Method and composition for treating tumors

Abstract

To provide immune cell therapy targeting and recognizing a tumor antigen, triggering the activation of immune effector cells, and exerting anti-tumor effects.SOLUTION: A method for treating CLD18-positive tumors comprises administering a first dose of immune effector cells to a subject in need thereof. The immune effector cells express a chimeric antigen receptor (CAR) and specifically recognizes CLD18. The first dose does not exceed about 2×1010 cells / kg of the subject's body weight, or the total number of cells does not exceed about 1×1012 cells.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to the field of immunotherapy, specifically to immune cell therapy that targets and identifies tumor antigens, induces the activation of immune effector cells, and exerts anti-tumor effects. [Background technology]

[0002] Recently, tumor immunotherapy has been widely studied and applied, and CAR-T technology in particular has achieved historic progress in human tumor control.

[0003] Cancer cells in solid tumors have formed a tumor microenvironment around them to support the growth and metastasis of cancer cells. The tumor microenvironment is the cellular environment in which the tumor resides, including surrounding blood vessels, immune cells, fibroblasts, other cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote tumor transformation, support tumor growth, invasion, protect tumors from host immunity, cultivate resistance to therapy, and provide a microenvironment for dormant transformation and rapid growth. Because tumors are closely associated with and constantly interact with their surrounding microenvironment, tumors can affect these microenvironments by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance. This is described by Swarts et al., “Tumor Microenvironment Complexity: "Emerging Roles in Cancer Therapy" Cancer Res, Vol. 72, pp. 2473-2480, 2012. Therefore, CAR-T technology is generally difficult to achieve when applied to the treatment of solid tumors. For example, most current literature reports that CD19 CAR T cell treatment in hematological tumors can induce very good clinical therapeutic effects for patients. However, CAR T cell treatment has not yet achieved satisfactory therapeutic effects in the treatment of solid tumors.

[0004] Therefore, there is an urgent need in the art for technical solutions that can have significant anti-tumor potential in the tumor microenvironment of solid tumors. Summary of the Invention

[0005] The objective of the present invention is to provide a technical means that can have an excellent killing effect against CLD18-positive solid tumors.

[0006] In a first aspect, the present invention provides a method of treating a CLD18-positive tumor, comprising administering to a subject in need thereof an initial dose of immune effector cells, said immune effector cells expressing a chimeric antigen receptor (CAR) and specifically recognizing CLD18, said initial dose being about 2×10 10 Not to exceed 1 x 10 cells / kg subject weight, or the total number of cells is about 1 x 10 12 Preferably, the initial dose is about 2×10 cells. 9 Not to exceed 1 x 10 cells / kg subject weight, or the total number of cells is about 1 x 10 11 Preferably, the initial dose is about 2.5×10 cells. 8 cells / kg covered The total number of cells should not exceed about 1×10 10 Preferably, the initial dose is about 5×10 7 Not to exceed 1 x 10 cells / kg subject weight, or the total number of cells is about 1 x 10 10 More preferably, the initial dose is about 3×10 7 Not to exceed 2 x 10 cells / kg subject body weight, or the total number of said cells is about 2 x 10 9 Does not exceed the cell size.

[0007] In a preferred embodiment, the initial dose is 1×10 5 Not less than cells / kg subject body weight.

[0008] In a specific embodiment, said CLD18 is CLD18A2.

[0009] In a specific embodiment, the initial dose of immune effector cells is administered followed by at least one re-administration of subsequent doses of immune effector cells that express a chimeric antigen receptor (CAR) and specifically recognize CLD18.

[0010] In a preferred embodiment, the subsequent dose of immune effector cells is administered under safe conditions to the patient.

[0011] In a preferred embodiment, the immune effector cells in said first dose and said subsequent doses of immune effector cells are the same.

[0012] In a preferred embodiment, the immune effector cells of the first dose and the immune effector cells of the subsequent doses all recognize CLD18A2 but comprise different chimeric antigen receptors, e.g., the chimeric antigen receptors have different extracellular segments but all recognize CLD18A2, or the chimeric antigen receptors have the same extracellular segment but different transmembrane or intracellular domains.

[0013] In a specific embodiment, said subsequent dose of immune effector cells is readministered after said first dose of immune effector cells is no longer detectable in vivo.

[0014] In a preferred embodiment, for example, it is measured by qPCR.

[0015] In a specific embodiment, said subsequent dose of immune effector cells is administered about 21 to 80 days after said initial dose is administered, preferably, said subsequent dose of immune effector cells is administered about 25 to 60 days after said initial dose is administered, and more preferably, said subsequent dose of immune effector cells is administered about 25 to 50 days after said initial dose is administered.

[0016] In a preferred embodiment, said subsequent doses are less than, the same as, or greater than the initial dose.

[0017] In a preferred embodiment, said subsequent doses correspond to said initial dose.

[0018] In a preferred embodiment, the subsequent dose is less than the initial dose.

[0019] In a preferred embodiment, the subsequent dose is greater than the initial dose.

[0020] In specific embodiments, said subsequent dose is greater than the initial dose, preferably said subsequent dose is at least 2-fold, 5-fold, 7-fold or 10-fold greater than said initial dose.

[0021] In a specific embodiment, said subsequent dose is about 2×10 10 Not to exceed 1 x 10 cells / kg subject weight, or the total number of cells is about 1 x 10 12 Does not exceed the cell size.

[0022] In a preferred embodiment, the subsequent dose is about 2×10 9 Not to exceed 1 x 10 cells / kg subject body weight or a total of approximately 1 x 10 11 Does not exceed the cell size.

[0023] In a preferred embodiment, the subsequent dose is about 2.5×10 8 cells / kg subject weight or the total number of cells is about 1×10 10 Does not exceed the cell size.

[0024] In a preferred embodiment, the subsequent dose is about 5×10 7 Not to exceed 1 x 10 cells / kg subject body weight or a total of approximately 1 x 10 10 Does not exceed the cell size.

[0025] In a preferred embodiment, the subsequent dose is about 3×10 7 Not to exceed 5 x 10 cells / kg subject weight or total number 9 or about 2 x 10 9 Does not exceed the cell size.

[0026] In a preferred embodiment, the subsequent dose is 1×10 5 Not less than cells / kg subject body weight.

[0027] In a specific embodiment, said initial dose or subsequent doses are administered in N separate doses within 20 days, where N is a whole number not less than 1.

[0028] In a preferred embodiment, the initial dose or subsequent doses are administered in N doses within 3 to 15 days, where N is a natural number not smaller than 1.

[0029] In preferred embodiments, N is 1, 2, 3 or 4.

[0030] In a preferred embodiment, N is 2 or 3.

[0031] In a specific embodiment, when a subsequent dose is administered, the subject receives: (i) the subject's serum level of a factor indicative of cytokine release syndrome (CRS) is about 10-fold, about 25-fold, and / or about 50-fold lower than the level in the subject immediately prior to administration of the first dose; (ii) The characteristic of not exhibiting grade 3 or higher neurotoxicity; (iii) the level of neurotoxicity or CRS is reduced compared to the peak level of neurotoxicity or CRS after administration of the first dose of immune effector cells; or (iv) the subject exhibits no detectable humoral or cell-mediated immune response to a CAR expressed by the first dose of cells.

[0032] In a specific embodiment, when said subsequent dose is administered, if said subject has characteristic (iii), the CRS level is reduced by at least 50%, preferably reduced by at least 20%, more preferably reduced by at least 5%, compared to the peak level of CRS after administration of the first dose of immune effector cells, or the CRS level corresponds to the CRS level before administration of the first dose of immune effector cells.

[0033] In a preferred embodiment, the CRS level can be assessed based on information such as fever, hypotension, hypoxia, neuropathy, or serum levels of inflammatory cytokines or C-reactive protein (CRP).

[0034] In preferred embodiments, the clinical risk of neurotoxicity and / or symptoms associated with grade 3 or higher neurotoxicity are selected from confusion, delirium, expressive aphasia, unresponsiveness, myoclonus, lethargy, altered mental status, convulsions, epileptiform activity, epilepsy (optionally confirmed by electroencephalogram (EEG)), elevated amyloid beta (Aβ) levels, elevated glutamate levels, and elevated oxygen free radical levels.

[0035] In a specific embodiment, the method further comprises a step of pre-treating the subject prior to administering the immune effector cells, wherein the pre-treatment comprises administering a chemotherapeutic agent to the subject, or administering total body radiation to the subject, or administering localized radiation therapy to the subject, or a combination thereof.

[0036] In a preferred embodiment, the pretreatment is performed before administration of the first dose or between administration of the first and subsequent doses, more preferably, the pretreatment is performed between administration of the first and subsequent doses.

[0037] In a preferred embodiment, said pretreatment is performed prior to administration of a first dose of immune effector cells, and there is no need to perform said pretreatment prior to administration of subsequent doses of immune effector cells.

[0038] In a preferred embodiment, the pretreatment should be performed prior to administration of the first dose of immune effector cells and prior to administration of any subsequent doses of immune effector cells.

[0039] In a preferred embodiment, said pretreatment is carried out to deplete lymphocytes.

[0040] In a preferred embodiment, the pretreatment is performed to stabilize or reduce tumor burden, particularly to maintain a stabilized or reduced tumor burden prior to immune effector cell therapy.

[0041] In a preferred embodiment, the subject is administered a lymphocyte depleting agent to deplete lymphocytes, such as cyclophosphamide, fludarabine, or the like.

[0042] In a specific embodiment, the pretreatment is performed 4 to 12 days prior to administration of the immune effector cells.

[0043] In a specific embodiment, the chemotherapeutic agent is selected from any one of cyclophosphamide, fludarabine, a taxane compound, a pyrimidine anti-tumor drug, or a combination thereof.

[0044] In a preferred embodiment, the taxane compound includes, but is not limited to, paclitaxel, albumin-bound paclitaxel, or docetaxel, and is preferably albumin-bound paclitaxel, and the pyrimidine antitumor drug is 5-fluorouracil, gimeracil, ottiracil potassium, or the like. These include, but are not limited to, potassium, diflufurouracil, carmofur, deoxyfluridine, and capecitabine.

[0045] In a specific embodiment, said chemotherapeutic agent is a combination of cyclophosphamide and fludarabine, or a combination of cyclophosphamide, fludarabine and albumin-bound paclitaxel.

[0046] In a preferred embodiment, the dosage of fludarabine is about 10-50 mg / m 2 / day, or approximately 15-40 mg / m 2 / day, or about 15-30 mg / m 2 / day, or about 20-30 mg / m 2 / day.

[0047] In a preferred embodiment, the dosage of cyclophosphamide is about 300 to 700 mg / m 2 / day, or approximately 400-650 mg / m 2 / day, or about 450-600 mg / m 2 / day, or approximately 450-550 mg / m 2 / day, or approximately 490-550 mg / m 2 / day.

[0048] In a preferred embodiment, the albumin-bound paclitaxel dosage is about 300 mg / m 2 / day or no more than about 200 mg / m 2 / day or about 150 mg / m 2 / day or no more than about 100 mg / m 2 / day or about 80 mg / m 2 / day or about 70 mg / m 2 Not more than / day.

[0049] In a specific embodiment, the period of continuous use of said chemotherapeutic agent does not exceed four days.

[0050] In a specific embodiment, the period of continuous use of said cyclophosphamide and / or said fludarabine does not exceed four days, and said albumin-bound paclitaxel is administered once.

[0051] In a preferred embodiment, pretreatment is performed, particularly prior to administration of the initial and subsequent doses, which may result in a corresponding reduction in the volume of said immune effector cells subsequently administered.

[0052] In a specific embodiment, the tumor is a CLD18A2-positive gastrointestinal tumor, preferably, the gastrointestinal tumor is an adenocarcinoma, more preferably, the gastrointestinal tumor is a pancreatic cancer or gastric adenocarcinoma.

[0053] In a specific embodiment, said chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain is an antibody or fragment thereof that specifically binds to CLD18A2.

[0054] In a preferred embodiment, the transmembrane domain of said chimeric antigen receptor is the transmembrane domain of CD28 or CD8.

[0055] In a preferred embodiment, the intracellular domain of the chimeric antigen receptor is a costimulatory signal domain of CD28 and a CD3 zeta fusion peptide, or a costimulatory signal domain of CD137 and a CD3 zeta fusion peptide, or a costimulatory signal domain of CD28, a costimulatory signal domain of CD137 and a CD3 zeta fusion peptide.

[0056] In a specific embodiment, the chimeric antigen receptor comprises an antibody or fragment thereof that specifically binds to CLD18A2, a transmembrane domain and an intracellular domain, wherein the antibody HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, HCDR3 as shown in SEQ ID NO:3, LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, LCDR3 as shown in SEQ ID NO:6, or HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:7, HCDR3 as shown in SEQ ID NO:3, LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, LCDR3 as shown in SEQ ID NO:6, or It has an HCDR1 shown in SEQ ID NO:8, an HCDR2 shown in SEQ ID NO:9 or 68, an HCDR3 shown in SEQ ID NO:10, an LCDR1 shown in SEQ ID NO:11, an LCDR2 shown in SEQ ID NO:12, and an LCDR3 shown in SEQ ID NO:13.

[0057] In a specific embodiment, the antibody or fragment thereof comprises: a heavy chain variable region set forth in SEQ ID NO:14 and a light chain variable region set forth in SEQ ID NO:16; or The heavy chain variable region shown in SEQ ID NO:18 and the heavy chain variable region shown in SEQ ID NO:16 a light chain variable region as indicated, or a heavy chain variable region set forth in SEQ ID NO:22 and a light chain variable region set forth in SEQ ID NO:20; or The antibody or fragment thereof has a heavy chain variable region as shown in SEQ ID NO:53 and a light chain variable region as shown in SEQ ID NO:52. Having the sequence shown in NO:54, 56, 57, or 58.

[0058] In a preferred embodiment, the antibody or fragment thereof has a heavy chain variable region as shown in SEQ ID NO:14 and a light chain variable region as shown in SEQ ID NO:16.

[0059] In a specific embodiment, the chimeric antigen receptor has an amino acid sequence set forth in any one of SEQ ID NOs:24, 25, 26, 27, 28, 29, 30, 31, 32, preferably the sequence set forth in SEQ ID NO:24, 25 or 26.

[0060] In a preferred embodiment, the chimeric antigen receptor has an amino acid sequence as set forth in any one of SEQ ID NOs:24, 25, 26.

[0061] In a preferred example, the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO:24.

[0062] In a specific embodiment, said immune effector cell is a T lymphocyte, a NK cell, or a NKT lymphocyte.

[0063] In a preferred embodiment, said preferred immune effector cells are T lymphocytes.

[0064] In a preferred embodiment, the T lymphocytes are derived from the subject himself.

[0065] In a preferred embodiment, said T lymphocytes are of allogeneic origin.

[0066] In specific embodiments, the subsequent doses of immune effector cells are administered two or more times, with the time interval between each administration of the subsequent doses of immune effector cells being between about 21 days and about 80 days, or between about 25 days and about 60 days, or between about 25 days and 50 days, inclusive.

[0067] In a preferred approach, the subsequent dose of immune effector cells administered later stabilizes or reduces the tumor burden in said subject relative to the prior dose of immune effector cells.

[0068] In a specific embodiment, the number of immune cells in each subsequent dose administered is essentially the same.

[0069] In a specific embodiment, the number of immune effector cells in the subsequently administered subsequent dose is greater than the number of immune cells in the previously administered subsequent dose.

[0070] In a specific embodiment, the number of immune effector cells in the subsequently administered subsequent dose is less than the number of immune cells in the previously administered subsequent dose.

[0071] In a specific embodiment, prior to administration of said immune effector cells, said subject has not been treated with immune cells that express a chimeric antigen receptor that targets CLD18.

[0072] In a specific embodiment, said subsequent dose is a number of cells sufficient to stabilize or reduce the tumor burden in said subject.

[0073] In a specific embodiment, prior to being administered said first dose, said subject has already undergone surgical treatment, chemotherapy or immunotherapy different from the method of the first aspect.

[0074] In a specific embodiment, prior to administration of the first dose, or after administration of the first dose and prior to administration of subsequent doses, the subject is assessed for serum levels of factors indicative of CRS, factors indicative of neurotoxicity, factors indicative of tumor burden, and / or factors indicative of a host anti-CAR immune response.

[0075] In specific embodiments, the factor indicative of said tumor burden is the total number of tumor cells in said subject, or the total number of tumor cells in an organ of said subject, or the total number of tumor cells in a tissue of said subject, or the tumor mass or volume, or the extent of tumor metastasis, or the number of tumors.

[0076] In a specific embodiment, the method of treating a tumor comprises: i) assessing factors indicative of tumor burden prior to administering a subsequent dose; ii) determining a subsequent dosage of cells to be administered to the subject based on the results of said evaluation; and and iii) upon determination via evaluation that the subject's tumor mass or volume has stabilized or decreased, administering to the subject a subsequent dose comprising less than, more than, or about the same number of CAR-expressing cells as in the initial dose.

[0077] In a specific embodiment, the number of cells administered in said initial dose or said subsequent dose is about 1.1 x 10 6 Cells / kg subject body weight or 2.8 x 10 7 cells / kg, approximately 2.9×1 0 6 Cells / kg or 2.6 x 10 7 cells / kg or approximately 3.3 x 10 6 Cells / kg to 1.3 x 10 7 cells / kg or approximately 1.3 x 10 7 Cells / kg or 1.8 x 10 7 cell / kg, inclusive.

[0078] In a specific embodiment, the number of cells in the subsequent dose of CAR-expressing cells administered is about 1. 1×10 7 Cells / kg (cells / kg) body weight or approximately 5.1 x 10 7 cells / kg, approximately 1.3×10 7 Cells / kg to approximately 3.7 x 10 7 cells / kg, approximately 1.6×10 7 Cells / kg to about 2.2 x 10 7 cells / kg, approximately 1.9×10 7 Cells / kg to about 2.2 x 10 7 cell / kg, approx. 2.2 x 10 7 Cells / kg to approximately 2.7 x 10 7 cells / kg, including the extreme values.

[0079] In a specific embodiment, after administration of the initial or subsequent doses of immune effector cells, the subject did not exhibit cytokine release syndrome (CRS), did not exhibit severe CRS, did not exhibit neurotoxicity, did not exhibit severe neurotoxicity, or did not exhibit greater than grade 3 neurotoxicity.

[0080] In a specific embodiment, after administration of the initial or subsequent doses of immune effector cells, said immune effector cells are expanded in said subject.

[0081] In a preferred embodiment, the proliferation is indicated by (i) an increase in serum CRP levels after administration of the initial dose and / or subsequent doses compared to immediately before said administration, and / or (ii) an increase in serum levels of CAR-encoding nucleic acid after administration of the initial dose and / or subsequent doses compared to immediately before said administration, e.g., as measured by qPCR.

[0082] In a specific embodiment, following administration of the initial dose of immune effector cells, the subject has a stabilized or reduced tumor burden, hi a specific embodiment, the stabilized or reduced tumor burden is expressed as a stabilization or reduction in one or more factors indicative of tumor burden.

[0083] In a preferred embodiment, the tumor burden is further stabilized or reduced after administration of the subsequent dose of immune effector cells compared to the tumor burden after administration of the initial dose of immune effector cells.

[0084] In a specific embodiment, when subsequent doses are administered, the subject has not yet relapsed and / or one or more factors indicative of tumor burden have decreased and then not increased.

[0085] In specific embodiments, the stabilization or reduction in burden and / or further reduction in burden comprises a stabilization or reduction in the total number of diseased cells in said subject, in said subject's organs, in said subject's tissues, or in said subject's bodily fluids, and / or a stabilization or reduction in tumor mass or volume, and / or a stabilization or reduction in the number and / or extent of metastases, and / or a stabilization or reduction in tumor markers, and / or the disappearance or attenuation of complications commonly seen in mid- to late-stage cancer.

[0086] In specific embodiments, the tumor markers include alpha fetal protein (AFP), CA125, CA15-3, squamous cell carcinoma antigen (SCC), soluble fragment of cytokeratin 19 (CYFRA21-1), carcinoembryonic antigen (CEA), CA199, CA724, and the like.

[0087] In a specific embodiment, common complications of mid- to late-stage cancer include cancer pain, infection, and cancerous pleural and ascites.

[0088] In a specific embodiment, tumor mass or volume is measured via PET (positron emission tomography) and CT (computed tomography).

[0089] In a second aspect of the present invention, there is further provided a method for treating a tumour, comprising the step of the subject undergoing chemotherapy, radiation therapy, immunotherapy or a combination thereof prior to treatment with any one of the methods described above.

[0090] In a preferred solution, said immunotherapy comprises treatment with a checkpoint inhibitor or with immune effector cells that do not target CLD18.

[0091] In a preferred solution, said immunotherapy does not include immune effector cell therapy targeting CLD18.

[0092] In a preferred solution, the subject is administered an immune effector that does not target CLD18.2. He received cell therapy.

[0093] In a preferred embodiment, the tumor burden does not remain stable or decrease after receiving said chemotherapy, radiation therapy, other immunotherapy, or a combination thereof.

[0094] In a specific embodiment, the tumor is a recurrent, refractory tumor.

[0095] In a third aspect of the invention, in subjects previously treated with CLD18A2-CAR T cells The present invention provides a use of a composition comprising immune effector cells expressing a chimeric antigen receptor for specifically recognizing CLD18 in the preparation of a medicament for treating a CLD18A2-positive tumor, wherein, in use, an initial dose of immune effector cells is administered to a subject in need thereof, the immune effector cells expressing a chimeric antigen receptor (CAR) to specifically recognize CLD18, the initial dose being about 2×10 10 Not to exceed 1 x 10 cells / kg subject weight, or the total number of cells is about 1 x 10 12 Preferably, the initial dose is about 2×10 9 Not to exceed 1 x 10 cells / kg subject weight, or the total number of cells is about 1 x 10 11 Preferably, the initial dose is about 2.5×10 cells. 8 Cells / kg test subject The total number of cells does not exceed about 1×10 10 Preferably, the initial dose is about 5×10 7 Not to exceed 1 x 10 cells / kg subject weight, or the total number of cells is about 1 x 10 10 More preferably, the initial dose is about 3×10 7 Not to exceed 2 x 10 cells / kg subject body weight, or the total number of said cells is about 2 x 10 9 Does not exceed the cell size.

[0096] In a specific embodiment, said CLD18 is CLD18A2.

[0097] In a specific embodiment, the initial dose of immune effector cells is administered followed by at least one re-administration of subsequent doses of immune effector cells that express a chimeric antigen receptor (CAR) and specifically recognize CLD18.

[0098] In a preferred embodiment, the subsequent dose of immune effector cells is administered under safe conditions to the patient.

[0099] In a preferred embodiment, the immune effector cells in said first dose and said subsequent doses are the same.

[0100] In a preferred embodiment, the immune effector cells of the first dose and the immune effector cells of the subsequent doses all recognize CLD18A2 but comprise different chimeric antigen receptors, e.g., the extracellular segments of the chimeric antigen receptors are different but all recognize CLD18A2, or the extracellular segments of the chimeric antigen receptors are the same but the transmembrane or intracellular domains are different.

[0101] In a specific embodiment, said subsequent dose of immune effector cells is readministered after said first dose of immune effector cells is no longer detectable in vivo.

[0102] In a preferred embodiment, e.g., as measured by qPCR. In a specific embodiment, the subsequent dose of immune effector cells is administered about 21 to 80 days after the initial dose is administered, preferably, the subsequent dose of immune effector cells is administered about 25 to 60 days after the initial dose is administered, and more preferably, the subsequent dose of immune effector cells is administered about 25 to 50 days after the initial dose is administered.

[0103] In a preferred embodiment, said subsequent doses correspond to said initial dose.

[0104] In a preferred embodiment, the subsequent dose is less than the initial dose.

[0105] In a preferred embodiment, the subsequent dose is greater than the initial dose.

[0106] In specific embodiments, said subsequent dose is greater than the initial dose, preferably said subsequent dose is at least 2-fold, 5-fold, 7-fold or 10-fold greater than said initial dose.

[0107] In a specific embodiment, said subsequent dose is about 2×10 10 Not to exceed 1 x 10 cells / kg subject weight, or the total number of cells is about 1 x 10 12 Does not exceed the cell size.

[0108] In a preferred embodiment, the subsequent dose is about 2×10 9 Not to exceed 1 x 10 cells / kg subject body weight or a total of approximately 1 x 10 11 Does not exceed the cell size.

[0109] In a preferred embodiment, the subsequent dose is about 2.5×10 8 cells / kg subject weight or the total number of cells is about 1×10 10 Does not exceed the cell size.

[0110] In a preferred embodiment, the subsequent dose is about 5×10 7 Not to exceed 1 x 10 cells / kg subject body weight or a total of approximately 1 x 10 10 Does not exceed the cell size.

[0111] In a preferred embodiment, the subsequent dose is about 3×10 7 Not to exceed 5 x 10 cells / kg subject weight or total number 9 or about 2 x 10 9 Does not exceed the cell size.

[0112] In a specific embodiment, said initial dose or subsequent doses are administered in N separate doses within 20 days, where N is a whole number not less than 1.

[0113] In a preferred embodiment, the initial dose or subsequent doses are administered in N doses within 3 to 15 days, where N is a natural number not smaller than 1.

[0114] In preferred embodiments, N is 1, 2, 3 or 4.

[0115] In a preferred embodiment, N is 2 or 3.

[0116] In a specific embodiment, when a subsequent dose is administered, the subject receives: (i) the subject's serum level of a factor indicative of cytokine release syndrome (CRS) is about 10-fold, about 25-fold, and / or about 50-fold lower than the level in the subject immediately prior to administration of the first dose; (ii) The characteristic of not exhibiting grade 3 or higher neurotoxicity; (iii) the level of neurotoxicity or CRS is reduced compared to the peak level of neurotoxicity or CRS after administration of the first dose of immune effector cells; or (iv) the subject exhibits no detectable humoral or cell-mediated immune response to a CAR expressed by the first dose of cells.

[0117] In a specific embodiment, when said subsequent dose is administered, if said subject has characteristic (iii), the CRS level is reduced by at least 50%, preferably reduced by at least 20%, more preferably reduced by at least 5%, compared to the peak level of CRS after administration of the first dose of immune effector cells, or the CRS level corresponds to the CRS level before administration of the first dose of immune effector cells.

[0118] In a preferred embodiment, the CRS level can be assessed based on information such as fever, hypotension, hypoxia, neuropathy, or serum levels of inflammatory cytokines or C-reactive protein (CRP).

[0119] In a preferred embodiment, the clinical risk of neurotoxicity and / or symptoms associated with grade 3 or higher neurotoxicity include confusion, delirium, expressive aphasia, unresponsiveness, myoclonus, lethargy, altered mental status, convulsions, epileptiform activity, epilepsy (optionally confirmed by electroencephalogram (EEG)), elevated amyloid beta (Aβ) levels, elevated glutamate levels, and and elevated oxygen free radical levels.

[0120] In a specific embodiment, the method further comprises a step of pre-treating the subject prior to administering the immune effector cells, wherein the pre-treatment comprises administering a chemotherapeutic agent to the subject, or administering total body radiation to the subject, or administering localized radiation therapy to the subject, or a combination thereof.

[0121] In a preferred embodiment, said pretreatment is performed prior to the administration of the first dose or between the first and subsequent doses, more preferably said pretreatment is performed between the administration of the first and subsequent doses.

[0122] In a preferred embodiment, said pretreatment is performed prior to administration of a first dose of immune effector cells, and there is no need to perform said pretreatment prior to administration of subsequent doses of immune effector cells.

[0123] In a preferred embodiment, the pretreatment should be performed prior to administration of the first dose of immune effector cells and prior to administration of any subsequent doses of immune effector cells.

[0124] In a preferred embodiment, said pretreatment is carried out to deplete lymphocytes.

[0125] In a preferred embodiment, the pretreatment is performed to stabilize or reduce tumor burden, particularly to maintain a stabilized or reduced tumor burden prior to immune effector cell therapy.

[0126] In a preferred embodiment, the subject is administered a lymphocyte depleting agent to deplete lymphocytes, such as cyclophosphamide, fludarabine, or the like.

[0127] In a specific embodiment, the pretreatment is performed 4 to 12 days prior to administration of the immune effector cells.

[0128] In a specific embodiment, the chemotherapeutic agent is selected from any one of cyclophosphamide, fludarabine, taxane compounds, pyrimidine anti-tumor drugs, or a combination thereof. In a specific embodiment, the chemotherapeutic agent is a combination of cyclophosphamide and fludarabine, or a combination of cyclophosphamide, fludarabine, and albumin-bound paclitaxel.

[0129] In a preferred embodiment, the dosage of fludarabine is about 10-50 mg / m 2 / day, or approximately 15-40 mg / m 2 / day, or about 15-30 mg / m 2 / day, or about 20-30 mg / m 2 / day.

[0130] In a preferred embodiment, the dosage of cyclophosphamide is about 300 to 700 mg / m 2 / day, or approximately 400-650 mg / m 2 / day, or about 450-600 mg / m 2 / day, or approximately 450-550 mg / m 2 / day, or approximately 490-550 mg / m 2 / day.

[0131] In a preferred embodiment, the albumin-bound paclitaxel dosage is about 300 mg / m 2 / day or no more than about 200 mg / m 2 / day or about 150 mg / m 2 / day or no more than about 100 mg / m 2 Not more than or about 80 mg / day / m 2 / day or about 70 mg / m 2 Not more than / day.

[0132] In a specific embodiment, the period of continuous use of said chemotherapeutic agent does not exceed four days.

[0133] In a specific embodiment, the period of continuous use of said cyclophosphamide and said fludarabine does not exceed four days, and said albumin-bound paclitaxel is administered once.

[0134] In a specific embodiment, the tumor is a CLD18A2-positive tumor, preferably, the tumor is a CLD18A2-positive gastrointestinal tumor, more preferably, the gastrointestinal tumor is an adenocarcinoma, and most preferably, the gastrointestinal tumor is pancreatic cancer or gastric adenocarcinoma.

[0135] In a specific embodiment, said chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain is an antibody or fragment thereof that specifically binds to CLD18A2.

[0136] In a preferred embodiment, the transmembrane domain of said chimeric antigen receptor is the transmembrane domain of CD28 or CD8.

[0137] In a preferred embodiment, the intracellular domain of the chimeric antigen receptor is a costimulatory signal domain of CD28 and a CD3 zeta fusion peptide, or a costimulatory signal domain of CD137 and a CD3 zeta fusion peptide, or a costimulatory signal domain of CD28, a costimulatory signal domain of CD137 and a CD3 zeta fusion peptide.

[0138] In a specific embodiment, the chimeric antigen receptor comprises an antibody or fragment thereof that specifically binds to CLD18A2, a transmembrane domain and an intracellular domain, wherein the antibody HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:2, HCDR3 as shown in SEQ ID NO:3, LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, LCDR3 as shown in SEQ ID NO:6, or HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:7, HCDR3 as shown in SEQ ID NO:3, LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, LCDR3 as shown in SEQ ID NO:6, or It has an HCDR1 shown in SEQ ID NO:8, an HCDR2 shown in SEQ ID NO:9 or 68, an HCDR3 shown in SEQ ID NO:10, an LCDR1 shown in SEQ ID NO:11, an LCDR2 shown in SEQ ID NO:12, and an LCDR3 shown in SEQ ID NO:13.

[0139] In a specific embodiment, the antibody or fragment thereof comprises: a heavy chain variable region set forth in SEQ ID NO:14 and a light chain variable region set forth in SEQ ID NO:16; or a heavy chain variable region set forth in SEQ ID NO:18 and a light chain variable region set forth in SEQ ID NO:16; or It has a heavy chain variable region set forth in SEQ ID NO:22 and a light chain variable region set forth in SEQ ID NO:20.

[0140] In a preferred embodiment, the antibody or fragment thereof has a heavy chain variable region as shown in SEQ ID NO:14 and a light chain variable region as shown in SEQ ID NO:16.

[0141] In a specific embodiment, the chimeric antigen receptor is selected from SEQ ID NOs: 24, 25, 26, 27, 28, 29, 30, 31, or 32.

[0142] In a preferred embodiment, the chimeric antigen receptor has an amino acid sequence as set forth in any one of SEQ ID NOs:24, 25, 26.

[0143] In a preferred example, the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO:24.

[0144] In a specific embodiment, said immune effector cell is a T lymphocyte, a NK cell, or a NKT lymphocyte.

[0145] In a preferred embodiment, said preferred immune effector cells are T lymphocytes.

[0146] In a preferred embodiment, the T lymphocytes are derived from the subject himself.

[0147] In a preferred embodiment, said T lymphocytes are of allogeneic origin.

[0148] In specific embodiments, the subsequent doses of immune effector cells are administered two or more times, with the time interval between each administration of the subsequent doses of immune effector cells being between about 21 days and about 80 days, or between about 25 days and about 60 days, or between about 25 days and 50 days, inclusive.

[0149] In a preferred approach, the subsequent dose of immune effector cells administered later stabilizes or reduces the tumor burden in said subject relative to the prior dose of immune effector cells.

[0150] In a specific embodiment, the number of immune cells in each subsequent dose administered is essentially the same.

[0151] In a specific embodiment, the number of immune effector cells in the subsequently administered subsequent dose is greater than the number of immune cells in the previously administered subsequent dose.

[0152] In a specific embodiment, the number of immune effector cells in the subsequently administered subsequent dose is less than the number of immune cells in the previously administered subsequent dose.

[0153] In a specific embodiment, prior to administration of said immune effector cells, said subject has not been treated with immune cells that express a chimeric antigen receptor that targets CLD18.

[0154] In a specific embodiment, said subsequent dose is a number of cells sufficient to stabilize or reduce the tumor burden in said subject.

[0155] In a specific embodiment, prior to being administered said first dose, said subject has already undergone surgical treatment, chemotherapy or immunotherapy different from the method of the first aspect.

[0156] In a specific embodiment, prior to administration of the first dose, or after administration of the first dose and prior to administration of subsequent doses, the subject is assessed for serum levels of factors indicative of CRS, factors indicative of neurotoxicity, factors indicative of tumor burden, and / or factors indicative of a host anti-CAR immune response.

[0157] In specific embodiments, the factor indicative of said tumor burden is the total number of tumor cells in said subject, or the total number of tumor cells in an organ of said subject, or the total number of tumor cells in a tissue of said subject, or the tumor mass or volume, or the extent of tumor metastasis, or the number of tumors.

[0158] In a specific embodiment, the method of treating a tumor comprises: i) assessing factors indicative of tumor burden prior to administering a subsequent dose; ii) determining a subsequent dosage of cells to be administered to the subject based on the results of said evaluation; and and iii) upon determination via evaluation that the subject's tumor mass or volume has stabilized or decreased, administering to the subject a subsequent dose comprising less than, more than, or about the same number of CAR-expressing cells as in the initial dose.

[0159] In a specific embodiment, the number of cells administered in said initial dose or said subsequent dose is about 1.1 x 10 6 Cells / kg subject body weight or 2.8 x 10 7 cells / kg, approximately 2.9×1 0 6 Cells / kg or 2.6 x 10 7 cells / kg or approximately 3.3 x 10 6 Cells / kg to 1.3 x 10 7 cells / kg or approximately 1.3 x 10 7 Cells / kg or 1.8 x 10 7 cell / kg, inclusive.

[0160] In a specific embodiment, the number of cells in the subsequent dose of CAR-expressing cells administered is about 1. 1×10 7 Cells / kg (cells / kg) body weight or approximately 5.1 x 10 7 cells / kg, approximately 1.3×10 7 Cells / kg to approximately 3.7 x 10 7 cells / kg, approximately 1.6×10 7Cells / kg to about 2.2 x 10 7 cells / kg, approximately 1.9×10 7 Cells / kg to about 2.2 x 10 7 cell / kg, approx. 2.2 x 10 7 Cells / kg to approximately 2.7 x 10 7 cells / kg, including the extreme values.

[0161] In a specific embodiment, after administration of the initial or subsequent doses of immune effector cells, the subject did not exhibit cytokine release syndrome (CRS), did not exhibit severe CRS, did not exhibit neurotoxicity, did not exhibit severe neurotoxicity, or did not exhibit greater than grade 3 neurotoxicity.

[0162] In a specific embodiment, after administration of the initial or subsequent doses of immune effector cells, said immune effector cells are expanded in said subject.

[0163] In a preferred embodiment, the proliferation is indicated by (i) an increase in serum CRP levels after administration of the initial dose and / or subsequent doses compared to immediately before said administration, and / or (ii) an increase in serum levels of CAR-encoding nucleic acid after administration of the initial dose and / or subsequent doses compared to immediately before said administration, e.g., as measured by qPCR.

[0164] In a specific embodiment, following administration of the initial dose of immune effector cells, the subject has a stabilized or reduced tumor burden, hi a specific embodiment, the stabilized or reduced tumor burden is expressed as a stabilization or reduction in one or more factors indicative of tumor burden.

[0165] In a preferred embodiment, the tumor burden is further stabilized or reduced after administration of the subsequent dose of immune effector cells compared to the tumor burden after administration of the initial dose of immune effector cells.

[0166] In a specific embodiment, when a subsequent dose is administered, the subject has not yet relapsed. and / or one or more factors indicative of tumor burden have decreased and then not increased.

[0167] In specific embodiments, the stabilization or reduction in burden and / or further reduction in burden comprises a stabilization or reduction in the total number of diseased cells in said subject, in said subject's organs, in said subject's tissues, or in said subject's bodily fluids, and / or a stabilization or reduction in tumor mass or volume, and / or a stabilization or reduction in the number and / or extent of metastases, and / or a stabilization or reduction in tumor markers, and / or the disappearance or attenuation of complications commonly seen in mid- to late-stage cancer.

[0168] In specific embodiments, the tumor markers include alpha fetal protein (AFP), CA125, CA15-3, squamous cell carcinoma antigen (SCC), soluble fragment of cytokeratin 19 (CYFRA21-1), carcinoembryonic antigen (CEA), CA199, CA724, and the like.

[0169] In a specific embodiment, common complications of mid- to late-stage cancer include cancer pain, infection, and cancerous pleural and ascites.

[0170] In a specific embodiment, tumor mass or volume is measured via PET (positron emission tomography) and CT (computed tomography).

[0171] In a fourth aspect of the present invention there is further provided a composition comprising cyclophosphamide and fludarabine and a taxane and / or a pyrimidine antitumor drug.

[0172] In a preferred embodiment, the taxane compounds include, but are not limited to, paclitaxel, albumin-bound paclitaxel or docetaxel, preferably albumin-bound paclitaxel, and the pyrimidine antitumor drugs include, but are not limited to, 5-fluorouracil, gimeracil, oteracil potassium, diflufloracil, carmoful, deoxyfluridine, and capecitabine.

[0173] In a specific embodiment, said composition comprises cyclophosphamide, fludarabine and paclitaxel.

[0174] In a specific embodiment, said composition comprises cyclophosphamide, fludarabine and albumin-bound paclitaxel.

[0175] In a preferred embodiment, the composition is administered to a subject receiving immune effector cell therapy before the subject receives the immune effector cells, preferably before each administration of immune effector cells to a subject receiving immune effector cell therapy.

[0176] In a specific embodiment, the dosage of fludarabine is about 10-50 mg / m 2 / day, or approximately 15-40 mg / m 2 / day, or about 15-30 mg / m 2 / day, or about 20-30 mg / m 2 / day, and / or the dose of cyclophosphamide is about 300-700 mg / m 2 / day, or approximately 400-650 mg / m 2 / day, or about 450-600 mg / m 2 / day, or approximately 450-550 mg / m 2 / day, or approximately 490-550 mg / m 2 / day, and / or the albumin-bound paclitaxel dose is about 300 mg / m 2 / day or about 200 mg / m 2 / day or about 150 mg / m 2 / day or about 100 mg / m2 / day or about 80 mg / m 2 / day or about 70 mg / m 2 Not to exceed / day.

[0177] In a specific embodiment, the composition is administered about 30 days, preferably about 20 days, more preferably about 12 days, and most preferably about 7 days, prior to administering immune effector cell therapy.

[0178] In a specific embodiment, the duration of continuous use of said cyclophosphamide, fludarabine and / or albumin-bound paclitaxel does not exceed four days, and preferably said albumin-bound paclitaxel is administered once.

[0179] In a specific embodiment, it is used in anti-tumor therapy, preferably in targeted anti-tumor therapy, and most preferably in chimeric antigen receptor targeted anti-tumor therapy.

[0180] In a specific embodiment, said immune effector cells are CAR T cells.

[0181] In a specific embodiment, the CAR T cells specifically recognize CLD18A2.

[0182] In a specific embodiment, the tumor is a CLD18A2-positive gastrointestinal tumor, more preferably, the gastrointestinal tumor is an adenocarcinoma, and most preferably, the gastrointestinal tumor is a pancreatic cancer or gastric adenocarcinoma.

[0183] In a fifth aspect of the present invention, there is further provided a method for treating a tumor with a combination of immune effector cells, comprising the step of first pretreating a subject with cyclophosphamide, fludarabine and a taxane compound prior to administering immune effector cell therapy to the subject.

[0184] In a preferred embodiment, the tumor is a solid tumor.

[0185] In a preferred embodiment, the solid tumor is breast cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer, gastric cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, anal cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, Hodgkin's lymphoma, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal adenocarcinoma, soft tissue sarcoma, urethral cancer, penile cancer, bladder cancer, kidney or ureter cancer, renal pelvic cancer, central nervous system (CNS) tumor, primary CNS lymphoma, spinal tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma.

[0186] In a preferred embodiment, the taxane compound is selected from paclitaxel, albumin-bound paclitaxel, or docetaxel.

[0187] In a preferred embodiment, the taxane compound is selected from albumin-bound paclitaxel.

[0188] In a specific embodiment, the dosage of fludarabine is about 10-50 mg / m 2 / day, or approximately 15-40 mg / m 2 / day, or about 15-30 mg / m 2 / day, or about 20-30 mg / m 2 / day, and / or the dose of cyclophosphamide is about 300-700 mg / m 2 / day, or approximately 400-650 mg / m 2 / day, or about 450-600 mg / m 2 / day, or approximately 450-550 mg / m 2 / day, or approximately 490-550 mg / m 2 / day, and / or albumin-bound paclitaxel dose is approximately 300 mg / m 2 / day or no more than about 200 mg / m 2 / day or about 150 mg / m 2 / day or no more than about 100 mg / m 2 / day or about 80 mg / m 2 / day or about 70 mg / m 2 Not more than / day.

[0189] In a specific embodiment, pretreatment occurs about 30 days, preferably about 20 days, more preferably about 12 days, and most preferably about 7 days prior to administering immune effector cell therapy.

[0190] In a specific embodiment, the duration of continuous use of said cyclophosphamide, fludarabine and / or albumin-bound paclitaxel does not exceed four days, and preferably said albumin-bound paclitaxel is administered once.

[0191] In a specific embodiment, said immune effector cells are chimeric antigen receptor modified immune effector cells, such as, for example, CAR-T cells, CAR-NK cells, or CAR-NKT cells.

[0192] In a sixth aspect of the present invention, there is further provided an application of immune effector cells comprising CLD18A2-CAR in the preparation of a medicament, said medicament comprising said cells and cyclophosphamide, fludarabine and a taxane compound (preferably paclitaxel, more preferably albumin-bound paclitaxel) for treating CLD18-positive tumors, said cells and cyclophosphamide, fludarabine and a taxane compound being prepared so as to provide a therapeutic effect greater than the sum of the effects of each of said agents.

[0193] In a specific embodiment, the method comprises administering an initial dose of said immune effector cells to a subject in need thereof, said initial dose being about 3×10 7 Not to exceed 2 x 10 cells / kg subject body weight, or the total number of said cells is about 2 x 10 9 Does not exceed the cell size.

[0194] In a specific embodiment, the initial dose of immune effector cells is followed by at least one subsequent dose of the immune effector cells, wherein the subsequent dose is about 3×10 7Not to exceed 5×10 cells / kg subject weight, or the total number of cells is about 5×10 9 Does not exceed the cell size.

[0195] In a specific embodiment, said subsequent dose of immune effector cells is administered about 21 to 80 days after said initial dose is administered, preferably, said subsequent dose of immune effector cells is administered about 25 to 60 days after said initial dose is administered, and more preferably, said subsequent dose of immune effector cells is administered about 25 to 50 days after said initial dose is administered.

[0196] In a specific embodiment, said cyclophosphamide, fludarabine and taxane compound are administered 4 to 12 days prior to administration of the immune effector cells.

[0197] In a specific embodiment, the dosage of fludarabine is about 10-50 mg / m 2 / day, or approximately 15-40 mg / m 2 / day, or about 15-30 mg / m 2 / day, or about 20-30 mg / m 2 / day, and / or The dose of cyclophosphamide is approximately 300-700 mg / m 2 / day, or approximately 400-650 mg / m 2 / day, or about 450-600 mg / m 2 / day, or approximately 450-550 mg / m 2 / day, or approximately 490-550 mg / m 2 / day, and / or The dosage of the taxane compound was approximately 300 mg / m 2 / day or about 200 mg / m 2 / day not to exceed about 150 mg / m 2 / day or about 100 mg / m 2 / day or about 80 mg / m 2 / day, or approximately 70 mg / m 2 Not to exceed / day.

[0198] In a specific embodiment, the period of continuous use of said cyclophosphamide and / or said fludarabine does not exceed four days, and said taxane compound is administered once.

[0199] In a specific embodiment, the chimeric antigen receptor has an amino acid sequence set forth in any one of SEQ ID NOs:24, 25, 26, 27, 28, 29, 30, 31, 32, preferably the sequence set forth in SEQ ID NO:24, 25 or 26.

[0200] Corresponding to a sixth aspect, the present invention provides a kit for treating a CLD18-positive tumor, the kit comprising: 1) immune effector cells containing a CLD18A2-CAR; and 2) Cyclophosphamide, 3) Fludarabine, 4) a taxane compound, preferably paclitaxel, more preferably albumin-bound paclitaxel; 5) A container for containing the substance 1)-4) above; and 6) instructions for administering the kit to treat a CLD18-positive tumor; Here, the cells, cyclophosphamide, fludarabine and taxane compounds are formulated to provide a therapeutic effect that is greater than the sum of the effects of each of the agents.

[0201] In a specific embodiment, said instructions for administration comprise administering an initial dose of said immune effector cells to a subject in need thereof, said initial dose being about 3×10 7 Not to exceed 2 x 10 cells / kg subject body weight, or the total number of said cells is about 2 x 10 9 Does not exceed the cell size.

[0202] In a specific embodiment, the instructions for administration include administering at least one subsequent dose of the immune effector cells after administering the initial dose of immune effector cells, wherein the subsequent dose is about 3×10 7Not to exceed 5×10 cells / kg subject weight, or the total number of cells is about 5×10 9 Does not exceed the cell size.

[0203] In a specific embodiment, the instructions include administering the subsequent dose of immune effector cells about 21 to 80 days after the initial dose is administered, preferably about 25 to 60 days after the initial dose is administered, and more preferably about 25 to 50 days after the initial dose is administered.

[0204] In a specific embodiment, the instructions state that the drug is to be administered 4 to 12 days prior to administration of the cyclophosphamide, fludarabine and taxane compound.

[0205] In a specific embodiment, the instructions for administration include: The dosage of fludarabine is approximately 10-50 mg / m 2 / day, or approximately 15-40 mg / m 2 / day, or about 15-30 mg / m 2 / day, or about 20-30 mg / m 2 / day, and / or The dose of cyclophosphamide is approximately 300-700 mg / m 2 / day, or approximately 400-650 mg / m 2 / day, or about 450-600 mg / m 2 / day, or approximately 450-550 mg / m 2 / day, or approximately 490-550 mg / m 2 / day, and / or The dosage of the taxane compound was approximately 300 mg / m 2 / day or about 200 mg / m 2 / day or about 150 mg / m 2 / day or about 100 mg / m 2 / day or about 80 mg / m 2 / day or about 70 mg / m 2 The contents were stated to not exceed / days.

[0206] In a specific embodiment, the instructions for administration include: The duration of continuous use of said cyclophosphamide and / or said fludarabine is more than 4 days. Instead, the taxane compound is administered once.

[0207] In a specific embodiment, the chimeric antigen receptor has an amino acid sequence set forth in any one of SEQ ID NOs:24, 25, 26, 27, 28, 29, 30, 31, 32, preferably the sequence set forth in SEQ ID NO:24, 25 or 26.

[0208] Those skilled in the art will know that the kit can contain each component in a dosage amount adapted to the dosage instructions.

[0209] According to one aspect of the present invention, a method of treating a tumor is provided, comprising administering to an individual suffering from a tumor an immune effector cell and a tubulin inhibitor, wherein the immune effector cell expresses a receptor for recognizing a tumor antigen. In some embodiments, the present invention provides a method of reducing cancer cell growth, survival and / or activity, comprising administering to an individual suffering from a tumor an immune effector cell and a tubulin inhibitor, wherein the immune effector cell expresses a receptor for recognizing a tumor antigen. In some embodiments, the tubulin inhibitor is a taxane compound. In some embodiments, the taxane compound is selected from paclitaxel, albumin-bound paclitaxel (nab-paclitaxel, Abraxane), docetaxel and derivatives having a taxane skeleton structure.

[0210] In another embodiment, the antibody that specifically recognizes the tumor antigen has HCDR1 shown in SEQ ID NO:1, HCDR2 shown in SEQ ID NO:2, HCDR3 shown in SEQ ID NO:3, LCDR1 shown in SEQ ID NO:4, LCDR2 shown in SEQ ID NO:5, LCDR3 shown in SEQ ID NO:6, LCDR4 shown in SEQ ID NO:7, LCDR5 shown in SEQ ID NO:8, LCDR6 shown in SEQ ID NO:9, LCDR7 shown in SEQ ID NO:10, LCDR8 shown in SEQ ID NO:11, LCDR9 shown in SEQ ID NO:12, LCDR10 shown in SEQ ID NO:13, LCDR11 shown in SEQ ID NO:14, LCDR12 shown in SEQ ID NO:15, LCDR13 shown in SEQ ID NO:16, LCDR14 shown in SEQ ID NO:17, LCDR15 shown in SEQ ID NO:18, LCDR16 shown in S LCDR2 as shown in SEQ ID NO:5, LCDR3 as shown in SEQ ID NO:6, or HCDR1 as shown in SEQ ID NO:1, HCDR2 as shown in SEQ ID NO:7, HCDR3 as shown in SEQ ID NO:3, LCDR1 as shown in SEQ ID NO:4, LCDR2 as shown in SEQ ID NO:5, LCDR3 as shown in SEQ ID NO:6, or It has an HCDR1 shown in SEQ ID NO:8, an HCDR2 shown in SEQ ID NO:9 or 68, an HCDR3 shown in SEQ ID NO:10, an LCDR1 shown in SEQ ID NO:11, an LCDR2 shown in SEQ ID NO:12, and an LCDR3 shown in SEQ ID NO:13.

[0211] In another embodiment, the antibody that specifically recognizes the tumor antigen comprises a heavy chain variable region set forth in SEQ ID NO:14 and a light chain variable region set forth in SEQ ID NO:16, or a heavy chain variable region set forth in SEQ ID NO:18 and a light chain variable region set forth in SEQ ID NO:16; or a heavy chain variable region set forth in SEQ ID NO:22 and a light chain variable region set forth in SEQ ID NO:20; or It has a heavy chain variable region set forth in SEQ ID NO:53 and a light chain variable region set forth in SEQ ID NO:52.

[0212] In another embodiment, the antibody that specifically recognizes the tumor antigen has an amino acid sequence set forth in the sequence set forth in SEQ ID NO:54, 56, 57, or 58, preferably the sequence set forth in SEQ ID NO:54.

[0213] In another embodiment, the subject is not subjected to pretreatment or lymphodepletion. Further provided is the use of immune effector cells expressing a receptor that recognizes a tumor antigen in the preparation of a drug, the drug comprising the immune effector cells and a taxane compound, the drug being used for tumor treatment. In some embodiments, the immune effector cells and taxane compound contained in the drug provide a therapeutic effect greater than the sum of their respective effects. In one aspect of the present invention, there is provided a kit for treating tumors, comprising: 1) immune effector cells expressing a receptor that recognizes a tumor antigen; 2) a taxane compound; 3) a container for containing the substances described in 1) and 2) above; and 4) an administration manual for treating tumors using the kit.

[0214] It should be understood that within the scope of the present invention, the above technical features of the present invention and the technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which will not be repeated here due to space limitations. [Brief description of the drawings]

[0215] [Figure 1] Shows CAR-T copy number detection. [Figure 2A] This shows the change in C-reactive protein (CRP) in the subject's body after the first dose of CAR-T was injected (first treatment course). [Figure 2B] The change in CRP in the subject after injection of a subsequent dose of CAR-T (second course of treatment) is shown. [Figure 3A] 1 shows changes in tumor lesions during the course of CAR-T treatment in subjects with pancreatic cancer. [Figure 3B] Shows changes in tumor markers during CAR-T treatment in pancreatic cancer subjects. [Figure 4A] This shows changes in tumor lesions during CAR-T treatment in subjects with gastric adenocarcinoma. [Figure 4B] This shows changes in new lesions during CAR-T treatment in gastric adenocarcinoma subjects. [Figure 4C] Shows changes in the pleura and ascites during the course of CAR-T treatment in a subject with gastric adenocarcinoma. [Figure 4D] 1 shows the change in CA125 levels during the course of CAR-T treatment in subjects with gastric adenocarcinoma. [Figure 4E] 1 shows changes in CA199 levels during the course of CAR-T treatment in subjects with gastric adenocarcinoma. [Diagram 5] 1 is a plasmid map of the recombinant carrier MSCV-8E5-2I-mBBZ. [Figure 6A] These are CCK8 experiments detecting the toxic effects of Abraxane on pancreatic cancer cells and CAR-T cells, respectively. [Figure 6B] These are CCK8 experiments detecting the toxic effects of Abraxane on pancreatic cancer cells and CAR-T cells, respectively. [Figure 7A] This shows an in vivo experiment observing the inhibitory effect of combining Abraxane and CAR T cells on mouse pancreatic cancer, as well as changes in mouse body weight. [Figure 7B] This shows an in vivo experiment observing the inhibitory effect of combining Abraxane and CAR T cells on mouse pancreatic cancer, as well as changes in mouse body weight. [Figure 8A] 1 shows survival times of mice after treatment with the method of the present invention. [Figure 8B] 1 shows survival times of mice after treatment with the method of the present invention. [Figure 8C] 1 shows survival times of mice after treatment with the method of the present invention. [Figure 9] 1 is a graph of weight change in a mouse model after treatment with the method of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0216] The inventors, through extensive and thorough research, have unexpectedly discovered that administering immune effector cells expressing a chimeric antigen receptor (CAR) or other genetically engineered receptor, such as a T cell receptor (TCR), in a specific amount and according to specific time parameters can significantly improve the therapeutic efficacy of using immune effector cells to treat tumors, thereby improving the application of immune effector cells in solid tumors. The present invention is based, at least in part, on the above discovery.

[0217] Unless otherwise defined, all terms, symbols and other techniques used herein and scientific terms and proprietary vocabulary have the same meaning as commonly understood by those of ordinary skill in the art. In some cases, the specification further limits terms having a commonly understood meaning for purposes of description and / or reference, but these additional limitations contained herein should not be understood as indicating a substantial difference as commonly understood in the art.

[0218] All publications mentioned in this invention, including patent documents, journal articles, and databases, are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. To the extent that the definitions set forth herein differ or otherwise conflict with those set forth in the patents, published applications, and other publications incorporated herein by reference, the definitions in the documents incorporated herein by reference shall control.

[0219] As used herein, the singular forms "a," "one," and "said" include references to multiple subjects unless the context clearly dictates otherwise. For example, "a" or "one" refers to "at least one or one" or "one (one) or more (one or more)."

[0220] In this disclosure, all aspects of the claimed subject matter are presented in a range format. It is to be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Thus, the description of a range should be considered to specifically disclose all possible subranges within that range and each individual value within the range. For example, when a range of values ​​is provided, it is to be understood that each intermediate value between the upper and lower limits of the range, and any other said value or intermediate value within the range, is included in the claimed subject matter, and that the upper and lower limits of the range also belong to the claimed subject matter. The smaller ranges can independently include the upper and lower limits of these smaller ranges, and unless the upper and lower limits of a range are expressly excluded, they also belong to the scope of the claimed subject matter. If a stated range includes one or two limits, the claimed subject matter also includes ranges excluding the one or two limits. This principle applies regardless of the range.

[0221] The term "about" as used herein refers to the normal error range of each value, which is readily known in the art. Reference to "about" a value or parameter herein includes (and describes) the embodiment that refers to said value or said parameter itself. For example, a description of "about X" includes a description of "X". For example, "about" may mean within 1 or more than 1, depending on the practical standard deviation in the art. Alternatively, "about" may mean within a range of up to 10% (i.e., ±10%). For example, about 5 mg may include any number between 4.5 mg and 5.5 mg. When specific values ​​or compositions are provided in the scope of applications and patent applications, unless otherwise specified, "about" should be assumed to be within the acceptable error range of the specific value or composition.

[0222] When describing an amino acid or nucleic acid sequence, "having" a particular sequence should be understood to encompass variants of the particular sequence. In some embodiments, an amino acid or nucleic acid sequence having a particular sequence means that the amino acid or nucleic acid sequence has greater than 80%, 85%, 90%, 95%, or 99% sequence identity with the particular sequence.

[0223] Unless otherwise specified, any concentration range, percentage range, ratio range, or integer range set forth herein should be understood to include any integer within said range, and fractions thereof, where appropriate (e.g., tenths of integers and hundredths of integers).

[0224] As used herein, a "dosing interval" refers to the time between multiple courses of immune effector cell therapy (e.g., the administration of an initial dose of immune effector cells and a subsequent dose of immune effector cells). The term "interval between administrations" refers to the time elapsed between administrations of a pretreatment drug (including multiple courses of treatment including administration of a pretreatment drug), and between administrations of a pretreatment drug. Thus, the administration interval can be shown as a range. In some embodiments of the invention, the invention comprises administering multiple courses of immune effector cell therapy to an individual, each course being administered at a dosage (e.g., an initial dosage and subsequent dosages) determined by a physician. In some embodiments of the invention, a particular dose of immune effector cell therapy according to the invention can be administered in two or more split dosages, with the total dosage administered in the split dosages being equal to the total dosage in the course of treatment as determined by a physician.

[0225] "Dosage" as referred to herein can be expressed as a dose calculated based on weight or a dose calculated based on body surface area (BSA). A dose calculated based on weight is the dose administered to a patient calculated based on the patient's weight, such as mg / kg, number of immune effector cells / kg, etc. A dose calculated based on BSA is the dose calculated based on mg / m 2 , number of immune effector cells / m 2 The dose to be administered to the patient is calculated based on the patient's surface area, such as:

[0226] "Dosage frequency" as used herein refers to the frequency of administration of doses of immune effector cells or pretreatment drug within a given time.Dosage frequency can be indicated as the number of doses per given time.For example, fludarabine can be administered once a day for 4 consecutive days, once a day for 3 consecutive days, once a day for 2 consecutive days, or once a day for 1 day.Cyclophosphamide can be administered once a day for 4 consecutive days, once a day for 3 consecutive days, once a day for 2 consecutive days, or once a day for 1 day.Albumin-bound paclitaxel can be administered once a day for 4 consecutive days, once a day for 3 consecutive days, once a day for 2 consecutive days, or once a day for 1 day.

[0227] A composition, as used herein, refers to any mixture of two or more products, substances or compounds (including cells). It may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0228] As used herein, "a cell or cell population is 'positive' for a particular marker" refers to the detectable presence of a particular marker (usually a surface marker) on or within a cell. When referring to a surface marker, the term means that the presence of surface expression is detected, for example, by flow cytometry, by staining with an antibody that specifically binds to the marker and detecting the antibody, where the staining can be detected by flow cytometry to be significantly higher than the level of staining detected when performing the same steps under the same other conditions using an isotype-matched control, and / or is substantially similar to the level of cells known to be positive for the marker, and / or is significantly higher than the level of cells known to be negative for the marker.

[0229] As used herein, a "cell or cell population is 'negative' for a particular marker" means that the cell or cell population does not have a particular marker, such as a surface marker, on or within the cell, or that the cell or cell population is substantially undetectable despite having the particular marker. When referring to a surface marker, the term means that the surface expression is undetectable by flow cytometry, or, despite the presence of surface expression, for example, when stained with an antibody that specifically binds to the marker, and when an antibody is detected to detect surface expression, the staining is significantly lower than the level of staining detected when performing the same steps under the same other conditions using an isotype-matched control, and / or is significantly lower than the level of cells known to be positive for the marker, and / or is substantially similar to the level of cells known to be negative for the marker, as detected by flow cytometry.

[0230] The term "carrier" as used herein refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes carriers in the form of self-replicating nucleic acid structures as well as vectors introduced into the genome of a host cell to which it is introduced. Certain carriers are capable of inducing the expression of nucleic acids operably linked to them. The carriers are also referred to herein as "expression vectors".

[0231] This application relates to adoptive or immune effector cells for treating solid tumors, and methods of use, compositions, and articles of manufacture that include multiple or repeated administration of the cells. The cells generally express chimeric antigen receptors (CARs) or other genetically engineered receptors, such as T cell receptors (TCRs).

[0232] Claudin 18 (CLD18) molecules (Genbank accession numbers: splice variant 1 (CLD18A1): NP_057453, NM016369, and splice variant 2 (CLD18A2): NM_001002026, NP_001002026) are integral transmembrane proteins with molecular weights of approximately 27.9 / 27.72 kD. Claudins are integral membrane proteins found at the tight junctions of epithelia and endothelia. Tight junctions are interconnected networks of intramembrane granules between adjacent cells. At tight junctions, occludin and claudins are the most important transmembrane protein components. Due to their strong intercellular adhesion properties, they prevent and control the paracellular transport of solutes and limit the lateral diffusion of membrane lipids and proteins, creating a primary barrier that maintains cell polarity. Proteins that form tight junctions are critically involved in the structure of epithelial tissues.

[0233] CLD18A1 is selectively expressed in normal lung and gastric epithelium, whereas CLD18A2 is expressed exclusively in gastric cells. CLD18A2 is also restricted to differentiated short-lived gastric epithelial cells, but is absent from the gastric stem cell region. The two variants are strongly expressed in a variety of cancer types, including gastric, esophageal, pancreatic and lung tumors and human cancer cell lines. Expression is predominantly in the adenocarcinoma subtype of these indications.

[0234] The present invention provides therapeutic methods and compositions for treating diseases associated with CLD18 expression, such as tumors.

[0235] The present invention provides a method of treating a tumor, particularly a solid tumor, in a subject using adoptive cells or immune effector cells expressing a genetically engineered chimeric receptor. The method includes a single course of reinfusion of adoptive cells or immune effector cells, or multiple courses of reinfusion. As used herein, "dosage" refers to the total amount of adoptive cells or immune effector cells administered or reinfused in one course of treatment. In some embodiments, when the method includes multiple courses of treatment, the dosage for each course of treatment is the same. In some embodiments, when the method includes multiple courses of treatment, the dosage for each course of treatment is different. "Fractional dose" refers to the dosage administered in one treatment when the total course of treatment is administered in multiple doses to a subject in one treatment. In some embodiments, when the dosage for one course of treatment is administered in multiple doses to a subject, the fractional dose administered each time is the same. In some embodiments, when the dosage for one course of treatment is administered in multiple doses to a subject, the fractional dose administered each time is different. As used herein, unless otherwise specified, all doses refer to the total amount of adoptive cells or immune effector cells administered or reinfused during a course of treatment.

[0236] In some embodiments, the methods herein include re-injecting the adoptive cells or immune effector cells in a single course of treatment. A single course of treatment means re-injecting a constant total amount of adoptive cells or immune effector cells at a constant time point. In some embodiments, the constant total amount of adoptive cells or immune effector cells is re-injected once in the course of treatment. In some embodiments, the constant total amount of adoptive cells or immune effector cells is re-injected two or more times. In some embodiments, the constant total amount of adoptive cells or immune effector cells is re-injected three or more times. In some embodiments, the constant total amount of adoptive cells or immune effector cells is re-injected four or more times. In some embodiments, the constant total amount of adoptive cells or immune effector cells is re-injected five or more times. In some embodiments, the constant total amount of adoptive cells or immune effector cells is re-injected six or more times. In some embodiments, the constant total amount of adoptive cells or immune effector cells is re-injected seven or more times. In some embodiments, a total amount of adoptive cells or immune effector cells is reinfused 8 or more times. In some embodiments, a total amount of adoptive cells or immune effector cells is reinfused 9 or more times. In some embodiments, a total amount of adoptive cells or immune effector cells is reinfused 10 or more times. In some embodiments, the adoptive cells or immune effector cells reinfused each time are an equal amount of the adoptive cells or immune effector cells to be reinfused. In some embodiments, the adoptive cells or immune effector cells reinfused each time are an unequal amount of the adoptive cells or immune effector cells to be reinfused. In some embodiments, the amount of adoptive cells or immune effector cells reinfused each time is determined by a physician according to the specific circumstances of the subject.The specific condition of the subject may be, for example, the subject's overall health condition, the severity of the disease, the response to the previous dose of the same treatment course, the response to the previous treatment course, the subject's drug concomitant condition, the degree or possibility of a toxic reaction, complications, the cancer metastasis condition, and any other factors that the physician believes may affect the amount of adoptive cells or immune effector cells reinfused into the subject. In some embodiments, during the process of reinjecting the constant total amount of adoptive cells or immune effector cells multiple times, the amount of adoptive cells or immune effector cells reinfused each time gradually increases. In some embodiments, during the process of reinjecting the constant total amount of adoptive cells or immune effector cells multiple times, the amount of adoptive cells or immune effector cells reinfused each time gradually decreases. In some embodiments, during the process of reinjecting the constant total amount of adoptive cells or immune effector cells multiple times, the amount of adoptive cells or immune effector cells reinfused each time gradually increases at first and then gradually decreases. In some embodiments, during the course of multiple re-infusions of said constant total amount of adoptive cells or immune effector cells, the amount of adoptive cells or immune effector cells re-infused each time is first gradually decreased and then gradually increased.

[0237] Reinfusion of a plurality of therapeutic courses means comprising a number of said time points, with a quantity of adoptive cells or immune effector cells reinfused within each time point. In some embodiments, the length of the plurality of time points is consistent. In some embodiments, the length of the plurality of time points is unequal. In some embodiments, the plurality of therapeutic courses means comprising at least two said time points. In some embodiments, the plurality of therapeutic courses means comprising at least three said time points. In some embodiments, the plurality of therapeutic courses means comprising at least four said time points. In some embodiments, the plurality of therapeutic courses means comprising at least five said time points. In some embodiments, the plurality of therapeutic courses means comprising at least six said time points. In some embodiments, the plurality of therapeutic courses means comprising at least seven said time points. In some embodiments, the plurality of therapeutic courses means comprising at least eight said time points. In some embodiments, the plurality of therapeutic courses means comprising at least nine of the above time points. In some embodiments, the plurality of therapeutic courses means comprising at least nine of the above time points. In some embodiments, the plurality of treatment courses refers to comprising at least 10 of said time points. In some embodiments, the plurality of treatment courses refers to comprising 10 or more of said time points.

[0238] In some embodiments, a total amount of adoptive cells or immune effector cells is re-infused once during one of the multiple treatment courses. In some embodiments, a total amount of adoptive cells or immune effector cells is re-infused two or more times during one of the multiple treatment courses. In some embodiments, a total amount of adoptive cells or immune effector cells is re-infused three or more times during one of the multiple treatment courses. In some embodiments, a total amount of adoptive cells or immune effector cells is re-infused four or more times during one of the multiple treatment courses. In some embodiments, a total amount of adoptive cells or immune effector cells is re-infused five or more times during one of the multiple treatment courses. In some embodiments, a total amount of adoptive cells or immune effector cells is re-infused six or more times during one of the multiple treatment courses. In some embodiments, a total amount of adoptive cells or immune effector cells is re-infused seven or more times during one of the multiple treatment courses. In some embodiments, a total amount of adoptive cells or immune effector cells is re-infused 8 or more times during one treatment course of the plurality of treatment courses. In some embodiments, a total amount of adoptive cells or immune effector cells is re-infused 9 or more times during one treatment course of the plurality of treatment courses. In some embodiments, a total amount of adoptive cells or immune effector cells is re-infused 10 or more times during one treatment course of the plurality of treatment courses. In some embodiments, the adoptive cells or immune effector cells re-infused at each treatment course of the plurality of treatment courses are an equal amount of the adoptive cells or immune effector cells to be re-infused. In some embodiments, the adoptive cells or immune effector cells re-infused at each treatment course of the plurality of treatment courses are an unequal amount of the adoptive cells or immune effector cells to be re-infused during each treatment course of the plurality of treatment courses.In some embodiments, the amount of adoptive cells or immune effector cells reinfused each time during a course of treatment in the multiple treatment courses is determined by the physician according to the specific circumstances of the subject. The specific circumstances of the subject can be, for example, the subject's overall health status, the severity of the disease, the response to the previous dose of the same course of treatment, the response to the previous course of treatment, the subject's drug combination status, the degree or possibility of a toxic reaction, complications, the cancer metastasis status, and any other factors that the physician believes may affect the amount of adoptive cells or immune effector cells reinfused into the subject. In some embodiments, a constant total amount of adoptive cells or immune effector cells is reinfused the same number of times during each treatment course of the multiple treatment courses. In some embodiments, a constant total amount of adoptive cells or immune effector cells is reinfused different times during each treatment course of the multiple treatment courses. In some embodiments, a constant total amount of adoptive cells or immune effector cells is reinfused the same number of times during each treatment course of the multiple treatment courses. In some embodiments, the same constant total amount of adoptive cells or immune effector cells is reinfused during each treatment course of the multiple treatment courses. In some embodiments, a different, constant total amount of adoptive cells or immune effector cells is reinfused during each of the multiple treatment courses.

[0239] In some embodiments, with each treatment step in the plurality of treatment courses, the total amount of adoptive cells or immune effector cells increases incrementally. In some embodiments, with each treatment step in the plurality of treatment courses, the total amount of adoptive cells or immune effector cells decreases incrementally. In some embodiments, with each treatment step in the plurality of treatment courses, the total amount of adoptive cells or immune effector cells first increases incrementally and then decreases incrementally. With each treatment step in the plurality of treatment courses, the total amount of adoptive cells or immune effector cells first decreases incrementally and then increases incrementally.

[0240] In some embodiments, the initial dose is a relatively low dose and / or a regulated or reduced dose, and / or the subsequent dose is an enhanced dose and / or a regulated or reduced dose. Also provided are cells, compositions, and products that can be used in these methods. In some embodiments, the chimeric receptor is an engineered antigen receptor, such as, for example, a functional non-TCR antigen receptor, but also other recombinant antigen receptors, such as, for example, chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs). The receptor further includes a receptor that includes an extracellular portion and an intracellular portion that is specifically bound to the same ligand or receptor or other binding partner as, for example, the intracellular signaling portion of the CAR. In some embodiments, the dosage comprises a relatively low initial dose.

[0241] In some embodiments, the method includes (a) administering to a subject suffering from a tumor a single course of cells expressing a chimeric antigen receptor (e.g., a CAR), and (b) administering to the subject a plurality of courses of cells expressing a chimeric antigen receptor (e.g., a CAR), which may be administered in one or more subsequent doses in other embodiments.

[0242] The term "CLD18" refers to claudin-18, but includes any CLD18 variants (including CLD18A1 and CLD18A2), conformations, isoforms, and species homologs expressed by cells naturally or transfected with the CLD18 gene. Preferably, "CLD18" refers to human CLD18, but in particular to CLD18A2 (SEQ ID NO:1, SEQ ID NO:2) and / or CLD18A1 (SEQ ID NO:7, SEQ ID NO:8), and more preferably to CLD18A2.

[0243] The term "CLD18A1" includes any post-translationally modified variants, isoforms, and species homologs of human CLD18A1 expressed by cells naturally expressed or transfected with the CLD18A1 gene.

[0244] The term "CLD18A2" includes any post-translationally modified variants, isoforms, and species homologs of human CLD18A2 expressed by cells naturally expressed or transfected with the CLD18A2 gene.

[0245] The term "CLD18 variant" should include (i) CLD18 splice variants, (ii) post-translationally altered variants of CLD18, in particular variants differing in N-glycosylation status, (iii) CLD18 sequence variants, in particular CLD18-sequence-1, CLD18-sequence-2 and CLD18-sequence-3, (iv) free CLD18 at cell-cell tight junctions and homo / allo-associated variants, (v) CLD18 cancer-associated variants and CLD18 non-cancer-related variants.

[0246] Methods of Treating Tumors Using Cells Expressing Chimeric Antigen Receptors The present invention provides methods, compositions, and products for use in cell therapy to treat diseases or conditions, including various solid tumors. The methods involve administration of immune effector cells expressing chimeric receptors that target and recognize and / or specifically bind to tumor antigens, activating the immune effector cells. The receptors include chimeric receptors, such as chimeric antigen receptors (CARs), T cell receptors (TCRs), T cell fusion proteins (TFPs) and T cell antigen couplers (TACs), and the like. nothing.

[0247] In some embodiments, the method includes administering one or more subsequent doses of cells to the subject. The doses are generally administered in specific amounts and based on specific time parameters. In some embodiments, the method generally includes administering an initial dose of cells followed by administering subsequent doses of cells at specific times relative to the initial dose. In some embodiments, the number of cells administered and the timing of multiple doses are designed to improve one or more outcomes, such as reducing the degree or likelihood of toxicity to the subject and / or improving the efficacy of the treatment.

[0248] In some embodiments, the methods provided herein can improve the efficacy and outcome of immune effector cell therapy based on the increased degree of subject exposure of administered cells (e.g., increased cell numbers or time over time) observed herein. In multiple clinical trials, subjects suffering from various CLD18A2-positive tumors have been administered different CAR-T cells targeting CLD18A2, followed by preliminary analysis, showing an association between higher and / or longer degree of CAR-T cell exposure and outcome of therapy. These outcomes include patient survival, palliation, or stable disease. In some embodiments, the methods provided herein include monitoring the degree of subject exposure of the adoptive or immune effector cells, and determining the dosage and time interval of subsequent doses or subsequent courses of therapy depending on the degree of exposure. In some embodiments, the methods herein include monitoring the subject's exposure to the adoptive cells or immune effector cells, and, upon reaching or exceeding a certain level of exposure, maintaining or decreasing the dosage of subsequent separate administrations or subsequent treatment courses, and / or maintaining or increasing the time interval between subsequent treatment courses. In some embodiments, the methods herein include monitoring the subject's exposure to the adoptive cells or immune effector cells, and, upon reaching or exceeding a certain level of exposure, maintaining or increasing the dosage of subsequent separate administrations or subsequent treatment courses, and / or maintaining or decreasing the time interval between subsequent treatment courses.

[0249] In some embodiments, the methods herein include monitoring the subject's level or risk of a toxic reaction to the adoptive cells or immune effector cells, and determining the dosage and time interval of subsequent doses or subsequent treatment courses depending on the level or risk of toxicity, which in some embodiments includes, but is not limited to, CRS, neurotoxicity, macrophage activation syndrome, tumor lysis syndrome, and the like.

[0250] In some embodiments, subsequent doses are generally administered if the risk of a toxic reaction or its symptoms or biochemical indices (e.g., CRS or neurotoxicity, macrophage activation syndrome or tumor lysis syndrome) is lower than or equal to an acceptable level after administration of the first dose or a previous dose. In some embodiments, the toxic reaction or its symptoms or biochemical indices include any one or more of fever, hypotension, hypoxia, neuropathy, inflammatory cytokines, and serum levels of C-reactive protein (CRP). In some embodiments, an acceptable level of risk of a toxic reaction or its symptoms or biochemical indices means 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the peak level after administration of the first dose or a previous dose.

[0251] In some embodiments, if the toxic reaction reaches a peak level but declines after administration of the initial dose or a previous dose, a subsequent dose is administered. In some embodiments, a subsequent dose is administered after the toxic reaction has decreased to a level lower than or equal to an acceptable level after administration of the first or previous dose. In some embodiments, a subsequent dose is administered if the risk of a toxic reaction or its symptoms or biochemical index is lower than or equal to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the peak level displayed after administration of the first or previous dose. Thus, in some embodiments, the first or previous dose is at least 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, Subsequent doses are administered at 7, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80 days, followed by administration of subsequent doses. In some embodiments, the presence of one or more symptoms or risks associated with toxic effects is monitored and / or evaluated to determine whether the symptoms or risks are below an acceptable level, followed by infusion of subsequent doses to determine the appropriate time.

[0252] In some embodiments, subsequent doses are administered if the serum level of a factor indicative of cytokine-release syndrome (CRS) from the subject after administration of the first dose is not more than 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold, 26-fold, 27-fold, 28-fold, 29-fold, or 30-fold the serum level in said subject before administration of the first dose.

[0253] In some embodiments, the timing of administration of the subsequent doses is selected to prevent the host immune response generated by the first dose or previous doses already administered from inducing a decrease in the effectiveness of the subsequent doses. In some aspects, the host immune response, e.g., the adaptability and specificity of the administered cells and / or the chimeric antigen receptor expressed thereby, is determined by administering the subsequent doses before, for example, humoral or cell-mediated immune responses are displayed. In some embodiments, the subsequent doses can be administered before these responses are detected, e.g., using one or a variety of specialized detection methods. Generally, one or more subsequent doses are administered when the host adaptive immune response of the cells has not been detected, has not yet been built, and / or has not yet reached a certain level or degree or stage. Thus, in some embodiments, the period of administration may be at most 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 15 Subsequent doses are administered 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 days later.

[0254] Thus, in some embodiments, the methods provided herein include administering one or more subsequent doses after a first dose has stabilized or reduced tumor burden after the time of risk of toxicity and before an adaptive host immune response (i.e., immune rejection of the organism of the CAR-T cells) has occurred. In such conditions, the subsequent doses can safely and effectively provide immune surveillance, elimination, or prevention against the growth or metastasis of residual tumor cells. Thus, in some embodiments, the subsequent doses are potentiated doses of the disease.

[0255] Tumor burden includes the size of tumor volume, degree of differentiation, or type, stage, and / or occurrence or disappearance of complications such as cancerous pleural fluid, which are often seen in any late stage cancer, and / or the production or change in expression levels of tumor markers, and / or the likelihood or incidence of toxic outcomes such as CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, etc. in the subject, and / or the host immune response to the administered cells and / or chimeric antigen receptor. In some embodiments, tumor size is measured by PET (positron emission tomography) and CT (computed tomography) scalers.

[0256] The tumor marker is also called tumor label, and refers to a substance that is characteristically present in malignant tumor cells, or abnormally produced by malignant tumor cells, or produced by the host's stimulation response to tumors, which can reflect tumor development and development, and monitor tumors for therapeutic response. Tumor markers are present in the tissues, body fluids and excretions of tumor patients, and can be detected by immunological, biological and chemical methods, including alpha fetal protein (AFP), CA125, CA15-3, squamous cell carcinoma antigen (SCC), soluble fragment of sheet keratin 19 (CYFRA21-1), carcinoembryonic antigen (CEA), CA199, CA724, etc.

[0257] In some embodiments, the initial dose comprises a quantity of cells sufficient to reduce the subject's tumor burden, and subsequent doses are administered if the subject's serum levels of factors indicative of cytokine-release syndrome (CRS) are 10-fold or 25-fold higher than the subject's serum levels prior to administration of the initial dose, and / or if the subject's CRS-related outcomes peak levels begin to decline after administration of the initial dose, and if the subject has not generated a detectable adaptive host immune response specific for the chimeric receptor expressed by the cells of the initial dose.

[0258] In some embodiments, the initial dose is about 2.5×10 8 cells / kg subject body weight Less than or about 1 × 10 12The subsequent doses contain fewer than about 100 cells and are administered more than about 21 days but not more than about 80 days after administration of the initial dose.

[0259] Cell Administration in Immune Effector Cell Therapy The methods provided by the present invention include administering to a subject with a solid tumor expressing a tumor antigen multiple doses of immune effector cells expressing a chimeric antigen receptor, e.g., a CAR, TCR, TFP, TAC chimeric antigen receptor, that recognizes the tumor antigen.

[0260] "Tumor antigen" as used herein includes, but is not limited to, thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin-13 receptor subunit alpha (IL-13Rα); interleukin-11 receptor alpha (IL-11Rα); prostate stem cell antigen (PSCA); prostate specific membrane antigen (PSMA); carcinoembryonic antigen (CEA); NY-ESO-1; HIV-1 Gag; MART-1; gp100; tyrosinase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-β); stage-specific embryonic antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and its mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFR FRvIII; neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrin A receptor 2 (EphA2); fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer; TGS5; high molecular weight melanoma-associated antigen (HMWMAA); O-acetyl-GD2 ganglioside (OAcGD2); folate receptor; tumor endothelial marker marker 1 (TEM1 / CD248); tumor endothelial marker 7-related (TEM7R); claudin 6, claudin 18.2, claudin 18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B-cell maturation antigen (BCMA); CA9; kappa light chain chain);CSPG4;EGP2, EGP40;FAP;FAR;FBP;embryonic AchR;HLA-A1, HLA-A2;MAGEA1, MAGE3;KDR;MCSP;NKG2D ligand;PSC1;ROR1;Sp17;SURVIVIN;TAG72;TEM1;fibronectin;tenascin;tumor necrotic lesions carcinoma embryonic variants;G protein-coupled receptor class C group 5 member D (GPRC5D);X chromosome open reading frame 61 (CXORF61);CD97;CD179a;anaplastic lymphoma kinase (ALK);polysialic acid;placenta specific 1 (PLAC1);globoH glycoceramide hexose moiety (globoH);breast differentiation antigen (NY-BR-1);uroplakin 2 (uroplakin 2, UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor β3 (ADRB3); pannexin 3 (pannexin 3, PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex locus K9 (LY6K); olfactory receptor 51E2 (OR51E2); TCR gamma alternate reading frame protein (TARP); Wilms tumor protein (WT1); ETS translocation variant gene 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family member 1A (XAGE1); angiopoietin cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; p53 mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of cell death (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17);pairing box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myelosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen 3 recognized by T cells (SART3); pairing box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); I The tumor antigens include, but are not limited to, Fc fragment of gA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); mucin-like hormone receptor-like 2 containing an EGF-like module (EMR2); lymphocyte antigen 75 (LY75); phosphatidyl sarcoglycan-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like peptide 1 (IGLL1). In some embodiments, the tumor antigen is EGFR, EGFRvIII, proteoglycan 3, claudin 18.2, or BCMA.

[0261] As used herein, a "subject" is a mammal, such as a human or other animal, but generally a person. In some embodiments, the subject has already undergone tumor chemotherapy or radiation therapy before administering the first dose and / or subsequent doses. In some aspects, the subject is refractory or non-responsive to other therapeutic agents.

[0262] In some embodiments, the tumor is refractory to other treatments, including, for example, chemotherapy and radiation therapy. In some embodiments, administration as described above effectively treats a subject, regardless of whether the subject is resistant to other therapies.

[0263] In some embodiments, the subject is treated with a therapeutic agent that is responsive to other therapeutic agents to reduce the tumor burden. In some aspects, the subject is initially responsive to the therapeutic agent, but over time, the tumor recurs. In some of these embodiments, once the subject is detected to be at risk of recurrence, e.g., once an increased risk of recurrence is detected, cells are administered prophylactically to reduce the likelihood of recurrence or to prevent recurrence.

[0264] The disease includes a tumor or other proliferative disease or condition. The tumors include, but are not limited to, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal adenocarcinoma, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, bladder cancer, kidney or ureter cancer, renal pelvic cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of the above cancers, and metastatic lesions of cancer. .

[0265] In some embodiments, the size of the dose and the reinfusion time are determined by the subject's initial tumor burden. For example, in some situations, the number of cells in the initial dose administered to the subject is generally relatively small, and when the tumor burden is relatively low, e.g., solid tumors can be detected using tumor markers to assess the size of the tumor burden and / or microscopic residual lesions, the initial dose can be relatively large. In other situations, in subjects with a relatively high tumor burden, the initial dose can be divided and injected continuously, but the initial dose is administered in 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses, preferably in 1 to 5 doses, more preferably in 2 to 3 doses. The interval between each dose can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, or no interval. In some circumstances, the subsequent doses can be the same as the first dose, or greater than the first dose, or less than the first dose.

[0266] The term "treatment" as used herein means to completely or partially alleviate or reduce a tumor or associated symptoms. Desired therapeutic effects include, but are not limited to, prevention of tumor development or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of a tumor, prevention of metastasis, slowing down the rate of tumor progression, improving or alleviating the tumor condition, and alleviating or improving prognosis. As used herein, "delaying tumor growth" means delaying, inhibiting, mitigating, slowing down, stabilizing, suppressing, and / or limiting the growth of said tumor. The delay can have different lengths of time, which are determined by the history of the disease and / or the individual awaiting treatment. One skilled in the art will appreciate that delay can include prevention (in individuals who have not developed said tumor). For example, late-stage cancer, such as the development of metastases, can be delayed.

[0267] In some embodiments, the provided cells and compositions slow tumor development or attenuate tumor progression.

[0268] As used herein, "inhibition" of a function or activity means that the function or activity is decreased relative to the same condition otherwise, or relative to another condition.

[0269] An "effective amount" from the administration of a reagent, such as a drug formulation, cell, or composition, means an amount effective when used at a given dosage and for a length of time necessary to obtain a desired result, e.g., a therapeutic or prophylactic effect.

[0270] A "therapeutically effective amount" of a reagent, such as a drug formulation or cells, refers to an amount effective to obtain a desired therapeutic result, e.g., treatment of a tumor, and / or the pharmacokinetic or pharmacodynamic effects of said treatment, at the dosage and time course required. A therapeutically effective amount can be subject to variation of various factors, e.g., disease state, age, sex, and weight of the subject, and the population of cells administered. In some embodiments, the methods provided relate to administration of an effective amount (e.g., a therapeutically effective amount) of said cells and / or compositions.

[0271] A "prophylactically effective amount" refers to an amount that is advantageous for obtaining a desired result at a given dosage and over the necessary time course. Generally, since a prophylactic dose is used in subjects prior to or at an early stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount. In some embodiments, when the tumor burden is relatively low, the prophylactically effective amount can be higher than the therapeutically effective amount.

[0272] Methods for administering cells used in immune effector cell therapy are known and can be used in conjunction with the methods and compositions provided herein.

[0273] In some embodiments, the cell therapy, e.g., adoptive T cell or immune effector cell therapy, proceeds via its own reinfusion regime. Thus, in some aspects, the cells are derived from a subject in need of therapy and the cells are isolated, processed, and then administered to the same subject.

[0274] In some embodiments, the cell therapy, e.g., adoptive T cell or immune effector cell therapy, proceeds via an allogeneic reinfusion approach, where the cells have been isolated and / or otherwise extracted and prepared from a donor, and the donor and the subject into which the cells are reinfused are different. In these embodiments, the cells are administered to a genetically and histocompatible subject. In some embodiments, the donor and subject are genetically identical. In some embodiments, the donor and subject are genetically similar. In some embodiments, the subject and donor belong to the same HLA class or supertype.

[0275] The cells can be administered in any suitable manner, for example, by injection, such as intravenous or subcutaneous injection, intraocular injection, fundus injection, subretinal injection, intravitreal injection, counter-interval injection, subscleral injection, intrachoroidal injection, anterior chamber injection, subconjectval injection, subconjuntival injection, suprascleral injection, retrobulbar injection, periocular injection or peribulbar delivery. In some embodiments, they are administered parenterally, intrapulmonary, and intranasally, followed by intralesional administration if localized treatment is desired. Extraperitoneal injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the cells are administered at a specified dose via a single intravenous injection. In some embodiments, the cells are administered via multiple intravenous injections, for example, not to exceed 20 days, or via continuous infusion.

[0276] The appropriate dosage in preventing or treating a disease will depend on the type of disease to be treated, the type of chimeric antigen receptor or cells, the severity and course of the disease, whether the cells are administered for prophylactic or therapeutic purposes, previous treatments, the clinical history of the subject and the response of the cells, and This can be determined by the attending physician's discretion, hi some embodiments, it may be preferred that the compositions and cells be administered to the subject at a single time point or over a series of treatments.

[0277] In some embodiments, the immune effector cells are administered as part of a combination therapy, e.g., in combination with other therapeutic agents or other therapeutic methods, such as other interventional therapies, e.g., antibodies, engineered immune effector cells, receptors or reagents, cytotoxic drugs, etc., simultaneously or sequentially, in any order. In some embodiments, the immune effector cells are administered in combination with one or more other therapies or in combination with other interventional therapeutic methods, simultaneously or sequentially, in any order. In some situations, the immune effector cells are administered in sufficient proximity to the other therapy to produce a greater therapeutic effect than the immune effector cell population or one or more other therapeutic drugs or methods, or vice versa. In some embodiments, the immune effector cells are administered prior to administration of one or more other therapeutic agents. In some embodiments, the immune effector cells are administered after administration of one or more other therapeutic agents. In some embodiments, the one or more other therapeutic agents include cytokines, such as IL-2, IL-12, to enhance persistence.

[0278] The other therapeutic agents can be administered in any suitable manner, for example, by injection, such as intravenous or subcutaneous injection, intraocular injection, fundus injection, subretinal injection, intravitreal injection, counter-interval injection, subscleral injection, intrachoroidal injection, anterior chamber injection, subconjectval injection, subconjuntival injection, suprascleral injection, retrobulbar injection, periocular injection or peribulbar delivery. In some embodiments, they are administered parenterally, intrapulmonary, and intranasally, and can proceed with intralesional administration if localized treatment is desired. Extraperitoneal injection includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, the therapeutic agent is administered at a specified dose via a single intravenous injection. In some embodiments, cells are administered via multiple intravenous injections, for example, not to exceed 20 days, or via continuous infusion.

[0279] In some embodiments, the method of the invention comprises administering to the subject a tubulin inhibitor and the adoptive cells or immune effector cells. In some embodiments, the method of the invention comprises administering to the subject a tubulin inhibitor and immune effector cells in one course of treatment. In some embodiments, the method of the invention comprises administering to the subject a tubulin inhibitor and then immune effector cells. In some embodiments, the method of the invention comprises administering to the subject first immune effector cells and then a tubulin inhibitor. In some embodiments, the method of the invention comprises administering to the subject a tubulin inhibitor and immune effector cells at about the same time. In some embodiments, the method of the invention comprises administering to the subject alternating between a tubulin inhibitor and immune effector cells. In multiple courses of treatment, the tubulin inhibitor and immune effector cells can be the same or different.

[0280] The tubulin inhibitors according to the present specification include tubulin polymerization promoters and tubulin polymerization inhibitors. The tubulin polymerization promoters include, but are not limited to, for example, taxanes, epothilones, sponge lactones, and laulimalide. The tubulin polymerization inhibitors include colchicine, combretastatin A-4, BPR0L075, plinabulin (NPI-2358), nakiterpiosin, vincristine, nocodazole, podophyllotoxin, doratidine, and the like. These include, but are not limited to, Statins 10 (Dolastatins 10), Indibulin (D-24851) and Eribulin.

[0281] Taxane compounds include paclitaxel (trade name: Taxol), albumin-bound paclitaxel (nab-paclitaxel, Abraxane), docetaxel, etc., and their derivatives. Taxane tubulin inhibitors promote the assembly of tubulin into microtubules and inhibit the depolymerization of microtubules, resulting in abnormal arrangement of microtubules and the formation of asteroid bodies, which may cause the mitotic spindle to lose its normal function and ultimately lead to cell death.

[0282] In some embodiments, the methods of the invention include administering to a subject a taxane drug and immune effector cells in a course of treatment. In some embodiments, the methods of the invention include administering to a subject a taxane drug and immune effector cells in a course of treatment. In some embodiments, the methods of the invention include administering to the subject first immune effector cells and then a taxane drug in a course of treatment. In some embodiments, the methods of the invention include administering to the subject a taxane drug and immune effector cells approximately simultaneously in a course of treatment. In some embodiments, the methods of the invention include administering to the subject alternating taxane drug and immune effector cells in a course of treatment.

[0283] In some embodiments, the methods of the invention comprise administering to the subject a taxane drug and immune effector cells over multiple treatment courses. In some embodiments, the methods of the invention comprise administering to the subject a taxane drug and then immune effector cells over multiple treatment courses. In some embodiments, the methods of the invention comprise administering to the subject first immune effector cells and then a taxane drug over multiple treatment courses. In some embodiments, the methods of the invention comprise administering to the subject a taxane drug and immune effector cells at approximately the same time over multiple treatment courses. In some embodiments, the methods of the invention comprise alternating administration of a taxane drug and immune effector cells to the subject over multiple treatment courses. In some embodiments, the methods of the invention comprise administering to the subject the same amount of taxane drug and / or immune effector cells over different treatment courses. In some embodiments, the methods of the invention comprise administering to the subject different amounts of taxane drug and / or immune effector cells over different treatment courses. The term "taxanes drug" refers to a drug that contains a taxane compound as a main component, and the taxane compound has a bridged methylene benzocyclodecene core structure similar to taxanes. In some embodiments, the bridged methylene benzocyclodecene core structure of the taxane compound includes an unsaturated bond. In some embodiments, the bridged methylene benzocyclodecene core structure of the taxane compound does not include an unsaturated bond. In some embodiments, a carbon atom of the bridged methylene benzocyclodecene core structure of the taxane is replaced by a heteroatom selected from N, O, S, and P. In some embodiments, the taxane compound is administered by injection. In some embodiments, the method of the present invention can increase, enhance, or prolong the activity and / or number of immune cells in the course of cancer treatment, or cause a medically effective response.The term "increasing" or "enhancing" immune cell activity means permitting a subject or tumor cells to have an improved ability to respond to a treatment disclosed herein. For example, an enhanced response can be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%. , 75%, 80%, 85%, 90%, 95% or 98% or more enhanced reactivity. As used herein, "enhancement" can also refer to an increase in the number of subjects treated for a response, such as, for example, treatment of immune effector cells. For example, an enhanced response can refer to a percentage of the total number of subjects treated for a response, where the percentage is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98% or more. In some embodiments, the dosage of a taxane compound, such as Abraxane, is 300 mg / m 2 Not more than 200 mg / m 2 Not more than 100 mg / m 2 / time~200mg / m 2 In some embodiments, the dose of a taxane compound, such as Abraxane, is about 200 mg / m 2 / dose, 195mg / m 2 / times, 190mg / m 2 / dose, 185mg / m 2 / times, 180mg / m 2 / dose, 175mg / m 2 / time, 170mg / m 2 / dose, 165mg / m 2 / time, 160mg / m 2 / dose, 155mg / m 2 / time, 150mg / m 2 / dose, 145mg / m 2 / dose, 140mg / m 2 / dose, 135mg / m 2 / dose, 130mg / m 2 / dose, 125mg / m 2 / time, 120mg / m 2 / dose, 115mg / m 2 / dose, 110mg / m 2 / time, 105mg / m 2 / times, 100mg / m 2 / times, 95mg / m 2 / dose, 90mg / m 2 / dose, 85mg / m 2 / dose, 80mg / m 2 / dose, 75mg / m 2 / dose, 70mg / m 2 / dose, 65mg / m 2 / time, 60mg / m 2 / dose, 55mg / m 2 / time, 50mg / m 2 / dose, 45mg / m 2 / dose, 40mg / m 2 / dose, 35mg / m 2 / times, 30mg / m 2 / dose, 25mg / m 2 / times, 20mg / m 2 / dose, 15mg / m 2 / time, 10mg / m 2 / dose, 8 mg / m 2 / dose, 6mg / m 2 / dose, 4mg / m 2 / times, 3mg / m 2 / times, 2mg / m 2 / dose or 1 mg / m 2 / time, preferably 100 mg / m 2 / time~200mg / m 2In some embodiments, the taxane compound, such as Abraxane, is intravenously infused once every 6 weeks, 5 weeks, 4 weeks, 3 weeks, 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day. In some embodiments, the taxane compound, such as Abraxane, is intravenously infused once during each course of treatment. In some embodiments, the immune effector cell treatment is performed 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 month, or any combination thereof, prior to administration of the taxane compound. In some embodiments, the treatment of immune effector cells is performed 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 month, or any combination thereof, after administration of the taxane compound.

[0284] In some embodiments, the methods of the present invention include a step of performing a pretreatment, such as, for example, a chemotherapy drug (chemotherapeutic agent), total body radiation, localized radiation therapy, or the like, or a combination thereof, prior to administration of the initial or subsequent doses. In some embodiments, the methods include a step of performing a pretreatment on the subject with one or more chemotherapeutic agents. In some embodiments, the methods include a step of performing a pretreatment on the subject with a tubulin inhibitor and one or more other chemotherapeutic agents. It is understood that, without being limited by theory, the effects of pretreatment include, but are not limited to, lymphocyte depletion, reduction of tumor burden, and the like. In some embodiments, the methods herein include a step of not performing said pretreatment prior to administration of the initial or subsequent doses. In some embodiments, the not performing said pretreatment includes, but is not limited to, non-administration of lymphocyte depleting agents, total body radiation, localized radiation therapy, or the like. This includes cases where pretreatment is performed with radiation therapy or a combination thereof, or by other means, where the lymphocyte depletion rate in the subject after said pretreatment is still less than 50%, 55%, 60%, 65% or 70%.

[0285] In some embodiments, the chemotherapeutic agent according to the present invention refers to a drug used in chemotherapy, which is a chemical drug that has a preventive or therapeutic effect against microbial infections, parasitic diseases, and malignant tumors. Chemotherapeutic agents include, but are not limited to, synthetic antibacterial agents, antibiotics, antiparasitic agents, antifungal agents, antiviral agents, alkylating agents, antimetabolites, antituberculous agents, and antineoplastic agents. For example, diterpene alkaloid compounds (e.g., taxanes), cyclophosphamide, fludarabine, cyclosporine, rapamycin, mycophenolic acid, steroids, melphalan, bendamustine, asparaginase, busulfan, carboplatin,Cisplatin, daunorubicin, doxorubicin, fluorouracil, hydroxyurea, methotrexate, rituximab, vinblastine, and / or vincristine. In some embodiments, the antimetabolite is carmoful, tegafur, pentostatin, deoxyfluridine, trimexate, fludarabine, capecitabine, gallopitabine, cytarabine stearyl sodium phosphate, fosteabine sodium hydrate, or the like. hydrate), raltitrexed, paltitrexid, dipyriteflu, tiazofurin, norlatrexed, pemetrexed, nelzarabine, 2'-deoxy-2'-methylenecytidine, 2'-fluoromethylene-2'-deoxycytidine, N-[5-(2,3-dihydrogen-benzofuranyl)sulfonyl]-N'-(3,4-dichlorophenyl)urea4-dichlorophenyl)urea), N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycerol-BL-mannose-heptanosylpyranosyl]adenine, Aplidin ine), ascidin, 4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-b]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamic acid acid), aminopterin, 5-fluorouracil, alanosine, 11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazate, tracycline (7.4.1.0.0)-tetradecano-2,4,6-triene-9-yl acetate), swainsonine, lometrisol, dexrazoxane, methioninase, 2'-cyano-2'-deoxy-N4-palmitoyl-1-BD-arabinofuranosyl cytosine, and 3-aminopyridine-2-aldehyde thiosemicarbazone. In some embodiments, the alkylating agents include, but are not limited to, dacarbazine, chlorambucil, cyclophosphamide, temozolomide, chlorambucil, busulfan, chlorambucil, and nitrosourea.

[0286] Prior to administration of the first or subsequent doses, subjects may be pretreated to improve the therapeutic efficacy of immune effector cells. The day of the first infusion of immune effector cells (e.g., CAR T cells) in each course of treatment is designated as day 0. Pretreatment occurs before immune effector cell infusion is performed. In some embodiments, pretreatment occurs at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days prior to administration of adoptive or immune effector cells. In some embodiments, pretreatment occurs with one chemotherapeutic agent. In some embodiments, pretreatment occurs with two or more chemotherapeutic agents. In some embodiments, the pretreatment is performed using one microtubule inhibitor and one or more other chemotherapeutic agents. In some embodiments, the pretreatment is performed using two microtubule inhibitors and one or more other chemotherapeutic agents. In some embodiments, the pretreatment is performed using one microtubule inhibitor, such as a tubulin polymerization promoter, one or more alkylating agents, and one or more antimetabolites. In some embodiments, the pretreatment is performed using one or more paclitaxel-based agents, one or more alkylating agents, and one or more antimetabolites. In some embodiments, the pretreatment is performed using one microtubule inhibitor (e.g., paclitaxel, particularly albumin-bound paclitaxel) and two other chemotherapeutic agents (e.g., fludarabine and cyclophosphamide). For the purpose of convenience of explanation, a specific embodiment will be described in which fludarabine and cyclophosphamide are used as representative chemotherapeutic agents, (albumin-bound) paclitaxel is used as a representative microtubule inhibitor, and one microtubule inhibitor and two other chemotherapeutic agents are used for pretreatment.For example, in some embodiments, fludarabine, or cyclophosphamide and albumin-bound paclitaxel, either alone or in combination, or in combination, are used as pretreatment to infuse CAR-T cells for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 days. , 24, 25, 26, 27, 28, 29, or 30 days prior to infusion of the CAR-T cells, preferably at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days prior to infusion of the CAR-T cells, and more preferably at least 2, 3, 4, 5, 6, 7, or 8 days prior to infusion of the CAR-T cells. In some embodiments, the pretreatment comprises administering fludarabine and cyclophosphamide 6 days prior to infusion of the CAR-T cells. In some embodiments, the pretreatment comprises administering fludarabine and cyclophosphamide 7 days prior to infusion of the CAR-T cells. In some embodiments, the pretreatment comprises administering fludarabine and cyclophosphamide 5 days prior to infusion of the CAR-T cells. In some embodiments, the pretreatment comprises CA. In some embodiments, the pretreatment comprises administering fludarabine, cyclophosphamide, and albumin-bound paclitaxel 6 days prior to injecting the CAR-T cells. In some embodiments, the pretreatment comprises administering albumin-bound paclitaxel 4 days prior to injecting the CAR-T cells.

[0287] As described above, the day of CAR T cell therapy during each course of treatment for a patient is designated as day 0. In some embodiments, fludarabine, cyclophosphamide, and albumin-bound paclitaxel can be administered at any time before CAR T cell therapy is administered. In some embodiments, fludarabine, cyclophosphamide, or albumin-bound paclitaxel is administered at least 7 days, at least 6 days, at least 5 days, at least 4 days, at least 3 days, at least 2 days, or at least 1 day before CAR T cell infusion. In some embodiments, fludarabine, cyclophosphamide, or albumin-bound paclitaxel is administered at least 12 days, at least 11 days, at least 10 days, at least 9 days, or at least 8 days before CAR T cell infusion. In some embodiments, fludarabine, cyclophosphamide, or albumin-bound paclitaxel is administered at least 7 days before CAR T cell infusion. In some embodiments, fludarabine, cyclophosphamide, or albumin-bound paclitaxel is administered at least 6 days before CAR T cell infusion. In some embodiments, administration of fludarabine, cyclophosphamide or albumin-bound paclitaxel begins 12 days prior to infusion of CAR T cells. In some embodiments, administration of fludarabine, cyclophosphamide or albumin-bound paclitaxel begins 5 days prior to infusion of CAR T cells. In some embodiments, administration of fludarabine, cyclophosphamide or albumin-bound paclitaxel begins 4 days prior to infusion of CAR T cells.

[0288] In some embodiments, fludarabine is administered 7 days prior to infusion of the CAR T cells, and cyclophosphamide is administered 7 days prior to infusion of the CAR T cells. In some embodiments, fludarabine is administered 6 days prior to infusion of the CAR T cells, and cyclophosphamide is administered 6 days prior to infusion of the CAR T cells. In some embodiments, fludarabine is administered 5 days prior to infusion of the CAR T cells, and cyclophosphamide is administered 5 days prior to infusion of the CAR T cells. In some embodiments, fludarabine is administered 12 days prior to infusion of the CAR T cells, and cyclophosphamide is administered 12 days prior to infusion of the CAR T cells. In some embodiments, fludarabine is administered 5 days prior to infusion of the CAR T cells, cyclophosphamide is administered 5 days prior to infusion of the CAR T cells, and albumin-bound paclitaxel is administered 4 days prior to infusion of the CAR T cells. In some embodiments, fludarabine is administered beginning 6 days prior to infusion of the CAR T cells, cyclophosphamide is administered beginning 6 days prior to infusion of the CAR T cells, and albumin-bound paclitaxel is administered beginning 5 days prior to infusion of the CAR T cells. In some embodiments, fludarabine is administered beginning 12 days prior to infusion of the CAR T cells, cyclophosphamide is administered beginning 12 days prior to infusion of the CAR T cells, and albumin-bound paclitaxel is administered beginning 11 days prior to infusion of the CAR T cells.

[0289] The time when the pretreatment components can be adjusted is the time when the therapeutic effect of CAR T is greatest. Generally, fludarabine, cyclophosphamide, and / or albumin-bound paclitaxel can be administered daily. In some embodiments, fludarabine, cyclophosphamide, and albumin-bound paclitaxel are administered daily for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In some embodiments, fludarabine is administered daily for 4 days and cyclophosphamide is administered daily for 2 days. In some embodiments, fludarabine is administered daily for 2 days and cyclophosphamide is administered daily for 3 days. In some embodiments, fludarabine is administered daily for 2 days and cyclophosphamide is administered daily for 4 days. In some embodiments, fludarabine is administered daily for 1 day ...2 days. In some embodiments, fludarabine is administered daily for one day, and cyclophosphamide is administered daily for four days. In some embodiments, fludarabine is administered daily for four days, and cyclophosphamide is administered daily for two days. In some embodiments, fludarabine is administered daily for one day, cyclophosphamide is administered daily for three days, and albumin-bound paclitaxel is administered daily for one day. In some embodiments, fludarabine is administered daily for one day, cyclophosphamide is administered daily for four days, and albumin-bound paclitaxel is administered daily for one day.

[0290] As described above, day 0 is designated as the day on which the patient will receive CAR T cell therapy. In some embodiments, the patient is administered fludarabine on the fourth day prior to day 0 (i.e., day -4). In some embodiments, the patient is administered fludarabine on day -5. In some embodiments, the patient is administered fludarabine on day -6. In some embodiments, the patient is administered fludarabine on day -12. In some embodiments, the patient is administered fludarabine on days -6 and -5. In some embodiments, the patient is administered fludarabine on days -5 and -4. In some embodiments, the patient is administered fludarabine on days -6, -5, -4 and -3. In some embodiments, the patient is administered fludarabine on days -7, -6, -5 and -4. In some embodiments, the patient is administered cyclophosphamide on days -6, -5, -4 and -3. In some embodiments, the patient is administered cyclophosphamide on day -6, day -5, and day -4. In some embodiments, the patient is administered cyclophosphamide on day -5, day -4, and day -2. In some embodiments, the patient is administered cyclophosphamide on day -5, day -4, and day -3. In some embodiments, the patient is administered cyclophosphamide on day -12, day -11, and day 10. In some embodiments, the patient is administered cyclophosphamide on day -7 and day -6. In some embodiments, the patient is administered cyclophosphamide on day -6 and day -5. In some embodiments, the patient is administered albumin-bound paclitaxel on day -4. In some embodiments, the patient is administered albumin-bound paclitaxel on day -5. In some embodiments, the patient is administered albumin-bound paclitaxel on day -11. In some embodiments, the patient is administered albumin-bound paclitaxel on day -5 and day -4.

[0291] Fludarabine, cyclophosphamide and albumin-bound paclitaxel can be administered on the same or different days. If fludarabine, cyclophosphamide and albumin-bound paclitaxel are administered on the same day, cyclophosphamide and / or albumin-bound paclitaxel can be administered before or after fludarabine, or fludarabine and / or albumin-bound paclitaxel can be administered before or after cyclophosphamide, or cyclophosphamide and / or fludarabine can be administered before or after albumin-bound paclitaxel. In some embodiments, the patient is administered fludarabine on days -6, -5, -4 and -3, and cyclophosphamide is administered to the patient on days -6 and -5. In some embodiments, the patient is administered fludarabine on days -7, -6, -5 and -4, and cyclophosphamide is administered to the patient on days -7 and -6. In some embodiments, the patient is administered fludarabine on day -5 and -4, and cyclophosphamide on day -5, -4, and -2. In some embodiments, the patient is administered fludarabine on day -6 and -5, and cyclophosphamide on day -6, -5, -4, and -3. In some embodiments, the patient is administered fludarabine on day -5, and cyclophosphamide on day -5, -4, and -3. In some embodiments, the patient is administered fludarabine on day -6, and cyclophosphamide on day -6, -5, and -4. In some embodiments, the patient is administered fludarabine on day -6, and cyclophosphamide on day -6, -5, -4, and -3. In some embodiments, the patient is administered fludarabine on day -6, and cyclophosphamide on day -6, -5, -4, and -3. In some embodiments, the patient is administered fludarabine on day -6, and cyclophosphamide on day -6, -5, -4, and -3. In some embodiments, the patient is administered fludarabine on day -5. On day -5, day -4 and day -3, the patient is administered fludarabine, on day -6, day -5 and day -4, the patient is administered cyclophosphamide, and on day -4, the patient is administered albumin-bound paclitaxel. In some embodiments, on day -6, the patient is administered fludarabine, on day -6, day -5 and day -4, the patient is administered cyclophosphamide, and on day -5, the patient is administered albumin-bound paclitaxel. In some embodiments, on day -12, the patient is administered fludarabine, on day -12, day -11 and day -10, the patient is administered cyclophosphamide, and on day -11, the patient is administered albumin-bound paclitaxel. In some embodiments, on day -6, the patient is administered fludarabine, on day -6, day -5, day -4 and day -3, the patient is administered cyclophosphamide, and on day -5, the patient is administered albumin-bound paclitaxel.

[0292] In some embodiments, fludarabine, cyclophosphamide, and albumin-bound paclitaxel can be administered simultaneously or sequentially. In some embodiments, the patient is administered cyclophosphamide before administering fludarabine. In some embodiments, the patient is administered cyclophosphamide after administering fludarabine. In some embodiments, the patient is administered albumin-bound paclitaxel before administering fludarabine. In some embodiments, the patient is administered albumin-bound paclitaxel before or after administering fludarabine. In some embodiments, the patient is administered albumin-bound paclitaxel before administering cyclophosphamide. In some embodiments, the patient is administered albumin-bound paclitaxel after administering cyclophosphamide.

[0293] Fludarabine, cyclophosphamide and albumin-bound paclitaxel can be administered via any route, including intravenous (IV). In some embodiments, fludarabine is administered via IV at about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 90 minutes, or about 120 minutes. In some embodiments, cyclophosphamide is administered via IV at about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 90 minutes, or about 120 minutes. In some embodiments, albumin-bound paclitaxel is administered via IV at about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 90 minutes, about 120 minutes.

[0294] In some embodiments, the patient is administered T cell therapy during administration of fludarabine and cyclophosphamide or fludarabine, cyclophosphamide and albumin-bound paclitaxel. In some embodiments, the T cell therapy comprises adoptive cell therapy or immune effector cell therapy. In some embodiments, the adoptive cell therapy or immune effector cell therapy is selected from tumor infiltrating lymphocyte (TIL) immunotherapy, autologous cell therapy, engineered autologous cell therapy (eACT), and allogeneic T cell transplantation. In some embodiments, the eACT comprises administration of engineered antigen-specific chimeric antigen receptor (CAR T) positive T cells. In some embodiments, the eACT comprises administration of engineered antigen-specific T cell receptor (TCR) positive T cells. In some embodiments, the engineered T cells treat the patient's tumor.

[0295] In some embodiments, the pretreatment is about 500 mg / m 2 / day, 490mg / m 2 / day, 480mg / m 2 / day, 470mg / m 2 / day, 460mg / m 2 / day, 450mg / m 2 / day, 440mg / m 2 / day, 430mg / m 2 / day, 420mg / m 2 / day, 410mg / m 2 / day, 400mg / m 2 / day, 390mg / m 2 / day, 380mg / m 2 / day, 370mg / m 2 / day, 360mg / m 2 / day, 350mg / m 2 / day, 340mg / m 2 / day, 330mg / m 2 / day, 320mg / m 2 / day, 310mg / m 2 / day, 300mg / m 2 / day, 290mg / m 2 / day, 280mg / m 2 / day, 270mg / m 2 / day, 260mg / m 2 / day, 250mg / m 2 / day, 240mg / m 2 / day, 230mg / m 2 / day, 220mg / m 2 / day, 210mg / m 2 / day, 200mg / m 2 / day, 190 mg / m 2 / day, 180mg / m 2 / day, 170mg / m 2 / day, 160mg / m 2 / day, 150mg / m 2 / day, 140mg / m 2 / day, 130mg / m 2 / day, 120mg / m 2 / day, 110mg / m 2 / day, 100mg / m 2 / day, 90mg / m 2 / day, 85mg / m 2 / day, 70mg / m 2 / day, 65mg / m 2 / day, 60mg / m 2 / day, 55mg / m 2 / day, 50mg / m 2 / day, 49mg / m 2 / day, 48mg / m 2 / day, 47mg / m 2 / day, 46mg / m2 / day, 45mg / m 2 / day, 44mg / m 2 / day, 43mg / m 2 / day, 42mg / m 2 / day, 41mg / m 2 / day, 40mg / m 2 / day, 39mg / m 2 / day, 38mg / m 2 / day, 37mg / m 2 / day, 36mg / m 2 / day, 35mg / m 2 / day, 34mg / m 2 / day, 33mg / m 2 / day, 32mg / m 2 / day, 31mg / m 2 / day, 30mg / m 2 / day, 29mg / m 2 / day, 28mg / m 2 / day, 27mg / m 2 / day, 26mg / m 2 / day, 25mg / m 2 / day, 24mg / m 2 / day, 23mg / m 2 / day, 22mg / m 2 / day, 21mg / m 2 / day, 20mg / m 2 / day, 19mg / m 2 / day, 18mg / m 2 / day, 17mg / m 2 / day, 16mg / m 2 / day, 15mg / m 2 / day, 14mg / m 2 / day, 13mg / m 2 / day, 12mg / m 2 / day, 11mg / m 2 / day, 10mg / m 2 / day, 9mg / m 2 / day, 8mg / m 2 / day, 7mg / m 2 / day, 6mg / m 2 / day, 5mg / m 2 / day, 4mg / m 2 / day, 3mg / m 2 / day, 2mg / m 2 / day, 1mg / m 2 / day and / or approximately 1000 mg / m 2 / day, 950mg / m 2 / day, 900mg / m 2 / day, 850mg / m 2 / day, 800mg / m 2 / day, 750mg / m 2 / day, 700mg / m 2 / day, 690mg / m 2 / day, 680mg / m 2 / day, 670mg / m 2 / day, 660mg / m 2 / day, 650mg / m 2 / day, 640mg / m 2 / day, 630mg / m 2 / day, 620mg / m 2 / day, 610mg / m 2 / day, 600mg / m 2 / day, 595mg / m 2 / day, 590mg / m 2 / day, 585mg / m 2 / day, 580mg / m 2 / day, 579 mg / m 2 / day, 578 mg / m 2 / day, 576mg / m 2 / day, 575mg / m 2 / day, 574mg / m 2 / day, 573mg / m 2 / day, 572 mg / m 2 / day, 571mg / m 2 / day, 570mg / m 2 / day, 569mg / m 2 / day, 568 mg / m 2 / day, 567mg / m 2 / day, 566mg / m 2 / day, 565mg / m 2 / day, 564mg / m 2 / day, 563mg / m 2 / day, 562mg / m 2 / day, 561mg / m 2 / day, 560mg / m 2 / day, 559mg / m 2 / day, 558mg / m 2 / day, 557mg / m2 / day, 556mg / m 2 / day, 555mg / m 2 / day, 554mg / m 2 / day, 553mg / m 2 / day, 552mg / m 2 / day, 551mg / m 2 / day, 550mg / m 2 / day, 549mg / m 2 / day, 548mg / m 2 / day, 547mg / m 2 / day, 546mg / m 2 / day, 545mg / m 2 / day, 544mg / m 2 / day, 543mg / m 2 / day, 542mg / m 2 / day, 541mg / m 2 / day, 540mg / m 2 / day, 539mg / m 2 / day, 538mg / m 2 / day, 537mg / m 2 / day, 536mg / m 2 / day, 535mg / m 2 / day, 534mg / m 2 / day, 533mg / m 2 / day, 532mg / m 2 / day, 531mg / m 2 / day, 530mg / m 2 / day, 529mg / m 2 / day, 528mg / m 2 / day, 527mg / m 2 / day, 526mg / m 2 / day, 525mg / m 2 / day, 524mg / m 2 / day, 523mg / m 2 / day, 522mg / m 2 / day, 521mg / m 2 / day, 520mg / m 2 / day, 519mg / m 2 / day, 518mg / m 2 / day, 517mg / m 2 / day, 516mg / m 2 / day, 515mg / m 2 / day, 514mg / m 2 / day, 513mg / m 2 / day, 512mg / m 2 / day, 511mg / m 2 / day, 510mg / m 2 / day, 509mg / m 2 / day, 508mg / m 2 / day, 507mg / m 2 / day, 506mg / m 2 / day, 505mg / m 2 / day, 504mg / m 2 / day, 503mg / m 2 / day, 502mg / m 2 / day, 501mg / m 2 / day, 500mg / m 2 / day, 490mg / m 2 / day, 480mg / m 2 / day, 470mg / m 2 / day, 460mg / m 2 / day, 450mg / m 2 / day, 440mg / m 2 / day, 430mg / m 2 / day, 420mg / m 2 / day, 410mg / m 2 / day, 400mg / m 2 / day, 390mg / m 2 / day, 380mg / m 2 / day, 370mg / m 2 / day, 360mg / m 2 / day, 350mg / m 2 / day, 340mg / m 2 / day, 330mg / m 2 / day, 320mg / m 2 / day, 310mg / m 2 / day, 300mg / m 2 / day, 290mg / m 2 / day, 280mg / m 2 / day, 270mg / m 2 / day, 260mg / m 2 / day, 250mg / m 2 / day, 240mg / m 2 / day, 230mg / m 2 / day, 220mg / m 2 / day, 210mg / m 2 / day, 200mg / m 2 / day, 190mg / m 2 / day, 180mg / m 2 / day, 170mg / m 2 / day, 160mg / m 2 / day, 150mg / m 2 / day, 140mg / m 2 / day, 130mg / m 2 / day, 120mg / m 2 / day, 110mg / m 2 / day, 100mg / m 2 / day, 90mg / m 2 / day, 80mg / m 2 / day, 70mg / m 2 / day, 60mg / m 2 / day, 50mg / m 2 / day, 40mg / m 2 / day, 30mg / m 2 / day, 20mg / m 2 / day or 10 mg / m 2 Cyclophosphamide administered not more than 500 mg / m2 / day and / or 2 / day, 450mg / m 2 / day, 400mg / m 2 / day, 350mg / m 2 / day, 300mg / m 2 / day, 290mg / m 2 / day, 280mg / m 2 / day, 270mg / m 2 / day, 265mg / m 2 / day, 260mg / m 2 / day, 255mg / m 2 / day, 250mg / m 2 / day, 245mg / m 2 / day, 240mg / m 2 / day, 235mg / m 2 / day, 230mg / m 2 / day, 225mg / m 2 / day, 220mg / m 2 / day, 215mg / m 2 / day, 210mg / m 2 / day, 205mg / m 2 / day, 200mg / m 2 / day, 195mg / m2 / day, 190mg / m 2 / day, 185mg / m 2 / day, 180mg / m 2 / day, 175mg / m 2 / day, 170mg / m 2 / day, 165mg / m 2 / day, 160mg / m 2 / day, 155mg / m 2 / day, 150mg / m 2 / day, 145mg / m 2 / day, 140mg / m 2 / day, 135mg / m 2 / day, 130mg / m 2 / day, 125mg / m 2 / day, 120mg / m 2 / day, 115mg / m 2 / day, 110mg / m 2 / day, 105mg / m 2 / day, 100mg / m 2 / day, 99mg / m 2 / day, 98mg / m 2 / day, 97mg / m 2 / day, 96mg / m 2 / day, 95mg / m 2 / day, 94mg / m 2 / day, 93mg / m 2 / day, 92mg / m 2 / day, 91mg / m 2 / day, 90mg / m 2 / day, 89mg / m 2 / day, 88mg / m 2 / day, 87mg / m 2 / day, 86mg / m 2 / day, 85mg / m 2 / day, 84mg / m 2 / day, 83mg / m 2 / day, 82mg / m 2 / day, 81mg / m 2 / day, 80mg / m 2 / day, 79mg / m 2 / day, 78mg / m 2 / day, 77mg / m 2 / day, 76mg / m 2 / day, 75 mg / m2 / day, 74mg / m 2 / day, 73mg / m 2 / day, 72 mg / m 2 / day, 71mg / m 2 / day, 70mg / m 2 / day, 69mg / m 2 / day, 68mg / m 2 / day, 67 mg / m 2 / day, 66mg / m 2 / day, 65mg / m 2 / day, 64mg / m 2 / day, 63mg / m 2 / day, 62 mg / m 2 / day, 61 mg / m 2 / day, 60mg / m 2 / day, 59mg / m 2 / day, 58mg / m 2 / day, 57mg / m 2 / day, 56mg / m 2 / day, 55mg / m 2 / day, 54mg / m 2 / day, 53mg / m 2 / day, 52mg / m 2 / day, 51mg / m 2 / day, 49 mg / m 2 / day, 48mg / m 2 / day, 47mg / m 2 / day, 46mg / m 2 / day, 45mg / m 2 / day, 44mg / m 2 / day, 43mg / m 2 / day, 42 mg / m 2 / day, 41mg / m 2 / day, 40mg / m 2 / day, 35mg / m 2 / day, 30mg / m 2 / day, 25mg / m 2 / day, 20mg / m 2 / day, 15mg / m 2 / day, 10mg / m 2 / day, 5mg / m 2 / day or 1 mg / m 2 Includes administration of albumin-bound paclitaxel not more than once per day.

[0296] In some embodiments, the pretreatment is at about 10 mg / m 2 / day~50mg / m 2 Fludarabine was administered at a dose of approximately 300 mg / m 2 / day~700mg / m 2 This involves administering cyclophosphamimid at a dose of 100 mg / day.

[0297] In some embodiments, the pretreatment is at about 10 mg / m 2 / day~50mg / m 2 Fludarabine is administered at a dose of approximately 300 mg / m 2 / day~700mg / m 2 Cyclophosphamide is administered at a dose of approximately 300 mg / m 2 / day, no more than about 200 mg / m 2 / day, no more than about 150 mg / m 2 / day, not more than 100 mg / m 2 / day, not more than 80 mg / m 2 / day or no more than 70 mg / m 2 The method includes administering albumin-bound paclitaxel at a dose not more than 1 mg / day.

[0298] In some embodiments, the pretreatment is about 15 mg / m 2 / day~40mg / m 2 Fludarabine was administered at a dose of approximately 400 mg / m 2 / day~650mg / m 2 This involves administering cyclophosphamimid at a dose of 100 mg / day.

[0299] In some embodiments, the pretreatment is about 15 mg / m 2 / day~40mg / m 2 Fludarabine was administered at a dose of approximately 400 mg / m 2 / day~650mg / m 2 Cyclophosphamimid is administered at a dose of approximately 300 mg / m 2 / day, no more than about 200 mg / m 2 / day, no more than about 150 mg / m 2 / day, not more than 100 mg / m 2 / day, not more than 80 mg / m 2 / day or no more than 70 mg / m 2 The method includes administering albumin-bound paclitaxel in an amount not more than 100 mg / day.

[0300] In some embodiments, the pretreatment is about 15 mg / m 2 / day~30mg / m 2 Fludarabine was administered at a dose of approximately 450 mg / m 2 / day~600mg / m 2 / day, 450mg / m 2 / day~550mg / m 2 / day or 490 mg / m 2 / day~550mg / m 2 The study involves administering cyclophosphamide at a dose of 100 mg / day.

[0301] In some embodiments, the pretreatment is about 15 mg / m 2 / day~30mg / m 2 Fludarabine was administered at a dose of approximately 450 mg / m 2 / day~600mg / m 2 / day, 450mg / m 2 / day~550mg / m 2 / day or 490 mg / m 2 / day~550mg / m 2 Cyclophosphamide was administered at a dose of approximately 300 mg / m 2 / day, no more than about 200 mg / m 2 / day, no more than about 150 mg / m 2 / day, not more than 100 mg / m 2 / day, not more than 80 mg / m 2 / day or no more than 70 mg / m 2 The method includes administering albumin-bound paclitaxel in an amount not more than 100 mg / day.

[0302] In some embodiments, the pretreatment is about 20 mg / m 2 / day~30mg / m 2Fludarabine was administered at a dose of approximately 450 mg / m 2 / day~600mg / m 2 / day, 450mg / m 2 / day~550mg / m 2 / day or 490 mg / m 2 / day~550mg / m 2 The study involves administering cyclophosphamide at a dose of 100 mg / day.

[0303] In some embodiments, the pretreatment is about 20 mg / m 2 / day~30mg / m 2 Fludarabine was administered at a dose of approximately 450 mg / m 2 / day~600mg / m 2 / day, 450mg / m 2 / day~550mg / m 2 / day or 490 mg / m 2 / day~550mg / m 2 Cyclophosphamide is administered at a dose of approximately 300 mg / m 2 / day, no more than about 200 mg / m 2 / day, no more than about 150 mg / m 2 / day, not more than 100 mg / m 2 / day, not more than 80 mg / m 2 / day or no more than 70 mg / m 2 The method includes administering albumin-bound paclitaxel in an amount not more than 100 mg / day.

[0304] In some embodiments, the pretreatment is about 20 mg / m 2 / day~25mg / m 2 Fludarabine was administered at a dose of approximately 450 mg / m 2 / day~600mg / m 2 / day, 450mg / m 2 / day~550mg / m 2 / day or 490 mg / m 2 / day~550mg / m 2 The study involves administering cyclophosphamide at a dose of 100 mg / day.

[0305] In some embodiments, the pretreatment is about 15 mg / m 2 / day~25mg / m 2 Fludarabine was administered at a dose of approximately 450 mg / m 2 / day~600mg / m 2 / day, 450mg / m 2 / day~550mg / m 2 / day or 490 mg / m 2 / day~550mg / m 2 Cyclophosphamide is administered at a dose of approximately 300 mg / m 2 / day, no more than about 200 mg / m 2 / day, no more than about 150 mg / m 2 / day, not more than 100 mg / m 2 / day, not more than 80 mg / m 2 / day or no more than 70 mg / m 2 The method includes administering albumin-bound paclitaxel in an amount not more than 100 mg / day.

[0306] In some embodiments, the invention includes a method of pretreating immune effector cells prior to infusion, the method comprising administering to the patient a dose of about 20 mg / m 2 / day and fludarabine at a dose of approximately 500 mg / m 2 / day, where fludarabine is administered on days -6, -5, -4, and -3, and cyclophosphamide is administered on days -6 and -5. In some embodiments, the invention includes a method of pre-treating prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 / day and fludarabine at a dose of approximately 514 mg / m 2 / day, where fludarabine is administered on days -7, -6, -5, and -4, and cyclophosphamide is administered on days -7 and -6. In some embodiments, the invention includes a method of pre-treating prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 / day and fludarabine at a dose of approximately 514 mg / m 2 / day, where fludarabine is administered on days -5 and -4, and cyclophosphamide is administered on days -5, -4, and -2. In some embodiments, the invention includes a method of pre-treating prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 / day and fludarabine at a dose of approximately 514 mg / m 2 / day, where fludarabine is administered on days -6 and -5, and cyclophosphamide is administered on days -6, -5, -4, and -3. In some embodiments, the invention includes a method of pre-treating prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 / day and fludarabine at a dose of approximately 510 mg / m 2 / day, where fludarabine is administered on days -6 and -5, and cyclophosphamide is administered on days -6, -5, -4, and -3. In some embodiments, the invention includes a method of pre-treating prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 / day and fludarabine at a dose of approximately 500 mg / m 2 / day, where fludarabine is administered on day -5, and cyclophosphamide is administered on days -5, -4, and -3. In some embodiments, the invention includes a method of pre-treating prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 / day and fludarabine at a dose of approximately 540 mg / m 2 / day, where fludarabine is administered on day -6, and cyclophosphamide is administered on days -6, -5, and -4. In some embodiments, the invention includes a method of pre-treating prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 / day and fludarabine at a dose of approximately 500 mg / m 2 / day, where fludarabine is administered on day -6, and cyclophosphamide is administered on days -6, -5, and -4. In some embodiments, the invention includes a method of pre-treating prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 / day and fludarabine at a dose of approximately 500 mg / m 2 / day, where fludarabine is administered on day -6, and cyclophosphamide is administered on days -6, -5, -4, and -3. In some embodiments, the invention includes a method of pre-treating prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 / day and fludarabine at a dose of approximately 500 mg / m 2 / day, where fludarabine is administered on day -12, and cyclophosphamide is administered on days -12, -11, and 10. In some embodiments, the invention includes a method of pre-treating prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 / day, and fludarabine at a dose of approximately 500 mg / m 2 / day and cyclophosphamide at a dose of approximately 100 mg / m 2 / day, wherein on day -5, cyclophosphamide on days -5, -4 and -3, and albumin-bound paclitaxel on day -4. In some embodiments, the invention includes a method of pre-treating prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 / day, and fludarabine at a dose of approximately 540 mg / m 2 / day and cyclophosphamide at a dose of approximately 71 mg / m 2 / day, where fludarabine is administered on day -6, cyclophosphamide is administered on days -6, -5 and -4, and albumin-bound paclitaxel is administered on day -5. In some embodiments, the invention includes a method of pre-treating prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 / day, and fludarabine at a dose of approximately 500 mg / m 2 / day and cyclophosphamide at a dose of approximately 71 mg / m 2 / day, where fludarabine is administered on day -12, cyclophosphamide is administered on days -12, -11 and 10, and albumin-bound paclitaxel is administered on day -11. In some embodiments, the invention includes a method of pre-treating prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 / day, and fludarabine at a dose of approximately 500 mg / m 2 / day and cyclophosphamide at a dose of approximately 100 mg / m 2 / day, where fludarabine is administered on day -6, cyclophosphamide is administered on days -6, -5 and -4, and albumin-bound paclitaxel is administered on day -5. In some embodiments, the invention includes a method of pre-treating prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 / day, and fludarabine at a dose of approximately 507 mg / m 2 / day and cyclophosphamide at a dose of approximately 68 mg / m 2 / day, where fludarabine is administered on day -6, cyclophosphamide is administered on days -6, -5, -4 and -3, and albumin-bound paclitaxel is administered on day -5. In some embodiments, the invention includes a method of pre-treating prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 137 mg / m 2 / day, where albumin-bound paclitaxel is administered on day -4.

[0307] Here, the method may include various other interventions. For example, cyclophosphamide and fludarabine can cause adverse effects in patients after administration. The scope of the present invention includes administering a composition to the patient to reduce the effects of some of these adverse effects. In some embodiments, the method includes administering saline to the patient. Saline can be administered to the patient before or after administration of cyclophosphamide and / or fludarabine, or before and after administration of cyclophosphamide and / or fludarabine. In some embodiments, saline is administered to the patient before administration of cyclophosphamide and / or fludarabine and after administration of cyclophosphamide and / or fludarabine on each infusion day. Adjuvants and excipients can also be administered to the patient. For example, Mesna (sodium 2-mercaptoethane sulfonate) can also be administered. Exogenous cytokines can also be administered to the patient.

[0308] In some embodiments, pretreatment improves the outcome of treatment before injecting the first or subsequent dose. For example, in some aspects, pretreatment improves the efficacy of treatment with the first or subsequent dose or increases the persistence of chimeric antigen receptor-expressing immune effector cells (e.g., CAR-expressing immune effector cells, such as CAR-expressing T cells) in the subject. In some embodiments, pretreatment treatment increases the plateau of disease.

[0309] Once the immune effector cells are administered to a subject (e.g., a human), in some embodiments, the biological activity of the engineered immune effector cell population is measured by any of a variety of known methods. Parameters used for evaluation include specific binding of antigen to engineered or natural T cells or other immune cells, either in vivo (e.g., via imaging) or ex vivo (e.g., via ELISA or flow cytometry analysis). In some embodiments, the ability of the engineered immune effector cells to destroy target cells can be detected by any suitable method known in the art, for example through cytotoxicity experiments as described in Kochenderfer et al., J. Immunotherapy, 32 (7): 689-702 (2009) and Herman et al., J. Immunological Methods, 285 (1): 25-40 (2004). In some embodiments, the expression and / or secretion of several cytokines, such as CD107a, IFNγ, IL-2, and TNF, can also be measured to measure the biological activity of the immune effector cells. In some embodiments, biological activity is measured by assessing clinical outcomes, such as, for example, reduction in tumor burden or burden. In some embodiments, reduction in tumor markers is assessed. In some embodiments, toxicity outcomes, persistence and / or proliferation of cells, and / or the presence or absence of a host immune response are assessed.

[0310] Administration In the present invention, the administration time and magnitude of multiple doses of immune effector cells is generally designed to reduce the risk or minimize toxic consequences and / or improve efficacy, e.g., provide increased exposure of the subject to immune effector cells over time. The methods generally include administering an initial dose prior to one or more subsequent doses, within a specific time range between the different doses.

[0311] In the case of immune effector cell therapy, administering a specified "dosage" includes a single composition and / or a single continuous administration, but also includes administering the specified amount or number of immune effector cells as, for example, a single injection or continuous infusion, and also includes administering the specified amount or number of immune effector cells in divided doses, in multiple single composition or infusions, within a particular time point not exceeding 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. Thus, an initial dose or subsequent doses is a single or continuous dose of a specified number of immune effector cells administered or initiated at a single time point. However, in some circumstances, the first dose or subsequent doses are injected or infused multiple times within a time period not exceeding 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days, e.g., once each day for 3 or 2 days, or multiple daily infusions. In some aspects, the first dose of immune effector cells is administered in a single drug composition. In some embodiments, the subsequent doses of immune effector cells are administered in a single drug composition. In some embodiments, the first dose of immune effector cells is administered in multiple compositions that contain the entire first dose of immune effector cells. In some embodiments, the subsequent doses of immune effector cells are administered in multiple compositions that contain the entire subsequent doses of immune effector cells. In some embodiments, multiple additional subsequent doses of the composition may be administered within a time period not exceeding 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days.

[0312] The term "fractional dose" refers to a single dosage, or to portions of that single dose that are administered in multiple doses, such that the single dose is administered to a subject over a period of time that exceeds one day. In some embodiments, the first dose and / or each subsequent dose can be administered in two or more divided doses. For example, in some embodiments, the doses (wherein the doses are the total dose of the first dose or the total dose of the subsequent doses) can be administered to the subject within 3, 5, 14, or 15 days. An exemplary method of administering divided doses includes administering a 50% divided dose of the total dose on day 0 (designating day 0 as the day on which the patient is administered each time CAR T cell therapy is administered) and administering a 50% divided dose of the total dose on day 2. In other embodiments, the dose can be administered at 10% of the total dose on day 0, 30% of the total dose on day 2, and 60% of the total dose on day 4. In some embodiments, the dose is administered at 10% of the total dose on day 0, 30% of the total dose on day 1, and 60% of the total dose on day 2. In some embodiments, a one-third total dose is administered on day 0, a one-third total dose is administered on day 11, and a one-third total dose is administered on day 14. In some embodiments, a 46% total dose is administered on day 0, an 18% total dose is administered on day 1, and a 36% total dose is administered on day 2. In some embodiments, a 25% total dose is administered on day 0, a 25% total dose is administered on day 2, a 25% total dose is administered on day 9, and a 25% total dose is administered on day 13. In some embodiments, the full time spread of administering a single dose in separate doses does not exceed 15 days.

[0313] As used herein, the term "initial dose" is intended to describe the total dose administered in a first course of treatment according to the methods herein. The dose may be the same as the total dose administered in a single course of treatment, or the total dose administered in a first course of treatment in a multi-course treatment. The term does not refer to a dose in which the subject has not been treated with immune effector cells prior to treatment with the methods herein, or in which the subject has not previously been administered immune effector cells expressing the same chimeric antigen receptor or targeting the same antigen.

[0314] In comparison to the initial dose, the term "subsequent dose" refers to a single dose administered at each subsequent treatment course after administration of the initial dose to perform multiple treatment courses, or the total doses administered at each treatment course. In some embodiments, the method includes administering multiple treatment courses, i.e., administering one or more subsequent doses, where the first subsequent dose is referred to as the second treatment course dose, the second subsequent dose is referred to as the third treatment course dose, and so forth. Also, in a series of doses, the previous dose is the dose immediately preceding the subsequent dose that is administered directly thereafter. In some embodiments, the subsequent doses are administered with a similar administration time and manner to the initial dose. In some embodiments, the interval between the initial dose and the first subsequent dose or the second treatment course dose and the interval between the multiple subsequent doses are the same or different, as described above.

[0315] The amount or size of the dose Generally, the dose size of the initial dose and / or one or more subsequent doses is designed to provide improved efficacy and / or reduced risk of toxicity. In some embodiments, the number of immune effector cells in the initial dose, or in a single subsequent dose, is about 1×10 6 Cells / kg subject body weight ~ approx. 3 x 10 7 More than, less than, or equal to, e.g., about 1 x 10 cells / kg subject body weight 5 , 1.5×10 5 , 2×105 、2.5×10 5 、3×10 5 、3.5×10 5 、4×10 5 、4.5×10 5 、5×10 5 、5.5×10 5 、 6×10 5 、6.5×10 5 、7×10 5 、7.5×10 5 、8×10 5 、8.5×10 5 、9×10 5 、9.5×10 5 、1×10 6 、1.5×10 6 、2×10 6 、2.5×10 6 、3×10 6 、3.5×10 6 、4×10 6 、4.5×10 6 、5×10 6 、5.5×1 0 6 、6×10 6 、6.5×10 6 、7×10 6 、7.5×10 6 、8×10 6 、8.5× 10 6 、9×10 6 、9.5×10 6 、1×10 7 、1.5×10 7 、2×10 7 、2.5 ×10 7 、3×10 7 、3.5×10 7 、4×10 7 、4.5×10 7 、5×10 7 、6×10 7 、6.5×10 7 、7×10 7 、7.5×10 7 、8×10 7 、8.5×10 7、9 ×10 7 、9.5×10 7 、1×10 8 、1.5×10 8 、2×10 8 、2.5×10 8 、 3×10 8 、3.5×10 8 、4×10 8 、4.5×10 8 、5×10 8 、5.5×10 8 、6×10 8 、6.5×10 8 、7×10 8 、7.5×10 8 、8×10 8 、8.5×10 8 、9×10 8 、9.5×10 8 、1×10 9 、1.5×10 9 、2×10 9 、2.5×1 0 9 、3×10 9 、3.5×10 9 、4×10 9 、4.5×10 9 、5×10 9 、5.5× 10 9 、6×10 9 、6.5×10 9 、7×10 9 、7.5×10 9 、8×10 9 、8.5 ×10 9 、9×10 9 、9.5×10 9 、1×10 10 、1.5×10 10 、2×10 10 、2.5×10 10 、3×10 10 、3.5×10 10 、4×10 10 、4.5×10 10 、5×10 10, 5.5×10 10 , 6×10 10 , 6.5×10 10 , 7×10 10 , 7.5×10 10 , 8×10 10 , 8.5×10 10 , 9×10 10 , 9.5×10 10 , 1 ×10 11 More than, less than, or equal to cells / kg subject body weight.

[0316] In a specific embodiment, the number and / or concentration of immune effector cells refers to the number of chimeric antigen receptor (e.g., CAR)-expressing immune effector cells. In other embodiments, the number and / or concentration of immune effector cells refers to the number or concentration of all cells, T cells, or peripheral blood mononuclear cells (PBMCs) administered.

[0317] In some embodiments, the number of immune effector cells, chimeric antigen receptor (e.g., CAR)-expressing cells, T cells, or peripheral blood mononuclear cells (PBMCs) in the initial dose is about 1×10 6 More than about 1 x 10 of these cells / kg subject body weight, but for example, about or at least about 1 x 10 6 , 1.1×10 6 , 2×10 6 , 2.9×10 6 , 3×10 6 , 3.3×10 6 , 5 ×10 6 , 1×10 7 , 1.3×10 7 , 1.6×10 7 , 2.6×10 7 , 2.8×10 7 , 3×10 7 , 5×10 7 , 2.5×10 8 , 1×10 9 , 1×10 10 , 5×10 10 these cells / kg body weight, or a range between any two of the previously mentioned values.

[0318] In some embodiments, the number of immune effector cells administered in a subsequent dose is the same, similar, or greater than the number of immune effector cells administered in the initial dose according to any embodiment herein, e.g., about 1×10 6 or about 1 x 10 6 , 1×10 7 or about 1 x 10 7 , 1.3×10 7 or about 1.3 x 10 7 , 1.6×10 7 or about 1.6 x 10 7 , 1.9×10 7 or about 1.9 x 10 7 , 2×10 7 or about 2 x 10 7 , 2.2×10 7 or about 2.2 x 10 7 , 3.7×10 7 or about 3.7 x 10 7 , 5×10 7 or about 5 x 10 7 , or 5.1 × 10 7 or about 5.1 x 10 7 , or 1 × 10 8 or about 1 x 10 8 , or 2.5 × 10 8 or about 2.5 x 10 8 , or 1 × 10 9 or about 1 x 10 9 , or 1 × 10 10 or about 1 x 10 10 , or 5 × 10 10 or about 5 x 10 10 these cells / kg subject body weight, or a range between any two of the previously mentioned values.

[0319] With respect to immune effector cell numbers, in some embodiments these values ​​refer to the number of chimeric antigen receptor-expressing (e.g., CAR-expressing) cells, and in other embodiments, the number of these administered T cells or PBMCs or total cells.

[0320] In some aspects, the subsequent dose is greater than, equal to, or less than the initial dose. For example, in some embodiments, the subsequent dose is about 1×10 6 More than about 1×10 cells, chimeric antigen receptor (e.g., CAR)-expressing cells, T cells and / or PBMCs / kg subject body weight, for example, about or at least about 1×10 6 , 1×10 7 , 1.3×10 7 , 1.6×10 7 , 1.9×10 7 , 2×10 7 , 2.2×10 7 , 3.7×10 7 , 5×10 7 , 5.1×10 7 , 1×10 8 , 2.5×10 8 , 1×10 9 , 1×10 10 , 5×10 10 pieces of these cells / kg subject body weight, or a value within the range between any two of the aforementioned values, Each is inclusive of the extreme values.

[0321] In some embodiments, the amount or magnitude of the subsequent dose is sufficient to reduce the tumor burden or index thereof and / or one or more symptoms of the disease or condition. In some embodiments, the dose is sized to effectively improve the survival of the subject, e.g., to induce survival in the subject and to improve survival in the subject, such as recurrence-free survival or event-free survival sustained for at least 1 month or at least 1, 2, 3, 4, or 5 years.

[0322] In some embodiments, the tumor burden, including tumor size, tumor volume and / or tumor mass, is reduced by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more after administration of a subsequent dose compared to the time before administration of the first or subsequent dose.

[0323] In other embodiments, the number of immune effector cells administered in the subsequent dose is less than the number of immune effector cells administered in the initial dose.

[0324] In some embodiments, the size of the initial dose or subsequent doses is determined based on one or more criteria, for example, the subject's response to previous treatments such as chemotherapy, tumor burden such as tumor volume, size, extent, or type, stage, and / or complications such as cancerous pleura and ascites that commonly appear in either mid- to late-stage cancer, and / or the likelihood or incidence of the subject experiencing toxic outcomes such as CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and / or the host immune response to the administered cells and / or chimeric antigen receptor.

[0325] Based on the teachings of the present invention, those skilled in the art should understand that the dosages specifically disclosed in the present invention are safe and effective dosages obtained through research by the inventors, but should also understand that a clinician can determine the specific initial dosage according to various actual circumstances, such as factors such as the patient's tumor burden and the patient's own physical condition, and if further subsequent dosages are required, those skilled in the art can determine the subsequent dosages according to, for example, changes in the tumor burden after administration of immune effector cells.

[0326] Those skilled in the art will also understand that the immune effector cells of the present invention, such as CLD18A2-CAR-T cells, can be used in combination with other CAR-Ts. For example, since the patient's tumor may express some tumor antigens, the patient may receive the CLD18A2-CAR-T cell therapy of the present invention after previously receiving a CAR-T cell therapy targeting the other tumor antigens, and the patient may receive the CLD18A2-CAR-T cell therapy of the present invention after previously receiving another treatment different from the CLD18A2-CAR-T cell therapy of the present invention if the antibody has weak specificity or the antibody displays mousegenic rejection. Therefore, in such cases, those skilled in the art should understand that, for example, a clinician can determine the number and dosage of the CLD18A2-CAR-T cells of the present invention depending on the situation of the previous treatment. For example, the safe and effective dosage and number of administrations disclosed herein can be referred to, i.e., about 2×10 10 The total amount of cells / kg of subject body weight should not exceed approximately 1 × 10 12 Cells; preferably about 2 x 10 9 The total amount of cells / kg of subject body weight should not exceed approximately 2 × 10 11 Not more than, and more preferably, about 2.5 x 10 cells 8 cells / kg subject body weight, or 5 x 10 7 Cells / kg subject weight, or or 3×10 7 Cells / kg subject body weight not to exceed 5 × 10 9 or Approximately 1×10 10 Not beyond the cells.

[0327] In some aspects, the size of the initial dose and / or subsequent doses is determined by the disease or condition burden of the subject. For example, in some aspects, the number of immune effector cells administered in the initial dose is determined based on the tumor burden present in the subject prior to administration of the initial dose. In some embodiments, the size of the initial dose and / or subsequent doses is inversely proportional to the tumor burden.

[0328] In some aspects, the number of immune effector cells to be administered in subsequent doses is determined based on the tumor burden present in the subject prior to administration of the first dose, e.g., where the first dose reduces or lowers the tumor burden or is already below a certain threshold or level, e.g., where the risk of displaying a higher than expected toxic outcome increases, e.g., about 1×10 7 More than about 2.0×10 cells (e.g., total cells, receptor-expressing cells, T cells, or PBMCs) per kg body weight, e.g., about 2.0×10 7 , 2.5×10 7 , 3.0×10 7 , 5.0×10 7 , 1.0×10 8 , 2.5×10 8 > cells / kg, and / or more than the initial dose.

[0329] In some circumstances, subsequent doses can be increased even if the tumor burden in subjects treated with the first dose does not decrease, e.g., to approximately 1 × 10 7 More than about 2.0×10 cells (e.g., total cells, receptor-expressing cells, T cells, or PBMCs) per kg body weight, e.g., about 2.0×10 7 , 2.5×10 7 , 3.0×10 7 , 5.0×10 7 , 1×10 8 , 2.5×10 8 More than cells / kg and / or greater than the initial dose.

[0330] Any route can be used to administer the T cells (e.g., CAR T cells), including intravenous (IV) administration. In some embodiments, the CAR T cells are administered via IV within about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60 minutes.

[0331] In some embodiments, the initial dose includes immune effector cells in an amount capable of causing or reducing a toxic or adverse outcome, such as, for example, cytokine release syndrome (CRS), severe CRS (sCRS), macrophage activation syndrome, tumor lysis syndrome, fever of at least 38° C. or about 38° C. sustained for 3 or more days, CRP plasma levels of at least about 20 mg / dL, and / or neurotoxicity. In some aspects, the number of cells administered in the initial dose is determined based on the likelihood that the subject will display a toxicity or toxic outcome (e.g., CRS, sCRS, and / or a CRS-related outcome) after administering the cells. For example, in some embodiments, the likelihood that the subject will have progressed to a toxic outcome is predicted based on tumor burden. In some embodiments, the method includes detecting or assessing a toxic outcome and / or tumor burden prior to administering the dose.

[0332] In some embodiments, if the serum level of a biochemical indicator, such as a CRS indicator, or other indicator of a toxicity outcome after administration of the subsequent dose is not increased to a level higher than a specified level, such as an acceptable level, e.g., about 10-fold, 15-fold, 20-fold, 25-fold, 50-fold, 75-fold, or 100-fold higher than or equal to the serum level of the indicator at the time of administration prior to administration of the first dose, or is already increased to or above the acceptable level, then the subsequent dose is administered to the subject. In some embodiments, the indicator is administered 10, 11, 22, 24, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 100, 100, 100, 100, 150, 100, 100, 150, If the indicator level falls below an acceptable level within 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 40 days, a subsequent dose will be administered, and if the indicator level does not fall to an acceptable level within said time period, a subsequent dose will not be administered.

[0333] In some embodiments, a subsequent dose is administered when the clinical risk of developing cytokine release syndrome (CRS), macrophage activation syndrome, or tumor lysis syndrome, or neurotoxicity is absent, has passed, or has disappeared after administration of the initial dose, e.g., such a situation generally occurs in about 60%, 70%, 80%, 90% or 95% of subjects with a particular disease or condition after a critical time that has been eliminated and / or is unlikely to occur.

[0334] In some embodiments, whether to administer a subsequent dose, the time at which the subsequent dose is administered, and / or the number of cells administered in the subsequent dose are determined by the presence, absence, or extent of an immune response or detectable immune response to the cells or the chimeric antigen receptor expressed thereby administered to the subject in the initial dose. In some aspects, subsequent doses containing cells expressing the receptor of the cells of the initial dose are not administered to subjects with a detectable host adaptive immune response or immune response that has already been established or reached a certain level, stage, or degree.

[0335] Administration time In some embodiments, the time to administer a subsequent dose begins to be calculated after the first dose is completed (the day that infusion of the total volume of the first dose is completed is designated as day 0).

[0336] In some embodiments, a subsequent dose is administered if the subject's serum level of a factor indicative of CRS is greater than about 10-fold, 25-fold, 50-fold, or 100-fold the serum level of said indicator in the subject at the time prior to administration of the first dose.

[0337] In some embodiments, subsequent doses are administered after the subject's outcomes associated with CRS (e.g., serum factors associated with or indicative of CRS) or their clinical signs or symptoms, such as fever, hypoxia, hypotension, or neuropathy, have already reached peak levels and begun to decrease after administration of the initial dose. In some embodiments, subsequent doses are administered when a decrease in levels is observed after administration, or after reaching a maximum or level of the outcome after administration.

[0338] In some embodiments, a subsequent dose is administered if the level of a toxic outcome indicator (e.g., a serum indicator of CRS) is reduced to about 25-fold the indicator level at the time before the first dose, hi some aspects, a subsequent dose is administered if the subject does not exhibit CRS or does not exhibit severe CRS.

[0339] In some aspects, the subsequent dose is administered at a time when the tumor burden has decreased relative to the patient's tumor burden prior to administration of the first dose, hi some embodiments, the subsequent dose is administered when the tumor burden or an indicator thereof, such as the size or number or percentage of diseased (e.g., tumor) cells in the subject's blood, other bodily fluids, tissues or tissues, or tumor size has already decreased by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more than 90% after administration of the first dose.

[0340] In some embodiments, a subsequent dose is administered if the subject's disease or condition does not recur after a decrease in response to the initial dose or previous doses. In some embodiments, a decrease in tumor burden is indicated by a decrease in one or more factors, such as the level of expression of a tumor marker in the subject or in a body fluid or tissue, the tumor cell burden or number, the mass or volume of the tumor, or the extent to which it has spread. In some embodiments, the recurrence is general or one or more factors or tumor burden. A subsequent dose is administered to a subject when the disease burden or factors thereof, as compared to the nadir measured or reached after the initial or previous dose, is still less than the amount administered at the beginning before the initial dose was administered. In some embodiments, a subsequent dose is administered to a subject when the tumor burden or factors thereof remain unchanged, e.g., when the tumor burden ceases to increase.

[0341] In some embodiments, subsequent doses are administered when the host adaptive immune response has not been detected, built up, or reached a certain level, extent, or stage, hi some aspects, subsequent doses are administered before the subject's memory immune response has developed.

[0342] In some aspects, the time between administration of the first dose and administration of the subsequent dose is about 21 to about 80 days, about 25 to about 60 days, or 25 to 55 days. In some embodiments, the time of administration of the subsequent dose is not more than about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days after administration of the first dose. In some embodiments, the time for administration of the subsequent dose is not more than about 44, 45, 46, 47, or 48 days after administration of the first dose.

[0343] In some embodiments, a subsequent dose (i.e., referred to as a first subsequent dose, or a second course of treatment dose) is followed by additional or more subsequent doses, e.g., a second subsequent dose (a third course of treatment dose), a third subsequent dose (a fourth course of treatment dose), and so forth. In some aspects, an additional subsequent dose is administered within at least about 21 to about 80 days after administration of a previous subsequent dose (e.g., a first subsequent dose). In some embodiments, an exemplary dosing scheme includes a timeline of when or about when to administer the chimeric antigen receptor expressing cells (e.g., CAR expressing cells, e.g., CAR T cells). In some embodiments, an additional dose (a second subsequent dose) is administered within about 49 days after administration of the first subsequent dose, and so forth. In some embodiments, the time of administration of the subsequently administered subsequent dose is not more than about 21 to about 80 days, about 25 to about 60 days, or 25 to 55 days after administration of the immediately preceding subsequent dose. In some embodiments, the time of administration of the subsequently administered subsequent dose is not more than about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days after administration of the immediately preceding subsequent dose.

[0344] In any embodiment, in some circumstances, the method includes administering an initial dose or a previous dose and a subsequent dose, and in other circumstances, the method includes administering a subsequent dose to a subject who has already previously been administered an initial dose or a previous dose, the subject having previously been administered an initial dose or a previous dose, but not including the initial dose or the previous dose itself. Thus, in some cases, the method includes administering a subsequent subsequent dose of a consolidation treatment to a subject who has previously been administered a dose (e.g., a reduced dose) of chimeric antigen receptor-expressing (e.g., CAR-expressing) cells. In some embodiments, the prior dose of cells expressing a receptor such as a CAR sufficiently reduces the burden of a disease or condition in the subject such that the efficacy and / or safety of administering a subsequent dose of cells is improved compared to administration to a subject not treated with the initial dose.

[0345] In some embodiments, the tumor burden, including tumor markers, tumor size, tumor volume and / or tumor mass, is reduced by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or greater after administration of a subsequent dose compared to the time before administration of the first or subsequent dose.

[0346] Host immune response to reinjected cells In some embodiments, one or more doses, e.g., subsequent doses, are administered when the subject's immune response (e.g., adaptive or specific immune response of the engineered receptor or cells) is absent, undetectable, or undetectable above a certain level. The presence or extent of the specific immune response of the engineered receptor is generally related to the immunogenicity of the receptor (e.g., CAR or engineered TCR expressed by the cells) and / or the time the subject is exposed to the cells. For example, in some embodiments, an immune response to the receptor, such as a specific humoral and / or cell-mediated immune response, is not detected before 28, 35, or 42 days after the subject first contacts the cells expressing the receptor. Thus, in some embodiments, the subsequent doses are administered before the subject develops an immune response, adaptive or specific immune response, detectable immune response, and / or memory response to the chimeric antigen receptor or cells. In this aspect, the subsequent doses improve cell proliferation and / or ability to continue to reside in the subject compared to other methods in which the subsequent doses are administered at a time relatively later than the previous or first dose. The methods include, for example, detecting the presence or absence, or level, of these immune responses or indicators thereof after administration of the initial or subsequent dose and before administration of the subsequent or next subsequent dose.

[0347] In some embodiments, when and / or whether to administer a subsequent dose is determined by whether the subject displays these immune responses or detectable readings, e.g., detectable specific or adaptive host immune responses specific to the cells or chimeric antigen receptor, e.g., the CAR expressed by the cells of the first dose, and / or whether such responses are detected at any level. In some embodiments, if such responses are detected, the subject is not administered a subsequent dose. If the subject does not display a specific or adaptive (e.g., humoral or cell-mediated) immune response to the receptor (e.g., the CAR expressed by the cells of the first dose), or if such responses or indicators are not displayed at a detectable level or higher than an acceptable level, the subsequent dose is administered. In some aspects, when administered in a subsequent dose, the subject displays a reduced humoral or cell-mediated immune response to the CAR expressed by the cells of the first dose compared to when the first dose is administered at a relatively high level.

[0348] In any one of the above embodiments, a detectable immune response refers to a detectable amount of any of a number of known methods for assessing specific immune responses of a cell to a particular antigen. For example, in some embodiments, a particular type of immune response is detected by detecting the presence of an epitope, e.g., an epitope bound to a chimeric antigen receptor, e.g., an antigen of a CAR, that is specifically bound to and / or neutralized by an antigen present on the cell, e.g., by performing an ELISPOT, ELISA, or antibody detection method on the cell (e.g., through flow cytometry analysis) on the subject's serum. Some such measurements include the isotype of the antibody detected, and Determine the type of instruction to give the response and / or whether the response is a memory response.

[0349] In some embodiments, the presence or absence of these host immune responses and / or their number, extent, etc., is detected or measured, eg, after administration of an initial dose or subsequent doses.

[0350] In some embodiments, a reduction has been observed in comparative methods using alternative administration methods, e.g., where a subject is administered a single dose (e.g., a single large dose of cells), but the total number of cells administered in the initial and subsequent doses, e.g., uniformly in the methods provided, compared to replacing the single dose or administering multiple larger or multiple doses less than about 21 days or more than about 80 days apart from each other, such that the methods reduce the burden of disease or illness to a greater extent, e.g., number of tumor cells, tumor size, duration of patient survival or case-free survival, and longer time periods. In some embodiments, the subject's survival period, survival over a certain time period, survival range, the presence or duration of event-free or event-free survival, or recurrence-free survival is evaluated.

[0351] In some aspects, the disease burden is measured or detected before administration of the first dose, after administration of the first dose but before administration of subsequent doses, and / or after administration of subsequent doses. In the case of multiple subsequent doses, the disease burden in some embodiments can be measured before or after administration of any subsequent dose, or during administration of the subsequent doses.

[0352] In some embodiments, after administration of the initial dose, the burden is reduced by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100%. In some embodiments, administration of a subsequent dose can result in a greater reduction in disease burden, tumor burden, for example, a reduction in burden that is equal to or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 100% compared to the time before the subsequent dose was administered or compared to the time before the first dose was administered.

[0353] In some embodiments, the method improves event-free survival or overall survival of a subject relative to other methods. For example, in some embodiments, within one month after administration of the initial dose, the rate or probability of progression-free event survival of a subject treated with the method is greater than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. In some aspects, the overall survival rate is greater than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. In some embodiments, subjects treated with the method exhibit relapse-free survival, recurrence-free survival, or survival of at least about 1 month, or at least about 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or survival of 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years. In some embodiments, the time to progression is improved, for example, by greater than about 1 month, or by at least about 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or by 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years.

[0354] In some embodiments, treatment with the methods reduces the chance of recurrence compared to other methods, e.g., in some embodiments, the chance of recurrence or progression within one month after the first dose is less than about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 9%, about 8%, about 7%, or about 6%.

[0355] Cell exposure and persistence In some embodiments, the dosage and / or the time is designed to facilitate exposure of the subject to the cells, e.g., to facilitate their proliferation and / or persistence over an increased period of time.

[0356] In some embodiments, the methods provided increase the subject's exposure to the cells used (e.g., increased cell numbers or time over time) and / or the efficacy and outcome of immune cell therapy. In some aspects, the advantages of the methods include a greater degree of exposure of cells expressing a CAR antigen receptor (e.g., CAR-expressing cells) and / or improved outcome of therapy to a greater extent than other methods. These outcomes can include patient survival and palliation, even in cases with severe tumor burden.

[0357] In some embodiments, the presence and / or amount of cells expressing the chimeric antigen receptor (e.g., CAR-expressing cells) is detected in the subject after administration of the initial dose and / or subsequent doses. In some aspects, quantitative PCR (qPCR) is used to assess the amount of cells expressing the chimeric antigen receptor (e.g., CAR-T) in the subject's blood or serum or organ or tissue (e.g., disease site). In some aspects, persistence is quantified by copies of DNA or plasmid encoding a receptor such as a CAR per microgram of DNA, or the number of cells expressing the receptor, e.g., CAR-expressing cells, per microliter of a sample such as blood or serum, or the total number of peripheral blood mononuclear cells (PBMCs) or white blood cells or T cells per microliter of a sample.

[0358] In some embodiments, cells from a subject are detected at or at least the 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th, 20th, 21st, 22nd, 23rd, 24th, 25th, 26th, 27th, 28th, 29th, or 30th day after administration of the first dose. In some embodiments, cells are detected 2 weeks, 4 weeks, or 6 weeks, or 3 months, 6 months, or 12 months, 18 months, or 24 months, or 30 months, or 36 months, or 1 year, 2 years, 3 years, 4 years, 5 years, or more than 5 years after administration of the first or at least a subsequent dose.

[0359] In some embodiments, alternative methods, such as administering a single dose, include, for example, a method that includes more cells than the initial dose, using the total dose of cells as a single dose, administering subsequent doses of cells to subjects who were not administered the initial dose, and / or administering subsequent doses at times other than the specified time, e.g., at or after a defined time that is later than the time that the subject's immune response to the receptor (e.g., CAR) is manifested.

[0360] Based on the maximum number of cells exposed to the subject, the duration of detectable cells higher than a certain number or percentage, the area under the curve of the number of cells as a function of time, and / or combinations and indices thereof, the contact can be displayed, for example, indicating cell number, proliferation, and / or persistence. These results can be evaluated using known methods such as qPCR to detect the number of copies of the nucleic acid encoding the chimeric antigen receptor, and / or flow cytometry analysis measurements, which generally detect cells expressing the receptor with receptor-specific antibodies, relative to the total amount of nucleic acid or DNA in a particular sample, such as blood or serum. Based on the measurement of cells, the number or percentage of functional cells can also be detected, but for example, a reaction (e.g., a cytotoxic reaction) that induces a disease or pathology or binds to and / or is neutralized by cells expressing the antigen recognized by the receptor.

[0361] In some aspects, the increased exposure of the subject's cells includes increased cell proliferation. In some embodiments, after administration of the initial dose and / or subsequent doses, the receptor- (e.g., CAR-) expressing cells proliferate in the subject. In some aspects, the method further increases cell proliferation compared to other methods, e.g., administering cells in a single dose, administering a relatively large initial dose, administering subsequent doses but not administering the initial dose, and / or administering subsequent doses before or after a specified time or time point to generate an immune response, e.g., before administering the subsequent doses.

[0362] In some aspects, the method significantly increases the in vivo proliferation of the administered cells, as measured, for example, via flow cytometry analysis. In some aspects, a higher peak ratio of cells is detected. For example, in some embodiments, after administration of the first or subsequent dose, when the subject's blood or disease site or its white blood cell portion (e.g., PBMC portion or T cell portion) reaches a peak or maximum level, 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%, or at least about 90% of the cells express a chimeric antigen receptor such as a CAR.

[0363] In some embodiments, the method provides for a maximum concentration of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 1500, 2000, 5000, 10000, or 15000 replicated or receptor-encoding nucleic acids in the subject's blood or serum or other bodily fluid or tissue, e.g., per microgram of DNA, or at least 0.1 mg / mL of CAR. In some embodiments, the cells expressing a CAR-like receptor are detected as being at least 10%, 20%, 30%, 40%, 50% or 60% of the total PBMCs from the subject's blood and / or persisting for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, or 52 weeks after administration of the initial or subsequent doses at said levels, or persisting for 1, 2, 3, 4, or 5 years or more after administration in these ways.

[0364] In some embodiments, the methods provide for replication of the nucleic acid encoding the chimeric antigen receptor, e.g., a CAR, such that there is at least a 2-fold, at least a 4-fold, at least a 10-fold, or at least a 20-fold increase in DNA per microgram.

[0365] In some embodiments, cells expressing the receptor are detected in the blood or serum of the subject, e.g., using a specified method, such as qPCR, or a detection method based on flow cytometry analysis, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36 days after administration of the first dose or after administration of a subsequent dose. , 37 days, 38 days, 39 days, 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, or 60 days or more, or for at least or about 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, or 24 weeks or more after administration of the first dose or any subsequent doses. do.

[0366] In some embodiments, at least about 1×10 2 , at least about 1 x 10 3 , at least about 1 x 10 4 , at least about 1 x 10 5 , or at least about 1 × 10 6 or at least about 5 × 10 6 or at least about 1 × 10 7 or at least about 5 × 10 7 or at least about 1 × 10 8 or at least about 2.5 × 10 8 Chimeric antigen receptor-expression, e.g. For example, CAR-expressing cells and / or at least 10, 25, 50, 100, 200, 300, 400, or 500, or 1000 receptor-expressing cells per microliter, e.g., at least 10 per microliter, can be detected or present in a subject or a fluid, tissue, or compartment thereof, e.g., in the blood, e.g., peripheral blood, or at a disease site thereof. In some embodiments, after administration of the first dose or after administration of a subsequent dose, cell numbers or concentrations can be detected in a subject for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 40 days, or at least about 60 days, or at least about 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months, or at least 2 years or 3 years. These cell numbers can be detected based on flow cytometry analysis or using quantitative PCR-based methods, and total cell numbers can be estimated using known methods.

[0367] In some embodiments, the copy number of a nucleic acid encoding a chimeric antigen receptor per 100 cells, e.g., the copy number of the carrier, e.g., in peripheral blood or bone marrow or other compartment, is at least 0.01, at least 0.1, at least 1, or at least 10, e.g., measured through immunohistochemistry, PCR and / or flow cytometry analysis, e.g., at about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or at least about 6 weeks, or at least about 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months, or at least 2 or 3 years, after administration of the initial dose or subsequent doses of cells. In some embodiments, the copy number of the carrier expressing a receptor such as CAR from genomic DNA per microgram is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, at least 1000, at least 5000, or at least 10000, or at least 15000, or at least 20000, when measured at about 1 week, about 2 weeks, about 3 weeks, or at least about 4 weeks after administration of the initial dose or subsequent doses of cells expressing a receptor such as CAR, or at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months, or at least 2 years or 3 years after administration in these ways.

[0368] In some embodiments, a receptor such as CAR expressed by the cells is detected in the blood of a subject and / or at the disease site via quantitative PCR (qPCR) or flow cytometry analysis, and is detected at least about 3 days, at least about 6 days, at least about 10 days, at least about 15 days, at least about 20 days, at least about 30 days, at least about 40 days, at least about 50 days, at least about 60 days, at least about 80 days, 3 months, at least about 6 months, at least about 12 months, at least about 1 year, at least about 2 years, at least about 3 years, or more than 3 years after administration of the cells, e.g., starting with an initial dose or subsequent doses, or after administration of any subsequent doses thereafter.

[0369] Chimeric antigen receptor expressed by a cell Cells typically express chimeric antigen receptors, which include antigen receptors that are genetically engineered T cell receptors (TCRs), chimeric antigen receptors (CARs) such as T cell fusion proteins (TFPs) and T cell antigen couplers (TACs), and functional non-TCR antigen receptors, such as receptors that bind other antigens.

[0370] Exemplary antigen receptors include CARs, and methods for engineering and introducing receptors into cells include those described, for example, in Chinese patent applications having publication numbers CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN106397593A, CN106467573A, and international patent applications having publication numbers WO2018006882A1, WO2015172339A8.

[0371] The chimeric receptor includes a chimeric antigen receptor (CAR). A chimeric receptor such as a CAR generally comprises an extracellular antigen binding domain, for example, a portion of an antibody molecule, generally a heavy chain variable (VH) region and / or a light chain variable (VL) region of an antibody, such as an scFv antibody fragment. In some embodiments, the chimeric receptor comprises splice variant 1 (CLD18A1, CLD18.1) of Claudin 18 (CLD18) (Accession Nos. NP_057453, NM016369) or variants thereof, such as the amino acid sequence set forth in SEQ ID NO:34 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:34, and splice variant 2 (CLD18A2, CLD18.2) (Accession Nos. NM_001002026, NP_001002027, NP_001002029 ... 002026) or variants thereof can be identified, for example, as comprising an amino acid sequence as set forth in SEQ ID NO:33 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:33. In some embodiments, the chimeric receptor specifically recognizes CLD18A2 but not CLD18A1.

[0372] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) comprises an scFv antibody fragment, such as, for example, an amino acid sequence containing an HCDR or LCDR as set forth in SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7, or a variant thereof, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:1 or SEQ ID NO:2 or SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 or SEQ ID NO:7.

[0373] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) is selected from the group consisting of, for example, SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:12 or SEQ ID or a variant thereof, which has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 11109, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 13 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater amino acid sequence identity to the scFv antibody fragment.

[0374] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) is selected from the group consisting of, for example, SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:12 or SEQ ID SEQ ID NO:13 or variants thereof, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8 or SEQ ID NO:9 or SEQ ID NO:10 or SEQ ID NO:11 or SEQ ID NO:12 or SEQ ID NO:13.

[0375] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) is selected from the group consisting of, for example, SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18 or SEQ and scFv antibody fragments, such as an amino acid sequence comprising a heavy or light chain variable region as set forth in SEQ ID NO:19, or a variant thereof, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:14 or SEQ ID NO:15 or SEQ ID NO:16 or SEQ ID NO:17 or SEQ ID NO:18 or SEQ ID NO:19.

[0376] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) comprises an scFv antibody fragment, such as, for example, an amino acid sequence comprising a heavy or light chain variable region as set forth in SEQ ID NO:20 or SEQ ID NO:21 or SEQ ID NO:22 or SEQ ID NO:23, or a variant thereof, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:20 or SEQ ID NO:21 or SEQ ID NO:22 or SEQ ID NO:23.

[0377] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) comprises, for example, an amino acid sequence comprising a heavy or light chain variable region as set forth in SEQ ID NO:20 or SEQ ID NO:21 or SEQ ID NO:22 or SEQ ID NO:23, or a variant thereof, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:20 or SEQ ID NO:21 or SEQ ID NO:22 or SEQ ID NO:23.

[0378] In some embodiments, the chimeric antigen receptor (e.g., CAR) is selected from the group consisting of, for example, SEQ ID NO:24 or SEQ ID NO:25 or SEQ ID NO:26 or SEQ ID NO:27 or SEQ ID NO:28 or SEQ ID NO:29 or SEQ ID NO:30 or SEQ ID NO:31 or SEQ ID The amino acid sequence set forth in SEQ ID NO:32 or a variant thereof, or SEQ ID NO:24 or SEQ ID NO:25 or SEQ ID NO:26 or SEQ ID SEQ ID NO:27 or SEQ ID NO:28 or SEQ ID NO:29 or SEQ ID NO:30 or SEQ ID NO:31 or SEQ ID NO:32 and at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, It includes amino acid sequences that exhibit 3%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity.

[0379] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) further comprises a connecting sequence that may be or include at least a portion of an immunoglobulin constant region, such as a hinge region, such as an IgG4 hinge region and / or CH1 / CL and / or Fc region, or a variant or modification thereof. In some embodiments, the constant region or portion is of a human IgG, such as IgG4 or IgG1. In some aspects, the portion of the constant region is an antigen-recognizing portion that is the same as the connecting sequence between the scFv and the transmembrane domain. The connecting sequence can have a length that increases the reactivity of the cell after binding to the antigen compared to when the connecting sequence is not present. In some implementations, the length of the connecting sequence is about 12 amino acids in length or is no more than 12 amino acids in length. Exemplary connecting sequences include lengths of at least about 10-229 amino acids, about 10-200 amino acids, about 10-175 amino acids, about 10-150 amino acids, about 10-125 amino acids, about 10-100 amino acids, about 10-75 amino acids, about 10-50 amino acids, about 10-40 amino acids, about 10-30 amino acids, about 10-20 amino acids, or about 10-15 amino acids, including integers between any endpoint of the above ranges. In some embodiments, the connecting sequence region comprises about 12 amino acids or less in length, about 119 amino acids or less in length, or about 229 amino acids or less in length. Exemplary connecting sequences include a single IgG4 hinge, an IgG4 hinge connected to a CH2 and CH3 domain, or an IgG4 hinge connected to a CH3 domain.

[0380] The antigen generally identifies a domain that is connected to one or more intracellular signaling moieties, e.g., in the case of CARs, a portion of the mimicked effective signaling through an antigen receptor complex (e.g., a TCR complex) and / or a signaling through other cell surface receptors. Thus, in some embodiments, an antigen binding moiety (e.g., an antibody) is connected to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain described above. In one embodiment, a transmembrane domain is used that is one of the domains in a naturally associated receptor (e.g., a CAR). In some situations, the transmembrane domain is selected or modified using amino acid substitutions to prevent the domain from being bound to the transmembrane domain of the same or a different surface membrane protein, thereby minimizing interaction with other members of the receptor complex.

[0381] In some embodiments, the transmembrane domain is natural or synthetic. Naturally, in some aspects, the domain is derived from any membrane-bound or transmembrane protein. The transmembrane domain comprises the α, β, or ζ chain derived from the T-cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and / or the transmembrane domain comprises a functional variant thereof (e.g., a structural portion thereof (e.g., a transmembrane structural portion), a property of which is essentially retained), etc. (i.e., a transmembrane domain comprising at least these). In some embodiments, the transmembrane domain is a transmembrane domain derived from CD4, CD28, or CD8, e.g., CD8α, or a functional variant thereof. Or, in some embodiments, the transmembrane domain is synthetic. In some embodiments, the synthetic transmembrane domain comprises primarily hydrophobic residues such as leucine and valine. In some embodiments, a trimer of phenylalanine, tryptophan, and valine appears at each end of the synthetic transmembrane domain. In some embodiments, the connection is implemented via a linker, a spacer and / or a transmembrane domain.

[0382] The intracellular signaling domains include signals that mimic or approximate signals through natural antigen receptors, signals that are coupled through these receptors to costimulatory receptors, and / or signals through costimulatory receptors alone, etc. In some embodiments, a short oligopeptide or polypeptide connector is present, e.g., a linker that is 2-10 amino acids in length that includes glycine and serine, e.g., a glycine-serine doublet linker, to form the connection between the cell signaling domain and the transmembrane domain of the CAR.

[0383] The receptors, such as CARs, generally comprise at least one intracellular signaling moiety or moieties. In some embodiments, the receptors comprise intracellular components of the TCR complex, such as the TCRCD3+ chain, which mediates T-cell activation and cytotoxicity. Thus, in some aspects, the antigen-binding moiety is connected to one or more cellular signaling modules. In some embodiments, the cellular signaling module comprises a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, the above receptors, such as CARs, further comprise portions of one or more other molecules, such as Fc receptor gamma, CD8, CD4, CD25, or CD16. For example, in some aspects, the CAR or other chimeric receptor comprises a chimeric molecule between CD3zeta (CD3-zeta) or Fc receptor gamma and CD8, CD4, CD25, or CD16.

[0384] In some embodiments, when bound to a CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor enables at least one of the normal effector functions or responses of an immune cell, e.g., engineering a T cell to express the CAR. For example, in some situations, the CAR induces a function of the T cell, e.g., cytolytic activity or auxiliary T cell activity, e.g., secretion of cytokines or other factors. In some embodiments, e.g., when transducing a signal for effector function, a full immunostimulatory chain is substituted for a portion of the antigen receptor or a truncated portion of the intracellular signaling domain of a costimulatory molecule. In some embodiments, the intracellular signaling domain comprises the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects further comprises a receptor that acts in concert with a naturally occurring co-receptor to initiate signaling after being bound to the antigen receptor.

[0385] For native TCR, full activation generally requires not only signaling through TCR but also costimulatory signal.Thus, in some embodiments, the CAR also includes a component for generating a second or costimulatory signal to promote full activation.In other embodiments, the CAR does not include a component for generating a costimulatory signal.In some aspects, the other CAR is expressed in the same cell and provides the source for generating a second or costimulatory signal.

[0386] In some embodiments, T cell activation is described as being mediated through two cell signaling sequences, one that is initially activated in a TCR-initiated antigen dependent manner (the initial cell signaling sequence), and one that acts in an antigen-independent manner to provide a second or costimulatory signal (the second cell signaling sequence). In some embodiments, the CAR comprises either or both of these signaling components.

[0387] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) further comprises a signal peptide, such as a signal peptide comprising CD8 or a variant thereof, e.g., the amino acid sequence set forth as SEQ ID NO:35, or SEQ ID NO:35. and further including a signal peptide comprising an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the

[0388] In some aspects, the CAR comprises a key cell signaling sequence that regulates the initial activation of the TCR complex. The initial cell signaling sequence that acts in a stimulatory manner can comprise an immune receptor tyrosine-based activation motif or a signaling motif known as ITAM. Examples of ITAM implementations include initial cell signaling sequences including those derived from TCRzeta, FcRgamma, FcRbeta, CD3gamma, CD3delta, CD3epsilon, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cell signaling molecule in the CAR comprises a cell signaling domain, a portion thereof, or a sequence derived from CD3zeta.

[0389] In some embodiments, the CAR comprises the transmembrane portion and / or signaling domain of a costimulatory receptor such as CD28, CD137, OX40, DAP10, and ICOS. In some aspects, the same CAR comprises both an active and a costimulatory portion.

[0390] In some embodiments, the activation domain is included in a single CAR, but the costimulatory component is provided by another CAR to recognize other antigens. In some embodiments, the CARs include an activating or stimulatory CAR, a costimulatory CAR, all expressed in the same cell (see WO2014 / 055668). In some aspects, the cell includes one or more stimulatory or activating CARs and / or costimulatory CARs. In some embodiments, the cell further includes an inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine, 5(215) (December 2013)), e.g., by identifying an excluding disease or disease-related and / or specific CAR, the stimulatory signal delivered via the disease-targeting CAR is reduced or inhibited via binding of the inhibitory CAR and its ligand, e.g., reducing off-target effects.

[0391] In some embodiments, the intracellular signaling portion of the chimeric antigen receptor, such as a CAR, comprises a costimulatory signaling region with a CD3 zeta intracellular domain. In some embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain connected to a CD3 (e.g., CD3-zeta) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and / or CD137 (4-1BB, TNFRSF9) costimulatory domain connected to a CD3 zeta intracellular domain.

[0392] In some embodiments, the CAR comprises one or more, e.g., two or more, costimulatory domains and an activation domain, such as an early activation domain, in the cellular portion. Exemplary CARs include the intracellular portions of CD3-zeta, CD28, and CD137.

[0393] In some situations, CARs are referred to as first generation, second generation and / or third generation CARs. In some embodiments, first generation CARs provide only CARs that induce CD3 chain signals upon antigen binding, in some embodiments, second generation CARs provide co-stimulatory signals with these signals, such as CARs that contain intracellular signaling domains derived from co-stimulatory receptors (e.g., CD28 or CD137), and in some embodiments, third generation CARs are CARs that contain multiple co-stimulatory domains of different co-stimulatory receptors.

[0394] In some embodiments, the chimeric antigen receptor comprises an extracellular portion that contains an antibody or an antibody fragment. In some aspects, the chimeric antigen receptor comprises an extracellular portion that contains an antibody or an antibody fragment. In some embodiments, the chimeric antigen receptor comprises an extracellular portion and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises an scFv, and the intracellular domain comprises an ITAM. In some aspects, the intracellular signaling domain comprises the signaling domain of the zeta chain of the CD3-zeta chain. In some embodiments, the chimeric antigen receptor comprises a transmembrane domain connected to the extracellular domain and the intracellular signaling domain. In some aspects, the transmembrane domain comprises the transmembrane portion of CD28. In some embodiments, the chimeric antigen receptor comprises an intracellular domain of a T cell costimulatory molecule. The extracellular domain and the transmembrane domain can be connected directly or indirectly. In some embodiments, the extracellular domain and the transmembrane domain are connected via a connecting sequence. In some embodiments, the receptor comprises an extracellular portion that is the same as the extracellular portion of CD28, a molecule from which the transmembrane domain is derived. In some embodiments, the chimeric antigen receptor comprises an intracellular domain from a T cell costimulatory molecule or a functional variant thereof, e.g., between the transmembrane domain and the intracellular signaling domain. In some aspects, the T cell costimulatory molecule is CD28 or 41BB. For example, in some embodiments, the CAR comprises an antibody, such as an antibody fragment, or comprises a transmembrane domain of a transmembrane portion of CD28 or a functional variant thereof, an intracellular signaling domain of a signaling portion of CD28 or a functional variant thereof, and a signaling portion of CD3ζ or a functional variant thereof. In some embodiments, the CAR comprises an antibody, such as an antibody fragment, comprises a transmembrane domain of a transmembrane portion of CD28 or a functional variant thereof, an intracellular signaling domain of a signaling portion of CD137 or a functional variant thereof, and a signaling portion of CD3ζ or a functional variant thereof. In some embodiments, the receptor further comprises a connecting sequence comprising a portion of an Ig molecule (e.g., a human Ig molecule), e.g., an Ig hinge, e.g., an IgG4 hinge, e.g., just a hinge connecting sequence.In some embodiments, the chimeric antigen receptor comprises (i) an antibody that specifically recognizes a tumor antigen, a transmembrane domain of CD28 or CD8, a costimulatory domain of CD28 and CD3ζ; or (ii) an antibody that specifically recognizes a tumor antigen, a transmembrane domain of CD28 or CD8, a costimulatory domain of CD137 and CD3ζ; or (iii) an antibody that specifically recognizes a tumor antigen, a transmembrane domain of CD28 or CD8, a costimulatory domain of CD28, a costimulatory domain of CD137 and CD3ζ.

[0395] In some embodiments, the transmembrane domain of the chimeric antigen receptor (e.g., CAR) is, or comprises, the transmembrane domain of human CD28 (e.g., Accession No.: P017471) or a variant thereof, for example, a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:39, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:39.

[0396] In some embodiments, the transmembrane domain of the chimeric antigen receptor (e.g., CAR) is, or comprises, the transmembrane domain of human CD8 or a variant thereof, e.g., a transmembrane domain comprising the amino acid sequence set forth in SEQ ID NO:45, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:45.

[0397] In some embodiments, the intracellular signaling portion of the chimeric antigen receptor (e.g., CAR) comprises an intracellular costimulatory signaling domain of human CD28 or a functional variant or portion thereof. For example, the intracellular signaling domain has an amino acid sequence as set forth in SEQ ID NO:41, or a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 17 , 97%, 98%, 99% or greater sequence identity.

[0398] In some embodiments, the intracellular domain comprises an intracellular costimulatory signaling domain of CD137 (e.g., Accession No: Q07011.1) or a functional variant or portion thereof, e.g., an amino acid sequence as set forth in SEQ ID NO:47, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:47.

[0399] In some embodiments, the intracellular signaling domain of the chimeric antigen receptor (e.g., CAR) comprises a human CD3 zeta stimulatory signaling domain or a functional variant thereof. For example, in some embodiments, the intracellular signaling domain comprises an amino acid sequence set forth in SEQ ID NO:43, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:43.

[0400] In some aspects, the chimeric antigen receptor comprises a hinge, such as a CD8 hinge. For example, in some embodiments, the CD8 hinge comprises the amino acid sequence set forth in SEQ ID NO:37, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:37.

[0401] For example, in some embodiments, the CAR comprises an antibody, such as an antibody fragment, e.g., an scFv, a connecting sequence comprising, e.g., a portion of an immunoglobulin molecule, e.g., a hinge region, and / or a constant region of one or more heavy chain molecules, e.g., a connecting sequence comprising an Ig-hinge, a transmembrane domain comprising all or a portion of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3 zeta signaling domain. In some embodiments, the CAR comprises an antibody or fragment, e.g., an scFv, a connecting sequence comprising, e.g., an Ig-hinge, a transmembrane domain comprising, e.g., a CD28-derived intracellular signaling domain, and a CD3 zeta signaling domain.

[0402] The terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides, including designated receptors and other polypeptides (e.g., linkers, or peptides), can contain amino acid residues, including natural and / or non-natural amino acid residues. The terms further include post-expression modifications of the polypeptide, such as, for example, glycosylation, sialylation, acetylation, and phosphorylation. In some embodiments, the polypeptide can further include modifications of the original or native sequence, so long as the protein retains the requisite activity. Such modifications can be significant, via site-directed mutagenesis, or unexpected, through the occurrence of host mutations in the protein or errors resulting from PCR amplification.

[0403] In some embodiments, the methods herein include administering to the subject one type of adoptive cell or immune effector cell. In some embodiments, the methods herein include administering to the subject two or more types of adoptive cells or immune effector cells against the same tumor antigen in different courses of treatment. In some embodiments, the methods herein include administering to the subject two or more adoptive cells or immune effector cells directed to different tumor antigens during different treatment courses. In some embodiments, the methods herein include administering to the subject two or more adoptive cells or immune effector cells directed to the same epitope of the same tumor antigen during different treatment courses. In some embodiments, the methods herein include administering to the subject two or more adoptive cells or immune effector cells directed to different epitopes of the same tumor antigen during different treatment courses. In some embodiments, the methods herein include administering to the subject two or more adoptive cells or immune effector cells directed to different epitopes of the same tumor antigen during different treatment courses to treat tumors at the same site. In some embodiments, the methods herein include administering to the subject two or more adoptive cells or immune effector cells during different treatment courses to treat tumors at different sites. In some embodiments, at least one of the adoptive cells or immune effector cells used in the methods herein is a CAR-T cell targeting CLD18A2. In some embodiments, at least one of the adoptive cells or immune effector cells used in the methods herein is a CAR-T cell targeting CLD18A2 according to the present invention.

[0404] The chimeric antigen receptor (e.g., CAR) expressed by the cells administered to the subject at various doses typically identifies or specifically binds to a molecule expressed by, associated with, and / or specific to the disease or condition or cells being treated. When specifically bound to a molecule such as an antigen, the receptor typically promotes an immune response targeting the disease or condition by transmitting an immunostimulatory signal (e.g., an ITAM-transmitted signal) to the cell. For example, in some embodiments, the cells in the initial dose express a CAR that specifically binds to an antigen expressed by a cell or tissue of the disease or condition or an antigen associated with the disease or condition.

[0405] The receptor (e.g., CAR) expressed by the cells of the subsequent dose generally specifically binds to the same antigen as the CAR of the first dose, and generally is the same receptor or a very similar receptor as the receptor of the cells of the first dose. In some embodiments, the receptor on the cells of the subsequent dose is the same as or very similar to the receptor on the cells of the first dose.

[0406] In some embodiments, the CAR expressed by the subsequent dose of cells contains the same scFv, the same signaling domain, and / or the same connection as the CAR expressed by the first dose of cells. In some embodiments, it further contains the same costimulatory, stimulatory, transmembrane, and / or other domains as the first dose. In some embodiments, one or more components of the CAR of the subsequent dose are different from the CAR of the first dose.

[0407] Immune effector cells In the present invention, the cells expressing the chimeric receptor and provided using the methods herein are immune effector cells. The cells are generally eukaryotic cells, such as mammalian cells, which are human cells. In some embodiments, the cells are cells of the immune system, such as cells of innate or adaptive immunity, such as myeloid or lymphoid cells, which include lymphocytes, generally T cells and / or NK cells, derived from blood, bone marrow, lymph, or lymphoid tissues. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). The cells are generally primary cells, such as cells isolated directly from a subject and / or isolated and frozen from a subject. In some embodiments, the cells include one or more subgroups of T cells or other cell types, such as pre-T cell populations, CD4+ cells, CD8+ cells and subgroups thereof, such as those capable of function, efficacy, maturation, differentiation, etc. The characteristics of the cells include, but are not limited to, the ability to amplify, recirculate, localize, and / or persist, antigen specificity, antigen receptor type, presence in a particular organ or compartment, marker or cytokine secretion status, and / or degree of differentiation. For subjects awaiting treatment, the cells can be allogeneic and / or autologous. The method includes existing methods. In some aspects, in the prior art, the cells are pluripotent and / or multipotent stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the method includes the steps of producing, processing, culturing, and / or manipulating cells isolated from a subject and introducing them into the same patient before or after cryopreservation.

[0408] Subtypes and subgroups of T cells and / or CD4+ and / or CD8+ T cells include original T (TN) cells, effector T cells (TEFF), memory T cells and subtypes thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosal-associated invariant T (MAIT) cells, naturally generated and adoptive regulatory T (Treg) cells, helper T cells include, for example, TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and δ / γ T cells.

[0409] In some embodiments, the cells are natural killer (NK) cells. In some embodiments, the cells are mononuclear cells or granular leukocytes, such as myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils.

[0410] In some embodiments, the cells contain one or more engineered nucleic acids that have been introduced, thereby expressing recombinant or engineered products of the nucleic acids. In some embodiments, the nucleic acids are heterologous, i.e., not generally present in the cells or samples obtained from the cells, e.g., not generally found in other organisms or cells, e.g., not generally found in the engineered cells and / or organisms from which these cells are derived. In some embodiments, the nucleic acids are not naturally occurring, e.g., the nucleic acids are found in nature, including chimeric combinations that include nucleic acids encoding domains derived from a variety of different cell types.

[0411] Methods and carriers for genetic engineering The present invention further provides methods, compositions, and kits for generating genetically engineered cells expressing chimeric antigen receptors. Genetic engineering generally refers to the introduction of nucleic acids encoding the recombinant or engineered moieties into cells, e.g., via retroviral transduction, transfection, transformation.

[0412] In some embodiments, gene transfer proceeds by first stimulating the cells, e.g., combining them with a stimulus that induces a response such as proliferation, survival and / or activation, as detected, e.g., by expression of a cytokine or activation marker, and then transducing the activated cells and expanding them from culture to numbers sufficient for clinical application.

[0413] In some situations, overexpression of a stimulatory factor (e.g., a lymphokine or a cytokine) can be toxic to a subject. Thus, in some situations, the engineered cells can be administered, for example, in a therapeutic immune response, to negatively select for gene segments to which the cells are sensitive in the body. For example, in some embodiments, the engineered cells can be administered in a therapeutic immune response to negatively select for gene segments to which the cells are sensitive in the body. By using a gene that confers sensitivity to an administered agent (e.g., a chemical compound), they can be eliminated by changes in conditions within the body of the subject to which they are administered. Negatively selectable phenotypes can be created using genes that provide sensitivity to an administered agent (e.g., a chemical compound). Negatively selectable genes include the herpes simplex virus type I thymidine kinase (HSV-I TK) gene, which provides sensitivity to ganciclovir (Wigler et al., Cell II:223, 1977), the cellular hypoxanthine phosphoribosyl transferase (HPRT) gene, the cellular adenine phosphoribosyl transferase (APRT) gene, and bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89: 33 (1992)).

[0414] In some embodiments, the cells are also engineered to promote the expression of cytokines or other factors. A variety of methods for introducing engineered components, such as antigen receptors (e.g., CARs), are known and can be used with the methods and compositions provided herein. Exemplary methods include methods for transferring nucleic acids encoding receptors, including methods via viruses, such as retroviruses or lentiviruses, transduction, transposable elements, and electroporation.

[0415] In some embodiments, recombinant infectious viral particles are used to transfer the recombinant nucleic acid to cells, for example, carriers derived from simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, recombinant lentiviral carriers, or retroviral carriers such as gamma-retroviral carriers are used to transfer the recombinant nucleic acid to T cells (see, for example, Koste et al. (2014) Gene Therapy April 3, 2014. doi:10.1038 / gt.2014.25; Carlens et al. (2000) Exp Hematol 28(10):1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. November 2011, 29(11):550-557).

[0416] In some embodiments, the retroviral carrier comprises a long terminal repeat (LTR), such as a retroviral carrier derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen foci forming virus (SFFV) or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retroviruses include those derived from any avian or mammalian cell source. Retroviruses are usually amphipathic, meaning that they can infect many types of host cells, including human. Lentiviral transduction methods are well known. Exemplary methods are described, for example, in Wang et al. (2012) J. Immunother. 35(9):689-701; Cooper et al. (2003) Blood. 101:1637-1644; Verhoeyen et al. (2009) Methods Mol Biol. 506:97-114; and Cavalieri et al. ( 2003) Blood. 102(2):497-505.

[0417] In some embodiments, the recombinant nucleic acid is transferred to the T cell via electroporation (see, e.g., Chicaybam et al., (2013) PLoS ONE 8(3):e60298 and Van Tedeloo et al., (2000) Gene Therapy 7(16):1431-1437). In some embodiments, the recombinant nucleic acid is transferred to the T cell via translocation (see, e.g., Manuri et al., (2010) Hum Gene Ther 21(4):427-437; Sharma et al., (2013) Molec Ther Nucl Acids 2, e74; and Huang et al., (2009) Methods Mol Biol 506:115-126). Other methods of introducing and expressing genetic material in immune cells include calcium phosphate transfection (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY), protoplast fusion, cationic liposome-mediated transfection, particle bombardment facilitated by tungsten particles (Johnston, Nature, 346:776-777 (1990)), and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)).

[0418] Other methods and carriers for the transfer of nucleic acids encoding recombinant products are described, for example, in International Patent Application having Publication No. WO2014055668, and U.S. Patent having Publication No. 7,446,190.

[0419] Other nucleic acids (e.g., genes) for introduction include those that enhance the activity and / or function of the transferred cells and improve therapeutic efficacy; genes that provide genetic markers to select and / or evaluate cells, e.g., genes that evaluate survival or positioning in the body; safety genes, e.g., genes that allow cells to negatively select sensitive gene segments in the body, as described, for example, in Lupton S.D. et al., Mol. and Cell Biol., 11:6 (1991); and Riddell et al., Human Gene Therapy 3:319-338 (1992); see also Lupton, Publication Nos. PCT / US91 / 08442 and PCT / US94 / 05601, which describe the application of selectable fusion genes with dual functions by fusion induction of positive and negative selectable markers. See, for example, columns 14-17 of Riddell et al., U.S. Patent No. 6,040,177.

[0420] Preparation of immune effector cells In some embodiments, the manufacture of engineered cells includes one or more culture and / or one or more preparation steps. Cells for introducing a nucleic acid encoding a genetically engineered receptor (e.g., CAR) can be isolated from a sample (e.g., a biological sample, e.g., obtained from or derived from a subject). In some embodiments, the subject from which the cells are isolated is a subject suffering from a certain disease or condition or in need of cell therapy or undergoing cell therapy. In some embodiments, the subject is a specific therapeutically mediated human, e.g., a human in need of adoptive cell therapy or effector cell therapy, and the cells used in the therapy have been isolated, processed and / or manipulated. In some embodiments, the cells are primary cells, such as primary human cells. The samples include samples obtained directly from tissues, body fluids and other samples of a subject, and samples obtained from one or more processing steps, e.g., separation, centrifugation, genetic engineering (e.g., transduction with a viral carrier), washing and / or incubation. The biological samples can be isolated from a subject, e.g., a sample obtained ... The sample may be obtained directly from a biological sample or from a processed sample, including, but not limited to, samples of body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissues, and organ samples, including processed samples derived therefrom.

[0421] In some embodiments, the sample from which the cells are derived or isolated is a blood or blood-derived sample, or the product of apheresis or leukapheresis. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), white blood cells, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil or other organ, and / or cells therefrom. In the case of cell therapy (e.g., adoptive cell therapy or immune effector cell therapy), the sample includes an original or allogeneic sample.

[0422] In some embodiments, the cells are from a cell line, such as a T cell line, hi some embodiments, the cells are from xenogeneic sources, such as mouse, rat, non-human primate, and pig.

[0423] In some embodiments, the separation of cells includes one or more preparative and / or non-specific cell separation steps. In some implementations, cells are washed, centrifuged, and / or incubated in the presence of one or more substances, e.g., to remove unwanted components, enrich for desired components, or to disaggregate or remove cells sensitive to a particular substance. In some implementations, cells are separated based on one or more properties, e.g., density, adhesive properties, size, sensitivity and / or resistance to a particular component. In some implementations, cells are obtained from the subject's circulating blood, e.g., via apheresis or leukapheresis. In some aspects, the sample includes lymphocytes, T cells, mononuclear cells, granular leukocytes, B cells, other erythrocytes, red blood cells, and / or platelets, and in some aspects includes cells other than red blood cells and platelets.

[0424] In some embodiments, blood cells collected from the subject are washed, e.g., by removing the plasma portion and placing the cells in an appropriate buffer or medium for use in subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution is deficient in calcium and / or magnesium and / or many or all divalent cations. In some aspects, the washing step is accomplished via semi-automated "flow-through" centrifugation (e.g., COBE 2991 Cell Processor, BaXter) according to the manufacturer's instructions. In some aspects, the washing step is accomplished via tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, after washing, the cells are washed with, e.g., Ca++ / Mg++ PBS free PBS (Ca++ / Mg++ PBS In some embodiments, the blood cell sample components are removed and the cells are resuspended directly in medium.

[0425] In some embodiments, the method includes a density-based cell separation method, for example, centrifuging peripheral blood through a Percoll or Ficoll gradient with or without lysis of red blood cells, or performing apheresis or leukapheresis on the sample to obtain peripheral blood mononuclear cells (PBMCs).

[0426] In some embodiments, the separation method involves determining the expression or presence of one or more specific molecules, e.g., surface markers, e.g., surface proteins, intracellular markers, or nucleic acids, in cells. In some embodiments, the separation includes separating different types of cells based on the expression or expression level of one or more markers (generally cell surface markers) of the cells, e.g., by incubating with an antibody or binding partner that is specifically bound to such marker, typically followed by a washing step to separate cells already bound to the antibody or binding partner from cells that are not yet bound to the antibody or binding partner.

[0427] These separation steps can be based on positive and / or negative selection, where positive selection retains and further applies cells already bound to the reagent, and negative selection retains cells that have not yet bound to the antibody or binding partner. In some implementations, both parts are retained and further applied. In some embodiments, it is desirable to proceed and separate based on markers expressed by cells distinct from the desired population, where negative selection can be very useful in the absence of antibodies that can specifically identify cell types in a heterogeneous population.

[0428] The separation does not necessarily enrich or eliminate a particular cell population, or 100% of cells expressing a particular marker. For example, positively selecting or enriching for a particular type of cell means that, for example, expressing a marker, the number or percentage of said cells is increased, but it does not necessarily have to be completely devoid of cells that do not express the marker. Similarly, negatively selecting, depleting or consuming a particular type of cell means that, for example, expressing a marker, the number or percentage of said cells is decreased, but it does not necessarily have to be completely removed of all these cells.

[0429] In some implementations, multiple separation steps are performed, where a portion of the positive or negative selection from one step is used to perform another separation step, e.g., to perform a subsequent positive or negative selection. In some implementations, a single separation step consumes cells expressing multiple markers simultaneously, e.g., by incubating the cells with multiple antibodies or binding partners specific for the markers that target the cells for self-negative selection via incubation. Similarly, cells can be incubated with different antibodies or binding partners expressed on different cell types, allowing for simultaneous positive selection against multiple cell types.

[0430] For example, in some embodiments, specific subpopulations of T cells, e.g., one or more surface marker positive cells, or cells expressing high levels of one or more surface markers, e.g., CD3+, CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+ and / or CD45RO+ T cells, are isolated by positive or negative selection techniques.

[0431] For example, CD3+,CD28+ T cells can be positively selected using magnetic beads coupled to CD3 / CD28 (e.g., DYNA Beads M-450 CD3 / CD28 T cell amplifier).

[0432] In some embodiments, separation is accomplished by enriching for a particular cell population through positive selection, or by depleting a particular cell population through negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding reagents that specifically bind to one or more surface markers, which bind to the positively or negatively selected cells, respectively. These proteins are either expressed in the cells (marker +) or at relatively high levels (marker high).

[0433] In some embodiments, T cells are isolated from PBMC samples using voice selection against markers (e.g., CD14) expressed on non-T cells (e.g., B cells, monocytes or other blood leukocytes). In some aspects, a CD4+ or CD8+ selection step is used to separate helper CD4+ and CD8+ cytotoxic T cells. These CD4+ and CD8+ populations can be further separated into subgroups using positive or negative selection against markers expressed or expressed to a relatively high extent on one or more subgroups of primary, memory and / or effector T cells.

[0434] In some embodiments, CD8+ cells further enrich or consume primary, central memory, effector memory and / or central memory stem cells, e.g., undergo positive or negative selection based on surface antigens associated with the corresponding subgroups. In some embodiments, to increase efficacy, e.g., enrich for central memory T (TCM) cells to improve long-term survival, expansion and / or engraftment after administration, which in some aspects is particularly strong in these subgroups. Terakura et al. (2012) Blood. 1: 72-82; Wang et al. (2012) J Immunother. 3 5(9): 689-701. In some embodiments, TCM-enriched CD8+ T cells are combined with CD4+ T cells to further enhance efficacy.

[0435] In an embodiment, memory T cells reside in the CD62L+ and CD62L- subgroups of CD8+ peripheral blood lymphocytes. PBMCs can be enriched or depleted of the CD62L-CD8+ and / or CD62L+CD8+ fractions, for example, using anti-CD8 and anti-CD62L antibodies.

[0436] In some embodiments, enrichment of central memory T (TCM) cells is performed by CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127 positivity or surface overexpression, and in some aspects, this is negatively selected based on CD45RA and / or granzyme B expression or overexpression. In some aspects, separation of the CD8+ population to enrich for TCM cells is performed using a method of depletion of cells expressing CD4, CD14, CD45RA, and positive selection or enrichment of cells expressing CD62L. Meanwhile, enrichment of central memory T (TCM) cells is performed using a scheme of negative selection based on CD14 and CD45RA expression, and positive selection based on CD62L, including a negative portion of cells selected based on CD4 expression. In some aspects, such selections are performed simultaneously, and in other aspects, such selections are performed sequentially in a specific order. In some embodiments, the same CD4 expression-based selection step is used to prepare the CD8+ cell population or subgroup and to generate the CD4+ cell population or subgroup, so that the positive and negative fractions from the CD4-based separation are retained and used in subsequent steps of the method, optionally after performing one or more additional positive or negative selection steps.

[0437] In a specific implementation, a PBMC sample or other blood leukocyte sample undergoes CD4+ cell selection, where a negative and positive portion is reserved, and then a negative selection is performed on the negative portion based on expression of CD14 and CD45RA or CD19, and a positive selection is performed based on central memory T cell characteristic markers such as CD62L or CCR7, where the positive and negative selections are performed in a specific order.

[0438] By examining cell populations bearing cell surface antigens, we can identify helper CD4+ T cells, primary, central memory and effector cells. CD4+ lymphocytes can be obtained via standard methods. In some embodiments, primary CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO-.

[0439] In one implementation, to enrich for CD4+ cells via negative selection, a mixture of single clone antibodies typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding partners are bound to a solid support or substrate, such as, for example, magnetic or paramagnetic beads, to allow for the separation of cells for positive and / or negative selection.

[0440] In some embodiments, the preparation method includes steps such as cryopreserving the cells before or after separation, incubation and / or manipulation. In some embodiments, the freezing and subsequent lysis step removes granular leukocytes and, to some extent, mononuclear cells in the cell population. In some embodiments, for example, a washing step is performed to remove plasma and platelets, followed by suspension of the cells in a freezing solution. In some aspects, any of a variety of known freezing solutions and parameters can be used. In one implementation, PBS or other suitable cell freezing medium containing 20% ​​DMSO and 8% human serum albumin (HAS) is used. The medium is then diluted 1:1 so that the final concentrations of DMSO and HAS are 10% and 4%, respectively. The cells are then frozen to -80°C or -90°C based on the same principles as a programmable cooling device, typically at a rate of 1° / min, according to a previously defined program or stored in the vapor phase of a liquid nitrogen storage tank.

[0441] In some embodiments, the methods provided include culturing, incubating, and / or engineering steps. For example, some embodiments provide methods of incubating and / or manipulating the consumed cell population and the culture starting composition.

[0442] In some embodiments, the cells are incubated with a culture starter composition, incubated and / or manipulated in a vessel, such as a cell, chamber, well, column, tube, tube set, valve, vial, petri dish, bag, or other vessel for culturing cells.

[0443] In some embodiments, the cells are incubated and / or cultured prior to or in conjunction with the genetic engineering. The incubation step can include culturing, stimulating, activating, and / or expanding. In some embodiments, the cells or compositions are incubated under stimulatory conditions or in the presence of stimulatory agents. These conditions include conditions designed to induce cell proliferation, reproduction, activation, and / or survival in populations, mimicking contact with antigens, and / or inducing cells to be used for genetic engineering, such as introduction of recombinant antigen receptors.

[0444] The conditions can include a particular medium, temperature, oxygen content, carbon dioxide content, time, reagents such as nutrients, amino acids, antibiotics, ions, and / or stimulatory factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinantly delivered receptors, and any other substances that can be engineered to maintain an activated state of the cells.

[0445] In some embodiments, the stimulatory condition or agent induces intracellular signaling of the TCR complex. The amplification method includes one or more substances, such as a ligand, capable of activating the TCR / CD3 domain. In some embodiments, the substances initiate or initiate a TCR / CD3 intracellular signaling cascade in the T cell. These substances can include, for example, antibodies specific for components of the TCR and / or co-stimulatory receptors, but can also include, for example, anti-CD3 antibodies, anti-CD28 antibodies, and / or one or more cytokines, bound to a solid support, such as beads. Optionally, the amplification method includes adding anti-CD3 antibodies and / or anti-CD28 antibodies to the medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulatory agent comprises IL-2, such as IL-2 and / or IL-15 and / or IL-7 and / or IL-21, at a concentration of at least about 10 units / mL.

[0446] In some embodiments, incubation proceeds based on some of the techniques described, for example, in U.S. Patent No. 6,040,177 to Riddell et al., Klebanoff et al., (2012) J Immunother. 35(9):651-660, Terakura et al., (2012) Blood. 1:72-82, and / or Wang et al., (2012) J Immunother. 35(9):689-701.

[0447] In some embodiments, the T cell population is expanded by expanding on a culture starting composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs) (e.g., the resulting cell population for each T lymphocyte in the initial population to be expanded comprises at least about 5, 10, 20, or 40 or more PBMC feeder cells) and incubating the culture (e.g., for a time sufficient to expand the number of T cells). In some embodiments, the non-dividing feeder cells can comprise γ-irradiated PBMC feeder cells. In some embodiments, the PBMCs are prevented from cell division via γ-irradiation in the range of about 3000-3600 rads. In some embodiments, the feeder cells are added to the medium prior to adding the T cells.

[0448] In some embodiments, the stimulatory conditions include a temperature suitable for the growth of human T lymphocytes, e.g., about 25° C., typically at least about 30° C., and typically or about 37° C. Optionally, the incubation can further include the step of adding non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. The LCL can be irradiated with gamma radiation in the range of about 6,000-10,000 rads. In some aspects, the LCL feeder cells are provided in any suitable amount, e.g., the ratio of LCL feeder cells to primary T lymphocytes is at least about 10:1.

[0449] In some embodiments, antigen-specific or naive T lymphocytes are stimulated with an antigen to obtain antigen-specific T cells, such as antigen-specific CD4+ and / or CD8+ T cells. For example, antigen-specific T cell lines or clones can be generated against a cytomegalovirus antigen by isolating T cells from an infected subject and stimulating the cells ex vivo with the same antigen.

[0450] Compositions and Formulations The present invention provides pharmaceutical compositions and formulations for the methods of the present invention. In some embodiments, the pharmaceutical compositions and formulations of the present invention include compositions of cells for administration, for example, pharmaceutical compositions and formulations that include unit dosage compositions from a number of cells for administration that includes a specified dosage or a fraction thereof. The pharmaceutical compositions and formulations generally include one or more optional pharma- ceutically acceptable carriers or excipients. In some embodiments, the compositions include at least one other therapeutic agent.

[0451] The term "drug formulation" refers to a formulation in such a form that allows the biological activity of the active ingredient contained therein to be effective and that does not contain additional ingredients that would have unacceptable toxicity to the subject to which the formulation is to be administered.

[0452] "Pharmaceutically acceptable carrier" means any component of a drug formulation that is not an active ingredient and is not toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0453] In some embodiments, the selected portion of the carrier is determined by the particular cell and / or the method of administration. Thus, there are various suitable preparation methods. For example, the drug composition may include a preservative. Suitable preservatives may include, for example, methyl paraben, propyl paraben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. Suitable preservatives may include, for example, methyl paraben, propyl paraben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The preservative or mixtures thereof are generally present in an amount of about 0.0001% to about 2% (calculated by total weight of the composition). Carriers are described, for example, in Remington's Pharmaceutical Sciences, 16th Edition, edited by Osol, A. (1980). Pharmaceutically acceptable carriers, at the dosages and concentrations employed, are generally non-toxic to subjects and may include, but are not limited to, buffers such as phosphate, citrate, and other organic acid buffers; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexaalkyl quaternary ammonium chlorides); and tertiary ammonium chlorides. quaternary ammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; p-hydroxy alkyl benzoates, such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol; polypeptides of low molecular weight (fewer than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; glycine, glutamine, and asparagine. These include, but are not limited to, amino acids such as asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other sugars, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[0454] In some embodiments, the composition includes a buffering agent. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffering agents is used. The amount of a buffering agent or mixtures thereof is generally about 0.001% to about 4% (calculated by total weight of the composition). Methods for preparing administrable drug compositions are known. Exemplary methods are specifically described, for example, in "Remington: The Science and Practice of Pharmacy", Lippincott Williams & Wilkins; 21st Edition (May 1, 2005).

[0455] The formulation may include an aqueous solution. The formulation or composition may further include one or more active ingredients, which may be used for the particular indication, disease or condition for which the cell therapy is applied, preferably those that provide the cell replenishing activity, where the corresponding active agents do not adversely affect each other. These active ingredients are appropriately combined and present in an effective dosage for the required purpose. Thus, in some embodiments, the pharmaceutical composition may further include other pharmacologic active substances or drugs, such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine.

[0456] In some embodiments, the pharmaceutical composition includes an amount of cells that effectively treats or prevents a disease or condition, e.g., a therapeutically effective amount or a prophylactically effective amount. In some embodiments, the treated subject is periodically evaluated to monitor the effectiveness of the treatment or prophylaxis. The required dosage can be administered by administering the cells via a single bolus, by administering the cells via multiple boluses, or by continuously infusing the cells.

[0457] In some embodiments, the compositions comprise an amount of cells that effectively reduces the burden of a disease or condition and / or does not cause CRS or significant CRS in a subject and / or implements any other outcome of the methods herein.

[0458] The cells and compositions can be administered using standard administration techniques, formulations and / or devices. Administration of the cells can be autologous or heterologous. For example, immunosuppressive cells or progenitor cells can be obtained from the same subject and administered to the same subject or to a different compatible subject. Peripheral blood derived immunosuppressive cells or their progeny (e.g., derived internally, ex vivo or ex vivo) can be administered via catheter administration, systemic injection, local injection, intravenous injection, or local injection, including parenteral administration. When administering a therapeutic composition (e.g., a drug composition containing genetically modified immunosuppressive cells), it is generally prepared in a unit dosage injectable form (solution, suspension, emulsion).

[0459] Formulations include those used for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, mucosal, sublingual, or suppository administration. In some embodiments, the cell population is administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to a subject using peripheral systemic delivery via intravenous, intraperitoneal, or subcutaneous injection.

[0460] In some embodiments, the composition is provided in the form of a sterile liquid formulation, for example, as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, some aspects of which may be buffered to a selected pH. Liquid formulations are generally easier to manufacture than gels, other viscous compositions, and solid compositions. Furthermore, liquid compositions are more easily administered, particularly via injection. In another aspect, viscous compositions can be prepared within an appropriate viscosity range to provide longer contact time with certain tissues. The liquid or viscous composition can include a carrier, which can be a solvent or dispersion medium, including, for example, water, saline, phosphate buffered saline, polyhydroxy compounds (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and suitable mixtures thereof. For example, the cells can be placed in a suitable carrier, diluent, or vehicle, such as sterile water, saline, glucose, dextrose, or other vehicle, to prepare a sterile injectable solution. The compositions may contain auxiliary substances such as wetting, dispersing or emulsifying agents (e.g., methyl cellulose), pH buffers, gelling or thickening additives, preservatives, flavorings, and / or dyes, as determined by the required route of administration and preparation. In some embodiments, standard textbooks may be consulted to prepare suitable preparations.

[0461] Various additives can be added, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, which enhance the stability and sterility of the composition. Antibacterial and antifungal agents, such as various parabens, chlorobutanol, phenol, and sorbic acid, can also be used to ensure the prevention of the action of microorganisms. Absorption of the injectable drug form can be prolonged by the use of substances that delay absorption, such as aluminum monostearate and gelatin.

[0462] Preparations for internal administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes.

[0463] product The invention provides articles of manufacture, e.g., kits and devices, for administering cells to a subject and storing the cells and compositions according to the adoptive cell therapy or immune effector cell therapy methods provided.

[0464] The article of manufacture includes one or more containers, and is typically a number of containers, packaging materials, and one or more container and / or packaging combinations, or a label attached thereto, or a package insert page that includes instructions for administering the cells to a subject.

[0465] The container typically contains, for example, one or more unit doses of cells to be administered. The product typically contains multiple containers containing a single unit dose of cells. A unit dose can be twice (or more than twice) the amount or number of cells administered to a subject in a first dose or the number of cells administered in a first or subsequent dose. It can be the smallest dose or the smallest possible amount of cells to be administered to a subject according to the method of administration. In some embodiments, the unit dose is the number of cells or the lowest number of cells administered to any subject suffering from a particular disease or condition, or any subject, in a unit dose according to the methods of the invention. For example, in some aspects, the unit dose can include the smallest amount of cells to be administered to a subject with a relatively low body weight and / or a relatively low disease burden, e.g., administering more than one unit dose to a subject designated for a first dose in one or some circumstances, but administering one or more unit doses to a subject designated for one or more subsequent doses, e.g., administering according to the methods provided. In some embodiments, the chimeric antigen receptor-expressing cells of a subject derived from, for example, cells. Or the number of cells in a unit dose, such as the number of CAR-expressing or cell numbers, should be administered to a particular subject at the initial dose. In some embodiments, the cells are derived from a subject to be treated or in need of treatment with the methods provided herein.

[0466] In some embodiments, each container independently contains a unit dose of cells, e.g., contains the same or essentially the same number of cells. Thus, in some embodiments, each container contains the same, approximately, or essentially the same number of cells or chimeric antigen receptor-expressing cells. In some embodiments, a unit dose is about 1×10 10 , about 1×10 9 , about 1×10 8 or about 1 x 10 7 The present invention includes subjects treated with and / or derived from fewer than 10 engineered cells, whole cells, T cells or PBMC / kg.

[0467] Suitable containers include, for example, bottles, vials, syringes, and flexible bags such as freezer bags. In certain embodiments, the container is a bag, but is not limited to, for example, a flexible bag, such as a bag suitable for injecting cells into a subject, for example, a flexible plastic or PVC bag or EVA or ULPDE, and / or an IV solution bag. In some embodiments, the bag is a sealable and / or sterilizable bag for providing the sterile solution, cells, and composition. In some embodiments, the volume of the container, e.g., bag, is equal to, about, or at least about 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml, or 1000 ml, for example, about 10 ml to 100 ml or about 100 ml, or about 10 ml to 500 ml or about 500 ml. In some embodiments, the container, such as a bag, is made of a material that is stable at one or more different temperatures and / or provides and / or maintains stable storage of the cells, e.g., at low temperatures, e.g., less than or equal to about -20°C, -80°C, -120°C, 135°C, -196°C, and / or temperatures suitable for frozen storage, and / or other temperatures, e.g., temperatures suitable for freezing and thawing the cells and body temperature, e.g., about 37°C or -38°C, or -39°C, or -40°C, for immediate freezing and thawing prior to treatment, e.g., at the subject's site or treatment site, e.g., at a bed.

[0468] The container can be made of a variety of materials, such as, for example, glass or plastic. In some embodiments, the container includes one or more ports, such as a sterile entry port, which is connected to one or more tubes, for example, through a tube or a cage, for other infusions, for example, of intravenous torn, and / or for the purpose of exiting from other containers, such as, for example, cell culture and / or storage bags or other containers, and transferring to another container. Exemplary containers include freezer bags, intravenous solution bags, and vials, including those with membranes that can be pierced via a syringe needle.

[0469] The product may further include a packaging insert or label displaying one or more usage information and / or instructions. In some embodiments, the information or instructions may indicate and / or provide instructions that a particular disease or condition may or should be treated. The label or packaging insert may display product instructions for treating a disease or condition. In some embodiments, the label or packaging insert may provide instructions for treating a subject, e.g., administering cells to a subject derived from the cells in an initial and one or more subsequent doses, e.g., in any of the methods described herein. In some embodiments, the instructions specify administering one unit dose of an initial dose, e.g., the contents of a single, single container of the product, followed by administration of one or more subsequent doses at a specified time or within a specified time period and / or after detecting the presence or absence or amount or extent of one or more factors or results in the subject.

[0470] In some embodiments, the instructions specify an initial administration and subsequent administrations in multiple unit doses to the subject, in some embodiments, the initial administration comprises providing any of said unit doses to the subject, and subsequent administrations comprise administering one or more of said unit doses to the subject.

[0471] In some embodiments, the instructions specify that the subsequent dose is administered at a time that is equal to or is about 21 to 80 days, or about 25 to 60 days, e.g., about 50 days, after the first dose, e.g., after the start of the first or previous dose. In some embodiments, the instructions specify that the subsequent dose is administered after determining that a serum level of a factor indicative of cytokine release syndrome (CRS) from the subject is about 10-fold, about 25-fold, and / or about 50-fold lower than the serum level of the indicator in said subject immediately prior to said first dose, and / or that an index of CRS has already reached a peak level and is declining, and / or that a detectable adaptive host immune response specific for a receptor (e.g., CAR) expressed by cells of said subject is not displayed.

[0472] In some embodiments, the label or packaging insert or package includes indicia to indicate the identity of the subject from which the cells are derived and / or the subject to whom they are administered. In the case of autologous transplantation, the identity of the subject from whom the cells are derived is the same as the identity of the subject to whom the cells are administered. Thus, identifying information can designate that the cells, such as the cells of their original source, are to be administered to a particular patient. This information can be present on the packaging material and / or label in the form of a bar code or other coded indicia, or can indicate the name and / or other identifying characteristic of the subject.

[0473] In some embodiments, the article of manufacture includes multiple containers containing one or more compositions, typically including cells, but further includes one or more other containers containing compositions therein, e.g., including a single unit dosage form thereof, which may include other reagents, e.g., cytotoxic or other therapeutic agents, that may be combined with the cells and administered simultaneously or once in any order. Alternatively, the article of manufacture may further include other or the same containers containing a pharma- ceutically acceptable buffer, which may further include other components, such as other buffers, diluents, filters, tubing, needles, and / or syringes.

[0474] The term "packaging insert page" refers to instructions that are often included in commercial packaging for therapeutic products, said instructions including information such as instructions for using these therapeutic products, methods of use, dosage, administration, concomitant therapy, contraindications and / or warnings.

[0475] Treating cancer patients with initial and subsequent doses of autologous T cells expressing a CAR The method of the present invention can be summarized as follows.

[0476] Based on the "mononuclear cell isolation technique," peripheral blood mononuclear cells (PBMCs) or T cells are isolated from a human subject suffering from cancer, and the cells are cultured and transduced with a viral carrier encoding a chimeric antigen receptor (CAR), which specifically binds to an antigen that is a tumor-associated or tumor-specific antigen expressed by the subject's cancer. The cells are stored frozen in infusion medium in a single flexible freezer bag, each of approximately 1 × 10 6 cells ~5×10 7 The initial dose injected into each subject was approximately 1 × 10 12 Not more than 1 x 10 cells, preferably about 1 x 10 11 Not more than about 1 x 10 cells, more preferably about 1 x 10 10 cells or approximately 5 x 10 9 or about 2 x 10 9 Prior to injection, the cells are maintained at a temperature below about -130°C or about -175°C.

[0477] Prior to initiating cell therapy, blood is obtained from the subject and serum factors indicative of serum cytokine release syndrome (CRS), such as one or more of tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), IL-10, and IL-6, are assessed using ELISA and / or MSD and / or CBA methods as appropriate. Prior to initiating therapy, solid tumor size and mass can be measured, for example, by PET or CT scan, to assess tumor burden, as appropriate.

[0478] After resuscitation by warming to approximately 38°C, the subject is administered an initial dose of cells via multiple injections, each administered intravenously (IV) as a continuous infusion over a period of approximately 3-30 minutes.

[0479] After administration of the first dose, the subject undergoes a physical examination to monitor for any toxicity or symptoms of toxicity consequences, such as fever, hypotension, hypoxia, neuropathy or elevated serum levels of inflammatory cytokines or C-reactive protein (CRP). Optionally, after administration of the first dose, if one or more doses are administered, blood is obtained from the patient to assess the levels of serum factors indicative of CRS using ELISA and / or MSD and / or CBA methods. Compare the levels of serum factors to those obtained just before administration of the first dose. If necessary, anti-IL6 or other CRS treatment is administered to reduce symptoms of CRS.

[0480] After administration of the initial dose, e.g., to selectively detect the presence or absence of an anti-CAR immune response from the subject at 1 week, 2 weeks, 3 weeks, and / or 4 weeks after initiating dosing, e.g., via qPCR, ELISA, ELISPOT, cell-based antibody measurements and / or mixed lymphocyte reaction.

[0481] The percentage reduction in tumor burden implemented using the initial dose can be measured one or more times after administration of the initial dose to solid tumor patients via scans (e.g., PET and CT scans) as appropriate and / or via quantification of disease positive cells in the blood or at the tumor site.

[0482] Subsequent doses are administered. In some subjects, subsequent doses are administered within about 80 days, preferably within about 60 days, more preferably within about 50 days after administration of the first dose. In some circumstances, subsequent doses are administered only if the CRS-related results or serum factor levels tested are lower than acceptable levels and the subject's anti-CAR immune response is not detected about 60 days after administration of the first dose. In other subjects, subsequent doses are administered if the subject is considered to have no CRS or significant CRS for more than 30 days after administration of the first dose, or if 20% of CRS is observed when all tested serum factor levels indicative of CRS are lower than the peak levels after administration of the first dose and the subject's anti-CAR immune response is not detected. In some embodiments, one or more subsequent doses of cells are administered. The interval between each dose is 21 to 80 days, preferably 25 to 60 days, more preferably 25 to 55 days.

[0483] The size of the subsequent doses is patient-specific and based on the tumor burden, the presence of anti-CAR immune responses, and the level of CRS-related outcomes. Some subjects will receive one, two, three, or even more subsequent doses of cells. The subsequent doses infused into each subject will be approximately 1 × 10 12 Not more than 1 x 10 cells, or approximately 1 x 10 11 Not more than 1 x 10 cells, or approximately 1 x 10 10 Not more than 5 x 10 cells, or approximately 5 x 10 9 No more than about 2 x 10 cells 9 Administer not more than 100 cells, with subsequent doses administered within approximately 3-30 minutes via continuous intravenous injection.

[0484] Beginning with the first dose, subjects will be monitored periodically for several years at patient visits to measure tumor burden and / or to measure in vivo proliferation and persistence of the administered cells using flow cytometric analysis and quantitative polymerase chain reaction (qPCR) to detect CAR-expressing cells and / or to assess the progression of the anti-CAR immune response.

[0485] The advantages of the present invention are as follows:

[0486] 1. The present invention significantly improves the therapeutic effect of proceeding with the treatment of tumors using immune effector cells expressing a chimeric antigen receptor, i.e., reduces the number or volume of tumors, and / or reduces the number and / or extent of metastases, and / or reduces tumor markers, and / or eliminates or worsens complications commonly seen in any late-stage cancer.

[0487] 2. The antitumor effect against solid tumors is remarkable, and the treatment is safe and effective, providing a therapeutic means that can stabilize or even cure CLA18A2-positive mid- to late-stage gastrointestinal adenocarcinoma.

[0488] 3. A pretreatment composition for improving immune effector cell anti-tumor therapy is provided. By administering a fludarabine, cyclophosphamide, and albumin-bound paclitaxel composition before administering immune effector cells, the anti-tumor therapeutic effect of immune effector cells can be significantly promoted, the tumor burden of patients can be significantly reduced, and the quality of life and survival period of patients can be improved. EXAMPLES

[0489] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are for the purpose of illustrating the present invention and do not limit the scope of the present invention. In the following examples, the experimental methods that do not specify specific conditions are generally carried out based on the conditions, for example, those in J. Sambrook et al., Molecular Cloning Experimental Guide, 3rd Edition, Science Press, 2002, or those suggested by the manufacturer.

[0490] material and method: material: The various materials, including the reagents, used in the present invention are all commercially available.

[0491] Exemplary antigen receptors of the present invention include CARs, and methods for engineering and introducing receptors into cells are described, for example, in Chinese patent applications having publication numbers CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN106397593A, CN106467573A, and international patent applications having publication numbers WO2018006882A1 and WO2015172339A8, all of which are incorporated herein by reference.

[0492] In an embodiment of the invention, the scFv portion of the chimeric antigen receptor comprises a heavy chain variable region as set forth in SEQ ID NO:14 and a light chain variable region as set forth in SEQ ID NO:18, the chimeric antigen receptor comprises a sequence as set forth in SEQ ID NO:24, the amino acid sequence of the scFv as set forth in SEQ ID NO:54, and the nucleic acid sequence as set forth in SEQ ID NO:55, and the CDR regions utilized are HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:7, HCDR3 as set forth in SEQ ID NO:3, LCDR1 as set forth in SEQ ID NO:4, LDR2 as set forth in SEQ ID NO:5, and LDR3 as set forth in SEQ ID NO:6. CDR2, LCDR3 as shown in SEQ ID NO:6.

[0493] The method for constructing CAR-T cells is as follows. First, a CAR gene is constructed. For example, the construction of hu8E5-2I-BBZ sequentially contains, from the 5' end to the 3' end, a CD8α signal peptide (nucleotide sequence shown in SEQ ID NO:36), hu8E5-2I The CAR gene is then cloned into a shuttle plasmid to obtain a target plasmid containing the CAR gene. The constructed target plasmid and packaging plasmid are co-transfected into 293T cells to produce a lentiviral carrier, and the lentiviral carrier is transduced into T cells obtained from the patient's peripheral blood to obtain CAR-T cells. For example, hu8E5-2I-BBZ is used in the following examples. EXAMPLES

[0494] Example 1: Treatment of cancer patients with initial and subsequent doses of CAR-expressing autologous T cells Patients with CLD18A2-positive gastrointestinal tumors (e.g., gastric adenocarcinoma, pancreatic cancer) are administered autologous T cells expressing anti-CLD18A2 chimeric antigen receptor (CAR). Before administering the cells, the patient undergoes a pretreatment treatment with apheresis isolation technology of "mononuclear cell isolation technology". To obtain autologous CAR-expressing T cells, PBMCs are obtained by isolation based on apheresis samples from individual subjects, transduced via a viral carrier encoding anti-CLD18A2 CAR, and then expanded in large quantities, after which the autologous CAR-expressing T cells in the formulation are frozen and stored in a single flexible freezer bag infusion medium. The initial dose to be infused into each subject is approximately 1 × 10 12 Not more than 1 x 10 cells, preferably about 1 x 10 11Not more than 1 x 10 cells, preferably about 1 x 10 10 Not more than 5 x 10 cells, or approximately 5 x 10 9 No more than 2 x 10 cells 9 Prior to injection, cells are maintained at a temperature below -175°C.

[0495] Prior to initiating cell therapy, blood is obtained from the subject and serum levels of one or more factors indicative of cytokine release syndrome (CRS), such as tumor necrosis factor alpha (TNFα), interferon gamma (IFNγ), IL-10, and IL-6, are assessed using ELISA and / or MSD and / or CBA methods as appropriate. Prior to initiating therapy, tumor burden can be assessed as appropriate by measuring the size or characteristics of solid tumors, for example, by PET or CT scan, and tumor burden can also be assessed by detecting tumor markers and / or observing tumor complication occurrence and severity.

[0496] The subject is resuscitated by warming to about 38° C. and administered an initial dose of cells via multiple injections over a period not exceeding 20 days, for example 1-6, preferably 1-5, preferably 1-4, more preferably 1-3, more preferably 2-3 injections of the initial dose. Each injection is administered via continuous intravenous (IV) infusion over a period of about 3-30 minutes, preferably 5-25 minutes.

[0497] After administration of the initial dose, the subject undergoes a physical examination to monitor for any toxicity or symptoms of toxicity consequences, e.g., fever, hypotension, hypoxia, neuropathy, or elevated serum levels of inflammatory cytokines or C-reactive protein (CRP). Optionally, if one or more doses are administered after administration of the initial dose, blood is obtained from the patient to assess levels of serum factors indicative of CRS using ELISA and / or MSD and / or CBA methods. Compare serum factor levels obtained just prior to administration of the first dose with serum factor levels obtained just prior to administration of the first dose. If necessary, administer anti-IL6 or other CRS treatment to reduce CRS symptoms.

[0498] After administration of the initial dose, e.g., to selectively detect the presence or absence of an anti-CAR immune response from the subject at 1 week, 2 weeks, 3 weeks, and / or 4 weeks after initiating dosing, e.g., via qPCR, ELISA, ELISPOT, cell-based antibody measurements and / or mixed lymphocyte reaction.

[0499] The percentage reduction in tumor burden implemented using the initial dose can be measured one or more times after administration of the initial dose to solid tumor patients via scans (e.g., PET and CT scans) as appropriate and / or via quantification of disease positive cells in the blood or at the tumor site.

[0500] Subsequent doses are administered. In some subjects, the initial dose is administered within about 80 days, preferably within about 60 days, more preferably within about 50 days after administration of the initial dose. In some circumstances, subsequent doses are administered only if the CRS-related results or serum factor levels tested are lower than acceptable levels and the subject's anti-CAR immune response is not detected about 60 days after administration of the initial dose. In other subjects, subsequent doses are administered if the subject is considered to have no CRS or severe CRS for more than 30 days after administration of the initial dose, or if 20% of CRS is observed when all tested serum factor levels indicative of CRS are lower than the peak levels after administration of the initial dose and the subject's anti-CAR immune response is not detected. In some embodiments, one or more subsequent doses of cells are administered. The interval between each dose is 21 to 80 days, preferably 25 to 60 days, more preferably 25 to 55 days.

[0501] The size of the subsequent doses is patient-specific and based on the tumor burden, the presence of anti-CAR immune responses, and the level of CRS-related outcomes. Some subjects will receive 1, 2, 3, 4, or even more subsequent doses. The number of subsequent doses infused into each subject will be approximately 1 x 10 12 Not more than 1 x 10 cells, preferably about 1 x 10 11 Not more than about 1 x 10 cells, more preferably about 1 x 10 10 Not more than about 5 x 10 cells, more preferably about 5 x 10 9 No more than about 2 x 10 cells 9 Subsequent doses are administered in multiple doses over a period not exceeding about 20 days, for example, 1-5 doses, preferably 1-4 doses, preferably 1-3 doses, more preferably 2-3 doses. Each dose is administered via continuous intravenous (IV) infusion within a period of about 3-30 minutes, preferably 5-25 minutes.

[0502] Subjects will be monitored periodically, starting from the first dose and continuing for several years, during patient visits to measure tumor burden and / or to measure in vivo proliferation and persistence of the administered cells using flow cytometric analysis and quantitative polymerase chain reaction (qPCR) to detect CAR-expressing cells and / or to assess the progression of the anti-CAR immune response. EXAMPLES

[0503] Example 2: Evaluation of neurotoxicity of CAR-T cell therapy in subjects with pre-treatment morphological disease Imaging was used to detect target lesions, evaluate pretreatment tumor burden, and determine the number and size of target lesions. The evaluation criteria for treatment efficacy are shown in Table 1.

[0504] [Table 1]

[0505] Within approximately 3 to 30 minutes, one, two, or three consecutive intravenous (IV) infusions were administered, with a total dose of approximately 1.1 × 10 6 pieces CAR-T cells / KG ~ approx. 2.8×10 7 Total amount of CAR-T / KG administered to subjects The CAR-expressing T cells are administered to the subject 1-30 days, preferably 1-20 days, more preferably 4-12 days prior to infusion of the CAR-T cells, with pre-conditioning chemotherapy of fludarabine, or cyclophosphamide, or albumin-bound paclitaxel, or fludarabine and albumin-bound paclitaxel, or cyclophosphamide and albumin-bound paclitaxel, or fludarabine and cyclophosphamide, or a combination of fludarabine, cyclophosphamide, and albumin-bound paclitaxel.

[0506] After administration of CAR-T cells, assess the subject's disease status and evaluate response to treatment. After treatment, evaluate the subject and monitor for neurotoxicity (symptoms of confusion, neurological complications including aphasia, epileptic seizures, convulsions, drowsiness, and / or altered mental status) and grade according to severity (use a 1-5 rating scale, e.g., Guido Cavaletti and Paola Marmiroli Nature Reviews Neurology 6, 657-666 (December 2010)), where grade 3 (severe symptoms), grade 4 (life-threatening symptoms) or grade 5 (death) are considered severe neurotoxicity.

[0507] (1) Identification and monitoring of Cytokine Release Syndrome (CRS): Grade 1 (mild) - not life-threatening and only requires systemic treatment such as antipyretics and antiemetics (e.g., fever, area, fatigue, headache, muscle pain, discomfort); Level 2 (Intermediate) - Moderate interference and response required: Oxygen gas requirement <40%, or Hypotension in response to fluid or low doses of a single vasoconstrictor, or Grade 2 organ toxicity (through CTCAE v4.0); Grade 3 (Severe) - Requires active intervention and response: Oxygen gas requirement ≥ 40%, or Hypotension requiring a test dose of a single vasoconstrictor (e.g., norepinephrine ≥ 20ug / kg / min, dopamine ≥ 10g / kg / min, phenylephrine ≥ 200ug / kg / min, or epinephrine ≥ 10ug / kg / min), or Various vasoconstrictors (e.g., antidiuretic plus any of the above agents, or ≥ 20ug Hypotension requiring a combination of vasopressor drugs (norepinephrine / kg / min) or Grade 3 organ toxicity or grade 4 transaminase inflammation (CTCAE v4.0) Tongham); Grade 4 (life-threatening) - ventilator supported or Grade 4 organ toxicity (excluding transaminase inflammation); Grade 5 (Fatal) - Death.

[0508] (2) Exemplary classification criteria for neurotoxicity: Grade 1 (no or mild clinical symptoms) - mild symptoms or no clinical symptoms; Grade 2 (Intermediate) - Symptoms are present that limit active activities of daily living (ADLs), for example cooking, shopping, doing laundry, using the telephone, or managing money; Grade 3 (severe) - Symptoms are present that limit self-care ADLs, e.g., bathing, dressing or undressing, eating, using the toilet, taking medications, etc.; Grade 4 (life-threatening) - there are symptoms that are life-threatening and require urgent intervention; Grade 5 (Fatal) - Death.

[0509] In subjects separated based on disease burden before treatment, the response after treatment with a single infusion of different doses of CAR-T cells compared to the dose was evaluated, the presence of severe CRS, and the presence of severe neurotoxicity. The results are shown in Table 2. Table 2 lists the specific usage of cells administered in various multiple doses, the first dose and subsequent doses, and the time interval between the adjacent previous subsequent dose and the adjacent later subsequent dose. Table 2 also lists the response after each treatment of CAR T cells evaluated using the time before administration of the first dose as the tumor burden baseline based on the criteria in Example 2. Table 2 also lists the results of the response to each administration for six patients (the column corresponding to "Response" lists the tumor burden after administration, and the response to treatment compared to this tumor burden baseline), the presence or absence of severe CRS and severe neurotoxicity (Y / N). As shown in Table 2, SD was observed in subjects treated with CAR-T cells at all doses tested, regardless of the dise...

Claims

1. 1. A method of treating a CLD18 positive tumor, comprising: administering to a subject in need thereof an initial dose of immune effector cells, wherein the immune effector cells express a chimeric antigen receptor (CAR) and specifically recognize CLD18, the initial dose being about 2×10 １０ The total number of cells is not more than about 1×10 １２ Without exceeding the cell Preferably, the initial dose is about 2×10 ９ The total number of cells is not more than about 1×10 １１ Without exceeding the cell Preferably, the initial dose is about 2.5×10 ８ cells / kg subject body weight, Or the total number of cells is about 1 x 10 １０ Without exceeding the cell Preferably, the initial dose is about 5×10 ７ The total number of cells is not more than about 1×10 １０ Without exceeding the cell More preferably, the initial dose is about 3×10 ７ cells / kg subject body weight, or the total number of said cells is about 2×10 ９ The method does not exceed the cell size.

2. The CLD18 is characterized in that it is CLD18A2. The method of claim 1.

3. After administering the initial dose of immune effector cells, a subsequent dose of immune effector cells expressing a chimeric antigen receptor (CAR) and specifically recognizing CLD18 is administered at least once again. The method according to claim 1 or 2.

4. and re-administering the subsequent dose of immune effector cells after the first dose of immune effector cells is no longer detectable in vivo. The method according to claim 3.

5. The subsequent dose of immune effector cells is administered about 21 to 80 days after the initial dose is administered, preferably about 25 to 60 days after the initial dose is administered, and more preferably about 25 to 50 days after the initial dose is administered. The method according to claim 3.

6. The subsequent dose is greater than the initial dose, preferably the subsequent dose is 2, 5, 7 or 10 times the initial dose. The method according to claim 5.

7. The subsequent dose is about 2×10 １０ The total number of cells is not more than about 1×10 １２ Without exceeding the cell Preferably, the subsequent dose is about 2×10 ９ The total number of cells is not more than about 1×10 １１ Without exceeding the cell Preferably, the subsequent dose is about 2.5×10 ８ cells / kg subject body weight, Or the total number of cells is about 1 x 10 １０ Without exceeding the cell Preferably, the subsequent dose is about 5×10 ７ The total number of cells is not more than about 1×10 １０ Without exceeding the cell More preferably, the subsequent dose is about 3×10 ７ cells / kg subject body weight, or the total number of said cells is about 2×10 ９ Characterized by not exceeding the cell size The method according to claim 3.

8. The initial dose or subsequent doses are administered in N separate doses within 20 days, where N is a natural number not smaller than 1, and preferably within 3 to 15 days. The method according to claim 1 or 3.

9. When a subsequent dose is administered, the subject (i) the subject's serum level of a factor indicative of cytokine release syndrome (CRS) is about 10-fold, about 25-fold, and / or about 50-fold lower than the level in the subject immediately prior to administration of the first dose; (ii) the characteristic of not exhibiting grade 3 or higher neurotoxicity; (iii) a decrease in neurotoxicity or CRS levels compared to the peak level of neurotoxicity or CRS levels after administration of the first dose of immune effector cells; or (iv) the subject has any one of the following characteristics: and (ii) the subject does not exhibit a detectable humoral or cell-mediated immune response to the CAR expressed by the first dose of cells. The method according to claim 3.

10. In the above (iii), the CRS level is reduced by at least 50%, preferably by at least 20%, more preferably by at least 5% compared to the peak CRS level after administration of the first dose of immune effector cells, or the CRS level corresponds to the CRS level before administration of the first dose of immune effector cells.

10. The method of claim 9.

11. The method further comprises a step of pre-treating the immune effector cells prior to administration, the pre-treatment comprising administering a chemotherapeutic agent to the subject, or administering total body radiation to the subject, or administering localized radiation therapy to the subject, or a combination thereof. The method according to any one of claims 1 to 10.

12. The pretreatment is carried out 4 to 12 days before administration of immune effector cells. The method of claim 11.

13. The chemotherapeutic agent is selected from any one of cyclophosphamide, fludarabine, taxane compounds, and pyrimidine anti-tumor drugs, or a combination thereof. The method of claim 11.

14. The chemotherapeutic agent is characterized in that it is cyclophosphamide and fludarabine, or cyclophosphamide, fludarabine and albumin-bound paclitaxel. The method of claim 13.

15. The dosage of fludarabine is about 10-50 mg / m ２ / day, or about 15-40 mg / m ２ / day, or about 15-30 mg / m ２ / day, or about 20-30 mg / m ２ / days, and / or The dosage of cyclophosphamide is about 300-700 mg / m ２ / day, or about 400 650 mg / m ２ / day, or about 450-600 mg / m ２ / day, or about 450-550 mg / m ２ / day, or about 490-550 mg / m ２ / days, and / or The albumin-bound paclitaxel dose is approximately 300 mg / m ２ / day, or no more than about 200 mg / m ２ / day or no more than about 150 mg / m ２ / day, or no more than about 100 mg / m ２ / day, or about 80 mg / m ２ / day, or about 70 mg / m ２ Characterized by not more than / day 15. The method according to claim 13 or 14.

16. The continuous use period of the chemotherapeutic agent is not more than 4 days. The method of claim 15.

17. The duration of continuous use of said cyclophosphamide and / or said fludarabine does not exceed 4 days, and said albumin-bound paclitaxel is administered once.

17. The method of claim 16.

18. The tumor is a CLD18A2-positive gastrointestinal tumor, preferably, the gastrointestinal tumor is an adenocarcinoma, and more preferably, the gastrointestinal tumor is pancreatic cancer or gastric adenocarcinoma. The method of claim 1.

19. The chimeric antigen receptor comprises an antibody or fragment thereof that specifically binds to CLD18A2, a transmembrane domain and an intracellular domain, the antibody comprising: HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:2, HCDR3 as set forth in SEQ ID NO:3, LCDR1 as set forth in SEQ ID NO:4, LCDR2 as set forth in SEQ ID NO:5, LCDR3 as set forth in SEQ ID NO:6, or HCDR1 as set forth in SEQ ID NO:1, HCDR2 as set forth in SEQ ID NO:7, HCDR3 as set forth in SEQ ID NO:3, LCDR1 as set forth in SEQ ID NO:4, LCDR2 as set forth in SEQ ID NO:5, LCDR3 as set forth in SEQ ID NO:6, or The present invention is characterized in that it has an HCDR1 shown in SEQ ID NO: 8, an HCDR2 shown in SEQ ID NO: 9 or 68, an HCDR3 shown in SEQ ID NO: 10, an LCDR1 shown in SEQ ID NO: 11, an LCDR2 shown in SEQ ID NO: 12, and an LCDR3 shown in SEQ ID NO:

13. The method of claim 2.

20. The antibody or fragment thereof a heavy chain variable region set forth in SEQ ID NO:14 and a light chain variable region set forth in SEQ ID NO:16; or a heavy chain variable region set forth in SEQ ID NO:18 and a light chain variable region set forth in SEQ ID NO:16; or a heavy chain variable region set forth in SEQ ID NO:22 and a light chain variable region set forth in SEQ ID NO:20; or having a heavy chain variable region as set forth in SEQ ID NO:53 and a light chain variable region as set forth in SEQ ID NO:52; Preferably, the antibody or fragment thereof is characterized in that it has a sequence as shown in SEQ ID NO: 54, 56, 57, or 58.

20. The method of claim 19.

21. The chimeric antigen receptor has an amino acid sequence shown in any one of SEQ ID NOs: 24, 25, 26, 27, 28, 29, 30, 31, and 32, and preferably has an amino acid sequence shown in SEQ ID NO: It is characterized in that it has a sequence shown in ID NO: 24, 25 or 26.

21. The method of claim 20.

22. The immune effector cells are T lymphocytes, NK cells or NKT lymphocytes, preferably T lymphocytes, and preferably the T cells are derived from the subject himself.

22. The method according to any one of claims 1 to 21.

23. The subsequent doses of immune effector cells are administered two or more times, and the time interval between each administration of the subsequent doses of immune effector cells is about 21 to about 80 days, about 25 to about 60 days, or about 25 to about 50 days, each of which is inclusive. The method according to claim 3.

24. The number of immune cells in each subsequent dose is essentially the same, or the number of immune effector cells in each subsequent dose is greater than the number of immune cells in the previous subsequent dose.

24. The method of claim 23.

25. The number of immune effector cells in the subsequent dose administered later is less than the number of immune cells in the previous dose administered later.

24. The method of claim 23.

26. Prior to administering the immune effector cells, the subject has not been treated with immune cells expressing a chimeric antigen receptor that targets CLD18. The method of claim 1.

27. the subsequent dose is a number of cells sufficient to stabilize or reduce the tumor burden in the subject. The method according to claim 3.

28. Prior to receiving the first dose, the subject has already undergone a surgical treatment, chemotherapy, or immunotherapy other than that described in claim 1. The method of claim 1.

29. assessing the subject's serum levels of a factor indicative of CRS, a factor indicative of neurotoxicity, a factor indicative of tumor burden, and / or a factor indicative of a host anti-CAR immune response prior to administration of a first dose, or after administration of a first dose and prior to administration of a subsequent dose. The method according to claim 1 or 3.

30. the factor indicative of tumor burden is the total number of tumor cells in the subject, or the total number of tumor cells in an organ of the subject, or the total number of tumor cells in a tissue of the subject, or tumor mass or volume, or the extent of tumor metastasis, or the number of tumors.

30. The method of claim 29.

31. i) assessing factors indicative of tumor burden prior to administration of a subsequent dose; ii) determining a subsequent dosage of cells to be administered to the subject based on the results of said evaluation; and iii) if the subject's tumor mass or volume is determined to be stable or reduced via evaluation, the subject is administered a lower or higher number of CAR-expressing cells than at the initial dose. or a subsequent dose comprising about the same number of CAR-expressing cells. The method of claim 30.

32. The number of cells administered in the initial dose or in the subsequent doses is about 1.1×10 ６ cell / kg subject body weight or 2.8 x 10 ７ cells / kg, approximately 2.9×10 ６ Cells / kg to 2.6 x 10 ７ cells / kg or approximately 3.3 x 10 ６ Cells / kg to 1.3 x 10 ７ cells / k g or about 1.3 x 10 ７ Cells / kg to 1.8 x 10 ７ cells / kg, each of which is inclusive of the extreme values. The method according to claim 1 or 3.

33. The number of cells administered in the subsequent dose of CAR-expressing cells is about 1.1 x 10 ７ cells / kg ( cells / kg body weight to approximately 5.1 x 10 ７ cells / kg, approximately 1.3×10 ７ Cells / kg to about 3.7 x 10 ７ cells / kg, approximately 1.6×10 ７ Cells / kg to about 2.2 x 10 ７ cell / kg, about 1.9 x 10 ７ Cells / kg to about 2.2 x 10 ７ cells / kg, approximately 2.2×10 ７ cells / kg to about 2.7 x 10 ７ cells / kg, each of which includes the extreme values. Let's say The method according to claim 3.

34. After administration of the initial or subsequent doses of immune effector cells, the subject does not exhibit cytokine release syndrome (CRS), does not exhibit severe CRS, does not exhibit neurotoxicity, does not exhibit severe neurotoxicity, or does not exhibit neurotoxicity greater than grade 3. The method according to claim 1 or 3.

35. After administration of an initial dose or subsequent doses of immune effector cells, the immune effector cells expand in the subject. The method according to claim 1 or 3.

36. 1. A method for treating a tumor, comprising: A method for treating said tumor, comprising the step of subjecting said subject to chemotherapy, radiation therapy or immunotherapy prior to treatment with the method of any one of claims 1 to 35.

37. 1. A composition comprising: The composition comprising cyclophosphamide and fludarabine or a combination thereof with a taxane compound and / or a pyrimidine antitumor drug.

38. The composition is characterized in that it contains cyclophosphamide, fludarabine and paclitaxel.

38. The composition of claim 37.

39. The composition is characterized in that it comprises cyclophosphamide, fludarabine and albumin-bound paclitaxel.

39. The composition of claim 38.

40. The dosage of fludarabine is about 10-50 mg / m ２ / day, or about 15-40 mg / m ２ / day, or about 15-30 mg / m ２ / day, or about 20-30 mg / m ２ / days, and / or The dosage of cyclophosphamide is about 300-700 mg / m ２ / day, or about 400-650 mg / m ２ / day, or about 450-600 mg / m ２ / day, or about 450-550 mg / m ２ / day, or about 490-550 mg / m ２ / days, and / or The albumin-bound paclitaxel dose is approximately 300 mg / m ２ / day or about 200 mg / m ２ / day or about 150 mg / m ２ / day or not more than about 100 mg / m ２ / day or about 80 mg / m ２ / day or about 70 mg / m ２ Characterized by not exceeding / days 40. The composition of claim 39.

41. The composition is administered about 30 days, preferably about 20 days, more preferably about 12 days, and most preferably about 7 days, prior to administering immune effector cell therapy. The composition according to any one of claims 37 to 40.

42. The continuous administration time of the cyclophosphamide, fludarabine and / or albumin-bound paclitaxel does not exceed 4 days, and preferably, the albumin-bound paclitaxel is administered once. The composition according to any one of claims 37 to 41.

43. It is characterized in that it is used for antitumor therapy, preferably for targeted therapy of antitumor, and most preferably for chimeric antigen receptor targeted therapy of antitumor. The composition according to any one of claims 37 to 42.

44. The immune effector cells are CAR T cells. The composition according to any one of claims 41 to 43.

45. The CAR T cells are characterized by specifically identifying CLD18A2.

45. The composition of claim 44.

46. The tumor is a CLD18A2-positive gastrointestinal tumor, more preferably, the gastrointestinal tumor is an adenocarcinoma, and most preferably, the gastrointestinal tumor is pancreatic cancer or gastric adenocarcinoma. The composition according to any one of claims 43 to 45.

47. The use of immune effector cells comprising CLD18A2-CAR in the preparation of a medicament, The application, wherein the medicament comprises the cells and cyclophosphamide, fludarabine and a taxane compound (preferably paclitaxel, more preferably albumin-bound paclitaxel) for treating CLD18-positive tumors, and the cells and cyclophosphamide, fludarabine and taxane compound are formulated to provide a therapeutic effect greater than the sum of the effects of each of the agents.

48. administering an initial dose of the immune effector cells to a subject in need thereof, the initial dose being about 3×10 ７ cells / kg subject body weight, or the total number of said cells is about 2×10 ９ Characterized by not exceeding the cell size 48. The application of claim 47.

49. After administering the initial dose of immune effector cells, at least one subsequent dose of the immune effector cells is administered, the subsequent dose comprising at least about 3×10 ７ cells / kg subject body weight, or the total number of said cells is about 5×10 ９ cells, preferably the total number of cells is about 2×10 ９ Characterized by not exceeding the cell size 49. The application according to claim 47 or 48.

50. The subsequent dose of immune effector cells is administered about 21 to 80 days after the initial dose is administered, preferably about 25 to 60 days after the initial dose is administered, and more preferably about 25 to 50 days after the initial dose is administered.

50. The application according to any one of claims 47 to 49.

51. The cyclophosphamide, fludarabine and taxane compound are administered 4 to 12 days prior to administration of immune effector cells. An application according to any one of claims 47 to 50.

52. The dosage of fludarabine is about 10-50 mg / m ２ / day, or about 15-40 mg / m ２ / day, or about 15-30 mg / m ２ / day, or about 20-30 mg / m ２ / days, and / or The dosage of cyclophosphamide is about 300-700 mg / m ２ / day, or about 400-650 mg / m ２ / day, or about 450-600 mg / m ２ / day, or about 450-550 mg / m ２ / day, or about 490-550 mg / m ２ / days, and / or The taxane compound dosage is about 300 mg / m ２ / day or about 200 mg / m ２ / day not to exceed about 150 mg / m ２ / day or not more than about 100 mg / m ２ / day or about 80 mg / m ２ / day, or about 70 mg / m ２ Characterized by not exceeding / days An application according to any one of claims 47 to 51.

53. The duration of continuous use of the cyclophosphamide and / or the fludarabine does not exceed 4 days, and the taxane compound is administered once. An application according to any one of claims 47 to 52.

54. The chimeric antigen receptor has an amino acid sequence shown in any one of SEQ ID NOs: 24, 25, 26, 27, 28, 29, 30, 31, and 32, and preferably has an amino acid sequence shown in SEQ ID NO: It is characterized in that it has a sequence shown in ID NO: 24, 25 or 26. An application according to any one of claims 47 to 53.

55. A kit for treating a CLD18-positive tumor, comprising: The kit comprises: 1) an immune effector cell comprising a CLD18A2-CAR; and 2) cyclophosphamide, 3) fludarabine, 4) a taxane compound, preferably paclitaxel, more preferably albumin-bound paclitaxel; 5) A container for containing the substance described in 1)-4) above; and 6) instructions for administering the kit to treat a CLD18-positive tumor; The kit is characterized in that the cells, cyclophosphamide, fludarabine and taxane compound are formulated to provide a therapeutic effect greater than the sum of the effects of each of the agents.

56. the instructions for administration include instructions for administering an initial dose of the immune effector cells to a subject in need thereof; The initial dose is about 3×10 ７ cells / kg subject body weight, or the total number of said cells is about 5×10 ９ cells, preferably the total number of cells is about 2×10 ９ Characterized by not exceeding the cell size 56. The kit of claim 55.

57. The instructions include administering the initial dose of immune effector cells followed by at least one subsequent dose of the immune effector cells, the subsequent dose being about 3×10 ７ cells / kg subject body weight, or the total number of said cells is about 5×10 ９ Characterized by not exceeding the cell size 57. The kit of claim 55 or 56.

58. The instructions for administration include instructions for administering the subsequent dose of immune effector cells about 21 to 80 days after administration of the initial dose, preferably about 25 to 60 days after administration of the initial dose, and more preferably about 25 to 50 days after administration of the initial dose.

58. The kit of claim 55 or 57.

59. The administration instructions state that the drug should be administered 4 to 12 days before administration of the cyclophosphamide, fludarabine and taxane compound.

58. The kit of claim 55 or 57.

60. The administration instructions may include: The dosage of fludarabine is about 10-50 mg / m ２ / day, or about 15-40 mg / m ２ / day, or about 15-30 mg / m ２ / day, or about 20-30 mg / m ２ / days, and / or The dosage of cyclophosphamide is about 300-700 mg / m ２ / day, or about 400-650 mg / m ２ / day, or about 450-600 mg / m ２ / day, or about 450-550 mg / m ２ / day, or about 490-550 mg / m ２ / days, and / or The taxane compound dosage is about 300 mg / m ２ / day or about 200 mg / m ２ / day or about 150 mg / m ２ / day or not more than about 100 mg / m ２ / day or about 80 mg / m ２ / day or about 70 mg / m ２ The content is not more than 10 days.

60. The kit of claim 55 or 59.

61. The administration instructions may include: The continuous use period of the cyclophosphamide and / or the fludarabine does not exceed 4 days, and the taxane compound is administered once.

61. The kit of claim 55 or 60.

62. The chimeric antigen receptor has an amino acid sequence shown in any one of SEQ ID NOs: 24, 25, 26, 27, 28, 29, 30, 31, and 32, and preferably has an amino acid sequence shown in SEQ ID NO: It is characterized in that it has a sequence shown in ID NO: 24, 25 or 26.

62. The kit of claim 55 or 61.

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