] Tumor antigen peptides may contain at least one MHC-I epitope, at least one MHC-II epitope, or both (multiple) MHC-I epitopes and (multiple) MHC-II epitopes. In some embodiments, a plurality of tumor antigen peptides includes at least one peptide containing an MHC-I epitope. In some embodiments, a plurality of tumor antigen peptides includes at least one peptide containing an MHC-II epitope. In some embodiments, at least one tumor antigen peptide among a plurality of tumor antigen peptides contains both an MHC-I epitope and an MHC-II epitope.

[0203] Specialized design strategies can be applied to the sequences of tumor antigen peptides (including neoantigen peptides) to optimize the immune response to dendritic cells (DCs) carrying tumor antigen peptides. Generally, longer peptides with more precise epitopes can increase peptide uptake into DCs. In some embodiments, based on the native sequence of the protein containing the epitope, an MHC-I or MHC-II epitope sequence is extended at the N-terminus, C-terminus, or both ends to obtain an extended sequence, wherein the extended sequence is suitable for presentation by both class I and class II MHC molecules, and by different subtypes of MHC molecules from different individuals. In some embodiments, the epitope sequence is extended at one or both ends by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20 amino acid residues to generate an extended epitope. In some embodiments, the peptide containing the MHC-I or MHC-II epitope further includes additional amino acids flanked by epitopes at the N-terminus, C-terminus, or both ends. In some embodiments, each of the plurality of tumor antigen peptides has an amino acid length of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100. Different tumor antigen peptides among the plurality of tumor antigen peptides may have the same length or different lengths. In some embodiments, each of the plurality of tumor antigen peptides has an amino acid length of about 20 to 40.

[0204] In some embodiments, the amino acid sequence of one or more epitope peptides used to design tumor antigen peptides in this application is based on sequences known in the art or sequences available from public databases such as the Peptide Database (Vigneron N. et al. Cancer Immunity, 13:15 (2013)).

[0205] In some embodiments, bioinformatics tools for T-cell epitope prediction are used to predict the amino acid sequences of one or more epitope peptides based on the sequence of an antigen protein. Exemplary bioinformatics tools for T-cell epitope prediction are known in the art, for example, see Yang X. and Yu X. (2009), “An introduction to epitope prediction methods and software”, Rev. Med. Virol. 19(2):77-96. In some embodiments, the sequence of the antigen protein is known in the art or is available from a public database. In some embodiments, the sequence of the antigen protein is determined by sequencing a sample (such as a tumor sample) of an individual receiving treatment.

[0206] This application envisions tumor antigen peptides derived from any tumor antigens and epitopes (including neoantigens and neoepitaxes) known in the art, or tumor antigen peptides specially developed or predicted by the inventors using bioinformatics tools.

[0207] In some embodiments, a plurality of tumor antigen peptides include general tumor antigen peptides of a first core group. In some embodiments, a plurality of tumor antigen peptides further include cancer type-specific antigen peptides of a second group. In some embodiments, a plurality of tumor antigen peptides include one or more neoantigen peptides. In some embodiments, the neoantigen peptides are cancer type-specific antigen peptides. In some embodiments, a plurality of tumor antigen peptides are composed of general tumor antigen peptides of the first core group. In some embodiments, a plurality of tumor antigen peptides are composed of general tumor antigen peptides of the first core group and cancer type-specific antigen peptides of the second group. In some embodiments, a plurality of tumor antigen peptides are composed only of neoantigen peptides. In some embodiments, a plurality of tumor antigen peptides include general tumor antigen peptides of the first core group and one or more neoantigen peptides. In some embodiments, a plurality of tumor antigen peptides are composed of general tumor antigen peptides of the first core group, cancer type-specific antigen peptides of the second group, and one or more neoantigen peptides.

[0208] In some embodiments, a plurality of tumor antigen peptides include general tumor antigen peptides of a first core group. In some embodiments, a plurality of tumor antigen peptides further include cancer type-specific antigen peptides of a second group. In some embodiments, a plurality of tumor antigen peptides include one or more neoantigen peptides. In some embodiments, the neoantigen peptides are cancer type-specific antigen peptides. In some embodiments, a plurality of tumor antigen peptides are composed of general tumor antigen peptides of the first core group. In some embodiments, a plurality of tumor antigen peptides are composed of general tumor antigen peptides of the first core group and cancer type-specific antigen peptides of the second group. In some embodiments, a plurality of tumor antigen peptides are composed only of neoantigen peptides. In some embodiments, a plurality of tumor antigen peptides include general tumor antigen peptides of the first core group and one or more neoantigen peptides. In some embodiments, a plurality of tumor antigen peptides are composed of general tumor antigen peptides of the first core group, cancer type-specific antigen peptides of the second group, and one or more neoantigen peptides.

[0209] The general tumor antigen peptides of the first core group are derived from tumor antigens that are often overexpressed by various types of cancer. Therefore, the general tumor antigen peptides of the first core group are used to prepare dendritic cells and / or activate T cells for the treatment of individuals with different types of cancer. For example, in some embodiments, the general tumor antigen peptides of the first core group are used in the methods described herein for treating various cancers such as lung cancer, colon cancer, gastric cancer, prostate cancer, melanoma, lymphoma, pancreatic cancer, ovarian cancer, breast cancer, glioma, esophageal cancer, nasopharyngeal cancer, cervical cancer, kidney cancer, or hepatocellular carcinoma. Exemplary tumor antigen peptides of the first core group include, but are not limited to, peptides derived from: hTERT, p53, survivin, NY-ESO-1, CEA, CCND1, MET, MUC1, Her2, MAGEA1, MAGEA3, WT-1, RGS5, MMP7, VEGFR (such as VEGFR1 and VEGFR2), and CDCA1. The first core group may include peptides derived from at least about 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, or more tumor antigens. The first core group may also include at least about 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, or more common tumor antigen peptides. In some embodiments, the first core group includes more than one common tumor antigen peptide. In some embodiments, the first core group includes about 10 to about 20 common tumor antigen peptides.

[0210] The second group of cancer type-specific antigenic peptides are derived from tumor antigens that are overexpressed only in one or a limited number of cancer types. Therefore, the second group of cancer type-specific antigenic peptides is intended for the preparation of dendritic cells and / or activated T cells for the treatment of individuals with specific cancer types. Exemplary cancer type-specific antigenic peptides for the treatment of hepatocellular carcinoma (HCC) include, but are not limited to, peptides derived from SSX, AFP, and GPC3. In some embodiments, one or more cancer-specific antigenic peptides are virus-specific antigenic peptides derived from viruses that can induce cancer or are involved in cancer development within an individual upon infection. In some embodiments, the virus-specific antigenic peptides are specific to the viral subtype of the infected individual. Exemplary virus-specific antigenic peptides for the treatment of HCC patients with HBV co-infection include, but are not limited to, peptides derived from HBV core antigen (HBcAg) and HBV DNA polymerase. In some embodiments, the second group includes virus-specific antigenic peptides derived from HBV antigens, wherein the method is used to treat hepatocellular carcinoma in an individual. In some embodiments, the second group comprises virus-specific antigenic peptides derived from HPV antigens, wherein the method is used to treat cervical cancer in an individual. In some embodiments, the second group comprises virus-specific antigenic peptides derived from EBV antigens, wherein the method is used to treat nasopharyngeal carcinoma in an individual. The cancer type-specific antigenic peptides in the second group may comprise peptides derived from at least about 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, or more cancer type-specific antigenic peptides. The second group of cancer type-specific antigenic peptides may comprise at least about 1, 2, 5, 10, 15, 20, 25, 30, 40, 50, or more cancer type-specific antigenic peptides. In some embodiments, the second group comprises more than one cancer type-specific antigenic peptide. In some embodiments, the second group comprises about 1 to about 10 cancer type-specific antigenic peptides. In some embodiments, the cancer types targeted by the cancer type-specific antigenic peptide are selected from the group consisting essentially of the following: hepatocellular carcinoma, cervical cancer, nasopharyngeal carcinoma, endometrial cancer, colorectal cancer, breast cancer, endometrial cancer, and lymphoma.

[0211] In some embodiments, a plurality of tumor antigen peptides comprises one or more (such as any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) neoantigen peptides. In some embodiments, a plurality of tumor antigen peptides consists of neoantigen peptides. In some embodiments, a plurality of tumor antigen peptides comprises neoantigen peptides and does not comprise general tumor antigen peptides. In some embodiments, a plurality of tumor antigen peptides comprises one or more general tumor antigen peptides and one or more neoantigen peptides. In some embodiments, a plurality of tumor antigen peptides comprises one or more general tumor antigen peptides, one or more cancer type-specific antigen peptides, and one or more neoantigen peptides. Neoantigen peptides are derived from neoantigens. Neoantigens are recently acquired and expressed antigens present in the tumor cells of an individual (such as an individual undergoing cancer treatment). In some embodiments, neoantigens are derived from mutant protein antigens that are present only in cancer cells but not in normal cells. Neoantigens may be uniquely present in the tumor cells (such as all or some tumor cells) of an individual undergoing cancer treatment, or may be present in an individual with a similar type of cancer undergoing treatment. In some embodiments, the neoantigen is a clonal neoantigen. In some embodiments, the neoantigen is a subclonal neoantigen. In some embodiments, the neoantigen is present in tumor cells of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more in an individual. In some embodiments, the neoantigen peptide comprises an MHC-I restricted neoeptope. In some embodiments, the neoantigen peptide comprises an MHC-II restricted neoeptope. In some embodiments, the neoantigen peptide is designed to promote the presentation of neoeptopes in both class I and class II MHC molecules, for example by extending the neoeptope at both the N-terminus and C-terminus. Exemplary neoantigen peptides include, but are not limited to, neoeptopes derived from: mutant KRAS (e.g., KRASG12A), PARP4 (e.g., PARP4T1170I), MLL3 (e.g., MLL3C988F), and MTHFR (e.g., MTHFRA222V).

[0212] Neoantigen peptides can be selected based on the genetic profile of one or more tumor sites in an individual receiving treatment, and the neoantigens are not expressed in normal tissues. In some embodiments, the genetic profile of the tumor sample includes the sequence information of the full-length genome. In some embodiments, the genetic profile of the tumor sample includes the sequence information of exons. In some embodiments, the genetic profile of the tumor sample includes the sequence information of cancer-related genes.

[0213] The neoantigen peptides applicable to this application may be derived from any mutated protein in tumor cells, such as mutated proteins encoded by mutated cancer-associated genes. In some embodiments, the neoantigen peptide comprises a single novel epitope derived from a cancer-associated gene. In some embodiments, the neoantigen peptide comprises more than one (such as 2, 3, or more) novel epitopes derived from cancer-associated genes. In some embodiments, the neoantigen peptide comprises more than one (such as 2, 3, or more) novel epitopes derived from more than one (such as 2, 3, or more) cancer-associated genes. In some embodiments, multiple tumor antigens comprise multiple neoantigen peptides derived from a single cancer-associated gene. In some embodiments, multiple tumor antigens comprise multiple neoantigen peptides derived from more than one (such as any one of 2, 3, 4, 5, or more) cancer-associated genes.

[0214] Cancer-associated genes are those that are overexpressed in cancer cells, but are expressed at low levels in normal cells. Exemplary cancer-associated genes include, but are not limited to, ABL1, AKT1, AKT2, AKT3, ALK, ALOX12B, APC, AR, ARAF, ARID1A, ARID1B, ARID2, ASXL1, ATM, ATRX, AURKA, AURKB, AXL, B2M, BAP1, BCL2, BCL2L1, BCL2L12, BCL6, BCOR, BCORL1, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD4, BRIP1, BUB1B, CADM2, and CARD1. 1. CBL, CBLB, CCND1, CCND2, CCND3, CCNE1, CD274, CD58, CD79B, CDC73, CDH1, CDK1, CDK2, CDK4, CDK5, CDK6, CDK9, CDKN1A, CDKN1B , CDKN1C, CDKN2A, CDKN2B, CDKN2C, CEBPA, CHEK2, CIITA, CREBBP, CRKL, CRLF2, CRTC1, CRTC2, CSF1R, CSF3R, CTNNB1, CUX1, CYLD,DDB2, DDR2, DEPDC5, DICER1, DIS3, DMD, DNMT3A, EED, EGFR, EP300, EP HA3, EPHA5, EPHA7, ERBB2, ERBB3, ERBB4, ERCC2, ERCC3, ERCC4, ERCC5 ESR1, ETV1, ETV4, ETV5, ETV6, EWSR1, EXT1, EXT2, EZH2, FAM46C, FANC A. FANCC, FANCD2, FANCE, FANCF, FANCG, FAS, FBXW7, FGFR1, FGFR2, FGF R3, FGFR4, FH, FKBP9, FLCN, FLT1, FLT3, FLT4, FUS, GATA3, GATA4, GAT A6, GLI1, GLI2, GLI3, GNA11, GNAQ, GNAS, GNB2L1, GPC3, GSTM5, H3F3A. HNF1A, HRAS, ID3, IDH1, IDH2, IGF1R, IKZF1, IKZF3, INSIG1, JAK2, JA K3, KCNIP1, KDM5C, KDM6A, KDM6B, KDR, KEAP1, KIT, KRAS, LINC00894, L MO1、LMO2、LMO3、MAP2K1、MAP2K4、MAP3K1、MAPK1、MCL1、MDM2、MDM4、M ECOM, MEF2B, MEN1, MET, MITF, MLH1, MLL(KMT2A), MLL2(KTM2D), MPL SH2, MSH6, MTOR, MUTYH, MYB, MYBL1, MYC, MYCL1(MYCL), MYCN, MYD88. NBN, NEGR1, NF1, NF2, NFE2L2, NFKBIA, NFKBIZ, NKX2-1, NOTCH1, NOTCH 2. NPM1, NPRL2, NPRL3, NRAS, NTRK1, NTRK2, NTRK3, PALB2, PARK2, PAX 5. PBRM1, PDCD1LG2, PDGFRA, PDGFRB, PHF6, PHOX2B, PIK3C2B, PIK3CA. PIK3R1, PIM1, PMS1, PMS2, PNRC1, PRAME, PRDM1, PRF1, PRKAR1A, PRKC I, PRKCZ, PRKDC, PRPF40B, PRPF8, PSMD13, PTCH1, PTEN, PTK2, PTPN11.PTPRD, QKI, RAD21, RAF1, RARA, RB1, RBL2, RECQL4, REL, RET, RFWD2, RHEB, RHPN2, ROS1, RPL26, RUNX1, SBDS, SDHA, SDH AF2, SDHB, SDHC, SDHD, SETBP1, SETD2, SF1, SF3B1, SH2B3, SLITRK6, SMAD2, SMAD4, SMARCA4, SMARCB1, SMC1A, SMC3, SM O, SOCS1, SOX2, SOX9, SQSTM1, SRC, SRSF2, STAG1, STAG2, STAT3, STAT6, STK11, SUFU, SUZ12, SYK, TCF3, TCF7L1, TCF7L 2. TERC, TERT, TET2, TLR4, TNFAIP3, TP53, TSC1, TSC2, U2AF1, VHL, WRN, WT1, XPA, XPC, XPO1, ZNF217, ZNF708, and ZRSR2.

[0215] In some embodiments, the plurality of tumor antigen peptides comprises at least one (e.g., at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) tumor antigen peptide, each comprising one or more epitopes encoded by a cancer-related gene selected from the group consisting of: hTERT, p53, survivin, NY-ESO-1, CEA, CCND1, RGS5, MMP7, VEGFR1, VEGFR2, MUC1, HER2, MAGE-A1, MAGE-A3, CDCA1, WT1, KRAS, PARP4, MLL3, MTHFR, HPV16-E6, HPV16-E7, HPV18-E6, HPV18-E7, HPV58-E6, HPV58-E7, HBcAg, HBV polymerase, GPC3, SSX, and AFP. In some embodiments, the plurality of tumor antigen peptides includes at least 10 tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides include tumor antigen peptides derived from the following: hTERT, p53, survivin, NY-ESO-1, CEA, CCND1, MUC1, Her2, MAGEA1, MAGEA3, WT-1, RGS5, VEGFR1, VEGFR2, and CDCA1.

[0216] In some embodiments, one or more tumor antigen peptides are present in a composition having at least about 95%, 96%, 97%, 98%, 99%, 99.9%, or higher percentages of tumor antigen peptides. In some embodiments, the purity of the one or more tumor antigen peptides is at least about 98%. In some embodiments, for pulsed delivery of the tumor antigen peptides to DC, the solubility of the one or more tumor antigen peptides in the culture medium is at least about 80%, 85%, 90%, 95%, 98%, 99%, 99.9%, or higher. In some embodiments, for pulsed delivery of the tumor antigen peptides to APC, the one or more tumor antigen peptides are about 100% soluble in the culture medium. [MASCT]

[0217] The TCRs described herein are obtained from PBMCs or T cells of individuals who have clinically benefited from MASCT. In some embodiments, such as as determined by ELISPOT, the individual has developed a specific response to the (multiple) target tumor antigen peptides or fragments thereof used in the methods described herein.

[0218] As used herein, "MASCT" or "Multiple Antigen Specific Cell Therapy" refers to a method of adoptive T-cell therapy that involves administering an effective amount of activated T cells to an individual, which are prepared by co-culturing a population of T cells with a population of dendritic cells (DCs) carrying multiple tumor antigen peptides. MASCT methods have been described, for example, in International Patent Application Publication No. WO2016145578A1, International Patent Application Nos. PCT / CN2018 / 081338 and PCT / CN2019 / 080535, the entire contents of which are incorporated herein by reference. First-generation MASCT, precision MASCT, PBMC-based MASCT, customized MASCT, neoantigen-based MASCT, modified MASCT, and combination therapies with MASCT (e.g., immune checkpoint inhibitors and MASCT) are all within the scope of MASCT in this application. Any suitable features and parameters described in this application or international patent applications No. WO2016145578A1, No. PCT / CN2018 / 081338, and No. PCT / CN2019 / 080535 for the preparation of antigen-loaded DCs, the preparation of activated T cells, the enrichment step, and the co-culture step may be combined in MASCT treatment.

[0219] An individual may have received a single type of MASCT or a combination of different types of MASCT, such as a customized MASCT and a modified MASCT. An individual may have received one or more cycles of MASCT. In some embodiments, an individual has received at least about 2, 5, 10, 15, 20, or more cycles of MASCT. In some embodiments, an individual has received MASCT after at least about 3 months, 6 months, 9 months, 12 months, 2 years, 3 years, or longer.

[0220] In some embodiments, the MASCT includes: (i) co-culturing a population of dendritic cells (DCs) carrying a plurality of tumor antigen peptides with a population of T cells to obtain a population of activated T cells, the plurality of tumor antigen peptides including the target tumor antigen peptide; and (ii) administering an effective amount of the activated T cells to an individual. In some embodiments, the MASCT includes administering an effective amount of DCs carrying the plurality of tumor antigen peptides to the individual.

[0221] In some embodiments, the MASCT comprises: (i) co-culturing a population of dendritic cells (DCs) loaded with multiple tumor antigen peptides and a population of T cells in an initial co-culture medium to provide a co-culture, the multiple tumor antigen peptides including the target tumor antigen peptide, the initial co-culture medium containing one or more cytokines (e.g., multiple cytokines) and an immune checkpoint inhibitor; (ii) adding an anti-CD3 antibody to the co-culture approximately 3 to 7 days after the start of the co-culture to obtain the activated T cell population; and (iii) administering an effective amount of the activated T cells to the individual. In some embodiments, the MASCT comprises administering an effective amount of DCs loaded with the multiple tumor antigen peptides to the individual.

[0222] In some embodiments, the MASCT includes: (i) contacting a population of dendritic cells (DCs) with a plurality of tumor antigen peptides to obtain a population of DCs loaded with the plurality of tumor antigen peptides, the plurality of tumor antigen peptides including the target tumor antigen peptide; (ii) culturing the population of DCs loaded with the plurality of tumor antigen peptides in a DC maturation medium containing MPLA; (iii) co-culturing the population of DCs loaded with the plurality of tumor antigen peptides with a population of T cells to obtain an activated T cell population; and (iv) administering an effective amount of the activated T cells to the individual. In some embodiments, the DC maturation medium contains INFγ, MPLA, and PGE2. In some embodiments, the MASCT includes administering an effective amount of DCs loaded with the plurality of tumor antigen peptides to the individual.

[0223] In some embodiments, the MASCT includes: (i) contacting a population of dendritic cells (DCs) with a plurality of tumor antigen peptides to obtain a population of DCs loaded with the plurality of tumor antigen peptides, the plurality of tumor antigen peptides including the target tumor antigen peptide; (ii) culturing the population of DCs loaded with the plurality of tumor antigen peptides in a DC maturation medium containing MPLA; (iii) co-culturing the population of DCs loaded with the plurality of tumor antigen peptides with a population of T cells in an initial co-culture medium containing one or more cytokines (e.g., a plurality of cytokines) and an immune checkpoint inhibitor; (iv) adding an anti-CD3 antibody to the co-culture approximately 3 to 7 days after the start of the co-culture to obtain the activated T cell population; and (v) administering an effective amount of the activated T cells to the individual. In some embodiments, the DC maturation medium contains INFγ, MPLA, and PGE2. In some embodiments, the MASCT includes administering an effective amount of DCs loaded with the plurality of tumor antigen peptides to the individual.

[0224] In some embodiments, the MASCT includes administering an effective amount of activated T cells to the individual, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of antigen-presenting cells (such as DCs) carrying multiple tumor antigen peptides. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at least three times. In some embodiments, the individual has previously been administered an effective amount of antigen-presenting cells carrying multiple tumor antigen peptides. In some embodiments, the method includes administering an effective amount of antigen-presenting cells (such as DCs) carrying the multiple tumor antigen peptides to the individual. In some embodiments, antigen-presenting cells are administered approximately 7 to 21 days prior to the administration of activated T cells (e.g., approximately 7 to 14 days, or approximately 14 to 21 days). In some embodiments, antigen-presenting cells are administered at least three times. In some embodiments, antigen-presenting cells are administered subcutaneously, intradermally, or intravenously. In some embodiments, the activated T cells and the antigen-presenting cell population are from the same individual. In some embodiments, the activated T cells and / or the antigen-presenting cell population are from an individual receiving treatment. In some embodiments, the antigen presents as a population of dendritic cells (DCs), B cells, or macrophages. In some embodiments, the antigen presents as a DC cell line. In some embodiments, MASCT further comprises administering an effective amount of an immune checkpoint inhibitor to the individual. In some embodiments, activated T cells and the immune checkpoint inhibitor are administered simultaneously, such as with the same composition. In some embodiments, activated T cells and the immune checkpoint inhibitor are administered sequentially.

[0225] In some embodiments, the MASCT comprises: (a) administering an effective amount of dendritic cells (DCs) loaded with multiple tumor antigen peptides to the individual; (b) co-culturing the DC population loaded with the multiple tumor antigen peptides with a T cell population to obtain an activated T cell population; and (c) administering an effective amount of the activated T cells to the individual. In some embodiments, the interval between DC administration and activated T cell administration is approximately 7 days to approximately 21 days (e.g., approximately 7 days to approximately 14 days, approximately 14 days to approximately 21 days, approximately 10 days, or approximately 14 days). In some embodiments, the DCs loaded with multiple tumor antigen peptides are administered subcutaneously. In some embodiments, the DCs loaded with multiple tumor antigen peptides are administered at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, a population of T cells is co-cultured with a population of dendritic cells (DCs) carrying multiple tumor antigen peptides for approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 10 days, approximately 10 to approximately 15 days, approximately 15 to approximately 21 days, approximately 14 to approximately 21 days, or approximately 10 days). In some embodiments, the T cell population is derived from the non-adhesive portion of a population of peripheral blood mononuclear cells (PBMCs). In some embodiments, co-culture further comprises contacting activated T cells with multiple cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, a population of DCs carrying multiple tumor antigen peptides is prepared by contacting the DC population with multiple tumor antigen peptides. In some embodiments, the T cell population and the DC population are derived from the same individual. In some embodiments, the T cell population, DC population, PBMC population, or any combination thereof is derived from the individual receiving treatment. In some embodiments, MASCT further comprises administering an effective amount of an immune checkpoint inhibitor to the individual. In some embodiments, activated T cells and immune checkpoint inhibitors are administered simultaneously, such as with the same composition. In some embodiments, activated T cells and immune checkpoint inhibitors are administered sequentially.

[0226] In some embodiments, the MASCT comprises: (a) inducing mononuclear spheroids to differentiate into dendritic (DC) populations; (b) contacting the DC populations with multiple tumor antigen peptides to obtain DC populations loaded with the multiple tumor antigen peptides; (c) administering an effective amount of DCs loaded with the multiple tumor antigen peptides to the individual; (d) co-culturing the DC populations loaded with the multiple tumor antigen peptides with non-adhesive PBMC populations to obtain activated T cell populations; and (e) administering an effective amount of the activated T cells to the individual, wherein the mononuclear spheroids and the non-adhesive PBMC populations are derived from PBMC populations. In some embodiments, the interval between DC administration and activated T cell administration is approximately 7 days to approximately 21 days (e.g., approximately 7 days to approximately 14 days, approximately 14 days to approximately 21 days, approximately 10 days, or approximately 14 days). In some embodiments, DCs loaded with the multiple tumor antigen peptides are administered subcutaneously. In some embodiments, DCs loaded with the multiple tumor antigen peptides are administered at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, co-culture lasts from about 7 days to about 21 days (e.g., from about 7 days to about 14 days, from about 14 days to about 21 days, or about 10 days). In some embodiments, co-culture further includes contacting activated T cells with a plurality of cytokines (e.g., IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, non-adhesive PBMCs are contacted with an immune checkpoint inhibitor (e.g., an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, the PBMCs are obtained from an individual receiving treatment. In some embodiments, MASCT further includes administering an effective amount of an immune checkpoint inhibitor to the individual. In some embodiments, activated T cells and immune checkpoint inhibitors are administered simultaneously, such as with the same composition. In some embodiments, activated T cells and immune checkpoint inhibitors are administered sequentially.

[0227] In some embodiments, MASCT comprises: contacting a population of peripheral blood mononuclear cells (PBMCs) with a plurality of tumor antigen peptides to obtain an activated PBMC population; and administering an effective amount of activated PBMCs to an individual. In some embodiments, the PBMC population is contacted with the plurality of tumor antigen peptides in the presence of a composition that promotes the uptake of the plurality of tumor antigen peptides by antigen-presenting cells (such as dendritic cells) in the PBMCs. In some embodiments, the PBMC population is contacted with the plurality of tumor antigen peptides in the presence of an immune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, and LAG-3. In some embodiments, the activated PBMC population is contacted with IL-2. In some embodiments, the activated PBMCs are administered at least three times. In some embodiments, the interval between each administration of activated PBMCs is from about 2 weeks to about 5 months (e.g., about 3 months). In some embodiments, the activated PBMCs are administered intravenously. In some embodiments, the PBMC population is obtained from an individual receiving treatment. In some embodiments, MASCT further comprises administering an effective amount of an immune checkpoint inhibitor to an individual. In some embodiments, activated T cells and immune checkpoint inhibitors are administered simultaneously, such as with the same composition. In some embodiments, activated T cells and immune checkpoint inhibitors are administered sequentially.

[0228] In some embodiments, the MASCT comprises: (a) co-culturing a population of dendritic cells (DCs) loaded with multiple tumor antigen peptides with a population of T cells in an initial co-culture medium to provide a co-culture, the initial co-culture medium containing one or more cytokines (e.g., multiple cytokines) and an immune checkpoint inhibitor; (b) adding an anti-CD3 antibody to the co-culture approximately 3 to 7 days after the start of the co-culture to obtain the activated T cell population; and (c) administering an effective amount of the activated T cells to the individual. In some embodiments, the multiple cytokines include IL-2, IL-7, IL-15, and IL-21. In some embodiments, IL-2 is present in the initial co-culture medium at a concentration of at least about 500 IU / mL. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is present in the initial co-culture medium at a concentration of at least about 10 μg / mL. In some embodiments, the anti-CD3 antibody is added to the co-culture approximately 5 days after the start of the co-culture. In some embodiments, a population of dendritic cells (DCs) carrying multiple tumor antigen peptides is co-cultured with a population of T cells in the presence of an anti-CD3 antibody for at least about 10 days. In some embodiments, the T cell population is present in a population of PBMCs. In some embodiments, the DC and T cell populations are obtained from an individual receiving treatment. In some embodiments, activated T cells are administered to the individual at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the method further comprises administering an effective amount of DCs carrying the multiple tumor antigen peptides to the individual. In some embodiments, DCs carrying multiple tumor antigen peptides are administered at least three times. In some embodiments, DCs carrying multiple tumor antigen peptides are administered subcutaneously, intradermally, or intravenously.

[0229] In some embodiments, the MASCT comprises: a) contacting a population of dendritic cells (DCs) with a plurality of tumor antigen peptides to obtain DCs loaded with the plurality of tumor antigen peptides; b) culturing the DCs loaded with the plurality of tumor antigen peptides in a DC maturation medium containing MPLA; c) co-culturing the DCs loaded with the plurality of tumor antigen peptides with a population of T cells to obtain a population of activated T cells; and d) administering an effective amount of the activated T cells to the individual. In some embodiments, step c) comprises co-culturing the DCs loaded with the plurality of tumor antigen peptides with the population of T cells in a co-culture medium containing a mixture of interleukins, an immune checkpoint inhibitor, and an anti-CD3 antibody. In some embodiments, the DCs loaded with the plurality of tumor antigen peptides and the population of T cells are co-cultured in the presence of the anti-CD3 antibody for at least about 10 days. In some embodiments, the DC maturation medium contains INFγ and MPLA. In some embodiments, the DC maturation medium further contains PGE2. In some embodiments, MPLA is present in the DC maturation medium at a concentration of at least about 0.5 μg / mL. In some embodiments, INFγ is present in the DC maturation medium at a concentration of at least about 100 IU / mL. In some embodiments, PGE2 is present in the DC maturation medium at a concentration of at least about 0.1 μg / mL. In some embodiments, a plurality of cytokines include IL-2, IL-7, IL-15, and IL-21. In some embodiments, IL-2 is present in the co-culture medium at a concentration of at least about 500 IU / mL. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is present in the co-culture medium at a concentration of at least about 10 μg / mL. In some embodiments, a population of DCs carrying a plurality of tumor antigen peptides and a population of T cells are co-cultured in the presence of an anti-CD3 antibody for at least about 10 days. In some embodiments, the T cell population is present in a population of PBMCs. In some embodiments, the DC population and the T cell population are obtained from an individual receiving treatment. In some embodiments, activated T cells are administered to the individual at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, the method further comprises administering an effective amount of DCs carrying the plurality of tumor antigen peptides to the individual. In some embodiments, a dendritic cell (DC) carrying multiple tumor antigen peptides is administered at least three times. In some embodiments, the DC carrying multiple tumor antigen peptides is administered subcutaneously, intradermally, or intravenously.

[0230] In some embodiments, the MASCT comprises: a) contacting a population of dendritic cells (DCs) with a plurality of tumor antigen peptides to obtain DCs loaded with the plurality of tumor antigen peptides; b) co-culturing the DCs loaded with the plurality of tumor antigen peptides with a population of T cells in an initial co-culture medium containing one or more cytokines (e.g., a plurality of cytokines) and an immune checkpoint inhibitor; c) adding an anti-CD3 antibody to the co-culture approximately 3 to 7 days after the start of the co-culture to obtain the activated T cell population; and d) administering an effective amount of the activated T cells to the individual. In some embodiments, step (a) further comprises culturing the DCs loaded with the plurality of tumor antigen peptides in a DC maturation medium containing a TLR receptor (TLR) agonist. In some embodiments, the TLR agonist is selected from the group consisting of MPLA, Poly I:C, rethimod, gademod, and CL075. In some embodiments, the DC maturation medium contains PGE2. In some embodiments, a plurality of cytokines include IL-2, IL-7, IL-15, and IL-21. In some embodiments, IL-2 is present in the initial co-culture medium at a concentration of at least about 500 IU / mL. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is present in the initial co-culture medium at a concentration of at least about 10 μg / mL. In some embodiments, an anti-CD3 antibody is added to the co-culture approximately 5 days after the start of co-culture. In some embodiments, a population of dendritic cells (DCs) carrying a plurality of tumor antigen peptides is co-cultured with a population of T cells in the presence of an anti-CD3 antibody for at least about 10 days. In some embodiments, the DCs and T cells are obtained from an individual receiving treatment. In some embodiments, activated T cells are administered to the individual at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, the method further comprises administering an effective amount of DCs carrying the plurality of tumor antigen peptides to the individual. In some embodiments, DCs carrying the plurality of tumor antigen peptides are administered at least three times. In some embodiments, DCs carrying multiple tumor antigen peptides are administered subcutaneously, intradermally, or intravenously.

[0231] In some embodiments, the MASCT comprises: a) contacting a population of dendritic cells (DCs) with a plurality of tumor antigen peptides to obtain DCs loaded with the plurality of tumor antigen peptides; b) culturing the DCs loaded with the plurality of tumor antigen peptides in a DC maturation medium containing MPLA; c) co-culturing the DCs loaded with the plurality of tumor antigen peptides with a population of T cells in an initial co-culture medium containing one or more cytokines (e.g., a plurality of cytokines) and an immune checkpoint inhibitor; d) adding an anti-CD3 antibody to the co-culture to obtain the activated T cell population; and e) administering an effective amount of the activated T cells to the individual. In some embodiments, the anti-CD3 antibody is added to the co-culture at the start of the co-culture. In some embodiments, the anti-CD3 antibody is added to the co-culture after the start of the co-culture. In some embodiments, the DC maturation medium contains INFγ and MPLA. In some embodiments, the DC maturation medium further contains PGE2. In some embodiments, MPLA is present in the DC maturation medium at a concentration of at least about 0.5 μg / mL. In some embodiments, INFγ is present in the DC maturation medium at a concentration of at least about 100 IU / mL. In some embodiments, PGE2 is present in the DC maturation medium at a concentration of at least about 0.1 μg / mL. In some embodiments, a plurality of cytokines include IL-2, IL-7, IL-15, and IL-21. In some embodiments, IL-2 is present in the initial co-culture medium at a concentration of at least about 500 IU / mL. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the anti-PD-1 antibody is present in the initial co-culture medium at a concentration of at least about 10 μg / mL. In some embodiments, DC populations loaded with a plurality of tumor antigen peptides are co-cultured with T cell populations in the presence of anti-CD3 antibodies for at least about 10 days. In some embodiments, the DC populations and T cell populations are obtained from individuals receiving treatment. In some embodiments, activated T cells are administered to the individual at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, the method further comprises administering an effective amount of a DC carrying the plurality of tumor antigen peptides to the individual. In some embodiments, the DC carrying the plurality of tumor antigen peptides is administered at least three times. In some embodiments, the DC carrying the plurality of tumor antigen peptides is administered subcutaneously, intradermally, or intravenously.

[0232] In some embodiments, the MASCT comprises: a) contacting a population of dendritic cells (DCs) with a plurality of tumor antigen peptides to obtain DCs loaded with the plurality of tumor antigen peptides; b) culturing the DCs loaded with the plurality of tumor antigen peptides in a DC maturation medium containing MPLA, INFγ, and PGE2; c) co-culturing the DCs loaded with the plurality of tumor antigen peptides with a population of T cells in an initial co-culture medium containing a plurality of cytokines and an anti-PD-1 antibody, the plurality of cytokines including IL-2, IL-7, IL-15, and IL-21; d) adding an anti-CD3 antibody to the co-culture approximately 3 to 7 days (e.g., approximately 5 days) after the start of the co-culture to obtain the activated T cell population; and e) administering an effective amount of the activated T cells to the individual. In some embodiments, MPLA is present in the DC maturation medium at a concentration of at least approximately 0.5 μg / mL. In some embodiments, INFγ is present in the DC maturation medium at a concentration of at least about 100 IU / mL. In some embodiments, PGE2 is present in the DC maturation medium at a concentration of at least about 0.1 μg / mL. In some embodiments, IL-2 is present in the initial co-culture medium at a concentration of at least about 500 IU / mL. In some embodiments, anti-PD-1 antibody is present in the initial co-culture medium at a concentration of at least about 10 μg / mL. In some embodiments, a population of DCs carrying multiple tumor antigen peptides is co-cultured with a population of T cells in the presence of an anti-CD3 antibody for at least about 10 days. In some embodiments, the DC population and the T cell population are obtained from an individual receiving treatment. In some embodiments, activated T cells are administered to the individual at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, the method further comprises administering an effective amount of DCs carrying the multiple tumor antigen peptides to the individual. In some embodiments, DCs carrying multiple tumor antigen peptides are administered at least three times. In some embodiments, DCs carrying multiple tumor antigen peptides are administered subcutaneously, intradermally, or intravenously.

[0233] Generally, the dosage, duration, and route of administration of the activated T cells and dendritic cells (DCs) carrying multiple tumor antigen peptides described herein can be determined based on the individual's size and condition, and according to standard medical practice. Illustrative routes of administration include intravenous, intra-arterial, intraperitoneal, intrapulmonary, intravesicular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, or percutaneous administration. In some embodiments, DCs carrying multiple tumor antigen peptides are administered subcutaneously. In some embodiments, activated T cells are administered intravenously.

[0234] The cell dose administered to an individual may vary depending on factors such as the specific type of cells administered, the route of administration, and the specific type and stage of cancer being treated. The dose should be sufficient to produce the desired response, such as a therapeutic response to cancer, without serious toxicity or adverse events. In some embodiments, the amount of activated T cells or dendritic cells (DCs) to be administered is a therapeutically effective dose. In some embodiments, the amount of cells (such as multiantigen-loaded DCs or activated T cells) is sufficient to reduce tumor size, cancer cell number, or tumor growth rate, with a difference of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, compared to the corresponding tumor size, cancer cell number, or tumor growth rate in the same individual before treatment, or compared to the corresponding activity in other untreated individuals. Standard methods may be used to measure the magnitude of this effect, such as in vitro assays using purified enzymes, cell-based assays, animal models, or human trials.

[0235] In some embodiments, antigen-loaded dendritic cell lines are administered at a dose of at least about any of the following: 1×10⁵, 5×10⁵, 1×10⁶, 1.5×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶, 7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, or 5×10⁷ cells / individual. In some embodiments, antigen-loaded dendritic cell lines are administered at a dose of about any of the following: 1×10⁵ to 5×10⁵, 5×10⁵ to 1×10⁶, 1×10⁶ to 2×10⁶, 2×10⁶ to 3×10⁶, 3×10⁶ to 4×10⁶, 4×10⁶ to 5×10⁶, 5×10⁶ to 6×10⁶, 6×10⁶ to 7×10⁶, 7×10⁶ to 8×10⁶, 8×10⁶ to 1×10⁸, 1×10⁶ to 3×10⁶, 3×10⁶ to 5×10⁶, 5×10⁶ to 7×10⁶, 2×10⁶ to 2×10⁷, 5×10⁶ to 2×10⁷, or 1×10⁶ to 2×10⁷ cells / individual. In some embodiments, antigen-loaded dendritic cell lines are administered at a dose of at least about 1×10⁶ cells / individual. In some embodiments, antigen-loaded dendritic cell lines are administered at a dose of about 1.5 × 10⁶ to about 1.5 × 10⁷ cells / individual.

[0236] In some embodiments, the antigen-loaded dendritic cell line is administered at a dose of at least about any of the following: 1×10⁴, 2.5×10⁴, 5×10⁴, 1×10⁵, 2×10⁵, 2.5×10⁵, 4×10⁵, 6×10⁵, 8×10⁵, 1×10⁶, 2×10⁶, or 1×10⁷ cells / kg. In some embodiments, the antigen-loaded dendritic cell line is administered at a dose of about any of the following: 1×10⁴ to 5×10⁴, 5×10⁴ to 1×10⁵, 1×10⁵ to 2×10⁵, 2×10⁵ to 4×10⁵, 4×10⁵ to 6×10⁵, 6×10⁵ to 8×10⁵, 8×10⁵ to 1×10⁶, 1×10⁶ to 2×10⁶, 2×10⁶ to 1×10⁷, 1×10⁴ to 1×10⁵, 1×10⁵ to 1×10⁶, 1×10⁶ to 1×10⁷, 1×10⁴ to 1×10⁶, or 1×10⁵ to 1×10⁷ cells / kg. In some embodiments, the antigen-loaded dendritic cell line is administered at a dose of at least about 2×10⁵ cells / kg. In some embodiments, the antigen-loaded dendritic cell line is administered at a dose of about 2.5 × 10⁴ to about 2.5 × 10⁵ cells / kg.

[0237] In some embodiments, the activated T cell line is administered at a dose of at least about any of the following: 1×10⁸, 5×10⁸, 1×10⁹, 2×10⁹, 3×10⁹, 4×10⁹, 5×10⁹, 6×10⁹, 7×10⁹, 8×10⁹, 9×10⁹, 1×10¹⁰, 1.5×10¹⁰, 2×10¹⁰, or 5×10¹⁰ cells / individual. In some embodiments, the activated T cell line is administered at a dose of about any of the following: 1×10⁸ to 5×10⁸, 5×10⁸ to 1×10⁹, 1×10⁹ to 5×10⁹, 5×10⁹ to 1×10¹⁰, 3×10⁹ to 7×10⁹, 1×10¹⁰ to 2×10¹⁰, or 1×10⁹ to 1×10¹⁰ cells / individual. In some embodiments, the activated T cell line is administered at a dose of at least about 3 × 10⁹ cells / individual. In some embodiments, the activated T cell line is administered at a dose of about 1 × 10⁹ to about 1 × 10¹⁰ cells / individual.

[0238] In some embodiments, activated T cell lines are administered at a dose of at least about any of the following: 1×10⁷, 2×10⁷, 4×10⁷, 6×10⁷, 8×10⁷, 1×10⁸, 2×10⁸, 4×10⁸, 6×10⁸, 8×10⁸, or 1×10⁹ cells / kg. In some embodiments, activated T cell lines are administered at a dose of about any of the following: 1×10⁷ to 1×10⁸, 1×10⁷ to 5×10⁷, 2×10⁷ to 4×10⁷, 5×10⁷ to 1×10⁸, 1×10⁸ to 2×10⁸, 5×10⁷ to 1×10⁸, 1×10⁸ to 2×10⁸, 2×10⁸ to 5×10⁸, 1×10⁸ to 1×10⁹, or 1×10⁷ to 1×10⁹ cells / kg. In some embodiments, the activated T cell line is administered at a dose of at least about 6 × 10⁷ cells / kg. In some embodiments, the activated T cell line is administered at a dose of about 1.5 × 10⁷ to about 2 × 10⁸ cells / kg.

[0239] MASCT can be used as a monotherapy or in combination with another agent. For example, any of the treatments described herein can be combined with the administration of one or more immune checkpoint inhibitors (such as 1, 2, 3, 4, or more). In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of inhibitors of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, and LAG-3.

[0240] In some embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody. Exemplary anti-PD-1 antibodies include, but are not limited to, nivorumab, pembrolizumab, pilizumab, BMS-936559, and atezolizumab, pembrolizumab, MK-3475, AMP-224, AMP-514, STI-A1110, and TSR-042. In some embodiments, the immune checkpoint inhibitor is nivorumab (e.g., OPDIVO®). In some embodiments, the immune checkpoint inhibitor is pembrolizumab (e.g., KEYTRUDA®). In some embodiments, the immune checkpoint inhibitor is SHR-1210.

[0241] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1. In some embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody. Exemplary anti-PD-L1 antibodies include, but are not limited to, KY-1003, MCLA-145, RG7446, BMS935559, MPDL3280A, MEDI4736, avelumab, or STI-A1010.

[0242] In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody. Exemplary anti-CTLA-4 antibodies include, but are not limited to, ipilimumab, trimemumab, and KAHR-102. In some embodiments, the immune checkpoint inhibitor is ipilimumab (e.g., YERVOY®).

[0243] In some embodiments, activated T cells and immune checkpoint inhibitors are administered simultaneously. In some embodiments, activated T cells and immune checkpoint inhibitors are administered as a single component. In some embodiments, the immune checkpoint inhibitor is present in a first, second, or third co-culture. In some embodiments, activated T cells and immune checkpoint inhibitors are mixed prior to administration (e.g., immediately prior to administration). In some embodiments, activated T cells and immune checkpoint inhibitors are administered simultaneously via different components.

[0244] In some embodiments, activated T cells and immune checkpoint inhibitors are administered sequentially. In some embodiments, the immune checkpoint inhibitor is administered before the activated T cells. In some embodiments, the immune checkpoint inhibitor is administered after the activated T cells.

[0245] Exemplary routes of administration for immune checkpoint inhibitors include, but are not limited to, intratumoral, intrabladder, intramuscular, intraperitoneal, intravenous, intraarterial, intracranial, intrapleural, subcutaneous, and epidermal routes, or delivery to lymph nodes, body spaces, organs, or tissues known to contain such live cancer cells. In some embodiments, immune checkpoint inhibitors are administered intravenously. In some embodiments, immune checkpoint inhibitors are administered by infusion. In some embodiments, immune checkpoint inhibitors are infused over at least about 10 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, or longer. In some embodiments, immune checkpoint inhibitors are administered via the same route of administration as activated T cells. In some embodiments, immune checkpoint inhibitors are administered via a different route of administration than activated T cells.

[0246] Suitable doses of immune checkpoint inhibitors include, but are not limited to, any of the following: 1 mg / m2, 5 mg / m2, 10 mg / m2, 20 mg / m2, 50 mg / m2, 100 mg / m2, 200 mg / m2, 300 mg / m2, 400 mg / m2, 500 mg / m2, 750 mg / m2, 1000 mg / m2, or greater. In some embodiments, the dose of the immune checkpoint inhibitor is any of the following: about 1 to about 5 mg / m2, about 5 to about 10 mg / m2, about 10 to about 20 mg / m2, about 20 to about 50 mg / m2, about 50 to about 100 mg / m2, about 100 mg / m2 to about 200 mg / m2, about 200 to about 300 mg / m2, about 300 to about 400 mg / m2, about 400 to about 500 mg / m2, about 500 to about 750 mg / m2, or about 750 to about 1000 mg / m2. In some embodiments, the dose of the immune checkpoint inhibitor is approximately any of the following: 1 μg / kg, 2 μg / kg, 5 μg / kg, 10 μg / kg, 20 μg / kg, 50 μg / kg, 0.1 mg / kg, 0.2 mg / kg, 0.3 mg / kg, 0.4 mg / kg, 0.5 mg / kg, 1 mg / kg, 2 mg / kg, 5 mg / kg, 10 mg / kg, 20 mg / kg, 50 mg / kg, 100 mg / kg, or greater. In some embodiments, the dose of the immune checkpoint inhibitor is any of the following: about 1 μg / kg to about 5 μg / kg, about 5 μg / kg to about 10 μg / kg, about 10 μg / kg to about 50 μg / kg, about 50 μg / kg to about 0.1 mg / kg, about 0.1 mg / kg to about 0.2 mg / kg, about 0.2 mg / kg to about 0.3 mg / kg, about 0.3 mg / kg to about 0.4 mg / kg, about 0.4 mg / kg to about 0.5 mg / kg, about 0.5 mg / kg to about 1 mg / kg, about 1 mg / kg to about 5 mg / kg, about 5 mg / kg to about 10 mg / kg, about 10 mg / kg to about 20 mg / kg, about 20 mg / kg to about 50 mg / kg, about 50 mg / kg to about 100 mg / kg, or about 1 mg / kg to about 100 mg / kg.

[0247] In some embodiments, immune checkpoint inhibitors are administered daily. In some embodiments, immune checkpoint inhibitors are administered at least once, twice, three times, four times, five times, six times, or seven times per week (i.e., daily). In some embodiments, immune checkpoint inhibitors are administered weekly. In some embodiments, immune checkpoint inhibitors are administered weekly without interruption; weekly (two out of three weeks); weekly (three out of four weeks); every two weeks; every three weeks; every four weeks; every six weeks; every eight weeks; monthly; or every two to twelve months. In some embodiments, the interval between administrations is less than about any of the following: 6 months, 3 months, 1 month, 20 days, 15 days, 12 days, 10 days, 9 days, 8 days, 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the interval between administrations is more than about any of the following: 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 8 months, or 12 months. In some embodiments, immune checkpoint inhibitors are administered every 3 months. In some embodiments, the dosing schedule is uninterrupted. In some embodiments, the interval between each administration is no more than about one week. In some embodiments, immune checkpoint inhibitors are administered at the same dosing schedule as activated T cells. In some embodiments, immune checkpoint inhibitors are administered at a different dosing schedule than activated T cells.

[0248] In some embodiments, immune checkpoint inhibitors are administered in each MASCT treatment cycle. For example, an immune checkpoint inhibitor may be administered approximately 1, 2, 3, 4, 5, 6, or more times per MASCT treatment cycle. In some embodiments, immune checkpoint inhibitors are not administered in each MASCT treatment cycle. For example, an immune checkpoint inhibitor may be administered approximately once every 1, 2, 3, 4, 5, or more MASCT treatment cycles.

[0249] Immune checkpoint inhibitors may be administered for extended periods (such as from about one month to up to about seven years). In some embodiments, immune checkpoint inhibitors are administered for at least one of the following periods: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 18, 24, 30, 36, 48, 60, 72, or 84 months. In some embodiments, immune checkpoint inhibitors are administered as a single dose. In some embodiments, immune checkpoint inhibitors are administered repeatedly. In some embodiments, immune checkpoint inhibitors are administered repeatedly until disease progression.

[0250] In some embodiments, MASCT is particularly suitable for individuals with a low total mutational burden in their cancer. In some embodiments, MASCT is particularly suitable for individuals with a low mutational burden in cancer-related genes in their cancer. In some embodiments, MASCT is particularly suitable for individuals with a low mutational burden in T-cell response-related immune genes in their cancer. In some embodiments, MASCT is particularly suitable for individuals with a low mutational burden in MHC genes in their cancer. Mutational burden may refer to the mutational burden of all cancer cells, or a subset of cancer cells, such as cells in primary or metastatic tumor sites, for example, cells in a tumor biopsy sample.

[0251] In some embodiments, a low mutational load of one or more genes refers to a low number of accumulated mutations in those one or more genes. In some embodiments, a low mutational load is indicated by the total number of mutations not exceeding any one of about 500, 400, 300, 200, 100, 50, 40, 30, 20, 10, 5, or fewer. In some embodiments, a low mutational load of one or more MHC genes is indicated by mutations not exceeding any one of about 50, 40, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In some embodiments, a low mutational load of one or more genes is a low ratio between the number of accumulated mutations in those one or more genes (such as MHC genes) and the total number of mutations in a selected set of genes (such as cancer-related genes) or the entire genome.

[0252] In some embodiments, one or more MHC genes comprise MHC class I genes (or loci). In some embodiments, one or more MHC genes comprise MHC class II genes (or loci). In some embodiments, in a particular human individual, one or more MHC genes are selected from the group consisting of HLA-A, HLA-B, HLA-C, and B2M.

[0253] Exemplary mutations include, but are not limited to, deletions, frameshifts, insertions, indels, missense mutations, meaningless mutations, point mutations, copy number variations, single nucleotide variations (SNVs), silencing mutations, splice site mutations, splice variants, gene fusions, and translocations. In some embodiments, copy number variations in MHC genes are caused by structural rearrangements of the genome, including deletions, duplications, inversions, and translocations of chromosomes or segments thereof. In some embodiments, mutations in one or more MHC genes are selected from point mutations, frameshift mutations, gene fusions, and copy number variations. In some embodiments, the mutation is located in the protein-coding region of an MHC gene. In some embodiments, the mutation is a nonsynonymous mutation. In some embodiments, the mutation is not polymorphic. In some embodiments, the mutation is present in normal cells of an individual. In some embodiments, the mutation is not present in normal cells of an individual. In some embodiments, the mutation affects the physiological, chemical, or functional properties of the MHC molecule encoded by the affected gene, such as stability or binding affinity. In some embodiments, the mutation results in an irreversible defect in the MHC molecule. In some embodiments, the mutation reduces the binding affinity of the MHC molecule to T cell epitopes and / or T cell receptors. In some embodiments, the mutation is a loss-of-function mutation. In some embodiments, the mutation results in a reversible defect in the MHC molecule. In some embodiments, the mutation does not affect the binding affinity of the MHC molecule to T cell epitopes and / or T cell receptors. In some embodiments, the mutation is a somatic mutation. In some embodiments, the mutation is a germline mutation.

[0254] The mutations included in the mutational burden may be present in all cancer cells or in a subset of cancer cells. In some embodiments, the mutation is present in all cancer cells of an individual. In some embodiments, the mutation is present in all cancer cells at the tumor site. In some embodiments, the mutation is proliferative. In some embodiments, the mutation is subproliferative. In some embodiments, the mutation is present in at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more of the cancer cells of an individual.

[0255] Mutations in certain MHC genes and / or certain domains or locations of one or more MHC genes may have a more profound impact on an individual's clinical response to the treatments described herein. For example, loss-of-function mutations can occur in the leader peptide sequence, α3 domain (which binds to the CD8 coreceptor on T cells), α1 peptide-binding domain, or α2 peptide-binding domain of HLA molecules; see, for example, Shukla S. et al. Nature Biotechnology 33, 1152-1158 (2015), which is incorporated herein by reference. Mutations in the B2M (β2-macroglobulin) gene can also initiate a tumor escape phenotype. See, for example, Monica B et al. Cancer Immunol. Immu., (2012) 61: 1359-1371. In some embodiments, the presence of any number (such as 1, 2, 3, 4, 5, or more) of mutations in functional regions of one or more MHC genes, such as leader peptide sequences, α1 domains, α2 domains, or α3 domains, indicates a high mutational burden. In some embodiments, the presence of any number (e.g., 1, 2, 3, 4, 5, or more) of loss-of-function mutations in one or more MHC genes (such as HLA-A, HLA-B, or HLA-C genes in human individuals) indicates a high mutational load. In some embodiments, a low mutational load in one or more MHC genes does not contain mutations in functional regions including the leader peptide sequence, α1 domain (e.g., residues directly contacting the CD8 coreceptor), α2 domain, and α3 domain (e.g., residues directly contacting epitopes) of the one or more MHC genes. In some embodiments, the presence of any number of mutations (such as loss-of-function mutations) in a B2M gene indicates a high mutational load. In some embodiments, a low mutational load in one or more MHC genes does not contain mutations in the B2M gene.

[0256] The mutation load of one or more genes (such as MHC genes) can be determined by any known method in the art, including but not limited to genomic DNA sequencing, exon sequencing, or other DNA sequencing-based methods using Sanger sequencing or next-generation sequencing platforms; polymerase chain reaction assays; in situ hybridization assays; and DNA microarrays.

[0257] In some embodiments, mutational burden of one or more MHC genes is determined by sequencing tumor samples from an individual. In some embodiments, the sequencing is next-generation sequencing. In some embodiments, the sequencing is whole-genome sequencing. In some embodiments, the sequencing is exon sequencing, such as whole exome sequencing (WES). In some embodiments, the sequencing is RNA sequencing. In some embodiments, the sequencing is targeted sequencing of candidate genes (such as cancer-related genes plus HLA genes). For example, ONCOGXONE™ Plus (Admera Health) can be used to sequence cancer-related genes and HLA loci with high sequencing depth. In some embodiments, the same sequencing data can be used to determine mutational burden of one or more MHC genes and identify neoantigens in an individual.

[0258] In some embodiments, the tumor sample is a tissue sample. In some embodiments, the tumor sample is a tumor biopsy sample, such as tumor cells obtained by fine-needle aspiration or tumor cells obtained by laparoscopy (e.g., including tumor stroma). In some embodiments, the tumor sample is freshly obtained. In some embodiments, the tumor sample is frozen. In some embodiments, the tumor sample is a formaldehyde fixed-paraffin embedded (FFPE) sample. In some embodiments, the tumor sample is a cell sample. In some embodiments, the tumor sample contains circulating metastatic cancer cells. In some embodiments, the tumor sample is obtained by classifying circulating tumor cells (CTCs) from the blood. In some embodiments, nucleic acids (such as DNA and / or RNA) are extracted from the tumor sample for sequencing analysis. In some embodiments, the sequencing data of the tumor sample is compared with the sequencing data of a reference sample (such as a healthy tissue sample from the same individual, or a sample from a healthy individual) to identify mutations and determine the mutational load in the tumor cells. In some embodiments, the sequencing data of the tumor sample is compared with a reference sequence from a genomic database to identify mutations and determine the mutational load in the tumor cells.

[0259] Any MASCT method may include one or more neoantigen peptides used among a plurality of tumor antigen peptides. In some embodiments, the MASCT further includes the step of selecting an individual for the treatment method based on the presence of one or more (such as at least 5) neoantigens in the individual, and / or the following steps: (i) identifying the neoantigen of the individual; and (ii) incorporating the neoantigen peptide derived from the neoantigen into the plurality of tumor antigen peptides for use in the treatment method.

[0260] In some embodiments, the MASCT includes: (a) recognizing a neoantigen of the individual; (b) incorporating a neoantigen peptide into a plurality of tumor antigen peptides, wherein the neoantigen peptide contains a novel epitope of the neoantigen; (c) optionally administering an effective amount of DCs loaded with the plurality of tumor antigen peptides; (d) preparing an activated T cell population by co-culturing antigen-loaded DCs with a T cell population; and (e) administering an effective amount of activated T cells to the individual, wherein the individual has one or more neoantigens.

[0261] Individuals may possess any number (such as at least about 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 100, or more) of neoantigens to benefit from the MASCT method, which uses multiple tumor antigen peptides comprising neoantigen peptides. In some embodiments, the MASCT method is particularly suitable for individuals possessing at least about 4, 5, 6, 7, 8, 10, 15, 20, 50, 100, or more neoantigens. In some embodiments, the neoantigen comprises one or more novel epitopes. In some embodiments, the MASCT method is particularly suitable for individuals possessing at least about 4, 5, 6, 7, 8, 10, 15, 20, 50, 100, or more novel epitopes. In some embodiments, the T-cell epitope is an MHC-I restricted epitope. In some embodiments, the novel epitope has a higher affinity for the individual's MHC molecules compared to the corresponding wild-type T-cell epitope. In some embodiments, the novel epitope exhibits higher affinity for model T cell receptors compared to the corresponding wild-type T cell epitope. In some embodiments, the neoantigen (or neoepitaxy) is a colony-specific neoantigen. In some embodiments, the neoantigen (or neoepitaxy) is a subcolony-specific neoantigen. In some embodiments, the neoantigen (or neoepitaxy) is present in tumor cells of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more in an individual.

[0262] The number of neoantigens can be combined with other biomarkers or selection criteria to select individuals for any of the MASCT methods described herein. In some embodiments, the MASCT method is particularly suitable for individuals who have a low mutational burden in cancer cells (such as in one or more MHC genes) and / or have at least about 4, 5, 6, 7, 8, 10, or more neoantigens (such as neoantigens with high-affinity MHC-I-restricted neoepitopes).

[0263] Any number (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) of neoantigen peptides can be designed based on an individual's neoantigens and incorporated into a plurality of tumor antigen peptides for any of the treatments described herein. In some embodiments, the plurality of tumor antigen peptides comprises a single neoantigen peptide. In some embodiments, the plurality of tumor antigen peptides comprises a plurality of neoantigen peptides. Each neoantigen peptide may contain one or more novel epitopes derived from the individual's neoantigen. In some embodiments, the novel epitopes are T-cell epitopes. The method for designing neoantigen peptides based on neoantigens is described in the section "A Plurality of Tumor Antigen Peptides".

[0264] Neoantigens in an individual can be identified using any method known in the art. In some embodiments, neoantigens are identified based on the genetic profile of a tumor sample from the individual. Each neoantigen contains one or more novel epitopes. In some embodiments, one or more novel epitopes in a neoantigen are identified based on the genetic profile of a tumor sample. Any known gene profiling method, such as next-generation sequencing (NGS), microarrays, or proteomics methods, can be used to provide the genetic profile of a tumor sample.

[0265] In some embodiments, neoantigens are identified by sequencing tumor samples from an individual. In some embodiments, the sequencing is next-generation sequencing. In some embodiments, the sequencing is whole-genome sequencing. In some embodiments, the sequencing is exon sequencing, such as whole exome sequencing (WES). In some embodiments, the sequencing is RNA sequencing. In some embodiments, the sequencing is targeted sequencing of candidate genes, such as cancer-related genes. Many commercially available NGS cancer sequences (e.g., ONCOGXONE™ Plus (Admera Health)) are available for sequencing cancer-related genes and offer high sequencing depth.

[0266] In some embodiments, the tumor sample is a tissue sample. In some embodiments, the tumor sample is a tumor biopsy sample, such as tumor cells obtained by fine-needle aspiration or tumor cells obtained by laparoscopy (e.g., including tumor stroma). In some embodiments, the tumor sample is freshly obtained. In some embodiments, the tumor sample is frozen. In some embodiments, the tumor sample is a formaldehyde fixed-paraffin embedded (FFPE) sample. In some embodiments, the tumor sample is a cell sample. In some embodiments, the tumor sample contains circulating metastatic cancer cells. In some embodiments, the tumor sample is obtained by sorting circulating tumor cells (CTCs) from the blood. In some embodiments, nucleic acids (such as DNA and / or RNA) are extracted from the tumor sample for sequencing analysis. In some embodiments, proteins are extracted from the tumor sample for sequencing analysis.

[0267] In some embodiments, the gene profile of a tumor sample is compared with the gene profile of a reference sample (such as a healthy tissue sample from the same individual, or a sample from a healthy individual) to identify candidate mutated genes in tumor cells. In some embodiments, the gene profile of a tumor sample is compared with a reference sequence from a genome database to identify candidate mutated genes in tumor cells. In some embodiments, the candidate mutated genes are cancer-related genes. In some embodiments, each candidate mutated gene includes one or more mutations, such as nonsynonymous substitutions, insertions or deletions, or gene fusions, which can produce neoantigens. Common single nucleotide polymorphisms (SNPs) are excluded from the candidate mutations.

[0268] In some embodiments, novel epitopes in neoantigens are identified from candidate mutant proteins. In some embodiments, novel epitopes are predicted using computer simulations. Exemplary bioinformatics tools for T-cell epitope prediction are known in the art, for example, see Yang X. and Yu X. (2009), “An introduction to epitope prediction methods and software”, Rev. Med. Virol. 19(2):77-96. Factors considered in T-cell epitope prediction algorithms include, but are not limited to, an individual’s MHC subtype, the sequence-derived physiochemical properties of the T-cell epitope, MHC-binding motifs, proteasome cleavage patterns, transport efficiency of transporters associated with antigen processing (TAPs), MHC binding affinity, peptide-MHC stability, and T-cell receptor binding affinity. In some embodiments, the novel epitope is an MHC-I restricted epitope. In some embodiments, the novel epitope is an MHC-II restricted epitope.

[0269] In some embodiments, the novel epitope has a high affinity for an individual's MHC molecules. In some embodiments, the method further includes determining the individual's MHC subtype (e.g., self-sequencing data determination) to identify one or more MHC molecules of the individual. In some embodiments, the method further includes determining the affinity of the novel epitope for MHC molecules (such as MHC class I molecules). In some embodiments, the method includes determining the affinity of the novel epitope for one or more MHC molecules (such as MHC class I) of the individual. In some embodiments, the affinity of the novel epitope for one or more MHC molecules of the individual is compared with the affinity of a corresponding wild-type epitope for one or more MHC molecules of the individual. In some embodiments, a novel epitope is selected that has a higher affinity (e.g., at least about 1.5, 2, 5, 10, 15, 20, 25, 50, 100, or more times) for one or more MHC molecules (such as MHC-I molecules) of the individual compared to a corresponding wild-type epitope. In some embodiments, MHC binding affinity is predicted by computer simulation using any tools or methods known in the art. In some embodiments, MHC binding affinity is determined experimentally, such as by using in vitro binding assays.

[0270] In some embodiments, the MASCT further includes determining the affinity of a complex for a T-cell receptor, the complex comprising a novel epitope and an MHC molecule (such as an individual's MHC class I molecule). In some embodiments, the affinity of a complex comprising the novel epitope and an MHC molecule for the T-cell receptor is compared to the affinity of a complex comprising a corresponding wild-type epitope and an MHC molecule. In some embodiments, the MHC molecule is derived from the individual. In some embodiments, the T-cell receptor is located on the surface of one or more T cells of the individual. In some embodiments, a novel epitope is selected that has a higher affinity (e.g., at least about 1.5, 2, 5, 10, 15, 20, 25, 50, 100, or more times) for the T-cell receptor model in the complex compared to the corresponding wild-type epitope, the complex comprising the novel epitope and an MHC molecule. In some embodiments, TCR binding affinity is predicted by computer simulation using any tools or methods known in the art. In some embodiments, TCR binding affinity is determined experimentally, for example by determining the T-cell response to the novel epitope.

[0271] In some embodiments, the neoantigen (or neoepitope) is further identified based on its expression level in the tumor sample. Any known mRNA or protein level quantification method in the art, such as RT-PCR, antibody-based assays, and mass spectrometry, can be used to determine the expression level of the neoantigen (or neoepitope). In some embodiments, the expression level of the neoantigen (or neoepitope) is determined from sequencing data of the tumor sample. In some embodiments, the neoantigen (or neoepitope) is expressed in tumor cells at a level of at least about 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 104, or more copies / cell. In some embodiments, the neoantigen (or neoepitope) is expressed in tumor cells at a level more than about 1.5, 2, 5, 10, 20, 50, 100, or more times the corresponding wild-type protein (or corresponding wild-type epitope).

[0272] In some embodiments, a neoantigen peptide is selected or identified by comprising the following steps: (a) sequencing a tumor sample from an individual to identify a neoantigen; (b) identifying a novel epitope in the neoantigen; optionally (c) determining the individual's MHC subtype (e.g., using sequencing data) to identify the individual's MHC molecules; optionally (d) determining the affinity of the novel epitope for the individual's MHC molecules; optionally (e) determining the affinity of a complex for a T-cell receptor, the complex comprising the novel epitope and the MHC molecule; and (f) obtaining a peptide comprising the novel epitope to provide the neoantigen peptide. In some embodiments, the novel epitope has a higher affinity for the individual's MHC molecules (such as MHC-I molecules) than a complex comprising a corresponding wild-type T-cell epitope and an MHC molecule, and / or has a higher affinity for the TCR in a complex comprising the novel epitope and the MHC molecule. In some embodiments, based on the natural sequence of a neoantigen having an epitope, the new epitope is extended at the N-terminus, C-terminus, or both ends to obtain an extended sequence, wherein the extended sequence is adapted to be presented by both class I and class II MHC molecules. Any treatment method described herein using one or more neoantigen peptides may further include one or more neoantigen selection / recognition steps.

[0273] Any treatment methods and MASCT methods described herein can be further incorporated into post-treatment monitoring procedures. Post-treatment monitoring can help adjust an individual's treatment regimen to optimize treatment outcomes.

[0274] For example, the multiple tumor antigen peptides described herein can be adjusted or customized based on an individual's specific immune response to each of the multiple tumor antigen peptides and / or an individual's clinical response to activated T cells, to provide multiple customized tumor antigen peptides that can be used for repeated treatment. In some embodiments, they can be used to prepare a pool of antigen peptides for future pulsed DCs or activated T cells, removing tumor antigen peptides that do not elicit a strong specific immune response.

[0275] Any method known in the art can be used, for example, by measuring the levels of cytotoxic factors (such as perforin or granzyme B) or cytokines (such as IFNγ or TNFα) released from T cells (or PBMCs) after stimulation with an individual tumor antigen peptide, to determine a specific immune response to one or more tumor antigen peptides. Antibody-based assays (such as ELISPOT) can be used to quantify the levels of cytotoxic factors or cytokines (such as IFNγ). In some embodiments, the level of cytokines (such as IFNγ) released from T cells (or PBMCs) in response to the tumor antigen peptide is normalized to a reference (such as baseline cytokines release level, or nonspecific cytokines release from T cells (or PBMCs) in response to an unrelated peptide) to provide a fold change value for cytokines (such as IFNγ). In some embodiments, in the ELISPOT assay, a fold change value for cytokines (such as IFNγ) greater than about 1.2, 1.5, 2, 2.5, 3, 4, 5, 6, 8, 10, or more indicates a strong specific immune response to the tumor antigen peptide. In some embodiments, tumor antigen peptides with fold change values ​​of cytokines (such as IFNγ) less than about 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5, 1.2, or less in the ELISPOT assay are removed from the plurality of tumor antigen peptides to provide a plurality of customized tumor antigen peptides for future treatment.

[0276] Physicians can assess an individual's clinical response to the treatments described herein using methods known in the art, such as imaging techniques, blood tests, biomarker assessments, and biopsies. In some embodiments, clinical response is monitored by determining the number of circulating tumor cells (CTCs) in an individual before and after receiving activated T cells. In some embodiments, CTCs have detached from the primary tumor and are circulating in body fluids. In some embodiments, CTCs have detached from the primary tumor and are circulating in the bloodstream. In some embodiments, CTCs are indicators of metastasis. The number of CTCs can be determined by various methods known in the art, including but not limited to CellSearch, Epic Science, IsoFlux, and maintrac. In some embodiments, the number of individual CTCs (including specific subtypes of CTCs) in an individual's blood sample is determined. In some embodiments, when an individual has more than about 10, 20, 50, 100, 150, 200, 300, or more individual CTCs per mL of blood sample after treatment, it indicates an increased risk of metastasis and / or a poor clinical response to the treatment. In some embodiments, an increase in the number of single CTCs after treatment (such as an increase of at least about 1.5, 2, 3, 4, 5, 10, or more times) in an individual after treatment compared to before treatment indicates a poor clinical response to the treatment. In some embodiments, the number of CTC clusters in an individual's blood sample is determined. In some embodiments, the detection of at least about 1, 5, 10, 50, 100, or more CTC clusters in an individual's blood sample after treatment indicates an increased risk of metastasis and / or a poor clinical response to treatment. In some embodiments, an increase in the number of CTC clusters after treatment (such as an increase of at least about 1.5, 2, 3, 4, 5, 10, or more times) in an individual after treatment compared to before treatment indicates a poor clinical response to treatment. [III. Tumor-specific TCR]

[0277] This document also provides tumor-specific TCRs, which are obtained using any of the methods described in Section II. In some embodiments, the tumor-specific TCR specifically recognizes CEA, RSG-5, or HPV18-E7. Nucleic acids and vectors encoding tumor-specific TCRs, as well as engineered immune cells expressing tumor-specific TCRs, are also within the scope of this application. [Example of tumor-specific TCR]

[0278] The exemplary TCRs identified using the methods described herein are shown in Table 1 below. The V, J, ...

Claims

1. A tumor-specific T-cell receptor (TCR) that specifically binds to a major histocompatibility complex (MHC) / HPV18-E7 epitope complex, the tumor-specific TCR comprising: (1) a TCRα chain comprising: CDR1 containing the amino acid sequence of SEQ ID NO: 278, CDR2 containing the amino acid sequence of SEQ ID NO: 279, and CDR3 containing the amino acid sequence of SEQ ID NO: 70; and a TCRβ chain comprising: CDR1 containing the amino acid sequence of SEQ ID NO: 280, CDR2 containing the amino acid sequence of SEQ ID NO: 281, and CDR3 containing the amino acid sequence of SEQ ID NO: 73; (2) a TCRα chain comprising: CDR1 containing the amino acid sequence of SEQ ID NO: 274, CDR2 containing the amino acid sequence of SEQ ID NO: 275, and CDR3 containing the amino acid sequence of SEQ ID NO:

73. CDR3 of the amino acid sequence NO: 58; and a TCRβ chain comprising: CDR1 of the amino acid sequence SEQ ID NO: 276, CDR2 of the amino acid sequence SEQ ID NO: 277, and CDR3 of the amino acid sequence SEQ ID NO: 61; or (3) a TCRα chain comprising: CDR1 of the amino acid sequence SEQ ID NO: 282, CDR2 of the amino acid sequence SEQ ID NO: 283, and CDR3 of the amino acid sequence SEQ ID NO: 76; and a TCRβ chain comprising: CDR1 of the amino acid sequence SEQ ID NO: 284, CDR2 of the amino acid sequence SEQ ID NO: 285, and CDR3 of the amino acid sequence SEQ ID NO:

79.

2. The tumor-specific TCR of claim 1, comprising a TCRα chain comprising: CDR1 composed of the amino acid sequence of SEQ ID NO: 278, CDR2 composed of the amino acid sequence of SEQ ID NO: 279, and CDR3 composed of the amino acid sequence of SEQ ID NO: 70; and a TCRβ chain comprising: CDR1 composed of the amino acid sequence of SEQ ID NO: 280, CDR2 composed of the amino acid sequence of SEQ ID NO: 281, and CDR3 composed of the amino acid sequence of SEQ ID NO:

73.

3. Tumor-specific TCRs as requested in item 1 or 2, wherein: (1) The HPV18-E7 epitope contains the amino acid sequence of any of SEQ ID NO: 84-86; and / or (2) The MHC is HLA-DRA / DRB1*09:01, HLA-DRA / DRB4*01:03, HLA-DPA1*02:02 / DPB1*05:01, HLA-DPA1*01:03 / DPB1*02:01, HLA-DPA1*01:03 / DPB1*05:01, or HLA-II.

4. The tumor-specific TCR of claim 1, which specifically binds to the major histocompatibility complex (MHC) / HPV18-E7 epitope complex, the tumor-specific TCR comprising: (1) a TCRα chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 59; and a TCRβ chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 62; (2) a TCRα chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 71; and a TCRβ chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 74; or (3) a TCRα chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 74; The amino acid sequence of SEQ ID NO: 77 has at least 80% identity; and the TCRβ chain contains an amino acid sequence that has at least 80% identity with the amino acid sequence of SEQ ID NO:

80.

5. The tumor-specific TCR of claim 3, which specifically binds to the major histocompatibility complex (MHC) / HPV18-E7 epitope complex, the tumor-specific TCR comprising: (1) a TCRα chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 59; and a TCRβ chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 62; (2) a TCRα chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 71; and a TCRβ chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 74; or (3) a TCRα chain comprising an amino acid sequence having at least 80% identity with the amino acid sequence of SEQ ID NO: 74; The amino acid sequence of SEQ ID NO: 77 has at least 80% identity; and the TCRβ chain contains an amino acid sequence that has at least 80% identity with the amino acid sequence of SEQ ID NO:

80.

6. A single nucleic acid encoding the TCRα chain and / or the TCRβ chain of a tumor-specific TCR as claimed in any one of claims 1 to 5.

7. An engineered immune cell comprising a tumor-specific TCR as claimed in any one of claims 1 to 5, wherein the immune cell line is a T cell.

8. A pharmaceutical composition comprising engineered immune cells as claimed in claim 7, and a pharmaceutically acceptable carrier.

9. Use of a pharmaceutical composition as claimed in claim 8, for the preparation of a medicament for treating cancer in an individual.

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