Methods of promoting persistence of cell therapy

Abstract

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

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the priority benefit of international patent application No. PCT / CN2022 / 109129 filed on July 29, 2022, and the contents of that international patent application are incorporated herein by reference in their entirety.

[0003] Sequence listings are submitted as ASCII text files

[0004] The following content submitted as an ASCII text file is incorporated herein by reference in its entirety: Sequence Listing in Computer Readable Form (CRF) (File Name: IEC230075PCT-seql.xml, Record Date: July 26, 2023, Size: 50KB). Technical Field

[0006] The present application generally relates to the combined use of lymphodepleting agents and S-phase inhibitors (eg, methotrexate) to promote persistence of cellular therapies in patients. Background Art

[0008] Cell therapy, such as adoptive cell therapy or adoptive cell transfer (ACT), is becoming an increasingly important treatment paradigm for a variety of diseases, such as in the treatment of cancer. Cell therapy involves transferring cells, most typically immune cells, into a patient. These cells can 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 trigger an immune response against the cancer. Although T cells are most commonly used in cell therapy, other immune cell types, such as NK cells, lymphocytes (e.g., tumor infiltrating lymphocytes or TILs), dendritic cells, and myeloid cells have also been used.

[0009] Ideally, immune cells infused (or re-infused in the case of autologous therapy) into a patient receiving cell therapy would expand and persist in that patient. However, the immune system has developed sophisticated and effective mechanisms to respond to foreign agents. The patient's immune response is the biggest obstacle to the success of cell therapy, especially when the immune cells come from other individuals. There is still a need for effective strategies to reduce the unwanted immune responses associated with cell therapy.

[0010] 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

[0012] The present application provides methods for promoting the persistence of cell therapy in individuals receiving cell therapy (e.g., adoptive cell therapy, such as allogeneic CAR-T cells) by administering a lymphodepleting agent and an S phase inhibitor (e.g., methotrexate).

[0013] One aspect of the present application provides 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, the optional lymphodepleting agent is administered prior to administering the cell therapy, and b) an S phase inhibitor, wherein the S phase inhibitor is administered more than once, wherein the cell therapy comprises immune cells resistant to the S phase inhibitor, and wherein the lymphodepleting agent is different from the S phase inhibitor. In some embodiments, the method comprises administering an S phase inhibitor prior to administering the cell therapy. In some embodiments, the method further comprises administering an S phase inhibitor after administering the cell therapy.

[0014] In some embodiments according to any of the methods above, the S phase inhibitor is an antifolate. In some embodiments, the antifolate is a DHFR inhibitor.

[0015] In some embodiments according to any of the methods above, the method comprises administering the S phase inhibitor within about 5 days prior to administering the immune cells.

[0016] In some embodiments according to any of the methods above, the method comprises administering the S phase inhibitor within about 10 days after administering the immune cells, optionally wherein the method comprises administering the S phase inhibitor within about 5 days before administering the immune cells and within about 10 days after administering the immune cells.

[0017] In some embodiments according to any of the methods above, the method comprises administering the S phase inhibitor and the lymphodepleting agent on the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour), optionally wherein the S phase inhibitor and the lymphodepleting agent are administered concurrently or simultaneously, optionally wherein the method comprises administering the S phase inhibitor concurrently or simultaneously with the lymphodepleting agent within 5 days prior to the immunotherapy.

[0018] In some embodiments according to any of the above methods, the method includes administering an S phase inhibitor once every 1-28 days during a period of about 5 days before administering the immune cells to about 90 days after administering the immune cells. In some embodiments, the method includes administering an S phase inhibitor once every 1-10 days during a period of about 5 days before administering the immune cells to about 90 days after administering the immune cells.

[0019] In some embodiments according to any of the above methods, the S phase inhibitor is methotrexate (MTX). In some embodiments, methotrexate is administered once every 3-7 days or every 2-7 days for at least three cycles, optionally wherein methotrexate is administered once every 3-7 days or every 2-7 days during a period of about 5 days before administration of the immune cells to about 90 days after administration of 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, at about 5 mg / m 2 About 3000mg / m 2 In some embodiments, methotrexate is administered in an amount of about 5 mg / m 2 / day to about 3000mg / m 2 / day dose of methotrexate. In some embodiments, at about 3 mg / m 2 About 3000mg / m 2 In some embodiments, methotrexate is administered in an amount of about 3 mg / m 2 / day to about 3000mg / m 2 In some embodiments, the methotrexate is administered at a dose of 100 mg / d. In some embodiments, the plasma concentration of methotrexate in the individual does not exceed about 2 μM within about 1-3 hours after administration of methotrexate. In some embodiments, the plasma concentration of methotrexate in the individual exceeds about 0.001 μM within about 1-3 hours after administration of methotrexate. In some embodiments, the plasma concentration of methotrexate in the individual exceeds about 0.01 μM within about 1-3 hours after administration of methotrexate. In some embodiments, the plasma concentration of methotrexate in the individual exceeds about 0.1 μM within about 1-3 hours after administration of methotrexate.

[0020] In some embodiments according to any of the methods above, the S phase inhibitor is administered each time orally, subcutaneously, intramuscularly, intravenously, intraarterially, or intrathecally.

[0021] In some embodiments according to any of the above methods, the lymphodepleting agent comprises fludarabine ("Flu") and cyclophosphamide ("Cy"). In some embodiments, the lymphodepleting agent is administered at about 25 mg / m 2 About 30 mg / m 2 Fludarabine was administered at a dose of approximately 250 mg / m 2 About 1000 mg / m 2Cyclophosphamide is administered at a dose of 100 mg / kg. In some embodiments, fludarabine and cyclophosphamide are administered about 1-3 times during a period of about 10 days before the administration of the immune cells to about 90 days after the administration of the immune cells. In some embodiments, fludarabine and cyclophosphamide are administered for about 3-4 consecutive days before the administration of the immune cells, optionally wherein: a) fludarabine and cyclophosphamide are administered for 3 consecutive days, b) fludarabine and cyclophosphamide are administered for 4 consecutive days, or c) fludarabine and cyclophosphamide are administered for 3 consecutive days, and then fludarabine is administered alone for 1 day.

[0022] In some embodiments according to any of the methods above, the immune cells are allogeneic.

[0023] In some embodiments according to any of the above methods, the immune cell comprises a T cell. In some embodiments, the T cell comprises an exogenous Nef protein. In some embodiments, the T cell is engineered to not express or express reduced levels of endogenous TCRα or TCRβ. In some embodiments, the T cell is a γ-δ T cell.

[0024] In some embodiments according to any of the methods above, the immune cells comprise NK cells.

[0025] In some embodiments according to any of the above methods, the immune cell comprises 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, kappa-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, p53, MART1, GP100, proteinase-3 (PR3), tyrosinase, survival protein, 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 anti-BCMA CAR, anti-CD20 CAR, anti-claudin 18.2 CAR or anti-CLL1 CAR. In some embodiments, the immune cell further comprises a second engineered receptor that converts a negative signal into a positive signal, optionally wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. In some embodiments, the immune cell comprises an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound.

[0026] In some embodiments according to any of the above methods, the immune cells comprise a methotrexate resistance transgene. In some embodiments, the methotrexate resistance transgene comprises a mutant DHFR gene. In some embodiments, the mutant DHFR gene comprises an L22F mutation and a F31S mutation.

[0027] In some embodiments according to any of the methods above, the immune cells overexpress the DHFR gene encoding dihydrofolate reductase.

[0028] In some embodiments according to any of the methods above, the immune cells are administered more than once (eg, twice, three times, four times, five times).

[0029] In some embodiments according to any of the methods above, the immune cells comprise about 30 million to about 900 million immune cells, or about 100,000 to about 50 million immune cells per kilogram of a human subject.

[0030] In some embodiments according to any of the methods above, the immune cells are administered about 2-5 times within about 90 days after the first administration of the immune cells.

[0031] In some embodiments according to any of the methods above, the individual has cancer. In some embodiments, the cancer is a blood cancer. In some embodiments, the cancer is a solid tumor.

[0032] In some embodiments according to any of the methods above, the host T cells measured as the number of host T cells in PBMCs do not exceed about 500, 400, 300, 200, 100, or 50 cells / μL during a time period between about 2-7 days prior to administration of the immune cells and about 10, 20, 30, 40, 50, 60, 70, 80, or 90 days after administration of the immune cells.

[0033] In some embodiments according to any of the methods above, the host leukocytes do not exceed about 3000, 2000, 1500, 1000, 900, 800, 700, 600, 500, 400, 300, 200, or 100 cells / μL during a time period between about 2-7 days prior to administration of the immune cells and about 10, 20, 30, 40, 50, 60, 70, 80, or 90 days after administration of the immune cells.

[0034] In some embodiments according to any of the methods above, the method further comprises monitoring the number of host WBCs (white blood cells) or host T cells in the individual.

[0035] In another aspect, the present application provides a method of treating a disease or disorder (eg, cancer) in a human individual, the method comprising any of the above methods for promoting persistence of cell therapy in the human individual.

[0036] On the other hand, the application provides immune cells or their colonies for use in treating cancer, wherein these immune cells are administered in combination with lymphodepleting agents and S phase inhibitors (e.g., methotrexate). In some embodiments, the S phase inhibitor is administered before administering the immune cells. In some embodiments, the S phase inhibitor is administered after administering the immune cells. In some embodiments, the immune cells comprise a methotrexate-resistant transgene.

[0037] Also provided are compositions, uses, kits, and articles of manufacture comprising any of the S phase inhibitors (eg, methotrexate), any of the lymphodepleting agents (eg, Flu and Cy), and / or the immune cells described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1A-1B The effect of different methotrexate (MTX) concentrations on the proliferation of UCAR-T cells in vitro is shown. LUCAR-20SD cells and CAR-γδT cells were prepared and cultured at MTX concentrations of 0μM, 0.01μM, 0.05μM, 0.1μM, 1μM, and 2μM. The LUCAR-20SD cells ( Figure 1A ) and CAR-γδT cells ( Figure 1B )'s proliferation curve.

[0039] Figure 2A-2D The effect of MTX and LUCAR-20SD cell combination on the allogeneic T cell in vitro mixed lymphocyte (MLR) model was shown. 6 Untransduced T cells (UnT, as host cells, T cell donor A), 1×10 6 LUCAR-20SD cells (as transplant cells, T cell donor B) and 5×10 6 Raji lymphoma cells were cultured together. AOPI counting and FACS detection were performed to calculate the Figure 2A )、0.05μM( Figure 2B )、1μM( Figure 2C ) or 2μM ( Figure 2D ) of MTX concentrations.

[0040] Figure 3A-3C The effect of MTX and CAR-γδT combination on the in vitro MLR system of allogeneic γδT cells was shown. 6 Untransduced T cells (UnT, as host cells, T cell donor A), 1×10 6 CAR-γδT (as transplant cells, T cell donor B) and 5×10 6 The number of lymphoma cells U937 was cultured together. AOPI counting and FACS detection were used to calculate the Figure 3A )、0.05μM( Figure 3B ) or 0.5μM ( Figure 3C ) Proliferation curves of UnT and CAR-γδT cells under different MTX concentrations.

[0041] Figure 4A-4CThe in vivo efficacy evaluation of the combination regimen of MTX and LUCAR-GC cells in a subcutaneous xenograft model of gastric cancer is shown. Tumor-bearing mice received a single intravenous injection of HBSS (G1, G5), untransduced T cells (UnT) (G2, at 3.6×10 6 cells / mouse) or LUCAR-GC cells (G3, G4, at 1×10 6 Mice in the G4 and G5 groups were given 22.635 mg / kg MTX by intraperitoneal injection on days 5, 10, 15, 20, 25, 30, and 35, respectively. Figure 4A Treatment of mice in G4 is shown. The tumor volume of mice was measured and counted ( Figure 4B ) and proliferation of transplanted cells in mouse peripheral blood ( Figure 4C ).

[0042] Figure 5A-Figure 5B The effect of the FC+MTX combination regimen is shown. 6 PBMC cells (HLA-A2+) were re-infused to establish the host immune system; on day 0, 3×10 6 LUCAR-BCMA cells (HLA-A2-) were re-infused as transplant cells. The mice in the G1 group were HBSS control groups, the mice in the G2 group were treated with fludarabine ("Flu") and cyclophosphamide ("Cy") (the combination was called "FC"), the mice in the G3 group were treated with 15.15 mg / kg MTX, and the mice in the G4 group were treated with fludarabine, cyclophosphamide and 15.15 mg / kg MTX. G5 mice were treated with 75.45 mg / kg MTX, and G6 mice were treated with fludarabine, cyclophosphamide and 75.45 mg / kg MTX. The total number of host cells (HLA-A2+) in the peripheral blood of mice on day 13 was counted, and the effects of different drug combinations on the proliferation capacity of host cells were evaluated ( Figure 5B The drug administration schedule for G4 and G6 is also shown in Figure 5A The combination of fludarabine and cyclophosphamide is abbreviated as "FC". Figure 5B The “ / ” in means that the drug was not administered.

[0043] Figure 6A-6B The effect of MTX dosing interval in the combination of fludarabine, cyclophosphamide, and MTX using an in vivo evaluation model is shown. C57BL / 6 mice in both G1 and G2 groups received a single intravenous injection of LUCAR-20SD cells (5×10 6The mice in the G2 group were pretreated with fludarabine and cyclophosphamide on days -5, -4, and -3, and with MTX treatment on days -3, 1, 6, and 11. Fig. 6A During the experiment, blood was collected regularly to count the number of white blood cells (WBC) ( Figure 6B ).

[0044] Figure 7A-7C The effect of MTX dose in the combination of fludarabine, cyclophosphamide and MTX using an in vivo evaluation model is shown. C57BL / 6 mice in the G1, G2 and G3 groups received a single intravenous injection of LUCAR-20SD cells (5×10 6 The mice in the G2 and G3 groups were treated with fludarabine and cyclophosphamide on days -5, -4, and -3, and each group was administered with different doses of MTX on days -3, 1, 6, and 11, respectively. Fig. 7A During the experiment, blood was collected regularly to count the number of white blood cells (WBC) in each group and compared with the control G1 group ( Figure 7B-7C ).

[0045] Figure 8A-B The results showed that the FC+MTX combination regimen has a protective effect on allogeneic transplanted cells. Fig. 8A In vivo evaluation models are shown. Transduced MPC-11 cells were derived from the mouse strain BALB / c and expressed DHFR L22F / F31S. Transduced MPC-11 cells were transplanted into the BALB / c strain as an autologous model (Group G1) or into C57BL / 6 mice as an allogeneic model (Groups G2, G3, and G4). Group G3 was treated with fludarabine and cyclophosphamide. Group G4 was treated with fludarabine, cyclophosphamide, and MTX, and the dosing regimen was shown in Fig. 8A middle. Figure 8B The tumor volume of each group is shown, indicating the cytoprotective effect of the fludarabine, cyclophosphamide and MTX combination regimen on xenograft cells.

[0046] Fig. 9 An exemplary clinical study design showing the protective effect of the FC+MTX combination regimen on allogeneic adoptive cells ("UCAR") is shown.

[0047] Figure 10A-10BIn vitro evaluation of the efficiency of MB12 armoring is shown. Cells armored with NextGen UCD20A or NextGen UCD20 M12 were prepared and either untreated cells were used as controls (unstimulated groups) or cells were stimulated with anti-CD3 / CD28 beads (stimulated groups). After 17-24 hours of incubation, cell culture supernatants were collected for determination of IL12p40 ( Fig. 10A ) and IL-23( Fig. 10B ) release. ** means p<0.05. DETAILED DESCRIPTION

[0048] The present application provides a method for promoting the persistence of cell therapy in patients by coordinating the administration of lymphoclearing agents (e.g., fludarabine and / or cyclophosphamide) and S phase inhibitors (e.g., antifolates, such as methotrexate), so as to achieve the effect of maintaining the number of host immune cells (e.g., T cells) at a threshold level for a long period of time (e.g., at least 30 days, 60 days, or 90 days after the administration of cell therapy). Cell therapy can include immune cells resistant to S phase inhibitors. S phase inhibitors (e.g., methotrexate) can be administered more than once. In certain embodiments, the present application provides a method for promoting the persistence of cell therapy by administering lymphoclearing agents before administering cell therapy and administering S phase inhibitors (e.g., methotrexate) before and after administering cell therapy. In certain embodiments, the present application provides a method for promoting the persistence of cell therapy by administering lymphoclearing agents and S phase inhibitors (e.g., methotrexate) within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour) (e.g., in parallel or simultaneously). In another aspect, the present application provides clinical strategies for using methotrexate in combination with CAR-T cell therapy (e.g., allogeneic CAR-T cell therapy), wherein the CAR-T cells are resistant to methotrexate.

[0049] The claimed method is based at least in part on the following discovery of the inventors: the use of an S phase inhibitor (e.g., methotrexate) targeting cells under robust proliferation in combination with a lymphodepleting agent effectively inhibits the proliferation of host immune cells (e.g., T cells) in individuals receiving cell therapy, and this inhibitory effect lasts for a long period of time. It is also demonstrated that the combined use achieves the desired effect of promoting the persistence / effectiveness of cell therapy.

[0050] Thus, in some embodiments, a method of promoting persistence of a cell therapy in a human individual is provided, the method comprising administering to the individual a) a lymphodepleting agent, the lymphodepleting agent being administered prior to administering the cell therapy, and b) an S phase inhibitor, wherein the S phase inhibitor is administered more than once, wherein the cell therapy comprises immune cells resistant to the S phase inhibitor, and wherein the lymphodepleting agent is different from the S phase inhibitor. The S phase inhibitor may be administered before and after administering the cell therapy. The S phase inhibitor may be an antifolate. The antifolate may be methotrexate.

[0051] I. Definitions

[0052] As used herein, "treatment (treatment or treating)" is a method for obtaining beneficial or desired results (including clinical results). For the purpose of the present application, beneficial or desired clinical results include but are not limited to one or more of the following: alleviating one or more symptoms caused by the disease, weakening the degree of the disease, stabilizing the disease (for example, preventing or delaying the deterioration of the disease), preventing or delaying the spread of the disease (for example, metastasis), preventing or delaying the recurrence of the disease, delaying or slowing down the progress of the disease, improving the disease state, providing the alleviation of the disease (partial or complete), reducing the dosage of one or more other drugs required for the treatment of the disease, delaying the progress of the disease, improving the quality of life, and / or prolonging the survival period." treatment " also encompasses the pathological consequences of reducing the disease. The method of the present application considers any one or more of these treatment aspects.

[0053] The term "prevent" and similar words such as "prevented" and "preventing" refer to methods for preventing, inhibiting or reducing the likelihood of a disease or condition (e.g., cancer) recurring. It also refers to delaying the recurrence of a disease or condition or delaying the recurrence of symptoms of a disease or condition. As used herein, "prevention" and similar words also include reducing the intensity, effects, symptoms and / or burden of a disease or condition before the disease or condition recurs.

[0054] As used herein, the development of "delayed" disease means to postpone, hinder, slow down, slow down, stabilize and / or delay the development of disease. This delay can have different time lengths, which depends on the history of the disease and / or the individual being treated. Compared with not using the method, the method of "delayed" disease development is to reduce the possibility of disease development within a given time frame and / or reduce the method of disease degree within a given time frame. Such a comparison is typically based on clinical studies conducted using statistically significant numbers of individuals. Standard methods can be used to detect disease development, and these standard methods include but are not limited to computerized axial tomography (CAT scan), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation test, arteriography or biopsy. Development can also refer to disease progression that may not be detected initially, and includes occurrence, recurrence and onset.

[0055] The term "effective amount" as used herein refers to an amount of a medicament sufficient to treat a particular disorder, condition or disease (e.g., to improve, alleviate, reduce and / or delay one or more symptoms thereof). When referring to cancer, an effective amount includes an amount sufficient to shrink a tumor and / or reduce the growth rate of the tumor (e.g., inhibit tumor growth) or prevent or delay other undesirable cell proliferation. An effective amount may be an amount sufficient to delay development. An effective amount may be an amount sufficient to prevent or delay recurrence. An effective amount may be administered in one or more administrations. An effective amount of a drug or composition may: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, slow, slow down and preferably prevent cancer cells from infiltrating peripheral organs to a certain extent; (iv) inhibit (i.e., slow down and preferably prevent) tumor metastasis to a certain extent; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of a tumor; and / or (vii) alleviate one or more symptoms associated with cancer to a certain extent.

[0056] 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 can be a human.

[0057] As used herein, "in conjunction with" or "in combination with" means administering a therapeutic modality in addition to another therapeutic modality. Thus, "in conjunction with" or "in combination with" means administering another therapeutic modality before, during, or after administering one therapeutic modality to an individual.

[0058] As used herein, "chimeric antigen receptor" or "CAR" refers to a genetically engineered receptor that can be used to transplant one or more antigen-specifics to 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". CAR may include an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain of T cells and / or other receptors that are specific for one or more antigens (eg, tumor antigens). "CAR-T" refers to a T cell expressing CAR.

[0059] As used herein, the term "simultaneous administration" means that the first therapy and the second therapy in the combination therapy are administered with a time interval of no more than about 15 minutes (e.g., no more than about any of 10, 5, or 1 minute). When the first therapy and the second therapy are administered simultaneously, the first therapy and the second therapy can be included in the same composition (e.g., a composition comprising the first therapy and the second therapy at the same time) or in separate compositions (e.g., the first therapy is included in one composition and the second therapy is included in another composition).

[0060] As used herein, the term "sequential administration" means that the first therapy and the second therapy in the combination therapy are administered at a time interval of more than about 15 minutes (e.g., more than about any of 20, 30, 40, 50, 60 or more minutes). The first therapy or the second therapy can be administered first. The first therapy and the second therapy are contained in separate compositions, which can be contained in the same or different packages or kits.

[0061] As used herein, the term "concurrent administration" means that the administration of a first therapy and the administration of a second therapy in the combination therapy overlap with each other.

[0062] As used herein, the term "binding", "specific binding" or "specific for..." refers to a measurable and reproducible interaction, such as the binding between a molecule (e.g., CAR) and a target, which determines the presence of a target in the presence of a heterogeneous population of molecules (including biomolecules). For example, a CAR that binds or specifically binds to a target (which may be an epitope) is a CAR that binds to this target, and its affinity, avidity, readiness and / or duration are superior to binding to other targets. In one embodiment, the degree of binding of CAR to an unrelated target is less than about 10% of the binding of the CAR to the target, as measured, for example, by radioimmunoassay (RIA). In certain embodiments, the CAR that specifically binds to the target has a dissociation constant (Kd) of ≤1 μM, ≤100nM, ≤10nM, ≤1nM, or ≤0.1nM. In certain embodiments, CAR specifically binds to a conserved protein epitope in proteins from different species. In another embodiment, specific binding may include but does not require exclusive binding.

[0063] "UCART" or "universal CAR-T cells" refers to off-the-shelf CAR-modified T cells that can be used to treat allogeneic patients in need. UCARTs include those that contain genetic modifications in addition to the CAR construct, and those that do not contain genetic modifications in addition to the CAR construct.

[0064] As used herein, "T cell receptor" or "TCR" refers to an endogenous or recombinant T cell receptor comprising an extracellular antigen binding domain that binds to a specific antigen peptide bound in an MHC molecule. The TCR may comprise a TCR alpha polypeptide chain and a TCR beta polypeptide chain. The TCR may specifically bind to a tumor antigen. "TCR-T" refers to a T cell expressing a recombinant TCR.

[0065] As used herein, "T cell antigen conjugate receptor" or "TAC receptor" refers to an engineered receptor that comprises an extracellular antigen binding domain that binds to a specific antigen and a T cell receptor (TCR) binding domain, a transmembrane domain, and an intracellular domain of a co-receptor molecule. TAC receptors specify the endogenous TCR of T cells expressing TAC receptors to elicit antigen-specific T cell responses against target cells.

[0066] As used herein, "TCR fusion protein" or "TFP" refers to an engineered receptor comprising an extracellular antigen binding domain that binds to a specific antigen that is fused to a subunit or portion thereof of a TCR complex (including a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, CD3 epsilon, CD3 delta, or CD3 gamma). A subunit or portion thereof of a TCR complex comprises a transmembrane domain and at least a portion of an intracellular domain of a naturally occurring TCR subunit. TFP may comprise an extracellular domain or portion thereof of a TCR subunit. TFP may not comprise an extracellular domain of a TCR subunit.

[0067] "Percentage (%) of amino acid sequence identity" and "homology" with respect to peptide, polypeptide or antibody sequences are defined as the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues in a particular peptide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity and not considering any conservative substitutions as part of the sequence identity. For the purpose of determining the percentage of amino acid sequence identity, alignment can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or MEGALIGN TM(DNASTAR) software. One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. For example, polypeptides having at least 70%, 85%, 90%, 95%, 98% or 99% identity to a particular polypeptide described herein and preferably exhibiting substantially the same function, and polynucleotides encoding such polypeptides are contemplated.

[0068] When a nucleic acid is in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably linked". For example, if the DNA of a presequence or secretory leader sequence is expressed as a preprotein that participates in the secretion of a polypeptide, the DNA of the presequence or secretory leader sequence is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the sequence; or if a ribosome binding site is positioned to facilitate translation, the ribosome binding site is operably linked to a coding sequence. Generally, "operably linked" means that the DNA sequence being linked is continuous, and in the case of a secretory leader sequence, continuous and in the reading frame. However, enhancers do not have to be continuous. Linking is accomplished by ligation at a convenient restriction site. If such a site does not exist, a synthetic oligonucleotide adapter or joint is used according to conventional practice.

[0069] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0070] As used herein, the term "transfected" or "transformed" or "transduced" refers to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary host cell and its progeny.

[0071] As used herein, the expressions "cell", "cells", "cell line" and "cell culture" are used interchangeably, and all such references include progeny. Thus, the terms "transfectants" and "transfected cells" include the primary host cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that the DNA content of all progeny may not be exactly the same due to intentional or unintentional mutations. Variant progeny are included that have the same function or biological activity as screened in the originally transformed cell.

[0072] "Primary cells" refer to cells that are taken directly from living tissue (i.e., biopsy material) and established for in vitro growth, which undergo infrequent population doublings compared to continuously tumorigenic cell lines or artificially immortalized cell lines and are therefore more representative of the major functional components and characteristics of the tissue from which they are derived.

[0073] The term "in vivo" means within the body of the organism from which the cell was obtained. "Ex vivo" or "in vitro" means outside the body of the organism from which the cell was obtained.

[0074] As used herein, the term "autologous" refers to any material derived from the same individual, wherein the material is subsequently reintroduced into the individual.

[0075] "Allogeneic" refers to a transplant that originates from a different individual of the same species.

[0076] The term "recombinant" refers to a biological molecule, such as a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or part of a polynucleotide with which the gene is found in nature, (3) is operably linked to a polynucleotide to which it is not linked in nature, or (4) does not occur in nature. The term "recombinant" may be used to refer to cloned DNA isolates, chemically synthesized polynucleotide analogs or polynucleotide analogs biologically synthesized by heterologous systems, and proteins and / or mRNA encoded by such nucleic acids.

[0077] The term "expression" refers to transcribing DNA into RNA (eg, mRNA) or translating nucleic acid into protein. Proteins may be expressed and retained intracellularly, become components of the cell surface membrane, or be secreted into the extracellular matrix or medium.

[0078] As used herein, "pharmaceutically acceptable" or "pharmacologically compatible" means a material that is not biologically or otherwise undesirable, for example, the material can be incorporated into a pharmaceutical composition administered to a patient without causing any significant undesirable biological effect or interacting in a deleterious manner with any other component of the composition containing it. Pharmaceutically acceptable carriers or excipients preferably meet the required standards of toxicology and manufacturing testing and / or are included in the Inactive Ingredient Guide compiled by the US Food and Drug Administration.

[0079] It should be understood that the embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" the embodiments.

[0080] References herein to "about" a value or parameter include (and describe) variations with respect to that value or parameter itself. For example, description referring to "about X" includes description of "X".

[0081] As used herein, reference to a value or parameter that is "not" generally means and describes a value or parameter that is "different from." For example, a method is not for treating cancer type X, meaning that the method is for treating cancer other than type X.

[0082] As used herein, the term "about XY" has the same meaning as "about X to about Y."

[0083] When used in the context of administering an agent once every XY days, the term "every XY days" encompasses situations where the agent need not be administered at equal intervals. For example, a description of administering an S phase inhibitor once every 1-28 days encompasses administering the S phase inhibitor 10 days after the first administration and then administering the S phase inhibitor 5 days after the second administration.

[0084] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0085] II. Treatment Methods

[0086] The present application provides methods for promoting the persistence of cell therapy comprising immune cells, increasing the efficacy of cell therapy, treating host versus graft (HvG) disorders, reducing unwanted immune responses associated with cell therapy, and / or treating diseases or disorders in individuals who have or are about to undergo cell therapy.

[0087] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering to the individual a) an optional lymphodepleting agent, and b) an S phase inhibitor (e.g., an antifolate, e.g., a DHFR inhibitor, e.g., methotrexate) before administering the cell therapy, wherein the S phase inhibitor is administered before and after administering the cell therapy, wherein the lymphodepleting agent is different from the S phase inhibitor. Cell therapy may include immune cells resistant to S phase inhibitors. Immune cells may be allogeneic. Immune cells may include T cells. T cells may include exogenous Nef proteins, and / or T cells are engineered to not express or express reduced levels of endogenous TCRα or TCRβ. T cells may be γ-δT cells. Immune cells may include NK cells. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (e.g., CARs targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal into a positive signal, optionally wherein the second engineered receptor includes 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.

[0088] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering a) an optional lymphodepleting agent to the individual within the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours or 1 hour) before administering the cell therapy, and b) an S phase inhibitor (e.g., an antifolate, e.g., methotrexate), wherein the lymphodepleting agent is different from the S phase inhibitor. Cell therapy may include immune cells resistant to S phase inhibitors. The method may further include administering an S phase inhibitor after administering cell therapy. The method may include administering an S phase inhibitor at least 2, 3, 4, 5, 6, 7 times after administering cell therapy. The immune cell may be allogeneic. The immune cell may include a T cell. The T cell may include an exogenous Nef protein, and / or the T cell is engineered to not express or express a reduced level of endogenous TCRα or TCRβ. The T cell may be a γ-δT cell. The immune cell may include an NK cell. The immune cell may include 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, 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.

[0089] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering a) an optional lymphodepleting agent to the individual in parallel or simultaneously before administering the cell therapy, and b) an S phase inhibitor (e.g., an antifolate, e.g., methotrexate), wherein the lymphodepleting agent is different from the S phase inhibitor. Cell therapy may include immune cells resistant to S phase inhibitors. Immune cells may be allogeneic. Immune cells may include T cells. T cells may include exogenous Nef proteins, and / or T cells may be engineered to not express or express reduced levels of endogenous TCRα or TCRβ. T cells may be γ-δT cells. Immune cells may include NK cells. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (e.g., CARs targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal into a positive signal, optionally wherein the second engineered receptor includes 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.

[0090] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method including applying a) fludarabine, b) cyclophosphamide, and c) methotrexate to the individual, wherein the cell therapy includes immune cells resistant to methotrexate, wherein methotrexate is applied before and after the cell therapy is applied. The method may include applying methotrexate within about 5 days before applying immune cells. The method may include applying methotrexate within about 10 days after applying immune cells, optionally wherein the method includes applying methotrexate within about 5 days before applying immune cells and within about 10 days after applying immune cells. The method may include applying MTX and one or both of Flu and Cy within the same day (for example, within about 12 hours, 8 hours, 4 hours, 2 hours or 1 hour). The method may include applying MTX and one or both of Flu and Cy in parallel or simultaneously. MTX can be administered once every 1-28 days (e.g., every 1-10 days, for example, every 2-7 days, for example, every 3-7 days, for example, every 4-6 days) during a period of 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 not exceed about 2 μM. 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. It can be administered at about 3 mg / m 2 About 3000mg / m 2(e.g., 5 mg / m 2 About 3000mg / 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 ) is administered in an amount of methotrexate. The immune cell may be allogeneic. The immune cell may include a T cell. The T cell may include an exogenous Nef protein, and / or the T cell is engineered to not express or express a reduced level of endogenous TCRα or TCRβ. The T cell may be a γ-δT cell. The immune cell may include an NK cell. The immune cell may include a heterologous nucleic acid sequence encoding an engineered receptor (eg, a CAR targeting a tumor antigen). The immune cell may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cell may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cell may include a methotrexate-resistant transgenic. The methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0091] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method including applying a) fludarabine, b) cyclophosphamide, and c) methotrexate to the individual, wherein the cell therapy includes immune cells resistant to methotrexate, wherein at least three methotrexate are applied after applying the cell therapy. The method may include applying methotrexate within about 5 days before applying immune cells. The method may include applying methotrexate within about 10 days after applying immune cells, optionally wherein the method includes applying methotrexate within about 5 days before applying immune cells and within about 10 days after applying immune cells. The method may include applying MTX and one or both of Flu and Cy in the same day (for example, within about 12 hours, 8 hours, 4 hours, 2 hours or 1 hour). The method may include applying MTX and one or both of Flu and Cy in parallel or simultaneously. MTX can be administered once every 1-28 days (e.g., every 1-10 days, for example, every 2-7 days, for example, every 3-7 days, for example, every 4-6 days) during a period of 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 not exceed about 2 μM. 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. It can be administered at about 3 mg / m 2 About 3000mg / m 2 (e.g., 5 mg / m 2 About 3000mg / 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) is administered in an amount of methotrexate. The immune cell may be allogeneic. The immune cell may include a T cell. The T cell may include an exogenous Nef protein, and / or the T cell is engineered to not express or express a reduced level of endogenous TCRα or TCRβ. The T cell may be a γ-δT cell. The immune cell may include an NK cell. The immune cell may include a heterologous nucleic acid sequence encoding an engineered receptor (eg, a CAR targeting a tumor antigen). The immune cell may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cell may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cell may include a methotrexate-resistant transgenic. The methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0092] In certain embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method including applying a) fludarabine, b) cyclophosphamide, and c) methotrexate to the individual, wherein the cell therapy includes immune cells resistant to methotrexate, wherein the method includes concurrent or simultaneous administration of fludarabine, cyclophosphamide and methotrexate, optionally wherein the concurrent or simultaneous administration occurs within about 7 days (for example, within about 5 days) before the cell therapy is administered. The method may include administering methotrexate within about 10 days after administering immune cells, optionally wherein the method includes administering methotrexate within about 5 days before administering immune cells and within about 10 days after administering immune cells. MTX may be administered once every 1-28 days (for example, every 1-10 days, for example, every 2-7 days, for example, every 3-7 days, for example, every 4-6 days) during the time period of about 90 days after administering immune cells from about 5 days before administering immune cells. Within about 1-3 hours after administering methotrexate, the plasma concentration of methotrexate in an individual may not exceed about 2 μM. Within about 1-3 hours after administration of methotrexate, the plasma concentration of methotrexate in a subject may exceed about 0.001 μM. Within about 1-3 hours after administration of methotrexate, the plasma concentration of methotrexate in a subject may exceed about 0.01 μM. Within about 1-3 hours after administration of methotrexate, the plasma concentration of methotrexate in a subject may exceed about 0.1 μM. Methotrexate may be taken at about 3 mg / m 2 About 3000mg / m 2 (e.g., 5 mg / m 2 About 3000mg / 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 ) is administered in an amount of methotrexate. The immune cell may be allogeneic. The immune cell may include a T cell. The T cell may include an exogenous Nef protein, and / or the T cell is engineered to not express or express a reduced level of endogenous TCRα or TCRβ. The T cell may be a γ-δT cell. The immune cell may include an NK cell. The immune cell may include a heterologous nucleic acid sequence encoding an engineered receptor (eg, a CAR targeting a tumor antigen). The immune cell may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cell may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cell may include a methotrexate-resistant transgenic. The methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0093] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method including applying a) fludarabine, b) cyclophosphamide, and c) methotrexate to the individual, wherein the cell therapy includes immune cells resistant to methotrexate, wherein methotrexate is applied once every 1-7 days for at least 30 days. The method may include applying methotrexate within about 5 days before applying immune cells. The method may include applying methotrexate within about 10 days after applying immune cells, optionally wherein the method includes applying methotrexate within about 5 days before applying immune cells and within about 10 days after applying immune cells. The method may include applying MTX and one or both of Flu and Cy in the same day (for example, within about 12 hours, 8 hours, 4 hours, 2 hours or 1 hour). The method may include applying MTX and one or both of Flu and Cy in parallel or simultaneously. MTX can be administered once every 2-7 days or every 3-7 days (e.g., every 4-6 days) for at least about 40, 50, 60, 70, 80 or 90 days, optionally wherein MTX is administered during a time period of about 5 days before administering immune cells to about 90 days after administering immune cells. Within about 1-3 hours after administering methotrexate, the plasma concentration of methotrexate in an individual may not exceed about 2 μM. Within about 1-3 hours after administering methotrexate, the plasma concentration of methotrexate in an individual may exceed about 0.001 μM. Within about 1-3 hours after administering methotrexate, the plasma concentration of methotrexate in an individual may exceed about 0.01 μM. Within about 1-3 hours after administering methotrexate, the plasma concentration of methotrexate in an individual may exceed about 0.1 μM. It can be administered at about 3 mg / m 2 About 3000mg / m 2(e.g., 5 mg / m 2 About 3000mg / 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 ) is administered in an amount of methotrexate. The immune cell may be allogeneic. The immune cell may include a T cell. The T cell may include an exogenous Nef protein, and / or the T cell is engineered to not express or express a reduced level of endogenous TCRα or TCRβ. The T cell may be a γ-δT cell. The immune cell may include an NK cell. The immune cell may include a heterologous nucleic acid sequence encoding an engineered receptor (eg, a CAR targeting a tumor antigen). The immune cell may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cell may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cell may include a methotrexate-resistant transgenic. The methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0094] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method including applying a) fludarabine to the individual, b) cyclophosphamide, and c) methotrexate, wherein the cell therapy includes immune cells resistant to methotrexate, wherein methotrexate is applied at a frequency of once every 1-28 days, for at least about 90 days. The method may include applying methotrexate within about 5 days before applying immune cells. The method may include applying methotrexate within about 10 days after applying immune cells, optionally wherein the method includes applying methotrexate within about 5 days before applying immune cells and within about 10 days after applying immune cells. The method may include applying MTX and one or both of Flu and Cy within the same day (for example, within about 12 hours, 8 hours, 4 hours, 2 hours or 1 hour). The method may include applying MTX and one or both of Flu and Cy in parallel or simultaneously. MTX can be administered once 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). Within about 1-3 hours after administration of methotrexate, the plasma concentration of methotrexate in the individual may not exceed about 2 μM. Within about 1-3 hours after administration of methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.001 μM. Within about 1-3 hours after administration of methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.01 μM. Within about 1-3 hours after administration of methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.1 μM. It can be administered at about 3 mg / m 2 About 3000mg / m 2 (e.g., 5 mg / m 2 About 3000mg / 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) is administered in an amount of methotrexate. The immune cell may be allogeneic. The immune cell may include a T cell. The T cell may include an exogenous Nef protein, and / or the T cell is engineered to not express or express a reduced level of endogenous TCRα or TCRβ. The T cell may be a γ-δT cell. The immune cell may include an NK cell. The immune cell may include a heterologous nucleic acid sequence encoding an engineered receptor (eg, a CAR targeting a tumor antigen). The immune cell may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cell may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cell may include a methotrexate-resistant transgenic. The methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0095] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering a) fludarabine, b) cyclophosphamide, and c) methotrexate to the individual, wherein the cell therapy comprises immune cells resistant to methotrexate, wherein the method further comprises administering the immune cells more than once (e.g., about 2, 3, 4, 5 times), and wherein the first administration of the immune cells to the last administration of the immune cells is at least about 10 days (e.g., at least about 15 days, 20 days, 25 days, or 30 days) after the time period of the immune cells. Methotrexate is administered once every 1-10 days. The method may include 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 include 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 not exceed about 2 μM. Within about 1-3 hours after administration of methotrexate, the plasma concentration of methotrexate in a subject may exceed about 0.001 μM. Within about 1-3 hours after administration of methotrexate, the plasma concentration of methotrexate in a subject may exceed about 0.01 μM. Within about 1-3 hours after administration of methotrexate, the plasma concentration of methotrexate in a subject may exceed about 0.1 μM. Methotrexate may be taken at about 3 mg / m 2 About 3000mg / m 2 (e.g., 5 mg / m 2 About 3000mg / 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 ) is administered in an amount of methotrexate. The immune cell may be allogeneic. The immune cell may include a T cell. The T cell may include an exogenous Nef protein, and / or the T cell is engineered to not express or express a reduced level of endogenous TCRα or TCRβ. The T cell may be a γ-δT cell. The immune cell may include an NK cell. The immune cell may include a heterologous nucleic acid sequence encoding an engineered receptor (eg, a CAR targeting a tumor antigen). The immune cell may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The immune cell may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The immune cell may include a methotrexate-resistant transgenic. The methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0096] In some embodiments, a method for promoting the persistence of a cell therapy in a human individual is provided, 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 for about 3-4 consecutive days prior to administering the immune cells, and wherein methotrexate is administered at least once concurrently or simultaneously with fludarabine and / or cyclophosphamide. Within about 1-3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may not exceed about 2 μM. 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. It may be administered at about 3 mg / m 2 About 3000mg / m 2 (e.g., 5 mg / m 2 About 3000mg / 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) is administered in an amount of methotrexate. Immune cells may be allogeneic. Immune cells may include T cells. T cells may include exogenous Nef proteins, and / or T cells are engineered to not express or express reduced levels of endogenous TCRα or TCRβ. T cells are γ-δT cells. Immune cells include NK cells. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (e.g., CARs targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal into a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. Immune cells may include a methotrexate-resistant transgenic. The methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0097] In some embodiments, a method for promoting the persistence of a cell therapy in a human individual is provided, 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 for about 3-4 consecutive days prior to administering the immune cells, and wherein after administering the immune cells, methotrexate is administered once every 1-7 days for at least 30 days. Within about 1-3 hours after administering methotrexate, the plasma concentration of methotrexate in the individual may not exceed about 2 μM. 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. It may be administered at about 3 mg / m 2 About 3000mg / m 2 (e.g., 5 mg / m 2 About 3000mg / 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) is administered in an amount of methotrexate. Immune cells may be allogeneic. Immune cells may include T cells. T cells may include exogenous Nef proteins, and / or T cells are engineered to not express or express reduced levels of endogenous TCRα or TCRβ. T cells may be γ-δT cells. Immune cells may include NK cells. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (e.g., CARs targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal into a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells may include a methotrexate-resistant transgene. Immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The methotrexate-resistant transgene may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0098] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering a) lympholysis agent to the individual, and b) methotrexate, wherein the cell therapy comprises immune cells resistant to methotrexate, wherein the immune cells comprise T cells (e.g., allogeneic T cells) containing exogenous Nef proteins. Lympholysis agent may comprise Flu and Cy, wherein Flu and Cy are administered before cell therapy is administered. The method may include administering methotrexate after cell therapy is administered. Methotrexate may be administered before and after cell therapy is administered. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells, optionally wherein the method includes administering methotrexate within about 5 days before administering immune cells and within about 10 days after administering immune cells. The method may include 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 include the concurrent or simultaneous administration of MTX and one or both of Flu and Cy. MTX may be administered once every 1-28 days (e.g., every 1-10 days, for example, every 2-7 days, for example, every 3-7 days, for example, every 4-6 days) during a time period of about 5 days before administering immune cells to about 90 days after administering immune cells. Within about 1-3 hours after administering methotrexate, the plasma concentration of methotrexate in an individual may not exceed about 2 μM. Within about 1-3 hours after administering methotrexate, the plasma concentration of methotrexate in an individual may exceed about 0.001 μM. Within about 1-3 hours after administering methotrexate, the plasma concentration of methotrexate in an individual may exceed about 0.01 μM. Within about 1-3 hours after administering methotrexate, the plasma concentration of methotrexate in an individual may exceed about 0.1 μM. It may be administered at about 3 mg / m2 About 3000mg / m 2 (e.g., 5 mg / m 2 About 3000mg / 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 ) is administered in an amount of methotrexate. Immune cells can be allogeneic. Immune cells can include heterologous nucleic acid sequences encoding engineered receptors (e.g., CARs targeting tumor antigens). Immune cells can further include a second engineered receptor that converts a negative signal into a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells can include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. Immune cells can include a methotrexate-resistant transgene. The methotrexate-resistant transgene can include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0099] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering a) an optional lymphodepleting agent to the individual, and b) methotrexate, wherein the cell therapy comprises immune cells resistant to methotrexate, wherein the immune cells comprise T cells (e.g., allogeneic T cells) engineered to not express or express reduced levels of endogenous TCR α or TCR β. The lymphodepleting agent may comprise Flu and Cy, wherein Flu and Cy are administered before administering the cell therapy. The method may include administering methotrexate after administering the cell therapy. Methotrexate may be administered before and after administering the cell therapy. The method may include administering methotrexate within about 5 days before administering the immune cells. The method may include administering methotrexate within about 10 days after administering the immune cells, optionally wherein the method includes administering methotrexate within about 5 days before administering the immune cells and within about 10 days after administering the immune cells. The method may include applying one or both of MTX and Flu and Cy in the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours or 1 hour). The method may include applying one or both of MTX and Flu and Cy in parallel or simultaneously. MTX may be applied once every 1-28 days (e.g., every 1-10 days, for example, every 2-7 days, for example, every 3-7 days, for example, every 4-6 days) during the time period of about 5 days to about 90 days after applying immune cells before applying immune cells. In about 1-3 hours after applying methotrexate, the plasma concentration of methotrexate in an individual may not exceed about 2 μM. In about 1-3 hours after applying methotrexate, the plasma concentration of methotrexate in an individual may exceed about 0.001 μM. In about 1-3 hours after applying methotrexate, the plasma concentration of methotrexate in an individual may exceed about 0.01 μM. Within about 1-3 hours after administration of methotrexate, the plasma concentration of methotrexate in a subject may exceed about 0.1 μM. 2 About 3000mg / m 2 (e.g., 5 mg / m 2 About 3000mg / 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) is administered in an amount of methotrexate. Immune cells can be allogeneic. Immune cells can include heterologous nucleic acid sequences encoding engineered receptors (e.g., CARs targeting tumor antigens). Immune cells can further include a second engineered receptor that converts a negative signal into a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells can include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. Immune cells can include a methotrexate-resistant transgene. The methotrexate-resistant transgene can include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0100] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering a) an optional lymphodepleting agent to the individual, and b) methotrexate, wherein the cell therapy comprises immune cells resistant to methotrexate, wherein the immune cells comprise γ-δT cells. Lymphodepleting agents may comprise Flu and Cy, wherein Flu and Cy are administered before cell therapy is administered. The method may include administering methotrexate after cell therapy is administered. Methotrexate may be administered before and after cell therapy is administered. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells, optionally wherein the method includes administering methotrexate within about 5 days before administering immune cells and within about 10 days after administering immune cells. The method may include 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 include administering MTX and one or both of Flu and Cy in parallel or simultaneously. MTX can be administered once 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 a period of 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 not exceed about 2 μM. 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 exceeds about 0.1 μM. It can be administered at about 3 mg / m 2 About 3000mg / m 2 (e.g., 5 mg / m 2 About 3000mg / m 2 , for example, about 5 mg / m 2 About 100 mg / m2 , for example, about 20 mg / m 2 About 500 mg / m 2 , for example, about 100 mg / m 2 About 500 mg / m 2 ) is administered in an amount of methotrexate. Immune cells can be allogeneic. Immune cells can include heterologous nucleic acid sequences encoding engineered receptors (e.g., CARs targeting tumor antigens). Immune cells can further include a second engineered receptor that converts a negative signal into a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells can include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. Immune cells can include a methotrexate-resistant transgene. The methotrexate-resistant transgene can include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0101] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering a) an optional lymphodepleting agent to the individual, and b) methotrexate, wherein the cell therapy comprises immune cells resistant to methotrexate, wherein the immune cells comprise NK cells. Lymphodepleting agents may comprise Flu and Cy, wherein Flu and Cy are administered before cell therapy is administered. The method may include administering methotrexate after cell therapy is administered. Methotrexate may be administered before and after cell therapy is administered. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells, optionally wherein the method includes administering methotrexate within about 5 days before administering immune cells and within about 10 days after administering immune cells. The method may include 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 include administering MTX and one or both of Flu and Cy in parallel or simultaneously. MTX can be administered once every 1-28 days (e.g., every 1-10 days, for example, every 2-7 days, for example, every 3-7 days, for example, every 4-6 days) during a period of 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 not exceed about 2 μM. 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. It can be administered at about 3 mg / m 2About 3000mg / m 2 (e.g., 5 mg / m 2 About 3000mg / 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 ) is administered in an amount of methotrexate. Immune cells can be allogeneic. Immune cells can include heterologous nucleic acid sequences encoding engineered receptors (e.g., CARs targeting tumor antigens). Immune cells can further include a second engineered receptor that converts a negative signal into a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells can include a methotrexate-resistant transgene. Immune cells can include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The methotrexate-resistant transgene can include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0102] In certain embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method including applying methotrexate to the individual before and after applying the cell therapy. Other lymphodepleting agents may not be added. Cell therapy may include immune cells resistant to MTX. MTX may be applied within 5 days before applying immune cells. MTX may be applied at a frequency of about 1-28 days (e.g., 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 5 days), for at least about 30 days, 40 days, 50 days, 60 days, 70 days, 80 days or 90 days. During the time period between about 5 days before applying immune cells and about 90 days after applying immune cells, MTX may be applied at a frequency of about 1-28 days. Immune cells may be allogeneic. Immune cells may include T cells. T cells may include exogenous Nef proteins, and / or T cells may be engineered to not express or express reduced levels of endogenous TCR α or TCR β. T cells can be γ-δT cells. Immune cells can include NK cells. Immune cells can include heterologous nucleic acid sequences encoding engineered receptors (e.g., CARs targeting tumor antigens). Immune cells can further include a second engineered receptor that converts a negative signal into a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells can include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound.

[0103] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) cell therapy comprising immune cells resistant to methotrexate to the individual, wherein fludarabine and cyclophosphamide are administered for about 3-4 consecutive days before administering these immune cells, and wherein after administering the immune cells, methotrexate is administered once every 1-7 days (e.g., 3-7 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 exogenous Nef proteins. Fludarabine and cyclophosphamide can be administered for 3 consecutive days. Fludarabine and cyclophosphamide can be administered for 4 consecutive days. Fludarabine and cyclophosphamide can be administered for 3 consecutive days, and then fludarabine is administered alone for 1 day. Methotrexate can be administered before and after administering cell therapy. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells. The method may include administering MTX and one or both of Flu and Cy within the same day (for example, within about 12 hours, 8 hours, 4 hours, 2 hours or 1 hour). The method may include administering MTX and one or both of Flu and Cy in parallel or simultaneously. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (such as CARs targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells may include a methotrexate-resistant transgenic. Immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0104] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering a) fludarabine, b) cyclophosphamide, c) methotrexate to the individual, and d) cell therapy comprising immune cells resistant to methotrexate, wherein before administering these immune cells, fludarabine and cyclophosphamide are administered for about 3-4 consecutive days, and wherein after administering the immune cells, methotrexate is administered once every 1-7 days (e.g., 3-7 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) that are engineered to not express or express reduced levels of endogenous TCR α or TCR β. Fludarabine and cyclophosphamide can be administered for 3 consecutive days. Fludarabine and cyclophosphamide can be administered for 4 consecutive days. Fludarabine and cyclophosphamide can be administered for 3 consecutive days, and then fludarabine is administered alone for 1 day. Methotrexate can be administered before and after administering cell therapy. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells. The method may include 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 include administering MTX and one or both of Flu and Cy in parallel or simultaneously. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (e.g., CAR targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. Immune cells may include a methotrexate-resistant transgenic. The methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0105] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) cell therapy comprising immune cells resistant to methotrexate to the individual, wherein before administering these immune cells, fludarabine and cyclophosphamide are administered for about 3-4 consecutive days, and wherein after administering the immune cells, methotrexate is administered once every 1-7 days (e.g., 3-7 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 can be administered for 3 consecutive days. Fludarabine and cyclophosphamide can be administered for 4 consecutive days. Fludarabine and cyclophosphamide can be administered for 3 consecutive days, and then fludarabine is administered alone for 1 day. Methotrexate can be administered before and after administering cell therapy. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells. The method may include administering MTX and one or both of Flu and Cy within the same day (for example, within about 12 hours, 8 hours, 4 hours, 2 hours or 1 hour). The method may include administering MTX and one or both of Flu and Cy in parallel or simultaneously. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (such as CARs targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. Immune cells may include a methotrexate-resistant transgenic. The methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0106] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) cell therapy comprising immune cells resistant to methotrexate to the individual, wherein before administering these immune cells, fludarabine and cyclophosphamide are administered for about 3-4 consecutive days, and wherein after administering these immune cells, methotrexate is administered once every 1-7 days (e.g., 3-7 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 can be administered for 3 consecutive days. Fludarabine and cyclophosphamide can be administered for 4 consecutive days. Fludarabine and cyclophosphamide can be administered for 3 consecutive days, and then fludarabine is administered alone for 1 day. Methotrexate can be administered before and after administering cell therapy. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells. The method may include administering MTX and one or both of Flu and Cy in the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may include administering MTX and one or both of Flu and Cy in parallel or simultaneously. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (e.g., CARs targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. Immune cells may include a methotrexate-resistant transgenic. A methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0107] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) cell therapy comprising immune cells resistant to methotrexate, wherein before administering these immune cells, fludarabine and cyclophosphamide are administered for about 3-4 consecutive 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 exogenous Nef proteins. Fludarabine and cyclophosphamide can be administered for 3 consecutive days. Fludarabine and cyclophosphamide can be administered for 4 consecutive days. Fludarabine and cyclophosphamide can be administered for 3 consecutive days, and then fludarabine is administered alone for 1 day. Methotrexate can be administered before and after administering cell therapy. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells. The method may include administering MTX and one or both of Flu and Cy within the same day (for example, within about 12 hours, 8 hours, 4 hours, 2 hours or 1 hour). The method may include administering MTX and one or both of Flu and Cy in parallel or simultaneously. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (such as CARs targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. Immune cells may include a methotrexate-resistant transgenic. The methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0108] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering a) fludarabine, b) cyclophosphamide, c) methotrexate to the individual, and d) a cell therapy comprising immune cells resistant to methotrexate, wherein before administering these immune cells, fludarabine and cyclophosphamide are administered for about 3-4 consecutive 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) that are engineered to not express or express reduced levels of endogenous TCRα or TCRβ. Fludarabine and cyclophosphamide can be administered for 3 consecutive days. Fludarabine and cyclophosphamide can be administered for 4 consecutive days. Fludarabine and cyclophosphamide can be administered for 3 consecutive days, and then fludarabine is administered alone for 1 day. Methotrexate can be administered before and after administering cell therapy. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells. The method may include 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 include administering MTX and one or both of Flu and Cy in parallel or simultaneously. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (e.g., CAR targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. Immune cells may include a methotrexate-resistant transgenic. The methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0109] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) cell therapy comprising immune cells resistant to methotrexate to the individual, wherein fludarabine and cyclophosphamide are administered for about 3-4 consecutive days before administering these immune cells, 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 are γ-δ T cells. Fludarabine and cyclophosphamide can be administered for 3 consecutive days. Fludarabine and cyclophosphamide can be administered for 4 consecutive days. Fludarabine and cyclophosphamide can be administered for 3 consecutive days, and then fludarabine is administered alone for 1 day. Methotrexate can be administered before and after administering cell therapy. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells. The method may include administering MTX and one or both of Flu and Cy within the same day (for example, within about 12 hours, 8 hours, 4 hours, 2 hours or 1 hour). The method may include administering MTX and one or both of Flu and Cy in parallel or simultaneously. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (such as CARs targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. Immune cells may include a methotrexate-resistant transgenic. The methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0110] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) cell therapy comprising immune cells resistant to methotrexate, wherein before administering these immune cells, fludarabine and cyclophosphamide are administered for about 3-4 consecutive 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 for 3 consecutive days. Fludarabine and cyclophosphamide may be administered for 4 consecutive days. Fludarabine and cyclophosphamide may be administered for 3 consecutive days, and then fludarabine may be administered alone for 1 day. Methotrexate may be administered before and after administering cell therapy. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells. The method may include administering MTX and one or both of Flu and Cy in the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may include administering MTX and one or both of Flu and Cy in parallel or simultaneously. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (e.g., CARs targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. Immune cells may include a methotrexate-resistant transgenic. A methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0111] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) cell therapy comprising immune cells resistant to methotrexate, wherein before administering these immune cells, fludarabine and cyclophosphamide are administered for about 3-4 consecutive 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 exogenous Nef proteins. Fludarabine and cyclophosphamide can be administered for 3 consecutive days. Fludarabine and cyclophosphamide can be administered for 4 consecutive days. Fludarabine and cyclophosphamide can be administered for 3 consecutive days, and then fludarabine is administered alone for 1 day. Methotrexate can be administered before and after administering cell therapy. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells. The method may include administering MTX and one or both of Flu and Cy in the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may include administering MTX and one or both of Flu and Cy in parallel or simultaneously. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (e.g., CARs targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. Immune cells may include a methotrexate-resistant transgenic. A methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0112] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) cell therapy comprising immune cells resistant to methotrexate, wherein fludarabine and cyclophosphamide are administered for about 3-4 consecutive days before administering these immune cells, and wherein methotrexate is administered before or after administering these immune cells, wherein these immune cells comprise T cells (e.g., allogeneic T cells) that are engineered to not express or express reduced levels of endogenous TCR α or TCR β. Fludarabine and cyclophosphamide can be administered for 3 consecutive days. Fludarabine and cyclophosphamide can be administered for 4 consecutive days. Fludarabine and cyclophosphamide can be administered for 3 consecutive days, and then fludarabine is administered alone for 1 day. Methotrexate can be administered before and after administering cell therapy. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells. The method may include administering MTX and one or both of Flu and Cy in the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may include administering MTX and one or both of Flu and Cy in parallel or simultaneously. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (e.g., CARs targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells may include a methotrexate-resistant transgenic. Immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0113] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) cell therapy comprising immune cells resistant to methotrexate, wherein before administering these immune cells, fludarabine and cyclophosphamide are administered for about 3-4 consecutive 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 can be administered for 3 consecutive days. Fludarabine and cyclophosphamide can be administered for 4 consecutive days. Fludarabine and cyclophosphamide can be administered for 3 consecutive days, and then fludarabine is administered alone for 1 day. Methotrexate can be administered before and after administering cell therapy. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells. The method may include administering MTX and one or both of Flu and Cy in the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may include administering MTX and one or both of Flu and Cy in parallel or simultaneously. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (e.g., CARs targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. Immune cells may include a methotrexate-resistant transgenic. A methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0114] In some embodiments, a method for promoting the persistence of cell therapy in human individuals is provided, the method comprising administering to the individual a) fludarabine, b) cyclophosphamide, c) methotrexate, and d) cell therapy comprising immune cells resistant to methotrexate, wherein fludarabine and cyclophosphamide are administered for about 3-4 consecutive days before administering these immune cells, 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 for 3 consecutive days. Fludarabine and cyclophosphamide may be administered for 4 consecutive days. Fludarabine and cyclophosphamide may be administered for 3 consecutive days, and then fludarabine may be administered alone for 1 day. Methotrexate may be administered before and after administering cell therapy. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells. The method may include administering MTX and one or both of Flu and Cy in the same day (e.g., within about 12 hours, 8 hours, 4 hours, 2 hours, or 1 hour). The method may include administering MTX and one or both of Flu and Cy in parallel or simultaneously. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (e.g., CARs targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. Immune cells may include a methotrexate-resistant transgenic. A methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0115] In some embodiments, a method for treating a disease or condition (e.g., cancer) of a human individual is provided, 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 resistant to the S phase inhibitor. In some embodiments, a method for treating a disease or condition (e.g., cancer) of a human individual is provided, 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 before administering these immune cells, fludarabine and cyclophosphamide are administered for about 3-4 consecutive days, and wherein after administering these immune cells, methotrexate is administered once every 1-7 days (e.g., 3-7 days), for at least 30 days (e.g., at least 60 days, e.g., at least 90 days). Immune cells can be T cells (e.g., allogeneic T cells). T cells can include exogenous Nef proteins. T cells can be engineered to not express or express reduced levels of endogenous TCRα or TCRβ. T cells may be γ-δT cells. Immune cells may be NK cells. Cancer may be a solid tumor. Cancer may be a blood cancer. Fludarabine and cyclophosphamide may be administered for 3 consecutive days. Fludarabine and cyclophosphamide may be administered for 4 consecutive days. Fludarabine and cyclophosphamide may be administered for 3 consecutive days, and then fludarabine may be administered alone for 1 day. Methotrexate may be administered before and after cell therapy is administered. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells. The method may include 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 include administering MTX and one or both of Flu and Cy in parallel or simultaneously. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (e.g., CARs targeting tumor antigens). The immune cell may further comprise a second engineered receptor that converts a negative signal into a positive signal, 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, optionally wherein the mutant DHFR gene comprises an L22F mutation and an F31S mutation.

[0116] In some embodiments, a method for treating a disease or condition (e.g., cancer) of a human individual is provided, 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 fludarabine and cyclophosphamide are administered for about 3-4 consecutive days before administering the immune cells, 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 cell may be a T cell (e.g., an allogeneic T cell). The T cell may comprise an exogenous Nef protein. The T cell may be engineered to not express or express reduced levels of endogenous TCRα or TCRβ. The T cell may be a γ-δT cell. The immune cell may be an NK cell. The cancer may be a solid tumor. The cancer may be a blood cancer. Fludarabine and cyclophosphamide may be administered for 3 consecutive days. Fludarabine and cyclophosphamide may be administered for 4 consecutive days. Fludarabine and cyclophosphamide can be administered for 3 consecutive days, and then fludarabine is administered alone for 1 day. Methotrexate can be administered before and after cell therapy is administered. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells. The method may include administering MTX and one or both of Flu and Cy within the same day (for example, within about 12 hours, 8 hours, 4 hours, 2 hours or 1 hour). The method may include administering MTX and one or both of Flu and Cy in parallel or simultaneously. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (such as CARs targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal into a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. Immune cells may include a methotrexate-resistant transgene. The methotrexate resistance transgene may comprise a mutant DHFR gene, optionally wherein the mutant DHFR gene comprises a L22F mutation and a F31S mutation.

[0117] In some embodiments, a method for treating a disease or disorder (e.g., cancer) of a human individual is provided, 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 before administering the immune cells, fludarabine and cyclophosphamide are administered for about 3-4 consecutive days, and wherein the method comprises administering methotrexate before and after administering the cell therapy. Immune cells may be T cells (e.g., allogeneic T cells). T cells may comprise exogenous Nef proteins. T cells may be engineered to not express or express reduced levels of endogenous TCRα or TCRβ. T cells may be γ-δT cells. Immune cells may be NK cells. Cancer may be a solid tumor. Cancer may be a blood cancer. Fludarabine and cyclophosphamide may be administered for 3 consecutive days. Fludarabine and cyclophosphamide may be administered for 4 consecutive days. Fludarabine and cyclophosphamide may be administered for 3 consecutive days, and then fludarabine may be administered alone for 1 day. Methotrexate may be administered before and after administering the cell therapy. The method may include administering methotrexate within about 5 days before administering immune cells. The method may include administering methotrexate within about 10 days after administering immune cells. The method may include 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 include administering MTX and one or both of Flu and Cy in parallel or simultaneously. Immune cells may include heterologous nucleic acid sequences encoding engineered receptors (e.g., CAR targeting tumor antigens). Immune cells may further include a second engineered receptor that converts a negative signal to a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. Immune cells may include an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. Immune cells may include a methotrexate-resistant transgenic. The methotrexate-resistant transgenic may include a mutant DHFR gene, optionally wherein the mutant DHFR gene includes an L22F mutation and an F31S mutation.

[0118] In some embodiments, a method for treating cancer (e.g., hematological cancer, e.g., solid tumor) of a human individual is provided, the method comprising administering a) cell therapy, b) methotrexate to the individual, wherein after the cell therapy, the methotrexate is administered at least more than once. Methotrexate can be administered once every day to seven days, for at least about 2, 3, 4, 5, 6 or 7 times. Cell therapy can include immune cells, optionally wherein these immune cells are resistant to methotrexate. The method may further include administering a lymphoproliferator before administering cell therapy. The method may include administering one or both of MTX and Flu and Cy in 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 include administering one or both of MTX and Flu and Cy in parallel or simultaneously. MTX can be administered about every 1-7 days (eg, about 3-7 days) for at least 30 days (eg, for at least 60 days, eg, for at least 90 days) after administration of the immune cells.

[0119] In some embodiments, a method for reducing an undesirable immune response associated with cell therapy in a human individual is provided, the method including applying a) lymphoclearance agent to the individual, b) S phase inhibitor (e.g., methotrexate), and c) cell therapy comprising immune cells resistant to S phase inhibitors. In some embodiments, a method for reducing an undesirable immune response associated with cell therapy in a human individual is provided, the method including applying a) lymphoclearance agent (e.g., Flu and Cy) to the individual, and b) methotrexate, wherein after the cell therapy, methotrexate is applied at least more than once. Methotrexate can be applied once every day to seven days, for at least about 2, 3, 4, 5, 6 or 7 times. Cell therapy can include immune cells, optionally wherein these immune cells are resistant to methotrexate. The method can include applying one or both of MTX and Flu and Cy in 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 include concurrent or simultaneous administration of one or both of MTX and Flu and Cy. MTX may be administered once approximately every 1-7 days (e.g., approximately 3-7 days) after administration of immune cells for at least 30 days (e.g., for at least 60 days, e.g., for at least 90 days).

[0120] In some embodiments, a method for increasing the efficacy of cell therapy in human individuals is provided, the method including applying a) an optional lymphoproliferative agent to the individual, 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, a method for increasing the efficacy of cell therapy in human individuals is provided, the method including applying a) a lymphoproliferative agent (e.g., Flu and Cy) to the individual, and b) methotrexate, wherein after the cell therapy, methotrexate is applied at least more than once. Methotrexate can be applied once every day to seven days, for at least about 2, 3, 4, 5, 6 or 7 times. Cell therapy can include immune cells, and optionally these immune cells are resistant to methotrexate. The method may include applying MTX and one or both of Flu and Cy in 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 include concurrent or simultaneous administration of one or both of MTX and Flu and Cy. MTX may be administered once approximately every 1-7 days (e.g., approximately 3-7 days) after administration of immune cells for at least 30 days (e.g., for at least 60 days, e.g., for at least 90 days).

[0121] In some embodiments, a method for treating a host-versus-graft (HvG) disorder in a human individual receiving cell therapy is provided, 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 resistant to the S phase inhibitor. In some embodiments, a method for treating a host-versus-graft (HvG) disorder in a human individual receiving cell therapy is provided, 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 at least more than once. Methotrexate may be administered once every day to seven days for at least about 2, 3, 4, 5, 6 or 7 times. Cell therapy may include immune cells, optionally wherein these immune cells are resistant to methotrexate. The method may include 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 include concurrent or simultaneous administration of MTX and one or both of Flu and Cy. MTX may be administered approximately every 1-7 days (e.g., approximately 3-7 days) after administration of immune cells for at least 30 days (e.g., for at least 60 days, e.g., for at least 90 days). It may be administered at about 3 mg / m 2 About 3000mg / m 2 (e.g., 5 mg / m 2 About 3000mg / m2 Methotrexate can be administered in an amount of about 3 mg / m 2 / day to about 3000mg / m 2 / day (e.g., 5 mg / m 2 / day to about 3000mg / m 2 / day) dose of methotrexate.

[0122] It can be taken at about 25 mg / m 2 To about 30mg / m 2 Fludarabine is administered at a dose of about 250 mg / m 2 About 1000 mg / m 2 Fludarabine and cyclophosphamide may be administered about 1-3 times during a period of about 10 days before administration of the immune cells to about 90 days after administration of the immune cells.

[0123] The S phase inhibitor (eg, methotrexate) can be administered orally, subcutaneously, intramuscularly, intravenously, intraarterially, or intrathecally.

[0124] The methods described herein are applicable to both autologous immune cells and allogeneic immune cells. Allogeneic immune cells can be readily available immune cells. Allogeneic immune cells can have genetic modifications that reduce the immunogenicity or alloreactivity of the immune cells. Allogeneic immune cells can be genetically modified to reduce the immunogenicity or alloreactivity of the immune cells.

[0125] The immune cell may include a heterologous nucleic acid sequence encoding an engineered receptor. The immune cell may be a CAR-T cell. The immune cell may be an allogeneic CAR-T cell. The immune cell may be a universal CART cell ("UCART cell"). The immune cell may be a TCR-T cell. The immune cell may include one or more engineered receptors selected from the group consisting of: CAR, TCR, TAC receptor, and TFP. The engineered receptor can 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, kappa-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, p53, MART1, GP100, proteinase-3 (PR3), tyrosinase, survival protein, 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 can be anti-BCMA CAR, anti-CD20 CAR or anti-claudin 18.2 CAR.

[0126] Immune cells can overexpress the DHFR gene, which encodes dihydrofolate reductase.

[0127] The immune cells can be administered more than once. The immune cells can be administered about 2-5 times within about 90 days after the first administration of the immune cells.

[0128] The immune cells may comprise about 30 million to about 900 million immune cells, or about 100,000 to about 50 million immune cells per kilogram of a human individual.

[0129] During a time period between about 2-7 days prior to administration of the immune cells and about 10, 20, 30, 40, 50, 60, 70, 80, or 90 days after administration of the immune cells, the host T cells as measured by the number of host T cells in the PBMCs may not exceed about 500, 400, 300, 200, 100, or 50 cells / μL.

[0130] The method may further comprise monitoring the number of host T cells in the individual.

[0131] The subject to be treated can 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 subject can be a human.

[0132] The individual mentioned herein can be any individual that is considered suitable for a cell therapy comprising immune cells. The individual can suffer from cancer (e.g., a blood cancer, e.g., a solid tumor, e.g., a lymphoma or a leukemia). The individual can be male. The individual can be female. The individual can be at least 50, 55, 60, 65, 70, 75, 80, 85, or 90 years old.

[0133] A. Cell therapy and lymphodepletion

[0134] Cell therapies, such as chimeric antigen receptor (CAR) T cells and CAR-NK therapies, show promise in treating diseases such as hematological tumors and solid tumors. Before infusion, lympholysis is often performed. Lympholysis causes lymphopenia, affects T cells, B cells, and NK cells, and lympholysis has a variety of positive effects before, for example, CAR T cell therapy, such as suppressing the host immune system to reduce immunogenicity and increase the persistence of infused CAR T cells. However, it is also important to note that lympholysis also has a variety of negative effects. For example, lympholysis can cause neutropenia, anemia, thrombocytopenia, and immunosuppression, leading to a greater risk of infection. In particular, fludarabine can cause fever and neurotoxicity. Cyclophosphamide can cause hemorrhagic cystitis, pericarditis, and neurotoxicity. In addition, the use of these agents can increase the risk of developing secondary malignancies.

[0135] BS phase inhibitors, antifolates (eg, DHFR inhibitors), and methotrexate

[0136] The methods described herein include administering an S phase inhibitor (e.g., an antifolate, e.g., methotrexate). The S phase inhibitor described herein refers to an agent that arrests cells in the S phase of the cell cycle. The S phase inhibitor can be an antifolate, which refers to a class of inhibitors of targets in the folate biosynthetic pathway.

[0137] The antifolate may be a DHFR inhibitor.

[0138] The antifolate may be selected from the group consisting of methotrexate, raltitrexed (e.g. ), pemetrexed (e.g., Pemfexy TM , Ciambra), pralatrexate (e.g., ), trimethoprim (e.g., Triprim, ) and pyrimethamine

[0139] The antifolate can be methotrexate. Exemplary methotrexates include Otrexup TM , Trexall TM , methotrexate, methotrexate sodium.

[0140] Methotrexate may be used to treat certain types of cancer (such as acute lymphoblastic leukemia, non-Hodgkin lymphoma) or to control severe psoriasis or rheumatoid arthritis that has not responded to other treatments. Methotrexate may also be used to control juvenile rheumatoid arthritis. Methotrexate belongs to a class of drugs called antimetabolites. It works by slowing or stopping the growth of cancer cells and suppressing the immune system.

[0141] Methotrexate is a folic acid antagonist. Methotrexate inhibits dihydrofolate reductase (DHFR), which reduces folic acid to tetrahydrofolate. Tetrahydrofolate must be regenerated via DHFR catalytic reactions to maintain intracellular pools of tetrahydrofolate one-carbon derivatives for thymidylate and purine nucleotide biosynthesis. The inhibition of DHFR by folic acid antagonists (methotrexate) results in a lack of cellular pools of thymidylate and purine, resulting in reduced nucleic acid synthesis. Therefore, methotrexate interferes with DNA synthesis, repair, and cell replication.

[0142] Methotrexate is most active against rapidly proliferating cells because its cytotoxic effects occur primarily during the S phase of the cell cycle. Since cell proliferation in malignant tissue is greater than that in most normal tissues, methotrexate may impair 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 intestinal mucosa, and bladder cells) are generally more sensitive to the DHFR inhibitory effects of methotrexate.

[0143] The cytotoxicity of methotrexate is caused by three important effects: inhibition of DHFR, inhibition of thymidylate synthase, and alteration of the transport of reduced folate. The affinity of DHFR for methotrexate is much greater than its affinity for folic acid or dihydrofolate, and therefore, simultaneous administration of high doses of folic acid will not reverse the effects of methotrexate. However, if calcium folinate (a derivative of tetrahydrofolate) is given shortly after the antineoplastic agent, it may block the effects of methotrexate. High doses of methotrexate followed by folinic acid rescue are used as part of the treatment of patients with non-metastatic osteosarcoma.

[0144] Methotrexate (MTX) is commonly used to prevent graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). It inhibits the production of dihydrofolate reductase and thymidylate and purine, thereby inhibiting T cell response and proliferation and the expression of adhesion molecules. See, for example, Nassar et al., J Transplant. 2014; 2014: 980301.

[0145] The method may include administering an S phase inhibitor (e.g., an antifolate, e.g., a DHFR inhibitor, e.g., methotrexate) within about 10, 9, 8, 7, 6, or 5 days prior to administering the immune cells. The method may include administering an S phase inhibitor (e.g., an antifolate, e.g., a DHFR inhibitor, e.g., methotrexate) within about 4, 3, 2, or 1 day prior to administering the immune cells.

[0146] The method may include administering an S phase inhibitor (e.g., an antifolate, e.g., a DHFR inhibitor, e.g., methotrexate) within about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 days after administering the immune cells. The method may include administering an S phase inhibitor (e.g., an antifolate, e.g., a DHFR inhibitor, e.g., methotrexate) within about 4, 3, 2, or 1 day after administering the immune cells.

[0147] The method can include administering an S phase inhibitor (eg, an antifolate, eg, a DHFR inhibitor, eg, methotrexate) concurrently or simultaneously with the immune cells.

[0148] The method can include administering an S phase inhibitor (e.g., an antifolate, e.g., a DHFR inhibitor, e.g., methotrexate) within about 10, 9, 8, 7, 6, or 5 days prior to 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.

[0149] The method may include administering an S phase inhibitor (e.g., an antifolate, e.g., a DHFR inhibitor, e.g., methotrexate) concurrently or simultaneously with a lymphodepleting agent, optionally the method includes administering an S phase inhibitor concurrently or simultaneously with a lymphodepleting agent within 5 days prior to immunotherapy. The method may include administering an S phase inhibitor (e.g., an antifolate, e.g., a DHFR inhibitor, e.g., methotrexate) and a lymphodepleting agent within 3 days, 2 days, or 1 day of each other. The method may include administering an S phase inhibitor (e.g., an antifolate, e.g., a DHFR inhibitor, e.g., methotrexate) and a lymphodepleting agent within 24 hours, 16 hours, 8 hours, 4 hours, 2 hours, or 1 hour.

[0150] The method may include administering an S phase inhibitor (e.g., an antifolate, e.g., a DHFR inhibitor, e.g., methotrexate) 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 an S phase inhibitor (e.g., an antifolate, e.g., a DHFR inhibitor, e.g., methotrexate) for 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 can include administering an S phase inhibitor (e.g., an antifolate, e.g., a DHFR inhibitor, e.g., methotrexate) 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 starting about 10, 9, 8, 7, 6, or 5 days prior to administration of the immune cells.

[0151] The method may include administering an S phase inhibitor (e.g., an antifolate, e.g., a DHFR inhibitor, e.g., methotrexate) 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 a time period of about 5 days before administering the immune cells to about 90 days after administering the immune cells. The method may include administering an S phase inhibitor (e.g., an antifolate, e.g., a DHFR inhibitor, e.g., methotrexate) every 1-10 days (e.g., every 2-9 days, every 2-8 days, every 3-7 days, or every 4-6 days) during a time period of about 90 days after administering the immune cells. Methotrexate may be administered on the 3rd, 5th, 10th, and 17th days after each administration of the immune cells.

[0152] It can be taken at about 5 mg / m2 About 3000mg / m 2 Methotrexate can be administered in an amount of about 3 mg / m 2 About 3000mg / m 2 Methotrexate is administered in an amount of .

[0153] It can be taken at about 5 mg / m 2 / day to about 3000mg / m 2 Methotrexate can be administered at a dose of about 3 mg / m 2 / day to about 3000mg / m 2 Methotrexate was administered at a dose of / day.

[0154] For each individual, 3.1 mg / m 2 Up to 15.6 mg / m 2 Or MTX can be administered once every 2-7 days (e.g., every 3-5 days) at a dose of 5 mg to 25 mg. For each individual, 15.6 mg / m 2 Up to 62.5 mg / m 2 Or MTX can be administered once every 2-7 days (e.g., every 3-5 days) at a dose of 25 mg to 100 mg. For each individual, 62.5 mg / m 2 Up to 187.5 mg / m 2 Or MTX can be administered once every 2-7 days (e.g., every 3-5 days) at a dose of 100 mg to 300 mg. For each individual, 15.6 mg / m 2 MTX can be administered once every 2-7 days (e.g., every 3-5 days) at a dose of 25 mg or 31.25 mg / m 2 MTX can be administered once every 2-7 days (e.g., every 3-5 days) at a dose of 63 mg / m 2 MTX may be administered once every 2-6 days (e.g., every 4-5 days) (e.g., for at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 days) at a dose of 100 mg or 100 mg. 2 MTX may be administered once every 2-6 days (e.g., every 4-5 days) (e.g., for at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 days) at a dose of 188 mg / m 2 MTX may be administered once every 2-6 days (e.g., every 4-5 days) (e.g., for at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 days) at a dose of 250 mg / m 2MTX may be administered once every 2-6 days (e.g., every 4-5 days) (e.g., for at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 days) at a dose of 313 mg / m 2 Or MTX is administered once every 2-6 days (e.g., every 4-5 days) at a dose of 500 mg (e.g., for at least 10, 20, 30, 40, 50, 60, 70, 80, or 90 days).

[0155] Within about 24 hours after administration of methotrexate, the plasma concentration of methotrexate in the subject may not exceed about 2 μM (e.g., not more than about 1.5 μM, not more than about 1 μM, not more than about 0.5 μM, or not more than about 0.2 μM). Within about 12 hours after administration of methotrexate, the plasma concentration of methotrexate in the subject may not exceed about 2 μM (e.g., not more than about 1.5 μM, not more than about 1 μM, not more than about 0.5 μM, or not more than about 0.2 μM). Within about 6 hours after administration of methotrexate, the plasma concentration of methotrexate in the subject may not exceed about 2 μM (e.g., not more than about 1.5 μM, not more than about 1 μM, not more than about 0.5 μM, or not more than about 0.2 μM). Within about 3 hours after administration of methotrexate, the plasma concentration of methotrexate in the subject may not exceed about 2 μM (e.g., not more than about 1.5 μM, not more than about 1 μM, not more than about 0.5 μM, or not more than about 0.2 μM). Within about 2 hours after administration of methotrexate, the plasma concentration of methotrexate in the subject may not exceed about 2 μM (e.g., not more than about 1.5 μM, not more than about 1 μM, not more than about 0.5 μM, or not more than about 0.2 μM). Within about 1 hour after administration of methotrexate, the plasma concentration of methotrexate in the subject may not exceed about 2 μM (e.g., not more than about 1.5 μM, not more than about 1 μM, not more than about 0.5 μM, or not more than about 0.2 μM).

[0156] Within about 24 hours after administration of methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.1 μM (e.g., exceeding about 0.2 μM, for example, exceeding about 0.5 μM, for example, exceeding about 1 μM). Within about 12 hours after administration of methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.1 μM (e.g., exceeding about 0.2 μM, for example, exceeding about 0.5 μM, for example, exceeding about 1 μM). Within about 6 hours after administration of methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.1 μM (e.g., exceeding about 0.2 μM, for example, exceeding about 0.5 μM, for example, exceeding about 1 μM). Within about 3 hours after administration of methotrexate, the plasma concentration of methotrexate in the individual may exceed about 0.1 μM (e.g., exceeding about 0.2 μM, for example, exceeding about 0.5 μM, for example, exceeding about 1 μM). Within about 2 hours after administration of methotrexate, the plasma concentration of methotrexate in the subject 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 the subject 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).

[0157] The S phase inhibitor (e.g., an antifolate, e.g., a DHFR inhibitor, e.g., methotrexate) can be administered orally, subcutaneously, intramuscularly, intravenously, intraarterially, or intrathecally each time. The S phase inhibitor can be methotrexate (MTX). For each individual, 15.6 mg / m2 can be administered on days 3, 5, 10, and 17 after each administration of immune cells. 2 For each individual, MTX can be administered at a dose of 31.25 mg / m on the 3rd, 5th, 10th, and 17th days after each administration of immune cells. 2 Or MTX was administered at a dose of 50 mg.

[0158] C. Lymphatic clearing agent

[0159] The methods described herein include, for example, administering a lymphodepleting agent (e.g., a chemotherapeutic agent for lymphodepleting) prior to administering the immune cells provided herein. Pre-treatment or pre-conditioning of the patient with a lymphodepleting agent (e.g., a chemotherapeutic agent for lymphodepleting, such as cyclophosphamide and / or fludarabine) prior to cell therapy improves the efficacy of cell therapy by reducing the number of endogenous host lymphocytes in the subject, thereby providing a better environment for the proliferation of the administered cells after administration to the subject.

[0160] The lymphodepleting agent used in the presently disclosed methods can be a biological lymphodepleting agent, a chemotherapeutic lymphodepleting agent, or a combination thereof. One or more biological and / or chemotherapeutic lymphodepleting agents can be included in the lymphodepleting regimen.

[0161] The biological lymphatic scavenger can be, for example, any biological material, such as an antibody, an antibody fragment, an antibody conjugate, etc., which can be administered as part of a lymphatic scavenging regimen to reduce endogenous lymphocytes in a subject for immunotherapy. Such a biological lymphatic scavenger can include, for example, a monoclonal antibody or a fragment thereof. In some instances, the biological lymphatic scavenger is specific for T cell antigens (i.e., antigens expressed on the cell surface of T cells). Examples of such antigens include, but are not limited to, CD52 and CD3. In a specific instance, the biological lymphatic scavenger is an antibody, such as a monoclonal antibody, that is specific for CD52. Such antibodies can include, for example, alemtuzumab (i.e., CAMPATH), ALLO-647 (Allogene Therapeutics, San Francisco, CA), a derivative thereof that binds CD52, or any other CD52 antibody. In another specific instance, the biological lymphatic scavenger is an antibody, such as a monoclonal antibody, that is specific for CD3. In some cases, the anti-CD3 antibody can be muromonab-CD3 (Orthoclone OKT3 TM ), otelizumab, telizumab, forerunner, visilizumab, or their derivatives specific for CD3.

[0162] The lympholysis scheme of the present application may include the administration of one or more chemotherapeutic lympholysis agents.Chemotherapeutic lympholysis agents may refer to non-biological materials, such as small molecules, which may be administered as part of a lympholysis scheme to reduce endogenous lymphocytes in a subject for use in immunotherapy. In some instances, chemotherapeutic lympholysis agents may be lympholysis agents, but non-myeloablative agents.Chemotherapeutic lympholysis agents may include those known in the art, including but not limited to purine analogs (e.g., fludarabine, pentostatin, azathioprine, mercaptopurine (e.g., 6-mercaptopurine), clofarabine, cladribine, and thiopurine (e.g., thioguanine)), and compounds capable of inducing DNA intrachain crosslinks (e.g., cisplatin, mitomycin C, carmustine, psoralen, or nitrogen mustard-derived alkylating agents (e.g., cyclophosphamide, ifosfamide, chlorambucil, uramustine, melphalan, and bendamustine)). Other non-limiting examples of chemotherapeutic lymphodepleting agents that can be used in the presently disclosed methods include daunorubicin, L-asparaginase, prednisone, dexamethasone, and nelarabine.

[0163] Examples of lymphoproliferative agents also include, but are not limited to, 5-fluorouracil, gemcitabine, dacarbazine, melphalan, doxorubicin, vinblastine, oxaliplatin, paclitaxel, docetaxel, irinotecan, etoposide phosphate, mitoxantrone, cladribine, denileukin, carboxysin or DAB-IL2. See, for example, WO 2018027135A1 and US10253086 B2, which are incorporated herein by reference. Table 1 below lists exemplary lymphoproliferative agents and their half-lives. In some cases, the lymphoproliferative agents discussed herein may be accompanied by low-dose radiation. The lymphoproliferative effects of the conditioning regimen can be monitored via conventional practice.

[0164] Table 1. Exemplary lymphodepleting agents.

[0165] The one or more lymphodepleting agents may be selected from the group consisting of cladribine, vinblastine, gemcitabine, doxorubicin, fluorouracil, irinotecan, paclitaxel, oxaliplatin, dacarbazine, melphalan, fludarabine, cyclophosphamide, and combinations thereof.

[0166] The one or more lymphodepleting agents may include fludarabine and cyclophosphamide. The method may include administering about 20-50 mg / m 2 (e.g., 25–30 mg / m 2 ) administer fludarabine intravenously to a subject for about 3 to about 5 days. The method may include administering about 250-1000 mg / m 2 Cyclophosphamide is administered intravenously to the subject for about 3 to about 5 days.

[0167] The method may include administering about 30 mg / m 2 Fludarabine is administered intravenously to the subject for 3 days, and starting with the first dose of fludarabine, approximately 300 mg / m2 per day. 2 Cyclophosphamide is administered intravenously to an individual for about 3 days. The first dose of fludarabine and cyclophosphamide can start about 5-7 days (e.g., about 5 days, about 6 days, or about 7 days) before administering the immune cells. The first dose of fludarabine and cyclophosphamide can start about 5 days before administering the immune cells. During the time period between about 7 days before administering the immune cells and about 90 days after administering the immune cells, fludarabine and / or cyclophosphamide can be administered at least twice or three times.

[0168] The method may include administering about 30 mg / m 2 Fludarabine is administered intravenously to the subject for 4 days, and starting with the first dose of fludarabine, approximately 300 mg / m2 per day. 2Cyclophosphamide is administered intravenously to the individual for about 4 days. The first dose of fludarabine and cyclophosphamide can start about 5-7 days (e.g., about 5 days, about 6 days, or about 7 days) before administering the immune cells. The first dose of fludarabine and cyclophosphamide can start about 5 days before administering the immune cells. During the time period between about 7 days before administering the immune cells and about 90 days after administering the immune cells, fludarabine and / or cyclophosphamide can be administered at least twice or three times.

[0169] The method may include administering about 30 mg / m 2 Fludarabine is administered intravenously to the subject for 3 days, and starting with the first dose of fludarabine, approximately 500 mg / m2 per day. 2 Cyclophosphamide is administered intravenously to an individual for about 3 days. The first dose of fludarabine and cyclophosphamide can start about 5-7 days (e.g., about 5 days, about 6 days, or about 7 days) before administering the immune cells. The first dose of fludarabine and cyclophosphamide can start about 5 days before administering the immune cells. During the time period between about 7 days before administering the immune cells and about 90 days after administering the immune cells, fludarabine and / or cyclophosphamide can be administered at least twice or three times.

[0170] The method may include administering about 30 mg / m 2 Fludarabine is administered intravenously to the subject for 4 days, and starting with the first dose of fludarabine, approximately 530 mg / m2 per day. 2 Cyclophosphamide is administered intravenously to an individual for about 3 days. The first dose of fludarabine and cyclophosphamide can start about 5-7 days (e.g., about 5 days, about 6 days, or about 7 days) before administering the immune cells. The first dose of fludarabine and cyclophosphamide can start about 5 days before administering the immune cells. During the time period between about 7 days before administering the immune cells and about 90 days after administering the immune cells, fludarabine and / or cyclophosphamide can be administered at least twice or three times.

[0171] An effective dose of one or more chemotherapeutic lymphodepleting agents can result in a decrease in one or more endogenous lymphocytes (e.g., B cells, T cells and / or NK cells) in a subject relative to a control (e.g., relative to a starting amount in a treated subject, relative to a predetermined threshold, or relative to an untreated 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%.

[0172] The lymphodepletion regimen administered during the methods of the present application can be administered prior to administering the pharmaceutical composition in an effective amount (i.e., an effective dose) that depletes or reduces the number of endogenous lymphocytes in the subject, for example, 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., relative to the starting amount in a subject receiving treatment, relative to a predetermined threshold, or relative to an untreated subject). The reduction in lymphocyte counts can be monitored using conventional techniques known in the art, such as by flow cytometry analysis of cells expressing characteristic lymphocyte surface antigens in blood samples drawn from a subject at different intervals during treatment with the antibody.

[0173] The lymphodepleting chemotherapeutic agent may comprise melphalan. Suitable 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 single dose (see (NDA) prescribing information). Each individual dose can be administered 1, 2, 3, 4 or more times per day for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or longer. Each individual dose can be repeated every 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or longer.

[0174] The lymphodepleting chemotherapeutic agent may comprise bendamustine. Suitable administration of bendamustine is known in the art. It may be administered at about 10 mg / m 2 / day to about 200 mg / m 2 Bendamustine may be administered in an amount of 1, 2, 3, 4 or more times per day for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or longer. 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 longer.

[0175] The lymphodepleting chemotherapeutic agent may comprise mercaptopurine. Suitable 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 Each individual dose can be administered 1, 2, 3, 4 or more times per day for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or longer. Each individual dose can be repeated every 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or longer.

[0176] The lymphodepleting chemotherapeutic agent may include daunorubicin. Suitable administration of daunorubicin is known in the art. It may be administered at about 10 to about 500 mg / m 2 The amount of daunorubicin (see the prescription information of daunorubicin) can be administered daily for 1, 2, 3, 4 or more times for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or longer. Each individual dose can be repeated every 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or longer.

[0177] The lymphodepleting chemotherapeutic agent may comprise cytarabine. Suitable administration of cytarabine is known in the art (see Prescribing information for cytarabine. 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 for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or longer. 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 longer.

[0178] The lymphodepleting chemotherapeutic agent may comprise L-asparaginase. Suitable administration of L-asparaginase is known in the art (see Prescribing information for the invention). L-asparaginase may be administered in an amount of about 100 I.U. / kg / day to about 1,500 IU / kg / day. Each individual dose may be administered 1, 2, 3, 4 or more times per day for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or longer. 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 longer. The lymphodepleting chemotherapeutic agent may comprise prednisolone or prednisone. Suitable administration of methotrexate is known in the art (see Prescribing information for the invention). Prednisolone or prednisone 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 for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or longer. 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 longer.

[0179] The lymphodepleting chemotherapeutic agent may comprise prednisolone or prednisone. Suitable administration of prednisolone or prednisone is known in the art (see Prescribing information for the invention). Prednisolone or prednisone 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 for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or longer. 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 longer.

[0180] The lymphodepleting chemotherapeutic agent may comprise nelarabine. Suitable administration of nelarabine is known in the art (see can be taken at about 500 mg / m 2 / day to about 2000mg / m 2 Nelarabine can be administered in an amount of 1, 2, 3, 4 or more times per day. Each individual dose can be administered 1, 2, 3, 4 or more times per day for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or longer. Each individual dose can be repeated every 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or longer.

[0181] The lymphodepleting agent may comprise rituximab. Suitable administration of rituximab is known in the art (see can be taken at about 100 mg / m 2 / day to about 3000mg / m 2 Rituximab may be administered in an amount of 1, 2, 3, 4 or more times per day. Each individual dose may be administered 1, 2, 3, 4 or more times per day for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or longer. 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 longer.

[0182] The lymphodepleting agent may comprise alemtuzumab.

[0183] Suitable administration of alemtuzumab is known in the art (see Prescribing information for the invention). Alemtuzumab may be administered in an amount of about 1 mg / day to about 30 mg / day. Each individual dose may be administered 1, 2, 3, 4 or more times per day for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month or longer. 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 longer.

[0184] As used herein, the terms "effective dose", "effective amount", "therapeutically effective dose" or "therapeutically effective amount", as used herein, refer to an amount sufficient to achieve a beneficial or desired biological and / or clinical outcome. In some cases, an effective dose of a lymphodepleting agent is sufficient to reduce endogenous lymphocytes in a subject; for example, to reduce one or more lymphocytes (e.g., B cells, T cells and / or NK cells) relative to a control (e.g., relative to a starting amount in a subject receiving treatment for a disease, condition or disorder, relative to a predetermined threshold, or relative to an untreated 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%. An effective dose may be equivalent to a suggested dose, a recommended dose, or a permitted dose (for adults or children) of a lymphodepleting agent provided in a drug label.

[0185] D. Immune cells

[0186] Methods described herein include administering immune cells to individuals in need. Immune cells can be used as adoptive cell therapy to treat individual diseases or disorders, such as cancer. Exemplary adoptive cell therapy includes, but is not limited to, tumor infiltrating lymphocytes (TIL), T cells (TCR-T) modified by T cell receptors (TCR), T cells modified by chimeric antigen receptors (CAR), natural killer (NK) cells, and hematopoietic stem cells (HSC), and dendritic cells (DC) or myeloid cell therapy. Immune cells can be resistant to S phase inhibitors (e.g., methotrexate). Immune cells can be engineered to express heterologous nucleic acids comprising methotrexate-resistant transgenes. Immune cells can express mutant DHFR genes encoding dihydrofolate reductase. Mutated DHFR genes can include L22F mutations and F31S mutations. Mutated DHFR genes can encode polypeptides comprising the amino acid sequence of SEQ ID NO:2. Immune cells can overexpress DHFR genes encoding dihydrofolate reductase.

[0187] Immune cells can be derived from a variety of cell types and cell sources. Cells from any mammalian species are contemplated herein, including but not limited to mice, rats, guinea pigs, rabbits, dogs, monkeys and people. Immune cells can be human cells. Immune cells can be autologous, that is, cells derived from individuals receiving immune cells. Immune cells can be isogenic (that is, donors and recipients are different individuals, but are identical twins). Immune cells can be allogeneic, that is, cells are obtained from or derived from donors belonging to the same species but different from individuals receiving immune cells. Allogeneic immune cells can be ready-made immune cells, which are pre-made, characterized, and can be used to apply immediately to patients. Allogeneic immune cells can be "universal" immune cells, which are derived from cells obtained from one or more donors or cell lines, and are used for adoptive cell therapy for other individuals of the same species.

[0188] Immune cells can be derived from primary cells. Immune cells can be primary cells separated from an individual. Immune cells can be propagated (e.g., proliferated and / or differentiated) from primary cells separated from an individual. Immune cells can belong to the hematopoietic lineage. Primary cells can be obtained from the thymus. Primary cells can be obtained from lymph or lymph nodes (e.g., tumor draining lymph nodes). Primary cells can be obtained from the spleen. Primary cells can be obtained from bone marrow. Primary cells can be obtained from blood, such as peripheral blood. Primary cells can be peripheral blood mononuclear cells (PBMC). Primary cells can be derived from plasma. Primary cells can be derived from tumors. Primary cells can be obtained from the mucosal immune system. Primary cells can be obtained from biopsy samples.

[0189] Immune cells can be derived from cell lines. Immune cells can be obtained from commercial cell lines. Immune cells can be propagated (e.g., proliferated and / or differentiated) from cell lines isolated from primary cells of individuals. Cell lines can be short-lived. Cell lines can be immortalized. Cell lines can be tumor cell lines, such as leukemia or lymphoma cell lines. Cell lines can be cell lines derived from PBMC. Cell lines can be stem cell lines. Cell lines can be NK-92NK92 ​​(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).

[0190] Immune cell can be immune cell or its progenitor cell.Exemplary immune cells that can be used for the present application include but are not limited to dendritic cells (including immature dendritic cells and mature dendritic cells), T lymphocytes (such as initial T cells, effector T cells, memory T cells, cytotoxic T lymphocytes, T helper 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.Immune cell subpopulations can be defined by the presence or absence of one or more cell surface markers known in the art (for example, CD3, CD4, CD8, CD19, CD20, CD11c, CD123, CD56, CD34, CD14, CD33, etc.).In the case where the pharmaceutical composition includes multiple immune cells, these immune cells can be a specific subpopulation of immune cell types, a combination of a subpopulation of immune cell types, or a combination of two or more immune cell types.Immune cells can be present in a homogeneous cell population. The immune cell may be present in a heterogeneous cell population that is enhanced in the immune cell. The immune cell may be a lymphocyte. The immune cell may not be a lymphocyte. The immune cell may be suitable for adoptive cell therapy. The immune cell may be a PBMC. The immune cell may be an immune cell derived from a PBMC. The immune cell may be a T cell. The immune cell may contain a CD4 + T cells (also called helper T cells). Immune cells can contain CD8 + T cells (also called cytotoxic T cells). Immune cells may include T cells expressing TCR α and TCR β chains (i.e., αβ T cells). Immune cells may include T cells expressing TCR γ and TCR δ chains (i.e., γδ T cells). Immune cells may include B cells. Immune cells may include NK cells. Immune cells may include NK-T cells. Immune cells may include dendritic cells (DC). Immune cells may include DC-activated T cells.

[0191] The immune cell may be a stem cell or derived from a stem cell. The stem cell may be a totipotent stem cell. The stem cell may be a pluripotent stem cell. The stem cell may be a unipotent stem cell. The stem cell may be a progenitor cell. The stem cell may be an embryonic stem cell. The stem cell may be a hematopoietic stem cell (HSC). The stem cell may be a mesenchymal stem cell. The stem cell may be an induced pluripotent stem cell (iPSC).

[0192] The immune cell can be a modified cell comprising one or more heterologous nucleic acid sequences. The modified immune cell can comprise 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 cell can comprise a single copy of a heterologous nucleic acid sequence. The modified immune cell can comprise multiple copies of a heterologous nucleic acid sequence.

[0193] Immune cells can be modified immune cells, and these modified immune cells include heterologous nucleic acids containing sequences encoding engineered receptors. Immune cells can be modified immune cells expressing two or more engineered receptors. Modified immune cells can express engineered receptors selected from the group consisting of: CAR, recombinant TCR, TAC receptors, TCR fusion proteins (TFPs) and combinations thereof. Modified immune cells can express CAR and TFP. Modified immune cells can express CAR and recombinant TCR. Modified immune cells can express CAR and TAC receptors. Modified immune cells can express recombinant TCR and TAC receptors.

[0194] The modified immune cell may further comprise a second engineered receptor that converts a negative signal into a positive signal, optionally wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain. For example, a signal switch or signal converter has been described, for example, in PCT / CN2022 / 087016 filed on April 15, 2022, which is incorporated herein 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 with SEQ ID NO: 11.

[0195] The modified immune cell may comprise an IL12p40 polypeptide, wherein the IL12p40 polypeptide is membrane-bound. The 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.

[0196] The modified immune cell may further include at least one other heterologous nucleic acid sequence, for example, a second heterologous nucleic acid sequence encoding an immunomodulator (e.g., a co-stimulatory molecule, a cytokine, a chemokine, and / or an immune checkpoint inhibitor). The heterologous nucleic acid sequence encoding an immunomodulator and the heterologous nucleic acid sequence encoding an engineered receptor may be operably connected to the same promoter or different promoters.

[0197] Immunomodulators expressed by heterologous nucleic acids include any protein-based or peptide-based agents that regulate (e.g., inhibit or activate) the immune system. Immunomodulators can target specific molecules, such as checkpoint molecules, or non-specifically regulate immune responses. Activators can include molecules that activate antigen presenting cells to stimulate cellular immune responses. For example, activators can be immunostimulatory peptides. Activators can include but are not limited to toll-like receptors TLR-2, 3, 4, 6, 7, 8 or 9, granulocyte macrophage colony stimulating factor (GM-CSF), TNF, CD40L, CD28, FLT-3 ligands or cytokines (e.g., IL-1, IL-2, IL-4, IL-7, IL-12, IL12p40, IL-15, IL-23 or IL-21). Activators can include agonists of receptors (including co-stimulatory receptors) on activation T cells, such as agonists (e.g., agonists of CD28, OX40, GITR, CD137, CD27, CD40 or HVEM (e.g., agonist antibodies). Activators can also include proteins that suppress the activity of immunosuppressants, such as inhibitors of immunosuppressants IL-10, IL-35, TGF-β, IDO, or proteins that suppress the activity of immune checkpoints, such as CTLA-4, PD-1, PD-L1, PD-L2, LAG-3, B7-1, B7-H3, B7-H4, BTLA, VISTA, KIR, A2aR or TIM-3 antagonists (e.g., antagonistic antibodies). Activators can also include costimulatory molecules, such as CD40, CD80 or CD86. Immunomodulators can also include agents that lower the immune system, such as antibodies for 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 optimal immune response.

[0198] The modified immune cells may comprise a heterologous nucleic acid encoding an exogenous negative regulator (Nef) protein, e.g., wild-type Nef (e.g., wild-type SIV Nef) or a mutant Nef (e.g., SIV Nef M116). Nef-containing immune cells have been described, for example, in PCT / CN2020 / 112181 and PCT / CN2020 / 112182, both filed on August 28, 2020, which are incorporated herein by reference in their entirety. The Nef protein may comprise 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 to SEQ ID NO: 18. SEQ ID NO: 18 (SIV Nef M116 sequence) MGSSNSKRQQQGLLKLWRGLRGKPGADWVLLSDPLIGQSSTVQEECGKALKKSWGKGKMTPDGRRLQEGDTFDEWDDDEEEVGFPVQPRVPLRQMTYKLAVDFSHFLKSKGGLDGIYYSERREKILNLYALNEWGIIDDWQAYSPGPGIRYPRVFGFCFKLVPVDLHEEARNCERHC AA HPAQMGEDPDGIDHGEVLVWKFDPKLAVEYRPDMFKDMHEHAKR

[0199] Immune cells may not have any genetic modification (e.g., genomic modification) that reduces the immunogenicity of immune cells in individuals. Immune cells may have one or more genetic modifications (e.g., genomic modification) that reduce the immunogenicity of immune cells in individuals. Genetic modification may include destroying the TCR genes and / or HLA class I loci of allogeneic T cells in terms of genes. Genetic modification may include knocking out endogenous TCR genes, for example, TRAC (i.e., TCRα), TRBC (i.e., TCRβ), TCRG (i.e., TCRγ), and / or TCRD (i.e., TCRδ) genes. Genetic modification may include knocking out b2-microglobulin (B2M). Genetic modification may include knocking out immune checkpoint molecules, such as PD-1 or CTLA-4 (also referred to as CD52). Genetic modification may make immune cells resistant to CD25 inhibitors. For example, genetic modification knocks out IL2RA (also referred to as CD25). Immune cells may have a functional IL2RA gene. The IL2RA gene in the genome of immune cells may not be modified. Immune cells may have modifications to the IL2RA gene.

[0200] The immune cell may be a T cell, such as an allogeneic T cell. The immune cell may comprise a TCR α β +T cells. The immune cell may be a CAR-T cell. The immune cell may express an anti-BCMA CAR. The immune cell may be a CAR-T cell expressing an anti-BCMA CAR and a Nef protein. The immune cell may express an anti-CD20 CAR. The immune cell may be a CAR-T cell expressing an anti-CD20 CAR and a Nef protein. The immune cell may express an anti-CLL1 CAR. The immune cell may be a CAR-T cell expressing an anti-CLL1 CAR and a Nef protein. The immune cell may be a TCR-T cell. The immune cell may be a T cell expressing a TAC receptor. The immune cell may be a T cell expressing TFP. The immune cell may be a T cell expressing a combination of engineered receptors selected from the group consisting of: CAR, TCR, TAC receptor, and TFP. The therapeutic T cell may include an endogenous TCR. The therapeutic T cell may not have a genetic modification that reduces its immunogenicity. The endogenous TCR genes, HLA genes (e.g., B2M) and immune checkpoint molecules (e.g., PD-1, CTLA-4, etc.) of the therapeutic T cells may not be genetically modified. The therapeutic T cells may not have genetic modifications other than the engineered receptor constructs.

[0201] The immune cell may be an allogeneic CAR-T cell, such as a UCART cell. The UCART cell may contain more than 90%, such as more than about 95% or more than about 97% TCRαβ - T cells. UCART cells may contain less than about 10%, or less than about 5%, or less than about 3% TCRαβ + T cells. UCART cells have been described in, for example, WO 2013 / 176915 A1, WO2016069283 A1, WO 2019 / 129850 A1, WO 2019 / 089650A1, and CA 2874609A1, which are incorporated herein by reference in their entirety.

[0202] The immune cell may be a γδ T cell. The immune cell may be a γδ T cell expressing one or more engineered receptors selected from the group consisting of: CAR, TCR, TAC receptor and TFP.

[0203] The immune cell may be an HSC, such as an allogeneic HSC. The immune cell may be an HSC without genetic modification. The immune cell may be an HSC expressing one or more therapeutic agents. The immune cell may be an HSC expressing one or more engineered receptors selected from the group consisting of: CAR, TCR, TAC receptor, and TFP. The immune cell may be an HSC expressing one or more therapeutic agents other than an engineered receptor. The immune cell may comprise CD34 +HSC. Immune cells can contain CD34 - HSC. The immune cells may comprise at least about 80%, 85%, 90% or 95% CD34 + HSC. The immune cells may contain no more than about 20%, 15%, 10% or 5% CD34 - HSC. The immune cell may be an HSC transduced with a viral vector, such as a retrovirus or lentivirus transduced HSC.

[0204] The immune cell may be a NK cell. The immune cell may be a NK cell expressing one or more engineered receptors selected from the group consisting of: CAR, TCR, TAC receptor and TFP.

[0205] The immune cell may be a NK-T cell. The immune cell may be a NK-T cell expressing one or more engineered receptors selected from the group consisting of: CAR, TCR, TAC receptor and TFP.

[0206] Nucleic acids comprising heterologous nucleic acid sequences as described herein can be transiently or stably incorporated into modified immune cells. Nucleic acids can be transiently expressed in modified immune cells. For example, nucleic acids can be present in the nucleus of modified immune cells in the form of extrachromosomal arrays. Any transfection or transduction method known in the art (including viral or non-viral methods) can be used to introduce nucleic acids into modified immune cells. Exemplary non-viral transfection methods include, but are not limited to, chemical-based transfection, such as using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethyleneimine); non-chemical methods, such as electroporation, cell extrusion, sonoporation, optical transfection, puncture infection, protoplast fusion, hydrodynamic delivery, or transposon; particle-based methods, such as using gene guns, magnetic transfection or magnetic-assisted transfection, particle bombardment; and hybrid methods, such as nuclear transfection.

[0207] The heterologous nucleic acid sequence may be present in the genome of the modified immune cell. For example, the nucleic acid comprising the heterologous nucleic acid sequence may be integrated into the genome of the modified immune cell by any method known in the art, including but not limited to virus-mediated integration, random integration, homologous recombination methods, and site-directed integration methods, such as using site-specific recombinases or integrases, transposases, transcription activator-like effector nucleases, or other methods. CRISPR / Cas9 and zinc finger nucleases. The heterologous nucleic acid sequence can be integrated into a specifically designed locus of the modified immune cell genome. The heterologous nucleic acid sequence can be integrated into an integration hotspot of the modified immune cell genome. The heterologous nucleic acid sequence can be integrated into a random locus of the modified immune cell genome. In the case where multiple copies of the heterologous nucleic acid sequence are present in a single modified immune cell, the heterologous nucleic acid sequence can be integrated into multiple loci of the modified immune cell genome.

[0208] Precursor immune cells can be prepared using a variety of methods known in the art. For example, primary immune cells (such as T cells) can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue and tumors. Precursor immune cells can be prepared using a variety of techniques known in the art (such as FICOLL TM Immune cells (such as T cells) are obtained from blood units collected from individuals by separation. Cells from individual circulating blood can be obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells and platelets. Cells collected by apheresis can be washed to remove the plasma portion, and the cells are placed in an appropriate buffer or culture medium for subsequent processing steps. Cells can be washed with phosphate buffered saline (PBS) or a washing solution lacking divalent cations (such as calcium and magnesium). As will be readily understood by those of ordinary skill in the art, the washing step can be completed by methods known to those skilled in the art, such as by 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 Ca-free 2+ , Mg-free 2+ Alternatively, undesirable components of the aliquot can be removed and the cells resuspended directly in culture medium.

[0209] This can be done, for example, by PERCOLL TM Primary T cells are isolated from peripheral blood lymphocytes by gradient centrifugation or by counterflow centrifugal elutriation to lyse erythrocytes and deplete monocytes. Specific subsets of T cells, such as CD3 + 、CD28 + 、CD4 + 、CD8 +, CD45RA and CD45RO cells. For example, in one embodiment, by conjugating beads such as anti-CD3 / anti-CD28 (i.e., 3x28) T cells are isolated by incubating with M-450 CD3 / CD28 T cells for a period of time sufficient to positively select the desired T cells.

[0210] The T cell population can be further enriched by negative selection using a combination of antibodies against surface markers unique to the negatively selected cells. For example, one method involves cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using 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 cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich or positively select for cells that typically express CD4 + 、CD25 + 、CD62L hi GITR + and FoxP3 + of regulatory T cells.

[0211] Methods for introducing vectors or nucleic acids into therapeutic cells (such as precursor immune cells) are known in the art. Vectors or nucleic acids can be transferred into therapeutic cells by physical, chemical or biological methods.

[0212] Physical methods for introducing vectors or nucleic acids into therapeutic cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. 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.

[0213] Biological methods for introducing vectors or nucleic acids into therapeutic cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, such as human, cells.

[0214] Chemical methods for introducing vectors or nucleic acids into therapeutic cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles and liposomes. An exemplary colloidal system used as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0215] Transduced or transfected precursor immune cells can be propagated in vitro after the introduction of heterologous nucleic acid. Transduced or transfected precursor immune cells can be cultured to propagate 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 no more than 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 further evaluated or screened to select modified immune cells.

[0216] Immune cells can be propagated in culture in the presence of IL-2. The culture may include IL-2 at a concentration of at least about 50 IU / mL (including, for example, at least about 75, 100, 125, 150, 200, 250, 300, 350, 400, 500 or any one of higher IU / mL). The culture may include 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 include IL-2 at a concentration of about 300 IU / mL. Clinical data show that 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.

[0217] Reporter gene can be used for identifying the cell that may be transfected and the function of evaluating the regulatory sequence.Usually, reporter gene is a gene that does not exist or is not expressed in a receptor organism or tissue, and the reporter gene encodes a polypeptide, and the expression of the polypeptide is expressed by some characteristics (e.g., enzymatic activity) that are easy to detect.The expression of reporter gene is measured at the appropriate time after DNA is introduced into the receptor cell.Suitable reporter gene can include a gene or a green fluorescent protein gene (e.g., Ui-Tei et al. FEBS Letters 479:79-82 (2000)) encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase.

[0218] Other methods for confirming the presence of heterologous nucleic acid in precursor immune cells include, for example, molecular biological assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; biochemical assays, such as detecting the presence or absence of specific peptides by immunological methods (such as ELISA and Western blotting).

[0219] These methods may include administering immune cells as described herein. These methods may include administering immune cells more than once. In such cases, unless otherwise stated, the relative time description in this application (e.g., about 5 days before administering immune cells, such as within 90 days of administering immune cells) refers to the time point of administering immune cells for the first time. The method may include administering immune cells at least about twice, three times, four times or five times. An individual may undergo at least two, three, four or five administrations of immune cells. Multiple administrations of immune cells may occur within about 20 days, 30 days, 40 days, 50 days, 60 days, 70 days, 80 days, 90 days, 100 days, 110 days or 120 days.

[0220] The immune cells can be administered intravenously. An effective amount of immune cells can be about 10 5 to about 10 9 cells / kg. 4 , 10 5 , 10 6 , 10 7 , 10 8 or 10 9 The immune cells may be administered at a dose of about 10 cells / kg body weight. 4 to about 10 5 , about 10 5 to about 10 6 , about 10 6 to about 10 7 , about 10 7 to about 10 8 , about 10 8 to about 10 9 , about 10 4 to about 10 9 , about 10 4 to about 10 6 , about 10 6 to about 10 8 or about 10 5 to about 10 7 The immune cells may be administered at a dose of about 0.5×10 cells / kg body weight. The immune cells may be modified immune cells. The effective amount of immune cells may be about 0.5×10 6 to 1.5×10 6 The effective amount of immune cells can 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.

[0221] Immune cells (e.g., for each administration) can include about 30 million to about 900 million cells. Immune cells (e.g., for each administration) can include 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. Immune cells (e.g., for each administration) can include 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. Immune cells (e.g., for each administration) can include no more than about 30 million, 40 million, 50 million, 60 million, 70 million, 80 million, 90 million or 100 million cells. The immune cells (e.g., for each administration) can comprise no more than 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.

[0222] The application also provides immune cells as described herein. Immune cells can be resistant to S phase inhibitors (e.g., methotrexate). Immune cells can be engineered to express heterologous nucleic acids comprising a mutant DHFR gene encoding dihydrofolate reductase. The mutant DHFR gene can include L22F mutations and F31S mutations. Immune cells can include engineered receptors. Immune cells can further include a second engineered receptor that converts a negative signal into a positive signal, optionally wherein the second engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain.

[0223] The present application also provides immune cells as described herein. The immune cell may comprise an IL12p40 polypeptide. The immune cell may be engineered by introducing an IL-12p40 polypeptide into a cell, wherein the IL-23p19 subunit is not introduced into the cell. The immune cell may express an exogenously introduced IL-23p40 polypeptide, but not an exogenously introduced IL-23p19 subunit. The immune cell may be engineered to comprise an IL-12p40 polypeptide, but not an IL-23p19 subunit. The immune cell may produce IL-23 upon activation. The IL-23 produced by the immune cell may not be secreted. The IL12p40 polypeptide may be membrane-bound. The 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 higher) sequence identity with SEQ ID NO: 12.

[0224] The application also provides nucleic acids described herein. The nucleic acid may include a mutant DHFR gene encoding a dihydrofolate reductase. The nucleic acid may include a polynucleotide sequence encoding an engineered receptor (eg, CAR). The nucleic acid may include a polynucleotide sequence encoding an engineered receptor that converts a negative signal into a positive signal, optionally wherein the engineered receptor includes a TGFβR extracellular domain and an IL-23 receptor intracellular domain. The nucleic acid may include a polynucleotide sequence encoding an IL12p40 polypeptide described herein. The nucleic acid may include a nucleic acid sequence shown in any one of SEQ ID NOs: 4-7, 13, and 15. The application also provides a vector comprising a nucleic acid described herein.

[0225] E. Engineered receptors

[0226] The immune cells described herein may express one or more engineered receptors. Exemplary engineered receptors include, but are not limited to, CAR, recombinant TCR, TAC receptors, and TFP. The engineered receptor may include an extracellular domain, a transmembrane domain, and an intracellular signaling domain that specifically binds to an antigen (e.g., a tumor antigen). The intracellular signaling domain may include a primary intracellular signaling domain and / or a co-stimulatory domain. The intracellular signaling domain may include an intracellular signaling domain of a TCR co-receptor. The engineered receptor may be encoded by a heterologous nucleic acid operably linked to a promoter (e.g., a constitutive promoter or an inducible promoter). The protein may be inserted into the cell membrane while the cell is passed through a microfluidic system (e.g., CELL ), introducing engineered receptors into modified immune cells (see, for example, U.S. Patent Application Publication No. 20140287509). Engineered receptors can enhance the function of immune cells, for example, by targeting immune cells (e.g., modified immune cells), by transducing signals, and / or by enhancing the cytotoxicity of immune cells (e.g., modified immune cells). Immune cells may not express engineered receptors, such as CAR, TCR, TAC receptors or TFP.

[0227] The engineered receptor may comprise 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.

[0228] The engineered receptor can 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. CAR can also be constructed to be specific to any cell surface marker by using, for example, an antigen binding fragment of an antibody molecule or an antibody variable domain. Any method for producing CAR can be used herein. See, for example, US 6,410,319, US 7,446,191, US 7,514,537, US 9765342B2, WO 2002 / 077029, WO 2015 / 142675, US2010 / 065818, US2010 / 025177, US2007 / 059298, WO 2017 / 025038 A1 and Berger C et al., J.Clinical Investigation 118: 1294-308 (2008), these documents are hereby incorporated by reference.

[0229] CAR may include an extracellular domain, a transmembrane domain, and an intracellular signaling domain, the extracellular domain including at least one targeting domain specifically bound to at least one tumor antigen. The intracellular signaling domain may generate a signal that promotes the immune effector function of CAR-containing cells (e.g., CAR-modified T cells (including αβT or γδT cells), NK cells, or NKT cells). "Immune effector function or immune effector response" refers to a function or response of, for example, immune effector cells that enhance or promote the immune attack of target cells. For example, immune effector function or response may refer to the characteristics of T or NK cells that promote killing or inhibiting the growth or proliferation of target cells. Examples of immune effector functions, such as in CAR-T cells, include cytolytic activity (such as antibody-dependent cellular toxicity, or ADCC) and auxiliary activity (such as secretion of cytokines). CAR may have an intracellular signaling domain having a weakened immune effector function. Compared to a CAR with full-length and wild-type CD3ζ and optionally one or more costimulatory domains, the CAR may have the following intracellular signaling domain, which has no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of any one of the immune effector functions (e.g., cytolytic function for target cells). The intracellular signaling domain can generate a signal that promotes the proliferation and / or survival of CAR-containing cells. CAR may include 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 (intracellular) (i.e., intracellular (cytoplasmic)) portion of a molecule or its fragment or derivative or the entire native intracellular signaling domain.

[0230] The intracellular signaling domain of CAR may include a primary intracellular signaling domain. "Primary intracellular signaling domain" refers to an intracellular signaling sequence that acts in a stimulatory manner to induce immune effector 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 a 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 a non-functional or attenuated 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. A non-functional or attenuated signaling domain may be a mutant signaling domain having a point mutation, insertion, or deletion that attenuates or eliminates one or more immune effector functions, such as cytolytic activity or auxiliary activity, including antibody-dependent cellular toxicity (ADCC). CAR may include a non-functional or attenuated CD3ζ (ie, CD3ζ or CD3z) signaling domain. The intracellular signaling domain may not include a primary intracellular signaling domain. The attenuated primary intracellular signaling domain may induce no more than about 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10% or less of any of the immune effector functions (e.g., cytolytic function against target cells) compared to a CAR having the same construct but having a wild-type primary intracellular signaling domain.

[0231] The intracellular signaling domain of CAR may include one or more (e.g., any one of 1, 2, 3 or more) costimulatory domains. A "costimulatory domain" may be the intracellular portion of a costimulatory molecule. The term "costimulatory molecule" refers to a cognate binding partner on an immune cell (such as a T cell) that specifically binds to a costimulatory ligand to mediate a costimulatory response of an immune cell, such as, but not limited to, proliferation and survival. Costimulatory molecules are cell surface molecules that contribute to an efficient immune response other than an antigen receptor or its ligand. Costimulatory molecules may be represented by the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors. Costimulatory molecules include, but are not limited to, MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). Additional examples of such co-stimulatory molecules include 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, L FA-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, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a and ligands that specifically bind to CD83.

[0232] CAR may include a single costimulatory domain. CAR may include two or more costimulatory domains. The intracellular signaling domain may include a functional primary intracellular signaling domain and one or more costimulatory domains. CAR may not include a functional primary intracellular signaling domain (e.g., CD3ζ). CAR may include an intracellular signaling domain consisting of one or more costimulatory domains or consisting essentially of one or more costimulatory domains. CAR may include an intracellular signaling domain consisting of a non-functional or weakened primary intracellular signaling domain (e.g., mutant CD3ζ) and one or more costimulatory domains or consisting essentially of a non-functional or weakened primary intracellular signaling domain (e.g., mutant CD3ζ) and one or more costimulatory domains. After the targeting domain binds to the tumor antigen, the costimulatory domain of CAR can transduce signals to enhance the proliferation, survival and differentiation of engineered immune cells (such as T cells) with CAR, and inhibit activation-induced cell death. The one or more co-stimulatory signaling domains can be derived from one or more molecules selected from the group consisting of 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 a ligand that specifically binds to CD83.

[0233] The intracellular signaling domain of CAR may include a costimulatory signaling domain derived from CD28. The intracellular signaling domain may include an intracellular signaling domain of CD3ζ and a costimulatory signaling domain of CD28. The intracellular signaling domain in the chimeric receptor of the present application may include a costimulatory signaling domain derived from 4-1BB (i.e., CD137). The intracellular signaling domain may include an intracellular signaling domain of CD3ζ and a costimulatory signaling domain of 4-1BB.

[0234] The intracellular signaling domain of CAR may include a costimulatory signaling domain of CD28 and a costimulatory signaling domain of 4-1BB. The intracellular signaling domain may include an intracellular signaling domain of CD3ζ, a costimulatory signaling domain of CD28, and a costimulatory signaling domain of 4-1BB. The intracellular signaling domain may include a polypeptide comprising, from N-terminus to C-terminus: a costimulatory signaling domain of CD28, a costimulatory signaling domain of 4-1BB, and an intracellular signaling domain of CD3ζ.

[0235] The CAR may comprise a polypeptide comprising, from N-terminus to C-terminus: a CD8 leader sequence, an extracellular binding domain, a CD8 hinge, a CD8 transmembrane, a 4-1BB intracellular co-stimulatory domain, and a CD3ζ intracellular signaling domain.

[0236] CAR can be a chimeric antigen receptor containing a chimeric signaling domain ("CMSD"), wherein the CMSD comprises an ITAM (also referred to herein as a "CMSD ITAM") and an optional linker (also referred to herein as a "CMSD linker"), and these ITAMs and optional linkers are arranged in a configuration different from any naturally occurring parent molecule containing ITAMs. For example, the CMSD may comprise two or more ITAMs directly connected to each other. The CMSD may comprise an ITAM connected by one or more "heterologous linkers", the one or more "heterologous linkers" being the following linker sequences, which are not derived from a parent molecule containing ITAMs (e.g., a G / S linker), or derived from a parent molecule containing ITAMs different from a parent molecule containing ITAMs from which one or more CMSD ITAMs are derived. The CMSD may comprise two or more (such as 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. CMSD may not include CD3ζITAM1 and / or CD3ζITAM2. At least one of CMSDITAMs may be CD3ζITAM3. CMSD may not include any ITAM from CD3ζ. At least two of CMSD ITAMs may be derived from the same parent molecule containing ITAM. CMSD may include two or more (such as 2, 3, 4 or more) ITAMs, wherein at least two of CMSD ITAMs are each derived from different parent molecules containing ITAM. At least one of CMSDITAMs may be derived from a parent molecule containing ITAM selected from the group consisting of: CD3ε, CD3δ, CD3γ, Igα (CD79a), Igβ (CD79b), FcεRIβ, FcεRIγ, DAP12, CNAIP / NFAM1, STAM-1, STAM-2 and membrane spike protein.

[0237] The CAR may comprise a polypeptide comprising, from N-terminus to C-terminus, a CD8 leader sequence, an extracellular binding domain, a CD8 hinge, a CD8 transmembrane, a 4-1BB intracellular co-stimulatory domain, and one or more ITAM sequences.

[0238] The targeting domain of CAR can be an antibody or antibody fragment, such as scFv, Fv, Fab, (Fab')2, single domain antibody (sdAb), or V H H domain. The targeting domain of CAR can be a ligand or extracellular portion of a receptor that specifically binds to an antigen (e.g., a tumor antigen). One or more targeting domains of CAR can specifically bind to a single tumor antigen. CAR can be a bispecific or multispecific CAR with a targeting domain that binds to two or more tumor antigens. The antigen (e.g., tumor antigen) can be selected from the group consisting of: 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, fetal acetylcholine receptor, folate receptor-α, GD3, hTERT, IL-13R-α2, kappa-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, p53, MART1, GP100, proteinase-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 clinically significant tumor antigens, and combinations thereof.

[0239] Many CARs targeting different tumor antigens have been widely disclosed in the art, such as anti-CD19CAR, anti-BCMACAR, anti-claudin 18.2CAR or anti-CD20CAR. The extracellular antigen binding domain of CD19 CAR can be or include CD19 binding fragments (e.g., FMC63, SJ25C1 or those disclosed in different patents such as WO 2022 / 012683, etc.). BCMACAR has also been fully described, and related patents include but are not limited to WO 2016 / 014789, WO 2016 / 014565, WO 2013 / 154760 and WO 2018 / 028647, etc.

[0240] The CAR may be an anti-BCMA CAR. A variety of antigen binding domain sequences may be used as the targeting domain of the CAR. See, for example, WO 2018028647 and WO 2021121228, which are incorporated herein in their entirety. The extracellular antigen binding domain of the 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, such as a V H H. The BCMA binding fragment may 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.

[0241] The CAR may be an anti-claudin 18.2 CAR. A variety of antigen binding domain sequences may be used as the targeting domain of the CAR. See, e.g., WO 2021 / 129765, which is incorporated herein in its entirety. The CAR may comprise an anti-claudin 18.2 scFv. The CAR may comprise an anti-claudin 18.2 sdAb, e.g., V H H. The CAR may be an ITAM-modified claudin 18.2 CAR comprising the sequence of SEQ ID NO:9.

[0242] The CAR may be an anti-CD20 CAR. The CAR may comprise an anti-CD20 scFv. The anti-CD20 scFv may be derived from an anti-CD20 antibody, such as rituximab (e.g., ) or Leu16. The CAR may be an ITAM-modified CD20 CAR comprising the sequence of SEQ ID NO: 8.

[0243] SEQ ID NO: 8 (ITAM010 modified CD20 CAR; CD8αSP-CD20 scFv (Leu16)-CD8α hinge-CD8αTM-4-1BB-ITAM010 amino acid sequence; CD8αSP is italicized, CD8α hinge is squared, CD8αTM is italicized, intracellular 4-1BB is underlined, and ITAM010 is bold)

[0244] The transmembrane domain of the CAR may comprise a transmembrane domain selected from the group consisting of: α, β or ζ chain of a 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, CD 19. IL-2Rβ, IL-2Rγ, IL-7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGA M, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D and / or NKG2C transmembrane domain. The transmembrane domain of CAR can be CD4, CD3, CD8α or CD28 transmembrane domain. The transmembrane domain of CAR can include the transmembrane domain of CD8α.

[0245] The extracellular domain can be connected to the transmembrane domain via a hinge region. In one embodiment, the hinge region comprises the hinge region of CD8α.

[0246] The CAR may comprise a signal peptide, such as CD8αSP.

[0247] Any CAR known in the art or developed by the inventors, including CARs described in PCT / CN2017 / 096938 and PCT / CN2016 / 094408 (the contents of these documents are incorporated herein by reference in their entirety), can be used in the methods described herein. An exemplary structure of CAR is shown in Figures 15A-15D of PCT / CN2017 / 096938.

[0248] The engineered receptor can be a recombinant T cell receptor. The recombinant TCR can be specific for an antigen (e.g., a tumor antigen). The antigen (e.g., a tumor antigen) can 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, kappa-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, p53, MART1, GP100, proteinase-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. Tumor antigens can be derived from intracellular proteins of tumor cells. Many TCRs with specificity for tumor antigens (including tumor-associated antigens) have been described, including, for example, NY-ESO-1 cancer-testis antigen, p53 tumor suppressor antigen, TCRs for tumor antigens in melanoma (e.g., MARTI, gp 100), leukemia (e.g., WT1, minor histocompatibility antigens) and breast cancer (e.g., HER2, NY-BR1). Any TCR known in the art can be used in the present application. The TCR can have an enhanced affinity for tumor antigens. For example, exemplary TCRs and methods for introducing these TCRs into immune cells have been described in US 5830755 and Kessels et al. Immunotherapy through TCR gene transfer. Nat. Immunol. 2, 957-961 (2001). The immune cell can be a TCR-T cell.

[0249] The TCR receptor complex is an octameric complex formed by the 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. The ionizable residues in the transmembrane domain of each subunit form an interacting polar network that holds the complex together. The TCR complex has the function of activating the signaling cascade in T cells.

[0250] The engineered receptor can be an engineered TCR comprising one or more T cell receptor (TCR) fusion proteins (TFP). For example, exemplary TFPs have been described in US20170166622 A1, which is incorporated herein by reference. TFP may include the extracellular domain of a TCR subunit, which includes an extracellular domain or a portion thereof of a protein selected from the group consisting of: TCR α chain, TCR β chain, CD3εTCR subunit, CD3γTCR subunit, CD3δTCR subunit, its functional fragment and its amino acid sequence with at least one but no more than 20 modifications. TFP may include a transmembrane domain, which includes a transmembrane domain of a protein selected from the group consisting of: TCR α chain, TCR β chain, CD3εTCR subunit, CD3γTCR subunit, CD3δTCR subunit, its functional fragment and its amino acid sequence with at least one but no more than 20 modifications. The TFP may comprise a transmembrane domain comprising a transmembrane domain of a protein selected from the group consisting of: TCRα chain, TCRβ chain, TCRζ chain, CD3εTCR subunit, CD3γTCR subunit, CD3δTCR subunit, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, functional fragments thereof, and amino acid sequences thereof having at least one but no more than 20 modifications.

[0251] The TFP may comprise a TCR subunit and an antigen binding domain, wherein the TCR subunit comprises at least a portion of a TCR extracellular domain and a TCR intracellular domain comprising a stimulatory domain from an intracellular signaling domain of CD3ε; 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.

[0252] The TFP may comprise a TCR subunit and an antigen binding domain, the TCR subunit comprising at least a portion of a TCR extracellular domain and a TCR intracellular domain comprising a stimulatory domain from an 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.

[0253] The TFP may comprise a TCR subunit and an antigen binding domain, the TCR subunit comprising at least a portion of a TCR extracellular domain and a TCR intracellular domain comprising a stimulatory domain from an 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.

[0254] The TFP may comprise a TCR subunit and an antigen binding domain, the TCR subunit comprising at least a portion of a TCR extracellular domain and a TCR intracellular domain containing a stimulatory domain from an 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.

[0255] The TFP may comprise a TCR subunit and an antigen binding domain, wherein the TCR subunit comprises at least a portion of a TCR extracellular domain and a TCR intracellular domain comprising a stimulatory domain from an 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.

[0256] The engineered receptor may be a T cell antigen conjugate (TAC) receptor. Exemplary TAC receptors have been described, for example, in US 20160368964A1, which is incorporated herein by reference. The TAC may comprise 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 HH. The targeting domain can be a designed ankyrin repeat (DARPin) polypeptide. The antigen (e.g., a tumor antigen) can 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, kappa-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, p53, MART1, GP100, proteinase-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. The protein associated with the TCR complex can be CD3, such as CD3ε. The TCR binding domain can be a single chain antibody, such as scFv or V H H. The TCR binding domain can be derived from UCHT1. The TAC receptor can include a cytoplasmic domain and a transmembrane domain. The T cell receptor signaling domain can include a cytoplasmic domain derived from a TCR co-receptor. Exemplary TCR co-receptors include but are not limited to CD4, CD8, CD28, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD 154. The TAC receptor can include a transmembrane domain and a cytoplasmic domain derived from CD4. The TAC receptor can include a transmembrane domain and a cytoplasmic domain derived from CD8 (e.g., CD8α).

[0257] T cell co-receptors are expressed as membrane proteins on T cells. They provide stability to the TCR:peptide:MHC complex and facilitate signal transduction. The two subtypes of T cell co-receptors, CD4 and CD8, show strong specificity for specific MHC types. The CD4 co-receptor can only stabilize the TCR:MHC II complex, while the CD8 co-receptor can only stabilize the TCR:MHC I complex. The differential expression of CD4 and CD8 on different T cell types results in different functional subsets of T cells. + The T cells are cytotoxic T cells.

[0258] CD4 is a glycoprotein expressed on the surface of immune cells (such as helper T cells, monocytes, macrophages and dendritic cells). CD4 has four immunoglobulin domains (D1 to D4) exposed to the extracellular cell surface. CD4 contains a specific amino acid sequence on its short intracellular (cytoplasmic / intracellular) tail, which allows the CD4 tail to recruit tyrosine kinase Lck and interact with the tyrosine kinase Lck. When the TCR complex and CD4 each bind to different regions of the MHC II molecule, the close proximity between the TCR complex and CD4 allows the Lck bound to the intracellular tail of CD4 to tyrosine phosphorylate the immunoreceptor tyrosine activation motif (ITAM) on the intracellular domain of CD3, thereby amplifying the signal generated by TCR.

[0259] CD8 is a glycoprotein that is a homodimer of two α chains (less common) or a heterodimer of one α chain and one β chain (more common), each of which contains an immunoglobulin variable (IgV)-like extracellular domain and an intracellular tail that are attached to the membrane by a thin stalk. CD8 is primarily expressed 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, which phosphorylates the intracellular CD3 and ζ chains of the TCR complex once the TCR binds to its specific antigen. Tyrosine phosphorylation on the intracellular CD3 and ζ chains initiates a phosphorylation cascade that ultimately leads to gene transcription.

[0260] Immune cells can express more than one engineered receptor, such as any combination of CAR, recombinant TCR, TAC receptor, and TFP.

[0261] 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 can trigger an immune response, particularly a T cell-mediated immune response. The selection of the targeted antigen of the present application will depend on the specific type of cancer to be treated. Exemplary tumor antigens include, e.g., glioma-associated antigen, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-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 enzymes, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostaglandins, PSMA, HER2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2 (ephrinB2), CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, and mesothelin.

[0262] Tumor antigens can include one or more antigen cancer epitopes associated with malignant tumors. Malignant tumors express many proteins that can be used as target antigens for immune attack. 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 transformation-related molecule groups, such as oncogenes HER2 / Neu / ErbB-2. Another group of target antigens are carcinoembryonic antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphoma, tumor-specific idiotype immunoglobulins constitute true tumor-specific immunoglobulin antigens unique to individual tumors. B-cell differentiation antigens (such as CD19, CD20 and CD37) are other candidates for target antigens in B-cell lymphoma.

[0263] Tumor antigens can be tumor-specific antigens (TSAs) or tumor-associated antigens (TAAs). TSAs are specific to tumor cells and are not present on other cells in the body. TAAs are not specific to tumor cells, but rather are expressed on normal cells under conditions that do not induce a state of immune tolerance to the antigen. Expression of antigens on tumors can occur under conditions that enable the immune system to respond to the antigen. TAAs may be antigens expressed on normal cells during embryonic development, when the immune system is immature and unable to respond, or they may be antigens that are normally present at very low levels on normal cells but expressed at much higher levels on tumor cells.

[0264] Non-limiting examples of TSA or TAA antigens include the following: differentiation antigens, such as MART-1 / MelanA (MART-I), gp 100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens, such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER2 / neu; unique tumor antigens generated 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 protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23HI, PSA, TAG-72, CA19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-catenin, CDK4, Mum-1, p 15, p 16, 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, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP and TPS.

[0265] F. Nucleic Acids

[0266] The immune cells described herein comprise one or more nucleic acids comprising a heterologous nucleic acid sequence encoding any of the engineered receptors, therapeutic agents, and / or immunomodulators described herein. The nucleic acid can be DNA. The nucleic acid can be RNA. The nucleic acid can be linear. The nucleic acid can be circular.

[0267] Heterologous nucleic acid sequences can be operably connected to one or more regulatory sequences.Exemplary regulatory sequences for controlling transcription and / or translation of coding sequences are known in the art and may include, but are not limited to, promoters; suitable initiation, regulation and / or termination (e.g., poly A transcription termination sequence) for transcription, mRNA transport (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., initiation Met, triplet leader sequence, IRES ribosome binding site, signal peptide, etc.) of additional elements; and insertion sites for introducing inserts into viral vectors. Regulatory sequences may be promoters, transcription enhancers, and / or sequences that allow engineered receptors to be correctly expressed.

[0268] The term "regulatory sequence" or "control sequence" refers to a DNA sequence that affects the expression of a coding sequence to which it is operably linked. The nature of such regulatory sequences varies depending on the host organism. In prokaryotes, regulatory sequences typically include promoters, ribosome binding sites, and terminators. In eukaryotes, regulatory sequences include promoters, terminators, and in some cases enhancers, transactivators, or transcription factors.

[0269] The term "operably linked" refers to a juxtaposition wherein the components are in a relationship permitting them to function in their intended manner. A regulatory sequence "operably linked" to a coding sequence is linked in such a way that expression of the coding sequence is achieved under conditions compatible with the regulatory sequences.

[0270] As used herein, "promoter" or "promoter region" refers to a DNA or RNA segment that controls the transcription of a DNA or RNA to which it is operably linked. The promoter region includes specific sequences involved in RNA polymerase recognition, binding, and transcription initiation. In addition, the promoter includes sequences that regulate RNA polymerase recognition, binding, and transcription initiation activity (i.e., the binding of one or more transcription factors). These sequences can be cis-acting, or can respond to trans-acting factors. Depending on the nature of the regulation, the promoter can be a constitutive promoter or a regulated promoter. Regulated promoters can be inducible or responsive to the environment (e.g., responsive to inducers such as pH, anaerobic conditions, osmotic agents, temperature, light, or cell density). Many such promoter sequences are known in the art. See, e.g., U.S. Pat. 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 ed., Cold Spring Harbor Press (1989).

[0271] The promoter can be an endogenous promoter. For example, any method known in the art (such as CRISPR / Cas9 method) can be used to knock the nucleic acid sequence encoding the engineered receptor into the genome of the modified immune cell downstream of the endogenous promoter. The endogenous promoter can be the promoter of an abundance protein (such as β-actin). The endogenous promoter can be an inducible promoter, for example, induced by an endogenous activation signal of an immune cell (for example, a modified immune cell). Wherein these immune cells are T cells, and the promoter can be a T cell activation-dependent promoter (such as an IL-2 promoter, a NFAT promoter, or a NFκB promoter). The promoter can be a heterologous promoter.

[0272] Various promoters have been explored to express genes in mammalian cells, and any promoter known in the art can be used in the present application. Promoters can be roughly divided into constitutive promoters or regulated promoters, such as inducible promoters. The heterologous nucleic acid sequence encoding the engineered receptor can be operably connected to the constitutive promoter. The heterologous nucleic acid sequence encoding the engineered receptor can be operably connected to the inducible promoter.

[0273] Constitutive promoters allow heterologous genes (also referred to as transgenes) to be constitutively expressed in host cells. Exemplary constitutive promoters contemplated herein include, but are not limited to, cytomegalovirus (CMV) promoters, human elongation factor-1α (hEF1α), ubiquitin C promoter (UbiC), phosphoglycerol kinase promoter (PGK), simian virus 40 early promoter (SV40), and chicken beta-actin promoter (CAGG) coupled to CMV early enhancer. In many studies, such constitutive promoters have been extensively compared for their efficiency in driving transgenic expression. The promoter may be the hEF1α promoter.

[0274] The promoter can be an inducible promoter. Inducible promoters belong to the category of regulated promoters. Inducible promoters can be induced by one or more conditions, such as physical conditions, microenvironment of immune cells (e.g., modified immune cells) or physiological state of immune cells (e.g., modified immune cells), inducers (i.e., inducers), or combinations thereof. Inducing conditions can not induce the expression of endogenous genes in immune cells (e.g., modified immune cells) and / or subjects receiving pharmaceutical compositions. Inducing conditions can be selected from the group consisting of: inducers, radiation (e.g., ionizing radiation, light), temperature (e.g., heat), redox state, tumor environment and activation state of immune cells (e.g., modified immune cells).

[0275] The promoter can be induced by an inducer. The inducer can be a small molecule, such as a chemical compound. The small molecule can be selected from the group consisting of doxycycline, tetracycline, alcohol, metal or steroid. Chemically induced promoters have been most widely explored. Such promoters include promoters whose transcriptional activity is regulated by the presence or absence of small molecule chemicals (such as doxycycline, tetracycline, alcohol, steroid, metal and other compounds). The doxycycline inducible system with a reverse tetracycline-controlled transactivator (rtTA) and a tetracycline response element promoter (TRE) is the most mature system at present. WO9429442 describes the strict control of gene expression in eukaryotic cells by tetracycline-responsive promoters. WO 9601313 discloses a transcriptional regulator regulated by tetracycline. In addition, Tet technology (such as the Tet-open system) is described, for example, on the website of TetSystems.com. Any known chemically regulated promoter can be used to drive the expression of the therapeutic protein of the present application.

[0276] The inducer can be a polypeptide, such as a ligand of a growth factor, a hormone or a cell surface receptor, such as a polypeptide that specifically binds to a tumor antigen. Many polypeptide inducers are also known in the art, and they can be applied to the present application. For example, a gene switch based on an ecdysone receptor, a gene switch based on a progesterone receptor, and a gene switch based on an estrogen receptor belong to gene switches using transactivators derived from steroid receptors (WO 9637609 and WO 9738117, etc.).

[0277] Inducer can include small molecule components and one or more polypeptides at the same time. For example, inducible promoters that rely 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 used FK1012 (a derivative of drug FK506) to induce homodimerization of FKBP. By using a similar strategy, Wu et al. successfully made CAR-T cells titrated by turning on the switch (ON-switch) by using rapamycin analogs (Rapalog) / FKPB-FRB* and gibberelline (Gibberelline) / GID1-GAI dimerization-dependent gene switches (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).

[0278] The promoter can be a light-inducible promoter, and the inducing 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)). Based on such gene regulation systems, (1) DNA binding or (2) the regulation of transcriptional activation domains recruited to DNA binding proteins, such gene regulation systems can be roughly divided into two categories. For example, a synthetic mammalian blue light-controlled transcription system based on opsin was developed and tested in mammalian cells, which responds to blue light (480nm) and triggers an increase in intracellular calcium, thereby causing calcineurin-mediated NFAT mobilization. Recently, Motta-Mena et al. described a new inducible gene expression system developed from the naturally occurring EL222 transcription factor, which confers high levels of blue light sensitivity control of transcription initiation in human cell lines and zebrafish embryos (Nat. Chem. Biol. 10 (3): 196-202 (2014)). In addition, the red light-induced gene expression regulation of Arabidopsis thaliana (Arabidopsisthaliana) photoreceptor phytochrome B (PhyB) and phytochrome interaction factor 6 (PIF6) is performed using the interaction of red light-induced Arabidopsis thaliana. In addition, an ultraviolet B (UVB) inducible gene expression system has been developed and demonstrated to be effective in target gene transcription in mammalian cells (Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, Chapter 25, Fourth 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.

[0279] The promoter can be a light-inducible promoter induced by a combination of a light-inducible molecule and light. For example, a light-caged group (photocaged group) on a chemical inducer keeps the inducer inactive unless the light-caged group is removed by radiation or other means. Such light-inducible molecules include small molecule compounds, oligonucleotides and proteins. For example, caged ecdysone, caged IPTG used with the lac operon, caged toyogamycin for ribozyme-mediated gene expression, caged doxycycline used with the Tet-open system, and caged rapamycin analogs for light-mediated FKBP / FRB dimerization have been developed (see, for example, Curr Opin Chem Biol. 16 (3-4): 292-299 (2012)).

[0280] The promoter can be a radiation-inducible promoter, and the inducing condition is radiation, such as ionizing radiation. Radiation-inducible promoters are also known in the art for controlling transgenic expression. Changes in gene expression occur after irradiating cells. For example, a group of genes called "immediate early genes" can respond rapidly 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 radiation response sequences in their promoter regions. The consensus sequence CC(A / T)6GG has been found in the Erg-1 promoter and is referred to as a serum response element or as a CArG element. The combination of radiation-inducible promoters and transgenics has been extensively studied and has been shown to have efficient therapeutic benefits. See, e.g., 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.

[0281] The promoter can be a heat-inducible promoter, and the inducing condition is heat. Heat-inducible promoters that drive transgene expression have also been widely studied in the art. Heat shock or stress proteins (HSPs) including Hsp90, Hsp70, Hsp60, Hsp40, Hsp10, etc. play an important role in protecting cells under heat or other physical and chemical stresses. Several heat-inducible promoters have been tried in preclinical studies, including heat shock protein (HSP) promoters and growth arrest and DNA damage (GADD) 153 promoters. The promoter of the human hsp70B gene, first described in 1985, appears to be one of the most efficient heat-inducible promoters. Huang et al. reported that after introducing hsp70B-EGFP, hsp70B-TNFα and hsp70B-IL12 coding sequences, tumor cells expressed extremely high transgenic expression after heat treatment, while in the absence of heat treatment, no transgenic expression was detected. In the group of mice treated with IL12 transgenic heat, tumor growth in vivo was significantly delayed (Cancer Res. 60: 3435 (2000)). Another group of scientists connected the HSV-tk suicide gene to the hsp70B promoter and tested the system in nude mice carrying mouse breast cancer. Compared with the control without heat treatment, mice to which the hsp70B-HSVtk coding sequence was applied and heat treated showed tumor regression and significant survival rate (Hum. Gene Ther. 11: 2453 (2000)). Other heat-inducible promoters known in the art can be found in, for example, Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, Chapter 25, Fourth Edition, CRC Press, January 20, 2015. Any heat-inducible promoter discussed herein can be used to drive the expression of the therapeutic protein of the present application.

[0282] The promoter can be induced by redox state. Exemplary promoters that can be induced by redox state include inducible promoters and hypoxia inducible promoters. For example, Post DE et al. developed a hypoxia inducible factor (HIF) responsive promoter that specifically and strongly induces transgene expression in HIF-active tumor cells (Gene Ther. 8: 1801-1807 (2001); Cancer Res. 67: 6872-6881 (2007)).

[0283] The promoter can be induced by the physiological state (e.g., endogenous activation signal) of an immune cell (e.g., a modified immune cell). Wherein these immune cells are T cells, the promoter can be a T cell activation-dependent promoter, which can be induced by an endogenous activation signal of a modified T cell. The modified T cells can be activated by inducers such as phorbol myristate acetate (PMA), ionomycin, or lectin. The modified T cells can be activated by recognizing tumor antigens on tumor cells via an engineered receptor (e.g., CAR, TCR, or TAC). The T cell activation-dependent promoter can be an IL-2 promoter. The T cell activation-dependent promoter can be an NFAT promoter. The T cell activation-dependent promoter can be an NFκB promoter.

[0284] The heterologous nucleic acid sequence described herein may be present in a heterologous gene expression cassette, which comprises one or more protein coding sequences and optionally one or more promoters. The heterologous gene expression cassette may comprise a single protein coding sequence. The heterologous gene expression cassette may comprise two or more protein coding sequences (i.e., polycistronic) driven by a single promoter. The heterologous gene expression cassette may further comprise one or more regulatory sequences (e.g., 5'UTR, 3'UTR, enhancer sequence, IRES, transcription termination sequence), recombination site, one or more selection markers (e.g., antibiotic resistance gene, reporter gene, etc.), signal sequence, or a combination thereof. The first heterologous nucleic acid sequence encoding the engineered receptor may be fused to a second heterologous nucleic acid sequence encoding an immunomodulator (e.g., Nef protein) via a third nucleic acid sequence encoding a self-cleavable joint (e.g., P2A, T2A, E2A or F2A peptide).

[0285] The immune cell may comprise a vector comprising a heterologous nucleic acid sequence encoding an engineered receptor. The vector may further comprise a second heterologous nucleic acid sequence encoding an immunomodulator.

[0286] 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 in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.

[0287] Many virus-based systems have been developed for transferring 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 in retroviral particles using techniques known 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 can be used. Slow virus vectors can be used. Self-inactivating slow virus vectors can be used. For example, self-inactivating slow virus vectors can be packaged using protocols known in the art. The resulting slow virus vectors can be used to transduce mammalian cells (e.g., human T cells) using methods known in the art.

[0288] The vector may be a non-viral vector (eg, a plasmid) or an episomal expression vector.

[0289] The vector can be an expression vector." expression vector " is a construct that can be used to transform a selected host and provide a coding sequence to be expressed in the selected host. The expression vector can be, for example, a cloning vector, a binary vector or an integration vector. Expression includes preferably transcribing the nucleic acid molecule into a translatable mRNA. The regulatory elements that ensure expression in eukaryotic cells are well known to those skilled in the art. In the case of eukaryotic cells, they generally include a promoter that ensures transcription initiation and optionally a poly A signal that ensures transcription termination and transcript stability. The example of the regulatory element allowed to be expressed in eukaryotic host cells is CMV-, SV40-, RSV-promoter (Rous sarcoma virus (Rous sarcoma virus)), CMV-enhancer, SV40-enhancer or globin intron in AOX1 or GAL1 promoter in yeast or mammals and other animal cells. In addition, according to the expression system used, the leader sequence that can guide the polypeptide to the cell compartment or secrete it into the medium can 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 the translation, start and stop sequences at the appropriate stage, and preferably, the leader sequence is capable of directing secretion of the translated protein or a portion thereof to the periplasmic space or the extracellular medium. Optionally, the nucleic acid sequence may encode a fusion protein comprising an N-terminal identification peptide that confers a desired characteristic (e.g., stabilization of the expressed recombinant product or simplified purification). Suitable expression vectors are known in the art, such as the Okayama-Berg cDNA expression vectors 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).

[0290] G. Disease or illness

[0291] The disease or condition to be treated by the methods described herein can be any disease or condition considered to be appropriately treated by cell therapy involving immune cells.

[0292] The disease or condition can be cancer (e.g., a blood cancer, e.g., a solid tumor). Cancers that can be treated using any of the methods described herein include any type of cancer. Types of cancers to be treated with agents as described in this application include, but are not limited to, epithelial cancers, blastomas, sarcomas, benign and malignant tumors, and malignant tumors, such as sarcomas, epithelial cancers, and melanomas. Adult tumors / cancers and pediatric tumors / cancers are also included.

[0293] In various embodiments, the cancer is an early stage cancer, a non-metastatic cancer, a primary cancer, an advanced stage cancer, a locally advanced cancer, a metastatic cancer, a cancer in remission, a recurrent cancer, a cancer being treated with the adjuvant therapy, a cancer being treated with the neoadjuvant therapy, or a cancer that is substantially refractory to treatment.

[0294] Examples of cancers that can be treated by the methods of the present application include, but are not limited to, anal cancer, astrocytoma (e.g., cerebellar and cerebral), basal cell carcinoma, bladder cancer, bone cancer (e.g., osteosarcoma and malignant fibrous histiocytoma), brain tumors (e.g., glioma, brain stem 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 (gastric) cancer, gastrointestinal stromal tumor (GI stromal tumor), and ovarian cancer. ST), head and neck cancer, hepatocellular (liver) cancer (e.g., hepatoepithelial 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 carcinoma, neuroblastoma, ovarian cancer, pancreatic cancer, parathyroid cancer, peritoneal cancer, pituitary tumors, rectal cancer, kidney cancer, renal pelvis and ureter cancer (transitional cell carcinoma), rhabdomyosarcoma, skin cancer (e.g., non-melanoma (e.g., squamous cell epithelial carcinoma), melanoma, and Merkel cell carcinoma), small intestine cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, and tuberous sclerosis. Additional examples 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 website); and SEER Program Coding and Staging Manual 2016, each of which is incorporated by reference in its entirety for all purposes.

[0295] The cancer may be a solid tumor.

[0296] The disease or disorder to be treated or prevented by the methods described herein can be a host-versus-graft (HvG) disorder in a human individual receiving cell therapy.

[0297] III. Compositions, Kits, and Articles of Manufacture

[0298] Also provided are compositions (eg, pharmaceutical compositions), kits, unit doses, and articles of manufacture comprising an S phase inhibitor (eg, methotrexate), a lymphodepleting agent (eg, Flu and Cy), and / or immune cells described herein.

[0299] In some embodiments, compositions are provided that include any of the immune cells described herein. The composition may include any number of immune cells. The composition may include a single copy of a therapeutic cell. The composition may include 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, a pharmaceutical composition is provided, which comprises an effective amount of immune cells (e.g., allogeneic CAR-T cells) and a pharmaceutically acceptable carrier.

[0300] In some embodiments, a pharmaceutical composition is provided, comprising an effective amount of an S phase inhibitor as described herein (e.g., methotrexate), a lymphodepleting agent (e.g., Flu and Cy), and a pharmaceutically acceptable carrier. The pharmaceutical composition can be a lyophilized composition comprising methotrexate, Flu, and Cy, potassium dihydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, sucrose, mannitol, and glycine.

[0301] Also provided are compositions for use in any of the methods described herein, and the use of such compositions in the preparation of a medicament for use in any of the methods described herein.

[0302] As used herein, "carrier" includes a pharmaceutically acceptable carrier, excipient or stabilizer that is nontoxic to cells or individuals exposed thereto at the dosages and concentrations employed. Physiologically acceptable carriers are typically pH buffered aqueous solutions. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions (such as oil / water emulsions), various types of wetting agents, sterile solutions, etc. Acceptable carriers, excipients or stabilizers are nontoxic to the receptor at the dosages and concentrations employed.

[0303] Pharmaceutical compositions comprising such carriers can be formulated by well-known conventional methods. The solvent or diluent is preferably isotonic, hypotonic or weakly hypertonic and has a relatively low ionic strength. Representative examples include sterile water, physiological saline (e.g., sodium chloride), Ringer's solution, glucose, trehalose or sucrose solution, Hank's solution and other physiologically balanced saline solutions (see, e.g., the latest edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins).

[0304] The pharmaceutical compositions described herein can be administered via any suitable route. The pharmaceutical compositions can be administered parenterally, percutaneously (into the dermis), intracavitary, intraarterially (into the artery), intramuscularly (into the muscle), intrathecally or intravenously. The pharmaceutical compositions can be administered subcutaneously (under the skin). The pharmaceutical compositions can be administered intravenously. The pharmaceutical compositions can be administered to an individual via infusion or injection. The pharmaceutical compositions can be administered directly to the target site, for example, by gene gun delivery to an internal or external target site or by catheter administration to a site in an artery. The pharmaceutical compositions can be administered topically (e.g., intratumorally). Administration can use a conventional syringe and needle or any compound or device available in the art that can promote or improve the delivery of an active agent in a subject.

[0305] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil) and injectable organic esters (such as ethyl oleate). Aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's solution or fixed oils. Intravenous vehicles include fluid and nutritional supplements, electrolyte supplements (such as those based on Ringer's dextrose), etc. Preservatives and other additives may also be present, such as antimicrobial agents, antioxidants, chelating agents and inert gases, etc. In addition, the pharmaceutical composition of the present disclosure may include protein carriers, such as serum albumin or immunoglobulin, preferably human-derived.

[0306] The pharmaceutical composition can be appropriately buffered for human use. Suitable buffers include, but are not limited to, phosphate buffer (e.g., PBS), bicarbonate buffer, and / or Tris buffer capable of maintaining a physiological or weakly alkaline pH (e.g., about pH 7 to about pH 9). The pharmaceutical composition can also be made isotonic with blood by adding a suitable tension modifier (e.g., glycerol).

[0307] The pharmaceutical composition can be contained in a single-use vial (e.g., a single-use sealed vial). The pharmaceutical composition can be contained in a multiple-use vial. The pharmaceutical composition can be contained in a container in bulk.

[0308] The pharmaceutical composition may have to meet certain standards for administration to an individual. For example, the U.S. Food and Drug Administration has issued regulatory guidelines for setting standards for cell-based immunotherapy products, including 21CFR 610 and 21CFR610.13. Methods for evaluating the appearance, characteristics, purity, safety and / or efficacy of pharmaceutical compositions are known in the art. The pharmaceutical composition may be substantially free of foreign proteins that can produce allergic effects, such as proteins of animal origin other than immune cells used in cell culture. "Substantially free" can be less than about 10%, 5%, 1%, 0.1%, 0.01%, 0.001%, 1ppm or less of the total volume or weight of the pharmaceutical composition. The pharmaceutical composition can be prepared in a GMP-level workshop. For parenteral administration, the pharmaceutical composition may contain endotoxins less than about 5EU / kg body weight / hour. For intravenous administration, at least about 70% of the immune cells in the pharmaceutical composition may be alive. When evaluated using the 14-day direct inoculation test method as described in the United States Pharmacopeia (USP), the pharmaceutical composition may have a "no growth" result. In some embodiments, prior to administration of the pharmaceutical composition, a sample including both immune cells and pharmaceutically acceptable excipients should be removed for sterility testing approximately 48-72 hours prior to final harvest (or concurrently with the final 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 disease pathogens, such as HIV type I, HIV type II, HBV, HCV, human T-lymphotropic virus type I; and human T-lymphotropic virus type II.

[0309] In some embodiments, a kit containing any of the pharmaceutical compositions described herein is provided, and preferably instructions for use thereof are provided. In some embodiments, a kit is provided, comprising: (a) any of the S phase inhibitors described herein (e.g., methotrexate); (b) any of the lymphodepleting agents described herein (e.g., Flu and Cy); and (c) immune cells described herein; and (d) instructions for use in any of the methods described herein. The immune cells may be allogeneic immune cells.

[0310] In some embodiments, a kit is provided, comprising: (a) any one of the S phase inhibitors described herein (e.g., methotrexate); (b) any one of the lymphodepleting agents described herein (e.g., Flu and Cy); and (c) allogeneic CAR-T cells; and (d) instructions for treating a disease or disorder (e.g., cancer) in an individual in need. Allogeneic CAR-T cells can target BCMA, claudin 18.2, CLL1, or CD20. Allogeneic CAR-T cells may not be genetically modified to reduce the immunogenicity of allogeneic cells in an individual. Allogeneic CAR-T cells may be genetically modified to be resistant to MTX. Allogeneic CAR-T cells may not have genetic modifications other than CAR.

[0311] The kit may further comprise one or more lymphodepleting agents. The kit may further comprise fludarabine and cyclophosphamide. In addition to immune cells, the kit may further comprise a second cancer therapy, such as chemotherapy, hormone therapy, and / or immunotherapy. The kit may be customized for an individual's specific cancer and comprise a corresponding second cancer therapy for the individual.

[0312] The kit may contain one or more additional components, such as containers, reagents, culture media, inducers, cytokines, buffers, antibodies, etc., to allow the propagation or induction of immune cells. The kit may also contain a device for administering the pharmaceutical composition.

[0313] The kit of the present application is in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), etc. The kit may optionally provide additional components, such as buffer and interpretative information. Therefore, the present application also provides articles, which include vials (e.g., sealed vials), bottles, jars, flexible packaging, etc. Some components of the kit may be packaged in aqueous media or in lyophilized form.

[0314] Articles can include containers and labels or package inserts on or associated with the containers. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed of a variety of materials (such as glass or plastic). Typically, the container holds a composition that effectively treats a disease or disorder described herein (such as cancer), and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The label or package insert indicates that the composition is used to treat a specific condition of an individual. The label or package insert will further include instructions for applying the composition to an individual. The label can indicate guidance for reconstruction and / or use. The container holding the pharmaceutical composition can be a vial that can be used multiple times, which allows repeated administration (for example, 2-6 administrations). Package inserts refer to instructions that are usually included in the commercial packaging of therapeutic products, which contain information about the indications, usage, dosage, administration, contraindications and / or warnings for using such therapeutic products. In addition, the article of manufacture may further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0315] The kit or article of manufacture may include multiple unit doses of the pharmaceutical composition and instructions for use, packaged in quantities sufficient for storage and use in pharmacies (e.g., hospital pharmacies and compounding pharmacies).

[0316] Examples

[0317] Example 1. Evaluation of the effects of different MTX concentrations on the proliferation of UCAR-T cells in vitro

[0318] 1) Construction of LUCAR expression vector

[0319] The pLVX-Puro vector purchased from Clontech was digested with ClaI and EcoRI restriction enzymes, and the CMV promoter was replaced with the human EF1α promoter (GenBank: J04617.1) to obtain the pLVX-hEF1α vector. The gene of DHFRL22F / F31S (SEQ ID NO: 1) was inserted between SIV NEF M116 and CD20 CAR of LCAR-UL186S, which is disclosed in PCT patent application Nos. PCT / CN2020 / 112181 and PCT / CN2020 / 112182. The fusion gene sequence of SIV Nef_M116-P2A-DHFR L22F / F31S-IRES-CD8αSP-CD20 scFv(Leu16)-CD8α hinge-CD8αTM-4-1BB-ITAM010 (hereinafter referred to as "LUCAR-20SD" (SEQ ID NO: 4)) was cloned into the expression plasmid pLVX-hEF1α to form a LUCAR-20SD expression plasmid named M1586. The recombinant expression plasmid M1586 was extracted, mixed with psPAX2 and pMD2.G auxiliary plasmids in a certain proportion, and co-transfected into HEK 293T cells. After 60h of transfection, the cell culture supernatant containing the virus was collected and centrifuged at 4°C and 3000rpm for 5min. After the supernatant was filtered through a 0.45 μm filter, it was further concentrated using a 500 KD hollow fiber membrane column tangential flow technology to prepare a lentiviral concentrate, which was stored at -80°C for later use.

[0320] 2) Preparation of UCAR-T cells

[0321] 50 mL of fresh peripheral blood from volunteers was drawn and mixed with lymphocyte separation medium. Peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation. The cells were labeled with magnetic beads using the Pan T cell isolation kit (Miltenyi Biotech), and T lymphocytes were separated and purified, and then activated by CD3 / CD28 magnetic beads. After culturing in a 37°C, 5% CO2 incubator for 24 hours, 5×10 6 Activated T lymphocytes were infected with LUCAR-20SD lentiviral vectors. The cell suspension was transferred to a 6-well plate and incubated in a 37°C, 5% CO2 incubator for CAR-T cell proliferation. After 17 days of transduction, TCRα / β negative cells were enriched and sorted with a TCRα / β sorting kit to obtain T cells expressing LUCAR-20SD (hereinafter referred to as LUCAR-20SD cells).

[0322] 50mL of fresh peripheral blood from volunteers was extracted and mixed with lymphocyte separation medium, and then peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation. The isolated lymphocytes were cultured overnight in RPMI 1640 medium supplemented with L-glutamine (2mM, Sigma-Aldrich), penicillin (100IU / mL) / streptomycin (100μg / mL) (Sigma-Aldrich, Missouri, USA), 10% serum (Gibco, Massachusetts, USA), zoledronic acid (5μM, Actavis, New Jersey, USA), IL-2 (100IU / mL; Aldesleukin, Novartis, Frimley, UK). The above-selected γδT cells were transduced with a lentiviral vector expressing anti-CLL1 CAR and DHFR L22F / F31S at a multiplicity of infection (MOI) of 5. 48 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).

[0323] Methotrexate (MTX) was added at a concentration of 0 μM, 0.01 μM, 0.05 μM, 0.1 μM, 1 μM or 2 μM for culture, and the cell number of LUCAR-20SD cells on day 0, day 3, day 6, day 9, day 12 and the cell number of CAR-γδT cells on day 0, day 2, day 5, day 7 were counted by AOPI. Cell proliferation curves were drawn separately.

[0324] Figure 1A The proliferation of LUCAR-20SD cells cultured with different concentrations of MTX is shown. On day 12, there was no significant difference in cell proliferation of LUCAR-20SD cells in 0.01 μM to 1 μM MTX compared with 0 μM MTX culture (p>0.05), while in 2 μM MTX, cell proliferation was significantly decreased (p<0.05), indicating that MTX ≥ 2 μM affects the proliferation of LUCAR-20SD cells. Figure 1B The proliferation of CAR-γδT cells cultured with different concentrations of MTX is shown. On day 7, there was no significant difference in the cell proliferation of CAR-γδT cells in 0.01μM to 0.1μM MTX compared with 0μM MTX culture (p>0.05), while cell proliferation was significantly reduced in a dose-dependent manner in 1μM MTX and 2μM MTX (p<0.05), indicating that MTX ≥ 1μM affects the proliferation of CAR-γδT cells. The above results show that in order to ensure the proliferation of UCAR-T cells, the concentration of MTX should be lower than 2μM.

[0325] Example 2: Evaluation of the protective effect of MTX on allogeneic UCAR-T cells

[0326] To evaluate the ability of LUCAR-20SD cells to tolerate allogeneic T cell rejection, an in vitro mixed lymphocyte (MLR) model was established.

[0327] Using T cells from a random healthy donor (B donor), LUCAR-20SD cells were prepared as transplant cells according to the method in Example 1. Untransduced T cells (UnT) from another random healthy donor (A donor) were also prepared as host cells.

[0328] The cell density of CD20 positive cells (lymphoma cell line Raji, ATCC, CCL-86) was adjusted to 1×10 6 Raji cells were treated with 20 μg / mL mitomycin at 37°C for 3 h, washed three times with 10 mL DPBS, and resuspended in culture medium. 6 Untransduced T cells (UnT, T cell donor A), 1×10 6 LUCAR-20SD cells (T cell donor B) and 5×10 6 The treated lymphoma cells Raji were mixed and cultured in the culture medium. The cell mixture was divided into 4 groups, and 0 μM MTX, 0.05 μM MTX, 1 μM MTX or 2 μM MTX were added respectively. The co-culture system was centrifuged on the 1st day, the 4th day, the 7th day, the 10th day and the 13th day to replace the culture medium, and samples (labeled as K0, K1, K2, K3, K4) were taken at the same time for cell counting, and CD5 antibody (Biolegend, catalog number 364008) and CD20 CAR antibody (GenScript, catalog number LGBUADAb-1) were used for FACS detection of LUCAR-20SD and UnT cells. LUCAR-20SD cells were repeatedly stimulated with Raji cells (CD20 positive lymphoma cells) at an E / T ratio of 1: 1 on the 0th day, the 4th day, the 7th day and the 11th day. The cell proliferation curves of UnT and LUCAR-20SD cells were plotted according to the following formula: fold change in cell number (UnT) = [number of cells counted by AOPI × (CAR-CD5+%)] / 20, fold change in cell number (LUCAR-20SD cells) = [number of cells counted by AOPI × (CAR+CD5+%)] / 1.

[0329] The results are shown in Figure 2A-2D In the presence of 0μM MTX ( Figure 2A), UnT cells (T cell donor A) showed a proliferation advantage, and the number of LUCAR-20SD cells (T cell donor B) gradually decreased. Figure 2B )、1μM( Figure 2C )、2μM( Figure 2D )) under the culture conditions, the proliferation of UnT cells was significantly inhibited, and the multiple change of the number of cells was always less than 1. LUCAR-20SD cells showed strong drug resistance and had significant advantages in proliferation ability and expansion multiple. The above results show that the administration of MTX can significantly inhibit the host cell's rejection of LUCAR-20SD cells and has a good protective effect on allogeneic LUCAR-20SD cells.

[0330] Example 3. Evaluation of the protective effect of MTX on allogeneic γδT cells

[0331] To evaluate the ability of γδ T cells to tolerate allogeneic T cell rejection, an in vitro mixed lymphocyte (MLR) model was established.

[0332] Using T cells from a random healthy donor (B donor), CAR-γδT cells were prepared as transplant cells according to the method in Example 1. Untransduced T cells (UnT) from another random healthy donor (T cell donor A) were also prepared as host cells.

[0333] The density of CLL1-positive human lymphoma cells (U937) was adjusted to 1×10 6 U937 cells were treated with 20 μg / mL mitomycin at 37°C for 3 h, washed three times with 10 mL DPBS, and suspended in culture medium. 6 Untransduced T cells (UnT, T cell donor A), 1×10 6 CAR-γδT cells (T cell donor B) and 5×10 6The treated human lymphoma cells U937 were mixed and cultured in culture medium. The cell mixture was divided into 3 groups, and 0 μM MTX, 0.05 μM MTX or 0.5 μM MTX were added respectively. The co-culture system was centrifuged on the 1st day, the 4th day, the 7th day and the 10th day to replace the culture medium, and samples (labeled as K0, K1, K2, K3, respectively) were taken for cell counting and detection of CD3 and TCR Vγ9 expression by FACS. CAR-γδT cells were repeatedly stimulated with U937 cells at an E / T ratio of 1: 1 on days 0, 4 and 7. According to the following formula: the fold change of the number of cells (UnT) = [the number of cells counted by AOPI × (CD3- / TCR Vγ9+%)] / 20, the fold change of the number of cells (CAR-γδT) = [the number of cells counted by AOPI × (CD3+ / TCR Vγ9+%)] / 1, and the cell proliferation curves of UnT and CAR-γδT cells were drawn.

[0334] The results of the study showed Figure 3A-3C In the presence of 0μM MTX ( Figure 3A ), UnT cells (T cell donor A) showed a proliferation advantage, and the number of CAR-γδT cells (T cell donor B) gradually decreased. Figure 3B ) and 0.5μM( Figure 3C )) Under the culture conditions, the proliferation of UnT cells was significantly inhibited, while CAR-γδT cells showed strong drug resistance and had significant advantages in proliferation ability and expansion multiples. The above results show that the administration of MTX can significantly inhibit the host cell's rejection of CAR-γδT cells and has a good protective effect on allogeneic CAR-γδT cells.

[0335] Example 4. In vivo efficacy evaluation of MTX on LUCAR-GC cells

[0336] 1) Preparation of LUCAR-GC cells

[0337] With reference to Example 1, the fusion gene sequence (SEQ ID NO: 5) of SIV Nef M116-IRES-CD8αSP claudin 18.2VHH-CD8α hinge-CD8αTM-4-1BB-ITAM010 is cloned into the expression plasmid pLVX-hEF1α to form the M1645 expression plasmid. Anti-claudin 18.2VHH and the antigen binding domain of CAR have been disclosed in PCT / CN2020 / 139143. TGB23-6-IRES-DHFR L22F / F31S (SEQ ID NO: 7) is cloned into the expression plasmid pLVX-hEF1α to form the M1647 expression plasmid. The fusion protein of TGB23-6 is a signal converter and has been described in PCT / CN2022 / 087016. In order to obtain LUCAR-GC cells, M1645 and M1647 are transduced into T cells. The production of viral vectors and transduction of primary T cells were completed as described in Example 1.

[0338] 2) Construction of NUGC4 mouse model and tumor monitoring

[0339] In vivo efficacy evaluation was performed using severely immunodeficient NCG mice. On day -14 before treatment, tumor cells (3 × 10 6 Human gastric cancer cells (NUGC4 / mice) were subcutaneously inoculated into NCG immunodeficient mice. On day 0, the xenografted mice were divided into five groups (G1, G2, G3, G4, and G5) and received HBSS (G1, G5), untransduced T cells (UnT) (G2, at 3.6 × 10 6 cells / mouse), LUCAR-GC cell preparations (G3 and G4, at 1×10 6 The mice in the G4 and G5 groups were additionally given 22.635 mg / kg MTX by intraperitoneal injection on days 5, 10, 15, 20, 25, 30, and 35, respectively. Figure 4A Treatment of mice in G4 is shown. During the experiment, animal status, survival rate, tumor volume and body weight were measured twice a week. Blood was collected from mouse eyeballs to separate cells every week and then resuspended in DPBS with 1 μL of FITC anti-human CD45 (Biolegend, 304038). The proportion of transplanted cells (hCD45+% / living cells) in mouse peripheral blood was counted.

[0340] The results of the study showed Figure 4B-4CThe G5 group was given 22.635 mg / kg MTX alone, and the tumor volume was not significantly different from that of the control G1 and G2 (p>0.05), indicating that MTX alone was ineffective in controlling tumor growth. The average tumor volume of mice transplanted with LUCAR-GC cells in group G3 on day 16 was 240.67 mm 3 , which was significantly lower than the 734.83 mm of the control group G2 3 (p<0.05), indicating that infusion of LUCAR-GC cells alone can effectively inhibit tumor cell proliferation. However, after day 30, the tumor volume showed a slow increase, indicating that the tumor recurred in the mice. The average tumor volume of mice treated with MTX and LUCAR-GC cells in G4 on day 16 was 223.62 mm 3 , which was significantly lower than the 734.83 mm of the control group G2 3 (p<0.05), indicating that the combined administration of MTX and LUCAR-GC cells effectively inhibited tumor cell proliferation, and tumor volume growth did not occur until the 42nd day. The above results show that 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. Figure 4C As shown, there was no significant difference in the expansion of mouse peripheral blood LUCAR-GC cells in the presence or absence of MTX, indicating that 22.635 mg / kg MTX did not affect the expansion and efficacy of LUCAR-GC cells. In conclusion, the combination therapy of MTX and LUCAR-GC cells was significantly superior to MTX or LUCAR-GC cells alone in tumor control.

[0341] Example 5. Evaluation of the combined effects of fludarabine, cyclophosphamide and MTX

[0342] 1) Preparation of LUCAR-BCMA cells

[0343] 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-CD8αSP-anti-BCMA antibody-CD8α hinge-CD8αTM-4-1BB-ITAM010 (hereinafter referred to as "LUCAR-BCMA" (SEQ ID NO: 6)) was cloned into the expression plasmid pLVX-hEF1α to form a LUCAR-BCMA expression plasmid named M1588. The anti-BCMA antibodies contained herein have been disclosed in PCT / CN2020 / 136570. LUCAR-BCMA cells were prepared by referring to the viral vector production and primary T transduction in Example 1.

[0344] 2) Evaluation of the effect of the combination of fludarabine, cyclophosphamide and MTX on the host immune system in vivo

[0345] In vivo efficacy evaluation was performed using severely immunodeficient NCG mice. Specific examples are as follows: On day -5.5, mice were intravenously inoculated with 20×10 6 PBMC cells (HLA-A2+) were used to construct host immune cells, and on day 0, mice were inoculated intravenously with 3×10 6 LUCAR-BCMA cells (HLA-A2-) were used as transplanted cells. The modeling mice were then treated with the following different combinations of fludarabine, cyclophosphamide and MTX to evaluate the inhibitory effect of the combination regimen on the host immune system. The combination and dosage of fludarabine, cyclophosphamide and MTX are shown in Table 2. The " / " in Table 2 means that the drug was not administered. The drug administration schedule for G4 and G6 is also shown in Figure 5AIn each experimental group, the dosage of fludarabine was 0.1623mg / mouse, the dosage of cyclophosphamide was 1.623mg / mouse, and the dosage of MTX was 15.15mg / kg (G3 and G4) or 75.45mg / kg (G5 and G6). On the 13th day (experimental end point), peripheral blood leukocytes (WBC) of mice were measured. Mouse peripheral blood cells were resuspended in DPBS, and then 1 μL FITC anti-human CD45 (Biolegend, 304038) and 1 μL PE anti-human HLA-A2 (Biolegend, 343306) were added to the cell resuspension, and incubated at 4 ° C for 30min. The stained cells were washed twice with 1mL DPBS, and then centrifuged at room temperature to discard the supernatant. Finally, the cells were suspended in DPBS to detect the expression of hCD45 and HLA-A2 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 peripheral blood of mice was evaluated to reflect the effects of different drug combinations on the host immunity in vivo.

[0346] Table 2. Combinations of fludarabine, cyclophosphamide, and MTX

[0347] The results of the study showed Figure 5B On day 13 (end point of the experiment), the number of host cells in the peripheral blood of the G2 group (fludarabine, cyclophosphamide), the G3 group (low-dose 15.15 mg / kg MTX), and the G5 group (high-dose 75.45 mg / kg MTX) were lower than that of the G1 blank group, indicating that the combination of fludarabine and cyclophosphamide or MTX alone can inhibit the proliferation of host cells, but the inhibitory effect is limited. Comparing the results of the G3 group and the G5 group, the inhibitory effect of MTX on host immune cells is dose-dependent. The combination of fludarabine, cyclophosphamide and MTX achieves a synergistic effect (see, for example, G4). Figure 5B The combination of fludarabine, cyclophosphamide and MTX effectively suppressed host immune cells at a significantly lower host cell ratio compared to the MTX-only group or the FC-only group. In conclusion, the combination of fludarabine, cyclophosphamide and MTX significantly suppressed the host immune system.

[0348] Example 6. Effect of the MTX dosing interval in the combination of fludarabine, cyclophosphamide, and MTX

[0349] The effect of the MTX dosing interval in the combination of fludarabine, cyclophosphamide, and MTX was further evaluated in the immunocompetent C57BL / 6 mouse model. Combination administration of fludarabine, cyclophosphamide, and MTX was performed in group G2, see also Fig. 6A On day 0, mice in both G1 and G2 groups were intravenously inoculated with 5 × 10 6 LUCAR-20SD cells were added, and the blood routine of mice was regularly tested during the experiment. In this test, the dosage of fludarabine was 0.1623 mg / mouse, the dosage of cyclophosphamide was 1.623 mg / mouse, and the dosage of MTX was 75.45 mg / kg. The schedule of dosing frequency is shown in Table 3. " / " in Table 3 means that no drug is administered.

[0350] Table 3. Combination administration of fludarabine, cyclophosphamide and MTX (different dosing intervals)

[0351] The results are shown in Figure 6B The mice in the G1 group that were not treated with drugs served as the control group, and the peripheral blood white blood cells (WBC) of the mice were stable during the experiment, with an average value of 8.51×10 9 For mice in G2 (MTX was administered on days -3, 1, 6, and 11, with dosing intervals of 4d, 5d, and 5d), the average WBC of mice during the experimental period from day 0 to day 14 was 2.47 × 10 9 / L, which was significantly lower than the mean WBC of the G1 control group.

[0352] Example 7. Effect of MTX dose in combination administration of fludarabine, cyclophosphamide and MTX

[0353] The effect of MTX dose in the combination of fludarabine, cyclophosphamide, and MTX was further evaluated in an immunocompetent C57BL / 6 mouse model. Each experimental group was inoculated intravenously on day 0 with 5 × 10 6 LUCAR-20SD cells were used, and the blood routine of the mice was tested regularly during the experiment. In this experiment, the dose of fludarabine was 0.1623 mg / mouse, and the dose of cyclophosphamide was 1.623 mg / mouse. The doses of fludarabine, cyclophosphamide and MTX are shown in Table 4. The " / " in Table 4 means that the drug was not administered. The dosing schedule is shown in Fig. 7A middle.

[0354] Table 4. Combination administration of fludarabine, cyclophosphamide and MTX (different doses)

[0355] The results are shown in Figure 7B-7C The mice in the G1 group that were not treated with drugs served as the control group, and the WBC count of the mice was stable during the experiment, with an average of 8.51×10 9 / L. During the experimental period from day 0 to day 14, the average WBC count of mice injected with 30.179 mg / kg MTX in group G2 was 3.4×10 9 / L, which was significantly lower than the average WBC of the control group. During the experimental period from day 0 to day 14, the average WBC of mice injected with 75.447 mg / kg MTX in group G3 was 2.47×10 9 / L, which was significantly lower than the mean WBC of the control group.

[0356] Example 8. Evaluation of the protective effect of the combination of fludarabine, cyclophosphamide and MTX on allogeneic transplanted cells

[0357] 1) Construction of MPC-11 drug-resistant cell line

[0358] The MPC-11 mouse plasmacytoma cell line was derived from the BALB / c mouse strain. 5×10 6 MPC-11 mouse plasmacytoma cells were infected with a lentiviral vector (M1647: TGB23-6-IRES-DHFR L22F / F31S), and then the cell suspension was transferred to a 6-well plate and cultured in a 37°C, 5% CO2 incubator for 3 days. Transduced cells were selected with 0.05 μM MTX until the positive cells were greater than 95% and used as transplant cells.

[0359] 2) Evaluation of the protective effect of the combination of fludarabine, cyclophosphamide and MTX on allogeneic transplanted cells

[0360] MPC-11 cells (1×10 6 The doses of fludarabine, cyclophosphamide, and MTX are shown in Table 5. The combined dosing regimen of the G4 group is also shown in Table 5. Fig. 8A In this experiment, 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. Tumor volume was measured three times a week to evaluate the effect of the combination of drugs on allogeneic transplanted cells.

[0361] Table 5. Combination administration of fludarabine, cyclophosphamide and MTX

[0362] The results of the study showed Figure 8BIn the autologous transplant model of the G1 group, MPC-11 tumors were successfully implanted. In the allogeneic transplant model of the G2 group, MPC-11 tumor cells could not be effectively expanded, and tumors could not be measured, indicating that the C57BL / 6 immune system in the allogeneic transplant model significantly suppressed the tumorigenesis of MPC-11 cells with a BALB / c genetic background. In the allogeneic transplant model of the G3 group, the combination of fludarabine and cyclophosphamide promoted weak tumor expansion of MPC-11 within 8 days (less than 8.62 mm 3 ), followed by tumor disappearance, indicating that fludarabine and cyclophosphamide temporarily suppress the host immune system in allogeneic transplantation, but cannot 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 maximum tumor volume reached 132.69 mm 3 , indicating that the combination of fludarabine, cyclophosphamide and MTX significantly inhibited immune rejection in allogeneic transplantation, and the combination showed a significant protective effect on allogeneic transplanted cells.

[0363] Example 9. Exemplary clinical study design showing the protective effect of FC+MTX combination regimen on allogeneic adoptive cells ("UCAR")

[0364] In clinical studies, the patient's immune system is continuously suppressed by using a combination of FC+MTX to protect the effectiveness and persistence of UCAR cells in vivo after transplantation. Fludarabine and cyclophosphamide are combined for lymphocyte depletion. At 25-30 mg / m 2 Fludarabine was administered daily for 3-4 consecutive days and at a dose of 250-1000 mg / m 2 Cyclophosphamide is administered for 3-4 consecutive days per day. The combination is administered 1-3 times between the -7th day and the 90th day. The MTX administration cycle is -5 days to 90 days, and MTX is administered at a frequency of 3 mg / m2 for about 1-28 days (e.g., 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). 2 / day to 3000mg / m 2 / day (e.g., 5 mg / m 2 / day to 3000mg / m 2 MTX can be administered orally or intravenously. 2 Up to 15.6 mg / m 2 Or MTX can be administered at a dose of 5 mg to 25 mg every 3-5 days. 2 Up to 62.5 mg / m 2Or MTX can be administered at a dose of 25 mg to 100 mg every 3-5 days. 2 Up to 187.5 mg / m 2 Or MTX can be administered at a dose of 100 mg to 300 mg every 3-5 days. For each individual, 15.6 mg / m 2 Or 25 mg of MTX is administered every 2-7 days. For each individual, 31.25 mg / m 2 Or 50mg MTX every 2-7 days. For each individual, 63mg / m 2 Or 100 mg of MTX every 4 days. For each individual, 125 mg / m 2 Or 200 mg of MTX once every 4 days. For each individual, 188 mg / m 2 MTX can be administered at a dose of 250 mg / m 2 Or 400 mg of MTX once every 5 days. For each individual, 313 mg / m 2 MTX can be administered once every 5 days at a dose of 15.6 mg / m2 or 500 mg. For each individual, MTX can be administered at a dose of 15.6 mg / m2 on the 3rd, 5th, 10th, and 17th days after each administration of UCAR cells. 2 For each individual, MTX can be administered at a dose of 31.25 mg / m on days 3, 5, 10, and 17 after each administration of UCAR cells. 2 MTX was administered at a dose of 50 mg or 10 mg. UCAR cells were administered 1-5 times from day 0 to day 90. When the MTX concentration in plasma was lower than 2 μM, the dose of UCAR cells could be fixed at 30×10 6 Up to 900×10 6 or 0.1×10 6 / kg to 50×10 6 / kg. Fig. 9 An exemplary clinical protocol for allogeneic adoptive cell transplantation is shown.

[0365] Example 10. In vitro evaluation of the efficiency of MB12 armor

[0366] 1) Construction of NextGen UCD20A expression vector

[0367] This example shows the construction of cells expressing MB12, which is an exemplary membrane-bound IL12p40 polypeptide having the structure IL12p40-CD8α hinge-CD8α transmembrane domain-CD8α intracellular domain, see SEQ ID NO: 12. The pLVX-Puro vector purchased from Clontech was digested with ClaI and EcoRI restriction enzymes, 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-CD8αSP-CD20 scFv (Leu16)-CD8α hinge-CD8αTM-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 psPAX2 and pMD2.G auxiliary plasmids in a certain ratio, and co-transfected into HEK 293T cells. After incubation for 60 hours, the cell culture supernatant containing the virus was collected and centrifuged at 4°C and 3000rpm for 5min. 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 prepare a lentiviral concentrate, which was stored at -80°C for later use. Similarly, an expression vector for secreting IL12p40 was constructed as a control. The fusion gene sequence of SIVNef_M116-T2A-CD8αSP-CD20 scFv (Leu16)-CD8α hinge-CD8αTM-4-1BB-ITAM045-P2A-DHFRL22F / F31S-P2A-IL12p40 (hereinafter referred to as "NextGen UCD20 M12" (SEQ ID NO: 15)) was cloned into the expression plasmid pLVX-hEF1α to form a NextGen UCD20 M12 expression plasmid named M2335. Concentrated lentivirus was prepared and then stored at -80°C for later use.

[0368] 2) Preparation of NextGen UCD20A cells

[0369] 50 mL of fresh peripheral blood from volunteers was drawn and mixed with lymphocyte separation medium. Peripheral blood mononuclear cells (PBMC) were separated by density gradient centrifugation. The cells were labeled with magnetic beads using the Pan T cell isolation kit (Miltenyi Biotech), and T lymphocytes were separated and purified, and then activated by CD3 / CD28 magnetic beads. After culturing in a 37°C, 5% CO2 incubator for 24 hours, 5×10 6 Activated T lymphocytes were infected with NextGen UCD20A or NextGenUCD20 M12 lentiviral vectors. The cell suspension was transferred to a 6-well plate and incubated in a 37°C, 5% CO2 incubator for CAR-T cell proliferation. After 17 days of transduction, TCRα / β negative cells were enriched and sorted with a TCRα / β sorting kit to obtain T cells expressing NextGen UCD20A or NextGen UCD20 M12 (hereinafter referred to as NextGen UCD20A cells and NextGen UCD20 M12 cells, respectively).

[0370] Each of UnT (untransduced T cells), NextGen UCD20A cells, and NextGen UCD20 M12 cells received no treatment as a control (unstimulated group) or was 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 determined using the ELISA kit (Abcam, catalog number ab220656), and IL-23 was tested using the human IL-23 kit (PerkinElmer, catalog number 62HIL23PEG).

[0371] like Fig. 10AAs shown, the IL12p40 levels from UnT, NextGen UCD20A cells or NextGen UCD20M12 cells reached 538pg / mL, 368pg / mL and 7760pg / 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 28pg / mL, 1312pg / mL and 15574pg / mL, respectively, revealing that the IL12p40 levels released by activated NextGen UCD20A cells were significantly lower than those of activated NextGen UCD20 M12 cells. Furthermore, IL-23 release from NextGen UCD20 M12 cells following CD3 / CD28 stimulation was significantly elevated compared to untreated cells (p<0.05), and IL12p40 levels released from NextGen UCD20A cells were again significantly lower than those released from activated NextGen UCD20 M12 cells (p<0.05).

[0372] Contemplated is the use of immune cells (eg, T cells) armored with MB12 in any of the methods described herein.

[0373] 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. Amino acids of ITAM-modified CD20 CAR MALPVTALLLPLALLLHAARPEVQLQQSGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGQGLEWIGAIYPGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSADYYCARSNYYGSSYWFFDVWGAGTTVTVSSGGGGSGGGGSGGGGSDIVLTQSPAILSASPGEKVTMTCRASSSVNYMDWYQKKPGSSPKPWIYATSNLASGVPARFSGSGSGTSYSLTISRVEAEDAATYYCQQWSFNPPTFGGGTKLEIKTSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELGENLYFQSGGDTQALLRNDQVYQPLRDRDDAQYSHLGGNGGSGERPPPVPNPDYEPIRKGQRDLYSGLNQRGGSGDKQTLLPNDQLYQPLKDREDDQYSHLQGNGGSGRKQRITETESPYQELQGQRSDVYSDLNTQGGSG SEQ ID NO:9. Amino acids of ITAM-modified Claudin 18.2 CAR 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 of promoting persistence of a cell therapy in a human individual, the method comprising administering to the individual a) a lymphodepleting agent, the lymphodepleting agent being administered prior to administering the cell therapy, and b) an S phase inhibitor, wherein the S phase inhibitor is administered more than once, wherein the cell therapy comprises immune cells that are resistant to the S phase inhibitor, and wherein the lymphodepleting agent is different from the S phase inhibitor.

2. The method of claim 1, wherein the method comprises administering the S phase inhibitor prior to administering the cell therapy.

3. The method of claim 2, wherein the method further comprises administering the S phase inhibitor after administering the cell therapy.

4. The method of any one of claims 1-3, wherein the S phase inhibitor is an antifolate.

5. The method of claim 4, wherein the antifolate is a DHFR inhibitor.

6. The method of claim 5, wherein the antifolate is methotrexate.

7. The method of claims 1-6, wherein the method comprises administering the S phase inhibitor within about 5 days prior to administering the immune cells.

8. The method of claims 1-7, wherein the method comprises administering the S phase inhibitor within about 10 days after administering the immune cells, optionally wherein the method comprises administering the S phase inhibitor within about 5 days before administering the immune cells and within about 10 days after administering the immune cells.

9. The method of any one of claims 1 to 8, wherein the method comprises administering the S phase inhibitor and the lymphodepleting agent on the same day, optionally wherein the S phase inhibitor and the lymphodepleting agent are administered concurrently or simultaneously, optionally wherein the method comprises administering the S phase inhibitor concurrently or simultaneously with the lymphodepleting agent within 5 days prior to immunotherapy.

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

11. The method of claim 10, wherein the method comprises administering the S phase inhibitor once every 1-10 days during a period of about 5 days before administration of the immune cells to about 90 days after administration of the immune cells.

12. The method of any one of claims 6-11, wherein methotrexate is administered once every 3-7 days or every 2-7 days for at least three cycles, optionally wherein methotrexate is administered once every 3-7 days or every 2-7 days during a time period from about 5 days before administration of the immune cells to about 90 days after administration of the immune cells, optionally wherein methotrexate is administered on days 3, 5, 10, and 17 after each administration of the immune cells.

13. The method of any one of claims 6-12, wherein the dosage is about 3 mg / m 2 About 3000mg / m 2 or about 3 mg / m 2 / day to about 3000mg / m 2 Methotrexate was administered at a dose of / day.

14. The method of any one of claims 6-13, wherein the dosage is about 5 mg / m 2 About 3000mg / m 2 or about 5 mg / m 2 / day to about 3000mg / m 2 Methotrexate was administered at a dose of / day.

15. The method of any one of claims 6-14, wherein the plasma concentration of methotrexate in the subject does not exceed about 2 μM within about 1-3 hours after administration of methotrexate.

16. The method of any one of claims 6-15, wherein the plasma concentration of methotrexate in the subject is greater than about 0.001 μM, greater than about 0.01 μM, or greater than about 0.1 μM within about 1-3 hours after administration of methotrexate.

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

18. The method of any one of claims 1-17, wherein the lymphodepleting agent comprises fludarabine and cyclophosphamide.

19. The method of claim 18, wherein the dosage is about 25 mg / m 2 To about 30mg / m 2 Fludarabine was administered at a dose of approximately 250 mg / m 2 About 1000 mg / m 2 Cyclophosphamide was administered at a dose of 20. The method of claim 18 or claim 19, wherein fludarabine and cyclophosphamide are administered about 1-3 times during a period of about 10 days before administration of the immune cells to about 90 days after administration of the immune cells.

21. The method of any one of claims 18-20, wherein fludarabine and cyclophosphamide are administered for about 3-4 consecutive days prior to administration of the immune cells, optionally wherein: a) Fludarabine and cyclophosphamide were administered for 3 consecutive days, b) Fludarabine and cyclophosphamide for 4 consecutive days, or c) Fludarabine and cyclophosphamide were administered for 3 consecutive days, followed by fludarabine alone for 1 day.

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

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

24. The method of claim 23, wherein the T cells comprise exogenous Nef protein.

25. The method of claim 23 or claim 24, wherein the T cells are engineered to not express or to express reduced levels of endogenous TCRα or TCRβ.

26. The method of claim 23, wherein the T cells are gamma-delta T cells.

27. The method of any one of claims 1-22, wherein the immune cells comprise NK cells.

28. The method of any one of claims 1-27, wherein the immune cell comprises a heterologous nucleic acid sequence encoding an engineered receptor.

29. The method of 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.

30. The method of claim 28 or claim 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, E GP-2, EGP-40, EpCAM, ERBB2, ERBB3, ERBB4, FBP, fetal acetylcholine receptor, folate receptor-α, GD3, hTERT, IL-13R-α2, kappa-light chain, KDR, LeY, L1 cell adhesion molecule, MAGE-A1, p53, MART1, GP100, proteinase-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.

31. The method of claim 30, wherein the engineered receptor is an anti-BCMACAR, an anti-CD20CAR, an anti-claudin 18.2CAR, or an anti-CLL1CAR, optionally wherein the CAR has a V comprising H The targeting domain of H.

32. The method of any one of claims 28-31, wherein the immune cell further comprises a second engineered receptor that converts a negative signal into a positive signal, optionally wherein the second engineered receptor comprises a TGFβR extracellular domain and an IL-23 receptor intracellular domain.

33. The method of any one of claims 28-31, wherein the immune cell further comprises an IL12p40 polypeptide, optionally wherein the IL12p40 polypeptide is membrane-bound.

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

12.

35. The method of any one of claims 1-34, wherein the immune cell comprises a methotrexate resistance transgene.

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

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

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

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

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

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

42. The method of claim 41, wherein the cancer is a hematological cancer.

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

44. The method of any one of claims 1-43, wherein the host T cells measured as the number of host T cells in PBMCs do not exceed about 500, 400, 300, 200, 100, or 50 cells / μL during a time period between about 2-7 days prior to administration of the immune cells and about 10, 20, 30, 40, 50, 60, 70, 80, or 90 days after administration of the immune cells.

45. The method of any one of claims 1-44, wherein the method further comprises monitoring the number of WBCs (white blood cells) in the individual.

46. ​​A method of treating a disease or condition in a human subject, the method comprising the method of any one of claims 1-45.

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

48. A vector comprising the nucleic acid of claim 47.

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

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

Images