Artificially synthesized tumor infiltrating lymphocytes

Abstract

Compositions and methods for preparing modified cells (e.g., synthetic tumor-infiltrating lymphocytes or TILs) are disclosed. The modified cells may be used to prepare a pharmaceutical composition for administration to a subject in need thereof. The modified cell may be a polyclonal T cell receptor (TCR)-T cell that expresses two or more different subject specific T cell receptors (TCRs).

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims international application No. PCT / CN2022 / 081501, filed on March 17, 2022, the entire contents of which are incorporated herein by reference in their entirety. Background of the Invention

[0002] New treatments for cancer are constantly being developed. Adoptive cell therapies (ACTs) are effective treatments not only for cancer, but also for other diseases such as infection and graft-versus-host disease. Adoptive cell transfer is the passive transfer of cells grown in vitro (most commonly immune-derived cells) into a host with the goal of transferring the immune function and properties of the transplant. Adoptive cell transfer can be autologous, which is common in adoptive T-cell therapy, or allogeneic, which is a typical approach for treating infection or graft-versus-host disease. Clinically, common embodiments of this approach may include transferring immune-promoting cells or tolerogenic cells (such as lymphocytes) to patients to enhance immunity to viruses and cancer or to promote tolerance in autoimmune diseases such as type I diabetes or rheumatoid arthritis.

[0003] Adoptive transfer of tumor infiltrating lymphocytes (TILs), especially autologous TILs, or genetically redirected peripheral blood mononuclear cells has been used to treat patients with advanced solid tumors, such as melanoma, as well as patients with hematological malignancies that express CD19. In ACT, the most commonly used cell type is T cells, sometimes sorted as CD8+, but other variations include CD4+ cells, NK cells, delta-gamma T cells, regulatory T cells, and peripheral blood mononuclear cells. Cells can be unmodified, such as in TIL therapy, or genetically modified. SUMMARY OF THE INVENTION

[0004] However, the efficacy of conventional TIL therapy in moderately immunogenic tumors (e.g., most epithelial tumors) remains inconsistent. It is clear that there is a need to develop improved TIL therapies with improved efficacy to combat various tumors (e.g., solid tumors). New compositions and methods are provided herein to produce modified cells expressing polyclonal subject-specific TCRs for the treatment of cancer. The compositions and methods described herein can provide fast turnaround time and low-cost personalized T cell therapy for subjects in need of solid tumors.

[0005] In one aspect, the present disclosure provides a method for preparing a plurality of synthetic tumor infiltrating lymphocytes (TILs), the method comprising: (a) obtaining a resection or biopsy sample comprising a plurality of T cells from a solid tumor lesion of a subject; (b) sequencing the plurality of T cells of the resection or biopsy sample to obtain naturally paired T cell receptor (TCR) α chain and β chain sequences of TCRs of the plurality of T cells; (c) synthesizing a plurality of polynucleotide sequences encoding the TCR or a subset thereof from the naturally paired TCR α chain and β chain sequences obtained in (b), the plurality of polynucleotide sequences comprising a first polynucleotide sequence encoding a first naturally paired TCR of a first T cell of the plurality of T cells and a second naturally paired TCR of a second T cell of the plurality of T cells, and wherein The first polynucleotide sequence is different from the second polynucleotide sequence; and (d) delivering the multiple polynucleotide sequences or their derivatives into the multiple recipient cells by contacting a mixture comprising the multiple polynucleotide sequences or their derivatives with the multiple recipient cells, thereby producing the multiple synthetic TILs, wherein a first portion of the multiple synthetic TILs exogenously expresses the first polynucleotide sequence encoding the first natural paired TCR, a second portion of the multiple synthetic TILs exogenously expresses the second polynucleotide sequence encoding the second natural paired TCR, and wherein the fraction of TILs in the third portion of the multiple synthetic TILs that exogenously express the first and second polynucleotide sequences is less than the product of the fractions of the first portion and the second portion.

[0006] In some embodiments, the multiple synthetic TILs cells (i) exogenously express only one polynucleotide sequence in the multiple polynucleotide sequences or (ii) do not exogenously express any of the multiple polynucleotide sequences or their derivatives. In some embodiments, none of the multiple synthetic TILs exogenously express the first and second polynucleotide sequences at the same time. In some embodiments, a cell in the multiple synthetic TILs that exogenously express the first polynucleotide sequence does not exogenously express the second polynucleotide sequence. In some embodiments, the method further comprises obtaining the expression levels of at least 10 genes in (b).

[0007] In another aspect, the present patent application discloses a method for preparing a plurality of synthetic tumor infiltrating lymphocytes (TILs), the method comprising: (a) obtaining a resection or biopsy sample containing a plurality of T cells from a solid tumor lesion of a subject; (b) sequencing the plurality of T cells of the resection or biopsy sample to obtain the naturally paired T cell receptor (TCR) α chain and β chain sequences of the TCRs of the plurality of T cells; (c) synthesizing a plurality of polynucleotide sequences encoding the TCRs or a subset thereof from the naturally paired T cell receptor (TCR) α chain and β chain sequences obtained in (b), the plurality of polynucleotide sequences comprising sequences encoding the plurality of (d) delivering the multiple polynucleotide sequences or their derivatives to the multiple recipient cells by contacting a mixture comprising the multiple polynucleotide sequences or their derivatives with the multiple recipient cells, thereby producing the multiple synthetic TILs, wherein the cells of the multiple synthetic TILs that exogenously express the first polynucleotide sequence do not exogenously express the second polynucleotide sequence.

[0008] In some embodiments, a first portion of the plurality of synthetic TILs exogenously expresses the first polynucleotide sequence, a second portion of the plurality of synthetic TILs exogenously expresses the second polynucleotide sequence, and wherein the fraction of the third portion of the plurality of synthetic TILs that exogenously expresses the first and second polynucleotide sequences is less than the product of the fractions of the first portion and the second portion. In some embodiments, the method further comprises obtaining expression levels of at least 10 genes in (b).

[0009] In another aspect, the present patent application discloses a method for preparing a plurality of synthetic tumor infiltrating lymphocytes (TILs), the method comprising: (a) obtaining a resection or biopsy sample containing a plurality of T cells from a solid tumor lesion of a subject; (b) sequencing the plurality of T cells of the resection or biopsy sample to obtain (i) expression levels of at least 10 genes and (ii) naturally paired T cell receptor (TCR) α chain and β chain sequences of the TCRs of the plurality of T cells; (c) synthesizing a plurality of polynucleotide sequences encoding the TCR or a subset thereof from the naturally paired TCR α chain and β chain sequences obtained in (b), the plurality of polynucleotide sequences comprising a first polynucleotide sequence encoding a first naturally paired TCR of a first T cell of the plurality of T cells and a second naturally paired TCR of a second T cell of the plurality of T cells, and wherein the first polynucleotide sequence is different from the second polynucleotide sequence; and (d) delivering the plurality of polynucleotide sequences or their derivatives into a plurality of receptor cells by contacting a mixture comprising the plurality of polynucleotide sequences or their derivatives with a plurality of receptor cells, thereby producing the plurality of synthetic TILs.

[0010] In some embodiments, a first portion of the plurality of synthetic TILs exogenously expresses the first polynucleotide sequence, a second portion of the plurality of synthetic TILs exogenously expresses the second polynucleotide sequence, and wherein the fraction of the third portion of the plurality of synthetic TILs that exogenously express the first and second polynucleotide sequences is less than the product of the fractions of the first portion and the second portion.

[0011] In some embodiments, the cells of the plurality of synthetic TILs (i) exogenously express only one of the plurality of polynucleotide sequences or (ii) do not exogenously express any of the plurality of polynucleotide sequences or their derivatives. In some embodiments, none of the plurality of synthetic TILs exogenously express the first and second polynucleotide sequences.

[0012] In some embodiments, cells of the plurality of synthetic TILs that exogenously express a first polynucleotide sequence do not exogenously express a second polynucleotide sequence.

[0013] In some embodiments, the fraction of the first portion, the second portion, or the third portion is determined by sequencing or pMHC multimer staining. In some embodiments, sequencing comprises single-cell sequencing, and wherein the single-cell sequencing comprises barcoding each cell in a single compartment. In some embodiments, pMHC multimer staining comprises using a plurality of pMHC multimers, each pMHC multimer specifically binding to a TCR encoded by a polynucleotide sequence of the plurality of polynucleotide sequences in (d) or a derivative thereof.

[0014] In some embodiments, the multiple polynucleotide sequences or their derivatives are delivered to the multiple receptor cells in (d) including site-specific knocking of the polynucleotide sequences into the genomic sites of the receptor cells in the multiple receptor cells. In some embodiments, the genomic sites of the receptor cells produce monoallelic expression of the polynucleotide sequences. In some embodiments, the polynucleotide sequences are site-specifically knocked into the frame of the genomic sites of the receptor cells in the multiple receptor cells. In some embodiments, the genomic sites are located on sex chromosomes. In some embodiments, the genomic sites are T cell receptor alpha constant regions (TRAC). In some embodiments, the site-specific knock-in genomic sites in the polynucleotide sequences include using a gene editing method selected from clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector (TALE) nucleases (TALENs), homing endonucleases (meganuclease), zinc finger nucleases (ZFN) or megaTAL nuclease methods.

[0015] In some embodiments, the gene editing method of the polynucleotide sequence site-specific knock-in genome includes using CRISPR method. In some embodiments, the CRISPR method includes delivering a ribonucleoprotein (RNP) complex comprising Cas9 protein and single guide RNA (sgRNA) into a recipient cell. In some embodiments, sgRNA targets TRAC site. In some embodiments, the method of knocking the polynucleotide sequence site-specific knock-in genome includes cutting the genome site using gene editing method, and inserting the polynucleotide sequence into the genome site by homologous recombination.

[0016] In some embodiments, endogenous TCR is inactivated in a receptor cell in multiple receptor cells. In some embodiments, endogenous TCR is knocked down or knocked out in a receptor cell of multiple receptor cells. In some embodiments, endogenous T cell receptor β constant region (TRBC) is inactivated in a receptor cell in multiple receptor cells. In some embodiments, endogenous TRBC gene is knocked down or knocked out in the receptor cells of multiple receptor cells. In some embodiments, endogenous TCR is inactivated in at least about 80% of multiple receptor cells. In some embodiments, endogenous TCR is knocked down or knocked out in at least about 80% of multiple receptor cells. In some embodiments, a gene editing method of a group consisting of clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector (TALE) nucleases (TALENs), homing endonucleases (meganuclease), zinc finger nucleases (ZFN) or megaTAL nucleases is selected to inactivate endogenous TCR.

[0017] In some embodiments, before, simultaneously or after delivering the plurality of polynucleotide sequences or derivatives thereof into the plurality of recipient cells in (d), the endogenous TCR is inactivated. In some embodiments, while delivering the plurality of polynucleotide sequences or derivatives thereof into the plurality of recipient cells in (d), the endogenous TCR is inactivated.

[0018] In some embodiments, the endogenous TCR is inactivated using a CRISPR method. In some embodiments, the CRISPR method includes delivering a ribonucleoprotein (RNP) complex comprising a Cas9 protein and a single guide RNA (sgRNA) into a recipient cell. In some embodiments, the sgRNA includes an sgRNA targeting the TRBC locus. In some embodiments, by delivering an RNP comprising a Cas9 protein and sgRNAs (the sgRNAs include an sgRNA targeting the TRBC locus and an sgRNA targeting the TRAC locus), the endogenous TCR is inactivated while specifically knocking in a polynucleotide sequence at the genomic site.

[0019] In some embodiments, at least about 50% of the plurality of synthetic TILs exogenously express only a single copy of a polynucleotide sequence encoding a naturally occurring pairing TCR of the plurality of T cells' TCRs.

[0020] In certain embodiments, the method further includes after (d), amplification of multiple synthetic TILs. In certain embodiments, the mixture comprises a polynucleotide sequence encoding at least 5 different natural pairing TCRs, and a unique TCR in the at least 5 different natural pairing TCRs has a first frequency in the mixture. In certain embodiments, after amplification, this unique TCR has a second frequency in multiple synthetic TILs. In certain embodiments, the second frequency is within 0.1 times to 10 times of the first frequency. In some embodiments, the polynucleotide sequence encoding at least 5 different natural pairing TCRs is mixed with an equimolar ratio. In certain embodiments, after amplification, each unique TCR maintains an equal ratio in the multiple synthetic TILs, or has a ratio of 0.1 times or no more than 10 times of the ratio of any other unique TCR in the multiple synthetic TILs that is not less than.

[0021] In some embodiments, the method further comprises administering at least one cell of the plurality of synthetic TILs or its progeny to a subject with a solid tumor. In some embodiments, prior to administration, the plurality of synthetic TILs are not subjected to any selection or screening to determine the tumor reactivity of the plurality of synthetic TILs or its subset.

[0022] In some embodiments, the method further comprises determining the tumor reactivity of the multiple synthetic TILs. In some embodiments, determining the tumor reactivity comprises: (i) providing a tumor cell or a derivative thereof from a subject; (ii) contacting the multiple synthetic TILs with the tumor cell or a derivative thereof; (iii) determining the tumor reactivity of the multiple synthetic TILs to the tumor cell or a derivative thereof; and (iv) determining whether to administer the multiple synthetic TILs or a subset thereof to the subject. In some embodiments, the method further comprises administering at least one cell of the multiple synthetic TILs or a progeny thereof to a subject with a solid tumor.

[0023] In some embodiments, the method further comprises, before synthesis in (c), calculating a score using the expression levels of at least 3 genes of the at least 10 genes obtained in (b); grading the plurality of T cells based on the score; and selecting at least two TCRs from the top 50% of the plurality of T cells according to the score. In some embodiments, the plurality of polynucleotide sequences encoding the TCRs or a subset thereof synthesized in (c) comprises polynucleotide sequences encoding at least two selected TCRs.

[0024] In some embodiments, the time interval between obtaining a resection or biopsy sample and using at least one cell of a plurality of synthetic TILs in a subject is less than eight weeks. In some embodiments, the time interval between obtaining a resection or biopsy sample in (a) and delivering in (d) is no more than 7 weeks.

[0025] In some embodiments, multiple synthetic TILs exogenously express at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000 or more different TCRs. In some embodiments, two or more cells of multiple synthetic TILs or their progeny are administered to a subject, and the two or more cells administered exogenously express at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000 or more different TCRs.

[0026] In some embodiments, the plurality of synthetic TILs includes a subpopulation of cells expressing TCRs that recognize a tumor antigen and a subpopulation of cells expressing TCRs that do not recognize a tumor antigen.

[0027] In some embodiments, the volume of the resection or biopsy sample is less than about 100 mm 3 (μL), or the weight of the resection or biopsy sample is less than about 0.1 g.

[0028] In some embodiments, multiple polynucleotide sequences or derivatives thereof are delivered to multiple recipient cells by a vector. In some embodiments, the vector is a non-viral vector. In some embodiments, the non-viral vector is a plasmid, a transposon, a self-amplification RNA, or any combination thereof. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is an adenoviral vector, an adeno-associated virus (AAV) vector, a herpes simplex virus vector, a lentiviral vector, a retroviral vector, or any combination thereof. In some embodiments, the viral vector is an AAV vector. In some embodiments, the vector does not include a promoter. In some embodiments, multiple recipient cells are multiple T cells.

[0029] In some embodiments, multiple receptor cells are separated from a subject. In some embodiments, the subject is the same subject in which multiple T cells in (a) are obtained. In some embodiments, the multiple receptor cells are primary T cells. In some embodiments, the multiple receptor cells are peripheral T cells.

[0030] In some embodiments, the mixture comprises at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000, or more different polynucleotide sequences encoding at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000, or more different naturally paired TCRs.

[0031] In some embodiments, the subject has failed a prior line of therapy.In some embodiments, the prior line of therapy is a checkpoint blocker therapy.

[0032] In another aspect, the present patent application discloses a method for producing multiple synthetic tumor infiltrating lymphocytes (TILs), the method comprising: (a) obtaining a resection or biopsy sample containing multiple T cells from a solid tumor lesion of a subject; (b) synthesizing multiple polynucleotide sequences encoding the natural paired TCRs or a subset thereof of the multiple T cells obtained in (a), the multiple polynucleotide sequences comprising a first polynucleotide sequence encoding a first natural paired TCR of a first T cell of the multiple T cells and a second polynucleotide sequence encoding a second natural paired TCR of a second T cell of the multiple T cells; and wherein the first polynucleotide sequence is different from the second polynucleotide sequence; and (c) delivering the multiple polynucleotide sequences or their derivatives to multiple receptor cells by contacting a mixture containing the multiple polynucleotide sequences or their derivatives with multiple receptor cells, thereby producing the multiple synthetic TILs.

[0033] In some embodiments, the method further comprises, before (b), sequencing a plurality of T cells of the resection or biopsy sample to obtain the naturally paired T cell receptor (TCR) α chain and β chain sequences of the TCR of the plurality of T cells. In some embodiments, the method further comprises obtaining the expression levels of at least 10 genes from sequencing.

[0034] In another aspect, the present invention provides a method for producing multiple synthetic tumor infiltrating lymphocytes (TILs), the method comprising: (a) delivering multiple polynucleotide sequences or their derivatives into multiple recipient cells by contacting a mixture comprising multiple polynucleotide sequences or their derivatives with multiple recipient cells, wherein each polynucleotide sequence in the multiple polynucleotide sequences encodes a homologous pair of TCRα chain and TCRβ chain, and wherein each polynucleotide sequence is knocked into a genomic site for monoallelic expression of the polynucleotide sequence; and (b) inactivating the endogenous TCR gene of each of the multiple receptor cells, or the endogenous TCR gene of each of the multiple receptor cells has been inactivated.

[0035] In some embodiments, at least about 50% of multiple synthetic TILs only exogenously express a single copy of a polynucleotide sequence encoding a natural pairing TCR of the TCRs of multiple T cells. In some embodiments, the genomic site is a TRAC site. In some embodiments, endogenous TCR genes are destroyed by knocking down or knocking out the TRBC locus. In some embodiments, the mixture comprises at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000 or more different polynucleotide sequences, which encode at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000 or more different natural pairing TCRs.

[0036] In some embodiments, the method further comprises, before (a), synthesizing a plurality of polynucleotide sequences encoding different homologous pairs of TCR α chains and TCR β chains. In some embodiments, the synthesis comprises assembling a plurality of polynucleotide sequences encoding different homologous pairs of TCR α chains and TCR β chains in the same mixture. In some embodiments, the synthesis comprises chemically synthesizing a plurality of polynucleotide sequences separately.

[0037] In some embodiments, before delivery in (a), the endogenous TCR gene of each receptor cell in the plurality of receptor cells has been inactivated. In some embodiments, before, simultaneously with or after delivery in (a), the endogenous TCR gene of each receptor cell in the plurality of receptor cells in (b) is inactivated.

[0038] In some embodiments, the method further comprises, before (a), obtaining a resection or biopsy sample comprising a plurality of T cells from a solid tumor lesion of the subject, and sequencing the plurality of T cells of the resection or biopsy sample to obtain naturally paired T cell receptor (TCR) α chain and β chain sequences of the TCR of the plurality of T cells.

[0039] In some embodiments, the method further comprises selecting from the sequencing a plurality of sequences encoding a plurality of naturally paired TCRs, wherein the selected plurality of sequences or a subset thereof are the plurality of polynucleotide sequences delivered in (a).

[0040] In some embodiments, the inactivation in (b) is performed simultaneously with, before, or after the delivery in (a).

[0041] In some embodiments, the method further comprises expanding a plurality of synthetic TILs. In some embodiments, the frequency of unique TCRs in the mixture before delivery is within 0.1 to 10 times the frequency of unique TCRs in the plurality of synthetic TILs after expansion.

[0042] In some embodiments, the method further comprises administering to a subject at least one cell of the plurality of synthetic TILs or its progeny. In some embodiments, after administration, the synthetic TILs may persist in the subject in need for at least 30 days. In some embodiments, after administration, the percentage of the synthetic TILs or their progeny in the subject's peripheral T cells is at least about 20%. In some embodiments, the CD62L of the synthetic TILs or their progeny is + The percentage of cells in the subject's peripheral T cells is at least about 50%. In some embodiments, this percentage persists for at least about 30 days. In some embodiments, the concentration of synthetic TILs in the subject's peripheral blood sample is at least about 0.2×10 6 In some embodiments, the concentration is sustained for at least about 30 days with or without IL-2 infusion.

[0043] In another aspect, the present invention discloses a pharmaceutical composition comprising one or more synthetic TILs described herein, and a pharmaceutically acceptable carrier.

[0044] In another aspect, the invention provides a mixture comprising at least 2, 5, 10, 15, 20, 25, 30, 50, 100, 200, 500 or 1000 modified cells, each modified cell in the mixture exogenously expressing a polynucleotide sequence encoding a different homologous pair of a TCR alpha chain and a TCR beta chain, wherein the polynucleotide sequence is knocked into a genomic locus for monoallelic expression of the polynucleotide sequence, and wherein the endogenous TCR gene of the modified cell is inactivated (e.g., knocked out).

[0045] In another aspect, the present invention provides a cell mixture comprising a plurality of modified cells, each of the plurality of modified cells exogenously expressing polynucleotide sequences encoding different homologous pairs of TCR α chains and TCR β chains, wherein the polynucleotide sequences are knocked into genomic sites to achieve monoallelic expression of the polynucleotide sequences. In some embodiments, the endogenous TCR genes of the modified cells are inactivated.

[0046] In some embodiments, each different cognate pair of TCR alpha chain and TCR beta chain is endogenously expressed in T cells from a subject having a solid tumor or in a subject having a solid tumor lesion, and wherein the subject is to receive administration of the modified cell mixture or a pharmaceutical composition comprising the modified cell mixture.

[0047] In some embodiments, the mixture comprises at least 10 modified cells, each modified cell of the mixture exogenously expressing a polynucleotide sequence encoding a different cognate pair of a TCR alpha chain and a TCR beta chain.

[0048] In some embodiments, the genomic site is a TRAC site. In some embodiments, the endogenous TCR gene of the modified cell is knocked out or knocked down. In some embodiments, the endogenous TCR gene comprises a TRBC locus. In some embodiments, the modified cell is a T cell. In some embodiments, the T cell is an allogeneic T cell or an autologous T cell.

[0049] In some embodiments, different homologous pairs of TCR alpha chains and TCR beta chains of each modified cell are selected by sequencing TCRs of multiple T cells from a subject. In some embodiments, different homologous pairs of TCR alpha chains and TCR beta chains of each modified cell are selected by a pre-designed scoring method based on the sequencing data of TCRs of multiple T cells from a subject. In some embodiments, different homologous pairs of TCR alpha chains and TCR beta chains of each modified cell have been confirmed to be functional. In some embodiments, different homologous pairs of TCR alpha chains and TCR beta chains of each modified cell are tumor reactive. In some embodiments, the mixture includes functional and non-functional TCRs. In some embodiments, the mixture contains tumor reactive and tumor non-reactive TCRs. In some embodiments, the mixture of modified cells has been amplified in vitro.

[0050] On the other hand, the present invention provides a cell mixture comprising a plurality of modified cells, each modified cell in the plurality of modified cells exogenously expresses a homologous pair of TCR from a subject, wherein the cell mixture expresses at least 10 different homologous pairs of TCR, wherein each TCR in the at least 10 different TCR homologous pairs is expressed in at least 2% of the cell mixture, and wherein at least 50% of the cell mixtures each exogenously express only one TCR homologous pair. In some embodiments, the subject intends to receive administration of a mixture or a pharmaceutical composition comprising a mixture. In some embodiments, a polynucleotide sequence encoding a TCR homologous pair is knocked into a genomic site so that the polynucleotide sequence is expressed in a monoallele in each modified cell of at least 50% of a mixture of only exogenously expressing a pair of TCR homologous pairs. In some embodiments, the modified cells can survive in the subject for at least about 30 days after being injected into the subject.

[0051] In another aspect, the present invention provides a pharmaceutical composition comprising a cell mixture containing modified cells as described herein and a pharmaceutically acceptable carrier.

[0052] In another aspect, the present invention provides a composition for preparing a medicament for treating a subject with cancer, comprising a cell mixture containing modified cells as described herein.

[0053] In another aspect, the present invention discloses a method of treating cancer in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein or a pharmaceutical composition comprising a mixture described herein.

[0054] In another aspect, the present invention discloses a method for treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) a synthetic TIL prepared according to the methods described herein or a mixture of two or more synthetic TILs prepared according to the methods described herein; and (b) a pharmaceutically acceptable carrier.

[0055] In another aspect, the present invention discloses a method for treating cancer in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising a cell mixture to a subject in need thereof, wherein the cell mixture exogenously expresses at least 10 different TCR homologous pairs from the subject, and wherein at least 50% of the cell mixture each exogenously expresses only one pair of homologous TCRs.

[0056] In some embodiments, the cell mixture is a mixture of modified cells. In some embodiments, the cell mixture includes multiple modified cells and multiple unmodified cells. In some embodiments, each TCR in the at least 10 different TCR homologous pairs is expressed in at least 2% of the modified cell mixture. In some embodiments, the polynucleotide sequence is knocked into the genomic site for monoallelic expression of the polynucleotide sequence. In some embodiments, the subject obtaining the TCR is the same as the subject receiving treatment. In some embodiments, the endogenous TCR gene of the modified cell is inactivated.

[0057] In some embodiments, the modified cell is a T cell. In some embodiments, the T cell is a CD4 + T cells, CD8 + T cells, CD4 + / CD8 + T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβT cells or γδT cells. In some embodiments, the T cells are allogeneic T cells or autologous T cells. In some embodiments, the modified cells are isolated from a subject to whom the composition is administered. In some embodiments, the unique homologous pair of each modified cell is from a subject in need or is endogenously expressed by a T cell of a subject in need.

[0058] In some embodiments, the mixture comprises at least 10 modified cells. In some embodiments, the genomic site is a TRAC site. In some embodiments, the endogenous TCR gene of the modified cell is knocked out or knocked down. In some embodiments, the endogenous TCR gene comprises a TRBC locus. In some embodiments, the subject in need has received one or more front-line treatments, or one or more front-line treatments have failed. In some embodiments, the cancer is metastatic or refractory. In some embodiments, the cancer is a solid tumor.

[0059] In some embodiments, the method further comprises administering IL-2 to the subject after administering the pharmaceutical composition. In some embodiments, prior to administration, the cell mixture is tested for tumor reactivity.

[0060] In some embodiments, the mixture of modified cells survives in the subject for at least about 30 days. In some embodiments, after administration, the percentage of modified cells or their progeny in the subject's peripheral T cells is at least about 20%. In some embodiments, the CD62L of synthetic TILs or their progeny +The percentage of cells in the subject's peripheral T cells is at least about 50%. In some embodiments, this percentage persists for at least about 30 days. In some embodiments, the concentration of synthetic TILs in the subject's peripheral blood sample is at least about 0.2×10 6 In some embodiments, the concentration is sustained for at least about 30 days with or without IL-2 infusion.

[0061] In another aspect, the present invention provides use of the modified cells described herein (e.g., synthetic TILs) in the preparation of a medicament for treating cancer (e.g., solid tumors).

[0062] Those skilled in the art will readily appreciate additional aspects and advantages of the present disclosure from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be appreciated, the present disclosure is capable of other different embodiments, and its several details are capable of modification in various obvious respects, all without departing from the present disclosure. Therefore, the drawings and description should be regarded as illustrative in nature, and not restrictive. Incorporated by Reference

[0063] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. If a publication or patent or patent application incorporated by reference contradicts the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] The novel features of the present invention are set forth in detail in the appended claims. The features and advantages of the present invention will be better understood with reference to the following illustrative embodiments and accompanying drawings (also referred to herein as "Figures," "Fig." and "FIGURE"), in which the principles of the present invention are applied. Among them:

[0065] Figure 1 Experimental data showing the quality of 48 individually amplified TCR genes assessed by a fragment analyzer are described.

[0066] Figure 2 An example of tetramer staining showing single copy insertion of the TCR is depicted.

[0067] Figure 3 An example of flow cytometric analysis of polyclonal synthetic TILs is described.

[0068] Figure 4 An example protocol for sequencing mRNA transcripts carrying an exogenous TCR is described.

[0069] Figures 5A-5C An example of processing a tumor fine needle biopsy specimen is described. Figure 5A A fine needle biopsy sample in tissue storage solution is depicted. Figure 5B Quality control data for single-cell suspensions obtained using the Bio-Rad TC20 automated cell counter are described. Figure 5C Phase contrast microscopy images of the resulting single-cell suspensions are depicted.

[0070] Figure 6 This paper describes the experimental quality control data of single-cell RNA-seq of fine-needle biopsy samples. Gene expression profiles of 7983 cells (including tumor cells and non-tumor cells in the samples) were obtained.

[0071] Figure 7 Experimental quality control data for single-cell RNA-seq of fine-needle biopsy samples are described. TCR sequences were detected for 4528 cells, of which 3462 cells contained prolific VJ transpairs.

[0072] Figure 8 Experimental quality control data for single-cell RNA-Seq (gene expression profiling) is depicted. From left to right, the distribution of feature counts (e.g., gene counts), transcript counts (e.g., UMI counts), percentage of mitochondrial-derived transcripts, and percentage of ribosome-derived transcripts are shown.

[0073] Fig. 9 Depicted are experimental data showing the Uniform Manifold Approximation and Projection (UMAP) displaying the gene expression profiles of all T cells in a fine needle biopsy sample. The grayscale shows the labeling fraction for each T cell.

[0074] Fig.10 Experimental data showing the UMAP coordinates of all T cells containing TCR2 ('CAVI' TCR) are depicted.

[0075] Fig.11 Experimental data showing the UMAP coordinates of all T cells containing TCR1 ('CAVH' TCR) are depicted.

[0076] Fig.12 Depicted are experimental data showing UMAP coordinates of all T cells containing TCR3 ('CADS' TCR).

[0077] Fig.13 Experimental data showing tumor reactivity of TCR1, TCR2, and TCR3 as measured by IFNg secretion when TCR-bearing T cells are co-cultured with primary tumors are described.

[0078] Fig.14Experimental data described showed that lung lesions in a patient with hepatocellular carcinoma (patient 1) were reduced after infusion of synthetic TILs.

[0079] Fig.15 A real-time cell analysis (RTCA) assay discovering 3 TCRs from patient 2 is described.

[0080] Fig.16 Depicted are experimental data showing that lesions in a patient with urothelial carcinoma (patient 3) were reduced in size following infusion of synthetic TILs. The horizontal bar graphs show the size of tumor lesions.

[0081] Figures 17A-17C Exemplary protocols for generating nucleic acid constructs encoding T cell receptors are described.

[0082] Figures 18A-18C A schematic diagram of the TCR gene is depicted. Fig.18A Schematic diagram depicting the germline genomic DNA of the TCR V gene. Fig.18B Schematic diagram depicting genomic DNA rearrangements of TCR VJ genes. Fig. 18C Schematic representation of the rearranged genomic DNA depicting the TCR VDJ genes.

[0083] Fig.19 Schematic diagram depicting potential challenges associated with joining a CDR3-J gene segment to a correct V gene segment. Dashed arrows indicate possible joining between a CDR3-J gene segment and an incorrect V gene segment.

[0084] Fig. 20 A scheme is depicted for connecting a CDR3-J polynucleotide (grey box connected to a white box) to a specified, presynthesized V gene germline polynucleotide (black box connected to a grey box indicated by a thin arrow) by overlapping primer extension. The top thick arrow (603) indicates hybridization between a linker sequence on a presynthesized V gene germline polynucleotide (601) and a linker sequence on a CDR3-J polynucleotide (602). The bottom thick arrow (604) indicates primer extension. 601 may be referred to as a linker sequence and 602 may be referred to as an anti-linker sequence (or vice versa).

[0085] Fig.21 The use of arbitrary linker (701) and anti-linker (702) sequences to link a CDR3-J polynucleotide and a specified V gene germline polynucleotide is described.

[0086] Fig. 22The general principle of TCR gene self-assembly is described. 801: Pre-synthesized V gene germline polynucleotide. 802: Polynucleotide comprising a CDR3-J sequence (e.g., a CDR3-J polynucleotide). 803: Nucleic acid sequence comprising a V gene germline polynucleotide sequence and a CDR3-J sequence. X is the number of polynucleotides, each of which is part of a different V gene germline polynucleotide. Y is the number of CDR3-J polynucleotides. Y can be much larger than X. Arrows represent batch reactions in which each CDR3-J polynucleotide is linked to a specified, pre-synthesized V gene germline polynucleotide.

[0087] Fig.23A and Fig. 23B An exemplary protocol for generating a TCR gene library is described, in which individual TCR genes can be selectively amplified by PCR using primers corresponding to the Zip sequence of the TCR gene.

[0088] Fig.24A and Fig. 24B Flow cytometric analysis of modified cells infected with lentiviral particles containing exogenous TCR is described.

[0089] Figures 25A-25D Single-cell transcriptome data from fine-needle biopsy of patient CMI-0102 are described. Fig.25A Describes the module E score, calculated from the gene expression levels of approximately 30 genes associated with T cell exhaustion. Fig.25B Automated cell clustering is described. Fig.25C The modular T score, calculated from the gene expression levels of approximately 30 genes related to secondary lymphoid structures and T-cell activation, is described. Fig.25D Clonality is described.

[0090] Figures 26A-26D Single-cell transcriptomic data from fine-needle biopsies of patient CMI-0102 are depicted. The expression levels of CD3D, CD8A, CD4, and Foxp3 in each cell are shown. Fig.26A , 26B , 26C and 26D.

[0091] Fig. 27 Depicted are single-cell transcriptomic data from a fine-needle biopsy of patient CMI-0102, showing the location of T cells carrying the TCR selected for making synthetic TIL cells, as visualized by UMAP.

[0092] Fig.28The abundance of each TCR in the R&D grade plasmid mixture (Figure A), GMP grade plasmid mixture (Figure B), GMP grade AAV mixture (Figure C) and synthetic TIL cell mixture (Figure D) of patient CMI-0102 is described. The unit of the displayed value is %. The dotted line represents the position 1 / N*100, where N is the number of TCRs mixed in equal proportions.

[0093] Fig.29A Depicted are levels of the tumor biomarker CEA in patient CMI-0102 at different days after infusion of the T cell product. Fig.29B = represents body temperature. For example, D10 represents the 10th day after infusion. Baseline refers to the day before infusion. LD-0 and LD-5 represent the day of clearing and the 5th day after clearing, respectively.

[0094] Fig.30 Serum levels of IL-6, CRP, and ferritin at different days after infusion of the T cell product are depicted for patient CMI-0102. The upper limit of the normal range is indicated by the dotted line.

[0095] Figures 31A-31D Describes blood cell count ( Fig.31A and 31B ) and the pharmacokinetics of synthetic TIL cells of CMI-0102 in patients ( Fig.31C and 31D ). WBC: white blood cell count. LYMPH: lymphocyte count. Fig.31A and Fig.31B The lower limit of the normal range is shown as a dotted line. Fig.31C and Fig.31D In this article, “disTIL” is used to describe synthetic TIL cell products. Fig.31C It showed that synthetic TIL cells accounted for all CD3 + The percentage of T cells. Fig.31D The absolute number of synthetic TIL cells is shown on a log10 scale.

[0096] Fig.32 Depicted is a single-cell transcriptome analysis of peripheral T cells from patient CMI-0102 at days 10, 14, and 21 after infusion of synthetic TIL cells. The expression levels of several markers are highlighted.

[0097] Figures 33A-33D Single-cell transcriptomic and clonotypic analysis of synthetic TIL cells in peripheral T cells at days 10, 14, and 21 after infusion of synthetic TIL cells in patient CMI-0102 is described. Fig.33A Depicted are the exhaustion scores calculated from the expression levels of approximately 30 genes associated with T cell exhaustion. Fig.33BThe relative abundance of different exogenous TCRs (used to generate synthetic TIL cells) is described. Fig.33C (Day 10), Fig.33D (Day 14) and highlighted on UMAP in Figure 33E (Day 21).

[0098] Figures 34A-34D Single-cell transcriptome data from fine-needle biopsy of patient CMI-0104 are described. Fig.34A Described is the module E score, calculated from the gene expression levels of approximately 30 genes associated with T cell exhaustion. Fig.33B Automated cell clustering is described. Fig.33C The modular T score, calculated from the gene expression levels of approximately 30 genes related to secondary lymphoid structures and T cell activation, is described. Fig.33D Clonality is described.

[0099] Figures 35A-35C Single-cell transcriptome data from fine-needle biopsies of patient CMI-0104 are described. The expression levels of CD8A, CD4, and Foxp3 in each cell were Fig.35A , 35B and 35C.

[0100] Fig.36 Depicted are single-cell transcriptome data from a fine-needle biopsy of patient CMI-0104, showing the location of T cells carrying the TCR selected for making synthetic TIL cells, as visualized by UMAP.

[0101] Fig.37 The abundance of each TCR in the R&D grade plasmid mixture (Figure A), GMP grade plasmid mixture (Figure B), GMP grade AAV mixture (Figure C) and synthetic TIL cell mixture (Figure D) of patient CMI-0104 is depicted. The unit of the displayed value is %. The dotted line represents the position 1 / N*100, where N is the number of TCRs mixed in equal proportions.

[0102] Fig.38 Body temperature of patient CMI-0104 on different days after infusion of T cell product is described.

[0103] Fig.39 Serum levels of IL-6, CRP, and ferritin at different days after infusion of the T cell product of patient CMI-0104 are depicted. The upper limit of the normal range is indicated by the dotted line.

[0104] Figures 40A-40D Describes blood cell count ( Fig.40A and 40B ) and the pharmacokinetics of CMI-0104-synthetic TIL cells in patients ( Fig.40C and40D ). WBC: white blood cell count. LYMPH: lymphocyte count. In A and B, the lower limit of the normal range is indicated by a dotted line. Fig.40C and Fig.40D In this article, “disTIL” is used to describe synthetic TIL cell products. Fig.40C Shows the percentage of synthetic TIL cells to all CD3+ T cells. Fig.40D The absolute number of synthetic TIL cells is shown. The duration of IL-2 infusion is indicated on each graph. Detailed description of the invention

[0105] In this disclosure, unless otherwise specifically stated, the use of the singular includes the plural. Furthermore, unless otherwise stated, the use of "or" means "and / or". Similarly, "includes", "comprising", "including" and "comprising" are not limiting.

[0106] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, according to the practice in the art, "about" can mean within 1 or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly for biological systems or processes, the term can mean within an order of magnitude, such as within 5 times or within 2 times a value. Where specific values ​​are described in the application and claims, the term "about" should be assumed to mean within an acceptable error range for the specific value unless otherwise indicated.

[0107] Whenever the term "at least", "greater than", or "greater than or equal to" precedes the first value in a series of two or more values, the term "at least", "greater than", or "greater than or equal to" applies to each value in the series. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0108] Whenever the term "not greater than," "less than," or "less than or equal to" precedes the first value in a series of two or more values, the term "not greater than," "less than," or "less than or equal to" applies to every value in the series. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.

[0109] The terms "polynucleotide", "nucleic acid" and "oligonucleotide" are used interchangeably in the present disclosure. They can refer to polymeric forms of nucleotides of various lengths. They can include deoxyribonucleotides and / or ribonucleotides, or analogs thereof. Polynucleotides can include one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) or variants thereof. Nucleotides can include nucleosides and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphate (PO3) groups. Nucleotides can include a nucleobase, a pentose (ribose or deoxyribose) and one or more phosphate groups. Polynucleotides can have any three-dimensional structure and can perform various functions. Polynucleotides can have various configurations, such as linear, circular, stem-loop and branched. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, gene loci determined by linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), circular RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. The polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, the nucleotide structure may be modified before or after polymer assembly. The nucleotide sequence may be interrupted by non-nucleotide components. The polynucleotide may be further modified after polymerization, for example by conjugation with a labeling component. The polynucleotide may include one or more nucleotide variants, including non-standard nucleotides, non-natural nucleotides, nucleotide analogs and / or modified nucleotides.

[0110] The term "sequence" as used herein refers to the order of nucleotides in a nucleic acid molecule, or the order of amino acid residues of a peptide. A nucleic acid sequence can be a deoxyribonucleic acid (DNA) sequence or a ribonucleic acid (RNA) sequence; it can be linear, circular or branched; and it can be single-stranded or double-stranded. The sequence can be mutated to make it different from a reference sequence (e.g., a wild-type sequence). The sequence can be of any length, such as a length of 2 and 1,000,000 or more amino acids or nucleotides (or any integer value therebetween or above), such as about 100 to about 10,000 nucleotides or about 200 to about 500 amino acids or nucleotides. In some cases, a given nucleic acid sequence can include sequence information of a given nucleic acid sequence and a reverse complementary sequence of a given nucleic acid sequence. In some cases, a DNA sequence can include sequence information of a corresponding RNA sequence transcribed from a DNA. The sequence can be an alphabetic representation of a polynucleotide or polypeptide molecule. The sequence can be a piece of information that can be used by a computer processor. In some cases, a nucleic acid sequence can be used to refer to the physical nucleic acid molecule itself.

[0111] The term "blunt end" as used herein refers to the end of a double-stranded nucleic acid molecule, wherein almost all of the nucleotides at the end of one strand of the nucleic acid molecule are base-paired with the opposite nucleotides of the other strand of the same nucleic acid molecule. If the end of a nucleic acid molecule includes a single-stranded portion of at least one nucleotide in length (referred to herein as an "overhang" or "sticky end"), the nucleic acid molecule is not blunt-ended.

[0112] The term "TCR V gene" as used herein refers to the genomic nucleic acid sequence of T cell receptor variable (V) gene, which, in germline configuration, comprises a sequence encoding the first part of the leader peptide (e.g., L-PART1 defined in IMGT), an intron (e.g., V-INTRON defined in IMGT) and an exon (e.g., V-EXON defined in IMGT), and 5'UTR and 3'UTR (including a recombination signal sequence). The recombination signal sequence may comprise a heptamer (e.g., V-HEPTAMER defined in IMGT) and a nonamer (e.g., V-NONAMER defined in IMGT), which are separated by a spacer element (e.g., V-SPACER defined in IMGT). V-EXON includes a sequence encoding the second part (L-PART2) of the leader peptide and V-REGION. The example of TCR V gene includes TCRalpha variable (TRAV) gene, TCRbeta variable (TRBV) gene, TCR gamma variable (TRGV) gene and TCR delta variable (TRDV) gene. Nucleic acid as described herein can include a sequence derived from a TCR V gene. "Derived from" refers to a sequence with a sequence identity of at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or 100% with a reference sequence. The sequence derived from a TCR V gene can be the full-length sequence of the genomic nucleic acid sequence of a TCR V gene as described above. The sequence derived from a TCR V gene can be a part of a TCR V gene, comprising at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more nucleotides of a TCRV gene. The sequence derived from a TCRV gene can be a codon-optimized (or codon-diverse) nucleic acid sequence. The codon-optimized sequence of a given nucleic acid sequence refers to a modified nucleic acid sequence of an amino acid sequence identical to the protein coding region of a given nucleic acid encoded by its protein coding region. The modified nucleic acid sequence can have a sequence different from a given nucleic acid sequence, or can be derived from a given nucleic acid. Codon optimization can be used to remove restriction sites, remove unwanted secondary structures in polynucleotide sequences, promote the correct connection of CDR3-J polynucleotides with the specified presynthesized parts of TCR V genes or for other purposes. Codon optimization or codon diversification can be achieved by changing one or more nucleotides of a given nucleic acid sequence. For example, codon optimization or codon diversification can be achieved by computational methods. Codon optimization and codon diversification are used interchangeably in the present disclosure.

[0113] The term "V-region" as used herein refers to the coding region of a TCR V gene in germline genomic DNA or cDNA (including 1 or 2 nucleotides preceding the V-HEPTAMER, if present), or the variable (V) region that is typically trimmed at the 3' end by rearrangement of the V-(D)-J rearrangement in the genomic DNA or cDNA.

[0114] The term "D-region" as used herein refers to the coding region of a TCR D gene in a germline genomic DNA or cDNA (including 1 or 2 nucleotides after the 5' D-heptamer and / or before the 3' D-heptamer, if present), or the diversity (D) region that is trimmed at the 5' and / or 3' end in a partially rearranged or rearranged genomic DNA or cDNA, typically by DJ or VDJ rearrangement.

[0115] The term "J-region" as used herein refers to the coding region of a TCR J gene in germline genomic DNA or cDNA (including 1 or 2 nucleotides after the J-heptamer, if present), or the joining (J) region that is typically trimmed at the 5' position by V-(D)-J rearrangement in the rearranged genomic DNA or cDNA.

[0116] The term "VJ-region" as used herein refers to the coding region of the TCR chain containing the V-region and the J-region in the rearranged genomic DNA or cDNA.

[0117] The term "VDJ-region" as used herein refers to the coding region of the TCR chain containing the V-region, D-region and J-region in the rearranged genomic DNA or cDNA.

[0118] The terms "link" or "connection" are used interchangeably in the present disclosure. They refer to physically connecting two or more nucleic acid molecules. The two or more nucleic acid molecules can be connected so that the two or more nucleic acid molecules form a continuous nucleic acid molecule. Two or more nucleic acid molecules can be covalently linked or non-covalently linked. Connection can be accomplished in a variety of ways, including the formation of hydrogen bonds, ionic bonds, covalent bonds or van der Waals forces. In some cases, two or more nucleic acid molecules can be connected to form a continuous nucleic acid molecule by a phosphodiester bond. In some cases, two or more nucleic acid molecules can be operably connected to form a continuous nucleic acid molecule. In some cases, two or more nucleic acid molecules can be effectively connected to form a continuous nucleic acid molecule so that the resulting molecule can be expressed in a cell.

[0119] The percentage (%) of sequence identity relative to a reference nucleic acid sequence (or peptide sequence) is the percentage of nucleotides (or amino acid residues in a peptide sequence) identical to the nucleotides (or amino acid residues) in the candidate sequence, if necessary, after alignment and introduction of gaps, to obtain maximum percentage sequence identity, and without considering any conservative substitutions as part of sequence identity. Alignment for determining percentage sequence identity can be achieved in various ways within the skill of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, CLUSTALW, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithm required for achieving maximum alignment over the full length of the compared sequence.

[0120] As used herein, the term "endogenous" refers to a substance that is native to an organism, cell, tissue or system.

[0121] As used herein, the term "exogenous" refers to a substance present in an organism, cell, tissue or system other than its own natural source. The substance may be introduced into the organism, cell, tissue or system from another source.

[0122] As used herein, the term "expression" is defined as the transcription and / or translation of a specific nucleic acid sequence driven by its promoter.

[0123] The term "exogenous expression" or "exogenous expression" refers to the expression of a polypeptide from an exogenous polynucleotide sequence (e.g., a polynucleotide sequence not derived from or derived from a host cell) introduced into a host cell. An exogenous protein can be a protein expressed from an exogenous polynucleotide sequence not derived from or derived from a host cell.

[0124] The term "expression vector" refers to a vector comprising a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. The expression vector comprises sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or an in vitro expression system. Expression vectors include, but are not limited to, cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., Sendai virus, lentivirus, retrovirus, adenovirus, and adeno-associated virus) that incorporate recombinant polynucleotides.

[0125] The term "homologous pair" as used herein refers to an original or natural pair of two nucleic acid molecules or proteins encoded by these two nucleic acid molecules contained in or derived from a single cell. A homologous pair can be a natural pairing chain in a single cell. For example, a homologous pair of a T cell receptor (TCR) can be a natural pairing TCR α and β chain in or derived from a single cell. For another example, a homologous pair of a T cell receptor (TCR) can be a natural pairing TCR γ and δ chain in or derived from a single cell.

[0126] As used herein, the term "knockdown" refers to a decrease in gene expression of one or more genes.

[0127] As used herein, the term "knockout" refers to the removal of genetic expression of one or more genes.

[0128] The term "modification" as used herein refers to a change in the state or structure of a molecule or cell. The term "modification" can be used interchangeably with "engineered", "synthetic" or "artificial". Molecules can be modified in a variety of ways, including chemical modification, structural modification, and functional modification. Cells can be modified by introducing exogenous nucleic acids or polypeptides. In some cases, the modified cells described herein express exogenous T cell receptors (TCRs). Modified cells that exogenously express TCRs (the TCRs are endogenous TCRs of tumor infiltrating lymphocytes (TILs) isolated from a tumor sample from a subject) may be referred to as "synthetic TILs" in the present disclosure.

[0129] The term "polyclonal T cell receptor (TCR)" describes a population of different TCR molecules capable of binding or reacting with different specific antigenic determinants from the same or different antigens. The term "polyclonal TCR-T cell" refers to a population of cells expressing a polyclonal TCR.

[0130] The term "highly efficient expression" as used herein means that the sequence can be translated into a protein, and if the protein is a TCR chain, it can form a homologous pair with another TCR chain. The protein can be a functional protein. The protein can be expressed on the surface of a cell. In some cases, the highly efficiently expressed TCR can form a TCR complex with a CD3 subunit (e.g., CD3ε, CD3γ, CD3δ, and CD3ζ).

[0131] As used herein, the term "line of treatment" refers to treatment consisting of one or more complete treatment cycles using a single agent, surgery, or dosing therapy, a regimen consisting of a combination of several drugs, surgery, or radiation therapy, or a planned sequential treatment of various regimens. A treatment is considered a new line of treatment if either of the following two conditions is met: (i) Starting a new line of treatment after stopping the previous line of treatment: If a treatment regimen is stopped for any reason and a different regimen is started, it should be considered a new line of treatment. A regimen is considered stopped if all drugs, radiation therapy, or surgery in the regimen have been stopped. A regimen is not considered stopped if some drugs, radiation therapy, or surgery in the regimen (but not all) have been stopped. (ii) Unplanned addition or substitution of one or more drugs, radiotherapy or surgery to an existing treatment plan: The unplanned addition of a new drug, new radiotherapy or new surgery, or the unplanned switch to a different drug (or drug combination), different radiotherapy or different surgery for any reason, is considered a new treatment.

[0132] In the present disclosure, polynucleotide sequences may be described at the domain level. Each domain name may correspond to a specific polynucleotide sequence. For example, domain "A" may have a 5'-TATTCCC-3' sequence, domain "B" may have a 5'-AGGGAC-3' sequence, and domain "C" may have a 5'-GGGAAGA-3' sequence. In this case, a polynucleotide having a sequence of domains A, B, and C in series may be written as [A|B|C}. The symbol "[" represents the 5' end, the symbol "}" represents the 3' end, and the symbol "|" separates the domain names. An ssDNA or ssDNA portion having a sequence "X" may be referred to as [X}. The asterisk indicates sequence complementarity. For example, domain [X*} is the reverse complement of domain [X}. The symbol ds[X} may be used to describe a double-stranded DNA formed by [X} and [X*}. In some cases, particularly where it is not necessary to distinguish between dsDNA and ssDNA, a dsDNA having a sequence [X} in one chain may also be generally referred to as [X}. A single-stranded RNA molecule or fragment with the same sequence as [X} (except that U replaces T) may also be referred to as [X}. Depending on the context, a domain name may refer to an exact sequence or describe a general function of the DNA or domain. For example, [RBS} may be used to describe a ribosome binding site, although the exact sequence of [RBS} may vary. Brackets may be used to group domain connections, and the reverse complement operation (denoted by "*") may be applied to a connection by adding "*" after the right bracket. For example, [(X|Y)*} is the same as [Y*|X*}. A double-stranded DNA formed by two strands [X} and [X*} may be written as [X}:[X*}. A double-stranded fragment of a double-stranded DNA may be written in a similar manner. For example, a dsDNA formed by [X|Y} and [Y*|X*} may be said to have double-stranded fragments [X}:[X*} and [Y}:[Y*}. A double-stranded fragment [X}:[X*} may also be referred to as "double-stranded DNA [X}" or "dsDNA [X}" without ambiguity. Overview

[0133] Adoptive cell therapy (ACT) is a promising approach for treating cancers such as solid tumors. In particular, tumor-infiltrating lymphocyte (TIL) therapy, in which tumor-infiltrating T cells (or TITs) can be harvested from surgical resections, expanded ex vivo, and infused back into patients, is close to receiving FDA approval (as of January 2022) for the treatment of melanoma. However, the efficacy of conventional TIL therapy in moderately immunogenic tumors (such as most epithelial tumors) remains inconsistent. A key limitation of TIL therapy may be that only a small fraction of the TIT population may be tumor-reactive, and these T cells may be in a terminally differentiated and / or dysfunctional state, while the vast majority of T cells are tumor-non-reactive cells with a proliferative phenotype (such as naive, central memory, or stem cell memory). Therefore, when these T cells are expanded in vitro, tumor-non-reactive, more proliferative T cells can grow faster and dominate the infused cell population. In addition, a large number of TIL cells may be required to initiate ex vivo TIL expansion, which in turn results in the need for large amounts of tumor material that can typically only be obtained from surgical specimens. However, surgery may not be recommended for patients with advanced disease because it is highly invasive and may not result in better patient outcomes.

[0134] To address these problems, the present disclosure provides compositions and methods for producing synthetic forms of TIL (or TIT) cells, wherein the proportion of tumor-reactive T cells can be enriched, and only a small amount of tumor samples (e.g., available from biopsies) is sufficient. The novelty of the method provided herein may include: (1) a novel minimally invasive sampling method to obtain the T cell receptor (TCR) sequence and transcriptome information of each TIL cell, (2) a novel algorithm to select TCRs that may be tumor-reactive, (3) a novel method for preparing hundreds or thousands or more therapeutic-grade TCR genes at low cost and fast turnaround time, and (4) a novel method for introducing hundreds, thousands or more TCR gene mixtures into primary T cells, wherein most T cells effectively express only one exogenous TCR copy. In various embodiments, the method described herein includes delivering a mixture of sequences encoding different exogenous TCRs to a cell population. In this case, if the cell obtains more than one copy of the sequence, adverse consequences may result, such as TCR chain mismatches, loss of the expected function of TCRs, and display of unexpected functions (e.g., activity against self-antigens). The compositions and methods provided herein can ensure that each cell receives only a single copy of a sequence encoding an exogenous TCR when contacted with a mixture of TCR-encoding sequences and cells. T cell receptor (TCR)

[0135] The present disclosure provides compositions and methods for producing modified cells (e.g., modified T cells) comprising exogenous TCR. Exogenous TCR can be from a subject in need thereof, which will administer a composition as described herein. In some cases, these exogenous TCRs can be referred to as autologous TCRs.

[0136] TCRs can be used to endow T cells with the ability to recognize antigens associated with various cancers or infectious organisms. TCRs are composed of two chains, for example, an alpha chain and a beta chain or a gamma chain and a delta chain. The proteins that make up these chains are encoded by DNA, which employs a unique mechanism to generate the enormous diversity of TCRs. This multi-subunit immune recognition receptor associates with the CD3 complex and binds to peptides presented by MHC class I and class II proteins on the surface of antigen presenting cells (APCs). Binding of the TCR to antigenic peptides on the APC may be a central event in T cell activation, occurring at the immunological synapse, the point of contact between the T cell and the APC.

[0137] TCR can recognize T cell epitopes in the context of MHC class I molecules. MHC class I proteins can be expressed in all nucleated cells of higher vertebrates. MHC class I molecules are heterodimers composed of 46kDa heavy chains, which are non-covalently bound to 12kDa light chain beta-2 microglobulin. In humans, there are multiple MHC alleles, such as HLA-A2, HLA-Al, HLA-A3, HLA-A24, HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45 and HLA-Cw8. In some embodiments, the MHC class I allele is the HLA-A2 allele, which is expressed by about 50% of the population in some populations. In some embodiments, the HLA-A2 allele can be the HLA-A*0201, *0202, *0203, *0206 or *0207 gene product. In some cases, there may be differences in the subtype frequencies between different populations. For example, in some embodiments, more than 95% of HLA-A2 positive Caucasians are HLA-A*0201, while in the Chinese population, the frequency is reported to be approximately 23% HLA-A*0201, 45% HLA-A*0207, 8% HLA-A*0206, and 23% HLA-A*0203.

[0138] In some embodiments, the TCR can recognize T cell epitopes in the context of MHC class II molecules. MHC class II proteins can be expressed in a subpopulation of APCs. In humans, there are several MHC class II alleles, such as DR1, DR3, DR4, DR7, DR52, DQ1, DQ2, DQ4, DQ8, and DPI. In some embodiments, the MHC class II allele is a HLA-DRB1*0101, HLA-DRB*0301, HLA-DRB*0701, HLA-DRB*0401, or HLA-DQB1*0201 gene product.

[0139] The TCR chain may include a variable domain (or variable region) and a constant domain (or constant region). The variable domain may be a V-domain defined by the IMGT unique numbering system. The variable domain may correspond to a VJ-domain or a VDJ-domain of a TCR chain. The constant domain may be a C-domain defined by the IMGT unique numbering system. In some cases, the constant domain may be a part of a constant region. For example, a full-length constant region may include a constant domain (extracellular region), a connecting region, a transmembrane region, and a cytoplasmic region.

[0140] The variable domains of TCRα or TCRδ chains can be encoded by multiple variable (V) and connecting (J) gene segments in the germline, while the variable domains of TCRβ or TCRγ chains are also encoded by diversity (D) gene segments. Each gene segment can be flanked by a recombination signal sequence. The recombination signal can contain heptamers and nonamers, separated by spacer elements. The length of the spacer element can be 12 or 23bp. During V(D)J recombination, one random allele of each gene segment is recombined with other gene segments to form a functional variable domain. The recombination of variable domains with constant (C) gene segments can produce functional TCR chain transcripts. In addition, random nucleotides can be added and / or deleted at the junction sites between gene segments. This process can lead to strong combinatorial diversity (depending on which gene regions will be recombined) and connection diversity (depending on which nucleotides will be added / deleted and how many nucleotides will be added / deleted), thereby generating a large and highly variable TCR library, which can ensure the recognition of a large number of antigens. Additional diversity is achieved by pairing (also called “assembling”) α and β or γ and δ chains to form a functional TCR. Through recombination, random insertions, deletions, and substitutions, it is possible to generate 10 15 to 10 20TCR clonotypes. As used herein, "clonotype" refers to a population of immune cells carrying the same immune receptor. For example, a clonotype refers to a population of T cells carrying the same TCR, or a population of B cells carrying the same BCR (or antibody). "Diversity" in immune receptor diversity refers to the number of clonotypes of immune receptors (e.g., TCR, BCR, and antibodies) in a population. As used herein, "homologous pair combination" refers to a natural combination of two chains (e.g., TCRα and TCRβ, or TCRγ and TCRδ) of a TCR from a T cell. The same homologous pair combination of two chains can produce the same TCR. For example, T cells with the same clonotype have the same homologous pair combination of TCRα and TCRβ chains. The higher the diversity of clonotypes, the higher the diversity of homologous pair combinations may indicate.

[0141] Each TCR chain may contain three highly variable loops in its structure, called complementarity determining regions (CDR1-3). CDR1 and CDR2 may be encoded by V genes and may be required for the interaction of the TCR with the MHC complex. However, the CDR3 portion is encoded by (1) the joining region between the V and J genes (in the case of TCRα or TCRγ) or (2) the joining region between the V and D genes and the joining region between the D and J genes (in the case of TCRβ or TCRδ) and may therefore be highly variable. CDR3 may be the region of the TCR that makes direct contact with the peptide antigen. CDR3 can be used as a target region for determining T cell clonotypes. The sum of all TCRs of an individual's T cells is called the TCR repertoire or TCR repertoire. The TCR repertoire may change with the onset and progression of the disease. Therefore, determining the state of the immune repertoire under different disease conditions, such as cancer, autoimmune diseases, inflammation, and infectious diseases may aid in the diagnosis and prognosis of the disease.

[0142] TCR should be understood to include full-length TCR and its antigen binding portion or antigen binding fragment (also referred to as MHC-peptide binding fragment). In some embodiments, TCR is a complete or full-length TCR. In some embodiments, TCR is an antigen binding portion, which is smaller than the full-length TCR, but binds to a specific antigen peptide bound to an MHC molecule (e.g., MHC-peptide complex). In some cases, the antigen binding portion or fragment of TCR may only include a portion of the domain of the full-length or complete TCR, but still be able to bind to the epitope (e.g., MHC-peptide complex) bound by the complete TCR. In some cases, the antigen binding portion or fragment of TCR includes the variable domain of TCR, such as the variable α chain and variable β chain of TCR, which is sufficient to form a binding site bound to a specific MHC-peptide complex, such as usually each chain includes three complementary determining regions. Including a polypeptide or protein with a binding domain, the binding domain is an antigen binding domain or is homologous to the antigen binding domain.

[0143] TCR molecules can be formed by an α chain (α chain or TCRα chain, encoded by the TRA gene / sequence) and a β chain (β chain or TCRβ chain, encoded by the TRB gene / sequence), or a γ chain (γ chain or TCRγ chain, encoded by the TRG gene / sequence) and a δ chain (δ chain or TCRδ chain, encoded by the TRD gene / sequence). These immune receptor chains can have variable domains (e.g., encoded by rearranged VDJ or VJ regions). Parts of the variable domains can be highly variable. Hypervariable regions may include complementary determining regions (CDRs), such as CDR1, CDR2, and CDR3. In some cases, only one functional α chain sequence and one functional β chain sequence may be expressed in a T cell. In some cases, only one functional γ chain sequence and one functional δ chain sequence may be expressed in a T cell. Methods for generating modified cells expressing exogenous TCRs

[0144] The present invention provides a method for producing a variety of modified cells called synthetic tumor infiltrating lymphocytes (TIL). In various cases, the method described herein can produce a mixture containing multiple synthetic TILs. The method can include obtaining a sample (e.g., excision or biopsy sample) containing multiple T cells from a subject. The sample can be obtained from a solid tumor lesion of a subject. The term "patient" or "subject" refers to an animal as a treatment, observation or experimental subject. By way of example only, subjects include but are not limited to mammals, including but not limited to humans or non-human mammals, such as non-human primates, cattle, horses, dogs, sheep or cats. In some cases, the subject is a cancer patient. In some cases, multiple T cells of a sample can be sequenced to obtain natural paired T cell receptor (TCR) information. For example, multiple T cells can be sequenced to obtain the natural TCR α chain and TCR β chain sequence pairs of the TCR of multiple T cells. Sequencing can be performed by various sequencing methods, including but not limited to Sanger sequencing, high-throughput sequencing, synthetic sequencing, single-molecule sequencing, single-cell sequencing, ligation sequencing, RNA-Seq, next-generation sequencing (NGS), digital gene expression, clonal single microarray, shotgun sequencing, Maxim-Gilbert sequencing, and massively parallel sequencing. In some cases, sequencing includes single-cell sequencing.

[0145] After obtaining the TCR information of natural pairing, the sequences encoding different homologous pairs of TCRs can be synthesized. In some cases, multiple polynucleotide sequences encoding TCR or a subset thereof from the natural pairing TCR α chain and β chain sequences obtained in sequencing can be synthesized. In some cases, all TCRs obtained in sequencing can be synthesized. In some cases, a subset of the TCR obtained in sequencing can be synthesized. Multiple polynucleotide sequences encoding TCR or a subset thereof can include a first polynucleotide sequence encoding a first natural pairing TCR of a first T cell in multiple T cells and a second polynucleotide sequence encoding a second natural pairing TCR of a second T cell in multiple T cells, wherein the first polynucleotide sequence is different from the second polynucleotide sequence. The plurality of polynucleotide sequences encoding TCRs or subsets thereof may comprise at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1200, 1,500, 1,800, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000 or more different sequences, each encoding a different cognate TCR pair. The plurality of polynucleotide sequences encoding TCRs or subsets thereof may encode at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1200, 1,500, 1,800, 2,000, 5,000, 10,000, 20,000, 50,000, 100,000 or more different cognate TCR pairs.

[0146] A plurality of polynucleotide sequences or derivatives thereof (e.g., copies thereof) can be delivered to a plurality of recipient cells to produce a plurality of synthetic TILs. In some cases, delivery is carried out by contacting a mixture (e.g., a pool) containing a plurality of polynucleotide sequences or derivatives thereof with a plurality of recipient cells. In some cases, each polynucleotide in a plurality of polynucleotide sequences or derivatives thereof is not delivered to a recipient cell in a single reaction. For example, before delivery to a recipient cell, a sequence encoding a TCR may not be separated from other different sequences encoding different TCRs. Delivery can be performed using various gene delivery methods described herein. In some cases, a recipient cell acquires and / or expresses only a single copy of a sequence encoding a TCR. For example, in some cases, after delivery, a first portion of a plurality of synthetic TILs exogenously expresses a first polynucleotide sequence encoding a first natural pairing TCR, and a second portion of a plurality of synthetic TILs exogenously expresses a second polynucleotide sequence encoding a second natural pairing TCR, and the fraction of the third portion of a plurality of synthetic TILs in which the first and second polynucleotide sequences are exogenously expressed is less than the product of the fractions of the first and second portions. For example, in a total of at least about 1×10 4 In the plurality of synthetic TILs, the number of cells exogenously expressing the first polynucleotide sequence encoding the first natural paired TCR (regardless of whether the second polynucleotide is expressed) can be at least about 1×10 3 The number of cells exogenously expressing the second polynucleotide sequence encoding the second naturally paired TCR (regardless of whether the first polynucleotide is expressed) can be at least about 1×10 3 , and the number of cells expressing both can be less than about 1×10 2 In some cases, cells in the plurality of synthetic TILs (i) exogenously express only one polynucleotide sequence in the plurality of polynucleotide sequences or (ii) do not exogenously express any of the plurality of polynucleotide sequences or derivatives thereof. In some cases, none of the plurality of synthetic TILs exogenously express the first and second polynucleotide sequences. In some cases, cells in the plurality of synthetic TILs that exogenously express the first polynucleotide sequence do not exogenously express the second polynucleotide sequence.

[0147] In some cases, the expression level of one or more genes in multiple T cells can also be obtained by sequencing. The one or more genes can be used to determine whether the cell is a potential tumor-reactive T cell and carries a tumor-reactive TCR. The one or more genes can include at least about 3, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more genes. The one or more genes may include genes selected from the group consisting of CXCL13, CTLA4, DUSP4, ENTPD1, ​​RBPJ, TNFSF4, GK, PDCD1, LAG3, FKBP1A, MYO7A, FABP5, GAPDH, TIGIT, CDCA7, ZBED2, RGS1, HAVCR2, MKI67, GZMB, SPRY1, PHLDA1, ASPM, MYO1E, TOX, LSP1HMGN2, and any combination thereof. In some cases, multiple T cells from a sample of a subject may be sequenced to obtain (i) expression levels of one or more genes (e.g., at least 10 genes) and (ii) natural paired TCR α chain and β chain sequences of the TCR of multiple T cells. In some cases, before synthesizing multiple polynucleotide sequences encoding homologous pairs of TCRs, the expression levels of one or more genes obtained from multiple T cells from a subject may be used to calculate a score. For example, the expression levels of at least 3 genes obtained may be used to calculate a score. A score can be calculated for each individual cell in the plurality of T cells. Next, the plurality of T cells can be ranked based on the score. At least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 300, 400, 500 or more TCRs can be selected from the plurality of T cells that are ranked in the top 50% according to the score. In some cases, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 300, 400, 500 or more TCRs can be selected from the top 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of multiple T cells according to the score. The selected TCR can then be synthesized and delivered to the recipient cell as a mixture or library. The selected TCR can be a functional TCR. The selected TCR can be a tumor-reactive TCR.The selected TCR can show better tumor reactivity or cytotoxicity than those unselected TCRs. The selected TCR can be synthesized and collected in a mixture to be delivered to a receptor cell. A plurality of polynucleotide sequences of synthetic coding TCR or its subset can include polynucleotide sequences encoding at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150, 200, 300, 400, 500 or more selected TCRs.

[0148] In some cases, the first part of multiple synthetic TILs exogenously expresses a first polynucleotide sequence encoding a first natural paired TCR, and the second part of multiple synthetic TILs exogenously expresses a second polynucleotide sequence encoding a second natural paired TCR, wherein the score of the third part of multiple synthetic TILs that exogenously express the first and second polynucleotide sequences is less than the product of the first and second part scores. The scores of the first part, the second part, or the third part can be determined by various methods. For example, the cell part can be determined by sequencing (e.g., single cell sequencing). Single cell sequencing can include barcoding each cell in a separate compartment. In some cases, the CDR3β of the exogenous TCR can be amplified and sequenced. The number of reads corresponding to the first TCR divided by the total number of reads can be a measured value of the first part score. The number of reads corresponding to the second TCR divided by the total number of reads can be a measured value of the second part score. The number of reads can be corrected by a unique molecular identifier (UMI). Another example is that the cell score can be determined by pMHC polymer staining. pMHC multimer staining can include the use of a plurality of pMHC multimers, each of which specifically binds to a TCR encoded by a polynucleotide sequence.

[0149] Receptor cells may obtain and / or express a single copy of a sequence encoding an exogenous TCR (e.g., a homologous TCR identified by sequencing of a sample obtained from a subject). In some cases, a receptor cell may obtain two or more sequences, each of which encodes different TCRs, but only expresses one sequence. For example, only one sequence may be knocked into a genomic site for expression. Expression may be controlled by an endogenous promoter. Other sequences that are not knocked into a genomic site may not be expressed. Other sequences may be degraded in the cell. For example, a vector containing a sequence may not include a promoter. In some cases, multiple polynucleotide sequences or derivatives thereof may be delivered to multiple receptor cells and may include knocking a polynucleotide sequence site-specifically into the genomic site of a receptor cell in multiple receptor cells. The genomic site of a receptor cell may result in monoallelic expression of a polynucleotide sequence. Monoallelic expression may include gene expression from a single allele in the context of a diploid heterozygous genome. Monoallelic expression may include gene expression from a single locus. In some cases, the monoallelic expression of homologous TCR pairs with TCR α chains and TCR β chains is controlled by a single promoter at the knock-in locus. The sequence of two chains encoding homologous TCR can be expressed continuously in frame from a single promoter. The sequence of two chains encoding homologous TCR can be connected by the sequence encoding self-cleavage peptide. In some cases, the monoallelic expression of homologous TCR pairs with TCR α chain and TCR β chain can be controlled by two promoters knocked into the locus. For example, two promoters may include a first promoter for TCR α chain and a second promoter for TCR β chain. The polynucleotide sequence can be site-specifically inserted into the genomic site of the receptor cell in multiple receptor cells in a manner that meets the reading frame. The genomic site can be located on a sex chromosome. The genomic site can be a T cell receptor α constant region (TRAC).

[0150] The polynucleotide sequence can be knocked into the genome site using gene editing methods as described herein. In some cases, gene editing methods may include clustered regularly spaced short palindromic repeats (CRISPR), transcription activator-like effectors (TALE) nucleases (TALENs), homing endonucleases (meganuclease), zinc finger nucleases (ZFN), homing endonucleases (meganuclease) methods or any combination thereof. In some cases, the polynucleotide sequence is knocked into the genome site using the CRISPR method. The CRISPR method may include delivering the ribonucleoprotein (RNP) complex comprising Cas9 protein and single guide RNA (sgRNA) to the recipient cell. sgRNA can target the locus on the sex chromosome for monoallelic expression. In some cases, sgRNA can target TRAC sites. The polynucleotide sequence is knocked into the genome site using the gene editing method to cut the genome site, and the polynucleotide sequence is inserted into the genome site by homologous recombination.

[0151] In various cases, endogenous TCR can be inactivated in recipient cells. Endogenous TCR can be knocked down (e.g., by RNA interference) or knocked out (e.g., by gene editing methods described herein) in recipient cells. In some cases, endogenous T cell receptor β constant region (TRBC) loci are inactivated in recipient cells. Endogenous TRBC loci can be knocked down or knocked out in recipient cells.

[0152] In some cases, the endogenous TCR is inactivated in at least about 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100% of the plurality of recipient cells.

[0153] Endogenous TCR can be knocked down or knocked out in at least about 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% of multiple receptor cells. Endogenous TCR can be inactivated using the gene editing method described herein. For example, endogenous TCR can be selected from clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector (TALE) nuclease (TALENs), homing nuclease (meganuclease), zinc finger nuclease (ZFN), megaTAL nuclease method and any combination thereof.

[0154] Endogenous TCR can be inactivated before, simultaneously or after multiple polynucleotide sequences or derivatives thereof are delivered to multiple receptor cells in (d). In some cases, endogenous TCR can be inactivated while multiple polynucleotide sequences or derivatives thereof are delivered to multiple receptor cells in (d). For example, a vector containing a sequence encoding exogenous TCR and a reagent (e.g., nuclease or RNA) for gene editing can be delivered to the receptor cell simultaneously. Endogenous TCR can be inactivated using CRISPR methods. CRISPR methods may include delivering a ribonucleoprotein (RNP) complex comprising Cas9 protein and single guide RNA (sgRNA) to the receptor cell. sgRNA may include sgRNA targeting TRBC locus. Endogenous TCR can be inactivated while polynucleotide sequence site-specific knock-in genomic locus by delivering RNP comprising Cas9 protein and sgRNA (including sgRNA targeting TRBC locus and sgRNA targeting TRAC locus).

[0155] Each cell in multiple synthetic TILs can exogenously express only a single type of TCR. Each cell of multiple synthetic TILs can exogenously express a single copy of the polynucleotide sequence encoding TCR. In some cases, at least about 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% of multiple synthetic TILs only exogenously express a single copy of the polynucleotide sequence of the natural pairing TCR encoding multiple T cells. In other words, at least about 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% of each cell in multiple synthetic TILs only exogenously expresses a single copy of the polynucleotide sequence of the TCR encoding natural pairing.

[0156] The synthetic TILs provided herein can be expanded prior to being administered to a subject from which a cognate TCR pair was obtained.The "activation and expansion" methods described in the present disclosure can be applied to expand synthetic TILs.

[0157] Unique homology TCR is to (such as clonal type) before being delivered to receptor cell and expressing, can have initial frequency in mixture, and this mixture comprises multiple polynucleotide sequences of multiple different homology TCR pairs (such as TCR selected from sequencing data) of encoding.In the process of producing synthetic TILs, the frequency of unique TCR can be maintained in 0.1 times to 10 times of initial frequency in mixture (for example, in 0.2 times to 5 times, or in 0.5 times to 2 times).In some cases, the frequency of unique TCR can be maintained in 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, 4 times, 3 times, 2 times or lower scope of initial frequency.In some cases, after sequence is delivered to receptor cell to express and amplify synthetic TILs, the frequency of unique homology TCR can be close to or in 10 times, 9 times, 8 times, 7 times, 6 times, 5 times, 4 times, 3 times, 2 times or lower scope of initial frequency.In other words, method described herein or synthetic TILs provided herein can resist growth deviation. In the manufacturing processes described herein, cells expressing one or more unique TCRs may not grow better or outcompete other unique TCRs in the mixture.

[0158] The frequency described herein refers to the relative abundance, ratio or copy number of a unique TCR in a TCR mixture. For example, if a mixture of 10 TCRs is selected for producing synthetic TILs, and each sequence encoding a unique TCR is mixed with other sequences in an equimolar ratio, the frequency of the unique TCR in the mixture is 1 / 10 or 10% (see Example 9). After the synthetic TILs are amplified, the frequency can be determined by various methods including sequencing. Taking sequencing as an example, the frequency can be calculated by dividing the sequencing reads of a specific TCR by the total sequencing reads of the TCR library. In some cases, the frequency can be determined by dividing the unique molecular index (UMI) count of a specific TCR by the total UMI count of the TCR library. When preparing a sequencing library using a standard protocol step, UMI can be added to the nucleic acid molecule to be sequenced.

[0159] In various embodiments, the synthetic TILs prepared herein can be administered to a subject. The subject can be the same subject of multiple source T cells. The subject can be a cancer patient. The subject can have a solid tumor lesion. The subject can have an advanced or metastatic solid tumor. Examples of cancer include, but are not limited to, melanoma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, head and neck cancer, colon cancer, gallbladder cancer, laryngeal cancer, liver cancer (e.g., hepatocellular carcinoma), thyroid cancer, gastric cancer, salivary gland cancer, prostate cancer, pancreatic cancer, urothelial carcinoma or Merkel cell carcinoma.In some cases, the cancer can be adrenocortical carcinoma, adrenocortical carcinoma, AIDS-related cancer, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoid tumor, basal cell carcinoma, bile duct cancer, extrahepatic, bladder cancer, bone cancer (including Ewing's sarcoma and osteosarcoma and malignant fibrous histiocytoma), brain tumor, breast cancer, carcinoid tumor (gastrointestinal tract), cancer of unknown primary site, cervical cancer, bile duct cancer, chronic myeloproliferative neoplasms, colorectal cancer, cutaneous T-cell lymphoma, ductal carcinoma in situ (dcis), endometrial cancer, esophageal cancer, extragonadal germ cell tumor, eye cancer, intraocular melanoma, optic plexus, Omentoblastoma, fallopian tube cancer, fibrous histiocytoma of bone, malignant and osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, extragonadal, ovarian, testicular, gestational trophoblastic disease, glioma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Langerhans cell histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor, pancreatic neuroendocrine tumor, laryngeal cancer, lip and oral cancer, liver cancer (primary), lung cancer, lymphoma, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma of bone and osteosarcoma, intraocular melanoma leukoma, Merkel cell carcinoma, mesothelioma, malignant, metastatic squamous neck cancer occult primary, oral cancer, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, paraganglioma, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, plasma cell neoplasms / multiple myeloma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, primary In some cases, the method may further include administering at least one cell of a plurality of synthetic TILs to a subject having a solid tumor lesion.

[0160] In some cases, prior to administration, the plurality of synthetic TILs have not been subjected to any selection or screening to determine the tumor reactivity of the plurality of synthetic TILs or a subset thereof. For example, a plurality of synthetic TILs each exogenously expressing a different TCR may not have been contacted with a tumor antigen, and any subset that can bind to a tumor antigen may not be selected for administration. In some cases, a plurality of synthetic TILs may contain both tumor-reactive cells and tumor-non-reactive cells.

[0161] In some cases, the tumor reactivity of multiple synthetic TILs can be determined before administration. The tumor reactivity of multiple synthetic TILs can be determined after amplification and before administration. Multiple synthetic TILs can be a mixture, and the tumor reactivity of multiple synthetic TILs can be determined together. In this case, multiple synthetic TILs may include tumor reactive cells and tumor non-reactive cells. In some cases, determining tumor reactivity may include: (i) providing tumor cells or derivatives thereof (e.g., progeny or cell components of tumor cells) from a subject; (ii) contacting multiple synthetic TILs with tumor cells or derivatives thereof; (iii) determining the tumor reactivity of multiple synthetic TILs to tumor cells or derivatives thereof; and (iv) determining whether multiple synthetic TILs or subsets thereof are administered to a subject. Tumor reactivity can be determined by a variety of methods. For example, a mixture of multiple synthetic TILs can be co-cultured with tumor cells or derivatives thereof, and cytokine release can be tested using methods such as ELISA and ELISPOT. In some cases, after determining the tumor reactivity of the mixture, at least one cell or mixture in a plurality of synthetic TILs can be administered to a subject with a solid tumor lesion.

[0162] Methods described herein can provide a rapid turnaround time from sample separation to the preparation of a pharmaceutical composition for administration. In some cases, the time interval from obtaining a sample (for example, excision or biopsy sample) to at least one cell mass in multiple synthetic TILs being applied to the subject can be less than 2,3,4,5,6,7,8 or longer. In some cases, the time interval from obtaining a sample (for example, excision or biopsy sample) and at least one cell mass in multiple synthetic TILs being given to the subject can be no more than 10,9,8,7,6,5,4,3 or less time. In some cases, the time interval from obtaining a sample to at least one cell mass in multiple synthetic TILs being applied to the subject can be less than 8 weeks. In some cases, the time interval from obtaining a sample to applying at least one cell mass in multiple synthetic TILs to the subject can be less than 4 weeks. In some cases, the time interval between obtaining a sample and using the synthetic TILs comprising at least about 10,20,30,40,50,60,70,80,90,100 or more different TCRs to an experimenter can be less than 2,3,4,5,6,7,8 weeks or longer. In some cases, the time interval between obtaining a sample to using the synthetic TILs comprising at least about 10,20,30,40,50,60,70,80,90,100 or more different TCRs to an experimenter can be less than 8 weeks. In some cases, the time interval between obtaining a sample to using the synthetic TILs comprising at least about 10,20,30,40,50,60,70,80,90,100 or more different TCRs to an experimenter can be less than 4 weeks.

[0163] In some cases, the time interval between the synthetic sequence encoding TCRs and the recipient cells from obtaining the sample may be no more than 8,7,6,5,4,3 weeks or shorter. In some cases, the time interval between the synthetic sequence encoding TCRs and the recipient cells from obtaining the sample may be no more than 7 weeks. In some cases, the time interval between the synthetic sequence encoding TCRs and the recipient cells from obtaining the sample may be no more than 3 weeks. In some cases, the time interval between the synthetic sequence encoding TCRs and the recipient cells from obtaining the sample may be no more than 8,7,6,5,4,3 weeks or shorter. In some cases, the time interval between the synthetic sequence encoding TCRs and the recipient cells from obtaining the sample may be no more than 7 weeks. In some cases, the time interval between obtaining the sample and delivering the synthetic sequences encoding at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more different TCRs to the recipient cells may be no more than 3 weeks.

[0164] The methods described herein can produce a pool of synthetic TILs expressing different TCRs. The synthetic TILs pool can be a polyclonal TCR-T cell. Synthetic TILs can exogenously express at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000, 10000, 100000 or more different TCRs.

[0165] Two or more cells in multiple synthetic TILs can be administered in a subject, and two or more cells administered can express at least about 2,3,4,5,10,20,30,40,50,100,500,1000,10000,100000 or more different exogenous TCRs.Multiple synthetic TILs can include a cell subgroup expressing the TCR identifying tumor antigens and a cell subgroup expressing the TCR not identifying tumor antigens.In various cases, IL-2 (e.g., administered intravenously by infusion) can be administered before, simultaneously or afterwards using synthetic TILs as described herein.

[0166] The sample may be a tumor sample. The tumor sample may be a biopsy sample, such as a core biopsy or fine needle biopsy sample. These samples may be small in size (e.g., <1000 mm 3 (μL), <500mm 3 、<100mm 3 、<50mm 3), even small volumes of tumor samples may contain sufficient cells for sequencing. In some cases, the volume of a tumor sample can be equal to or up to about 2000 mm 3 , 1000mm 3 , 800mm 3 , 500mm 3 , 100mm 3 , 50mm 3 , 20mm 3 or less. Thus, the method is suitable for situations where it is difficult to obtain a large surgical tumor sample. For example, the volume of the resection or biopsy sample can be less than about 200, 150, 100, 50, 20 or less mm 3 In some cases, the volume of the resection or biopsy sample may be less than about 100 mm 3 The weight of the excision or biopsy sample may be less than about 1 gram (g), 0.9g, 0.8g, 0.7g, 0.6g, 0.5g, 0.4g, 0.3g, 0.2g, 0.1g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g or less. In some cases, the weight of the excision or biopsy sample may be less than about 0.1g. In some cases, the weight of the excision or biopsy sample may be less than about 0.03g. In some cases, the weight of the excision or biopsy sample may not exceed 0.1g, 0.08g, 0.05g, 0.03g or less.

[0167] The coding sequence of exogenous TCRs can be delivered to the receptor cells for expression. These sequences can be delivered as a mixture. The mixture contains at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000, 10000, 100000 or more different polynucleotide sequences. The polynucleotide sequence encodes at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000, 10000, 100000 or more different natural paired TCRs. Delivery can be carried out by various methods described herein. For example, multiple polynucleotide sequences or derivatives thereof can be delivered to multiple receptor cells by a carrier. The carrier can be a non-viral vector. The non-viral vector can be a plasmid, a transposon, a self-amplification RNA or any combination thereof. The carrier can be a viral vector. The viral vector can be an adenoviral vector, an adeno-associated virus (AAV) vector, a herpes simplex virus vector, a lentiviral vector, a retroviral vector or any combination thereof. The viral vector may be an AAV vector. The viral vector may be a lentiviral vector. The vector may not contain a promoter.

[0168] Receptor cells can be obtained from a variety of sources, including cell line cells or primary cells isolated from a subject. These receptor cells can be multiple T cells. Receptor cells can be isolated from the same subject to obtain multiple source T cells (e.g., those cells from which natural paired TCRs are obtained). Receptor cells can be primary T cells or peripheral T cells.

[0169] The subject described herein may have failed a previous treatment. The previous treatment may be a blocker treatment under investigation.

[0170] In some cases, before synthesizing the sequence encoding TCRs, sequencing as described herein may be optional. For example, the method of producing multiple synthetic tumor infiltrating lymphocytes (TILs) may include obtaining a resection or biopsy sample containing multiple T cells from a subject's solid tumor lesion. Next, multiple polynucleotide sequences of multiple T cells obtained may be synthesized, and these sequences encode natural paired TCRs or a subset thereof. Multiple polynucleotide sequences may include a first polynucleotide sequence encoding a first natural paired TCR of a first T cell in multiple T cells and a second polynucleotide sequence encoding a second natural paired TCR of a second T cell in multiple T cells, wherein the first polynucleotide sequence is different from the second polynucleotide sequence. Next, multiple polynucleotide sequences or their derivatives may be delivered to multiple receptor cells by contacting a mixture containing multiple polynucleotide sequences or their derivatives with multiple receptor cells, thereby producing multiple synthetic TILs. In some cases, sequencing may be performed to obtain the natural paired T cell receptor (TCR) α chain and β chain sequences of the TCR of multiple T cells. In some cases, the expression level of one or more genes (e.g., at least 10 genes) may be obtained from sequencing.

[0171] In some aspects, the present invention discloses a method for producing multiple synthetic tumor infiltrating lymphocytes (TILs), the method comprising contacting a mixture containing multiple polynucleotide sequences or derivatives thereof with multiple receptor cells, and delivering multiple polynucleotide sequences or derivatives thereof into multiple receptor cells. Each polynucleotide sequence in a plurality of polynucleotide sequences can encode paired sequence TCR alpha chain and TCR beta chain. Each polynucleotide sequence can be knocked into a genomic site to achieve monoallelic expression of a polynucleotide sequence. In some cases, the endogenous TCR gene of each receptor cell in a plurality of receptor cells can be inactivated. In some cases, the endogenous TCR gene of each receptor cell in a plurality of receptor cells can be inactivated before, simultaneously or after the expression of a sequence encoding paired sequence TCR. In some cases, the endogenous TCR gene of each receptor cell in a plurality of receptor cells may have been inactivated before the expression of a sequence encoding a homologous TCR pair. In some cases, at least about 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% of the synthetic TILs only exogenously express a single copy of a polynucleotide sequence that encodes a plurality of naturally paired TCRs of T cells. The genomic locus may be a TRAC gene. The endogenous TCR gene may be destroyed by knocking down or knocking out the TRBC site. The mixture may comprise at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1,000, 10,000, 100,000 or more different polynucleotide sequences encoding at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000, 10000, 100000 or more different naturally paired TCRs.

[0172] The polyclonal synthetic TILs produced herein can be used to treat subjects in need, or can be used to prepare drugs for treating diseases. The disease can be cancer. The cancer can be a solid tumor. In some cases, the cancer is esophageal cancer, gastric cancer, colorectal cancer, head and neck cancer, lung cancer, liver cancer, ovarian cancer or cervical cancer. In some cases, the cancer is melanoma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, head and neck cancer, colon cancer, gallbladder cancer, laryngeal cancer, liver cancer (e.g., hepatocellular carcinoma), thyroid cancer, gastric cancer, salivary gland cancer, prostate cancer, pancreatic cancer, urothelial carcinoma or Merkel cell carcinoma. Subjects in need can be subjects from which natural paired TCRs are obtained. The methods or pharmaceutical compositions provided herein can be used to carry out personalized treatment of subjects in need. Source T cells or T cell receptors

[0173] T cells can be isolated from a sample of a subject, from which a naturally paired TCR (or a paired sequence of TCR) can be obtained and sequenced. In the method for producing synthetic TILs described herein, T cells can be used as a source for providing TCR. For example, multiple T cells can be isolated from a tissue sample (e.g., a tumor sample or a solid tumor sample) of a subject. The TCR of a T cell can be sequenced to determine the paired sequence of a TCR alpha chain and a beta chain or a TCR gamma chain and a delta chain.

[0174] T cells obtained from a subject may express a subject-derived TCR or a subject-specific TCR. A subject-derived TCR or a subject-specific TCR may be specific to a subject or a subject's tumor, and the subject is intended to be treated with a pharmaceutical composition described herein.

[0175] In some cases, the T cell can be a CD4+T cell, a CD8+T cell, or a CD4+ / CD8+T cell. In some cases, the T cell can be a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a natural killer T cell, an αβT cell, or a γδT cell.

[0176] T cells can be obtained from tissue samples, and the tissue samples include solid tissues, and specific examples include but are not limited to tissues from brain, liver, lung, kidney, prostate, ovary, spleen, lymph nodes (such as tonsils), thyroid, thymus, pancreas, heart, skeletal muscle, intestine, larynx, esophagus and stomach. Other sources include but are not limited to bone marrow, umbilical cord blood, tissue from infection site, ascites, pleural effusion, spleen tissue and tumor. In some cases, T cells can be obtained from experimenter's solid tumor lesions. T cells can be derived from or taken from healthy donors, patients diagnosed as cancer or patients diagnosed as infection.

[0177] T cells can be isolated from a sample and T cells with certain characteristics can be selected by various methods. When isolating T cells from tissue (e.g., isolating tumor-infiltrating T cells from tumor tissue), the prepared tissue is minced or crushed to separate the cells before lysing red blood cells or consuming monocytes. Specific subsets of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, T cells can be isolated by beads conjugated with anti-CD3 / anti-CD28 (e.g., 3×28), such as DYNABEADS TMM-450CD3 / CD28 T, continue enough to positively select the desired T cell time period. In some cases, the time period is about 30 minutes. In some cases, the range of the time period is from 30 minutes to 36 hours or longer, and all integer values ​​therebetween. In some cases, the time period is at least or equal to about 1, 2, 3, 4, 5 or 6 hours. In some cases, the time period is 10 to 24 hours. In some cases, the incubation time is about 24 hours. In any case where T cells are less than other cell types, for example, when separating tumor infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals, longer incubation times can be used to separate T cells. In addition, using longer incubation times can increase the capture efficiency of CD8+T cells. Therefore, by simply shortening or extending the time allowing T cells to bind to anti-CD3 / anti-CD28 beads and / or by increasing or reducing the ratio of beads to T cells, a subpopulation of T cells can be selected at the beginning of cultivation or at other time points in the process. In addition, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on beads or other surfaces, T cell subsets can be selected at the start of culture or other required time points. In some cases, multiple rounds of selection can be used. In some aspects, a selection procedure can be performed, and "unselected" cells (cells that may not be combined with anti-CD3 / anti-CD28 beads) can be used for activation and amplification processes. "Unselected" cells can also be further selected.

[0178] Enrichment of T cell populations by negative selection can be completed with a combination of antibodies for surface markers unique to negatively selected cells. An exemplary method can be cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, which uses a monoclonal antibody mixture for cell surface markers present on cells selected by negative selection. For example, in order to enrich CD4+ cells by negative selection, a monoclonal antibody mixture generally includes antibodies for CD14, CD20, CD11b, CD16, HLA-DR and CD8. In some aspects, enrichment or positive selection generally express regulatory T cells of CD4+, CD25+, CD62LHI, GITR+ and FoxP3+ and may be useful. Or, in some aspects, T regulatory cells are removed by anti-C25 conjugated beads or other similar selection methods.

[0179] T cell populations expressing one or more of the following molecules can be selected: IFN-γ, TNF-α, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B and perforin, or other molecules, such as other cytokines and transcription factors, such as T-bet, Eomes, Tcf1 (TCF7 in humans). The method of screening cell expression can be determined by the method described in PCT Publication No.: WO 2013 / 126712.

[0180] In order to separate cell populations by positive or negative selection, the concentration of cell or particle surface (for example, particles such as sorting magnetic beads) can be changed. In some aspects, the volume (for example, increasing the concentration of cells) that beads and cells are mixed together can be reduced to ensure the maximum contact of cells and beads. For example, in some cases, a concentration of 2 billion cells / ml is used. In another aspect, a concentration of 1 billion cells / ml is used. In some cases, greater than 100 million cells / ml is used. In some cases, a cell concentration of at least about 10, 15, 20, 25, 30, 35, 40, 45 or 50 million cells / ml is used. In some cases, a cell concentration of at least about 75, 80, 85, 90, 95 or 100 million cells / ml is used. In some cases, a cell concentration of at least about 125 million or 150 million cells / ml can be used. Using high concentration can cause cell yield to increase, cell activation and cell expansion. In addition, using high cell concentrations can more effectively capture cells of interest that may weakly express cell surface markers, such as CD28-negative T cells, or from samples where many tumor cells are present (e.g., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value. For example, using high concentrations of cells can more effectively select CD8+ T cells with weak CD28 expression.

[0181] In some cases, lower concentrations of cells can be used. By significantly diluting the mixture of T cells, surface interactions between the particles and the cells can be minimized. This can select cells that express large amounts of the desired antigen for binding to the particles. For example, CD4+ T cells may express higher levels of CD28 and may be captured more efficiently than CD8+ T cells at dilute concentrations. In some aspects, the concentration of cells used is at least about 5×10 5 / mL, 5×10 6 / mL or more. In other aspects, the concentration used can be about 1×10 5 / ml to 1×10 6 / ml, and any integer value therebetween. In other aspects, the cells can be incubated on a rotator at 2-10°C or room temperature for varying lengths of time and at varying speeds.

[0182] T cell source can be tumor infiltrating lymphocytes (TILs), such as tumor infiltrating T cells (TIT). TILs can be separated from organs suffering from cancer. One or more cells can be separated from organs suffering from cancer, and the organ can be brain, heart, lung, eye, stomach, pancreas, kidney, liver, intestine, uterus, bladder, skin, hair, nail, ear, gland, nose, mouth, lip, spleen, gums, teeth, tongue, salivary gland, tonsil, pharynx, esophagus, large intestine, small intestine, rectum, anus thyroid gland, thymus, bone, cartilage, tendon, ligament, adrenal gland membrane, skeletal muscle, smooth muscle, blood vessel, blood, spinal cord, trachea, ureter, urethra, hypothalamus, pituitary gland, pylorus, adrenal gland, ovary, fallopian tube, uterus, vagina, mammary gland, testis, seminal vesicle, penis, lymph, lymph node or lymphatic vessel. One or more TILs can be from brain, heart, liver, skin, intestine, lung, kidney, eye, small intestine or pancreas. TILs can be from pancreas, kidney, eye, liver, small intestine, lung or heart. The one or more cells can be islet cells, such as pancreatic beta cells. In some cases, TILs may be from gastrointestinal cancer. TILs culture can be prepared in a variety of ways. For example, tumors can be trimmed from non-cancerous tissue or necrotic areas. The tumor can then be broken into pieces to about 2-3mm long. In some cases, the size of the tumor can be about 0.5mm to about 5mm, about 1mm to about 2mm, about 2mm to about 3mm, about 3mm to about 4mm or about 4mm to about 5mm. Tumor fragments can then be cultured in vitro using culture medium and cell stimulants such as cytokines. In some cases, IL-2 can be used to amplify TILs from tumor fragments. IL-2 concentration can be about 6000IU / ml. The concentration of IL-2 can also be about 2000IU / ml, 3000IU / ml, 4000IU / ml, 5000IU / ml, 6000IU / ml, 7000IU / ml, 8000IU / ml, 9000IU / ml or up to about 10000IU / ml. Once TILs are amplified, they can be subjected to in vitro analysis to determine tumor reactivity. For example, CD3, CD4, CD8 and CD58 expression of TILs can be assessed by FACS. TILs can also be subjected to co-cultivation, cytotoxicity, ELISA or ELISPOT analysis. In some cases, TILs cultures can be cryopreserved or rapidly amplified. Cells, such as TILs, can be separated from donors at developmental stages, including but not limited to fetuses, newborns, youth and adults.

[0183] In some cases, the tumor sample can be a surgically removed tumor sample (or excision sample). T cells can be TITS separated from the excision sample. For example, after the removal of fat and connective tissue, the tumor tissue can be cut into 3-5mm2 fragments and decomposed using a gentle mechanical crushing in cold RPMI 1640 using a Seward Stomacher device (Fisher, Pittsburgh, PA). The method can quickly prepare a single cell suspension without enzymatic digestion. The cell suspension can be filtered through a sieve (BD Biosciences, San Jose CA) with a 75 μm pore size and washed in a culture medium. A portion of cells for immediate staining and analysis by flow cytometry can be washed in a culture medium, and a cell suspension can be layered on a discontinuous 70%, followed by 100% Ficoll Isopaque gradient centrifugation to separate tumor cells (70% interface) and enriched cells (100% interface). Then wash the enriched TITS in D-PBS, 1% BSA, and then process. In some cases, TITs from tumor samples can be expanded in TIT medium (TILs-CM), which contains RPMI 1640 with Glutamax (Invitrogen), 10% human AB serum (Sigma, St. Louis, MI), 50 mM 2-mercaptoethanol (Invitrogen), 1 mM pyruvate, 1X penicillin / streptomycin (Invitrogen) and 3000 IU / ml recombinant IL-2.

[0184] In some cases, the tumor sample can be a biopsy sample, such as a core needle biopsy or fine needle biopsy sample. These samples can have a small volume (e.g., <1000mm 3 (μL), <500mm 3 , <100mm 3 , <50mm 3 ), even a small volume of tumor sample may contain enough cells for sequencing. In some cases, the volume of the tumor sample can be equal to or at most about 2000 mm 3 , 1000mm 3 , 800mm 3 , 500mm 3 , 100mm 3 , 50mm 3 , 20 mm3 or less. Therefore, the method can be applied to situations where it is difficult to obtain a large tumor sample surgically. For example, the volume of the resection or biopsy sample can be less than about 200 mm 3 、150mm 3 , 100mm 3 , 50mm 3, 20mm 3 In some cases, the volume of the excision or biopsy sample can be less than about 100 mm 3. The weight of the excision or biopsy sample can be less than about 1 gram (g), 0.9g, 0.8g, 0.7g, 0.6g, 1.5g, 0.5g, 0.3g, 0.2g, 0.1g, 0.08g, 0.07g, 0.06g, 0.05g, 0.04g, 0.03g, 0.02g, 0.01g or less. In some cases, the weight of the excision or biopsy sample can be less than about 0.1g. The T cell can be a tumor infiltrating cell from a biopsy sample.

[0185] In some cases, fresh tumors may not be available, in which case cell nuclei can be isolated from frozen or fixed tissues. These cell nuclei can also be used as input for identifying paired sequences of TCR and preparing synthetic TILs described herein. These cells and cell nuclei can be used without further selection. Alternatively, specific populations of cells or cell nuclei can be isolated to enrich tumor-specific TCRs. For example, the expression of cell surface markers CD39, CD69, CD103, CD25, PD-1, Tim-3, OX-40, 4-1BB may be associated with tumor reactivity. Cell surface markers can be used to separate / enrich tumor-reactive TCRs by FACS. In other words, cells that highly express one or more of these markers can be used as input for identifying paired sequences of TCRs. Sequencing

[0186] Various sequencing methods can be used. Various sequencing methods include but are not limited to Sanger sequencing, high-throughput sequencing, synthetic sequencing, single molecule sequencing, connection sequencing, RNA-Seq, second-generation sequencing (NGS), digital gene expression, cloned single microarray, shotgun sequencing, Maxim-Gilbert sequencing, or large-scale parallel sequencing. Cells expressing TCR can be used as inputs for single-cell RNA-Seq methods (such as InDrop or DropSeq). For example, sequencing can use single-cell barcodes (for example, cells are assigned to separate compartments, nucleic acids released from single cells are barcoded, nucleic acids are sequenced, and TCR chains from single cells are paired based on the same barcode). If the sequence encoding the paired TCR chains in the cell has been fused or connected into a single continuous polynucleotide chain, sequencing may not include the use of barcodes.

[0187] The sequencing described herein may be single-cell sequencing. Single-cell sequencing refers to obtaining sequence information from a single cell and detecting the genome, transcriptome, and other multi-omics of a single cell. In single-cell sequencing, a group of cells can be made into a single-cell suspension and compartmentalized into separate partitions. Within each partition, the sequences released from a single cell can be barcoded and then sequenced. Various single-cell sequencing methods can be used for TCR reconstruction (see De Simone M, Rossetti Gand Pagani M (2018) Single Cell T Cell Receptor Sequencing: Techniques and Future Challenges. Front. Immunol. 9: 1638).

[0188] In some cases, the source T cells separated from the sample of the subject can be sequenced to obtain information about the natural pairing sequence of the TCR alpha chain and the TCR beta chain or the TCR gamma chain and the TCR delta chain. In some cases, the source T cells can be sequenced to obtain the gene expression levels of certain gene markers. Gene markers include but are not limited to CXCL13, CTLA4, DUSP4, ENTPD1, ​​RBPJ, TNFSF4, GK, PDCD1, LAG3, FKBP1A, MYO7A, FABP5, GAPDH, TIGIT, CDCA7, ZBED2, RGS1, HAVCR2, MKI67, GZMB, SPRY1, PHLDA1, ASPM, MYO1E, TOX, LSP1, MCM10, MAD2L1SAMSN1, TSHZ2 and HMGN2. Oligonucleotide synthesis

[0189] The polynucleotides encoding various TCR pairing sequences can be chemically synthesized. In some cases, these polynucleotides can be synthesized by phosphoramide chemistry. For example, based on sequencing information, the sequence of the pairing sequence encoding TCR can be obtained, and each sequence can be synthesized by a chemical method, such as by phosphoramide chemistry. In some cases, sequence fragments encoding TCR pairing sequences can be chemically synthesized, and each homologous pair of fragments can be assembled in a separate compartment, while other sequences encoding different TCRs are assembled in other compartments.

[0190] The polynucleotides encoding different paired TCRs can also be synthesized in large quantities or mixed (e.g., the same mixture) using the method disclosed in International Application No. PCT / US2020 / 026558, the entire contents of which are incorporated herein by reference. In this method, each polynucleotide comprising a sequence derived from a TCR V gene or a portion thereof can be pre-synthesized and mixed as a pool. The sequence derived from the TCR V gene can be a part of the TCRV gene. The sequence derived from the TCR V gene can be a codon-optimized sequence or comprise one or more modified nucleotides. For example, a sequence derived from a TCR V gene can be pre-synthesized, comprising L-PART1 (the first part of the leader peptide), L-PART2 (the second part of the leader peptide), FR1, CDR1, FR2, CDR2 and FR3 encoding sequence, referred to as LV-REGION. For another example, a sequence derived from a TCR V gene can be pre-synthesized, comprising FR1, CDR1, FR2, CDR2 and FR3 encoding sequence, referred to as V-REGION. The nucleic acid sequence segments of L-PART1, L-PART2, FR1, CDR1, FR2, CDR2 or FR3 can be defined according to the IMGT unique numbering system (http: / / www.imgt.org). In some cases, the sequence derived from the TCR V gene may include a sequence starting with a sequence encoding L-PART1 and ending with a codon encoding a second conserved cysteine ​​(e.g., 2nd-Cys as defined by IMGT corresponds to the codon for the 104th conserved cysteine ​​of the V-domain). Since there are about 80 or more TCRV genes (e.g., TRAV and TRBV genes) in the human genome, synthesizing such a "V gene germline polynucleotide library" (e.g., Fig. 22801 and brackets X) are feasible. In some cases, a subset of the TCR V genes of a species (e.g., human or mouse) is used to generate a "V gene germline polynucleotide library". All identified TCR V genes or subsets of TCRV genes can be synthesized. 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 or more TCR V genes of the species can be synthesized to generate a library. In some cases, the TCR V genes of all identifications of a species are synthesized to produce a library. The TCR V gene can be TRAV, TRBV, TRGV or TRDV. As described herein, in some cases, " V ​​gene germline polynucleotides " refers to a part of the genome or codon-optimized polynucleotides of the TCR V gene. The sequence derived from the TCRV gene can be a V gene germline polynucleotide. The sequence between FR3 and the constant region (for example, CDR3 plus the remainder in J district, referred to herein as " CDR3-J ") can be at least about 10,20,30,40,50,60,70,80,90 or more nucleotides long, or in some cases, can be up to about 90 nucleotides long. The CDR3-J sequence of the α chain and β chain of TCR can be at least about 50,60,70,80,90,100,120,150,180 or more nucleotides long. The CDR3-J sequences of the α and β chains of a TCR (in some cases, up to about 180 nucleotides in total) can be contained in oligonucleotides (referred to as "paired CDR3-J oligonucleotides," "paired CDR3-J oligonucleotides," or "paired CDR3-J polynucleotides," which can be used interchangeably) that can be amenable to chip-based synthesis (e.g., Fig. 22As shown, 802 and brackets Y containing 802). In some cases, the paired CDR3-J polynucleotides may include the CDR3-J sequences of the TCRγ chain and the CDR3-J polynucleotides of the TCRδ chain. As used herein, the terms "CDR3-J polynucleotides", "CDR3-J oligonucleotides" and "CDR3-J oligonucleotides" (which can be used interchangeably) refer to polynucleotide sequences comprising one or more CDR3-J sequences. CDR3-J polynucleotides can be paired CDR3-J polynucleotides (e.g., including CDR3-J sequences from paired TCR chains). CDR3-J polynucleotides (e.g., non-paired) may contain only the CDR3-J sequences from one of the paired TCR chains. For example, CDR3-J polynucleotides may contain only the CDR3-J sequences from TCRα chains, TCRβ chains, TCRγ chains or TCRδ chains. A remaining challenge may be to convert such paired CDR3-J oligonucleotides into a high-throughput construction of expressible TCRs (e.g., construction of >1,000 TCRs in one batch). Using the methods described herein, paired CDR3-J oligonucleotides can be ligated to their corresponding V gene germline polynucleotides in a bulk reaction (see Fig. 22 803). In some cases, a CDR3-J polynucleotide library (e.g., paired or unpaired) can include at least about 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 12000, 15000, 20000, 100000, 10000 34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80 or more TCR V genes. When using the methods described herein, a plurality of at least 2, 5, 10, 20, 50, 100, 200, 300, 400, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 12000, 15000, 20000, 100000, 1000000 or more encoding different natural paired TCRs can be produced. Natural paired TCRs can be produced in batches in a single compartment.

[0191] Examples of germline or rearranged gene constructs of nucleic acid molecules comprising the TCRV gene sequence are shown in Figures 18A-18C For example, Fig.18A The germline genomic DNA of the TCR V genes is shown, which includes L-PART1, V-INTRON, V-EXON and recombination signal sequences (V-HEPTAMER, V-SPACER and V-NONAMER). Fig.18A Two conserved cysteines are also shown in FIG. After V-(D)-J recombination, an exemplary construct of rearranged genomic DNA is shown in FIG. Fig.18B or Fig. 18C CDR3 may be encoded by (i) the junction (or junction region) between the V-region and the J-region or (ii) the junction between the V-region and the D-region and the junction between the D-region and the J-region.

[0192] TCR V genes can be very diverse. In humans, more than 40 functional TRAV genes have been identified, including, for example, TRAV1-1, TRAV1-2, TRAV2, TRAV3, TRAV4, TRAV5, TRAV6, TRAV7, TRAV8-1, TRAV8-2, TRAV8-3, TRAV8-4, TRAV8-6, TRAV9-1, TRAV9-2, TRAV10, TRAV12-1, TRAV12-2, TRAV12-3, TRAV13-1, TRAV13-2, TRAV In some embodiments, TRAV1-1 and TRAV1-2 belong to the same subgroup.As used herein, " group " is to have identical " gene type " (for example V, D, J or C type) and may participate in the synthetic group of identical " chain type " polypeptide.By extension, a colony comprises relevant pseudogene and orphan. "Subgroup" refers to a group of genes that belong to the same group in a given species and share at least 75% identity at the nucleotide level (in the germline configuration of V, D, and J).

[0193] In humans, more than 40 functional TRB V genes have been identified, including, for example, TRBV2, TRBV3-1, TRBV4-1, TRBV4-2, TRBV4-3, TRBV5-1, TRBV5-4, TRBV5-5, TRBV5-6, TRBV5-8, TRBV6-1, TRBV6-2, TRBV6-3, TRBV6-4, TRBV6-5, TRBV6-6, TRBV6-8, TRBV6-9, TRBV7-2, TRBV7-3, TRBV7-4, TRBV7-6, TRBV7-7, TRBV7-8. 8, TRBV7-9, TRBV9, TRBV10-1, TRBV10-2, TRBV10-3, TRBV11-1, TRBV11-2, TRBV11-3, TRBV12-3, TRBV12-4, TRBV12-5, TRBV13, TRBV14, TRBV15, TRBV16, TRBV18, TRBV19, TRBV20-1, TRBV24-1, TRBV25-1, TRBV27, TRBV28, TRBV29-1, and TRBV30. V genes from other species (e.g., mouse) can be found in the IMGT database. Diversified connection sequence

[0194] Linking the V gene germline polynucleotide and the CDR3-J polynucleotide can be achieved by molecular biology techniques such as ligation and overlapping primer extension ( Fig. 20 However, to fully exploit the power of chip-based oligonucleotide synthesis, thousands or even more CDR3-J oligonucleotides can be ligated to their corresponding V gene germline polynucleotides in an overall reaction (e.g. Fig. 22 The main challenge in doing this may be the V gene germline polynucleotide (see arrow 803). Fig. 20 601) and CDR3-J (Appendix Fig. 20 602) may be a conserved FR3 region. Therefore, in the overall reaction, it may be difficult to control the connection of a specific V gene germline polynucleotide to a specific CDR3-J (such as the attached Fig.19As shown). Wherein the solid arrow indicates connection to the correct V gene germline polynucleotide, and the dotted arrow indicates connection to the incorrect V gene germline polynucleotide). For example, the TCR sequence can be formed by TRBV4-1 connected to a specific CDR3-jβ sequence. In the overall reaction, there may be V gene germline polynucleotides of TRBV4-1 and TRBV4-2, and the FR3 regions of these TRBV genes are highly similar. Therefore, the CDR3-J oligonucleotide of the TCR may be incorrectly connected to the TRBV4-2 germline polynucleotide. In order to alleviate this problem, codon diversification can be used to create differences between different FR3 sequences. For example, linker sequences (also referred to as "ZIP" sequences in the present disclosure) can be codon-diverse so that they can have different nucleic acid sequences, even if they can encode the same amino acid sequence. Codon diversification can be achieved by computational methods. By assigning codons to amino acids randomly or according to arbitrary rules, multiple nucleic acid sequences can be generated, each of which can encode the same amino acid sequence. Next, multiple nucleic acid sequences can be evaluated by calculation to assign scores according to arbitrary rules. Arbitrary rules can take into account factors such as restriction sites, tendency to hybridize with unwanted sequences, tendency to hybridize with a given sequence, or unwanted secondary structures in the sequence. Next, based on the score, a nucleic acid sequence can be selected from a plurality of nucleic acid sequences as a codon-diverse linker sequence. Codon-diverse linker sequences can be used to achieve the correct connection of CDR3-J polynucleotides with a specified TCR V gene presynthetic part. In a "V gene germline polynucleotide library" comprising some or all known TCR V genes, for example, each different TCR V gene can have a different linker sequence, which can be used to correctly connect corresponding CDR3-J oligonucleotides to form TCR chains according to a reference sequence. Reference sequences can be produced by sequencing the paired sequences of TCR chains. However, in some cases, it may be unclear to what extent linker sequences can be diversified, and to what extent the connection between V gene germline polynucleotides and CDR3-J oligonucleotides can be correct in the overall reaction. By diversifying the FR3 region of human T cell receptor V genes, the "wrong connection probability" of any given CDR3-J sequence can be made almost undetectable.

[0195] Different linker sequences or Zips used in one homogeneous assembly reaction can have different lengths or GC contents, but can have similar melting temperatures. In some cases, hundreds to thousands of Zips sequences are used in a homogeneous assembly reaction. Designing Zips sequences can follow similar rules as designing primers for PCR reactions, for example: all Zips used in one assembly reaction can have similar melting temperatures, a Zip cannot form a strong hairpin at 5°C below the melting temperature, a Zip cannot strongly hybridize with another Zip at 5°C below the melting temperature, and a Zip may not strongly hybridize with the complementary sequence of another Zip at 5°C below the melting temperature.

[0196] For example, to generate a set of 1,000 Zips that can be used for the same assembly reaction, one million random 50-mer sequences can be generated first. Next, a desired melting temperature (e.g., 60°C) can be selected. Then, the shortest subsequence of each 30-mer sequence (starting from the 5 end) with a melting temperature higher than the desired melting temperature can be retained, while the remaining bases can be removed. The resulting one million sequences (with different lengths) can be referred to as trimmed random sequences. Next, the secondary structure of each trimmed random sequence can be evaluated and ranked based on the Gibbs free energy of the minimum free energy (MFE) structure at 5°C below the desired melting temperature. The top 10,000 trimmed random sequences with the highest (e.g., the least negative) Gibbs free energy can be retained. Each of these retained sequences can be referred to as a Zips candidate. If a restriction enzyme is used in the assembly reaction, the Zips candidate sequence containing such a restriction site can be removed. Next, it can be evaluated based on the strength of the primer dimer formed by each Zip candidate with all other Zips candidates and their complements. If a strong primer dimer is formed, a penalty can be specified. The penalty is positively correlated with the strength of the primer dimer. The sum of all penalty scores can be the final penalty score for each zipper. The top 3000 Zip candidates with the lowest final penalty scores can be retained and are called ZIP finalists. The primer dimer evaluation process can then be repeated for the 3000 finalists to select the top 1000 sequences with the lowest final penalty scores, which can be used as Zips. Many web-based and stand-alone software packages, such as Primer3, UNAfold, NUPACK, PrimerROC, Pythia, Multiple Primer Analyzer (Thermo Fisher), and OligoEvaluator (Sigma-Aldrich) can be used to implement this process.

[0197] Once a set of different linker sequences are found, many methods using molecular biology techniques (e.g., ligation, restriction digestion, cyclization) can be used to convert the CDR3-J oligonucleotide library into a full-length, expressible TCR library. Embodiment 1 provides an example workflow. The method provided herein can also be used to produce a pool of a single TCR chain (e.g., a non-paired chain) in an overall reaction. For example, in order to produce a library of TCRα chains, each individual CDR3-J oligonucleotide can include a CDR3 and J region from a TCRα chain, but may not include another CDR3 and J region from a TCRβ chain, and then the CDR3-J oligonucleotide can be used to connect to the corresponding TRAV gene to form a TCRα chain. Methods for constructing nucleic acid molecules encoding TCR

[0198] The nucleic acid molecules encoding TCR described herein can be constructed from two or more nucleic acid fragments. In some embodiments, the two or more nucleic acid fragments can be referred to as the first nucleic acid molecule, the second nucleic acid molecule, the third nucleic acid molecule, the fourth nucleic acid molecule, etc. When constructing nucleic acid molecules, standard molecular biology techniques can be used, including but not limited to hybridization, extension, connection and enzyme digestion / cutting.

[0199] Nucleic acid fragments described herein can encode TCR chains or parts thereof.For example, the TCR chain portion encoded by nucleic acid fragments may include greater than or equal to about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 200, 250 or more amino acids.The nucleic acid fragments may include sequences encoding functional TCR chains.Functional TCR chains may be or may not be full-length TCR chains.Functional TCR chains may include one or more mutations or modifications.In some cases, when expressed in a host cell, a functional TCR chain may be integrated into a TCR complex (for example, a complex with TCR α, TCR β, CD3 γ, CD3 δ, CD3 ε and ζ chains).In some cases, a functional TCR may be combined with a target ligand.In some cases, when a functional TCR is expressed in a host cell, it may be integrated into a cell membrane. In some cases, functional TCR chains can be expressed in host cells.

[0200] The nucleic acid fragment used to construct a nucleic acid molecule encoding TCR or a portion thereof may contain a sequence encoding CDR3.

[0201] The nucleic acid fragment for constructing a nucleic acid molecule encoding TCR or part thereof may include a sequence encoding the first CDR3 of the first TCR chain and the second CDR3 of the second TCR chain, wherein the first CDR3 and the second CDR3 are derived from a pair of homologous TCR chains. In some embodiments, the sequence encoding the first CDR3 and the sequence encoding the second CDR3 are separated by at most about 100, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 5 nucleotides.

[0202] The nucleic acid fragment for building TCR can include all or part of TCR V gene sequence.The nucleic acid fragment for building TCR can include the sequence derived from TCR V gene.The sequence derived from TCRV gene can include V region nucleic acid sequence.The sequence derived from TCRV gene can include the nucleic acid sequence encoding FR1, CDR1, FR2, CDR2 and / or FR3.The sequence derived from TCR V gene can include the sequence encoding leader peptide.The sequence derived from TCRV gene can include the nucleic acid sequence encoding L-PART1, L-PART2, FR1, CDR1, FR2, CDR2 and / or FR3.The sequence derived from TCRV gene can be a part of TCR V gene.The length of TCRV gene part can be at least 10 nucleotides. For example, the length of the TCRV gene portion may be greater than or equal to about 10, 20, 30, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more nucleotides. The sequence derived from the TCR V gene may include one or more modified nucleotides. The sequence derived from the TCR V gene may be codon optimized (or codon diversified) so that it has a sequence different from the TCR V gene or a different part thereof, but it can encode the same amino acid sequence. The sequence derived from the TCR V gene may not include a sequence encoding a part of CDR3. The sequence derived from the TCR V gene may not include the sequence of the junction area of ​​the rearranged gene.

[0203] The nucleic acid fragment for constructing a nucleic acid molecule encoding TCR or its portion may include a sequence encoding a constant domain or its portion. The nucleic acid fragment for constructing a nucleic acid molecule encoding TCR or its portion may include a sequence encoding a constant region or its portion. In some cases, the constant domain or constant region is a TCR alpha constant domain or constant region, a TCR beta constant domain or constant region, a TCR gamma constant domain or constant region, or a TCR delta constant domain and a constant region. In some cases, the constant region includes a constant domain. In some cases, the constant region also includes a transmembrane region, a connecting region, a cytoplasmic region, or a combination thereof.

[0204] The nucleic acid fragment used to construct the nucleic acid molecule encoding TCR or part thereof may include a linker sequence. A linker sequence can be used to connect a nucleic acid molecule to another nucleic acid molecule. A linker sequence of a nucleic acid molecule can be hybridized with (to form a base pair or base pair with a reverse linker sequence of another nucleic acid molecule). A reverse linker sequence can be complementary to a linker sequence (e.g., completely or substantially complementary). A reverse linker sequence can hybridize with a linker sequence under certain conditions, (e.g., temperature, buffer conditions, pH, etc.). A reverse linker sequence can be a reverse complementary sequence (or complementary sequence) of a linker sequence. When a linker sequence hybridizes with a reverse linker sequence, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100 or more base pairs are formed. The base pair formed between the linker sequence and the reverse linker sequence can be continuous or discontinuous. For example, in the case of forming a discontinuous base pair, there may be an unpaired region or a region that separates the paired region. If the first nucleic acid molecule comprises a linker sequence, the complementary sequence of the linker sequence on the second nucleic acid molecule can be referred to as a reverse linker sequence. The linker sequence (or reverse linker sequence) can have various lengths. For example, the length of the linker sequence (or reverse linker sequence) can be greater than or equal to about 2,3,4,5,6,7,8,9,10,11,12,13,14,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,100 or more nucleotides. The connexon sequence (or reverse connexon sequence) can be less than or equal to about 300, 250, 200, 150, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 nucleotides. The connexon sequence (or reverse connexon sequence) can be located at the 5' end or 3' end of the nucleic acid molecule. The connexon sequence (or reverse connexon sequence) can also be the internal sequence of the nucleic acid molecule. For example, the connexon sequence can be an internal connexon sequence, and can be exposed at the 5' end or 3' end by cutting the internal sequence of the nucleic acid molecule (for example, a sequence adjacent to the internal connexon sequence). The example of the internal connexon sequence is provided in embodiment 1, i.e., an interchain connector (ICC). In some cases, the linker sequence and the reverse linker sequence are used to connect the nucleic acid molecule encoding the CDR3 of the TCR chain or its part to another nucleic acid molecule comprising a TCR V gene or its part. In some cases, the linker sequence and the reverse linker sequence are used to connect the nucleic acid molecule comprising the J region of the TCR to another nucleic acid molecule comprising a TCR V gene or its part.In some cases, the linker sequence and the reverse linker sequence are used to connect a nucleic acid molecule comprising a sequence encoding a CDR3 or a portion thereof and a J region of a TCR with another nucleic acid molecule comprising a TCRV gene or a portion thereof. In some cases, the linker sequence and the reverse linker sequence are used to connect a nucleic acid molecule comprising a sequence encoding a CDR3 or a portion thereof, a J region and a TCRV gene or a portion thereof with another nucleic acid molecule encoding a constant region of a TCR or a portion thereof.

[0205] The linker sequence (or reverse linker sequence) can be a sequence encoding a portion of a TCRV gene (e.g., a portion of a TCR V gene adjacent to a sequence encoding CDR3 in a rearranged gene). In this case, the linker sequence and one or more other linker sequences in the linker sequence library can encode the same amino acid sequence (e.g., a conserved portion of a TCR V gene adjacent to CDR3). When the linker sequence encodes a conserved portion of a TCR V gene, the linker sequence can be codon-diverse, so that the linker sequence can be used to specifically connect a nucleic acid molecule to another nucleic acid molecule, generating a construction nucleic acid molecule encoding a paired sequence of a TCR. In certain embodiments, the linker sequence (or reverse linker sequence) comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 150, 200 or more nucleotides, which are located in the TCR V gene next to the sequence encoding CDR3 in the rearranged gene. Due to the specificity of the linker sequence and the reverse linker sequence, the nucleic acid molecule library with different sequences encoding different TCRs can be constructed in the overall reaction (for example, in the same compartment). According to the reference sequence (for example, the natural sequence of TCR determined by sequencing), the linker sequence can specify which TCR V gene the sequence encoding CDR3 should be connected to. The linker sequence (or reverse linker sequence) can be any predefined sequence and does not necessarily encode a part of the TCR V gene. In this case, any sequence can be removed after the two nucleic acid fragments are connected together.

[0206] Fig.21An example of using any linker (701) and reverse linker (702) sequences to connect a CDR3-J polynucleotide to a specified V gene germline polynucleotide (thin arrow) is shown. Each V gene germline polynucleotide here has a partially double-stranded structure. The 3' end of the top chain in the figure is on its right side, and its 3' end has a single-stranded region. The linker and reverse linker sequences can be single-stranded and can hybridize with each other. The linker and reverse linker sequences are only used for specific hybridization and may not be related to TCR, so they are arbitrary. After the linker and reverse linker hybridize, the 3' end of the top chain of the V gene germline polynucleotide can hybridize with the CDR3-J polynucleotide and can be extended by a DNA polymerase. The number of nucleotides at the 3' end of the top chain of the V gene germline polynucleotide hybridized with the CDR3-J polynucleotide can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, up to 20.

[0207] The nucleic acid fragment used to construct the nucleic acid molecule encoding TCR or part thereof may comprise a self-cleaving peptide. The self-cleaving peptide may be a 2A peptide, an intein or a hedgehog peptide. Examples of 2A peptides include, but are not limited to, a P2A peptide (sequence: ATNFSLLKQAGDVEENPGP), an E2A peptide (sequence QCTNYALLKLAGDVESNPGP), a F2A (sequence VKQTLNFDLLKLAGDVESNPGP), and a T2A peptide (sequence EGRGSLLTCGDVEENPGP).

[0208] The nucleic acid fragment used to construct the coding TCR or its partial nucleic acid molecule may include a restriction enzyme recognition site. The restriction enzyme recognition site may be a recognition site of an IIS type restriction enzyme. The IIS type restriction enzyme that can be used for the present invention includes but is not limited to EarI, MnlI, PleI, AlwI, BbsI, BbvI, BcoDI, BsaI, BseRI, BsmAI, BsmBI, BspMI, Esp3I, HgaI, SapI, SfaNI, BbvI, BsmFI, BsrDI, BtsI, FokI, BseRI, HphI, MlyI and MboII. In some cases, two or more different restriction enzymes can be used in the nucleic acid construction process. In some cases, a restriction enzyme (e.g., BbsI, BbvI, BcoDI, BsaI, BsmBI, FokI, etc.) that produces a 4-bp 5' overhang can be used. In some cases, restriction enzymes that produce blunt ends or 3' overhangs (eg, BseRI, BsrDI, BtsI, MlyI, etc.) may be used.

[0209] The nucleic acid fragments used to construct the nucleic acid molecules encoding TCR or parts thereof can be cyclized. For example, the nucleic acid fragments can be cyclized by connecting the two ends of the nucleic acid fragments. The connection can be a blunt end connection. The connection can be carried out after using 5'-3' exonucleases (for example, Gibson assembly), 3'-5' exonucleases (for example, cloning or SLIC independent of sequence and ligase) or user enzyme mixtures (for example, user-friendly DNA recombination or USEREC) to produce sticky ends. Other cyclization methods include but are not limited to circular polymerase extension cloning (CPEC) and seamless connection clone extract (slice) assembly. Alternatively, the two ends can be connected by overlapping PCR. A variety of ligases can be used for connection, including but not limited to T4 DNA ligase, T4 RNA ligase, Escherichia coli DNA ligase.

[0210] The nucleic acid fragments for constructing the nucleic acid molecules encoding TCR chains or parts thereof can be chemically synthesized. For example, the nucleic acid fragments can be pre-synthesized by chip-based synthesis. In some cases, the length of the synthesized nucleic acid fragments can be equal to or greater than about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400 or more nucleotides. In some cases, the length of the synthesized nucleic acid fragments can be equal to or less than about 500, 450, 400, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20 or 10 nucleotides.

[0211] The two nucleic acid sequences encoding the two peptide chains of the TCR can be constructed in several directions, such as head to head, head to tail, and tail to tail. As described herein, "head" refers to the "5' end" of the sense nucleic acid chain, and "tail" refers to the "3' end" of the sense nucleic acid chain. In some cases, the direction is head to tail, and the order of the paired nucleic acid sequences encoding the TCR can be controlled (for example, TRA is followed by TRB, or TRB is followed by TRA).

[0212] Any nucleic acid molecule described herein can be a double-stranded nucleic acid molecule or a single-stranded nucleic acid molecule. In some cases, a nucleic acid molecule can include a double-stranded region and a single-stranded region. For example, a nucleic acid molecule with a linker sequence or a reverse linker sequence can be a double-stranded nucleic acid molecule with a linker sequence or a reverse linker sequence region as a single-stranded region (e.g., an overhang or a sticky end). The overhang can be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides long. The overhang can be at the 5' end or 3' end of the nucleic acid molecule.

[0213] Any nucleic acid molecule described herein may comprise one or more modified nucleotides. Modified nucleotides include, but are not limited to, diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluridine, dihydrouracil, β-D-galactosylqueosine, hypoxanthine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, In some cases, the nucleotides may include modifications in the phosphate moiety thereof, including modifications to the triphosphate moiety. Non-limiting examples of such modifications include longer phosphate chains (e.g., phosphate chains with 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e.g., α-thiotriphosphate and β-thiotriphosphate). Nucleic acid molecules may also be modified in the base moiety (e.g., on one or more atoms that are typically available for hydrogen bonding with complementary nucleotides, and / or on one or more atoms that are typically unable to hydrogen bond with complementary nucleotides), sugar moieties, or phosphate backbones. Nucleic acid molecules may also contain amine-modified groups, such as aminoallyl 1-dUTP (AA-dUTP) and aminohexyl acrylamide-dCTP (AHA-dCTP), to allow covalent attachment of amine-reactive moieties (e.g., N-hydroxysuccinimide ester (NHS)). Substitutes for standard DNA base pairs or RNA base pairs in the oligonucleotides disclosed herein may provide higher bit density per cubic millimeter, higher safety (against accidental or purposeful synthesis of natural toxins), and easier to distinguish or lower secondary structures in light-programmed polymerases. Such alternative base pairs may be compatible with natural and mutant polymerases for de novo synthesis and / or amplified synthesis.

[0214] An exemplary workflow for constructing a nucleic acid molecule encoding a TCR is as follows Figures 17A-17CAs shown. Before using the methods described herein to construct nucleic acid molecules encoding TCRs, various existing methods (e.g., single-cell barcodes and sequencing) can be used to predetermine the paired sequences of multiple TCRs. A variety of sequencing methods can be used to determine the sequences of paired TCR chains, such as Sanger sequencing, high-throughput sequencing, synthetic sequencing, single-molecule sequencing, connection sequencing, RNA-Seq (Illumina), next-generation sequencing, digital gene expression (Helicos), cloned single microarrays (Solexa), shotgun sequencing, Maxim-Gilbert sequencing, or massive parallel sequencing. Paired sequences from sequencing libraries can be used as reference sequences for paired sequences of TCR chains, so that which CDR3 is paired with which V gene can be known by the specific interaction between the linker sequence and the reverse linker sequence. Multiple nucleic acid molecules encoding different TCRs can be constructed in large quantities using the methods described herein, but the construction of one molecule is shown in Figure 1 A-1C. A first nucleic acid molecule comprising a sequence encoding a first CDR3 (e.g., CDR3α) and a second CDR3 (e.g., CDR3α) (e.g., CDR3β) may be contacted with a second nucleic acid molecule comprising a sequence derived from a first TCR V gene (e.g., TRAV). A linker sequence (e.g., ConA#*) of a first nucleic acid molecule may be hybridized with a reverse linker sequence (ConA#) of a second nucleic acid molecule to connect two nucleic acid molecules. Extension and connection may be performed to produce a third nucleic acid molecule comprising a sequence derived from a first TCR V gene and a sequence encoding a first CDR3 and a second CDR3. Fig.17A) can be used to produce the overhang (or sticky end) of the third nucleic acid molecule.Next, the third nucleic acid molecule can be contacted with the fourth nucleic acid molecule comprising the sequence encoding the first constant region or constant region domain (for example, TRBC).Then the third nucleic acid molecule can be connected to the fourth nucleic acid molecule to produce the fifth nucleic acid molecule by the overhang, which comprises the sequence derived from the first TCR V gene, the sequence encoding the first CDR3 and the second CDR3, and the sequence encoding the first constant region.The fifth nucleic acid molecule can be cyclized and cut with a restriction enzyme (for example, TIISRE3) to expose an internal connector sequence (for example, ICC).Next, the fifth nucleic acid molecule can be contacted with the sixth nucleic acid molecule comprising the sequence derived from the second TCR V gene (for example, TRBV).The fifth nucleic acid molecule can be connected to the sixth nucleic acid molecule by the interaction between the linker sequence and the reverse linker sequence.Next, the sixth nucleic acid molecule can be cut by a restriction enzyme (for example, TIISRE2) to produce an overhang.Next, the sixth nucleic acid molecule can be contacted with the seventh nucleic acid molecule comprising the sequence encoding the second constant region or constant region domain (for example TRAC). The 6th nucleic acid molecule and the 7th nucleic acid molecule can be connected to form the 8th nucleic acid molecule, which comprises all regions encoding paired TCR chains. The 8th nucleic acid molecule can be further constructed into the expression vector for expressing TCR chains in a host cell. It should be understood that the nucleic acid fragment comprising the sequence deriving from TCR V gene can be single-stranded, and in this case, the 3 ends of the linker sequence of the nucleic acid fragment encoding CDR3 can be extended when hybridizing with the reverse linker sequence.

[0215] The methods described herein can be used to generate pools of single TCR chains, such as pools of TCR alpha chains or TCR beta chains.

[0216] The method for producing multiple nucleic acid molecules as described herein may include providing a first plurality of nucleic acid molecules (or nucleic acid fragments). The nucleic acid molecules in the first plurality of nucleic acid molecules may include a sequence encoding the first CDR3 of the first T cell receptor (TCR) chain and the second CDR3 of the second TCR chain. The first CDR3 and the second CDR3 may be from the paired sequence of the TCR chain. Next, a second plurality of nucleic acid molecules may be provided. The nucleic acid molecules in the second plurality of nucleic acid molecules may include a sequence derived from a TCR V gene. The nucleic acid molecules may not include a sequence encoding a constant domain. Next, the first plurality of nucleic acid molecules and the second plurality of nucleic acid molecules may be contacted. The nucleic acid molecules in the first plurality of nucleic acid molecules may be connected to the nucleic acid molecules in the second plurality of nucleic acid molecules to form a nucleic acid molecule comprising a sequence encoding the first CDR3 and the second CDR3 and a sequence derived from a TCR V gene. The sequence encoding the first CDR3 and the second CDR3 and the TCR V gene may be derived from the paired sequence of the TCR chain.

[0217] The method for producing multiple nucleic acid molecules, each nucleic acid molecule encoding T cell receptor (TCR) chain or its region in the multiple nucleic acid molecules, can include making the first multiple nucleic acid molecules and the second multiple nucleic acid molecules contact, to produce the third multiple nucleotide molecules comprising at least two different nucleic acid molecules.Each of the at least two different nucleic acid molecules can have different sequences encoding different TCR chains or their regions.The at least two different nucleic acid molecules can be produced in the same compartment.In some cases, at least about 5,10,20,50,100,200,300,400,500,1000,1500,2000,2500,3000,3500,4000,5000,6000,7000,8000,9000,10000,12000,15000,20000,1000000,1000000 or more encoding different TCR sequences can be produced in the same compartment.

[0218] The method for producing multiple nucleic acid molecules as described herein may include providing a first plurality of nucleic acid molecules. The nucleic acid molecules in the first plurality of nucleic acid molecules may include the sequence of the first CDR3 encoding the first T cell receptor (TCR) chain and the second CDR3 of the second TCR chain. The first CDR3 and the second CDR3 may be from the paired sequence of the TCR chain. Next, a second plurality of nucleic acid molecules may be provided. The nucleic acid molecules in the second plurality of nucleic acid molecules may include the sequence derived from the TCR V gene. Next, the first plurality of nucleic acid molecules and the second plurality of nucleic acid molecules may be contacted. The nucleic acid molecules of the first plurality of nucleic acid molecules may be connected to the nucleic acid molecules of the second plurality of nucleic acid molecules to form a linear nucleic acid molecule, which includes the sequence encoding the first CDR3 and the second CDR3 and the sequence derived from the TCRV gene. The sequence encoding the first CDR3 and the second CDR3 and the TCRV gene may be derived from the paired sequence of the TCR chain.

[0219] The method for producing multiple nucleic acid molecules may include providing a first plurality of nucleic acid molecules. The nucleic acid molecules in the first plurality of nucleic acid molecules may include a synthetic sequence of the first CDR3 of the first T cell receptor (TCR) chain and the second CDR3 of the second TCR chain (I) and a synthetic sequence of the third CDR3 of the third T cell receptor (TCR) chain and the fourth CDR3 of the fourth TCR chain (II). The first CDR3 and the second CDR3 may be from the first paired sequence of the TCR chain, and the third CDR3 and the fourth CDR3 may be from the second paired sequence of the TCR chain. Next, a second plurality of nucleic acid molecules may be provided. The nucleic acid molecules in the second plurality of nucleic acid molecules may include a sequence derived from a TCR V gene. Next, the first plurality of nucleic acid molecules and the second plurality of nucleic acid molecules may be contacted. The nucleic acid molecules in the first plurality of nucleic acid molecules may be connected to the nucleic acid molecules in the second plurality of nucleic acid molecules to form a nucleic acid molecule comprising a sequence encoding the first CDR3 and the second CDR3 and a sequence derived from a TCR V gene. The sequence encoding the first CDR3 and the second CDR3 and the TCR V gene may be derived from the paired sequence of the TCR chain.

[0220] The method for producing the nucleic acid molecule encoding T cell receptor (TCR) chain or its part may include providing at least one nucleic acid molecule comprising the sequence of the CDR3 encoding TCR chain. Next, a plurality of nucleic acid molecules may be provided. Each nucleic acid molecule in the plurality of nucleic acid molecules may include a sequence derived from TCR V gene. The plurality of nucleic acid molecules may include at least two different sequences derived from at least two different TCR V genes. In some cases, the plurality of nucleic acid molecules may include at least 2, 5, 10, 15, 20, 25, 30, 35, 40 or more different sequences derived from at least 2, 5, 10, 15, 20, 25, 30, 35, 40 or more different TCR V genes. Next, the at least one nucleic acid molecule comprising the sequence of the CDR3 encoding TCR chain may be contacted with the plurality of nucleic acid molecules in the same compartment, and the plurality of nucleic acid molecules each include a sequence derived from TCR V gene. The at least one nucleic acid molecule comprising a sequence encoding CDR3 of the TCR chain can be connected to a nucleic acid molecule among the multiple nucleic acid molecules to produce a third nucleic acid molecule, wherein the third nucleic acid molecule comprises a sequence encoding the CDR3 and a sequence derived from one of at least two different TCR V genes, thereby producing a nucleic acid molecule encoding the TCR chain or a portion thereof.

[0221] The compositions described herein that can be used for the methods described herein may include a first plurality of nucleic acid molecules. Each nucleic acid molecule in the first plurality of nucleic acid molecules may include a sequence encoding the first CDR3 of the first T cell receptor (TCR) chain and the second CDR3 of the second TCR chain. The first CDR3 and the second CDR3 may be from the paired sequence of the TCR chain. The composition may also include a second plurality of nucleic acid molecules. Each nucleic acid molecule in the second plurality of nucleic acid molecules may include a sequence derived from a TCRV gene. Each nucleic acid molecule in the second plurality of nucleic acid molecules may not include a sequence encoding the first CDR3 and the second CDR3. In the composition, (I) each nucleic acid molecule in the first plurality of nucleic acid molecules may include a sequence encoding different first CDR3 and / or second CDR3, and / or (II) each nucleic acid molecule in the second plurality of nucleic acid molecules includes a sequence derived from different TCR V genes.

[0222] The compositions described herein that can be used for the methods described herein may include multiple nucleic acid molecules. Each of the multiple nucleic acid molecules may include a sequence derived from a T cell receptor (TCR) V gene. The multiple nucleic acid molecules may include a first nucleic acid molecule having a first linker sequence and a second nucleic acid molecule having a second linker sequence. The first linker sequence may be different from the second linker sequence.

[0223] The compositions described herein that can be used for the methods described herein may include multiple nucleic acid molecules. Each nucleic acid molecule in the multiple nucleic acid molecules may encode the CDR3 of a T cell receptor (TCR) chain. The first nucleic acid molecule in the multiple nucleic acid molecules may include a first linker sequence, and the second nucleic acid molecule in the multiple nucleic acid molecules may include a second linker sequence. The first linker sequence may be different from the second linker sequence.

[0224] The compositions described herein that can be used for the methods described herein may include multiple nucleic acid molecules. Each nucleic acid molecule in the multiple nucleic acid molecules may include a sequence of at least 10 amino acids encoding a T cell receptor (TCR) chain. The first nucleic acid molecule in the multiple nucleic acid molecules may include a first linker sequence, and the second nucleic acid molecule in the multiple nucleic acid molecules may include a second linker sequence. The first linker sequence may be different from the second linker sequence. The first linker sequence or the second linker sequence may encode a portion of a TCR chain. The first linker sequence or the second linker sequence may be in frame with a sequence of at least 10 amino acids encoding a TCR chain.

[0225] Compositions described herein that can be used for methods described herein may include multiple nucleic acid molecules.Each nucleic acid molecule in the multiple nucleic acid molecules may include a sequence derived from a T cell receptor (TCR) V gene, and may not include a CDR3 sequence.The first nucleic acid molecule in the multiple nucleic acid molecules may include a first reverse connection subsequence, and the second nucleic acid molecule in the multiple nucleic acid molecules may include a second reverse connection subsequence.The first reverse connection subsequence may be different from the second reverse connection subsequence.The sequence derived from TCR V genes of the first nucleic acid molecule and the second nucleic acid molecule may be derived from different TCR V genes.The composition may further include at least one nucleic acid molecule, and the nucleic acid molecule includes a sequence of the CDR3 encoding TCR chains.The at least one nucleic acid molecule may further include a first connector sequence complementary to the first reverse connector sequence.

[0226] The present invention provides compositions and methods for assembling or synthesizing a TCR library comprising multiple TCRs. In some cases, it may be useful to separate or purify a specific TCR sequence (e.g., a target TCR) from a TCR library for further characterization or operation. For this reason, a barcode may be included in a nucleic acid molecule or fragment for constructing a sequence encoding a TCR or a portion thereof. In some cases, a nucleic acid fragment comprising a sequence encoding a CDR3 comprises a barcode. In some cases, a nucleic acid fragment comprising a sequence encoding a first CDR3 of a first TCR chain and a second CDR3 of a second TCR chain comprises a barcode. For example, a CDR3-J oligonucleotide or a paired CDR3-J oligonucleotide may comprise a barcode. A linker sequence (or in some cases, a reverse linker sequence) may comprise a barcode. The interchain connector (or ICC) of a CDR3-J oligonucleotide may comprise a barcode. A barcode may be a primer binding site, such as a TCR specific primer binding site or DOPBS.

[0227] For example, each sequence in the paired CDR3-J oligonucleotide pool encodes a unique paired CDR3-J (e.g., Fig.17A) can include a unique barcode (or unique DOPBS). The sequence of DOPBSes can be arbitrarily designed. The sequence of DOPBSes can be designed to avoid common defects, such as unwanted secondary structures, restriction sites, similarities with other sequences in TCR genes, or similarities between primer binding sites. The barcode (or DOPBS) can be at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides. DOPBS can be an additional sequence included in each sequence of paired CDR3-J pools. DOPBS can be a sequence already included in each sequence of paired CDR3-J pools. For example, a linker sequence or a portion thereof can be used as a DOPBS. The sequences listed in Table 3 can be used as DOPBSes. The product of step (9) of Figure 17-C can be used as a template for PCR amplification using a forward primer corresponding to T2A-3 and a reverse primer corresponding to a DOPBS associated with the target TCR. PCR products are obtained according to steps (10) and (11) of Figure 17-C. The final product mainly contains the target TCR. TCR expression

[0228] The present invention provides sequences encoding exogenous TCR.For example, in various embodiments, the method provided herein includes delivering multiple polynucleotide sequences encoding natural paired TCR or derivatives thereof to multiple receptor cells to produce multiple synthetic TILs.Multiple polynucleotide sequences or derivatives thereof can be synthesized using the methods described herein.The derivative of a polynucleotide sequence can be a copy of a polynucleotide sequence (for example, an amplified product or a chemically synthesized copy).Each polynucleotide sequence in a plurality of polynucleotide sequences can encode the TCR of natural pairing.By contacting a library or mixture containing a plurality of polynucleotide sequences or derivatives thereof with a plurality of receptor cells, a plurality of polynucleotide sequences or derivatives thereof can be delivered in a library or mixture.

[0229] The exogenous TCR of the present disclosure can be obtained from a subject (e.g., separated from a sample from a subject), and can be referred to as a subject-derived or subject-specific TCR. The cell may include a TCR alpha chain and a TCR beta chain or a TCR gamma chain and a TCR delta chain. Therefore, the present invention provides nucleic acids encoding TCR alpha chains (or TCR gamma chains), and nucleic acids encoding TCR beta chains (or TCR delta chains). In some embodiments, nucleic acids encoding TCR alpha chains (or TCR gamma chains) are separated from nucleic acids encoding TCR beta chains (or TCR delta chains). In some embodiments, nucleic acids encoding TCR alpha chains (or TCR gamma chains) and nucleic acids encoding TCR beta chains (or TCR delta chains) are present in the same nucleic acid.

[0230] The polynucleotide sequence encoding the derived or subject-specific TCR of the subject can be delivered to the cell for expression. The polynucleotide sequence encoding the derived or subject-specific TCR of the subject can be delivered to the cell as a linear or circular nucleic acid molecule to produce modified cells. In some cases, the polynucleotide can be delivered (for example, electroporation, transfection, transduction or conversion) to the cell by electroporation. In some cases, the polynucleotide can be delivered to the cell by a carrier such as a cationic polymer. In some cases, the vector comprising the sequence encoding the derived or subject-specific TCR of the subject can be delivered to the cell. In some cases, the derived or subject-specific TCR of the subject can be expressed in the cell. TCR can be expressed by a carrier (or expression vector), such as a plasmid, a transposon (for example, Sleeping Beauty, Piggy Bac) and a viral vector (for example, an adenoviral vector, an adeno-associated virus (AAV) vector, a retroviral vector and a lentiviral vector). Other examples of carriers include shuttle vectors, phagemids, cosmids and expression vectors. Non-limiting examples of plasmid vectors include pUC, pBR322, pET, pBluescript and variants thereof. In addition, the vector may include additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selection marker sequences (e.g., antibiotic resistance genes), replication origins, etc. In some cases, the vector is a nucleic acid molecule introduced into a cell to produce a transformed cell (e.g., an engineered cell). The vector may include a nucleic acid sequence that allows it to replicate in a cell, such as a replication origin. The vector may also include one or more selection marker genes and other genetic elements. The vector may be an expression vector comprising a paired TCR encoding polynucleotide operably connected to a sequence that allows TCR expression according to the present disclosure. The vector may be a viral or non-viral vector, such as a retroviral vector (including a lentiviral vector), an adenoviral vector (including its replication competence, replication defective type, and intestinal type), an AAV vector, a simian virus 40 (SV-40) vector, a bovine papilloma virus vector, an Epstein-Barr vector, a herpes virus vector, a vaccinia virus vector, a Moloney murine leukemia vector, a Harvey murine sarcoma virus vector, a mouse mammary tumor virus vector, a Rous sarcoma virus vector, and a non-viral plasmid.

[0231] In some cases, the vector is an AAV vector (e.g., an AAV6 vector). In addition to the sequence encoding the exogenous TCR, the AAV vector may also include a pair of homology arms (HAs) flanking the sequence encoding the exogenous TCR. The HAs of the AAV vector may also be flanked by upstream and downstream inverted terminal repeats (ITRs).

[0232] In some cases, the vector is a lentiviral vector. Recipient cells (e.g., peripheral T cells of a patient) can be delivered with a mixture of lentiviral particles at a low multiplicity of infection (MOI). The dilution factor of the lentiviral particles can first be determined so that the functional MOI (e.g., the T cell portion expressing one or more exogenous TCRs) is about 5% to 15%. In this case, the recipient cells can only express a single copy of the sequence encoding the TCR.

[0233] In some cases, the vector is a self-amplifying RNA replicon, also referred to as self-replicating (m) RNA, self-replicating (m) RNA, self-amplifying (m) RNA or RNA replicon. The self-amplifying RNA replicon is a kind of RNA that can self-replicate. In some embodiments, the self-amplifying RNA replicon can self-replicate in the cell. In some embodiments, the self-amplifying RNA replicon encodes RNA polymerase and target molecule. The RNA polymerase can be RNA dependent RNA polymerase (RDRP or RdRp). The self-amplifying RNA replicon can also encode protease or RNA capping enzyme. In some embodiments, the self-amplifying RNA replicon vector belongs to or is derived from a virus of the family Togaviridae known as alphaviruses, which may include Eastern Equine Encephalitis Virus (EEE), Venezuelan Equine Encephalitis Virus (VEE), Everglades Virus, Mukambo Virus, Pixuna Virus, Western Equine Encephalitis Virus (WEE), Sindbis Virus, South African Arbovirus No. 86, Semliki Forest Virus, Middelburg Virus, Chikungunya Virus, Oneonian Virus, Ross River Virus, Balma Forest Virus, Geta Virus, Lushan Virus, Bebaru Virus, Mayaro Virus, Una Virus, Ora Virus, Wataroa Virus, Babangi Virus, Kizilagachi Virus, Highland J Virus, Fort Morgan Virus, Ndumu Virus, Badji Creek Virus, and any other virus classified as an alphavirus by the International Committee on Taxonomy of Viruses (ICTV). In some embodiments, the self-amplifying RNA replicon is or contains a portion from an attenuated form of an alphavirus, such as the VEE TC-83 vaccine strain. In some embodiments, the self-amplification RNA replicon vector is an attenuated form of a virus, which allows expression of a target molecule without causing cytopathic or apoptotic effects on cells. In some embodiments, the self-amplification RNA replicon vector has been transformed or selected in vitro, in vivo, in vitro or silica, to have a specific function (e.g., TCR expression extended or increased) in a host cell, a target cell or an organism. For example, the host cell colony carrying the different variants of the self-amplification RNA replicon can be selected based on the expression level of one or more target molecules (encoded in the self-amplification RNA replicon or host genome) at different time points. In some embodiments, the self-amplification RNA replicon selected or engineered has been modified to reduce type I interferon response, innate antiviral response or the adaptive immune response from a host cell or an organism, and it causes the protein expression of the RNA replicon to continue longer or to express with higher levels in a host cell, a target cell or an organism. In some embodiments, the optimized self-amplifying RNA replicon sequence is obtained from a single cell or a population of cells having a desired phenotypic trait, such as higher or more sustained expression of a molecule of interest, or reduced innate antiviral immune response to the vector, compared to a wild-type strain or a vaccine strain.In some embodiments, cells carrying a desired or selected self-amplifying RNA replicon sequence are obtained from a subject (e.g., a human or animal) and have a beneficial response feature (e.g., an elite responder or subject with complete remission) after treatment with a therapeutic agent comprising a self-amplifying RNA replicon. In some embodiments, the self-amplifying RNA replicon vector can express additional agents. In some embodiments, additional agents include cytokines, such as IL-2, IL-12, IL-15, IL-10, GM-CSF, TNFα, granzyme B, or a combination thereof. In some embodiments, the additional agent can be amplified by directly affecting the expression of TCR or by regulating the host cell phenotype (e.g., inducing apoptosis or). In some embodiments, the self-amplifying RNA replicon may contain one or more subgenomic sequences to produce one or more subgenomic polynucleotides. In some embodiments, subgenomic polynucleotides are functional mRNA molecules translated by a cellular translation mechanism. Subgenomic polynucleotides can be produced by the function of a determined sequence element (e.g., a subgenomic promoter or SGP) on a self-amplifying RNA replicon, and the self-amplifying RNA replicon instructs a polymerase to produce subgenomic polynucleotides from a subgenomic sequence. In some embodiments, SGP is recognized by RNA-dependent RNA polymerase (RDRP or RdRp). In some embodiments, multiple SGP sequences are present in a single self-amplification RNA replicon, and can be located upstream of the subgenomic sequence of the composition of encoding TCR, TCR or other reagents. In some embodiments, the nucleotide length or composition of the SGP sequence can be modified to change the expression characteristics of the subgenomic polynucleotides. In some embodiments, the non-identical SGP sequence is located on the self-amplification RNA replicon, so that the ratio of the corresponding subgenomic polynucleotides is different from the situation that the SGP sequence is identical. In some embodiments, different SGP sequences guide TCR and other reagents (production, such as cytokines) so that they are produced relative to each other in a certain ratio, and the ratio causes the expression of TCR to increase, the amplification of target cells to increase or faster, and there is no cytotoxic effect on target cells or hosts, or weakens the innate or adaptive immune response for RNA replicons. In some embodiments, the position of subgenomic sequences and SGP sequences relative to each other and the genome sequence itself can be used to change the ratio of subgenomic polynucleotides relative to each other. In some embodiments, the SGP and subgenomic sequences encoding the TCR may be located downstream of the SGP and subgenomic regions encoding other agents, so that the expression of the TCR is significantly increased relative to the other agents. In some embodiments, the RNA replicon or SGP has been selected or modified to express an optimal amount of cytokines so that the cytokines promote the expansion of T cells or enhance the therapeutic effect of the TCR, but do not cause serious side effects, such as cytokine release syndrome, cytokine storm or neurotoxicity.

[0234] In some embodiments, provided herein are carriers of polynucleotides encoding TCRs in pairs comprising coding TCR α chains and TCR β chains. In some embodiments, provided herein are carriers of polynucleotides encoding TCRs in pairs comprising coding TCR γ chains and TCR δ chains. In some embodiments, the carrier is a self-amplifying RNA replicon, plasmid, bacteriophage, transposon, clay, virus or virion. In some embodiments, the carrier is a viral vector. In some embodiments, the carrier is derived from a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, a herpes virus, a poxvirus, an alpha virus, a vaccinia virus, a hepatitis B virus, a human papillomavirus or its pseudotype. In some embodiments, the carrier is a non-viral vector. In some embodiments, the non-viral vector can be formulated into nanoparticles, cationic lipids, cationic polymers, metal nanopolymers, nanorods, liposomes, micelles, microvesicles, cell penetrating peptides or lipid globules.

[0235] The expression of two TCR chains can be driven by two promoters or one promoter. In some cases, two promoters are used. In some cases, the protein coding sequences of two promoters and their respective two chains can be arranged in a head-to-head, head-to-tail or tail-to-tail direction. In some cases, a promoter is used. Two protein coding sequences can be connected, optionally in accordance with the reading frame, so that a promoter can be used to express two chains. In this case, the two protein coding sequences can be arranged in a head-to-tail direction, and can be with a ribosome binding site (e.g., internal ribosome binding site or IRES), a protease cleavage site, or a sequence of a self-processing cleavage site (such as encoding a 2A peptide) to promote bicistronic expression. In some cases, the two chains can be connected with a peptide linker so that the two chains can be expressed as a single-chain polypeptide. Each expression chain can contain a complete variable domain sequence, including rearranged V (D) J genes. Each expression chain can contain a complete variable domain sequence, including CDR1, CDR2 and CDR3. Each expressed chain may contain a complete variable domain sequence, including FR1, CDR1, FR2, CDR2, FR3 and CDR3. In some cases, each expressed chain may further comprise a constant domain sequence.

[0236] To generate an expression vector, additional sequences can be added to the sequence encoding the target gene, such as TCR. These additional sequences include a vector backbone (e.g., an element for vector replication in a target cell or temporary host, such as E. coli), a promoter, an IRES, a sequence encoding a self-cleaving peptide, a terminator, an auxiliary gene (e.g., a cargo), and a partial sequence of a paired TCR encoding polynucleotide (e.g., a portion of a sequence encoding a constant domain).

[0237] Protease cleavage sites include, but are not limited to, enterokinase cleavage sites: (ASP)4Lys; factor Xa cleavage sites: Ile-Glu-Gly-Arg; thrombin cleavage sites, such as Leu-Val-Pro-Arg-Gly-Ser; renin cleavage sites, such as His-Pro-Phe-His-Leu-Val-Ile-His; collagenase cleavage sites, such as X-Gly-Pro (wherein X is any amino acid); trypsin cleavage sites, such as arginine-lysine; viral protease cleavage sites, such as viral 2A or 3C protease cleavage sites, including but not limited to protease 2A cleavage sites from picornaviruses, hepatitis A virus 3C cleavage sites, human rhinovirus 2A protease cleavage sites, picornavirus 3 protease cleavage sites; and caspase cleavage sites, such as DEVD, which is recognized and cleaved by activated caspase-3, wherein cleavage occurs after the second aspartic acid residue. In some embodiments, the present disclosure provides expression vectors comprising a protease cleavage site, wherein the protease cleavage site comprises a cellular protease cleavage site or a viral protease cleavage site. In some embodiments, the first protein cleavage site includes a site recognized by furin; VP4 of IPNV; tobacco etch virus (TeV) protease; rhinovirus 3C protease; PC5 / 6 protease; Space protease, LPC / PC7 protease; enterokinase; factor Xa protease; thrombin; gene enzyme I; MMP protease; turnip mosaic virus nuclear inclusion protein A (N1A) research dengue 4 type flavivirus NS2B / NS3, yellow fever virus NS3 protease; cauliflower mosaic virus ORF V; Kex2 protease; CB2; or 2A. In some embodiments, the protein cleavage site is a virus internal cleavable signal peptide cleavage site. In some embodiments, the virus internal cleavable signal peptide cleavage site includes a site from influenza C virus, hepatitis C virus, Hantavirus, flavivirus or rubella virus.

[0238] Suitable IRES elements included in the vectors of the present invention may include RNA sequences that can bind to eukaryotic ribosomes. In some embodiments, the IRES elements of the present disclosure are at least about 250 base pairs, at least about 350 base pairs, or at least about 500 base pairs. The IRES elements of the present disclosure may be derived from the DNA of an organism, including but not limited to viruses, mammals, and fruit flies. In some cases, the viral DNA derived from the IRES element includes but is not limited to small RNA virus complementary DNA (cDNA), encephalomyocarditis virus (EMCV) cDNA, and poliovirus cDNA. Examples of mammalian DNA derived from IRES elements include but are not limited to DNA encoding immunoglobulin heavy chain binding protein (BiP) and DNA encoding basic fibroblast growth factor (bFGF). Examples of fruit fly DNA derived from IRES elements include but are not limited to the antennapedal gene from Drosophila melanogaster. In addition, examples of poliovirus IRES elements include, for example, poliovirus IRES, encephalomyocarditis virus IRES, or hepatitis A virus IRES. Examples of Flavivirus IRES elements include a Hepatitis C virus IRES, a GB virus BIRES, or a Pestivirus IRES, including but not limited to a Bovine Viral Diarrhea Virus IRS or a Classical Swine Fever Virus IRS.

[0239] Examples of self-processing cleavage sites include, but are not limited to, intein sequences; modified inteins; hedgehog sequences; other HOG family sequences; 2A sequences, such as the 2A sequence derived from foot-and-mouth disease virus (FMDV); and variants of each of these.

[0240] The vector for recombinant gene expression (e.g., TCR expression) may include any number of promoters, wherein the promoter is constitutive, regulatable or inducible, cell type specific, tissue specific or species specific. Further examples include tetracycline responsive promoters. The vector may be a replicon adapted to the host cell in which the TCR is to be expressed, and it may also be included in a replicon functional in a bacterial cell (e.g., Escherichia coli). The promoter may be constitutive or inducible, wherein the induction is associated with a specific cell type or a specific level of maturity. Alternatively, a variety of viral promoters may be used. Examples of promoters include β-actin promoters, SV40 early and late promoters, immunoglobulin promoters, human cytomegalovirus promoters, retroviral promoters, elongation factor 1A (EF-1A) promoters, phosphoglycerol kinase (PGK) promoters, and Friend spleen focus forming virus promoters. These promoters may be associated or unassociated with enhancers, wherein enhancers may be naturally associated with a specific promoter or may be associated with different promoters.

[0241] Promoters for mammalian cells can be constitutive (herpes virus TK promoter; SV40 early promoter; Rous sarcoma virus promoter; cytomegalovirus promoter; mouse mammary tumor virus promoter) or regulated (metallothionein promoter, for example). Vectors can be based on viruses that infect specific mammalian cells, such as retroviruses, vaccinia viruses, and adenoviruses and their derivatives. Promoters can include, but are not limited to, cytomegalovirus, adenovirus late, and vaccinia 7.5K promoters. Enolase is an example of a constitutive yeast promoter, and alcohol dehydrogenase is an example of a regulated promoter. The choice of specific promoters, transcription termination sequences, and other optional sequences (such as sequences encoding tissue-specific sequences) can be determined by the cell type in which expression is to be performed.

[0242] In some cases, the vector used for recombinant gene expression (e.g., TCR expression) does not include a promoter. In this case, the target gene can be expressed under the control of an endogenous promoter at the genomic site. For example, the vector may include homology arms (HAs) flanking the target gene so that the target gene is inserted into the genomic site by homologous recombination after cutting the genomic site using a selected gene editing method. The target gene can be inserted into the genomic site in frame and expressed by an endogenous promoter. Gene delivery

[0243] Various methods for delivering (or introducing) and expressing genes or genetic materials (e.g., nucleic acid molecules encoding target proteins) into cells can be used. The target protein described herein can be an exogenous TCR chain. In the context of an expression vector, the expression vector can be easily transferred to a host cell, for example, a T cell, by physical, chemical or biological methods. Physical methods for introducing nucleic acid molecules into host cells include but are not limited to calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, etc. Biological methods for introducing target nucleic acid molecules into host cells include but are not limited to the use of DNA and RNA vectors. Viral vectors such as retroviral vectors, lentiviral vectors, adenoviral vectors and adeno-associated viral vectors can be used to deliver genes into mammalian cells such as human cells. Chemical methods for introducing nucleic acid molecules into host cells can include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads and lipid-based systems, including water-in-oil emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). Other methods may include but are not limited to delivering nucleic acids with targeted nanoparticles or other suitable submicron delivery systems.

[0244] In the case of using a non-viral delivery system, the example of a delivery vector is a liposome. It is contemplated that a lipid formulation is used to import nucleic acid into a host cell (in vitro, in vitro or in vivo). In another aspect, nucleic acid can be combined with lipids. Nucleic acids combined with lipids can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, connected to liposomes by connecting molecules combined with liposomes and oligonucleotides, embedded in liposomes, compounded with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, containing or compounded with micelles, or otherwise associated with lipids. Lipid, lipid / DNA or lipid / expression vector related compositions are not limited to any specific structure in solution. For example, they can exist as a double-layer structure, as micelles, or have a "collapse" structure. They can also be simply dispersed in solution, and may form aggregates of uneven size or shape. Lipid is a fatty substance, which can be a naturally occurring or synthetic lipid. For example, lipids include fat droplets naturally present in the cytoplasm as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0245] Lipids suitable for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; c. dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Lipid stock solutions in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform can be used as a solvent because it evaporates more easily than methanol. "Liposome" is a general term that includes various monolayer and multilayer lipid carriers formed by producing closed lipid bilayers or aggregates. Liposomes are characterized by having a vesicle structure having a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in an excess of aqueous solution, they can form spontaneously. The lipid components can undergo self-rearrangement before forming a closed structure and trap water and dissolved solutes between the lipid bilayers. However, compositions having structures different from normal vesicle structures in solution are also included. For example, lipids can present micellar structures or exist only as heterogeneous aggregates of lipid molecules. Also contemplated herein may include lipofectamine-nucleic acid complexes. Modified cells

[0246] In various embodiments, modified cells of one or more exogenous paired TCRs generated using the methods described herein are provided. These modified cells can be engineered cells or synthetic cells. The modified cells can be synthetic tumor infiltrating lymphocytes (TILs). In the disclosure including the accompanying drawings, synthetic TILs can be referred to as InfiniTILs or DisTILs. Synthetic TILs can be prepared by delivering and / or expressing the polynucleotides encoding the paired sequences of TCR from the TILs obtained from the subject to the receptor cells (or host cells). The receptor cells can be T cells. In some cases, T cells can be CD4+T cells, CD8+T cells or CD4+ / CD8+T cells. In some cases, T cells can be helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, αβT cells or γδT cells. The modified cells can be modified T cells.

[0247] Recipient cells can be isolated from a subject to be administered (e.g., a subject with a tumor). In some cases, recipient cells can be obtained from a healthy donor.

[0248] In some cases, the present invention provides at least 2,5,10,15,20,25,30,35,40,45,50,55,60,65,70,75,80,85,90,95,100,150,200,250,300,350,400,450,500,550,600,650,700,750,800,850 each modified cell exogenously expresses the polynucleotide sequence of the different paired sequences encoding TCR α chain and TCR β chain. The polynucleotide sequence can be knocked into the genomic site for the monoallelic expression of the polynucleotide sequence. In some cases, the endogenous TCR of the modified cell can be knocked out. Each different paired sequence of TCR α chain and TCR β chain can be a subject-specific TCR or can be from a subject with a solid tumor lesion.

[0249] The receptor cells described herein can be various cell types. The receptor cells can be T cells, B cells, NK cells, macrophages, neutrophils, granulocytes, eosinophils, erythrocytes, platelets, stem cells, IPSCs or mesenchymal stem cells. In addition, the receptor cells can be cell line cells. The cell line can be a tumorigenic cell line or an artificial immortalized cell line. The cell line includes but is not limited to CHO-K1 cells, HEK293 cells, Caco2 cells, U2-OS cells, NIH3T3 cells, NSO cells, SP2 cells, CHO-S cells, DG44 cells, K-562 cells, U-937 cells, MRC5 cells, IMR90 cells, Jurkat cells, HepG2 cells, HeLa cells, HT-1080 cells, HCT-116 cells, Hu-H7 cells, HUVEC cells and Molt 4 cells. Recipient cells can be autologous T cells (e.g., patient-derived T cells) or allogeneic T cells (e.g., donor T cells from healthy subjects). Recipient cells can be genetically modified or engineered cells. In some cases, the recipient cells are autologous to the subject in need of treatment (e.g., cancer treatment). In other cases, the recipient cells are allogeneic to the subject in need of treatment.

[0250] The recipient cells can be primary T cells isolated from a subject. The subject can be the same subject from which the source T cells were obtained. The recipient cells can be primary T cells isolated from a blood sample. The recipient cells can be obtained from a peripheral blood mononuclear cell (PBMC) sample. The recipient cells can be peripheral T cells. The recipient cells can be isolated using a variety of techniques (e.g., Ficoll TMSeparation) is obtained from the blood of the subject. Cells from the circulating blood of an individual can be obtained by apheresis. Apheresis products may contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells and platelets. The cells collected by apheresis can be washed to remove the plasma portion, and the cells are placed in an appropriate buffer or culture medium for subsequent processing steps. In some cases, the cells can be washed with phosphate buffered saline (PBS). The washing solution may lack calcium or magnesium or other divalent cations. The initial activation step in the absence of calcium can lead to amplified activation. According to the manufacturer's instructions, the washing step can be completed by methods such as using a semi-automatic "flow-through" centrifuge (e.g., COBE 2991 cell processor, Baxter Cytomate or Haemonetics Cell Saver 5). After washing, the cells can be resuspended in various biocompatible buffers, such as PBS, Plasmalyte A without Ca, magnesium or other saline solutions containing or not containing buffer. Alternatively, the components not needed in the apheresis sample can be removed and the cells can be directly resuspended in the culture medium.

[0251] In one aspect, T cells are isolated from peripheral blood lymphocytes or tissue by lysing erythrocytes and depleting monocytes, e.g., by PERCOLL TM Gradient centrifugation or elution by countercurrent centrifugation. Specific subsets of T cells, such as CD3+, CD28+, CD4+, CD8+, CD45RA+, and CD45RO+ T cells, can be further isolated by positive or negative selection techniques. For example, T cells can be isolated by incubation with anti-CD3 / anti-CD28 (e.g., 3×28)-conjugated beads, such as DYNABEADS TMM-450CD3 / CD28 T, continue enough to positively select the desired T cell time period. In some cases, the time period is about 30 minutes. In some cases, the range of the time period is from 30 minutes to 36 hours or longer, and all integer values ​​therebetween. In some cases, the time period is at least or equal to about 1, 2, 3, 4, 5 or 6 hours. In some cases, the time period is 10 to 24 hours. In some cases, the incubation time is about 24 hours. In any case where T cells are less than other cell types, for example, when separating tumor infiltrating lymphocytes (TILs) from tumor tissue or immunocompromised individuals, longer incubation times can be used to separate T cells. In addition, using longer incubation times can increase the capture efficiency of CD8+T cells. Therefore, by simply shortening or extending the time allowing T cells to bind to anti-CD3 / anti-CD28 beads and / or by increasing or reducing the ratio of beads to T cells, a subpopulation of T cells can be selected at the beginning of cultivation or at other time points in the process. In addition, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on beads or other surfaces, T cell subsets can be selected at the start of culture or other required time points. In some cases, multiple rounds of selection can be used. In some aspects, a selection procedure can be performed, and "unselected" cells (cells that may not be combined with anti-CD3 / anti-CD28 beads) can be used for activation and amplification processes. "Unselected" cells can also be further selected.

[0252] Enrichment of T cell populations by negative selection can be accomplished with a combination of antibodies for surface markers specific to negatively selected cells. An exemplary method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, which uses a monoclonal antibody mixture for cell surface markers present on negatively selected cells. For example, in order to enrich CD4+ cells by negative selection, a monoclonal antibody mixture generally includes antibodies for CD14, CD20, CD11b, CD16, HLA-DR and CD8. In some aspects, enrichment or positive selection generally expresses regulatory T cells of CD4+, CD25+, CD62Lhi, GITR+ and FoxP3+ and may be useful. Alternatively, in some aspects, T regulatory cells are removed by anti-C25 conjugated beads or other similar selection methods.

[0253] Receptor cells can be obtained in a manner similar to source T cells. For example, receptor cells can be TILs, such as tumor infiltrating T cells (TITs). TILs can be separated from organs suffering from cancer. One or more cells can be separated from organs suffering from cancer, and the organ can be brain, heart, lung, eye, stomach, pancreas, kidney, liver, intestine, uterus, bladder, skin, hair, nail, ear, gland, nose, mouth, lip, spleen, gums, teeth, tongue, salivary gland, tonsil, pharynx, esophagus, large intestine, small intestine, rectum, anus thyroid gland, thymus, bone, cartilage, tendon, ligament, adrenal gland membrane, skeletal muscle, smooth muscle, blood vessel, blood, spinal cord, trachea, ureter, urethra, hypothalamus, pituitary gland, pylorus, adrenal gland, ovary, fallopian tube, uterus, vagina, breast, testis, seminal vesicle, penis, lymph, lymph node or lymphatic vessel. TILs can be from the brain, heart, liver, skin, intestine, lung, kidney, eye, small intestine or pancreas. TILs can be from the pancreas, kidney, eye, liver, small intestine, lung or heart. The one or more cells can be islet cells, such as pancreatic beta cells. In some cases, TILs may be from gastrointestinal cancer. TILs culture can be prepared in a variety of ways. For example, tumors can be trimmed from non-cancerous tissue or necrotic areas. The tumor can then be broken into about 2-3mm long. In some cases, the size of the tumor can be about 0.5mm to about 5mm, about 1mm to about 2mm, about 2mm to about 3mm, about 3mm to about 4mm or about 4mm to about 5mm. Tumor fragments can then be cultured in vitro using culture medium and cell stimulants such as cytokines. In some cases, IL-2 can be used to amplify TILs from tumor fragments. IL-2 concentration can be about 6000IU / ml. The concentration of IL-2 can also be about 2000IU / ml, 3000IU / ml, 4000IU / ml, 5000IU / ml, 6000IU / ml, 7000IU / ml, 8000IU / ml, 9000IU / ml or up to about 10000IU / ml. Once TILs are amplified, they can be subjected to in vitro analysis to determine tumor reactivity. For example, CD3, CD4, CD8 and CD58 expression of TILs can be assessed by FACS. TILs can also be subjected to co-cultivation, cytotoxicity, ELISA or ELISPOT analysis. In some cases, TILs cultures can be cryopreserved or rapidly amplified. Cells, such as TILs, can be separated from donors at developmental stages, including but not limited to fetuses, newborns, youth and adults.

[0254] Recipient cells can be isolated from a surgically removed tumor. For example, after removing fat and connective tissue, the tumor tissue can be cut into 3-5 mm 2The fragments were separated and disintegrated using gentle mechanical crushing in cold RPMI 1640 using a Seward Stomacher apparatus (Fisher, Pittsburgh, PA). This method allows rapid preparation of single cell suspensions without enzymatic digestion. The cell suspension can be filtered through a 75 μm pore size mesh (BD Biosciences, San Jose CA) and washed in culture medium. A portion of the cells for immediate staining and analysis by flow cytometry can be washed in culture medium, and the cell suspension can be layered on a discontinuous 70%, followed by 100% FicollIsopaque gradient centrifugation to separate tumor cells (70% interface) from TITs (100% interface). The enriched TITs can then be washed in D-PBS, 1% BSA and then processed. In some cases, TITs from tumor samples can be expanded in TIT medium (TILs-CM) containing RPMI 1640 and Glutamax (Invitrogen), 10% human AB serum (Sigma, St. Louis, MI), 50 mM 2-mercaptoethanol (Invitrogen), 1 mM pyruvate, 1X penicillin / streptomycin (Invitrogen), using 3,000 IU / ml recombinant IL-2. Activation and amplification

[0255] Whether before or after delivery of the sequence encoding the exogenous TCR to the recipient T cells, the cells can generally be activated and expanded using methods such as those described in U.S. Pat. Nos. 6,352,694, 6,534,055, 6,905,680, 6,692,964, 5,858,358, 6,887,466, 6,905,681, 7,144,575, 7,067,318, 7,172,869, 7,232,566, 7,175,843, 5,883,223, 6,905,874, 6,797,514, 6,867,041; and U.S. Patent Application Publication No. 20060121005. T cells can be expanded in vitro or in vivo.

[0256] T cells can be amplified by contacting with reagents stimulating CD3 TCR complexes and co-stimulatory molecules on the surface of T cells to produce activation signals of T cells. For example, chemical substances such as calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA) or mitogenic lectins such as phytohemagglutinin (PHA) can be used to produce activation signals of T cells. As a non-limiting example, T cell colonies can be stimulated in vitro, for example, by contacting with anti-CD3 antibodies or their antigen-binding fragments, or anti-CD2 antibodies fixed on the surface, or by contacting with protein kinase C activators (e.g., bryostatin) and calcium ionophores. The ligand co-stimulation of the auxiliary molecules on the surface of T cells is used to bind auxiliary molecules. For example, under conditions suitable for stimulating T cell proliferation, T cell colonies can be contacted with anti-CD3 antibodies and anti-CD28 antibodies. The reagents of anti-CD3 antibodies and anti-CD28 antibody signals can be in solution or coupled to the surface to stimulate the proliferation of CD4+T cells or CD8+T cells. The ratio of particles to cells can depend on the particle size relative to the target cell. In a further embodiment, cells such as T cells are combined with the beads of the reagent coating, beads and cells are separated subsequently, and then the cells are cultured. In an alternative embodiment, before cultivation, the beads of the reagent coating are cultivated together with the cells, not separated. Conditions suitable for T cell culture include suitable culture medium (for example, minimal essential medium or RPMI culture medium 1640 or X-vivo 5, (Lonza)), which may contain factors necessary for proliferation and survival, including serum (for example, fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-G, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGF β and TNF-α or any other additives for cell growth. Other additives for cell growth include but are not limited to surfactants, plasma acid salts and reducing agents such as N-acetyl-cysteine ​​and 2-mercaptoethanol etc. The culture medium may include RPMI 1640, A1M-V, DMEM, MEM, A-MEM, F-12, X-vivo 1 and X-vivo 20, Optimizer, with the addition of amino acids, sodium pyruvate and vitamins, without serum or supplemented with an appropriate amount of serum (or plasma) or a set of determined hormones, and / or cytokines sufficient to grow and expand T cells. The target cells may be maintained under conditions necessary to support growth, such as an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2). T cells exposed to different stimulation times may exhibit different characteristics.

[0257] In another embodiment, cells can be activated or expanded by co-culturing with tissues or cells. Cells used to activate T cells can be APCs or artificial APCs (aAPCs). APCs can be professional APCs, such as dendritic cells, macrophages, or B cells. APCs can be monocytes or monocyte-derived dendritic cells. aAPCs can express ligands for T cell receptors and co-stimulatory molecules, and can activate and expand T cells for transfer, while improving their effectiveness and function in some cases. aAPCs can be modified to express any gene for T cell activation. aAPCs can be modified to express any gene for T cell expansion. aAPCs can be beads, cells, proteins, antibodies, cytokines, or any combination. aAPCs can transmit signals to cell populations that may undergo genome transplantation. For example, aAPCs can transmit signal 1, signal 2, signal 3, or any combination. Signal 1 can be an antigen recognition signal. For example, signal 1 can be connected to TCR or combined with an agonistic antibody for CD3 through a peptide-MHC complex, which can lead to the activation of the CD3 signal transduction complex. Signal 2 can be a co-stimulatory signal. For example, the costimulatory signal can be anti-CD28, inducible costimulator (ICOS), CD27 and 4-1BB (CD137), which bind to ICOS-L, CD70 and 4-1BBL, respectively. Signal 3 can be a cytokine signal. The cytokine can be any cytokine. The cytokine can be IL-2, IL-7, IL-12, IL-15, IL-21 or any combination thereof.

[0258] In some cases, aAPC can be used to activate and / or expand cell populations. In some cases, artificial allospecificity may not be induced. In some cases, aAPC may not express HLA. aAPC can be genetically modified to stably express genes that can be used for activation and / or stimulation. In some cases, K562 cells can be used for activation. K562 cells can also be used for expansion. K562 cells can be human erythroleukemia cell lines. K562 cells can be transformed to express target genes. K562 cells may not express endogenous HLA class I, class II or CD1d molecules, but may express ICAM-1 (CD54) and LFA-3 (CD58). K562 can be modified to deliver signals 1 to T cells. For example, K562 cells can be transformed to express class I HLA. In some cases, K562 cells can be engineered to express other molecules, such as B7, CD80, CD83, CD86, CD32, CD64, 4-1BBL, anti-CD3, anti-CD3 mAb, anti-CD28, anti-CD28 mAb, CD1d, anti-CD2, membrane-bound IL-15, membrane-bound IL-17, membrane-bound IL-21, membrane-bound IL-2, truncated CD19, or any combination. In some cases, the engineered K562 cells can express a membrane-based anti-CD3 mAb, i.e., clone OKT3, in addition to CD80 and CD83. In some cases, the engineered K562 cells can express a membrane-based anti-CD3 mAb, clone OKT3, or a membrane-based anti-CD28 mAb, in addition to CD80 and CD83.

[0259] In some cases, the restimulation of cells can be performed with antigens and irradiated histocompatible APCs (e.g., feeder PBMCs). In some cases, nonspecific mitogens (e.g., PHA) and allogeneic feeder cells can be used to culture cells. Feeder PBMCs can be irradiated with 40 Gy. Feeder PBMCs can be irradiated with about 10 Gy to about 15 Gy, about 15 Gy to about 20 Gy, about 20 Gy to about 25 Gy, about 25 Gy to about 30 Gy, about 30 Gy to about 35 Gy, about 35 Gy to about 40 Gy, about 40 Gy to about 45 Gy, about 45 Gy to about 50 Gy. In some cases, a control bottle of irradiated feeder cells is stimulated only with anti-CD3 and IL-2.

[0260] aAPC can be a bead. Spherical polystyrene beads can be coated with antibodies against CD3 and CD28 and used for T cell activation. The beads can be of any size. In some cases, the beads can be or can be about 3 and 6 microns. The size of the beads can be or can be about 4.5 microns. The beads can be used in any ratio of cells to beads. For example, a 3 to 1 ratio of beads to cells of 1 million cells per ml can be used. aAPC can also be a rigid spherical particle, a polystyrene latex microbead, a magnetic nano- or micro-particle, a nano-sized quantum dot, a poly(lactic-co-glycolic acid) (PLGA) microsphere, a non-spherical particle, a carbon nanotube bundle, an ellipsoidal PLGA microparticle, a nanoworm, a 2D-supported lipid bilayer containing a fluid lipid bilayer system (2D-SLB), a liposome, a liposome / microdomain liposome, an SLB particle, or any combination thereof.

[0261] In some cases, aAPCs can expand CD4 T cells. For example, aAPCs can be engineered to mimic the antigen processing and presentation pathways of HLA class II restricted CD4 T cells. K562 can be engineered to express HLA-D, DPα, DPβ chain, Ii, DMα, DMβ, CD80, CD83, or any combination thereof. For example, engineered K562 cells can be pulsed with HLA restricted peptides to expand HLA restricted antigen specific CD4 T cells.

[0262] In some cases, the use of aAPCs can be combined with exogenously introduced cytokines for T cell activation, expansion, or any combination. The cells can also be expanded in vivo, for example in the blood of a subject after the genome-transplanted cells are administered to the subject.

[0263] Cells (eg, recipient cells) can also be expanded in vivo, such as in the blood of a subject following administration of the cells to the subject. Gene Editing

[0264] The receptor cells disclosed herein can be delivered together with the sequence of the exogenous paired sequence of the TCR for expression. The sequence can be knocked into the genomic site for expression. The knock-in can be site-specific knock-in. For example, the sequence of the exogenous paired sequence encoding TCR can be knocked into the genomic site by site-specific knock-in for monoallelic expression. The genomic site can be located on a sex chromosome. The genomic site can be a tracer locus. The knock-in can be performed by various gene editing methods described herein. For example, the knock-in can be performed by clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector (TALE) nucleases (TALENs), homing nucleases (meganuclease), zinc finger nucleases (ZFN), megaTAL nucleases or any combination thereof. A vector containing a sequence encoding an exogenous TCR homologous pair can be delivered to a receptor cell together with a gene editing agent (e.g., nucleases such as Cas9 and / or sgRNA). The gene editing agent can introduce site-specific cutting at a genomic site, and the sequence encoding an exogenous TCR homologous pair can be inserted into a specific genomic site by homologous recombination.

[0265] Receptor cells disclosed herein may be modified to inactivate one or more endogenous TCR chains. Endogenous TCR genes encoding TCR alpha chains, TCR beta chains or TCR alpha chains and TCR beta chains may be inactivated in modified cells described herein (e.g., modified T cells). In some cases, endogenous TCR genes encoding TCR gamma chains, TCR delta chains or TCR gamma chains and TCR delta chains may be inactivated in modified cells described herein (e.g., modified T cells). The inactivation of endogenous TCR genes in receptor cells may be carried out before, simultaneously or after the delivery of polynucleotides or sequences encoding exogenous TCR. In some cases, the endogenous TCR genes in receptor cells are inactivated while delivering polynucleotides or sequencing encoding exogenous TCR. In some cases, the endogenous TCR genes in receptor cells are inactivated, and the sequences encoding exogenous TCR are knocked into genomic sites by site-specificity. For example, the receptor cell can be delivered with a vector containing a sequence encoding an exogenous TCR and a nuclease protein or a nucleic acid sequence encoding a nuclease protein targeting an endogenous gene encoding a TCR α chain, a TCR β chain or a TCR α chain and a TCR β chain. The nuclease protein can introduce an incision in a genomic site. The vector may further include a pair of HAS flanking the sequence encoding an exogenous TCR, which promotes the site-specific knock-in of the sequence by homologous recombination. The nuclease can be a homing endonuclease (meganuclease), a zinc finger nuclease (ZFN), a transcription activator-like effector nuclease (TALENs), a CRISPR / CAS nuclease or a megaTAL nuclease.

[0266] Inactivation may include the destruction of genomic sites, gene silencing, inhibition or reduction of transcription or inhibition or reduction of translation. In some cases, endogenous TCR genes may be knocked down. Endogenous TCR genes may be knocked down (or silenced), for example, by inhibitory nucleic acids such as siRNA and shRNA. The antisense RNA that RNase-H depends on can inhibit the expression of endogenous TCR genes. The translation of endogenous TCR genes may be inhibited by inhibitory nucleic acids such as microRNA. In some embodiments, gene editing techniques are used to destroy endogenous TCR genes or knock out endogenous genes. For example, CRISPR / Cas9 editing of TRAC or TRBC loci can be used to knock out endogenous TCR genes. In some embodiments, gene editing techniques pave the way for multiple genome editing, which allows the simultaneous destruction of multiple genomic sites in endogenous TCR genes. In some embodiments, multiple genome editing techniques are applied to produce genetically disrupted T cells, which are defective in the expression of endogenous TCR and / or human leukocyte antigen (HLA) and / or programmed cell death protein 1 (PD1) and / or other genes.

[0267] The genes encoding endogenous TCR chains can be inactivated using gene editing techniques (e.g., clustered regularly interspaced short palindromic repeats (CRISPRs) See, e.g., U.S. Pat. No. 8697,359), transcription activator-like effector (TALE) nucleases (TALENs, see, e.g., U.S. Pat. No. 9393,257), homing endonucleases (deoxyribonuclease endonucleases with large recognition sites for double-stranded DNA sequences containing 12 to 40 base pairs), zinc finger nucleases (ZFNs, see, e.g., Urnov et al., Nat. Rev. Genetics (2010) v11, 636-646) or megaTAL nucleases (fusion proteins of homing endonucleases and TAL repeat sequences). Alternatively, a technique such as RNA interference (RNAi) is used to knock down a gene of interest as described herein.

[0268] These gene editing technologies may share a common mode of action in terms of incorporating user-defined DNA sequences and mediating double-stranded DNA breaks (DSBs). DSBs can then be repaired by nonhomologous end joining (NHEJ) or (when donor DNA is present) homologous recombination (HR), an event that introduces homologous sequences from the donor DNA fragment. In addition, nicking nucleases generate single-stranded DNA breaks (SSBs). Double-stranded breaks can be repaired by single-stranded DNA incorporation (ssDI) or single-stranded template repair (ssTR), an event that introduces homologous sequences from the donor DNA.

[0269] Genetic modification of genomic DNA can be carried out using site-specific, rare-cutting endonucleases that are engineered to recognize the DNA sequence in the target locus. Methods for producing engineered site-specific endonucleases are known in the art. For example, zinc finger nucleases (ZFNs) can be modified to recognize and cut predetermined sites in the genome. ZFN is a chimeric protein comprising a zinc finger DNA binding domain fused to the nuclease domain of the Fok1 restriction enzyme. The zinc finger domain can be redesigned by rational or experimental methods to produce a protein that binds to a predetermined DNA sequence (e.g., a sequence of 18 base pairs in length). By fusing the engineered protein domain with the Fok1 nuclease, it is possible to target DNA breaks at the genome level. ZFN can be used to add, remove and replace targeted genes in a wide range of eukaryotic organisms. Similarly, TAL effector nucleases (TALENs) can be produced to cut specific sites in genomic DNA. Like ZFN, TALEN comprises an engineered site-specific DNA binding domain fused to the Fok1 nuclease domain. However, in this case, the DNA binding domain includes a tandem array of TAL-effector domains, each of which specifically recognizes a single DNA base pair. Compact TALENs have an alternative nuclease structure that avoids the need for dimerization. Compact TALENs may contain an engineered, site-specific TAL effector DNA binding domain fused to the nuclease domain from the I-TevI ​​homing endonuclease. Unlike Fokl, I-TevI ​​may not dimerize to produce double-stranded DNA breaks, so compact TALENs can function as monomers.

[0270] Engineered nucleases based on the CRISPR / Cas9 system can also be used. CRISPR gene editing technology may include nuclease proteins whose DNA targeting specificity and cleavage activity can be programmed by short guide RNA or double-stranded crRNA / TracrRNA. CRISPR nucleases include two components: (1) caspase effector nuclease, usually microbial Cas9; and (2) a short "guide RNA" or RNA duplex containing a targeting sequence of 18 to 20 nucleotides that guides the nuclease to the target location in the genome. By expressing multiple guide RNAs in the same cell, each with a different targeting sequence, DNA breaks can be simultaneously targeted to multiple sites in the genome (multiple genome editing).

[0271] There are two classes of CRISPR systems, each containing multiple CRISPR types. Class I contains type I and type III CRISPR systems commonly found in archaea. And, class II contains type II, IV, V, and VI CRISPR systems. Although the most widely used CRISPR / Cas system is the type II CRISPR-Cas9 system, CRISPR / Cas systems have been repurposed for genome editing. In the past few years, more than 10 different CRISPR / Cas proteins have been engineered. For example, the Cas12a (Cpf1) protein from Acidaminococcus (AsCpf1) or Lachnospiraceae (LbCpf1) can be used.

[0272] Homing endonucleases are a group of naturally occurring nucleases that recognize 15-40 base pair cleavage sites commonly found in plant and fungal genomes. They may be associated with parasitic DNA elements, such as group 1 self-splicing introns and inteins. They can naturally promote homologous recombination or gene insertion at specific locations in the host genome by generating double-strand breaks in chromosomes and recruiting cellular DNA repair mechanisms. Specific amino acid substitutions can reprogram the DNA cleavage specificity of homing nucleases. Homing endonucleases (MNs) are monomeric proteins with natural nuclease activity that are derived from bacterial homing endonucleases and engineered for unique target sites. In some cases, the homing endonuclease is a modified I-CreI homing endonuclease. In other cases, the homing endonuclease is an engineered I-SceI homing endonuclease.

[0273] In addition to the above gene editing technologies, chimeric proteins comprising fusions of homing endonucleases, ZFNs, and TALENs can be engineered to generate novel monomeric enzymes that exploit the binding affinity of ZFNs and TALENs and the cleavage specificity of homing endonucleases. For example, megaTAL is a single chimeric protein that combines the easily customizable DNA binding domain from TALENs with the high cleavage efficiency of homing endonucleases.

[0274] In order to perform gene editing technology, nuclease and gRNA in the case of CRISPR / Cas9 system can be delivered to the target cell. Delivery methods include but are not limited to physical, chemical and viral methods. In some cases, physical delivery methods can be selected from methods including but not limited to electroporation, microinjection or using ballistic particles. On the other hand, chemical delivery methods can use molecules such as calcium phosphate, lipids or proteins. In some embodiments, viral delivery methods can use viruses, such as adenovirus, slow virus or retrovirus.

[0275] The modified cells described herein (e.g., synthetic TILs) can be administered to a subject (e.g., a human) suffering from a disease such as cancer. The modified cells can proliferate or amplify in the subject. The modified cells (or their progeny) can persist in the subject. For example, after infusion, the proportion of the modified cells in all peripheral T cells of the subject can be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more. In some cases, after infusion, the proportion of the modified cells in all peripheral T cells of the subject can be at least about 30%, 40%, 50%, 60% or more. CD62L of modified cells + The proportion of cells in the peripheral T cells of the subject may be at least about 50%, 60%, 70%, 80%, 90% or more. The proportion of modified cells described herein can persist in the subject. For example, the proportion of modified cells can last for at least 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days or longer; or in some cases, for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months 13 months, 14 months, 15 months, 16 months, 17 months or more. In some other cases, the proportion of modified cells can last for at least 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or longer. In some other cases, the proportion of modified cells can persist for at least 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months or longer. In some cases, the proportion of modified cells in all peripheral T cells in a peripheral blood mononuclear cell (PBMC) sample of a subject is at least about 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In some cases, after infusion, the modified cells in each milliliter of peripheral blood in a subject (or a PBMC sample of a subject) can be maintained at least about 0.2×10 6 , 0.3×10 6 , 0.4×10 6 , 0.5×10 6 , 0.6×10 6 , 0.7×10 6 , 0.8×10 6 , 0.9×10 6 , 1×10 6 , 1.5×106 , 2×10 6 , 2.5×10 6 , 3×10 6 , 3.5×10 6 , 4×10 6 , 4.5×10 6 , 5×10 6 , 5.5×10 6 , 6×10 6 , 6.5×10 6 ,7×10 6 , 7.5×10 6 ,8×10 6 ,9×10 6 ,1×10 7 In some cases, after infusion, at least about 0.2×10 modified cells per milliliter of peripheral blood in the subject (or a sample of PBMCs from the subject) can be maintained for at least 10 days, 15 days, 20 days, 25 days, 30 days, 35 days, 40 days, 50 days, 60 days, 70 days, 80 days, or longer. 6 , 0.3×10 6 , 0.4×10 6 , 0.5×10 6 , 0.6×10 6 , 0.7×10 6 , 0.8×10 6 , 0.9×10 6 , 1×10 6 , 1.5×10 6 , 2×10 6 , 2.5×10 6 , 3×10 6 , 3.5×10 6 , 4×10 6 , 4.5×10 6 , 5×10 6 , 5.5×10 6 , 6×10 6 , 6.5×10 6 ,7×10 6 , 7.5×10 6 ,8×10 6 ,9×10 6 ,1×10 7Or more cells; or in some cases, maintain at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months or more. As described herein, with or without IL-2 infusion, the ratio or concentration of modified cells can remain unchanged. The detection of modified cells can be based on markers unique to the modified cells, rather than other endogenous T cells of the subject. For example, recombinant marker proteins can be expressed exogenously by modified cells. For another example, RNA-FISH methods can be used to detect exogenous TCR transcripts in modified cells.

[0276] The modified cells can proliferate at a substantially equivalent rate compared to comparable cells (e.g., immune cells, T cells, or TILs).

[0277] The present invention also provides a cell mixture comprising a plurality of modified cells. The mixture may comprise at least 2, 5, 10, 15, 20, 25, 30, 50, 100, 200, 500 or 1000 modified cells, each modified cell of the mixture exogenously expressing polynucleotide sequences encoding different associated pairs of TCR α chains and TCR β chains. The polynucleotide sequence may be knocked into a genomic locus for monoallelic expression of the polynucleotide sequence. The endogenous TCR gene of the modified cell may be inactivated.

[0278] In some cases, the cell mixture may include a plurality of modified cells, each of which exogenously expresses a polynucleotide sequence encoding a different pairing sequence of a TCR α chain and a TCR β chain. The polynucleotide sequence may be knocked into a genomic site for monoallelic expression of the polynucleotide sequence. The endogenous TCR gene of the modified cell may be inactivated.

[0279] Each different pairing sequence of a TCR alpha chain and a TCR beta chain can be from a subject with a solid tumor lesion, or can be endogenously expressed in a T cell from a subject with a solid tumor lesion. The subject (e.g., a cancer patient) may be intended to receive treatment with a pharmaceutical composition comprising a modified cell mixture or a modified cell mixture. The mixture may comprise at least 10, 20, 30, 50 or more modified cells, each of which exogenously expresses polynucleotide sequences encoding different associated pairs of TCR alpha chains and TCR beta chains.

[0280] The genomic locus may be a tracer locus. The endogenous TCR gene of the modified cell may be knocked out or knocked down. The endogenous TCR gene may comprise a TRBC locus.

[0281] The modified cell can be a T cell. The T cell can be an allogeneic T cell or an autologous T cell. The different paired sequences of the TCR α chain and the TCR β chain of each modified cell can be selected by sequencing the TCR of multiple T cells from the subject. The different paired sequences of the TCR α chain and the TCR β chain of each modified cell can be selected by a pre-designed scoring method, which is based on the sequencing data of the TCR of multiple T cells from the subject. The different paired sequences of the TCR α chain and the TCR β chain of each modified cell may have been confirmed to be functional. The unique paired sequences of the TCR α chain and the TCR β chain of each modified cell may be tumor reactive, and the tumor reactivity of the mixture may or may not be tested. The mixture may include functional and non-functional TCRs. The mixture may contain tumor reactive and tumor non-reactive TCRs. The mixture of modified cells may have been amplified in vitro.

[0282] The cell mixture provided herein may include multiple modified cells, each of which exogenously expresses a pairing sequence of the TCR from a subject. The mixture or a pharmaceutical composition comprising the mixture may be administered to a subject. The cell mixture may express at least 10 different TCR pairing sequences. Each TCR in the at least 10 different TCR pairing sequences may be expressed in at least 2% of the cell mixture. The relative abundance of each unique TCR in the mixture may be similar to any other TCR. The cells expressing a given TCR may not grow beyond the cells expressing any other TCR. In some cases, at least 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% of the cell mixture each exogenously expresses only a pair of homologous TCRs.

[0283] The polynucleotide sequence encoding the TCR pairing sequence can be knocked into the genomic locus to mono-allelically express the polynucleotide sequence in each modified cell that exogenously expresses at least 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% of the mixture of only one pair of TCR pairing sequences. When administered to a subject, the modified cells can be expressed in the subject at a rate of at least about 0.2×10 per milliliter of peripheral blood. 6 (In some cases, 0.4×10 6 Up to 1×10 6 ) cells for at least about 30 days (e.g., 40 days, 45 days, 50 days, 55 days, 60 days, or more). Treatment options

[0284] Disclosed herein may be cells (e.g., synthetic TILs) used in a treatment regimen. For example, a subject may receive cells as part of a treatment regimen for a cancer or disease. Treatment regimens may include: surgery, chemotherapy, radiotherapy, immunosuppressants, immunostimulants, antifungals, antivirals, antibiotics, or antiemetics, etc. In some cases, the cell composition may be combined with (e.g., before, at the same time, or after) bone marrow transplantation, T cell ablation therapy using chemotherapeutics such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or Campath. In some cases, the expanded cells may be administered before or after surgery. In some cases, the surgery may be a tumor resection. Surgery may be performed to separate TILs or TITs.

[0285] In some cases, the present disclosure provides a method of treating cancer in a subject in need thereof. The method may include administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein or a pharmaceutical composition comprising a mixture of modified cells described herein.

[0286] In some cases, the present disclosure provides a method of treating cancer in a subject in need thereof. The method may include administering to the subject a pharmaceutical composition comprising (a) a synthetic TIL prepared according to the methods described herein; and (b) a pharmaceutically acceptable carrier.

[0287] In some cases, the disclosure provides a method for treating cancer in a subject in need. The method may include administering a pharmaceutical composition comprising a cell mixture to a subject in need. The cell mixture may exogenously express at least 10 (e.g., 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1500, 1800, 2000, 5000, 100000, 20000, 50000, 100000 or more) different paired sequences of TCRs from a subject. In some cases, at least 40%, 50%, 60%, 70%, 80%, 90%, 99% or 100% of the cell mixture may each only exogenously express a pair of homologous TCRs. The cell mixture may be a mixture of modified cells. The cell mixture may include multiple modified cells and multiple unmodified cells. Each TCR in the at least 10 different TCR pairing sequences may be expressed in at least 2% of the modified cell mixture. The polynucleotide sequence may be knocked into a genomic site for monoallelic expression of the polynucleotide sequence. The subject from which the TCR is obtained may be the same as the subject to be treated.

[0288] In some cases, the endogenous TCR gene of the modified cell is inactivated. The modified cell can be a T cell. The T cell can be a CD4+T cell, a CD8+T cell, a CD4+ / CD8+T cell, a helper T cell, a cytotoxic T cell, a memory T cell, a regulatory T cell, a natural killer T cell, an αβT cell or a γδT cell. The T cell can be an allogeneic T cell or an autologous T cell.

[0289] The modified cells can be isolated from a subject to which the composition is administered. The different paired sequences of each modified cell can be from a subject in need thereof, or can be endogenously expressed by T cells from a subject in need thereof. The mixture can contain at least 10 modified cells. The genomic locus can be a tracer locus. The endogenous TCR gene of the modified cell can be knocked out or knocked down. The endogenous TCR gene can include a TRBC locus.

[0290] A subject in need thereof may have received one or more lines of treatment (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more lines), or may have failed to respond to one or more prior lines of treatment. The cancer may be metastatic or refractory. The cancer may be a solid tumor.

[0291] The method may further include, after administering the pharmaceutical composition, optionally administering IL-2 to the subject. Prior to administration, the cell mixture may be tested for tumor reactivity. The mixture of modified cells may persist in the subject for at least about 30 days. After administration, the proportion of the modified cells or their progeny in the subject's peripheral T cells may be at least about 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more). CD62L in the subject's peripheral T cells of synthetic TILs or their progeny + The proportion of cells can be at least about 50% (60%, 70%, 80%, 90% or more). This proportion can last for at least about 30 days. In some cases, the concentration of synthetic TILs in the peripheral blood sample of the subject can be at least about 0.2×10 per milliliter of peripheral blood. 6 This concentration can be sustained for at least about 30 days with or without IL-2 infusion.

[0292] In some cases, lymphodepletion may be performed. Pretreatment with lymphodepleting chemotherapy can enhance the effect of adoptive transfer T cell therapy. Chemotherapy pretreatment before infusion of T cell products can significantly enhance the duration and therapeutic effect of T cells in vivo, and can be used in clinical studies of gene-modified T cells. For example, fludarabine and cyclophosphamide can be used for chemotherapy pretreatment of T cells (including TCR-T cells, CAR-T cells, and TIL cells) in the treatment of malignant tumors. For example, subjects can be given cyclophosphamide for 2 consecutive days on day 6 before TCR-T cell infusion, followed by 3 days of fludarabine lymphodepleting chemotherapy drug pretreatment (see Table 3).

[0293] A therapeutically effective amount of cells may be administered. In some cases, about 5×10 10 In some cases, about 5×10 10 cells represents the median number of cells administered to a subject. In some embodiments, about 5×10 10 cells are necessary to achieve a therapeutic response in a subject. In some embodiments, at least about 1×10 6 cells, at least about 2×10 6 cells, at least about 3×10 6 cells, at least about 4×10 6 cells, at least about 5×10 6 cells, at least about 6×10 6 cells, at least about 6×10 6 cells, at least about 8×10 6 cells, at least about 9×10 6 cells, 1×10 7 cells, at least about 2×10 7 cells, at least about 3×10 7 cells, at least about 4×10 7 cells, at least about 5×10 7 cells, at least about 6×10 7 cells, at least about 6×10 7 cells, at least about 8×10 7 cells, at least about 9×10 7 cells, at least about 1×10 8 cells, at least about 2×10 8 cells, at least about 3×10 8 cells, at least about 4×10 8 cells, at least about 5×10 8 cells, at least about 6×10 8 cells, at least about 6×10 8cells, at least about 8×10 8 cells, at least about 9×10 8 cells, at least about 1×10 9 cells, at least about 2×10 9 cells, at least about 3×10 9 cells, at least about 4×10 9 cells, at least about 5×10 9 cells, at least about 6×10 9 cells, at least about 6×10 9 cells, at least about 8×10 9 cells, at least about 9×10 9 cells, at least about 1×10 10 cells, at least about 2×10 10 cells, at least about 3×10 10 cells, at least about 4×10 10 cells, at least about 5×10 10 cells, at least about 6×10 10 cells, at least about 6×10 10 cells, at least about 8×10 10 cells, at least about 9×10 10 cells, at least about 1×10 11 cells, at least about 2×10 11 cells, at least about 3×10 11 cells, at least about 4×10 11 cells, at least about 5×10 11 cells, at least about 6×10 11 cells, at least about 6×10 11 cells, at least about 8×10 11 cells, at least about 9×10 11 cells or at least about 1×10 12 For example, about 5×10 cells may be administered to a subject. 10 In another example, from 3×10 6 Starting from 10 cells, the cells can be expanded to about 5×10 10 In some cases, the cells are expanded to a number sufficient for treatment. For example, 5×10 7 The cells can be rapidly expanded to produce sufficient numbers for therapeutic use. In some cases, the number of cells required for therapeutic use may be 5 × 10 10 Any number of cells may be injected for therapeutic purposes. For example, 1×10 6 Up to 5×10 12The number of cells between (inclusive). As many cells as possible can be injected into the patient as long as they can be generated. In some cases, not all of the cells injected into the patient are engineered. For example, at least 90% of the cells injected into the patient can be engineered. In other cases, at least 40% of the cells injected into the patient can be engineered. For a patient, the number of cells required for effective treatment may vary depending on the viability of the cells and the efficiency with which the cells are genetically modified. In some cases, the product of the viability of cells after genetic modification (e.g., doubling) may correspond to an aliquot of therapeutic cells that can be used for administration to a subject. In some cases, an increase in cell viability after genetic modification may correspond to a reduction in the number of cells required for effective administration of treatment to a patient.

[0294] In some cases, a method may include calculating and / or administering to a subject a number of engineered cells sufficient to produce a therapeutic response in the subject. In certain embodiments, calculating the number of engineered cells required to produce a therapeutic response includes determining the viability of the engineered cells. In certain embodiments, in order to produce a therapeutic response in a subject, the cells administered to the subject are living cells. In certain embodiments, in order to produce a therapeutic response in a subject, at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20%, at least about 15%, at least about 10% of the cells are living cells. In certain embodiments, to produce a therapeutic response in a subject, at least about 95%, at least about 90%, at least about 85%, at least about 80%, at least about 75%, at least about 70%, at least about 65%, at least about 60%, at least about 55%, at least about 50%, at least about 45%, at least about 40%, at least about 35%, at least about 30%, at least about 25%, at least about 20%, at least about 15%, at least about 10% of the cells have been introduced with a polynucleotide encoding a TCR cognate pair.

[0295] In some cases, adoptively transplanted cells can be monitored by quantitative PCR (qPCR). qPCR analysis of adoptively transplanted cells can indicate the level of modified cells present in the subject after introduction. In some cases, adoptively transferred cells can be monitored using flow cytometry. For example, flow cytometry can determine the level of 4-1BB relative to TCR. In some cases, single cell TCR PCR can be performed. The level of adoptively transferred cells can be identified on the 40th day after infusion. The levels of adoptively transferred cells (e.g., modified cells) can be assessed on day 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or up to day 200 following infusion. Immunostimulants

[0296] In some cases, an immunostimulatory agent can be introduced into a cell or subject. An immunostimulatory agent can be specific or nonspecific. A specific immunostimulatory agent can provide antigen specificity, such as a vaccine or an antigen. A nonspecific immunostimulatory agent can enhance an immune response or stimulate an immune response. A nonspecific immunostimulatory agent can be an adjuvant. An immunostimulatory agent can be a vaccine, a colony stimulating agent, an interferon, an interleukin, a virus, an antigen, a co-stimulatory agent, an immunogenic agent, an immunomodulator, or an immunotherapeutic agent. An immunostimulatory agent can be a cytokine such as an interleukin. One or more cytokines can be introduced together with the cells disclosed herein. Cytokines can be used to promote the proliferation of cytotoxic T lymphocytes (including adoptively transferred tumor-specific cytotoxic T lymphocytes) in a tumor microenvironment. In some cases, IL-2 can be used to promote the proliferation of cells described herein. Cytokines such as IL-15 can also be used. Other related cytokines in the field of immunotherapy, such as IL-2, IL-7, IL-12, IL-15, IL-21, or any combination thereof, can also be used. In some cases, IL-2, IL-7 and IL-15 are used to cultivate the cells of the present disclosure. Interleukin can be IL-2 or aldesleukin. Aldesleukin can be administered in low doses or high doses. High-dose aldesleukin regimens can include intravenous administration of aldesleukin every 8 hours when tolerated, with about 0.037mg / kg (600,000IU / kg) for up to about 14 doses. Immunostimulants (e.g., aldesleukin) can be administered within 24 hours after cell administration. Immunostimulants (e.g., aldesleukin) can be infused for about 15 minutes every 8 hours after cell infusion for about 4 days. The immunostimulant (e.g., aldesleukin) can be administered at a dose of about 100,000 IU / kg, 200,000 IU / kg, 300,000 IU / kg, 400,000 IU / kg, 500,000 IU / kg, 600,000 IU / kg, 700,000 IU / kg, 800,000 IU / kg, 900,000 IU / kg, or up to about 1,000,000 IU / kg. In some cases, aldesleukin can be administered at a dosage of about 100,000 IU / kg to 300,000 IU / kg, 300,000 IU / kg to 500,000 IU / kg, 500,000 IU / kg to 700,000 IU / kg, 700,000 IU / kg to about 1,000,000 IU / kg. Immunostimulants (e.g., aldesleukin) can be administered from 1 dose to about 14 doses. Immunostimulants (e.g., aldesleukin) can be administered from at least about 1 dose, 2 doses, 3 doses, 4 doses, 5 doses, 6 doses, 7 doses, 8 doses, 9 doses, 10 doses, 11 doses, 12 doses, 13 doses, 14 doses, 15 doses, 16 doses, 17 doses, 18 doses, 19 doses or up to about 20 doses.In some cases, immunostimulants such as aldesleukin can be administered from about 1 dose to 3 doses, from 3 doses to 5 doses, from 5 doses to 8 doses, from 8 doses to 10 doses, from 10 doses to 14 doses, from 14 doses to 20 doses. In some cases, the dosage of Aldeskukin exceeds 20 doses. In some cases, immunostimulants such as aldesleukin can be administered in sequence or simultaneously with cell administration. For example, immunostimulants can be administered from about -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 days or until about the 14th day. In some cases, immunostimulants, such as aldesleukin, are administered from the 0th day to the 4th day after the cell population is administered. In some cases, an immunostimulant (e.g., aldesleukin) is administered in about 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 1 hour, 2 hours, or at most about 3 hours. In some cases, an immunostimulant (e.g., aldesleukin) can be administered from about 24 hours before the engineered cells are administered to about 4 days after the engineered cells are administered. An immunostimulant (e.g., aldesleukin) can be administered from -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 days or up to about 20 days after the engineered cells are administered.

[0297] Immunostimulants such as aldesleukin are available as disposable vials containing 22 million IU (1.3 mg) of IL-2, which is a sterile white to off-white lyophilized cake plus 50 mg mannitol and 0.18 mg sodium lauryl sulfate, buffered to a pH of 7.5 (range 7.2 to 7.8) with about 0.17 mg monobasic base and 0.89 mg disodium hydrogen phosphate. The vial can be reconstituted with 1.2 ml of sterile water for injection (USP) to a concentration of 18 million IU / ml or 1.1 mg / ml. The diluent should be directly against the side of the vial to avoid excessive foaming. Since the vial does not contain a preservative, the reconstituted solution should be used within 24 hours. The reconstituted aldesleukin can be further diluted with 50 ml of 5% human serum albumin (HSA). HSA can be added to the diluent before adding RIL-2. Dilutions of more than 1000-fold (i.e., 1 mg / mL to 1 mcg / mL) of the reconstituted solution are acceptable in glass bottles or polyvinyl chloride bags. Aldesleukin is chemically stable for 48 hours when refrigerated and at room temperature (2°–30°C). Aldesleukin dosing can be calculated based on total body weight. The final dilution of aldesleukin can be infused within 15 minutes.

[0298] In some cases, the immunostimulant is a colony stimulating factor. The colony stimulating factor can be G-CSF (filgrastim). Filgrastim can be stored in 300mcg / mL and 480UG / 1.6mL vials. Filgrastim can be administered by subcutaneous injection daily. Filgrastim administration can be about 5mcg / kg / day. Filgrastim dosing may start at about 1mcg / kg / day, filgrastim dosing may start at about 2mcg / kg / day, filgrastim dosing may start at about 3mcg / kg / day, filgrastim dosing may start at about 4mcg / kg / day, filgrastim dosing may start at about 5mcg / kg / day, filgrastim dosing may start at about 6mcg / kg / day, filgrastim dosing may start at about 7mcg / kg / day, filgrastim dosing may start at about 8mcg / kg / day, filgrastim dosing may start at about 9mcg / kg / day, and filgrastim dosing may start at about 10mcg / kg / day. In some cases, filgrastim can be administered at about 0.5 mcg / kg / day to about 1.0 mcg / kg / day, about 1.0 mcg / kg / day to 1.5 mcg / kg / day, about 1.5 μg / kg / day to about 2.0 μg / kg / day, about 2.0 μg / kg / day to about 3.0 mcg / kg / day, from about 3.5 mcg / kg / day to about 4.0 mcg / kg / day, from about 4.0 mcg / kg / day to about 4.5 mcg / kg / day. Administration of filgrastim can continue daily until the neutrophil count is at least about 1.0×10 9 / LX 3 days or at least about 5.0×10 9 / L. An immunostimulant such as filgrastim can be administered on day -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to about day 20 after administration of the engineered cells. Chemotherapy drugs

[0299] Chemotherapeutic drugs or compounds can be chemical substances used to treat cancer. Chemotherapeutic cancer agents that can be used in combination with modified cells include, but are not limited to, mitotic inhibitors (vinca alkaloids). These drugs include vincristine, vinblastine, vindesine, and Navelbine TM (vinorelbine, 5-noranhydrovinblastine). In other cases, chemotherapeutic cancer agents include topoisomerase I inhibitors, such as camptothecin compounds. As used herein, "camptothecin compounds" include Camptosar TM (Irinotecan hydrochloride), Hycamtin TM(topotecan hydrochloride) and other compounds derived from camptothecin and its analogs. Another class of chemotherapeutic cancer drugs that can be used in the methods and compositions disclosed herein are podophyllotoxin derivatives, such as etoposide, teniposide and mitopozine. The present invention also includes other chemotherapeutic cancer agents known as alkylating agents, which alkylate the genetic material in tumor cells. These include, but are not limited to, cisplatin, cyclophosphamide, nitrogen mustard, trimethylenethiophosphoramide, carmustine, busulfan, chlorambucil, belulastine, uracil nitrogen mustard, clomiphene and dacarbazine. The present invention includes antimetabolites as chemotherapeutic agents. Examples of these types of agents include cytarabine, fluorouracil, methotrexate, mercaptopurine, azathioprine and procarbazine. Another class of chemotherapeutic cancer drugs that can be used in the methods and compositions disclosed herein include antibiotics. Examples include, but are not limited to, doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin, mitomycin C and daunorubicin. There are many commercially available liposomal preparations of these compounds. The present invention also encompasses other chemotherapeutic cancer drugs, including but not limited to anti-tumor antibodies, dacarbazine, azacytidine, amsacrine, melphalan, ifosfamide, and mitoxantrone.

[0300] The modified cells described herein can be administered in combination with other anti-tumor agents, including cytotoxic agents / antitumor agents and anti-angiogenic agents. Cytotoxic / antitumor drugs can be defined as drugs that attack and kill cancer cells. Some cytotoxic / antitumor agents can be alkylating agents, which alkylate the genetic material in tumor cells, such as cisplatin, cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide, carmustine, busulfan, chlorambucil, belulastine, uracil nitrogen mustard, clomazine and dacarbazine. Other cytotoxic / antitumor agents can be antimetabolites of tumor cells, such as cytarabine, fluorouracil, methotrexate, mercaptopurine, thiopurine and procarbazine. Other cytotoxic / antitumor agents can be antibiotics, such as doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin, mitomycin C and daunorubicin. These compounds have many commercially available liposome preparations. There are also other cytotoxic / antitumor agents that can be mitotic inhibitors (vinca alkaloids). These drugs include vincristine, vinblastine, and etoposide. Various cytotoxic / antineoplastic drugs include paclitaxel and its derivatives, L-asparaginase, antitumor antibodies, dacarbazine, azacytidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine.

[0301] Anti-angiogenic agents may also be used. Suitable anti-angiogenic agents for the disclosed methods and compositions include anti-VEGF antibodies, including humanized antibodies and chimeric antibodies, anti-VEGF aptamers and antisense oligonucleotides. Other angiogenesis inhibitors include angiostatin, endostatin, interferon, interleukin 1 (including α and β), interleukin 12, retinoic acid and tissue inhibitors of metalloproteinases-1 and -2. (TIMP-1 and -2). Small molecules may also be used, including topoisomerases such as razoxane, a topoisomerase II inhibitor with anti-angiogenic activity.

[0302] Other anticancer agents that can be used in combination with the disclosed engineered cells can include, but are not limited to, Acivicin; Aclarubicin; Acodazole hydrochloride; Aclonine; Adozelexin; Aldesleukin; Hexamethylmelamine; Ambromycin; Amenthranol acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Aspergillus; Avastin; Azacitidine; Azetep; Azomycin; Batimastat; Bendipa; Bicalutamide; Bisantrin hydrochloride; Bisnafide dimesylate; Bizelexin; Bleomycin sulfate; Blequina sodium; Bropiridamine; Busulfan; Carbotamycin; Calustamide; Calusetamide; Cabetimer; Carboplatin; Carmustine; Carrubicin hydrochloride; Cazelexin; Sildenafil; Chlorambucil; Ciromycin; Cisplatin; Cladribine; Crisnadol mesylate; Cyclophosphamide; Cytarabine; Dacarbazine; Actinomycin; Daunorubicin hydrochloride; Decitabine; Dextroplatin; Dezaguanine; Desaguanide mesylate; Diazoquinone; Docetaxel; Doxorubicin; Doxorubicin hydrochloride; Droloxifene; Droloxifene citrate; Drostranolone propionate; Dizolin; Edatrexate; Eflornithine hydrochloride; Examitriptyline; Enloplatin; Enpromide; Epiprodine; Epirubicin hydrochloride; Erbulazole; Esorubicin hydrochloride; Estramustine; Estramustine sodium phosphate; Etanidazole; Etoposide; Etoposide phosphate; Etopopurin; Fadrozole hydrochloride; Fazarabine; Fenretinib; Floxuridine; Fludarabine phosphate; Fluorouracil; Flucitabine; Fosquinone; Fostrexone sodium; Gemcitabine; Hydrochloride gemcitabine; hydroxyurea; idarubicin hydrochloride; ifosfamide; imofosine; interleukin II (including recombinant interleukin II or rIL2), interferon α-2a; interferon α-2b; interferon α-N1; interferon α-N3; interferon β-Ⅰa; interferon γ-IB; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; lirazo; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprodol; maytansine; nitrogen mustard hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menolipir; mercaptopurine; methotrexate; methotrexate sodium; metoprolol; metuledopal; mitodamide; mitokacin; mitociramine; mitocillin; mitomycin; mitosporin; mitociramphenicol; mitosporin; mitociramphenicol tan; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogamycin; ormaplatin; oxisolan; paclitaxel; pegaspargase; pelinomycin; pentamustine; peplomycin sulfate; perphosphamide; pibobroman; piposufan; pyroxantrone hydrochloride; plicamycin; plomestan; porfimer sodium; bofilmycin; strong nimustine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofuran; ribofuranoside; rosiglitazone; safengo; safengo hydrochloride; semustine; octoterone; spavulin sodium; sparse mycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptozocin; streptozocin; suprofen; talisomycin; teicogalan sodium; tegafur; tiloxantrone hydrochloride; temoporfin; teniposide; trosilon; testolactone; timipril; thioguanine;Thiotepa; Thiazofurine; Tirapazamine; Toremifene citrate; Tristolone acetate; Triclosan phosphate; Trimetrexate; Trimetrexate glucuronide; Triptorelin; Tubazoline hydrochloride; Uracil mustard; Uradep; Vapreotide; Verteporfin; Vinblastine sulfate; Vincristine sulfate; Vindesine; Vindesine sulfate; Vinpitidine sulfate; Vitamin glycine sulfate; Vincristine sulfate; Vinorelbine tartrate; Vinprodine sulfate; Vinazolidine sulfate; Vorozole; Zeniplatin; Zinostatin ; Other anticancer drugs of zorubicin hydrochloride include but are not limited to: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; decenol; adozellexin; aldesleukin; ALL-TK antagonist; hexamethylmelamine; anmustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; anteri g; anti-dorsal morphogenetic protein-1; anti-androgen, prostate cancer; anti-estrogen; anti-angiogenic; antisense oligonucleotide; aphidicolin glycinate; apoptosis gene modulator; apoptosis modulator; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulix; atamestane; atramustine; aspirin 1; aspirin 2; aspirin 3; azasetron; azatoxin; azatyrosine; baccatin III derivative; barano; batimastat; B CR / ABL antagonists; benzoclorin; benzoylstaurosporine; β-lactam derivatives; β-opioids; beta-clarithromycin B; betulinic acid; bFGF inhibitors; bicalutamide; bisantrene; diaziridine spermine; binefat; bitrastatin A; bisezolexin; brifolin; bropiridamine; budotentan; buthionine sulfoxide; calcipotriol; calcipotriol C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide aminotriazole; carboxamide triazole; CaRest M3; CARN 700; cartilage-derived inhibitor; carzelesin; casein kinase inhibitor (ICOS); castanospermine; cecropin B; cetrorelix; clorin; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomiphene analogs; clotrimazole; colimycin A; colimycin B; combretastatin A4; combretastatin analogs; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthrone; cycloplatin; cypemycin; cytarabine octaphosphate; cytolytic factor; cytostatin; daximab; decitabine; dehydrodiazepine B; deslorelin; dexamethasone; dextrophosphamide; dexrazoxane; dexverapamil; diazinon; diazepine B; nitrogen dioxide; diethylnorspermidine; dihydro-5-azacytidine; dihydropaclitaxel, 9-; dioxin; diphenylspiromustine; docetaxel; docosanol; dolasetron; doxifluridine;droloxifene; dronabinol; duocarmycin SA; ebselen; icomustine; edilfosine; edrecolomab; eflornithine; elemene; imipenem; epirubicin; eprenolide; estramustine analogs; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrolazole; fazarabine; fenretinoic acid; filgrastim; finasteride; flavopyridine; flustatin; flusterone; fludarabine; fludaunorubicin hydrochloride; fufenimex; formestane; fostramesin; fostramustine; gadotolium teksafillin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylenebisacetamide; hyperforin; ibandronic acid; idarubicin; idoxifene; idarucifen; idarucifen; ilomastat; imidazoacridone; imiquimod; immunostimulatory peptides; insulin-like growth factor-1 receptor inhibitors; interferon agonists; interferons; interleukins; iobenguanidine; iodoxorubicin; 4-isopropanol; iroplact; irsogladine; isobenzazole; isohexylhalide B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenoglostim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibitory factor; interferon-alpha; leuprolide + estrogen + progesterone; leuprolide; levamisole; lialorazole; linear polyamine analogs; lipophilic disaccharide peptides; lipophilic platinum compounds; lisoclinamide 7; loplatin; lobulin; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lutetol ... lysozyme inhibitor; matrix metalloproteinase inhibitor; menogali; mebarone; mitrelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mimustine; mismatched double-stranded RNA; mitoxantrone; mitolactol; mitomycin analog; mitonadamide; mitochondrial toxin fibroblast growth factor-saporin; mitoxantrone; mofaratin; muramustine; monoclonal antibody, human chorionic gonadotropin; monophosphoryl lipid A+ mycobacterial cell wall sk; mopiridamole; multidrug resistance gene inhibitor; multiple tumor suppressor 1 therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; naglitazone; naloxone + pentazocine; napalene; nalidone; narostamicin; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitric oxide antioxidants; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron;ondansetron; olacin; oral cytokine inducers; ormaplatin; oxaliplatin; oxamycin; paclitaxel; paclitaxel analogs; paclitaxel derivatives; palauamide; palmitoyl rhizoxin; pamidronate; panaxytriol; panomifen; parahydroxybenzoic acid; parilitine; pegaspargase; perdesin; pentosan polysulfate sodium; pentastine; pentrazol; paflon; perfosfamide; perillyl alcohol; phenazinemycin; phenylacetic acid; phosphatase inhibitors; pilsibanil; pilocarpine hydrochloride; pirarubicin; pirexine; placenta A; placenta B; plasminogen activator inhibitors; platinum complex ; platinum compounds; platinum-triamine complexes; porfimer sodium; porfiromycin; prednisone; propyl diacridone; prostaglandin J2; proteasome inhibitors; protein A-based immunomodulators; protein kinase C inhibitors; protein kinase C inhibitors, microalgae; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurin; pyrazoloacridine; pyridoxalyl hemoglobin polyoxyethylene conjugates; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitors; demethylated retiliptin; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinoic acid; rosiglitamide; roxitoin; romotilide; roquimecetide; rubiginone B1; ruboxyl; safenol; santopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 analog; semustine; senescence-derived inhibitor 1; sense oligonucleotide; signal transduction inhibitor; signal transduction regulator; single-chain antigen binding protein; cizofilen; sobzoxan; sodium borate; sodium phenylacetate; solvelol; growth factor binding protein; sonermin; spadronic acid; spikemycin D; spiromustine; splenopentin; spongestatin 1; squalamine; stem cell inhibitor; stem cell division inhibitor; stipimide; stromelysin inhibitor; sulfinositol; superactive vasoactive intestinal peptide antagonist; suradis; suramin; swainsonine; synthetic aminodextran; tamustine; tamoxifen methyl iodide; tauromustine; tazarotene; teicogalan sodium; tegafur; Tellopyran; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecoxide; tetrazolamine; thalidomide; sulcoraline; thrombopoietin; thrombopoietin mimetics; thymalfasin; thymopoietin receptor agonist; thymine; thyroid stimulating hormone; stannopurpurin; tirapazamine; titanium dichloride; topesentine; toremifene; totipotent stem cell factor; translation inhibitor; retinoic acid; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; tolusamide; tyrosine kinase inhibitors; tyrosine phosphorylation inhibitors; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonist; vapreotide; variolin B; erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin;Vinorelbine; vinblastine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; and zinotatin stimalamer. Any of the above chemotherapeutic agents may be administered at a clinically effective dose. The chemotherapeutic agent may also be administered on or about day -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or up to about day 14 after administration of the cell population. In some cases, the subject may have a refractory cancer that is unresponsive to chemotherapy. ; Antifungal drugs

[0303] In some cases, an antifungal treatment is administered to a subject receiving a receptor cell programmed with an immunoreceptor. An antifungal agent may be a drug that kills or prevents fungal growth. Targets of antifungal agents include sterol biosynthesis, DNA biosynthesis, and beta-glucan biosynthesis. An antifungal agent may also be a folate synthesis inhibitor or a nucleic acid crosslinker. A folate synthesis inhibitor may be a sulfonamide-based drug. For example, a folate synthesis inhibitor may be an agent or competitive inhibitor that inhibits the synthesis of folate by fungi. A sulfonamide-based drug or folate synthesis inhibitor may be methotrexate or sulfamethoxazole. In some cases, an antifungal agent may be a nucleic acid crosslinker. A crosslinker may inhibit DNA or RNA processes in fungi. For example, a crosslinker may be 5-fluorocytosine, which may be a fluorinated analog of cytosine. 5-fluorocytosine may inhibit DNA and RNA synthesis by conversion to 5-fluorouracil in the cytoplasm. Other antifungal agents may be griseofulvin. Griseofulvin is an antifungal antibiotic produced by Penicillium griseofulvin. Griseofulvin inhibits mitosis of fungi and may be considered a crosslinker. Other cross-linking agents may be allylamines (naftifine and terbinafine), which inhibit ergosterol synthesis at the level of squalene epoxidase; a morpholine derivative (amorolfine) which inhibits subsequent steps in the ergosterol pathway.

[0304] In some cases, the antifungal agent can be from polyenes, azoles, allylamines or echinocandins. In some embodiments, the polyene antifungal agent is amphotericin B, bacteriocin, philipinocin, hamycin, natamycin, nystatin or remoxetine. In some cases, the antifungal agent can be from the azole family. Azoles antifungal agents can inhibit lanosterol 14α-demethylase. Azoles antifungal agents can be imidazoles, such as bifonazole, butoconazole, clotrimazole, econazole, fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole or tioconazole. Azoles antifungal agents can be triazoles, such as albaconazole, ifenconazole, oxepiconazole, fluconazole, isavorazole, itraconazole, posaconazole, propiconazole, ravuconazole, terconazole or voriconazole. In some cases, the azoles can be thiazoles, such as abafungin. The antifungal agent can be an allylamine, such as amorolfine, butenafine, naftifine or terbinafine. The antifungal agent can also be an echinocandin, such as anidulafungin, caspofungin or micafungin. Other agents that can be used as antifungal agents can be aurones, benzoic acid, ciclopirox, flucytosine, griseofulvin, haloprogin, tolnaftate, undecylenic acid, crystal violet or Peruvian balsam.

[0305] Those skilled in the art can appropriately determine which known antifungal drug to use based on the fungus that infects the individual. In some cases, the subject can receive a combination of fluconazole and engineered cells. Antifungal therapy can be administered in advance.

[0306] Fluconazole can be made into 200 mg tablets. In some cases, fluconazole can be administered as 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg or up to about 400 mg tablets. For intravenous administration to subjects who cannot tolerate oral formulations, fluconazole is administered with an injection solution of 2 mg / mL. It should be administered at a maximum intravenous injection rate of 200 mg / hr. In some cases, the infusion rate can be from about 50 mg / hr to about 500 mg / hr. The infusion rate can also be about 20 mg / hr to about 30 mg / hr, about 30 mg / hr to about 40 mg / hr, about 40 mg / hr to about 50 mg / hr, about 50 mg / hr to about 60 mg / hr, about 60 mg / hr to about 70 mg / hr, about 70 mg / hr to about 80 mg / hr, about 100 mg / hr to about 120 mg / hr, about 120 mg / hr to about 140 mg / hr, about 140 mg / hr to about 160 mg / hr, about 160 mg / hr to about 180 mg / hr, about 180 mg / hr to about 200 mg / hr, about 180 mg / hr to about 220 mg / hr, about 220 mg / hr to about 240 mg / hr, or about 240 mg / hr to about 275 mg / hr.

[0307] Antifungal drugs can be administered in a therapeutically effective dose. A therapeutically effective dose is a dose that treats or prevents fungal infections but is ineffective for treating cancer. For example, antifungal agents such as fluconazole can be administered from about 10 mg to about 1000 mg. Fluconazole can be administered from about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg or about 1000 mg at the most. The dosage of fluconazole is 400 mg. In some cases, antifungal administration can be before cell therapy, during cell therapy, or after cell therapy has been given. For example, the administration of fluconazole can be about the 0th day (the day when cell therapy is introduced into the subject) after cell therapy administration to about the 4th day. Antifungal agents can be administered from about 14 days before cell therapy administration to about 14 days after cell therapy is completed. Antifungal agents can be administered from about -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 days or until about the 14th day. Immunosuppressants

[0308] In some cases, the subject can accept immunosuppressants as part of the treatment regimen. Immunosuppressants can refer to radiotherapeutic agents, biological agents or chemical agents. In some cases, immunosuppressants can include chemical agents. For example, chemical agents can include at least one selected from cyclophosphamide, mechlorethamine, chlorambucil, melphalan, ifosfamide, thiotepa, hexamethylmelamine, busulfan, fludarabine, nitrosoureas, platinum, methotrexate, azathioprine, mercaptopurine, procarbazine, dacarbazine, temozolomide, carmustine, lomustine-streptozotocin, fluorouracil, dactinomycin, anthracycline, mitomycin C, bleomycin and mithramycin. Chemical agents can be cyclophosphamide or fludarabine.

[0309] In addition, immunosuppressants may include glucocorticoids, cytostatics, antibodies, anti-immunophilins or any derivatives thereof. Glucocorticoids may suppress allergic reactions, inflammation and autoimmune diseases. Glucocorticoids may be prednisone, dexamethasone and hydrocortisone. Immunosuppressive therapy may include any treatment that suppresses the immune system. Immunosuppressive therapy may help alleviate, minimize or eliminate transplant rejection in recipients. For example, immunosuppressive therapy may include immunosuppressive drugs. Immunosuppressive drugs that may be used before, during and / or after transplantation include but are not limited to MMF (mycophenolate mofetil (Cellcept)), ATG (anti-thymocyte globulin), anti-CD154 (CD4OL), anti-CD40 (2C10, ASKP1240, CCFZ533X2201), alemtuzumab (Campath), anti-CD20 (rituximab), anti-IL-6R antibodies (tocilizumab, Actemra), anti-IL-6 antibodies (sarilumab, olokizumab), CTLA4-Ig (Abatacept / Orencia ), belatacept (LEA29Y), rapamycin (Rapimune), everolimus, tacrolimus (Prograf), daclizumab (Ze-napax), basiliximab (Simulect), infliximab (Remicade), cyclosporine, deoxyspergualin, soluble complement receptor 1, cobra venom factor, compstatin, anti-C5 antibody (eculizumab / Soliris), methylprednisolone, FTY720, everolimus, leflunomide, anti-IL-2R-Ab, rapamycin, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40 antibody, anti-CD122 antibody. In addition, one or more immunosuppressants / drugs can be used in combination or sequentially. One or more immunosuppressants / drugs can be used for induction therapy or maintenance therapy. The same or different drugs can be used for induction and maintenance. In some cases, daclizumab (Zenapax) can be used for induction therapy, and tacrolimus (Prograf) and rapamycin (Rapimune) can be used for maintenance therapy. Daclizumab (Zenapax) can also be used for induction therapy, and low-dose tacrolimus (Prograf) and low-dose rapamycin (Rapimune) can be used for maintenance therapy. Immunosuppression can also be achieved through non-drug therapies, including but not limited to total body irradiation, thymic irradiation, and total and / or partial splenectomy.

[0310] In some cases, a cytostatic agent can be administered to perform immunosuppression. The cytostatic agent can inhibit cell division. The cytostatic agent can be a purine analog. The cytostatic agent can be an alkylating agent, an antimetabolite, such as methotrexate, azathioprine or mercaptopurine. The cytostatic agent can be at least one of cyclophosphamide, mechlorethamine, chlorambucil, melphalan, ifosfamide, thiotepa, hexamethylmelamine, busulfan, fludarabine, nitrosoureas, platinum, methotrexate, azathioprine, mercaptopurine, procarbazine, dacarbazine, temozolomide, carmustine, lomustine, streptozotocin, fluorouracil, actinomycin, anthracycline, mitomycin C, bleomycin and mithramycin.

[0311] In some cases, an immunosuppressant such as fludarabine may be administered as part of the treatment regimen. Fludarabine phosphate may be a synthetic purine nucleoside that differs from physiological nucleosides in that the sugar moiety may be arabinose rather than ribose or deoxyribose. Fludarabine may be a purine antagonist antimetabolite. Fludarabine may be provided as a white lyophilized solid cake of fludarabine phosphate powder in 50 mg vials. The pH of the solution may be 7.7 after reconstitution with 2 ml of sterile water for injection to a concentration of 25 mg / ml. Fludarabine powder is stable for at least 18 months at 2-8°C; after reconstitution, fludarabine is stable for at least 16 days at room temperature. Due to the absence of preservatives, reconstituted fludarabine may be administered within 8 hours. For specific compatibility information, please refer to the dedicated references. Fludarabine may be dephosphorylated in serum, transported intracellularly and converted to the nucleotide fludarabine triphosphate; this 2-fluoro-Ara-ATP molecule is thought to be necessary for the drug's cytotoxic effects. Fludarabine inhibits DNA polymerase, ribonucleotide reductase, DNA primase, and may interfere with chain extension and RNA and protein synthesis. Fludarabine can be administered as an intravenous infusion in 100 mL of 0.9% sodium chloride, USP, over 15 to 30 minutes. Dosages can be based on body surface area (BSA). If the patient is obese (BMI>35), actual body weight can be used to calculate the drug dose. In some cases, immunosuppressants such as fludarabine can be administered at about 20 mg / m 2 About 30 mg / m 2 In some cases, an immunosuppressant such as fludarabine may be administered at about 5 mg / m 2 About 10 mg / m 2 of the subject's body surface area, approximately 10 mg / m 2 About 15 mg / m 2 of the subject's body surface area, approximately 15 mg / m 2 About 20 mg / m 2 Body surface area: about 20 mg / m 2 About 25 mg / m 2of the subject's body surface area, approximately 25 mg / m 2 About 30 mg / m 2 of the subject's body surface area, about 30 mg / m 2 Up to about 40 mg / m 2 In some cases, immunosuppressants such as fludarabine can be administered at about 1 mg / m 2 , 2mg / m 2 , 3mg / m 2 , 4mg / m 2 , 5mg / m 2 , 6mg / m 2 , 7mg / m 2 , 8mg / m 2 , 9mg / m 2 , 10mg / m 2 , 11mg / m 2 , 12mg / m 2 , 13mg / m 2 , 14mg / m 2 , 15mg / m 2 , 16mg / m 2 , 17mg / m 2 , 18mg / m 2 , 19mg / m 2 , 20mg / m 2 , 21mg / m 2 , 22mg / m 2 , 23mg / m 2 , 24mg / m 2 , 25mg / m 2 , 26mg / m 2 , 27mg / m 2 , 28mg / m 2 , 29mg / m 2 , 30mg / m 2 , 31mg / m 2 , 32mg / m 2 , 33mg / m 2 , 34mg / m 2 , 35mg / m 2 , 36mg / m 2 , 37mg / m 2 , 38mg / m 2 , 39mg / m 2 , 40mg / m 2 41mg / m 2 42mg / m 2 43mg / m 2 44mg / m 2、45mg / m 2 、46mg / m 2 、47mg / m 2 、48mg / m 2 、49mg / m 2 、50mg / m 2 、51mg / m 2 、52mg / m 2 、53mg / m 2 、54mg / m 2 、55mg / m 2 、56mg / m 2 、57mg / m 2 、58mg / m 2 、59mg / m 2 、60mg / m 2 、61mg / m 2 、62mg / m 2 、63mg / m 2 、64mg / m 2 、65mg / m 2 、66mg / m 2 、67mg / m 2 、68mg / m 2 、69mg / m 2 、70mg / m 2 、71mg / m 2 、72mg / m 2 、73mg / m 2 、74mg / m 2 、75mg / m 2 、76mg / m 2 、77mg / m 2 、78mg / m 2 、79mg / m 2 、80mg / m 2 、81mg / m 2 、82mg / m 2 、83mg / m 2 、84mg / m 2 、85mg / m 2 、86mg / m 2 、87mg / m 2 、88mg / m 2 、89mg / m 2 、90mg / m 2 、91mg / m 2 、92mg / m 2 、93mg / m 2 、94mg / m 2, 95mg / m 2 , 96mg / m 2 , 97mg / m 2 , 98mg / m 2 , 99mg / m 2 , up to about 100 mg / m 2 In some cases, immunosuppressants such as fludarabine are dosed at 25 mg / m in 100 ml of 0.9% sodium chloride, USP. 2 , and infused over about 15 to about 30 minutes.

[0312] In some cases, immunosuppressants such as cyclophosphamide may be administered as part of the treatment regimen. Cyclophosphamide may be a nitrogen mustard derivative alkylating agent. After conversion to active metabolites in the liver, cyclophosphamide acts as an alkylating agent; the drug also has potent immunosuppressive activity. The serum half-life after intravenous injection is 3-12 hours; the drug and / or its metabolites may be detected in serum up to 72 hours after administration. Cyclophosphamide is stable for 24 hours at room temperature and for 6 days at 2-8°C after reconstitution with sterile water for injection as directed. Cyclophosphamide may be diluted in 250 mL of D5W and infused over 1 hour. Dosages may be based on the subject's weight. If the subject is obese (BMI>35), actual weight may be used to calculate drug doses, as described in. In some cases, the dosage of immunosuppressants such as cyclophosphamide can be about 1 mg / kg to about 3 mg / kg, about 3 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, 20 mg / KG to about 30 mg / KG, about 50 mg / kg to about 60 mg / kg, about 60 mg / kg to about 70 mg / kg, about 70 mg / kg to about 80 mg / kg, about 80 mg / kg to about 90 mg / kg, about 90 mg / kg to about 100 mg / kg. In some cases, the dosage of immunosuppressants such as cyclophosphamide exceeds 50 mg / kg of the subject.In some cases, an immunosuppressant such as cyclophosphamide may be administered at about 1 mg / kg, 2 mg / kg, 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, 9 mg / kg, 10 mg / kg, 11 mg / kg, 12 mg / kg, 13 mg / kg, 14 mg / kg, 15 mg / kg, 16 mg / kg, 17 mg / kg, 18 mg / kg, 19 mg / kg, 20 mg / kg, 21 mg / kg, 22 mg / kg, 23 mg / kg, 24 mg / kg, 25 mg / kg, 5mg / kg, 26mg / kg, 27mg / kg, 28mg / kg, 29mg / kg, 30mg / kg, 31mg / kg, 32mg / kg, 33mg / kg, 34mg / kg, 35mg / kg, 36mg / kg, 37mg / kg , 38mg / kg, 39mg / kg, 40mg / kg, 41mg / kg, 42mg / kg, 43mg / kg, 44mg / kg, 45mg / kg, 46mg / kg, 47mg / kg, 48mg / kg, 49mg / kg, 50mg / k g, 51mg / kg, 52mg / kg, 53mg / kg, 54mg / kg, 55mg / kg, 56mg / kg, 57mg / kg, 58mg / kg, 59mg / kg, 60mg / kg, 61mg / kg, 62mg / kg, 63mg / kg, 64mg / kg, 65mg / kg, 66mg / kg, 67mg / kg, 68mg / kg, 69mg / kg, 70mg / kg, 71mg / kg, 72mg / kg, 73mg / kg, 74mg / kg, 75mg / kg, 76m In some cases, immunosuppressants such as cyclophosphamide may be administered in an amount of about 100 mg / kg to about 300 mg / kg, 300 mg / kg to about 1 ...

[0313] The immunosuppressant may be, for example, a cyclophosphamide and fludarabine regimen. For example, a cyclophosphamide fludarabine regimen may be administered to a subject receiving engineered cell therapy. The cyclophosphamide fludarabine regimen may be administered at 60 mg / kg QD for 2 days and 25 mg / m 2 The chemotherapeutic regimen, such as cyclophosphamide fludarabine, can be administered 1 hour to 14 days before the engineered cells of the present disclosure are administered. The chemotherapeutic regimen can be administered at different doses. For example, the subject can receive a higher initial dose followed by a lower dose. The subject can receive a lower initial dose followed by a higher dose.

[0314] In some cases, the immunosuppressant may be an antibody. The antibody may be administered at a therapeutically effective dose. The antibody may be a polyclonal antibody or a monoclonal antibody. The polyclonal antibody that may be administered may be an anti-lymphocyte or anti-thymocyte antigen. The monoclonal antibody may be an anti-IL-2 receptor antibody, an anti-CD25 antibody, or an anti-CD3 antibody. Anti-CD20 antibodies may also be used. B cell ablation therapy, such as agents that react with CD20, such as rituximab, may also be used as an immunosuppressant.

[0315] The immunosuppressant may also be an anti-immunophilin. The anti-immunophilin may be cyclosporine, tacrolimus, everolimus or sirolimus. Other immunosuppressants may be interferons, such as IFN-β, opioids, anti-TNF binders, mycophenolate mofetil or fingolimod.

[0316] Immunosuppressants may also refer to radiotherapy drugs. Radiotherapy may include radiation. The whole body radiation dose is 12Gy. The radiation dose may include a cumulative dose of 12Gy to the whole body (including healthy tissue). The radiation dose may include from 5Gy to 20Gy. The radiation dose may be 5Gy, 6Gy, 7Gy, 8Gy, 9Gy, 10Gy, 11Gy, 12Gy, 13Gy, 14Gy, 15Gy, 16Gy, 17Gy, 18Gy, 19Gy or up to 20Gy. Radiation may be whole body radiation or local radiation. In the case where radiation is whole body radiation, it may be uniform or uneven. For example, when radiation may be uneven, a narrower area of ​​the body (such as the neck) may receive a higher dose than a wider area (such as the buttocks). For example, in one embodiment, the subject may receive standard treatment with high-dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, after transplantation, the subject receives an infusion of the expanded cells of the present disclosure (e.g., immune receptor programmed recipient cells). The dosage of the above treatment given to the patient can vary depending on the exact nature of the condition being treated and the recipient of the treatment. The scaling of the dosage for human administration can be performed according to practices acceptable in the art. For example, for adult patients, the dosage of CAMPATH can be in the range of 1 to about 100 mg, usually administered daily for 1 to 30 days. The daily dosage can be 1 to 10 mg per day, although larger doses of up to 40 mg per day can be used in some cases (described in U.S. Patent No. 6,120,766). Antibiotic preparations

[0317] Antibiotics can be given to a subject as part of a treatment regimen. Antibiotics can be administered at therapeutically effective doses. Antibiotics can kill bacteria or inhibit bacterial growth. Antibiotics can be broad-spectrum antibiotics against a variety of bacteria. Third-generation or fourth-generation broad-spectrum antibiotics can be cephalosporins or quinolones.

[0318] Antibiotics can also be narrow spectrum antibiotics, which can target specific types of bacteria. Antibiotics can target the bacterial cell wall, such as penicillins and cephalosporins. Antibiotics can target the cell membrane, such as polymyxins. Antibiotics can interfere with enzymes essential to bacteria, such as the antibiotics: rifamycin, ionomycin, quinolones, and sulfonamides. Antibiotics can also be protein synthesis inhibitors, such as macrolides, lincosamides, and tetracyclines. Antibiotics can also be cyclic lipopeptides such as daptomycin, glycylcyclines such as tigecycline, oxazolidinones such as linezolid, and ionomycins such as fidaxomicin.

[0319] In some cases, antibiotics can be the 1st, 2nd, 3rd, 4th or 5th generation. First generation antibiotics can have a narrow spectrum. Examples of first generation antibiotics can be penicillins (penicillin G or penicillin V), cephalosporins (cefazolin, cephalothin, cefepime, cefethyn, cephradine or cefuroxime). In some cases, antibiotics can be second generation. Second generation antibiotics can be penicillins (amoxicillin or ampicillin), cephalosporins (cefuroxime, cefmandol, cefoxitin, cefaclor, cefuroxime, loracarb). In some cases, antibiotics can be third generation. Third generation antibiotics can be penicillins (carbenicillin and ticarcillin) or cephalosporins (cefotaxime, ceftriaxone, cefotaxime, ceftizoxime and ceftazidime). Antibiotics can also be fourth generation antibiotics. Fourth generation antibiotics can be cefepime. Antibiotics can also be the 5th generation. Fifth-generation antibiotics may be ceftriaxone or cefoperazone.

[0320] In some cases, antibiotics can be bacterial wall targeting agents, cell membrane targeting agents, bacterial enzyme interfering agents, bactericides, protein synthesis inhibitors or antibacterial agents. Bacterial wall targeting agents can be penicillin derivatives (penicillanes), cephalosporins (cephems), monocyclic lactams and carbapenems. β-lactam antibiotics have bactericidal or antibacterial effects, and play a role by inhibiting the synthesis of the peptidoglycan layer of the bacterial cell wall. In some cases, antibiotics may be protein synthesis inhibitors. Protein synthesis inhibitors can be ampicillin, which is an irreversible inhibitor of transpeptidase, which is necessary for bacteria to make cell walls. It inhibits the third and final stage of bacterial cell wall synthesis in binary fission, ultimately leading to cell lysis. Therefore, ampicillin usually has a lytic effect. In some cases, bactericides can be cephalosporins or quinolones. In other cases, antibacterial agents are trimethoprim, sulfamethoxazole or pentamidine.

[0321] In some cases, agents for preventing PCP pneumonia may be administered. For example, trimethoprim and sulfamethoxazole can be used to prevent pneumonia. The dosage of trimethoprim and sulfamethoxazole (TMP / SMX; an example of a sulfonamide drug) can be 1 tablet three times a week, daily, at the time of or after the first administration of chemotherapy, on non-consecutive days, for at least about 6 months, until the CD4 count is greater than 200 in at least 2 consecutive laboratory studies. In some cases, the dosage of trimethoprim may be 160 mg. Trimethoprim may be administered from about 100 to about 300 mg. Trimethoprim may be administered from about 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, or up to about 300 mg. In some cases, the dosage of sulfamethoxazole is 800 mg. Sulfamethoxazole may be administered from about 500 mg to about 1000 mg. The dosage of sulfamethoxazole can be about 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg or up to about 1000 mg. In some cases, the TMP / SMX regimen can be administered in a therapeutically effective amount. TMP / SMX can be administered about 1 time to about 10 times a day. TMP / SMX can be administered 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times or up to about 20 times a day. In some cases, TMP / SMX can be administered once a week. For example, TMP / SMX can be administered once, twice, three times, four times, five times, six times, or up to about seven times per week. The TMP / SMX regimen can be administered on about day -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or up to about day 14 after administration of the cell therapy, as described herein, to the recipient cells.

[0322] In some cases, subjects with sulfa allergies can receive pentamidine. Pentamidine can be administered by aerosol. Pentamidine 300 mg / nebulizer within one week before admission, continued once a month until the CD4 count exceeds 200 in two consecutive laboratory follow-up studies and at least 6 months after chemotherapy. Pentamidine can be used to prevent the occurrence of PCP infection. It is available in a 300 mg lyophilized powder vial and can be administered by nebulizer. Pentamidine can be administered from about 300 mg to about 500 mg. In some cases, pentamidine can be administered from about 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg or up to about 800 mg.

[0323] In some cases, bacteriostatic agents such as antibiotics can be administered before, simultaneously with, or after the administration of immune receptor programmed recipient cells. In some cases, bacteriostatic agents can be administered about 14 days before the administration of immune receptor programmed recipient cells to about 6 months after the administration of these cells. Antiviral agents

[0324] In some cases, antiviral agents can be used as part of a treatment regimen. In some cases, herpes virus preventive agents can be given to subjects as part of a treatment regimen. The herpes virus preventive agent can be valacyclovir (Valtrex). Oral Valtrex can prevent herpes virus infection in HSV serologically positive subjects. It can be supplied in 500 milligrams of tablets. Valalacyclovir can be administered in a therapeutically effective amount. For example, valacyclovir can be from about 50mg, 75mg, 100mg, 125mg, 150mg, 175mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375mg, 400mg, 425mg, 450mg, 475mg, 500mg, 525mg, 550mg, 575mg, 600mg, 625mg, 650mg or up to about 700mg tablets. If the subject can tolerate oral intake, valacyclovir can be taken starting the day after the last dose of fludarabine at a dose of 500 mg orally daily. Antiviral therapy can be administered on about day -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or up to about day 14 after administration of the cell therapy.

[0325] In some cases, the subject may not be able to take oral medications for preventing herpes. In these cases, acyclovir can be used. Acyclovir can be provided in the form of powder injection, 500 mg per bottle. In some cases, acyclovir can be administered in a therapeutically effective amount. Acyclovir can be from about 50mg, 75mg, 100mg, 125mg, 150mg, 175mg, 200mg, 225mg, 250mg, 275mg, 300mg, 325mg, 350mg, 375mg, 400mg, 425mg, 450mg, 475mg, 500mg, 525mg, 550mg, 575mg, 600mg, 625mg, 650mg, or up to about 700 mg. Acyclovir can be administered once, twice, three times, four times, five times, six times or about seven times at most every day. Acyclovir can be administered on about day -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or up to about day 14 after administration of the cell therapy. In some cases, acyclovir can be administered intravenously. For example, the dosage of acyclovir can be 1mg / kg to about 3mg / kg, about 3mg / kg to about 5mg / kg, about 5mg / kg to about 10mg / kg, about 10mg / kg to about 20mg / kg, 20mg / kg to about 30mg / kg, about 30mg / kg to about 40mg / kg, about 40mg / kg to about 50mg / kg, about 50mg / kg to about 60mg / kg, about 60mg / kg to about 70mg / kg, about 70mg / kg to about 80mg / kg, about 80mg / kg to about 90mg / kg, about 90mg / kg to about 100mg / kg. In some cases, the dosage of acyclovir exceeds 50mg / kg. Acyclovir can be diluted to 50mg / mL concentration with 10mL sterile water for injection. The diluted solution should be used within 12 hours. The intravenous solution can be diluted to 7mg / mL or lower concentrations and infused within 1 hour to avoid kidney damage. Patient population

[0326] The modified cells described herein, synthetic TILs or pharmaceutical compositions comprising such cells can be applied to subjects in need thereof. Subjects can be patients with diseases or conditions. Subjects can be human subjects. In certain embodiments, patients or patient populations treated with modified cells, synthetic TILs or pharmaceutical compositions of the present disclosure have solid tumors. In certain embodiments, solid tumors are melanoma, renal cell carcinoma, lung cancer, bladder cancer, breast cancer, cervical cancer, colorectal cancer, head and neck cancer, colon cancer, gallbladder cancer, laryngeal cancer, liver cancer (e.g., hepatocellular carcinoma), thyroid cancer, gastric cancer, salivary gland cancer, prostate cancer, pancreatic cancer or Merkel cell carcinoma. In certain embodiments, patients or patient populations treated with pharmaceutical compositions of the present disclosure have blood cancers.

[0327] In various cases, the subject described herein may have received a first-line treatment for cancer prior to administering the compositions described herein. In some cases, the subject may have received two or more cancer treatments prior to administering the compositions described herein. The cancer may be a cancer that recurred after one or more front-line treatments. The cancer may be highly resistant or highly resistant to previous first-line or multi-line treatments.

[0328] Provided herein is a method for treating a subject in need after a first-line or multi-line existing therapy for treating cancer. The method may include administering to the subject a colony of modified cells described herein or synthetic TILs of one or more doses, or a pharmaceutical composition comprising such cells as described herein. In various embodiments, t...

Claims

1. A method for preparing a plurality of synthetic tumor infiltrating lymphocytes (TILs), the method comprising: (a) obtaining a resection or biopsy sample containing a plurality of T cells from a solid tumor lesion of a subject; (b) sequencing a plurality of T cells from the resection or biopsy sample to obtain naturally paired T cell receptor (TCR) α chain and β chain sequences of the TCRs of the plurality of T cells; (c) synthesizing a plurality of polynucleotide sequences encoding TCRs or subsets thereof from the naturally paired TCR α chain and β chain sequences obtained in (b), the polynucleotide sequences comprising a first polynucleotide sequence encoding a first naturally paired TCR of a first T cell among the plurality of T cells and a second polynucleotide sequence encoding a second naturally paired TCR of a second T cell among the plurality of T cells, wherein the first polynucleotide sequence is different from the second polynucleotide sequence; and (d) delivering the plurality of polynucleotide sequences or their derivatives into a plurality of recipient cells by contacting a mixture comprising the plurality of polynucleotide sequences or their derivatives with a plurality of recipient cells, thereby producing a plurality of synthetic TILs, wherein a first portion of the plurality of synthetic TILs exogenously expresses a first polynucleotide sequence encoding a first natural paired TCR, and a second portion of the plurality of synthetic TILs exogenously expresses a second polynucleotide sequence encoding a second natural paired TCR, and wherein the fraction of the third portion of the plurality of synthetic TILs that exogenously express the first and second polynucleotide sequences is less than the product of the fractions of the first portion and the second portion.

2. The method of claim 1, wherein the plurality of cells synthesizing TILs (i) exogenously express only one polynucleotide sequence among the plurality of polynucleotide sequences or (ii) do not exogenously express any one of the plurality of polynucleotide sequences or their derivatives.

3. The method of claim 1 or 2, wherein none of the plurality of synthetic TILs exogenously expresses the first and second polynucleotide sequences.

4. The method of any one of claims 1-3, wherein the cells of the plurality of synthetic TILs that exogenously express the first polynucleotide sequence do not exogenously express the second polynucleotide sequence.

5. The method of any one of claims 1-4, further comprising obtaining the expression levels of at least 10 genes in (b).

6. A method for preparing a plurality of synthetic tumor infiltrating lymphocytes (TILs), the method comprising: (a) obtaining a resection or biopsy sample containing a plurality of T cells from a solid tumor lesion of a subject; (b) sequencing a plurality of T cells from the resection or biopsy sample to obtain naturally paired T cell receptor (TCR) α chain and β chain sequences of the TCR of the plurality of T cells; (c) synthesizing a plurality of polynucleotide sequences encoding the TCR or a subset thereof from the naturally paired TCR α chain and β chain sequences obtained in (b), the polynucleotide sequences comprising a first polynucleotide sequence encoding a first naturally paired TCR of a first T cell among the plurality of T cells and a second polynucleotide sequence encoding a second naturally paired TCR of a second T cell among the plurality of T cells, wherein the first polynucleotide sequence is different from the second polynucleotide sequence; and (d) delivering the multiple polynucleotide sequences or their derivatives into multiple recipient cells by contacting a mixture comprising the multiple polynucleotide sequences or their derivatives with multiple recipient cells, thereby producing multiple synthetic TILs, wherein the cells of the multiple synthetic TILs that exogenously express the first polynucleotide sequence do not exogenously express the second polynucleotide sequence.

7. The method of claim 6, wherein a first portion of the plurality of synthetic TILs exogenously expresses the first polynucleotide sequence, and a second portion of the plurality of synthetic TILs exogenously expresses the second polynucleotide sequence, and wherein the fraction of a third portion of the plurality of synthetic TILs that exogenously express the first polynucleotide sequence and the second polynucleotide sequence is less than the product of the fractions of the first portion and the second portion.

8. The method according to claim 6 or 7, further comprising obtaining expression levels of at least 10 genes in (b).

9. A method for preparing a plurality of synthetic tumor infiltrating lymphocytes (TILs), the method comprising: (a) obtaining a resection or biopsy sample containing a plurality of T cells from a solid tumor lesion of a subject; (b) sequencing a plurality of T cells from the resection or biopsy sample to obtain (i) expression levels of at least 10 genes and (ii) naturally paired T cell receptor (TCR) α chain and β chain sequences of the TCR of the plurality of T cells; (c) synthesizing a plurality of polynucleotide sequences encoding the TCR or a subset thereof from the naturally paired TCR α chain and β chain sequences obtained in (b), including a first polynucleotide sequence encoding a first naturally paired TCR of a first T cell in the plurality of T cells and a second polynucleotide sequence encoding a second naturally paired TCR of a second T cell in the plurality of T cells, wherein the first polynucleotide sequence is different from the second polynucleotide sequence; and (d) delivering the multiple polynucleotide sequences or their derivatives into multiple recipient cells by contacting a mixture comprising the multiple polynucleotide sequences or their derivatives with multiple recipient cells, thereby producing multiple synthetic TILs.

10. The method of claim 9, wherein a first portion of the plurality of synthetic TILs exogenously expresses the first polynucleotide sequence, and a second portion of the plurality of synthetic TILs exogenously expresses the second polynucleotide sequence, and wherein the fraction of a third portion of the plurality of synthetic TILs that exogenously express the first polynucleotide sequence and the second polynucleotide sequence is less than the product of the fractions of the first portion and the second portion.

11. The method of claim 9 or 10, wherein the plurality of cells synthesizing TILs (i) exogenously express only one polynucleotide sequence among the plurality of polynucleotide sequences or (ii) do not exogenously express any one of the plurality of polynucleotide sequences or their derivatives.

12. The method of any one of claims 9-11, wherein none of the plurality of synthetic TILs exogenously expresses the first and second polynucleotide sequences.

13. The method according to any one of claims 9 to 12, wherein the plurality of synthetic TILs cells that exogenously express the first polynucleotide sequence do not exogenously express the second polynucleotide sequence.

14. The method of any one of claims 1-5, 7, 8, and 10-13, wherein the fraction of the first portion, the second portion, or the third portion is determined by sequencing or pMHC multimer staining.

15. The method of claim 14, wherein the sequencing comprises single-cell sequencing, and wherein the single-cell sequencing comprises barcoding each cell in a single compartment.

16. The method of claim 14, wherein the pMHC multimer staining comprises using a plurality of pMHC A multimer, each pMHC multimer specifically binds to a TCR encoded by a polynucleotide sequence of the multiple polynucleotide sequences or derivatives thereof described in (d).

17. The method according to any one of claims 1-16, wherein delivering the multiple polynucleotide sequences or their derivatives into the multiple recipient cells in (d) comprises site-directed knock-in of the polynucleotide sequences into a genomic site of a recipient cell among the multiple recipient cells.

18. The method of claim 17, wherein the genomic locus of the recipient cell results in monoallelic expression of the polynucleotide sequence.

19. The method of claim 17 or 18, wherein the polynucleotide sequence is site-specifically knocked into a genomic site of a recipient cell in the plurality of recipient cells in a manner consistent with the reading frame.

20. The method of any one of claims 17-19, wherein the genomic locus is located on a sex chromosome.

21. The method of any one of claims 17-20, wherein the genomic locus is T cell receptor alpha Constant region (TRAC) site.

22. The method of any one of claims 17 to 21, wherein: Site-directed knock-in of the polynucleotide sequence into the genomic site includes the use of a gene editing method selected from clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector (TALE) nucleases (TALENs), homing nucleases (meganuclease), zinc finger nucleases (ZFN) or megaTAL nuclease methods.

23. The method of claim 22, wherein site-directed knock-in of the polynucleotide sequence into a genomic site comprises using a CRISPR approach.

24. The method of claim 23, wherein the CRISPR method comprises delivering a ribonucleoprotein (RNP) complex comprising a Cas9 protein and a single guide RNA (sgRNA) into the recipient cell.

25. The method of claim 24, wherein the sgRNA targets the TRAC gene site.

26. The method of any one of claims 22-25, wherein the site-specific knock-in of the polynucleotide sequence into the genomic site comprises cutting the genomic site using the gene editing method and inserting the polynucleotide sequence into the genomic site by homologous recombination.

27. The method of any one of claims 1-26, wherein an endogenous TCR is inactivated in a receptor cell among the plurality of receptor cells.

28. The method of claim 27, wherein the endogenous TCR is knocked down or knocked out in the receptor cells of the plurality of receptor cells.

29. The method of any one of claims 1-28, wherein an endogenous T cell receptor beta constant region (TRBC) site is inactivated in the recipient cells of the plurality of recipient cells.

30. The method of claim 29, wherein the endogenous TRBC locus is knocked down or knocked out in the recipient cells of the plurality of recipient cells.

31. The method of any one of claims 1-30, wherein endogenous TCR is inactivated in at least about 80% of the plurality of receptor cells.

32. The method of claim 31, wherein the endogenous TCR is knocked down or knocked out in at least about 80% of the plurality of receptor cells.

33. The method of any one of claims 29-32, wherein the endogenous TCR is inactivated by using a gene editing method selected from clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector (TALE) nucleases (TALENs), homing endonucleases (meganuclease), zinc finger nuclease (ZFN), or megaTAL nuclease method.

34. The method of any one of claims 1-33, wherein the endogenous TCR is inactivated before, simultaneously with, or after delivering the plurality of polynucleotide sequences or derivatives thereof to the plurality of recipient cells in (d).

35. The method of claim 34, wherein in (d) delivering the plurality of polynucleotide sequences or derivatives thereof to the plurality of recipient cells simultaneously inactivates the endogenous TCR.

36. The method of any one of claims 33-35, wherein the endogenous TCR is inactivated using a CRISPR approach.

37. The method of claim 36, wherein the CRISPR method comprises delivering a ribonucleoprotein (RNP) complex comprising a Cas9 protein and a single guide RNA (sgRNA) into the recipient cell.

38. The method of claim 37, wherein the sgRNA comprises a sgRNA targeting the TRBC locus.

39. The method of any one of claims 35 to 38, wherein: The endogenous TCR is inactivated and a multinucleotide sequence is knocked into the genomic locus by delivering RNPs containing Cas9 protein and sgRNA, wherein the sgRNA includes an sgRNA targeting the TRBC locus and an sgRNA targeting the TRAC locus.

40. The method of any one of claims 1-39, wherein at least about 50% of the plurality of synthetic TILs exogenously express only a single copy of a polynucleotide sequence encoding a naturally-paired TCR of the TCR of the plurality of T cells.

41. The method of any one of claims 1-40, further comprising amplifying the plurality of synthetic TILs after (d).

42. The method of any one of claims 1-41, wherein the mixture comprises polynucleotide sequences encoding at least 5 different naturally paired TCRs, and wherein a unique TCR among the at least 5 different naturally paired TCRs has a first frequency in the mixture.

43. The method of claim 41 or 42, wherein after amplification, the unique TCR has a second frequency in the plurality of synthetic TILs.

44. The method according to claim 43, wherein the second frequency is between 0.1 and 10 times the first frequency. Times within.

45. The method according to any one of claims 42-44, wherein the polynucleotide sequences encoding the at least 5 different naturally paired TCRs are mixed in an equimolar ratio.

46. ​​The method of claim 45, wherein after amplification, each unique TCR is expressed in the plurality of synthetic TILs, or have no less than any other unique A ratio of 0.1 times or not more than 10 times the ratio of TCR.

47. The method of any one of claims 1-46, further comprising administering at least one cell of the plurality of synthetic TILs or its progeny into a subject having a solid tumor lesion.

48. The method of claim 47, wherein: Prior to administration, the plurality of synthetic TILs are not subjected to any selection or screening to determine the tumor reactivity of the plurality of synthetic TILs or a subset thereof.

49. The method according to any one of claims 1-47, further comprising determining the plurality of synthetic TILs of tumor reactivity.

50. The method of claim 49, wherein determining the tumor responsiveness comprises: (i) providing tumor cells or their derivatives from a subject; (ii) contacting the plurality of synthetic TILs with the tumor cells or derivatives thereof; (iii) determining the tumor reactivity of the plurality of synthetic TILs to the tumor cells or derivatives thereof; and (iv) determining whether to administer the plurality of synthetic TILs or a subset thereof to the subject.

51. The method of claim 49 or 50, further comprising administering at least one cell of the plurality of synthetic TILs or a progeny thereof to the subject having the solid tumor lesion.

52. The method according to any one of claims 5 and 8-51 further comprises, before synthesizing in (c), calculating a score using the expression levels of at least 3 genes out of the at least 10 genes obtained in (b); sorting the multiple T cells based on the score; and selecting at least two TCRs from the top 50% of the multiple T cells based on the score.

53. The method of claim 52, wherein the plurality of polynucleotide sequences encoding TCRs or subsets thereof synthesized in (c) include polynucleotide sequences encoding at least two selected TCRs.

54. The method of any one of claims 41, 48, and 51-53, wherein the time interval between obtaining the resection or biopsy sample and administering the at least one cell of the plurality of synthetic TILs to the subject is less than eight weeks.

55. The method of any one of claims 1-54, wherein the time interval between obtaining the resection or biopsy sample in (a) and delivering in (d) is no more than 7 weeks.

56. The method of any one of claims 1-54, wherein the plurality of synthetic TILs exogenously express at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000 or more different TCRs.

57. The method of any one of claims 41, 48, and 51-56, wherein two or more cells of the plurality of synthetic TILs or progeny thereof are administered to the subject, and the two or more cells administered exogenously express at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000 or more different TCRs.

58. The method of any one of claims 1-57, wherein the plurality of synthetic TILs comprises a subpopulation of cells expressing a TCR that recognizes a tumor antigen and a subpopulation of cells expressing a TCR that does not recognize a tumor antigen.

59. The method of any one of claims 1-58, wherein the volume of the resection or biopsy sample is less than about 100 mm 3 (μL), or the weight of the resection or biopsy sample is less than about 0.1 g.

60. The method of any one of claims 1-59, wherein the plurality of polynucleotide sequences or derivatives thereof are delivered to the plurality of recipient cells via a vector.

61. The method of claim 60, wherein the vector is a non-viral vector.

62. The method of claim 61, wherein the non-viral vector is a plasmid, a transposon, a self-amplifying RNA, or any combination thereof.

63. The method of claim 60, wherein the vector is a viral vector.

64. The method of claim 63, wherein the viral vector is an adenoviral vector, an adeno-associated viral (AAV) vector, a herpes simplex viral vector, a lentiviral vector, a retroviral vector, or any combination thereof.

65. The method of claim 64, wherein the viral vector is an AAV vector.

66. The method of any one of claims 60-65, wherein the vector does not comprise a promoter.

67. The method of any one of claims 1-64, wherein the plurality of recipient cells are a plurality of T cells.

68. The method of any one of claims 1-67, wherein the plurality of recipient cells are isolated from a subject.

69. The method of claim 68, wherein: The subject is the same as the subject from which the plurality of T cells in (a) are obtained.

70. The method of any one of claims 1-69, wherein the plurality of recipient cells are primary T cells.

71. The method of any one of claims 1-70, wherein the plurality of recipient cells are peripheral T cells.

72. The method of any one of claims 1-71, wherein the mixture comprises at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1,000 or more different polynucleotide sequences encoding at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1,000 or more different naturally paired TCRs.

73. The method of any one of claims 1-72, wherein the subject has failed prior line therapy.

74. The method of claim 73, wherein: The frontline therapy is checkpoint blockade therapy.

75. A method for preparing a plurality of synthetic tumor infiltrating lymphocytes (TILs), the method comprising: (a) obtaining a resection or biopsy sample containing a plurality of T cells from a solid tumor lesion of a subject; (b) synthesizing a plurality of polynucleotide sequences encoding the naturally paired TCRs or a subset thereof of the plurality of T cells obtained in (a), including a polynucleotide sequence encoding a first naturally paired TCR of a first T cell among the plurality of T cells; and a second polynucleotide sequence encoding a second naturally paired TCR of a second T cell in the plurality of T cells, wherein the first polynucleotide sequence is different from the second polynucleotide sequence; and (c) delivering the multiple polynucleotide sequences or their derivatives into multiple recipient cells by contacting a mixture comprising the multiple polynucleotide sequences or their derivatives with multiple recipient cells, thereby producing multiple synthetic TILs.

76. The method of claim 75, wherein: The method further comprises, before (b), sequencing the plurality of T cells of the resection or biopsy sample to obtain naturally paired T cell receptor (TCR) α chain and β chain sequences of the TCR of the plurality of T cells.

77. The method of claim 76, further comprising obtaining expression levels of at least 10 genes from the sequencing.

78. A method for preparing a plurality of synthetic tumor infiltrating lymphocytes (TILs), the method comprising: (a) delivering a plurality of polynucleotide sequences or derivatives thereof into a plurality of recipient cells by contacting a mixture comprising the plurality of polynucleotide sequences or derivatives thereof with a plurality of recipient cells, wherein each polynucleotide sequence in the plurality of polynucleotide sequences encodes a pair of homologous TCR α chain and TCR β chain, and wherein each polynucleotide sequence is knocked into a genomic site to achieve monoallelic expression of the polynucleotide sequence; and (b) the endogenous TCR gene of each receptor cell in the plurality of receptor cells is inactivated or the endogenous TCR gene of each receptor cell in the plurality of receptor cells has been inactivated.

79. The method of claim 78, wherein: wherein at least about 50% of the plurality of synthetic TILs exogenously express only a single copy of a polynucleotide sequence encoding a naturally-paired TCR of the TCR of the plurality of T cells.

80. The method of claim 78 or 79, wherein the genomic site is a TRAC site.

81. The method of any one of claims 78-80, wherein the endogenous TCR gene is inactivated by knocking down or knocking out the TRBC gene.

82. The method of any one of claims 78-81, wherein the mixture comprises at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1,000, or more different polynucleotide sequences encoding at least about 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1,000, or more different polynucleotide sequences. 1,000 or more different naturally paired TCRs.

83. The method of any one of claims 78-82, further comprising synthesizing the plurality of polynucleotide sequences encoding different homologous pairs of TCR alpha chains and TCR beta chains prior to (a).

84. The method of claim 83, wherein: The synthesizing comprises assembling the plurality of polynucleotide sequences encoding different cognate pairs of TCR alpha chains and TCR beta chains in the same mixture.

85. The method of claim 83, wherein: The synthesizing includes chemically synthesizing the plurality of polynucleotide sequences separately.

86. The method of any one of claims 78-85, wherein the endogenous TCR gene of each recipient cell in the plurality of recipient cells has been inactivated prior to delivery in (a).

87. The method according to any one of claims 78-85, wherein inactivating the endogenous TCR gene of each of the plurality of receptor cells in (b) is performed before, simultaneously with or after the delivery in (a).

88. The method as described in any one of claims 78-87 further comprises, before (a), obtaining a resection or biopsy sample containing multiple T cells from a solid tumor lesion of the subject, and sequencing the multiple T cells of the resection or biopsy sample to obtain naturally paired T cell receptor (TCR) α chain and β chain sequences of the TCR of the multiple T cells.

89. The method of claim 88, wherein: Also included is selecting a plurality of sequences encoding a plurality of naturally paired TCRs from the sequencing, wherein the selected plurality of sequences or a subset thereof are the plurality of polynucleotide sequences delivered in (a).

90. The method of any one of claims 78-89, wherein the inactivation in (b) is performed simultaneously with, before, or after the delivery in (a).

91. The method according to any one of claims 75-90, further comprising expanding the plurality of synthetic TILs cells.

92. The method of claim 91, wherein the frequency of unique TCRs in the mixture prior to delivery is within 0.1 to 10 times the frequency of unique TCRs in the plurality of synthetic TILs after expansion.

93. The method of any one of claims 75-77 and 88-92, further comprising administering to the subject at least one cell of the plurality of synthetic TILs or a progeny thereof.

94. The method of any one of claims 1-93, wherein after administration, the synthetic TILs persist in a subject in need thereof for at least about 30 days.

95. The method of claim 94, wherein after administration, the percentage of the synthetic TILs or their progeny in the subject's peripheral T cells is at least about 20%.

96. The method of claim 95, wherein the CD62L of the synthetic TILs or their progeny + The percentage of cells among the subject's peripheral T cells is at least about 50%.

97. The method of claim 95 or 96, wherein the percentage persists for at least about 30 days.

98. The method of any one of claims 94-97, wherein the concentration of the synthetic TILs in the peripheral blood sample of the subject is at least about 0.2×10 6 cells / mL peripheral blood.

99. The method of claim 98, wherein the concentration is sustained for at least about 30 days with or without IL-2 infusion.

100. A pharmaceutical composition comprising one or more synthetic TILs according to any one of claims 1-99 and a pharmaceutically acceptable carrier.

101. A mixture comprising at least 2, 5, 10, 15, 20, 25, 30, 50, 100, 200, 500 or 1000 modified cells, each modified cell in the mixture exogenously expressing a protein encoding a TCR α chain and a TCR The invention relates to a polynucleotide sequence for different homologous pairs of beta chains, wherein the polynucleotide sequence is knocked into a genomic locus to achieve monoallelic expression of the polynucleotide sequence, and wherein the endogenous TCR gene of the modified cell is inactivated.

102. A cell mixture comprising a plurality of modified cells, each of the plurality of modified cells exogenously expressing a polynucleotide sequence encoding a different homologous pair of TCR α chain and TCR β chain, wherein the polynucleotide sequence is knocked into a genomic site to achieve monoallelic expression of the polynucleotide sequence.

103. A mixture according to claim 102, wherein the endogenous TCR genes of the modified cells are inactivated.

104. A mixture according to any one of claims 101-103, wherein each different cognate pair of TCR alpha chain and TCR beta chain is from a subject with a solid tumor lesion or is endogenously expressed in a T cell from a subject with a solid tumor lesion, and wherein the subject is intended to administer the mixture of modified cells or a pharmaceutical composition comprising the mixture of modified cells.

105. The mixture according to any one of claims 101-104, wherein the mixture comprises at least 10 modified cells, each modified cell of the mixture exogenously expressing a polynucleotide sequence encoding a different homologous pair of TCR α chain and TCR β chain.

106. The mixture of any one of claims 101-105, wherein the genomic site is a TRAC site.

107. A mixture according to any one of claims 101-106, wherein the endogenous TCR gene of the modified cells is knocked out or knocked down.

108. The mixture of any one of claims 101-107, wherein the endogenous TCR genes comprise TRBC genes.

109. The mixture of any one of claims 101-108, wherein the modified cells are T cells.

110. The mixture of claim 109, wherein the T cells are allogeneic T cells or autologous T cells.

111. The mixture according to any one of claims 101-110, wherein the The TCRs of the cells are sequenced to select different cognate pairs of TCR alpha chains and TCR beta chains for each modified cell.

112. A mixture according to any one of claims 101-111, wherein different homologous pairs of TCR α chain and TCR β chain of each modified cell are selected by a pre-designed scoring method based on sequencing data of TCRs of multiple T cells from a subject.

113. The mixture of any one of claims 101-112, wherein a different cognate pair of TCR alpha chain and TCR beta chain of each modified cell has been demonstrated to be functional.

114. The mixture of any one of claims 101-113, wherein a different cognate pair of TCR alpha chain and TCR beta chain of each modified cell is tumor reactive.

115. A mixture according to any one of claims 101-110, wherein the mixture comprises functional TCRs and non-functional TCRs.

116. A mixture according to any one of claims 101-110, wherein the mixture comprises tumor-reactive TCRs and tumor-non-reactive TCRs.

117. The mixture of any one of claims 101-116, wherein the mixture of modified cells has been expanded in vitro.

118. A cell mixture comprising a plurality of modified cells, each of the plurality of modified cells exogenously expressing a cognate pair of TCRs from a subject, wherein the cell mixture expresses at least 10 different cognate pairs of TCRs, wherein each TCR of the at least 10 different cognate pairs of TCRs is expressed in at least 2% of the cell mixture, and wherein at least 50% of the cell mixtures each exogenously express only one cognate pair of TCRs.

119. The mixture of claim 118, wherein the subject is intended to receive administration of the mixture or a pharmaceutical composition comprising the mixture.

120. A mixture according to claim 118 or 119, wherein a polynucleotide sequence encoding a TCR homologous pair is knocked into a genomic site so that the polynucleotide sequence is monoallelically expressed in each modified cell of at least 50% of the mixture that only exogenously expresses one TCR homologous pair.

121. The mixture of any one of claims 101-120, wherein when administered to a subject, the modified cells survive in the subject for at least about 30 days.

122. A pharmaceutical composition comprising the mixture of any one of claims 101-121 and a pharmaceutically acceptable carrier.

123. A composition for preparing a medicament for treating a subject with cancer, comprising the mixture of claims 101-121.

124. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 100 or a pharmaceutical composition comprising the mixture of claims 101-121.

125. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject a pharmaceutical composition comprising (a) synthetic TILs produced according to the method of any one of claims 1-94 or a mixture of two or more synthetic TILs produced according to the method of any one of claims 1-94; and (B) a pharmaceutically acceptable carrier.

126. A method for treating cancer in a subject in need thereof, the method comprising administering a pharmaceutical composition comprising a cell mixture to the subject in need thereof, wherein the cell mixture exogenously expresses at least 10 different TCR homology pairs from the subject, and wherein at least 50% of the cell mixture each exogenously expresses only one TCR homology pair.

127. The method of claim 126, wherein the cell mixture is a mixture of modified cells.

128. The method of claim 126, wherein the cell mixture comprises a plurality of modified cells and a plurality of unmodified cells.

129. The method according to any one of claims 126-128, wherein: The at least 10 different TCRs Each TCR of a cognate pair is expressed in at least 2% of the modified cell mixture.

130. The method of any one of claims 126-129, wherein the polynucleotide sequence is knocked into a genomic locus for monoallelic expression of the polynucleotide sequence.

131. The method of any one of claims 126-130, wherein the subject from which the TCR is obtained is the same as the subject to be treated.

132. The method of any one of claims 126-131, wherein the endogenous TCR of the modified cell The gene is inactivated.

133. The method of claim 126 or 132, wherein the modified cell is a T cell.

134. The method of claim 133, wherein: The T cells are CD4 + T cells, CD8 + T cells, CD4 + / CD8 + T cell, helper T cell, cytotoxic T cell, memory T cell, regulatory T cell, natural killer T cell, αβ T cell or γδ T cell.

135. The method of claim 133 or 134, wherein the T cells are allogeneic T cells or autologous T cells. cell.

136. The method of any one of claims 126-135, wherein the modified cells are isolated from a subject to which the composition is administered.

137. The method of any one of claims 126-136, wherein each unique cognate pair of modified cells is derived from a subject in need thereof or is endogenously expressed by a T cell from the subject in need thereof.

138. The method of any one of claims 126-137, wherein the mixture comprises at least 10 modified cells.

139. The method of any one of claims 126-138, wherein the genomic site is a TRAC site.

140. The method of any one of claims 126-139, wherein the endogenous The TCR gene is knocked out or knocked down.

141. The method of any one of claims 126-140, wherein the endogenous TCR gene comprises TRBC locus.

142. The method of any one of claims 126-141, wherein: The subject in need thereof has received one or more prior lines of treatment or has failed one or more prior lines of treatment.

143. The method of any one of claims 126-142, wherein the cancer is metastatic or refractory.

144. The method of claim 143, wherein the cancer is a solid tumor.

145. The method of any one of claims 126-144, further comprising administering IL-2 to the subject after administering the pharmaceutical composition.

146. The method of any one of claims 126-145, wherein prior to administration, the cell mixture is tested for tumor reactivity.

147. The method of any one of claims 127-146, wherein the mixture of modified cells persists in the subject for at least about 30 days.

148. The method of claim 147, wherein after administration, the percentage of the modified cells or their progeny in the subject's peripheral T cells is at least about 20%.

149. The method of claim 148, wherein the CD62L of the synthetic TILs or their progeny + The percentage of cells among the subject's peripheral T cells is at least about 50%.

150. The method of claim 148 or 149, wherein the percentage persists for at least about 30 days.

151. The method of any one of claims 147-150, wherein the concentration of the synthetic TILs in the peripheral blood sample of the subject is at least about 0.2×10 6 cells / mL peripheral blood.

152. The method of claim 151, wherein the concentration is sustained for at least about 30 days with or without IL-2 infusion.

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