Methods and compositions for modulating arginine concentration in immune cells

Abstract

To provide a method and composition for modulating arginine concentration in immune cells. [Solution] Genetically modified T cells and CAR-T cells are provided in which the ability to process the essential amino acid arginine is increased, for example, by overexpressing an amino acid transporter, particularly an arginine transporter. Genetically modified T cells and CAR-T cells can often survive well in the harsh tumor microenvironment because of their increased ability to process arginine. Methods and compositions are also provided for providing CAR-T cells that can be effective in treating solid tumors by increasing the amount of arginine available to T cells and thus surviving in the tumor microenvironment.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefits and priority of U.S. Provisional Application No. 62 / 979,805, filed on 21 February 2020, the entirety of which is incorporated herein by reference. Sequence List (0001.1) This application includes an electronically submitted sequence listing in ASCII format, the entirety of which is incorporated herein by reference. The ASCII copy, created on 18 February 2021, is named SKP-001WO_SL.txt and has a size of 312,657 bytes. Technical field

[0002] The present invention relates to compositions and methods for modulating arginine levels in immune cells, for example, to extend cell survival in the tumor microenvironment. [Background technology]

[0003] Chimeric antigen receptor (CAR) T-cell therapy has emerged as a major breakthrough in cancer treatment. In CAR-T therapy, a patient's T cells are harvested and genetically engineered to produce CARs that bind to pre-selected specific antigens, such as transmembrane receptors on cancer cells. The CAR-T cells are then reintroduced into the patient's body, enabling them to attack designated targets, such as cancer cells. When the CAR receptor binds to its target antigen, the CAR-T cell is activated and initiates an immune response against the cell presenting the target antigen. CAR-T cell therapy has induced successful patient responses and, in some cases, remission in patients who had previously failed to respond to standard treatments. For example, in some forms of leukemia, CAR-T therapy has shown remission rates as high as 94%.

[0004] Existing CAR-T cell therapies are currently approved for use only in hematological malignancies. Currently, there are two FDA-approved CAR-T cell therapies on the market: Kymriah (tisagenlecleucel) and Yescarta (axicabutagensilolucel), which are used in several specialized research hospitals to treat hematological malignancies. These treatments have resulted in complete and long-term remission in several subjects, including those with cancers that were previously resistant to standard treatment regimens.

[0005] Previous CAR developments focused on targeting the B lymphocyte antigen CD19, a transmembrane protein that recruits cytoplasmic signaling proteins to the membrane and lowers the threshold of the B cell receptor signaling pathway. Due to these required functions, CD19 is ubiquitous on all B cells and is used as a biomarker for malignancies originating from B cells, particularly B-cell lymphoma, acute lymphoblastic leukemia, and chronic lymphocytic leukemia. Other domains targeted by CAR-T therapies include CD22, a sugar-binding transmembrane protein found on the surface of mature B cells; CD123, an interleukin-3 signaling molecule expressed across acute myeloid leukemia subtypes; and B cell maturation antigens, cell surface receptors of the tumor necrosis factor receptor superfamily that recognize B cell activators associated with various leukemias, lymphomas, and multiple myeloma.

[0006] The tumor microenvironment (TME) of solid tumors is harsh for all effector T cells, including engineered CAR-T cells. Immunosuppressive signals and deficiencies in essential nutrients within the TME lead to T cell exhaustion. Therefore, the ability of CAR-T cells to invade and function within the TME remained limited.

[0007] Therefore, there is a need for CAR-T cells and pharmaceutical compositions containing CAR-T cells that are resistant to TME challenges and capable of functioning in the destruction of cancer cells within TMEs. There is also a need for methods of effectively treating cancer with CAR-T cells, as well as pharmaceutical compositions containing CAR-T cells that are effective in inducing patient responsiveness and remission, in patients who are resistant to other forms of cancer treatment or other methods of treatment with CAR-T cells. Furthermore, there is a need for methods of treating cancer by administering superior CAR-T cells that are effective in destroying cancer cells within TMEs without becoming exhausted. [Overview of the Initiative] [Means for solving the problem]

[0008] This disclosure relates, at least in part, to T cells expressing amino transporter proteins, such as arginine transporter proteins, and CARs that specifically bind to cell surface antigens on target cells. Such CAR-T cells are useful for treating malignant tumors such as cancer. The genetically modified T cells and expression vectors described herein may have enhanced robustness and / or survival in the tumor microenvironment and resource-depleted microenvironments, such as arginine-depleted microenvironments, compared to, for example, non-genetically modified T cell populations. The genetically modified T cells and expression vectors described herein are useful for treating cancers and other diseases that require the targeting of T cells to specific cell populations.

[0009] In another aspect, this disclosure relates to genetically modified T cells expressing an amino acid transporter, for example, an arginine transporter. The amino acid transporter may be the product of a recombinant amino acid transporter nucleotide sequence. T cells described herein, genetically modified to express or overexpress an amino acid transporter, may have improved robustness and / or viability in the tumor microenvironment and resource depletion, such as arginine depletion, compared to T cell populations that are not genetically modified to express or overexpress an amino acid transporter, for example. The genetically modified T cells and expression vectors described herein are useful for treating cancers and other diseases that require the targeting of T cells to a specific cell population, or that require enhanced robustness of T cells to survive in a biological environment depleted of one or more amino acids, such as arginine.

[0010] In one embodiment, genetically modified T cells are disclosed herein that express an arginine transporter and a chimeric antigen receptor (CAR). In some embodiments, the CAR has at least one antigen-specific targeting region that specifically binds to a cell surface antigen present on a target cell population, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR has at least an antigen-specific targeting region that specifically binds to a cell surface antigen present on a target cell population, a transmembrane domain, at least one costimulatory domain, and an intracellular signaling domain.

[0011] Expression vectors comprising nucleotide sequences encoding CARs and / or amino acid transporters, such as arginine transporters, are also described herein. In some embodiments, transcription of the expression vectors described herein results in the production of messenger RNA (mRNA) sequences encoding ribonucleic acid (RNA), such as CARs and / or amino acid transporters, such as arginine transporter nucleotide sequences. In some embodiments, the expression vectors described herein can express CARs and / or amino acid transporters, such as arginine transporters. In some embodiments, expression vectors comprising isolated nucleic acids encoding an antigen-specific targeting region, a transmembrane domain, optionally at least one costimulatory domain, an intracellular signaling domain, and an arginine transporter are described herein. In some embodiments, expression vectors comprising isolated nucleic acids encoding an antigen-specific targeting region, a transmembrane domain, optionally at least one costimulatory domain, and an intracellular signaling domain are described herein. In some embodiments described herein, the expression vector comprises an isolated nucleic acid sequence encoding an arginine transporter. In some embodiments, expression vectors comprising isolated nucleic acids encoding an antigen-specific targeting region, a transmembrane domain, optionally at least one costimulatory domain, an intracellular signaling domain, and an amino acid transporter are described herein. In some embodiments described herein, the expression vector comprises an isolated nucleic acid sequence encoding an amino acid transporter.

[0012] Expression vectors comprising nucleotide sequences encoding amino acid transporters, such as arginine transporters, are also described herein. In some embodiments, transcription of the expression vectors described herein results in the production of messenger RNA (mRNA) sequences encoding ribonucleic acid (RNA), such as amino acid transporters, such as arginine transporter nucleotide sequences. In some embodiments, the expression vectors described herein can express amino acid transporters, such as arginine transporters. In some embodiments described herein, the expression vector comprises an isolated nucleic acid sequence encoding an arginine transporter. In some embodiments described herein, the expression vector comprises an isolated nucleic acid sequence encoding an arginine transporter. In some embodiments described herein, the expression vector comprises an isolated nucleic acid sequence encoding an amino acid transporter. In some embodiments described herein, the expression vector comprises an isolated nucleic acid sequence encoding an amino acid transporter. In some embodiments described herein, the nucleic acid sequence may be, for example, a ribonucleic acid (RNA) sequence, a deoxyribonucleic acid (DNA) sequence, or a mixed DNA and RNA sequence.

[0013] In some embodiments, the expression vector described herein comprises nucleotide sequences encoding a CAR and an amino acid transporter, and the CAR and amino acid transporter nucleotide sequences are transcribed into separate mRNA transcripts. In some embodiments, the expression vector described herein comprises nucleotide sequences encoding a CAR and an amino acid transporter, and the CAR and amino acid transporter nucleotide sequences are transcribed together into a single mRNA transcript. In embodiments where the CAR and amino acid transporter nucleotide sequences are transcribed together into a single mRNA transcript, the expression vector nucleotide sequences encoding the CAR and amino acid transporter mRNA transcripts may include an internal ribosome entry sequence (IRES). In some embodiments, the IRES is positioned between the portion of the nucleotide sequence encoding the CAR and the portion of the nucleotide sequence encoding the amino acid transporter. Thus, in embodiments, the CAR nucleotide sequence and the amino acid transporter nucleotide sequence are separated by the IRES sequence. In embodiments where the CAR and amino acid transporter nucleotide sequences are transcribed together into a single mRNA transcript, the expression vector nucleotide sequence encoding the CAR and the amino acid transporter mRNA transcript may include a 2A self-cleavage sequence positioned between the portion of the nucleotide sequence encoding the CAR and the portion of the nucleotide sequence encoding the amino acid transporter. Therefore, in some embodiments, the CAR nucleotide sequence and the amino acid transporter nucleotide sequence are separated by the 2A autocleavage sequence. In some embodiments, the peptide translated from mRNA containing the CAR nucleotide sequence, the 2A autocleavage sequence, and the amino acid transporter nucleotide sequence is cleaved at the 2A autocleavage site post-translation.

[0014] Genetically modified T cells modified to express a CAR encoded by an expression vector are also described herein. Genetically modified T cells modified to express a CAR encoded by an expression vector and an amino acid transporter, such as an arginine transporter, are also described herein. Genetically modified T cells modified to express an amino acid transporter, such as an arginine transporter, encoded by an expression vector are also described herein. Genetically modified T cells modified to express a CAR encoded by a first expression vector and an amino acid transporter, such as an arginine transporter, encoded by a second expression vector are also described herein. In the embodiments described herein, the CAR encoded by the expression vector may include an antigen-specific targeting region, a transmembrane domain, optionally at least one costimulatory domain, and an intracellular signaling domain. Genetically modified T cells modified to express an amino acid transporter, such as an arginine transporter, encoded by an expression vector may include genetically modified T cells modified to express a recombinant amino acid transporter, such as a recombinant arginine transporter.

[0015] Genetically modified T cells that have been modified to express a CAR encoded by a virus-derived transgene are also described herein. Genetically modified T cells that have been modified to express a CAR encoded by a virus-derived transgene and an amino acid transporter, such as an arginine transporter, are also described herein. Genetically modified T cells that have been modified to express an amino acid transporter, such as an arginine transporter, encoded by a virus-derived transgene are also described herein. Genetically modified T cells that have been modified to express a CAR encoded by a first virus-derived transgene and an amino acid transporter, such as an arginine transporter, encoded by a second virus-derived transgene are also described herein. In the embodiments described herein, the CAR encoded by a virus-derived transgene may include an antigen-specific targeting region, a transmembrane domain, optionally at least one costimulatory domain, and an intracellular signaling domain. Genetically modified T cells that have been modified to express an amino acid transporter, such as an arginine transporter, encoded by a virus-derived transgene may include genetically modified T cells that have been modified to express a recombinant amino acid transporter, such as a recombinant arginine transporter.

[0016] The genetically modified T cells described herein can express specific arginine transporters. In some embodiments, the arginine transporter protein comprises a single arginine transporter protein. In some embodiments, the arginine transporter protein comprises two arginine transporter proteins. For example, the genetically modified T cells described herein express CAT-1, CAT-2, CAT-3, CAT-4, y + LAT1, 4F2hc, y + LAT2, y + LAT1 and 4F2hc, y + LAT2 and 4F2hc, b 0,+ AT, rBAT, b 0,+ AT and rBAT, as well as ATB 0,+It may express an arginine transporter selected from the group consisting of or combinations thereof.

[0017] In some embodiments, the expression vectors described herein comprise an isolated nucleic acid sequence encoding an arginine transporter. In some embodiments, the expression vectors described herein comprise two or more isolated nucleic acid sequences encoding proteins that together comprise an arginine transporter. In some embodiments, the arginine transporter nucleic acid sequence(s) is / are selected from the group consisting of CAT-1, CAT-2, CAT-3, CAT-4, y + LAT1, 4F2hc, y + LAT2, y + LAT1 and 4F2hc, y + LAT2 and 4F2hc, b 0,+ AT, rBAT, b 0,+ AT and rBAT, and ATB 0,+ or nucleic acid sequence(s) of combinations thereof.

[0018] In some embodiments, the virus-derived transgenes described herein comprise an isolated nucleic acid sequence encoding an arginine transporter. In some embodiments, the virus-derived transgenes described herein comprise two or more isolated nucleic acid sequences encoding proteins that together comprise an arginine transporter. In some embodiments, the arginine transporter nucleic acid sequence(s) is / are selected from the group consisting of CAT-1, CAT-2, CAT-3, CAT-4, y + LAT1, 4F2hc, y + LAT2, y + LAT1 and 4F2hc, y + LAT2 and 4F2hc, b 0,+ AT, rBAT, b 0,+ AT and rBAT, and ATB 0,+ or are selected from the group consisting of nucleic acid sequence(s) of combinations thereof.

[0019] Expression vectors containing nucleic acid sequences encoding amino acid transporter sequences, and genetically modified T cells containing recombinant nucleic acid sequences encoding amino acid transporters are also described herein. For example, expression vectors containing nucleic acid sequences, or fragments or variants thereof, selected from the group consisting of SEQ ID NOs: 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246 are described herein. Expression vectors containing any one nucleotide sequence of SEQ ID NOs: 220-222 and any one nucleotide sequence of SEQ ID NOs: 227-230 are also described herein. Expression vectors containing any one nucleotide sequence of SEQ ID NOs: 214 and 215, and any one nucleotide sequence of SEQ ID NOs: 227-230 are also described herein. Expression vectors containing any one nucleotide sequence of SEQ ID NOs: 234-236 and the nucleotide sequence of SEQ ID NO: 242 are also described herein.

[0020] Genetically modified T cells containing recombinant nucleic acid sequences, or fragments or variants thereof, containing sequences selected from the group consisting of SEQ ID NOs: 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246 are also described herein. Genetically modified T cells containing recombinant nucleic acids containing any one nucleotide sequence of SEQ ID NOs: 220-222 and any one nucleotide sequence of SEQ ID NOs: 227-230 are also described herein. Genetically modified T cells containing recombinant nucleic acids containing any one nucleotide sequence of SEQ ID NOs: 214 and 215, and any one nucleotide sequence of SEQ ID NOs: 227-230 are also described herein. Genetically modified T cells containing recombinant nucleic acids containing any one nucleotide sequence of SEQ ID NOs: 234-236 and the nucleotide sequence of SEQ ID NO: 242 are also described herein.

[0021] In some embodiments, the genetically modified T cells described herein are genetically modified to include a recombinant nucleic acid sequence, or a fragment or variant thereof, comprising a sequence selected from the group consisting of SEQ ID NOs: 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246. For example, in some embodiments, the genetically modified T cells described herein are modified to include one or more additional copies of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246, or a fragment or variant thereof. Genetically modified T cells comprising an expression vector containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246, or a fragment or variant thereof, are also described herein. In some embodiments, an expression vector, genetically modified T cell, or genetically modified T cell comprising an expression vector described herein comprises a combination of nucleic acid sequences selected from the group consisting of SEQ ID NOs: 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246, or a fragment or variant thereof. In some embodiments described herein, an expression vector, a genetically modified T cell containing a recombinant nucleic acid sequence, a genetically modified T cell modified to contain a recombinant nucleic acid sequence, a genetically modified T cell modified to contain one or more additional copies of a nucleic acid sequence, or a genetically modified T cell containing an expression vector containing a nucleic acid sequence comprises at least two nucleic acid sequences, or fragments or variants thereof, selected from the group consisting of SEQ ID NOs: 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246.For example, in some embodiments described herein, an expression vector, a genetically modified T cell containing a recombinant nucleic acid sequence, a genetically modified T cell modified to contain a recombinant nucleic acid sequence, a genetically modified T cell modified to contain one or more additional copies of a nucleic acid sequence, or a genetically modified T cell containing an expression vector containing a nucleic acid sequence comprises one of the following nucleotide sequence pairs: one nucleotide sequence of any of SEQ ID NOs. 220-222 and one nucleotide sequence of any of SEQ ID NOs. 227-230; one nucleotide sequence of any of SEQ ID NOs. 214 and 215, and one nucleotide sequence of any of SEQ ID NOs. 227-230; one nucleotide sequence of any of SEQ ID NOs. 234-236 and the nucleotide sequence of SEQ ID NOs. 242.

[0022] The genetically modified T cells described herein may contain recombinant nucleic acid sequences that share similarity with any of the nucleic acid sequences SEQ ID NOs: 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246. For example, in some embodiments, the genetically modified T cells described herein include nucleic acid sequences having an identity percentage of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, about 90% to about 95%, about 95% to about 99%, or about 90% to about 99% with one of sequence numbers 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246. In some embodiments, the genetically modified T cells described herein contain nucleic acid sequences having approximately 90%, 95%, or 99% identity with one of sequence numbers 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246. In some embodiments, the genetically modified T cells described herein include an expression vector comprising a nucleic acid sequence having an identity percentage of about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, about 90% to about 95%, about 95% to about 99%, or about 90% to about 99% with one of SEQ ID NOs: 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246. In some embodiments, the genetically modified T cells described herein include an expression vector comprising a nucleic acid sequence having approximately 90%, 95%, or 99% identity with one of SEQ ID NOs: 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246.

[0023] In another embodiment, a pharmaceutically acceptable composition comprising genetically modified T cells and pharmaceutically acceptable excipients described herein is described herein.

[0024] A priming medium containing L-arginine for priming genetically modified T cells, such as the genetically modified T cells described herein, is also described herein. The priming medium described herein can increase the intracellular arginine concentration in genetically modified T cells, such as genetically modified T cells expressing an arginine transporter. The priming medium described herein can be used to prime genetically modified T cells for treatment. For example, the priming medium described herein can increase the intracellular arginine concentration in genetically modified T cells before administering them to a patient who needs them, such as a patient who needs cancer treatment. In some embodiments described herein, the priming medium contains the genetically modified T cells and L-arginine described herein.

[0025] Pharmaceutical compositions containing CAR-T cells are also described herein. For example, pharmaceutical compositions containing CAR-T cells expressing recombinant arginine transporter and chimeric antigen receptor protein are described herein. In some embodiments, the pharmaceutical compositions of the present invention include CAR-T cells, each containing one or more expression vectors comprising nucleic acid sequences encoding arginine transporter and / or chimeric antigen receptor protein. In some embodiments, the pharmaceutical compositions described herein include CAR-T cells expressing arginine transporter. In some embodiments, the pharmaceutical compositions of the present invention include CAR-T cells, each containing one or more recombinant nucleic acid sequences encoding arginine transporter and / or chimeric antigen receptor protein. In some embodiments, the pharmaceutical compositions described herein include CAR-T cells expressing arginine transporter, for example, recombinant protein arginine transporter. In various embodiments, the arginine transporter is CAT-1, CAT-2, CAT-3, CAT-4, y + LAT1, 4F2hc, y + LAT2, y + LAT1 and 4F2hc, y + LAT2 and 4F2hc, b0,+ AT, rBAT, b 0,+ AT and rBAT, as well as ATB 0,+ , or a combination thereof, is selected from the group. In some embodiments, one or more nucleic acid sequences encoding an arginine transporter are one or more recombinant arginine transporter nucleic acid sequences, e.g., recombinant CAT-1 nucleic acid sequence, recombinant CAT-2 nucleic acid sequence, recombinant CAT-3 nucleic acid sequence, recombinant CAT-4 nucleic acid sequence, recombinant y + LAT1 nucleic acid sequence, recombinant 4F2hc nucleic acid sequence, recombinant Y + LAT2 nucleic acid sequence, recombinant Y + LAT1 nucleic acid sequence and recombinant 4F2hc nucleic acid sequence, recombinant y + LAT2 nucleic acid sequence and recombinant 4F2hc nucleic acid sequence, recombinant b 0,+ AT nucleic acid sequence, recombinant rBAT nucleic acid sequence, recombinant b 0,+ AT nucleic acid sequence and recombinant rBAT nucleic acid sequence, or recombinant ATB 0,+ It includes a nucleic acid sequence. In some embodiments, the arginine transporter protein is a recombinant arginine transporter protein, e.g., recombinant CAT-1, recombinant CAT-2, recombinant CAT-3, recombinant CAT-4, recombinant y + LAT1, recombinant 4F2hc, recombinant y + LAT2, recombinant y + LAT1 and recombinant 4F2hc, recombinant y + LAT2 and recombinant 4F2hc, recombinant b 0,+ AT, recombinant rBAT, recombinant b 0,+ AT and recombinant rBAT, or recombinant ATB 0,+ That is the case.

[0026] In other embodiments, the pharmaceutical compositions described herein are packaged as kits. For example, in some embodiments, a pharmaceutical composition comprising CAR-T cells expressing an arginine transporter and a chimeric antigen receptor protein (e.g., genetically modified CAR-T cells expressing an arginine transporter and a chimeric antigen receptor protein) is packaged as a kit. In some embodiments, a pharmaceutical composition comprising T cells expressing an arginine transporter (e.g., genetically modified T cells expressing an arginine transporter, e.g., recombinant arginine transporter protein) is packaged as a kit. The kits described herein may include instructions for administering the CAR-T cells to a patient in need of treatment. The kits described herein may include instructions for priming the CAR-T cells for administration to a patient in need of treatment. In some embodiments, the kit may include at least one buffer (e.g., a buffer containing a sufficient level of L-arginine to prime the T cells), reagents, and detailed instructions for producing, administering, and / or priming the CAR-T cells. In some embodiments, the kit described herein may include an activator for producing CAR-T cells, comprising an expression vector, a viral construct, cells, transfection reagents and culture media, an activator for cell selection (e.g., an antibody) and / or growth medium.

[0027] Methods for treating cancer using the pharmaceutical compositions described herein are also described herein. For example, a method for treating a solid tumor cancer in a patient requiring treatment of the solid tumor cancer is described herein, comprising administering an effective amount of the pharmaceutical composition described herein to the patient. For example, a method for treating a solid tumor cancer in a patient requiring treatment of the solid tumor cancer is described herein, comprising administering an effective amount of the pharmaceutical composition described herein, comprising administering to the patient a genetically modified T cell (e.g., CAR-T cell or T cell genetically modified to express an amino acid transporter) and a pharmaceutically acceptable excipient.

[0028] Methods for treating hematological cancers using the pharmaceutical compositions described herein are also described herein. For example, a method for treating hematological cancers in a patient requiring treatment is described herein, comprising administering an effective amount of the pharmaceutical composition described herein to the patient. For example, a method for treating hematological cancers in a patient requiring treatment is described herein, comprising administering an effective amount of the pharmaceutical composition described herein, comprising administering to the patient an effective amount of the pharmaceutical composition comprising genetically modified T cells (e.g., CAR-T cells or genetically modified T cells expressing amino acid transporters) and pharmaceutically acceptable excipients.

[0029] Methods for modulating intracellular arginine levels (e.g., intracellular T cell arginine levels) to induce a T cell-mediated immune response in patients requiring treatment are also described herein. For example, a method for modulating intracellular arginine levels to induce a T cell-mediated immune response in patients requiring modification of intracellular arginine levels to induce a T cell-mediated immune response is described herein, which includes modulating the intracellular arginine levels of genetically modified T cells. In some embodiments, a method for modulating intracellular arginine levels to achieve a T cell-mediated immune response in patients requiring modification of intracellular arginine levels to achieve a T cell-mediated immune response further includes administering to the patient an effective amount of the pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising genetically modified T cells described herein and pharmaceutically acceptable excipients, wherein the genetically modified T cells are subjected to conditions effective in increasing intracellular arginine levels). In some embodiments, a method for modulating intracellular arginine levels to induce a T cell-mediated immune response in a patient requiring such modulation comprises modulating the intracellular arginine levels of genetically modified T cells and administering to the patient an effective amount of a pharmaceutical composition comprising the genetically modified T cells and a pharmaceutically acceptable excipient.

[0030] In yet another embodiment, a method for treating a condition in a human patient requiring treatment of the condition is described herein, comprising administering to the human patient a composition comprising a therapeutically effective amount of CAR-T cells expressing an arginine transporter (e.g., recombinant arginine transporter) and a chimeric antigen receptor protein (e.g., genetically modified CAR-T cells). In some embodiments, a method for treating a condition in a human patient requiring treatment of the condition comprises administering to the human patient a composition comprising a therapeutically effective amount of the CAR-T cells described herein, e.g., genetically modified CAR-T cells described herein. For example, in some embodiments, a method for treating a condition in a human patient requiring treatment of the condition comprises administering to the human patient a composition comprising a therapeutically effective amount of CAR-T cells, the CAR-T cells comprising one or more recombinant nucleic acid sequences encoding an arginine transporter and / or a chimeric antigen receptor protein. In some embodiments described herein, a method for treating a condition in a human patient requiring treatment of the condition comprises administering to the human patient a therapeutically effective amount of a composition comprising genetically modified T cells genetically modified to express or overexpress an amino acid transporter, e.g., an arginine transporter.

[0031] Methods for modulating a T cell-mediated immune response to a target cell population expressing a cell surface antigen in patients requiring modulation of such a response include administering a therapeutically effective dose of genetically modified T cells to the patient. In some embodiments, the T cells are genetically modified to a) express a chimeric antigen receptor, the chimeric antigen receptor comprising at least one antigen-specific targeting region that specifically binds to a cell surface antigen present on the target cell population, a transmembrane domain, and an intracellular signaling domain, and b) express an arginine transporter (e.g., recombinant arginine transporter). For example, in some embodiments, T cells for administration include one or more recombinant nucleic acid sequences encoding a chimeric antigen receptor, the chimeric antigen receptor comprising at least one antigen-specific targeting region that specifically binds to cell surface antigens present on the target cell population, a transmembrane domain, an intracellular signaling domain, and an arginine transporter. In some embodiments, T cells for administration include a recombinant chimeric antigen receptor protein, the recombinant chimeric antigen receptor protein comprising at least one antigen-specific targeting region that specifically binds to cell surface antigens present on the target cell population, a transmembrane domain, an intracellular signaling domain, and a recombinant arginine transporter protein. In some embodiments, T cells for administration include one or more recombinant nucleic acid sequences encoding an arginine transporter. In some embodiments, T cells for administration include a recombinant arginine transporter protein.

[0032] In another embodiment, the present disclosure relates to a method for increasing T cell survival in a low-arginine environment, comprising administering T cells comprising a recombinant arginine transporter to a low-arginine environment. In certain embodiments, the method comprises transfecting the T cells with a DNA construct comprising a nucleotide sequence encoding the recombinant arginine transporter prior to the administration step. In certain embodiments, the T cells comprise a DNA construct comprising a chimeric antigen receptor and / or a nucleotide sequence encoding the chimeric antigen receptor. In certain embodiments, the T cells are CAR-T cells. In certain embodiments, prior to the administration step, the method comprises culturing the T cells or CAR-T cells in a culture medium containing arginine until, for example, the intracellular arginine level of the T cells or CAR-T cells accumulates to a certain level. In certain embodiments, the low-arginine environment is a cell culture medium. In certain embodiments, the low-arginine environment is a tumor microenvironment.

[0033] In embodiments described herein, the disclosed methods may include the step of culturing T cells in a culture medium containing arginine before administration (e.g., administration to a patient in need of treatment). For example, a method for treating a condition in a human patient in need of treatment is described herein, which includes culturing T cells in a culture medium containing arginine before administering a composition containing a therapeutically effective amount of T cells to the human patient. Also described herein is a method for modulating a T cell-mediated immune response to a target cell population expressing a cell surface antigen in a patient in need of modulation of a T cell-mediated immune response to a target cell population expressing a cell surface antigen, which includes culturing genetically modified T cells in a culture medium containing arginine before administering a therapeutically effective amount of T cells to the patient.

[0034] In the method described herein, the arginine transporter is CAT-1, CAT-2, CAT-3, CAT-4, y + LAT1, 4F2hc, y + LAT2, y +LAT1 and 4F2hc, y + LAT2 and 4F2hc, b 0,+ AT, rBAT, b 0,+ AT and rBAT, as well as ATB 0,+ Selected from a group consisting of, or combinations thereof. For example, a method for treating a condition in a human patient requiring treatment of the condition, comprising CAR-T cells expressing a chimeric antigen receptor protein and CAT-1, CAT-2, CAT-3, CAT-4, y + LAT1, 4F2hc, y + LAT2, y + LAT1 and 4F2hc, y + LAT2 and 4F2hc, b 0,+ AT, rBAT, b 0,+ AT and rBAT, as well as ATB 0,+ This specification describes a method comprising administering to a human patient a therapeutically effective amount of a composition comprising a chimeric antigen receptor and an arginine transporter (e.g., a recombinant arginine transporter) selected from the group consisting of , or a combination thereof. A method for modulating a T cell-mediated immune response against a target cell population expressing a cell surface antigen in a patient requiring modulation of such a response, comprising a chimeric antigen receptor and CAT-1, CAT-2, CAT-3, CAT-4, y + LAT1, 4F2hc, y + LAT2, y + LAT1 and 4F2hc, y + LAT2 and 4F2hc, b 0,+ AT, rBAT, b 0,+ AT and rBAT, as well as ATB 0,+ Methods also described herein include administering a therapeutically effective amount of T cells genetically modified to express an arginine transporter (e.g., recombinant arginine transporter) selected from the group consisting of , or combinations thereof. In some embodiments, the arginine transporter protein is recombinant arginine transporter protein, e.g., recombinant CAT-1, recombinant CAT-2, recombinant CAT-3, recombinant CAT-4, recombinant y+ LAT1, recombinant 4F2hc, recombinant y + LAT2, recombinant y + LAT1 and recombinant 4F2hc, recombinant y + LAT2 and recombinant 4F2hc, recombinant b 0,+ AT, recombinant rBAT, recombinant b 0,+ AT and recombinant rBAT, or recombinant ATB 0,+ In certain embodiments, the arginine transporter comprises a nucleic acid sequence, or a fragment or variant thereof, selected from the group consisting of SEQ ID NOs: 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246. In certain embodiments, the arginine transporter comprises a nucleic acid expressing a sequence having approximately 90%, 95%, or 99% identity with one of SEQ ID NOs: 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246.

[0035] In some embodiments, the method further includes the step of administering a second therapeutic agent to a human patient. For example, a method for treating a condition in a human patient requiring treatment of the condition is described herein, comprising administering a composition containing a therapeutically effective amount of CAR-T cells and administering a second therapeutic agent to the human patient. In some embodiments, the method described herein includes administering the second therapeutic agent before, during, or after administering the composition containing CAR-T cells. Also described herein is a method for modulating a T cell-mediated immune response to a target cell population expressing a cell surface antigen in a patient requiring modulation of the T cell-mediated immune response to a target cell population expressing a cell surface antigen, comprising administering a therapeutically effective amount of genetically modified T cells to the patient and administering a second therapeutic agent to the human patient. In some embodiments, the method described herein includes administering the second therapeutic agent before, during, or after administering a composition containing a therapeutically effective amount of T cells.

[0036] In some embodiments, the second therapeutic agent is a checkpoint protein inhibitor, for example, a checkpoint protein inhibitor that inhibits checkpoint protein activity or checkpoint protein signaling, for example, an antibody that inhibits checkpoint protein or checkpoint protein signaling. For example, in some embodiments, the second therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody. In some embodiments, the second therapeutic agent is a DNA damage and repair inhibitor. For example, in some embodiments, the DNA damage and repair inhibitor is an ATM / ATR inhibitor, a PARP inhibitor, a WEE1 inhibitor, a Chk1 inhibitor, a Chk2 inhibitor, or a DNA-dependent protein kinase (DNA-PK) inhibitor.

[0037] In embodiments described herein, a composition comprising CAR-T cells is administered to a human patient once every week, once every two weeks, once every three weeks, or once every four weeks. For example, a method for treating a condition in a human patient who requires treatment of the condition is described herein, comprising administering a therapeutically effective amount of a composition comprising CAR-T cells to the human patient once every week, once every two weeks, once every three weeks, or once every four weeks. Also described herein is a method for modulating a T cell-mediated immune response to a target cell population expressing a cell surface antigen in a patient who requires modulation of the T cell-mediated immune response to a target cell population expressing a cell surface antigen, comprising administering a therapeutically effective amount of genetically modified T cells to the patient once every week, once every two weeks, once every three weeks, or once every four weeks.

[0038] In some embodiments, the methods described herein include administering a specified number of CAR-T cells based on the patient's body weight, or a specified range of CAR-T cells based on the patient's body weight. In some embodiments, the methods described herein include administering about 10 per kilogram of the patient. 2 pieces, about 10 3 pieces, about 104 cells, about 10 5 cells, about 10 6 cells, about 10 7 cells, about 10 8 cells, about 10 9 cells, about 10 10 cells, about 10 11 cells, about 10 12 cells, about 10 13 cells, about 10 14 cells, about 10 15 cells, about 10 16 cells, about 10 17 cells, about 10 18 cells, about 10 19 cells, about 10 20 cells, about 10 25 cells, about 10 30 cells, about 10 35 cells, about 10 40 cells, about 10 45 cells, or about 10 50 cells of CAR-T cells. In some embodiments, the methods described herein are about 10 2 ~10 7 cells, about 10 2 ~10 10 cells, about 10 3 ~10 10 cells, about 10 4 ~10 10 cells, about 10 5 ~10 10 cells, about 10 6 ~10 10 cells, about 10 7 ~10 10 cells, about 10 8 ~10 11 cells, about 10 9 ~10 12 cells, about 10 10 ~10 13 cells, about 10 7 ~10 15 cells, about 10 5 ~10 15 cells, about 10 10 ~10 20 cells, about 10 10 ~10 25 cells, about 10 10 ~10 30 cells, about 10 7 ~1020 pieces, about 10 7 ~10 25 pieces, about 10 10 ~10 50 10, or about 10 7 ~10 50 This involves administering 10 CAR-T cells per kilogram of patient. For example, in some embodiments, the method described herein involves administering about 10 CAR-T cells per kilogram of patient. 7 ~10 10 This includes administering individual CAR-T cells.

[0039] Embodiments described herein include a method for producing genetically modified CAR-T cells expressing an arginine transporter, comprising transfecting T cells with a DNA construct containing nucleotide sequences for a specific chimeric antigen receptor and an arginine transporter to produce genetically modified CAR-T cells expressing both the chimeric antigen receptor and the arginine transporter, and culturing the genetically modified CAR-T cells in a culture medium containing arginine. Embodiments described herein also include a method for producing genetically modified CAR-T cells expressing an arginine transporter, comprising transducing T cells with a virus containing a nucleotide construct containing nucleotide sequences for a specific chimeric antigen receptor and an arginine transporter to produce genetically modified CAR-T cells expressing both the chimeric antigen receptor and the arginine transporter, and culturing the genetically modified CAR-T cells in a culture medium containing arginine. In some embodiments, the genetically modified CAR-T cells express a recombinant arginine transporter nucleotide sequence. In some embodiments, genetically modified CAR-T cells express recombinant arginine transporter protein. In some embodiments, culturing involves culturing the genetically modified CAR-T cells in culture medium until the intracellular arginine levels of the CAR-T cells accumulate to a certain level. In some embodiments, the intracellular arginine level of the CAR-T cells is the intracellular arginine level that allows the CAR-T cells to survive in the tumor microenvironment. For example, in some embodiments, culturing involves culturing genetically modified CAR-T cells in culture medium until the intracellular arginine levels of the CAR-T cells reach approximately 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1,000 μM, 1,100 μM, 1,200 μM, 1,300 μM, 1,400 μM, 1,500 μM, 1,600 μM, 1,700 μM, 1,800 μM, 1,900 μM, 2,000 μM, 2,500 μM, 3,000 μM, 3,500 μM, or 4,000 μM.In some embodiments, culturing involves culturing genetically modified CAR-T cells in culture medium until the intracellular arginine levels of the CAR-T cells reach approximately 500 μM to 1,000 μM, 800 μM to 1,200 μM, 1,000 μM to 1,500 μM, 1,000 μM to 2,000 μM, 1,500 μM to 2,000 μM, 700 μM to 900 μM, 900 μM to 1,100 μM, 900 μM to 1,200 μM, or 1,300 μM to 1,500 μM.

[0040] The embodiments described herein also include a method for producing genetically modified T cells expressing an arginine transporter, comprising transfecting T cells with a DNA construct containing a nucleotide sequence for an arginine transporter to produce genetically modified T cells expressing an arginine transporter, and culturing the genetically modified T cells in a culture medium containing arginine. The embodiments described herein also include a method for producing genetically modified T cells expressing an arginine transporter, comprising transfecting T cells with a virus containing a nucleotide construct containing a nucleotide sequence for an arginine transporter to produce genetically modified T cells expressing an arginine transporter, and culturing the genetically modified T cells in a culture medium containing arginine. In some embodiments, culturing comprises culturing the genetically modified T cells in a culture medium until the intracellular arginine level of the T cells accumulates to a certain level. In some embodiments, the intracellular arginine level of the T cells is the intracellular arginine level that allows the T cells to survive in a tumor microenvironment or an arginine-depleted environment. For example, in some embodiments, culturing involves culturing genetically modified T cells in culture medium until the intracellular arginine levels of the T cells reach approximately 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1,000 μM, 1,100 μM, 1,200 μM, 1,300 μM, 1,400 μM, 1,500 μM, 1,600 μM, 1,700 μM, 1,800 μM, 1,900 μM, 2,000 μM, 2,500 μM, 3,000 μM, 3,500 μM, or 4,000 μM. In some embodiments, culturing involves culturing genetically modified T cells in culture medium until the intracellular arginine levels of the T cells reach approximately 500 μM to 1,000 μM, 800 μM to 1,200 μM, 1,000 μM to 1,500 μM, 1,000 μM to 2,000 μM, 1,500 μM to 2,000 μM, 700 μM to 900 μM, 900 μM to 1,100 μM, 900 μM to 1,200 μM, or 1,300 μM to 1,500 μM. The present invention provides, for example, the following items: (Item 1) Genetically modified T cells, a) Recombinant arginine transporter, and b) Genetically modified T cells that express a chimeric antigen receptor having at least one antigen-specific targeting region that specifically binds to a cell surface antigen present on a target cell population, a transmembrane domain, and an intracellular signaling domain. (Item 2) An expression vector comprising a) an antigen-specific targeting region, b) a transmembrane domain, c) optionally at least one costimulatory domain, d) an intracellular signaling domain, and e) an isolated nucleic acid encoding an arginine transporter. (Item 3) Genetically modified T cells that have been modified to express a chimeric antigen receptor encoded by the expression vector described in item 2. (Item 4) The genetically modified T cell described in item 1 or 3, wherein the arginine transporter is selected from the group consisting of CAT-1, CAT-2, CAT-3, CAT-4, y+LAT1 and 4F2hc, y+LAT2 and 4F2hc, b0,+AT and rBAT, and ATB0,+. (Item 5) The genetically modified T cells described in any one of items 1, 3, or 4, comprising a nucleic acid sequence, or a fragment or variant thereof, selected from the group consisting of SEQ ID NOs: 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246. (Item 6) The genetically modified T cells according to item 1, 3, or 4, wherein the genetically modified T cells contain nucleic acids expressing a sequence having approximately 90%, 95%, or 99% identity with one of sequence numbers 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246. (Item 7) A pharmaceutically acceptable composition comprising genetically modified T cells as described in item 1 or any one of items 3 to 6, and a pharmaceutically acceptable excipient. (Item 8) A priming medium comprising genetically modified T cells as described in item 1 or any one of items 3-6, and L-arginine. (Item 9) A pharmaceutical composition comprising a recombinant arginine transporter and chimeric antigen receptor T cells (CAR-T cells) expressing a chimeric antigen receptor protein. (Item 10) The pharmaceutical composition according to item 9, wherein the arginine transporter is CAT-1. (Item 11) The pharmaceutical composition according to item 9, wherein the arginine transporter is CAT-2. (Item 12) The pharmaceutical composition according to item 9, wherein the arginine transporter is CAT-3. (Item 13) The pharmaceutical composition according to item 9, wherein the arginine transporter is CAT-4. (Item 14) The pharmaceutical composition according to item 9, wherein the arginine transporters are y+LAT1 and 4F2hc. (Item 15) The pharmaceutical composition according to item 9, wherein the arginine transporters are y+LAT2 and 4F2hc. (Item 16) The pharmaceutical composition according to item 9, wherein the arginine transporter is b0,+AT and rBAT. (Item 17) The pharmaceutical composition according to item 9, wherein the arginine transporter is ATB0,+. (Item 18) The pharmaceutical composition according to item 9, wherein the pharmaceutical composition is packaged as a kit. (Item 19) A method for treating a solid tumor in a patient requiring treatment for a solid tumor, comprising administering an effective amount of the pharmaceutical composition described in item 7 or 9 to the patient. (Item 20) A method for treating hematological cancer in a patient requiring treatment for hematological cancer, comprising administering an effective amount of the pharmaceutical composition described in item 7 or 9 to the patient. (Item 21) A method for modulating intracellular arginine levels in a patient who requires modulation of intracellular arginine levels to achieve a T cell-mediated immune response, comprising administering an effective amount of the pharmaceutical composition described in item 7 or 9 to the patient. (Item 22) A method for treating a condition in a human patient requiring treatment, comprising administering to the human patient a therapeutically effective amount of a composition comprising a recombinant arginine transporter and chimeric antigen receptor T cells (CAR-T cells) expressing a chimeric antigen receptor protein. (Item 23) A method for modulating a T cell-mediated immune response against a target cell population expressing a cell surface antigen in a patient who requires modulation of such a response, comprising administering to the patient a therapeutically effective dose of T cells, wherein a) the T cells are genetically modified to express a chimeric antigen receptor, the chimeric antigen receptor comprising at least one antigen-specific targeting region that specifically binds to the cell surface antigen present on the target cell population, a transmembrane domain, and an intracellular signaling domain, and b) the T cells are genetically modified to express a recombinant arginine transporter. (Item 24) The method according to item 22 or 23, wherein the T cells are cultured in a culture medium containing arginine before administration. (Item 25) The method according to any one of items 22 to 24, wherein the arginine transporter is CAT-1. (Item 26) The method according to any one of items 22 to 24, wherein the arginine transporter is CAT-2. (Item 27) The method according to any one of items 22 to 24, wherein the arginine transporter is CAT-3. (Item 28) The method according to any one of items 22 to 24, wherein the arginine transporter is CAT-4. (Item 29) The arginine transporters are y+LAT1 and 4F2hc, item 22 The method described in any one of the following 24 items. (Item 30) The method according to any one of items 22 to 24, wherein the arginine transporters are y+LAT2 and 4F2hc. (Item 31) The method according to any one of items 22 to 24, wherein the arginine transporter is b0,+AT and rBAT. (Item 32) The method according to any one of items 22 to 24, wherein the arginine transporter is ATB0,+. (Item 33) The method according to any one of items 22-24, comprising administering T cells as described in item 1 and any one of items 3-6. (Item 34) The method according to any one of items 22 to 33, further comprising administering a second therapeutic agent to the human patient. (Item 35) The method according to item 34, wherein the second therapeutic agent is an anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibody. (Item 36) The method according to item 34 or 35, wherein the administration of the second therapeutic agent is performed before, during, or after the administration of the composition containing the CAR-T cells. (Item 37) The method according to item 34 or 35, wherein the administration of the second therapeutic agent is performed before, during, or after the administration of the therapeutically effective amount of T cells. (Item 38) The method according to any one of items 22 to 37, wherein the composition comprising the CAR-T cells is administered to the human patient once every week, once every two weeks, once every three weeks, or once every four weeks. (Item 39) The method according to any one of items 22 to 37, comprising administering 107 to 1010 CAR-T cells per kilogram of the patient. (Item 40) A method for producing genetically modified CAR-T cells that express recombinant arginine transporters, Transfecting T cells with a DNA construct containing nucleotide sequences for a specific chimeric antigen receptor and an arginine transporter, thereby producing genetically modified CAR-T cells that express both the chimeric antigen receptor and the arginine transporter, and The genetically modified CAR-T cells are cultured in a culture medium containing arginine. Methods that include... (Item 41) The method according to item 40, wherein the culturing includes culturing the genetically modified CAR-T cells in the culture medium until the intracellular arginine level of the CAR-T cells accumulates to a certain level. (Item 42) Genetically modified T cells that have been genetically altered to express recombinant arginine transporters. (Item 43) The aforementioned arginine transporters are CAT-1, CAT-2, CAT-3, CAT-4 Genetically modified T cells as described in item 42, selected from the group consisting of y+LAT1 and 4F2hc, y+LAT2 and 4F2hc, b0,+AT and rBAT, and ATB0,+. (Item 44) The genetically modified T cells described in item 42 or 43, comprising a nucleic acid sequence, or a fragment or variant thereof, selected from the group consisting of SEQ ID NOs: 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246. (Item 45) The genetically modified T cells according to item 42 or 43, wherein the genetically modified T cells contain nucleic acids expressing a sequence having approximately 90%, 95%, or 99% identity with one of sequence numbers 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246. (Item 46) A pharmaceutically acceptable composition comprising genetically modified T cells as described in any one of items 42 to 45 and a pharmaceutically acceptable excipient. (Item 47) A priming medium containing genetically modified T cells as described in any one of items 42-45, and L-arginine. (Item 48) A pharmaceutical composition containing T cells expressing recombinant arginine transporter protein. (Item 49) The pharmaceutical composition according to item 48, wherein the arginine transporter is CAT-1. (Item 50) The pharmaceutical composition according to item 48, wherein the arginine transporter is CAT-2. (Item 51) The pharmaceutical composition according to item 48, wherein the arginine transporter is CAT-3. (Item 52) The pharmaceutical composition according to item 48, wherein the arginine transporter is CAT-4. (Item 53) The pharmaceutical composition according to item 48, wherein the arginine transporters are y+LAT1 and 4F2hc. (Item 54) The pharmaceutical composition according to item 48, wherein the arginine transporters are y+LAT2 and 4F2hc. (Item 55) The pharmaceutical composition according to item 48, wherein the arginine transporter is b0,+AT and rBAT. (Item 56) The pharmaceutical composition according to item 48, wherein the arginine transporter is ATB0,+. (Item 57) The pharmaceutical composition according to item 48, wherein the pharmaceutical composition is packaged as a kit. (Item 58) A method for treating a solid tumor in a patient requiring treatment for a solid tumor, comprising administering an effective amount of the pharmaceutical composition described in item 46 or 48 to the patient. (Item 59) A method for treating hematological cancer in a patient requiring treatment for hematological cancer, comprising administering an effective amount of the pharmaceutical composition described in item 46 or 48 to the patient. (Item 60) A method for modulating intracellular arginine levels in a patient who requires modulation of intracellular arginine levels to achieve a T cell-mediated immune response, comprising administering an effective amount of the pharmaceutical composition described in item 46 or 48 to the patient. (Item 61) A method for treating a condition in a human patient requiring treatment, comprising administering to the human patient a therapeutically effective amount of a composition containing T cells expressing recombinant arginine transporters. (Item 62) The method according to item 61, wherein the T cells are cultured in a culture medium containing arginine before administration. (Item 63) The method according to item 61 or 62, wherein the arginine transporter is CAT-1. (Item 64) The method according to item 61 or 62, wherein the arginine transporter is CAT-2. (Item 65) The method according to item 61 or 62, wherein the arginine transporter is CAT-3. (Item 66) The method according to item 61 or 62, wherein the arginine transporter is CAT-4. (Item 67) The method according to item 61 or 62, wherein the arginine transporters are y+LAT1 and 4F2hc. (Item 68) The method according to item 61 or 62, wherein the arginine transporters are y+LAT2 and 4F2hc. (Item 69) The method according to item 61 or 62, wherein the arginine transporters are b0,+AT and rBAT. (Item 70) The method according to item 61 or 62, wherein the arginine transporter is ATB0,+. (Item 71) The method described in item 61 or 62, comprising administering T cells as described in any one of items 42 to 45. (Item 72) The method according to any one of items 58 to 71, further comprising administering a second therapeutic agent to the human patient. (Item 73) The method according to item 72, wherein the second therapeutic agent is an anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibody. (Item 74) The method according to item 72 or 73, wherein the administration of the second therapeutic agent is performed before, during, or after the administration of the composition containing the T cells. (Item 75) The administration of the second therapeutic agent is performed before and during the administration of the therapeutically effective amount of T cells. or the method described in item 72 or 73, performed after the administration. (Item 76) A method for producing genetically modified T cells that express recombinant arginine transporters, Transfecting T cells with a DNA construct containing a nucleotide sequence for an arginine transporter, thereby producing genetically modified T cells that express the arginine transporter, and The genetically modified T cells are cultured in a culture medium containing arginine. Methods that include... (Item 77) The method according to item 76, wherein the culturing includes culturing the genetically modified T cells in the culture medium until the intracellular arginine level of the T cells accumulates to a certain level. (Item 78) A method for increasing T cell survival in a low-arginine environment, comprising administering T cells containing a recombinant arginine transporter to a low-arginine environment. (Item 79) The method according to item 78, comprising transfecting the T cells with a DNA construct comprising a nucleotide sequence encoding the recombinant arginine transporter prior to the administration step. (Item 80) The method according to item 78 or item 79, wherein the T cells comprise a DNA construct comprising a chimeric antigen receptor and / or a nucleotide sequence encoding the chimeric antigen receptor. (Item 81) The method according to any one of items 78 to 80, comprising culturing the T cells in a culture medium containing arginine prior to the administration step. (Item 82) The method according to item 81, wherein the culturing includes culturing the T cells in the culture medium until the intracellular arginine level of the T cells accumulates to a certain level. (Item 83) The method according to any one of items 78 to 82, wherein the arginine transporter is selected from the group consisting of CAT-1, CAT-2, CAT-3, CAT-4, y+LAT1 and 4F2hc, y+LAT2 and 4F2hc, b0,+AT and rBAT, and ATB0,+. (Item 84) The method according to any one of items 78 to 82, wherein the DNA construct comprising the nucleotide sequence encoding the recombinant arginine transporter comprises a nucleic acid sequence, or a fragment or variant thereof, selected from the group consisting of SEQ ID NOs: 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246. (Item 85) The DNA construct comprising a nucleotide sequence encoding the recombinant arginine transporter comprises a nucleic acid expressing a sequence having approximately 90%, 95%, or 99% identity with one of SEQ ID NOs: 180, 184-188, 204, 205, 210, 214, 215, 220-222, 227-230, 234-236, 242, and 246, or the method according to any one of items 78-82. (Item 86) The method according to any one of items 78 to 85, wherein the low-arginine environment is a cell culture medium. (Item 87) The method according to any one of items 78 to 85, wherein the low-arginine environment is the tumor microenvironment. [Brief explanation of the drawing]

[0041] [Figure 1] Figure 1 is a map of the pBCTex01G expression vector. Figure 1 discloses "(G4S)3" as sequence number 30.

[0042] [Figure 2] Figure 2 is a map of the pBCTex02mini expression vector.

[0043] [Figure 3A] Figure 3A is a schematic diagram showing the transfection and arginine depletion steps of the experiment described in Example 1.

[0044] [Figure 3B] Figure 3B is a schematic diagram showing the cell filtering and counting steps of the experiment described in Example 1.

[0045] [Figure 3C] Figure 3C is a set of graphs showing the estimated change in the percentage of cells transfected with an expression construct (control, CAT, or ASS) after 72 hours in an arginine-rich environment (left) or an arginine-depleted environment (right). Each data point represents the estimated percentage change in the number of cells in one isolated well of independently transfected cells.

[0046] [Figure 4] Figure 4 is a set of graphs showing the estimated change in percentage of primary human T cells transfected with control (mNeonGreen) or CAT (arginine transporter) mRNA in control (top) or arginine-depleted (bottom) medium. After 24 hours in arginine-rich medium, an increase in cell percentage was observed in both GFP control (approximately 100%) and CAT (approximately 200%) transfected cells. In contrast, in arginine-depleted medium, a net decrease was observed in GFP control cells, but an increase of approximately 15% was observed in cells transfected with CAT mRNA. [Modes for carrying out the invention]

[0047] definition As used herein, the term “chimeric antigen receptor” (CAR) generally refers to a genetically engineered receptor designed to bind to a specific antigen, such as an antigen presented on the surface of cancer cells. CARs can be introduced into immune cells to help them identify and kill cancer cells that express the specific antigen.

[0048] As used herein, the terms “T lymphocyte” or “T cell” generally refer to a type of immune cell distinguished from other lymphocytes by the presence of T cell receptors on its cell surface. Differentiated T cells play many important roles, including regulating and shaping the immune response through several immune-related functions (e.g., immune-mediated cell death), recruiting cells to initiate the immune response via cytokines, determining whether and how other parts of the immune system respond to certain recognized threats, influencing regulatory B cells, and, among other functions, distinguishing foreign cells from themselves.

[0049] As used herein, the term “costimulatory signaling region” refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are necessary for the efficient response of lymphocytes to an antigen. Examples of costimulatory signaling molecules include CD28, ICOS (CD278), 4-1BB (CD137), OX40 (CD134), CD27, CD40, CD40L, TLR (e.g., TLR2), DAP10, IL-2RB, IL-2RA, and MYD88.

[0050] As used herein, the term “CAR-T cell therapy” generally refers to genetically modified T cells (CAR-T cells) in which receptor proteins are genetically incorporated into existing lymphocytes. Such receptor proteins can confer the ability of the modified CAR-T cells to target specific proteins. “CAR-T cell therapy” may also refer to a method of treatment comprising the administration of CAR-T cells or CAR-T cell pharmaceutical compositions.

[0051] As used herein, the term “host cell” means any cell of an organism that is selected, modified, transformed, grown, used or handled for the production of substances by the cell, such as genes, DNA or RNA sequences, proteins or enzymes. The host cells of the present invention include T cells and NK cells that contain DNA or RNA sequences encoding chimeric receptors and express chimeric receptors on their cell surface. Host cells may be used to enhance T lymphocyte activity in the treatment of cancer.

[0052] As used herein, the terms “express” and “expression” mean revealing information within a gene or DNA sequence, for example, enabling or causing the production of proteins such as CARs or amino acid transporters by activating cellular functions involved in the transcription and translation of the corresponding gene or DNA sequence. As used herein, the terms “overexpression” and “to overexpress” generally refer to the enhancement of protein expression by engineered ectopic expression, which results in the artificial induction or enhancement of a gene and subsequent protein expression of the target modality at levels higher than normal. A DNA sequence is expressed intracellularly or by a cell to form “expression products,” such as proteins. The expression product itself, for example, the resulting protein, may also be said to be “expressed” by a cell. Expression products can be characterized as intracellular, extracellular, or transmembrane. The term “intracellular” means something inside a cell. The term “extracellular” means something outside a cell. The term transmembrane means something having an extracellular domain, a portion embedded in the cell membrane, and an intracellular domain.

[0053] As used herein, the terms “encoding an expression construct” or “expression vector manipulation” refer to plasmids designed for gene expression in cells. These vectors are used to introduce a specific gene(s) into target cells and can manipulate the cell’s mechanisms for protein synthesis to produce the protein encoded by that gene. Vectors are typically engineered to contain regulatory sequences that act as enhancer and promoter regions, resulting in efficient transcription of the gene supported on the expression vector. Expression vectors can efficiently produce the desired protein(s) by producing modalities such as messenger RNA, which can be translated into the protein(s).

[0054] As used herein, the term "amino acid" generally means at least one amino group, -NH2 (which may exist in its ionized form), -NH3 + ), and one carboxyl group, -COOH (which may exist in its ionized form, -COOH) - ) refers to organic compounds containing carboxylic acids, which are deprotonated at neutral pH and have the basic formula NH2CHRCOOH. Amino acids, and therefore peptides, have an N (amino) terminal residue region and a C (carboxy) terminal residue region. The types of amino acids include at least 20 amino acids that are considered "natural" because they make up the majority of mammalian biological proteins, and include amino acids such as lysine, cysteine, tyrosine, and threonine. Amino acids can also be grouped based on their side chains, such as those having a carboxylic acid group including aspartic acid or aspartate (Asp;D) and glutamic acid or glutamate (Glu;E); as well as basic amino acids (neutral pH) including lysine (Lys;L), arginine (Arg;N), and histidine (His;H).

[0055] As used herein, the term “amino acid transporter” (AAT) refers to membrane transport proteins capable of transporting amino acids, such as arginine. More specifically, these are membrane transport proteins that mediate the movement of amino acids into and out of cells or organelles. As used herein, the term “arginine transporter” refers to membrane transport proteins capable of transporting arginine across the cell membrane. “Arginine transporters” may transport other amino acids in addition to arginine. A non-limiting example of arginine transporters is shown in Table 1. They play diverse functional roles in various biological systems, among others, that can modulate metabolic reprogramming, acid-base balance, and anabolic and catabolic reactions.

[0056] As used herein, the term “tumor microenvironment” (TME) generally refers to the intracellular and circumcellular environment of a solid tumor, including blood vessels, immune cells, fibroblasts, signaling molecules, and the extracellular matrix. Tumor progression is largely influenced by the interaction between cancer cells and this microenvironment, which can determine metastasis, growth, and disease progression. The TME can shape therapeutic responses and resistances by physically or chemically inhibiting therapeutic factors or by contributing to metastasis.

[0057] As used herein, the term “T cell metabolic reprogramming” refers to the metabolic reprogramming of T cells during activation, which is associated with the acquisition of a distinct differentiation profile. During antigen encounter and activation, T cells have increased bioenergy and anabolic needs to support their rapid replication and production of soluble factors. To meet these needs, T cells increase glucose and amino acid uptake for utilization through a variety of processes including, but not limited to, glycolysis, glutaminolysis, branched-chain amino acid catabolism, fatty acid uptake, lipid synthesis, and fatty acid oxidation. The role of amino acids as important metabolic regulators of T cell differentiation and functional fate is well demonstrated. Amino acids can function both as fuel sources during these metabolic demands and as precursors for the synthesis of proteins and nucleic acids.

[0058] As used herein, the term “myelo-derived suppressor cells” refers to a heterogeneous group of immune cells from myeloid lineages. These cells proliferate and expand rapidly in pathological situations as a result of altered hematopoiesis. These cells possess potent immunosuppressive activity and interact with other immune cell types, such as T cells, dendritic cells, macrophages, and natural killer cells, to modulate their functions. These cells are particularly relevant to cancers in which their presence and upmodulation are associated with poor patient prognosis and treatment resistance.

[0059] As used in the specification and claims of this application, the terms “administer” include any method effective in bringing about the expression of a chimeric antigen receptor and / or arginine transporter in T lymphocytes of the subject organism. Therefore, one method for administering a chimeric antigen receptor is by ex vivo transfection or transduction of peripheral blood T cells or hematopoietic progenitor cells (which will ultimately be allogeneic) using the nucleic acid construct according to the present invention, and by returning the transfected or transduced cells to the subject organism, preferably after expansion. In embodiments described herein, administering a drug, for example, administering CAR-T cells or CAR expression vectors, may include contacting the body fluids of a patient containing cells. For example, administering a drug may include ex vivo contacting the body fluids of a patient containing cancer cells (e.g., tumor cells) with the drug. In embodiments described herein, "administering, giving," for example, administering CAR-T cells or CAR expression vectors, may include ex vivo contacting the body fluids of a patient containing cancer cells (e.g., tumor cells) with the drug. This may include contact in vivo.

[0060] As used herein, the terms “administered in combination,” “combination administration,” or “co-administered” mean that when two or more drugs are administered to a subject simultaneously or within a certain interval, and both are administered as part of the same treatment regimen, there may be an additive or improved therapeutic effect of each drug on the patient. Two or more drugs administered in combination may be administered simultaneously or nearly simultaneously. Two or more drugs administered in combination do not need to be administered together. In some embodiments, the drugs are administered within 90 days (e.g., 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 day), within 28 days (e.g., 14, 7, 6, 5, 4, 3, 2, or 1 day), within 24 hours (e.g., 12, 6, 5, 4, 3, 2, or 1 hour), or within approximately 60, 30, 15, 10, 5, or 1 minute. In some embodiments, the administration of the drugs is carried out at intervals close enough to each other so that the combination effect is achieved.

[0061] The term "cancer" refers to any disease caused by the proliferation of malignant neoplastic cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias, and lymphomas. "Solid tumor cancer" refers to cancers that are abnormal tissue masses, such as sarcomas, carcinomas, and lymphomas. As used interchangeably herein, "blood cancer" or "fluid cancer" refers to cancers that exist in bodily fluids, such as lymphomas and leukemias.

[0062] The term "refractory cancer" refers to a form of cancer that is or may be unresponsive to treatment with currently used anticancer drugs or current anticancer regimens. Refractory cancers may initially respond to anticancer drug treatment and then become unresponsive. For example, refractory cancers may include forms of cancer in which cancer cells fail to halt growth in response to treatment, or initially halt growth in response to treatment but resume growth despite further anticancer drug treatment. Apparent regression with a high frequency of recurrence is also considered refractory. Refractory cancers may not respond to specific anticancer treatments with current first-line, second-line, or even third-line treatments. Patients with refractory cancer may be referred to herein as "patients with refractory cancer." The methods of the present invention described herein can be used to treat, prevent, or improve refractory cancer, or to treat patients with refractory cancer.

[0063] As used herein, the term “effective dose” of a drug (e.g., genetically modified T cells) refers to a quantity sufficient to produce a beneficial or desired outcome, such as a clinical result, and therefore the “effective dose” depends on the context in which it is applied.

[0064] As used herein, the term “pharmaceutical composition” refers to a composition containing the compounds described herein, formulated with pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical compositions are manufactured or marketed with the approval of a government regulatory body as part of a treatment regimen for treating diseases in mammals. The pharmaceutical compositions may be formulated, for example, parenterally, orally, pulmonaryly, intratracheally, intranasally, transdermally, or intraduodenally. In various embodiments, the pharmaceutical compositions described herein may be administered by one or more routes, including parenterally, for example, subcutaneously or intravenously. As used herein, the term parenteral includes subcutaneous injection, intrapancreatic administration, and intravenous, intramuscular, intraperitoneal, and intrasternal injection or infusion techniques. In embodiments described herein, the pharmaceutical compositions may be administered intravenously to patients requiring treatment for cancer.

[0065] As used herein, “pharmaceutically acceptable excipients” refers to any component (e.g., a vehicle capable of suspending or dissolving an active compound) that is non-toxic and non-inflammatory in the patient. Examples of excipients include antifouling agents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film-forming agents or coatings, flavors, fragrances, lubricants (flow enhancers), lubricants, preservatives, printing inks, radioprotective agents, adsorbents, suspending agents or dispersants, sweeteners, or hydration water. Examples of excipients include, but are not limited to, ascorbic acid, histidine, phosphate buffer, butylated hydroxytoluene (BHT), calcium carbonate, dibasic calcium phosphate, calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0066] As used herein, the term “polypeptide” refers to a string of at least two amino acids linked to one another by peptide bonds. In some embodiments, a polypeptide may contain at least three to five amino acids, each of which is linked to another amino acid by at least one peptide bond. Those skilled in the art will understand that a polypeptide may contain one or more “unnatural” amino acids or other entities that can nevertheless be incorporated into a polypeptide chain. In some embodiments, a polypeptide may be glycosylated, for example, a polypeptide may contain one or more covalently bonded sugar moieties. In some embodiments, a single “polypeptide” (e.g., an antibody polypeptide) may contain two or more individual polypeptide chains, which in some cases may be linked to one or more disulfide bonds or other means.

[0067] "Patient" or "subject" means human or non-human animal (e.g., mammal). In some embodiments described herein, the patient is in need of cancer treatment. Such patients may also be referred to as "cancer patients."

[0068] "Substantially identical" means that the two polypeptides or nucleotide sequences have the same reference sequence, or that they have a certain percentage of amino acid residues or nucleotides that are the same at corresponding positions in the reference sequence when the two sequences are optimally aligned. For example, an amino acid sequence that is "substantially identical" to a reference sequence has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the reference amino acid sequence. For polypeptides, the length of the comparison sequence is generally at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 100, 150, 200, 250, 300, or 350 consecutive amino acids (e.g., full-length sequence). Similarly, a nucleotide sequence that is "substantially identical" to a reference sequence has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity with the reference nucleotide sequence. For nucleotides, the length of the comparison sequence is generally at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 100, 150, 200, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 10000, or more than 10,000 consecutive nucleotides (e.g., full-length sequence). Sequence identity can be measured using sequence analysis software with default settings (e.g., the sequence analysis software package of the Genetic Computing Group, Center for Biotechnology, University of Wisconsin, 53705, Madison, Wisconsin, 1710 University Avenue). Such software can match similar sequences by assigning a degree of homology to various substitutions, deletions, and other modifications.

[0069] As used herein and as is well understood in the art, “treating” a condition or “treatment” a condition (e.g., cancer, as described herein) is an approach to obtain beneficial or desired outcomes, such as clinical outcomes. Beneficial or desired outcomes may include, but are not limited to, the alleviation or recovery of one or more symptoms or conditions, whether detectable or not; a reduction in the severity of a disease, disorder or condition; a stabilized (i.e., non-aggravating) state of a disease, disorder or condition; prevention of the spread of a disease, disorder or condition; delay or slow the progression of a disease, disorder or condition; improvement or mitigation of a disease, disorder or condition; and remission (partial or completely unchanged). “Mitiating” a disease, disorder or condition means that the severity and / or clinical manifestation of the disease, disorder or condition is reduced and / or the time of progression compared to the degree or course of the condition without treatment. This means that the process will be slow or prolonged.

[0070] As used herein, the terms “decrease,” “decreased,” “increase,” “increased,” or “reduction,” “reduced” (for example, with respect to therapeutic outcomes or effects) have meaning in relation to a reference level. In some embodiments, the reference level is the level determined by the use of the method with an experimental animal model or a control in a clinical trial. In some embodiments, the reference level is the level in the same subject before treatment or at the start of treatment. In some embodiments, the reference level is the mean level in a population not treated by the treatment method.

[0071] The term "DNA damage and repair inhibitors" (DDRi) refers to drugs that prevent the repair of cellular DNA damage caused by endogenous or exogenous chromosomal injury, acting by inhibiting normally occurring DNA repair mechanisms and related processes necessary for maintaining cell viability.

[0072] The term "checkpoint inhibitor," also known as "immune checkpoint inhibitor" or "ICI," refers to drugs that block the action of immune checkpoint proteins, for example, by blocking such immune checkpoint proteins from binding to their partner proteins. Cancer cells are known to express immune checkpoint proteins, and as a result, T cells cannot recognize such cancer cells as targets for destruction. Generally, checkpoint inhibitors promote the destruction of cancer cells by T cells by blocking the interaction between specific immune checkpoint proteins on T cells and target cells, otherwise such interaction acts as a signal that inhibits the destruction of target cells by T cells. Checkpoint inhibitors include drugs that block the interaction between PD-1 and PD-L1, or drugs that block the interaction between CTLA-4 and B7-1 / B7-2. Examples of specific checkpoint inhibitors include the following antibody-based drugs: ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and semiprimab.

[0073] As used herein, the terms "tumor-associated antigen" or "tumor-associated antigen" mean an antigen that is present on tumor cells in significantly higher amounts than on normal cells.

[0074] As used herein, the terms "tumor-specific antigen" or "tumor-specific antigen" refer to an antigen that is endogenously present only on tumor cells.

[0075] As used herein, the term “cancer cell antigen” means an antigen present on cells that form part of cancer (e.g., malignant neoplasm cells, e.g., tumors, neoplasms, carcinomas, sarcomas, leukemias, and lymphomas). Individual cancer cells may express one or more cancer cell antigens. Preferred cancer cell antigens for targeting are those that have significantly differential expression on cancer cells compared to healthy cells in the target.

[0076] As used herein, the terms “binding” or “binding” refer to, for example, an antibody, its antigen-binding fragment, or the antigen-specific binding domain of a CAR, and mean at least a transient interaction or association with or to a target antigen. For example, “binding” or “binding” may refer to the process by which the antigen-binding portion of a CAR comes into transient or persistent contact with a cancer cell expressing a cancer cell antigen. In some embodiments described herein, a CAR can bind to a cancer cell antigen. In such embodiments, binding occurs via an interaction between the cancer cell antigen and the antigen-specific binding region of the CAR.

[0077] As used herein, “primary tumor” refers to the original tumor growth at the primary site and not a product of metastasis.

[0078] As used herein, “secondary tumor” refers to tumor growth that has spread from the primary site to secondary anatomical sites, often through metastasis.

[0079] A “solid tumor” is an abnormal mass of tissue, such as sarcoma, cancer, and lymphoma. As used herein, a “liquid tumor” is cancer present in body fluids, such as lymphoma and leukemia.

[0080] As used herein, "cold tumor" is characterized by the absence of T cell infiltration. This refers to tumors characterized by the absence of T-cell infiltration. Non-inflammatory tumors are also characterized by the fact that checkpoint inhibitors are ineffective in terms of treatment efficacy when used as monotherapy. Examples of non-inflammatory tumors include, but are not limited to, glioblastoma, ovarian cancer, prostate cancer, pancreatic cancer, and breast cancer tumors, all of which are characterized by the absence of T-cell infiltration. Modes for carrying out the invention Chimeric antigen receptor

[0081] Chimeric antigen receptors (CARs) are genetically engineered cell surface receptor proteins designed to bind to specific antigens, such as those presented on the surface of cancer cells. CARs may be expressed in immune cells, such as T lymphocytes (T cells or T-cells), to direct T cells to target cells expressing CAR-binding antigens and to target antigen-expressing cells for destruction. The CARs described herein can bind to, for example, proteins, carbohydrates, or glycolipid antigens. For example, the CARs described herein may bind to any one of the following antigens: α-folate receptor, CAIX, CD19, CD20, CD22, CD24, CD30, CD33, CD38, CD44v7 / 8, carcinoembryonic antigen (CEA), EGFRvIII, EGP-2, EGP-40, EphA2, EphA3, Erb-B2, Erb-B2, 3,4, FBP, fetal acetylcholine receptor, G D2 , G D3HER2, HMW-MAA, IL-11Rα, IL-13Rα2, KDR, κ light chain, Lewis Y, L1 cell adhesion molecule, melanoma-associated antigen (MAGE), mesothelin, mouse CMV-infected cells, MUC1, MUC16, NKG2D, NY-ESO-1 / LAGE-1, tumor embryo antigen, PSCA, PSMA, ROR1, mAb IgE, TAG-72, VEGF-R2, insulin-like growth factor 1 receptor (IGF-1R), tumor endothelial marker 1 (TEM-1), alpha-fetoprotein (AFP), cancer antigen 125 (CA125), cancer antigen 15-3 (CA15-3), carbohydrate antigen 19-9 (CA19-9), human chorionic gonadotropin (hCG or β-hCG), prostate-specific antigen (PSA), epithelial tumor antigen (ETA), immature laminin receptor, HPV E6, HPV E7, BING-4, calcium-activated chloride channel 2, cyclin-B1, 9D7, Ep-CAM, telomerase, mesothelin, SAP-1, survivalin, livin, BAGE family proteins, CAGE family proteins, GAGE ​​family proteins, MAGE family proteins, SAGE family proteins, XAGE family proteins, PRAME, SSX-2, Melan-A / MART-1, MART-2, Gp100 / pmel17, tyrosinase, TRP-1 / -2, P. polypeptide, MC1R, β-catenin, β-catenin-m, β-actin / 4 / m, myosin / m, HSP70-2 / m, GM2, sTn, globo-H, HLA-A2-R17OJ, BRCA1 / 2, CDK4, CML66, fibronectin, p53, Ras, TGF-βRII, or mammoglobin-A. The CARs described herein can bind to cancer cell antigens, including tumor-associated antigens or tumor-specific antigens.

[0082] The CARs described herein include at least the following components: an antigen-binding fragment, a transmembrane domain component, and a cytoplasmic activation domain. CARs may also include one or more cytoplasmic costimulatory domains. An example CAR is, for example, Feins et al. (2018) "An introduction to chimeric antigen receptor (CAR) T-cell immunotherapy for human cancer'' Am.J.Hematol.94:S3-9;Stoiber et al. (2019)''Limitations in the Design of Chimeric Antigen Receptors for Cancer Therapy'' Cells,8(472):1-26; and Sadelain et al. (2013)''The Basic Principles "of Chimeric Antigen Receptor Design" is described in Cancer Discovery, 3(4):388-98.

[0083] In the embodiments described herein, the CAR may include a hinge or spacer region. The CAR antigen-binding fragment is generally ligated to the CAR transmembrane domain via a hinge or spacer region. The hinge region may be an amino acid sequence of immunoglobulin G (IgG) or the CD8α or CD28 extracellular domain, or an amino acid sequence derived therefrom. An exemplary hinge domain is described, for example, in Stoiber et al. (2019) "Limitations in the Design of Chimeric Antigen Receptors for Cancer Therapy," Cells, 8(472):1-26. CAR antigen-binding fragment or domain

[0084] CAR antigen-binding fragments can be single-strand variable fragments (scFv), antigen-binding fragments (Fab), F(ab')2s fragments, or ligands, such as natural, artificial, or engineered ligands. scFv are the heavy chains (V) of immunoglobulins. H ) and light chain (V L It is a fusion protein composed of a variable region and held together by a peptide linker. The linker of scFv can be, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residue lengths, for example, 10-20, 15-20, 15-25, or 10-25 residue lengths. scFv can be expressed as a single-chain peptide in mammalian or bacterial cells. It is also possible to create bivalent and trivalent scFv by cloning scFv in tandem with the linker region. Furthermore, two or more V H and V L It is possible to express pairs, and each V H and V L The pairs are joined by short linkers, each V H is another connected V H and V L Paired V L And it dimerizes into a diamond body (i.e., V H / V L Two scFv') or triabodies (i.e., V) are formed by dimerization. H / V L Dimerization forms three scFv' units. The diabody and tribody may contain a short linker, e.g., about 5 amino acids long. The scFv linker may contain glycine and serine repeats, e.g., pentapeptides (Gly4Ser) (SEQ ID NO: 275), (Gly4Ser)2 (SEQ ID NO: 276), (Gly4Ser)3 (SEQ ID NO: 30), or (Gly4Ser)4 (SEQ ID NO: 277). The scFv amino acid sequence may be a mouse antibody sequence, a human antibody sequence, or a humanized antibody sequence. In some embodiments, the CAR may contain two or three antigen-specific targeting regions, e.g., two or three scFv, Fab, F(ab')2, or ligands that bind to different cell surface antigens (e.g., mutaines).

[0085] Fab consists of the constant and variable domains of the heavy and light chains of the antibody. Fab can be prepared by direct cleavage of the antibody using enzymes such as papain, pepsin, or IdeS.

[0086] Examples of naturally occurring ligands that may be included in CARs include, but are not limited to, CD8, CD4, CD25, and CD16.

[0087] CARs contain antigen-binding fragments or domains that recognize and bind to specific cell surface antigens. Examples of CD33 antigen-recognition domain nucleotide sequences include: GAAGTGCAGCTGGTGCAGAGCGGAGCAGAAGTGAAGAAGCCCGGAAGCAGCGTGAAGGTGTCTTGCAAGGCCAGCGGCTACACCATCACCGACAGCAACATCCATTGGGTCCGGCAGGCTCCAGGACAGTCTCTGGAGTGGATCGGCTACATCTACCCCTACAACGGCGGCACC GACTACAACCAGAAGTTCAAGAACCGGGCCACCCTGACCGTGGATAACCCCACCAACACCGCCTACATGGAGCTGAGCAGCCTGAGAAGCGAGGACACCGCCTTCTACTATTGCGTGAACGGCAACCCTTGGCTGGCCTATTGGGGACAGGGAACACTGGTGACCGTGTCCTCT (SEQ ID NO: 1); and GACATCCAGCTGACCCAGTCTCCTAGCACCCTGAGCGCTAGCGTGGGAGATAGAGTGACCATCACTTGCAGAGCCAGCGAGAGCCTGGACAACTACGGCATCCGGTTCCTGACTTGGTTCCAGCAGAAACCCGGCAAGGCCCCTAAACTGCTGATGTACGCCGCCTCTAAC CAGGGAAGCGGAGTGCCTAGCAGATTCAGCGGCAGCGGAAGCGGAACCGAGTTCACCCTGACCATCAGCTCTCTGCAGCCAGACGACTTCGCCACCTACTACTGCCAGCAGACCAAGGAGGTGCCTTGGAGCTTCGGCCAGGGAACCAAGGTGGAAGTGAAGCGGACAGTG (Sequence number 2).

[0088] Examples of CD33 antigen-binding fragment amino acid sequences include the following: Anti-CD33 heavy chain variable domain:EVQLVQSGAEVKKPGSSVKVSCKASGYTITDSNIHWVRQAPGQSLEWIGYIYPYNGGTDYNQKFKNRATLTVDNPTNTAYMELSSLRSEDTAFYYCVNGNPWLAYWGQGTLVTVSS (Sequence ID 3); and Anti-CD33 light chain variable domain:DIQLTQSPSTLSASVGDRVTITCRASESLDNYGIRFLTWFQQKPGKAPKLLMYAASNQGSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQTKEVPWSFGQGTKVEVKRTV (Sequence ID 4). CAR hinge or spacer area

[0089] Exemplary CD8α-derived hinge region nucleotide sequences include the following: ACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 5); and GCGAAGCCCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCATCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 6).

[0090] Examples of CD8α-derived hinge region amino acid sequences include the following: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (Sequence ID 7); and AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (Sequence ID 8).

[0091] An example of a CD28-derived hinge region nucleotide sequence is the sequence of Sequence ID No. 9: ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC.

[0092] An example of the hinge region amino acid sequence derived from CD 28 is the sequence of SEQ ID NO: 10: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP.

[0093] An example of an IgG1-derived hinge region nucleotide sequence is the sequence of Sequence ID No. 11: GAGCCCAAGAGCTGCGACAAGACCCACACCTGCCCCCCCTGCCCC.

[0094] An example of an IgG1-derived hinge region amino acid sequence is the sequence of SEQ ID NO: 12:EPKSCDKTHTCPPCP.

[0095] An exemplary IgG2-derived hinge region nucleotide sequence is the sequence of Sequence ID No. 13: ATTGAAGTTATGTATCCTCCTCCTTACCTAGACAATGAGAAGAGCAATGGAACCATTATCCATGTGAAAGGGAAACACCTTTGTCCAAGTCCCCTATTTCCCGGACCTTCTAAGCCC.

[0096] An example of an IgG2-derived hinge region amino acid sequence is the sequence of SEQ ID NO: 14: ERKCCVECPPCP.

[0097] An exemplary IgG3-derived hinge region nucleotide sequence is the sequence of Sequence ID No. 15: GAGCTCAAAACCCCACTTGGTGACACAACTCACACATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCATGCCCACGGTGCCCAGAGCCCAAATCTTGTGACACACCTCCCCCGTGCCCAAGGTGCCCA.

[0098] An exemplary IgG3-derived hinge region amino acid sequence is the sequence of SEQ ID NO: 16: ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP.

[0099] Exemplary IgG4-derived hinge region nucleotide sequences include the following: GAGTCCAAATATGGTCCCCCATGCCCATCATGCCCA (Sequence ID 17); GAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCA (Sequence ID 18); GGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGCTGGCTGGCTGGTCCAAAGGCCTTCTCACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACACTACAAGACCACGCCTCCCGTGCTGCGTGCACTCCGACGGCTCCTTCTCTCTACAGCAGCTCACCGTGACAAGAGCAGGTGGCAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTTGCACAACCACTACACACACACAGAGACCTTCCCGTTCCTTGGGTAAA (sequence number 19); GAGAGCAAGTACGGCCCCCCCTGCCCCCCCCCCCGGCGCGGCAGCAGCGGCGGCGGCAGCGGCGGCCAGCGAGAGCCCCAGGTGTACACCCTGCCCCCCAGGAGGATGACCAAGAACCAGGTGAGCCTGGCCTGGTGGAAGGCCAGAACAACTACAAGACCACCCCCCCCGTGGCTGGACAGCGACGGCCACTTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAGGGGCAACGTGTTCAGCTGCACGGTGATGCACGGAGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCTGGCAAG (sequence number 20); GAGAGCAAGTACGGCCCCCCCTGCCCCAGCTGCCCCGCCCCCGAGTTCGAGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAGGACCCCGAGGTGCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCACCAGGCCAAGACCAAGCCCAGAGAGGAGCAGTTCAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCCAGCAGCATCGAGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAGGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCTGGGCAAG (SEQ ID NO: 21); and GAGAGCAAGTACGGCCCCCCCTGCCCCCTGCCCCGCCCCCGAGTTCGAGGGCGGCCCCAGCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATCAGCAGAACCCCGGGTGACCTGCGTGGTGGTGGACGTGAGCCAGGAGGACCCCGAGGTGCAGTTC AACTGGTACGTGGACGGCGTGGAGGTGCACCAGGCCAAGACCAAGCCCAGAGAGGAGCAGTTCAACAGCACCTACAGAGTGGTGAGCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGAGCAACAAGGGCCTGCCCAGCATCG AGAAGACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGTGTACACCCTGCCCCCAGCCAGGAGGAGATGACCAAGAACCAGGTGAGCCTGACCTGCCTGGTGAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAA CTACAAGACCACCCCCCCCGTGCTGGACAGCGACGGCAGCTTCTTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGATGGCAGGAGGGCAACGGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGAGCCTGAGCCTGGGCAAG (SEQ ID NO: 22).

[0100] Exemplary IgG4-derived hinge region amino acid sequences include the following: ESKYGPPCPSCP (Sequence ID 23); ESKYGPPCPSCPA (Sequence ID 24); GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Sequence ID 25); ESKYGPPCPPCPGGGSSGGGSGGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Sequence ID 26); ESKYGPPCPSCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHQAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Sequence ID 27); and ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHQAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (Sequence ID 28).

[0101] In the embodiments described herein, the CAR may include a spacer region. An exemplary spacer region nucleotide sequence is as follows: GGCGGGAGGATCTGGCGGAGGTGGAAGCGGAGGCGGTGGAAGC (SEQ ID NO: 29).

[0102] An example of a spacer region amino acid sequence is as follows: GGGGSGGGGSGGGGS (sequence number) No. 30). CAR transmembrane domain

[0103] The transmembrane domains of CARs described herein link an antigen-binding domain with an intracellular signaling domain. The CAR transmembrane domain may be an amino acid sequence derived, for example, from CD4, CD8α, CD28, CD3ζ, or an inducible T cell costimulator (ICOS). The transmembrane domain may contribute to CAR dimerization and CAR surface expression by the T cell receptor (TCR) complex. Exemplary CAR transmembrane domains are described, for example, in Stoiber et al. (2019) "Limitations in the Design of Chimeric Antigen Receptors for Cancer Therapy," Cells, 8(472):1-26.

[0104] As used herein, “CD4” (also known as T cell surface glycoprotein CD4 and CD4mut) refers to the gene identified by Entrez gene ID number 920, its allele variants, its orthologues, its protein products, and mRNA transcripts encoded by the gene, including the nucleotide sequences of NCBI reference sequences: NM_000616.5, NM_001195014.3, NM_001195015.3, NM_001195016.3, and NM_001195017.3. CD4 protein products include proteins encoded by CD4, such as proteins containing the amino acid sequences of NCBI reference sequences: NP_000607.1, NP_001181943.1, NP_001181944.1, NP_001181945.1, or NP_001181946.1. The transmembrane region of CD4 includes amino acids 397-418 of the amino acid sequence of the NCBI reference sequence NP_000607.1, which is encoded by the following nucleotide sequence: ATGGCCCTGATTGTGCTGGGGGGCGTCGCCGGCCTCCTGCTTTTCATTGGGCTAGGCATCTTCTTC (Sequence No. 42).

[0105] As used herein, “CD8α” (also known as the CD8a molecule, T cell surface glycoprotein CD8, p32, Leu2, and CD8a) refers to the gene identified by Entrez gene number 925, its allele variants, its orthologues, its protein products, and mRNA transcripts encoded by the gene, including the nucleotide sequences of NCBI reference sequences: NM_001145873.1, NM_001768.6, and NM_171827.3. CD8 protein products include proteins encoded by CD8, such as proteins containing the amino acid sequences of NCBI reference sequences: NP_001139345.1, NP_001759.3, or NP_741969.1. The transmembrane region of CD8α includes, for example, amino acids 183-203, 183-205, or 183-206 of the amino acid sequence of NCBI reference sequence NP_001139345.1, respectively: IYIWAPLAGTCGVLLLSLVIT (Sequence ID 49); IYIWAPLAGTCGVLLLSLVITLY (Sequence ID 50); and IYIWAPLAGTCGVLLLSLVITLYC (Sequence ID 51).

[0106] The transmembrane region of CD8α is encoded by the following nucleotide sequence. ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACC (Sequence code 52); ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTAC (Sequence No. 53); and ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTATCACCCTTTACTGC (array Number 54).

[0107] As used herein, “CD28” (also known as T cell-specific surface glycoprotein CD28, CD28 molecule, or Tp44) refers to the gene identified by Entrez gene number 940, its allele variants, its orthologues, its protein products, and mRNA transcripts encoded by the gene, including the nucleotide sequences of NCBI reference sequences: NM_001243077.2, NM_001243078.1, and NM_006139.4. CD28 protein products include proteins encoded by CD28, such as proteins containing the amino acid sequences of NCBI reference sequences: NP_001230006.1, NP_001230007.1, or NP_006130.1, or mature CD28 proteins containing amino acids 19–220 of the amino acid sequence of NCBI reference sequence NP_006130.1. The transmembrane region of CD28 includes, for example, amino acids 153-179 of the amino acid sequence of the NCBI reference sequence NP_006130.1:FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 62).

[0108] The transmembrane region of CD28 is encoded by the following nucleotide sequence: TTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCTTGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTG (SEQ ID NO: 63).

[0109] As used herein, “CD3ζ” (also known as CD247, CD247 molecule, T cell surface glycoprotein CD3 zeta chain, T3Z, CD3H, CD3Q, CD3Z, TCRZ, IMD25, and CD3-ZETA) refers to the gene identified by Entrez gene number 919, its allele variants, its orthologues, its protein products, and mRNA transcripts encoded by the gene, including the nucleotide sequences of NCBI reference sequences: NM_000734.4 and NM_198053.2. CD3ζ protein products include proteins encoded by CD3ζ, e.g., proteins containing the amino acid sequence of NCBI reference sequence: NP_000725.1 or NP_932170.1. The transmembrane region of CD3ζ includes, for example, amino acids 31-51 of the amino acid sequence of NCBI reference sequence NP_000725.1:LCYLLDGILFIYGVILTALFL(SEQ ID NO: 68) ).

[0110] The transmembrane region of CD3ζ is encoded by the following nucleotide sequence: CTCTGCTACCTGCTGGATGGAATCCTCTTCATCTATGGTGTCATTCTCACTGCCTTGTTCCTG (SEQ ID NO: 69).

[0111] As used herein, "ICOS" (also known as Inducible T Cell Costimulation, Inducible T Cell Costimulation Precursor, AILIM, CD278, and CVID1) refers to the gene identified by Entrez gene number 29851, its allele variants, its orthologues, its protein products, and the mRNA transcript encoded by the gene, including the nucleotide sequence of the NCBI reference sequence NM_012092.4. ICOS protein products include proteins encoded by ICOS, such as proteins containing the amino acid sequence of the NCBI reference sequence NP_036224.1 (SEQ ID NO: 71), or mature ICOS proteins containing amino acids 21-199 of the amino acid sequence of the NCBI reference sequence NP_036224.1 (SEQ ID NO: 72). The transmembrane region of ICOS contains amino acids 141-161 of the amino acid sequence of the NCBI reference sequence NP_036224.1: fwlpigcaafvvvcilgcili (SEQ ID NO: 73).

[0112] In some embodiments described herein, the CAR may comprise all or part of the transmembrane domains described herein, for example, the CD4, CD8α, CD28, CD3ζ, or ICOS transmembrane domains described herein. For example, in some embodiments described herein, the CAR comprises transmembrane domains of about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, or about 25 amino acids, or about 15 to about 20, about 15 to about 25, about 15 to about 22, about 18 to about 20, about 18 to about 22, or about 18 to about 25 amino acids. For example, in some embodiments described herein, the CAR comprises a transmembrane domain of approximately 15, approximately 16, approximately 17, approximately 18, approximately 19, approximately 20, approximately 21, approximately 22, approximately 23, approximately 24 or approximately 25 amino acids of the CD4, CD8α, CD28, CD3ζ or ICOS transmembrane domain described herein, or a transmembrane domain of approximately 15 to approximately 20, approximately 15 to approximately 25, approximately 15 to approximately 22, approximately 18 to approximately 20, approximately 18 to approximately 22 or approximately 18 to approximately 25 amino acids.

[0113] In some embodiments described herein, the CAR includes a transmembrane domain having an amino acid sequence that is approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, approximately 90% to approximately 95%, approximately 95% to approximately 100%, or approximately 90% to approximately 100% identical to the CD4, CD8α, CD28, CD3ζ, or ICOS transmembrane domain described herein. CAR intracellular signaling and co-stimulatory domains

[0114] The intracellular portion of the CAR described herein may include an intracellular signaling domain and, optionally, one or more co-stimulatory domains. Exemplary intracellular signaling domains include, for example, amino acid sequences of the Fc receptor γ chain subunit (FcRγ) or CD3ζ signaling domain. Exemplary co-stimulatory domains include, for example, amino acid sequences from or derived from the intracellular domains of 4-1BB (C137;TNFRS9), CD27, CD28, CD40, CD40L, TLR2, DAP10, OX40 (CD134), IL-2RB, IL-2RA, MYD88, or ICOS (CD278). For example, the CARs described herein may include, but are not limited to, the following combinations of signaling domains and co-stimulatory domains: 4-1BB / CD3ζ, CD27 / CD3ζ, CD28 / CD3ζ, DAP10 / CD3ζ, OX40 / CD3ζ, ICOS / CD3ζ, 4-1BB / FcRγ, CD27 / FcRγ, CD28 / FcRγ, DAP10 / FcRγ, OX40 / FcRγ, ICOS / FcRγ, 4-1BB / CD28 / CD3ζ, 4-1BB / CD28 / FcRγ, OX40 / CD28 / CD3ζ, OX40 / CD28 / FcRγ, ICOS / 4-1BB / CD3ζ, and ICOS / 4-1BB / FcRγ.

[0115] The CD3ζ signaling domains described herein may include, for example, proteins containing amino acids 52-163 of the amino acid sequence of the NCBI reference sequence NP_000725.1: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (Sequence ID 74).

[0116] The nucleotide sequence encoding the CD3ζ signaling domain is as follows: AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (Sequence ID 75).

[0117] An example of an FcRγ protein is the Fc fragment of the IgE receptor Ig (also known as FCER1G, FCRG, and high-affinity immunoglobulin epsilon receptor subunit γ) identified by Entrez gene ID number 2207. The FcRγ nucleotide sequences described herein may include allele variants, orthologues, and mRNA transcripts encoded by Entrez gene ID number 2207, for example, the nucleotide sequence of the NCBI reference sequence NM_004106.2. FcRγ protein products include proteins encoded by the nucleotide sequence of the NCBI reference sequence NM_004106.2, for example, proteins containing the amino acid sequence of the NCBI reference sequence NP_004097.1, or mature FcRγ proteins containing amino acids 19-86 of the amino acid sequence of the NCBI reference sequence NP_004097.1. The signaling domain of FcRγ contains, for example, amino acids 45-86 of the amino acid sequence of the NCBI reference sequence NP_001552.2: cspcppnsfssaggqrtcdicrqckgvfrtrkecsstsnaec (Sequence ID 79).

[0118] The FcRγ subunit and CD3ζ contain multiple YXXL immunoreceptor-activating tyrosine motif ("ITAM") sequences. While not theoretically bound, tyrosine phosphorylation of ITAM after cell surface antigen binding by the antigen-binding moiety of CAR is thought to promote T cell activation. An example of a CD3ζ amino acid sequence containing an ITAM sequence is SEQ ID NO: 74. An example of a CD3ζ nucleotide sequence encoding an ITAM sequence is SEQ ID NO: 75.

[0119] An example of a CD3ζ amino acid sequence containing an ITAM sequence is as follows: RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (Sequence ID 80).

[0120] In some embodiments described herein, the CAR may comprise all or part of a signaling domain described herein, for example, a CD3ζ or FcRγ signaling domain described herein. For example, in some embodiments described herein, the CAR comprises a signaling domain of about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or about 130 amino acids, or a signaling domain of about 20 to about 40, about 30 to about 50, about 40 to about 50, about 40 to about 60, about 100 to about 120, about 110 to about 120 or about 110 to about 130 amino acids. For example, in some embodiments described herein, the CAR comprises about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, or about 130 amino acids, or about 20 to about 40, about 30 to about 50, about 40 to about 50, about 40 to about 60, about 100 to about 120, about 110 to about 120, or about 110 to about 130 amino acids, and these amino acids comprise the CD3ζ or FcRγ signaling domain described herein, or a portion thereof.

[0121] In some embodiments described herein, the CAR includes a transmembrane domain having an amino acid sequence that is approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, approximately 90% to approximately 95%, approximately 95% to approximately 100%, or approximately 90% to approximately 100% identical to the CD3ζ or FcRγ signaling domain described herein.

[0122] As used herein, “4-1BB” (also known as TNFRSF9, TNF receptor superfamily member 9, CD137, ILA, CDw137, tumor necrosis factor receptor superfamily member 9, and TNFRS9) refers to the gene identified by Entrez gene number 3604, its allele variants, its orthologues, its protein products, and mRNA transcripts encoded by the gene, including the nucleotide sequence of the NCBI reference sequence NM_001561.6. 4-1BB protein products include proteins encoded by 4-1BB, such as proteins containing the amino acid sequence of the NCBI reference sequence NP_001552.2, or mature 4-1 BB proteins containing amino acids 24-255 of the amino acid sequence of the NCBI reference sequence NP_001552.2.

[0123] The transmembrane region of 4-1BB includes, for example, the following amino acid sequence: IISFFLALTSTALLFLLFFLTLRFSVV (Sequence No. 84).

[0124] The transmembrane region of 4-1BB is encoded by the following nucleotide sequence: ATCATCTCCTTCTTTCTTGCGCTGACGTCGACTGCGTTGCTCTTCCTGCTGTTCTTCCTCACGCTCCGTTTCTCTGTTGTT (SEQ ID NO: 85).

[0125] The 4-1BB co-stimulatory domain includes, for example, amino acids 214-255 of the amino acid sequence of the NCBI reference sequence NP_001552.2: KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (Sequence No. 86).

[0126] The 4-1BB co-stimulatory domain is encoded by the following nucleotide sequence. AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAACCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGATGGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGATGTGAACTG (SEQ ID NO: 87).

[0127] As used herein, “CD27” (also known as the CD27 molecule, T14, S152, Tp55, TNFRSF7, S152, LPFS2, and CD27 antigen) refers to the gene identified by Entrez gene number 939, its allele variants, its orthologues, its protein products, and the mRNA transcripts encoded by the gene, including the nucleotide sequence of the NCBI reference sequence NM_001242.4. CD27 protein products include proteins encoded by CD27, e.g., proteins containing the amino acid sequence of the NCBI reference sequence NP_001233.1, or mature CD27 proteins containing amino acids 21–260 of the amino acid sequence of the NCBI reference sequence NP_001233.1. The co-stimulatory domain of CD27 contains, for example, amino acids 213–260 of the amino acid sequence of the NCBI reference sequence NP_001233.1: qrrkyrsnkgespvepaepcryscpreeegstipiqedyrkpepacsp (Sequence ID 91).

[0128] The co-stimulatory domain of CD28 includes, for example, amino acids 180-220 of the amino acid sequence of the NCBI reference sequence NP_006130.1: RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (Sequence ID 92).

[0129] The CD28 costimulatory domains described herein also include: RSKRSRGGHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (Sequence ID 93).

[0130] The nucleotide sequence encoding the CD28 co-stimulatory domain includes the following: AGGAGTAAGAGGAGCAGGCTCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 94); and AGGAGTAAGAGGAGCAGGGGCGGCCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCAGCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCC (SEQ ID NO: 95).

[0131] As used herein, “CD40” (also known as the CD40 molecule, p50, Bp50, CDW40, TNFRSF5, and tumor necrosis factor receptor superfamily member 5) refers to the gene identified by Entrez gene number 958, its allele variants, its orthologues, its protein products, and mRNA transcripts encoded by the gene, including the nucleotide sequence of the NCBI reference sequence: NM_001250.6, NM_001302753.2, NM_001322421.2, NM_001322422.2, NM_001362758.2, or NM_152854.4. CD40 protein products include proteins encoded by CD40, such as proteins containing the amino acid sequences of NCBI reference sequences: NP_001241.1, NP_001289682.1, NP_001309350.1, NP_001309351.1, NP_001349687.1, NP_690593.1, or mature CD40 proteins containing amino acids 21-277 of the amino acid sequence of NCBI reference sequence NP_001241.1, for example. The co-stimulatory domain of CD40 contains amino acids 216-277 of the amino acid sequence of NCBI reference sequence NP_001241.1, for example: kkvakkptnkaphpkqepqeinfpddlpgsntaapvqetlhgcqpvtqedgkesrisvqerq (Sequence No. 109).

[0132] As used herein, “CD40L” (also known as CD40 ligand, CD40LG, IGM, IMD3, TRAP, gp39, CD154, HIGM1, T-BAM, TNFSF5, and hCD40L) refers to the gene identified by Entrez gene number 959, its allele variants, its orthologues, its protein products, and mRNA transcripts encoded by the gene, including the nucleotide sequence of the NCBI reference sequence NM_000074.3. CD40L protein products include proteins encoded by CD40L, e.g., proteins containing the amino acid sequence of the NCBI reference sequence NP_000065.1. The co-stimulatory domain of CD40L includes, for example, amino acids 1-22 of the amino acid sequence of the NCBI reference sequence NP_000065.1: mietynqtsprsaatglpismk (Sequence ID 112).

[0133] As used herein, "TLR2" (also known as Toll-like receptor 2, TIL4, and CD282) refers to the gene identified by Entrez gene number 7097, its allele variants, its orthologues, its protein products, and the mRNA transcripts encoded by the gene, including the nucleotide sequences of the NCBI reference sequences: NM_001318787.2, NM_001318789.2, NM_001318790.2, NM_001318791.2, NM_001318793.2, NM_001318795.2, NM_001318796.2, and NM_003264.5. TLR 2 protein products include proteins encoded by TLR2, such as proteins containing the amino acid sequences of the NCBI reference sequences: NP_001305716.1, NP_001305718.1, NP_001305719.1, NP_001305720.1, NP_001305722.1, NP_001305724.1, NP_001305725.1, NP_003255.2, or mature TLR2 proteins containing amino acids 21-784 of the amino acid sequence of the NCBI reference sequence NP_001305716.1. The TLR 2 co-stimulatory domain contains, for example, amino acids 610-784 of the NCBI reference sequence NP_001305716.1: hrfhglwymkmmwawlqakrkprkapsrnicydafvsyserdaywvenlmvqelenfnppfklclhkrdfipgkwiidniidsiekshktvfvlsenfvkewckyeldfshfrlfdenndaailillepiekkaipqrfcklrkimntktylewpmdeaqregfwvnlraaiks (Sequence ID 130); or

[0134] Amino acids 640-784 of the amino acid sequence of NCBI reference sequence NP_001305716.1: cydafvsyserdaywvenlmvqelenfnppfklclhkrdfipgkwiidniidsiekshktvfvlsenfvksewckyeldfshfrlfdenndaailillepiekkaipqrfcklrkimntktylewpmdeaqregfwvnlraaiks (Sequence ID 131).

[0135] As used herein, “DAP10” (also known as HCST, hematopoietic cell signaling molecule, KAP10, and PIK3AP) refers to the gene identified by Entrez gene number 10870, its allele variants, its orthologues, its protein products, and mRNA transcripts encoded by the gene, including the nucleotide sequence of NCBI reference sequence: NM_001007469.2 or NM_014266.4. DAP10 protein products include proteins encoded by DAP10, such as proteins containing the amino acid sequence of NCBI reference sequence: NP_001007470.1 or NP_055081.1, or mature DAP10 proteins containing amino acids 20–92 of the amino acid sequence of NCBI reference sequence NP_001007470.1. The co-stimulatory domain of DAP10 includes, for example, amino acids 70-92 of the amino acid sequence of the NCBI reference sequence NP_001007470.1: carprrspaqdgkvyinmpgrg (SEQ ID NO: 137).

[0136] As used herein, “OX40” (also known as TNFRSF4, TNF receptor superfamily member 4, CD134, ACT35, IMD16, tumor necrosis factor receptor superfamily member 4, and TXGP1L) refers to the gene identified by Entrez gene number 7293, its allele variants, its orthologues, its protein products, and mRNA transcripts encoded by the gene, including the nucleotide sequence of the NCBI reference sequence NM_003327.4. OX40 protein products include proteins encoded by OX40, such as proteins containing the amino acid sequence of the NCBI reference sequence NP_003318.1, or mature OX40 proteins containing amino acids 29–277 of the amino acid sequence of the NCBI reference sequence NP_003318.1. The co-stimulatory domain of OX40 includes, for example, amino acids 236-277 of the amino acid sequence of the NCBI reference sequence NP_003318.1: ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI (Sequence ID 141).

[0137] The OX40 co-stimulatory domain is encoded by the following nucleotide sequence: GCCCTGTACCTGCTCCGGAGGGACCAGAGGCTGCCCCCCGATGCCCACAAGCCCCCTGGGGGAGGCAGTTTCCGGACCCCCATCCAAGAGGAGCAGGCCGACGCCCACTCCACCCTGGCCAAGATC (Sequence ID 142).

[0138] The ICOS co-stimulatory domain includes amino acids 162-199 or 165-199 of the amino acid sequence of the NCBI reference sequence NP_036224.1, for example: TKKKYSSSYHDPNGEYMFMRAVNTAKKSRLTDVTL (SEQ ID NO: 143).

[0139] The ICOS co-stimulatory domain is encoded by the following nucleotide sequence: ACAAAAAAGAAGTATTCATCCAGTGTGCACGACCCTAACGGTGAATACATGTTCATGAGAGCAGTGAACACAGCCAAAAAATCTAGACTCACAGATGTGACCCTA (SEQ ID NO: 144).

[0140] As used herein, “IL-2Rβ” (also known as IL2RB, interleukin-2 receptor subunit β, CD122, IMD63, IL15RB, and P70-75) refers to the gene identified by Entrez gene number 3560, its allele variants, its orthologues, its protein products, and the mRNA transcripts encoded by the gene, including the nucleotide sequences of the NCBI reference sequences: NM_000878.5, NM_001346222.1, or NM_001346223.2. IL-2Rβ protein products include proteins encoded by IL-2Rβ, such as proteins containing the amino acid sequences of the NCBI reference sequences: NP_000869.1, NP_001333151.1, or NP_001333152.1, or mature IL-2Rβ proteins containing amino acids 27-551 of the NCBI reference sequence NP_000869.1. The IL-2Rβ co-stimulatory domain contains, for example, amino acids 266-551 of the NCBI reference sequence NP_000869.1: ncrntgpwlkkvlkcntpdpskffsqlssehggdvqkwlsspfpsssfspgglapeisplevlerdkvtqlllqqdkvpepaslssnhsltscftnqgyfffhlpdaleieacqvyftydpyseedpdegvagaptgsspqplqplsgeddayctfpsrddlllfspsllggpsppstapggsgageermppslqervprdwdpqplgpptpgvpdlvdfqpppelvlreageevpdagpregvsfpwsrppgqgefralnarlplntdaylslqelqgqdpthlv (Sequence ID 152).

[0141] As used herein, “IL2RA” (also known as IL2RA, interleukin-2 receptor subunit α, p55, CD25, IL2R, IMD41, TCGFR, and IDDM10) refers to the gene identified by Entrez gene number 3559, its allele variants, its orthologues, its protein products, and mRNA transcripts encoded by the gene, including the nucleotide sequence of NCBI reference sequence: NM_000417.3, NM_001308242.2, or NM_001308243.2. IL2RA protein products include proteins encoded by IL2RA, such as proteins containing the amino acid sequence of NCBI reference sequence: NP_000408.1, NP_001295171.1, or NP_001295172.1, or mature IL2RA proteins containing amino acids 22–272 of the amino acid sequence of NCBI reference sequence NP_000408.1. The co-stimulatory domain of IL2RA includes, for example, amino acids 260-272 of the amino acid sequence of the NCBI reference sequence NP_000408.1:

[0142] As used herein, "MYD88" (also known as MYD88 Innate Immune Signaling Adapter, Myeloid Differentiation Primary Response Protein MyD88 and MYD88D) refers to the gene identified by Entrez gene number 4615, its allele variants, its orthologues, its protein products, and the mRNA transcript encoded by the gene, which includes the nucleotide sequence of the NCBI reference sequence: NM_001172566.2, NM_001172567.2, NM_001172568.2, NM_001172569.3, NM_001365876.1, NM_001365877.1, NM_001374787.1, NM_001374788.1, or NM_002468.5. MYD88 protein products include proteins encoded by MYD88, such as proteins containing the amino acid sequences of NCBI reference sequences: NP_001166037.2, NP_001166038.2, NP_001166039.2, NP_001166040.2, NP_001352805.1, NP_001352806.1, NP_001361716.1, NP_001361717.1 and NP_002459.3. The co-stimulatory domain of MYD 88 contains, for example, amino acids 160-304 of the amino acid sequence of NCBI reference sequence NP_001166038.2: rfdaficycpsdiqfvqemirqleqtnyrlklcvsdrdvlpgtcvwsiaseliekrlarrprggcrrmvvvvsddylqskecdfqtkfalslspgahqkrlipikykamkkefpsilrfitvcdytnpctkswfwtrlakalslp (Sequence ID 179).

[0143] In some embodiments described herein, CAR may include all or part of the co-stimulatory domains described herein, such as 4-1BB, CD27, CD28, CD40, CD40L, TLR2, DAP10, OX40, IL-2RB, IL-2RA, MYD88, or ICOS co-stimulatory domains described herein. For example, in some embodiments described herein, the CAR comprises a co-stimulatory domain of about 10, about 12, about 15, about 20, about 22, about 25, about 30, about 35, about 37, about 40, about 41, about 45, about 47, about 50, about 60, about 61, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 134, about 140, about 144, about 150, about 160, about 170, about 174, about 180, about 190, about 200, about 225, about 250, about 275, about 285, about 290, or about 300 amino acids in length. For example, in some embodiments described herein, the CAR includes the 4-1BB, CD27, CD28, CD40, CD40L, TLR2, DAP10, OX40, IL-2RB, IL-2RA, MYD88, or ICOS costimulatory domains, or parts thereof, in lengths of approximately 10, approximately 12, approximately 15, approximately 20, approximately 22, approximately 25, approximately 30, and approximately 3. 5, contains a co-stimulatory domain of approximately 37, 40, 41, 45, 47, 50, 60, 61, 70, 80, 90, 100, 110, 120, 130, 134, 140, 144, 150, 160, 170, 174, 180, 190, 200, 225, 250, 275, 285, 290, or 300 amino acids.

[0144] In some embodiments described herein, the CAR includes a co-stimulatory domain having an amino acid sequence that is approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, approximately 90% to approximately 95%, approximately 95% to approximately 100%, or approximately 90% to approximately 100% identical to the 4-1BB, CD27, CD28, CD40, CD40L, TLR2, DAP10, OX40, IL-2RB, IL-2RA, MYD88, or ICOS co-stimulatory domain described herein.

[0145] Orthologues of genes, nucleotide sequences (e.g., mRNA sequences), and proteins described herein include, but are not limited to, mammalian orthologues, including mouse (i.e., Mus musculus) orthologues. CAR-T cells

[0146] CAR-expressing T cells (CAR-T cells) are T lymphocyte cells (T cells) that have been isolated and genetically engineered to express one or more CARs. CAR-T cells may be T cells isolated from a patient, for example, a patient requiring cancer treatment, that have been genetically engineered to express one or more CARs. The cells can be collected from the patient using any appropriate method, such as leukocyte apheresis or apheresis, followed by elutriation to remove bone marrow cells and other contaminating cells, and enrichment of T cells. Once isolated, T cells These T cells can be expanded and genetically modified by any suitable means, such as viral transduction (e.g., lentiviral or gamma-retroviral transduction), or transfection or electroporation with a suitable expression vector. The genetically modified T cells can then be cultured and expanded ex vivo before administration to a patient.

[0147] While not constrained by theory, CAR expression is thought to enable CAR-T cell targeting of target cell populations, such as cancer cells expressing specific cell surface antigens to which the antigen-specific targeting region of the CAR (e.g., scFv, Fab fragment, F(ab')2 fragment, or ligand) specifically binds. CAR binding to these specific cell surface antigens in complex with major histocompatibility complex molecules is thought to result in activation of a signaling cascade via intracellular signaling domains (e.g., FcRγ or CD3ζ signaling domains) and, if present, one or more costimulatory domains of the CAR (e.g., 4-1BB, CD27, CD28, CD40, CD40L, TLR2, DAP10, OX40, IL-2RB, IL-2RA, MYD88, and / or ICOS costimulatory domains). Therefore, the introduction of CARs into T cells is thought to enable T cells to target and kill target cell populations expressing cell surface antigens recognized by the CAR, via the same effector functions (e.g., FcRγ, CD3ζ, or costimulatory protein signaling) used by wild-type T cells to eliminate infected or transformed cells. For example, the introduction of CARs into T cells as described herein is effective in enabling T cells to target and kill target cancer cell populations expressing cell surface antigens recognized by the CAR (e.g., cancer cell antigens, tumor-associated antigens, or tumor-specific antigens) via FcRγ, CD3ζ, and / or costimulatory protein signaling.

[0148] In some embodiments described herein, the CAR-T cells of the present invention may be produced by introducing one or more viral vectors into isolated T cells or an isolated population of T cells. In some embodiments, the viral vector delivers a transgene encoding a CAR nucleotide sequence to the T cells. In some embodiments, the CAR nucleotide sequence includes nucleotide sequences encoding an antigen-specific targeting region, a transmembrane domain, and an intracellular signaling domain. In some embodiments, the CAR nucleotide sequence also includes nucleotide sequences encoding one or more costimulatory domains, hinge domains, spacer domains, and / or amino acid transporter domains, such as an arginine transporter domain.

[0149] CAR-T cells expressing arginine transporter and chimeric antigen receptor proteins (hereinafter referred to herein as "arg+CAR-T cells") that can be used for the treatment of solid tumor cancers and hematological cancers are provided herein.

[0150] In addition to CAR expression, CAR-T cells can also be genetically modified to co-express one or more distinct costimulatory proteins, including cytokines that enhance the function and persistence of CARs. For example, CAR-T cells can be programmed to co-express CD28, CD80, 4-1BB, 4-1BBL, CD86, OX40L, IL-12, IL-15, IL-18, and / or CD70 proteins. Furthermore, in some embodiments, CAR-T cells can be genetically modified to express two or more CARs targeting different cell surface antigens. In some embodiments, CAR-T cells can be genetically modified to express a single CAR targeting a single cell surface antigen.

[0151] The CAR-T cells of the present invention include first, second, third, fourth, and fifth generation CARs. CAR-T technology is described, for example, in Petersen and Krenciute, (2019) "Next Generation CAR T Cells for the Immunotherapy of High-Grade Glioma," Frontiers in Oncology, 9:1-9.

[0152] First-generation CARs include fusions of antigen-binding protein domains (e.g., CD8, CD4, CD25, CD16, or antibody-derived scFv), hinge / spacer domains, transmembrane domains, and signaling domains such as CD3ζ or FcRγ intracellular signaling domains.

[0153] Second-generation CARs include an antigen-binding protein domain, a hinge / spacer domain, a transmembrane domain, and a CD3ζ or FcRγ signaling domain, as well as an intracellular co-stimulatory domain (e.g., 4-1BB, CD27, CD28, CD40, CD40L, TLR2, DAP10, OX40, IL-2RB, IL-2RA, MYD88, or ICOS intracellular co-stimulatory domain).

[0154] Third-generation CARs contain all the components found in second-generation CARs, but also include multiple co-stimulatory domains (e.g., more than one 4-1BB, CD27, CD28, CD40, CD40L, TLR2, DAP10, OX40, IL-2RB, IL-2RA, MYD88, and / or ICOS intracellular co-stimulatory domains).

[0155] Fourth and fifth generation CARs (also known as armored CARs or TRUCKs) are further genetically engineered to express CARs and transgenes encoding one or more signaling proteins, such as cytokines or cytokine receptor proteins. For example, in some embodiments, CAR-T cells are genetically engineered to overexpress IL-12, IL-15, IL-18, IL-7R, CD28, CD80, 4-1BB, 4-1BBL, CD86, OX40L, or CD70. In some embodiments, overexpression of signaling proteins such as cytokines or cytokine receptor proteins is effective in providing CAR-T cells with enhanced persistence, proliferation, or antitumor activity.

[0156] Furthermore, CAR-T cells can be genetically engineered, for example using CRISPR / Cas9 gene editing tools, to delete genes that inhibit cell-specific checkpoints, such as PD-1 or CTLA4. Thus, in some embodiments, the CAR-T cells described herein are genetically engineered to delete or reduce PD-1 gene expression or CTLA4 gene expression. CAR-T cells can also be genetically engineered to delete diacylglycerol kinase (DGK). Thus, in some embodiments, the CAR-T cells described herein are genetically engineered to delete or reduce DGK gene expression, for example, DGKα and / or DGKζ isotype expression.

[0157] In some embodiments, CAR-T cells can be genetically engineered using, for example, a CRISPR / Cas9 gene editing tool to enable targeted CAR transgene insertion into the T cell genome. In some embodiments, CAR transgene insertion is mediated by an adeno-associated virus (AAV) vector encoding a CAR nucleotide sequence, such as the AAV6 vector. For example, in some embodiments, CAR-T cells are genetically engineered to insert a CAR transgene into the endogenous TCR gene sequence, such as the TCR alpha chain locus. In some embodiments, CAR-T cells can be genetically engineered using, for example, a CRISPR / Cas9 gene editing tool to replace the native T cell gene sequence with a mutant gene sequence. For example, in some embodiments, the CAR-T cells described herein are genetically engineered to replace the PD-1 gene sequence or the CXCR4 gene sequence with a mutant PD-1 gene sequence or the CXCR4 gene sequence, respectively.

[0158] In some embodiments, the CAR-T cells described herein include an episome encoding a CAR. In some embodiments, the CAR-T cells described herein include an embedded transgene encoding a CAR.

[0159] CAR-T cells genetically engineered to express amino acid transporter proteins, such as arginine transporter protein, are also described herein. In some embodiments described herein, CAR-T cells are CAT-1, CAT-2, CAT-3, CAT-4, y + LAT1, 4F2hc, y + LAT2, y + LAT1 and 4F2hc, y + LAT2 and 4F2hc, b 0,+ AT, rBAT, b 0,+ AT and rBAT, as well as ATB 0,+The cells are genetically engineered to express an arginine transporter protein selected from the group consisting of, or a combination thereof. For example, in some embodiments, the CAR-T cells described herein are genetically engineered to include a nucleotide sequence encoding an amino acid transporter, for example, a nucleotide sequence encoding an arginine transporter. In some embodiments, the CAR-T cells described herein include an episome encoding an amino acid transporter, for example, an arginine transporter. In some embodiments, the CAR-T cells described herein include a transgene encoding an amino acid transporter, for example, an arginine transporter.

[0160] CAR-T cells genetically engineered to express CAR and amino acid transporter proteins, such as arginine transporter proteins, are also described herein. For example, in some embodiments, the CAR-T cells described herein are genetically engineered to include nucleotide sequences encoding CAR and amino acid transporters, such as nucleotide sequences encoding arginine transporters. In some embodiments, the CAR-T cells described herein include episomes encoding CAR and amino acid transporters, such as arginine transporters. In some embodiments, the CAR-T cells described herein include transgenes encoding CAR and amino acid transporters, such as arginine transporters. In some embodiments, the CAR-T cells described herein include episomes encoding CAR and episomes encoding amino acid transporters, such as arginine transporters. In some embodiments, the CAR-T cells described herein include transgenes encoding CAR and transgenes encoding amino acid transporters, such as arginine transporters. Amino acid transporter proteins

[0161] This specification describes CAR-T cells genetically modified to express one or more amino acid transporter proteins (AATs). Amino acid transporters are membrane transport proteins that play a vital role in regulating energy metabolism, protein synthesis, gene expression, redox balance signaling pathways, and growth at the cellular and whole organism levels through amino acid transport. Because amino acids do not readily diffuse across lipid membranes, transmembrane transporter proteins are necessary to move amino acids across cells and between membrane-bound intracellular compartments. Amino acid transport is carried out by Na + H + , K + , and / or Cl - The movement of ions containing AAT may be coupled with the movement of other amino acids via antiportation. Dysregulation of AAT leads to metabolic reprogramming that alters intracellular amino acid levels contributing to pathogenesis. Dysregulation of AAT is involved in various pathological conditions, including autophagy and tumor cell proliferation, mediated by metabolic reprogramming and hereditary human metabolic disorders such as cystinuria. Due to these metabolic capacities, AAT may offer potential targets for anticancer drugs.

[0162] Amino acid transporter proteins are encoded by genes belonging to several families, including solute carrier (SLC) proteins; amino acid-polyamine-organic cation (APC) superfamily; amino acid / auxin permease (AAAP) family; dicarboxylate / amino acid:cation (Na+ or H+) sinporter (DAACS) family; branched-chain amino acid:cation sinporter (LIVCS) family; hydroxy / aromatic amino acid permease (HAAAP) family; branched-chain amino acid exporter (LIV-E) family; 6TMS neutral amino acid transporter (NAAT) family; basic amino acid antiporter (ArcD) family; and putative amino acid permease (PAAP) family. SLC proteins constitute the largest group of amino acid transporter proteins, containing more than 400 proteins distributed among 65 families.

[0163] Amino acid transporter proteins include sodium-dependent neutral amino acid transporters, sodium-independent neutral amino acid transporters, and sodium-dependent anionic amino acid transporter system X. - AG Sodium-independent anionic amino acid transporter system x C - They can be classified into sodium-dependent cationic amino acid transporters and sodium-independent cationic amino acid transporters. Amino acid transporter proteins regulate the transport of amino acids across the cell membrane, including arginine, glutamine, and leucine, as well as signaling compounds such as gamma-aminobutyric acid (GABA). Examples of amino acid transporter proteins are described, for example, in Ren et al., (2017) "Amino-acid transporters in T-cell activation and differentiation," Cell Death and Disease, 8, e2655. Arginine transporter protein

[0164] CAR-T cells genetically modified to express one or more arginine transporter proteins are also described herein. Arginine transporter proteins are encoded by genes belonging to the solute carrier gene (SLC) family. Most SLCs encode proteins localized to the cell membrane, but some members localize to mitochondria or other intracellular organelles. SLC family protein products can transport, for example, charged organic molecules, uncharged organic molecules, inorganic ions and / or ammonia across the cell membrane. SLC families that specifically encode transporter proteins capable of transporting arginine across the cell membrane include the SLC3, SLC6, and SLC7 families.

[0165] In mammals, cellular arginine availability is largely regulated by members of the SLC7 family, but the solute carrier gene superfamily includes six major families of AATs. The protein products of these transporter genes are characterized by having multiple transmembrane domains organized around a central pore region. Their efficiency and capacity in the plasma membrane greatly determine arginine availability in cells.

[0166] The SLC7 family is divided into two subgroups: cationic amino acid transporters (CATs) and L-type amino acid transporters (LATs). CATs function as monomers in the plasma membrane, while LATs are obligate heterodimers that transport transporters to the plasma membrane and form disulfide-bonded dimers with single transmembrane glycoproteins (SLC3s), which aid in protein stability. The CAT and LAT families exhibit various differences in their interactions with the SLC3 family, substrate specificity, and transport mechanisms. CATs are specific to cationic amino acids, including arginine. Initially, system y + CAT, which was called Na +They mediate the independent uptake of cationic amino acids with high affinity. In mammals, CATs act as exchangers or promoters. Arginine metabolism involves these cationic amino acid transporters. + It is significantly regulated by the expression of the system.

[0167] Arginine transporter proteins include CAT-1, CAT-2, CAT-3, CAT-4, and y + LAT2, 4F2hc, y + LAT1, b 0,+ AT, rBAT, and ATB 0,+ This includes. In some embodiments described herein, the arginine transporter is a single SLC family protein, e.g., CAT-1, CAT-2, CAT-3, CAT-4, or ATB 0,+ It is composed of. In some embodiments, the arginine transporter is a combination of SLC family proteins, for example y + LAT2 and 4F2hc, y + LAT1 and 4F2hc, or b 0,+ It consists of AT and rBAT.

[0168] Arginine transporter proteins can be sodium- and chloride-dependent or sodium-independent amino acid transporter proteins. An example of a sodium-independent amino acid transporter protein is y + (For example, CAT-1, CAT-2, CAT-3), y + L (for example, y + LAT1 or y + 4F2hc) combined with LAT2, and b 0,+ Examples of transport system members include sodium-dependent amino acid transporter proteins, such as B 0,+ Members of the transport system include: The arginine transporter system, which consists of a single protein, is y + and B 0,+ This includes members of the transporter system. In contrast, y + L and b0,+ The arginine transporter system is composed of glycoproteins (e.g., 4F2hc) and proteins.

[0169] As used herein, “SLC7A1” (also known as Solute Carrier Family 7 Member 1, ERR, ATRC1, CAT-1, HCAT1, and REC1L) refers to the gene identified by Entrez gene number 6541, its allele variants, its orthologs, and the mRNA transcript encoded by the gene, including the nucleotide sequence of the NCBI reference sequence NM_003045.5 (SEQ ID NO: 180).

[0170] The cationic amino acid transporter 1 (CAT-1) proteins described herein include the protein sequence encoded by SLC7A1, the amino acid sequence of the NCBI reference sequence NP_003036.1, and the amino acid sequence of the NCBI consensus coding sequence (CCDS) ID number CCDS9333.1. MGCKVLLNIGQQMLRRKVVDCSREETRLSRCLNTFDLVALGVGSTLGAGVYVLAGAVARENAGPAIVISFLIAALASVLAGLCYGEFGARVPKTGSAYLYSYVTVGELWAFIITGWNLILSYIIGTSSVARAWSATFDELIGRPIGEFSRTHMTLNAP GVLAENPDIFAVIIILILTGLLTLGVKESAMVNKIFTCINVLVLGFIMVSGFVKGSVKNWQLTEEDFGNTSGRLCLNNDTKEGKPGVGGFMPFGFSGVLSGAATCFYAFVGFDCIATTGEEVKNPQKAIPVGIVASLLICFIAYFGVSAALTLMMPY FCLDNNSPLPDAFKHVGWEGAKYAVAVGSLCALSASLLGSMFPMPRVIYAMAEDGLLFKFLANVNDRTKTPIIATLASGAVAAVMAFLFDLKDLVDLMSIGTLLAYSLVAACVLVLRYQPEQPNLVYQMASTSDELDPADQNELASTNDSQLGFLPEAEMFSLKTILSPKNMEPSKISGLIVNISTSLIAVLIITFCIVTVLGREALTKGALWAVFLLAGSALLCAVVTGVIWRQPESKTKLSFKVPFLPVLPILSIFVNVYLMMQLDQGTWVRFAVWMLIGFIIYFGYGLWHSEEASLDADQARTPDGNLDQCK (Sequence ID 182).

[0171]

[0172] As used herein, “SLC7A2” (also known as Solute Carrier Family 7 Member 2, CAT2, ATRC2, and HCAT2) refers to the gene identified by Entrez gene number 6542, its allele variants, its orthologs, and the mRNA transcripts encoded by the gene, including the nucleotide sequences of the NCBI reference sequences: NM_001008539.4 (SEQ ID NO: 184), NM_001164771.2 (SEQ ID NO: 185), NM_001370337.1 (SEQ ID NO: 186), NM_001370338.1 (SEQ ID NO: 187), or NM_003046.6 (SEQ ID NO: 188).

[0173] The cationic amino acid transporter 2 (CAT-2) proteins described herein include the amino acid sequences of the protein sequence encoded by SLC7A2 and the NCBI reference sequences: NP_001008539.3, NP_001158243.1, NP_001357266.1, NP_001357267.1, or NP_003037.4. CAT-2 can be represented as multiple isoforms, including CAT-2A (identified as the amino acid sequence of NCBI CCDS ID number CCDS6002.2): MKIETSGYNSDKLICRGFIGTPAPPVCDSKFLLSPSSDVRMIPCRAALTFARCLIRRKIVTLDSLEDTKLCRCLSTMDLIALGVGSTLGAGVYVLAGEVAKADSGPSIVVSFLIAALASVMAGLCYAEFGARVPKTGSAYLYTYVTVGELWAFITGWNLILSYVIGTSSVARAWSGTFDELLSKQIGQFLRTYFRMNYTGLAEYPDFFAVCLILLLAGLLSFGVKESAWVNKVFTAVNILVLLFVMVAGFVKGNVANWKISEEFLKNISASAREPPSENGTSIYGAGGFMPYGFTGTLAGAATCFYAFVGFDCIATTGEEVRNPQKAIPIGIVTSLLVCFMAYFGVSAALTLMMPYYLLDEKSPLPVAFEYVGWGPAKYVVAAGSLCALSTSLLGSMFPLPRILFAMARDGLLFRFLARVSKRQSPVAATLTAGVISALMAFLFDLKALVDMMSIGTLMAYSLVAACVLILRYQPGLSYDQPKCSPEKDGLGSSPRVTSKSESQVTMLQRQGFSMRTLFCPSLLPTQQSASLVSFLVGFLAFLVLGLSVLTTYGVHAITRLEAWSLALLALFLVLFVAIVLTIWRQPQNQQKVAFMVPFLPFLPAFSILVNIYLMVQLSADTWVRFSIWMAIGFLIYFSYGIRHSLEGHLRDENNEEDAYPDNVHAAAEEKSAIQANDHHPRNLSSPFIFHEKTSEF (SEQ ID NO: 194)); and CAT-2B (identified as the amino acid sequence of NCBI CCDS ID number CCDS34852.1): MIPCRAALTFARCLIRRKIVTLDSLEDTKLCRCLSTMDLIALGVGSTLGAGVYVLAGEVAKADSGPSIVVSFLIAALASVMAGLCYAEFGARVPKTGSAYLYTYVTVGELWAFITGWNLILSYVIGTSSVARAWSGTFDELLSKQIGQFLRTYFRMNYTGLAEYPDFFAVCLILLLAGLLSFGVKESAWVNKVFTAVNILVLLFVMVAGFVKGNVANWKISEEFLKNISASAREPPSENGTSIYGAGGFMPYGFTGTLAGAATCFYAFVGFDCIATTGEEVRNPQKAIPIGIVTSLLVCFMAYFGVSAALTLM MPYYLLDEKSPLPVAFEYVGWGPAKYVVAAGSLCALSTSLLGSIFPMPRVIYAMAEDGLLFKCLAQINSKTKTPIIATLSSGAVAALMAFLFDLKALVDMMSIGTLMAYSLVAACVLILRYQPGLSYDQPKCSPEKDGLGSSPRVTSKSESQVTMLQRQGFSMRTLFCPSLLPTQQSASLVSFLVGFLAFLVLGLSVLTTYGVHAITRLEAWSLALLALFLVLFVAIVLTIWRQPQNQQKVAFMVPFLPFLPAFSILVNIYLMVQLSADTWVRFSIWMAIGFLIYFSYGIRHSLEGHLRDENNEEDAYPDNVHAAAEEKSAIQANDHHPRNLSSPFIFHEKTSEF (Sequence ID 195).

[0174]

[0175] (Sequence ID 197).

[0176] CAT-2A proteins containing one or more naturally occurring or engineered amino acid mutations are also described herein. For example, CAT-2A proteins containing substitution and / or insertion mutations are described herein. CAT-2A amino acid sequences may include, for example, the amino acid mutations R369E, N381i or R369E and N381i. Examples of CAT-2A amino acid sequences containing the R369E, N381i, and R369E / N381i mutations include: MIPCRAALTFARCLIRRKIVTLDSLEDTKLCRCLSTMDLIALGVGSTLGAGVYVLAGEVAKADSGPSIVVSFLIAALASVMAGLCYAEFGARVPKTGSAYLYTYVTVGELWAFITGWNLILSYVIGTSSVARAWSGTFDELLSKQIGQFLRTYFRMNYTGLAEYPDFFAVCLILLLAGLLSFGVKESAWVNKVFTAVNILVLLFVMVAGFVKGNVANWKISEEFLKNISASAREPPSENGTSIYGAGGFMPYGFTGTLAGAATCFYAFVGFDCIATTGEEVRNPQKAIPIGIVTSLLVCF MAYFGVSAALTLMMPYYLLDEKSPLPVAFEYVGWGPAKYVVAAGSLCALSTSLLGSMFPLPRILFAMAEDGLLFRFLARVSKRQSPVAATLTAGVISALMAFLFDLKALVDMMSIGTLMAYSLVAACVLILRYQPGLSYDQPKCSPEKDGLGSSPRVTSKSESQVTMLQRQGFSMRTLFCPSLLPTQQSASLVSFLVGFLAFLVLGLSVLTTYGVHAITRLEAWSLALLALFLVLFVAIVLTIWRQPQNQQKVAFMVPFLPFLPAFSILVNIYLMVQLSADTWVRFSIWMAIGFLIYFSYGIRHSLEGHLRDENNEEDAYPDNVHAAAEEKSAIQANDHHPRNLSSPFIFHEKTSEF (Sequence ID 198); MIPCRAALTFARCLIRRKIVTLDSLEDTKLCRCLSTMDLIALGVGSTLGAGVYVLAGEVAKADSGPSIVVSFLIAALASVMAGLCYAEFGARVPKTGSAYLYTYVTVGELWAFITGWNLILSYVIGTSSVARAWSGTFDELLSKQIGQFLRTYFRMNYTGLAEYPDFFAVCLILLLAGLLSFGVKESAWVNKVFTAVNILVLLFVMVAGFVKGNVANWKISEEFLKNISASAREPPSENGTSIYGAGGFMPYGFTGTLAGAATCFYAFVGFDCIATTGEEVRNPQKAIPIGIVTSLLVCFMAYFGVSAALTLMMPYYLLDEKSPLPVAFEYVGWGPAKYVVAAGSLCALSTSLLGSMFPLPRILFAMARDGLLFRFLARVNSKRQSPVAATLTAGVISALMAFLFDLKALVDMMSIGTLMAYSLVAACVLILRYQPGLSYDQPKCSPEKDGLGSSPRVTSKSESQVTMLQRQGFSMRTLFCPSLLPTQQSASLVSFLVGFLAFLVLGLSVLTTYGVHAITRLEAWSLALLALFLVLFVAIVLTIWRQPQNQQKVAFMVPFLPFLPAFSILVNIYLMVQLSADTWVRFSIWMAIGFLIYFSYGIRHSLEGHLRDENNEEDAYPDNVHAAAEEKSAIQANDHHPRNLSSPFIFHEKTSEF (SEQ ID NO: 199); and MIPCRAALTFARCLIRRKIVTLDSLEDTKLCRCLSTMDLIALGVGSTLGAGVYVLAGEVAKADSGPSIVVSFLIAALASVMAGLCYAEFGARVPKTGSAYLYTYVTVGELWAFITGWNLILSYVIGTSSVARAWSGTFDELLSKQIGQFLRTYFRMNYTGLAEY PDFFAVCLILLLAGLLSFGVKESAWVNKVFTAVNILVLLFVMVAGFVKGNVANWKISEEFLKNISASAREPPSENGTSIYGAGGFMPYGFTGTLAGAATCFYAFVGFDCIATTGEEVRNPQKAIPIGIVTSLLVCFMAYFGVSAALTLMMPYYLLDEKSPLPVAF EYVGWGPAKYVVAAGSLCALSTSLLGSMFPLPRILFAMAEDGLLFRFLARVNSKRQSPVAATLTAGVISALMAFLFDLKALVDMMSIGTLMAYSLVAACVLILRYQPGLSYDQPKCSPEKDGLGSSPRVTSKSESQVTMLQRQGFSMRTLFCPSLLPTQQSASLVSFLVGFLAFLVLGLSVLTTYGVHAITRLEAWSLALLALFLVLFVAIVLTIWRQPQNQQKVAFMVPFLPFLPAFSILVNIYLMVQLSADTWVRFSIWMAIGFLIYFSYGIRHSLEGHLRDENNEEDAYPDNVHAAAEEKSAIQANDHHPRNLSSPFIFHEKTSEF (Sequence ID 200).

[0177]

[0178] As used herein, “SLC7A3” (also known as Solute Carrier Family 7 Member 3, CAT3, ATRC3, and CAT-3) refers to the gene identified by Entrez gene number 84889, its allele variants, its orthologs, and the mRNA transcripts encoded by the gene, including the nucleotide sequences of the NCBI reference sequence: NM_001048164.3 (SEQ ID NO: 204) or NM_032803.6 (SEQ ID NO: 205).

[0179] The cationic amino acid transporter 3 (CAT-3) proteins described herein include the protein sequence encoded by SLC7A3, the amino acid sequences of NCBI reference sequences NP_001041629.1 and NP_116192.4, and the amino acid sequence of NCBI CCDS ID number CCDS14404.1. MPWQAFRRFGQKLVRRRTLESGMAETRLARCLSTLDLVALGVGSTLGAGVYVLAGEVAKDKAGPSIVICFLVAALSSVLAGLCYAEFGARVPRSGSAYLYSYVTVGELWAFTTGWNLILSYVIGTASVARAWSSAFDNLIGNHISKTLQGSIAL HVPHVLAEYPDFFALGLVLLLTGLLALGASESALVTKVFTGVNLLVLGFVMISGFVKGDVHNWKLTEEDYELAMAELNDTYSLGPLGSGGFVPFGFEGILRGAATCFYAFVGFDCIATTGEEAQNPQRSIPMGIVISLSVCFLAYFAVSSALTLM MPYYQLQPESPLPEAFLYIGWAPARYVVAVGSLCALSTSLLGSMFPMPRVIYAMAEDGLLFRVLARIHTGTRTPIIATVVSGIIAAFMAFLFKLTDLVDLMSIGTLLAYSLVSICVLILRYQPDQETKTGEEVELQEEAITTESEKLTLWGLFFPLNSIPTPLSGQIVYVCSSLLAVLLTALCLVLAQWSVPLLSGDLLWTAVVVLLLLLIIGIIVVIWRQPQSSTPLHFKVPALPLLPLMSIFVNIYLMMQMTAGTWARFGVWMLIGFAIYFGYGIQHSLEEIKSNQPSRKSRAKTVDLDPGTLYVHSV (Sequence ID 208).

[0180]

[0181] As used herein, “SLC7A4” (also known as solute carrier family 7 member 4, VH, CAT4, CAT-4, and HCAT3) refers to the gene identified by Entrez gene number 6545, its allele variants, its orthologs, and the mRNA transcript encoded by the gene, including the nucleotide sequence of the NCBI reference sequence NM_004173.3 (SEQ ID NO: 210).

[0182] The cationic amino acid transporter 4 (CAT-4) proteins described herein include the protein sequence encoded by SLC7A4, the amino acid sequence of the NCBI reference sequence NP_004164.2, and the amino acid sequence of the NCBI CCDS ID number CCDS33608.1. MARGLPTIASLARLCQKLNRLKPLEDSTMETSLRRCLSTLDLTLLGVGGMVGSGLYVLTGAVAKEVAGPAVLLSFGVAAVASLLAALCYAEFGARVPRTGSAYLFTYVSMGELWAFLIGWNVLLEYIIGGAAVARAWSGYLDSMFSHSIRNFTETHVGSWQVPLLGHYPDFLAAGIILLASAFVSCGARVSSWLNHTFSAISLLVILFIVILGFILAQPHNWSADEGGFAPFGFSGVMAGTASCFYAFVGFDVIAASSEEAQNPRRSVPLAIAISLAIAAGAYILVSTVLTLMVPWHSLDPDSALADAFYQRGYRWA GFIVAAGSICAMNTVLLSLLFSLPRIVYAMAADGLFFQVFAHVHPRTQVPVAGTLAFGLLTAFLALLLDLESLVQFLSLGTLLAYTFVATSIIVLRFQKSSPPSSPGPASPGPLTKQQSSFSDHLQLVGTVHASVPEPGELKPALRPYLGFLDGYSPGAVVTWALGVMLASAITIGCVLVFGNSTLHLPHWGYILLLLLTSVMFLLSLLVLGAHQQQYREDLFQIPMVPLIPALSIVLNICLMLKLSYLTWVRFSIWLLMGLAVYFGYGIRHSKENQRELPGLNSTHYVVFPRGSLEETVQAMQPPSQAPAQDPGHME (Sequence ID 212).

[0183]

[0184] The term "SLC7A6" used herein refers to members 6, LAT3, LAT-2 and y of the solute carrier family 7. + LAT-2 (also known as LAT-2) refers to the mRNA transcript encoded by the gene, which includes the gene identified by Entrez gene ID number 9057, its allele variants, its orthologues, and the nucleotide sequences of the NCBI reference sequence: NM_001076785.3 (SEQ ID NO: 214) or NM_003983.6 (SEQ ID NO: 215).

[0185] y + L amino acid transporter 2(Y) + The LAT2) protein includes the protein sequence encoded by SLC7A6, the amino acid sequences of the NCBI reference sequences NP_001070253.1 and NP_003974.3, and the amino acid sequence of NCBI CCDS ID number CCDS 32470.1. MEAREPGRPTPTYHLVPNTSQSQVEEDVSSPPQRSSETMQLKKEISLLNGVSLVVGNMIGSGIFVSPKGVLVHTASYGMSLIVWAIGGLFSVVGALCYAELGTTITKSGASYAYILEAFGGFIAFIRLWVSLLVVEPTGQAIIAITFANYIIQPSFPSCDPPYLACRLLAAACICLLTFVNCAYVKWGTRVQDTFTYAKVVALIAIIVMGLVKLCQGHSEHFQDAFEGSSWDMGNLSLALYSALFSYSGWDTLNFVT EEIKNPERNLPLAIGISMPIVTLIYILTNVAYYTVLNISDVLSSDAVAVTFADQTFGMFSWTIPIAVALSCFGGLNASIFASSRLFFVGSREGHLPDLLSMIHIERFTPIPALLFNCTMALIYLIVEDVFQLINYFSFSYWFFVGLSVVGQLYLRWKEPKRPRPLKLSVFFPIVFCICSVFLVIVPLFTDTINSLIGIGIALSGVPFYFMGVYLPESRRPLFIRNVLAAITRGTQQLCFCVLTELDVAEEKKDERKTD (Sequence No. 218).

[0186] y +

[0187] The term "SLC7A7" used herein refers to members 7 of the solute carrier family 7, LPI, LAT3, MOP-2, Y+LAT1 and y + LAT-1 (also known as LAT-1) refers to the mRNA transcript encoded by the gene, which includes the gene identified by Entrez gene number 9056, its allele variants, its orthologues, and the nucleotide sequences of the NCBI reference sequences: NM_001126105.3 (SEQ ID NO: 220), NM_003982.4 (SEQ ID NO: 221), and NM_001126106.4 (SEQ ID NO: 222).

[0188] y described herein + L amino acid transporter 1(y) +The LAT1) protein includes the amino acid sequences of the protein encoded by SLC7A7, NCBI reference sequences NP_001119578.1 and NP_003973.3, and NCBI CCDS ID number CCDS9574.1. MVDSTEYEVASQPEVETSPLGDGASPGPEQVKLKKEISLLNGVCLIVGNMIGSGIFVSPKGVLIYSASFGLSLVIWAVGGLFSVFGALCYAELGTTIKKSGASYAYILEAFGGFLAFIRLWTSLLIIEPTSQAIIAITFANYMVQPLFPSCFAPYAASRLLAAACICLLTFINCAYVKWGTLVQDIFTYAKVLALIAVIVAGIVRLGQGASTHFENSFEGSSFAVGDIALALYSALFSYSGWDTLNYVTEEIKNP ERNLPLSIGISMPIVTIIYILTNVAYYTVLDMRDILASDAVAVTFADQIFGIFNWIIPLSVALSCFGGLNASIVAASRLFFVGSREGHLPDAICMIHVERFTPVPSLLFNGIMALIYLCVEDIFQLINYYSFSYWFFVGLSIVGQLYLRWKEPDRPRPLKLSVFFPIVFCLCTIFLVAVPLYSDTINSLIGIAIALSGLPFYFLIIRVPEHKRPLYLRRIVGSATRYLQVLCMSVAAEMDLEDGGEMPKQRDPKSN (Sequence ID 225).

[0189] y +

[0190] As used herein, “SLC3A2” (also known as solute carrier family 3 member 2, 4F2, CD98, MDU1, 4F2HC, 4T2HC, NACAE, and CD98HC) refers to the gene identified by Entrez gene number 6520, its allele variants, its orthologues, and mRNA transcripts encoded by the gene, including the nucleotide sequences of the NCBI reference sequences: NM_001012662.3 (SEQ ID NO: 227), NM_001012664.3 (SEQ ID NO: 228), NM_001013251.3 (SEQ ID NO: 229), or NM_002394.6 (SEQ ID NO: 230).

[0191] The 4F2 cell surface antigen heavy chain (4F2hc) protein described herein includes the protein encoded by SLC3A2, as well as the amino acid sequence of the NCBI reference sequence NP_002385.3 and NCBI CCDS ID number CCDS8039.2. MELQPPEASIAVVSIPRQLPGSHSEAGVQGLSAGDDSELGSHCVAQTGLELLASGDPLPSASQNAEMIETGSDCVTQAGLQLLASSDPPALASKNAEVTGTMSQDTEVDMKEVELNELEPEKQPMNAASGAAMSLAGAEKNGLVKIKVAEDEAEAAAAAKFTGLSKEELLKVAGSPGWVRTRWALLLLFWLGWLGMLAGAVVIIVRAPRCRELPAQKWWHTGALYRIGDLQAFQGHGAGNLAGLKGRLDYLSSLKVKGLVLGPIHKNQKDDVAQTDLLQIDPNFGSKEDFDSLLQSAKKKSIRVILDLTPNYRGE NSWFSTQVDTVATKVKDALEFWLQAGVDGFQVRDIENLKDASSFLAEWQNITKGFSEDRLLIAGTNSSDLQQILSLLESNKDLLLTSSYLSDSGSTGEHTKSLVTQYLNATGNRWCSWSLSQARLLTSFLPAQLLRLYQLMLFTLPGTPVFSYGDEIGLDAAALPGQPMEAPVMLWDESSFPDIPGAVSANMTVKGQSEDPGSLLSLFRRLSDQRSKERSLLHGDFHAFSAGPGLFSYIRHWDQNERFLVVLNFGDVGLSAGLQASDLPASASLPAKADLLLSTQPGREEGSPLELERLKLEPHEGLLLRFPYAA (arrangement number) No. 232).

[0192]

[0193] As used herein, “SLC7A9” (also known as solute carrier family 7 member 9, BAT1, and CSNU3) refers to the gene identified by Entrez gene ID number 11136, its allele variants, its orthologues, and the mRNA transcripts encoded by the gene, including the nucleotide sequences of the NCBI reference sequences NM_001126335.2 (SEQ ID NO: 234), NM_001243036.2 (SEQ ID NO: 235), and NM_014270.5 (SEQ ID NO: 236).

[0194] The sodium-dependent neutral amino acid transporter BAT1(b) described herein 0,+ The AT) protein includes the protein encoded by SLC7A9, as well as the amino acid sequences of the NCBI reference sequences NP_001119807.1, NP_001229965.1, NP_055085.1 and NCBI CCDS ID number CCDS12425.1. MGDTGLRKRREDEKSIQSQEPKTTSLQKELGLISGISIIVGTIIGSGIFVSPKSVLSNTEAVGPCLIIWAACGVLATLGALCFAELGTMITKSGGEYPYLMEAYGPIPAYLFSWASLIVIKPTSFAIICLSFSEYVCAPFYVGCKPPQIVVKCLAAAAILFISTVNSLSVRLGSYVQNIFTAAKLVIVAIIIISGLVLLAQGNTKNFDNSFEGAQLSVGAISLAFYNGLWAYDGWNQLNYITE ELRNPYRNLPLAIIIGIPLVTACYILMNVSYFTVMTATELLQSQAVAVTFGDRVLYPASWIVPLFVAFSTIGAANGTCFTAGRLIYVAGREGHMLKVLSYISVRRLTPAPAIIFYGIIATIYIIPGDINSLVNYFSFAAWLFYGLTILGLIVMRFTRKELERPIKVPVVIPVLMTLISVFLVLAPIISKPTWEYLYCVLFILSGLLFYFLFVHYKFGWAQKISKPITMHLQMLMEVVPPEEDPE (Sequence ID 240).

[0195] b 0,+

[0196] As used herein, "SLC3A1" (also known as solute carrier family 3 member 1, D2H, ATR1, NBAT, RBAT, and CSNU1) refers to the gene identified by Entrez gene ID number 6519, its allele variants, its orthologs, and the mRNA transcript encoded by the gene, including the nucleotide sequence of the NCBI reference sequence NM_000341.4 (SEQ ID NO: 242).

[0197] The neutral and basic amino acid transport protein rBAT (rBAT) proteins described herein include the protein encoded by SLC3A1, as well as the amino acid sequences of NCBI reference sequence NP_000332.2 and NCBI CCDS ID number CCDS1819.1. MAEDKSKRDSIEMSMKGCQTNNGFVHNEDILEQTPDPGSSTDNLKHSTRGILGSQEPDFKGVQPYAGMPKEVLFQFSGQARYRIPREILFWLTVASVLVLIAATIAIIALSPKCLDWWQEGPMYQIYPRSFKDSNKDGNGDLKGIQDKLDYITALNIKTVWITSFYKSSLK DFRYGVEDFREVDPIFGTMEDFENLVAAIHDKGLKLIIDFIPNHTSDKHIWFQLSRTRTGKYTDYYIWHDCTHENGKTIPPNNWLSVYGNSSWHFDEVRNQCYFHQFMKEQPDLNFRNPDVQEEIKEILRFWLTKGVDGFSLDAVKFLLEAKHLRDEIQVNKTQIPDTVTQ YSELYHDFTTTQVGMHDIVRSFRQTMDQYSTEPGRYRFMGTEAYAESIDRTVMYYGLPFIQEADFPFNNYLSMLDTVSGNSVYEVITSWMENMPEGKWPNWMIGGPDSSRLTSRLGNQYVNVMNMLLFTLPGTPITYYGEEIGMGNIVAANLNESYDINTLRSKSPMQWDNSSNAGFSEASNTWLPTNSDYHTVNVDVQKTQPRSALKLYQDLSLLHANELLLNRGWFCHLRNDSHYVVYTRELDGIDRIFIVVLNFGESTLLNLHNMISGLPAKMRIRLSTNSADKGSKVDTSGIFLDKGEGLIFEHNTKNLLHRQTAFRDRCFVSNRACYSSVLNILYTSC (Sequence ID 244).

[0198]

[0199] As used herein, "SLC6A14" (also known as solute carrier family 6 member 14 and BMIQ11) refers to the gene identified by Entrez gene ID number 11254, its allele variants, its orthologs, and the mRNA transcript encoded by the gene, including the nucleotide sequence of the NCBI reference sequence NM_007231.5 (sequence number 246).

[0200] Sodium and chloride-dependent neutral and basic amino acid transporter B described herein 0、+ (ATB 0、+The protein includes the protein encoded by SLC6A14, as well as the amino acid sequences of the NCBI reference sequence NP_009162.1 and NCBI CCDS ID number CCDS14570.1. MDKLKCPSFFKCREKEKVSASSENFHVGENDENQDRGNWSKKSDYLLSMIGYAVGLGNVWRFPYLTYSNGGGAFLIPYAIMLALAGLPLFFLECSLGQFASLGPVSVWRILPLFQGVGITMVLISIFVTIYYNVIIAYSLYYMFASFQSELPWKNCSSWSDKNCSRSPIVTHCNVSTVNKGIQEIIQMNKSWVDINNFTCINGSEIYQPGQLPSEQYWNKVALQRSSGMNETGVIVWYLALCLLLAWLIVGAALFKGIKSSGKVVYFTALFPYVVLLILLVRGATLEGASKGISYYIGAQSNFTKLKEAEVWKDAATQIFY SLSVAWGGLVALSSYNKFKNNCFSDAIVVCLTNCLTSVFAGFAIFSILGHMAHISGKEVSQVVKSGFDLAFIAYPEALAQLPGGPFWSILFFFMLLTLGLDSQFASIETITTTIQDLFPKVMKKMRVPITLGCCLVLFLLGLVCVTQAGIYWVHLIDHFCAGWGILIAAILELVGIIWIYGGNRFIEDTEMMIGAKRWIFWLWWRACWFVITPILLIAIFIWSLVQFHRPNYGAIPYPDWGVALGWCMIVFCIIWIPIMAIIKIIQAKGNIFQRLISCCRPASNWGPYLEQHRGERYKDMVDPKKEADHEIPTVSGSRKPE (Sequence ID 248).

[0201] ATB 0,+

[0202] Activated T cells dramatically increase arginine transport via upregulation of cationic amino acid transporters. Upregulation of CAT promotes T cell proliferation. Arginine deficiency is fundamental in immunosuppression associated with inflammation and cancer and causes severe impairment of T cell function. In response to arginine deficiency, T cells induce autophagy to increase access to arginine within the cell. This cellular defense mechanism maintains T cell viability but cannot maintain cell proliferation.

[0203] Bone marrow-derived suppressor cells can directly promote immune dysfunction by depriving T cells of essential metabolites such as arginine, or by inhibiting T cell viability, migration, or activation. MDSCs can also indirectly suppress T cells by inducing other immunomodulatory cells, such as T regulatory cells and tumor-associated macrophages, thereby increasing competition for resources. Arginine availability modulates many of these activities. Polymorphonuclear MDSCs, the primary source of arginase-1 in tumor-bearing hosts, secrete arginase-1 and reduce extracellular arginine by enhancing arginine uptake via cationic amino acid transporters. Reorganizing adaptive immune function in situations of arginine-mediated tumor immunity evasion is a potential therapeutic strategy for boosting immunological antitumor responses.

[0204] Methods for rescuing T cell proliferation and activity in environments where arginine availability is limited (e.g., in TME) when bone marrow cells and cancer cells overcome T cells for arginine are described herein. In some embodiments, CAR-T cells overexpressing a specific amino acid transporter or combination of amino acid transporters are described herein. In some embodiments, CAR-T cells overexpressing an arginine transporter are described herein. In some embodiments, CAR-T cells expressing or overexpressing an amino acid transporter capable of transporting arginine from the extracellular space into the cytosol of the CAR-T cell are described herein. In some embodiments, the amino acid transporter is a human amino acid transporter for reducing immunogenicity, but may be modified from another species. For example, in some embodiments, the amino acid transporter is a humanized amino acid transporter. Table 1 lists human amino acid transporters capable of bidirectional transport of cationic amino acids such as arginine. [Table 1]

[0205] Members of the CAT family transport cationic amino acids by promoting diffusion through different trans-facilitations by intracellular substrates. In some cells, they can regulate the rate of NO synthesis by controlling the uptake of L-arginine as a substrate for nitric oxide synthase. At normal physiological concentrations, the biochemical system is primarily represented by cationic amino acid transporter type 1 (CAT-1). + Carriers are the major cellular transporter systems that cross the plasma membrane. CAT-1 is encoded by the SLC7A1 gene, is widely distributed in many systems, and is important for various cellular functions. CAT-1 is Na + It is an independent transporter and has the highest affinity for arginine (lowest K MIt possesses ), enabling efficient transport even at low arginine concentrations. The strong transform-enhancing properties of CAT-1 indicate that it performs better in exchange than uniport mode.

[0206] This disclosure also relates to CAT-2A R369E CAT-2A N381i and CAT-2A R369E / N381i We intend to create artificial variants of CAT-2A isoforms such as the apparent K of cationic amino acids. M The values ​​and the sensitivity of CAT-1, CAT-2B, and CAT-3 to transform enhancement are in system y + While characteristic of CAT-1, CAT-2A exhibits 10 times lower substrate affinity and is largely independent of the substrate on the trans side of the membrane. This variant is artificially created by transplanting two amino acids from the intracellular domain of CAT-1 to the homologous domain of CAT-2A. Specifically, the Arg residue at position 369 is replaced with a Glu residue (R369E), while an Asn residue is inserted at position 381. The resulting variant does not undergo trans-enhancement but exhibits K2 affinity comparable to CAT-1. M It has.

[0207] CAR-T cells exhibit target specificity comparable to monoclonal antibodies, demonstrate effector function of cytotoxic T cells, and make CAR-T therapy attractive for a variety of diseases. These characteristics enable antigen recognition independent of the major histocompatibility complex and allow for design to specifically target conserved essential epitopes of antigens.

[0208] TMEs in solid tumors present a harsh environment where a barrage of immunosuppressive signals and a deficiency of essential nutrients lead to T cell exhaustion. In particular, arginine is rapidly consumed by active cancer cells and degraded by various arginases secreted by infiltrating bone marrow-derived suppressor cells. Furthermore, T cells cannot regenerate arginine from other amino acids and depend on exogenous arginine supply. We have discovered that by enhancing CAR-T cells with arginine transporters, these cells can better compete for arginine in these harsh microenvironments.

[0209] Overexpression of arginine transporters can also be used, for example, to prime enlarged CAR-T cells before reinjection into a patient. CAR-T cells expressing arginine transporters can be cultured ex vivo under arginine-rich conditions until they acquire sufficient arginine to maintain expression and subsequent antitumor activity within the TME. Intracellular arginine enrichment via in vitro priming of T cells can enhance the survival, lifespan, activity, and therapeutic efficacy of CAR-T cells.

[0210] Exemplary arginine transporters include CAT-1, CAT-2, CAT-3, and ATB. 0,+ These include arginine transporters, y + LAT1+4F2hc, y + LAT2+4F2hc, or b 0,+ AT+rBAT is also intended. For example, CAT-1, CAT-2 and CAT-3 are Na + or Cl - If it does not cotransport and is overexpressed, the effect on membrane potential may be minimal. CAT-1 has high affinity for arginine (i.e., the lowest K m ) possesses a property that may enable efficient transport even at low arginine concentrations. CAT-2 activity may not be affected by trans-promoting.

[0211] arg+CAR-T cells may express arginine transporters containing one or more mutations. Appropriate amino acid modifications to improve arginine transporter expression can be conservative or non-conservative mutations. Mutations can be induced so that the encoded transporter is modified into a polar, nonpolar, basic, or acidic amino acid transporter. Manipulated CAR-T cells can be generated from the whole blood of a subject, where T cells are isolated from whole blood products and remanufactured in the laboratory by inserting genes into the cells via a vector to produce chimeric antigen receptors on their surface that specifically target the antigen of interest. These modified T cells are increased and returned to the subject's bloodstream, where they continue to increase. While not bound by theory, it is thought that CAR-T cells, when administered to a subject, are attracted to targets on the surface of cancer cells. While not bound by theory, it is thought that CAR-T cells identify cells expressing the target antigen and kill them. CAR-T cells may remain in the body after an acute attack, preventing the target cells from returning. Method for producing CAR-T cells

[0212] The CAR-T cells described herein may be produced from immune cells, such as CD4+ and CD8+ T cells, collected from a subject, such as a patient requiring treatment. A suitable T cell population can be collected and isolated from whole blood using apheresis / leukocyte apheresis in combination with a cell separation method, such as countercurrent centrifugation. Methods for isolating T cell populations are known in the art and can be carried out using suitable equipment, such as Haemonetics Cell Saver (Haemonetics, Boston, Massachusetts) and / or CliniMACS Prodigy (Miltenyi Biotec, Germany). The isolated T cells can be expanded, proliferated, and stimulated using methods known in the art, such as culturing them in the presence of, for example, anti-CD3 antibody, anti-CD28 antibody, magnetic bead conjugate anti-CD3 antibody, magnetic bead conjugate anti-CD28 antibody, growth factors (e.g., IL-2) and artificial antigen-presenting cells, using, for example, feeder cells and / or bioreactors. Suitable bioreactor systems include CliniMACS Prodigy (Miltenyi Biotec, Germany), WAVE Bioreactor (GE Healthcare Life Sciences, Pittsburgh, Pennsylvania), and G-Rex (Wilson Wolf Manufacturing, St. Paul, Minnesota). For example, isolated T cells can be expanded and grown at 37°C and 5% CO2 in TexMACS medium (Miltenyi Biotec, Germany) supplemented with 200 IU / mL of IL-2 and TransAct beads (Miltenyi Biotec, Germany). Methods for isolating and expanding T cell populations are described, for example, in Levine et al., (2017) "Global Manufacturing of CAR T Cell Therapy" Mol Ther Methods Clin Dev. 4:92-101.

[0213] The methods for producing CAR-T cells described herein may also include the step of transfecting an expanding T cell population with one or more expression vectors encoding a CAR, an amino acid transporter, or a CAR and an amino acid transporter. Suitable transfection methods are known in the art and include, for example, calcium phosphate transfection, lipofection, polymer transfection, Fugene product-based transfection (Promega Corporation, Madison, Wisconsin), and electroporation using, for example, a CliniMACS Electroporator (Miltenyi Biotec, Germany). In some embodiments described herein, the methods for producing CAR-T cells may include the step of transfecting an expanding T cell population with one or more transposon-containing plasmids, for example, the Sleeping Beauty transposon, and a CAR, an amino acid transporter, or a plasmid encoding a CAR and an amino acid transporter.

[0214] In some embodiments described herein, a method for producing CAR-T cells may include the step of transducing an expanded and proliferating T cell population with one or more expression vectors encoding a CAR, an amino acid transporter, or a CAR and an amino acid transporter using a virus (e.g., a lentivirus, retrovirus, adenovirus, or adeno-associated virus).

[0215] T cells transfected or transduced with an appropriate nucleotide construct can be further nourished in an appropriate medium (e.g., TexMACS medium (Miltenyi Biotec, Germany) supplemented with 1 mM L-arginine (Sigma-Aldrich, USA)) and assayed for viability.

[0216] T cell purity and the helper T cell to killer T cell ratio can be determined using flow cytometry and fluorescence-assisted cell sorting (FACS) methods with appropriate antibodies (e.g., anti-CD19, CD14, CD45, CD3, CD4, and CD8 antibodies). CAR and arginine transporter protein expression can be determined using custom antibodies specific to the antigen recognition domain of the CAR or to the arginine transporter.

[0217] The arginine content in CAR-T cells can be determined using the L-arginine ELISA kit (ALPCO, USA).

[0218] The methods described herein may include the step of harvesting CAR-T cells for downstream application based on the number of cells obtained. For example, in some embodiments, the amount of CAR-T cells to be harvested is about 1 × 10⁶ cells per kg of body weight of the subject. 3 pieces, about 1×10 4 pieces, about 1×10 5 pieces, about 1×10 6 pieces, about 1×10 7 pieces, about 1×10 8 pieces, about 1×10 9 pieces, about 1×10 10 pieces, approximately 2×10 10 pieces, about 3 x 10 10 pieces, about 4×10 10 pieces, about 5×10 10 pieces, about 6×10 10 pieces, about 7×10 10 pieces, about 8×10 10 pieces, approximately 9×10 10 pieces, about 1×10 11 pieces, about 1×10 12 pieces, about 1×10 13 pieces, about 1×10 14 pieces, about 1×10 15 pieces, about 1×10 3 ~Approx. 3×10 10 pieces, about 1×10 5 ~Approx. 3×10 10 pieces, about 1×10 3 ~Approx. 1×10 5 pieces, about 1×105 ~Approx. 1×10 15 pieces, about 1×10 5 ~Approx. 1×10 10 pieces, about 1×10 7 ~Approx. 1×10 12 pieces, about 1×10 5 ~Approx. 1×10 7 pieces, about 1×10 10 ~Approx. 9×10 10 pieces, or approximately 1 x 10 9 ~Approx. 1×10 11 Examples include a number of cells. In some embodiments, the amount of CAR-T cells to be harvested is approximately 1 × 10⁶ 5 pieces, about 1×10 6 pieces, about 1×10 7 pieces, about 1×10 8 pieces, about 1×10 9 pieces, about 1×10 10 pieces, about 1×10 11 pieces, about 1×10 12 pieces, about 1×10 5 ~Approx. 1×10 12 pieces, about 1×10 5 ~Approx. 1×10 10 pieces, about 1×10 5 ~Approx. 1×10 7 pieces, about 1×10 7 ~Approx. 1×10 10 pieces, about 1×10 7 ~Approx. 1×10 12 pieces, about 1×10 9 ~Approx. 1×10 10 pieces, about 1×10 6 ~Approx. 1×10 8 pieces, about 1×10 7 ~Approx. 1×10 9 pieces, or approximately 1 x 10 9 ~Approx. 1×10 11 Individual cells are cited as examples.

[0219] The methods described herein may include the step of harvesting CAR-T cells for downstream application based on the arginine content of the obtained cells. For example, in some embodiments, the CAR-T cells for harvesting may have arginine content per cell of approximately 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1000 μM, 1500 μM, 2000 μM, 2500 μM, 3000 μM, and 35 μM. This includes cells having an intracellular arginine content of 00 μM, approximately 4000 μM, approximately 100 μM to approximately 4000 μM, approximately 100 μM to approximately 1000 μM, approximately 100 μM to approximately 2000 μM, approximately 1000 μM to approximately 2000 μM, approximately 1000 μM to approximately 3000 μM, approximately 1000 μM to approximately 4000 μM, approximately 500 μM to approximately 1000 μM, approximately 3000 μM to approximately 4000 μM, approximately 2000 μM to approximately 4000 μM, or approximately 500 μM to approximately 2000 μM.

[0220] The CAR-T cells described herein are genetically modified to express a specific CAR and / or amino acid transporter protein, such as an arginine transporter protein. In some embodiments, an expression cassette encoding the arginine transporter is introduced into the T cells (e.g., by genetic engineering) before, after, or simultaneously with the expression cassette encoding the CAR. The nucleotide sequence encoding the amino acid transporter can be placed together with the nucleotide sequence encoding the CAR on the same vector (e.g., one vector for both the CAR and the transporter). This allows both the CAR and the transporter to be introduced into the same cell simultaneously, resulting in all resulting arg+CAR-T cells being enhanced by the transporter. In some embodiments, the nucleotide construct encoding the amino acid transporter is placed in a separate vector from the one encoding the CAR (e.g., separate vectors for both the CAR and the transporter).

[0221] CAR-T cells as described herein can be produced by transfection, electroporation, or transformation of T cells with one or more specific expression vectors encoding nucleic acid sequences for CAR and / or amino acid transporter proteins, such as arginine transporter protein. CAR-T cells as described herein can also be produced by transduction of T cells with one or more viruses having specific expression vectors encoding nucleic acid sequences for CAR and / or amino acid transporter proteins, such as arginine transporter protein. Isolated T cells can be transduced with one or more retroviral vectors, such as embedded γ-retroviral vectors or lentiviral vectors. The γ-retroviral vectors and lentiviral vectors are randomly integrated into the T cell genome. Isolated T cells can also be transformed with one or more embedded artificial transposons or via transfection with non-embedded RNA molecules. In some embodiments, isolated T cells can be electroporated with a CRISPR / Cas-9 expression construct and transfected with one or more adenovirus or AAV vectors encoding specific CAR and / or amino acid transporter proteins, such as arginine transporter proteins.

[0222] For example, a method for producing genetically modified T cells (e.g., CAR-T cells) is described herein, comprising transfecting T cells with an expression vector comprising a nucleic acid sequence encoding a CAR and a nucleic acid sequence encoding an amino acid transporter, such as an arginine transporter. A method for producing genetically modified T cells (e.g., CAR-T cells) is also described herein, comprising transfecting T cells with a first expression vector comprising a nucleic acid sequence encoding a CAR and a second expression vector comprising a nucleic acid sequence encoding an amino acid transporter, such as an arginine transporter. In some embodiments, transfection is performed by a chemical method of transfection (e.g., calcium phosphate transfection, lipofection, polymer transfection (e.g., DEAE-dextran or polyethyleneimine (PEI) transfection)), or by a transfection reagent developed by Fugene (Promega FuGENE Corporation, Madison, Wisconsin; e.g., FuGENE HD or FuGENE 6 transfection reagents), non-chemical methods of transfection (e.g., electroporation, cell compression, sonoporation, phototransfection, protoplast fusion, impalefection, or fluid This can be carried out by biomechanical delivery, particle-based transfection (gene gun transfection, magnetic-assisted transfection), nucleofection, or heat shock transfection. In some embodiments, the method involves transfecting T cells with a first expression vector and a second expression vector simultaneously or sequentially.

[0223] Methods for producing genetically modified T cells (e.g., CAR-T cells) are also described herein, comprising transducing T cells with a virus (e.g., adenovirus, AAV, lentivirus, or retrovirus) possessing a nucleic acid sequence encoding a CAR and a nucleic acid sequence encoding an amino acid transporter, such as an arginine transporter. Methods for producing genetically modified T cells (e.g., CAR-T cells) are also described herein, comprising transducing T cells with a first virus (e.g., adenovirus, adeno-associated virus, lentivirus, or retrovirus) possessing a nucleic acid sequence encoding a CAR, and transducing T cells with a second virus (e.g., adenovirus, adeno-associated virus, lentivirus, or retrovirus) possessing a nucleic acid sequence encoding an amino acid transporter, such as an arginine transporter. In some embodiments, the method comprises transducing T cells with the first virus and the second virus simultaneously or sequentially.

[0224] In some embodiments, if the method for producing genetically modified T cells involves transfecting T cells with an expression vector or transducing cells with a virus, the method may also include the step of selecting transfectants, for example, by antibiotic resistance.

[0225] In some embodiments, if a method for producing genetically modified T cells involves sequentially transfecting T cells with a first expression vector and a second expression vector, the method may also include a step of selecting transfectants by expressing a selection marker suitable for FACS, such as antibiotic resistance or a fluorescent protein. For example, such a method may include a step of selecting transfectants for the first expression vector. Such a method may further include a step of selecting transfectants for the second expression vector. Such a method may further include a step of selecting transfectants for both the first and second expression vectors. In some embodiments, the selection of transfectants for the first expression vector is performed before transfection by the second expression vector.

[0226] In some embodiments, if a method for producing genetically modified T cells involves sequentially transducing T cells with a first virus and then a second virus, the method may also include a step of selecting the transduced cells, for example, by antibiotic resistance or FACS. For example, such a method may include a step of selecting cells transduced with the first virus. Such a method may further include a step of selecting cells transduced with the second virus. Such a method may further include a step of selecting cells transduced with both the first and second viruses. In some embodiments, the selection of cells transduced with the first virus is performed before transduction with the second virus.

[0227] In some embodiments, a method for producing genetically modified T cells includes transducing T cells with a virus and transfecting T cells with an expression vector, wherein the transduction and transfection can be performed in either order (e.g., transfecting after transduction, or transfecting after transduction). For example, in some embodiments, a method for producing genetically modified T cells includes transducing T cells with a virus having a nucleic acid sequence encoding a CAR, and transfecting T cells with an expression vector containing a nucleic acid sequence encoding an amino acid transporter, such as an arginine transporter. CAR and amino acid transporter expression vectors and transgenes

[0228] The CAR-T nucleotide constructs described herein (e.g., nucleotide expression vectors and viral nucleotide constructs) may include standard components, for example, but are not limited to, promoters, Kozak sequences, gene expression cassettes, autocleavage sites, markers for selection (e.g., fluorescent protein expression cassettes or antibiotic resistance cassettes), inverted tandem repeat sequences, transcription termination and poly(A) signal sequences.

[0229] Examples of promoter sequences include: PGK: GGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCG (SEQ ID NO: 251); CMV: CGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT (SEQ ID NO: 252); and CAG: GCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATGGGACTTTCCATTGACGTCATGGGTGGAGTATTTACGGTAAACT GCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCAT CGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCTCCCCACCCCCAATTTTGTATTTATTTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGCGCGCGCCAG GCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCG (SEQ ID NO: 253).

[0230] Examples of transcription termination and polyA signal sequences include: bGH pA:CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG (SEQ ID NO: 254); rbHBB pA: AATAAAAGATCTTTATTTTCATTAGATCTGTGTGTTGGTTTTTTGTGTG (Sequence ID 255); SV40 pA: CTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGATATGCA (SEQ ID NO: 256); and hGH pA: GACGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCT TGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTGGTAGAGA CGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTT (Sequence number 257).

[0231] Exemplary inverted tandem repeat (TIR) ​​sequences include the following pT4 left inverted repeat (LIR) and right inverted repeat (RIR) sequences: pT4 LIR: TACAGTTGAAGTCGGAAGTTTACATACACTTAAGTTGGAGTCATTAAAACTCGTTTTCAACTACTCCACAAATTTCTTGTTAACAAACAATAGTTTTGGCAAGTCAGTTAGGACATCTACTTTGTGCATGACACAAGTCATTTTTCCAACAATTGTTTACAGACAGATTATTTCACTTATAATTCACTGTATCACAATTCCAGTGGGTCAGAAGTGTACATACACGCGCTTGACTGTGCCTTT (SEQ ID NO: 258); and pT4 RIR: TTAAAACAATTTAAAGGCAATGCTACCAAATACTAAGCGCGTGTATGTACACTTCTGACCCACTGGGAATGTGATGAAAGAAATAAAAGCTGAAATGAATCATTCTCTCTACTATTATTCTGATATTTCACATT CTTAAAATAAAGTGGTGATCCTAACTGACCTTAAGACAGGGAATCTTTACTCGGATTAAATGTCAGGAATTGTGAAAAAGTGAGTTTAAATGTATTTGGCTAAGGTGTATGTAAACTTCCGACTTCAACTGTA (SEQ ID NO: 259).

[0232] Examples of self-cleavage site nucleotide sequences include the following: P2A: GCCACCAATTTCAGCCTGCTGAAACAGGCTGGCGACGTGGAAGAGAACCCTGGACCT (Sequence ID 260); T2A: GGCAGCGGCGAGGGCAGAGGCAGCCTGCTGACCTGCGGCGACGTGGAGGAGAACCCCGGCCCC (SEQ ID NO: 261); E2A: GGCAGCGGCCAGTGCACCAACTACGCCCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAAC (Sequence ID 262); and F2A: GGCAGCGGCGTGAAGCAGACCCTGAACTTCGACCTGCTGAAGCTGGCCGGCGACGTGGAGAGCAACCCCGGCCCC (SEQ ID NO: 263).

[0233] Examples of selectable marker nucleotide sequences include sequences encoding the following fluorescent proteins and antibiotic resistance proteins: mEGFP (fluorescent protein coding sequence): GTGTCCAAGGGCGAAGAACTGTTTACCGGCGTGGTGCCCATCCTGGTGGAACTGGATGGGGATGTGAACGGCCACAAGTTCAGCGTTAGCGGAGAAGGCGAAGGCGACGCCACATACGGAAAGCTGACACTGAAGTTCATCTGCACCACCGGCAAGCTGCCTG TGCCATGGCCAACACTGGTCACCACACTGACATACGGCGTGCAGTGCTTCAGCAGATACCCCGACCATATGAAGCAGCATGACTTCTTCAAGAGCGCCATGCCTGAGGGCTACGTGCAAGAGCGGACCATCTTCTTTAAGGACGACGGCAACTACAAGACCAGGGCCGAAGTGAAGTTCGAGGG CGACACCCTCGTGAACCGGATCGAGCTGAAGGGCATCGACTTCAAAGAGGACGGCAACATCCTGGGCCACAAGCTCGAGTACAACTACAACAGCCACAACGTGTACATCATGGCCGACAAGCAGAAAAACGGCATCAAAGTGAACTTCAAGATCCGGCACAACATCGAGGACGGCTCAGTGCA GCTGGCCGACCACTATCAGCAGAACACACCCATCGGAGATGGCCCCGTTCTGCTGCCCGATAACCACTACCTGAGCACACAGAGCAAGCTGAGCAAGGACCCCAACGAGAAGCGGGACCACATGGTCCTGCTGGAATTTGTGACAGCCGCCGGAATCACCCTCGGCATGGACGAGCTTTACAAA (SEQ ID NO: 264); mEmerald (fluorescent protein coding sequence): GTGAGCAAGGGCGAGGAGCTGTTCACCGGCGTGGTGCCCATCCTGGTGGAGCTGGACGGCGACGTGAACGGCCACAAGTTCAGCGTGAGCGGCGAGGGCGAGGGCGACGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTGGTGACCACCCTGACCTACGGCGTGCAGTGCTTCGCCAGATACCCCGACCACATGAAGCAGCACGACTTCTTCAAGAGCGCCATGCCCGAGGGCTACGTGCAGGAGAGAACCATCTTCTTCAAGGACGACGGCAACTACAAGACCAGAGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACAGAATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGCCACAAGCTGGAGTACAACTACAACAGCCACAAGGTGTACATCACCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGACCAGACACAACATCGAGGACGGCAGCGTGCAGCTGGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGAGCAAGCTGAGCAAGGACCCCAACGAGAAGAGAGACCACATGGTGCTGCTGGAGTTCGTGACCGCCGCCGGCATCACCCTGGGCATGGACGAGCTGTACAAG (SEQ ID NO: 265); mCherry 2 (fluorescent protein coding sequence): GTGTCTAAGGGCGAAGAGGACAACATGGCCATCATCAAAGAATTCATGCGGTTCAAGGTGCACATGGAAGGCAGCGTGAACGGCCACGAGTTCGAGATTGAAGGCGAAGGCGAGGGCAGACCTTACGAGGGAACACAGACCGCCAAGCTGAAAGTCACCAAAGGCGGCCCTCTGCCTTTTGCCTGGGACATTCTGAGCCCTCAGTTTATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGATATTCCCGACTATCTGAAGCTGAGCTTCCCCGAGGGCTTCAACTGGGAGCGCGTGATGAATTTCGAGGACGGCGGCGTGGTCACCGTGACTCAAGATAGCTCTCTGCAGGACGGCGAGTTCATCTACAAAGTGAAGCTGCGGGGCACAAACTTCCCCAGCGACGGACCTGTGATGCAGTGCAGAACAATGGGCTGGGAAGCCAGCACCGAGAGAATGTACCCAGAAGATGGCGCCCTGAAGGGCGAGATTAAGCAGCGGCTGAAACTCAAGGATGGCGGCCACTACGACGCCGAAGTGAAAACCACCTACAAGGCCAAGAAACCCGTGCAGCTGCCTGGCGCCTACAACGTGGACATCAAGCTGGATATCCTGAGCCACAATGAGGACTACACCATCGTCGAGCAGTACGAGAGAGCCGAGGGGAGACATTCTACCGGCGGAATGGACGAGCTGTACAAA (SEQ ID NO: 266); mScarlet-i (fluorescent protein coding sequence): GTGTCTAAGGGCGAAGCCGTGATCAAAGAATTCATGCGGTTCAAGGTGCACATGGAAGGCAGCATGAACGGCCACGAGTTCGAGATCGAAGGCGAAGGCGAGGGCAGACCTTATGAGGGAACACAGACCGCCAAGCTGAAAGTGACCAAAGGCGGCCCTCTGCCTTTCAGCTGGGACATTCTGAGCCCTCAGTTTATGTACGGCAGCCGGGCCTTCATCAAGCACCCTGCCGATATTCCCGACTACTACAAGCAGAGCTTCCCCGAGGGCTTCAAGTGGGAGAGAGTGATGAACTTCGAGGACGGCGGAGCCGTGACCGTGACACAGGATACAAGCCTGGAAGATGGCACCCTGATCTACAAAGTGAAGCTGCGGGGCACCAACTTTCCACCTGATGGCCCCGTGATGCAGAAAAAGACCATGGGCTGGGAAGCCAGCACCGAGAGACTGTATCCTGAGGATGGCGTGCTGAAGGGCGACATCAAGATGGCCCTGAGACTGAAGGATGGCGGCAGATACCTGGCCGACTTCAAGACCACCTACAAGGCCAAGAAACCCGTGCAGATGCCTGGCGCCTACAACGTGGACAGAAAGCTGGACATCACCAGCCACAACGAGGACTACACCGTGGTGGAACAGTACGAGCGGAGCGAAGGCAGACACTCTACAGGCGGAATGGACGAGCTGTACAAA (SEQ ID NO: 267); Puromycin N-acetyltransferase (puromycin resistance coding sequence): ACAGAGTACAAACCTACAGTGCGCCTGGCCACCAGGGACGATGTTCCTAGAGCCGTCAGAACTCTGGCCGCTGCCTTCGCCGATTATCCAGCCACAAGACACACCGTGGATCCCGACAGACACATCGAGAGAGTGACCGAGCTGCAAGAGCTGTTTCTGACCAGAGTCGGCCTGGACATCGGCAAAGTGTGGGTTGCAGATGATGGCGCCGCTGTGGCTGTGTGGACAACACCTGAATCTGTGGAAGCCGGCGCAGTGTTTGCCGAGATCGGACCT AGAATGGCCGAGCTGAGCGGATCTAGACTGGCTGTCCAACAGCAGATGGAAGGCCTGCTGGCTCCCCACAGACCAAAAGAGCCTGCTTGGTTTCTGGCCACCGTGGGCGTTAGCCCTGACCACCAAGGCAAAGGACTGGGATCTGCTGTGGTGCTGCCT GGCGTTGAAGCCGCTGAAAGAGCTGGCGTTCCAGCCTTCCTGGAAACAAGCGCCCCTCGGAACCTGCCTTTCTACGAGAGACTGGGCTTTACCGTGACCGCCGATGTGGAAGTGCCAGAGGGACCAAGAACCTGGTGCATGACCAGAAAGCCTGGCGCC (SEQ ID NO: 268); Aminoglycoside 3'-phosphotransferase II (G418 resistance coding sequence): ATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTC (SEQ ID NO: 269); and

[0234] For example, the CAR-T expression vectors described herein may include the following nucleotide components: a promoter sequence (e.g., EF1α, kmeto, CAG, CMV, UbC, or PGK promoter sequence), an antigen-specific targeting sequence, a transmembrane domain sequence (e.g., CD4, CD8α, CD28, CD3ζ, or ICOS nucleotide sequence), a transmembrane domain sequence (e.g., CD4, CD8α, CD28, CD3ζ, or ICOS transmembrane domain nucleotide sequence), and an intracellular signaling domain sequence (e.g., FcRγ or CD3ζ intracellular signaling domain sequence). The CAR-T expression vectors described herein may further comprise one or more of the following components: one or more costimulatory domain sequences (e.g., 4-1BB, CD27, CD28, CD40, CD40L, TLR2, DAP10, OX40, IL-2RB, IL-2RA, MYD88, or ICOS costimulatory domain sequences), arginine transporter sequences (e.g., SLC7A1, SLC7A2, SLC7A3, SLC7A4, SLC7A6, SLC7A7, SLC3A2, SLC3A1, SLC7A9, or SLC6A14 nucleotide sequences), and hinge or spacer domain sequences(s).

[0235] In some embodiments, the CAR-T expression vectors described herein include a promoter sequence (e.g., EF1α, cumate, CMV, CAG, UbC, or PGK promoter sequence) and an arginine transporter sequence (e.g., SLC7A1, SLC7A2, SLC7A3, SLC7A4, SLC7A6, SLC7A7, SLC3A2, SLC3A1, SLC7A9, or SLC6A14 nucleotide sequence).

[0236] In some embodiments, the CAR-T expression vectors described herein may also include one or more of the following: an antibiotic selection cassette (e.g., ampicillin, geneticin, zeosin, hygromycin, blastosidine, puromycin, or kanamycin resistance cassette), and a replication origin sequence (e.g., pUC, pMB1, pBR322, ColE1, R6K, p15A, pSC101, pMSCV, or F1 sequence). The lentiviral and γ-retroviral vectors described herein may also include one or more of the following: a 5' long-chain terminal repeat (LTR) sequence (containing one or more of the U3, R, and U5 sequences), a 3' LTR sequence (containing one or more of the U3, R, and U5 sequences), a psi(Ψ) sequence, a transactivation response (TAR) element sequence, a central polyprint lact (cPPT) sequence, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) sequence, and a Rev response element (RRE) sequence. The adenoviruses and AAV vectors described herein may also contain inverted terminal repeat (ITR) sequences.

[0237]

[0238] In some embodiments, the CAR-T expression vectors described herein may comprise components in the following order: a promoter sequence, a Kozak sequence, a start codon, one or more nucleotide sequences encoding the protein of interest (e.g., a CAR nucleotide sequence and / or an amino acid transporter nucleotide sequence, e.g., an arginine transporter nucleotide sequence), a stop codon, a termination and poly(A) signal nucleotide sequences.

[0239] Number 272).

[0240] The CAR-T integrated lentivirus-derived transgenes described herein may include the following nucleotide components: 5' long-chain terminal repeat (LTR) sequences (containing one or more of the U3, R, and U5 sequences), promoter sequences (e.g., EF1α, cumate, CAG, CMV, UbC, or PGK promoter sequences), antigen-specific targeting sequences, transmembrane domain sequences (e.g., CD4, CD8α, CD28, CD3ζ, or ICOS transmembrane domain nucleotide sequences), intracellular signaling domain sequences (e.g., FcRγ or CD3ζ intracellular signaling domain sequences), and 3' LTR sequences (containing one or more of the U3, R, and U5 sequences). The CAR-T transgenes described herein may further comprise one or more of the following components: a psi(Ψ) sequence, an RRE sequence, one or more costimulatory domain sequences (e.g., 4-1BB, CD27, CD28, CD40, CD40L, TLR2, DAP10, OX40, IL-2RB, IL-2RA, MYD88, or ICOS costimulatory domain sequences), an arginine transporter sequence (e.g., SLC7A1, SLC7A2, SLC7A3, SLC7A4, SLC7A6, SLC7A7, SLC3A2, SLC3A1, SLC7A9, or SLC6A14 nucleotide sequences), or a hinge or spacer domain sequence.

[0241] In some embodiments, the CAR-T transgenes described herein include the following nucleotide components: 5' long-chain terminal repeat (LTR) sequences (including one or more of U3, R, and U5 sequences), promoter sequences (e.g., EF1α, cumate, CMV, CAG, UbC, or PGK promoter sequences), arginine transporter sequences (e.g., SLC7A1, SLC7A2, SLC7A3, SLC7A4, SLC7A6, SLC7A7, SLC3A2, SLC3A1, SLC7A9, or SLC6A14 nucleotide sequences), and 3' LTR sequences (including one or more of U3, R, and U5 sequences). The CAR-T transgenes described herein may further include one or more psi(Ψ) sequences and RRE sequences.

[0242] In some embodiments, the expression cassette described herein may include a eukaryotic promoter that functions in T cells (e.g., EF-1α, PGK, CAG, or CMV promoter), a coding sequence for an amino acid transporter that includes or does not include a preceding Kozak sequence, and a eukaryotic transcription terminator and poly(A) signal (e.g., SV40, hGH, bGH, rbHBB, and rbGlob). The expression cassette can be embedded in a transposon (e.g., Sleeping Beauty, piggyBac, Tol2) to enable genome integration without the use of lentiviruses or retroviruses.

[0243] Antibiotic resistance genes (e.g., puromycin N-acetyltransferase), protein tags (e.g., 6×His (SEQ ID NO: 278), FLAG), and / or reporters, such as fluorescent proteins, may also be included in the expression vectors described herein, either in tandem with amino acid transporters (e.g., in the form of fusion proteins) or as separate entities (e.g., separated from amino acid transporter coding sequences by IRES or 2A cleavage sequences), to facilitate downstream selection.

[0244] In some embodiments, the amino acid transporter expression vector described herein may have components in the following order: IR / DR(SB)-P EF1α ::Kozak-transporter-P2A-PAC-(G4S)3-mEGFP-BGHpolyA-DR / IR(SB) (disclosed as Sequence ID No. 30, "(G4S)3"). Tumor microenvironment

[0245] Cancer cells create a tumor microenvironment (TME) that partially allows for tumor growth and proliferation by depleting essential nutrients from their surroundings. The metabolic status of the TME is regulated by the metabolic activity of cancer cells, which alters the availability of nutrients in the microenvironment, such as glucose, lipids, and amino acids. For example, the TME is characterized by low levels of the amino acid arginine. Arginine depletion is partially caused by the uptake of arginine from the TME by tumor cells. Arginine depletion is also mediated by the activation of arginase and inducible nitric oxide synthase (iNOS) in tumor cells, local macrophages, granulocytes, and bone marrow-derived suppressor cells.

[0246] In particular, naturally occurring T cells cannot synthesize arginine. Therefore, T cells depend on a sustained supply of exogenous arginine. However, T cell activation, survival, and persistence are impaired by relatively low levels of arginine in the tumor mesenteric environment (TME). Specifically, TME conditions containing low arginine levels impair T cell receptor signaling, glycolysis, amino acid uptake, and metabolism, impairing the antitumor effector function of tumor-specific T effector cells. Furthermore, Treg cells, which depend primarily on fatty acid oxidation in contrast to amino acid uptake, can survive under TME conditions and exert immunosuppressive effects against tumor-specific T effector cells. Therefore, TME conditions suppress the differentiation of T effector cells and promote immunosuppression. Currently available CAR-T cells are susceptible to the same TME adversities as their natural T cell counterparts, resulting in reduced efficacy of CAR-T treatment in solid tumors. The present invention provides CAR-T cells that can compete with cancer cells and MDSCs for arginine and increase their survival, persistence, and antitumor activity in solid tumors compared to CAR-T cells known in the art.

[0247] In particular, the present invention provides CAR-T cells with enhanced ability to transport amino acids, particularly arginine, from the extracellular space to the cytosol. For example, the CAR-T cells described herein are genetically engineered to express an amino acid transporter capable of transporting amino acids (e.g., arginine) into the CAR-T cell. The CAR-T cells described herein, genetically engineered to express an amino acid transporter, are characterized by higher T cell activation, persistence, proliferation, and / or antitumor activity compared to T cells and CAR-T cells that are not genetically engineered to express an amino acid transporter. Furthermore, the CAR-T cells described herein, genetically engineered to express an amino acid transporter, are characterized by higher survival rate and persistence in TME compared to T cells and CAR-T cells that are not genetically engineered to express an amino acid transporter. CAR-T cell priming

[0248] In one embodiment, the present invention includes a method for modulating intracellular arginine levels in CAR-T cells (e.g., CAR-T cells as described herein) to induce a T cell-mediated immune response in a patient in need. For example, in some embodiments, the present invention includes exposing CAR-T cells expressing an arginine transporter and CAR to an arginine-containing medium, and exposure of CAR-T cells to the medium is effective in increasing the intracellular arginine concentration of the CAR-T cells. Exposing CAR-T cells expressing an arginine transporter and CAR to an arginine-containing medium, for example, by in vitro culturing such CAR-T cells in an arginine-rich medium, can increase the intracellular arginine concentration of CAR-T cells compared to CAR-T cells that have not been exposed to the medium. Such intracellular arginine-enriched CAR-T cells can compete with cancer cells and MDSCs for extracellular arginine, for example, in the extracellular space of the TME. Accordingly, in some embodiments, the present invention comprises exposing CAR-T cells expressing the arginine transporter and CAR to a culture medium containing arginine, and exposure of CAR-T cells to the culture medium is effective in increasing the survival, lifespan, and functional activity of CAR-T cells. For example, in some embodiments, exposure of CAR-T cells to the culture medium is effective in increasing CAR-T antitumor activity (for example, exposure of CAR-T cells to the culture medium is effective in increasing CAR-T antitumor activity in the TME of solid tumors). Accordingly, a method for administering intracellular arginine-enriched CAR-T cells, which is effective in treating hematological malignancies and solid tumors, is also described herein.

[0249] In some embodiments, a medium effective in increasing the intracellular arginine concentration of CAR-T cells contains physiological levels of L-arginine (including, but not limited to, 0.2 g / L or 100 μmol / L), or, but not limited to, hyperphysiological levels of L-arginine such as 100 μmol / L, 200 μmol / L, 300 μmol / L, 400 μmol / L, 500 μmol / L, 600 μmol / L, 700 μmol / L, 800 μmol / L, 900 μmol / L, 1000 μmol / L, or greater than 1000 μmol / L. The medium may be RPMI-1640 with or without supplementation. The medium may be supplemented with serum and / or nutrients such as, but not limited to, fetal bovine serum, human AB serum, or human platelet lysate. Manipulated T cells can be cultured and primed in an L-arginine-rich medium until intracellular arginine accumulates to a sufficient level, but is not limited to, 20 μmol, 30 μmol, 40 μmol, 50 μmol, 60 μmol, 70 μmol, 80 μmol, 90 μmol, 100 μmol, 200 μmol, 2000 μmol, or more than 2000 μmol. In some embodiments, CAR-T cells are given intracellular arginine levels of approximately 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM, 700 μM, 800 μM, 900 μM, 1000 μM, 1500 μM, 2000 μM, 2500 μM, 3000 μM, 3500 μM, and 4000 μM per cell. L-arginine can be cultured and primed in a medium rich in arginine until it accumulates at concentrations of M, approximately 100 μM to 4000 μM, approximately 100 μM to 1000 μM, approximately 100 μM to 2000 μM, approximately 1000 μM to 2000 μM, approximately 1000 μM to 3000 μM, approximately 1000 μM to 4000 μM, approximately 500 μM to 1000 μM, approximately 3000 μM to 4000 μM, approximately 2000 μM to 4000 μM, or approximately 500 μM to 2000 μM. kit

[0250] Kits comprising the pharmaceutical compositions described herein are also described herein. For example, in some embodiments, a pharmaceutical composition comprising CAR-T cells expressing an arginine transporter and a CAR is packaged as a kit. The kits described herein may include instructions for administering the CAR-T cells to a patient in need of treatment. The kits described herein may include instructions for priming the CAR-T cells for administration to a patient in need of treatment. The kits described herein may include instructions for producing CAR-T cells expressing an arginine transporter and a CAR. In some embodiments, the kit may include at least one buffer (e.g., a buffer containing a sufficient level of L-arginine to prime T cells), reagents, and detailed instructions for producing, expanding, administering, and / or priming CAR-T cells.

[0251] The kit described herein for producing CAR-T cells may include an expression vector encoding a CAR, an expression vector encoding an arginine transporter, an expression vector encoding both a CAR and an arginine transporter, and / or an expression vector encoding a transposase for stable incorporation of the CAR and / or the arginine transporter. The kit may also include a polycistronic expression vector capable of expressing both a CAR and an arginine transporter.

[0252] The kits described herein for producing CAR-T cells may comprise a culture medium, cells, transfection reagents, buffers, and a nucleotide construct for producing a virus comprising a CAR, an arginine transporter, or a nucleotide construct encoding both a CAR and an arginine transporter. The kits may also comprise a polycistronic expression vector capable of expressing both a CAR and an arginine transporter.

[0253] The kits described herein may include reagents for assaying CAR and / or arginine transporter protein expression in CAR-T cells. For example, the kits described herein may include an antibody specific to the arginine transporter (e.g., a polyclonal antibody). The kits described herein may include an antibody specific to the CAR antigen recognition domain (e.g., a polyclonal antibody). Methods of treating cancer

[0254] The methods disclosed herein include methods for treating, preventing, stopping, reversing or restoring a disease. In some embodiments of the methods described herein, the disease is cancer. In some embodiments, the methods for treating, preventing, stopping, reversing or restoring a disease are achieved by administering a therapeutically effective dose of the CAR-T cells described herein, for example, the arg+CAR-T cells described herein. For example, a method for treating a solid tumor cancer in a patient requiring treatment of the solid tumor cancer is described herein, comprising administering to the patient an effective amount of the CAR-T cells described herein or a pharmaceutical composition comprising the CAR-T cells described herein. Also described herein is a method for treating a hematological cancer in a patient requiring treatment of the hematological cancer, comprising administering to the patient an effective amount of the CAR-T cells described herein or a pharmaceutical composition comprising the CAR-T cells described herein. Also described herein is a method for treating a condition in a human patient requiring treatment of the condition, comprising administering to the human patient a composition comprising a therapeutically effective dose of CAR-T cells expressing an arginine transporter and a chimeric antigen receptor protein, or a pharmaceutical composition comprising CAR-T cells expressing an arginine transporter and a chimeric antigen receptor protein.

[0255] The activity of multiple cells in the immune system can be modulated by arginine, for example, macrophages, B cells, T cells, natural killer cells, neutrophils, and dendritic cells. Modulation of intracellular arginine can result in a T cell-mediated immune response. Therefore, in patients who require modulation of intracellular arginine levels to induce a T cell-mediated immune response, a method for modulating intracellular arginine levels to induce a T cell-mediated immune response is described herein, comprising administering to the patient an effective amount of CAR-T cells as described herein or a pharmaceutical composition comprising CAR-T cells as described herein.

[0256] Methods for treating, preventing, stopping, reversing, or reversing a disease in subjects or patients requiring treatment, prevention, cessation, reversal, or mitigation of the disease are described herein. In embodiments described herein, patients and subjects may include humans, non-human primates such as chimpanzees, and other apes and monkey species; domestic animals such as cattle, horses, sheep, goats, and pigs; domestic animals such as rabbits, dogs, and cats; and laboratory animals such as rodents such as rats, mice, and guinea pigs. Subjects or patients may be of any age. Subjects and patients may be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, or infants.

[0257] Examples of diseases or conditions that can be treated with engineered CAR-T cells that overexpress the arginine transporter, including engineered CAR-T cells that overexpress the arginine transporter shown in Table 1, include hematological malignancies, solid tumor malignancies, metastatic cancers, benign tumors, non-inflammatory tumors, primary tumors, and secondary tumors.

[0258] In some embodiments, methods for treating cancer with arginine transporter-overexpressing CAR-T cells are disclosed herein, including engineered CAR-T cells described herein, for example, engineered CAR-T cells overexpressing the arginine transporter listed in Table 1. Methods for treating cancer described herein include, for example, methods for treating any of the following: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancer, AIDS-related lymphoma, anal cancer, appendiceal cancer, astrocytoma, neuroblastoma, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brain tumors, e.g., cerebellar astrocytoma, cerebral astrocytoma / gliomas, ependymoma, medulloblastoma, supratentorial primitive neuroectoderm tumor, optic tract and hypothalamic glioma, breast cancer, bronchial adenoma, Burkitt lymphoma, cancer of unknown primary origin, central nervous system lymphoma. Cancer, cerebellar astrocytoma, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colon cancer, cutaneous T-cell lymphoma, fibrinogenic round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumor, gallbladder cancer, stomach cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular carcinoma (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, pancreatic islet cell carcinoma, Kaposi's sarcoma, kidney cancer, laryngeal cancer, lip and oral cancer (oral) Cavity cancer, liposarcoma, liver cancer, lung cancer (e.g., non-small cell and small cell lung cancer), lymphoma, leukemia, macroglobulinemia, malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma, melanoma, mesothelioma, metastatic squamous cell carcinoma of the neck of unknown primary origin, oral cancer, multiple endocrine neoplasm syndrome, myelodysplastic syndrome, myeloid leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumor, pancreatic cancer, pancreatic islet cell carcinomaCell cancer, paranasal sinus cancer and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germ cell tumor, pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasm, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureteral transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer, Merkel cell carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric cancer, T-cell lymphoma, pharyngeal cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastoma (pregnancy-related), cancer of unknown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor.

[0259] In some embodiments, in a method for treating cancer that includes the step of administering CAR-T cells, the antigen-specific target region of the CAR can recognize and bind to a cell surface antigen. In some embodiments, the CAR can be used in a method for treating cancer in which a specific monoclonal antibody is present or capable of producing such an antibody. In particular, cancers such as neuroblastoma, small cell lung cancer, melanoma, ovarian cancer, renal cell carcinoma, colon cancer, Hodgkin lymphoma, and pediatric acute lymphoblastic leukemia have antigens that are recognized by the CAR described herein.

[0260] The treatment methods described herein may include treating subjects (e.g., patients with a disease and / or experimental animals with a condition) with genetically engineered CAR-T cells that overexpress amino acid transporters, including engineered CAR-T cells that overexpress arginine transporters. The disease may be a hematological malignancy. The disease may be a solid tumor malignancy. The subject may be a human. The treatment may be provided to the subject before the clinical onset of the disease. The treatment may be provided to the subject after the clinical onset of the disease.

[0261] Treatment may be provided to subjects approximately 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, or longer than that period following the clinical onset of the disease. Treatment may be provided to subjects at approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 15 hours, 18 hours after the clinical onset of the disease, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years or longer. Treatment may be provided to subjects for a period of time longer than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 15 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, or longer. Treatment may be provided to subjects less than 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 12 hours, 15 hours, 18 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 1 year, 2 years, 3 years, 4 years, 5 years, or longer than that period.The treatment may also include treating humans in a clinical trial. The treatment may include administering a pharmaceutical composition, for example, one or more pharmaceutical compositions described throughout this disclosure. The treatment may include modulating endogenous arginine levels in vivo.

[0262] Methods for reintroducing cellular components are known in the art and include procedures as exemplified in U.S. Patents No. 4,844,893 and No. 4,690,915. The amount of activated T cells used may vary between in vitro and in vivo use, as well as depending on the quantity and type of target cells. The dose administered should also vary depending on the patient's condition and should be determined by the practitioner taking all appropriate factors into consideration. Combination of manipulated CAR-T cells with immune checkpoint therapy

[0263] Also disclosed herein are combination therapies and methods of use thereof, including administering engineered CAR-T cells (or their pharmaceutical composition), for example, CAR-T cells overexpressing an amino acid transporter, for example, the arginine transporter disclosed herein, in combination with a second therapeutic agent. For example, methods of treating cancer, including administering genetically modified T cells modified to express a CAR and an amino acid transporter, for example, an arginine transporter, and immunotherapies targeting immune checkpoints (e.g., immune checkpoint inhibitors), are described herein. For example, methods of treating cancer, including administering genetically modified T cells modified to express a CAR and an amino acid transporter, for example, an arginine transporter, and an agent that blocks the interaction between PD-1 and PD-L1, or the interaction between CTLA-4 and B7-1 / B7-2, are described herein. For example, methods of treating cancer, including administering genetically modified T cells modified to express a CAR and an amino acid transporter, for example, an arginine transporter, and an anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibody, are described herein. Methods for treating cancer are also described herein, comprising administering genetically modified T cells modified to express CARs and amino acid transporters, such as arginine transporters, and compounds selected from the group consisting of ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and semiprimab. The combination therapies of this disclosure may be co-administered to subjects to improve cancer treatment outcomes. In some embodiments, the CAR-T cells and immune checkpoint inhibitors described herein are administered simultaneously or sequentially to patients requiring treatment.

[0264] Most immunological checkpoint molecules are members of the immunoglobulin superfamily and are often inhibitory receptors that prevent uncontrolled immune responses. Adaptive immune responses are regulated by such checkpoint molecules, which are crucial for maintaining self-tolerance and minimizing secondary tissue damage that may occur during the immune response. In some embodiments, combination therapies that target immune checkpoints and promote amino acid uptake, particularly arginine uptake, by CAR-T cells may result in better outcomes for subjects with solid and hematological malignancies.

[0265] Immune checkpoints are co-stimulatory and inhibitory elements inherent to the immune system. They help maintain self-tolerance and modulate the duration and amplitude of physiological immune responses to prevent tissue damage when the immune system responds to pathogenic infections. Immune responses can also be initiated when T cells recognize antigens characteristic of tumor cells. The equilibrium between co-stimulatory and inhibitory signals used to control immune responses from T cells can be modulated by immune checkpoint proteins. After T cells mature and are activated in the thymus, they can migrate to sites of inflammation and injury to perform repair functions. T cell function can occur through direct action or through the recruitment of cytokines and membrane ligands involved in the immune system. Steps involved in T cell maturation, activation, proliferation, and function can be regulated via co-stimulatory and inhibitory signals, i.e., immune checkpoint proteins. Tumors can dysregulate the function of checkpoint proteins as an immune resistance mechanism. Therefore, the development of checkpoint protein modulators may have therapeutic value. Non-limiting examples of immune checkpoint molecules include CTLA4 and PD-1. These checkpoint molecules can act upstream of IL-2 in the pathway. Checkpoint inhibitors include drugs that block the interaction between PD-1 and PD-L1, or drugs that block the interaction between CTLA-4 and B7-1 / B7-2. Examples of specific checkpoint inhibitors include the following antibody-based drugs: ipilimumab, nivolumab, pembrolizumab, atezolizumab, avelumab, durvalumab, and semiprimab.

[0266] In some cases, the present disclosure provides a method for treating a condition in a human subject, comprising (a) administering to the human subject a therapeutically effective amount of a composition comprising ectopically expressing arginine transporter(s) and chimeric antigen receptor proteins CAR-T cells, and (b) administering to the human subject a second therapeutic agent, wherein the second therapeutic agent is an anti-PD-1, anti-PD-L1, or anti-CTLA-4 antibody. The administration of the second therapeutic agent may be performed before, during, or after administration of the composition comprising the CAR-T cell composition.

[0267] PD1 is an inhibitory receptor belonging to the CD28 / CTLA-4 family and is expressed on the surface of activated T cells, B cells, monocytes, DCs, and natural killer (NK) cells. In contrast to CTLA-4, the primary role of PD-1 is to limit T cell activity in peripheral tissues and limit autoimmunity during inflammatory responses to infection. Chronic antigen exposure can result in persistently high levels of PD-1 expression, which can induce a state of antigen-specific T cell exhaustion or anergy, which can be at least partially reversed by PD-1 blockade. In some embodiments of this disclosure, engineered CAR-T cells and anti-PD-1 antibodies or anti-PD-L1 antibodies are co-administered to subjects suffering from a condition.

[0268] CTLA-4 (cytotoxic T lymphocyte antigen 4), also known as CD152 (differentiation antigen group 152), shares sequence homology and ligands (CD80 / B7-1 and CD86 / B7-2) with the costimulatory molecule CD28, but differs in that it delivers inhibitory signals to T cells expressing CTLA-4 as a receptor. CTLA-4 has a much higher overall affinity for both ligands and can compete with CD28 for binding when ligand density is limited. CTLA-4 is expressed on the surface of CD8+ effector T cells and plays a functional role in the early activation phase of both naive and memory T cells. CTLA-4 counteracts CD28 activity during the early stages of T cell activation by increasing its affinity for CD80 and CD86. Key functions of CTLA-4 include downmodulation of helper T cells and enhancement of regulatory T cell immunosuppressive activity.

[0269] CTLA-4 can also downmodulate immune system function by inhibiting IL-2 production and IL-2 receptor expression. CTLA-4 can inhibit the CD28-dependent upregulation of IL-2, and inhibition of IL-2 production can lead to cell cycle arrest. The decrease in IL-2 and subsequent cell cycle arrest may be responsible for the reduced T cell proliferation observed in the presence of CTLA-4. Other combination therapies

[0270] As described above, combination therapies and methods of using the same are also disclosed herein, including administering engineered CAR-T cells, for example, CAR-T cells overexpressing an amino acid transporter, such as the arginine transporter disclosed herein, or a pharmaceutical composition thereof, in combination with a second therapeutic agent. In some embodiments, the CAR-T cells described herein, or a pharmaceutical composition thereof, and the second therapeutic agent are administered simultaneously or sequentially to a patient in need of treatment. In some embodiments, the method includes administering the second therapeutic agent before, during, or after administration of a therapeutically effective amount of T cells or a composition containing a therapeutically effective amount of CAR-T cells.

[0271] For example, methods for treating cancer are described herein, comprising administering genetically modified T cells modified to express CARs and amino acid transporters, such as arginine transporters, or a pharmaceutical composition thereof, and a DNA damage response inhibitor (DDRi). In some embodiments, the DDRi is selected from the group consisting of ATM inhibitors, PARP inhibitors, ATR inhibitors, WEE1 inhibitors, Chk1 inhibitors, Chk2 inhibitors, and DNA-protein kinase inhibitors. In some embodiments, the DDRi is a PARP inhibitor (PARPi) selected from the group consisting of niraparib, olaparib, pamiparib, rucaparib (cansilat), talazoparib, veliparib and its analogs. In some embodiments, the DDRi is an ATM / ATR inhibitor. In some embodiments, the ATM / ATR inhibitor is selected from the group consisting of AZ20, AZD0156, AZD1390, AZD6738, BAY-1895344, EPT-46464, M3541, M4344, M6620 (formerly known as VE-922 or VX-970), NU6027, VE-821, and their analogs. In some embodiments, the PARPi is adavocertib, AZD2811, or their analogs. In some embodiments, the DDRi is a WEE1 inhibitor, a Chk1 inhibitor, or a Chk2 inhibitor. In some embodiments, DDRi is a DNA-dependent protein kinase (DNA-PK) inhibitor selected from the group consisting of AZD7648, KU-0060648, NU7026, NU7441 (KU-57788), PI-103, PIK-75 HCl, PP121, SF2523, and their analogues.

[0272] In some embodiments, the method includes administering genetically modified T cells modified to express CARs and amino acid transporters, such as arginine transporters, or a pharmaceutical composition thereof, as well as radiotherapy, chemotherapy, immunotherapy, hormone therapy, angiogenesis inhibitors, stem cell transplantation therapy, bone marrow transplantation therapy, or targeted therapy.

[0273] Examples of radiotherapy include external beam radiation therapy, internal beam radiation therapy, brachytherapy, and whole-body radiation therapy.

[0274] Examples of chemotherapeutic agents include alkylating agents (e.g., altretamine, bendamustine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, ifosfamide, lomustine, mechloretamine, melphalan, oxaliplatin, temozolomide, thiotepa, and trabectedin), nitrosoureas (e.g., carmustine, lomustine, and streptozocin), and antimetabolites (e.g., azacitidine, 5-fluorouracil (5- FU), 6-mercaptopurine (6-MP), capecitabine (Xeloda), cladribine, clofarabine, cytarabine (ARA-C), decitabine, phloxuridine, fludarabine, gemcitabine (Gemzar), hydroxyurea, methotrexate, nelarabine, pemetrexed (Alimta), pentostatin, pralatrexate, thioguanine and trifluridine (trifluridien) / tipiracil), anthracyclines (e.g., daunorubicin, doxorubicin) Adriamycin, doxorubicin liposomes, epirubicin, idarubicin, and barurubicin), non-anthracycline antitumor antibiotics (e.g., bleomycin, dactinomycin, mitomycin-C, and mitoxantrone), topoisomerase inhibitors (e.g., irinotecan, irinotecan liposomes, topotecan etoposide (VP-16), mitoxantrone, teniposide), mitotic inhibitors such as taxanes and vinca alkaloids (e.g., capazitaxel, docetaxel) Examples include nab-paclitaxel, paclitaxel, vinca alkaloids (including vinblastine, vincristine, vincristine liposomes, and vinorelbine), corticosteroids (e.g., prednisone, methylprednisolone, and dexamethasone), all-trans retinoic acid, arsenic trioxide, asparaginase, eribulin, hydroxyurea, ixabepyrone, mitotane, omacetaxin, pegasparaginase, procarbazine, romidepsin, and vorinostat.

[0275] Examples of immunotherapies include immune checkpoint inhibitors, cancer treatment vaccines (e.g., human papillomavirus vaccine, hepatitis B vaccine, Sipuleucel-T (Provenge), and Talimogene laherparepvec (T-VEC)), monoclonal antibodies (e.g., alemtuzumab, bevacizumab, cetuximab, gemtuzumab ozogamicin, ipilimumab, ofatumumab, panitumumab, pembrolizumab, ranibizumab, rituximab, and trastuzumab), and immune system modulators (e.g., interleukins (e.g., IL-2, IL-7, IL-21, and IL-12)), cytokines (e.g., Examples include interferons (IFN-α, IFN-β, and IFN-γ) and G-CSF), chemokines (e.g., CCL3, CCL26, and CXCL7), immunomodulatory imide drugs (e.g., thalidomide and its analogues (lenalidomide, pomalidomide, and apremilast)), imiquimod, Bacillus calmette guerin (BCG), cytosine phosphate guanosine, oligodeoxynucleotides, and glucans).

[0276] Examples of hormone therapies include abiraterone (Zytiga®), anastrozole (Arimidex®), exemestane (Aromasin®), fulvestrant (Faslodex®), letrozole (Femara®), leuprolide (Eligard®, Lupron Depot®), toremifene (Fareston®), fluoxymesterone (Halotestin®), megestrol acetate (Megace®), bicalutamide (Cased®), nilutamide (Nilandron®), flutamide (Eulexin®), goserelin (Zoladex®), degarelix (Firmagon®), and tamoxifen (Nolvadex®).

[0277] Examples of angiogenesis inhibitors include axitinib (Inlyta®), bevacizumab (Avastin®), cabozantinib (Cometriq®), everolimus (Afinitor®), lenalidomide (Revlimid®), lenvatinib mesylate (Lenvima®), pazopanib (Votrient®), ramucirumab (Cyramza®), regorafenib (Stivarga®), sorafenib (Nexavar®), sunitinib (Sutent®), thalidomide (Synovir, Thalomid®), vandetanib (Caprelsa®), and div-aflibercept (Zaltrap®).

[0278] Examples of targeted therapies include: EGFR inhibitors (e.g., cetuximab (Erbitux®) and panitumumab (Vectibix®)), HER2 inhibitors (e.g., trastuzumab (Herceptin®), pertuzumab (Perjeta®), and Ado-trastuzumab emtansine (Kadcyla®)), kinase inhibitors (e.g., axitinib (Inlyta®), bosutinib (Bosulif®), cabozantinib (Cometriq®), crizotinib (Xalkori®), dabrafenib (Tafinlara), dasatinib (Sprycel®), erlotinib Nib (Tarceva®), Ibrutinib (Imbruvica®), Imatinib (Gleevec®), Lapatinib (Tykerb®), Nilotinib (Tasigna®), Pazopanib (Votrient®), Ponatinib (Iclusig®), Regorafenib (Stivarga®), Sorafenib (Nexavar®), Sunitinib (Sutent®), Trametinib (Mekinist®), Vandetanib (Caprelsa®) and vemurafenib (Zelboraf®), mTOR inhibitors (e.g., sirolimus (Rapamune®), everolimus (Afinitor®), and temsirolimus (Toricel®)), hedgehog pathway inhibitors (e.g., bismodegib (Erivedge®)), immune system targeted inhibitors (e.g., alemtuzumab (Campath®), brentuximab vedotin (Adcetris®), i Pylimumab (Yervoy®), ibritumomab tiuxetan (Zevalin®), obinutuzumab (Gazyva®), ofatumumab (Azerra®), and rituximab (Rituxan®), VEGF receptor inhibitors (e.g., bevacizumab (Avastin®) and ziv-aflibercept (Zaltrap®)), estrogen-targeted inhibitors (e.g., anastrozole (Arimidex®), exemestane (Aromasin)) , fulvestrant (Faslodex®), letrozole (Femara®), raloxifene (Evista®), tamoxifen citrate and toremifene citrate (Fareston®), androgen-targeted inhibitors (e.g., abiraterone acetate (Zytiga®), bicalutamide (Casodex®), enzalutamide (Xtandi®), flutamide and nilutamide (Nilandron®), proteasome-targeted inhibitors (e.g.,Bortezomib (Velcade®) and carfilzomib (Kyprolis®), histone deacetylase-targeted inhibitors (e.g., romidepsin (Istodax®) and vorinostat (Zolinza®)), folate-targeted inhibitors (e.g., pralatrexate (Folotyn®)), and retinoic acid receptor-targeted inhibitors (e.g., isotretinoin, tretinoin, acitretin (Soriatane®), and bexarotene (Targretin®)).

[0279] In some embodiments, the methods described herein include administering genetically modified T cells modified to express CARs and amino acid transporters, such as arginine transporters, or a pharmaceutical composition thereof, and performing surgery on a patient. In some embodiments, the methods include administering genetically modified T cells modified to express CARs and amino acid transporters, such as arginine transporters, or a pharmaceutical composition thereof, and the administration is to a patient who has undergone anti-cancer surgery, a patient undergoing anti-cancer surgery, or a patient who is a candidate for anti-cancer surgery. Anti-cancer surgery includes, for example, cryosurgery, laser surgery, hyperthermia, photodynamic therapy, open surgery, and minimally invasive surgery. Pharmaceutical composition

[0280] The pharmaceutical composition of the present invention may be a combination of any arginine transporter that overexpresses CAR-T cells as described herein and other chemical components, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners and / or excipients. The pharmaceutical composition facilitates the administration of the manipulated CAR-T cells described herein to an organism. The pharmaceutical composition may be administered by various forms and routes, including, for example, intravenous, subcutaneous, intramuscular, rectal, aerosol, parenteral, ophthalmic, pulmonary, transdermal, vaginal, optic, nasal and topical administration. The drug can then be administered in a therapeutically effective dose. The pharmaceutical composition can be administered locally or systemically, for example, by direct injection of CAR-T cells into organs.

[0281] In some embodiments, the CAR-T pharmaceutical compositions described herein are administered intravenously, for example, by intravenous infusion. In some embodiments, the dose of the CAR-T pharmaceutical composition is administered over a period of about 20 to 30 minutes. In some embodiments, the dose of the CAR-T pharmaceutical composition is administered over a period of approximately 5 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 10 to 20 minutes, 10 to 30 minutes, 10 to 60 minutes, 30 to 60 minutes, 40 to 60 minutes, 20 to 30 minutes, 20 to 40 minutes, 1 to 2 hours, 1 to 3 hours, 1 to 4 hours, 1 to 5 hours, 1 to 6 hours, 2 to 3 hours, 2 to 4 hours, or 3 to 6 hours.

[0282] In some embodiments, the dose of the CAR-T pharmaceutical composition is administered to the subject daily over 1, 2, 3, 4, 5, 6, or 7 days. In some embodiments, the dose of the CAR-T pharmaceutical composition is administered over 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 1 to about 2 weeks, about 1 to about 3 weeks, about 2 to about 3 weeks, about 1 to about 4 weeks, about The drug is administered weekly to the target individual over a period of approximately 2-4 weeks, 3-4 weeks, 1-12 weeks, 4-12 weeks, 6-12 weeks, 8-12 weeks, 10-12 weeks, 6-24 weeks, 8-24 weeks, 10-24 weeks, 12-24 weeks, 6-18 weeks, 8-18 weeks, 10-18 weeks, 12-18 weeks, 14-18 weeks, or 16-18 weeks. In some embodiments, the dose of the CAR-T pharmaceutical composition is administered to the subject every two weeks over a period of 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 1 to about 2 weeks, about 4 to about 12 weeks, about 6 to about 12 weeks, about 8 to about 12 weeks, about 10 to about 12 weeks, about 6 to about 24 weeks, about 8 to about 24 weeks, about 10 to about 24 weeks, about 12 to about 24 weeks, about 6 to about 18 weeks, about 8 to about 18 weeks, about 10 to about 18 weeks, about 12 to about 18 weeks, about 14 to about 18 weeks, or about 16 to about 18 weeks.

[0283] When performing the treatment or use methods provided herein, a therapeutically effective dose of the arginine transporter that overexpresses CAR-T cells as described herein is administered in a pharmaceutical composition to a subject suffering from an immune system-affecting condition. In some embodiments, the subject is a mammal such as a human. The therapeutically effective dose may vary considerably depending on the severity of the disease, the subject's age and relative health status, the potency of the compound used, and other factors.

[0284] The pharmaceutical compositions described herein may include genetically modified living cells, such as CAR-T cells that overexpress the arginine transporter. The CAR-T pharmaceutical compositions described herein may be administered to a subject in individual doses. For example, the CAR-T cell pharmaceutical compositions described herein may include 10 4 ~10 11 Cells / kg, target body weight, 10 5 ~10 11 Cells / kg, target body weight, 10 6 ~10 11 Cells / kg, target body weight, 10 7 ~10 11 Cells / kg, target body weight, 10 8 ~10 11 Cells / kg, target body weight, 10 9 ~10 11 Cells / kg, target body weight, 10 10 ~10 11 Cells / kg, target body weight, 10 4 ~10 10 Cells / kg, target body weight, 10 5 ~10 10 Cells / kg, target body weight, 10 6 ~10 10 Cells / kg, target body weight, 10 7 ~10 10 Cells / kg, target body weight, 10 8 ~10 10 Cells / kg, target body weight, 10 9 ~10 10 Cells / kg, target body weight, 10 4 ~10 9 Cells / kg, target body weight, 10 5 ~10 9 Cells / kg, target body weight, 10 6 ~10 9 Cells / kg, target body weight, 10 7 ~10 9 Cells / kg, target body weight, 10 8 ~10 9 Cells / kg, target body weight, 10 4 ~10 8 Cells / kg, target body weight, 10 5 ~10 8 Cells / kg, target body weight, 10 6 ~10 8Cells / kg, target body weight, 10 7 ~10 8 Cells / kg, target body weight, 10 4 ~10 7 Cells / kg, target body weight, 10 5 ~10 7 Cells / kg, target body weight, 10 6 ~10 7 Cells / kg, target body weight, 10 4 ~10 6 Cells / kg, target body weight, 10 5 ~10 6 Cells / kg, target body weight, or 10 4 ~10 5 It can be administered at a dose based on the target body weight per kg of cells.

[0285] In some embodiments, the dose of the CAR-T cell pharmaceutical composition is approximately 1 × 10⁶ times the subject's body weight per kg. 3 pieces, about 1×10 4 pieces, about 1×10 5 pieces, about 1×10 6 pieces, about 1×10 7 pieces, about 1×10 8 pieces, about 1×10 9 pieces, about 1×10 10 pieces, approximately 2×10 10 pieces, about 3 x 10 10 pieces, about 4×10 10 pieces, about 5×10 10 pieces, about 6×10 10 pieces, about 7×10 10 pieces, about 8×10 10 pieces, approximately 9×10 10 pieces, about 1×10 11 pieces, about 1×10 12 pieces, about 1×10 13 pieces, about 1×10 14 pieces, about 1×10 15 pieces, about 1×10 3 ~Approx. 3×10 10 pieces, about 1×10 5 ~Approx. 3×10 10 pieces, about 1×10 3 ~Approx. 1×10 5 pieces, about 1×10 5 ~Approx. 1×10 15 pieces, about 1×10 5 ~Approx. 1×1010 pieces, about 1×10 7 ~Approx. 1×10 12 pieces, about 1×10 5 ~Approx. 1×10 7 pieces, about 1×10 10 ~Approx. 9×10 10 pieces, or approximately 1 x 10 9 ~Approx. 1×10 11 Contains individual cells.

[0286] In some embodiments, the dose of the CAR-T cell pharmaceutical composition is approximately 1 × 10⁻⁶ 5 pieces, about 1×10 6 pieces, about 1×10 7 pieces, about 1×10 8 pieces, about 1×10 9 pieces, about 1×10 10 pieces, about 1×10 11 pieces, about 1×10 12 pieces, about 1×10 13 pieces, about 1×10 14 pieces, about 1×10 15 pieces, about 1×10 5 ~Approx. 1×10 12 pieces, about 1×10 5 ~Approx. 1×10 10 pieces, about 1×10 5 ~Approx. 1×10 7 pieces, about 1×10 7 ~Approx. 1×10 10 pieces, about 1×10 7 ~Approx. 1×10 12 pieces, about 1×10 9 ~Approx. 1×10 10 pieces, about 1×10 6 ~Approx. 1×10 8 pieces, about 1×10 7 ~Approx. 1×10 9 pieces, about 1×10 5 ~Approx. 1×10 14 pieces, about 1×10 10 ~Approx. 1×10 15 pieces, or approximately 1 x 10 9 ~Approx. 1×10 11 Contains individual cells.

[0287] In some embodiments, the patient is administered an escalating dose of the CAR-T cell pharmaceutical composition. For example, in some embodiments, the treatment method includes administering an initial dose of the CAR-T pharmaceutical composition containing a specified number of cells per kg of body weight of the subject, and subsequent doses of the CAR-T pharmaceutical composition containing more CAR-T cells per kg of body weight of the subject compared to the initial dose. For example, in some embodiments, the treatment method involves administering approximately 1 × 10⁶ cells per kg of body weight of the subject. 5 The initial dose of a CAR-T drug composition containing individual cells is administered, and approximately 1 × 10⁶ cells are administered per kg of the subject's body weight. 6 pieces, about 1×10 7 pieces, about 1×10 8 pieces, about 1×10 9 pieces, about 1×10 10 pieces, about 2x10 10 pieces, about 3x10 10 pieces, about 4x10 10 pieces, about 5x10 10 pieces, about 6x10 10 pieces, about 7x10 10 pieces, about 8x10 10 pieces, approx. 9x10 10 pieces, about 1×10 11 pieces, about 1×10 12 pieces, about 1×10 13 pieces, about 1×10 14 pieces, about 1×10 15 pieces, about 1×10 6 ~about 3x10 10 pieces, about 1×10 6 ~about 3x10 10 pieces, about 1×10 6 ~Approx. 1×10 7 pieces, about 1×10 6 ~Approx. 1×10 15 pieces, about 1×10 6 ~Approx. 1×10 10 pieces, about 1×10 7 ~Approx. 1×10 12 pieces, about 1×10 6 ~Approx. 1×10 8 pieces, about 1×10 10 ~approximately 9x10 10 pieces, or approximately 1 x 10 9 ~Approx. 1×10 11This involves administering one or more subsequent doses of a CAR-T pharmaceutical composition containing individual cells.

[0288] In some embodiments, the initial dose of the CAR-T cell pharmaceutical composition is approximately 1 × 10¹⁶ units per kg of body weight of the subject. 3 pieces, about 1×10 4 pieces, about 1×10 5 pieces, about 1×10 6 pieces, about 1×10 7 pieces, about 1×10 8 pieces, about 1×10 9 pieces, about 1×10 10 pieces, approximately 2×10 10 pieces, about 3 x 10 10 pieces, about 4×10 10 pieces, about 5×10 10 pieces, about 6×10 10 pieces, about 7×10 10 pieces, about 8×10 10 pieces, approximately 9×10 10 pieces, about 1×10 11 pieces, about 1×10 12 pieces, about 1×10 13 pieces, about 1×10 14 pieces, about 1×10 15 pieces, about 1×10 3 ~Approx. 3×10 10 pieces, about 1×10 5 ~Approx. 3×10 10 pieces, about 1×10 3 ~Approx. 1×10 5 pieces, about 1×10 5 ~Approx. 1×10 15 pieces, about 1×10 5 ~Approx. 1×10 10 pieces, about 1×10 7 ~Approx. 1×10 12 pieces, about 1×10 5 ~Approx. 1×10 7 pieces, about 1×10 10 ~Approx. 9×10 10 pieces, or approximately 1 x 10 9 ~Approx. 1×10 11 Contains individual cells.

[0289] In some embodiments, the subsequent dose of the CAR-T cell pharmaceutical composition is approximately 1 × 10⁶ units per kg of body weight of the subject.3 pieces, about 1×10 4 pieces, about 1×10 5 pieces, about 1×10 6 pieces, about 1×10 7 pieces, about 1×10 8 pieces, about 1×10 9 pieces, about 1×10 10 pieces, approximately 2×10 10 pieces, about 3 x 10 10 pieces, about 4×10 10 pieces, about 5×10 10 pieces, about 6×10 10 pieces, about 7×10 10 pieces, about 8×10 10 pieces, approximately 9×10 10 pieces, about 1×10 11 pieces, about 1×10 12 pieces, about 1×10 13 pieces, about 1×10 14 pieces, about 1×10 15 pieces, about 1×10 3 ~Approx. 3×10 10 pieces, about 1×10 5 ~Approx. 3×10 10 pieces, about 1×10 3 ~Approx. 1×10 5 pieces, about 1×10 5 ~Approx. 1×10 15 pieces, about 1×10 5 ~Approx. 1×10 10 pieces, about 1×10 7 ~Approx. 1×10 12 pieces, about 1×10 5 ~Approx. 1×10 7 pieces, about 1×10 10 ~Approx. 9×10 10 pieces, or approximately 1 x 10 9 ~Approx. 1×10 11 Contains individual cells.

[0290] The pharmaceutical compositions containing CAR-T cells described herein may also be administered multiple times at these doses. The cells can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al, New Eng. J. of Med. 319:1676, 1988). Non-limiting examples of pharmaceutically acceptable excipients are described, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), each of which is incorporated in whole by reference. Method of administration

[0291] Pharmaceutical compositions containing CAR-T cells or functional fragments of CAR-T cells overexpressing arginine transporters, as described herein, may be administered for prophylactic and / or therapeutic purposes. For therapeutic use, the compositions may be administered to subjects already suffering from a disease or condition in an amount sufficient to cure or at least partially halt the symptoms of the disease or condition, or in an amount sufficient to cure, heal, improve, or restore the condition. Arginine transporter-overexpressing CAR-T cells may also be administered to reduce the likelihood of developing, becoming afflicted with, or worsening a condition. The effective dose for this use may be modified based on the severity and course of the disease or condition, previous treatments, the subject's health status, weight, and response to the drug, as well as the judgment of the treating physician.

[0292] The arginine transporter-overexpressing CAR-T cells described herein can be administered before, during, or after the onset of a disease or condition, and the timing of administration of compositions containing arginine transporter-overexpressing CAR-T cells can be modified. For example, arginine transporter-overexpressing CAR-T cells can be used as a prophylactic agent and can be administered sequentially to subjects prone to a condition or disease to reduce the likelihood of the onset of the disease or condition. Arginine transporter-overexpressing CAR-T cells can be administered to subjects as soon as possible during or after the onset of symptoms. Administration of arginine transporter-overexpressing CAR-T cells can be initiated immediately after the onset of symptoms, within the first three hours of symptom onset, within the first six hours of symptom onset, within the first 24 hours of symptom onset, within 48 hours of symptom onset, or within any period after the onset of symptoms. Initial administration can be carried out using any formulation described herein via any practical route, including any route described herein. Arginine transporter-overexpressing CAR-T cells can be administered as soon as possible after the onset of an immune disorder or condition, for the duration necessary to treat the immune disorder, for example, approximately 24 to 48 hours, approximately 48 hours to approximately 1 week, approximately 1 week to approximately 2 weeks, approximately 2 weeks to approximately 1 month, or approximately 1 month to approximately 3 months. In some embodiments, arginine transporter-overexpressing CAR-T cells can be administered for at least 24 hours, at least 48 hours, at least 72 hours, at least 96 hours, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 1 year, at least 2 years, at least 3 years, at least 4 years, or at least 5 years. The length of treatment may vary depending on the subject.

[0293] The homology of a reference nucleotide sequence to the recombinant nucleotide sequence of CAR-T cells described herein may be expressed as a percentage of sequence homology. In some embodiments, the homology of the reference sequence is about 60% to about 100% of the bases of the recombinant sequence. In some embodiments, the homology of the reference sequence is about 60% to about 70% of the bases, about 60% to about 80% of the bases, about 60% to about 90% of the bases, about 60% to about 100% of the bases, about 70% to about 80% of the bases, about 70% to about 90% of the bases, about 70% to about 100% of the bases, about 80% to about 90% of the bases, or about 90% to about 100% of the bases of the recombinant sequence. In some embodiments, the homology of the reference sequence is about 60% of the bases, about 70% of the bases, about 80% of the bases, about 90% of the bases, or about 100% of the bases of the recombinant sequence. In some embodiments, the homology of the sequence is at least about 60% of the bases, about 70% of the bases, about 80% of the bases, or about 90% of the bases of the recombinant sequence. In some embodiments, the homology of the reference sequence is about 70% or less of the bases, about 80% of the bases, about 90% of the bases, or about 100% of the bases of the recombinant sequence. [Examples]

[0294] This disclosure is further illustrated by the following embodiments. The embodiments are provided for illustrative purposes only and should not be construed as limiting the scope or content of this disclosure. Example 1. Effects of arginine transporter and arginine synthesis protein expression on T cell survival.

[0295]

[0296] Jurkat clone E 6-1 cells (ATCC, USA) were cultured at 37°C and 5% CO2 in RPMI-1640 medium containing 2 mM L-alanyl-L-glutamine (Transgen Biotech, China), 10% filtered unthermally inactivated fetal bovine serum (TransSerum EQ fetal bovine serum; Transgen Biotech, China), 100 U / mL penicillin, and 100 μg / mL streptomycin (Thermo Fisher Scientific, USA). After reaching approximately 80% confluence, the cells were cultured at 2 × 10⁶ cells. 5 The cells were resuspended in fresh complete medium at a density of cells / mL. The cells were then placed in a 24-well culture plate (500 μL / well, 1 × 10⁶ cells). 5 The cells were seeded in wells.

[0297] Four wells of cells were transfected with each construct (control, CAT, or ASS). 5 μg of each purified plasmid was diluted in 1 mL of OptiMEM reduced serum medium (Thermo Fisher Scientific, Waltham, Massachusetts) containing 1 μL of PLUS reagent. The mixture was incubated at room temperature for 15 minutes, then 2.75 μL of LTX reagent was added to initiate complex formation. The complexes were allowed to form at room temperature for 25 minutes. 100 μL of the vector liposome complex was added to each well. After adding the transfection complex, the plate was shaken for 2 minutes. The cells were then incubated at 37°C and 5% CO2 for 24 hours. Trypan blue staining (Thermo Fisher Cell viability (>90%) was measured using Scientific (Waltham, Massachusetts), and transfection efficiency was determined by analyzing fluorescent protein expression under a microscope (Zeiss Axio Observer, Germany).

[0298] The transfection efficiency of Jurkat E 6-1 cells was 7%–14% (average transfection efficiency 10%). After transfection, cells were cultured for 72 hours at 37°C and 5CO2 in the same medium, either supplemented with or without 400 ng / mL of BCT-100, to achieve arginine depletion in the medium (Figure 3A). The total number of viable transfected cells was counted in each sample. To count only fluorescent cells, cell counting was performed in each well using a Countess II FL (Thermo Fisher Scientific, Waltham, Massachusetts) with default gating parameters (Figure 3B).

[0299] For cells transfected with a control construct, a CAT construct, or an ASS construct, the percentage change in cell number after 72 hours of culture in arginine-rich and arginine-depleted media was calculated (Figure 3C). The percentage change in cell number was calculated by dividing the number of transfected cells after 72 hours of culture minus the initial number of transfected cells by the initial number of transfected cells, and multiplying all results by 100 (100 × ((number of transfected cells after 72 hours - number of transfected cells) / (number of transfected cells))). The initial number of transfected cells was estimated by multiplying the initial number of viable cells by the estimated transfection efficiency (initial number of viable cells × transfection efficiency). Each data point plotted in Figure 3C represents the estimated percentage change in cell number in one isolated well of independently transfected cells. Cell transfection with the control construct, CAT construct, or ASS construct all resulted in an increase in cell number after 72 hours in arginine-rich medium (Figure 3C, left). In contrast, cell transfection with the control construct resulted in an overall decrease in cell number after 72 hours in arginine-depleted medium, while cell transfection with either the CAT or ASS expression construct resulted in an overall increase in cell number after 72 hours in arginine-depleted medium (Figure 3C, right).

[0300] These results demonstrate that the expression of proteins that promote intracellular arginine uptake or intracellular arginine synthesis increases T cell survival and proliferation under conditions of low extracellular arginine concentrations. Example 2. Effect of arginine transporter on primary human T cell survival.

[0301] To evaluate the effects of exogenous arginine transporter proteins, human primary T cell survival was analyzed under low environmental arginine conditions. Frozen primary human CD4+ T cells were obtained from Stem Express (California, USA), thawed, and incubated in RPMI-1640 (Thermo Fisher Scientific) supplemented with GlutaMAX and HEPES (1 × 10⁶ cells). 6 The cells were resuspended at a concentration of cells / mL. The cells were stimulated with 25 μL / mL ImmunoCult Human CD3 / CD28 T Cell Activator (STEMCELL Technologies, Canada) and 10 ng / mL rIL-2 (Solarbio, China) at 37°C and 5% CO2 for 3 days. Activated T cells were collected and 1 × 10⁶ cells were extracted. 7 Opti-MEM I reduced serum medium (Thermo) at a density of cells / mL The cells were resuspended in Fisherbrand Electroporation Cuvettes. 100 microliters of cell suspension were added to Fisherbrand Electroporation Cuvettes. The samples were transferred to Plus (Fisher Scientific, Pennsylvania, USA), and in vitro transcribed mRNA encoding either mNeonGreen (SEQ ID NO: 274) or CAT (SEQ ID NO: 203) was electroporated using an ECM 830 square wave electroporation system (BTX, USA). The mNeonGreen nucleotide sequences used were as follows:

[0298] ATGGTGTCCAAGGGTGAAGAGGACAACATGGCTTCCTTGCCTGCCACCCATGAACTCCATATCTTCGGGTCTTATTAACGGAGTCGACTTTGATATGGTGGGGCAGGGTACGGGCAACCCTAACGACGGCTACGAAGAGCTGAACCTGAAGTCCACTAAGGGCGACCTCCAGTTTTCT CCTTGGATTCTGGTGCCACACATCGGTTATGGTTTTCATCAGTACCTTCCATACCCGGACGGCATGTCCCCGTTCCAGGCGGCTATGGTCGACGGATCTGCTACCAGGTGCACCGCACTATGCAGTTTGAAAGACGGCGCATCTCTGACCGTGAACTACCGTTACACTTATGAGGGC TCCCATATCAAGGGTGAGGCGCAAGTCAAGGGCACGGTTTCCCGGCGGATGGATCCAGTGATGACCAACAGTCTTACCGCAGCCGACTGGTGTCGCAGCAAAAAGACATATCCCAACGACAAGACCATTATCAGCCACCTTTAAATGGTCTTACAGCACCGGGAACGGTAAACGCTATAGGAGCCAGCCCGCACTACCTATACCTTTGCAAAACCTATGGCCGGAACTATCTGAAAAACCAGCCGAGTGACGTCTTCCGGAAGACCGAGCTGAAGCACAGTAAGACAGTAAGACAGCTGAACTTCAAAGAGTGGCAAAAAGCTTTTACGGACGGTGATGGGCATGGATGAATTGTACAAG (sequence number 274)

[0302] Electroporated cells were transferred to one well in a 6-well plate containing 900 μL of RPMI-1640 supplemented with GlutaMAX, HEPES, and rIL-2, and cultured overnight. Cell viability (50–60%) was measured using trypan blue staining (Thermo Fisher Scientific), and transfection efficiency was determined by analyzing fluorescent protein expression under a microscope (Zeiss Axio Observer). Transfection efficiency exceeded 80%. 500 microliters of culture were dispensed into an adjacent empty well, supplemented with 400 ng / ml of BCT-100, and arginine depletion was achieved. The plate was cultured overnight at 37°C and 5% CO2. Cell viability was determined again as described above.

[0303] For control or CAT mRNA-transfected cells, the percentage change in cell number after 24 hours of culture in arginine-rich and arginine-depleted media was calculated (Figure 4). The percentage change in cell number was calculated by subtracting the initial cell number from the cell number after 24 hours of culture, dividing by the initial cell number, and multiplying all calculations by 100.

[0304] Transfection of primary human T cells with either control or CAT mRNA resulted in an increase in cell number after 24 hours in arginine-rich medium (Figure 4, top). In contrast, transfection of cells with GFP control mRNA resulted in a net decrease in cell number after 24 hours in arginine-depleted medium, while transfection of cells with CAT mRNA resulted in an overall increase in cell number after 24 hours in arginine-depleted medium (Figure 4, bottom).

[0305] These results demonstrate that the expression of arginine transporter proteins, which promote intracellular arginine uptake, increases the survival and proliferation of primary human T cells under conditions of low extracellular arginine concentrations. Example 3. Production of CAR-T cells

[0306] This example aims to demonstrate a method for producing CAR-T cells as described herein.

[0307] CD4+ and CD8+ T cells are isolated from whole blood using CliniMACS Prodigy (Miltenyi Biotec, Germany) containing tube set TS520 and CD4 / CD8 microbeads. Approximately 1 × 10⁶ 8 The isolated cells were cultured for 3 days at 37°C and 5% CO2 in 70 mL of TexMACS medium supplemented with 200 IU / mL of IL-2 and TransAct beads (Miltenyi Biotec, Germany) to expand and grow.

[0308] Expanded cells are transfected with an expression vector encoding CAR, arginine transporter, or both CAR and arginine transporter using a CliniMACS Electroporator (Miltenyi Biotec, Germany). Expanded cells can also be co-transfected with a first expression vector encoding CAR and a second expression vector encoding arginine transporter. After transfection, cells are cultured in TexMACS medium (Miltenyi Biotec, Germany) supplemented with 1 mM L-arginine (Sigma-Aldrich, USA). Cells are sampled daily to measure cell number and viability using a Live / Dead Cell Double Staining Kit (Sigma-Aldrich, USA). Fresh medium is added daily, with a concentration of 2 × 10⁶ per mL. 5 ~1 × 10 6 Maintain the cell density of the cells. Half of the culture medium is replaced every other day.

[0309] T cell purity and the helper T cell to killer T cell ratio are determined using a BD FACSAria III flow cytometer and labeled anti-CD19, CD14, CD45, CD3, CD4, and CD8 antibodies (BD Biosciences, USA). CAR and arginine transporter protein expression are determined using custom antibodies specific to the antigen recognition domains of the CAR and arginine transporters, respectively (GenScript, USA).

[0310] The intracellular arginine content is approximately 1 × 10⁻⁶. 5 The number of CAR-T cells is determined by collecting aliquots. Cells are pelleted, washed twice in 10 mL of PBS, and then lysed in 100 μL of RIPA buffer. The arginine level of the cell lysates is determined using the L-arginine ELISA kit (ALPCO, USA). The total arginine level is normalized to the number of lysed cells.

[0311] Approximately 1 x 10 per kg of body weight of the subject 5 ~Approx. 3×10 10 Once individual cells are obtained, they are harvested for downstream application, at which point the cells have an intracellular arginine content of approximately 100 μM to 4000 μM per cell. Example 4. Ex vivo nutrition and priming of CAR-T cells overexpressing arginine transporters

[0312] In this embodiment, a priming method for treating genetically modified CAR-T cells expressing arginine transporter protein is envisioned.

[0313] Genetically modified CAR-T cells and CARs expressing arginine transporters are cultured in a culture medium containing or supplemented with L-arginine. This medium contains L-arginine at concentrations of 0.2 g / L to 1000 μmol / L. The engineered T cells are cultured in L-arginine medium until intracellular arginine levels reach 100 μmol to 4000 μmol.

[0314] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which this invention pertains.

[0315] Throughout this specification, where compositions and kits are described as having, encompassing, or containing certain components, or where processes and methods are described as having, encompassing, or containing certain steps, it is intended that there are compositions and kits of the present invention that are essentially composed of or consist of the listed components, and processes and methods of the present invention that are essentially composed of or consist of the listed processing steps.

[0316] In this application, when it is said that an element or component is included in a list of enumerated elements or components and / or selected from a list of enumerated elements or components, it should be understood that the element or component may be any one of the enumerated elements or components, or it may be selected from a group of two or more enumerated elements or components.

[0317] Furthermore, it should be understood that the elements and / or features of the compositions or methods described herein, whether expressly or implicitly, can be combined in various ways without departing from the spirit and scope of the invention. For example, where a particular compound is mentioned, that compound can be used in various embodiments of the compositions and / or methods of the invention unless otherwise understood from the context. In other words, while embodiments have been described and illustrated in this application in a manner that enables a clear and concise description and depiction of the application, it is intended and understood that embodiments can be combined and separated in various ways without departing from the teachings and the invention(s). For example, it will be understood that all features described and illustrated herein may be applicable to all embodiments of the invention(s) described and illustrated herein.

[0318] The articles “a” and “an” are used in this disclosure to refer to one or more (i.e., at least one) of the grammatical objects of the articles, unless the context is inappropriate. For example, “element” means one or more elements.

[0319] In this disclosure, the terms "and / or" are used to mean either "and" or "or" unless otherwise indicated.

[0320] The expression "at least one of ~" should be understood, unless otherwise understood from the context and usage, to include each of the enumerated objects that follow the expression, and various combinations of two or more of the enumerated objects individually. The expression "and / or" relating to three or more enumerated objects should be understood to have the same meaning, unless otherwise understood from the context.

[0321] The use of the terms “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including their grammatical equivalents, should generally be understood as unrestricted and non-limiting, and for example, not to exclude further elements or steps not listed unless otherwise specified or understood from the context.

[0322] Where the term "approximately" is used before a quantitative value, this disclosure also includes the specific quantitative value itself unless otherwise specified.

[0323] For example, when the molecular weight of a polymer is provided rather than its absolute value, that molecular weight should be understood to be the average molecular weight unless otherwise specified or understood from the context.

[0324] It should be understood that, as long as the present invention remains operable, the order of the steps or the order in which certain actions are performed is not important. Furthermore, two or more steps or actions may be performed simultaneously.

[0325] In various places in this specification, substituents are disclosed in groups or ranges. This specification is specifically intended to include all individual partial combinations of members of such groups and ranges. For example, integers in the range of 0 to 40 are specifically intended to individually disclose 0, 1, 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, and 40, and integers in the range of 1 to 20 are specifically intended to individually disclose 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, and 20.

[0326] Any use of examples or illustrative language herein, such as “such as” or “including,” is intended solely to better illustrate the invention and, unless otherwise claimed, does not limit the scope of the invention. No language herein should be construed as indicating an unclaimed element essential to the practice of the invention.

[0327] As a general rule, percentages for compositions are based on weight unless otherwise specified. Furthermore, if a variable is not defined, the previous definition of the variable takes precedence. Embedding by reference

[0328] All scientific papers, publications, and patent documents referenced herein are incorporated herein by reference in their entirety for any purpose as if each individual publication or patent were incorporated specifically and individually. In case of any conflict, the present application, including any definitions herein, shall prevail. Equivalents

[0329] While specific embodiments of the present invention have been considered, the above specification is illustrative and not limiting. Many variations of the present invention will become apparent to those skilled in the art by examining this specification. The full scope of the present invention should be determined by referring to the claims, the full scope of its equivalents, and the specification, as well as such variations.

[0330] Unless otherwise indicated, all figures used herein and in the claims to represent quantities of components, reaction conditions, etc., should be understood in all cases to be modified by the term "approximately." Therefore, unless otherwise indicated, the numerical parameters described herein and in the appended claims are approximations that may vary depending on the desired properties to be obtained by the present invention. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] Table 2-9 Table 2-10 Table 2-11 Table 2-12 Table 2-13 Table 2-14 Table 2-15 Table 2-16 Table 2-17 Table 2-18 Table 2-19 Table 2-20 Table 2-21 Table 2-22 Table 2-23 Table 2-24 Table 2-25 Table 2-26 Table 2-27 Table 2-28 Table 2-29 Table 2-30

Claims

1. A composition for use in a method for increasing the survival of T cells in a low-arginine environment, comprising T cells comprising a recombinant arginine transporter encoded by a nucleic acid sequence having at least 95% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 184-188, 196, 197, 201, 202, and 203, wherein the method comprises administering the T cells to the low-arginine environment.

2. The composition for use according to claim 1, wherein the nucleic acid sequence encoding the recombinant arginine transporter has at least 95% identity with SEQ ID NO: 201, 202, or 203.

3. The composition for use according to claim 2, wherein the nucleic acid sequence encoding the recombinant arginine transporter is sequence number 201, 202, or 203.

4. i) Prior to the administration step, the method comprises transfecting the T cells with a DNA construct comprising a nucleotide sequence encoding the recombinant arginine transporter; ii) The T cell comprises a DNA construct comprising a chimeric antigen receptor and / or a nucleotide sequence encoding the chimeric antigen receptor; and / or iii) Prior to the administration step, the method includes culturing the T cells in a culture medium containing arginine, A composition for use according to any one of claims 1 to 3.

5. The composition for use according to claim 4, wherein the culturing comprises culturing the T cells in the culture medium until the intracellular arginine level of the T cells accumulates to a certain level.

6. The composition according to any one of claims 1 to 5, wherein the low-arginine environment is a cell culture medium or a tumor microenvironment.

7. Genetically modified T cells, which are genetically modified to express a recombinant arginine transporter encoded by a nucleic acid sequence having at least 95% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 184-188, 196, 197, 201, 202, and 203.

8. The genetically modified T cell according to claim 7, further comprising a chimeric antigen receptor having at least one antigen-specific targeting region that specifically binds to a cell surface antigen present on a target cell population, a transmembrane domain, and an intracellular signaling domain.

9. The genetically modified T cell according to claim 7 or 8, wherein the nucleic acid sequence encoding the recombinant arginine transporter has at least 95% identity with SEQ ID NO: 201, 202, or 203.

10. The genetically modified T cell according to claim 9, wherein the nucleic acid sequence encoding the recombinant arginine transporter is sequence number 201, 202, or 203.

11. One or more expression vectors, wherein the one or more expression vectors a) antigen-specific targeting region; b) Transmembrane domain, c) Intracellular signaling domain, and d) Arginine transporter One or more expression vectors comprising one or more isolated nucleic acid sequences encoding the arginine transporter, wherein the isolated nucleic acid sequences encoding the arginine transporter have at least 95% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 184-188, 196, 197, 201, 202, and 203.

12. The one or more expression vectors according to claim 11, wherein the isolated nucleic acid sequence encoding the recombinant arginine transporter has at least 95% identity with SEQ ID NO: 201, 202, or 203.

13. The one or more expression vectors according to claim 12, wherein the isolated nucleic acid sequence encoding the recombinant arginine transporter is SEQ ID NO: 201, 202, or 203.

14. The one or more isolated nucleic acid sequences further encode at least one costimulatory domain in the expression vector according to any one of claims 11 to 13.

15. A pharmaceutical composition comprising a genetically modified T cell according to any one of claims 7 to 10, and a pharmaceutically acceptable excipient.

16. The pharmaceutical composition according to claim 15, wherein the pharmaceutical composition is packaged as a kit.

17. A pharmaceutical composition according to claim 15 for use in a method of treating a solid tumor cancer or hematological cancer in a patient, wherein the method comprises administering the pharmaceutical composition to the patient.

18. The pharmaceutical composition according to claim 15 for use in modulating intracellular arginine levels to achieve a T cell-mediated immune response.

19. A priming medium comprising genetically modified T cells according to any one of claims 7 to 10, and L-arginine.

20. A composition for use in a method of treating a condition in a human patient, wherein the composition comprises genetically modified T cells according to any one of claims 7 to 10, and the method comprises administering the composition to the human patient.

21. A composition for use in a method for modulating a T cell-mediated immune response against a target cell population expressing a cell surface antigen, wherein the composition comprises genetically modified T cells according to any one of claims 7 to 10, and the method comprises administering the genetically modified T cells.

22. The composition for use according to claim 20 or 21, wherein the genetically modified T cells are cultured in a culture medium containing arginine before administration.

23. The composition for use according to any one of claims 20 to 22, wherein the genetically modified T cells are CAR-T cells.

24. i) The composition comprising the CAR-T cells is administered to the human patient once every week; or ii) The composition for use according to claim 23, comprising administering 10⁷ to 10¹⁰ CAR-T cells per kilogram of the patient.

25. i) The composition comprising the CAR-T cells is administered to the human patient once every two weeks; or ii) The composition for use according to claim 23, comprising administering 10⁷ to 10¹⁰ CAR-T cells per kilogram of the patient.

26. i) The composition comprising the CAR-T cells is administered to the human patient once every three weeks; or ii) The composition for use according to claim 23, comprising administering 10⁷ to 10¹⁰ CAR-T cells per kilogram of the patient.

27. ​​i) The composition comprising the CAR-T cells is administered to the human patient once every four weeks; or ii) The composition for use according to claim 23, comprising administering 10⁷ to 10¹⁰ CAR-T cells per kilogram of the patient.

28. A method for producing genetically modified T cells that express a recombinant arginine transporter encoded by a nucleic acid sequence having at least 95% identity with a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 184-188, 196, 197, 201, 202, and 203, Transfecting T cells with a DNA construct containing a nucleotide sequence for an arginine transporter, thereby producing genetically modified T cells that express the arginine transporter, and The genetically modified T cells are cultured in a culture medium containing arginine. Methods that include...

29. The method according to claim 28, wherein the nucleic acid sequence encoding the recombinant arginine transporter has at least 95% identity with SEQ ID NO: 201, 202, or 203.

30. The method according to claim 29, wherein the nucleic acid sequence encoding the recombinant arginine transporter is SEQ ID NO: 201, 202, or 203.

31. The method according to claim 29 or 30, wherein the culturing comprises culturing the genetically modified T cells in the culture medium until the intracellular arginine level of the genetically modified T cells accumulates to a certain level.

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