Methods and compositions for treating tumors

Abstract

Provided herein are compositions of immune effector cells, wherein the compositions of immune effector cells comprise a first dose of immune effector cells and a subsequent dose of immune effector cells, as well as therapeutic kits comprising such compositions of immune effector cells.

Description

[0001] This application is a divisional application of the Chinese patent application with the application date of March 8, 2019, application number 201980018325.3, and invention name “Methods and compositions for treating tumors” (the application date of the corresponding PCT application is March 8, 2019, and the application number is PCT / CN2019 / 077529). Technical Field

[0002] The present invention belongs to the field of immunotherapy; specifically, it relates to immune cell therapy that targets and recognizes tumor antigens, triggers the activation of immune effector cells and exerts anti-tumor effects. Background Art

[0003] Tumor immunotherapy has received widespread attention and application in recent years, especially the emergence of CAR-T technology, which has made a milestone in human control of tumors.

[0004] Cancer cells in solid tumors can form 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 exists, including surrounding blood vessels, immune cells, fibroblasts, other cells, soluble factors, signal transduction molecules, extracellular matrix, and mechanical factors (mechanicalcue) that can promote tumor transformation, support tumor growth and invasion, protect tumors from host immunity, cultivate treatment resistance, and provide a microenvironment for dormant metastasis to thrive. Tumors and their surrounding microenvironments are closely related and constantly interact. Tumors can affect their microenvironments by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance. See Swarts et al., "Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy" Cancer Res, Vol. 72, pp. 2473-2480, 2012. Therefore, CAR-T technology is usually difficult to be effective in the treatment of solid tumors. For example, most of the literature currently reports that CD19 CAR T cell therapy can bring good clinical treatment effects to patients in hematological tumors. However, CART cell therapy has not yet achieved satisfactory results in the treatment of solid tumors.

[0005] Therefore, there is an urgent need in this field for technical means that can have significant anti-tumor capabilities in the tumor microenvironment of solid tumors. Summary of the invention

[0006] The purpose of the present invention is to provide a technical means that can have an excellent killing effect on CLD18-positive solid tumors.

[0007] In a first aspect, the present invention provides a method for treating a CLD18-positive tumor, the method comprising administering a first dose of immune effector cells to a subject in need thereof, the immune effector cells expressing a chimeric antigen receptor (CAR) and specifically recognizing CLD18, the first dose comprising no more than about 2×10 10 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 12 Preferably, the first dose contains no more than about 2 x 10 9 No more than approximately 1x10 cells / kg subject body weight or total volume 11 Preferably, the first dose contains no more than about 2.5 x 10 8 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 10 Preferably, the first dose contains no more than about 5 x 10 7 No more than approximately 1x10 cells / kg subject body weight or total volume 10 More preferably, the first dose comprises no more than about 3 x 10 7 No more than approximately 5 x 10 cells / kg of subject body weight or total volume 9 or about 2x10 9 cell.

[0008] In a preferred embodiment, the first dose is not less than 1*10 5 cells / kg subject body weight.

[0009] In a specific embodiment, the CLD18 is CLD18A2.

[0010] In a specific embodiment, after administering the first dose of immune effector cells, at least one subsequent dose of immune effector cells expressing a chimeric antigen receptor (CAR) and specifically recognizing CLD18 is administered.

[0011] In a preferred embodiment, the subsequent dose of immune effector cells is administered while being safe for the patient.

[0012] In a preferred embodiment, the immune effector cells of the first dose are the same as the immune effector cells of the subsequent doses.

[0013] In a preferred embodiment, the immune effector cells of the first dose and the immune effector cells of the subsequent dose both recognize CLD18A2, but contain different chimeric antigen receptors, such as the extracellular segments of the chimeric antigen receptors are different, but both recognize CLD18A2, or the extracellular segments of the chimeric antigen receptors are the same, but the transmembrane domains or intracellular domains are different.

[0014] In a specific embodiment, the subsequent dose of immune effector cells is administered after the first dose of immune effector cells is undetectable in vivo.

[0015] In a preferred embodiment, as measured by qPCR.

[0016] In a specific embodiment, the subsequent dose of immune effector cells is administered at a time point of about 21 to 80 days after the first dose; preferably, the subsequent dose of immune effector cells is administered at a time point of about 25 to 60 days after the first dose; more preferably, the subsequent dose of immune effector cells is administered at a time point of about -25 to 50 days after the first dose.

[0017] In a preferred embodiment, the subsequent dose is lower than, equal to, or higher than the initial dose.

[0018] In a preferred embodiment, the subsequent dose is comparable to the initial dose.

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

[0020] In a preferred embodiment, the subsequent dose is higher than the initial dose.

[0021] In a specific embodiment, the subsequent dose is higher than the initial dose; preferably, the subsequent dose is at least 2 times, 5 times, 7 times or 10 times the initial dose.

[0022] In a specific embodiment, the subsequent dose comprises no more than about 2 x 10 10 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 12 cell.

[0023] In a preferred embodiment, the subsequent dose comprises no more than about 2 x 10 9 No more than approximately 1x10 cells / kg subject body weight or total volume 11 cell.

[0024] In a preferred embodiment, the subsequent dose comprises no more than about 2.5 x 10 8 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 10 cell.

[0025] In a preferred embodiment, the subsequent dose comprises no more than about 5 x 10 7 No more than approximately 1x10 cells / kg subject body weight or total volume 10 cell.

[0026] In a preferred embodiment, the subsequent dose comprises no more than about 3 x 10 7 Cells / kg subject body weight or total amount does not exceed 5x10 9 or about 2x10 9 cell.

[0027] In a preferred embodiment, the subsequent dose is not less than 1×10 5 cells / kg subject body weight.

[0028] In a specific embodiment, the first dose or subsequent doses are administered in N times within 20 days, where N is a natural number not less than 1.

[0029] In a preferred embodiment, the first dose or subsequent doses are administered in N times within 3-15 days, where N is a natural number not less than 1.

[0030] In a preferred embodiment, N is 1, 2, 3 or 4.

[0031] In a preferred embodiment, N is 2 or 3.

[0032] In a specific embodiment, when the subsequent dose is administered, the subject has any of the following characteristics:

[0033] (i) a serum level in the subject of a factor indicative of cytokine release syndrome (CRS) that is about 10-fold less, about 25-fold less, and / or about 50-fold less than the level in the subject immediately prior to administration of the first dose;

[0034] (ii) did not show grade 3 or higher neurotoxicity;

[0035] (iii) the level of neurotoxicity or CRS is reduced compared to the peak level of neurotoxicity or CRS after the first dose of immune effector cells; or

[0036] (iv) the subject shows no detectable humoral or cell-mediated immune response to the CAR expressed by the cells of the first dose.

[0037] In a specific embodiment, when the subsequent doses are administered as described above, when the subject has characteristic (iii), the CRS level is reduced by at least 50% compared to the peak level of CRS after the first dose of immune effector cells, preferably, by at least 20%, more preferably, by at least 5%, or the CRS level is equivalent to the CRS level before the first dose of immune effector cells.

[0038] In a preferred embodiment, the CRS level can be assessed by the following information: fever, hypotension, hypoxia, neurological disorder, or serum levels of inflammatory cytokines or C-reactive protein (CRP).

[0039] In a preferred embodiment, the symptoms associated with clinical risk of neurotoxicity and / or grade 3 or higher neurotoxicity are selected from confusion, delirium, expressive aphasia, unresponsiveness, myoclonus, lethargy, altered mental status, convulsions, epileptiform activity, seizures (optionally confirmed by electroencephalogram (EEG)), increased levels of amyloid beta (Aβ), increased levels of glutamate, and increased levels of oxygen free radicals.

[0040] In a specific embodiment, the method further comprises pretreatment before administering the immune effector cells, wherein the pretreatment comprises administering a chemotherapeutic agent to the subject, or subjecting the subject to whole-body radiation, or subjecting the subject to local radiation therapy, or a combination thereof.

[0041] In a preferred embodiment, the pretreatment is performed before the first dose or between the first dose and subsequent doses; more preferably, the pretreatment is performed before administering the first dose and subsequent doses.

[0042] In a preferred embodiment, the pretreatment is performed before administering the first dose of immune effector cells, and the pretreatment is not required before administering subsequent doses of immune effector cells.

[0043] In a preferred embodiment, the pretreatment is required before administering the first dose of immune effector cells and before administering subsequent doses of immune effector cells.

[0044] In a preferred embodiment, the pretreatment is for lymphodepletion.

[0045] In a preferred embodiment, the pretreatment is to stabilize the tumor burden or reduce the tumor burden, in particular to maintain the tumor burden stable or reduce the tumor burden before administering immune effector cell therapy.

[0046] In a preferred embodiment, the subject is administered a lymphodepleting agent for lymphocyte clearance, such as cyclophosphamide, fludarabine, and the like.

[0047] In a specific embodiment, the pretreatment is performed 4-12 days prior to administering the immune effector cells.

[0048] In a specific embodiment, the chemotherapeutic agent is selected from any one of the following chemotherapeutic drugs or a combination thereof: cyclophosphamide, fludarabine, taxane compounds, and pyrimidine anti-tumor drugs.

[0049] In a preferred embodiment, the taxane compounds include but are not limited to paclitaxel, albumin-bound paclitaxel or docetaxel, preferably albumin-bound paclitaxel; the pyrimidine anti-tumor drugs include but are not limited to 5-fluorouracil, gimeracil, oteracil potassium, fufuroxidine, carmofur, doxifluridine, and capecitabine.

[0050] In a specific embodiment, the chemotherapeutic agent is a combination of cyclophosphamide and fludarabine; or a combination of cyclophosphamide, fludarabine, and nab-paclitaxel.

[0051] In a preferred embodiment, fludarabine is administered in an amount of 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 / sky.

[0052] In a preferred embodiment, cyclophosphamide is administered in an amount of 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 490-550mg / m 2 / sky.

[0053] In a preferred embodiment, nab-paclitaxel is administered in an amount of no more than about 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 no more than about 80 mg / m 2 / day, or no more than about 70 mg / m 2 / sky.

[0054] In a specific embodiment, the chemotherapeutic agent is administered for no more than 4 consecutive days.

[0055] In a specific embodiment, said cyclophosphamide and / or said fludarabine are administered continuously for no more than 4 days, and said nab-paclitaxel is administered once.

[0056] In a preferred embodiment, pretreatment is performed, especially before the first dose and subsequent doses, and the dose of the immune effector cells subsequently administered can be reduced accordingly.

[0057] In a specific embodiment, the tumor is a CLD18A2-positive tumor; preferably, the tumor is a CLD18A2-positive digestive tract tumor; more preferably, the digestive tract tumor is adenocarcinoma; most preferably, the digestive tract tumor is pancreatic cancer or gastric adenocarcinoma.

[0058] In a specific embodiment, the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular domain, and the extracellular domain is an antibody or a fragment thereof that specifically binds to CLD18A2.

[0059] In a preferred embodiment, the transmembrane domain of the chimeric antigen receptor is the transmembrane region of CD28 or CD8.

[0060] In a preferred embodiment, the intracellular domain of the chimeric antigen receptor is the costimulatory signaling domain of CD28 and a CD3ζ fusion peptide, or the costimulatory signaling domain of CD137 and a CD3ζ fusion peptide, or the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137 and a CD3ζ fusion peptide.

[0061] In a specific embodiment, the chimeric antigen receptor comprises an antibody or a fragment thereof that specifically binds to CLD18A2, a transmembrane domain, and an intracellular domain, wherein the antibody has:

[0062] 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; or

[0063] HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 7, 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; or

[0064] HCDR1 shown in SEQ ID NO: 8, HCDR2 shown in SEQ ID NO: 9 or 68, HCDR3 shown in SEQ ID NO: 10, LCDR1 shown in SEQ ID NO: 11, LCDR2 shown in SEQ ID NO: 12, LCDR3 shown in SEQ ID NO: 13.

[0065] In a specific embodiment, the antibody or fragment thereof has:

[0066] The heavy chain variable region set forth in SEQ ID NO: 14 and the light chain variable region set forth in SEQ ID NO: 16; or

[0067] The heavy chain variable region set forth in SEQ ID NO: 18 and the light chain variable region set forth in SEQ ID NO: 16; or

[0068] The heavy chain variable region set forth in SEQ ID NO: 22 and the light chain variable region set forth in SEQ ID NO: 20; or

[0069] The heavy chain variable region is shown in SEQ ID NO: 53 and the light chain variable region is shown in SEQ ID NO: 52. Preferably, the antibody or fragment thereof has the sequence shown in SEQ ID NO: 54, 56, 57, or 58.

[0070] In a preferred embodiment, the antibody or fragment thereof has a heavy chain variable region shown in SEQ ID NO:14 and a light chain variable region shown in SEQ ID NO:16.

[0071] In a specific embodiment, 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; preferably, it is a sequence shown in SEQ ID NO: 24, 25 or 26.

[0072] In a preferred embodiment, the chimeric antigen receptor has an amino acid sequence shown in any one of SEQ ID NOs: 24, 25, and 26.

[0073] In a preferred example, the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO:24.

[0074] In a specific embodiment, the immune effector cell is a T lymphocyte, a NK cell, or a NKT lymphocyte.

[0075] In a preferred embodiment, the preferred immune effector cells are T lymphocytes.

[0076] In a preferred embodiment, the T lymphocytes are derived from the subject's own body.

[0077] In a preferred embodiment, the T lymphocytes are of allogeneic origin.

[0078] In a specific embodiment, two or more subsequent doses of immune effector cells are administered, and the interval between each subsequent dose of immune effector cells is about 21 to about 80 days, or about 25 to about 60 days, or about 2550 days, each including the end values.

[0079] In a preferred embodiment, the subsequent dose of immune effector cells administered later stabilizes or reduces the tumor burden of the subject compared to the subsequent dose of immune effector cells administered earlier.

[0080] In a specific embodiment, each subsequent dose administered contains substantially the same number of immune cells.

[0081] In a specific embodiment, the number of immune effector cells in the subsequent dose administered later is greater than the number of immune cells in the subsequent dose administered earlier.

[0082] In a specific embodiment, the number of immune effector cells in a subsequent dose administered later is lower than the number of immune cells in a subsequent dose administered earlier.

[0083] In a specific embodiment, prior to administering the immune effector cells, the subject has not received treatment with immune cells expressing a chimeric antigen receptor targeting CLD18.

[0084] In a specific embodiment, said subsequent dose is a number of cells sufficient to stabilize or reduce said subject's tumor burden.

[0085] In a specific embodiment, prior to administration of the first dose, the subject has undergone surgery, chemotherapy, or immunotherapy different from that described in the first aspect.

[0086] In a specific embodiment, the subject's 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 are assessed prior to administration of the first dose, or after administration of the first dose and prior to administration of subsequent doses.

[0087] In a specific embodiment, the factor indicative of tumor load is: the total number of tumor cells in the subject, or the total number of tumor cells in the subject's organ, or the total number of tumor cells in the subject's tissue, or the mass or volume of the tumor, or the degree of tumor metastasis, or the number of tumors.

[0088] In a specific embodiment, the method for treating a tumor comprises:

[0089] i) evaluating factors indicative of tumor burden prior to administration of subsequent doses; and

[0090] ii) determining successive doses of cells to be administered to the subject based on the results of said evaluation, and

[0091] iii) if the evaluation determines that the subject's tumor mass or volume is stable or decreasing, administering to the subject a subsequent dose comprising less than, more than, or about the same number of CAR-expressing cells as in the first dose.

[0092] In a specific embodiment, the number of cells administered in the first dose or the subsequent dose is about 1.1 x 10 6 Cells / kg subject body weight up to 2.8x10 7 Cells / kg, about 2.9x10 6 Cells / kg to 2.6x10 7 cells / kg or approximately 3.3x10 6 Cells / kg to 1.3x10 7 cells / kg or about 1.3x10 7 Cells / kg to 1.8x10 7 cells / kg, each inclusive.

[0093] In a specific embodiment, the number of cells administered in subsequent doses of CAR-expressing cells comprises about 1.1 x 10 7 Cells / kg (cells / kg) body weight is about 5.1x10 7 Cells / kg, about 1.3x10 7 Cells / kg: about 3.7x10 7 Cells / kg, about 1.6x10 7 Cells / kg: about 2.2x10 7 Cells / kg, about 1.9x10 7 Cells / kg: about 2.2x10 7 Cells / kg, about 2.2x10 7 Cells / kg: about 2.7x10 7 cells / kg, each inclusive.

[0094] In a specific embodiment, following administration of the first 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.

[0095] In a specific embodiment, following administration of a first or subsequent dose of immune effector cells, said immune effector cells proliferate in said subject.

[0096] In a preferred embodiment, the proliferation is shown by: (i) an increase in serum CRP levels after administration of the first dose and / or subsequent doses compared to immediately before the administration, and / or (ii) an increase in the level of CAR encoding nucleic acid in the serum after administration of the first dose and / or subsequent doses compared to immediately before the administration, as measured by qPCR.

[0097] In a specific embodiment, after administration of the first dose of immune effector cells, the subject's tumor burden is stabilized or reduced. In a specific embodiment, the stabilization or reduction of tumor burden is represented by a stabilization or reduction of one or more factors indicative of tumor burden.

[0098] In preferred embodiments, administration of subsequent doses of immune effector cells results in stabilization or further reduction of tumor burden compared to the tumor burden after administration of the first dose of immune effector cells.

[0099] In a specific embodiment, the subject has not relapsed and / or one or more factors indicative of tumor burden have not increased after a decrease when the subsequent dose is administered.

[0100] In a specific embodiment, the stabilization or reduction of the load and / or further reduction of the load includes stabilization or reduction of the total number of disease cells in the subject, in the subject's organ, in the subject's tissue, or in the subject's body fluid, and / or stabilization or reduction of the mass or volume of the tumor, and / or stabilization or reduction of the number and / or extent of metastases, and / or stabilization or reduction of tumor markers, and / or disappearance or reduction of complications common to mid- and late-stage cancer.

[0101] In a specific embodiment, the tumor markers include: alpha-fetoprotein (AFP), CA125, CA15-3, squamous cell carcinoma antigen (SCC), soluble fragment of cytokeratin 19 (CYFRA21-1), carcinoembryonic antigen (CEA), CA199, CA724, etc.

[0102] In a specific implementation scheme, common complications of advanced cancer include: cancer pain, infection, and cancerous pleural effusion and ascites.

[0103] In specific embodiments, the mass or volume of the tumor is measured by PET (positron emission tomography) and CT (computed tomography).

[0104] In the second aspect of the present invention, another method for treating tumors is provided, comprising: before administering any of the above methods, the subject has received chemotherapy, radiotherapy, immunotherapy, or a combination thereof.

[0105] In a preferred embodiment, the immunotherapy includes administering a checkpoint inhibitor or administering immune effector cell therapy that does not target CLD18.

[0106] In a preferred embodiment, the immunotherapy is an immune effector cell therapy that does not include targeting CLD18.

[0107] In a preferred embodiment, the subject received immune effector cell therapy that did not target CLD18.2.

[0108] In a preferred embodiment, the tumor burden does not remain stable or decrease after receiving chemotherapy, radiotherapy, other immunotherapy, or a combination thereof.

[0109] In a specific embodiment, the tumor is a relapsed, refractory tumor.

[0110] In a third aspect of the present invention, there is provided a use of a composition comprising immune effector cells expressing a chimeric antigen receptor that specifically recognizes CLD18 in the preparation of a medicament for treating a CLD18A2-positive tumor in a subject previously treated with CLD18A2-CAR T cells, wherein, when used, a first dose of immune effector cells expressing a chimeric antigen receptor (CAR) and specifically recognizing CLD18 is administered to a subject in need thereof, the first dose comprising no more than about 2×10 10 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 12 Preferably, the first dose contains no more than about 2 x 10 9 No more than approximately 1x10 cells / kg subject body weight or total volume 11 Preferably, the first dose contains no more than about 2.5 x 10 8 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 10 Preferably, the first dose contains no more than about 5 x 10 7 No more than approximately 1x10 cells / kg subject body weight or total volume 10 More preferably, the first dose comprises no more than about 3 x 10 7 Cells / kg subject body weight or total amount does not exceed 5x10 9 or about 2x10 9 cell.

[0111] In a specific embodiment, the CLD18 is CLD18A2.

[0112] In a specific embodiment, after administering the first dose of immune effector cells, at least one subsequent dose of immune effector cells expressing a chimeric antigen receptor (CAR) and specifically recognizing CLD18 is administered.

[0113] In a preferred embodiment, the subsequent dose of immune effector cells is administered while being safe for the patient.

[0114] In a preferred embodiment, the immune effector cells of the first dose are the same as the immune effector cells of the subsequent doses.

[0115] In a preferred embodiment, the immune effector cells of the first dose and the immune effector cells of the subsequent dose both recognize CLD18A2, but contain different chimeric antigen receptors, such as the extracellular segments of the chimeric antigen receptors are different, but both recognize CLD18A2, or the extracellular segments of the chimeric antigen receptors are the same, but the transmembrane domains or intracellular domains are different.

[0116] In a specific embodiment, the subsequent dose of immune effector cells is administered after the first dose of immune effector cells is undetectable in vivo.

[0117] In a preferred embodiment, as measured by qPCR. In a specific embodiment, the subsequent dose of immune effector cells is administered at a time point of about 21 to 80 days after the first dose is administered; preferably, the subsequent dose of immune effector cells is administered at a time point of about 25 to 60 days after the first dose is administered; more preferably, the subsequent dose of immune effector cells is administered at a time point of about 25 to 50 days after the first dose is administered.

[0118] In a preferred embodiment, the subsequent dose is comparable to the initial dose.

[0119] In a preferred embodiment, the subsequent dose is lower than the initial dose.

[0120] In a preferred embodiment, the subsequent dose is higher than the initial dose.

[0121] In a specific embodiment, the subsequent dose is higher than the initial dose, the subsequent dose is at least 2 times, 5 times, 7 times or 10 times greater than the initial dose.

[0122] In a specific embodiment, the subsequent dose comprises no more than about 2 x 10 10 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 12 cell.

[0123] In a preferred embodiment, the subsequent dose comprises no more than about 2 x 10 9 No more than approximately 1x10 cells / kg subject body weight or total volume 11 cell.

[0124] In a preferred embodiment, the subsequent dose comprises no more than about 2.5 x 10 8 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 10 cell.

[0125] In a preferred embodiment, the subsequent dose comprises no more than about 5 x 10 7 No more than approximately 1x10 cells / kg subject body weight or total volume 10 cell.

[0126] In a preferred embodiment, the subsequent dose comprises no more than about 3 x 10 7 Cells / kg subject body weight or total amount does not exceed 5x10 9 or about 2x10 9 cell.

[0127] In a specific embodiment, the first dose or subsequent doses are administered in N times within 20 days, where N is a natural number not less than 1.

[0128] In a preferred embodiment, the first dose or subsequent doses are administered in N times within 3-15 days, where N is a natural number not less than 1.

[0129] In a preferred embodiment, N is 1, 2, 3 or 4.

[0130] In a preferred embodiment, N is 2 or 3.

[0131] In a specific embodiment, when the subsequent dose is administered, the subject has any of the following characteristics:

[0132] (i) a serum level in the subject of a factor indicative of cytokine release syndrome (CRS) that is about 10-fold less, about 25-fold less, and / or about 50-fold less than the level in the subject immediately prior to administration of the first dose;

[0133] (ii) did not show grade 3 or higher neurotoxicity;

[0134] (iii) the level of neurotoxicity or CRS is reduced compared to the peak level of neurotoxicity or CRS after the first dose of immune effector cells; or

[0135] (iv) the subject shows no detectable humoral or cell-mediated immune response to the CAR expressed by the cells of the first dose.

[0136] In a specific embodiment, when the subsequent doses are administered as described above, when the subject has characteristic (iii), the CRS level is reduced by at least 50% compared to the peak level of CRS after the first dose of immune effector cells, preferably, by at least 20%, more preferably, by at least 5%, or the CRS level is equivalent to the CRS level before the first dose of immune effector cells.

[0137] In a preferred embodiment, the CRS level can be assessed by the following information: fever, hypotension, hypoxia, neurological disorder, or serum levels of inflammatory cytokines or C-reactive protein (CRP).

[0138] In a preferred embodiment, the symptoms associated with clinical risk of neurotoxicity and / or grade 3 or higher neurotoxicity are selected from confusion, delirium, expressive aphasia, unresponsiveness, myoclonus, lethargy, altered mental status, convulsions, epileptiform activity, seizures (optionally confirmed by electroencephalogram (EEG)), increased levels of amyloid beta (Aβ), increased levels of glutamate, and increased levels of oxygen free radicals.

[0139] In a specific embodiment, the method further comprises pretreatment before administering the immune effector cells, wherein the pretreatment comprises administering a chemotherapeutic agent to the subject, or subjecting the subject to whole-body radiation, or subjecting the subject to local radiation therapy, or a combination thereof.

[0140] In a preferred embodiment, the pretreatment is performed before administering the first dose of immune effector cells, and the pretreatment is not required before administering subsequent doses of immune effector cells.

[0141] In a preferred embodiment, the pretreatment is required before administering the first dose of immune effector cells and before administering subsequent doses of immune effector cells.

[0142] In a preferred embodiment, the pretreatment is for lymphodepletion.

[0143] In a preferred embodiment, the pretreatment is to stabilize the tumor burden or reduce the tumor burden, in particular to maintain the tumor burden stable or reduce the tumor burden before administering immune effector cell therapy.

[0144] In a preferred embodiment, the subject is administered a lymphodepleting agent for lymphocyte clearance, such as cyclophosphamide, fludarabine, and the like.

[0145] In a specific embodiment, the pretreatment is performed 4-12 days prior to administering the immune effector cells.

[0146] In a specific embodiment, the chemotherapeutic agent is selected from any one of the following chemotherapeutic drugs or a combination thereof: cyclophosphamide, fludarabine, taxane compounds, pyrimidine anti-tumor drugs. In a specific embodiment, the chemotherapeutic agent is a combination of cyclophosphamide and fludarabine; or a combination of cyclophosphamide, fludarabine and albumin-bound paclitaxel.

[0147] In a preferred embodiment, fludarabine is administered in an amount of 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 / sky.

[0148] In a preferred embodiment, cyclophosphamide is administered in an amount of 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 490-550mg / m 2 / sky.

[0149] In a preferred embodiment, nab-paclitaxel is administered in an amount of no more than about 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 no more than about 80 mg / m 2 / day, or no more than about 70 mg / m 2 / sky.

[0150] In a specific embodiment, the chemotherapeutic agent is administered for no more than 4 consecutive days.

[0151] In a specific embodiment, said cyclophosphamide and said fludarabine are administered continuously for no more than 4 days, and said nab-paclitaxel is administered once.

[0152] In a specific embodiment, the tumor is a CLD18A2-positive tumor; preferably, the tumor is a CLD18A2-positive digestive tract tumor; more preferably, the digestive tract tumor is adenocarcinoma; most preferably, the digestive tract tumor is pancreatic cancer or gastric adenocarcinoma.

[0153] In a specific embodiment, the chimeric antigen receptor comprises an extracellular domain, a transmembrane domain and an intracellular domain, and the extracellular domain is an antibody or a fragment thereof that specifically binds to CLD18A2.

[0154] In a preferred embodiment, the transmembrane domain of the chimeric antigen receptor is the transmembrane region of CD28 or CD8.

[0155] In a preferred embodiment, the intracellular domain of the chimeric antigen receptor is the costimulatory signaling domain of CD28 and a CD3ζ fusion peptide, or the costimulatory signaling domain of CD137 and a CD3ζ fusion peptide, or the costimulatory signaling domain of CD28, the costimulatory signaling domain of CD137 and a CD3ζ fusion peptide.

[0156] In a specific embodiment, the chimeric antigen receptor comprises an antibody or a fragment thereof that specifically binds to CLD18A2, a transmembrane domain, and an intracellular domain, wherein the antibody has:

[0157] 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; or

[0158] HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 7, 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; or

[0159] HCDR1 shown in SEQ ID NO: 8, HCDR2 shown in SEQ ID NO: 9 or 68, HCDR3 shown in SEQ ID NO: 10, LCDR1 shown in SEQ ID NO: 11, LCDR2 shown in SEQ ID NO: 12, LCDR3 shown in SEQ ID NO: 13.

[0160] In a specific embodiment, the antibody or fragment thereof has:

[0161] The heavy chain variable region set forth in SEQ ID NO: 14 and the light chain variable region set forth in SEQ ID NO: 16; or

[0162] The heavy chain variable region set forth in SEQ ID NO: 18 and the light chain variable region set forth in SEQ ID NO: 16; or

[0163] The heavy chain variable region is shown in SEQ ID NO:22 and the light chain variable region is shown in SEQ ID NO:20.

[0164] In a preferred embodiment, the antibody or fragment thereof has a heavy chain variable region shown in SEQ ID NO:14 and a light chain variable region shown in SEQ ID NO:16.

[0165] In a specific embodiment, 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.

[0166] In a preferred embodiment, the chimeric antigen receptor has an amino acid sequence shown in any one of SEQ ID NOs: 24, 25, and 26.

[0167] In a preferred example, the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO:24.

[0168] In a specific embodiment, the immune effector cell is a T lymphocyte, a NK cell, or a NKT lymphocyte.

[0169] In a preferred embodiment, the preferred immune effector cells are T lymphocytes.

[0170] In a preferred embodiment, the T lymphocytes are derived from the subject's own body.

[0171] In a preferred embodiment, the T lymphocytes are of allogeneic origin.

[0172] In a specific embodiment, two or more subsequent doses of immune effector cells are administered, and the interval between each subsequent dose of immune effector cells is about 21 to about 80 days, or about 25 to about 60 days, or about 25-50 days, each including the end values.

[0173] In a preferred embodiment, the subsequent dose of immune effector cells administered later stabilizes or reduces the tumor burden of the subject compared to the subsequent dose of immune effector cells administered earlier.

[0174] In a specific embodiment, each subsequent dose administered contains substantially the same number of immune cells.

[0175] In a specific embodiment, the number of immune effector cells in the subsequent dose administered later is greater than the number of immune cells in the subsequent dose administered earlier.

[0176] In a specific embodiment, it is characterized in that the number of immune effector cells in the subsequent dose administered later is lower than the number of immune cells in the subsequent dose administered earlier.

[0177] In a specific embodiment, prior to administering the immune effector cells, the subject has not received treatment with immune cells expressing a chimeric antigen receptor targeting CLD18.

[0178] In a specific embodiment, said subsequent dose is a number of cells sufficient to stabilize or reduce said subject's tumor burden.

[0179] In a specific embodiment, prior to administration of the first dose, the subject has undergone surgery, chemotherapy, or an immunotherapy other than that described in claim 1.

[0180] In a specific embodiment, the subject's 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 are assessed prior to administration of the first dose, or after administration of the first dose and prior to administration of subsequent doses.

[0181] In a specific embodiment, the factor indicative of tumor load is: the total number of tumor cells in the subject, or the total number of tumor cells in the subject's organ, or the total number of tumor cells in the subject's tissue, or the mass or volume of the tumor, or the degree of tumor metastasis, or the number of tumors.

[0182] In a specific embodiment, the method for treating a tumor comprises:

[0183] i) evaluating factors indicative of tumor burden prior to administration of subsequent doses; and

[0184] ii) determining successive doses of cells to be administered to the subject based on the results of said evaluation, and

[0185] iii) if the evaluation determines that the subject's tumor mass or volume is stable or decreasing, administering to the subject a subsequent dose comprising less than, more than, or about the same number of CAR-expressing cells as in the first dose.

[0186] In a specific embodiment, the number of cells administered in the first dose or the subsequent dose is about 1.1 x 10 6 Cells / kg subject body weight up to 2.8x10 7 Cells / kg, about 2.9x10 6 Cells / kg to 2.6x10 7 cells / kg or approximately 3.3x10 6 Cells / kg to 1.3x10 7 cells / kg, each inclusive.

[0187] In a specific embodiment, the number of cells administered in subsequent doses of CAR-expressing cells comprises about 1.1 x 10 7 Cells / kg (cells / kg) body weight is about 5.1x10 7 Cells / kg, about 1.3x10 7 Cells / kg: about 3.7x10 7 Cells / kg, about 1.6x10 7 Cells / kg: about 2.2x10 7 Cells / kg, about 1.9x10 7 Cells / kg: about 2.2x10 7 cells / kg, each inclusive.

[0188] In a specific embodiment, following administration of the first 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.

[0189] In a specific embodiment, following administration of a first or subsequent dose of immune effector cells, said immune effector cells proliferate in said subject.

[0190] In a preferred embodiment, the proliferation is shown by: (i) an increase in serum CRP levels after administration of the first dose and / or subsequent doses compared to immediately before the administration, and / or (ii) an increase in the level of CAR encoding nucleic acid in the serum after administration of the first dose and / or subsequent doses compared to immediately before the administration, as measured by qPCR.

[0191] In a specific embodiment, after administration of the first dose of immune effector cells, the subject's tumor burden is stabilized or reduced. In a specific embodiment, the stabilization or reduction of tumor burden is represented by a stabilization or reduction of one or more factors indicative of tumor burden.

[0192] In preferred embodiments, administration of subsequent doses of immune effector cells results in stabilization or further reduction of tumor burden compared to the tumor burden after administration of the first dose of immune effector cells.

[0193] In a specific embodiment, the subject has not relapsed and / or one or more factors indicative of tumor burden have not increased after a decrease when the subsequent dose is administered.

[0194] In a specific embodiment, the stabilization or reduction of tumor burden and / or further reduction of burden includes stabilization or reduction of the total number of disease cells in the subject, in the subject's organ, in the subject's tissue, or in the subject's body fluid, and / or stabilization or reduction of the mass or volume of the tumor, and / or stabilization or reduction of the number and / or extent of metastasis, and / or stabilization or reduction of tumor markers, and / or disappearance or reduction of complications common to mid- and late-stage cancer.

[0195] In a specific embodiment, the tumor markers include: alpha-fetoprotein (AFP), CA125, CA15-3, squamous cell carcinoma antigen (SCC), soluble fragment of cytokeratin 19 (CYFRA21-1), carcinoembryonic antigen (CEA), CA199, CA724, etc.

[0196] In a specific implementation scheme, common complications of advanced cancer include: cancer pain, infection, and cancerous pleural effusion and ascites.

[0197] In specific embodiments, the mass or volume of the tumor is measured by PET (positron emission tomography) and CT (computed tomography).

[0198] In the fourth aspect of the present invention, a composition is provided, comprising cyclophosphamide, fludarabine and taxane compounds and / or pyrimidine anti-tumor drugs.

[0199] In a preferred embodiment, the taxane compounds include but are not limited to paclitaxel, albumin-bound paclitaxel or docetaxel, preferably albumin-bound paclitaxel; the pyrimidine anti-tumor drugs include but are not limited to 5-fluorouracil, gimeracil, oteracil potassium, fufuroxidine, carmofur, doxifluridine, and capecitabine.

[0200] In a specific embodiment, the composition comprises cyclophosphamide, fludarabine, and paclitaxel.

[0201] In a specific embodiment, the composition comprises cyclophosphamide, fludarabine, and nab-paclitaxel.

[0202] In a preferred embodiment, the composition is used to administer the composition before the subject receiving immune effector cell therapy receives immune effector cells, preferably before each time the subject receiving immune effector cell therapy receives immune effector cells.

[0203] In a specific embodiment, fludarabine is administered in an amount of 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 / day; and / or cyclophosphamide is administered in an amount of 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 490-550mg / m 2 / day; and / or nab-paclitaxel administered in an amount not exceeding about 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 no more than about 80 mg / m 2 / day, or no more than about 70 mg / m 2 / sky.

[0204] In a specific embodiment, pretreatment is administered about 30 days prior to administration of immune cell therapy; preferably, about 20 days; more preferably, about 12 days; and most preferably, about 7 days.

[0205] In a specific embodiment, the cyclophosphamide, fludarabine and / or nab-paclitaxel are used continuously for no more than 4 days; preferably, the nab-paclitaxel is administered once.

[0206] In a specific embodiment, it is used for anti-tumor therapy; preferably, it is used for anti-tumor targeted therapy; most preferably, it is used for anti-tumor chimeric antigen receptor targeted therapy.

[0207] In a specific embodiment, the immune effector cells are CAR T cells.

[0208] In a specific embodiment, the CAR T cells specifically recognize CLD18A2.

[0209] In a specific embodiment, the tumor is a CLD18A2-positive digestive tract tumor; more preferably, the digestive tract tumor is adenocarcinoma; most preferably, the digestive tract tumor is pancreatic cancer or gastric adenocarcinoma.

[0210] In the fifth aspect of the present invention, a method for combined immune effector cell therapy for tumors is provided, comprising: before administering immune effector cell therapy to a subject, first administering cyclophosphamide, fludarabine and taxane compounds for pretreatment.

[0211] In a preferred embodiment, the tumor is a solid tumor.

[0212] In a preferred embodiment, the solid tumor is breast cancer, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, lung cancer, stomach 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 lymphoma, non-Hodgkin lymphoma, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumor, primary CNS lymphoma, spinal tumor, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma.

[0213] In a preferred embodiment, the taxane compound is selected from paclitaxel, albumin-bound paclitaxel, or docetaxel.

[0214] In a preferred embodiment, the taxane compound is selected from albumin-bound paclitaxel.

[0215] In a specific embodiment, fludarabine is administered in an amount of 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 / day; and / or cyclophosphamide is administered in an amount of 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 490-550mg / m 2 / day; and / or nab-paclitaxel administered in an amount not exceeding about 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 no more than about 80 mg / m 2 / day, or no more than about 70 mg / m 2 / sky.

[0216] In a specific embodiment, pretreatment is administered about 30 days before administration of immune cell therapy; preferably, about 20 days; more preferably, about 12 days; most preferably, about 7 days.

[0217] In a specific embodiment, the cyclophosphamide, fludarabine and / or nab-paclitaxel are used continuously for no more than 4 days; preferably, the nab-paclitaxel is administered once.

[0218] In a specific embodiment, the immune effector cells are immune effector cells modified with chimeric antigen receptors, such as CAR-T cells, CAR-NK cells, CAR-NKT cells, etc.

[0219] In the sixth aspect of the present invention, there is also provided an application of an immune effector cell comprising CLD18A2-CAR in the preparation of a drug, wherein the drug contains the cell and cyclophosphamide, fludarabine and a taxane compound (preferably paclitaxel, more preferably albumin-bound paclitaxel) for treating CLD18-positive tumors, wherein the cell 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.

[0220] In a specific embodiment, a first dose of the immune effector cells is administered to a subject in need thereof, wherein the first dose does not exceed about 3 x 10 7 cells / kg of subject body weight or the total amount of said cells does not exceed about 2 x 10 9 cell.

[0221] In a specific embodiment, after administering the first dose of immune effector cells, at least one subsequent dose of the immune effector cells is administered, wherein the subsequent dose comprises no more than about 3×10 7 cells / kg of subject body weight or the total amount of said cells does not exceed about 5 x 10 9 cell.

[0222] In a specific embodiment, the subsequent dose of immune effector cells is administered at a time point of about 21 to 80 days after the first dose; preferably, the subsequent dose of immune effector cells is administered at a time point of about 25 to 60 days after the first dose; more preferably, the subsequent dose of immune effector cells is administered at a time point of about 25 to 50 days after the first dose.

[0223] In a specific embodiment, the cyclophosphamide, fludarabine and taxane compound are administered 4-12 days prior to the administration of immune effector cells.

[0224] In a specific embodiment, fludarabine is administered in an amount of 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 / day; and / or

[0225] The dose 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 490-550mg / m 2 / day; and / or

[0226] The taxane compound is administered in an amount not exceeding about 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 no more than about 80 mg / m 2 / day, or no more than about 70 mg / m 2 / sky.

[0227] In a specific embodiment, the cyclophosphamide and / or the fludarabine are used continuously for no more than 4 days, and the taxane compound is administered once.

[0228] In a specific embodiment, 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; preferably, it is a sequence shown in SEQ ID NO: 24, 25, or 26.

[0229] Corresponding to the sixth aspect, the present invention further provides a kit for treating CLD18-positive tumors, characterized in that the kit comprises:

[0230] 1) Immune effector cells containing CLD18A2-CAR;

[0231] 2) Cyclophosphamide;

[0232] 3) Fludarabine;

[0233] 4) Taxane compounds, preferably paclitaxel, more preferably albumin-bound paclitaxel;

[0234] 5) a container for containing the substances described in 1) to 4) above; and

[0235] 6) Instructions for use of the kit for treating CLD18-positive tumors;

[0236] The cells and cyclophosphamide, fludarabine and taxane compounds are formulated to provide a therapeutic effect greater than the sum of the effects of each of the agents.

[0237] In a specific embodiment, the instructions for administration state that a first dose of the immune effector cells is administered to a subject in need thereof, and the first dose does not exceed about 3×10 7 cells / kg of subject body weight or the total amount of said cells does not exceed about 2 x 10 9 cell.

[0238] In a specific embodiment, the administration instructions record that after the first dose of immune effector cells is administered, at least one subsequent dose of the immune effector cells is administered, and the subsequent dose contains no more than about 3×10 7 cells / kg of subject body weight or the total amount of said cells does not exceed about 5 x 10 9 cell.

[0239] In a specific embodiment, the administration instructions record that the subsequent dose of immune effector cells is administered at a time point of about 21 to 80 days after the administration of the first dose; preferably, the subsequent dose of immune effector cells is administered at a time point of about 25 to 60 days after the administration of the first dose; more preferably, the subsequent dose of immune effector cells is administered at a time point of about 25 to 50 days after the administration of the first dose.

[0240] In a specific embodiment, the administration instructions state that the cyclophosphamide, fludarabine and taxane compounds are administered 4-12 days before the administration of immune effector cells.

[0241] In a specific embodiment, the administration instructions state:

[0242] Fludarabine is administered in doses of approximately 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 / day; and / or

[0243] The dose 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 490-550mg / m 2 / day; and / or

[0244] The taxane compound is administered in an amount not exceeding about 300 mg / m 2 / day, or no more than about 200 mg / m 2 / day, or no more than about 150 mg / m2 / day, or no more than about 100 mg / m 2 / day, or no more than about 80 mg / m 2 / day, or no more than about 70 mg / m 2 / sky.

[0245] In a specific embodiment, the administration instructions state:

[0246] The cyclophosphamide and / or fludarabine are used continuously for no more than 4 days, and the taxane compound is administered once.

[0247] In a specific embodiment, 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; preferably, it is a sequence shown in SEQ ID NO: 24, 25 or 26.

[0248] Those skilled in the art will appreciate that the kit may contain each component in a dosage that is consistent with that described in the instructions for administration.

[0249] In one aspect of the present invention, a method for treating a tumor is provided, comprising administering immune effector cells and tubulin inhibitors to an individual with a tumor, wherein the immune effector cells express receptors that recognize tumor antigens. In some embodiments, the present invention provides a method for reducing cancer cell growth, survival, or viability or all, comprising administering immune effector cells and tubulin inhibitors to an individual with a tumor, wherein the immune effector cells express receptors that recognize tumor antigens. 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 (docetaxel) and derivatives having a taxane skeleton structure.

[0250] In another embodiment, the antibody that specifically recognizes a 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, and LCDR3 shown in SEQ ID NO: 6; or

[0251] HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 7, 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; or

[0252] HCDR1 shown in SEQ ID NO: 8, HCDR2 shown in SEQ ID NO: 9 or 68, HCDR3 shown in SEQ ID NO: 10, LCDR1 shown in SEQ ID NO: 11, LCDR2 shown in SEQ ID NO: 12, LCDR3 shown in SEQ ID NO: 13.

[0253] In another embodiment, the antibody that specifically recognizes a tumor antigen 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; or

[0254] The heavy chain variable region set forth in SEQ ID NO: 18 and the light chain variable region set forth in SEQ ID NO: 16; or

[0255] The heavy chain variable region set forth in SEQ ID NO: 22 and the light chain variable region set forth in SEQ ID NO: 20; or

[0256] The heavy chain variable region is shown in SEQ ID NO:53 and the light chain variable region is shown in SEQ ID NO:52.

[0257] In another embodiment, the antibody that specifically recognizes a tumor antigen has an amino acid sequence as shown in SEQ ID NO: 54, 56, 57, or 58, preferably, the sequence as shown in SEQ ID NO: 54.

[0258] In another embodiment, the subject is not subjected to pretreatment or lymphocyte clearance. The present invention also provides a use of an immune effector cell expressing a receptor that recognizes a tumor antigen in the preparation of a drug, the drug containing the immune effector cell and a taxane compound, and the drug is used to treat a tumor. In some embodiments, the immune effector cell and the 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, a kit for treating a tumor is provided, comprising: 1) an immune effector cell 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) instructions for administration of the kit for treating a tumor.

[0259] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0260] Figure 1 Displays CAR-T copy number detection.

[0261] Figure 2A It shows the changes of C-reactive protein (CRP) in subjects after the first dose of CAR-T injection (first course of treatment); Figure 2B The changes in CRP in the subjects after the subsequent dose of CAR-T injection (second course of treatment) are shown.

[0262] Figure 3A It shows the changes in tumor lesions during CAR-T treatment in pancreatic cancer subjects; Figure 3B Shows changes in tumor markers during CAR-T treatment in pancreatic cancer subjects.

[0263] Figure 4A It shows the changes in tumor lesions during CAR-T treatment in subjects with gastric adenocarcinoma; Figure 4B Shows changes in new lesions during CAR-T treatment in subjects with gastric adenocarcinoma; Figure 4C It shows the changes of pleural and abdominal fluid in gastric adenocarcinoma subjects during CAR-T treatment; Figure 4D The changes in CA125 levels during CAR-T treatment in subjects with gastric adenocarcinoma are shown; Figure 4E Shown are changes in CA199 levels during CAR-T therapy in subjects with gastric adenocarcinoma.

[0264] Figure 5 . is the plasmid map of the recombinant vector MSCV-8E5-2I-mBBZ.

[0265] Fig. 6A and Figure 6B CCK8 experiments were used to detect the toxic effects of Abraxane on pancreatic cancer cells and CAR-T cells.

[0266] Fig. 7A and Figure 7B The graph shows the inhibitory effect of Abraxane combined with CAR-T cells on pancreatic cancer in mice and the weight changes of mice observed in vivo.

[0267] Figures 8A to 8C Shown is the survival period in mice following treatment using the methods of the invention.

[0268] Fig. 9 This is a curve of body weight changes in a mouse model after treatment with the method of the present invention. DETAILED DESCRIPTION

[0269] After extensive and in-depth research, the inventors unexpectedly found that the administration of immune effector cells expressing chimeric antigen receptors (CAR) or other transgenic receptors such as T cell receptors (TCR) in a specific amount and according to specific time parameters significantly improved the efficacy of tumor treatment using immune effector cells, thereby improving the application effect of immune effector cells in solid tumors. The present invention is based at least in part on this discovery.

[0270] Unless otherwise defined, all professional terms, symbols and other technical and scientific terms or proprietary words used herein are intended to have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In some cases, the terms with the commonly understood meanings are further defined herein for the purpose of clarification and / or ease of reference, and such further definitions included herein should not be construed as representing a substantial difference from the commonly understood meanings in the art.

[0271] All publications, including patent documents, academic papers and databases, mentioned in the present invention are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication were independently incorporated by reference. If the definitions set forth herein are different from or otherwise inconsistent with the definitions set forth in the patents, published applications and other publications incorporated herein by reference, the definitions set forth herein shall prevail relative to the definitions in the documents incorporated herein by reference.

[0272] As used herein, the singular forms "a", "an", and "the" include plural references to subjects unless the context clearly indicates otherwise. For example, "a" or "an" means "at least one or one" or "one (one) or more (ones)".

[0273] In the present disclosure, various aspects of the subject matter for protection are presented in the form of ranges. It should be understood that the description in the form of ranges is only for convenience and brevity, and should not be interpreted as a hard limit to the scope of the subject matter for protection. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, in the case of providing a range of values, it should be understood that each intermediate value between the upper and lower limits of the range and any other described or intermediate values ​​within the range are included in the subject matter for protection, and the upper and lower limits of the range also belong to the scope of the subject matter for protection. The upper and lower limits of these smaller ranges can be independently included in the smaller range, and they also belong to the scope of the subject matter for protection, unless the upper and lower limits of the range are explicitly excluded. When the range is set to include one or two limits, the subject matter for protection also includes a range that excludes one or two of the limits. Regardless of the width of the range, this principle applies.

[0274] The term "about" as used herein refers to the usual error range of each value that is readily known to those skilled in the art. "About" values ​​or parameters are described herein, including (and describing) embodiments pointing to the value or 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 according to the actual standard deviation in the field. Or "about" may mean 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 a specific value or composition is provided in an application and a patent application, unless otherwise indicated, "about" should be assumed to be within an acceptable error range for the specific value or composition.

[0275] When describing an amino acid or nucleic acid sequence, "having" a specific sequence should be understood to encompass variants of the specific sequence. In some embodiments, an amino acid or nucleic acid sequence having a specific sequence means that the amino acid or nucleic acid sequence has more than 80%, 85%, 90%, 95% or 99% sequence identity with the specific sequence.

[0276] Any concentration range, percentage range, ratio range or integer range described herein should be understood to include any integer within the range, and, where appropriate, fractional values ​​thereof (e.g., tenths and hundredths of an integer), unless otherwise indicated.

[0277] "Dosing interval" as described herein refers to the time between multiple courses of immune effector cell therapy (such as multiple courses of immune effector cells including the administration of a first dose and subsequent doses) given to an individual and the time between the administration of pretreatment drugs. Therefore, the dosing interval can be indicated as a range. In some aspects of the invention, the present invention includes multiple courses of immune effector cell therapy given to an individual, and each course of treatment is given a dose (such as a first dose and a subsequent dose) determined by a physician. In some aspects of the invention, the specific dose of the immune effector cell therapy according to the present invention can be divided into two or more administrations, and the total dose given in multiples is equal to the total dose in the course of treatment determined by the physician.

[0278] The "dose" described herein may 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 a dose given to a patient calculated based on the patient's weight, such as mg / kg, immune effector cells / kg, etc. A dose calculated based on BSA is a dose given to a patient calculated based on the patient's surface area, such as mg / m 2 , and the number of immune effector cells / m 2 wait.

[0279] As used herein, "dosing frequency" refers to the frequency with which doses of immune effector cells or pretreatment drugs are administered within a given period of time. The dosing frequency may be indicated as the number of doses per given period of time. For example, fludarabine may be administered as follows: 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. Cyclophosphamide may be administered as follows: 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. Albumin-bound paclitaxel may be administered as follows: 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.

[0280] As used herein, a composition refers to any mixture of two or more products, substances or compounds (including cells). It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous or any combination thereof.

[0281] As used herein, a cell or population of cells that is "positive" for a particular marker refers to the detectable presence of a particular marker, typically a surface marker, on or in the cell. When referring to a surface marker, the term refers to the presence of surface expression detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting the antibody. Wherein the staining is detectable by flow cytometry at a level that is significantly higher than the level of staining detected when the same procedure is performed using an isotype-matched control under otherwise identical conditions, 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.

[0282] As used herein, "a cell or cell population is "negative" for a particular marker" means that the particular marker, such as a surface marker, is not present on or in the cell or is present but is substantially undetectable. When referring to a surface marker, the term means that surface expression is not detectable by flow cytometry, or that surface expression is detected, for example, when surface expression is detected by staining with an antibody that specifically binds to the marker and detecting the antibody, the staining is detectable by flow cytometry at a level that is significantly lower than the level of staining detected when the same procedure is performed using an isotype-matched control under otherwise identical conditions, and / or significantly lower than the level of cells known to be positive for the marker, and / or substantially similar to the level of cells known to be negative for the marker.

[0283] As used herein, the term "vector" refers to a nucleic acid molecule that is capable of propagating another nucleic acid to which it is attached. The term includes vectors in the form of self-replicating nucleic acid structures, as well as vectors that are introduced into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively attached. Such vectors are also referred to herein as "expression vectors."

[0284] The present application relates to adoptive cell or immune effector cell therapy for solid tumors, including administration of multiple or repeated doses of cells, and methods, compositions and products used therein. The cells generally express chimeric antigen receptors (CAR) or other transgenic receptors such as T cell receptors (TCR).

[0285] Claudin 18 (CLD18) molecules (Genbank accession numbers: splice variant 1 (CLD18A1): NP_057453, NM016369, and splice variant 2 (CLD18A2): NM_001002026, NP_001002026) are intrinsic transmembrane proteins with a molecular weight of approximately 27.9 / 27.72 kD. Claudin is an intrinsic membrane protein located in the tight junctions of epithelium and endothelium. Tight junctions organize a network of interconnected chains of particles within the membrane between adjacent cells. In tight junctions, occludin and claudin are the most important transmembrane protein components. Due to their strong intercellular adhesion properties, they create a primary barrier that prevents and controls the paracellular transport of solutes and limits the lateral diffusion of membrane lipids and proteins to maintain cell polarity. Proteins that form tight junctions are critically involved in organizing epithelial tissue structure.

[0286] CLD18A1 is selectively expressed in normal lung and gastric epithelia, while CLD18A2 is only expressed in gastric cells. Furthermore, CLD18A2 is localized in differentiated short-lived cells of the gastric epithelium but absent from the gastric stem cell compartment. Both variants are strongly expressed in several cancer types, including tumors of the stomach, esophagus, pancreas, and lung, as well as human cancer cell lines. Expression is primarily in adenocarcinoma subtypes of these indications.

[0287] The present invention provides therapeutic methods and compositions for treating diseases (such as tumors) associated with CLD18 expression.

[0288] The present invention provides a method for treating a tumor, particularly a solid tumor, in a subject using adoptive cells or immune effector cells expressing a genetically engineered (recombined) chimeric receptor. The method includes a single course of reinfusion or multiple courses of reinfusion of adoptive cells or immune effector cells. For the purposes of this article, "dose" 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 described herein includes multiple courses of treatment, the dose for each course of treatment is the same. In some embodiments, when the method described herein includes multiple courses of treatment, the dose for each course of treatment is different. "Split dose" refers to a dose for a single administration in one course of treatment, when the dose for the entire course of treatment is divided into multiple administrations to the subject. In some embodiments, when the dose in one course of treatment is divided into multiple administrations to the subject, the split doses for each administration are the same. In some embodiments, when the dose in one course of treatment is divided into multiple administrations to the subject, the split doses for each administration are different. For the purposes of this article, unless otherwise specified, the dose refers to the total amount of adoptive cells or immune effector cells administered or reinfused in one course of treatment.

[0289] In some embodiments, the methods described herein include reinfusing the adoptive cells or immune effector cells in a single course of treatment. A single course of treatment refers to reinfusing a certain total amount of adoptive cells or immune effector cells within a certain period of time. In some embodiments, a certain total amount of adoptive cells or immune effector cells is reinfused once in the course of treatment. In some embodiments, a certain total amount of adoptive cells or immune effector cells is reinfused twice or more. In some embodiments, a certain total amount of adoptive cells or immune effector cells is reinfused three or more times. In some embodiments, a certain total amount of adoptive cells or immune effector cells is reinfused four or more times. In some embodiments, a certain total amount of adoptive cells or immune effector cells is reinfused five or more times. In some embodiments, a certain total amount of adoptive cells or immune effector cells is reinfused six or more times. In some embodiments, a certain total amount of adoptive cells or immune effector cells is reinfused seven or more times. In some embodiments, a certain total amount of adoptive cells or immune effector cells is reinfused eight or more times. In some embodiments, a certain total amount of adoptive cells or immune effector cells is reinfused nine or more times. In some embodiments, a certain total amount of adoptive cells or immune effector cells are infused back ten times or more. In some embodiments, the adoptive cells or immune effector cells infused back each time are equal amounts of the adoptive cells or immune effector cells to be infused back. In some embodiments, the adoptive cells or immune effector cells infused back each time are non-equal amounts of the adoptive cells or immune effector cells to be infused back. In some embodiments, the amount of adoptive cells or immune effector cells infused back each time is determined by the physician according to the specific circumstances of the subject. The specific circumstances of the subject can be, for example, the overall health of the subject, 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 combined medication, the degree or possibility of toxic reactions, complications, cancer metastasis, and any other factors that the physician believes will affect the amount of adoptive cells or immune effector cells suitable for infusion in the subject. In some embodiments, in the multiple infusions of a certain total amount of adoptive cells or immune effector cells, the amount of adoptive cells or immune effector cells infused back each time is increasing. In some embodiments, in the multiple reinfusions of a certain total amount of adoptive cells or immune effector cells, the amount of adoptive cells or immune effector cells reinfused each time shows a decreasing trend. In some embodiments, in the multiple reinfusions of a certain total amount of adoptive cells or immune effector cells, the amount of adoptive cells or immune effector cells reinfused each time shows a trend of first increasing and then decreasing. In some embodiments, in the multiple reinfusions of a certain total amount of adoptive cells or immune effector cells, the amount of adoptive cells or immune effector cells reinfused each time shows a trend of first decreasing and then increasing.

[0290] Multi-course reinfusion refers to having multiple time periods, and a certain total amount of adoptive cells or immune effector cells are reinfused in each time period. In some embodiments, the lengths of the multiple time periods are consistent. In some embodiments, the lengths of the multiple time periods are unequal. In some embodiments, the multiple courses refer to having at least two time periods. In some embodiments, the multiple courses refer to having at least three time periods. In some embodiments, the multiple courses refer to having at least four time periods. In some embodiments, the multiple courses refer to having at least five time periods. In some embodiments, the multiple courses refer to having at least six time periods. In some embodiments, the multiple courses refer to having at least seven time periods. In some embodiments, the multiple courses refer to having at least eight time periods. In some embodiments, the multiple courses refer to having at least nine time periods. In some embodiments, the multiple courses refer to having at least ten time periods. In some embodiments, the multiple courses refer to having more than ten time periods.

[0291] In some embodiments, a certain total amount of adoptive cells or immune effector cells is infused back once in one of the multiple courses of treatment. In some embodiments, a certain total amount of adoptive cells or immune effector cells is infused back twice or more in one of the multiple courses of treatment. In some embodiments, a certain total amount of adoptive cells or immune effector cells is infused back three or more times in one of the multiple courses of treatment. In some embodiments, a certain total amount of adoptive cells or immune effector cells is infused back four or more times in one of the multiple courses of treatment. In some embodiments, a certain total amount of adoptive cells or immune effector cells is infused back five or more times in one of the multiple courses of treatment. In some embodiments, a certain total amount of adoptive cells or immune effector cells is infused back six or more times in one of the multiple courses of treatment. In some embodiments, a certain total amount of adoptive cells or immune effector cells is infused back seven or more times in one of the multiple courses of treatment. In some embodiments, a certain total amount of adoptive cells or immune effector cells is infused back eight or more times in one of the multiple courses of treatment. In some embodiments, a certain total amount of adoptive cells or immune effector cells is infused back nine or more times in one of the multiple courses. In some embodiments, a certain total amount of adoptive cells or immune effector cells is infused back ten or more times in one of the multiple courses. In some embodiments, in one of the multiple courses, the adoptive cells or immune effector cells infused back each time are equal amounts of the adoptive cells or immune effector cells to be infused back. In some embodiments, in one of the multiple courses, the adoptive cells or immune effector cells infused back each time are non-equal amounts of the adoptive cells or immune effector cells to be infused back. In some embodiments, in one of the multiple courses, the amount of adoptive cells or immune effector cells infused back each time is determined by the physician according to the specific circumstances of the subject. The specific circumstances of the subject may be, for example, the overall health of the subject, the severity of the disease, the response to the previous dose of the same course, the response to the previous course, the subject's combined medication, the degree or possibility of toxic reactions, complications, cancer metastasis, and any other factors that the physician believes will affect the amount of adoptive cells or immune effector cells suitable for infusion in the subject. In some embodiments, a total amount of adoptive cells or immune effector cells are infused back at the same number of times in each of the multiple courses of treatment. In some embodiments, a total amount of adoptive cells or immune effector cells are infused back at different numbers of times in each of the multiple courses of treatment. In some embodiments, a total amount of adoptive cells or immune effector cells are infused back at the same number of times in each of the multiple courses of treatment. In some embodiments, the same total amount of adoptive cells or immune effector cells are infused back in each of the multiple courses of treatment.In some embodiments, a different total amount of adoptive cells or immune effector cells is infused in each of the multiple courses of treatment.

[0292] In some embodiments, the total amount of the adoptive cells or immune effector cells in each of the multiple courses of treatment is increasing. In some embodiments, the total amount of the adoptive cells or immune effector cells in each of the multiple courses of treatment is decreasing. In some embodiments, the total amount of the adoptive cells or immune effector cells in each of the multiple courses of treatment is first increasing and then decreasing. The total amount of the adoptive cells or immune effector cells in each of the multiple courses of treatment is first decreasing and then increasing.

[0293] In some embodiments, the first dose is a lower dose and / or an adjustment or reduction dose and / or a subsequent dose is a consolidation dose and / or an adjustment or reduction dose. Cells, compositions and products that can be used for such methods are also provided. In some embodiments, a chimeric receptor is a genetically engineered antigen receptor, such as a functional non-TCR antigen receptor, for example, a chimeric antigen receptor (CAR) and other recombinant antigen receptors such as genetically engineered T cell receptors (TCR). The receptor also includes other recombinant chimeric receptors, such as those containing extracellular and intracellular portions that specifically bind to a ligand or receptor or other binding partner, such as those receptors of the intracellular signal transduction portion of CAR. In some embodiments, the dose includes a lower first dose.

[0294] In some embodiments, the method includes (a) giving a subject with a tumor a single course of expression of a chimeric antigen receptor (e.g., CAR) cells; and (b) giving a subject multiple courses of expression of a chimeric antigen receptor (e.g., CAR) cells. In other embodiments, one or more subsequent doses may be given.

[0295] The term "CLD18" refers to claudin-18, and includes any variants (including CLD18A1 and CLD18A2), conformations, isoforms and species homologs of CLD18 expressed naturally by cells or by cells transfected with the CLD18 gene. Preferably, "CLD18" refers to human CLD18, in particular CLD18A2 (SEQ ID NO: 1, 2) and / or CLD18A1 (SEQ ID NO: 7, 8), more preferably CLD18A2.

[0296] The term "CLD18A1" includes any post-translationally modified variants, isoforms and interspecies homologs of human CLD18A1 expressed naturally by a cell or by a cell transfected with the CLD18A1 gene.

[0297] The term "CLD18A2" includes any post-translationally modified variants, isoforms and interspecies homologs of human CLD18A2 expressed naturally by a cell or by a cell transfected with the CLD18A2 gene.

[0298] The term "CLD18 variant" shall include (i) CLD18 splice variants, (ii) CLD18 post-translational modification variants, in particular including variants with different N-glycosylation states, (iii) CLD18 conformational variants, in particular including CLD18-conformation-1, CLD18-conformation-2 and CLD18-conformation-3, (iv) free CLD18 and homotypic / heterotypic associated variants located in intercellular tight junctions, (v) CLD18 cancer-associated variants and CLD18 non-cancer-associated variants.

[0299] Method for treating tumors using cells expressing chimeric antigen receptors

[0300] The present invention provides methods, compositions and products for cell therapy, for treating diseases or conditions including various solid tumors. The method involves administering immune effector cells expressing chimeric receptors, which target recognition and / or specifically bind to tumor antigens and activate immune effector cells. The receptor includes chimeric receptors, such as chimeric antigen receptors (CAR), T cell receptors (TCR), T cell fusion proteins (T cell fusion protein, TFP) and T cell antigen couplers (T cell antigen coupler, TAC).

[0301] In some embodiments, the method comprises administering one or more subsequent doses of cells to a subject. The dose is typically administered in a specific amount and according to specific time parameters. In some embodiments, the method typically comprises administering an initial dose of cells, followed by administering subsequent doses of cells during a specific time window 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 a subject, and / or improving the efficacy of the treatment.

[0302] In some embodiments, the method provided is based on the observation herein that the subject's exposure to the administered cells increases (e.g., the number of cells or duration increases over time) can improve the efficacy and treatment outcomes in immune effector cell therapy. Preliminary analysis after administering different CAR-T cells targeting CLD18A2 to subjects with various CLD18A2-positive cancers in multiple clinical trials showed a correlation between higher and / or longer exposure to CAR-T cells and treatment outcomes. These results include patient survival, remission, or stability of the condition. In some embodiments, the method described herein includes monitoring the subject's exposure to the adoptive cells or immune effector cells, and determining the subsequent doses and intervals of the subsequent courses of treatment based on the exposure. In some embodiments, the method described herein includes monitoring the subject's exposure to the adoptive cells or immune effector cells, and maintaining or reducing the dose of the subsequent doses or subsequent courses of treatment based on the exposure reaching or exceeding a certain level and / or maintaining or extending the intervals between the subsequent doses or subsequent courses of treatment. In some embodiments, the methods described herein include monitoring the exposure level of the subject to the adoptive cells or immune effector cells, and maintaining or increasing the dosage of subsequent divided administrations or subsequent treatment courses and / or maintaining or shortening the interval between subsequent divided administrations or subsequent treatment courses based on the exposure level being below a certain level.

[0303] In some embodiments, the methods described herein include monitoring the degree or risk of toxicity of the subject to the adoptive cells or immune effector cells, and determining the dosage and interval of subsequent divided administration or subsequent courses of treatment according to the degree or risk of toxicity. In some embodiments, the degree or risk of toxicity includes, but is not limited to, for example, CRS, neurotoxicity, macrophage activation syndrome, and tumor lysis syndrome.

[0304] In some embodiments, a subsequent dose is usually given when the risk of toxic reaction or its symptoms or biochemical indicators (such as CRS or neurotoxicity, macrophage activation syndrome or tumor lysis syndrome) is equal to or lower than an acceptable level after the first dose or the previous dose. In some embodiments, the toxic reaction or its symptoms or biochemical indicators include one or more of the serum levels of fever, hypotension, hypoxia, neurological disorders, inflammatory cytokines, and C-reactive protein (CRP). In some embodiments, the acceptable level of the risk of the toxic reaction or its symptoms or biochemical indicators refers to 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15% or 10% of the peak level after the first dose or the previous dose.

[0305] In some embodiments, a subsequent dose is given when the toxic reaction reaches a peak and is decreasing after the first dose or the previous dose is given. In some embodiments, a subsequent dose is given after the toxic reaction drops to an acceptable level or less after the first dose or the previous dose is given. In some embodiments, after the first dose or the previous dose is given, a subsequent dose is given when the risk of toxic reaction or its symptoms or biochemical indicators is equal to or lower than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% of the peak level that occurs after the first dose or the previous dose is given. Thus, in some embodiments, a subsequent dose is administered 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, 80 days after the first or previous dose is administered. In some embodiments, the appropriate time is determined by monitoring and / or assessing the presence of one or more symptoms or risks associated with a toxic event and infusing a subsequent dose after determining that the symptoms or risks are at or below an acceptable level.

[0306] In some embodiments, subsequent doses are administered when, based on the first dose, the serum level of a factor indicative of cytokine-release syndrome (CRS) in a subject is no more than 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 times the subject's serum level prior to administration of the first dose.

[0307] In some embodiments, the timing of administering a subsequent dose is selected to avoid the possibility that a host immune response generated by the first dose or the previous dose that has been administered may induce a reduction in the efficacy of the subsequent dose. In some aspects, a subsequent dose is administered before a host immune response occurs, such as an adaptability or specificity to the administered cells and / or the chimeric antigen receptors expressed therein, such as a humoral or cell-mediated immune response. In some embodiments, a subsequent dose is administered before such a response can be detected, for example, by one or more specialized detection methods. One or more subsequent doses are typically administered when a host adaptive immune response to a cell is not detected, has not yet been established, and / or has not yet reached a certain level, degree, or stage. Thus, in some embodiments, a subsequent dose is administered up to 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, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 days after the first or previous dose.

[0308] Therefore, the method provided in some embodiments involves administering one or more subsequent doses after the window of toxicity risk, after stabilizing or reducing the tumor burden with the first dose, but before the adaptive host immune response (i.e., the immune rejection reaction produced by the body to the CAR-T cells) occurs. Under such conditions, subsequent doses can safely and effectively provide immune monitoring, clearance or prevention of proliferation or metastasis of residual tumor cells. Therefore, in some embodiments, the subsequent dose is a disease consolidation dose.

[0309] The so-called tumor burden includes the size or degree of differentiation of the tumor, or the type and stage of metastasis, and / or the appearance and disappearance of common complications of advanced cancer, such as cancerous pleural effusion, and / or the appearance or change of expression level of tumor markers, and / or the possibility or incidence of toxic results in the subject, such as CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity and / or host immune response to administered cells and / or chimeric antigen receptors. In some embodiments, the size of the tumor is measured by the ruler provided by PET (positron emission computed tomography) and CT (computerized X-ray tomography).

[0310] The tumor markers are also called tumor markers, which refer to substances that are characteristically present in malignant tumor cells, or abnormally produced by malignant tumor cells, or substances produced by the host in response to tumor stimulation, and can reflect the occurrence and development of tumors and monitor the tumor's response to treatment. Tumor markers exist in the tissues, body fluids and excretions of tumor patients and can be detected by immunological, biological and chemical methods, including alpha-fetoprotein (AFP), CA125, CA15-3, squamous cell carcinoma antigen (SCC), soluble fragment of cytokeratin 19 (CYFRA21-1), carcinoembryonic antigen (CEA), CA199, CA724, etc.

[0311] In some embodiments, the first dose includes an amount of cells sufficient to reduce the tumor burden in the subject, and subsequent doses are administered when the serum level of a factor indicative of cytokine-release syndrome (CRS) in the subject is no more than 10 or 25 times the serum level in the subject prior to administration of the first dose, and / or when the CRS-related outcome in the subject is at a peak level and begins to decline after administration of the first dose, and the subject has no detectable adaptive host immune response specific for the chimeric receptor expressed by the cells of the first dose.

[0312] In some embodiments, the first dose comprises less than about 2.5×10 8 cells / kg subject body weight, or less than approximately 1×10 12 cells, and subsequent doses are administered more than about 21 days and less than about 80 days after the start of administration of the initial dose.

[0313] Administration of cells in immune effector cell therapy

[0314] The method provided by the present invention comprises administering multiple doses of immune effector cells expressing chimeric antigen receptors that recognize the tumor antigen, such as CAR, TCR, TFP, and TAC, to a subject with a solid tumor that expresses a tumor antigen.

[0315] The "tumor antigens" described herein include, but are not limited to, thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD 22; CD 30; CD 70; CD 123; CD138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit α (IL-13Rα); interleukin 11 receptor α (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; proteinase serine 21 (PRSS21); vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; platelet-derived growth factor receptor β (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, EGFRvIII); neural cell adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrin type 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 1 (TEM1 / CD248); tumor endothelial marker 7 related (TEM7R); Claudin 6, Claudin18.2, Claudin18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B cell maturation antigen (BCMA); CA9; kappa light chain (kappa light 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; carcinoembryonic variant of tumor necrosis zone; 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);The hexose moiety of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cell receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); 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 binding 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 breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myelocytic virus oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (Rho C); cytochrome P4501B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS); squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired 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 (LA IR1); Fc fragment of IgA 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); EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); phosphatidylinositol proteoglycan-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like polypeptide 1 (IGLL1). In some embodiments, the tumor antigen is EGFR, EGFRvIII, phosphatidylinositol proteoglycan 3, claudin 18.2 or BCMA. ;

[0316] As used herein, a "subject" is a mammal, such as a human or other animal, and is typically a human. In some embodiments, the subject has been treated with tumor chemotherapy or radiation therapy prior to administering the first dose and / or prior to administering subsequent doses. In some aspects, the subject is refractory or non-responsive to other therapeutic agents.

[0317] In some embodiments, the tumor is persistent or recurrent, for example, after intervention with another treatment, including chemotherapy, radiation. In some embodiments, the administration is effective to treat the subject regardless of whether the subject has become resistant to another therapy.

[0318] In some embodiments, the subject is responsive to other therapeutic agents and treatment with the therapeutic agent reduces tumor burden. In some aspects, the subject is initially responsive to the therapeutic agent, but shows recurrence of the tumor over time. In some such embodiments, the subject is determined to be at risk of recurrence, e.g., at high risk of recurrence, and cells are thus administered prophylactically, e.g., to reduce the likelihood of recurrence or prevent recurrence.

[0319] The disease includes a tumor, or other proliferative disease or disorder. 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, cancer of the anal region, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, Hodgkin's disease, non-Hodgkin's lymphoma, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors of childhood, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphomas, tumor angiogenesis, spinal tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of the above cancers, and metastatic lesions of the above cancers.

[0320] In some embodiments, the size of the dose and the time of reinfusion are determined by the initial tumor load of the subject. For example, in some cases, the number of cells in the first dose generally given to the subject is low. In the case of a lower tumor load, such as solid tumors, the size of the tumor load and / or micro-residual lesions can be assessed by tumor marker detection, and the initial dose may be higher. In other cases, in subjects with a higher tumor load, the first dose can be divided into 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 administrations, preferably 1-5 administrations, and more preferably 2-3 administrations. Each administration is separated by 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 cases, subsequent doses may be the same as the first dose or higher than the first dose or lower than the first dose.

[0321] The term "treatment" as used herein refers to the complete or partial alleviation or reduction of a tumor or symptoms associated therewith. The desired therapeutic effect includes, but is not limited to, preventing the occurrence or recurrence of a tumor, alleviating symptoms, reducing any direct or indirect pathological results of a tumor, preventing metastasis, slowing the rate of tumor progression, improving or alleviating the state of the tumor, and alleviating or improving prognosis. "Delaying the development of a tumor" as used herein means postponing, hindering, slowing down, slowing down, stabilizing, curbing and / or delaying the development of the tumor. The delay may have different lengths of time, depending on the history of the disease and / or the individual to be treated. It will be appreciated by those skilled in the art that sufficient or significant delays may encompass prevention (for individuals who have not developed the tumor). For example, advanced cancer, such as the development of metastasis, may be delayed.

[0322] In some embodiments, provided cells and compositions are used to delay tumor development or slow tumor progression.

[0323] As used herein, "inhibiting" a function or activity means a decrease in the function or activity when compared to the same condition under other circumstances or compared to another condition.

[0324] An "effective amount" of an agent, e.g., a pharmaceutical agent, cell, or composition, in the context of administration, refers to an amount that is effective, at dosages and for periods of time necessary, to achieve a desired result, e.g., a therapeutic or preventive result.

[0325] A "therapeutically effective amount" of an agent, e.g., a pharmaceutical preparation or a cell, refers to an amount that is effective, at dosages and over the necessary time course, to achieve a desired therapeutic outcome, e.g., treatment of a tumor, and / or a pharmacokinetic or pharmacodynamic effect of the treatment. A therapeutically effective amount can vary according to a variety of factors, e.g., disease state, age, sex, and weight of the subject, as well as the population of cells being administered. In some embodiments, provided methods involve administering an effective amount (e.g., a therapeutically effective amount) of the cells and / or compositions.

[0326] "Prophylactically effective amount" refers to an amount that, for a given dose and necessary duration, is conducive to achieving the desired prophylactic outcome. Generally, since a prophylactic dose is used in a subject at an early stage or before the disease, the prophylactic effective amount will be less than the therapeutically effective amount. In the case of a lower tumor burden, in some aspects, the prophylactic effective amount may be higher than the therapeutically effective amount.

[0327] Methods for administering cells for immune effector cell therapy are known and can be used in conjunction with the methods and compositions provided herein.

[0328] In some embodiments, the cell therapy, e.g., adoptive T cell or immune effector cell therapy, is performed by: autologous reinfusion. Thus, in some aspects, the cells are derived from a subject in need of treatment and the cells are administered to the same subject after isolation and processing.

[0329] In some embodiments, the cell therapy, e.g., adoptive T cell or immune effector cell therapy, is performed by allogeneic transfusion, wherein the cells are isolated and / or otherwise extracted and prepared from a donor, and the donor and the subject to whom the cells are transfused are different. In such embodiments, the cells are then administered to a subject with high genetic tissue compatibility. In some embodiments, the donor and recipient are genetically identical. In some embodiments, the donor and recipient are genetically similar. In some embodiments, the recipient and donor have the same HLA class or supertype.

[0330] The cells can be given by any suitable means, for example, by injection, for example, intravenous or subcutaneous injection, intraocular injection, fundus injection, subretinal injection, intravitreal injection, reverse septal injection, subscleral injection, intrachoroidal injection, anterior chamber injection, subconjunctival (subconjectval) injection, subconjunctival (subconjuntival) injection, suprascleral injection, retrobulbar injection, periocular injection or peribulbar delivery. In some embodiments, they are by parenteral, intrapulmonary and intranasal, and if local treatment is desired, intralesional administration. Extraperitoneal infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In some embodiments, a given dose is given by a single bolus. In some embodiments, cells are given by multiple boluses, for example, multiple administrations are no more than 20 days, or cells are given by continuous infusion.

[0331] For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease being treated, the chimeric antigen receptor or cell type, the severity of the disease and the course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous treatments, the subject's clinical history and response to the cells, and the judgment of the attending physician. In some embodiments, the compositions and cells are suitable for administration to a subject at one time point or over a series of treatments.

[0332] In some embodiments, the immune effector cells are used as part of a combined therapy, for example, in combination with another interventional therapy, for example, in combination with another therapeutic agent such as an antibody, an engineered immune effector cell, a receptor or reagent, a cytotoxic drug or other therapeutic method, and are administered simultaneously or sequentially in any order. In some embodiments, immune effector cells are used in combination with one or more other therapeutic methods, or in combination with another interventional therapeutic method, and are performed sequentially simultaneously or in any order. In some cases, the immune effector cells are co-administered with another treatment at a sufficiently close time to produce a therapeutic effect greater than the above-mentioned immune effector cell group or one or more other therapeutic drugs or methods, and vice versa. In some embodiments, the immune effector cells are administered before one or more other therapeutic agents. In some embodiments, the immune effector cells are administered after one or more other therapeutic agents. In some embodiments, one or more other therapeutic drugs include cytokines, such as IL-2, IL-12, to enhance persistence.

[0333] The other therapeutic agent can be given by any suitable means, for example, by injection, for example, intravenous or subcutaneous injection, intraocular injection, fundus injection, subretinal injection, intravitreal injection, reverse septal injection, subscleral injection, intrachoroidal injection, anterior chamber injection, subconjunctival (subconjectval) injection, subconjunctival (subconjuntival) injection, suprascleral injection, retrobulbar injection, periocular injection or peribulbar delivery. In some embodiments, they are delivered by parenteral, intrapulmonary and intranasal delivery, and if local treatment is desired, can be given in the focus. Extraperitoneal infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. In some embodiments, a given dose is given by a single bolus to give the therapeutic agent. In some embodiments, given by multiple boluses, for example, multiple administrations no more than 20 days, or given by continuous infusion.

[0334] In some embodiments, the methods of the present invention include administering a tubulin inhibitor and the adoptive cells or immune effector cells to a subject. In some embodiments, the methods of the present invention include administering a tubulin inhibitor and immune effector cells to a subject within one course of treatment. In some embodiments, the methods of the present invention include administering a tubulin inhibitor to a subject and then administering immune effector cells. In some embodiments, the methods of the present invention include administering immune effector cells to the subject first and then administering a tubulin inhibitor. In some embodiments, the methods of the present invention include administering tubulin inhibitors and immune effector cells to the subject substantially simultaneously. In some embodiments, the methods of the present invention include alternately administering tubulin inhibitors and immune effector cells to the subject. In multiple courses of treatment, the tubulin inhibitors and immune effector cells may be the same or different.

[0335] Tubulin inhibitors as described herein include tubulin polymerization promoters and tubulin polymerization inhibitors. Tubulin polymerization promoters include but are not limited to, for example, taxanes, epothilones, spongolides, and laulimalide, etc. Tubulin polymerization inhibitors include but are not limited to, for example, colchicine, Combretastatin A-4, BPROL075, Plinabulin (NPI-2358), Nakiterpiosin, vincristine, Nocodazole, podophyllotoxin, Dolastatins10, Indibulin (D-24851), and Eribulin, etc.

[0336] Taxane compounds include paclitaxel (trade name: Taxol), albumin-bound paclitaxel (nab-paclitaxel, Abraxane), docetaxel, etc. and their derivatives. Taxane microtubule inhibitors can promote the assembly of tubulin into microtubules and inhibit the depolymerization of microtubules, resulting in abnormal arrangement of microtubule bundles and the formation of astrocytes, thereby causing the spindle to lose its normal function and ultimately leading to cell death.

[0337] In some embodiments, the methods of the present invention comprise administering a taxane and immune effector cells to a subject in one course of treatment. In some embodiments, the methods of the present invention comprise administering a taxane and then administering immune effector cells to a subject in one course of treatment. In some embodiments, the methods of the present invention comprise administering immune effector cells first and then administering a taxane to the subject in one course of treatment. In some embodiments, the methods of the present invention comprise administering a taxane and immune effector cells to the subject substantially simultaneously in one course of treatment. In some embodiments, the methods of the present invention comprise administering a taxane and immune effector cells to the subject alternately in one course of treatment.

[0338] In some embodiments, the method of the present invention comprises administering taxanes and immune effector cells to a subject in multiple treatment courses. In some embodiments, the method of the present invention comprises administering taxanes to a subject and then administering immune effector cells in multiple treatment courses. In some embodiments, the method of the present invention comprises administering immune effector cells to the subject first and then administering taxanes to the subject in multiple treatment courses. In some embodiments, the method of the present invention comprises administering taxanes and immune effector cells to the subject substantially simultaneously in multiple treatment courses. In some embodiments, the method of the present invention comprises administering taxanes and immune effector cells to the subject alternately in multiple treatment courses. In some embodiments, the method of the present invention comprises administering the same dose of taxanes and / or immune effector cells to the subject in different treatment courses. In some embodiments, the method of the present invention comprises administering different doses of taxanes and / or immune effector cells to the subject in different treatment courses. The term "taxanes drug" refers to a drug whose main component contains a taxane compound, and the taxane compound has a cross-methylene benzocyclodecene mother core structure similar to taxane. In some embodiments, the cross-methoxybenzocyclodecene core structure of the taxane compound contains an unsaturated bond. In some embodiments, the cross-methoxybenzocyclodecene core structure of the taxane compound does not contain an unsaturated bond. In some embodiments, the carbon atoms on the cross-methoxybenzocyclodecene core structure of the taxane are substituted by heteroatoms selected from N, O, S, and P. In some embodiments, the administration of the taxane compound is by injection. In some embodiments, the method of the present invention can increase, enhance or prolong the activity and / or number of immune cells, or produce a medically effective response during cancer treatment. The term "increase" or "enhance" the activity of immune cells refers to allowing a subject or tumor cell to improve its ability to respond to the treatment disclosed herein. For example, an enhanced response can include an increase in responsiveness of 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more. As used herein, "enhancement" can also refer to an increase in the number of subjects who respond to a treatment, such as an immune effector cell therapy. For example, an enhanced response can refer to the total percentage of subjects who respond to a treatment, wherein 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 certain embodiments, the dose of a taxane compound such as Abraxane administered is no greater than 300 mg / m 2 / time, or not more than about 200mg / m 2 / time, preferably, 100 mg / m 2 / time-200mg / m 2 In certain embodiments, the dosage of a taxane compound such as Abraxane is about 200, 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 8, 6, 4, 3, 2 or 1 mg / m 2 / time, preferably, 100 mg / m 2 / time-200mg / m 2 In certain 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 per treatment course. In certain embodiments, the immune effector cell therapy is administered 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 certain embodiments, the immune effector cell therapy is administered 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.

[0339] In some embodiments, the method of the present invention includes administering pretreatment before the first or subsequent doses, for example, chemotherapeutic drugs (chemotherapeutic agents), systemic radiation, local radiation therapy, etc. or a combination thereof. In some embodiments, the method includes pretreating the subject with one or more chemotherapeutic agents. In some embodiments, the method includes pretreating the subject with a tubulin inhibitor and one or more other chemotherapeutic agents. Without being limited by theory, the effects of pretreatment are believed to include, but are not limited to, lymphocyte clearance, reduction of tumor burden, etc. In some embodiments, the methods described herein include not performing the pretreatment before the first or subsequent doses are administered. In some embodiments, not performing the pretreatment includes not administering a lymphocyte clearance agent, systemic radiation therapy, or a combination thereof, or other means for pretreatment, and the lymphocyte clearance rate in the subject after the pretreatment is still less than 50%, 55%, 60%, 65% or 70%.

[0340] In some embodiments, the chemotherapeutic agent described herein refers to a drug used in chemotherapy, and refers to a chemical drug that has a preventive and therapeutic effect on microbial infection, parasitic disease and malignant tumor. Chemotherapeutic agents include, but are not limited to, synthetic antibiotics, antibiotics, antiparasitic drugs, antifungal drugs, antiviral drugs, alkylating agents, antimetabolites, antituberculosis drugs and antitumor drugs. For example, diterpene alkaloid compounds (such as taxanes), cyclophosphamide, fludarabine, cyclosporin, rapamycin, mycophenolic acid, steroids, melphalan, bendamustine, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, hydroxyurea, methotrexate, rituximab, vinblastine, and / or vincristine, etc. In some embodiments, the antimetabolite is, for example, but is not limited to, antimetabolites such as carmofur, tegafur, pentostatin, doxifluridine, trimesatide, fludarabine, capecitabine, galocitabine, cytarabine sodium octadecyl phosphate, fosteabine sodium hydrate, raltitrexed, paltitrexid, ethidium bromide, tiazofurin, nolatrexed, pemetrexed, nelzarabine, 2'-deoxy-2'-methylene cytidine, 2'-fluoromethylene-2'-deoxy cytidine, N-[5-(2,3-dihydro-benzofuranyl)sulfonyl]-N'-(3,4-dichlorophenyl)urea, N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycero-BL-mannose-pyrrolidone adenine, aplidine, ecteinascin, 4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-b]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamate, aminopterin, 5-fluorouracil, alanosine, 11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6 -methoxy-14-oxa-1,11-diazatetracyclo (7.4.1.0.0)-tetradecane-2,4,6-triene-9-yl acetate, swainsonine, lometrexol, dexrazoxane, methioninase, 2'-cyano-2'-deoxy-N4-palmitoyl-1-BD-arabinofuranosyl cytosine and 3-aminopyridine-2-aldehyde thiosemicarbazone, etc. In some embodiments, the alkylating agent includes, for example, but is not limited to, dacarbazine, phenylalanine nitrogen mustard, cyclophosphamide, temozolomide, chlorambucil, busulfan, nitrogen mustard and nitrosourea, etc.

[0341] Pretreatment of subjects before administration of the first dose or subsequent doses can improve the effect of immune effector cell therapy. The day of the first infusion of immune effector cells (eg, CAR T cells) in each course of treatment is designated as day 0. Pretreatment is given before the immune effector cell infusion is given. In some embodiments, 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 are pretreated before the adoptive cells or immune effector cells are administered. In some embodiments, a chemotherapeutic agent is used for pretreatment. In some embodiments, two or more chemotherapeutic agents are used for pretreatment. In some embodiments, a microtubule inhibitor is used for pretreatment with one or more additional chemotherapeutic agents. In some embodiments, two microtubule inhibitors are used for pretreatment with one or more additional chemotherapeutic agents. In some embodiments, a microtubule inhibitor such as a tubulin polymerization promoter is used for pretreatment with one or more alkylating agents and one or more antimetabolites. In some embodiments, one or more taxanes are used for pretreatment with one or more alkylating agents and one or more antimetabolites. In some embodiments, a microtubule inhibitor (e.g., paclitaxel, especially albumin-bound paclitaxel) is used for pretreatment with two other chemotherapeutic agents (e.g., fludarabine and cyclophosphamide). For the purpose of convenience of presentation, the following will use fludarabine and cyclophosphamide as representative chemotherapeutic agents, and (albumin-bound) paclitaxel as a representative microtubule inhibitor to describe the specific embodiments when pretreatment is performed with a microtubule inhibitor and two other chemotherapeutic agents. For example, in some embodiments, fludarabine, or cyclophosphamide and albumin-bound paclitaxel are used alone, in combination of two or three as pretreatment 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 before CAR-T cell infusion, 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, more preferably, at least 2, 3, 4, 5, 6, 7 or 8 days. In some embodiments, pretreatment includes administering fludarabine and cyclophosphamide 6 days before CAR-T cell infusion. In some embodiments, pretreatment comprises administering fludarabine and cyclophosphamide 7 days prior to CAR-T cell infusion. In some embodiments, pretreatment comprises administering fludarabine and cyclophosphamide 5 days prior to CAR-T cell infusion. In some embodiments, pretreatment comprises administering fludarabine, cyclophosphamide, and nab-paclitaxel 6 days prior to CAR-T cell infusion.In some embodiments, the conditioning comprises administering nab-paclitaxel 4 days prior to CAR-T cell infusion.

[0342] As described above, the day on which CAR T cell therapy is administered to the patient for each course of treatment is designated as Day 0. In some embodiments, fludarabine, cyclophosphamide, and albumin-bound paclitaxel may be administered at any time prior to the administration of CAR T cell therapy. 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 the administration of the 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 the administration of the CAR T cell infusion. In some embodiments, fludarabine, cyclophosphamide, or albumin-bound paclitaxel is administered 7 days prior to the administration of the CAR T cell infusion. In some embodiments, fludarabine, cyclophosphamide, or albumin-bound paclitaxel is administered 6 days prior to the administration of the CAR T cell infusion. In some embodiments, fludarabine, cyclophosphamide, or nab-paclitaxel is administered starting 12 days prior to administration of the CAR T cell infusion. In some embodiments, fludarabine, cyclophosphamide, or nab-paclitaxel is administered starting 5 days prior to administration of the CAR T cell infusion. In some embodiments, fludarabine, cyclophosphamide, or nab-paclitaxel is administered starting 4 days prior to administration of the CAR T cell infusion.

[0343] In some embodiments, fludarabine is started 7 days before the CAR T cell infusion is given and cyclophosphamide is started 7 days before the CAR T cell infusion is given. In some embodiments, fludarabine is started 6 days before the CAR T cell infusion is given and cyclophosphamide is started 6 days before the CAR T cell infusion is given. In some embodiments, fludarabine is started 5 days before the CAR T cell infusion is given and cyclophosphamide is started 5 days before the CAR T cell infusion is given. In some embodiments, fludarabine is started 12 days before the CAR T cell infusion is given and cyclophosphamide is started 12 days before the CAR T cell infusion is given. In some embodiments, fludarabine is started 5 days before the CAR T cell infusion is given, cyclophosphamide is started 5 days before the CAR T cell infusion is given, and nab-paclitaxel is started 4 days before the CAR T cell infusion is given. In some embodiments, fludarabine is started 6 days before the CAR T cell infusion, cyclophosphamide is started 6 days before the CAR T cell infusion, and nab-paclitaxel is started 5 days before the CAR T cell infusion. In some embodiments, fludarabine is started 12 days before the CAR T cell infusion, cyclophosphamide is started 12 days before the CAR T cell infusion, and nab-paclitaxel is started 11 days before the CAR T cell infusion.

[0344] The timing of administration of the pretreatment components can be adjusted to maximize the efficacy of CAR T therapy. Typically, fludarabine, cyclophosphamide, and / or nab-paclitaxel can be administered daily. In some embodiments, fludarabine, cyclophosphamide, and nab-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, and cyclophosphamide is administered daily for 3 days. In some embodiments, fludarabine is administered daily for 1 day, and cyclophosphamide is administered daily for 3 days. In some embodiments, fludarabine is administered daily for 1 day, and cyclophosphamide is administered daily for 4 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 1 day, and cyclophosphamide is administered daily for 3 days, and nab-paclitaxel is administered daily for 1 day. In some embodiments, fludarabine is administered daily for 1 day, and cyclophosphamide is administered daily for 4 days, and nab-paclitaxel is administered daily for 1 day.

[0345] As described above, the day on which each round of CAR T cell therapy is administered to a patient is designated as Day 0. In some embodiments, fludarabine is administered to a patient on the 4th day prior to Day 0 (i.e., Day -4). In some embodiments, fludarabine is administered to a patient on Day -5. In some embodiments, fludarabine is administered to a patient on Day -6. In some embodiments, fludarabine is administered to a patient on Day -12. In some embodiments, fludarabine is administered to a patient on Days -6 and -5. In some embodiments, fludarabine is administered to a patient on Days -5 and -4. In some embodiments, fludarabine is administered to a patient on Days -6, -5, -4, and -3. In some embodiments, fludarabine is administered to a patient on Days -7, -6, -5, and -4. In some embodiments, cyclophosphamide is administered to a patient on Days -6, -5, -4, and -3. In some embodiments, cyclophosphamide is administered to a patient on Days -6, -5, -4, and -4. In some embodiments, cyclophosphamide is administered to the patient on days -5, -4, and -2. In some embodiments, cyclophosphamide is administered to the patient on days -5, -4, and -3. In some embodiments, cyclophosphamide is administered to the patient on days -12, -11, and -10. In some embodiments, cyclophosphamide is administered to the patient on days -7 and -6. In some embodiments, cyclophosphamide is administered to the patient on days -6 and -5. In some embodiments, nab-paclitaxel is administered to the patient on day -4. In some embodiments, nab-paclitaxel is administered to the patient on day -5. In some embodiments, nab-paclitaxel is administered to the patient on day -11. In some embodiments, nab-paclitaxel is administered to the patient on days -5 and -4.

[0346] Fludarabine, cyclophosphamide, and nab-paclitaxel may be administered on the same day or on different days. If fludarabine, cyclophosphamide, and nab-paclitaxel are administered on the same day, cyclophosphamide and / or nab-paclitaxel may be administered before or after fludarabine; or fludarabine and / or nab-paclitaxel may be administered before or after cyclophosphamide; or cyclophosphamide and / or fludarabine may be administered before or after nab-paclitaxel. In some embodiments, fludarabine is administered to the patient on days -6, -5, -4, and -3, and cyclophosphamide is administered to the patient on days -6 and -5. In some embodiments, fludarabine is administered to the patient on days -7, -6, -5, and -4, and cyclophosphamide is administered to the patient on days -7 and -6. In some embodiments, fludarabine is administered to the patient on days -5 and -4, and cyclophosphamide is administered to the patient on days -5, -4, and -2. In some embodiments, fludarabine is administered to the patient on days -6 and -5, and cyclophosphamide is administered to the patient on days -6, -5, -4, and -3. In some embodiments, fludarabine is administered to the patient on day -5, and cyclophosphamide is administered to the patient on days -5, -4, and -3. In some embodiments, fludarabine is administered to the patient on day -6, and cyclophosphamide is administered to the patient on days -6, -5, and -4. In some embodiments, fludarabine is administered to the patient on day -6, and cyclophosphamide is administered to the patient on days -6, -5, -4, and -3. In some embodiments, fludarabine is administered to the patient on day -5, cyclophosphamide is administered to the patient on days -5, -4, and -3, and nab-paclitaxel is administered on day -4. In some embodiments, fludarabine is administered to the patient on day -6, cyclophosphamide is administered to the patient on days -6, -5, and -4, and nab-paclitaxel is administered on day -5. In some embodiments, fludarabine is administered to the patient on day -12, cyclophosphamide is administered to the patient on days -12, -11, and -10, and nab-paclitaxel is administered on day -11. In some embodiments, fludarabine is administered to the patient on day -6, cyclophosphamide is administered to the patient on days -6, -5, -4, and -3, and nab-paclitaxel is administered on day -5.

[0347] In certain embodiments, fludarabine, cyclophosphamide, and nab-paclitaxel may be administered simultaneously or sequentially. In some embodiments, cyclophosphamide is administered to the patient before fludarabine is administered. In some embodiments, cyclophosphamide is administered to the patient after fludarabine is administered. In some embodiments, nab-paclitaxel is administered to the patient before fludarabine is administered. In some embodiments, nab-paclitaxel is administered to the patient before fludarabine is administered. In some embodiments, nab-paclitaxel is administered to the patient before fludarabine is administered. In some embodiments, nab-paclitaxel is administered to the patient before cyclophosphamide is administered. In some embodiments, nab-paclitaxel is administered to the patient after cyclophosphamide is administered.

[0348] Fludarabine, cyclophosphamide, and nab-paclitaxel may be administered by any route, including intravenous (IV). In some embodiments, fludarabine is administered by IV over a period of 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. In some embodiments, cyclophosphamide is administered by IV over a period of 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. In some embodiments, nab-paclitaxel is administered by IV over 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.

[0349] In some embodiments, T cell therapy is administered to patients after fludarabine and cyclophosphamide, or fludarabine, cyclophosphamide and albumin-bound paclitaxel are administered. In some embodiments, the T cell therapy comprises adoptive cell therapy or immune effector cell therapy. In some embodiments, 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, eACT comprises administering engineered antigen-specific chimeric antigen receptor (CAR T) positive T cells. In some embodiments, eACT comprises administering engineered antigen-specific T cell receptor (TCR) positive T cells. In some embodiments, engineered T cells treat tumors in patients.

[0350] In some embodiments, pretreatment comprises administering no more than about 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130 ,120,110,100,90,85,70,65,60,55,50,49,48,47,46,45,44,43,42,41,40,39,38,37,36,35,34,33,32 ,31,30,29,28,27,26,25,24,23,22,21,20,19,18,17,16,15,14,13,12,11,10,9,8,7,6,5,4,3,2,1mg / m 2 / day; and / or administering no more than about 1000, 950, 900, 850, 800, 750, 700, 690, 680, 670, 660, 650, 640, 630, 620, 610, 600, 595, 590, 585, 580, 579, 578, 576, 575, 574, 573, 572, 571, 570, 569, 568, 567, 568 6, 565, 564, 563, 562, 561, 560, 559, 558, 557, 556, 555, 554, 553, 552, 551, 550, 549, 548, 547, 546, 545, 544, 543, 542, 541, 540, 539, 538, 537, 536, 535, 534, 533, 532, 531, 530, 529, 52 8, 527, 526, 525, 524, 523, 522, 521, 520, 519, 518, 517, 516, 515, 514, 513, 512, 511, 510, 509, 508, 507, 506, 505, 504, 503, 502, 501, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 40 0, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 mg / m 2500, 450, 400, 350, 300, 290, 280, 270, 265, 260, 255, 250, 245, 240, 235, 230, 225, 220, 215, 210, 205, 200, 195, 190, 185, 180, 175, 170, 165, 160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 35, 30, 25, 20, 15, 10, 5, or 1 mg / m 2 / day.

[0351] In some embodiments, the pretreatment comprises fludarabine administered at a dose of about 10 mg / m 2 / day to 50mg / m 2 / day and cyclophosphamide at a dose of approximately 300 to 700 mg / m 2 / day.

[0352] In some embodiments, the pretreatment comprises fludarabine administered at a dose of about 10 mg / m 2 / day to 50mg / m 2 / day, and cyclophosphamide at a dose of approximately 300 to 700 mg / m 2 / day, and nab-paclitaxel at a dose not exceeding approximately 300 mg / m 2 / day, not more than about 200mg / m 2 / day, not more than about 150mg / m 2 / day, 100mg / m 2 / day, not more than 80mg / m 2 / day or not higher than 70mg / m 2 / day.

[0353] In some embodiments, the pretreatment comprises fludarabine administered at a dose of about 15 mg / m 2 / day to 40mg / m 2 / day and cyclophosphamide at a dose of approximately 400 to 650 mg / m 2 / day.

[0354] In some embodiments, the pretreatment comprises fludarabine administered at a dose of about 15 mg / m 2 / day to 40mg / m 2 / day and cyclophosphamide at a dose of approximately 400 to 650 mg / m 2 / day, and nab-paclitaxel at a dose not exceeding approximately 300 mg / m 2 / day, not more than about 200mg / m 2 / day, not more than about 150mg / m 2 / day, 100mg / m 2 / day, not more than 80mg / m 2 / day or not higher than 70mg / m 2 / day.

[0355] In some embodiments, the pretreatment comprises fludarabine administered at a dose of about 15 mg / m 2 / day to 30mg / m 2 / day and cyclophosphamide at a dose of about 450 to 600, 450 to 550, or 490 to 550 mg / m 2 / day.

[0356] In some embodiments, the pretreatment comprises fludarabine administered at a dose of about 15 mg / m 2 / day to 30mg / m 2 / day and cyclophosphamide at a dose of about 450 to 600, 450 to 550, or 490 to 550 mg / m 2 / day, and nab-paclitaxel at a dose not exceeding approximately 300 mg / m 2 / day, not more than about 200mg / m 2 / day, not more than about 150mg / m 2 / day, 100mg / m 2 / day, not more than 80mg / m 2 / day or not higher than 70mg / m 2 / day.

[0357] In some embodiments, the pretreatment comprises fludarabine administered at a dose of about 20 mg / m 2 / day to 30mg / m 2 / day and cyclophosphamide at a dose of about 450 to 600, 450 to 550, or 490 to 550 mg / m 2 / day.

[0358] In some embodiments, the pretreatment comprises fludarabine administered at a dose of about 20 mg / m 2 / day to 30mg / m 2 / day and cyclophosphamide at a dose of about 450 to 600, 450 to 550, or 490 to 550 mg / m 2 / day, and nab-paclitaxel at a dose not exceeding approximately 300 mg / m 2 / day, not more than about 200mg / m 2 / day, not more than about 150mg / m 2 / day, 100mg / m 2 / day, not more than 80mg / m 2 / day or not higher than 70mg / m 2 / day.

[0359] In some embodiments, the pretreatment comprises fludarabine administered at a dose of about 20 mg / m 2 / day to 25mg / m 2 / day and cyclophosphamide at a dose of about 450 to 600, 450 to 550, or 490 to 550 mg / m 2 / day.

[0360] In some embodiments, the pretreatment comprises fludarabine administered at a dose of about 15 mg / m 2 / day to 25mg / m 2 / day and cyclophosphamide at a dose of about 450 to 600, 450 to 550, or 490 to 550 mg / m 2 / day, and nab-paclitaxel at a dose not exceeding approximately 300 mg / m 2 / day, not more than about 200mg / m 2 / day, not more than about 150mg / m 2 / day, 100mg / m 2 / day, not more than 80mg / m 2 / day or not higher than 70mg / m 2 / day.

[0361] In some embodiments, the present invention includes a method of pre-treating a patient prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 Fludarabine and a dose of approximately 500 mg / m 2 / day of cyclophosphamide, wherein fludarabine is administered on days -6, -5, -4, and -3, and cyclophosphamide is administered on days -6 and -5. In some embodiments, the present invention includes a method of pre-treatment prior to administering an immune effector cell infusion, the method comprising administering to the patient a dose of about 20 mg / m 2 / day of fludarabine and a dose of approximately 514 mg / m 2 / day of cyclophosphamide, wherein fludarabine is administered on days -7, -6, -5, and -4, and cyclophosphamide is administered on days -7 and -6. In some embodiments, the invention comprises a method of pre-treatment prior to administering an immune effector cell infusion, the method comprising administering to the patient a dose of about 20 mg / m 2 / day of fludarabine and a dose of approximately 514 mg / m 2 / day of cyclophosphamide, wherein fludarabine is administered on days -5 and -4, and cyclophosphamide is administered on days -5, -4, and -2. In some embodiments, the present invention includes a method of pre-treatment prior to administering an immune effector cell infusion, the method comprising administering to the patient a dose of about 20 mg / m 2 / day of fludarabine and a dose of approximately 514 mg / m 2 / day of cyclophosphamide, wherein fludarabine is administered on days -6, and -5, and cyclophosphamide is administered on days -6, -5, -4, and -3. In some embodiments, the present invention includes a method of pre-treatment prior to administering an immune effector cell infusion, the method comprising administering to the patient a dose of about 20 mg / m 2 Fludarabine and a dose of approximately 510 mg / m 2 / day of cyclophosphamide, wherein fludarabine is administered on days -6, and -5, and cyclophosphamide is administered on days -6, -5, -4, and -3. In some embodiments, the present invention includes a method of pre-treatment prior to administering an immune effector cell infusion, the method comprising administering to the patient a dose of about 20 mg / m 2 Fludarabine and a dose of approximately 500 mg / m 2 / day of cyclophosphamide, wherein fludarabine is administered on day -5, and cyclophosphamide is administered on days -5, -4, and -3. In some embodiments, the present invention includes a method of pre-treatment prior to administering an immune effector cell infusion, the method comprising administering to the patient a dose of about 20 mg / m 2 Fludarabine and a dose of approximately 540 mg / m 2 / day of cyclophosphamide, wherein fludarabine is administered on day -6, and cyclophosphamide is administered on days -6, -5, and -4. In some embodiments, the present invention includes a method of pre-treatment prior to administering an immune effector cell infusion, the method comprising administering to the patient a dose of about 20 mg / m 2 Fludarabine and a dose of approximately 500 mg / m 2 / day of cyclophosphamide, wherein fludarabine is administered on day -6, and cyclophosphamide is administered on days -6, -5, and -4. In some embodiments, the present invention includes a method of pre-treatment prior to administering an immune effector cell infusion, the method comprising administering to the patient a dose of about 20 mg / m 2 Fludarabine and a dose of approximately 500 mg / m 2 / day of cyclophosphamide, wherein fludarabine is administered on day -6, and cyclophosphamide is administered on days -6, -5, -4, and -3. In some embodiments, the present invention includes a method of pre-treatment prior to administering an immune effector cell infusion, the method comprising administering to the patient a dose of about 20 mg / m 2 Fludarabine and a dose of approximately 500 mg / m 2 / day of cyclophosphamide, wherein fludarabine is administered on day -12, and cyclophosphamide is administered on days -12, -11, and -10. In some embodiments, the present invention includes a method of pre-treatment prior to administering an immune effector cell infusion, the method comprising administering to the patient a dose of about 20 mg / m 2 Fludarabine, approximately 500 mg / m2 / day 2 Cyclophosphamide and a dose of approximately 100 mg / m 2 / day of nab-paclitaxel, wherein fludarabine is administered on day -5, cyclophosphamide is administered on days -5, -4, and -3, and nab-paclitaxel is administered on day -4. In some embodiments, the present invention comprises a method of pre-treating the patient prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 Fludarabine per day, dose approximately 540 mg / m 2 / day cyclophosphamide and a dose of approximately 71 mg / m 2 / day of nab-paclitaxel, wherein fludarabine is administered on day -6, and cyclophosphamide is administered on days -6, -5, and -4, and nab-paclitaxel is administered on day -5. In some embodiments, the present invention comprises a method of pre-treating the patient prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 Fludarabine, approximately 500 mg / m2 / day 2 / day cyclophosphamide and a dose of approximately 71 mg / m 2 / day of nab-paclitaxel, wherein fludarabine is administered on day -12, and cyclophosphamide is administered on days -12, -11, and -10, and nab-paclitaxel is administered on day -11. In some embodiments, the present invention comprises a method of pre-treating the patient prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 Fludarabine, approximately 500 mg / m2 / day 2 Cyclophosphamide and a dose of approximately 100 mg / m 2 / day of nab-paclitaxel, wherein fludarabine is administered on day -6, and cyclophosphamide is administered on days -6, -5, and -4, and nab-paclitaxel is administered on day -5. In some embodiments, the present invention comprises a method of pre-treating the patient prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 20 mg / m 2 Fludarabine per day, dose approximately 507 mg / m 2 / day cyclophosphamide and a dose of approximately 68 mg / m 2 / day of nab-paclitaxel, wherein fludarabine is administered on day -6, and cyclophosphamide is administered on days -6, -5, -4, and -3, and nab-paclitaxel is administered on day -5. In some embodiments, the present invention comprises a method of pre-treating the patient prior to infusion of immune effector cells, the method comprising administering to the patient a dose of about 137 mg / m 2 / day of nab-paclitaxel, with nab-paclitaxel being administered on day -4.

[0362] Various other interventions may be included in the methods described herein. For example, cyclophosphamide and fludarabine may cause adverse events in patients after administration. The scope of the present invention includes administering a composition to a patient to reduce certain of these adverse events. In certain embodiments, the method comprises administering normal saline to a patient. Normal saline may be administered to a patient before or after administration of cyclophosphamide and / or fludarabine, or before and after administration of cyclophosphamide and / or fludarabine. In some embodiments, normal saline is administered to a patient before administration of cyclophosphamide and / or fludarabine on each infusion day, and after administration of cyclophosphamide and / or fludarabine. In addition, adjuvants and excipients may also be administered to a patient. For example, mesna (sodium 2-mercaptoethane sulfonate). In addition, exogenous cytokines may also be administered to a patient.

[0363] In some embodiments, pretreatment is given before the first dose or subsequent doses are infused to improve the results of treatment. For example, in some aspects, pretreatment improves the efficacy of treatment with the first dose or subsequent doses, 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 subjects. In some embodiments, pretreatment treatment increases disease stabilization.

[0364] Once immune effector cells are given to a subject (e.g., a person), in some aspects, the biological activity of the engineered immune effector cell group is measured by any of a variety of known methods. The parameters used to evaluate include: engineered or natural T cells or other immune cells and antigen specific binding, in vivo mode (e.g., by imaging) or in vitro mode (e.g., by ELISA or flow cytometry). In some embodiments, the ability of engineered immune effector cells to destroy target cells can be detected by any suitable method known in the art, such as described in the cytotoxicity test of the following document, for example, 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 biological activity of immune effector cells can also be measured by measuring certain cytokines, such as CD107a, IFNγ, IL-2 and TNF expression and / or secretion. In some aspects, biological activity is by evaluating clinical results, such as tumor load or load reduction. In some aspects, a reduction in tumor markers is assessed. In some aspects, the cells are assessed for toxic outcome, persistence and / or proliferation, and / or the presence or absence of a host immune response.

[0365] Drug administration

[0366] In the present invention, the administration time and size of multiple doses of immune effector cells are generally designed to reduce the risk or minimize toxicity and / or improve efficacy, for example, by providing the subject with increased exposure to immune effector cells over time. The method includes administering a first dose, usually before one or more subsequent doses, within a specific time range between different doses.

[0367] In the case of immune effector cell therapy, giving a given "dose" includes administering a given amount or number of immune effector cells with a single composition and / or a single uninterrupted administration, such as a single injection or continuous infusion, and also includes administering a given amount or number of immune effector cells in a specific time period of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days, in divided doses, provided in multiple separate compositions or infusions. Therefore, the first dose or subsequent dose is a single or continuous administration of a specified number of immune effector cells given or started at a single time point. However, in some cases, the first dose or subsequent dose is multiple injections or infusions within a time period of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days, such as once a day for three or two days, or by multiple infusions within a day. In some aspects, the first dose of immune effector cells is administered with a single pharmaceutical composition. In some embodiments, subsequent doses of immune effector cells are administered with a single pharmaceutical composition. In some embodiments, the first dose of immune effector cells is administered with multiple compositions containing the first dose of immune effector cells as a whole. In some embodiments, subsequent doses of immune effector cells are administered with multiple compositions containing subsequent doses of immune effector cells as a whole. In some aspects, additional subsequent doses can be administered with multiple compositions within a period of no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 days.

[0368] The term "split dose" refers to a portion of a single dose administered in a single dose in divided doses, so that the single dose is administered to a subject over a period of more than 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 dose (which is the total dose of the first dose or the total dose of the subsequent dose) can be administered to the subject within 3 days, 5 days, 14 days, or 15 days. An exemplary method of separate administration includes administering 50% of the total dose on day 0 (the day on which CAR T cell therapy is administered to the patient for each round is designated as day 0) and administering 50% of the total dose on day 2. In other embodiments, 10% of the total dose can be administered on day 0, 30% of the total dose on day 2, and 60% of the total dose on day 4. In some embodiments, 10% of the total dose is administered on day 0, 30% of the total dose is administered on day 1, and 60% of the total dose is administered on day 2. In some embodiments, 1 / 3 of the total dose is administered in divided doses on day 0, 1 / 3 of the total dose is administered in divided doses on day 11, and 1 / 3 of the total dose is administered in divided doses on day 14. In some embodiments, 46% of the total dose is administered in divided doses on day 0, 18% of the total dose is administered in divided doses on day 1, and 36% of the total dose is administered in divided doses on day 2. In some embodiments, 25% of the total dose is administered in divided doses on day 0, 25% of the total dose is administered in divided doses on day 2, 25% of the total dose is administered in divided doses on day 9, and 25% of the total dose is administered in divided doses on day 13. In some embodiments, the total time span of divided administration of a single dose does not exceed 15 days.

[0369] As used herein, "first dose" is used to describe the total dose administered in the first course of treatment according to the methods described herein. This dose is equal to the total dose administered in the course of treatment in a single course of administration, or the total dose administered in the first course of treatment in multiple courses of administration. This term does not necessarily mean that the subject has never received immune effector cell therapy before receiving treatment according to the methods described herein, or that the subject has not previously received a dose of the same immune effector cells expressing the same chimeric antigen receptor or targeting the same antigen.

[0370] Relative to the first dose, the term "subsequent dose" refers to a single dose given in each course of treatment after the first dose in multiple courses of administration or the total dose in each course of treatment. In some embodiments, the method includes multiple courses of administration, i.e., administering one or more subsequent doses, wherein the first subsequent dose is also called the second course dose, the second subsequent dose is also called the third course dose, and so on. Also, in a series of doses, the previous dose is the previous dose of the subsequent dose that follows immediately thereafter. In some embodiments, the subsequent dose is administered at a time and in a manner similar to the first dose. In some embodiments, the interval between the first dose and the first subsequent dose or the second course dose and the interval between multiple subsequent doses may be the same or different, as described in detail above.

[0371] The amount or size of the dose

[0372] The dose size of the first dose and / or one or more subsequent doses is generally designed to provide improved efficacy and / or reduced risk of toxicity. In some embodiments, the number of immune effector cells in the first dose or a single subsequent dose is greater than, less than, or equal to about 1 x 10 6 Cells / kg subject body weight approximately 3x10 7 cells / kg subject body weight, such as greater than, less than, or equal to about 1×10 5 , 1.5×10 5 , 2×10 5 , 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 , 5.5×10 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 、2.5×10 9 、3×10 9 、3.5×10 9 、4×10 9 、4.5×10 9 、5×10 9 、5.5×109 , 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 cells / kg subject body weight.

[0373] In a particular embodiment, the number and / or concentration of immune effector cells refers to the number of chimeric antigen receptor (eg, 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 (PBMC) given.

[0374] 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 first dose is greater than about 1×10 6 Such cells / kg of subject body weight, such as about or at least about 1×10 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 , 2.6×107 , 2.8×10 7 , 3x10 7 , 5×10 7 , 2.5×10 8 , 1×10 9 , 1×10 10 , 5×10 10 Such cells / kg body weight, or a range between any two of the foregoing values.

[0375] In some embodiments, the number of immune effector cells administered in a subsequent dose is the same or similar or greater than the number of immune effector cells administered in the first dose in any embodiment herein, for example, equal to or about 1×10 6 , equal to or about 1×10 7 , equal to or about 1.3×10 7 , equal to or about 1.6×10 7 , equal to or about 1.9×10 7 , equal to or about 2×10 7 , equal to or about 2.2×10 7 , equal to or about 3.7×10 7 , equal to or about 5×10 7 , or equal to or about 5.1×10 7 , or equal to or about 1×10 8 , or equal to or about 2.5×10 8 , or equal to or about 1×10 9 , or equal to or about 1×10 10 , or equal to or about 5×10 10 Such cells / kg subject body weight, or a value within a range between any two of these values.

[0376] With respect to the number of immune effector cells, in some embodiments, these values ​​refer to the number of chimeric antigen receptor-expressing (e.g., CAR-expressing) cells; in other embodiments, they refer to the number of administered T cells or PBMCs or total cells.

[0377] 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 contains more than about 1×10 6 cells, chimeric antigen receptor (e.g., CAR)-expressing cells, T cells, and / or PBMCs / kg of subject body weight, e.g., 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 , 2.2×107 , 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 Such cells / kg subject body weight, or a value within a range between any two of the above values, each including the endpoints.

[0378] In some embodiments, the amount or size of the subsequent dose is sufficient to reduce the tumor burden or an indicator thereof, and / or one or more symptoms of the disease or condition. In some embodiments, the dose has a size effective to improve the survival of the subject, e.g., to improve the survival of the subject, e.g., to induce the survival of the subject, recurrence-free survival or event-free survival for at least 1 month or at least 1, 2, 3, 4 or 5 years.

[0379] In some embodiments, the tumor burden, including tumor size, tumor volume, and / or tumor mass, is reduced by at least 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 more after a subsequent dose compared to the time before the first or subsequent dose is administered.

[0380] In other embodiments, the number of immune effector cells administered in a subsequent dose is lower than the number of immune effector cells administered in the first dose.

[0381] In some aspects, the size of the first and / or subsequent doses is determined based on one or more criteria, such as the subject's response to prior treatment, e.g., chemotherapy, the subject's tumor burden, e.g., tumor volume, size or degree, extent, or metastasis type, stage and / or complications common to advanced cancer, such as cancerous pleural effusions and / or the subject's likelihood or incidence of toxic consequences, such as CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity and / or host immune response to the administered cells and / or chimeric antigen receptor.

[0382] Based on the teachings of the present invention, those skilled in the art should understand that the dosage specifically disclosed in the present invention is a safe and effective dosage obtained by the inventors after research, and those skilled in the art, such as clinical physicians, can determine the specific first dosage based on various actual conditions, such as the patient's tumor load, the patient's own physical condition and other factors; if further subsequent dosages are required, those skilled in the art can determine the subsequent dosages based on, for example, changes in tumor load after administration of immune effector cells.

[0383] Those skilled in the art should 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, a patient's tumor may express several tumor antigens, so the patient may receive CAR-T cells targeting other tumor antigens in the early stage, and then receive the CLD18A2-CAR-T cell therapy of the present invention; the patient may also receive other CLD18A2-CAR-T cell therapies different from the present invention in the early stage, and switch to the CLD18A2-CAR-T cell therapy of the present invention when the antibody specificity is not strong or the antibody is a mouse-derived rejection reaction. Therefore, in the above situation, those skilled in the art, such as clinicians, can determine the number of administrations and dosages of the CLD18A2-CAR-T cells of the present invention based on the previous treatment. For example, reference can be made to the safe and effective doses and number of administrations disclosed herein, that is, no more than about 2x10 10 No more than approximately 1x10 cells / kg subject body weight or total volume 12 cells; preferably, no more than about 2 x 10 9 No more than approximately 2 x 10 cells / kg subject body weight or total volume 11 cells; more preferably, no more than about 2.5 x 10 8 cells / kg subject body weight, or 5x10 7 cells / kg subject body weight, or 3x10 7 cells / kg subject body weight, or the total amount does not exceed 5x10 9 or about 1x10 10 cell.

[0384] In some aspects, the size of the first dose and / or subsequent doses is determined by the load of the disease or condition in the subject. For example, in some aspects, the number of immune effector cells administered with the first dose is determined based on the tumor load present in the subject at the moment before the first dose is administered. In some embodiments, the size of the first dose and / or subsequent doses is inversely proportional to the tumor load.

[0385] In some aspects, the number of immune effector cells to be administered in a subsequent dose is determined based on the tumor burden present in the subject prior to administration of the first dose. In some embodiments, for example, where the first dose results in a decrease or reduction in tumor burden, or is already below a particular threshold amount or level, such as above which indicates an increased risk of toxic outcomes, subsequent doses are increased, for example, by more than about 1x10 7 cells (e.g., total cells, receptor-expressing cells, T cells, or PBMCs) / kg body weight, e.g., greater than about 2.0 x 10 7 , 2.5x10 7 , 3.0x10 7 , 5.0x10 7 , 1.0x10 8 , 2.5x10 8 cells / kg, and / or greater than the first dose.

[0386] In some cases, even if the subject's tumor burden does not decrease after receiving the first dose, subsequent doses may be increased, for example, to more than about 1×10 7 cells (e.g., total cells, receptor-expressing cells, T cells or PBMCs) / kg body weight, e.g., more than about 2.0 x 10 7 , 2.5x10 7 , 3.0x10 7 , 5.0x10 7 , 1x10 8 , 2.5x10 8 cells / kg, and / or greater than the first dose.

[0387] T cells (e.g., CAR T cells) can be administered by any route, including intravenous injection (IV). In some embodiments, CAR T cells are administered by IV within the following time: about 3 minutes, about 4 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 60 minutes.

[0388] In some embodiments, the first dose includes immune effector cells that do not cause or reduce the possibility of toxicity or toxic results, such as cytokine release syndrome (CRS), severe CRS (sCRS), macrophage activation syndrome, tumor lysis syndrome, fever of at least 38 degrees Celsius or about 38 degrees Celsius for three or more days, CRP plasma levels are at least equal to about 20mg / dL, and / or neurotoxicity. In some aspects, the number of cells given in the first dose is determined based on the possibility that the subject will show toxicity or toxic results (such as CRS, sCRS and / or CRS related results) after giving cells. For example, in some embodiments, the possibility of developing toxic results in the subject is predicted based on tumor load. In some embodiments, the method includes detecting or evaluating toxic results and / or tumor load before administering the dose.

[0389] In some embodiments, a subject is administered a subsequent dose when a biochemical indicator or other indicator of a toxic outcome after the first dose, for example, a serum level of a CRS indicator, has not increased above a given level, for example, an acceptable level, for example, greater than or equal to or about 10, 15, 20, 25, 50, 75, or 100 times the serum level of the indicator at the time before the first dose was administered, or has increased above an acceptable level, but has dropped to equal to or below an acceptable level. In certain aspects, if the indicator drops below an acceptable level within 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, or 40 days of the first dose, a subsequent dose is administered, but if the level of the indicator does not drop below an acceptable level within that time period, a subsequent dose is not administered.

[0390] In some embodiments, subsequent doses are administered if the clinical risk of developing cytokine release syndrome (CRS), macrophage activation syndrome or tumor lysis syndrome, or neurotoxicity is not present or has passed or has resolved after the first dose, such as after a critical window in which such events typically have resolved and / or are unlikely to occur, for example in about 60%, 70%, 80%, 90% or 95% of subjects with a particular disease or condition.

[0391] In some embodiments, whether to administer a subsequent dose, when to administer a subsequent dose, and / or the number of cells administered in a subsequent dose is determined based on the presence, absence, or extent of an immune response or a detectable immune response in a subject to the cells of the first dose or the chimeric antigen receptors expressed by them. In some aspects, a subsequent dose of cells containing receptors expressing the cells of the first dose will not be administered to a subject with a detectable host adaptive immune response or an immune response that has been established or has reached a certain level, stage, or extent.

[0392] Time of administration

[0393] In some embodiments, the timing of subsequent dose administration is calculated from the completion of the first dose (the day when the total dose infusion of the first dose is completed is set as day 0).

[0394] In some embodiments, a subsequent dose is administered when the serum level of a factor indicative of CRS in the subject is no more than about 10, 25, 50, or 100 times the serum level of that indicator in the subject at a time prior to administration of the first dose.

[0395] In some embodiments, a result associated with CRS in a subject (e.g., a serum factor associated with or indicative of CRS) or a clinical sign or symptom thereof such as fever, hypoxia, hypotension, or neurological disorder has reached a peak level, and a subsequent dose is administered after the first dose begins to decline. In some embodiments, a subsequent dose is administered when a decrease is observed compared to the highest level of such a result after administration, or when the level decreases after reaching the maximum value or level of the result after administration.

[0396] In some embodiments, when the level of an indicator of a toxic outcome (e.g., a serum indicator of CRS) drops to less than about 25 times the level of the indicator at the time before the first dose, a subsequent dose is administered. In some aspects, a subsequent dose is administered when the subject does not show CRS or does not show severe CRS.

[0397] In some aspects, subsequent doses are given at a time point when the tumor load in the patient decreases compared to the tumor load before the first dose is given. In some embodiments, subsequent doses are given after the first dose is given, when the tumor load or its indicator, such as the volume or number or percentage of disease (e.g., tumor) cells in the subject's blood, other body fluids, organs or tissues, or the tumor size has been reduced 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.

[0398] In some embodiments, a subsequent dose is given when the subject's disease or illness does not recur after the response to the first dose or the previous dose is reduced. In some embodiments, reducing tumor load is indicated by the reduction of one or more factors, such as the expression level of tumor markers in the subject or its body fluids or organs or tissues, the load or quantity of tumor cells, the quality or volume of the tumor, the degree or degree of metastasis. In some embodiments, recurrence is generally or one or more factors, or in tumor load. In some aspects, a subject, a disease burden or its factor recurs compared to the lowest point measured or reached after the first or previous administration, but still less than the first dose before the moment to give a subsequent dose. In some embodiments, when tumor load or its indicator factors do not change, for example, when tumor load has been prevented from increasing, a subsequent dose is given to the subject.

[0399] In some embodiments, the subsequent dose is administered when the host adaptive immune response has not been detected, has not yet been established, or has not yet reached a certain level, extent or stage. In some aspects, the subsequent dose is administered before the subject's memory immune response develops.

[0400] 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 subsequent dose is administered no more than about 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 31 days, 32 days, 33 days, 34 days, 35 days, 36 days, 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 after the first dose is administered. In some embodiments, the subsequent dose is administered no more than about 44 days, 45 days, 46 days, 47 days, or 48 days after the first dose is administered.

[0401] In some embodiments, after a subsequent dose (i.e., the first subsequent dose, or referred to as the second course of treatment dose) is administered, an additional or further subsequent dose is administered, for example, a second subsequent dose (third course of treatment dose), a third subsequent dose (fourth course of treatment dose), and so on. In some aspects, an additional subsequent dose is administered at least about 21 days and less than about 80 days after a previous subsequent dose (e.g., the first subsequent dose). In some embodiments, an exemplary dosage regimen includes a schedule of when or about when chimeric antigen receptor expressing cells (e.g., CAR expressing cells, such as CAR T cells) are administered. In some embodiments, an additional dose (second subsequent dose) is administered about 49 days after the first subsequent dose, and so on. In some embodiments, the subsequent dose administered does not exceed about 21 to about 80 days, about 25 to about 60 days, or 25 to 55 days after the subsequent amount adjacent to the previous is administered. In some embodiments, the later subsequent dose is administered no 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 the administration of the adjacent online subsequent dose.

[0402] In any embodiment, in some cases, the method includes administering a first dose or a previous dose and a subsequent dose, and in other cases includes administering a subsequent dose to a subject who has previously received a first dose or a previous dose, and the subject has previously received a first dose or a previous dose, but does not include administering the first dose or the previous dose itself. Therefore, in some cases, the method involves administering consolidation therapy, such as by administering a consolidation subsequent dose to a subject of a chimeric antigen receptor-expressing (e.g., CAR-expressing) cell that has previously received a dose (e.g., a reduction dose). In some aspects, receptor expression, such as a previous dose of a CAR-expressing cell, has been sufficient to reduce the disease or condition load in the subject, so that the efficacy and / or safety of the cells administered in subsequent doses are improved relative to the dose administered to a subject who has not received the first dose.

[0403] In some embodiments, the tumor burden, including tumor markers, tumor size, tumor volume, and / or tumor mass, is reduced by at least 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 more after a subsequent dose compared to the time before the first or subsequent dose is administered.

[0404] Host immune response to infused cells

[0405] In some embodiments, one or more doses, such as subsequent doses, are not present in the subject's immune response (e.g., adaptive or specific immune response to transgenic receptors or cells), undetectable, or given when it is undetectable above a certain level. The presence or extent of a specific immune response to a transgenic is generally related to the immunogenicity of the receptor (e.g., CAR or transgenic TCR expressed by cells) and / or the time when the subject is exposed to the cell. 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 days, 35 days, or 42 days after the subject first contacts a cell expressing the receptor. Therefore, in some embodiments, a subsequent dose is administered before an immune response, adaptive or specific immune response, detectable immune response, and / or memory response to a chimeric antigen receptor or cell has been developed in the subject. In this regard, compared with other methods of administering subsequent doses at a later time point compared to the previous or first dose, the ability of subsequent doses to proliferate and / or persist in the subject is improved. The methods include detecting the presence or absence or level of such an immune response or an indicator thereof, for example, after administration of a first or subsequent dose and prior to administration of a subsequent or next subsequent dose.

[0406] In some embodiments, when and / or whether to give a subsequent dose depends on whether the subject shows such an immune response or its detectable reading, such as a detectable specific or adaptive host immune response specific to a cell or chimeric antigen receptor, such as a CAR expressed by a first dose of cells, and / or whether such a reaction is detected at a certain level. In some embodiments, in the case of detecting such a reaction, a subsequent dose is not given to the subject. When the subject does not show specificity or adaptability (such as humoral or cell-mediated) immune response to a receptor (such as CAR expressed by a first dose of cells), or does not show such a reaction or indicator at a detectable level or above an acceptable level, a subsequent dose is given. In some aspects, when a subsequent dose is given, the subject shows a reduced humoral or cell-mediated immune response to the CAR expressed by the cells of the first dose compared to when the initial dose is larger.

[0407] In any of the above embodiments, detectable immune response refers to any detectable amount by any of the many known methods for assessing specific immune responses to specific antigens and cells.For example, in some embodiments, by performing ELISPOT, ELISA or cell-based antibody detection methods (e.g., by flow cytometry) on the serum of the subject to detect specific binding and / or neutralization of antigens present on cells, such as binding to the epitope of a chimeric antigen receptor, such as the presence of antibodies of CAR to detect a specific type of immune response.In some such assays, the isotype of the antibody detected is determined and it can be indicated whether the type of response and / or response is a memory response.

[0408] In some aspects, the presence or absence of such a host immune response and / or its amount, extent or degree is detected or measured, e.g., after administration of an initial dose or subsequent doses.

[0409] In some embodiments, the method reduces the disease or condition load to a greater extent, such as the number of tumor cells, the size of the tumor, the duration of patient survival or event-free survival, and a longer time period, compared to the reduction observed with a comparison method using an alternative dosing regimen, such as where the subject receives a single dose (e.g., a single large dose of cells), for example, with the total number of cells uniformly given with the first dose and subsequent doses instead of a single dose, or with multiple large doses or multiple doses given less than about 21 or more than about 80 days apart from each other, and longer time periods. In some aspects, the subject's survival, survival over a certain time period, survival range, presence or duration of event-free or symptom-free survival, or recurrence-free survival are assessed.

[0410] In some aspects, the disease load is measured or detected before the first dose is administered, after the first dose is administered but before the subsequent dose is administered, and / or after the subsequent dose is administered. In the case of multiple subsequent doses, the disease load in some embodiments can be measured before or after any subsequent dose or between subsequent doses.

[0411] In some embodiments, after administration of the first dose, the load 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 aspects, administration of a subsequent dose further reduces disease burden, tumor burden, for example by 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% reduction in burden compared to the time before administration of the subsequent dose or compared to the time before the initial dose.

[0412] In some embodiments, compared with other methods, the event-free survival rate or overall survival rate of the subject is improved by this method. For example, in some embodiments, the progression-free survival rate or probability of the subject treated by this method 1 month after the first dose is greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90%, or greater than about 95%. In some aspects, the overall survival rate is greater than about 10%, greater than about 20%, greater than about 30%, greater than about 40%, greater than about 50%, greater than about 60%, greater than about 70%, greater than about 80%, greater than about 90% or greater than about 95%. In some embodiments, the subject treated with this method shows no progression event survival, no recurrence survival or survival of at least about 1 month, or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years. In some embodiments, the time to progression is improved, for example, greater than or about 1 month, or at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 months, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years.

[0413] In some embodiments, the treatment by this method reduces the probability of relapse compared to other methods. For example, in some embodiments, the probability of relapse or progression 1 month after the first dose is less than about 80%, less than about 70%, less than about 60%, less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%.

[0414] Cell exposure and persistence

[0415] In some embodiments, the dosage and / or the timing thereof are designed to promote exposure of the subject to the cells, for example by promoting their proliferation and / or persistence over time.

[0416] In some embodiments, the method provided increases the subject's exposure to the administered cells (e.g., the number of cells or duration increased over time) and / or improves the efficacy and treatment results in immune cell therapy. In some respects, the advantage of the method is that, compared with other methods, the exposure degree of cells expressing chimeric antigen receptors (e.g., CAR-expressing cells) is greater and / or improves the treatment results to a longer extent. These results may include the patient's survival and relief, even in individuals with severe tumor burden.

[0417] In some embodiments, the presence and / or amount of cells (e.g., CAR-expressing cells) expressing chimeric antigen receptors in a subject after the first dose and / or subsequent doses are detected. In some aspects, quantitative PCR (qPCR) is used to assess the amount of cells (e.g., CAR-T) expressing chimeric antigen receptors in the blood or serum or organs or tissues (e.g., disease sites) of the subject. In some aspects, persistence is quantified as a copy of the DNA or plasmid encoding a receptor, such as CAR, per microgram of DNA, or as a sample per microliter, for example, blood or serum receptor expression, such as the number of CAR expressing cells, or the total number of peripheral blood mononuclear cells (PBMC) or leukocytes or T cells per microliter of sample.

[0418] In some embodiments, the cells are detected in the subject after administration of the first dose or at least on day 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. In some aspects, the cells are detected at or at least 2, 4, or 6 weeks, or 3, 6, or 12, 18, or 24, or 30, or 36 months, or 1, 2, 3, 4, 5, or more years after administration of the first or subsequent doses.

[0419] In some embodiments, the total dose of cells is administered as a single dose, as opposed to by an alternative method, such as a method involving administration of a single dose, e.g., a method containing more cells than the initial dose, a subsequent dose of cells is administered to a subject who did not receive the initial dose, and / or a subsequent dose is administered at a time outside a specified time window, e.g., later than or subsequent to the time specified for the subject's immune response to the receptor (e.g., CAR) to occur.

[0420] Exposure, for example, indicating cell number, proliferation, and / or persistence, can be expressed according to the maximum number of cells to which the subject is exposed, the duration of detectable cells above a certain number or percentage, the area under the cell number curve over time, and / or combinations thereof, and indices thereof. Such results can be evaluated using known methods such as qPCR to detect the number of copies of nucleic acids encoding chimeric antigen receptors, and / or flow cytometry assays that typically use antibodies specific for the receptor to detect cells expressing the receptor, compared to the total amount of nucleic acids or DNA in a particular sample, such as blood or serum. Cell-based assays can also be used to detect the number or percentage of functional cells, such as those capable of binding and / or neutralizing and / or inducing a response (e.g., a cytotoxic response) to a disease or disorder or a cell expressing an antigen recognized by the receptor.

[0421] In some aspects, the subject's exposure to the increase of cells includes increasing the proliferation of cells. In some embodiments, after giving the first dose and / or giving subsequent doses, receptor-(eg CAR-) expressing cells proliferate in the subject. In some aspects, with other methods, such as involving giving cells with a single dose, giving a larger first dose, giving subsequent doses without giving those methods of the first dose, and / or giving subsequent doses before or after a specified time window or time point, so that, for example, an immune response is generated before giving subsequent doses, the method results in greater cell proliferation.

[0422] In some aspects, the method results in high in vivo proliferation of the cells administered, such as measured by flow cytometry. In some aspects, cells at a peak ratio are detected. For example, in some embodiments, after the first or subsequent administration, at a peak or maximum level in the subject's blood or disease site or its leukocyte portion (such as a PBMC portion or a T cell portion), 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 cells express chimeric antigen receptors, such as CAR.

[0423] In some embodiments, the method results in a maximum concentration of at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 1500, 2000, 5000, 10000 or 15000 copies of or nucleic acid encoding the receptor, e.g., CAR per microgram of DNA, or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9 of receptor expression, e.g., CAR expressing cells per total number of peripheral blood mononuclear cells (PBMCs), total number of monocytes, total number of T cells, or total number of microliters in the blood or serum or other bodily fluid or organ or tissue of the subject. In some embodiments, cells expressing the receptor are detected as at least 10%, 20%, 30%, 40%, 50% or 60% of the total PBMCs in the blood of the subject, and / or at this level for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 24, 36, 48, or 52 weeks after the first or subsequent administration, or for 1, 2, 3, 4 or 5 years or more after such administration.

[0424] In some aspects, the method results in, for example, an increase in the copies of a nucleic acid encoding a chimeric antigen receptor, e.g., CAR, per microgram of DNA by at least 2-fold, at least 4-fold, at least 10-fold, or at least 20-fold in the serum of the subject.

[0425] In some embodiments, cells expressing the receptor can be detected in the blood or serum of the subject, for example by a specified method, such as qPCR or flow cytometry-based detection method, 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, 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 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 or more weeks after administration of the first or subsequent doses.

[0426] In some aspects, at least about 1×10 2 , at least about 1×10 3 , at least about 1×10 4 , at least about 1×10 5 , or at least about 1×10 6 or at least about 5×10 6 or at least about 1×107 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-expressing, e.g., 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, are detectable or present in a subject or a fluid, tissue, or compartment thereof, e.g., in the blood, e.g., peripheral blood, or a disease site thereof. In some embodiments, the number or concentration of cells is detectable 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, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 2 or 3 years after administration of the first dose or after administration of subsequent doses. Such cell numbers can be detected by flow cytometry-based or quantitative PCR-based methods and extrapolated to total cell number using known methods.

[0427] In some aspects, the copy number of the nucleic acid encoding the chimeric antigen receptor, e.g., vector copy number, per 100 cells, e.g., in the peripheral blood or bone marrow or other compartments, is at least 0.01, at least 0.1, at least 1, or at least 10, as measured by immunohistochemistry, PCR and / or flow cytometry, when the cells are administered, 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, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months, or at least 2 or 3 years after the first or subsequent doses. In some embodiments, the number of copies of the vector expressing the receptor, e.g., CAR, per microgram of genomic DNA 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 at about 1 week, about 2 weeks, about 3 weeks, or at least about 4 weeks after administration of the first or subsequent doses of the receptor expressing, e.g., CAR expressing cells, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 2 or 3 years after such administration.

[0428] In some aspects, the receptor, e.g., CAR, expressed by the cells can be detected in the subject, its blood and / or its disease site by quantitative PCR (qPCR) or by flow cytometry after administration of the cells, e.g., 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 initiation of administration of the first dose or subsequent doses or subsequent subsequent doses.

[0429] Chimeric antigen receptor expressed by cells

[0430] The cells typically express chimeric antigen receptors, including antigen receptors such as functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs) and other antigen binding receptors such as transgenic T cell receptors (TCRs), T cell fusion proteins (TFPs), and T cell antigen couplers (TACs).

[0431] Exemplary antigen receptors, including CARs, and methods for engineering and introducing receptors into cells, include, for example, those described in Chinese Patent Application Publication Nos. CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN106397593A, CN106467573A, International Patent Application Publication Nos. WO2018006882A1, WO2015172339A8.

[0432] The chimeric receptor includes a chimeric antigen receptor (CAR). A chimeric receptor, such as a CAR, generally includes an extracellular antigen binding domain, such as a part of an antibody molecule, typically a variable heavy (VH) chain region and / or a variable light (VL) chain region of an antibody, for example, a scFv antibody fragment. In some embodiments, the chimeric receptor can recognize splice variant 1 (CLD18A1, CLD18.1) of claudin 18 (CLD18) (Accession Nos. NP_057453, NM016369) or a variant thereof, such as comprising the amino acid sequence shown in SEQ ID NO:34, 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:34, and can recognize splice variant 2 (CLD18A2, CLD18.2) (Accession Nos. NM_001002026, NP_001002026) or a variant thereof, such as comprising the amino acid sequence shown 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. NO:33 shows an amino acid sequence of at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity. In some embodiments, the chimeric receptor specifically recognizes CLD18A2 but not CLD18A1.

[0433] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) comprises a scFv antibody fragment, e.g., comprising the HCDR or LCDR amino acid sequence shown in SEQ ID NO: 1 or 2 or 3 or 4 or 5 or 6 or 7, or a variant thereof, 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: 1 or 2 or 3 or 4 or 5 or 6 or 7.

[0434] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) comprises a scFv antibody fragment, for example, comprising the HCDR or LCDR amino acid sequence shown in SEQ ID NO: 8 or 9 or 10 or 11 or 12 or 13, or a variant thereof, 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: 8 or 9 or 10 or 11 or 12 or 13.

[0435] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) comprises a scFv antibody fragment, for example, comprising the HCDR or LCDR amino acid sequence shown in SEQ ID NO: 8 or 9 or 10 or 11 or 12 or 13, or a variant thereof, 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: 8 or 9 or 10 or 11 or 12 or 13.

[0436] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) includes a scFv antibody fragment, such as a heavy chain or light chain variable region amino acid sequence shown in SEQ ID NO: 14 or 15 or 16 or 17 or 18 or 19, or a variant thereof, 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: 14 or 15 or 16 or 17 or 18 or 19.

[0437] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) includes a scFv antibody fragment, for example, comprising a heavy chain or light chain variable region amino acid sequence shown in SEQ ID NO: 20 or 21 or 22 or 23, or a variant thereof, 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 with SEQ ID NO: 20 or 21 or 22 or 23.

[0438] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) includes a scFv antibody fragment, for example, comprising a heavy chain or light chain variable region amino acid sequence shown in SEQ ID NO: 20 or 21 or 22 or 23, or a variant thereof, 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 with SEQ ID NO: 20 or 21 or 22 or 23.

[0439] In some embodiments, the chimeric antigen receptor (e.g., CAR), for example, comprises the amino acid sequence shown in SEQ ID NO: 24 or 25 or 26 or 27 or 28 or 29 or 30 or 31 or 32, or a variant thereof, 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: 24 or 25 or 26 or 27 or 28 or 29 or 30 or 31 or 32.

[0440] In some embodiments, the antibody portion of the chimeric antigen receptor (eg, CAR) further includes a linking sequence, which may be or include at least a portion of an immunoglobulin constant region or a variant thereof or a modified form thereof, such as a hinge region, for example, an IgG4 hinge region and / or CH1 / CL and / or Fc region. In some embodiments, the constant region or portion is human IgG, such as IgG4 or IgG1. In some aspects, the portion of the constant region serves as a linking sequence between an antigen-recognition portion, such as scFv, and a transmembrane domain. The linking sequence may have a length that provides increased cell responsiveness after antigen binding compared to the absence of the linking sequence. In some instances, the linking sequence length is or is about 12 amino acids or is no more than 12 amino acids in length. Exemplary linking sequences include those having a length 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, and including integers between any endpoints of the above-listed ranges. In some embodiments, the linking sequence region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less. Exemplary linking 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.

[0441] The antigen recognition domain is generally connected to one or more intracellular signal transduction parts, for example, in the case of CAR, by the antigen receptor complex (such as TCR complex) simulation activation of the signal transduction part, and / or by the signal of another cell surface receptor.Therefore, in some embodiments, antigen binding components (e.g., antibodies) are connected with one or more transmembrane and intracellular signal transduction domains.In some embodiments, the transmembrane domain is fused to the extracellular domain.In one embodiment, a transmembrane domain of one of the domains in a natural associated receptor (such as CAR) is used.In some cases, the transmembrane domain is selected or modified by amino acid substitution to avoid the domain from being bound to the transmembrane domain of the same or different surface membrane proteins, so as to minimize the interaction with other members of the receptor complex.

[0442] In some embodiments, the membrane-spanning domain is derived from a natural or synthetic source. When the source is a natural source, in some aspects, the domain is derived from any membrane-bound or transmembrane protein. The membrane-spanning region includes α, β or ζ chains derived from T-cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD 16, CD22, CD33, CD37, CD64, CD80, CD86, CD 134, CD137, CD 154, and / or the membrane-spanning region includes those (i.e., including at least their membrane-spanning regions) of its functional variants (e.g., those that substantially retain its structural portion (e.g., membrane-spanning portion), properties). In some embodiments, the membrane-spanning domain is a membrane-spanning domain derived from CD4, CD28 or CD8, for example, CD8α or its functional variant. Alternatively, in some embodiments, the membrane-spanning domain is synthetic. In some aspects, the synthetic membrane-spanning domain mainly includes hydrophobic residues such as leucine and valine. In some aspects, a trimer of phenylalanine, tryptophan and valine will appear at each end of the synthetic transmembrane domain. In some embodiments, the connection occurs via a linker, a spacer and / or a transmembrane domain.

[0443] The intracellular signal transduction domain includes those that simulate or approximate the signal of a natural antigen receptor, the signal of a co-stimulatory receptor combined with such a receptor, and / or the signal of a separate co-stimulatory receptor. In some embodiments, there is a short oligopeptide or polypeptide linker, for example, a linker of 2-10 amino acids in length, for example, comprising glycine and serine, for example, a linker of a glycine-serine doublet, and a connection is formed between the cytoplasmic signal transduction domain and the transmembrane domain of the CAR.

[0444] The receptor, for example, CAR, generally includes at least one or more intracellular signal transduction parts. In some embodiments, the receptor includes intracellular components of the TCR complex, such as TCR CD3+ chains that mediate T- cell activation and cytotoxicity, for example, CD3 ζ chains. Therefore, in some aspects, the antigen binding moiety is connected to one or more cell signal transduction modules. In some embodiments, the cell signal transduction module includes a CD3 transmembrane domain, a CD3 intracellular signal transduction domain, and / or other CD transmembrane domains. In some embodiments, the receptor, for example, CAR, also includes one or more other molecules, such as Fc receptor γ, CD8, CD4, CD25 or CD16. For example, in some aspects, CAR or other chimeric receptors include chimeric molecules between CD3 ζ (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25 or CD16.

[0445] In some embodiments, when CAR or other chimeric receptors bind, the cytoplasmic domain of the receptor or the intracellular signal transduction domain activates at least one of the normal effector functions or responses of immune cells, such as T cells engineered to express CAR. For example, in some cases, CAR induces the function of T cells, such as cytolytic activity or helper T cell activity, such as the secretion of cytokines or other factors. In some embodiments, a truncated portion of the intracellular signal transduction domain of an antigen receptor portion or a co-stimulatory molecule is used to replace the complete immunostimulatory chain, for example, if it transduces an effector function signal. In some embodiments, the intracellular signal transduction domain includes the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects, also includes those co-receptors present in natural conditions that act in concert with these receptors to start signal transduction after antigen receptor engagement.

[0446] In the case of natural TCR, full activation generally requires not only signal transduction by TCR, but also costimulatory signals. Therefore, in some embodiments, in order to promote full activation, components for generating a second or costimulatory signal are also included in the CAR. In other embodiments, CAR does not include components for generating costimulatory signals. In some aspects, other CARs are expressed in the same cell and are provided for generating components of a second or costimulatory signal.

[0447] In some aspects, T cell activation is described as being mediated by two types of cytoplasmic signal transduction sequences: those (first cytoplasmic signal transduction sequences) that initiate antigen-dependent first activation by TCR, and those (second cytoplasmic signal transduction sequences) that act in an antigen-independent manner to provide a second or costimulatory signal. In some aspects, the CAR includes one or both of such signal transduction components.

[0448] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) further includes a signal peptide, such as a signal peptide comprising CD8 or a variant thereof, such as an amino acid sequence comprising the amino acid sequence shown in SEQ ID NO:35, 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:35.

[0449] In some aspects, the CAR includes a major cytoplasmic signal transduction sequence that regulates the initial activation of the TCR complex. The first cytoplasmic signal transduction sequence acting in a stimulatory manner may include a signal transduction motif, which is known to be an immune receptor tyrosine-based activation motif or ITAM. Examples of ITAMs include the first cytoplasmic signal transduction sequence, which includes those derived from: TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cytoplasmic signal transduction molecule in the CAR includes a cytoplasmic signal transduction domain, which is partially or derived from a sequence of CD3ζ.

[0450] In some embodiments, the CAR includes a transmembrane portion and / or a signal transduction domain of a co-stimulatory receptor, such as CD28, CD137, OX40, DAP10, and ICOS. In some aspects, the same CAR includes both activation and co-stimulatory portions.

[0451] In some embodiments, the activation domain is included in a CAR, and the costimulatory component is provided by another CAR that recognizes another antigen. In some embodiments, the CAR includes activation or stimulation CAR, costimulatory CAR, which are all expressed on the same cell (see WO2014 / 055668). In some aspects, the cell includes one or more stimulation or activation CAR and / or costimulatory CAR. In some embodiments, the cell also includes inhibitory CAR (iCAR, see Fedorov et al., Sci. Transl. Medicine, 5 (215) (December 2013)), for example, identifying CAR related to and / or specific to a disease or illness, thereby reducing or inhibiting the activation signal delivered by the disease-targeted CAR by binding to its ligand by inhibitory CAR, for example, reducing off-target effects.

[0452] In some embodiments, the intracellular signal transduction portion of the chimeric antigen receptor, such as CAR, comprises a CD3 ζ intracellular domain and a costimulatory signal transduction region. In certain embodiments, the intracellular signal transduction domain comprises a CD28 transmembrane and a signal transduction domain, which is connected to a CD3 (e.g., CD3-ζ) intracellular domain. In some embodiments, the intracellular signal transduction domain comprises a chimeric CD28 and / or CD137 (4-1BB, TNFRSF9) costimulatory domain, which is connected to a CD3 ζ intracellular domain.

[0453] In some embodiments, the CAR encompasses one or more, e.g., two or more, costimulatory domains and activation domains, e.g., the initial activation domain in the cytoplasmic portion. Exemplary CARs include the intracellular portion of CD3-ζ, CD28 and CD137.

[0454] In some cases, CAR is referred to as the first, second and / or third generation CAR. In some aspects, the first generation CAR is a CAR that only provides CD3 chain induction signals when antigens bind; In some aspects, the second generation CAR is a CAR that provides such signals and costimulatory signals, such as including CARs from intracellular signal transduction domains of costimulatory receptors (such as CD28 or CD137); In some aspects, the third generation CAR is a CAR including multiple costimulatory domains of different costimulatory receptors.

[0455] In some embodiments, the chimeric antigen receptor includes an extracellular portion containing an antibody or antibody fragment. In some aspects, the chimeric antigen receptor includes an extracellular portion containing an antibody or fragment and an intracellular signal transduction domain. In some embodiments, the antibody or fragment includes scFv, and the intracellular domain includes ITAM. In some aspects, the intracellular signal transduction domain includes the signal transduction domain of the ζ chain of the CD3-ζ chain. In some embodiments, the chimeric antigen receptor includes a transmembrane domain that connects the extracellular domain and the intracellular signal transduction domain. In some aspects, the transmembrane domain includes the transmembrane portion of CD28. In some embodiments, the chimeric antigen receptor contains the intracellular domain of a T cell costimulatory molecule. The extracellular domain and the transmembrane domain can be directly or indirectly connected. In some embodiments, the extracellular domain and the transmembrane pass through a connecting sequence. In some embodiments, the receptor includes the extracellular portion of a molecule derived from a transmembrane domain, such as the extracellular portion of CD28. In some embodiments, the chimeric antigen receptor comprises an intracellular domain derived from a T cell co-stimulatory molecule or its functional variant, such as between a transmembrane domain and an intracellular signal transduction domain. In some aspects, the T cell co-stimulatory molecule is CD28 or 41BB. For example, in some embodiments, CAR contains an antibody, such as an antibody fragment, is or contains a transmembrane portion of CD28 or a transmembrane domain of its functional variant, and a signal transduction portion or a functional variant of CD28 containing a signal transduction portion, and a signal transduction portion or a functional variant of CD3ζ. In some embodiments, CAR contains an antibody, such as an antibody fragment, is or contains a transmembrane portion or a functional variant of CD28 containing a transmembrane domain, and a signal transduction portion or a functional variant of CD137 containing a signal transduction portion, and a signal transduction portion or a functional variant of CD3ζ. In some embodiments, the receptor further includes a connection sequence, which includes a portion of an Ig molecule (such as a human Ig molecule), such as an Ig hinge, such as an IgG4 hinge, such as only a hinge connection sequence. In some embodiments, the chimeric antigen receptor has: (i) an antibody that specifically recognizes a tumor antigen, a transmembrane region of CD28 or CD8, a costimulatory signaling domain of CD28, and CD3ζ; or (ii) an antibody that specifically recognizes a tumor antigen, a transmembrane region of CD28 or CD8, a costimulatory signaling domain of CD137, and CD3ζ; or (iii) an antibody that specifically recognizes a tumor antigen, a transmembrane region of CD28 or CD8, a costimulatory signaling domain of CD28, a costimulatory signaling domain of CD137, and CD3ζ.

[0456] In some embodiments, the transmembrane domain of the chimeric antigen receptor (e.g., CAR) is or includes the transmembrane domain of human CD28 (e.g., Accession No. P01747.1) or a variant thereof, such as a transmembrane domain comprising the amino acid sequence shown in SEQ ID NO: 39, 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: 39.

[0457] In some embodiments, the transmembrane domain of the chimeric antigen receptor (e.g., CAR) is or includes the transmembrane domain of human CD8 or a variant thereof, such as a transmembrane domain comprising the amino acid sequence shown 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.

[0458] In some embodiments, the intracellular signal transduction portion of the chimeric antigen receptor (eg, CAR) comprises an intracellular co-stimulatory signal transduction domain of human CD28 or a functional variant or portion thereof. For example, the intracellular signal transduction domain may comprise an amino acid sequence shown in SEQ ID NO: 41, or an amino acid sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 41.

[0459] In some embodiments, the intracellular domain comprises an intracellular co-stimulatory signal transduction domain of CD137 (e.g., (Accession No. Q07011.1) or a functional variant or a portion thereof, such as the amino acid sequence shown in SEQ ID NO: 47, 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: 47.

[0460] In some embodiments, the intracellular signal transduction domain of the chimeric antigen receptor (eg, CAR) comprises a human CD3 ζ stimulatory signal transduction domain or a functional variant thereof. For example, in some embodiments, the intracellular signal transduction domain comprises an amino acid sequence as shown in SEQ ID NO: 43 or an amino acid sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 43.

[0461] In some aspects, the chimeric antigen receptor comprises a hinge, such as a CD8 hinge. For example, in some embodiments, the CD8 hinge comprises an amino acid sequence as 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.

[0462] For example, in some embodiments, CAR includes an antibody, such as an antibody fragment, including scFv, a connecting sequence, such as a connecting sequence containing a portion of an immunoglobulin molecule, such as a hinge region and / or one or more heavy chain molecule constant regions, such as a connecting sequence containing an Ig-hinge, a transmembrane domain containing all or part of a CD28-derived transmembrane domain, a CD28-derived intracellular signaling domain, and a CD3ζ signaling domain. In some embodiments, CAR includes an antibody or fragment, such as an scFv, a connecting sequence, such as any connecting sequence containing an Ig-hinge, a CD28-derived transmembrane domain, a CD137-derived intracellular signaling domain, and a CD3ζ-derived signaling domain.

[0463] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides including provided receptors and other polypeptides (e.g., linkers or peptides) can include amino acid residues, including natural and / or non-natural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, and phosphorylation. In some aspects, the polypeptide may include modifications to the original or native sequence, as long as the protein retains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or may be accidental, such as by mutations in the host producing the protein or errors caused by PCR amplification.

[0464] In some embodiments, the methods described herein include administering one adoptive cell or immune effector cell to the subject. In some embodiments, the methods described herein include administering two or more adoptive cells or immune effector cells for the same tumor antigen to the subject in different courses of treatment. In some embodiments, the methods described herein include administering two or more adoptive cells or immune effector cells for different tumor antigens to the subject in different courses of treatment. In some embodiments, the methods described herein include administering two or more adoptive cells or immune effector cells for the same epitope of the same tumor antigen to the subject in different courses of treatment. In some embodiments, the methods described herein include administering two or more adoptive cells or immune effector cells for different epitopes of the same tumor antigen to the subject in different courses of treatment. In some embodiments, the methods described herein include administering two or more adoptive cells or immune effector cells to the subject in different courses of treatment to treat tumors at the same site. In some embodiments, the methods described herein include administering two or more adoptive cells or immune effector cells to the subject in different courses of treatment to treat tumors at different sites. In some embodiments, at least one of the adoptive cells or immune effector cells used in the methods described 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 described herein is a CAR-T cell targeting CLD18A2 as described herein.

[0465] The chimeric antigen receptor (eg, CAR) expressed by cells given to subjects at various doses generally recognizes or specifically binds to the disease or disorder or cells being treated, expressed, associated therewith and / or specific molecules. When specifically binding molecules such as antigens, receptors generally deliver immunostimulatory signals (eg, signals transduced by ITAMs) to cells, thereby promoting the immune response of targeted diseases or disorders. For example, in some embodiments, the cells in the first dose express specifically binding to antigens expressed by cells or tissues of diseases or disorders or CARs associated with diseases or disorders.

[0466] The receptor (e.g., CAR) expressed by the cell in a subsequent dose typically specifically binds to the same antigen as the CAR of the first dose, and is typically a receptor identical to the receptor of the cell of the first dose or an extremely similar receptor. In some embodiments, the receptor on the cell of the subsequent dose is identical to the receptor in the cell of the first dose or has a large homology with the receptor.

[0467] In some embodiments, the CAR expressed by the cells of the subsequent dose comprises the same scFv, the same signal transduction domain and / or the same connection as the CAR expressed by the cells of the first dose. In some embodiments, it also comprises 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.

[0468] Immune effector cells

[0469] In the present invention, the cells expressing chimeric receptors and provided by the methods described herein are immune effector cells. The cells are generally eukaryotic cells, such as mammalian cells, and are generally human cells. In some embodiments, cells are derived from blood, bone marrow, lymph or lymphoid organs, and are cells of the immune system, such as cells of innate or adaptive immunity, for example, bone marrow or lymphoid cells, including lymphocytes, generally T cells and / or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSC). Cells are generally primary cells, such as those directly separated from a subject and / or separated and frozen from a subject. In some embodiments, cells include one or more subgroups of T cells or other cell types, such as full T cell populations, CD4+ cells, CD8+ cells and their subgroups, such as those defined by function, activation state, maturation, differentiation potential, amplification, recirculation, positioning, and / or persistence, antigen specificity, antigen receptor type, presence in a specific organ or compartment, markers or cytokine secretion profiles, and / or degree of differentiation. Regarding the subject to be treated, the cell can be allogeneic and / or autologous. The method includes existing methods. In some aspects, such as for existing technologies, the cell is pluripotent and / or specialized, such as stem cells, such as induced pluripotent stem cells (iPSC). In some embodiments, the method includes isolating cells from the subject, preparing, processing, culturing and / or engineering them, and reintroducing them into the same patient before or after cryopreservation.

[0470] Subtypes and subpopulations of T cells and / or CD4+ and / or CD8+ T cells include: naive 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, mucosa-associated non-variant T (MAIT) cells, naturally occurring and adoptive regulatory T (Treg) cells, helper T cells, such as 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.

[0471] In some embodiments, the cell is a natural killer (NK) cell. In some embodiments, the cell is a monocyte or a granulocyte, for example, a bone marrow cell, a macrophage, a neutrophil, a dendritic cell, a mast cell, an eosinophil, and / or a basophil.

[0472] In some embodiments, the cell includes one or more nucleic acids introduced by genetic engineering, and thus expresses the recombinant or genetically engineered product of the nucleic acid. In some embodiments, the nucleic acid is heterologous, that is, it is not usually present in the cell or a sample obtained from the cell, such as a sample obtained from another organism or cell, which is, for example, not usually found in the cell being engineered and / or the organism from which such cell is derived. In some embodiments, the nucleic acid is not naturally occurring, such as the nucleic acid is not found in nature, including chimeric combinations of nucleic acids comprising encoding various domains from a variety of different cell types.

[0473] Methods and vectors for genetic engineering

[0474] The present invention also provides methods, compositions and kits for producing genetically engineered cells expressing chimeric antigen receptors.Genetic engineering generally involves introducing nucleic acids encoding the recombinant or engineered moieties into cells, for example, by retroviral transduction, transfection or transformation.

[0475] In some embodiments, gene transfer is performed by first stimulating the cells, e.g., by combining them with a stimulator that induces a response, e.g., proliferation, survival and / or activation, e.g., as detected by expression of cytokines or activation markers, and then transducing the activated cells and expanding them in culture to a number sufficient for clinical use.

[0476] In some cases, overexpression of stimulatory factors (e.g., lymphokines or cytokines) may be toxic to the subject. Therefore, in some cases, engineered cells include, for example, after administration in immune effector therapy, gene segments that make cells susceptible to negative selection in vivo. For example, in some aspects, the cells are engineered so that they can be eliminated due to changes in the in vivo conditions of the subject to which they are administered. Negatively selectable phenotypes can be produced by providing genes that provide sensitivity to the administered reagent (e.g., compound). Negatively selectable genes include: herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell II: 223, 1977), which provides ganciclovir sensitivity; cell hypoxanthine phosphoribosyltransferase (HPRT) gene, cell adenine phosphoribosyltransferase (APRT) gene, bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89: 33 (1992)).

[0477] In some aspects, cells are also engineered to promote the expression of cytokines or other factors. Various methods for introducing genetically engineered components, for example, antigen receptors (e.g., CAR) are well known, and methods and compositions provided herein can be used. Exemplary methods include those for transferring nucleic acids encoding receptors, including by viruses, for example, retroviruses or slow viruses, transduction, transposons, and electroporation.

[0478] In some embodiments, the recombinant nucleic acid is transferred into cells using recombinant infectious viral particles, e.g., vectors derived from simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV). In some embodiments, the recombinant nucleic acid is transferred into T cells using recombinant lentiviral vectors or retroviral vectors, e.g., γ-retroviral vectors (see, e.g., Koste et al., (2014) Gene Therapy 2014 Apr 3. 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. 2011 Nov; 29(11): 550–557).

[0479] In some embodiments, the retroviral vector has a long terminal repeat sequence (LTR), for example, a retroviral vector derived from Moloney murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), mouse embryonic stem cell virus (MESV), mouse stem cell virus (MSCV), spleen focus forming virus (SFFV) or adeno-associated virus (AAV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, the retrovirus includes those derived from any avian or mammalian cell source. Retroviruses are generally amphotropic, meaning that they are able to infect host cells of several species, including humans. Lentivirus transduction methods are known. Exemplary methods are described in, e.g., 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.

[0480] In some embodiments, the recombinant nucleic acid is transferred into T cells by 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 into T cells by transposition (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 for 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; microprojectile bombardment promoted by tungsten particles (Johnston, Nature, 346:776-777 (1990)); and strontium phosphate DNA coprecipitation (Brash et al., Mol. Cell Biol., 7:2031-2034 (1987)).

[0481] Other methods and vectors for transferring nucleic acids encoding recombinant products are those described in, for example, International Patent Application Publication No. WO2014055668 and US Patent No. 7,446,190.

[0482] Other nucleic acids (e.g., genes) for introduction include those for improving therapeutic efficacy, for example, by promoting the viability and / or function of transferred cells; genes for providing genetic markers for selection and / or evaluation of cells, for example, to assess survival or localization in vivo; genes for improving safety, for example, by making cells susceptible to negative selection in vivo, as described in Lupton SD et al., Mol. and Cell Biol., 11: 6 (1991); and Riddell et al., Human Gene Therapy 3: 319-338 (1992); also see Lupton's publications PCT / US91 / 08442 and PCT / US94 / 05601, etc., which describe the use of dual-function selectable fusion genes derived from the fusion of dominant positive selectable markers and negative selectable markers. See, for example, Riddell et al., U.S. Patent No. 6,040,177, columns 14-17.

[0483] Preparation of immune effector cells

[0484] In some embodiments, the preparation of engineered cells includes one or more cultures and / or one or more preparation steps. Cells for introducing nucleic acids encoding transgenic receptors (e.g., CAR) can be separated from samples (e.g., biological samples, such as samples obtained from or derived from subjects). In some embodiments, the subject from which the cell is separated is a subject suffering from a certain disease or condition or requiring cell therapy or to be given cell therapy. In some embodiments, the subject is a person who needs a specific therapeutic intervention, for example, a person who needs adoptive cell therapy or effector cell therapy, and the cells used for the therapy are separated, processed and / or engineered. In some embodiments, the cell is a primary cell, for example, a primary human cell. The sample includes tissues, body fluids and other samples taken directly from the subject, and samples obtained from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g., transduction using a viral vector), cleaning and / or incubation. The biological sample can be a sample directly obtained from a biological source or a processed sample. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

[0485] In some aspects, the sample from which cells are derived or separated is a blood or blood-derived sample or is or is derived from a single sampling or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMC), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, intestinal 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 organs, and / or cells derived therefrom. In the case of cell therapy (e.g., adoptive cell therapy or immune effector cell therapy), samples include samples from autologous and allogeneic sources.

[0486] In some embodiments, the cells are derived from a cell line, e.g., a T cell line. In some embodiments, the cells are obtained from a xenogeneic source, e.g., from a mouse, a rat, a non-human primate, and a pig.

[0487] In some embodiments, the separation of cells includes one or more preparations and / or not based on affinity cell separation steps. In some instances, cells are washed, centrifuged and / or incubated in the presence of one or more substances, for example, to remove unwanted components, enrich required components, crack or remove cells sensitive to specific substances. In some instances, cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity and / or resistance to specific components. In some instances, for example, by single sampling or leukocyte removal, cells from subject's circulating blood are obtained. In some aspects, the sample includes lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated blood leukocytes, red blood cells, and / or platelets, and, in some aspects, include cells different from red blood cells and platelets.

[0488] In some embodiments, the blood cells collected from the subject are washed, for example, to remove the plasma portion, and the cells are placed in a suitable buffer or culture medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the cleaning solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, the cleaning step is completed by semi-automated "flow-through" centrifugation (e.g., COBE 2991 cell processor, Baxter) according to the manufacturer's instructions. In some aspects, the cleaning step is completed by endo-flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, after cleaning, the cells are resuspended in a variety of biocompatible buffers, for example, without Ca++ / Mg++PBS. In certain embodiments, the blood cell sample components are removed, and the cells are directly resuspended in culture medium.

[0489] In some embodiments, the method comprises a density-based cell separation method, for example, obtaining peripheral blood mononuclear cells (PBMCs) by lysing red blood cells or without lysing red blood cells and centrifuging peripheral blood or apheresis samples or leukapheresis sample preparation through Percoll or Ficoll gradients.

[0490] In some embodiments, the separation method includes, based on one or more specific molecules in the cell, such as surface markers, for example, expression or presence of surface proteins, intracellular markers or nucleic acids to separate different cell types. In some embodiments, any known method for separation based on such markers can be adopted. In some embodiments, the separation is based on affinity or separation based on immunoaffinity. For example, in some aspects, the separation includes separation of cells and cell populations based on the expression or expression level of one or more markers (typically cell surface markers) of the cell, for example, by incubation with antibodies or binding partners that specifically bind to such markers, followed by typically a washing step, and separation of cells that have been bound to the antibody or binding partner from those cells that have not yet been bound to the antibody or binding partner.

[0491] Such separation steps can be based on positive selection, wherein cells that have bound the agent are retained for further use, and / or, negative selection, wherein cells that have not bound to the antibody or binding partner are retained. In some instances, both fractions are retained for further use. In some aspects, negative selection may be particularly useful when there are no antibodies available to specifically identify cell types in a heterogeneous population, so that separation is best performed based on markers expressed by cells that are different from the desired population.

[0492] The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, such as those expressing a marker, refers to an increase in the number or percentage of said cells, but need not result in the complete absence of cells that do not express said marker. Similarly, negative selection, removal or depletion of a particular type of cell, such as those expressing a marker, refers to a decrease in the number or percentage of said cells, but need not result in the complete removal of all such cells.

[0493] In some examples, multiple rounds of separation steps are performed, wherein the fraction of positive or negative selection from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step simultaneously depletes cells expressing multiple markers, such as by incubating the cells with multiple antibodies or binding partners that are each specific for the marker targeted by the negative selection. Similarly, multiple cell types can be simultaneously positively selected by incubating the cells with multiple antibodies or binding partners expressed on the various cell types.

[0494] For example, in some aspects, specific subpopulations of T cells, such as cells positive for one or more surface markers 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.

[0495] For example, CD3+, CD28+ T cells can be positively selected using CD3 / CD28 linked magnetic beads (e.g., DYNA beads M-450 CD3 / CD28 T cell expander).

[0496] In some embodiments, separation is performed by enriching a specific cell population by positive selection or depleting a specific cell population by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers, which are expressed (marker+) or expressed at relatively high levels (markerhigh) on cells that are positively or negatively selected, respectively.

[0497] In some embodiments, T cells are separated from PBMC samples by negative selection of markers (e.g., CD14) expressed on non-T cells (e.g., B cells, monocytes, or other blood leukocytes). In some aspects, CD4+ or CD8+ selection steps are used to separate auxiliary CD4+ and CD8+ cytotoxic T cells. By positive or negative selection for markers expressed or expressed at a relatively high degree on one or more original, memory, and / or effector T cell subsets, such CD4+ and CD8+ groups can be further sorted into subgroups.

[0498] In some embodiments, CD8+ cells are further enriched or depleted for naive, central memory, effector memory and / or central memory stem cells, for example by positive or negative selection based on surface antigens associated with the corresponding subpopulations. In some embodiments, enrichment for central memory T (TCM) cells is performed to increase efficacy, for example to improve long-term survival, expansion and / or post-administration engraftment, which in some aspects are particularly robust in such subpopulations. See Terakura et al., (2012) Blood. 1: 72–82; Wang et al., (2012) J Immunother. 35(9): 689-701. In some embodiments, TCM-enriched CD8+T cells are combined with CD4+T cells to further enhance efficacy.

[0499] In embodiments, memory T cells are present in CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes.PBMCs can be enriched or depleted for CD62L-CD8+ and / or CD62L+CD8+ fractions, for example using anti-CD8 and anti-CD62L antibodies.

[0500] In some embodiments, the enrichment for central memory T (TCM) cells is based on CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127 positive or high surface expression; in some aspects, it is based on CD45RA and / or granzyme B expression or highly expressed cells for negative selection. In some aspects, the separation of CD8+ groups enriched for TCM cells is carried out in the following manner: consuming cells expressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. On the one hand, the enrichment for central memory T (TCM) cells is carried out in the following manner: starting from the negative part of the cells selected based on CD4 expression, it is negatively selected based on the expression of CD14 and CD45RA, and positive selection is carried out based on CD62L. In some aspects, such selections are carried out simultaneously, and in other aspects, such selections are carried out in sequence in a certain order. In some aspects, the same selection step based on CD4 expression used to prepare a CD8+ cell population or subpopulation is also used to generate a CD4+ cell population or subpopulation, thereby retaining the positive and negative fractions from the CD4-based separation and, optionally after one or more further positive or negative selection steps, for use in subsequent steps of the method.

[0501] In a specific example, a PBMC sample or other blood leukocyte sample is subjected to CD4+ cell selection, wherein negative and positive fractions are retained. Then, the negative fraction is subjected to negative selection based on the expression of CD14 and CD45RA or CD19, and positive selection is performed based on markers characteristic of central memory T cells, such as CD62L or CCR7, wherein the positive and negative selections are performed in a certain order.

[0502] By identifying cell populations with cell surface antigens, helper CD4+T cells are sorted into naive, central memory and effector cells. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive 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-.

[0503] In one example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail 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 matrix, such as magnetic or paramagnetic beads, to allow separation of cells for positive and / or negative selection.

[0504] In some embodiments, the preparation method includes: a freezing step, for example, freezing the cells before or after separation, incubation and / or engineering. In some embodiments, the freezing and subsequent thawing steps remove granulocytes and, to some extent, remove monocytes in the cell population. In some embodiments, for example, after a washing step to remove plasma and platelets, the cells are suspended in a freezing solution. In some aspects, any of a variety of known freezing solutions and parameters may be used. One example involves using PBS or other suitable cell freezing medium containing 20% ​​DMSO and 8% human serum albumin (HAS). Then, it is diluted 1:1 with a culture medium so that the final concentrations of DMSO and HSA are 10% and 4%, respectively. Then, the cells are generally frozen to -80°C or -90°C in a program-controlled cooling device according to a predetermined procedure or principle such as 1° / minute, and stored in the vapor phase of a liquid nitrogen storage tank.

[0505] In some embodiments, provided methods include cultivating, incubating, culturing, and / or genetic engineering steps. For example, in some embodiments, provided methods are for incubating and / or engineering spent cell populations and culture initiation compositions.

[0506] In some embodiments, the cell population is incubated in a culture initiation composition. Incubation and / or engineering can be performed in a culture vessel, such as a unit, chamber, well, column, tube, tube set, valve, vial, culture dish, bag, or other container for culturing or growing cells.

[0507] In some embodiments, cells are incubated and / or cultured prior to or with genetic engineering. Incubation steps may include culturing, nurturing, stimulating, activating, and / or propagating. In some embodiments, cells or compositions are incubated in the presence of stimulating conditions or irritating agents. Such conditions include being designed to induce cell proliferation, propagation, activation, and / or survival in a population to simulate antigen contact, and / or to initiate cells for genetic engineering, such as those for importing recombinant antigen receptors.

[0508] The conditions may include one or more of the following: specific culture media, 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, recombinant soluble receptors, and any other substances designed to maintain an activated cell state.

[0509] In some embodiments, the stimulation condition or reagent includes one or more substances, for example, a ligand, which can activate the intracellular signal transduction domain of the TCR complex. In some aspects, the substance opens or starts the TCR / CD3 intracellular signal transduction cascade in T cells. Such substances may include antibodies, such as those with specificity to TCR components and / or costimulatory receptors, for example, anti-CD3, anti-CD28, for example, which are bound to a solid support, such as a bead, and / or one or more cytokines. Optionally, the amplification method may also include the steps of adding anti-CD3 and / or anti-CD28 antibodies to the culture medium (for example, with a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulant includes IL-2 and / or IL-15 and / or IL-7 and / or IL-21, for example, at least about 10 units / mL of IL-2.

[0510] In some aspects, incubation is performed according to techniques such as those described in U.S. Pat. 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.

[0511] In some embodiments, the T cell population is expanded by adding to the culture starting composition feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs), (e.g., so that for each T lymphocyte in the initial population to be expanded, the resulting cell population contains at least about 5, 10, 20 or 40 or more PBMC feeder cells); and incubating the culture (e.g., a time sufficient to expand the number of T cells). In some aspects, the non-dividing feeder cells may include γ-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with γ-rays in the range of about 3000-3600 rads to prevent cell division. In some aspects, the feeder cells are added to the culture medium before adding the population of T cells.

[0512] In some embodiments, the stimulation conditions include a temperature suitable for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally or about 37 degrees Celsius. Optionally, incubation may also include the addition of non-dividing EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000-10000 rad. In some aspects, LCL feeder cells are provided in any suitable amount, for example, the ratio of LCL feeder cells to initial T lymphocytes is at least about 10:1.

[0513] In one embodiment, antigen-specific T cells, such as antigen-specific CD4+ and / or CD8+ T cells, are obtained by stimulating natural or antigen-specific T lymphocytes with an antigen. For example, an antigen-specific T cell line or clone can be generated against a cytomegalovirus antigen by isolating T cells from an infected subject and stimulating the cells in vitro with the same antigen.

[0514] Compositions and preparations

[0515] The present invention also provides pharmaceutical compositions and preparations for the methods of the present invention. In some embodiments, the pharmaceutical compositions and preparations of the present invention include compositions of cells for administration, including pharmaceutical compositions and preparations, such as unit dosage compositions from a number of cells for administration containing a given dose or a portion thereof. The pharmaceutical compositions and preparations generally include one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition includes at least one other therapeutic agent.

[0516] The term "pharmaceutical formulation" refers to a preparation in such form that it permits the biological activity of the active ingredients contained therein to be effective, and does not contain additional ingredients that are unacceptably toxic to a subject to which the formulation would be administered.

[0517] "Pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical preparation that is not an active ingredient and is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers or preservatives.

[0518] In some aspects, the selection of carriers is determined in part by specific cells and / or methods of administration. Therefore, there are a variety of suitable formulations. For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methyl paraben, propyl paraben, sodium benzoate and benzalkonium chloride. In some aspects, a mixture of two or more preservatives is used. The amount of preservative or its mixture is generally about 0.0001% to about 2% (based on the total weight of the composition). Carriers are described in, for example, Remington's Pharmaceutical Sciences, 16th edition, Osol, A. (1980). Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acid buffers; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residual yl) polypeptide; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, 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 (such as Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG).

[0519] In some aspects, the composition comprises a buffer. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate and a variety of other acids and salts. In some aspects, a mixture of two or more buffers is used. The buffer or its mixture is typically present in an amount of about 0.001% to about 4% (based on the total weight of the composition). Methods for preparing a pharmaceutical composition that can be administered are known. Exemplary methods are specifically described in, for example, Remington: The Science and Practice of Pharmacy, LWW (Lippincott Williams & Wilkins); 21st edition (May 1, 2005).

[0520] The preparation may comprise an aqueous solution. The preparation or composition may also comprise more than one active ingredient, which may be used for a specific indication, disease or condition to be treated with the cell, preferably those with a supplementary activity to the cell, wherein the corresponding active agents do not negatively affect each other. Such active ingredients are suitable for being present in combination with an amount effective for the desired purpose. Therefore, in some embodiments, the pharmaceutical composition further comprises other pharmaceutically active substances or drugs, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine.

[0521] In some embodiments, the pharmaceutical composition comprises an amount effective to treat or prevent a disease or condition, such as a therapeutically effective or preventively effective amount of cells. In some embodiments, the therapeutic or preventive efficacy is monitored by regularly assessing the treated subject. The desired dose can be delivered by administering the cells as a single pill, by administering the cells as multiple pills, or by administering the cells as a continuous infusion.

[0522] In some embodiments, the composition comprises cells in an amount effective to reduce disease or disorder burden, and / or in an amount that does not cause CRS or severe CRS in a subject, and / or in an amount that achieves any other outcome of the methods described herein.

[0523] The cell and composition can be administered using standard administration techniques, preparations and / or devices. The administration of the cell can be autologous or heterologous. For example, immunosuppressive cells or progenitor cells can be obtained from a subject and administered to the same subject or different compatible subjects. Peripheral blood-derived immunosuppressive cells or their offspring (for example, in vivo, ex vivo or in vitro derived) can be administered by local injection, including catheter administration, systemic injection, local injection, intravenous injection or parenteral administration. When administering a therapeutic composition (for example, a pharmaceutical composition containing genetically modified immunosuppressive cells), it is usually formulated into an injectable form (solution, suspension, emulsion) of a unit dosage form.

[0524] Formulations include those for oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, mucosal, sublingual or suppository administration. In some embodiments, the cell population is administered parenterally. The term "parenteral" as used herein 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.

[0525] In some embodiments, the composition is provided in the form of a sterile liquid preparation, for example, an isotonic aqueous solution, a suspension, an emulsion, a dispersion or a viscous composition, which can be buffered to a selected pH in some aspects. Liquid preparations are generally easier to prepare than gels, other viscous compositions and solid compositions. In addition, liquid compositions are more or less convenient for administration, especially by injection. On the other hand, viscous compositions can be formulated within a suitable viscosity range to provide a longer contact time with a particular tissue. Liquid or viscous compositions can include a carrier, which can be a solvent or a dispersion medium, which contains, for example, water, saline, phosphate buffered saline, polyols (for example, glycerol, propylene glycol, liquid polyethylene glycol) and a suitable mixture thereof. Sterile injectable solutions can be prepared by incorporating the cells into a solvent, such as with a suitable carrier, diluent or excipient such as sterile water, saline, glucose, dextrose, etc. The composition may contain auxiliary substances, such as wetting, dispersing, or emulsifying agents (e.g., methylcellulose), pH buffers, gelling or viscosity enhancing additives, preservatives, flavoring agents, and / or pigments, depending on the desired route of administration and preparation. In some aspects, standard textbooks may be consulted to prepare suitable preparations.

[0526] Various additives may be added to enhance the stability and sterility of the composition, including antimicrobial preservatives, antioxidants, chelating agents and buffers. Prevention of the action of microorganisms may also be ensured by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Absorption of injectable pharmaceutical forms may be enhanced by the use of substances that delay absorption, such as aluminum monostearate and gelatin.

[0527] Preparations for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through sterile filtration membranes.

[0528] Products

[0529] The present invention also provides articles of manufacture, such as kits and devices, for administering cells to a subject according to the provided methods for adoptive cell therapy or immune effector cell therapy, and for storing and administering the cells and compositions.

[0530] The article of manufacture includes one or more containers, typically a plurality of containers, packaging material, and a label or package insert associated with or on the one or more containers and / or packaging that typically includes instructions for administering the cells to a subject.

[0531] The container generally contains cells to be administered, for example, one or more unit doses thereof. The article generally includes multiple containers, each containing a single unit dose of cells. The unit dose may be the amount or number of cells to be administered to the subject in the first dose or twice (or more) the number of cells to be administered in the first or subsequent dose. It may be the lowest dose or the lowest possible dose of cells administered to the subject in connection with the method of administration. In some embodiments, the unit dose is the number of cells or the minimum number of cells to be administered to any subject or any subject with a specific disease or condition in a unit dose according to the method of the present invention. For example, in some aspects, the unit dose may include the minimum amount of cells to be administered to a subject with a lower body weight and / or a lower disease load, such as administering one or more than one unit dose to a given subject in the first dose and administering one or more than one unit dose to a given subject in one or more subsequent doses, for example, according to the method provided. In some embodiments, the number of cells in a unit dose is the number or number of cells that need to be administered to a specific subject in the first dose, such as a chimeric antigen receptor-expressed or CAR-expressed by a subject derived from a cell. In some embodiments, the cell is derived from a subject to be treated or in need of the method provided herein.

[0532] In some embodiments, each container individually comprises a unit dose of cells, e.g., comprising the same or substantially the same number of cells. Thus, in some embodiments, each container comprises the same or approximately or substantially the same number of cells or chimeric antigen receptor-expressing cells. In some embodiments, a unit dose comprises less than about 1 x 10 10 , less than about 1x10 9 , less than about 1x10 8 or less than about 1x10 7 engineered cells, total cells, T cells, or PBMCs per kilogram of subject to be treated and / or from which the cells are derived.

[0533] Suitable containers include, for example, bottles, vials, syringes, and flexible bags such as freezing bags. In a specific embodiment, the container is a bag, for example, a flexible bag, such as those suitable for infusing cells to 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 sealable and / or sterilizable to provide a sterile solution and the delivery of cells and compositions. In some embodiments, the container, for example, the volume of the bag is equal to or about or at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, or 1000 ml volume, such as equal to or about 10 to equal to or about 100 ml or equal to or about 10 to equal to or about 500 ml volume. In some embodiments, the container, e.g., bag, is and / or is made of a material that is stable and / or provides stable storage and / or maintenance of cells at one or more different temperatures, such as at low temperatures, e.g., below or about or at or about -20°C, -80°C, -120°C, 135°C, -196°C and / or temperatures suitable for cryopreservation, and / or other temperatures, such as temperatures suitable for freezing and thawing cells and body temperature, such as at or about 37°C or -38°C, or -39°C, or -40°C, to allow freezing and thawing immediately prior to treatment, e.g., at the subject's site or treatment site, e.g., at the bedside.

[0534] The container can be made of various materials such as glass or plastic. In some embodiments, the container has one or more ports, e.g., a sterile immersion port, e.g., for connection to one or more tubes via a tube or catheter, e.g., for intravenous or other infusions and / or for connection for the purpose of transfer from and to other containers, such as cell culture and / or storage bags or other containers. Exemplary containers include frozen bags, intravenous solution bags, vials, including those with stoppers that can be pierced by injection needles.

[0535] The article may also include a package insert or label, one or more of which display information and / or instructions for use. In some embodiments, the information or instructions display the content that can or should be used to treat a specific disease or condition, and / or provide instructions for it. The label or package insert may display the content of the article to be used to treat a disease or condition. In some embodiments, the label or package insert provides instructions for treating a subject, e.g., a subject from which the cell has been derived, by including administering a first and one or more subsequent doses of cells, e.g., according to any of the embodiments of the provided methods. In some embodiments, the instructions specify that in the first dose, a unit dose, e.g., the contents of a single separate container of the article is administered, followed by administering one or more subsequent doses at a specified time point or within a specified time window and / or after detecting the presence or absence or amount or degree of one or more factors or results in the subject.

[0536] In some embodiments, the instructions specify administering multiple unit doses to the subject by performing a first administration and subsequent administrations. In some embodiments, the first administration comprises delivering one of the unit doses to the subject and subsequent administrations comprise administering one or more of the unit doses to the subject.

[0537] In some embodiments, the instructions specify that subsequent administration is to be performed after the first administration, for example, about 21 days to about 80 days or about 25 days to about 60 days after the first administration or the start of the previous administration, for example, equal to or about 50 days. In some embodiments, the instructions specify that subsequent doses will be given after the serum level of the factor indicating cytokine release syndrome (CRS) in the subject is determined to be about 10 times lower, about 25 times lower, and / or about 50 times lower than the serum level of the indicator of the subject immediately before the first administration, and / or the index of CRS has reached a peak and is declining, and / or the subject does not show a detectable adaptive host immune response specific to a receptor (e.g., CAR) expressed by a cell.

[0538] In some embodiments, the label or package insert or package contains an identifier to indicate the identity of the subject from which the cells are derived and / or the subject to be administered. In the case of an autologous transplant, the identity of the subject from which the cells are derived is the same as the identity of the subject to which the cells are to be administered. Thus, the identification information can specify that the cells are to be administered to a particular patient, such as the cells from which the cells were originally derived. Such information can be present in the packaging material and / or label in the form of a barcode or other coded identifier, or can indicate the name and / or other identifying features of the subject.

[0539] In some embodiments, the article of manufacture includes one or more, typically multiple containers containing a composition comprising cells, such as a single unit dosage form thereof, and also includes one or more other containers containing a composition, which composition includes other agents, such as cytotoxic or other therapeutic agents, which are, for example, to be combined with the cells, for example, administered at once, simultaneously or in any order. Alternatively or in addition, the article of manufacture may also include another or the same container containing a pharmaceutically acceptable buffer. It may also include other materials, such as other buffers, diluents, filters, tubes, needles, and / or syringes.

[0540] The term "package insert" refers to instructions customarily included in commercial packages of therapeutic products, that contain information about the use, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning such therapeutic products.

[0541] The method of the present invention for treating cancer patients with an initial dose and subsequent doses of autologous T cells expressing CAR can be summarized as follows:

[0542] Peripheral blood mononuclear cells (PBMCs) or T cells from human subjects with cancer are isolated by "mononuclear cell separation" and cultured and transduced with a viral vector encoding a chimeric antigen receptor (CAR) that specifically binds to an antigen expressed by the cancer in the subject, which is a tumor-associated or tumor-specific antigen. The cells are cryopreserved in an infusion medium in individual flexible freezing bags, each containing a single unit dose of cells, which is approximately 1×10 6 cells to 5×10 7 The first dose infused to each subject was no higher than approximately 1×10 12 cells, preferably no more than about 1×10 11 cells, more preferably, no more than about 1×10 10 cells or about 5x10 9 or about 2x10 9 Prior to infusion, the cells are maintained at a temperature of about below -130°C or about below -175°C.

[0543] Before starting cell therapy, blood is obtained from the subject, and the level of one or more serum factors indicating cytokine release syndrome (CRS) in the serum is optionally assessed by ELISA and / or MSD and / or CBA methods, such as tumor necrosis factor α (TNFα), interferon γ (IFNγ), IL-10 and IL-6. Before the start of treatment, the tumor burden can be optionally assessed by measuring the size or mass of solid tumors, for example, by PET or CT scanning.

[0544] Resuscitation is performed by warming to about 38°C, and the subject is given a first dose of cells via multiple infusions. Each infusion is given as a continuous infusion intravenously (IV) over a period of about 3-30 minutes.

[0545] After the first dose is given, the subject undergoes a physical examination and monitors the symptoms of any toxicity or toxic results, such as fever, hypotension, hypoxia, neurological disorders or inflammatory cytokines or C-reactive protein (CRP) serum levels. Optionally, after the first dose is given, in one or more cases, blood is obtained from the patient, and the level of serum factors indicating CRS is assessed by ELISA and / or MSD and / or CBA methods. The level of serum factors is compared with the level of serum factors obtained just before the first dose is given. If necessary, anti-IL6 or other CRS treatments are given to reduce the symptoms of CRS.

[0546] After administration of the first dose, e.g., 1, 2, 3, and / or 4 weeks after initiation of dosing, the subject is optionally tested for the presence or absence of an anti-CAR immune response, e.g., by qPCR, ELISA, ELISPOT, cell-based antibody assay, and / or mixed lymphocyte reaction.

[0547] The percent reduction in tumor burden achieved by the first dose can optionally be measured one or more times after the first dose in patients with solid tumors by scans (e.g., PET and CT scans), and / or by quantifying disease-positive cells in the blood or at the tumor site.

[0548] Subsequent doses are given. In some subjects, subsequent doses are given within about 80 days after the first dose is given, preferably about 60 days, more preferably about 50 days. In some cases, subsequent doses are given only when the tested CRS-related results or serum factor levels are below acceptable levels and no anti-CAR immune response is detected in the subject about 60 days after the first dose. In other subjects, more than 30 days after the first dose is given, and the subject is considered to have no CRS or severe CRS, or the level of all tested serum factors indicating CRS is less than 20% of the CRS observed at the peak after the first dose, and the subject is considered to have no detectable anti-CAR immune response. Subsequent doses are given. In some embodiments, one or more subsequent doses of cells are given. The time interval between each dose is 21-80 days, preferably 25-60 days, more preferably 25-55 days.

[0549] The size of subsequent doses is patient-specific and based on the level of tumor burden, the presence of anti-CAR immune responses, and CRS-related outcomes. Some subjects are given subsequent doses containing 1, 2, 3, or even more unit doses of cells. Subsequent doses infused per subject are no greater than approximately 1×10 12 cells, or no more than about 1×10 11 cells, or no more than about 1×10 10 cells, or no more than about 5×10 9 , or not more than about 2×10 9 Subsequent doses are given by continuous intravenous infusion over approximately 3 to 30 minutes.

[0550] Subjects were monitored regularly starting with the first dose and continuing for up to several years. During follow-up, tumor burden was measured, and / or CAR-expressing cells were detected by flow cytometry and quantitative polymerase chain reaction (qPCR) to measure the in vivo proliferation and persistence of the administered cells, and / or to assess the development of anti-CAR immune responses.

[0551] The present invention provides embodiments including but not limited to the following:

[0552] 1. A method for treating a CLD18-positive tumor, the method comprising administering a first dose of immune effector cells to a subject in need thereof, the immune effector cells expressing a chimeric antigen receptor (CAR) and specifically recognizing CLD18, the first dose comprising no more than about 2×10 10 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 12 cell,

[0553] Preferably, the first dose does not exceed about 2 x 10 9 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 11 cell,

[0554] Preferably, the first dose does not exceed about 2.5 x 10 8 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 10 cell,

[0555] Preferably, the first dose does not exceed about 5 x 10 7 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 10 cell,

[0556] More preferably, the first dose does not exceed about 3 x 10 7 cells / kg of subject body weight or the total amount of said cells does not exceed about 2 x 10 9 cell.

[0557] 2. The method as described in embodiment 1 is characterized in that the CLD18 is CLD18A2.

[0558] 3. The method as described in embodiment 1 or 2 is characterized in that after administering the first dose of immune effector cells, at least one subsequent dose of immune effector cells expressing a chimeric antigen receptor (CAR) and specifically recognizing CLD18 is administered.

[0559] 4. The method as described in embodiment 3 is characterized in that the subsequent dose of immune effector cells is administered after the immune effector cells administered at the first dose cannot be detected in vivo.

[0560] 5. The method as described in embodiment 3 is characterized in that the subsequent dose of immune effector cells is administered at a time point of about 21 to 80 days after the administration of the first dose; preferably, the subsequent dose of immune effector cells is administered at a time point of about 25 to 60 days after the administration of the first dose; more preferably, the subsequent dose of immune effector cells is administered at a time point of about 25 to 50 days after the administration of the first dose.

[0561] 6. The method described in embodiment 5 is characterized in that the subsequent dose is higher than the initial dose; preferably, the subsequent dose is 2 times, 5 times, 7 times or 10 times the initial dose.

[0562] 7. The method of embodiment 3, wherein the subsequent dose comprises no more than about 2×10 10 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 12 cell,

[0563] Preferably, the subsequent dose comprises no more than about 2 x 10 9 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 11 cell,

[0564] Preferably, the subsequent dose contains no more than about 2.5 x 10 8 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 10 cell,

[0565] Preferably, the subsequent dose comprises no more than about 5 x 10 7 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 10 cell,

[0566] More preferably, the subsequent dose comprises no more than about 3 x 10 7 cells / kg of subject body weight or the total amount of said cells does not exceed about 2 x 10 9 cell.

[0567] 8. The method as described in embodiment 1 or 3 is characterized in that the first dose or subsequent doses are administered in N times within 20 days, where N is a natural number not less than 1, and preferably, are administered in N times within 3-15 days.

[0568] 9. The method of embodiment 3, wherein when the subsequent dose is administered, the subject has any of the following characteristics:

[0569] (i) a serum level of a factor indicative of cytokine release syndrome (CRS) in the subject that is about 10-fold less, about 25-fold less, and / or about 50-fold less than the level in the subject immediately prior to administration of the first dose;

[0570] (ii) did not show grade 3 or higher neurotoxicity;

[0571] (iii) the level of neurotoxicity or CRS is reduced compared to the peak level of neurotoxicity or CRS after the first dose of immune effector cells; or

[0572] (iv) the subject shows no detectable humoral or cell-mediated immune response to the CAR expressed by the cells of the first dose.

[0573] 10. The method as described in embodiment 9 is characterized in that in (iii), the CRS level is reduced by at least 50% compared to the peak level of CRS after administration of the first dose of immune effector cells, preferably, by at least 20%, more preferably, by at least 5%, or the CRS level is equivalent to the CRS level before administration of the first dose of immune effector cells.

[0574] 11. The method according to any one of embodiments 1 to 10 is characterized in that the method further comprises performing pretreatment before administering the immune effector cells, wherein the pretreatment comprises administering a chemotherapeutic agent to the subject, or performing whole-body radiation therapy to the subject, or performing local radiation therapy to the subject, or a combination thereof.

[0575] 12. The method according to any one of embodiments 11, wherein the pretreatment is performed 4-12 days before administering the immune effector cells.

[0576] 13. The method according to embodiment 11 is characterized in that the chemotherapeutic agent is selected from any one or a combination of the following: cyclophosphamide, fludarabine, taxane compounds, and pyrimidine anti-tumor drugs.

[0577] 14. The method of embodiment 13, wherein the chemotherapeutic agents are cyclophosphamide and fludarabine; or cyclophosphamide, fludarabine, and albumin-bound paclitaxel.

[0578] 15. The method according to any one of embodiments 13 or 14, characterized in that

[0579] Fludarabine is administered in doses of approximately 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 / day; and / or

[0580] The dose 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 490-550mg / m 2 / day; and / or

[0581] The amount of nab-paclitaxel administered is no more than about 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 no more than about 80 mg / m 2 / day, or no more than about 70 mg / m 2 / sky.

[0582] 16. A method as described in embodiment 15, characterized in that the chemotherapeutic agent is used continuously for no more than 4 days.

[0583] 17. The method according to embodiment 16, characterized in that the cyclophosphamide and / or the fludarabine are used continuously for no more than 4 days, and the albumin-bound paclitaxel is administered once.

[0584] 18. The method described in embodiment 1 is characterized in that the tumor is a CLD18A2-positive digestive tract tumor; preferably, the digestive tract tumor is adenocarcinoma; more preferably, the digestive tract tumor is pancreatic cancer or gastric adenocarcinoma.

[0585] 19. The method according to embodiment 2, wherein the chimeric antigen receptor comprises an antibody or a fragment thereof, a transmembrane domain and an intracellular domain that specifically binds to CLD18A2, and the antibody has:

[0586] 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; or

[0587] HCDR1 shown in SEQ ID NO: 1, HCDR2 shown in SEQ ID NO: 7, 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; or

[0588] HCDR1 shown in SEQ ID NO: 8, HCDR2 shown in SEQ ID NO: 9 or 68, HCDR3 shown in SEQ ID NO: 10, LCDR1 shown in SEQ ID NO: 11, LCDR2 shown in SEQ ID NO: 12, LCDR3 shown in SEQ ID NO: 13.

[0589] 20. The method according to embodiment 19, wherein the antibody or fragment thereof has:

[0590] The heavy chain variable region set forth in SEQ ID NO: 14 and the light chain variable region set forth in SEQ ID NO: 16; or

[0591] The heavy chain variable region set forth in SEQ ID NO: 18 and the light chain variable region set forth in SEQ ID NO: 16; or

[0592] The heavy chain variable region set forth in SEQ ID NO: 22 and the light chain variable region set forth in SEQ ID NO: 20; or

[0593] The heavy chain variable region set forth in SEQ ID NO:53 and the light chain variable region set forth in SEQ ID NO:52;

[0594] Preferably, the antibody or fragment thereof has the sequence shown in SEQ ID NO: 54, 56, 57, or 58.

[0595] 21. The method according to embodiment 20, characterized in that 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; preferably, it is a sequence shown in SEQ ID NO: 24, 25 or 26.

[0596] 22. The method according to any one of embodiments 1 to 21, characterized in that the immune effector cells are T lymphocytes, NK cells or NKT lymphocytes; preferably T lymphocytes; preferably, the T cells are derived from the subject himself.

[0597] 23. The method according to embodiment 3, characterized in that:

[0598] Two or more subsequent doses of immune effector cells are administered, and the interval between each subsequent dose of immune effector cells is about 21 to about 80 days, or about 25 to about 60 days, or about 25 to 50 days, each including the end values.

[0599] 24. A method as described in embodiment 23, characterized in that the number of immune cells in each subsequent dose administered is substantially the same, or the number of immune effector cells in a subsequent dose administered later is higher than the number of immune cells in a subsequent dose administered earlier.

[0600] 25. A method as described in embodiment 23, characterized in that the number of immune effector cells in the subsequent dose administered later is lower than the number of immune cells in the subsequent dose administered earlier.

[0601] 26. The method as described in embodiment 1 is characterized in that, before administering the immune effector cells, the subject has not received treatment with immune cells expressing a chimeric antigen receptor targeting CLD18.

[0602] 27. A method as described in embodiment 3, characterized in that the subsequent dose is a number of cells sufficient to stabilize or reduce the tumor burden of the subject.

[0603] 28. The method described in embodiment 1 is characterized in that, before administering the first dose, the subject has undergone surgery, chemotherapy, or immunotherapy different from that described in embodiment 1.

[0604] 29. A method as described in embodiment 1 or 3, characterized in that the subject's serum levels of factors indicating CRS, factors indicating neurotoxicity, factors indicating tumor burden, and / or factors indicating host anti-CAR immune response are evaluated before the first dose is administered, or after the first dose is administered and before subsequent doses are administered.

[0605] 30. A method as described in embodiment 29, characterized in that the factor indicating tumor load is: the total number of tumor cells in the subject, or the total number of tumor cells in the subject's organ, or the total number of tumor cells in the subject's tissue, or the mass or volume of the tumor, or the degree of tumor metastasis, or the number of tumors.

[0606] 31. The method according to embodiment 30, comprising:

[0607] i) evaluating factors indicative of tumor burden prior to administration of subsequent doses; and

[0608] ii) determining a subsequent dose of cells to be administered to the subject based on the results of said evaluation, and

[0609] iii) if the evaluation determines that the tumor mass or volume of the subject is stable or decreasing, administering to the subject a subsequent dose comprising less than or more than or about the same number of CAR-expressing cells as in the first dose.

[0610] 32. The method of embodiment 1 or 3, wherein the number of cells administered in the first dose or the subsequent dose is about 1.1 x 10 6 Cells / kg subject body weight up to 2.8x10 7 Cells / kg, about 2.9x10 6 Cells / kg to 2.6x10 7 cells / kg or approximately 3.3x10 6Cells / kg to 1.3x10 7 cells / kg or about 1.3x10 7 Cells / kg to 1.8x10 7 cells / kg, each inclusive.

[0611] 33. The method of embodiment 3, wherein the number of cells administered in the subsequent dose of CAR-expressing cells comprises about 1.1 x 10 7 Cells / kg (cells / kg) body weight is about 5.1x10 7 Cells / kg, about 1.3x10 7 Cells / kg: about 3.7x10 7 Cells / kg, about 1.6x10 7 Cells / kg: about 2.2x10 7 Cells / kg, about 1.9x10 7 Cells / kg: about 2.2x10 7 Cells / kg, about 2.2x10 7 Cells / kg: about 2.7x10 7 cells / kg, each inclusive.

[0612] 34. A method as described in embodiment 1 or 3, characterized in that after administration of the first dose or subsequent doses of immune effector cells, the subject does not show cytokine release syndrome (CRS), does not show severe CRS, does not show neurotoxicity, does not show severe neurotoxicity, or does not show neurotoxicity greater than grade 3.

[0613] 35. A method as described in embodiment 1 or 3, characterized in that after administering a first dose or subsequent doses of immune effector cells, the immune effector cells proliferate in the subject.

[0614] 36. A method for treating tumors, the method comprising that the subject receives chemotherapy, radiotherapy, or immunotherapy before administering the method of any one of embodiments 1-35.

[0615] 37. A composition comprising cyclophosphamide and fludarabine or a combination thereof with a taxane compound and / or a pyrimidine anti-tumor drug.

[0616] 38. The composition of embodiment 37, wherein the composition comprises cyclophosphamide, fludarabine and paclitaxel.

[0617] 39. The composition of embodiment 38, wherein the composition comprises cyclophosphamide, fludarabine and albumin-bound paclitaxel.

[0618] 40. The composition of embodiment 39, wherein the amount of fludarabine administered 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 / day; and / or cyclophosphamide is administered in an amount of 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 490-550mg / m 2 / day; and / or nab-paclitaxel administered in an amount not exceeding about 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 no more than about 80 mg / m 2 / day, or no more than about 70 mg / m 2 / sky.

[0619] 41. A composition as described in any of embodiments 37-40, characterized in that the composition is administered about 30 days before administering immune effector cell therapy; preferably, about 20 days; more preferably, about 12 days; most preferably, about 7 days.

[0620] 42. A composition as described in any one of embodiments 37-41, characterized in that the cyclophosphamide, fludarabine and / or albumin-bound paclitaxel are used continuously for no more than 4 days; preferably, the albumin-bound paclitaxel is administered once.

[0621] 43. A composition as described in any one of embodiments 37-42, characterized in that it is used for anti-tumor treatment; preferably, it is used for anti-tumor targeted therapy; most preferably, it is used for anti-tumor chimeric antigen receptor targeted therapy.

[0622] 44. A composition as described in any one of embodiments 41-43, characterized in that the immune effector cells are CART cells.

[0623] 45. The composition as described in embodiment 44 is characterized in that the CAR T cells specifically recognize CLD18A2.

[0624] 46. ​​The composition of any one of embodiments 43-45, characterized in that the tumor is a CLD18A2-positive digestive tract tumor; more preferably, the digestive tract tumor is adenocarcinoma; most preferably, the digestive tract tumor is pancreatic cancer or gastric adenocarcinoma.

[0625] 47. Use of immune effector cells comprising CLD18A2-CAR in the preparation of a drug, the drug containing the cells and cyclophosphamide, fludarabine and a taxane compound (preferably paclitaxel, more preferably albumin-bound paclitaxel) for treating CLD18-positive tumors, wherein the cells and cyclophosphamide, fludarabine and taxane compounds are formulated to provide a therapeutic effect greater than the sum of the effects of each of the agents.

[0626] 48. The use according to embodiment 47, characterized in that it comprises administering a first dose of the immune effector cells to a subject in need thereof, wherein the first dose does not exceed about 3×10 7 cells / kg of subject body weight or the total amount of said cells does not exceed about 2 x 10 9 cell.

[0627] 49. The use according to embodiment 47 or 48, characterized in that after administering the first dose of immune effector cells, at least one subsequent dose of the immune effector cells is administered, wherein the subsequent dose contains no more than about 3×10 7 cells / kg of subject body weight or the total amount of said cells does not exceed about 5 x 10 9 cells, preferably, the total amount of cells does not exceed about 2 x 10 9 cell.

[0628] 50. The use as described in any one of embodiments 47-49 is characterized in that the subsequent dose of immune effector cells is administered at a time point of about 21 to 80 days after the administration of the first dose; preferably, the subsequent dose of immune effector cells is administered at a time point of about 25 to 60 days after the administration of the first dose; more preferably, the subsequent dose of immune effector cells is administered at a time point of about 25 to 50 days after the administration of the first dose.

[0629] 51. The use according to any one of embodiments 47-50, characterized in that the cyclophosphamide, fludarabine and taxane compounds are administered 4-12 days before the administration of immune effector cells.

[0630] 52. The use according to any one of embodiments 47 to 51, characterized in that:

[0631] Fludarabine is administered in doses of approximately 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 / day; and / or

[0632] The dose 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 490-550mg / m 2 / day; and / or

[0633] The taxane compound is administered in an amount not exceeding about 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 no more than about 80 mg / m 2 / day, or no more than about 70 mg / m 2 / sky.

[0634] 53. The use according to any one of embodiments 47-52, characterized in that the cyclophosphamide and / or the fludarabine are used continuously for no more than 4 days, and the taxane compound is administered once.

[0635] 54. The use according to any one of embodiments 47 to 53, characterized in that 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; preferably, it is a sequence shown in SEQ ID NO: 24, 25 or 26.

[0636] 55. A kit for treating CLD18-positive tumors, characterized in that the kit comprises:

[0637] 1) Immune effector cells containing CLD18A2-CAR;

[0638] 2) Cyclophosphamide;

[0639] 3) Fludarabine;

[0640] 4) Taxane compounds, preferably paclitaxel, more preferably albumin-bound paclitaxel;

[0641] 5) a container for containing the substances described in 1) to 4) above; and

[0642] 6) Instructions for use of the kit for treating CLD18-positive tumors;

[0643] The cells and cyclophosphamide, fludarabine and taxane compounds are formulated to provide a therapeutic effect greater than the sum of the effects of each of the agents.

[0644] 56. The kit according to embodiment 55, characterized in that the instructions for administration record that a first dose of the immune effector cells is administered to a subject in need thereof, and the first dose does not exceed about 3×10 7 cells / kg of subject body weight or the total amount of said cells does not exceed about 5 x 10 9 cells, preferably, the total amount of cells does not exceed about 2 x 10 9 cell.

[0645] 57. The kit of embodiment 55 or 56, wherein the instructions for administration state that after administering the first dose of immune effector cells, at least one subsequent dose of the immune effector cells is administered, wherein the subsequent dose contains no more than about 3×10 7 cells / kg of subject body weight or the total amount of said cells does not exceed about 5 x 10 9 cell.

[0646] 58. A kit as described in any one of embodiments 55-57, characterized in that the administration instructions record that the subsequent dose of immune effector cells is administered at a time point of about 21 to 80 days after the administration of the first dose; preferably, the subsequent dose of immune effector cells is administered at a time point of about 25 to 60 days after the administration of the first dose; more preferably, the subsequent dose of immune effector cells is administered at a time point of about 25 to 50 days after the administration of the first dose.

[0647] 59. The kit according to any one of embodiments 55-57, characterized in that the instructions for administration record that the cyclophosphamide, fludarabine and taxane compounds are administered 4-12 days before the administration of immune effector cells.

[0648] 60. The kit according to any one of embodiments 55-59, wherein the administration instructions contain:

[0649] Fludarabine is administered in doses of approximately 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 / day; and / or

[0650] The dose 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 490-550mg / m 2 / day; and / or

[0651] The taxane compound is administered in an amount not exceeding about 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 no more than about 80 mg / m 2 / day, or no more than about 70 mg / m 2 / sky.

[0652] 61. The kit according to any one of embodiments 55-60, wherein the administration instructions state:

[0653] The cyclophosphamide and / or fludarabine are used continuously for no more than 4 days, and the taxane compound is administered once.

[0654] 62. A kit as described in any one of embodiments 55-61, characterized in that the chimeric antigen receptor has an amino acid sequence shown in any one of SEQ ID NO: 24, 25, 26, 27, 28, 29, 30, 31, and 32; preferably, it is a sequence shown in SEQ ID NO: 24, 25 or 26.

[0655] Advantages of the present invention:

[0656] 1. The present invention significantly improves the therapeutic efficacy of tumor treatment using immune effector cells expressing chimeric antigen receptors: the number of tumors is reduced or the volume is reduced, and / or the number and / or degree of metastasis is reduced, and / or tumor markers are reduced, and / or the common complications of advanced cancer disappear or weaken.

[0657] 2. It has a significant anti-tumor effect on solid tumors, providing a safe and effective treatment method that can stabilize or even cure CLA18A2-positive advanced gastrointestinal adenocarcinoma.

[0658] 3. Provide a pretreatment composition for improving the anti-tumor therapy of immune effector cells. Administration of a fludarabine, cyclophosphamide, and albumin-bound paclitaxel composition before administering immune effector cells can significantly promote the anti-tumor efficacy of immune effector cells, significantly reduce the patient's tumor burden, improve the patient's quality of life, and prolong survival.

[0659] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd edition, Science Press, 2002, or according to the conditions recommended by the manufacturer.

[0660] Materials and Methods

[0661] Material:

[0662] Various materials, including reagents, used in the present invention can be purchased from commercial channels.

[0663] Exemplary antigen receptors of the present invention, including CARs, and methods for engineering and introducing receptors into cells, refer to, for example, the full contents disclosed in Chinese Patent Application Publication Nos. CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN106397593A, CN106467573A, International Patent Application Publication Nos. WO2018006882A1, WO2015172339A8.

[0664] In the following embodiments of the present invention, the scFv portion of the chimeric antigen receptor 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: 18, the chimeric antigen receptor has a sequence as shown in SEQ ID NO: 24, the amino acid sequence of the scFv is shown in SEQ ID NO: 54, the nucleic acid sequence is shown in SEQ ID NO: 55, and the CDR regions are 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, and LCDR3 as shown in SEQ ID NO: 6.

[0665] Method for constructing CAR-T cells: First, construct the CAR gene, such as the constructio...

Claims

1. A method for treating a CLD18-positive tumor, the method comprising administering a first dose of immune effector cells to a subject in need thereof, the immune effector cells expressing a chimeric antigen receptor (CAR) and specifically recognizing CLD18, the first dose comprising no more than about 2×10 10 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 12 cell, Preferably, the first dose does not exceed about 2 x 10 9 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 11 cell, Preferably, the first dose does not exceed about 2.5 x 10 8 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 10 cell, Preferably, the first dose does not exceed about 5 x 10 7 cells / kg of subject body weight or the total amount of said cells does not exceed about 1 x 10 10 cell, More preferably, the first dose does not exceed about 3 x 10 7 cells / kg of subject body weight or the total amount of said cells does not exceed about 2 x 10 9 cell.

2. The method according to claim 1, characterized in that The CLD18 is CLD18A2.

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

4. The method according to claim 3, characterized in that After the immune effector cells administered at the first dose are no longer detectable in vivo, the subsequent dose of immune effector cells is administered.

5. The method according to claim 3, characterized in that The subsequent dose of immune effector cells is administered at a time point of about 21 to 80 days after the first dose; preferably, the subsequent dose of immune effector cells is administered at a time point of about 25 to 60 days after the first dose; more preferably, the subsequent dose of immune effector cells is administered at a time point of about 25 to 50 days after the first dose.

6. The method according to claim 5, characterized in that The subsequent dose is higher than the initial dose; preferably, the subsequent dose is 2 times, 5 times, 7 times or 10 times the initial dose.

7. A method for treating tumors, comprising administering chemotherapy, radiotherapy, or immunotherapy to the subject before administering the method of any one of claims 1 to 6.

8. A composition comprising cyclophosphamide and fludarabine or a combination thereof with a taxane compound and / or a pyrimidine antitumor drug.

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

10. A kit for treating CLD18-positive tumors, characterized in that: The kit comprises: 1) Immune effector cells containing CLD18A2-CAR; 2) Cyclophosphamide; 3) Fludarabine; 4) Taxane compounds, preferably paclitaxel, more preferably albumin-bound paclitaxel; 5) A container for containing the substances described in 1) to 4) above; and 6) Instructions for use of the kit for treating CLD18-positive tumors; The cells and cyclophosphamide, fludarabine and taxane compounds are formulated to provide a therapeutic effect greater than the sum of the effects of each of the agents.

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