Methods for promoting the persistence of cell therapy

Abstract

The present application provides a method for promoting the persistence of cell therapy by using a combination regimen comprising both a lymphodepleting agent (e.g., fludarabine and / or cyclophosphamide) and an S-phase inhibitor (e.g., methotrexate) in an individual undergoing cell therapy such as allogeneic CAR-T cell therapy. Also provided are pharmaceutical compositions and kits comprising a lymphodepleting agent, an S-phase inhibitor, and / or immune cells used in cell therapy.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority of International Patent Application No. PCT / CN2022 / 109129, filed on July 29, 2022, the content of which is hereby incorporated by reference in its entirety into this specification.

[0002] Submission of the Sequence Listing as an ASCII Text File The content submitted in the following ASCII text file is hereby incorporated by reference in its entirety into this specification. Sequence Listing in computer - readable format (CRF) (File name: IEC230075PCT - seql.xml, Date of record: July 26, 2023, Size: 50KB).

[0003] Field of the Present Application This application generally relates to promoting the persistence of cell therapy in patients by using a lymphodepleting agent and an S - phase inhibitor (such as methotrexate) in combination.

Background Art

[0004] Background of the Present Application Cell therapy (such as adoptive cell therapy or adoptive cell transfer (ACT)) has become an increasingly important treatment paradigm for various diseases (such as in the treatment of cancer). Cell therapy involves introducing cells (typically immune cells) into a patient's body. These cells may be derived from the patient (i.e., autologous therapy) or from another individual of the same species (i.e., allogeneic therapy). In some cases, the goal of cell therapy is to improve the function and characteristics of the patient's immune system. In particular, in cancer immunotherapy, the goal is to induce an immune response against cancer. T cells are the most commonly used in cell therapy, but other immune cell types, such as NK cells, lymphocytes (such as tumor - infiltrating lymphocytes or TIL), dendritic cells, and myeloid cells, are also used.

[0005] Ideally, the immune cells injected into a patient receiving cell therapy (or reinjected in the case of autologous therapy) expand and persist in the patient's body. However, the immune system has developed complex and effective mechanisms against foreign substances. The patient's immune response is the greatest obstacle to the success of cell therapy, especially when the immune cells are derived from another individual. An effective strategy for reducing unwanted immune responses related to cell therapy is still needed.

[0006] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are hereby incorporated by reference in their entirety. SUMMARY OF THE INVENTION

[0007] Summary This application provides a method for promoting the persistence of cell therapy (e.g., adoptive cell therapy, e.g., allogeneic CAR-T cells) in an individual receiving cell therapy by administering a lymphodepleting agent and an S-phase inhibitor (e.g., methotrexate).

[0008] In one aspect of this application, a method for promoting the persistence of cell therapy in a human individual is provided, the method comprising administering to the individual a) an optional lymphodepleting agent, and b) an S-phase inhibitor, wherein the optional lymphodepleting agent is administered before the cell therapy is performed, the S-phase inhibitor is administered more than once, the cell therapy comprises immune cells that are resistant to the S-phase inhibitor, and the lymphodepleting agent is different from the S-phase inhibitor. In some aspects, the method comprises administering an S-phase inhibitor before performing the cell therapy. In some aspects, the method further comprises administering an S-phase inhibitor after performing the cell therapy.

[0009] In some aspects according to any one of the above methods, the S-phase inhibitor is an antimetabolite of folic acid. In some aspects, the antimetabolite of folic acid is a DHFR inhibitor.

[0010] In some embodiments according to any one of the above methods, the method comprises administering an S-phase inhibitor within about 5 days before administering the immune cells.

[0011] In some embodiments according to any one of the above methods, the method comprises administering an S-phase inhibitor within about 10 days after administering the immune cells, and optionally, wherein the method comprises administering an S-phase inhibitor within about 5 days before and within about 10 days after administering the immune cells.

[0012] In some embodiments according to any one of the above methods, the method comprises administering an S-phase inhibitor and a lymphodepleting agent on the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour), and optionally, wherein the S-phase inhibitor and the lymphodepleting agent are administered in parallel or simultaneously, and optionally, wherein the method comprises administering the S-phase inhibitor and the lymphodepleting agent in parallel or simultaneously within 5 days before immunotherapy.

[0013] In some embodiments according to any one of the above methods, the method comprises administering an S-phase inhibitor once every 1 to 28 days, or in some embodiments, once every 1 to 10 days, during the period from about 5 days before administering the immune cells to about 90 days after administering the immune cells.

[0014] In some embodiments according to any one of the above methods, the S-phase inhibitor is methotrexate (MTX). In some embodiments, methotrexate is administered once every 3 to 7 days or once every 2 to 7 days over at least 3 cycles, and optionally, wherein methotrexate is administered once every 3 to 7 days or once every 2 to 7 days during the period from about 5 days before administering the immune cells to about 90 days after administering the immune cells. In some embodiments, methotrexate is administered on the 3rd, 5th, 10th, and 17th days after each administration of the immune cells. In some embodiments, about 5 mg / m 2 ~ about 3000 mg / m2 Administer methotrexate in an amount of. In some embodiments, about 5 mg / m 2 / day to about 3000 mg / m 2 / day. In some embodiments, administer methotrexate at a dose of about 3 mg / m 2 to about 3000 mg / m 2 Administer methotrexate in an amount of. In some embodiments, about 3 mg / m 2 / day to about 3000 mg / m 2 / day. In some embodiments, within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual is about 2 μM or less. In some embodiments, within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual exceeds about 0.001 μM. In some embodiments, within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual exceeds about 0.01 μM. In some embodiments, within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual exceeds about 0.1 μM.

[0015] In some embodiments according to any one of the above methods, the S-phase inhibitor is administered orally, subcutaneously, intramuscularly, intravenously, intraarterially or intrathecally each time.

[0016] In some embodiments according to any one of the above methods, the lymphodepleting agent comprises fludarabine ("Flu") and cyclophosphamide ("Cy"). In some embodiments, administer fludarabine at a dose of about 25 mg / m 2 to about 30 mg / m 2 and about 250 mg / m 2 to about 1000 mg / m 2Administer cyclophosphamide at a dose of . In some embodiments, administer fludarabine and cyclophosphamide about 1 to 3 times during the period from about 10 days before to about 90 days after administering the immune cells. In some embodiments, before administering the immune cells, administer fludarabine and cyclophosphamide continuously for about 3 to 4 days, and optionally, where a) administer fludarabine and cyclophosphamide continuously for 3 days, b) administer fludarabine and cyclophosphamide continuously for 4 days, or c) administer fludarabine and cyclophosphamide continuously for 3 days, followed by administering fludarabine alone for 1 day.

[0017] In some embodiments according to any one of the above methods, the immune cells are allogeneic.

[0018] In some embodiments according to any one of the above methods, the immune cells include T cells. In some embodiments, the T cells include an exogenous Nef protein. In some embodiments, the T cells are engineered not to express or to express at a reduced level the endogenous TCRα or TCRβ. In some embodiments, the T cells are γ-δ T cells.

[0019] In some embodiments according to any one of the above methods, the immune cells include NK cells.

[0020] In some embodiments according to any one of the above methods, the immune cells comprise a heterologous nucleic acid sequence encoding an engineered receptor. In some embodiments, the engineered receptor is selected from the group consisting of a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), and a T cell antigen conjugate (TAC) receptor. In some embodiments, the engineered receptor targets an antigen (e.g., a tumor antigen) selected from the group consisting of CD19, BCMA, Claudin 18.2, NY-ESO-1, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR, GD2, HER2, IGF1R, Mesothelin, PSMA, GPC3, DLL3, GPRC5D, CLL1, ROR1, WT1, CD4, GU2CYC, MUC16, MUC1, CAIX, CD8, CD7, CD10, CD30, CD34, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD3, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, p53, MART1, GP100, Protease-3 (PR3), Tyrosinase, Survivin, EphA2, h5T4, PSCA, TAG-72, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, Claudin 6, NKG2D, CD70, ADGRE2, FcRH5, NKp80, NKp30, NKG2A, CD229, CS-1, and combinations thereof. In some embodiments, the engineered receptor is an anti-BCMA CAR, an anti-CD20 CAR, an anti-Claudin 18.2 CAR, or an anti-CLL1 CAR. In some embodiments, the immune cells further comprise a second engineered receptor that converts a negative signal into a positive signal, and optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. In some embodiments, the immune cells comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound.

[0021] In some embodiments according to any one of the above methods, the immune cells contain a methotrexate resistance transgene. In some embodiments, the methotrexate resistance transgene contains a mutant DHFR gene. In some embodiments, the mutant DHFR gene contains an L22F mutation and an F31S mutation.

[0022] In some embodiments according to any one of the above methods, the immune cells overexpress a DHFR gene encoding dihydrofolate reductase.

[0023] In some embodiments according to any one of the above methods, the immune cells are administered more than once (e.g., 2, 3, 4, 5 times).

[0024] In some embodiments according to any one of the above methods, the immune cells contain from about 30 million to about 900 million immune cells, or from about 100,000 to about 50 million immune cells per kilogram of human body weight.

[0025] In some embodiments according to any one of the above methods, within about 90 days after the first administration of the immune cells, the immune cells are administered about 2 to 5 times.

[0026] In some embodiments according to any one of the above methods, the individual has cancer. In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is a solid tumor.

[0027] In some embodiments according to any one of the above methods, during the period from about 2 to 7 days before the administration of the immune cells to about 10, 20, 30, 40, 50, 60, 70, 80, or 90 days after the administration of the immune cells, the host T cells measured by the number of host T cells in PBMC are 500, 400, 300, 200, 100, or 50 cells per μL or less.

[0028] In some embodiments according to any one of the above methods, during the period from about 2 to 7 days before the administration of immune cells to about 10, 20, 30, 40, 50, 60, 70, 80, or 90 days after the administration of immune cells, the host white blood cells are about 3000, 2000, 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 cells or less per μL.

[0029] In some embodiments according to any one of the above methods, the method further comprises monitoring the number of host WBC (white blood cells) or host T cells of an individual.

[0030] In another aspect, the present application provides a method for treating a disease or medical condition (e.g., cancer) in a human individual, the method comprising any one of the above methods for promoting the persistence of cell therapy in a human individual.

[0031] In another aspect, the present application provides immune cells or a population thereof for use in the treatment of cancer, wherein these immune cells are administered in combination with a lymphodepleting agent and an S-phase inhibitor (e.g., methotrexate). In some embodiments, the S-phase inhibitor is administered before the administration of immune cells. In some embodiments, the S-phase inhibitor is administered after the administration of immune cells. In some embodiments, the immune cells comprise a methotrexate resistance transgene.

[0032] Further provided are compositions, uses, kits, and manufactured articles comprising any one of the S-phase inhibitors (e.g., methotrexate), any one of the lymphodepleting agents (e.g., Flu and Cy), and / or the immune cells described herein.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0034] Detailed Description The present application provides a method for promoting the persistence of cell therapy in a patient by administering in combination a lymphodepleting agent (e.g., fludarabine and / or cyclophosphamide) and an S-phase inhibitor (e.g., an antimetabolite of folic acid, e.g., methotrexate), thereby achieving the effect of maintaining the number of host immune cells (e.g., T cells) below a threshold level for a long period (e.g., at least 30 days, 60 days, or 90 days after cell therapy administration). The cell therapy may include immune cells that are resistant to the S-phase inhibitor. The S-phase inhibitor (e.g., methotrexate) may be administered more than once. In some embodiments, the present application provides a method for promoting the persistence of cell therapy by administering a lymphodepleting agent before cell therapy and an S-phase inhibitor (e.g., methotrexate) before and after cell therapy. In some embodiments, the present application provides a method for promoting the persistence of cell therapy by administering a lymphodepleting agent and an S-phase inhibitor (e.g., methotrexate) (e.g., in parallel or simultaneously) within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). In another aspect, the present application uses a clinical strategy using a combination of methotrexate and CAR-T cell therapy (e.g., allogeneic CAR-T cell therapy), where these CAR-T cells are resistant to methotrexate.

[0035] The method described in the claims is based at least in part on the following discovery by the inventors: In an individual receiving cell therapy, a combination of an S-phase inhibitor (e.g., methotrexate) targeting strongly proliferating cells and a lymphodepleting agent is used to effectively suppress the proliferation of host immune cells (e.g., T cells) over a long period, and this inhibitory effect persists for a long time. It has also been demonstrated that the desired effect of promoting the persistence / effectiveness of cell therapy is achieved by combined administration.

[0036] Accordingly, in some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) a lymphodepleting agent, and b) an S-phase inhibitor, wherein the lymphodepleting agent is administered before the cell therapy is performed, the S-phase inhibitor is administered more than once, the cell therapy comprises immune cells that are resistant to the S-phase inhibitor, and the lymphodepleting agent is different from the S-phase inhibitor. The S-phase inhibitor may be administered before and after the cell therapy is performed. The S-phase inhibitor may be an antimetabolite of folic acid. The antimetabolite of folic acid may be methotrexate.

[0037] I. Definitions As used herein, "treatment" or "treating" is a method for obtaining a beneficial or desired result, including clinical results. For the purposes of this application, beneficial or desired clinical results include, but are not limited to, reducing one or more symptoms caused by a disease, reducing the degree of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread (e.g., metastasis) of the disease, preventing or delaying the recurrence of the disease, delaying or alleviating the progression of the disease, improving the condition of the disease, providing a remission (partial or complete) of the disease, reducing the dosage of one or more other drugs required for the treatment of the disease, delaying the progression of the disease, improving the quality of life, and / or extending the survival period. "Treatment" further encompasses reducing the pathological consequences of the disease. The methods of this application contemplate any one or more of these aspects of treatment.

[0038] The term "prevention" and like terms such as, for example, "prevented", "preventing", etc. refer to methods for preventing, inhibiting or reducing the likelihood of recurrence of a disease or medical condition (e.g., cancer). It further refers to delaying the recurrence of the disease or medical condition or delaying the recurrence of the symptoms of the disease or medical condition. As used herein, "prevention" and like terms further include reducing the intensity, effect, symptoms and / or burden of a disease or medical condition prior to recurrence of the disease or medical condition.

[0039] As used herein, "delaying" the progression of cancer means delaying, suppressing, decelerating, extending, stabilizing and / or slowing the progression of the disease. Such delay may have different time durations depending on the history of the disease and / or the individual being treated. A method of "delaying" the progression of cancer, as compared to not using the method, is a method that reduces the probability of disease progression within a given time frame and / or reduces the extent of the disease within a given time frame. Such comparisons are typically based on clinical trials using a statistically significant number of individuals. The progression of a disease can be detected using standard methods, which include, but are not limited to, computed axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Progression may refer to the progression of a disease that may initially be undetectable and includes occurrence, recurrence and onset.

[0040] As used herein, the term "effective amount" refers to an amount of an agent sufficient to treat a particular disorder, disease state or disease (e.g., ameliorate, mitigate, reduce and / or delay one or more of its symptoms). With respect to cancer, an effective amount includes an amount sufficient to shrink a tumor and / or reduce the rate of growth of the tumor (e.g., inhibit tumor growth) or prevent or delay other unwanted cell growth. An effective amount may be an amount sufficient to delay progression. An effective amount may be an amount sufficient to prevent or delay recurrence. An effective amount may be administered in a single or multiple administrations. The effective amount of a drug or composition can (i) reduce the number of cancer cells, (ii) reduce the tumor size, (iii) to some extent inhibit, slow down, mitigate, preferably prevent the invasion of cancer cells into surrounding organs, (iv) inhibit the metastasis of the tumor (i.e., to some extent mitigate, preferably prevent), (v) inhibit the growth of the tumor, (vi) prevent or delay the occurrence and / or recurrence of the tumor, and / or (vii) to some extent alleviate one or more symptoms associated with cancer.

[0041] As used herein, "individual" or "subject" refers to a mammal, including but not limited to humans, cows, horses, cats, dogs, rodents or primates. The individual may be a human.

[0042] As used herein, "administered in combination with" or "combined with" refers to administering one therapy in addition to another. Thus, "administered in combination with" or "combined with" refers to administering another therapy before, during or after administering one therapy to an individual.

[0043] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to specifically graft one or more antigens onto immune effector cells such as T cells. Some CARs are also referred to as "artificial T cell receptors", "chimeric T cell receptors" or "chimeric immune receptors". A CAR may include an extracellular antigen-binding domain specific for one or more antigens (e.g., tumor antigens), a transmembrane domain, and an intracellular signaling domain of a T cell and / or other receptor. "CAR-T" refers to T cells that express a CAR.

[0044] As used herein, the term "co-administration" means that in combination therapy, the first therapy and the second therapy are administered at an interval of about 15 minutes or less (e.g., any one of about 10, 5, or 1 minute or less). When the first therapy and the second therapy are co-administered, the first therapy and the second therapy may be included in the same composition (e.g., a composition containing both the first therapy and the second therapy), or may be included in separate compositions (e.g., the first therapy is included in one composition and the second therapy is included in another composition).

[0045] As used herein, the term "sequential administration" means that in combination therapy, the first therapy and the second therapy are administered at an interval exceeding about 15 minutes (e.g., exceeding any one of about 20, 30, 40, 50, 60 minutes or more). The first therapy or the second therapy may be administered first. The first therapy and the second therapy are included in separate compositions, which may be included in the same or different packages or sets.

[0046] As used herein, the term "parallel administration" means that in combination therapy, the administration of the first therapy and the administration of the second therapy overlap with each other.

[0047] As used herein, the terms "bind", "specifically bind", or "specific for" refer to interactions that are measurable and reproducible, such as the binding between a molecule (e.g., a CAR) and a target point, which determines the presence of the target point when a population of heteromolecules (including biomolecules) is present. For example, a CAR that binds or specifically binds to a target point (which may be an epitope) has a greater affinity, avidity for binding to this target point than to other target points, and binds to this target point more readily and / or for a longer duration. In one aspect, the degree of binding of the CAR to a target point not related to the CAR is less than about 10% of the binding of the CAR to the target point, as measured, for example, by a radioimmunoassay (RIA). In some aspects, a CAR that specifically binds to a target point has a dissociation constant (Kd) of ≦1 μM, ≦100 nM, ≦10 nM, ≦1 nM, or ≦0.1 nM. In some aspects, the CAR specifically binds to a conserved protein epitope in proteins from different species. In another aspect, specific binding may include, but is not limited to, exclusive binding.

[0048] "UCART" or "universal CAR-T cell" refers to an off-the-shelf T cell modified by a CAR that can be used for the treatment of allogeneic patients in need thereof. UCART includes those containing genetic modifications in addition to the CAR construct and those not containing genetic modifications except for the CAR construct.

[0049] As used herein, "T cell receptor" or "TCR" refers to an endogenous or recombinant T cell receptor that includes an extracellular antigen-binding domain that binds to a specific antigen peptide that binds to an MHC molecule. The TCR may include a TCRα polypeptide chain and a TCRβ polypeptide chain. The TCR can specifically bind to a tumor antigen. "TCR-T" refers to a T cell that expresses a recombinant TCR.

[0050] As used herein, the "T cell antigen conjugate receptor" or "TAC receptor" refers to an engineered receptor that includes an extracellular antigen-binding domain that binds a specific antigen, a T cell receptor (TCR)-binding domain, a transmembrane domain, and an intracellular domain of a co-receptor molecule. The TAC receptor identifies the endogenous TCR of a T cell expressing the TAC receptor and induces an antigen-specific T cell response against a target cell.

[0051] As used herein, the "TCR fusion protein" or "TFP" refers to an engineered receptor that includes an extracellular antigen-binding domain that binds a specific antigen, which is fused to a subunit or a portion thereof of a TCR complex (including TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ε, CD3δ, or CD3γ). The subunit or a portion thereof of the TCR complex includes the transmembrane domain and at least a portion of the intracellular domain of a naturally occurring TCR subunit. The TFP may include the extracellular domain or a portion thereof of a TCR subunit. The TFP may not include the extracellular domain of a TCR subunit.

[0052] "Percent amino acid sequence identity (%)" and "homology" with respect to a peptide, polypeptide or antibody sequence are defined as the percentage of amino acid residues in a candidate sequence that are the same as the amino acid residues in a particular peptide or polypeptide sequence when sequence alignment is performed to achieve maximum sequence identity and gaps are introduced where necessary, and any conservative substitutions are not considered as part of the sequence identity. For the purpose of determining the percent amino acid sequence identity, alignment can be achieved by various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN (trademark) (DNASTAR) software. One of ordinary skill in the art can include any algorithm that achieves maximum alignment within the full length range of the sequences being compared and can determine the appropriate parameters to use for the alignment measurement. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity with a particular polypeptide described herein and preferably exhibiting substantially the same function, and polynucleotides encoding such polypeptides were considered. <{

[0053] A nucleic acid is "functionally linked" when it is in a functional relationship with another nucleic acid sequence. For example, when the DNA of a presequence or a secretion leader sequence is expressed as a preprotein involved in polypeptide secretion, the DNA of the presequence or the secretion leader sequence is functionally linked to the DNA of the polypeptide, and when a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is functionally linked to the sequence, or when a ribosome binding site is positioned so as to be advantageous for translation, the ribosome binding site is functionally linked to the coding sequence. Usually, "functionally linked" means that the linked DNA sequences are contiguous and, in the case of a secretion leader sequence, are contiguous and within the reading frame. However, an enhancer does not necessarily have to be contiguous. Ligation is performed by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adapters or linkers are used based on common practice.

[0054] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid linked thereto. The term includes vectors that are self-replicating nucleic acid structures and vectors that have been integrated into the host cell genome into which the vector has been introduced. Some vectors are capable of inducing the expression of nucleic acids functionally linked thereto. Such vectors are referred to herein as "expression vectors".

[0055] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing foreign nucleic acid into a host cell. "Transfected" or "transformed" or "transduced" cells are cells that have already been transfected, transformed or transduced with exogenous nucleic acid. The cells include the primary cells of interest and their progeny.

[0056] As used herein, the terms "cell," "cells," "cell line," and "cell culture" may be used interchangeably, and all such references include progeny. Thus, "transformant" and "transformed cells" include the subject primary cells and cultures derived therefrom, without regard to the number of transfers. It should be understood that due to either intentional or unintentional mutations, the DNA content of all progeny may not be exactly the same. Mutant progeny having the same function or biological activity as screened from the originally transformed cells are included.

[0057] "Primary cells" refer to cells directly obtained from a living tissue (i.e., biopsy material) and established for in vitro growth. Compared with continuous tumorigenic cell lines or artificially immortalized cell lines, these cells have undergone few population doublings and can thus better represent the main functional components and characteristics of the tissue from which they are derived.

[0058] The term "in vivo" refers to the body of the organism from which the cells were obtained. "Ex vivo" or "in vitro" means outside the body of the organism from which the cells were obtained.

[0059] As used herein, the term "autologous" refers to any material derived from the same individual, where the material is later reintroduced into that individual.

[0060] "Allogeneic" refers to a graft derived from a different individual of the same species.

[0061] The term "recombinant" refers to a biomolecule, such as a gene or protein, that has been (1) removed from its natural environment, (2) is unrelated to all or part of the polynucleotide in which the gene is found in nature, (3) is functionally linked to a polynucleotide that is not linked in nature, or (4) does not exist in nature. The term "recombinant" may also be used to refer to cloned DNA isolates, chemically synthesized polynucleotide analogs, or biologically synthesized polynucleotide analogs from heterologous systems, and proteins and / or mRNAs encoded by such nucleic acids.

[0062] The term "expression" refers to the transcription of DNA into RNA (e.g., mRNA) or the translation of a nucleic acid into a protein. The protein may be expressed, retained within the cell, become a component of the cell surface membrane, or be secreted into the extracellular matrix or medium.

[0063] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means that the material is not a biologically or otherwise undesirable material; for example, the material may be incorporated into a pharmaceutical composition administered to a patient without causing a significant undesirable biological effect or interacting in a harmful manner with any other component of the composition. Pharmaceutically acceptable carrier agents or excipients preferably meet the requirements of toxicological and manufacturing tests and / or are included in the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.

[0064] It should be understood that the aspects of the present application described herein include "consisting of" and / or "consisting essentially of" aspects.

[0065] As used herein, reference to a "about" value or parameter includes (and describes) variations that are made to the value or parameter itself. For example, a description of "about X" includes a description of "X".

[0066] References to "not" values or parameters used herein typically mean and describe "different" values or parameters. For example, a method that is not for treating type X cancer means that the method is used for treating a cancer that is different from type X.

[0067] As used herein, the term "about X to Y" has the same meaning as "from about X to about Y".

[0068] When used in the context of administering a drug once every X to Y days, the term "every X to Y days" covers the case where the drug does not need to be administered at the same interval. For example, the description of administering an S-phase inhibitor once every 1 to 28 days covers administering the S-phase inhibitor 10 days after the first administration and administering the S-phase inhibitor 5 days after the second administration.

[0069] As used in this specification and the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly dictates otherwise.

[0070] II. Treatment Methods This application provides a method for promoting the persistence of a cell therapy comprising immune cells, enhancing the effectiveness of the cell therapy, treating a host-versus-graft (HvG) disease state, reducing an undesirable immune response associated with the cell therapy, and / or treating a disease or disease state in an individual who has received or will receive a cell therapy.

[0071] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual, prior to the cell therapy, a) an optional lymphocyte depleting agent, and b) an S-phase inhibitor (e.g., a folate antagonist, e.g., a DHFR inhibitor, e.g., methotrexate), wherein the S-phase inhibitor is administered before and after the cell therapy, and wherein the lymphocyte depleting agent is different from the S-phase inhibitor. The cell therapy may comprise immune cells that are resistant to the S-phase inhibitor. The immune cells may be allogeneic. The immune cells may comprise T cells. The T cells may comprise an exogenous Nef protein and / or the T cells are engineered to not express or express at reduced levels the endogenous TCRα or TCRβ. The T cells may be γ-δ T cells. The immune cells may comprise NK cells. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal to a positive signal, optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound.

[0072] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual, within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour), prior to the cell therapy, a) an optional lymphodepleting agent, and b) an S-phase inhibitor (e.g., a folate antagonist, e.g., methotrexate), wherein the lymphodepleting agent is different from the S-phase inhibitor. The cell therapy may comprise immune cells that are resistant to the S-phase inhibitor. The method may further comprise administering the S-phase inhibitor after the cell therapy. The method may comprise administering the S-phase inhibitor at least 2, 3, 4, 5, 6, 7 times after the cell therapy. The immune cells may be allogeneic. The immune cells may comprise T cells. The T cells may comprise an exogenous Nef protein and / or the T cells are engineered to not express or express at reduced levels the endogenous TCRα or TCRβ. The T cells may be γ-δ T cells. The immune cells may comprise NK cells. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal to a positive signal, optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound.

[0073] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual, prior to the cell therapy, a) an optional lymphodepleting agent, and b) an S-phase inhibitor (e.g., a folate antagonist, e.g., methotrexate) in parallel or simultaneously, wherein the lymphodepleting agent is different from the S-phase inhibitor. The cell therapy may comprise immune cells that are resistant to the S-phase inhibitor. The immune cells may be allogeneic. The immune cells may comprise T cells. The T cells may comprise an exogenous Nef protein and / or the T cells are engineered to not express or express at a reduced level an endogenous TCRα or TCRβ. The T cells may be γ-δ T cells. The immune cells may comprise NK cells. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal to a positive signal, optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound.

[0074] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human subject, the method comprising administering to the subject a) fludarabine, b) cyclophosphamide, and c) methotrexate, wherein the cell therapy comprises immune cells that are resistant to methotrexate, and wherein methotrexate is administered before and after the cell therapy is administered. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells, and optionally, wherein the method comprises administering methotrexate within about 5 days before and within about 10 days after administering the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. MTX may be administered every 1 - 28 days (e.g., every 1 - 10 days, e.g., every 2 - 7 days, e.g., every 3 - 7 days, e.g., every 4 - 6 days) during the period from about 5 days before administering the immune cells to about 90 days after administering the immune cells. Within about 1 - 3 hours after administering methotrexate, the plasma concentration of methotrexate in the subject may be about 2 μM or less. Within about 1 - 3 hours after administering methotrexate, the plasma concentration of methotrexate in the subject may exceed about 0.001 μM. Within about 1 - 3 hours after administering methotrexate, the plasma concentration of methotrexate in the subject may exceed about 0.01 μM. Within about 1 - 3 hours after administering methotrexate, the plasma concentration of methotrexate in the subject may exceed about 0.1 μM. About 3 mg / m 2 ~ about 3000 mg / m 2 (e.g., 5 mg / m 2 ~ about 3000 mg / m 2 , e.g., about 5 mg / m 2 ~ about 100 mg / m 2 , e.g., about 20 mg / m 2 ~ about 500 mg / m 2 , e.g., about 100 mg / m 2~about 500 mg / m 2 ) The methotrexate may be administered in an amount of. The immune cells may be allogeneic. The immune cells may include T cells. The T cells may include an exogenous Nef protein and / or the T cells are engineered to not express or express at a reduced level the endogenous TCRα or TCRβ. The T cells may be γ-δ T cells. The immune cells may include NK cells. The immune cells may include a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further include a second engineered receptor that converts a negative signal to a positive signal, and optionally, wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may include a methotrexate resistance transgene. The methotrexate resistance transgene may include a mutant DHFR gene, and optionally, wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0075] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human subject, the method comprising administering to the subject a) fludarabine, b) cyclophosphamide, and c) methotrexate, wherein the cell therapy comprises immune cells resistant to methotrexate, and wherein after the cell therapy is administered, methotrexate is administered at least 3 times. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells, and optionally, wherein the method comprises administering methotrexate within about 5 days before and within about 10 days after administering the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. MTX may be administered every 1 to 28 days (e.g., every 1 to 10 days, e.g., every 2 to 7 days, e.g., every 3 to 7 days, e.g., every 4 to 6 days) during the period from about 5 days before administering the immune cells to about 90 days after administering the immune cells. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the subject may be about 2 μM or less. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the subject may exceed about 0.001 μM. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the subject may exceed about 0.01 μM. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the subject may exceed about 0.1 μM. About 3 mg / m 2 ~ about 3000 mg / m 2 (e.g., 5 mg / m 2 ~ about 3000 mg / m 2 , e.g., about 5 mg / m 2 ~ about 100 mg / m 2 , e.g., about 20 mg / m 2 ~ about 500 mg / m 2 , e.g., about 100 mg / m 2~about 500 mg / m 2 ) Methotrexate may be administered in an amount of. The immune cells may be allogeneic. The immune cells may include T cells. The T cells may include an exogenous Nef protein and / or the T cells are engineered to not express or express at a reduced level an endogenous TCRα or TCRβ. The T cells may be γ-δ T cells. The immune cells may include NK cells. The immune cells may include a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further include a second engineered receptor that converts a negative signal to a positive signal, optionally where the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may include an IL12p40 polypeptide, where the IL12p40 polypeptide is membrane-bound. The immune cells may include a methotrexate resistance transgene. The methotrexate resistance transgene may include a mutant DHFR gene, optionally where the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0076] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, and c) methotrexate, wherein the cell therapy comprises immune cells resistant to methotrexate, wherein the method comprises administering fludarabine, cyclophosphamide and methotrexate in parallel or simultaneously, optionally, wherein the parallel or simultaneous administration occurs about 7 days (e.g., about 5 days) before the cell therapy is performed. The method may comprise administering methotrexate within about 10 days after administering the immune cells, optionally, wherein the method comprises administering methotrexate within about 5 days before and within about 10 days after administering the immune cells. MTX may be administered every 1 to 28 days (e.g., every 1 to 10 days, e.g., every 2 to 7 days, e.g., every 3 to 7 days, e.g., every 4 to 6 days) during the period from about 5 days before administering the immune cells to about 90 days after administering the immune cells. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may be about 2 μM or less. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.001 μM. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.01 μM. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.1 μM. About 3 mg / m 2 ~ about 3000 mg / m 2 (e.g., 5 mg / m 2 ~ about 3000 mg / m 2 , e.g., about 5 mg / m 2 ~ about 100 mg / m 2 , e.g., about 20 mg / m 2 ~ about 500 mg / m 2 , e.g., about 100 mg / m 2 ~ about 500 mg / m 2Methotrexate may be administered in an amount of ). The immune cells may be allogeneic. The immune cells may include T cells. The T cells may include an exogenous Nef protein, and / or the T cells are engineered to not express or express at a reduced level the endogenous TCRα or TCRβ. The T cells may be γ-δ T cells. The immune cells may include NK cells. The immune cells may include a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further include a second engineered receptor that converts a negative signal into a positive signal, optionally where the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may include an IL12p40 polypeptide, where the IL12p40 polypeptide is membrane-bound. The immune cells may include a methotrexate resistance transgene. The methotrexate resistance transgene may include a mutant DHFR gene, optionally where the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0077] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, and c) methotrexate, wherein the cell therapy comprises immune cells resistant to methotrexate, and wherein methotrexate is administered once every 1 to 7 days for at least 30 days. The method may comprise administering methotrexate within about 5 days before administration of the immune cells. The method may comprise administering methotrexate within about 10 days after administration of the immune cells, and optionally, wherein the method comprises administering methotrexate within about 5 days before and within about 10 days after administration of the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. MTX may be administered every 2 to 7 days or every 3 to 7 days (e.g., every 4 to 6 days) for at least about 40, 50, 60, 70, 80, or 90 days, and optionally, wherein MTX is administered for a period from about 5 days before administration of the immune cells to about 90 days after administration of the immune cells. Within about 1 to 3 hours after administration of methotrexate, the plasma concentration of methotrexate in the individual may be about 2 μM or less. Within about 1 to 3 hours after administration of methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.001 μM. Within about 1 to 3 hours after administration of methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.01 μM. Within about 1 to 3 hours after administration of methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.1 μM. About 3 mg / m 2 ~ about 3000 mg / m 2 (e.g., 5 mg / m 2 ~ about 3000 mg / m 2 , e.g., about 5 mg / m 2 ~ about 100 mg / m 2 , e.g., about 20 mg / m 2 ~ about 500 mg / m 2, for example, methotrexate may be administered in an amount of about 100 mg / m 2 ~ about 500 mg / m 2 ). The immune cells may be allogeneic. The immune cells may include T cells. The T cells may include an exogenous Nef protein and / or the T cells are engineered to not express or express at a reduced level the endogenous TCRα or TCRβ. The T cells may be γ-δ T cells. The immune cells may include NK cells. The immune cells may include a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further include a second engineered receptor that converts a negative signal into a positive signal, and optionally, wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may include a methotrexate resistance transgene. The methotrexate resistance transgene may include a mutant DHFR gene, and optionally, wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0078] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, and c) methotrexate, wherein the cell therapy comprises immune cells resistant to methotrexate, and wherein methotrexate is administered at a frequency of once every 1 to 28 days for at least about 90 days. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells, and optionally, wherein the method comprises administering methotrexate within about 5 days before and within about 10 days after administering the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. MTX is administered once every 1 to 28 days (e.g., once every 1 to 10 days, e.g., once every 2 to 7 days, e.g., once every 3 to 7 days, e.g., once every 4 to 6 days). Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may be about 2 μM or less. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.001 μM. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.01 μM. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.1 μM. About 3 mg / m 2 ~ about 3000 mg / m 2 (e.g., 5 mg / m 2 ~ about 3000 mg / m 2 , e.g., about 5 mg / m 2 ~ about 100 mg / m 2 , e.g., about 20 mg / m 2 ~ about 500 mg / m 2 , e.g., about 100 mg / m 2 ~ about 500 mg / m 2Methotrexate may be administered in an amount of ). The immune cells may be allogeneic. The immune cells may include T cells. The T cells may include an exogenous Nef protein and / or the T cells are engineered to not express or express at a reduced level an endogenous TCRα or TCRβ. The T cells may be γ-δ T cells. The immune cells may include NK cells. The immune cells may include a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further include a second engineered receptor that converts a negative signal into a positive signal, optionally where the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may include an IL12p40 polypeptide, where the IL12p40 polypeptide is membrane-bound. The immune cells may include a methotrexate resistance transgene. The methotrexate resistance transgene may include a mutant DHFR gene, optionally where the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0079] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, and c) methotrexate, wherein the cell therapy comprises immune cells resistant to methotrexate, wherein the method further comprises administering these immune cells more than once (e.g., about 2, 3, 4, 5 times), and wherein, from the first administration of these immune cells to at least about 10 days (e.g., at least about 15 days, 20 days, 25 days, or 30 days) after the last administration of these immune cells, methotrexate is administered once every 1 - 10 days. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. Within about 1 - 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may be about 2 μM or less. Within about 1 - 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.001 μM. Within about 1 - 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.01 μM. Within about 1 - 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.1 μM. About 3 mg / m 2 ~ about 3000 mg / m 2 (e.g., 5 mg / m 2 ~ about 3000 mg / m 2 , e.g., about 5 mg / m 2 ~ about 100 mg / m 2 , e.g., about 20 mg / m 2 ~ about 500 mg / m 2 , e.g., about 100 mg / m 2 ~ about 500 mg / m 2Methotrexate may be administered in an amount of ). The immune cells may be allogeneic. The immune cells may include T cells. The T cells may include an exogenous Nef protein and / or the T cells are engineered to not express or express at a reduced level the endogenous TCRα or TCRβ. The T cells may be γ-δ T cells. The immune cells may include NK cells. The immune cells may include a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further include a second engineered receptor that converts a negative signal to a positive signal, optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may include a methotrexate resistance transgene. The methotrexate resistance transgene may include a mutant DHFR gene, optionally, wherein the mutant DHFR gene comprises an L22F mutation and an F31S mutation.

[0080] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, and c) methotrexate, wherein the cell therapy comprises immune cells resistant to methotrexate, wherein fludarabine and cyclophosphamide are administered continuously for about 3 to 4 days prior to administering these immune cells, and wherein methotrexate and fludarabine and / or cyclophosphamide are administered in parallel or simultaneously at least once. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may be about 2 μM or less. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.001 μM. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.01 μM. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.1 μM. About 3 mg / m 2 ~ about 3000 mg / m 2 (e.g., 5 mg / m 2 ~ about 3000 mg / m 2 , e.g., about 5 mg / m 2 ~ about 100 mg / m 2 , e.g., about 20 mg / m 2 ~ about 500 mg / m 2 , e.g., about 100 mg / m 2 ~ about 500 mg / m 2Methotrexate may be administered in an amount of ). The immune cells may be allogeneic. The immune cells may include T cells. The T cells may include an exogenous Nef protein, and / or the T cells are engineered to not express or express at a reduced level the endogenous TCRα or TCRβ. The T cells are γ-δ T cells. The immune cells include NK cells. The immune cells may include a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further include a second engineered receptor that converts a negative signal into a positive signal, optionally, where the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may include an IL12p40 polypeptide, where the IL12p40 polypeptide is membrane-bound. The immune cells may include a methotrexate resistance transgene. The methotrexate resistance transgene may include a mutant DHFR gene, optionally, where the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0081] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, and c) methotrexate, wherein the cell therapy comprises immune cells resistant to methotrexate, wherein fludarabine and cyclophosphamide are administered continuously for about 3 to 4 days prior to administering these immune cells, and wherein methotrexate is administered once every 1 to 7 days for at least 30 days after administering the immune cells. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may be about 2 μM or less. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.001 μM. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.01 μM. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.1 μM. About 3 mg / m 2 ~ about 3000 mg / m 2 (e.g., 5 mg / m 2 ~ about 3000 mg / m 2 , for example, about 5 mg / m 2 ~ about 100 mg / m 2 , for example, about 20 mg / m 2 ~ about 500 mg / m 2 , for example, about 100 mg / m 2 ~ about 500 mg / m 2Methotrexate may be administered in an amount of ). The immune cells may be allogeneic. The immune cells may include T cells. The T cells may include an exogenous Nef protein and / or the T cells are engineered to not express or express at a reduced level the endogenous TCRα or TCRβ. The T cells may be γ-δ T cells. The immune cells may include NK cells. The immune cells may include a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further include a second engineered receptor that converts a negative signal to a positive signal, optionally where the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may include a methotrexate resistance transgene. The immune cells may include an IL12p40 polypeptide, where the IL12p40 polypeptide is membrane-bound. The methotrexate resistance transgene may include a mutant DHFR gene, optionally where the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0082] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) a lymphodepleting agent, and b) methotrexate, wherein the cell therapy comprises immune cells resistant to methotrexate, and wherein these immune cells comprise T cells (e.g., allogeneic T cells) containing an exogenous Nef protein. The lymphodepleting agent may comprise Flu and Cy, where Flu and Cy are administered before the cell therapy is performed. The method may comprise administering methotrexate after the cell therapy is performed. Methotrexate may be administered before and after the cell therapy is performed. The method may comprise administering methotrexate within about 5 days before the administration of the immune cells. The method may comprise administering methotrexate within about 10 days after the administration of the immune cells, and optionally, wherein the method comprises administering methotrexate within about 5 days before and within about 10 days after the administration of the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. MTX may be administered every 1 to 28 days (e.g., every 1 to 10 days, e.g., every 2 to 7 days, e.g., every 3 to 7 days, e.g., every 4 to 6 days) during the period from about 5 days before the administration of the immune cells to about 90 days after the administration of the immune cells. Within about 1 to 3 hours after the administration of methotrexate, the plasma concentration of methotrexate in the individual may be about 2 μM or less. Within about 1 to 3 hours after the administration of methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.001 μM. Within about 1 to 3 hours after the administration of methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.01 μM. Within about 1 to 3 hours after the administration of methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.1 μM. About 3 mg / m 2 ~ about 3000 mg / m 2 (e.g., 5 mg / m 2 ~ about 3000 mg / m2 , for example, about 5 mg / m 2 ~ about 100 mg / m 2 , for example, about 20 mg / m 2 ~ about 500 mg / m 2 , for example, about 100 mg / m 2 ~ about 500 mg / m 2 ) of methotrexate may be administered. The immune cells may be allogeneic. The immune cells may contain a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further contain a second engineered receptor that converts a negative signal into a positive signal, and optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may contain an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may contain a methotrexate resistance transgene. The methotrexate resistance transgene may contain a mutant DHFR gene, and optionally, wherein the mutant DHFR gene comprises an L22F mutation and an F31S mutation.

[0083] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) an optional lymphodepleting agent, and b) methotrexate, wherein the cell therapy comprises immune cells resistant to methotrexate, and wherein these immune cells comprise T cells (e.g., allogeneic T cells) engineered not to express or to express at a reduced level an endogenous TCRα or TCRβ. The lymphodepleting agent may comprise Flu and Cy, wherein Flu and Cy are administered prior to the cell therapy. The method may comprise administering methotrexate after the cell therapy. Methotrexate may be administered before and after the cell therapy. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells, and optionally, wherein the method comprises administering methotrexate within about 5 days before and within about 10 days after administering the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. MTX may be administered every 1 to 28 days (e.g., every 1 to 10 days, e.g., every 2 to 7 days, e.g., every 3 to 7 days, e.g., every 4 to 6 days) during the period from about 5 days before administering the immune cells to about 90 days after administering the immune cells. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may be about 2 μM or less. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.001 μM. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.01 μM. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.1 μM. About 3 mg / m 2 ~ about 3000 mg / m 2(For example, 5 mg / m 2 ~ about 3000 mg / m 2 , for example, about 5 mg / m 2 ~ about 100 mg / m 2 , for example, about 20 mg / m 2 ~ about 500 mg / m 2 , for example, about 100 mg / m 2 ~ about 500 mg / m 2 ) methotrexate may be administered in an amount. The immune cells may be allogeneic. The immune cells may contain a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further contain a second engineered receptor that converts a negative signal to a positive signal, and optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may contain an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may contain a methotrexate resistance transgene. The methotrexate resistance transgene may contain a mutant DHFR gene, and optionally, wherein the mutant DHFR gene comprises an L22F mutation and an F31S mutation.

[0084] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) an optional lymphocyte depleting agent, and b) methotrexate, wherein the cell therapy comprises immune cells resistant to methotrexate, and wherein these immune cells comprise γ-δ T cells. The lymphocyte depleting agent may comprise Flu and Cy, wherein Flu and Cy are administered prior to the cell therapy. The method may comprise administering methotrexate after the cell therapy. Methotrexate may be administered before and after the cell therapy. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells, and optionally, wherein the method comprises administering methotrexate within about 5 days before and within about 10 days after administering the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. MTX may be administered every 1 to 28 days (e.g., every 1 to 10 days, e.g., every 2 to 7 days, e.g., every 3 to 7 days, e.g., every 4 to 6 days) during the period from about 5 days before administering the immune cells to about 90 days after administering the immune cells. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may be about 2 μM or less. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.001 μM. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.01 μM. Within about 1 to 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual exceeds about 0.1 μM. About 3 mg / m 2 ~ about 3000 mg / m 2 (e.g., 5 mg / m 2 ~ about 3000 mg / m 2 , e.g., about 5 mg / m2 ~about 100 mg / m 2 , for example, about 20 mg / m 2 ~about 500 mg / m 2 , for example, about 100 mg / m 2 ~about 500 mg / m 2 ) Methotrexate may be administered in an amount of. The immune cells may be allogeneic. The immune cells may contain a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further contain a second engineered receptor that converts a negative signal into a positive signal, and optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may contain an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may contain a methotrexate resistance transgene. The methotrexate resistance transgene may contain a mutant DHFR gene, and optionally, wherein the mutant DHFR gene comprises an L22F mutation and an F31S mutation.

[0085] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) an optional lymphocyte depleting agent, and b) methotrexate, wherein the cell therapy comprises immune cells resistant to methotrexate, and wherein these immune cells comprise NK cells. The lymphocyte depleting agent may comprise Flu and Cy, wherein Flu and Cy are administered prior to the cell therapy. The method may comprise administering methotrexate after the cell therapy. Methotrexate may be administered before and after the cell therapy. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells, and optionally, wherein the method comprises administering methotrexate within about 5 days before and within about 10 days after administering the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. MTX may be administered every 1 - 28 days (e.g., every 1 - 10 days, e.g., every 2 - 7 days, e.g., every 3 - 7 days, e.g., every 4 - 6 days) during the period from about 5 days before administering the immune cells to about 90 days after administering the immune cells. Within about 1 - 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may be about 2 μM or less. Within about 1 - 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.001 μM. Within about 1 - 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.01 μM. Within about 1 - 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.1 μM. About 3 mg / m 2 ~ about 3000 mg / m 2 (e.g., 5 mg / m 2 ~ about 3000 mg / m 2 , e.g., about 5 mg / m2 ~about 100 mg / m 2 , for example, about 20 mg / m 2 ~about 500 mg / m 2 , for example, about 100 mg / m 2 ~about 500 mg / m 2 ) Methotrexate may be administered in an amount of. The immune cells may be allogeneic. The immune cells may contain a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further contain a second engineered receptor that converts a negative signal into a positive signal, and optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may contain a methotrexate resistance transgene. The immune cells may contain an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The methotrexate resistance transgene may contain a mutant DHFR gene, and optionally, wherein the mutant DHFR gene comprises an L22F mutation and an F31S mutation.

[0086] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering methotrexate to the individual before and after the cell therapy. Other lymphodepleting agents may not be added. The cell therapy may comprise immune cells that are resistant to MTX. Methotrexate may be administered within 5 days before administration of the immune cells. Methotrexate may be administered at a frequency of about 1-28 days (e.g., about 1-10 days, e.g., about 2-8 days, e.g., about 3-7 days, e.g., about 4-6 days, e.g., about 5 days) for at least about 30 days, 40 days, 50 days, 60 days, 70 days, 80 days or 90 days. Methotrexate may be administered at a frequency of about 1-28 days for a period from about 5 days before administration of the immune cells to about 90 days after administration of the immune cells. The immune cells may be allogeneic. The immune cells may comprise T cells. The T cells may comprise an exogenous Nef protein and / or the T cells are engineered to not express or express at reduced levels the endogenous TCRα or TCRβ. The T cells may be γ-δ T cells. The immune cells may comprise NK cells. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR that targets a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal to a positive signal, optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound.

[0087] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) a cell therapy comprising immune cells resistant to methotrexate, wherein prior to administering these immune cells, fludarabine and cyclophosphamide are administered continuously for about 3 to 4 days, and wherein after administering the immune cells, methotrexate is administered once every 1 to 7 days (e.g., 3 to 7 days) for at least 30 days (e.g., at least 60 days, e.g., at least 90 days), wherein these immune cells comprise T cells (e.g., allogeneic T cells) containing an exogenous Nef protein. Fludarabine and cyclophosphamide may be administered continuously for 3 days. Fludarabine and cyclophosphamide may be administered continuously for 4 days. Fludarabine and cyclophosphamide may be administered continuously for 3 days, followed by administration of fludarabine alone for 1 day. Methotrexate may be administered before and after the cell therapy. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may comprise a methotrexate resistance transgene. The immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The methotrexate resistance transgene may comprise a mutant DHFR gene, optionally wherein the mutant DHFR gene comprises an L22F mutation and an F31S mutation.

[0088] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) a cell therapy comprising immune cells resistant to methotrexate, wherein, prior to administering these immune cells, fludarabine and cyclophosphamide are administered continuously for about 3 to 4 days, and wherein, after administering the immune cells, methotrexate is administered once every 1 to 7 days (e.g., 3 to 7 days) for at least 30 days (e.g., at least 60 days, e.g., at least 90 days), wherein these immune cells comprise T cells (e.g., allogeneic T cells) engineered not to express or to express at reduced levels endogenous TCRα or TCRβ. Fludarabine and cyclophosphamide may be administered continuously for 3 days. Fludarabine and cyclophosphamide may be administered continuously for 4 days. Fludarabine and cyclophosphamide may be administered continuously for 3 days, followed by administration of fludarabine alone for 1 day. Methotrexate may be administered before and after the cell therapy. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells. The method may comprise administering one or both of MTX and one of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering one or both of MTX and one of Flu and Cy in parallel or simultaneously. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal to a positive signal, and optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may comprise a methotrexate resistance transgene.The methotrexate-resistant introduced gene may contain a mutant DHFR gene, and optionally, where the mutant DHFR gene contains an L22F mutation and an F31S mutation.

[0089] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) a cell therapy comprising immune cells resistant to methotrexate, wherein prior to administering these immune cells, fludarabine and cyclophosphamide are administered continuously for about 3 to 4 days, and wherein after administering the immune cells, methotrexate is administered once every 1 to 7 days (e.g., 3 to 7 days) for at least 30 days (e.g., at least 60 days, e.g., at least 90 days), wherein these immune cells comprise T cells (e.g., allogeneic T cells), and these T cells are γ-δ T cells. Fludarabine and cyclophosphamide may be administered continuously for 3 days. Fludarabine and cyclophosphamide may be administered continuously for 4 days. Fludarabine and cyclophosphamide may be administered continuously for 3 days, followed by administering fludarabine alone for 1 day. Methotrexate may be administered before and after the cell therapy. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells. The method may comprise administering one or both of MTX and one of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering one or both of MTX and one of Flu and Cy in parallel or simultaneously. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal to a positive signal, and optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may comprise a methotrexate resistance transgene.The methotrexate resistance-introducing gene may contain a mutant DHFR gene, and optionally, wherein the mutant DHFR gene contains an L22F mutation and an F31S mutation.

[0090] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) a cell therapy comprising immune cells resistant to methotrexate, wherein, prior to administering these immune cells, fludarabine and cyclophosphamide are administered continuously for about 3 to 4 days, and wherein, after administering these immune cells, methotrexate is administered once every 1 to 7 days (e.g., 3 to 7 days) for at least 30 days (e.g., at least 60 days, e.g., at least 90 days), wherein these immune cells comprise NK cells. Fludarabine and cyclophosphamide may be administered continuously for 3 days. Fludarabine and cyclophosphamide may be administered continuously for 4 days. Fludarabine and cyclophosphamide may be administered continuously for 3 days, followed by administering fludarabine alone for 1 day. Methotrexate may be administered before and after the cell therapy. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells. The method may comprise administering one or both of MTX and Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering one or both of MTX and Flu and Cy in parallel or simultaneously. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal into a positive signal, and optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may comprise a methotrexate resistance transgene. The methotrexate resistance transgene may comprise a mutant DHFR gene, and optionally, wherein the mutant DHFR gene comprises an L22F mutation and an F31S mutation.

[0091] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) a cell therapy comprising immune cells resistant to methotrexate, wherein, prior to administering these immune cells, fludarabine and cyclophosphamide are administered continuously for about 3 to 4 days, and wherein the method comprises administering methotrexate and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour), wherein these immune cells comprise T cells (e.g., allogeneic T cells) containing an exogenous Nef protein. Fludarabine and cyclophosphamide may be administered continuously for 3 days. Fludarabine and cyclophosphamide may be administered continuously for 4 days. Fludarabine and cyclophosphamide may be administered continuously for 3 days, followed by administering fludarabine alone for 1 day. Methotrexate may be administered before and after the cell therapy. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal to a positive signal, optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may comprise a methotrexate resistance transgene. The methotrexate resistance transgene may comprise a mutant DHFR gene, optionally, wherein the mutant DHFR gene comprises an L22F mutation and an F31S mutation.

[0092] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) a cell therapy comprising immune cells resistant to methotrexate, wherein, prior to administering these immune cells, fludarabine and cyclophosphamide are administered continuously for about 3 to 4 days, and wherein the method comprises administering methotrexate and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour), wherein these immune cells comprise T cells (e.g., allogeneic T cells) engineered not to express or to express at reduced levels endogenous TCRα or TCRβ. Fludarabine and cyclophosphamide may be administered continuously for 3 days. Fludarabine and cyclophosphamide may be administered continuously for 4 days. Fludarabine and cyclophosphamide may be administered continuously for 3 days followed by fludarabine alone for 1 day. Methotrexate may be administered before and after the cell therapy. The method may comprise administering methotrexate within about 5 days prior to administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may comprise a methotrexate resistance transgene.The methotrexate resistance-introducing gene may contain a mutant DHFR gene, and optionally, wherein the mutant DHFR gene contains an L22F mutation and an F31S mutation.

[0093] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) a cell therapy comprising immune cells resistant to methotrexate, wherein prior to administering these immune cells, fludarabine and cyclophosphamide are administered continuously for about 3 to 4 days, and wherein the method comprises administering methotrexate and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour), wherein these immune cells comprise T cells (e.g., allogeneic T cells), and these T cells comprise γ-δ T cells. Fludarabine and cyclophosphamide may be administered continuously for 3 days. Fludarabine and cyclophosphamide may be administered continuously for 4 days. Fludarabine and cyclophosphamide may be administered continuously for 3 days, followed by administering fludarabine alone for 1 day. Methotrexate may be administered before and after the cell therapy. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal to a positive signal, and optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may comprise a methotrexate resistance transgene. The methotrexate resistance transgene may comprise a mutant DHFR gene, and optionally, wherein the mutant DHFR gene comprises an L22F mutation and an F31S mutation.

[0094] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) a cell therapy comprising immune cells resistant to methotrexate, wherein, prior to administering these immune cells, fludarabine and cyclophosphamide are administered continuously for about 3 to 4 days, and wherein the method comprises administering methotrexate and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour), wherein these immune cells comprise NK cells. Fludarabine and cyclophosphamide may be administered continuously for 3 days. Fludarabine and cyclophosphamide may be administered continuously for 4 days. Fludarabine and cyclophosphamide may be administered continuously for 3 days, followed by administering fludarabine alone for 1 day. Methotrexate may be administered before and after the cell therapy. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may comprise a methotrexate resistance transgene. The methotrexate resistance transgene may comprise a mutant DHFR gene, optionally wherein the mutant DHFR gene comprises an L22F mutation and an F31S mutation.

[0095] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) a cell therapy comprising immune cells resistant to methotrexate, wherein prior to administering these immune cells, fludarabine and cyclophosphamide are administered continuously for about 3 to 4 days, and wherein methotrexate is administered before and after administering these immune cells, wherein these immune cells comprise T cells (e.g., allogeneic T cells) containing an exogenous Nef protein. Fludarabine and cyclophosphamide may be administered continuously for 3 days. Fludarabine and cyclophosphamide may be administered continuously for 4 days. Fludarabine and cyclophosphamide may be administered continuously for 3 days, followed by administering fludarabine alone for 1 day. Methotrexate may be administered before and after performing the cell therapy. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal to a positive signal, and optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may comprise a methotrexate resistance transgene. The methotrexate resistance transgene may comprise a mutant DHFR gene, and optionally, wherein the mutant DHFR gene comprises an L22F mutation and an F31S mutation.

[0096] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) a cell therapy comprising immune cells resistant to methotrexate, wherein prior to administering these immune cells, fludarabine and cyclophosphamide are administered continuously for about 3 to 4 days, and wherein methotrexate is administered before or after administering these immune cells, wherein these immune cells comprise T cells (e.g., allogeneic T cells) engineered not to express or to express at reduced levels endogenous TCRα or TCRβ. Fludarabine and cyclophosphamide may be administered continuously for 3 days. Fludarabine and cyclophosphamide may be administered continuously for 4 days. Fludarabine and cyclophosphamide may be administered continuously for 3 days followed by fludarabine alone for 1 day. Methotrexate may be administered before and after the cell therapy is administered. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may comprise a methotrexate resistance transgene. The immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The methotrexate resistance transgene may comprise a mutant DHFR gene, optionally wherein the mutant DHFR gene comprises L22F and F31S mutations.

[0097] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) a cell therapy comprising immune cells resistant to methotrexate, wherein prior to administering these immune cells, fludarabine and cyclophosphamide are administered continuously for about 3 to 4 days, and wherein methotrexate is administered before and after administering these immune cells, wherein these immune cells comprise T cells (e.g., allogeneic T cells), and these T cells are γ-δ T cells. Fludarabine and cyclophosphamide may be administered continuously for 3 days. Fludarabine and cyclophosphamide may be administered continuously for 4 days. Fludarabine and cyclophosphamide may be administered continuously for 3 days, followed by administering fludarabine alone for 1 day. Methotrexate may be administered before and after performing the cell therapy. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal into a positive signal, and optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may comprise a methotrexate resistance transgene. The methotrexate resistance transgene may comprise a mutant DHFR gene, and optionally, wherein the mutant DHFR gene comprises an L22F mutation and an F31S mutation.

[0098] In some embodiments, provided is a method for promoting the persistence of cell therapy in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) a cell therapy comprising immune cells resistant to methotrexate, wherein prior to administering these immune cells, fludarabine and cyclophosphamide are administered continuously for about 3 to 4 days, and wherein methotrexate is administered before and after administering these immune cells, wherein these immune cells comprise NK cells. Fludarabine and cyclophosphamide may be administered continuously for 3 days. Fludarabine and cyclophosphamide may be administered continuously for 4 days. Fludarabine and cyclophosphamide may be administered continuously for 3 days, followed by administering fludarabine alone for 1 day. Methotrexate may be administered before and after performing the cell therapy. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal into a positive signal, and optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may comprise a methotrexate resistance transgene. The methotrexate resistance transgene may comprise a mutant DHFR gene, and optionally, wherein the mutant DHFR gene comprises L22F and F31S mutations.

[0099] In some embodiments, provided is a method of treating a disease or medical condition (e.g., cancer) in a human subject, the method comprising administering to the subject a) an optional lymphodepleting agent, b) an S-phase inhibitor (e.g., methotrexate), and c) a cell therapy comprising immune cells that are resistant to the S-phase inhibitor. In some embodiments, provided is a method of treating a disease or medical condition (e.g., cancer) in a human subject, the method comprising administering to the subject a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) a cell therapy comprising immune cells that are resistant to methotrexate, wherein fludarabine and cyclophosphamide are administered consecutively for about 3 to 4 days prior to administration of these immune cells, and wherein methotrexate is administered once every 1 to 7 days (e.g., 3 to 7 days) for at least 30 days (e.g., at least 60 days, e.g., at least 90 days) after administration of these immune cells. The immune cells may be T cells (e.g., allogeneic T cells). The T cells may comprise an exogenous Nef protein. The T cells may be engineered to not express or express at a reduced level the endogenous TCRα or TCRβ. The T cells may be γ-δ T cells. The immune cells may be NK cells. The cancer may be a solid tumor. The cancer may be a hematological cancer. Fludarabine and cyclophosphamide may be administered consecutively for 3 days. Fludarabine and cyclophosphamide may be administered consecutively for 4 days. Fludarabine and cyclophosphamide may be administered consecutively for 3 days followed by fludarabine alone for 1 day. Methotrexate may be administered before and after the cell therapy. The method may comprise administering methotrexate within about 5 days before administration of the immune cells. The method may comprise administering methotrexate within about 10 days after administration of the immune cells. The method may comprise administering one or both of MTX and Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering one or both of MTX and Flu and Cy in parallel or simultaneously. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen).The immune cell may further comprise a second engineered receptor that converts a negative signal into a positive signal, and optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cell may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cell may comprise a methotrexate resistance transgene. The methotrexate resistance transgene may comprise a mutant DHFR gene, and optionally, wherein the mutant DHFR gene comprises an L22F mutation and an F31S mutation.

[0100] In some embodiments, provided is a method of treating a disease or medical condition (e.g., cancer) in a human individual, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) a cell therapy comprising immune cells resistant to methotrexate, wherein prior to administering these immune cells, fludarabine and cyclophosphamide are administered continuously for about 3 to 4 days, and wherein the method comprises administering methotrexate and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The immune cells may be T cells (e.g., allogeneic T cells). The T cells may comprise an exogenous Nef protein. The T cells may be engineered to not express or express at a reduced level the endogenous TCRα or TCRβ. The T cells may be γ-δ T cells. The immune cells may be NK cells. The cancer may be a solid tumor. The cancer may be a hematologic cancer. Fludarabine and cyclophosphamide may be administered continuously for 3 days. Fludarabine and cyclophosphamide may be administered continuously for 4 days. Fludarabine and cyclophosphamide may be administered continuously for 3 days followed by fludarabine alone for 1 day. Methotrexate may be administered before and after the cell therapy. The method may comprise administering methotrexate within about 5 days before administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain.The immune cells may contain the IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may contain a methotrexate resistance transgene. The methotrexate resistance transgene may contain a mutant DHFR gene, and optionally, wherein the mutant DHFR gene contains an L22F mutation and an F31S mutation.

[0101] In some embodiments, provided is a method of treating a disease or medical condition (e.g., cancer) in a human subject, the method comprising administering to the subject a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) a cell therapy comprising immune cells resistant to methotrexate, wherein fludarabine and cyclophosphamide are administered consecutively for about 3 - 4 days prior to administration of these immune cells, and wherein the method comprises administering methotrexate before and after administering the cell therapy. The immune cells may be T cells (e.g., allogeneic T cells). The T cells may comprise an exogenous Nef protein. The T cells may be engineered to not express or express at reduced levels the endogenous TCRα or TCRβ. The T cells may be γ-δ T cells. The immune cells may be NK cells. The cancer may be a solid tumor. The cancer may be a hematological cancer. Fludarabine and cyclophosphamide may be administered consecutively for 3 days. Fludarabine and cyclophosphamide may be administered consecutively for 4 days. Fludarabine and cyclophosphamide may be administered consecutively for 3 days, followed by administration of fludarabine alone for 1 day. Methotrexate may be administered before and after administering the cell therapy. The method may comprise administering methotrexate within about 5 days prior to administering the immune cells. The method may comprise administering methotrexate within about 10 days after administering the immune cells. The method may comprise administering one or both of MTX and Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering one or both of MTX and Flu and Cy in parallel or simultaneously. The immune cells may comprise a heterologous nucleic acid sequence encoding an engineered receptor (e.g., a CAR targeting a tumor antigen). The immune cells may further comprise a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cells may comprise a methotrexate resistance transgene.The methotrexate-resistant transgene may comprise a mutant DHFR gene, and optionally, wherein the mutant DHFR gene comprises an L22F mutation and an F31S mutation.

[0102] In some embodiments, a method of treating cancer in a human individual (e.g., a blood cancer, e.g., a solid tumor) is provided, the method comprising administering to the individual a) a cell therapy, b) methotrexate, wherein after the cell therapy, methotrexate is administered more than once, at least once. Methotrexate may be administered once every 1 to 7 days and administered at least about 2, 3, 4, 5, 6, or 7 times. The cell therapy may comprise immune cells, and optionally, wherein these immune cells are resistant to methotrexate. The method may further comprise administering a lymphodepleting agent prior to administering the cell therapy. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour) (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering MTX and one or both of Flu and Cy in parallel or simultaneously. After administering the immune cells, MTX may be administered once every about 1 to 7 days (e.g., about 3 to 7 days) for at least 30 days (e.g., at least 60 days, e.g., at least 90 days).

[0103] In some embodiments, provided is a method of reducing an undesirable immune response associated with cell therapy in a human individual, the method comprising administering to the individual a) a lymphodepleting agent, b) an S-phase inhibitor (e.g., methotrexate), and c) a cell therapy comprising immune cells resistant to the S-phase inhibitor. In some embodiments, provided is a method of reducing an undesirable immune response associated with cell therapy in a human individual, the method comprising administering to the individual a) a lymphodepleting agent (e.g., Flu and Cy), and b) methotrexate, wherein after the cell therapy, methotrexate is administered more than at least once. Methotrexate may be administered once every 1 to 7 days, and may be administered at least about 2, 3, 4, 5, 6, or 7 times. The cell therapy may comprise immune cells, and optionally, wherein these immune cells are resistant to methotrexate. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour) (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering one or both of MTX and Flu and Cy in parallel or simultaneously. After administering the immune cells, MTX may be administered once every about 1 to 7 days (e.g., about 3 to 7 days) for at least 30 days (e.g., at least 60 days, e.g., at least 90 days).

[0104] In some embodiments, provided is a method of enhancing the effectiveness of cell therapy in a human subject, the method comprising administering to the subject a) an optional lymphocyte depleting agent, b) an S-phase inhibitor (e.g., methotrexate), and c) a cell therapy comprising immune cells resistant to the S-phase inhibitor. In some embodiments, provided is a method of enhancing the effectiveness of cell therapy in a human subject, the method comprising administering to the subject a) a lymphocyte depleting agent (e.g., Flu and Cy), and b) methotrexate, wherein after the cell therapy, methotrexate is administered more than at least once. Methotrexate may be administered once every 1 - 7 days and administered at least about 2, 3, 4, 5, 6, or 7 times. The cell therapy may comprise immune cells, and optionally, wherein these immune cells are resistant to methotrexate. The method may comprise administering one or both of MTX and one of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour) (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering one or both of MTX and one of Flu and Cy in parallel or simultaneously. After administering the immune cells, MTX may be administered once every about 1 - 7 days (e.g., about 3 - 7 days) for at least 30 days (e.g., at least 60 days, e.g., at least 90 days).

[0105] In some embodiments, provided is a method of treating a host-versus-graft (HvG) disease state in a human individual undergoing cell therapy, the method comprising administering to the individual a) an optional lymphodepleting agent, b) an S-phase inhibitor (e.g., methotrexate), and c) a cell therapy comprising immune cells that are resistant to the S-phase inhibitor. In some embodiments, provided is a method of treating a host-versus-graft (HvG) disease state in a human individual undergoing cell therapy, the method comprising administering to the individual a) a lymphodepleting agent (e.g., Flu and Cy), and b) methotrexate, wherein after the cell therapy, methotrexate is administered more than at least once. Methotrexate may be administered once every 1 to 7 days, and may be administered at least about 2, 3, 4, 5, 6, or 7 times. The cell therapy may comprise immune cells, and optionally, wherein these immune cells are resistant to methotrexate. The method may comprise administering MTX and one or both of Flu and Cy within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may comprise administering one or both of MTX and Flu and Cy in parallel or simultaneously. After administering the immune cells, MTX may be administered once every about 1 to 7 days (e.g., about 3 to 7 days) for at least 30 days (e.g., at least 60 days, e.g., at least 90 days). About 3 mg / m 2 ~ about 3000 mg / m 2 (e.g., 5 mg / m 2 ~ about 3000 mg / m 2 ) of methotrexate may be administered. About 3 mg / m 2 / day to about 3000 mg / m 2 / day (e.g., 5 mg / m 2 / day to about 3000 mg / m 2 / day) of methotrexate may be administered.

[0106] Fluorouracil may be administered at a dose of about 25 mg / m 2 ~ about 30 mg / m 2 , and about 250 mg / m 2 ~ about 1000 mg / m 2Cyclophosphamide may be administered at the dosage of. From about 10 days before the administration of immune cells to about 90 days after the administration of immune cells, fludarabine and cyclophosphamide may be administered about 1 to 3 times.

[0107] An S-phase inhibitor (for example, methotrexate) may be administered orally, subcutaneously, intramuscularly, intravenously, intraarterially or intrathecally each time.

[0108] The methods described herein apply to both autologous immune cells and allogeneic immune cells. The allogeneic immune cells may be off-the-shelf immune cells. The allogeneic immune cells may have genetic modifications that reduce the immunogenicity or allogeneic reactivity of the immune cells. The allogeneic immune cells may not be genetically modified to reduce the immunogenicity or allogeneic reactivity of the immune cells.

[0109] The immune cells may contain a heterologous nucleic acid sequence encoding an engineered receptor. The immune cells may be CAR-T cells. The immune cells may be allogeneic CAR-T cells. The immune cells may be universal CART cells (“UCART cells”). The immune cells may be TCR-T cells. The immune cells may contain one or more engineered receptors selected from the group consisting of CAR, TCR, TAC receptor, and TFP. The engineered receptor may target an antigen (e.g., a tumor antigen) selected from the group consisting of CD19, BCMA, Claudin 18.2, NY-ESO-1, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR, GD2, HER2, IGF1R, mesothelin, PSMA, GPC3, DLL3, GPRC5D, CLL1, ROR1, WT1, CD4, GU2CYC, MUC16, MUC1, CAIX, CD8, CD7, CD10, CD30, CD34, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD3, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, p53, MART1, GP100, protease-3 (PR3), tyrosinase, survivin, EphA2, h5T4, PSCA, TAG-72, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, Claudin 6, NKG2D, CD70, ADGRE2, FcRH5, NKp80, NKp30, NKG2A, CD229, CS-1, and combinations thereof. The engineered receptor may be an anti-BCMA CAR, an anti-CD20 CAR, or an anti-Claudin 18.2 CAR.

[0110] The immune cells can overexpress the DHFR gene encoding dihydrofolate reductase.

[0111] The immune cells may be administered more than once. Within about 90 days after the first administration of the immune cells, these immune cells may be administered about 2 to 5 times.

[0112] The immune cells may contain about 30 million to about 900 million immune cells, or may contain about 100,000 to about 50 million immune cells per kilogram of human individual.

[0113] During the period from about 2 to 7 days before administering the immune cells to about 10, 20, 30, 40, 50, 60, 70, 80 or 90 days after administering the immune cells, the host T cells measured by the number of host T cells in PBMC may be 500, 400, 300, 200, 100 or 50 cells or less per μL.

[0114] The method may further include monitoring the number of host T cells of an individual.

[0115] The individual to be treated may be a mammal. Examples of mammals include, but are not limited to, humans, monkeys, rats, mice, hamsters, guinea pigs, dogs, cats, rabbits, pigs, sheep, goats, horses, cows, etc. The individual may be a human.

[0116] The individual referred to herein may be any individual considered suitable for cell therapy involving immune cells. The individual may be suffering from cancer (e.g., blood cancer, e.g., solid tumor, e.g., lymphoma or leukemia). The individual may be male. The individual may be female. The individual may be at least 50, 55, 60, 65, 70, 75, 80, 85 or 90 years old.

[0117] A. Cell Therapy and Lymphocyte Depletion Cell therapies (e.g., chimeric antigen receptor (CAR) T cells and CAR-NK therapies) are promising in the treatment of diseases such as hematological malignancies and solid tumors. Prior to infusion, lymphodepletion is usually performed. Lymphodepletion reduces lymphocytes, affects T cells, B cells, and NK cells, and has various positive effects, e.g., suppressing the host immune system, reducing immunogenicity, and increasing the persistence of infused CAR T cells, for example, before CAR T cell therapy. However, it is also important to note that lymphodepletion has many adverse effects. For example, lymphodepletion can pose a greater risk of infection by causing neutropenia, anemia, thrombocytopenia, and immunosuppression. In particular, fludarabine can cause fever and neurotoxicity. Cyclophosphamide can cause hemorrhagic cystitis, pericarditis, and neurotoxicity. Note that the use of these agents may increase the risk of developing secondary malignancies.

[0118] B. S-phase inhibitors, folate antagonists (e.g., DHFR inhibitors), and methotrexate The methods described herein include administering an S-phase inhibitor (e.g., a folate antagonist, e.g., methotrexate). The S-phase inhibitors described herein refer to agents that arrest cells in the S phase of the cell cycle. The S-phase inhibitor may be a folate antagonist, which is a class of inhibitors that target points in the folate biosynthesis pathway.

[0119] The folate antagonist may be a DHFR inhibitor.

[0120] The folate antagonist may be selected from the group consisting of methotrexate, raltitrexed (e.g., Tomudex®), pemetrexed (e.g., Alimta®, Pemfexy™, Ciambra), pralatrexed (e.g., FOLOTYN®), trimethoprim (e.g., PROLOPRIM®, Monotrim®, Triprim, Trimopan®), and pyrimethamine (Daraprim®).

[0121] The folic acid antagonist may be methotrexate. Exemplary methotrexates include Otrexup (trademark), Rasuvo (registered trademark), Rheumatrex (registered trademark), Trexall (trademark), methotrexate, and methotrexate sodium.

[0122] Methotrexate can be used for the treatment of several types of cancer (e.g., acute lymphoblastic leukemia, non-Hodgkin lymphoma) or the control of severe psoriasis or rheumatoid arthritis that has not responded to other treatments. Methotrexate can also be used for the control of juvenile idiopathic arthritis. Methotrexate belongs to a class of drugs called antimetabolites. It acts by slowing or stopping the growth of cancer cells and suppressing the immune system.

[0123] Methotrexate is a folic acid antagonist. Methotrexate inhibits dihydrofolate reductase (DHFR), which reduces folic acid to tetrahydrofolic acid. Tetrahydrofolic acid must be regenerated via the DHFR-catalyzed reaction to maintain the intracellular pool of tetrahydrofolic acid one-carbon derivatives for the biosynthesis of thymidylate and purine nucleotides. Inhibition of DHFR by the folic acid antagonist (methotrexate) results in a decrease in nucleic acid synthesis by causing a deficiency in the cellular pools of thymidylate and purines. Therefore, methotrexate interferes with DNA synthesis, repair, and cell replication.

[0124] Methotrexate is most active against rapidly proliferating cells because its cytotoxic action mainly occurs in the S phase of the cell cycle. Since cell proliferation in malignant tissues is greater than that in most normal tissues, methotrexate inhibits malignant growth without causing irreversible damage to normal tissues. As a result, actively proliferating tissues (e.g., malignant cells, bone marrow, fetal cells, oral and mucosal, and bladder cells) are usually more sensitive to the DHFR inhibitory action of methotrexate.

[0125] The cytotoxicity of methotrexate is caused by three important actions: inhibition of DHFR, inhibition of thymidylate synthase, and changes in the transport of reduced folates. Since the affinity of DHFR for methotrexate is much greater than its affinity for folic acid or dihydrofolic acid, simultaneous administration of large amounts of folic acid does not reverse the action of methotrexate. However, if leucovorin (a derivative of tetrahydrofolic acid) is administered immediately after the administration of the antitumor agent, it may block the action of methotrexate. High-dose methotrexate followed by folic acid rescue is used as part of the treatment for patients with non-metastatic osteosarcoma.

[0126] Methotrexate (MTX) is generally used to prevent graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). It suppresses T cell responses, proliferation, and adhesive molecule expression by inhibiting dihydrofolate reductase and the production of thymidylate and purines. See, for example, Nassar et al., J Transplant. 2014;2014:980301.

[0127] The method may include administering an S-phase inhibitor (e.g., a folic acid antagonist, e.g., a DHFR inhibitor, e.g., methotrexate) within about 10, 9, 8, 7, 6, or 5 days before administration of the immune cells. The method may include administering an S-phase inhibitor (e.g., a folic acid antagonist, e.g., a DHFR inhibitor, e.g., methotrexate) within about 4, 3, 2, or 1 day before administration of the immune cells.

[0128] The method may include administering an S-phase inhibitor (e.g., a folic acid antagonist, e.g., a DHFR inhibitor, e.g., methotrexate) within about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days after administration of the immune cells. The method may include administering an S-phase inhibitor (e.g., a folic acid antagonist, e.g., a DHFR inhibitor, e.g., methotrexate) within about 4, 3, 2, or 1 day after administration of the immune cells.

[0129] The method may include administering an S-phase inhibitor (e.g., a folic acid antagonist, e.g., a DHFR inhibitor, e.g., methotrexate) in parallel with or simultaneously with immune cells.

[0130] The method may include administering an S-phase inhibitor (e.g., a folic acid antagonist, e.g., a DHFR inhibitor, e.g., methotrexate) within about 10, 9, 8, 7, 6, or 5 days before administering the immune cells and within about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days after administering the immune cells.

[0131] The method may include administering an S-phase inhibitor (e.g., a folic acid antagonist, e.g., a DHFR inhibitor, e.g., methotrexate) in parallel with or simultaneously with a lymphodepleting agent, and optionally, the method includes administering the S-phase inhibitor in parallel with or simultaneously with the lymphodepleting agent within 5 days before the immunotherapy. The method may include administering the S-phase inhibitor (e.g., a folic acid antagonist, e.g., a DHFR inhibitor, e.g., methotrexate) and the lymphodepleting agent within 3, 2, or 1 day of each other. The method may include administering the S-phase inhibitor (e.g., a folic acid antagonist, e.g., a DHFR inhibitor, e.g., methotrexate) and the lymphodepleting agent within 24, 16, 8, 4, 2, or 1 hour.

[0132] The method may include administering a S-phase inhibitor (e.g., a folic acid antagonist, e.g., a DHFR inhibitor, e.g., methotrexate) once every 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, and / or 28 days. The method may include administering a S-phase inhibitor (e.g., a folic acid antagonist, e.g., a DHFR inhibitor, e.g., methotrexate) over a period of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 days. The method starts about 10, 9, 8, 7, 6, or 5 days before administering immune cells and administers a S-phase inhibitor (e.g., a folic acid antagonist, e.g., a DHFR inhibitor, e.g., methotrexate) once every 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, and / or 28 days.

[0133] The method may include administering a S-phase inhibitor (e.g., a folic acid antagonist, e.g., a DHFR inhibitor, e.g., methotrexate) once every 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, and / or 28 days during the period from about 5 days before administering immune cells to about 90 days after administering immune cells. The method may include administering a S-phase inhibitor (e.g., a folic acid antagonist, e.g., a DHFR inhibitor, e.g., methotrexate) once every 1 - 10 days (e.g., 2 - 9 days, 2 - 8 days, 3 - 7 days, or 4 - 6 days) during the period from about 5 days before administering immune cells to about 90 days after administering immune cells. Methotrexate may be administered on the 3rd, 5th, 10th, and 17th days after each administration of immune cells.

[0134] about 5 mg / m 2 ~ about 3000 mg / m 2 and methotrexate may be administered in an amount of. About 3 mg / m 2 ~ about 3000 mg / m 2Methotrexate may be administered in an amount of.

[0135] About 5 mg / m 2 / day to about 3000 mg / m 2 / day. Methotrexate may be administered at a dose of about 3 mg / m 2 / day to about 3000 mg / m 2 / day.

[0136] For each individual, MTX may be administered once every 2 to 7 days (for example, 3 to 5 days) at a dose of 3.1 mg / m 2 ~15.6 mg / m 2 or 5 mg to 25 mg. For each individual, MTX may be administered once every 2 to 7 days (for example, 3 to 5 days) at a dose of 15.6 mg / m 2 ~62.5 mg / m 2 or 25 mg to 100 mg. For each individual, MTX may be administered once every 2 to 7 days (for example, 3 to 5 days) at a dose of 62.5 mg / m 2 ~187.5 mg / m 2 or 100 mg to 300 mg. For each individual, MTX may be administered once every 2 to 7 days (for example, 3 to 5 days) at a dose of 15.6 mg / m 2 or 25 mg. For each individual, MTX may be administered once every 2 to 7 days (for example, 3 to 5 days) at a dose of 31.25 mg / m 2 or 50 mg. For each individual, MTX may be administered (for example, over at least 10, 20, 30, 40, 50, 60, 70, 80 or 90 days) at a dose of 63 mg / m 2 or 100 mg once every 2 to 6 days (for example, 4 to 5 days). For each individual, MTX may be administered (for example, over at least 10, 20, 30, 40, 50, 60, 70, 80 or 90 days) at a dose of 125 mg / m 2 or 200 mg once every 2 to 6 days (for example, 4 to 5 days). For each individual, MTX may be administered (for example, over at least 10, 20, 30, 40, 50, 60, 70, 80 or 90 days) at a dose of 188 mg / m 2Or it may be administered once every 2 to 6 days (for example, 4 to 5 days) at a dose of 300 mg. For each individual, MTX may be administered at 250 mg / m 2 Or it may be administered once every 2 to 6 days (for example, 4 to 5 days) at a dose of 400 mg. For each individual, MTX may be administered at 313 mg / m 2 Or it may be administered once every 2 to 6 days (for example, 4 to 5 days) at a dose of 500 mg.

[0137] Within about 24 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may be about 2 μM or less (for example, about 1.5 μM or less, about 1 μM or less, about 0.5 μM or less, or about 0.2 μM or less). Within about 12 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may be about 2 μM or less (for example, about 1.5 μM or less, about 1 μM or less, about 0.5 μM or less, or about 0.2 μM or less). Within about 6 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may be about 2 μM or less (for example, about 1.5 μM or less, about 1 μM or less, about 0.5 μM or less, or about 0.2 μM or less). Within about 3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may be about 2 μM or less (for example, about 1.5 μM or less, about 1 μM or less, about 0.5 μM or less, or about 0.2 μM or less). Within about 2 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may be about 2 μM or less (for example, about 1.5 μM or less, about 1 μM or less, about 0.5 μM or less, or about 0.2 μM or less). Within about 1 hour after administering methotrexate, the plasma concentration of methotrexate in the individual may be about 2 μM or less (for example, about 1.5 μM or less, about 1 μM or less, about 0.5 μM or less, or about 0.2 μM or less).

[0138] Within about 24 hours after administration of methotrexate, the plasma concentration of methotrexate in an individual may exceed about 0.1 μM (e.g., exceed about 0.2 μM, e.g., exceed about 0.5 μM, e.g., exceed about 1 μM). Within about 12 hours after administration of methotrexate, the plasma concentration of methotrexate in an individual may exceed about 0.1 μM (e.g., exceed about 0.2 μM, e.g., exceed about 0.5 μM, e.g., exceed about 1 μM). Within about 6 hours after administration of methotrexate, the plasma concentration of methotrexate in an individual may exceed about 0.1 μM (e.g., exceed about 0.2 μM, e.g., exceed about 0.5 μM, e.g., exceed about 1 μM). Within about 3 hours after administration of methotrexate, the plasma concentration of methotrexate in an individual may exceed about 0.1 μM (e.g., exceed about 0.2 μM, e.g., exceed about 0.5 μM, e.g., exceed about 1 μM). Within about 2 hours after administration of methotrexate, the plasma concentration of methotrexate in an individual may exceed about 0.1 μM (e.g., exceed about 0.2 μM, e.g., exceed about 0.5 μM, e.g., exceed about 1 μM). Within about 1 hour after administration of methotrexate, the plasma concentration of methotrexate in an individual may exceed about 0.1 μM (e.g., exceed about 0.2 μM, e.g., exceed about 0.5 μM, e.g., exceed about 1 μM).

[0139] An S-phase inhibitor (e.g., a folic acid antagonist, e.g., a DHFR inhibitor, e.g., methotrexate) may be administered orally, subcutaneously, intramuscularly, intravenously, intraarterially, or intrathecally each time. The S-phase inhibitor may be methotrexate (MTX). For each individual, MTX may be administered at a dose of 15.6 mg / m 2 or 25 mg on the 3rd, 5th, 10th, and 17th days after each administration of immune cells. For each individual, MTX may be administered at a dose of 31.25 mg / m 2 or 50 mg on the 3rd, 5th, 10th, and 17th days after each administration of immune cells.

[0140] C. Lymphocyte depleting agent The methods described herein include, for example, administering a lymphodepleting agent (e.g., a chemotherapeutic agent for lymphodepletion) prior to administering immune cells according to the present specification. Prior to cell therapy, a lymphodepleting agent (e.g., a chemotherapeutic agent for lymphodepletion, e.g., cyclophosphamide and / or fludarabine) is used to pre-treat or pre-condition a patient to reduce the number of endogenous host lymphocytes in the subject, thereby improving the effectiveness of cell therapy and thereby providing a better environment for the growth of the administered cells after administration to the subject.

[0141] The lymphodepleting agents used in the presently disclosed methods may be biological lymphodepleting agents, chemotherapeutic lymphodepleting agents, or combinations thereof. The lymphodepletion regimen may include one or more biological and / or chemotherapeutic lymphodepleting agents.

[0142] Biological lymphocyte depleting agents can be any biological material, such as, for example, antibodies, antibody fragments, antibody conjugates, etc., which can be administered as part of a lymphocyte depletion regimen to reduce endogenous lymphocytes in a subject and used in immunotherapy. Such biological lymphocyte depleting agents may include, for example, monoclonal antibodies or fragments thereof. In some instances, the biological lymphocyte depleting agent is specific for a T cell antigen (i.e., an antigen expressed on the cell surface of T cells). Examples of such antigens include, but are not limited to, CD52 and CD3. In certain instances, the biological lymphocyte depleting agent is an antibody specific for CD52, such as, for example, a monoclonal antibody. Such antibodies may include, for example, alemtuzumab (i.e., CAMPATH), ALLO-647 (Allogene Therapeutics, San Francisco, CA), derivatives thereof that bind CD52, or any other CD52 antibody. In another specific instance, the biological lymphocyte depleting agent is an antibody specific for CD3, such as, for example, a monoclonal antibody. In some cases, the anti-CD3 antibody may be muromonab-CD3 (Orthoclone OKT3 (trademark)), otrexup, teprotumumab, foralumab, bispecific anti-CD3 T cell engaging antibodies (BiTEs) or derivatives thereof specific for CD3.

[0143] The lymphodepletion regimen of the present application may include the administration of one or more chemotherapeutic lymphodepleting agents. The chemotherapeutic lymphodepleting agent may refer to a non-biological material, such as a small molecule, which is administered as part of a lymphodepletion regimen to reduce endogenous lymphocytes in a subject and can be used in immunotherapy. In some examples, the chemotherapeutic lymphodepleting agent may be a lymphodepleting agent but not a bone marrow clearance agent. The chemotherapeutic lymphodepleting agent may include those known in the art, including purine analogs (e.g., fludarabine, pentostatin, azathioprine, mercaptopurine (e.g., 6-mercaptopurine), clofarabine, cladribine and thiopurine (e.g., thioguanine)), and compounds capable of inducing interstrand cross-links in DNA (e.g., cisplatin, mitomycin C, carmustine, psoralen or alkylating agents derived from nitrogen mustard (e.g., cyclophosphamide, ifosfamide, chlorambucil, uracil mustard, melphalan and bendamustine)), but not limited thereto. Other non-limiting examples of chemotherapeutic lymphodepleting agents that can be used in the currently disclosed methods include daunorubicin, L-asparaginase, prednisone, dexamethasone and nelarabine.

[0144] Examples of lymphodepleting agents further include, but are not limited to, 5-fluorouracil, gemcitabine, dacarbazine, melphalan, doxorubicin, vinblastine, oxaliplatin, paclitaxel, docetaxel, irinotecan, etoposide phosphate, mitoxantrone, cladribine, denileukin diftitox, cyclophosphamide or DAB-IL2. For example, reference is made to WO 2018027135 A1 and US 10253086 B2, which are incorporated herein by reference. Table 1 below lists exemplary lymphodepleting agents and their half-lives. In some cases, the lymphodepleting agents discussed herein may be accompanied by low-dose radiation. The lymphodepleting effect of the conditioning regimen can be monitored by conventional practice.

[0145]

Table 1

[0146] One or more lymphocyte-depleting agents may be selected from the group consisting of cladribine, vinblastine, gemcitabine, doxorubicin, fluorouracil, irinotecan, paclitaxel, oxaliplatin, dacarbazine, melphalan, fludarabine, cyclophosphamide, and combinations thereof.

[0147] One or more lymphocyte-depleting agents may include fludarabine and cyclophosphamide. The method may include intravenously administering fludarabine to an individual at about 20-50 mg / m 2 (e.g., 25-30 mg / m 2 ) for about 3 to about 5 days. The method may include intravenously administering cyclophosphamide to an individual at about 250-1000 mg / m 2 for about 3 to about 5 days.

[0148] The method may include intravenously administering fludarabine to an individual at about 30 mg / m 2 for 3 days, and starting with the first dose of fludarabine, intravenously administering cyclophosphamide to an individual at about 300 mg / m 2 for about 3 days. The first doses of fludarabine and cyclophosphamide may be started about 5-7 days (e.g., about 5 days, about 6 days, or about 7 days) before administering immune cells. The first doses of fludarabine and cyclophosphamide may be started about 5 days before administering immune cells. Fludarabine and / or cyclophosphamide may be administered at least 2 or 3 times during the period from about 7 days before administering immune cells to about 90 days after administering immune cells.

[0149] The method may include intravenously administering fludarabine to an individual at about 30 mg / m 2 for 4 days, and starting with the first dose of fludarabine, intravenously administering cyclophosphamide to an individual at about 300 mg / m 2It is intravenously administered to the individual for about 4 days. The first doses of fludarabine and cyclophosphamide may be started about 5 to 7 days (for example, about 5 days, about 6 days, or about 7 days) before administering the immune cells. The first doses of fludarabine and cyclophosphamide may be started about 5 days before administering the immune cells. Fludarabine and / or cyclophosphamide may be administered at least 2 or 3 times during the period from about 7 days before administering the immune cells to about 90 days after administering the immune cells.

[0150] The method may include intravenously administering fludarabine to the individual at about 30 mg / m per day 2 for 3 days, starting from the first dose of fludarabine, and cyclophosphamide is intravenously administered to the individual at about 500 mg / m 2 for about 3 days. The first doses of fludarabine and cyclophosphamide may be started about 5 to 7 days (for example, about 5 days, about 6 days, or about 7 days) before administering the immune cells. The first doses of fludarabine and cyclophosphamide may be started about 5 days before administering the immune cells. Fludarabine and / or cyclophosphamide may be administered at least 2 or 3 times during the period from about 7 days before administering the immune cells to about 90 days after administering the immune cells.

[0151] The method may include intravenously administering fludarabine to the individual at about 30 mg / m per day 2 for 4 days, starting from the first dose of fludarabine, and cyclophosphamide is intravenously administered to the individual at about 530 mg / m 2 for about 3 days. The first doses of fludarabine and cyclophosphamide may be started about 5 to 7 days (for example, about 5 days, about 6 days, or about 7 days) before administering the immune cells. The first doses of fludarabine and cyclophosphamide may be started about 5 days before administering the immune cells. Fludarabine and / or cyclophosphamide may be administered at least 2 or 3 times during the period from about 7 days before administering the immune cells to about 90 days after administering the immune cells.

[0152] One or more chemotherapeutic lymphodepleting agents at an effective dose can reduce one or more endogenous lymphocytes (e.g., B cells, T cells, and / or NK cells) in a subject by at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or up to 100% relative to a control (e.g., starting amount in a subject receiving treatment, a pre-determined threshold, or an untreated subject).

[0153] The lymphodepletion regimen administered during the methods of the present application can be administered at an effective amount (i.e., effective dose) prior to administration of the pharmaceutical composition, and the amount can deplete or reduce the number of endogenous lymphocytes in the subject by 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more relative to a control (e.g., starting amount in a subject receiving treatment, a pre-determined threshold, or an untreated subject). The decrease in lymphocyte count can be monitored by conventional techniques known in the art, e.g., by flow cytometry, by analyzing cells expressing characteristic lymphocyte surface antigens in blood samples taken from the subject at different intervals during treatment with the antibody.

[0154] The lymphodepleting chemotherapeutic agent may include melphalan. Appropriate administration of melphalan is known in the art. Melphalan may be administered in an amount of about 1 mg / day to about 30 mg / day per individual dose (see the prescribing information for Alkeran® (NDA)). The individual dose may be administered 1, 2, 3, 4 or more times per day over 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more. Each individual dose may be repeated every 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more.

[0155] The lymphodepleting chemotherapeutic agent may contain bendamustine. Appropriate administration of bendamustine is known in the art. Bendamustine may be administered in an amount of about 10 mg / m 2 / day to about 200 mg / m 2 / day (see the prescribing information for bendamustine). Individual doses may be administered 1, 2, 3, 4 or more times per day over 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more. Each individual dose may be repeated every 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more.

[0156] The lymphodepleting chemotherapeutic agent may contain mercaptopurine. Appropriate administration of mercaptopurine is known in the art. Mercaptopurine may be administered in an amount of about 0.5 to about 5 mg / kg / day (see the prescribing information for Purinethol®). Individual doses may be administered 1, 2, 3, 4 or more times per day over 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more. Each individual dose may be repeated every 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more.

[0157] The lymphodepleting chemotherapeutic agent may contain daunorubicin. Appropriate administration of daunorubicin is known in the art. Daunorubicin may be administered in an amount of about 10 to about 500 mg / m 2 / day (see the prescribing information for daunorubicin). Individual doses may be administered 1, 2, 3, 4 or more times per day over 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more. Each individual dose may be repeated every 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more.

[0158] The lymphodepletion chemotherapeutic agent may contain cytarabine. Appropriate administration of cytarabine is known in the art (see the prescribing information for DepoCyt®). Cytarabine may be administered in an amount of about 1 mg / day to about 100 mg / day. Each individual dose may be administered 1, 2, 3, 4 or more times per day over 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more. Each individual dose may be repeated every 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more.

[0159] The lymphodepletion chemotherapeutic agent may contain L-asparaginase. Appropriate administration of L-asparaginase is known in the art (see the prescribing information for Elspar®). L-asparaginase may be administered in an amount of about 100 I.U. / kg / day to about 1,500 I.U. / kg / day. Individual doses may be administered 1, 2, 3, 4 or more times per day over 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more. Each individual dose may be repeated every 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more. The lymphodepletion chemotherapeutic agent may contain prednisone or prednisolone. Appropriate administration of methotrexate is known in the art (see the prescribing information for Prapred ODT®). Prednisone or prednisolone may be administered in an amount of about 1 mg / day to about 100 mg / day. Individual doses may be administered 1, 2, 3, 4 or more times per day over 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more. Each individual dose may be repeated every 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more.

[0160] The lymphocyte-depleting chemotherapeutic agent may contain prednisolone or prednisone. Appropriate administration of prednisolone or prednisone is known in the art (see the prescribing information for Oprapred ODT®). Prednisolone or prednisone may be administered in an amount of about 1 mg / day to about 100 mg / day. Individual doses may be administered 1, 2, 3, 4 times or more per day for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more. Each individual dose may be repeated every 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more.

[0161] The lymphocyte-depleting chemotherapeutic agent may contain nelarabine. Appropriate administration of nelarabine is known in the art (see the prescribing information for ARRANON®). Nelarabine may be administered in an amount of about 500 mg / m 2 / day to about 2000 mg / m 2 / day. Individual doses may be administered 1, 2, 3, 4 times or more per day for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more. Each individual dose may be repeated every 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more.

[0162] The lymphocyte-depleting agent may contain rituximab. Appropriate administration of rituximab is known in the art (see the prescribing information for Rituxan®). Rituximab may be administered in an amount of about 100 mg / m 2 / day to about 3000 mg / m 2 / day. Individual doses may be administered 1, 2, 3, 4 times or more per day for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more. Each individual dose may be repeated every 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more.

[0163] The lymphocyte-depleting agent may contain alemtuzumab.

[0164] Appropriate administration of alemtuzumab is known in the art (see the prescribing information for Campath®). Alemtuzumab may be administered in an amount of about 1 mg / day to about 30 mg / day. Individual doses may be administered once, twice, three times, four times or more per day for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more. Each individual dose may be repeated every 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or more.

[0165] As used herein, the terms “effective dose,” “effective amount,” “therapeutically effective dose,” or “therapeutically effective amount” refer to an amount sufficient to achieve a beneficial or desired biological and / or clinical result as used herein. In some cases, an effective dose of a lymphocyte depleting agent is sufficient to reduce endogenous lymphocytes in a subject, e.g., reduce one or more lymphocytes (e.g., B cells, T cells and / or NK cells) by at least about 1%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95% or up to 100% compared to a control (e.g., starting amount, pre-determined threshold, or untreated subject in a subject being treated for a disease, disease state or disorder). The effective dose may be equivalent to the recommended dose, recommended dose or allowable dose (for adults or children) of the lymphocyte depleting agent according to the drug label.

[0166] D. Immune cells The methods described herein include administering immune cells to an individual in need thereof. Immune cells can be used as adoptive cell therapy to treat a disease or medical condition of an individual, such as cancer. Exemplary adoptive cell therapies include, but are not limited to, tumor infiltrating lymphocytes (TIL), T cell receptor (TCR) engineered T cells (TCR-T), chimeric antigen receptor (CAR) engineered T cells, natural killer (NK) cells and hematopoietic stem cells (HSC), and dendritic cell (DC) or myeloid cell therapies. The immune cells may be resistant to S-phase inhibitors (e.g., methotrexate). The immune cells can be engineered to express a heterologous nucleic acid comprising a methotrexate resistance transgene. The immune cells can express a mutant DHFR gene encoding dihydrofolate reductase. The mutant DHFR gene may include an L22F mutation and an F31S mutation. The mutant DHFR gene can encode a polypeptide comprising the amino acid sequence of SEQ ID NO:2. The immune cells can overexpress a DHFR gene encoding dihydrofolate reductase.

[0167] Immune cells can be derived from a variety of cell types and cell sources. As used herein, cells from any mammalian species are contemplated, including, but not limited to, mouse, rat, guinea pig, rabbit, dog, monkey, and human. The immune cells may be human cells. The immune cells may be autologous, i.e., the cells are derived from the individual receiving the immune cells. The immune cells may be syngeneic (i.e., the donor and recipient are different individuals but are monozygotic twins). The immune cells may be allogeneic, i.e., the cells are obtained or derived from a donor different from the individual receiving the immune cells but belonging to the same species. Allogeneic immune cells may be off-the-shelf immune cells, which are pre-manufactured, characterized, and available for immediate administration to a patient. Allogeneic immune cells may be “universal” immune cells, which are derived from cells obtained from one or more donors or cell lines and used for adoptive cell therapy for other individuals of the same species.

[0168] The immune cells may be derived from primary cells. The immune cells may be primary cells isolated from an individual. The immune cells can proliferate (e.g., grow and / or differentiate) from primary cells isolated from an individual. The immune cells may belong to the hematopoietic system. The primary cells can be obtained from the thymus. The primary cells can be obtained from lymph or lymph nodes (e.g., tumor-draining lymph nodes). The primary cells can be obtained from the spleen. The primary cells can be obtained from the bone marrow. The primary cells can be obtained from blood, e.g., peripheral blood. The primary cells may be peripheral blood mononuclear cells (PBMC). The primary cells may be derived from plasma. The primary cells may be derived from a tumor. The primary cells can be obtained from the mucosal immune system. The primary cells can be obtained from a biopsy sample.

[0169] The immune cells may be derived from cell lines. The immune cells may be derived from commercial cell lines. The immune cells may be those that have proliferated (e.g., grown and / or differentiated) from cell lines established from primary cells isolated from an individual. The cell lines may have a short lifespan. The cell lines may be immortalized. The cell lines may be tumor cell lines, e.g., leukemia or lymphoma cell lines. The cell lines may be cell lines derived from PBMC. The cell lines may be stem cell lines. The cell lines may be NK-92 NK92 (Jiang-Hong Gong, 1994), HATAK (Takuji Katayama, 2013), IMC-1 (IM Chen, 2004), KHYG-1 (M Yagita, 2000), NKG (Min Cheng, 2011), NKL (Michael J. Robertson, 1996), NK-YS (Junjiro Tsuchiyama, 1998) or SNK-6 (Hiroshi Nagata, 2001).

[0170] The immune cells may be immune cells or their precursor cells. Exemplary immune cells that can be used in the present application include dendritic cells (including immature dendritic cells and mature dendritic cells), T lymphocytes (e.g., naive T cells, effector T cells, memory T cells, cytotoxic T lymphocytes, helper T cells, natural killer T cells, Treg cells, tumor-infiltrating lymphocytes (TIL) and lymphokine-activated killer (LAK) cells), B cells, natural killer (NK) cells, monocytes, macrophages, neutrophils, granulocytes, and combinations thereof, but are not limited thereto. Immune cell subgroups can be defined by the presence or absence of one or more cell surface markers known in the art (e.g., CD3, CD4, CD8, CD19, CD20, CD11c, CD123, CD56, CD34, CD14, CD33, etc.). When the pharmaceutical composition contains multiple immune cells, these immune cells may be a specific subgroup of immune cell types, a combination of subgroups of immune cell types, or a combination of two or more immune cell types. The immune cells may be present in a homogeneous cell population. The immune cells may be present in a heterogeneous cell population enhanced in immune cells. The immune cells may be lymphocytes. The immune cells may not be lymphocytes. The immune cells can be suitable for adoptive cell therapy. The immune cells may be PBMC. The immune cells may be immune cells derived from PBMC. The immune cells may be T cells. The immune cells may be CD4 + T cells (also called helper T cells). The immune cells may be CD8 + T cells (also called cytotoxic T cells). The immune cells may include T cells expressing TCRα and TCRβ chains (i.e., αβT cells). The immune cells may include T cells expressing TCRγ and TCRδ chains (i.e., γδT cells). The immune cells may include B cells. The immune cells may include NK cells. The immune cells may include NK-T cells. The immune cells may include dendritic cells (DC). The immune cells may include DC-activated T cells.

[0171] The immune cells may be stem cells or derived from stem cells. The stem cells may be totipotent stem cells. The stem cells may be pluripotent stem cells. The stem cells may be unipotent stem cells. The stem cells may be progenitor cells. The stem cells may be embryonic stem cells. The stem cells may be hematopoietic stem cells (HSCs). The stem cells may be mesenchymal stem cells. The stem cells may be induced pluripotent stem cells (iPSCs).

[0172] The immune cells may be modified cells containing one or more heterologous nucleic acid sequences. The modified immune cells may contain any number (e.g., any one of 1, 2, 3, 4, 5, 10, 50, 100, 1000 or more) of heterologous nucleic acid sequences. The modified immune cells may contain a single copy of a heterologous nucleic acid sequence. The modified immune cells may contain multiple copies of a heterologous nucleic acid sequence.

[0173] The immune cells may be modified immune cells, and these modified immune cells contain a heterologous nucleic acid encoding a manipulated receptor. The immune cells may be modified immune cells that express two or more manipulated receptors. The modified immune cells can express a manipulated receptor selected from the group consisting of CAR, recombinant TCR, TAC receptor, TCR fusion protein (TFP) and combinations thereof. The modified immune cells can express CAR and TFP. The modified immune cells can express CAR and recombinant TCR. The modified immune cells can express CAR and TAC receptor. The modified immune cells can express recombinant TCR and TAC receptor.

[0174] The modified immune cells may further comprise a second engineered receptor that converts a negative signal into a positive signal, and optionally, wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. For example, signal switches or signal converters are described, for example, in PCT / CN2022 / 087016 filed on April 15, 2022, which is hereby incorporated by reference in its entirety. The signal converter may comprise the amino acid sequence of SEQ ID NO:11, or an amino acid sequence having at least about 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:11.

[0175] The modified immune cells may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. Membrane-bound IL12p40 may comprise the amino acid sequence of SEQ ID NO:12, or an amino acid sequence having at least about 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:12.

[0176] The modified immune cells may further comprise at least one other heterologous nucleic acid sequence, for example, a second heterologous nucleic acid sequence encoding an immunomodulatory agent (e.g., a costimulatory molecule, a cytokine, a chemokine, and / or an immune checkpoint inhibitor). The heterologous nucleic acid sequence encoding the immunomodulatory agent and the heterologous nucleic acid sequence encoding the engineered receptor may be operably linked to the same promoter or different promoters.

[0177] Immune modulators expressed by heterologous nucleic acids include any agent based on a protein or peptide that modulates (e.g., inhibits or activates) the immune system. Immune modulators can target specific molecules, such as checkpoint molecules, or non-specifically modulate the immune response. The activator may include molecules that activate antigen-presenting cells to stimulate a cellular immune response. For example, the activator may be a peptide of an immune stimulant. The activator may include, but is not limited to, agonists of toll-like receptors TLR-2, 3, 4, 6, 7, 8 or 9, granulocyte macrophage colony-stimulating factor (GM-CSF), TNF, CD40L, CD28, FLT-3 ligand or cytokines (e.g., IL-1, IL-2, IL-4, IL-7, IL-12, IL12p40, IL-15, IL-23 or IL-21). The activator may include agonists (e.g., agonist antibodies) that activate receptors (including co-stimulatory receptors) in T cells, such as agonists of CD28, OX40, GITR, CD137, CD27, CD40 or HVEM. The activator may further include proteins that inhibit the activity of immune inhibitors, such as inhibitors of immune inhibitors IL-10, IL-35, TGF-β, IDO, or proteins that inhibit the activity of immune checkpoints, such as antagonists (e.g., antagonist antibodies) of CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, B7-1, B7-H3, B7-H4, BTLA, VISTA, KIR, A2aR or TIM-3. The activator may further include co-stimulatory molecules, such as CD40, CD80 or CD86. The immune modulator may further include agents that downregulate the immune system, such as antibodies against IL-12p70, antagonists of toll-like receptors TLR-2, 3, 4, 5, 6, 8 or 9, or general inhibitors of immune function. These agents (e.g., activators or downregulators) can be combined to achieve an optimal immune response.

[0178] The modified immune cells may contain a heterologous nucleic acid encoding an exogenous negative regulatory factor (Nef) protein (e.g., wild-type Nef (e.g., wild-type SIV Nef) or mutant Nef (e.g., SIV Nef M116)). Nef-containing immune cells are described, for example, in PCT / CN2020 / 112181 and PCT / CN2020 / 112182, both filed on August 28, 2020, which are hereby incorporated by reference in their entireties. The Nef protein may include the amino acid sequence of SEQ ID NO:18, or an amino acid sequence having at least about 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity with SEQ ID NO:18. SEQ ID NO:18 (SIV Nef M116 sequence) TIFF2025524921000003.tif21161

[0179] The immune cells may not have any genetic modifications (e.g., genomic modifications) that reduce the immunogenicity of the immune cells in an individual. The immune cells may have one or more genetic modifications (e.g., genomic modifications) that reduce the immunogenicity of the immune cells in an individual. The genetic modifications may include disruption of the TCR gene and / or the HLA class I locus of allogeneic T cells in the gene. The genetic modifications may include knockout of an endogenous TCR gene, e.g., TRAC (i.e., TCRα), TRBC (i.e., TCRβ), TCRG (i.e., TCRγ), and / or TCRD (i.e., TCRδ) genes. The genetic modifications may include knockout of b2-microglobulin (B2M). The genetic modifications may include knockout of an immune checkpoint molecule, e.g., PD-1 or CTLA-4 (also called CD52). The genetic modifications can confer resistance of the immune cells to CD25 inhibitors. For example, the genetic modifications knockout IL2RA (also called CD25). The immune cells may have a functional IL2RA gene. The IL2RA gene in the genome of the immune cells may not be modified. The immune cells may have modifications to the IL2RA gene.

[0180] The immune cells may be T cells, for example, allogeneic T cells. The immune cells may be TCRαβ + T cells may be included. The immune cells may be CAR-T cells. The immune cells can express an anti-BCMA CAR. The immune cells may be CAR-T cells that express an anti-BCMA CAR and a Nef protein. The immune cells can express an anti-CD20 CAR. The immune cells may be CAR-T cells that express an anti-CD20 CAR and a Nef protein. The immune cells can express an anti-CLL1 CAR. The immune cells may be CAR-T cells that express an anti-CLL1 CAR and a Nef protein. The immune cells may be TCR-T cells. The immune cells may be T cells that express a TAC receptor. The immune cells may be T cells that express a TFP. The immune cells may be T cells that express a combination of engineered receptors, and these engineered receptors are selected from the group consisting of CAR, TCR, TAC receptor, and TFP. The therapeutic T cells may include an endogenous TCR. The therapeutic T cells may not have a genetic modification that reduces their immunogenicity. The endogenous TCR gene, HLA gene (for example, B2M), and immune checkpoint molecule (for example, PD-1, CTLA-4, etc.) of the therapeutic T cells may not be genetically modified. The therapeutic T cells may not have a genetic modification other than the engineered receptor construct.

[0181] The immune cells may be allogeneic CAR-T cells, for example, UCART cells. The UCART cells may contain more than 90%, for example, more than about 95% or more than about 97% of TCRαβ - T cells may be included. The UCART cells may contain less than about 10%, or less than about 5%, or less than about 3% of TCRαβ + T cells may be included. The UCART cells are described, for example, in WO 2013 / 176915 A1, WO 2016069283 A1, WO 2019 / 129850 A1, WO 2019 / 089650 A1, and CA 2874609 A1, and these documents are hereby incorporated by reference in their entirety.

[0182] The immune cells may be γδ T cells. The immune cells may be γδ T cells expressing one or more engineered receptors, and the one or more engineered receptors are selected from the group consisting of CAR, TCR, TAC receptor, and TFP.

[0183] The immune cells may be HSCs, such as allogeneic HSCs. The immune cells may be HSCs without genetic modification. The immune cells may be HSCs expressing one or more therapeutic agents. The immune cells may be HSCs expressing one or more engineered receptors, and the one or more engineered receptors are selected from the group consisting of CAR, TCR, TAC receptor, and TFP. The immune cells may be HSCs expressing one or more therapeutic agents other than the engineered receptors. The immune cells may + contain HSCs. The immune cells may - contain HSCs. The immune cells may contain at least about 80%, 85%, 90%, or 95% of CD34 + HSCs. The immune cells may contain about 20%, 15%, 10%, or 5% or less of CD34 - HSCs. The immune cells may be HSCs transduced by a viral vector, such as HSCs transduced by a retrovirus or a lentivirus.

[0184] The immune cells may be NK cells. The immune cells may be NK cells expressing one or more engineered receptors, and the one or more engineered receptors are selected from the group consisting of CAR, TCR, TAC receptor, and TFP.

[0185] The immune cells may be NK-T cells. The immune cells may be NK-T cells expressing one or more engineered receptors, and the one or more engineered receptors are selected from the group consisting of CAR, TCR, TAC receptor, and TFP.

[0186] A nucleic acid comprising a heterologous nucleic acid sequence described herein can be transiently or stably incorporated into a modified immune cell. The nucleic acid can be transiently expressed in the modified immune cell. For example, the nucleic acid can be present in the cell nucleus of the modified immune cell in the form of an episomal array. The nucleic acid can be introduced into the modified immune cell using any transfection or transduction method known in the art, including viral or non-viral methods. Exemplary non-viral transfection methods include chemical-based transfection such as the use of calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethyleneimine), electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, hydrodynamic delivery, or non-chemical methods such as transposons, gene gun, magnetofection or magnet-assisted transfection, particle-based methods such as the use of particle bombardment, and hybrid methods such as nucleofection, but are not limited thereto.

[0187] The heterologous nucleic acid sequence can be present in the genome of the modified immune cell. For example, the nucleic acid comprising the heterologous nucleic acid sequence can be incorporated into the genome of the modified immune cell by any method known in the art, including viral-mediated integration, random integration, homologous recombination methods, and site-specific integration methods such as the use of site-specific recombinases or integrases, transposases, transcription activator-like effector nucleases (TALEN®), CRISPR / Cas9, and zinc finger nucleases, but are not limited thereto. The heterologous nucleic acid sequence can be incorporated into a specially designed locus of the modified immune cell genome. The heterologous nucleic acid sequence can be incorporated into an integration hot spot of the modified immune cell genome. The heterologous nucleic acid sequence can be incorporated into a random locus of the modified immune cell genome. When multiple copies of the heterologous nucleic acid sequence are present in a single modified immune cell, the heterologous nucleic acid sequence can be incorporated into multiple loci of the modified immune cell genome.

[0188] Precursor immune cells can be prepared by various methods known in the art. For example, primary immune cells (e.g., T cells) can be obtained from various sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue, and tumors. Immune cells (e.g., T cells) can be obtained from a blood unit collected from an individual using various techniques known in the art (e.g., FICOLL™ isolation). Cells from an individual's circulating blood can be obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis can be washed to remove the plasma portion and used in subsequent processing steps in an appropriate buffer or medium. The cells can be washed with phosphate-buffered saline (PBS) or a wash solution lacking divalent cations (e.g., calcium and magnesium). As will be readily understood by those skilled in the art, the washing step can be completed by methods well known to those skilled in the art, such as using a semi-automated "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in various biocompatible buffers, such as PBS without Ca 2+ free, Mg 2+ free PBS, PlasmaLyte A, or other saline solutions that may or may not contain a buffer. Alternatively, unwanted components of the apheresis sample can be removed and the cells can be resuspended directly in the medium.

[0189] For example, erythrocytes can be lysed and monocytes depleted by PERCOLL™ gradient centrifugation or counterflow centrifugal elutriation to isolate primary T cells from peripheral blood lymphocytes. By positive or negative selection techniques, CD3 + , CD28 + , CD4 + , CD8 +Specific sub - groups of T cells, such as CD45RA and CD45RO cells, can be further isolated. For example, in one embodiment, T cells are isolated by incubating for a time sufficient to perform positive selection on the desired T cells with anti - CD3 / anti - CD28 (i.e., 3x28) conjugation beads, such as DYNABEADS® M - 450 CD3 / CD28 T.

[0190] The T - cell population can be further enriched by negative selection using a combination of antibodies against surface markers specific to the negatively selected cells. For example, one method relates to performing cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry, and the method uses a mixture of monoclonal antibodies against cell - surface markers present on the negatively selected cells. For example, to enrich for CD4 + cells, the monoclonal antibody mixture typically includes antibodies against CD14, CD20, CD11b, CD16, HLA - DR, and CD8. In some embodiments, it may be desirable to enrich or positively select for regulatory T cells that typically express CD4 + , CD25 + , CD62L hi , GITR + and FoxP3. +

[0191] Methods for introducing a vector or nucleic acid into a therapeutic cell (e.g., a precursor immune cell) are known in the art. The vector or nucleic acid can be transferred into the therapeutic cell by physical, chemical, or biological methods.

[0192] Physical methods for introducing vectors or nucleic acids into therapeutic cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Vectors can be introduced into cells by electroporation.

[0193] Biological methods for introducing vectors or nucleic acids into therapeutic cells include using DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, e.g., human cells.

[0194] Chemical methods for introducing vectors or nucleic acids into therapeutic cells include colloidal dispersion systems such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems such as water-in-oil emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system as an in vitro delivery vehicle is liposomes (e.g., artificial membrane vesicles).

[0195] Transduced or transfected precursor immune cells can be expanded ex vivo after introducing heterologous nucleic acids. Transduced or transfected precursor immune cells can be cultured and propagated for any of at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days. Transduced or transfected precursor immune cells can be cultured for any of about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days or less. Transduced or transfected precursor immune cells can be further evaluated or screened to select modified immune cells.

[0196] Immune cells can proliferate in culture in the presence of IL-2. The culture may contain IL-2 at a concentration of at least about 50 IU / mL (for example, any one of at least about 75, 100, 125, 150, 200, 250, 300, 350, 400, 500 or higher IU / mL). The culture may contain IL-2 at a concentration of about 50-500, 50-150, 50-200, 50-300, 50-400, 100-400, 200-400, 200-300 or 200-500 IU / mL. The culture may contain IL-2 at a concentration of about 300 IU / mL. According to clinical data, the serum concentration of IL-2 in healthy individuals and individuals receiving CAR-T therapy is about 20 IU / mL to about 3000 IU / mL.

[0197] Reporter genes can be used for the identification of transfectable cells and the evaluation of the function of regulatory sequences. Usually, a reporter gene is a gene that is not present or not expressed in the recipient organism or tissue, the reporter gene encodes a polypeptide, and the expression of the polypeptide is represented by some easily detectable characteristics (such as enzyme activity). The expression of the reporter gene is measured at an appropriate time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (for example, Ui-Tei et al. FEBS Letters 479: 79-82 (2000)).

[0198] Other methods for the presence of heterologous nucleic acids in precursor immune cells include, for example, molecular biology assays well known to those skilled in the art such as DNA blotting and RNA blotting, RT-PCR and PCR, and biochemical assays that detect the presence or absence of specific peptides by immunological methods (such as ELISA and Western blotting).

[0199] These methods may include administering the immune cells described herein. These methods may include administering the immune cells more than once. In such cases, unless otherwise specified, the relevant time descriptions in this application (e.g., about 5 days before administering the immune cells, e.g., within 90 days after administering the immune cells) refer to the time point of the first administration of the immune cells. The method may include administering the immune cells at least about 2, 3, 4, or 5 times. An individual can receive at least 2, 3, 4, 5 administrations of immune cells. Multiple administrations of immune cells can occur within about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, or 120 days.

[0200] The immune cells may be administered intravenously. The effective amount of the immune cells may be about 10 5 ~ about 10 9 cells per kg. The immune cells may be administered at any one of the doses of at least about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 or 10 9 cells per kg body weight. The immune cells may be administered at any one of the doses of about 10 4 ~ about 10 5 , about 10 5 ~ about 10 6 , about 10 6 ~ about 10 7 , about 10 7 ~ about 10 8 , about 10 8 ~ about 10 9 , about 10 4 ~ about 10 9 , about 10 4 ~ about 10 6 , about 10 6 ~ about 10 8 or about 10 5 ~ about 10 7 cells per kg body weight. The immune cells may be modified immune cells. The effective amount of the immune cells is about 0.5×10 6 ~ 1.5×10 6It may also be. The effective amount of immune cells may be about 1.7×10 5 , 5×10 5 , 1.5×10 6 , 5×10 6 , 1×10 7 or 1.5×10 7 cells / kg.

[0201] The immune cells may contain about 30 million to about 900 million cells (for example, for each administration). The immune cells may contain at least about or about 10 million, 20 million, 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million or 100 million cells (for example, for each administration). The immune cells may contain at least about or about 150 million, 200 million, 250 million, 300 million, 350 million, 400 million, 450 million, 500 million, 550 million, 600 million, 650 million, 700 million, 750 million, 800 million, 850 million or 900 million cells (for example, for each administration). The immune cells may contain about 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million or 100 million cells or less (for example, for each administration). The immune cells may contain about 150 million, 200 million, 250 million, 300 million, 350 million, 400 million, 450 million, 500 million, 550 million, 600 million, 650 million, 700 million, 750 million, 800 million, 850 million or 900 million cells or less (for example, for each administration).

[0202] This application further includes the immune cells described herein. The immune cells may be resistant to S-phase inhibitors (for example, methotrexate). The immune cells can be engineered to express a heterologous nucleic acid containing a mutant DHFR gene encoding dihydrofolate reductase. The mutant DHFR gene may include an L22F mutation and an F31S mutation. The immune cells may include an engineered receptor. The immune cells may further include a second engineered receptor that converts a negative signal into a positive signal, and optionally, where the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain.

[0203] This application further comprises immune cells described herein. The immune cells may comprise an IL12p40 polypeptide. The immune cells can be engineered by introducing the IL-12p40 polypeptide into the cells, where the IL-23p19 subunit has not been introduced into the cells. The immune cells can express an exogenously introduced IL-23p40 polypeptide but do not express an exogenously introduced IL-23p19 subunit. The immune cells can be engineered to comprise the IL-12 p40 polypeptide but not the IL-23p19 subunit. The immune cells can produce IL-23 when activated. The IL-23 produced by the immune cells may not be secreted. The IL12p40 polypeptide may be membrane-bound. Membrane-bound IL12p40 may comprise the amino acid sequence of SEQ ID NO:12, or an amino acid sequence having at least about 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) sequence identity to SEQ ID NO:12.

[0204] This application further provides a nucleic acid described herein. The nucleic acid may comprise a mutant DHFR gene encoding dihydrofolate reductase. The nucleic acid may comprise a polynucleotide sequence encoding an engineered receptor (e.g., a CAR). The nucleic acid may comprise a polynucleotide sequence encoding an engineered receptor that converts a negative signal into a positive signal, optionally where the engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The nucleic acid may comprise a polynucleotide sequence encoding the IL12p40 polypeptide described herein. The nucleic acid may comprise a nucleic acid sequence shown in any one of SEQ ID NOs: 4-7, 13 and 15. This application further provides a vector comprising the nucleic acid described herein.

[0205] E. Engineered Receptor The immune cells described in this specification can express one or more engineered receptors. Exemplary engineered receptors include, but are not limited to, CARs, recombinant TCRs, TAC receptors, and TFP. The engineered receptor may include an extracellular domain that specifically binds to an antigen (e.g., a tumor antigen), a transmembrane domain, and an intracellular signaling domain. The intracellular signaling domain may include a primary intracellular signaling domain and / or a co-stimulatory domain. The intracellular signaling domain may include the intracellular signaling domain of a TCR co-receptor. The engineered receptor may be encoded by a heterologous nucleic acid that is operably linked to a promoter (e.g., a constitutive promoter or an inducible promoter). The engineered receptor can be introduced into the modified immune cells by inserting the protein into the cell membrane and passing the cells through a microfluidic system (e.g., CELL SQUEEZE®) (see, e.g., U.S. Patent Application Publication No. 20140287509). The engineered receptor can enhance the function of immune cells, for example, by targeting immune cells (e.g., modified immune cells), transducing signals, and / or enhancing the cytotoxicity of immune cells (e.g., modified immune cells). The immune cells may not express an engineered receptor, such as a CAR, TCR, TAC receptor, or TFP.

[0206] The engineered receptor may include one or more specific binding domains that target at least one tumor antigen, and one or more intracellular effector domains, such as one or more primary intracellular signaling domains and / or co-stimulatory domains.

[0207] The receptor that has been operated on may be a chimeric antigen receptor (CAR). Many chimeric antigen receptors are known in the art and can be applied to the immune cells of the present application. The CAR may be constructed to be specific for any cell surface marker, for example, by using an antigen-binding fragment of an antibody molecule or an antibody variable domain. Any method for producing the CAR may be used in this specification. For example, reference is made to US 6,410,319, US 7,446,191, US 7,514,537, US 9765342 B2, WO 2002 / 077029, WO 2015 / 142675, US 2010 / 065818, US 2010 / 025177, US 2007 / 059298, WO 2017 / 025038 A1 and Berger C et al., J. Clinical Investigation 118: 1294-308 (2008), and these documents are incorporated by reference.

[0208] A CAR may comprise an extracellular domain, a transmembrane domain, and an intracellular signaling domain, wherein the extracellular domain comprises at least one targeting domain that specifically binds to at least one tumor antigen. The intracellular signaling domain can produce a signal that promotes the immune effector function of CAR-containing cells (e.g., T cells modified by CAR (including αβT or γδT cells), NK cells, or NKT cells). "Immune effector function or immune effector response" refers to enhancing or promoting the immune attack on target cells, e.g., the function or response of immune effector cells. For example, immune effector function or response refers to the properties of T or NK cells that kill target cells or suppress their growth or proliferation. Examples of immune effector functions include, for example, in CAR-T cells, cytolytic activity (e.g., antibody-dependent cell cytotoxicity, or ADCC) and helper activity (e.g., cytokine secretion). A CAR may have an intracellular signaling domain that has a reduced immune effector function. Compared to a full-length CAR having wild-type CD3ζ and optionally one or more co-stimulatory domains, the CAR may have the following intracellular signaling domain, which has about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of any one immune effector function (e.g., cytolytic function against target cells). The intracellular signaling domain can produce a signal that promotes the proliferation and / or survival of CAR-containing cells. The CAR may comprise one or more intracellular signaling domains selected from the signaling domains of CD28, CD137, CD3, CD27, CD40, ICOS, GITR, and OX40. The signaling domain of a naturally occurring molecule may include the entire intracellular (i.e., cytoplasmic) portion of the molecule or its fragment or derivative, or the entire native intracellular signaling domain.

[0209] The intracellular signaling domain of the CAR may include a primary intracellular signaling domain. The "primary intracellular signaling domain" refers to an intracellular signaling sequence that acts stimulatory and induces an immune effector to function. The primary intracellular signaling domain may contain a signaling motif called an immunoreceptor tyrosine-based activation motif or ITAM. The primary intracellular signaling domain may include the functional signaling domain of a protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεRib), CD79a, CD79b, FcγRIIa, DAP10, and DAP12. The primary intracellular signaling domain may include the non-functional or reduced signaling domain of a protein selected from the group consisting of CD3ζ, CD3γ, CD3δ, CD3ε, common FcRγ (FCER1G), FcRβ (FcεRib), CD79a, CD79b, FcγRIIa, DAP10, and DAP12. The non-functional or reduced signaling domain may be a mutant signaling domain having a point mutation, insertion, or deletion that reduces or eliminates one or more immune effector functions (e.g., cytolytic activity or helper activity, including antibody-dependent cell cytotoxicity (ADCC)). The CAR may include a non-functional or reduced CD3ζ (i.e., CD3ζ or CD3z) signaling domain. The intracellular signaling domain may not include a primary intracellular signaling domain. Compared to a CAR having the same construct but a wild-type primary intracellular signaling domain, the reduced primary intracellular signaling domain can induce an immune effector function (e.g., cytolytic function against target cells) of about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less of any one of the following.

[0210] The intracellular signaling domain of the CAR may include one or more (e.g., any one of 1, 2, 3, or more) co-stimulatory domains. A "co-stimulatory domain" may be the intracellular portion of a co-stimulatory molecule. The term "co-stimulatory molecule" refers to a cognate binding partner in immune cells (e.g., T cells) that mediates a co-stimulatory response of the immune cell, such as, but not limited to, proliferation and survival, by specifically binding to a co-stimulatory ligand. A co-stimulatory molecule refers to a cell surface molecule other than an antigen receptor or its ligand that is useful for an effective immune response. Co-stimulatory molecules can be represented as a protein family including TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors. Co-stimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD27, CD28, CD8, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).Another example of such co-stimulatory molecules includes ligands that specifically bind to CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8α, CD8β, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0211] The CAR may comprise a single co-stimulatory domain. The CAR may comprise two or more co-stimulatory domains. The intracellular signaling domain may comprise a functional primary intracellular signaling domain and one or more co-stimulatory domains. The CAR may not comprise a functional primary intracellular signaling domain (e.g., CD3ζ). The CAR may comprise an intracellular signaling domain consisting of one or more co-stimulatory domains or substantially consisting of one or more co-stimulatory domains. The CAR may comprise a non-functional or reduced primary intracellular signaling domain (e.g., mutant CD3ζ) and one or more co-stimulatory domains or a non-functional or reduced primary intracellular signaling domain (e.g., mutant CD3ζ) and an intracellular signaling domain consisting of one or more co-stimulatory domains or substantially consisting of a non-functional or reduced primary intracellular signaling domain (e.g., mutant CD3ζ) and one or more co-stimulatory domains. After the targeting domain binds to the tumor antigen, the co-stimulatory domain of the CAR can enhance the proliferation, survival, and differentiation of engineered immune cells (e.g., T cells) having the CAR by transducing a signal and suppress activation-induced cell death. The one or more co-stimulatory signaling domains may be derived from one or more molecules selected from the group consisting of ligands that specifically bind to CD27, CD28, 4-1BB (i.e., CD137), OX40, CD30, CD40, CD3, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83.

[0212] The intracellular signaling domain of the CAR may comprise a co-stimulatory signaling domain derived from CD28. The intracellular signaling domain may comprise the intracellular signaling domain of CD3ζ and the co-stimulatory signaling domain of CD28. The intracellular signaling domain in the chimeric receptor of the present application may comprise a co-stimulatory signaling domain derived from 4-1BB (i.e., CD137). The intracellular signaling domain may comprise the intracellular signaling domain of CD3ζ and the co-stimulatory signaling domain of 4-1BB.

[0213] The intracellular signaling domain of the CAR may include the co-stimulatory signaling domain of CD28 and the co-stimulatory signaling domain of 4-1BB. The intracellular signaling domain may include the intracellular signaling domain of CD3ζ, the co-stimulatory signaling domain of CD28, and the co-stimulatory signaling domain of 4-1BB. The intracellular signaling domain may include a polypeptide, and the polypeptide includes, from the N-terminus to the C-terminus, the co-stimulatory signaling domain of CD28, the co-stimulatory signaling domain of 4-1BB, and the intracellular signaling domain of CD3ζ.

[0214] The CAR may include a polypeptide, and the polypeptide includes, from the N-terminus to the C-terminus, a CD8 leader sequence, an extracellular binding domain, a CD8 hinge, a CD8 transmembrane, a 4-1BB intracellular stimulatory domain, and a CD3ζ intracellular signaling domain.

[0215] The CAR may be a chimeric antigen receptor containing a chimeric signaling domain (the "CMSD"), where the CMSD includes an immunoreceptor tyrosine-based activation motif (herein also referred to as the "CMSD ITAM") and an optional linker (herein also referred to as the "CMSD linker"), and these ITAM and optional linker are arranged in an arrangement different from any ITAM-containing parent molecule that exists in nature. For example, the CMSD may include two or more ITAMs directly linked to each other. The CMSD may include ITAMs linked via one or more "heterologous linkers", where the one or more "heterologous linkers" are, namely, the following linker sequences, and these linker sequences are not derived from an ITAM-containing parent molecule (e.g., the G / S linker), or are derived from an ITAM-containing parent molecule different from the ITAM-containing parent molecule from which one or more CMSD ITAMs are derived. The CMSD may include two or more (e.g., 2, 3, 4 or more) identical ITAMs. At least two of the CMSD ITAMs may be different from each other. At least one of the CMSD ITAMs may not be derived from CD3ζ. At least one of the CMSD ITAMs may not be ITAM1 or ITAM2 of CD3ζ. The CMSD may not include CD3ζ ITAM1 and / or CD3ζ ITAM2. At least one of the CMSD ITAMs may be CD3ζ ITAM3. The CMSD may not include any ITAM from CD3ζ. At least two of the CMSD ITAMs may be derived from the same ITAM-containing parent molecule. The CMSD may include two or more (e.g., 2, 3, 4 or more) ITAMs, where at least two of the CMSD ITAMs are each derived from a different ITAM-containing parent molecule. At least one of the CMSD ITAMs may be derived from an ITAM-containing parent molecule selected from the group consisting of CD3ε, CD3δ, CD3γ, Igα (CD79a), Igβ (CD79b), FcεRIβ, FcεRIγ, DAP12, CNAIP / NFAM1, STAM-1, STAM-2, and moesin.

[0216] A CAR may comprise a polypeptide comprising, from N- to C-terminus, a CD8 leader sequence, an extracellular binding domain, a CD8 hinge, a CD8 transmembrane, a 4-1BB intracellular costimulatory domain, and one or more ITAM sequences.

[0217] The targeting domain of the CAR can be an antibody or antibody fragment, such as an scFv, Fv, Fab, (Fab'), single domain antibody (sdAb), or V H The targeting domain of a CAR may be an H domain. The targeting domain of a CAR may be a ligand or an extracellular portion of a receptor that specifically binds to an antigen (e.g., a tumor antigen). One or more targeting domains of a CAR can specifically bind to a single tumor antigen. A CAR may be a bispecific or multispecific CAR having targeting domains that bind to two or more tumor antigens. Antigens (e.g., tumor antigens) include CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR (e.g., EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, GPC3, DLL3, GPRC5D, CLL1, WT1, CD4, GU2CYC, MUC16, MUC1, CAIX, CD8, CD7, CD10, CD30, CD34, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, The antigen may be selected from the group consisting of fetal acetylcholine receptor, folate receptor-α, GD3, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, p53, MART1, GP100, protease-3 (PR3), tyrosinase, survivin, EphA2, h5T4, PSCA, TAG-72, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, claudin 6, NKG2D, CD70, ADGRE2, FcRH5, NKp80, NKp30, NKG2A, CD229, CS-1 and other tumor antigens of clinical significance, and combinations thereof.

[0218] Many CARs targeting different tumor antigens, such as anti-CD19 CAR, anti-BCMA CAR, anti-Claudin 18.2 CAR or anti-CD20 CAR, are widely disclosed in the art. The extracellular antigen-binding domain of a CD19 CAR may be or include a CD19-binding fragment (e.g., those disclosed in different patents such as FMC63, SJ25C1 or WO 2022 / 012683). BCMA CARs are also well described, and related patents include, but are not limited to, WO 2016 / 014789, WO 2016 / 014565, WO 2013 / 154760 and WO 2018 / 028647.

[0219] The CAR may be an anti-BCMA CAR. Various antigen-binding domain sequences can be used as the targeting domain of the CAR. See, for example, WO 2018028647 and WO 2021121228, which are incorporated herein by reference in their entireties. The extracellular antigen-binding domain of a BCMA CAR may be or include a BCMA-binding fragment. The CAR may include an anti-BCMA scFv. The CAR may include an anti-BCMA sdAb, e.g., V H H. The BCMA-binding fragment can bind to one or more epitopes on BCMA. The BCMA CAR may be a bivalent CAR comprising two anti-BCMA sdAbs targeting the same or different BCMA epitopes. The CAR may be an ITAM-modified BCMA CAR comprising the sequence of SEQ ID NO:10.

[0220] The CAR may be an anti-Claudin 18.2 CAR. Various antigen-binding domain sequences can be used as the targeting domain of the CAR. See, for example, WO 2021 / 129765, which is incorporated herein by reference in its entirety. The CAR may include an anti-Claudin 18.2 scFv. The CAR may include an anti-Claudin 18.2 sdAb, e.g., V HIt may contain H. The CAR may be an ITAM-modified Claudin 18.2 CAR containing the sequence of SEQ ID NO:9.

[0221] The CAR may be an anti-CD20 CAR. The CAR may contain an anti-CD20 scFv. The anti-CD20 scFv may be derived from an anti-CD20 antibody, such as rituximab (e.g., RITUXAN®, MABTHERA®) or Leu16. The CAR may be an ITAM-modified CD20 CAR containing the sequence of SEQ ID NO:8.

[0222] SEQ ID NO:8 (ITAM010-modified CD20 CAR, CD8α SP-CD20 scFv (Leu16)-CD8α hinge-CD8α TM-4-1BB-ITAM010 amino acid sequence, CD8α SP is shown in italics, CD8α hinge is boxed, CD8α TM is shown in italics, intracellular 4-1BB is underlined, and ITAM010 is in bold) TIFF2025524921000004.tif44161

[0223] The transmembrane domain of the CAR may comprise a transmembrane domain selected from the transmembrane domains of the α, β or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL-2Rβ, IL-2Rγ, IL-7Rα, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or NKG2C. The transmembrane domain of the CAR may be the transmembrane domain of CD4, CD3, CD8α or CD28. The transmembrane domain of the CAR may comprise the transmembrane domain of CD8α.

[0224] The extracellular domain may be linked to the transmembrane domain via a hinge region. In one embodiment, the hinge region comprises the hinge region of CD8α.

[0225] The CAR may comprise a signal peptide, such as CD8αSP.

[0226] Any CAR known in the art or developed by the inventors includes the CARs described in PCT / CN2017 / 096938 and PCT / CN2016 / 094408 (the contents of these documents are incorporated herein by reference in their entireties) and can be used in the methods described herein. Exemplary structures of the CARs are shown in FIGS. 15A-15D of PCT / CN2017 / 096938.

[0227] The receptor that has been operated on may be a recombinant T cell receptor. The recombinant TCR may be specific for an antigen (e.g., a tumor antigen). The antigen (e.g., a tumor antigen) may be selected from the group consisting of CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR (e.g., EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, GPC3, DLL3, GPRC5D, CLL1, WT1, CD4, GU2CYC, MUC16, MUC1, CAIX, CD8, CD7, CD10, CD30, CD34, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD3, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, p53, MART1, GP100, protease-3 (PR3), tyrosine kinase, survivin, EphA2, h5T4, PSCA, TAG-72, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, claudin 6, NKG2D, CD70, ADGRE2, FcRH5, NKp80, NKp30, NKG2A, CD229, CS-1 and other tumor antigens having clinical significance. The tumor antigen may be derived from an intracellular protein of a tumor cell. For example, many TCRs specific for tumor antigens (including tumor-associated antigens) are described, including TCRs for tumor antigens in testicular cancer antigen NY-ESO-1, p53 tumor suppressor antigen, melanoma (e.g., MARTI, gp100), leukemia (e.g., WT1, minor histocompatibility antigen) and breast cancer (e.g., HER2, NY-BR1). Any TCR known in the art may be used in this application. The TCR may have an enhanced affinity for the tumor antigen. For example, exemplary TCRs and methods for introducing these TCRs into immune cells are described in US 5830755 and Kessels et al. Immunotherapy through TCR gene transfer. Nat. Immunol. 2,957-961 (2001).The immune cell may be a TCR-T cell.

[0228] The TCR receptor complex is an octameric complex formed by variable TCR receptor α and β chains (γ and δ chains in the case of γδ T cells) and three dimeric signaling modules CD3δ / ε, CD3γ / ε, and CD247 (T cell surface glycoprotein CD3ζ chain) ζ / ζ or ζ / η. Ionizable residues in the transmembrane domain of each subunit form a polar network of interactions that hold the complex together. The TCR complex has the function of activating the signaling cascade in T cells.

[0229] The engineered receptor may be an engineered T cell receptor (TCR) that comprises one or more T cell receptor (TCR) fusion proteins (TFPs). For example, exemplary TFPs are described in US 20170166622 A1, which is incorporated herein by reference. The TFP may comprise an extracellular domain of a TCR subunit, where the TCR subunit is selected from the group consisting of a TCR α chain, a TCR β chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, functional fragments thereof, and amino acid sequences thereof having one or more modifications of 20 or fewer. The TFP may comprise a transmembrane domain, where the transmembrane domain is selected from the group consisting of a TCR α chain, a TCR β chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, functional fragments thereof, and transmembrane domains of amino acid sequences thereof having one or more modifications of 20 or fewer. The TFP may comprise a transmembrane domain, where the transmembrane domain is selected from the group consisting of a TCR α chain, a TCR β chain, a TCR ζ chain, a CD3ε TCR subunit, a CD3γ TCR subunit, a CD3δ TCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and transmembrane domains of amino acid sequences thereof having one or more modifications of 20 or fewer.

[0230] The TFP may comprise a TCR subunit and an antigen-binding domain, where the TCR subunit comprises at least a portion of the TCR extracellular domain and a TCR intracellular domain containing a stimulatory domain from the intracellular signaling domain of CD3ε, where the TCR subunit is functionally linked to the antigen-binding domain and where, when expressed in a T cell, the TFP incorporates into the TCR.

[0231] TFP may comprise a TCR subunit and an antigen-binding domain, wherein the TCR subunit comprises at least a portion of the TCR extracellular domain and a TCR intracellular domain containing a stimulatory domain from the intracellular signaling domain of CD3γ, wherein the TCR subunit is operably linked to the antigen-binding domain, and wherein, when expressed in a T cell, the TFP is incorporated into the TCR.

[0232] TFP may comprise a TCR subunit and an antigen-binding domain, wherein the TCR subunit comprises at least a portion of the TCR extracellular domain and a TCR intracellular domain containing a stimulatory domain from the intracellular signaling domain of CD3δ, wherein the TCR subunit is operably linked to the antigen-binding domain, and wherein, when expressed in a T cell, the TFP is incorporated into the TCR.

[0233] TFP may comprise a TCR subunit and an antigen-binding domain, wherein the TCR subunit comprises at least a portion of the TCR extracellular domain and a TCR intracellular domain containing a stimulatory domain from the intracellular signaling domain of TCRα, wherein the TCR subunit is operably linked to the antigen-binding domain, and wherein, when expressed in a T cell, the TFP is incorporated into the TCR.

[0234] TFP may comprise a TCR subunit and an antigen-binding domain, wherein the TCR subunit comprises at least a portion of the TCR extracellular domain and a TCR intracellular domain containing a stimulatory domain from the intracellular signaling domain of TCRβ, wherein the TCR subunit is operably linked to the antigen-binding domain, and wherein, when expressed in a T cell, the TFP is incorporated into the TCR.

[0235] The receptor that is operated on may be a T cell antigen conjugate (TAC) receptor. For example, exemplary TAC receptors are described in US 20160368964 A1, which is incorporated herein by reference. The TAC may include a targeting domain, a TCR binding domain that specifically binds to a protein associated with the TCR complex, and a T cell receptor signaling domain. The targeting domain may be an antibody fragment that specifically binds to an antigen (e.g., a tumor antigen), such as an scFv or V H H. The targeting domain may be a designed ankyrin repeat protein (DARPin) polypeptide. The antigen (e.g., a tumor antigen) may be selected from the group consisting of CD19, BCMA, NY-ESO-1, VEGFR2, MAGE-A3, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR (e.g., EGFRvIII), GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, GPC3, DLL3, GPRC5D, CLL1, WT1, CD4, GU2CYC, MUC16, MUC1, CAIX, CD8, CD7, CD10, CD30, CD34, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD3, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, p53, MART1, GP100, protease-3 (PR3), tyrosinase, survivin, EphA2, h5T4, PSCA, TAG-72, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, claudin 6, NKG2D, CD70, ADGRE2, FcRH5, NKp80, NKp30, NKG2A, CD229, CS-1, and other tumor antigens having clinical significance. The protein associated with the TCR complex may be CD3, such as CD3ε. The TCR binding domain may be a single-chain antibody, such as an scFv or V HIt may also be H. The TCR binding domain may be derived from UCHT1. The TAC receptor may include a cytoplasmic domain and a transmembrane domain. The T cell receptor signaling domain may include a cytoplasmic domain derived from a TCR coreceptor. Exemplary TCR coreceptors include, but are not limited to, CD4, CD8, CD28, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. The TAC receptor may include a transmembrane domain and a cytoplasmic domain derived from CD4. The TAC receptor may include a transmembrane domain and a cytoplasmic domain derived from CD8 (e.g., CD8α).

[0236] T cell coreceptors are expressed on T cells as membrane proteins. They can provide stability to the TCR:peptide:MHC complex and facilitate signal transduction. Two subtypes of T cell coreceptors, CD4 and CD8, exhibit strong specificity for specific MHC types. The CD4 coreceptor can only stabilize the TCR:MHC II complex, while the CD8 coreceptor can only stabilize the TCR:MHC I complex. Differential expression of CD4 and CD8 on different T cell types results in differences in functional subgroups of T cells. CD8 + The T cell is a cytotoxic T cell.

[0237] CD4 is a glycoprotein expressed on the surface of immune cells (e.g., helper T cells, monocytes, macrophages, and dendritic cells). CD4 has four immunoglobulin domains (D1 - D4) exposed on the extracellular cell surface. CD4 contains a specific amino acid sequence in its short intracellular tail, which enables the CD4 tail to recruit and interact with the tyrosine kinase Lck. When the TCR complex and CD4 bind to different regions of the MHC II molecule, the close proximity between the TCR complex and CD4 allows Lck, which binds to the intracellular tail of CD4, to tyrosine - phosphorylate the immunoreceptor tyrosine - based activation motif (ITAM) in the intracellular domain of CD3, thereby enhancing the signal generated by the TCR.

[0238] CD8 is a glycoprotein that is either a homodimer (less common) consisting of two α chains or a heterodimer (more common) consisting of one α chain and one β chain, which each contain an immunoglobulin variable (IgV) - like extracellular domain and an intracellular tail linked to the membrane via a thin stalk. CD8 is expressed mainly on the surface of cytotoxic T cells but can also be found on natural killer cells, cortical thymocytes, and dendritic cells. The CD8 intracellular tail interacts with Lck and phosphorylates the intracellular CD3 and ζ chains of the TCR complex when the TCR binds to its specific antigen. Tyrosine phosphorylation in the intracellular CD3 and ζ chains initiates a phosphorylation cascade that ultimately leads to gene transcription.

[0239] Immune cells can express more than one engineered receptor, such as any combination of CAR, recombinant TCR, TAC receptor, and TFP.

[0240] Engineered receptors (e.g., CAR, TCR, TAC, or TFP) expressed by immune cells can target one or more tumor antigens. Tumor antigens are proteins produced by tumor cells that induce an immune response, particularly a T cell-mediated immune response. The selection of the target antigen for this application depends on the specific type of cancer being treated. Exemplary tumor antigens include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, α-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CAIX, human telomerase reverse transcriptase, RU1, RU2(AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostate enzyme, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostaglandin, PSMA, HER2 / neu, survivin, and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin-like growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0241] Tumor antigens may contain one or more antigenic cancer epitopes associated with a malignant tumor. Malignant tumors express many proteins that can be used as target antigens for an immune challenge. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and gp100 in melanoma, and prostate acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to a group of molecules associated with transformation, such as the cancer gene HER2 / Neu / ErbB-2. Another group of target antigens are cancer fetal antigens such as carcinoembryonic antigen (CEA). In B cell lymphoma, the tumor-specific idiotype immunoglobulin constitutes a true tumor-specific immunoglobulin antigen unique to an individual tumor. B cell differentiation antigens (e.g., CD19, CD20, and CD37) are other candidates for target antigens in B cell lymphoma.

[0242] The tumor antigen may be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSA is unique to tumor cells and does not exist in other cells in the body. TAA is not unique to tumor cells and, conversely, is expressed even in normal cells under conditions where it cannot induce an immune tolerance state to the antigen. The expression of the antigen in the tumor can occur under conditions that allow the immune system to respond to the antigen. TAA can be an antigen that is expressed in normal cells during embryonic development when the immune system is immature and non-responsive, or an antigen that is normally present at very low levels in normal cells but is expressed at much higher levels in tumor cells.

[0243] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-system antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15, fetal antigens with overexpression such as CEA, oncogenes with overexpression such as p53, Ras, HER2 / neu, and mutated tumor suppressor genes, unique tumor antigens produced by chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large antigens based on proteins include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, β-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.29\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS 1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0244] F. Nucleic Acids The immune cells described herein contain one or more nucleic acids, and the one or more nucleic acids contain a heterologous nucleic acid sequence encoding any one of the engineered receptors, therapeutic agents, and / or immunomodulatory agents described herein. The nucleic acid may be DNA. The nucleic acid may be RNA. The nucleic acid may be linear. The nucleic acid may be circular.

[0245] Heterologous nucleic acid sequences can be operably linked to one or more regulatory sequences. Exemplary regulatory sequences that control the transcription and / or translation of a coding sequence are known in the art and include a promoter, proper initiation, regulation and / or termination of transcription (e.g., polyA transcription termination sequence), mRNA translocation (e.g., nuclear localization signal sequence), processing (e.g., splicing signal), stability (e.g., introns and non-coding 5' and 3' sequences), translation (e.g., start Met, triplet leader sequence, IRES ribosome binding site, signal peptide, etc.), and other elements for insertion into a viral vector, and may include, but are not limited to, an insertion site. The regulatory sequence may be a promoter, a transcriptional enhancer, and / or a sequence that enables the correct expression of the engineered receptor.

[0246] The term "regulatory sequence" or "control sequence" refers to a DNA sequence that affects the expression of a coding sequence operably linked thereto. The nature of such regulatory sequences varies depending on the host organism. In prokaryotes, regulatory sequences typically include a promoter, a ribosome binding site, and a terminator. In eukaryotes, regulatory sequences include a promoter, a terminator, and optionally an enhancer, a trans-activator, or a transcription factor.

[0247] The term "operably linked" refers to juxtaposition, where the components are in a relationship that enables them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence effects the expression of the coding sequence under the conditions adapted to the regulatory sequence by such linkage.

[0248] As used herein, "promoter" or "promoter region" refers to a DNA or RNA segment that controls the transcription of DNA or RNA operably linked thereto. The promoter region contains specific sequences related to RNA polymerase recognition, binding, and transcription initiation. Note that a promoter contains sequences that regulate the recognition, binding, and transcription initiation activity of RNA polymerase (i.e., the binding of one or more transcription factors). These sequences may act in cis or may respond to trans-acting factors. A promoter can be a constitutive promoter or a regulatable promoter depending on its regulatory nature. A regulatable promoter may be inducible or may respond to the environment (e.g., in response to stimuli such as pH, anaerobic conditions, osmotic regulators, temperature, light, or cell density). Many such promoter sequences are known in the art. See, for example, U.S. Patent Nos. 4,980,285, 5,631,150, 5,707,928, 5,759,828, 5,888,783, 5,919,670 and Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press (1989).

[0249] The promoter may be an endogenous promoter. For example, a nucleic acid sequence encoding an engineered receptor can be knocked into the genome of a modified immune cell downstream of an endogenous promoter using any method known in the art (e.g., the CRISPR / Cas9 method). The endogenous promoter may be a promoter of an abundant protein (e.g., β-actin). The endogenous promoter may be an inducible promoter, for example, induced by an endogenous activation signal of an immune cell (e.g., a modified immune cell). Here, these immune cells are T cells, and the promoter may be a T cell activation-dependent promoter (e.g., the IL-2 promoter, the NFAT promoter, or the NFκB promoter). The promoter may be a heterologous promoter.

[0250] Various promoters have been explored for gene expression in mammalian cells, and in the present application, any of the promoters known in the art can be used. Promoters can be broadly classified into constitutive promoters or regulatable promoters, such as inducible promoters. The heterologous nucleic acid sequence encoding the engineered receptor can be operably linked to a constitutive promoter. The heterologous nucleic acid sequence encoding the engineered receptor can be operably linked to an inducible promoter.

[0251] Constitutive promoters enable constitutive expression of heterologous genes (also called transgenic) in host cells. Exemplary constitutive promoters contemplated herein include, but are not limited to, the cytomegalovirus virus (CMV) promoter, human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerate kinase promoter (PGK), simian virus 40 early promoter (SV40), and the chicken β-actin promoter (CAGG) coupled to the CMV early enhancer. The efficiency of such constitutive promoters for driving transgenic expression has been widely compared in many studies. The promoter may be the hEF1α promoter.

[0252] The promoter may be an inducible promoter. Inducible promoters belong to the class of regulatable promoters. Inducible promoters can be induced by one or more conditions, which can be, for example, physical conditions, the microenvironment of immune cells (e.g., modified immune cells), or the physiological state of immune cells (e.g., modified immune cells), inducers (i.e., inducing agents), or combinations thereof. The inducing conditions may not induce the expression of endogenous genes in immune cells (e.g., modified immune cells) and / or the subject receiving the pharmaceutical composition. The inducing conditions may be selected from the group consisting of inducers, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, tumor environment, and the activation state of immune cells (e.g., modified immune cells).

[0253] The promoter can be induced by an inducer. The inducer may be a small molecule, for example, a chemical compound. The small molecule may be selected from the group consisting of doxycycline, tetracycline, alcohol, metal or steroid. Chemically inducible promoters have been the most widely explored. Such promoters include those whose transcriptional activity is regulated by the presence or absence of small molecule chemical substances (such as doxycycline, tetracycline, alcohol, steroid, metal and other compounds). The doxycycline-inducible system having a reverse tetracycline-regulated transactivator (rtTA) and a tetracycline response element promoter (TRE) is currently the most mature system. Tight control of gene expression in eukaryotic cells by tetracycline-responsive promoters is described in WO 9429442. Tetracycline-regulated transcriptional regulators are disclosed in WO 9601313. Also, Tet technology (for example, the Tet-On system) is described on the website of, for example, TetSystems.com. Any known chemically regulatable promoter can be used to drive the expression of the therapeutic protein of the present application.

[0254] The inducer may be a polypeptide, for example, a growth factor, a hormone or a ligand of a cell surface receptor, for example, a polypeptide that specifically binds to a tumor antigen. Many polypeptide inducers are known in the art and can be applied to the present application. For example, gene switches based on the ecdysone receptor, gene switches based on the progesterone receptor and gene switches based on the estrogen receptor belong to gene switches using transactivators derived from steroid receptors (such as WO 9637609 and WO 9738117).

[0255] The inducer may include both a small molecule component and one or more polypeptides. For example, inducible promoters that depend on polypeptide dimerization are known in the art and can be applied to the present application. The first small molecule CID system developed in 1993 uses FK1012 (a derivative of the drug FK506) to induce homodimerization of FKBP. Using a similar strategy, Wu et al. successfully made CAR-T cells titratable in an ON-switch manner using a rapamycin analog (Rapalog) / FKPB-FRB* and a gibberellin / GID1-GAI dimerization-dependent gene switch (C.-Y. Wu et al., Science 350, aab4077 (2015)). Other dimerization-dependent switch systems include coumermycin / GyrB-GyrB (Nature 383 (6596): 178-81) and HaXS / Snap-tag-HaloTag (Chemistry and Biology 20 (4): 549-57).

[0256] The promoter may be a light-inducible promoter, and the induction condition is light. Light-inducible promoters for regulating gene expression in mammalian cells are also well-known in the art (see, for example, Science 332, 1565-1568 (2011), Nat. Methods 9, 266-269 (2012), Nature 500: 472-476 (2013), Nature Neuroscience 18: 1202-1212 (2015)). Such gene regulatory systems can be broadly classified into two classes: (1) regulation of DNA-binding proteins from the recruitment of DNA-binding or (2) transcriptional activation domains. For example, in mammalian cells, a synthetic mammalian blue light-controlled transcription system based on melanopsin has been developed and tested, which results in the recruitment of NFAT mediated by calcineurin by inducing an increase in intracellular calcium in response to blue light (480 nm). Recently, Motta-Mena et al. described a novel inducible gene expression system developed from the naturally occurring EL222 transcription factor, which confers a high level of blue light-sensitive control over transcription initiation in human cell lines and zebrafish embryos (Nat. Chem. Biol. 10(3): 196-202 (2014)). In addition, red light-induced gene expression regulation is performed by utilizing the red light-induced interaction between phytochrome B (PhyB), a photoreceptor of Arabidopsis thaliana, and phytochrome interacting factor 6 (PIF6). An ultraviolet B (UVB)-inducible gene expression system has been developed and shown to be effective for target gene transcription in mammalian cells (Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, Chapter 25, 4th Edition, CRC Press, January 20, 2015). Any light-inducible promoter described herein can be used to drive the expression of the therapeutic protein of the present application.

[0257] The promoter may be a light-inducible promoter induced by the combination of a light-inducible molecule and light. For example, a photocaged group in a chemical inducer keeps the inducer inactive unless removed by irradiation or other means. Such light-inducible molecules include low molecular weight compounds, oligonucleotides, and proteins. For example, caged exons, caged IPTG used with the lac operon, caged toyocamycin for ribozyme-mediated gene expression, caged doxycycline used with the Tet-on system, and caged rapamycin analogs for light-mediated FKBP / FRB dimerization have already been developed (see, for example, Curr Opin Chem Biol. 16(3-4): 292-299 (2012)).

[0258] The promoter may be a radiation-inducible promoter, and the induction condition is radiation, for example, ionizing radiation. Radiation-inducible promoters are known in the art and are used to control transgenic expression. Changes in gene expression occur after cell irradiation. For example, a group of genes called "immediate early genes" can rapidly respond after ionizing radiation. Exemplary immediate early genes include, but are not limited to, Erg-1, p21 / WAF-1, GADD45α, t-PA, c-Fos, c-Jun, NF-κB, and AP1. Immediate early genes contain a radiation response sequence in their promoter region. The consensus sequence CC(A / T)6GG is found in the Erg-1 promoter and is called a serum response element or CArG element. The combination of radiation-inducible promoters and transgenics has been intensively studied and proven to have efficient therapeutic advantages. See, for example, Cancer Biol Ther. 6(7):1005-12 (2007) and Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, Chapter 25, 4th Edition, CRC Press, January 20, 2015.

[0259] The promoter may be a heat-inducible promoter, and the induction condition is heat. In the art, heat-inducible promoters driving transgenic expression have been widely studied. Heat shock or stress proteins (HSPs) including Hsp90, Hsp70, Hsp60, Hsp40, Hsp10, etc. play important roles in protecting cells under heat or other physical and chemical stresses. In preclinical studies, several attempts have been made with heat-inducible promoters, including the heat shock protein (HSP) promoter and the growth arrest and DNA damage (GADD) 153 promoter. The promoter of the human hsp70B gene, first described in 1985, is considered to be one of the most efficient heat-inducible promoters. Huang et al. reported that after the introduction of the hsp70B-EGFP, hsp70B-TNFα, and hsp70B-IL12 coding sequences, tumor cells expressed extremely high transgenic expression after heat treatment, but no transgenic expression was detected without heat treatment. In the group of IL12 transgenic heat-treated mice, tumor growth in vivo was significantly delayed (Cancer Res. 60:3435 (2000)). Another group of scientists linked the HSV-tk suicide gene to the hsp70B promoter and tested this system in nude mice with murine breast cancer. Mice administered the hsp70B-HSVtk coding sequence to their tumors and heat-treated showed tumor regression and significant survival rates compared to non-heat-treated controls (Hum. Gene Ther. 11:2453 (2000)). Another known heat-inducible promoter in the art can be found, for example, in Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, Chapter 25, 4th Edition, CRC Press, January 20, 2015. Any inducible promoter discussed herein can be used to drive the expression of the therapeutic protein of the present application.

[0260] The promoter can be induced by the redox state. Exemplary promoters that can be induced by the redox state include inducible promoters and hypoxia-inducible promoters. For example, Post DE et al. developed a hypoxia-inducible factor (HIF) response promoter that specifically and potently induces transgenic expression in HIF-active tumor cells (Gene Ther. 8: 1801-1807 (2001), Cancer Res. 67: 6872-6881 (2007)).

[0261] The promoter can be induced by the physiological state (e.g., endogenous activation signal) of immune cells (e.g., modified immune cells). Here, these immune cells are T cells, and the promoter may be a T cell activation-dependent promoter, which can be induced by the endogenous activation signal of the modified T cells. The modified T cells can be activated by inducers (e.g., phorbol myristate acetate (PMA), ionomycin, or plant lectin). The modified T cells can be activated by recognizing tumor antigens in tumor cells by engineered receptors (e.g., CAR, TCR, or TAC). The T cell activation-dependent promoter may be an IL-2 promoter. The T cell activation-dependent promoter may be an NFAT promoter. The T cell activation-dependent promoter may be an NFκB promoter.

[0262] The heterologous nucleic acid sequences described herein may be present in a heterologous gene expression cassette, which may include one or more protein coding sequences and optionally one or more promoters. The heterologous gene expression cassette may include a single protein coding sequence. The heterologous gene expression cassette may include two or more protein coding sequences (i.e., polycistronic) driven by a single promoter. The heterologous gene expression cassette may further include one or more regulatory sequences (e.g., 5'UTR, 3'UTR, enhancer sequence, IRES, transcription termination sequence), recombination sites, one or more selectable markers (e.g., antibiotic resistance gene, reporter gene, etc.), signal sequences or combinations thereof. The first heterologous nucleic acid sequence encoding an engineered receptor may be fused to a second heterologous nucleic acid sequence encoding an immunomodulatory agent (e.g., Nef protein) via a third nucleic acid sequence encoding a self-cleavable linker (e.g., P2A, T2A, E2A or F2A peptide).

[0263] The immune cell may include a vector containing a heterologous nucleic acid sequence encoding an engineered receptor. The vector may further include a second heterologous nucleic acid sequence encoding an immunomodulatory agent.

[0264] The vector may be a viral vector. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex virus vectors and derivatives thereof. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.

[0265] Many virus-based systems have been developed for introducing genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Heterologous nucleic acids can be inserted into vectors and packaged into retroviral particles using known techniques in the art. The recombinant virus can then be isolated and delivered to immune cells (e.g., modified immune cells) in vitro or ex vivo. Many retroviral systems are known in the art. Adenoviral vectors may be used. Lentiviral vectors may be used. Self-inactivating lentiviral vectors may be used. For example, self-inactivating lentiviral vectors can be packaged using known protocols in the art. The resulting lentiviral vectors may be used for the transduction of mammalian cells (e.g., human T cells) using known methods in the art.

[0266] The vector may be a non-viral vector (e.g., plasmid) or an episomal expression vector.

[0267] The vector may be an expression vector. An "expression vector" is a construct that can be used to transform a selected host and provide for the expression of a coding sequence in the selected host. The expression vector may be, for example, a cloning vector, a binary vector or an integration vector. Expression preferably includes transcribing the nucleic acid molecule into translatable mRNA. Ensure that regulatory elements for expression in eukaryotic cells are well known to those skilled in the art. In the case of eukaryotic cells, they usually include a promoter to ensure transcription initiation and an optional polyA signal to ensure transcription termination and transcript stability. Examples of regulatory elements that enable expression in eukaryotic host cells include the AOX1 or GAL1 promoter in yeast, or the CMV-, SV40-, RSV-promoters (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or globin intron in mammalian and other animal cells. Depending on the expression system used, a leader sequence that can direct the polypeptide into a cellular compartment or secrete it into the medium may be added to the coding sequence of the nucleic acid sequence, and these leader sequences are well known in the art. The leader sequence is assembled with translation, start and stop sequences at an appropriate stage and preferably can direct the secretion of the translated protein or a portion thereof into the periplasmic space or extracellular medium by the leader sequence. Optionally, the nucleic acid sequence can encode a fusion protein, which includes an N-terminal identification peptide that confers desired characteristics (e.g., stabilization of the recombinant product of expression or simplification of purification). Suitable expression vectors are known in the art and include, for example, the Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pEF-Neo, pCDM8, pRc / CMV, pcDNA1, pcDNA3 (Invitrogen), pEF-DHFR and pEF-ADA (Raum et al., Cancer Immunol Immunother (2001) 50(3), 141-150) or pSPORT1 (GIBCO BRL).

[0268] G. Disease or disease state The diseases or medical conditions treated by the methods described herein can be considered to be any disease or medical condition that is appropriately treatable by cell therapy involving immune cells.

[0269] The disease or medical condition may be cancer (e.g., blood cancer, e.g., solid tumor). The cancers treatable using any of the methods described herein include any type of cancer. The types of cancer treated with the agents described in the present application include, but are not limited to, epithelial cancer, blastoma, sarcoma, benign and malignant tumors and malignancies, e.g., sarcoma, epithelial cancer and melanoma. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0270] In each aspect, the cancer is early-stage cancer, non-metastatic cancer, primary cancer, advanced cancer, locally advanced cancer, metastatic cancer, remission cancer, recurrent cancer, cancer in adjuvant therapy, cancer in neoadjuvant therapy or substantially treatment-resistant cancer.

[0271] Examples of cancers that can be treated by the methods of the present application include anal cancer, astrocytoma (e.g., cerebellum and brain), basal cell cancer, bladder cancer, bone cancer (e.g., osteosarcoma and malignant fibrous histiocytoma), brain tumor (e.g., glioma, brainstem glioma, cerebellar or cerebral astrocytoma (e.g., astrocytoma, malignant glioma, medulloblastoma and glioblastoma)), breast cancer (e.g., TNBC), cervical cancer, colon cancer, colorectal cancer, endometrial cancer (e.g., uterine cancer), esophageal cancer, eye cancer (e.g., intraocular melanoma and retinoblastoma), gastric (stomach) cancer, gastrointestinal stromal tumor (GIST), head and neck cancer, hepatocellular (liver) cancer (e.g., hepatic epithelial cancer and hepatoma), liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma), medulloblastoma, melanoma, mesothelioma, myelodysplastic syndrome, nasopharyngeal cancer, neuroblastoma, ovarian cancer, pancreatic cancer, parathyroid cancer, peritoneal cancer, pituitary tumor, rectal cancer, renal cancer, renal pelvis cancer and ureteral cancer (transitional cell cancer), rhabdomyosarcoma, skin cancer (e.g., non-melanoma (e.g., squamous cell carcinoma), melanoma and Merkel cell carcinoma), small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer and tuberous sclerosis. Another example of cancer can be found in The Merck Manual of Diagnosis and Therapy, 19th Edition, § on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3), The Merck Manual of Diagnosis and Therapy, 20th Edition, § on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2018 (ISBN 978-0-911-91042-1) (2018 digital online version on the Merck Manuals internet site), and SEER Program Coding and Staging Manual 2016, and for all purposes, these documents are hereby incorporated by reference in their entireties respectively.

[0272] The cancer may be a solid tumor.

[0273] The disease or medical condition treated or prevented by the methods described herein may be a host-versus-graft (HvG) disease condition in a human individual receiving cell therapy.

[0274] III. Compositions, Kits, and Articles of Manufacture Also provided are compositions (e.g., pharmaceutical compositions), kits, unit doses, and articles of manufacture comprising an S-phase inhibitor (e.g., methotrexate), a lymphodepleting agent (e.g., Flu and Cy), and / or immune cells described herein.

[0275] In some embodiments, provided is a composition comprising any one of the immune cells described herein. The composition may comprise any number of immune cells. The composition may comprise a single copy of a therapeutic cell. The composition may comprise at least about 1, 10, 100, 1000, 10 4 、10 5 、10 6 、10 7 、10 8 or more copies of any one of the immune cells. In some embodiments, provided is a pharmaceutical composition comprising an effective amount of an immune cell (e.g., allogeneic CAR-T cells) and a pharmaceutically acceptable carrier agent.

[0276] In some embodiments, provided is a pharmaceutical composition comprising an effective amount of an S-phase inhibitor (e.g., methotrexate), a lymphodepleting agent (e.g., Flu and Cy), and a pharmaceutically acceptable carrier agent described herein. The pharmaceutical composition may be a lyophilized composition, and the lyophilized composition may comprise methotrexate, Flu and Cy, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, sucrose, mannitol, and glycine.

[0277] Provided are compositions for use in any one of the methods described herein, and the use of these compositions in the manufacture of a drug for use in any one of the methods described herein.

[0278] As used herein, "carrier agent" includes pharmaceutically acceptable carrier agents, excipients or stabilizers that are non-toxic to cells or individuals exposed thereto at the dosages and concentrations used. Physiologically acceptable carrier agents are usually pH buffered aqueous solutions. Examples of suitable drug carrier agents are well known in the art and include phosphate buffered saline aqueous solutions, water, emulsions (e.g., oil / water emulsions), various types of wetting agents, sterile solutions, and the like. Acceptable carrier agents, excipients or stabilizers are non-toxic to the recipient at the dosages and concentrations used.

[0279] Drug compositions containing such carrier agents can be formulated by well-known general methods. The solvent or diluent is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength. Representative examples include sterile water, saline (e.g., sodium chloride), Ringer's solution, glucose, trehalose or sucrose solutions, Hank's solution and other physiologically balanced saline aqueous solutions (see, for example, the latest edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins).

[0280] The drug compositions described herein can be administered via any suitable route. The drug compositions may be administered parenterally, transdermally (into the dermis), intracavity, intraarterially (into an artery), intramuscularly (into muscle), intrathecally or intravenously. The pharmaceutical composition may be administered subcutaneously (under the skin). The pharmaceutical composition may be administered intravenously. The pharmaceutical composition may be administered to an individual by infusion or injection. The pharmaceutical composition can be administered directly to the target site, for example, delivered to internal or external target sites by a gene gun or administered to a site in an artery by a catheter. The pharmaceutical composition may be administered locally (e.g., intratumorally). Administration may be by means of a conventional syringe and needle, or any compound or device available in the art that can facilitate or improve delivery of the activator to the subject.

[0281] Formulations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Aqueous carrier agents include water, alcohol / aqueous solutions, emulsions or suspensions containing saline and buffer media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or fixed oils. Intravenous vehicles include liquids and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, inert gases, etc. may also be present. It should be noted that the pharmaceutical composition of the present disclosure may include carrier proteins such as, for example, preferably serum albumin or immunoglobulins of human origin.

[0282] The pharmaceutical composition may be appropriately buffered for use in humans. Suitable buffers include, but are not limited to, phosphate buffers (such as PBS), bicarbonate buffers and / or Tris buffers that can maintain a physiological or weakly basic pH (such as about pH 7 to about pH 9). The pharmaceutical composition can also be made isotonic with blood by adding a suitable tonicity regulator (such as glycerol).

[0283] The pharmaceutical composition may be contained in a single-use vial (such as a sealed single-use vial). The pharmaceutical composition may be contained in a multi-use vial. The pharmaceutical composition may be contained in a container in bulk form.

[0284] A pharmaceutical composition may have to meet several criteria for administration to an individual. For example, the US Food and Drug Administration has issued regulatory guidelines for establishing criteria for cell-based immunotherapy products, including 21 CFR 610 and 21 CFR 610.13. Methods for evaluating the appearance, characteristics, purity, safety, and / or efficacy of a pharmaceutical composition are known in the art. The pharmaceutical composition may substantially exclude foreign proteins that can cause allergic reactions, such as proteins derived from animals other than immune cells used in cell culture. "Substantially exclude" may mean less than any one of about 10%, 5%, 1%, 0.1%, 0.01%, 0.001%, 1 ppm, or less of the total volume or weight of the drug composition. The pharmaceutical composition can be manufactured in a GMP-level workshop. For parenteral administration, the drug composition may contain endotoxin less than about 5 EU / kg body weight / hour. For intravenous administration, at least about 70% of the immune cells in the drug composition are viable. When evaluated using the 14-day direct inoculation test method described in the United States Pharmacopeia (USP), the drug composition may have a "no growth" result. In some embodiments, prior to administration of the pharmaceutical composition, a sample containing both immune cells and a pharmaceutically acceptable excipient should be removed for sterility testing about 48 - 72 hours before final collection (or simultaneously with the last re-feeding of the culture). The pharmaceutical composition may be free of mycoplasma contamination. The pharmaceutical composition may be free of detectable microbial agents. The pharmaceutical composition may be free of infectious agents such as HIV type I, HIV type II, HBV, HCV, human T-lymphotropic virus type I, and human T-lymphotropic virus type II.

[0285] In some embodiments, a kit containing any one of the pharmaceutical compositions described herein is provided, and preferably, its instructions for use are provided. In some embodiments, a kit is provided that comprises: (a) any one of the S-phase inhibitors (e.g., methotrexate) described herein; (b) any one of the lymphodepleting agents (e.g., Flu and Cy) described herein; (c) immune cells described herein; and (d) instructions for use in any one of the methods described herein. The immune cells may be allogeneic immune cells.

[0286] In some embodiments, a kit is provided that comprises: (a) any one of the S-phase inhibitors (e.g., methotrexate) described herein; (b) any one of the lymphodepleting agents (e.g., Flu and Cy) described herein; (c) allogeneic CAR-T cells; and (d) instructions for treating a disease or medical condition (e.g., cancer) of an individual in need thereof. The allogeneic CAR-T cells can target BCMA, Claudin 18.2, CLL1, or CD20. The allogeneic CAR-T cells may not be genetically modified to reduce the immunogenicity of allogeneic cells in an individual. The allogeneic CAR-T cells may be genetically modified to be resistant to MTX. The allogeneic CAR-T cells may not have genetic modifications other than the CAR.

[0287] The kit may further comprise one or more lymphodepleting agents. The kit may further comprise fludarabine and cyclophosphamide. In addition to the immune cells, the kit may further comprise a second cancer therapy, such as chemotherapy, hormonal therapy, and / or immunotherapy. The kit can be customized according to the specific cancer of an individual and includes the corresponding second cancer therapy for the individual.

[0288] The kit may contain one or more other components such as a container, reagents, media, inducers, cytokines, buffers, antibodies, etc. to enable the proliferation or induction of immune cells. The kit may further contain an apparatus for administering the pharmaceutical composition.

[0289] The kit of the present application is in a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (e.g., sealed polyester film (Mylar) or plastic bags). The kit can optionally provide other components such as buffers and explanatory information. Thus, the present application further provides a manufactured article, which includes vials (e.g., sealed vials), bottles, jars, flexible packages, etc. Some components of the kit may be packaged in an aqueous medium or in a lyophilized form.

[0290] The manufactured article may include a container and a label or packaging insert that is in or related to the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed of various materials (e.g., glass or plastic). Usually, the container contains a composition that effectively treats the diseases or disorders described herein (e.g., cancer) and may have a sterile inlet (e.g., the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic needle). The label or packaging insert indicates that the composition is used for the treatment of a specific disease state of an individual. The label or packaging insert further includes instructions for administering the composition to an individual. The label may indicate instructions regarding reconstitution and / or use. The container containing the pharmaceutical composition may be a multi-use vial, which allows for repeated dosing (e.g., 2 to 6 doses). The packaging insert refers to the instructions normally included in the commercial package of the therapeutic product, and these instructions include information regarding the indications, usage, dosage, administration, contraindications and / or warnings for the use of such a therapeutic product. Also, the manufactured article may further include a second container, which contains a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. It may include other materials that are desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0291] The kit or manufactured article may include a plurality of unit doses of a drug composition and handling instructions, which are packaged in an amount sufficient for storage and use in pharmacies (e.g., hospital pharmacies and dispensing pharmacies).

Examples

[0292] Example 1. Evaluation of the effect of different MTX concentrations on the in vitro proliferation of UCAR-T cells 1) Construction of the LUCAR expression vector The pLVX-Puro vector purchased from Clontech was digested using ClaI and EcoRI restriction endonucleases, and the CMV promoter was replaced with the human EF1α promoter (GenBank: J04617.1) to obtain the pLVX-hEF1α vector. The gene of DHFR L22F / F31S (SEQ ID NO: 1) was inserted between SIV NEF M116 of LCAR-UL186S and CD20 CAR, which is disclosed in PCT patent applications No. PCT / CN2020 / 112181 and PCT / CN2020 / 112182. The fusion gene sequence of SIV Nef_M116-P2A-DHFR L22F / F31S-IRES-CD8α SP-CD20 scFv (Leu16)-CD8α hinge-CD8α TM-4-1BB-ITAM010 (hereinafter referred to as "LUCAR-20SD" (SEQ ID NO: 4)) was cloned into the expression plasmid pLVX-hEF1α to form the LUCAR-20SD expression plasmid named M1586. The recombinant expression plasmid M1586 was extracted, mixed with psPAX2 and pMD2.G helper plasmids at a certain ratio, and co-transfected into HEK 293T cells. 60 hours after transfection, the cell culture supernatant containing the virus was collected and centrifuged at 4°C and 3000 rpm for 5 minutes. After filtering the supernatant through a 0.45 μm filter, it was further concentrated using a 500KD hollow fiber membrane column tangential flow technique to produce a lentivirus concentrate, which was stored at -80°C for later use.

[0293] 2) Production of UCAR-T cells 50 mL of fresh peripheral blood was collected from volunteers and mixed with lymphocyte separation medium. Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation. The cells were labeled with magnetic beads using a Pan T cell separation kit (Miltenyi Biotech), and T lymphocytes were separated, purified, and activated with CD3 / CD28 magnetic beads. After culturing in an incubator at 37°C and 5% CO2 for 24 hours, 5×10 6Activated T lymphocytes were infected with the LUCAR-20SD lentiviral vector. The cell suspension was transferred to a 6-well plate and incubated in an incubator at 37°C and 5% CO2 for use in CAR-T cell proliferation. Seventeen days after transduction, TCRα / β-negative cells were enriched and sorted using a TCRα / β selection set to obtain T cells expressing LUCAR-20SD (hereinafter referred to as LUCAR-20SD cells).

[0294] 50 mL of fresh peripheral blood was collected from volunteers, mixed with lymphocyte separation medium, and peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation. The separated lymphocytes were cultured overnight in RPMI 1640 medium supplemented with L-glutamine (2 mM, Sigma-Aldrich), penicillin (100 IU / mL) / streptomycin (100 μg / mL) (Sigma-Aldrich, Missouri, USA), 10% serum (Gibco, Massachusetts, USA), zoledronic acid (5 μM, Actavis, New Jersey, USA), and IL-2 (100 IU / mL; Aldesleukin, Novartis, Frimley, UK). The selected γδ T cells were transduced with a lentiviral vector expressing anti-CLL1 CAR and DHFR L22F / F31S at a multiplicity of infection (MOI) of 5. Forty-eight hours after transduction, IL-2 (100 IU / mL) was added every 2 - 3 days to expand T cells expressing anti-CLL1 CAR (hereinafter referred to as CAR-γδ T cells).

[0295] Cultures were performed with the addition of methotrexate (MTX) at concentrations of 0 μM, 0.01 μM, 0.05 μM, 0.1 μM, 1 μM, or 2 μM, and the cell numbers of LUCAR-20SD cells on days 0, 3, 6, 9, and 12 and CAR-γδ T cells on days 0, 2, 5, and 7 were counted using AOPI. Cell growth curves were plotted individually.

[0296] Figure 1A shows the growth of LUCAR-20SD cells cultured with different concentrations of MTX. On the 12th day, compared with the culture with 0 μM MTX, there was no significant difference in cell growth of LUCAR-20SD cells in MTX at 0.01 μM to 1 μM (p > 0.05). On the other hand, at 2 μM MTX, cell growth was significantly decreased (p < 0.05), indicating that MTX ≥ 2 μM affects the growth of LUCAR-20SD cells. Figure 1B shows the growth of CAR-γδT cells cultured with different concentrations of MTX. On the 7th day, compared with the culture with 0 μM MTX, there was no significant difference in cell growth of CAR-γδT cells in MTX at 0.01 μM to 0.1 μM (p > 0.05). On the other hand, at 1 μM MTX and 2 μM MTX, cell growth was significantly decreased in a dose-dependent manner (p < 0.05), indicating that MTX ≥ 1 μM affects the growth of CAR-γδT cells. According to the above results, in order to ensure the growth of UCAR-T cells, the concentration of MTX should be less than 2 μM.

[0297] Example 2: Evaluation of the protective effect of MTX on heterogeneous UCAR-T cells To evaluate the resistance of LUCAR-20SD cells to the rejection of allogeneic T cells, an in vitro mixed lymphocyte reaction (MLR) model was established.

[0298] Using T cells from a random healthy donor (Donor B), LUCAR-20SD cells were prepared as transplanted cells according to the method in Example 1. In addition, untransduced T cells (UnT) from another random healthy donor (Donor A) were prepared as host cells.

[0299] The cell density of CD20-positive cells (lymphoma cell line Raji, ATCC, CCL-86) was adjusted to 1×10 6 cells / mL. At 37 °C, Raji cells were treated with 20 μg / mL mitomycin for 3 hours, washed 3 times with 10 mL of DPBS, and resuspended in the medium. For 20×10 6 untransduced T cells (UnT, T cell donor A), 1×10 6 cells of LUCAR-20SD cells (T cell donor B) and 5×106 Individual processed lymphoma cells Raji were mixed and cultured in a medium. The cell mixture was divided into four groups, and 0 μM of MTX, 0.05 μM of MTX, 1 μM of MTX, or 2 μM of MTX was added thereto, respectively. On the 1st, 4th, 7th, 10th, and 13th days of the co-culture system, centrifugation was performed to change the medium, and sampling was performed together therewith (labeled as K0, K1, K2, K3, K4, respectively), and used for the detection of LUCAR-20SD and UnT cells by FACS using cell counting, CD5 antibody (Biolegend, catalog number 364008), and CD20 CAR antibody (GenScript, catalog number LGBUADAb-1). On the 0th, 4th, 7th, and 11th days, LUCAR-20SD cells were repeatedly stimulated with Raji cells (CD20-positive lymphoma cells) at an E / T ratio of 1:1. Based on the following formula: magnification of change in cell number (UnT) = [number of cells counted by AOPI × (CAR - CD5+ %)] / 20, magnification of change in cell number (LUCAR-20SD cells) = [number of cells counted by AOPI × (CAR+CD5+ %)] / 1, the cell growth curves of UnT and LUCAR-20SD cells were plotted.

[0300] The results are shown in FIGS. 2A to 2D. In the co-culture with 0 μM of MTX (FIG. 2A), UnT cells (T cell donor A) showed superiority in proliferation, and the number of LUCAR-20SD cells (T cell donor B) gradually decreased. Under the culture conditions of different concentrations of MTX (0.05 μM (FIG. 2B), 1 μM (FIG. 2C), 2 μM (FIG. 2D)), the proliferation of UnT cells was significantly inhibited, and the magnification of change in cell number was always less than 1. LUCAR-20SD cells showed strong drug resistance and had significant superiority in proliferation ability and magnification of increase. According to the above results, administration of MTX can significantly inhibit the rejection ability of host cells against LUCAR-20SD cells and has a good protective effect on heterologous LUCAR-20SD cells.

[0301] Example 3. Evaluation of the protective effect of MTX on heterologous γδ T cells To evaluate the resistance of γδ T cells to the rejection of allogeneic T cells, an in vitro mixed lymphocyte reaction (MLR) model was established.

[0302] Using T cells from a random healthy donor (Donor B), CAR-γδ T cells were produced as transplanted cells according to the method in Example 1. Also, untransduced T cells (UnT) from another random healthy donor (T cell donor A) were produced as host cells.

[0303] The density of CLL1-positive human lymphoma cells (U937) was adjusted to 1×10 6 cells / mL. The U937 cells were treated with 20 μg / mL of mitomycin at 37°C for 3 hours, washed 3 times with 10 mL of DPBS, and suspended in the medium. 20×10 6 untransduced T cells (UnT, T cell donor A), 1×10 6 CAR-γδ T cells (T cell donor B), and 5×10 6 treated human lymphoma cells U937 were mixed and cultured in the medium. The cell mixture was divided into three groups, and 0 μM of MTX, 0.05 μM of MTX, or 0.5 μM of MTX was added to each group, respectively. The co-culture system was centrifuged on day 1, day 4, day 7, and day 10 to replace the medium, and samples were taken simultaneously (labeled as K0, K1, K2, K3 respectively) and used for cell counting and detection of CD3 and TCR Vγ9 expression by FACS. On day 0, day 4, and day 7, CAR-γδ T cells were repeatedly stimulated with U937 cells at an E / T ratio of 1:1. Based on the following formula: magnification of change in cell number (UnT) = [number of cells counted by AOPI × (CD3- / TCR Vγ9+ %) ] / 20, magnification of change in cell number (CAR-γδT) = [number of cells counted by AOPI × (CD3+ / TCR Vγ9+ %) ] / 1, the cell growth curves of UnT and CAR-γδ T cells were plotted.

[0304] The results of the study are shown in FIGS. 3A-3C. In co-culture with 0 μM MTX (FIG. 3A), UnT cells (T cell donor A) showed superiority in proliferation, and the number of CAR-γδ T cells (T cell donor B) gradually decreased. Under the culture conditions of different concentrations of MTX (0.05 μM (FIG. 3B) and 0.5 μM (FIG. 3C)), the proliferation of UnT cells was significantly inhibited, while CAR-γδ T cells showed strong drug resistance and had significant superiority in proliferation ability and fold increase. According to the above results, the administration of MTX can significantly inhibit the rejection ability of host cells against CAR-γδ T cells and has a good protective effect on heterologous CAR-γδ T cells.

[0305] Example 4. Evaluation of the in vivo efficacy of MTX against LUCAR-GC cells 1) Production of LUCAR-GC cells Referring to Example 1, the fusion gene sequence of SIV Nef M116-IRES-CD8α SP Claudin 18.2 VHH-CD8α hinge-CD8α TM-4-1BB-ITAM010 (SEQ ID NO: 5) was cloned into the expression plasmid pLVX-hEF1α to form the M1645 expression plasmid. The anti-Claudin 18.2 VHH and the antigen-binding domain of CAR are disclosed in PCT / CN2020 / 139143. TGB23-6-IRES-DHFR L22F / F31S (SEQ ID NO: 7) was cloned into the expression plasmid pLVX-hEF1α to form the M1647 expression plasmid. The fusion protein of TGB23-6 is a signal converter and is described in PCT / CN2022 / 087016. To obtain LUCAR-GC cells, M1645 and M1647 were transduced into T cells. Referring to Example 1, the production of viral vectors and the transduction of primary T were completed.

[0306] 2) Construction of NUGC4 mouse model and tumor monitoring The in vivo efficacy was evaluated using severely immunodeficient NCG mice. On the 14th day before treatment, tumor cells (3×10 6Individual human gastric cancer cells NUGC4 / (mouse) were subcutaneously inoculated into NCG immunodeficient mice. The xenograft mice were divided into 5 groups (G1, G1, G3, G4, and G5) on day 0, and received intravenous injection of HBSS (G1, G5), untransduced T cells (UnT) (G2, 3.6×10 6 cells / mouse dose), and LUCAR-GC cell preparation (G3 and G4, 1×10 6 CAR+ cells / mouse dose) once. Mice in groups G4 and G5 were additionally administered 22.635 mg / kg of MTX by intraperitoneal injection on days 5, 10, 15, 20, 25, 30, and 35, respectively. Figure 4A shows the treatment of mice in G4. During the experiment, the animal status, survival rate, tumor volume, and body weight were measured twice a week. Blood was collected from the eyes of the mice weekly to isolate cells, and then resuspended in DPBS with 1 μL of FITC anti-human CD45 (Biolegend, 304038). The proportion of transplanted cells (hCD45+ % / live cells) in the peripheral blood of the mice was counted.

[0307] The results of the study are shown in Figures 4B to 4C. When 22.635 mg / kg of MTX was administered alone to group G5, the tumor volume was not significantly different from that of the control groups G1 and G2 (p>0.05), indicating that MTX alone was ineffective in controlling tumor growth. In group G3, the average tumor volume of the mice transplanted with LUCAR-GC cells on day 16 was 240.67 mm 3 which was significantly lower than that of the control group G2 (734.83 mm 3 (p<0.05), indicating that single injection of LUCAR-GC cells could effectively inhibit the growth of tumor cells. However, after 30 days, the tumor volume showed a slow increase, indicating that the tumor recurred in the mice. In G4, the average tumor volume of the mice treated with MTX and LUCAR-GC cells on day 16 was 223.62 mm 3 which was significantly lower than that of the control group G2 (734.83 mm 3Significantly lower (p < 0.05), indicating that the combined administration of MTX and LUCAR-GC cells effectively suppressed the proliferation of tumor cells, and no increase in tumor volume occurred until day 42. According to the above results, the combined administration of MTX and LUCAR-GC cells has a significantly higher tumor control ability than MTX alone or LUCAR-GC cells alone, that is, the combined administration of MTX and LUCAR-GC cells can significantly improve the in vivo tumor control effect of LUCAR-GC cells. As shown in Figure 4C, regardless of the presence or absence of MTX, there was no significant difference in the increase of mouse peripheral blood LUCAR-GC cells, indicating that 22.635 mg / kg of MTX does not affect the increase and effectiveness of LUCAR-GC cells. In short, the combination therapy of MTX and LUCAR-GC cells is significantly superior to MTX or LUCAR-GC cells alone in tumor control.

[0308] Example 5. Evaluation of the combined effects of fludarabine, cyclophosphamide, and MTX 1) Production of LUCAR-BCMA cells The pLVX-Puro vector purchased from Clontech was digested with ClaI and EcoRI restriction endonucleases. The CMV promoter was replaced with the human EF1α promoter (GenBank: J04617.1) to obtain the pLVX-hEF1α vector. The fusion gene sequence of SIV Nef_M116-P2A-DHFR L22F / F31S-IRES-CD8α SP-anti-BCMA antibody-CD8α hinge-CD8α TM-4-1BB-ITAM010 (hereinafter referred to as "LUCAR-BCMA" (SEQ ID NO: 6)) was cloned into the expression plasmid pLVX-hEF1α to form the LUCAR-BCMA expression plasmid named M1588. The anti-BCMA antibody included in this specification is disclosed in PCT / CN2020 / 136570. Referring to the production of the viral vector and the transduction of primary T in Example 1, LUCAR-BCMA cells were produced.

[0309] 2) Evaluation of the effect of the combination of fludarabine, cyclophosphamide, and MTX on the in vivo host immune system In vivo efficacy was evaluated using severely immunodeficient NCG mice. Specific embodiments are as follows: - On day -5.5, 20 × 10 6 PBMC cells (HLA - A2+) were intravenously inoculated into the mice to construct host immune cells. On day 0, 3 × 10 6 LUCAR - BCMA cells (HLA - A2 -) were intravenously inoculated as transplanted cells. Then, the modeled mice were treated with the following different combinations of fludarabine, cyclophosphamide, and MTX to evaluate the inhibitory effect of the combined regimen on the host immune system. The combinations and dosages of fludarabine, cyclophosphamide, and MTX are shown in Table 2. " / " in Table 2 means that the drug was not administered. The drug administration schedules for G4 and G6 are also shown in Figure 5A. In each experimental group, the dosage of fludarabine was 0.1623 mg / mouse, the dosage of cyclophosphamide was 1.623 mg / mouse, and the MTX dosage was 15.15 mg / kg (G3 and G4) or 75.45 mg / kg (G5 and G6). On day 13 (the end point of the experiment), the peripheral blood white blood cells (WBC) of the mice were measured. The mouse peripheral blood cells were resuspended in DPBS, and 1 μL of FITC anti - human CD45 (Biolegend, 304038) and 1 μL of PE anti - human HLA - A2 (Biolegend, 343306) were added to the cell resuspension and incubated at 4°C for 30 minutes. The stained cells were washed twice with 1 mL of DPBS and centrifuged at room temperature to discard the supernatant. Finally, the cells were suspended in DPBS, and the expression of hCD45 and HLA - A2 was detected by flow cytometry (FACS). According to the following formula: the number of host cells in the peripheral blood of each mouse = WBC * (HLA - A2+ / hCD45 + %). The proliferation of host PBMC cells in the mouse peripheral blood was evaluated, reflecting the effect of different drug combinations on the in vivo host immunity.

[0310]

Table 2

[0311] The results of the study are shown in Figure 5B. On the 13th day (experimental endpoint), in Group G2 (fludarabine, cyclophosphamide), Group G3 (low-dose 15.15 mg / kg MTX), and Group G5 (high-dose 75.45 mg / kg MTX), the number of host cells in peripheral blood was lower than that in the peripheral blood of the G1 blank group. Both the combined regimen of fludarabine and cyclophosphamide or MTX alone were shown to be able to suppress the proliferation of host cells, but the inhibitory effect was limited. Comparing the results of Group G3 and Group G5, the inhibitory effect of MTX on host immune cells is dose-dependent. The combination of fludarabine, cyclophosphamide, and MTX achieves a synergistic effect (for example, see G4). Compared with only the MTX group or only the FC group in Figure 5B, the combined use of fludarabine, cyclophosphamide, and MTX effectively suppressed host immune cells at a significantly lower host cell ratio. In short, the combination of fludarabine, cyclophosphamide, and MTX significantly suppressed the host immune system.

[0312] Example 6. Influence of MTX Administration Interval in Combined Administration of Fludarabine, Cyclophosphamide, and MTX In an immunocompetent C57BL / 6 mouse model, the influence of the MTX administration interval in the combined administration of fludarabine, cyclophosphamide, and MTX was further evaluated. In Group G2, combined administration of fludarabine, cyclophosphamide, and MTX was performed. See also Figure 6A. Mice in Group G1 and Group G2 were both intravenously inoculated with 5×10 6 individual LUCAR-20SD cells on day 0, and the blood of the mice was regularly examined during the experiment. In this test, the dose of fludarabine was 0.1623 mg / mouse, the dose of cyclophosphamide was 1.623 mg / mouse, and the dose of MTX was 75.45 mg / kg. The dosing schedule is shown in Table 3. " / " in Table 3 means that the drug was not administered.

[0313]

Table 3

[0314] According to the results, in Figure 6B, taking the mice not receiving drug treatment in Group G1 as the control group, the peripheral blood white blood cells (WBC) of the mice during the experiment were stable, and the average value was 8.51×10 9 / L. For the mice in G2 (MTX was administered on days -3, 1, 6, and 11, and the dosing intervals were 4 days, 5 days, and 5 days), during the experiment from day 0 to day 14, the average WBC of the mice was 2.47×10 9 / L, which was significantly lower than the average WBC of the G1 control group.

[0315] Example 7. Influence of MTX dosage in the combined administration of fludarabine, cyclophosphamide and MTX In an immunocompetent C57BL / 6 mouse model, the influence of MTX dosage in the combined administration of fludarabine, cyclophosphamide and MTX was further evaluated. Each experimental group was intravenously inoculated with 5×10 6 individual LUCAR-20SD cells on day 0, and the blood of the mice was regularly examined during the experiment. In this test, the dosage of fludarabine was 0.1623 mg / mouse, and the dosage of cyclophosphamide was 1.623 mg / mouse. The dosages of fludarabine, cyclophosphamide and MTX are shown in Table 4. " / " in Table 4 means that the drug was not administered. The dosing regimen is shown in Figure 7A.

[0316]

Table 4

[0317] The results are shown in Figures 7B to 7C. Taking the mice not receiving drug treatment in Group G1 as the control group, the WBC of the mice during the experiment was stable, and the average value was 8.51×10 9 / L. During the experiment from day 0 to day 14, in Group G2, the average WBC of the mice injected with 30.179 mg / kg of MTX was 3.4×10 9 / L, which was significantly lower than the average WBC of the control group. During the experiment from day 0 to day 14, in the G3 group, the average WBC of the mice injected with 75.447 mg / kg of MTX was 2.47×10 9 / L, which was significantly lower than the average WBC of the control group.

[0318] Example 8. Evaluation of the protective effect of the combination of fludarabine, cyclophosphamide and MTX on allogeneic transplanted cells 1) Construction of MPC-11 drug-resistant cell line The MPC-11 mouse myeloma cell line is derived from the mouse strain BALB / c. 5×10 6 Individual MPC-11 mouse myeloma cells were infected with a lentiviral vector (M1647:TGB23-6-IRES-DHFR L22F / F31S), and the cell suspension was transferred to a 6-well plate and cultured in an incubator at 37°C and 5% CO2 for 3 days. The transduced cells were selected with 0.05 μM of MTX until the positive cells exceeded 95% and used as transplanted cells.

[0319] 2) Evaluation of the protective effect of the combination of fludarabine, cyclophosphamide and MTX on allogeneic transplanted cells In the mouse strains of BALB / c and C57BL / 6, subcutaneous xenograft models were established by transducing the above-mentioned transduced MPC-11 cells (1×10 6 / mouse). The doses of fludarabine, cyclophosphamide and MTX are shown in Table 5. The combination administration regimen of the G4 group is also shown in Figure 8A. In this study, the dose of fludarabine was 0.1623 mg / mouse, the dose of cyclophosphamide was 1.623 mg / mouse, and the dose of MTX was 75.447 mg / kg. The tumor volume was evaluated three times a week to evaluate the effect of the drug combination on allogeneic transplanted cells.

[0320]

Table 5

[0321] The results of the study are shown in Figure 8B. In the autologous transplantation model of Group G1, transplantation of MPC-11 tumors was successful. In the allogeneic transplantation model of Group G2, MPC-11 tumor cells could not effectively increase and tumors could not be measured, indicating that the C57BL / 6 immune system in the allogeneic transplantation model significantly inhibited tumor formation of MPC-11 cells with BALB / c genetic background. In the allogeneic transplantation model of Group G3, the combination of fludarabine and cyclophosphamide promoted weak tumor expansion (less than 8.62 mm 3 within 8 days) of MPC-11, and then the tumors disappeared, indicating that fludarabine and cyclophosphamide temporarily suppressed the host immune system in allogeneic transplantation but could not provide long-term effective protection for allogeneic transplanted cells. In the allogeneic transplantation model of Group G4, the combination of fludarabine, cyclophosphamide and MTX significantly promoted the expansion of MPC-11 tumors within 15 days, and the tumor volume reached up to 132.69 mm 3 , indicating that the combination of fludarabine, cyclophosphamide and MTX significantly inhibited immune rejection in allogeneic transplantation and the combination had a significant protective effect on allogeneic transplanted cells.

[0322] Example 9. Exemplary clinical study design showing the protective effect of the FC + MTX combination regimen on allogeneic adoptive cells ("UCAR") In the clinical study, the immune system of patients was continuously suppressed by using the FC + MTX combination to protect the in vivo efficacy and persistence of transplanted UCAR cells. The combination of fludarabine and cyclophosphamide was used for lymphodepletion. Fludarabine was administered continuously at 25 - 30 mg / m 2 / day for 3 - 4 days, and cyclophosphamide was administered continuously at 250 - 1000 mg / m 2 / day for 3 - 4 days. This combination was administered 1 - 3 times during the period from day - 7 to day 90. The administration cycle of MTX was from day - 5 to day 90, and MTX was administered at a frequency of about 1 - 28 days (for example, about 1 - 10 days, for example, about 2 - 8 days, for example, about 3 - 7 days, for example, about 4 - 6 days, for example, about 4 days, for example, about 5 days) at 3 mg / m2 / day to 3000 mg / m 2 / day (for example, 5 mg / m 2 / day to 3000 mg / m 2 / day) and administered. As the administration method of MTX, it is orally or intravenously administered each time. For each individual, MTX is administered at a dose of 3.1 mg / m 2 ~15.6 mg / m 2 or 5 mg to 25 mg once every 3 to 5 days may also be administered. For each individual, MTX is administered at a dose of 15.6 mg / m 2 ~62.5 mg / m 2 or 25 mg to 100 mg once every 3 to 5 days may also be administered. For each individual, MTX is administered at a dose of 62.5 mg / m 2 ~187.5 mg / m 2 or 100 mg to 300 mg once every 3 to 5 days may also be administered. For each individual, MTX is administered at a dose of 15.6 mg / m 2 or 25 mg once every 2 to 7 days may also be administered. For each individual, MTX is administered at a dose of 31.25 mg / m 2 or 50 mg once every 2 to 7 days may also be administered. For each individual, MTX is administered at a dose of 63 mg / m 2 or 100 mg once every 4 days may also be administered. For each individual, MTX is administered at a dose of 125 mg / m 2 or 200 mg once every 4 days may also be administered. For each individual, MTX is administered at a dose of 188 mg / m 2 or 300 mg once every 4 days may also be administered. For each individual, MTX is administered at a dose of 250 mg / m 2 or 400 mg once every 5 days may also be administered. For each individual, MTX is administered at a dose of 313 mg / m 2 or 500 mg once every 5 days may also be administered. For each individual, MTX is administered at a dose of 15.6 mg / m on the 3rd, 5th, 10th, and 17th days after each administration of UCAR cells 2 or 25 mg may also be administered. For each individual, MTX is administered at a dose of 31.25 mg / m on the 3rd, 5th, 10th, and 17th days after each administration of UCAR cells 2It may also be administered at a dose of 50 mg. UCAR cells were administered 1 to 5 times from day 0 to day 90. When the MTX concentration in plasma is lower than 2 μM, the dose of UCAR cells is 30×10 6 ~900×10 6 or 0.1×10 6 / kg~50×10 6 / kg may be immobilized. Figure 9 shows an exemplary clinical regimen for allogeneic adoptive cell transfer.

[0323] Example 10. In Vitro Evaluation of the Efficiency of MB12 Armoring 1) Construction of the NextGen UCD20A Expression Vector This example shows the construction of cells expressing MB12, which is an exemplary membrane-bound IL12p40 polypeptide with a structure of IL12p40-CD8α hinge-CD8α transmembrane domain-CD8α intracellular domain. See SEQ ID NO:12. The pLVX-Puro vector purchased from Clontech was digested using ClaI and EcoRI restriction endonucleases, and the CMV promoter was replaced with the human EF1α promoter (GenBank:J04617.1) to obtain the pLVX-hEF1α vector. The fusion gene sequence of SIV Nef_M116-T2A-CD8α SP-CD20 scFv(Leu16)-CD8α hinge-CD8α TM-4-1BB-ITAM010-P2A-DHFR L22F / F31S-P2A-SP-MB12 (hereinafter referred to as "NextGen UCD20A" (SEQ ID NO:13)) was cloned into the expression plasmid pLVX-hEF1α to form the NextGen UCD20A expression plasmid named M1898. The recombinant expression plasmid M1898 was extracted, mixed with the psPAX2 and pMD2.G helper plasmids at a certain ratio, and co-transfected into HEK 293T cells. After incubating for 60 hours, the cell culture supernatant containing the virus was collected and centrifuged at 4°C and 3000 rpm for 5 minutes. The supernatant was filtered through a 0.45 μm filter and then further concentrated using a 500KD hollow fiber membrane column tangential flow technique to produce a lentivirus concentrate, which was stored at -80°C for later use. Similarly, an expression vector secreting IL12p40 was constructed as a control. The fusion gene sequence of SIV Nef_M116-T2A-CD8α SP-CD20 scFv(Leu16)-CD8α hinge-CD8α TM-4-1BB-ITAM045-P2A-DHFR L22F / F31S-P2A-IL12p40 (hereinafter referred to as "NextGen UCD20 M12" (SEQ ID NO:15)) was cloned into the expression plasmid pLVX-hEF1α to form the NextGen UCD20 M12 expression plasmid named M2335. The concentrated lentivirus was produced and stored at -80°C for later use.

[0324] 2) Production of NextGen UCD20A cells 50 mL of fresh peripheral blood was collected from volunteers and mixed with lymphocyte separation medium. Peripheral blood mononuclear cells (PBMCs) were separated by density gradient centrifugation. The cells were labeled with magnetic beads using a Pan T cell separation kit (Miltenyi Biotech), and T lymphocytes were separated, purified, and activated with CD3 / CD28 magnetic beads. After incubation in an incubator at 37 °C and 5% CO2 for 24 hours, 5×10 6 activated T lymphocytes were infected with the lentiviral vector of NextGen UCD20A or NextGen UCD20 M12. The cell suspension was transferred to a 6-well plate and incubated in an incubator at 37 °C and 5% CO2 for use in CAR-T cell proliferation. 17 days after transduction, TCRα / β-negative cells were concentrated and sorted using a TCRα / β selection set to obtain T cells expressing NextGen UCD20A or NextGen UCD20 M12 (hereinafter referred to as NextGen UCD20A cells and NextGen UCD20 M12 cells, respectively).

[0325] Each of UnT (untransduced T cells), NextGen UCD20A cells, and NextGen UCD20 M12 cells was used as a control (unstimulated group) without treatment or stimulated with anti-CD3 / CD28 beads (stimulated group). After incubation for 17 - 24 hours, the cell culture supernatant was collected. The level of IL12p40 was measured using a Human IL-12 / IL-23p40 SimpleStep ELISA(登録商標) kit (Abcam, catalog number ab220656), and IL-23 was tested using a Human IL-23 kit (PerkinElmer, catalog number 62HIL23PEG).

[0326] As shown in Figure 10A, the IL12p40 levels from UnT, NextGen UCD20A cells or NextGen UCD20 M12 cells reached 538 pg / mL, 368 pg / mL, and 7760 pg / mL, respectively, indicating that the baseline level of IL12p40 released by NextGen UCD20 M12 cells was much higher. After stimulation with anti-CD3 / CD28 beads, the IL12p40 levels from UnT, NextGen UCD20A or NextGen UCD20 M12 cells were 28 pg / mL, 1312 pg / mL and 15574 pg / mL, respectively, indicating that the IL12p40 level released by activated NextGen UCD20A cells was significantly lower compared to activated NextGen UCD20 M12 cells. In addition, compared to untreated cells, after CD3 / CD28 stimulation, the IL-23 released by NextGen UCD20 M12 cells increased significantly (p<0.05), and the IL12p40 level released by NextGen UCD20A cells was again significantly lower than the IL12p40 level released by activated NextGen UCD20 M12 cells (p<0.05).

[0327] Use in any method described herein of immune cells (e.g., T cells) and MB12 arming is contemplated.

[0328] Sequence Listing SEQ ID NO:1. DHFR L22F / F31S nucleic acid ATGGTCGGGTCCCTGAATTGTATCGTCGCAGTGTCACAGAATATGGGCATTGGCAAAAACGGAGATTTCCCTTGGCCCCCCCTGAGGAACGAGTCTCGGTACTTCCAGAGAATGACCACAACCAGCTCCGTGGAGGGCAAGCAGAATCTGGTGATCATGGGCAAGAAGACATGGTTTAGCATCCCTGAGAAGAACAGGCCACTGAAGGGCCGCATCAATCTGGTGCTGTCCAGGGAGCTGAAGGAGCCACCTCAGGGAGCACACTTCCTGTCCCGGAGCCTGGACGATGCCCTGAAGCTGACCGAGCAGCCCGAGCTGGCCAACAAGGTGGACATGGTGTGGATCGTGGGCGGCTCTAGCGTGTATAAGGAGGCCATGAATCACCCCGGCCACCTGAAGCTGTTCGTGACACGGATCATGCAGGACTTTGAGTCCGATACCTTCTTTCCTGAGATCGATCTGGAGAAGTACAAGCTGCTGCCAGAGTATCCCGGCGTGCTGAGCGATGTCCAGGAGGAGAAAGGGATTAAATACAAGTTTGAGGTCTATGAGAAGAACGACTGA SEQ ID NO:2. DHFR L22F / F31S amino acid MVGSLNCIVAVSQNMGIGKNGDFPWPPLRNESRYFQRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND SEQ ID NO:3. Wild-type DHFR amino acid MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVWIVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND SEQ ID NO:4. LUCAR-20SD nucleic acid SEQ ID NO:5. SIV Nef M116-IRES-Claudin 18.2 CAR nucleic acid SEQ ID NO:6. LUCAR-BCMA nucleic acid SEQ ID NO:7. TGB23-6-IRES-DHFR L22F / F31S nucleic acid SEQ ID NO:8. ITAM-modified CD20 CAR amino acids MALPVTALLLPLALLLHAARPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGENLYFQSGGDTQALLRNDQVYQPLRDRDDAQYSHLGGNGGSGERPPPVPNPDYEPIRKGQRDLYSGLNQRGGSGDKQTLLPNDQLYQPLKDREDDQYSHLQGNGGSGRKQRITETESPYQELQGQRSDVYSDLNTQGGSG SEQ ID NO:9. ITAM-modified Claudin 18.2 CAR amino acids MALPVTALLLPLALLLHAARPQVQLEESGGGSVQVGGSLRLSCAASGYRSSVCMGWFRQAPGKERERVAVIGRDGSTTYIDSVKGRFTISRDSAKNTLSLQMDNLKPEDTAMYSCAAGLGYWACEYNYWGQGTQVTVSSTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGENLYFQSGGDTQALLRNDQVYQPLRDRDDAQYSHLGGNGGSGERPPPVPNPDYEPIRKGQRDLYSGLNQRGGSGDKQTLLPNDQLYQPLKDREDDQYSHLQGNGGSGRKQRITETESPYQELQGQRSDVYSDLNTQGGSG SEQ ID NO:10. ITAM-modified BCMA CAR amino acids MALPVTALLLPLALLLHAARPQVQLVESGGGLVQPGGSLRLSCAASGRAFSTYFMAWFRQAPGKEREFVAGIAWSGGSTAYADSVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCARRGIEVEEFGAWGQGTMVTVSSGGGGSQVQLEESGGGLVQPGGSLRLSCAYTYSTYSNYYMGWFREAPGKGLTSVAIISSDTTITYKDAVKGRFTISKDNSKNTLYLQMNSLRAEDSAVYRCAAWTSDWSVAYWGQGTLVTVSSTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGENLYFQSGGDTQALLRNDQVYQPLRDRDDAQYSHLGGNGGSGERPPPVPNPDYEPIRKGQRDLYSGLNQRGGSGDKQTLLPNDQLYQPLKDREDDQYSHLQGNGGSGRKQRITETESPYQELQGQRSDVYSDLNTQGGSG SEQ ID NO:11. Amino acids of TGB23-6 MEAAVAAPRPRLLLLVLAAAAAAAAALLPGATALQCFCHLCTKDNFTCVTDGLCFVSVTETTDKVIHNSMCIAEIDLIPRDRPFVCAPSSKTGSVTTTYCCNQDHCNKIELPTTVKSSPGLGPVELVSDWLIFFASLGSFLSILLVGVLGYLGLNRAARHLCPPLPTPCASSAIEFPGGKETWQWINPVDFQEEASLQEALVVEMSWDKGERTEPLEKTELPEGAPELALDTELSLEDGDRCKAKMATNFSLLKQAGDVEENPGPMGRGLLRGLWPLHIVLWTRIASTIPPHVQKSVNNDMIVTDNNGAVKFPQLCKFCDVRFSTCDNQKSCMSNCSITSICEKPQEVCVAVWRKNDENITLETVCHDPKLPYHDFILEDAASPKCIMKEKKKPGETFFMCSCSSDECNDNIIFSEEYNTSNPDLLLVIFQVPQVTSKAFQHDTWNSGLTVASISTGHLTSDNRGDIGLLLGMIVFAVMLSILSLIGIFNRSFRTGIKRRILLLIPKWLYEDIPNMKNSNVVKMLQENSELMNNNSSEQVLYVDPMITEIKEIFIPEHKPTDYKKENTGPLETRDYPQNSLFDNTTVVYIPDLNTGYKPQISNFLPEGSHLSNNNEITSLTLKPPVDSLDSGNNPRLQKHPNFAFSVSSVNSLSNTIFLGELSLILNQGECSSPDIQNSVEEETTMLLENDSPSETIPEQTLLPDEFVSCLGIVNEELPSINTYFPQNILESHFNRISLLEK SEQ ID NO:12. Amino acids of MB12 IWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCLYCNHRNRRR SEQ ID NO:13. NextGen UCD20A nucleic acid SEQ ID NO:14. Human IL12p40 MCHQQLVISWFSLVFLASPLVAIWELKKDVYVVELDWYPDAPGEMVVLTCDTPEEDGITWTLDQSSEVLGSGKTLTIQVKEFGDAGQYTCHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEPKNKTFLRCEAKNYSGRFTCWWLTTISTDLTFSVKSSRGSSDPQGVTCGAATLSAERVRGDNKEYEYSVECQEDSACPAAEESLPIEVMVDAVHKLKYENYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVEVSWEYPDTWSTPHSYFSLTFCVQVQGKSKREKKDRVFTDKTSATVICRKNASISVRAQDRYYSSSWSEWASVPCS SEQ ID NO:15. NextGen UCD20 M12 nucleic acid SEQ ID NO:16. LCAR-UL186S nucleic acid SEQ ID NO:17. SP MDWTWILFLVAAATRVHS SEQ ID NO:18. SIV Nef M116 MGSSNSKRQQQGLLKLWRGLRGKPGADWVLLSDPLIGQSSTVQEECGKALKKSWGKGKMTPDGRRLQEGDTFDEWDDDEEEVGFPVQPRVPLRQMTYKLAVDFSHFLKSKGGLDGIYYSERREKILNLYALNEWGIIDDWQAYSPGPGIRYPRVFGFCFKLVPVDLHEEARNCERHCAAHPAQMGEDPDGIDHGEVLVWKFDPKLAVEYRPDMFKDMHEHAKR SEQ ID NO:19. ITAM010 amino acid GENLYFQSGGDTQALLRNDQVYQPLRDRDDAQYSHLGGNGGSGERPPPVPNPDYEPIRKGQRDLYSGLNQRGGSGDKQTLLPNDQLYQPLKDREDDQYSHLQGNGGSGRKQRITETESPYQELQGQRSDVYSDLNTQGGSG SEQ ID NO:20. IRES GCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACA

Claims

1. A method for promoting the persistence of cell therapy in a human individual, comprising administering to the individual a) a lymphocyte-depleting agent before the cell therapy, and b) an S-phase inhibitor, wherein the S-phase inhibitor is administered more than once, the cell therapy comprises immune cells that are resistant to the S-phase inhibitor, and the lymphocyte-depleting agent is different from the S-phase inhibitor.

2. The method according to claim 1, comprising administering the S-phase inhibitor before the cell therapy.

3. The method according to claim 2, further comprising administering the S-phase inhibitor after the cell therapy.

4. The method according to any one of claims 1 to 3, wherein the S-phase inhibitor is an antimetabolite of folic acid.

5. The method according to claim 4, wherein the antimetabolite of folic acid is a DHFR inhibitor.

6. The method according to claim 5, wherein the antimetabolite of folic acid is methotrexate.

7. The method according to any one of claims 1 to 6, comprising administering the S-phase inhibitor within about 5 days before administering the immune cells.

8. Comprising administering the S-phase inhibitor within about 10 days after administering the immune cells, optionally comprising administering the S-phase inhibitor both within about 5 days before administering the immune cells and within about 10 days after administering the immune cells. The method according to any one of claims 1 to 7.

9. Comprising administering the S-phase inhibitor and the lymphocyte-depleting agent on the same day, optionally administering the S-phase inhibitor and the lymphocyte-depleting agent in parallel or simultaneously, optionally comprising administering the S-phase inhibitor and the lymphocyte-depleting agent in parallel or simultaneously within 5 days before immunotherapy. The method according to any one of claims 1 to 8.

10. The method according to any one of claims 1 to 9, comprising administering the S-phase inhibitor once every 1 to 28 days during the period from about 5 days before administering the immune cells to about 90 days after administering the immune cells.

11. The method according to claim 10, comprising administering the S-phase inhibitor once every 1 to 10 days during the period from about 5 days before administering the immune cells to about 90 days after administering the immune cells.

12. Administering methotrexate once every 3 to 7 days or once every 2 to 7 days over at least 3 cycles. Optionally, methotrexate is administered once every 3 to 7 days or once every 2 to 7 days during the period from about 5 days before administration of the immune cells to about 90 days after administration of the immune cells. The method according to any one of claims 6 to 11, wherein methotrexate is administered on the 3rd, 5th, 10th, and 17th days after each administration of the immune cells.

13. About 3 mg / m ２ to about 3000 mg / m ２ in an amount of or about 3 mg / m ２ / day to about 3000 mg / m ２ The method according to any one of claims 6 to 12, wherein methotrexate is administered at a dosage of / day.

14. About 5 mg / m ２ to about 3000 mg / m ２ in an amount of or about 5 mg / m ２ / day to about 3000 mg / m ２ The method according to any one of claims 6 to 13, wherein methotrexate is administered at a dose of / day.

15. The method according to any one of claims 6 to 14, wherein the plasma concentration of methotrexate in the individual is about 2 μM or less within about 1 to 3 hours after administration of methotrexate.

16. The method according to any one of claims 6 to 15, wherein the plasma concentration of methotrexate in the individual exceeds about 0.001 μM, exceeds about 0.01 μM, or exceeds about 0.1 μM within about 1 to 3 hours after administration of methotrexate.

17. The method according to any one of claims 1 to 16, wherein the S-phase inhibitor is administered orally, subcutaneously, intramuscularly, intravenously, intraarterially, or intrathecally each time.

18. The method according to any one of claims 1 to 17, wherein the lymphocyte-depleting agent comprises fludarabine and cyclophosphamide.

19. About 25 mg / m ２ to about 30 mg / m ２ administer fludarabine at a dose of and about 250 mg / m ２ to about 1000 mg / m ２ administer cyclophosphamide at a dose of, the method according to claim 18.

20. The method according to claim 18 or 19, wherein fludarabine and cyclophosphamide are administered about 1 to 3 times during the period from about 10 days before administration of the immune cells to about 90 days after administration of the immune cells.

21. Fludarabine and cyclophosphamide are administered continuously for about 3 to 4 days before administration of the immune cells, and optionally, a) Fludarabine and cyclophosphamide are administered continuously for 3 days. b) Fludarabine and cyclophosphamide are administered continuously for 4 days, or c) Fludarabine and cyclophosphamide are administered continuously for 3 days, and then fludarabine is administered alone for 1 day. The method according to any one of claims 18 to 20.

22. The method according to any one of claims 1 to 21, wherein the immune cells are allogeneic.

23. The method according to any one of claims 1 to 22, wherein the immune cells comprise T cells.

24. The method according to claim 23, wherein the T cells comprise an exogenous Nef protein.

25. The method according to claim 23 or 24, wherein the T cells are engineered not to express or to express at a reduced level endogenous TCRα or TCRβ.

26. The method according to claim 23, wherein the T cell is a γ-δ T cell. **Claim 27** The method according to any one of claims 1 to 22, wherein the immune cell comprises an NK cell. **Claim 28** The method according to any one of claims 1 to 27, wherein the immune cell comprises a heterologous nucleic acid sequence encoding an engineered receptor. **Claim 29** The method according to claim 28, wherein the engineered receptor is selected from the group consisting of a chimeric antigen receptor (CAR), an engineered T cell receptor (TCR), and a T cell antigen conjugate (TAC) receptor. **Claim 30** The method according to claim 28 or 29, wherein the engineered receptor targets an antigen selected from the group consisting of CD19, BCMA, Claudin 18.2, NY-ESO-1, VEGFR2, MAGE-A3, CD20, CD22, CD33, CD38, CEA, EGFR, GD2, HER2, IGF1R, mesothelin, PSMA, ROR1, GPC3, DLL3, GPRC5D, CLL1, WT1, CD4, GU2CYC, MUC16, MUC1, CAIX, CD8, CD7, CD10, CD30, CD34, CD41, CD44, CD49f, CD56, CD74, CD133, CD138, EGP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD3, hTERT, IL-13R-α2, κ-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, p53, MART1, GP100, protease-3 (PR3), tyrosinase, survivin, EphA2, h5T4, PSCA, TAG-72, CD123, CD44V6, NKCS1, EGF1R, EGFR-VIII, Claudin 6, NKG2D, CD70, ADGRE2, FcRH5, NKp80, NKp30, NKG2A, CD229, CS-1, and combinations thereof. **Claim 31** The operated receptor is an anti-BCMA CAR, anti-CD20 CAR, anti-claudin 18.2 CAR or anti-CLL1 CAR, and optionally, the CAR has a targeting domain comprising V Ｈ H, the method according to claim 30. **Claim 32** The method according to any one of claims 28 to 31, wherein the immune cell further comprises a second engineered receptor that converts a negative signal into a positive signal, and optionally, the second engineered receptor comprises an extracellular domain of TGFβR and an intracellular domain of IL-23 receptor. **Claim 33** The method according to any one of claims 28 to 31, wherein the immune cells further comprise an IL12p40 polypeptide, and optionally, the IL12p40 polypeptide is membrane-bound.

34. The method according to claim 33, wherein the immune cells comprise membrane-bound IL12p40, and the membrane-bound IL12p40 comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least about 90% sequence identity with SEQ ID NO:

12.

35. The method according to any one of claims 1 to 34, wherein the immune cells comprise a methotrexate resistance transgene.

36. The method according to claim 35, wherein the methotrexate resistance transgene comprises a mutant DHFR gene, and optionally, the mutant DHFR gene comprises an L22F mutation and an F31S mutation.

37. The method according to any one of claims 1 to 34, wherein the immune cells overexpress a DHFR gene encoding dihydrofolate reductase.

38. The method according to any one of claims 1 to 37, wherein the immune cells are administered more than once.

39. The method according to any one of claims 1 to 38, wherein the immune cells comprise from about 30 million to about 900 million immune cells, or from about 100,000 to about 50 million immune cells per kilogram of the human individual.

40. The method according to claim 38 or 39, wherein the immune cells are administered about 2 to 5 times within about 90 days after the first administration of the immune cells.

41. The method according to any one of claims 1 to 40, wherein the individual has cancer.

42. The method according to claim 41, wherein the cancer is a blood cancer.

43. The method according to claim 41, wherein the cancer is a solid tumor.

44. During the period from about 2 to 7 days before administration of the immune cells to about 10, 20, 30, 40, 50, 60, 70, 80, or 90 days after administration of the immune cells, the host T cells measured by the number of host T cells in PBMC are about 500, 400, 300, 200, 100, or 50 cells or less per μL. The method according to any one of claims 1 to 43.

45. The method according to any one of claims 1 to 44, further comprising monitoring the number of WBC (white blood cells) in the individual.

46. A method for treating a disease or medical condition of a human individual, the method comprising the method according to any one of claims 1 to 45.

47. A nucleic acid comprising a nucleic acid sequence shown in any one of SEQ ID NOs: 4 to 7, 13 and 15.

48. A vector comprising the nucleic acid according to claim 47.

49. An engineered cell comprising the engineered receptor according to any one of claims 1 to 34, the nucleic acid according to claim 47 and / or the vector according to claim 48.

50. An immune cell or a population thereof for use in the treatment of cancer, wherein the immune cell is administered in combination with a lymphodepleting agent and an S-phase inhibitor.

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