Tumor treatment method and composition

Abstract

The present invention provides a composition of immune effector cells, wherein the composition of immune effector cells comprises an initial amount of immune effector cells and a subsequent amount of immune effector cells, and a therapeutic kit comprising the composition of such immune effector cells.

Description

Technology Field

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

[0002] Recently, tumor immunotherapy has been widely monitored and applied, and CAR-T technology, in particular, has made historic progress in human control of tumors.

[0003] Cancer cells in solid tumors form a tumor microenvironment in their surroundings, supporting their growth and metastasis. The tumor microenvironment is the cellular environment in which a tumor exists, comprising surrounding blood vessels, immune cells, fibroblasts, other cells, soluble factors, signaling molecules, extracellular matrix, and mechanical cues that promote tumor transformation, support tumor growth and invasion, protect the tumor from host immunity, cultivate resistance to treatment, and provide a microenvironment for dormant metastasis and rapid growth. Since tumors are closely related to and constantly interact with their surrounding microenvironment, they can influence this microenvironment by releasing extracellular signals, promoting tumor angiogenesis, and inducing peripheral immune tolerance. In this regard, refer to Swarts et al., "Tumor Microenvironment Complexity: Emerging Roles in Cancer Therapy," Cancer Res, Vol. 72, pp. 2473–2480, 2012. Consequently, CAR-T technology is generally difficult to apply effectively to the treatment of solid tumors. For example, in the majority of current literature, it has been reported that CD19 CAR T-cell therapy in hematological malignancies can produce very good clinical therapeutic effects in patients. However, CAR T-cell therapy has not yet achieved satisfactory therapeutic effects in the treatment of solid tumors.

[0004] Therefore, there is an urgent need for a technical solution in this field that can possess significant anti-tumor capabilities in the cancer microenvironment of solid tumors.

[0005] The objective of the present invention is to provide a technical solution capable of having an excellent lethal effect against CLD18-positive solid tumors.

[0006] According to a first aspect, the present invention provides a method for treating a CLD18-positive tumor, wherein the method comprises the step of administering an initial dose of immune effector cells to a subject requiring such cells, said immune effector cells express a chimeric antigen receptor (CAR) and specifically identify CLD18, and said initial dose is approximately 2 x 10 10 Cells / kg shall not exceed the subject's body weight, or the total amount of cells is approximately 1 x 10⁻⁶ 12 It does not exceed cells. Preferably, the initial usage is about 2 x 10 9 Cells / kg not exceeding the subject's body weight, or the total amount is approximately 1 x 10⁻⁶ 11 It does not exceed the cell. Preferably, the initial usage is about 2.5 x 10 8 Cells / kg shall not exceed the subject's body weight, or the total amount of cells is approximately 1 x 10⁻⁶ 10 It does not exceed cells. Preferably, the initial usage is about 5×10 7 Cells / kg not exceeding the subject's body weight, or the total amount is approximately 1 x 10⁻⁶ 10 It does not exceed cells. More preferably, the initial usage is about 3×10 7 Cells / kg not exceeding the subject's body weight, or the total amount is approximately 5×10 9 or about 2x10 9 It does not exceed the cell.

[0007] In a preferred embodiment, the initial usage is 1X10 5 Cells / kg is not less than the subject's body weight.

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

[0009] In a specific embodiment, after administering the first amount of immune effector cells, at least one subsequent amount of immune effector cells expressing a chimeric antigen receptor (CAR) and specifically identifying CLD18 is administered.

[0010] In a preferred embodiment, under the premise of safety for the patient, the subsequent amount of immune effector cells is administered.

[0011] In a preferred embodiment, the immune effector cells of the first amount and the immune effector cells of the subsequent amount are identical.

[0012] In a preferred embodiment, the immune effector cells of the first use and the immune effector cells of the subsequent use both identify CLD18A2 but contain different chimeric antigen receptors, for example, the extracellular segments of the chimeric antigen receptors are different but both identify CLD18A2, or the extracellular segments of the chimeric antigen receptors are the same but the transmembrane domain or the intracellular domain is different.

[0013] In a specific embodiment, after the immune effector cells administered in the first amount are not detected in the body, the subsequent amount of immune effector cells is administered.

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

[0015] In a specific embodiment, the subsequent amount of immune effector cells is administered at a time of about 21 to 80 days after the first amount is administered; preferably, the subsequent amount of immune effector cells is administered at a time of about 25 to 60 days after the first amount is administered; more preferably, the subsequent amount of immune effector cells is administered at a time of about 25 to 50 days after the first amount is administered.

[0016] In a preferred embodiment, the subsequent usage is less than, equal to, or greater than the initial usage.

[0017] In a preferred embodiment, the subsequent usage is equivalent to the initial usage.

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

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

[0020] In a specific embodiment, the subsequent usage is higher than the initial usage; preferably, the subsequent usage is at least 2, 5, 7, or 10 times the initial usage.

[0021] In a specific embodiment, the subsequent usage is approximately 2X10 10 Cells / kg shall not exceed the subject's body weight, or the total amount of cells is approximately 1 x 10⁻⁶ 12 It does not exceed the cell.

[0022] In a preferred embodiment, the subsequent usage is about 2X10 9 Cells / kg not exceeding the subject's body weight, or the total amount is approximately 1 x 10⁻⁶ 11 It does not exceed the cell.

[0023] In a preferred embodiment, the subsequent usage is about 2.5 x 10 8 Cells / kg shall not exceed the subject's body weight, or the total amount of cells is approximately 1 x 10⁻⁶ 10 It does not exceed the cell.

[0024] In a preferred embodiment, the subsequent usage is about 5×10 7 Cells / kg not exceeding the subject's body weight, or the total amount is approximately 1 x 10⁻⁶ 10 It does not exceed the cell.

[0025] In a preferred embodiment, the subsequent usage is about 3X10 7 Cells / kg must not exceed the subject's body weight, or the total amount is 5×109 or about 2x10 9 It does not exceed the cell.

[0026] In a preferred embodiment, the subsequent usage is 1X10 5 Cells / kg is not less than the subject's body weight.

[0027] In a specific embodiment, the first or subsequent amount is administered in N divided doses within 20 days, where N is a natural number not less than 1.

[0028] In a preferred embodiment, the first or subsequent amount is administered in N divided doses within 3 to 15 days, where N is a natural number not less than 1.

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

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

[0031] In a specific embodiment, when administered at a subsequent dosage, the subject,

[0032] (i) a feature in which the serum level of the factor indicating cytokine release syndrome (CRS) in the subject is about 10 times lower, about 25 times lower, and / or about 50 times lower than the level immediately prior to administration of the first dose in the subject;

[0033] (ii) Characteristics of not exhibiting grade 3 or higher neurotoxicity;

[0034] (iii) A characteristic in which the neurotoxicity or CRS level is reduced compared to the peak level of the neurotoxicity or CRS level after administration of the initial dose of immune effector cells; or

[0035] (iv) The subject has one of the following characteristics: not exhibiting a detectable humoral or cell-mediated immune response to the CAR expressed by the cells of the first amount used.

[0036] In a specific embodiment, when administered with the subsequent amount, if the subject has characteristic (iii), the CRS level is reduced by at least 50% compared to the peak level of CRS after administering the first amount 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 administering the first amount of immune effector cells.

[0037] In a preferred embodiment, the CRS level can be evaluated based on information such as fever, hypotension, hypoxia, neurological disorders, or serum levels of inflammatory cytokines or C-reactive protein (CRP).

[0038] In a preferred embodiment, symptoms associated with clinical risk of neurotoxicity and / or grade 3 or higher neurotoxicity are selected from confusion, delirium, expressive aphasia, insensitivity, myoclonus, somnolence, altered mental state, convulsions, epilepsy-like symptoms, epilepsy (optional, confirmed by electroencephalogram EEG), elevated beta-amyloid (Aβ) levels, elevated glutamate levels, and elevated oxygen free radical levels.

[0039] In a specific embodiment, the method further comprises the step of pre-treating the immune effector cells before administering them, wherein the pre-treatment includes administering a chemotherapy agent to the subject, or performing whole-body radiation therapy on the subject, or performing local radiation therapy on the subject, or a combination thereof.

[0040] In a preferred embodiment, the pretreatment is performed before administering the first dose or between administering the first dose and the subsequent dose; more preferably, the pretreatment is performed before administering the first dose and the subsequent dose.

[0041] In a preferred embodiment, the pretreatment is performed before administering the first amount of immune effector cells, and it is not necessary to perform the pretreatment before administering the subsequent amount of immune effector cells.

[0042] In a preferred embodiment, the above pretreatment must be performed both before administering the first amount of immune effector cells and before administering the subsequent amount of immune effector cells.

[0043] In a preferred embodiment, the pretreatment is performed to remove lymphocytes.

[0044] In a preferred embodiment, the pretreatment is performed to stabilize or reduce the tumor burden, and in particular to maintain the stabilization or reduction of the tumor burden before performing immune effector cell therapy.

[0045] In a preferred embodiment, lymphocytes are removed by administering a lymphocyte-removing agent to the subject. Lymphocyte-removing agents are, for example, cyclophosphamide, fludarabine, etc.

[0046] In a specific embodiment, the pretreatment is performed 4 to 12 days before administering immune effector cells.

[0047] In a specific embodiment, the chemotherapy agent is selected from one chemotherapy agent among cyclophosphamide, fludarabine, taxane compounds, pyrimidine antitumor drugs, or a combination thereof.

[0048] In a preferred embodiment, the taxane compound includes, but is not limited to, paclitaxel, albumin-bound paclitaxel or docetaxel, preferably, albumin-bound paclitaxel; and the pyrimidine antitumor drug includes, but is not limited to, 5-fluorouracil, gemepyrimidine, ottiracil potassium, difurfurouracil, carmofur, deoxyfluridine, and capecitabine.

[0049] In a specific embodiment, the chemotherapy agent is a combination of cyclophosphamide and fludarabine; or a combination of cyclophosphamide, fludarabine and albumin-bound paclitaxel.

[0050] In a preferred embodiment, the dosage of fludarabine is about 10 to 50 mg / m² 2 / day, or about 15–40 mg / m² 2 / day, or about 15 to 30 mg / m² 2 / day, or about 20–30 mg / m² 2 It is work.

[0051] In a preferred embodiment, the dosage of cyclophosphamide is about 300 to 700 mg / m² 2 / day, or about 400–650 mg / m² 2 / day, or about 450–600 mg / m² 2 / day, or about 450–550 mg / m² 2 / day, or about 490–550 mg / m² 2 It is work.

[0052] In a preferred embodiment, the dosage of albumin-bound paclitaxel is about 300 mg / m² 2 No more than / day, or about 200 mg / m² 2 Not more than / day, or 150 mg / m² 2No more than / day, or about 100 mg / m² 2 No more than / day, or about 80 mg / m² 2 No more than / day, or about 70 mg / m² 2 It is not more than work.

[0053] In a specific embodiment, the continuous use time of the above chemotherapy agent does not exceed 4 days.

[0054] In a specific embodiment, the continuous use time of the cyclophosphamide and / or fludarabine does not exceed 4 days, and the albumin-bound paclitaxel is administered once.

[0055] In a preferred embodiment, pretreatment is performed, and in particular, if pretreatment is performed before administration of the initial amount and subsequent amounts, the amount of immune effector cells subsequently administered can be correspondingly reduced.

[0056] In a specific embodiment, the tumor is a CLD18A2-positive tumor; preferably, the tumor is a CLD18A2-positive gastrointestinal tumor; more preferably, the gastrointestinal tumor is an adenocarcinoma; and most preferably, the gastrointestinal tumor is pancreatic cancer or gastric adenocarcinoma.

[0057] 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.

[0058] In a preferred embodiment, the transmembrane domain of the chimeric antigen receptor is the transmembrane domain of CD28 or CD8.

[0059] In a preferred embodiment, the intracellular domain of the chimeric antigen receptor is a CD28 co-stimulatory signal domain and a CD3ζ fusion peptide, or a CD137 co-stimulatory signal domain and a CD3ζ fusion peptide, or a CD28 co-stimulatory signal domain, a CD137 co-stimulatory signal domain and a CD3ζ fusion peptide.

[0060] 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, and the antibody,

[0061] HCDR1 indicated by SEQ ID NO: 1, HCDR2 indicated by SEQ ID NO: 2, HCDR3 indicated by SEQ ID NO: 3, LCDR1 indicated by SEQ ID NO: 4, LCDR2 indicated by SEQ ID NO: 5, LCDR3 indicated by SEQ ID NO: 6; or

[0062] HCDR1 indicated by SEQ ID NO: 1, HCDR2 indicated by SEQ ID NO: 7, HCDR3 indicated by SEQ ID NO: 3, LCDR1 indicated by SEQ ID NO: 4, LCDR2 indicated by SEQ ID NO: 5, LCDR3 indicated by SEQ ID NO: 6; or

[0063] It has HCDR1 marked with SEQ ID NO: 8, HCDR2 marked with SEQ ID NO: 9 or SEQ ID NO: 68, HCDR3 marked with SEQ ID NO: 10, LCDR1 marked with SEQ ID NO: 11, LCDR2 marked with SEQ ID NO: 12, and LCDR3 marked with SEQ ID NO: 13.

[0064] In a specific embodiment, the antibody or fragment thereof is,

[0065] Heavy chain variable region indicated by SEQ ID NO: 14 and light chain variable region indicated by SEQ ID NO: 16; or

[0066] Heavy chain variable region indicated by SEQ ID NO: 18 and light chain variable region indicated by SEQ ID NO: 16; or

[0067] Heavy chain variable region indicated by SEQ ID NO: 22 and light chain variable region indicated by SEQ ID NO: 20; or

[0068] It has a heavy chain variable region represented by SEQ ID NO: 53 and a light chain variable region represented by SEQ ID NO: 52. Preferably, the antibody or fragment thereof has a sequence represented by SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58.

[0069] In a preferred embodiment, the antibody or fragment thereof has a heavy chain variable region represented by SEQ ID NO: 14 and a light chain variable region represented by SEQ ID NO: 16.

[0070] In a specific embodiment, the chimeric antigen receptor has an amino acid sequence represented by any one of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32; preferably, has a sequence represented by SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

[0071] In a preferred embodiment, the chimeric antigen receptor has an amino acid sequence represented by any one of SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

[0072] In a preferred example, the chimeric antigen receptor has an amino acid sequence represented by SEQ ID NO: 24.

[0073] In a specific embodiment, the immune effector cell is a T lymphocyte, NK cell, or NKT lymphocyte.

[0074] In a preferred embodiment, the preferred immune effector cell is a T lymphocyte.

[0075] In a preferred embodiment, the T lymphocytes are derived from the subject themselves.

[0076] In a preferred embodiment, the T lymphocytes are derived from a homologous lineage.

[0077] In a specific embodiment, subsequent amounts of immune effector cells are administered two or more times, wherein the time interval between each administration of subsequent amounts of immune effector cells is about 21 to about 80 days, or about 25 to about 60 days, or about 25 to 50 days, and each includes an end value.

[0078] In a preferred solution, 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.

[0079] In a specific embodiment, the number of immune cells in the subsequent dose administered each time is basically the same.

[0080] 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.

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

[0082] 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 that targets CLD18.

[0083] In a specific embodiment, the subsequent usage amount is the number of cells that sufficiently stabilize or reduce the tumor burden of the subject.

[0084] In a specific embodiment, prior to administration of the initial dose, the subject had already received surgical treatment, chemotherapy, or immunotherapy different from the method according to the first embodiment.

[0085] In a specific embodiment, serum levels of factors indicating the subject's CRS, factors indicating neurotoxicity, factors indicating tumor burden, and / or factors indicating the host anti-CAR immune response are evaluated before administering the first dose, or after administering the first dose and also before administering the subsequent dose.

[0086] In a specific embodiment, the factor indicating the tumor burden is the total number of tumor cells of 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.

[0087] In a specific embodiment, the tumor treatment method is,

[0088] i) A step of evaluating factors indicating tumor burden before administering a subsequent dose;

[0089] ii) a step of determining the continuous amount of cells to be administered to the subject based on the results of the above evaluation; and

[0090] iii) If it is determined through evaluation that the tumor mass or volume of the subject is stable or reduced, the step of administering to the subject a subsequent amount containing a number of CAR-expressing cells that is less than, greater than, or nearly equal to the number of CAR-expressing cells in the initial amount is included.

[0091] In a specific embodiment, the number of cells administered at the initial amount or the subsequent amount is approximately 1.1 x 10⁻⁶ 6 Cells / kg subject body weight to 2.8×10 7 Cells / kg, approx. 2.9 x 10⁻⁶ 6 cells / kg to 2.6X10 7 Cells / kg or about 3.3X10 6 cells / kg to 1.3X10 7 Cells / kg or about 1.3X10 7 cells / kg to 1.8×10⁻¹⁰ 7 It is cells / kg, and each includes the end value.

[0092] In a specific embodiment, the number of cells to which CAR-expressing cells are administered for a subsequent use is approximately 1.1 x 10⁻⁶ 7 Cells / kg (cells / kg) Body weight to about 5.1 x 10⁻⁶ 7 Cells / kg, approx. 1.3 x 10⁻⁶ 7 cells / kg to about 3.7X10 7 Cells / kg, approx. 1.6 x 10⁻⁶ 7 cells / kg to about 2.2X10 7 Cells / kg, approx. 1.9 x 10⁻⁶ 7 cells / kg to about 2.2X10 7 Cells / kg, approx. 2.2X10 7 cells / kg to about 2.7X10 7 Includes cells / kg, and each includes an end value.

[0093] In a specific embodiment, after administering an initial dose or a subsequent dose 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 exceeding grade 3.

[0094] In a specific embodiment, after administering an initial or subsequent amount of immune effector cells, the immune effector cells proliferate in the subject.

[0095] In a preferred embodiment, the proliferation is indicated by (1) an increase in serum CRP levels after administration of the first and / or subsequent doses compared to immediately before administration, and / or (ii) an increase in serum CAR encoding nucleic acid levels after administration of the first and / or subsequent doses compared to immediately before administration, which is measured, for example, by qPCR.

[0096] In a specific embodiment, after administering an initial amount of immune effector cells, the tumor burden of the subject is stabilized or reduced. In a specific embodiment, the stabilization or reduction of the tumor burden is expressed as stabilization or reduction according to one or more factors indicating the tumor burden.

[0097] In a preferred embodiment, after administering a subsequent amount of immune effector cells, the tumor burden was further stabilized or reduced compared to the tumor burden after administering the first amount of immune effector cells.

[0098] In a specific embodiment, when administered at a subsequent dose, the subject has not yet relapsed, and / or one or more factors indicating tumor burden have not yet increased after being reduced.

[0099] In specific embodiments, the stabilization or reduction of the burden and / or further reduction of the burden includes the stabilization or reduction of the total number of disease cells in the subject, in the subject's organs, in the subject's tissues, or in the subject's body fluids, and / or the stabilization or reduction of the mass or volume of the tumor, and / or the stabilization or reduction of the number and / or degree of metastasis, and / or the stabilization or reduction of tumor markers, and / or the disappearance or attenuation of complications commonly seen in late-stage cancer.

[0100] In a specific embodiment, the tumor marker includes alpha-fetoprotein (AFP), CA125, CA15-3, squamous cell carcinoma antigen (SCC), soluble fragment of cytokeratin 19 (CYFRA21-1), carcinogen antigen (CEA), CA199, CA724, etc.

[0101] In a specific embodiment, complications commonly occurring in advanced cancer include cancer pain, infection, and cancerous pleural and abdominal distension.

[0102] In a specific embodiment, the mass or volume of the tumor is measured using PET (positron emission tomography) and CT (computed tomography).

[0103] According to a second aspect of the present invention, a tumor treatment method is further provided, comprising the step of having already received chemotherapy, radiation therapy, immunotherapy, or a combination thereof, before treatment according to any one of the above methods.

[0104] In a preferred solution, the immunotherapy includes a treatment involving the administration of a checkpoint inhibitor or an immune effector cell therapy that does not target CLD18.

[0105] In a preferred solution, the immunotherapy does not include immune effector cell therapy targeting CLD18.

[0106] In a preferred solution, the subject received immune effector cell therapy that does not target CLD18.2.

[0107] In a preferred embodiment, after receiving the chemotherapy, radiation therapy, other immunotherapy, or a combination thereof, the tumor burden did not remain stabilized or reduced.

[0108] In a specific embodiment, the tumor is a recurrent tumor or an intractable tumor.

[0109] According to a third aspect of the present invention, in the preparation of a drug for treating a CLD18A2-positive tumor in a subject previously treated with CLD18A2-CAR T cells, the use of a composition comprising an immune effector cell expressing a chimeric antigen receptor that specifically identifies CLD18 is provided, wherein, at use, an initial dose of immune effector cells is administered to a subject requiring the drug, said immune effector cell expresses a chimeric antigen receptor (CAR) and specifically identifies CLD18, and said initial dose is approximately 2 x 10 10 Cells / kg shall not exceed the subject's body weight, or the total amount of cells is approximately 1 x 10⁻⁶ 12 It does not exceed cells. Preferably, the initial usage is about 2 x 10 9 Cells / kg not exceeding the subject's body weight, or the total amount is approximately 1 x 10⁻⁶ 11 It does not exceed the cell. Preferably, the initial usage is about 2.5 x 10 8 Cells / kg shall not exceed the subject's body weight, or the total amount of cells is approximately 1 x 10⁻⁶ 10 It does not exceed cells. Preferably, the initial usage is about 5×10 7 Cells / kg not exceeding the subject's body weight, or the total amount is approximately 1 x 10⁻⁶ 10 It does not exceed cells. More preferably, the initial usage is about 3×10 7 Cells / kg must not exceed the subject's body weight, or the total amount is 5×10 9 or about 2x109 It does not exceed the cell.

[0110] In a specific embodiment, the above CLD18 is CLD18A2.

[0111] In a specific embodiment, after administering the first amount of immune effector cells, at least one subsequent amount of immune effector cells expressing a chimeric antigen receptor (CAR) and specifically identifying CLD18 is administered.

[0112] In a preferred embodiment, under the premise of safety for the patient, the subsequent amount of immune effector cells is administered.

[0113] In a preferred embodiment, the immune effector cells of the first amount and the immune effector cells of the subsequent amount are identical.

[0114] In a preferred embodiment, the immune effector cells of the first use and the immune effector cells of the subsequent use both identify CLD18A2 but contain different chimeric antigen receptors, for example, the extracellular segments of the chimeric antigen receptors are different but both identify CLD18A2, or the extracellular segments of the chimeric antigen receptors are the same but the transmembrane domain or the intracellular domain is different.

[0115] In a specific embodiment, after the immune effector cells administered in the first amount are not detected in the body, the subsequent amount of immune effector cells is administered.

[0116] In a preferred embodiment, it is measured, for example, by qPCR. In a specific embodiment, the subsequent amount of immune effector cells is administered at a time of about 21 to 80 days after the first amount is administered; preferably, the subsequent amount of immune effector cells is administered at a time of about 25 to 60 days after the first amount is administered; more preferably, the subsequent amount of immune effector cells is administered at a time of about 25 to 50 days after the first amount is administered.

[0117] In a preferred embodiment, the subsequent usage is equivalent to the initial usage.

[0118] In a preferred embodiment, the subsequent usage is less than the initial usage.

[0119] In a preferred embodiment, the subsequent usage is greater than the initial usage.

[0120] In a specific embodiment, the subsequent usage is higher than the initial usage, and the subsequent usage is at least 2, 5, 7, or 10 times the initial usage.

[0121] In a specific embodiment, the subsequent usage is approximately 2X10 10 Cells / kg shall not exceed the subject's body weight, or the total amount of cells is approximately 1 x 10⁻⁶ 12 It does not exceed the cell.

[0122] In a preferred embodiment, the subsequent usage is about 2X10 9 Cells / kg not exceeding the subject's body weight, or the total amount is approximately 1 x 10⁻⁶ 11 It does not exceed the cell.

[0123] In a preferred embodiment, the subsequent usage is about 2.5 x 10 8 Cells / kg shall not exceed the subject's body weight, or the total amount of cells is approximately 1 x 10⁻⁶ 10 It does not exceed the cell.

[0124] In a preferred embodiment, the subsequent usage is about 5×10 7 Cells / kg not exceeding the subject's body weight, or the total amount is approximately 1 x 10⁻⁶ 10 It does not exceed the cell.

[0125] In a preferred embodiment, the subsequent usage is about 3X10 7 Cells / kg must not exceed the subject's body weight, or the total amount is 5×10 9 or about 2x10 9 It does not exceed the cell.

[0126] In a specific embodiment, the first or subsequent amount is administered in N divided doses within 20 days, where N is a natural number not less than 1.

[0127] In a preferred embodiment, the first or subsequent amount is administered in N divided doses within 3 to 15 days, where N is a natural number not less than 1.

[0128] In a preferred embodiment, N is 1, 2, 3, or 4.

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

[0130] In a specific embodiment, when administered at a subsequent dosage, the subject,

[0131] (i) a feature in which the serum level of the factor indicating cytokine release syndrome (CRS) in the subject is about 10 times lower, about 25 times lower, and / or about 50 times lower than the level immediately prior to administration of the first dose in the subject;

[0132] (ii) Characteristics of not exhibiting grade 3 or higher neurotoxicity;

[0133] (iii) A characteristic in which the neurotoxicity or CRS level is reduced compared to the peak level of the neurotoxicity or CRS level after administration of the initial dose of immune effector cells; or

[0134] (iv) The subject has one of the following characteristics: not exhibiting a detectable humoral or cell-mediated immune response to the CAR expressed by the cells of the first amount used.

[0135] In a specific embodiment, when administered with the subsequent amount, if the subject has characteristic (iii), the CRS level is reduced by at least 50% compared to the peak level of CRS after administering the first amount 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 administering the first amount of immune effector cells.

[0136] In a preferred embodiment, the CRS level can be evaluated based on information such as fever, hypotension, hypoxia, neurological disorders, or serum levels of inflammatory cytokines or C-reactive protein (CRP).

[0137] In a preferred embodiment, the clinical risk of neurotoxicity and / or symptoms associated with grade 3 or higher neurotoxicity are confusion, delirium, expressive aphasia, insensitivity, myoclonus, somnolence, altered mental status, seizures, epilepsy-like symptoms, epilepsy (optionally, confirmed by electroencephalogram EEG), beta-amyloid (A It is selected from an increase in ) level, an increase in glutamic acid level, and an increase in oxygen free radical level.

[0138] In a specific embodiment, the method further comprises the step of pre-treating the immune effector cells before administering them, wherein the pre-treatment includes administering a chemotherapy agent to the subject, or performing whole-body radiation therapy on the subject, or performing local radiation therapy on the subject, or a combination thereof.

[0139] In a preferred embodiment, the pretreatment is performed before administering the first amount of immune effector cells, and it is not necessary to perform the pretreatment before administering the subsequent amount of immune effector cells.

[0140] In a preferred embodiment, the above pretreatment must be performed both before administering the first amount of immune effector cells and before administering the subsequent amount of immune effector cells.

[0141] In a preferred embodiment, the pretreatment is performed to remove lymphocytes.

[0142] In a preferred embodiment, the pretreatment is performed to stabilize or reduce the tumor burden, and in particular to maintain the stabilization or reduction of the tumor burden before performing immune effector cell therapy.

[0143] In a preferred embodiment, lymphocytes are removed by administering a lymphocyte-removing agent to the subject. Lymphocyte-removing agents are, for example, cyclophosphamide, fludarabine, etc.

[0144] In a specific embodiment, the pretreatment is performed 4 to 12 days before administering immune effector cells.

[0145] In a specific embodiment, the chemotherapy agent is selected from one chemotherapy agent among cyclophosphamide, fludarabine, taxane compounds, and pyrimidine antitumor drugs, or a combination thereof. In a specific embodiment, the chemotherapy agent is a combination of cyclophosphamide and fludarabine; or a combination of cyclophosphamide, fludarabine, and albumin-bound paclitaxel.

[0146] In a preferred embodiment, the dosage of fludarabine is about 10 to 50 mg / m² 2 / day, or about 15–40 mg / m² 2 / day, or about 15 to 30 mg / m² 2 / day, or about 20–30 mg / m² 2 It is work.

[0147] In a preferred embodiment, the dosage of cyclophosphamide is about 300 to 700 mg / m² 2 / day, or about 400–650 mg / m² 2 / day, or about 450–600 mg / m² 2 / day, or about 450–550 mg / m² 2 / day, or about 490–550 mg / m² 2 It is work.

[0148] In a preferred embodiment, the dosage of albumin-bound paclitaxel is about 300 mg / m² 2 No more than / day, or about 200 mg / m² 2 Not more than / day, or 150 mg / m² 2 No more than / day, or about 100 mg / m² 2 No more than / day, or about 80 mg / m² 2 No more than / day, or about 70 mg / m² 2 It is not more than work.

[0149] In a specific embodiment, the continuous use time of the above chemotherapy agent does not exceed 4 days.

[0150] In a specific embodiment, the continuous use time of the cyclophosphamide and the fludarabine does not exceed 4 days, and the albumin-bound paclitaxel is administered once.

[0151] In a specific embodiment, the tumor is a CLD18A2-positive tumor; preferably, the tumor is a CLD18A2-positive gastrointestinal tumor; more preferably, the gastrointestinal tumor is an adenocarcinoma; and most preferably, the gastrointestinal tumor is pancreatic cancer or gastric adenocarcinoma.

[0152] 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.

[0153] In a preferred embodiment, the transmembrane domain of the chimeric antigen receptor is the transmembrane domain of CD28 or CD8.

[0154] In a preferred embodiment, the intracellular domain of the chimeric antigen receptor is a CD28 co-stimulatory signal domain and a CD3ζ fusion peptide, or a CD137 co-stimulatory signal domain and a CD3ζ fusion peptide, or a CD28 co-stimulatory signal domain, a CD137 co-stimulatory signal domain and a CD3ζ fusion peptide.

[0155] 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, and the antibody,

[0156] HCDR1 indicated by SEQ ID NO: 1, HCDR2 indicated by SEQ ID NO: 2, HCDR3 indicated by SEQ ID NO: 3, LCDR1 indicated by SEQ ID NO: 4, LCDR2 indicated by SEQ ID NO: 5, LCDR3 indicated by SEQ ID NO: 6; or

[0157] HCDR1 indicated by SEQ ID NO: 1, HCDR2 indicated by SEQ ID NO: 7, HCDR3 indicated by SEQ ID NO: 3, LCDR1 indicated by SEQ ID NO: 4, LCDR2 indicated by SEQ ID NO: 5, LCDR3 indicated by SEQ ID NO: 6; or

[0158] It has HCDR1 marked with SEQ ID NO: 8, HCDR2 marked with SEQ ID NO: 9 or SEQ ID NO: 68, HCDR3 marked with SEQ ID NO: 10, LCDR1 marked with SEQ ID NO: 11, LCDR2 marked with SEQ ID NO: 12, and LCDR3 marked with SEQ ID NO: 13.

[0159] In a specific embodiment, the antibody or fragment thereof is,

[0160] Heavy chain variable region indicated by SEQ ID NO: 14 and light chain variable region indicated by SEQ ID NO: 16; or

[0161] Heavy chain variable region indicated by SEQ ID NO: 18 and light chain variable region indicated by SEQ ID NO: 16; or

[0162] It has a heavy chain variable region indicated by SEQ ID NO: 22 and a light chain variable region indicated by SEQ ID NO: 20.

[0163] In a preferred embodiment, the antibody or fragment thereof has a heavy chain variable region represented by SEQ ID NO: 14 and a light chain variable region represented by SEQ ID NO: 16.

[0164] In a specific embodiment, the chimeric antigen receptor has an amino acid sequence represented by any one of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32.

[0165] In a preferred embodiment, the chimeric antigen receptor has an amino acid sequence represented by any one of SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

[0166] In a preferred example, the chimeric antigen receptor has an amino acid sequence represented by SEQ ID NO: 24.

[0167] In a specific embodiment, the immune effector cell is a T lymphocyte, NK cell, or NKT lymphocyte.

[0168] In a preferred embodiment, the preferred immune effector cell is a T lymphocyte.

[0169] In a preferred embodiment, the T lymphocytes are derived from the subject themselves.

[0170] In a preferred embodiment, the T lymphocytes are derived from a homologous lineage.

[0171] In a specific embodiment, subsequent amounts of immune effector cells are administered two or more times, wherein the time interval between each administration of subsequent amounts of immune effector cells is about 21 to about 80 days, or about 25 to about 60 days, or about 25 to 50 days, and each includes an end value.

[0172] In a preferred solution, 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.

[0173] In a specific embodiment, the number of immune cells in the subsequent dose administered each time is basically the same.

[0174] 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.

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

[0176] 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 that targets CLD18.

[0177] In a specific embodiment, the subsequent usage amount is the number of cells that sufficiently stabilize or reduce the tumor burden of the subject.

[0178] In a specific embodiment, prior to administration of the initial dose, the subject had already received surgical treatment, chemotherapy, or immunotherapy different from the method according to the first embodiment.

[0179] In a specific embodiment, serum levels of factors indicating the subject's CRS, factors indicating neurotoxicity, factors indicating tumor burden, and / or factors indicating the host anti-CAR immune response are evaluated before administering the first dose, or after administering the first dose and also before administering the subsequent dose.

[0180] In a specific embodiment, the factor indicating the tumor burden is the total number of tumor cells of 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.

[0181] In a specific embodiment, the tumor treatment method is,

[0182] i) A step of evaluating factors indicating tumor burden before administering a subsequent dose;

[0183] ii) a step of determining the continuous amount of cells to be administered to the subject based on the results of the above evaluation; and

[0184] iii) If it is determined through evaluation that the tumor mass or volume of the subject is stable or reduced, the step of administering to the subject a subsequent amount containing a number of CAR-expressing cells that is less than, greater than, or nearly equal to the number of CAR-expressing cells in the initial amount is included.

[0185] In a specific embodiment, the number of cells administered at the initial amount or the subsequent amount is approximately 1.1 x 10⁻⁶ 6 Cells / kg subject body weight to 2.8×10 7 Cells / kg, approx. 2.9 x 10⁻⁶ 6cells / kg to 2.6X10 7 Cells / kg or about 3.3X10 6 cells / kg to 1.3X10 7 It is cells / kg, and each includes the end value.

[0186] In a specific embodiment, the number of cells to which CAR-expressing cells are administered for a subsequent use is approximately 1.1 x 10⁻⁶ 7 Cells / kg (cells / kg) Body weight to about 5.1 x 10⁻⁶ 7 Cells / kg, approx. 1.3 x 10⁻⁶ 7 cells / kg to about 3.7X10 7 Cells / kg, approx. 1.6 x 10⁻⁶ 7 cells / kg to about 2.2X10 7 Cells / kg, approx. 1.9 x 10⁻⁶ 7 cells / kg to about 2.2X10 7 Includes cells / kg, and each includes an end value.

[0187] In a specific embodiment, after administering an initial dose or a subsequent dose 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 exceeding grade 3.

[0188] In a specific embodiment, after administering an initial or subsequent amount of immune effector cells, the immune effector cells proliferate in the subject.

[0189] In a preferred embodiment, the proliferation is indicated by (1) an increase in serum CRP levels after administration of the first and / or subsequent doses compared to immediately before administration, and / or (ii) an increase in serum CAR encoding nucleic acid levels after administration of the first and / or subsequent doses compared to immediately before administration, which is measured, for example, by qPCR.

[0190] In a specific embodiment, after administering an initial amount of immune effector cells, the tumor burden of the subject is stabilized or reduced. In a specific embodiment, the stabilization or reduction of the tumor burden is expressed as stabilization or reduction according to one or more factors indicating the tumor burden.

[0191] In a preferred embodiment, after administering a subsequent amount of immune effector cells, the tumor burden was further stabilized or reduced compared to the tumor burden after administering the first amount of immune effector cells.

[0192] In a specific embodiment, when administered at a subsequent dose, the subject has not yet relapsed, and / or one or more factors indicating tumor burden have not yet increased after being reduced.

[0193] In specific embodiments, the stabilization or reduction of the tumor burden and / or further reduction of the burden includes the stabilization or reduction of the total number of disease cells in the subject, in the subject's organs, in the subject's tissues, or in the subject's body fluids, and / or the stabilization or reduction of the mass or volume of the tumor, and / or the stabilization or reduction of the number and / or degree of metastasis, and / or the stabilization or reduction of tumor markers, and / or the disappearance or attenuation of complications commonly seen in late-stage cancer.

[0194] In a specific embodiment, the tumor marker includes alpha-fetoprotein (AFP), CA125, CA15-3, squamous cell carcinoma antigen (SCC), soluble fragment of cytokeratin 19 (CYFRA21-1), carcinogen antigen (CEA), CA199, CA724, etc.

[0195] In a specific embodiment, complications commonly occurring in advanced cancer include cancer pain, infection, and cancerous pleural and abdominal distension.

[0196] In a specific embodiment, the mass or volume of the tumor is measured using PET (positron emission tomography) and CT (computed tomography).

[0197] According to a fourth aspect of the present invention, a composition comprising cyclophosphamide, fludarabine, a taxane-based compound and / or a pyrimidine-based antitumor drug is further provided.

[0198] In a preferred embodiment, the taxane compound comprises, but is not limited to, paclitaxel, albumin-bound paclitaxel, or docetaxel, preferably, albumin-bound paclitaxel; and the pyrimidine antitumor drug comprises, but is not limited to, 5-fluorouracil, gemepyrimidine, othiracil potassium, difurfuracil, carmofur, deoxyfluridine, and capecitabine.

[0199] In a specific embodiment, the composition comprises cyclophosphamide, fludarabine, and paclitaxel.

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

[0201] In a preferred embodiment, the composition is administered to a subject receiving immune effector cell therapy before receiving immune effector cells, preferably, before the subject receiving immune effector cell therapy receives immune effector cells each time.

[0202] In a specific embodiment, the dosage of fludarabine is about 10 to 50 mg / m² 2 / day, or about 15–40 mg / m² 2 / day, or about 15 to 30 mg / m² 2 / day, or about 20–30 mg / m² 2 / day; and / or the dosage of cyclophosphamide is about 300 to 700 mg / m² 2 / day, or about 400–650 mg / m² 2 / day, or about 450–600 mg / m² 2 / day, or about 450–550 mg / m² 2 / day, or about 490–550 mg / m² 2 / day; and / or the dose of albumin-bound paclitaxel is about 300 mg / m² 2 No more than / day, or about 200 mg / m² 2 Not more than / day, or 150 mg / m² 2 No more than / day, or about 100 mg / m² 2 No more than / day, or about 80 mg / m² 2 No more than / day, or about 70 mg / m² 2 It is not more than work.

[0203] In a specific embodiment, pretreatment is performed about 30 days before performing immune cell therapy; preferably, about 20 days before; more preferably, about 12 days before; and most preferably, about 7 days before.

[0204] In a specific embodiment, the continuous use time of the cyclophosphamide, fludarabine and / or albumin-bound paclitaxel does not exceed 4 days; preferably, the albumin-bound paclitaxel is administered once.

[0205] In specific embodiments, it is used for antitumor therapy; preferably, it is used for targeted antitumor therapy; most preferably, it is used for targeted antitumor therapy with chimeric antigen receptors.

[0206] In a specific embodiment, the immune effector cell is a CAR T cell.

[0207] In a specific embodiment, the CAR T cell specifically identifies CLD18A2.

[0208] In a specific embodiment, the tumor is a CLD18A2-positive gastrointestinal tumor; more preferably, the gastrointestinal tumor is an adenocarcinoma; and most preferably, the gastrointestinal tumor is pancreatic cancer or gastric adenocarcinoma.

[0209] According to the fifth aspect of the present invention, a method for combining immune effector cell therapy and tumor therapy is further provided, comprising the step of pre-treating by administering cyclophosphamide, fludarabine, and a taxane-based compound before performing immune effector cell therapy on a subject.

[0210] In a preferred embodiment, the tumor is a solid tumor.

[0211] In a preferred embodiment, the solid tumor is breast cancer, colon cancer, rectal cancer, kidney cancer, 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 cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) cancer, primary CNS lymphoma, spinal tumor, brainstem glioma, pituitary adenoma, Kaposi's sarcoma, epidermocarcinoma, squamous cell carcinoma.

[0212] In a preferred embodiment, the taxane compound is selected from paclitaxel, albumin-bound paclitaxel, or docetaxel.

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

[0214] In a specific embodiment, the dosage of fludarabine is about 10 to 50 mg / m² 2 / day, or about 15–40 mg / m² 2 / day, or about 15 to 30 mg / m² 2 / day, or about 20–30 mg / m² 2 / day; and / or the dosage of cyclophosphamide is about 300 to 700 mg / m² 2 / day, or about 400–650 mg / m² 2 / day, or about 450–600 mg / m² 2 / day, or about 450–550 mg / m² 2 / day, or about 490–550 mg / m² 2 / day; and / or the dose of albumin-bound paclitaxel is about 300 mg / m² 2 No more than / day, or about 200 mg / m² 2 Not more than / day, or 150 mg / m² 2 No more than / day, or about 100 mg / m² 2 No more than / day, or about 80 mg / m² 2 No more than / day, or about 70 mg / m² 2 It is not more than work.

[0215] In a specific embodiment, pretreatment is performed about 30 days before performing immune cell therapy; preferably, about 20 days before; more preferably, about 12 days before; and most preferably, about 7 days before.

[0216] In a specific embodiment, the continuous use time of the cyclophosphamide, fludarabine and / or albumin-bound paclitaxel does not exceed 4 days; preferably, the albumin-bound paclitaxel is administered once.

[0217] In a specific embodiment, the immune effector cell is an immune effector cell modified by a chimeric antigen receptor, such as a CAR-T cell, CAR-NK cell, or CAR-NKT cell.

[0218] According to a sixth aspect of the present invention, the application of an immune effector cell comprising CLD18A2-CAR in the manufacture of a drug is further provided, wherein the drug contains said cell and a cyclophosphamide, fludarabine, and a taxane compound (preferably paclitaxel, more preferably albumin-bound paclitaxel) for treating a CLD18-positive tumor, and wherein said cell and the cyclophosphamide, fludarabine, and taxane compound are prepared to provide a therapeutic effect greater than the sum of the effects of each of said reagents.

[0219] In a specific embodiment, the step of administering an initial amount of the immune effector cells to a subject requiring them is included, wherein the initial amount is approximately 3 x 10 7 Cells / kg shall not exceed the subject's body weight, or the total amount of cells is approximately 2×10 9 It does not exceed the cell.

[0220] In a specific embodiment, after administering the initial amount of immune effector cells, a subsequent amount of immune effector cells is administered at least once, wherein the subsequent amount is approximately 3 x 10 7 Cells / kg not exceeding the subject's body weight, or the total amount of cells is approximately 5×10 9 It does not exceed the cell.

[0221] In a specific embodiment, the subsequent amount of immune effector cells is administered at a time of about 21 to 80 days after the first amount is administered; preferably, the subsequent amount of immune effector cells is administered at a time of about 25 to 60 days after the first amount is administered; more preferably, the subsequent amount of immune effector cells is administered at a time of about 25 to 50 days after the first amount is administered.

[0222] In a specific embodiment, the cyclophosphamide, fludarabine, and taxane-based compounds are administered 4 to 12 days before administering immune effector cells.

[0223] In a specific embodiment, the dosage of fludarabine is about 10 to 50 mg / m² 2 / day, or about 15–40 mg / m² 2 / day, or about 15 to 30 mg / m² 2 / day, or about 20–30 mg / m² 2 / is or; and / or

[0224] The dosage of cyclophosphamide is approximately 300 to 700 mg / m² 2 / day, or about 400–650 mg / m² 2 / day, or about 450–600 mg / m² 2 / day, or about 450–550 mg / m² 2 / day, or about 490–550 mg / m² 2 / is or; and / or

[0225] The dosage of taxane compounds is approximately 300 mg / m² 2 No more than / day, or about 200 mg / m² 2 No more than / day, or about 150 mg / m² 2 No more than / day, or about 100 mg / m² 2 No more than / day, or about 80 mg / m² 2 No more than / day, or about 70 mg / m² 2 It is not more than work.

[0226] In a specific embodiment, the continuous use time of the cyclophosphamide and / or fludarabine does not exceed 4 days, and the taxane compound is administered once.

[0227] In a specific embodiment, the chimeric antigen receptor has an amino acid sequence represented by any one of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32; preferably, has a sequence represented by SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

[0228] Corresponding to the sixth aspect, the present invention further provides a kit for treating CLD18-positive tumors, wherein the kit is,

[0229] 1) Immuno-effector cells containing CLD18A2-CAR;

[0230] 2) Cyclophosphamide;

[0231] 3) Fludarabine;

[0232] 4) Taxane-based compounds, preferably paclitaxel, more preferably albumin-bound paclitaxel;

[0233] 5) A container comprising the material according to 1) to 4) above; and

[0234] 6) Includes an administration instruction manual for treating CLD18-positive tumors using the above kit, and

[0235] Here, the cell and the cyclophosphamide, fludarabine, and taxane-based compounds are prepared to provide a therapeutic effect greater than the sum of the effects of each of the reagents.

[0236] In a specific embodiment, the administration instructions describe administering the initial dose of the immune effector cells to a subject requiring them, and the initial dose is approximately 3 x 10 7 Cells / kg shall not exceed the subject's body weight, or the total amount of cells is approximately 2×10 9 It does not exceed the cell.

[0237] In a specific embodiment, the administration instructions describe administering the immune effector cells in a subsequent amount at least once after administering the initial amount, and the subsequent amount is approximately 3 x 10 7 Cells / kg not exceeding the subject's body weight, or the total amount of cells is approximately 5×10 9 Without exceeding the cells.

[0238] In a specific embodiment, the administration instructions describe administering the subsequent amount of immune effector cells at a time approximately 21 to 80 days after administering the initial amount; preferably, administering the subsequent amount of immune effector cells at a time approximately 25 to 60 days after administering the initial amount; and more preferably, administering the subsequent amount of immune effector cells at a time approximately 25 to 50 days after administering the initial amount.

[0239] In a specific embodiment, the administration instructions state that the cyclophosphamide, fludarabine, and taxane compounds are administered 4 to 12 days before administering immune effector cells.

[0240] In a specific embodiment, the above administration instructions include,

[0241] The dosage of fludarabine is approximately 10 to 50 mg / m² 2 / day, or about 15–40 mg / m² 2 / day, or about 15 to 30 mg / m² 2 / day, or about 20–30 mg / m² 2 / is or; and / or

[0242] The dosage of cyclophosphamide is approximately 300 to 700 mg / m² 2 / day, or about 400–650 mg / m² 2 / day, or about 450–600 mg / m² 2 / day, or about 450–550 mg / m² 2 / day, or about 490–550 mg / m² 2 / is or; and / or

[0243] The dosage of taxane compounds is approximately 300 mg / m² 2 No more than / day, or about 200 mg / m² 2 No more than / day, or about 150 mg / m² 2 No more than / day, or about 100 mg / m² 2 No more than / day, or about 80 mg / m² 2 No more than / day, or about 70 mg / m² 2 Content that is not more than one is recorded.

[0244] In a specific embodiment, the above administration instructions include,

[0245] The continuous use time of the above cyclophosphamide and / or the above fludarabine does not exceed 4 days, and the above taxane compound is described as being administered once.

[0246] In a specific embodiment, the chimeric antigen receptor has an amino acid sequence represented by any one of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32; preferably, has a sequence represented by SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26.

[0247] A person skilled in the art will know that the kit may contain each component in an amount corresponding to the dosage instructions.

[0248] According to one aspect of the present invention, a method for treating a tumor is provided, comprising the step of using immune effector cells and a tubulin inhibitor on an individual suffering from a tumor, wherein the immune effector cells express a receptor that identifies a tumor antigen. In some embodiments, the present invention provides a method for reducing the growth, survival, or activity, or both, of cancer cells, comprising the step of using immune effector cells and a tubulin inhibitor on an individual suffering from a tumor, wherein the immune effector cells express a receptor that identifies a tumor antigen. In some embodiments, the tubulin inhibitor is a taxane compound. In some embodiments, the taxane compound is selected from paclitaxel, albumin-bound paclitaxel (nab-paclitaxel, Abraxane), docetaxel, and derivatives having a taxane skeleton structure.

[0249] In another embodiment, the antibody specifically identifying the tumor antigen is HCDR1 represented by SEQ ID NO: 1, HCDR2 represented by SEQ ID NO: 2, HCDR3 represented by SEQ ID NO: 3, LCDR1 represented by SEQ ID NO: 4, LCDR2 represented by SEQ ID NO: 5, LCDR3 represented by SEQ ID NO: 6; or

[0250] HCDR1 indicated by SEQ ID NO: 1, HCDR2 indicated by SEQ ID NO: 7, HCDR3 indicated by SEQ ID NO: 3, LCDR1 indicated by SEQ ID NO: 4, LCDR2 indicated by SEQ ID NO: 5, LCDR3 indicated by SEQ ID NO: 6; or

[0251] It has HCDR1 marked with SEQ ID NO: 8, HCDR2 marked with SEQ ID NO: 9 or SEQ ID NO: 68, HCDR3 marked with SEQ ID NO: 10, LCDR1 marked with SEQ ID NO: 11, LCDR2 marked with SEQ ID NO: 12, and LCDR3 marked with SEQ ID NO: 13.

[0252] In another embodiment, the antibody specifically identifying the tumor antigen comprises a heavy chain variable region represented by SEQ ID NO: 14 and a light chain variable region represented by SEQ ID NO: 16; or

[0253] Heavy chain variable region indicated by SEQ ID NO: 18 and light chain variable region indicated by SEQ ID NO: 16; or

[0254] Heavy chain variable region indicated by SEQ ID NO: 22 and light chain variable region indicated by SEQ ID NO: 20; or

[0255] It has a heavy chain variable region indicated by SEQ ID NO: 53 and a light chain variable region indicated by SEQ ID NO: 52.

[0256] In another embodiment, the antibody that specifically identifies the tumor antigen has an amino acid sequence represented by the sequence represented by SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, or SEQ ID NO: 58, and preferably has the sequence represented by SEQ ID NO: 54.

[0257] In other embodiments, pretreatment or lymphocyte removal is not performed on the subject. The present invention further provides the application of immune effector cells expressing a receptor identifying a tumor antigen in the manufacture of a drug, wherein the drug contains said immune effector cells and a taxane compound, and said drug is intended to treat a tumor. In some embodiments, said immune effector cells and a taxane compound included in said drug may 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, said kit comprising: 1) immune effector cells expressing a receptor identifying a tumor antigen; 2) a taxane compound; 3) a container containing the substances according to 1) and 2); and 4) instructions for administering the kit to treat the tumor.

[0258] It should be understood that within the scope of the present invention, each of the technical features of the present invention and each of the technical features specifically described below (e.g., in the embodiments) may be combined with one another to form a new or desirable technical solution. Due to limitations of scope, they are not described in detail here. Brief explanation of the drawing

[0259] Figure 1 shows the detection of the number of CAR-T replicas. Figure 2A shows the change in C-reactive protein (CRP) in the body of a subject after injecting the first dose of CAR-T (first treatment course); Figure 2B shows the change in CRP in the body of a subject after injecting the subsequent dose of CAR-T (second treatment course). Figure 3A shows changes in tumor lesions during the CAR-T treatment process of a pancreatic cancer subject; Figure 3B shows changes in tumor markers during the CAR-T treatment process of a pancreatic cancer subject. Figure 4A shows changes in tumor lesions during the CAR-T treatment process of a gastric adenocarcinoma subject; Figure 4B shows changes in new lesions during the CAR-T treatment process of a gastric adenocarcinoma subject; Figure 4C shows changes in thoracoperitoneum during the CAR-T treatment process of a gastric adenocarcinoma subject; Figure 4D shows changes in CA125 levels during the CAR-T treatment process of a gastric adenocarcinoma subject; and Figure 4E shows changes in CA199 levels during the CAR-T treatment process of a gastric adenocarcinoma subject. Figure 5 is a plasmid map of the recombinant carrier MSCV-8E5-2I-mBBZ. Figures 6A and 6B are CCK8 experiments detecting the toxic effects of Abraxane on pancreatic cancer cells and CAR-T cells, respectively. Figures 7A and 7B show the in vivo experiment observing the inhibitory effect of the combination of Abraxane and CAR T cells on mouse pancreatic cancer and the change in mouse body weight. FIGS. 8A to 8C show the survival rate of mice after treatment with the method of the present invention. Figure 9 is a graph of the change in body weight of a mouse model after treatment with the method of the present invention. Specific details for implementing the invention

[0260] Through extensive and thorough research, the inventor unexpectedly discovered that administering immune effector cells expressing chimeric antigen receptors (CARs) or other genetically modified receptors, such as T cell receptors (TCRs), in specific amounts according to specific time parameters significantly enhances the therapeutic effect 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 partially on the above discovery.

[0261] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms or proper vocabulary used in this text have the same meaning as understood by a person skilled in the art to which this invention pertains. In some situations, this text additionally limits terms having the commonly understood meaning for the purpose of explanation and / or citation; however, such additional limitations contained herein should not be understood as representing a substantial difference as commonly understood in the art.

[0262] All publications mentioned in this invention include patent documents, academic papers, and databases, and all of these are cited and incorporated into the text for all purposes, to the same extent that each is cited and incorporated independently as a publication. If the definitions in the text and the definitions in the patents, public applications, and other publications cited and incorporated into the text are identical or differ in other respects, the definitions in the cited documents incorporated into the text shall be based primarily on the definitions in the text.

[0263] As used in the text, the singular forms "one," "one thing," and "the above" include the plural forms unless otherwise explicitly stated in the text. For example, "one thing" or "one" means "at least one thing or one" or "one thing (one) or several things (more than one)."

[0264] In the content of the present invention, each aspect of the subject to be protected is presented in the form of a range. It should be understood that descriptions in the form of a range are merely for convenience and brevity and should not be interpreted as forcibly limiting the scope of the subject to be protected. Accordingly, descriptions of the range should be interpreted as having already specifically disclosed all possible sub-ranges and individual values ​​within said ranges. For example, if a range of values ​​is provided, it should be understood that each intermediate value between the upper and lower limits of said range and any other such or intermediate value within said range are all included within the subject to be protected, and that the upper and lower limits of said range also fall within the scope of the subject to be protected. Unless the upper and lower limits of said range are explicitly excluded, said relatively small ranges may independently include the upper and lower limits of said relatively small ranges, and these also fall within the scope of the subject to be protected. If a range is set to include one or two limits, the subject to be protected also includes a range that excludes one or two of said limits. Regardless of the breadth or narrowness of the range, the above principles apply.

[0265] As used in the text, the term "about" refers to the ordinary range of error for each value that is readily known to a person skilled in the art. The "about" values ​​or parameters mentioned in the text include (describe) embodiments that refer to said values ​​or parameters themselves. For example, a description of "about X" includes a description of "X". For example, "about" may mean less than or equal to 1 or greater than or equal to 1 according to the actual standard deviation in the art. "About" may mean a range of up to 10% (i.e., ±10%). For example, about 5 mg may be included in any number between 4.5 mg and 5.5 mg. Where specific values ​​or compositions are provided in the application and patent application scope, "about" shall be assumed to be within the allowable range of said specific values ​​or compositions unless otherwise specified.

[0266] When describing an amino acid or nucleic acid sequence, "having a particular sequence" should be understood to include a variant of the particular sequence. In some embodiments, an amino acid or nucleic acid sequence having a particular sequence means that the amino acid or nucleic acid sequence has sequence homology exceeding 80%, 85%, 90%, 95%, or 99% with the particular sequence.

[0267] Unless otherwise specified, any concentration range, percentage range, ratio range, or integer range according to the text shall be understood as any integer included within the said range, and, where appropriate, fractions thereof (e.g., 1 / 10 and 1 / 100 of an integer).

[0268] The "administration interval" according to the text refers to the time consumed while performing a multi-treatment course of immune effector cell therapy on an individual (e.g., a multi-treatment course including the administration of an initial dose of immune effector cells and a subsequent dose of immune effector cells) and while administering a pretreatment drug. Accordingly, the administration interval may be expressed as a range. In some aspects of the present invention, the present invention comprises performing a multi-treatment course of immune effector cell therapy on an individual, and administering a dose determined by a physician in each treatment course (e.g., an initial dose and a subsequent dose). In some aspects of the present invention, a specific dose of the immune effector cell therapy of the present invention may be administered in two or more divided doses, and the total dose administered in divided doses is equal to the total dose in the treatment course determined by the physician.

[0269] The term "use amount" as used in this text may refer to a use amount calculated based on weight or a use amount calculated based on body surface area (BSA). A use amount calculated based on weight refers to the amount administered to the patient calculated based on the patient's body weight, e.g., mg / kg, number of immune effector cells / kg, etc. A use amount calculated based on BSA refers to the amount administered to the patient calculated based on the patient's surface area, e.g., mg / m² 2 , and number of immune effector cells / m 2 It is the back.

[0270] The "number of administrations" in the text refers to the frequency of administration of immune effector cells or pretreatment drugs within a specified time. The number of administrations can be expressed as the amount used within each specified time. For example, fludarabine may be administered by administering a once-daily dose for 4 consecutive days, a once-daily dose for 3 consecutive days, a once-daily dose for 2 consecutive days, or a once-daily dose for 1 day. Cyclophosphamide may be administered by administering a once-daily dose for 4 consecutive days, a once-daily dose for 3 consecutive days, a once-daily dose for 2 consecutive days, or a once-daily dose for 1 day. Albumin-bound paclitaxel may be administered by administering a once-daily dose for 4 consecutive days, a once-daily dose for 3 consecutive days, a once-daily dose for 2 consecutive days, or a once-daily dose for 1 day.

[0271] The term "composition" as used in the text means any mixture of two or more products, substances, or compounds (including cells). This may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0272] As used in the text, "cells or a group of cells show 'positive' for a specific marker" means the detectable presence of a specific marker (generally a surface marker) on or among the said cells. When relating to surface markers, the term means detecting the presence of surface expression by flow cytometry, for example, by staining with an antibody that specifically binds to said marker and detecting said antibody. Here, said staining may be detected at a certain level by flow cytometry, said level being significantly higher than the level of staining detected when the same step is performed under identical conditions with an isotype-matched control, and / or nearly similar to the level of known cells showing positive for said marker, and / or significantly higher than the level of known cells showing negative for said marker.

[0273] The phrase "cells or cell populations show 'negative' for a specific marker" as used in the text means that specific markers, such as surface markers, are not present on or among the said cells, or even if present, are hardly detected. In the case of surface markers, the term means that surface expression is not detected by flow cytometry, or even if surface expression is detected—for example, by staining with an antibody that specifically binds to said marker and detecting the antibody—and even if said staining is detected at a certain level by flow cytometry, said level is significantly lower than the level of staining detected when the same step is performed under identical conditions with an isotype-matched control, and / or is significantly lower than the level of known cells showing positive for said marker, and / or is nearly similar to the level of known cells showing negative for said marker.

[0274] As used in the text, the term "carrier" refers to a nucleic acid molecule, wherein said nucleic acid molecule can amplify other nucleic acids connected thereto. The term includes carriers in the form of self-replicating nucleic acid structures and carriers introduced into the host cell genome. Some carriers can induce the expression of nucleic acids operably connected thereto. Such carriers are also referred to as "expression carriers" in the text.

[0275] The present invention relates to adoptive cells or immune effector cells for treating solid tumors comprising cells administered in large or duplicate doses, and methods of use, compositions, and products thereof. The cells generally express other genetically modified receptors such as chimeric antigen receptors (CARs) or T cell receptors (TCRs).

[0276] The molecule Claudin 18 (CLD18) (Genbank registration numbers: splice variant 1 (CLD18A1): NP_057453, NM016369, and splice variant 2 (CLD18A2): NM_001002026, NP_001002026) is an intrinsic transmembrane protein with a molecular weight of approximately 27.9 / 27.72 kD. Claudin is an intrinsic membrane protein located at the tight junction sites of the epithelium and endothelium. A tight junction is a network of interconnected granules within the tissue membrane between adjacent cells. At tight junctions, occludin and claudin are the most major transmembrane protein components. Due to their strong intercellular adhesion properties, they create a primary barrier that maintains cell polarity by preventing and controlling paracellular transport of solutes and limiting the lateral diffusion of membrane lipids and proteins. Proteins that form tight junctions are critically involved in the structure of epithelial tissues.

[0277] CLD18A1 is selectively expressed in normal lung and gastric epithelium, whereas CLD18A2 is expressed only in gastric cells. Additionally, CLD18A2 is localized to already differentiated short-lived gastric epithelial cells but is not present in the gastric stem cell region. Both variants are intensely expressed in various cancer types, including tumors of the stomach, esophagus, pancreas, and lung, as well as human cancer cell lines. Expression occurs primarily in adenocarcinoma subtypes of these indications.

[0278] The present invention provides a treatment method and a composition for treating a disease (e.g., a tumor) associated with CLD18 expression.

[0279] The present invention provides a method for treating a subject's tumor, particularly a solid tumor, with adoptive cells or immune effector cells expressing a genetically engineered (recombined) chimeric receptor. The method comprises reinfusion of adoptive cells or immune effector cells in a single course of treatment or reinfusion of multiple courses of treatment. In the text, "dose" refers to the total amount of adoptive cells or immune effector cells administered or reinfused in a single course of treatment. In some embodiments, if the method according to the text includes multiple courses of treatment, the dose for each course of treatment is the same. In some embodiments, if the method according to the text includes multiple courses of treatment, the dose for each course of treatment is different. "Divided dose" refers to the amount administered in a single dose when the total dose of a single course of treatment is divided into multiple doses and administered to the subject. In some embodiments, if the dose of a single course of treatment is divided into multiple doses and administered to the subject, the divided dose administered in each dose is the same. In some embodiments, if the dose of a single course of treatment is divided into multiple doses and administered to the subject, the divided dose administered in each dose is different. In the text, unless otherwise specifically stated, usage refers to the total amount of adoptive cells or immune effector cells administered or re-infused during a single course of treatment.

[0280] In some embodiments, the method according to the text includes the step of re-injecting the adoptive cells or immune effector cells during a single treatment course. A single treatment course means re-injecting a fixed total amount of adoptive cells or immune effector cells at a fixed time interval. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is re-injected once during the treatment course. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is re-injected in two or more divided doses. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is re-injected in three or more divided doses. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is re-injected in four or more divided doses. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is re-injected in five or more divided doses. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is re-injected in six or more divided doses. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is re-injected in seven or more divided doses. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is re-injected in eight or more divided doses. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is re-injected in nine or more divided doses. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is re-injected in ten or more divided doses. In some embodiments, the adoptive cells or immune effector cells re-injected each time are equal amounts of the adoptive cells or immune effector cells to be re-injected. In some embodiments, the adoptive cells or immune effector cells re-injected each time are non-equal amounts of the adoptive cells or immune effector cells to be re-injected. In some embodiments, the amount of adoptive cells or immune effector cells re-injected each time is determined by a physician according to the specific condition of the subject.The specific circumstances of the subject may be, for example, the subject's overall health status, the severity of the disease, the response to the previous dose of the same treatment course, the response to the previous treatment course, the concomitant drug use status of the subject, the degree or likelihood of toxic reactions, complications, the status of cancer metastasis, and any other factors that the physician believes may influence the amount of adoptive cells or immune effector cells reinjected into the subject. In some embodiments, during the process of reinjecting the constant total amount of adoptive cells or immune effector cells multiple times, the amount of adoptive cells or immune effector cells reinjected each time is gradually increased. In some embodiments, during the process of reinjecting the constant total amount of adoptive cells or immune effector cells multiple times, the amount of adoptive cells or immune effector cells reinjected each time is gradually decreased. In some embodiments, during the process of reinjecting the constant total amount of adoptive cells or immune effector cells multiple times, the amount of adoptive cells or immune effector cells reinjected each time is first gradually increased and then gradually decreased. In some embodiments, during the process of re-injecting the constant total amount of adoptive cells or immune effector cells multiple times, the amount of adoptive cells or immune effector cells re-injected each time is first gradually decreased and then gradually increased.

[0281] Re-injection of a multiple treatment process means having a plurality of the above-mentioned time zones and re-injecting a certain total amount of adoptive cells or immune effector cells within each time zone. In some embodiments, the lengths of the plurality of time zones are equal. In some embodiments, the lengths of the plurality of time zones are unequal. In some embodiments, the multiple treatment process means having at least two of the above-mentioned time zones. In some embodiments, the multiple treatment process means having at least three of the above-mentioned time zones. In some embodiments, the multiple treatment process means having at least four of the above-mentioned time zones. In some embodiments, the multiple treatment process means having at least five of the above-mentioned time zones. In some embodiments, the multiple treatment process means having at least six of the above-mentioned time zones. In some embodiments, the multiple treatment process means having at least seven of the above-mentioned time zones. In some embodiments, the multiple treatment process means having at least eight of the above-mentioned time zones. In some embodiments, the multiple treatment process means having at least nine of the above-mentioned time zones. In some embodiments, the multiple treatment process means having at least ten of the above-mentioned time zones. In some embodiments, the multiple treatment process means having 10 or more of the above time periods.

[0282] In some embodiments, a fixed total amount of adoptive cells or immune effector cells is reinjected once during one treatment course in the multiple treatment course. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is reinjected in two or more divided doses during one treatment course in the multiple treatment course. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is reinjected in three or more divided doses during one treatment course in the multiple treatment course. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is reinjected in four or more divided doses during one treatment course in the multiple treatment course. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is reinjected in five or more divided doses during one treatment course in the multiple treatment course. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is reinjected in six or more divided doses during one treatment course in the multiple treatment course. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is reinjected in seven or more divided doses during one treatment course in the multiple treatment course. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is reinjected in eight or more divided doses during one treatment course of the multi-treatment course. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is reinjected in nine or more divided doses during one treatment course of the multi-treatment course. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is reinjected in ten or more divided doses during one treatment course of the multi-treatment course. In some embodiments, in one treatment course of the multi-treatment course, the adoptive cells or immune effector cells reinjected each time are equal amounts of the adoptive cells or immune effector cells to be reinjected.In some embodiments, in one treatment course of the multiple treatment course, the adoptive cells or immune effector cells reinjected each time are non-equivalent amounts of adoptive cells or immune effector cells to be reinjected. In some embodiments, in one treatment course of the multiple treatment course, the amount of adoptive cells or immune effector cells reinjected each time is determined by the physician based on the subject's specific circumstances. The subject's specific circumstances may be, for example, the subject's overall health status, the severity of the disease, the response to the previous dose of the same treatment course, the response to the previous treatment course, the subject's drug concomitant status, the degree or likelihood of toxic reactions, complications, the status of cancer metastasis, and any other factors that the physician believes may influence the amount of adoptive cells or immune effector cells reinjected to the subject. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is reinjected the same number of times in each treatment course of the multiple treatment course. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is reinjected the same number of times in each treatment course of the multiple treatment course. In some embodiments, a fixed total amount of adoptive cells or immune effector cells is reinjected the same number of times in each treatment course of the multiple treatment course. In some embodiments, the same fixed total amount of adoptive cells or immune effector cells is reinjected in each treatment course of the multiple treatment course. In some embodiments, different fixed total amounts of adoptive cells or immune effector cells are reinjected in each treatment course of the multiple treatment course.

[0283] In some embodiments, the total amount of adoptive cells or immune effector cells is gradually increased in each treatment course of the multiple treatment course. In some embodiments, the total amount of adoptive cells or immune effector cells is gradually decreased in each treatment course of the multiple treatment course. In some embodiments, the total amount of adoptive cells or immune effector cells is first gradually increased and then gradually decreased in each treatment course of the multiple treatment course. In each treatment course of the multiple treatment course, the total amount of adoptive cells or immune effector cells is first gradually decreased and then gradually increased.

[0284] In some embodiments, the initial use is a relatively low use and / or a controlled or reduced use, and / or the subsequent use is an enhanced use and / or a controlled or reduced use. Cells, compositions, and products that can be used in this method are further provided. In some embodiments, the 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 a genetically engineered T cell receptor (TCR). The receptor further comprises a receptor comprising an extracellular portion and an intracellular portion that specifically bind to a ligand or receptor or other binding partner, such as the intracellular signaling portion of a CAR. In some embodiments, the use includes a relatively low initial use.

[0285] In some embodiments, the method comprises: (a) administering a single course of treatment cells expressing a chimeric antigen receptor (e.g., CAR) to a subject suffering from a tumor; and (b) administering a multiple course of treatment cells expressing a chimeric antigen receptor (e.g., CAR) to the subject. In other embodiments, one or more subsequent doses may be administered.

[0286] The term "CLD18" means Claudin-18, but includes any variant of CLD18 (including CLD18A1 and CLD18A2), sequences, and interspecies homologs that are naturally expressed in cells or expressed by cells transfected with the CLD18 gene. Preferably, "CLD18" means human CLD18, in particular CLD18A2 (SEQ ID NO: 1, SEQ ID NO: 2) and / or CLD18A1 (SEQ ID NO: 7, SEQ ID NO: 8), and more preferably CLD18A2.

[0287] The term "CLD18A1" includes any post-translated modified variants, isoforms, and interspecies homologs of human CLD18A1 that are naturally expressed in cells or expressed by cells transfected with the CLD18A1 gene.

[0288] The term "CLD18A2" includes any post-translated modified variants, isoforms, and interspecies homologs of human CLD18A2 that are naturally expressed in cells or expressed by cells transfected with the CLD18A2 gene.

[0289] The term “CLD18 variant” must include (i) CLD18 splice variants, (ii) post-translated modified variants of CLD18, in particular variants with different N glycosylation states, (iii) CLD18 array variants, in particular CLD18-array-1, CLD18-array-2 and CLD18-array-3, (iv) free CLD18 located at intercellular tight junction sites and homo / allo-associated variants, (v) CLD18 cancer-associated variants and CLD18 non-cancer-related variants.

[0290] A method of treating tumors using cells that express chimeric antigen receptors

[0291] The present invention provides a method, composition, and product for treating diseases or pathologies including various solid tumors, used in cell therapy. The method relates to the administration of immune effector cells expressing a chimeric receptor, wherein the chimeric receptor targets and identifies a tumor antigen, and / or specifically binds to a tumor antigen and activates the immune effector cells. The receptor includes, for example, chimeric receptors such as a chimeric antigen receptor (CA v9cdR), a T cell receptor (TCR), a T cell fusion protein (TFP), and a T cell antigen coupler (TAC).

[0292] In some embodiments, the method comprises the step of administering one or more subsequent doses of cells to a subject. The doses are generally administered in specific amounts according to specific time parameters. In some embodiments, the method comprises the step of administering subsequent doses of cells at specific time intervals relative to the first dose, after administering the first dose of cells. In some embodiments, the number of cells administered and the time intervals for multiple doses are designed to improve one or more outcomes, for example, by reducing the degree or potential for toxicity to the subject, and / or improving the therapeutic effect.

[0293] In some embodiments, the provided method may improve the efficacy and treatment outcomes in immuno-effector cell therapy based on an increased degree of subject exposure to the administered cells observed in the text (e.g., an increased number of cells or duration over time). In several clinical trials, different CAR-T cells targeting CLD18A2 were administered to subjects suffering from various CLD18A2-positive tumors, and preliminary analyses were performed to indicate an association existing between a higher and / or longer degree of CAR-T cell exposure and treatment outcomes. These results include patient survival, remission, or stabilization of the disease. In some embodiments, the method according to the text includes the step of monitoring the degree of subject exposure to the adoptive cells or immuno-effector cells, and, based on the degree of exposure, subsequently administering them in divided doses or determining the dosage and time intervals of a subsequent course of treatment. In some embodiments, the method according to the text comprises the step of monitoring the subject’s exposure to the adoptive cell or immune effector cell, and, as the exposure level reaches or exceeds a certain level, subsequently administering in divided doses or maintaining or decreasing the dosage of a subsequent treatment course, and / or subsequently administering in divided doses or maintaining or extending the time interval between subsequent treatment courses. In some embodiments, the method according to the text comprises the step of monitoring the subject’s exposure to the adoptive cell or immune effector cell, and, as the exposure level is lower than a certain level, subsequently administering in divided doses or maintaining or increasing the dosage of a subsequent treatment course, and / or subsequently administering in divided doses or maintaining or shortening the time interval between subsequent treatment courses.

[0294] In some embodiments, the method according to the text includes the step of monitoring the degree or risk of a subject’s toxic response to the adoptive cell or immune effector cell, and, depending on the degree or risk of toxicity, subsequently administering the treatment in divided doses or determining the dosage and time interval of a subsequent treatment course. In some embodiments, the degree or risk of toxic response includes, but is not limited to, examples such as CRS, neurotoxicity, macrophage and tumor lysis syndrome.

[0295] In some embodiments, a subsequent dose is administered when the risk of a toxic reaction or symptoms thereof or biochemical index (e.g., CRS or neurotoxicity, macrophage activation syndrome or tumor lysis syndrome) is lower than or equal to an acceptable level after administration of the initial dose or a previous dose. In some embodiments, said toxic reaction or symptoms thereof or biochemical index includes one or more of fever, hypotension, hypoxia, neurological disorders, inflammatory cytokines, and serum levels of C-reactive protein (CRP). In some embodiments, the acceptable level of risk of the toxic reaction or its symptoms or biochemical index means 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, or 10% of the peak level after administration of the first dose or the previous dose.

[0296] In some embodiments, a subsequent dose is administered when, after administration of the first dose or previous dose, the toxic reaction reaches a peak level and is also decreasing. In some embodiments, a subsequent dose is administered when, after administration of the first dose or previous dose, the toxic reaction has decreased to a level lower than or equal to an acceptable level. In some embodiments, a subsequent dose is administered when, after administration of the first dose or previous dose, the risk of the toxic reaction or its symptoms or biochemical index is lower than or equal to 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the peak level that appeared after administration of the first dose or previous dose. Accordingly, in some embodiments, 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, After administering on days 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, and 80, a subsequent dose is administered. In some embodiments, the presence of one or more symptoms or risks associated with toxic effects is monitored and / or evaluated, and after determining that the symptoms or risks are lower than or equal to an acceptable level, a subsequent dose is administered to determine an appropriate time.

[0297] In some embodiments, after administering the first dose, if the serum level of the factor indicating cytokine-release syndrome (CRS) in the subject does not exceed 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 times the serum level of the subject prior to administering the first dose, a subsequent dose is administered.

[0298] In some embodiments, the timing of administration of a subsequent dose is selected to prevent the host immune response caused by the initial dose or previous dose already administered from inducing a reduction in the effect of the subsequent dose. In some embodiments, the subsequent dose is administered before a host immune response, e.g., the adaptability or specificity of the administered cells and / or the chimeric antigen receptors expressed thereby, e.g., a humoral or cell-mediated immune response, appears. In some embodiments, the subsequent dose may be administered before such response is detected, for example, through one or various specialized detection methods. Generally, one or more subsequent doses are administered when a host adaptive immune response to the cells has not been detected, has not yet been established, and / or has not yet reached a certain level, degree, or stage. Accordingly, in some embodiments, administered at the initial amount or the previous amount for 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, Administer as a subsequent dose after 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.

[0299] Accordingly, the method provided in some embodiments is administered with one or more subsequent doses after the tumor burden has been stabilized or reduced with the first dose following the time period of toxic risk, and before an adaptive host immune response (i.e., the organism's immune rejection of CAR-T cells) occurs. Under these conditions, the subsequent doses can safely and effectively provide immune monitoring, elimination, or prevention of proliferation or metastasis of residual tumor cells. Accordingly, in some embodiments, the subsequent dose is an enhanced dose for the disease.

[0300] Tumor burden includes tumor volume size or degree of differentiation, or type and stage of metastasis, and / or occurrence and disappearance of complications such as malignant pleura and ascites commonly seen in late-stage cancer, and / or changes in the production or expression levels of tumor markers, and / or the likelihood or incidence of toxic outcomes such as CRS, macrophage activation syndrome, tumor lysis syndrome, and neurotoxicity in subjects, and / or the host immune response to administered cells and / or chimeric antigen receptors. In some embodiments, tumor size is measured by a PET (positron emission tomography) and CT (computed tomography) scaler.

[0301] The above-mentioned tumor markers are also referred to as tumor labels and refer to substances that are characteristically present in or abnormally produced by malignant tumor cells, or substances produced by the host’s response to stimulation of the tumor, which can reflect tumor formation and development and monitor the tumor for treatment response. Tumor markers are present in the tissues, body fluids, and excretions of tumor patients and can be detected by immunological, biological, and chemical methods, and 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.

[0302] In some embodiments, the initial dose contains an amount of cells sufficient to reduce the tumor burden of the subject, and if the serum level of the factor indicating cytokine-releasing syndrome (CRS) in the subject is 10 or 25 times the serum level of the subject prior to administration of the initial dose, and / or if the CRS-related result peak level in the subject begins to decrease after administration of the initial dose, and also if no specific detectable adaptive host immune response occurs to the chimeric receptor expressed by the cells of the initial dose in the subject.

[0303] In some embodiments, the initial usage is about 2.5 x 10 8 Number of cells / kg is less than the subject's body weight, or about 1 x 10⁶ 12 It contains a smaller amount than the cells of dogs and is administered as a subsequent dose at a time exceeding about 21 days but less than about 80 days after the first dose is administered.

[0304] Cell administration in immune effector cell therapy

[0305] The method provided by the present invention comprises the step of administering a multiple amount of immune effector cells expressing a chimeric antigen receptor, such as a CAR, TCR, TFP, or TAC chimeric antigen receptor, to a subject with a solid tumor expressing a tumor antigen.

[0306] According to the text, "tumor antigens" are thyroid-stimulating hormone receptor (TSHR); CD171; CS-1; type C lectin-like molecule-1; ganglyxide GD3; Tn antigen; CD19; CD20; CD22; CD30; CD70; CD123; CD138; CD33; CD44; CD44v7 / 8; CD38; CD44v6; B7H3(CD276), B7H6; KIT(CD117); interleukin 13 receptor subunit 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; protease serine 21 (PRSS21); Vascular endothelial growth factor receptor; Lewis (Y) antigen; CD24; platelet-derived growth factor receptor (PDGFR- ); developmental stage-specific fetal antigen-4 (SSEA-4); cell surface-associated mucin 1 (MUC1), MUC6; epidermal growth factor receptor family and their mutants (EGFR, EGFR2, ERBB3, ERBB4, EGFRvIII); neuronal adhesion molecule (NCAM); carbonic anhydrase IX (CAIX); LMP2; ephrin receptor 2 type A (EphA2); fucosyl GM1; sialyllous adhesion molecule (sLe); Gangliasid GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); TGS5; High molecular weight melanoma-associated antigen (HMWMAA); O-acetyl GD2 gangliasid (OAcGD2); folate receptor; tumor vascular endothelial marker 1 (TEM1 / CD248); tumor vascular endothelial marker 7-associated (TEM7R); Claudin 6, Claudin 18.2, Claudin 18.1; ASGPR1; CDH16; 5T4; 8H9; αvβ6 integrin; B-cell maturation antigen (BCMA); CA9; 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; tenacin; carcinoembryo variant of tumor necrosis site; 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 gene 1 (PLAC1); hexose portion of glycoceramide (GloboH); breast differentiation antigen (NY-BR-1); Uroplakin 2 (UPK2); Hepatitis A virus cell receptor 1 (HAVCR1); Adrenergic receptor β3 (ADRB3); Pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); Lymphocyte antigen 6 complex locus K9 (LY6K); Olfactory receptor 51E2 (OR51E2); T cell receptor gamma alternative read frame protein (TARP); Wilms oncology 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 testicular antigen-1 (MAD-CT-1); Melanoma testicular antigen-2 (MAD-CT-2); Fos-associated antigen 1; p53 mutant; Human telomerase reverse transcriptase (hTERT); sarcoma translocation interruption point; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyl transferase V (NA17); pairbox protein Pax-3 (PAX3); androgen receptor; cyclin B1; V-myc avian myeloma virus oncogene neuroblastoma-derived homologue (MYCN);Ras homologous family member C (RhoC); cytochrome P450 1B1 (CYP1B1); CCCTC binding factor (zinc finger protein) similar (BORIS); squamous cell carcinoma antigen 3 identified by T cells (SART3); pairbox protein Pax-5 (PAX5); proacrosin binding protein sp32 (OYTES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase fixation protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR); leukocyte immunoglobulin-like receptor subfamily member 2 (LILRA2); CD300 molecule similar 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 2 (EMR2); lymphocyte antigen 75 (LY75); phosphatidyl muscle glycan-3 (GPC3); Fc receptor-like 5 (FCRL5); immunoglobulin lambda-like peptide 1 (IGLL1), but not limited thereto. In some embodiments, the tumor antigen is EGFR, EGFRvIII, glypican 3, Claudin 18.2, or BCMA.

[0307] As used in the text, "subject" is a mammal, such as a human or other animal, and is generally a human. In some embodiments, prior to administration of the first dose and / or subsequent doses, the subject has already received tumor chemotherapy or radiation therapy. In some embodiments, the subject is resistant or non-responsive to other therapeutic agents.

[0308] In some embodiments, the tumor persists or recurs after intervention with other treatments, such as chemotherapy or radiation therapy. In some embodiments, by administering as described above, the subject is effectively treated regardless of whether the subject has resistance to other treatment methods.

[0309] In some embodiments, the subject is responsive to another therapeutic agent and is treated with the therapeutic agent to reduce the tumor burden. In some embodiments, the subject is initially responsive to the therapeutic agent, but the tumor recurs over time. In these embodiments, if the subject is detected to be at risk of recurrence, for example, if the risk of recurrence is detected to be high, cells are administered prophylactically to reduce the likelihood of recurrence or prevent recurrence.

[0310] The above diseases include tumors, or other proliferative diseases or pathologies. The above tumors include colon cancer, rectal cancer, kidney cancer, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, pediatric solid tumors, bladder cancer, kidney or ureteral cancer, renal pelvis cancer, central nervous system (CNS) cancer, primary CNS lymphoma, neovascularization, spinal tumor, brainstem glioma, pituitary adenoma, Kaposi sarcoma, epidermal carcinoma, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancer, combinations of the above cancers, and metastatic lesions of the above cancers, but thereof It is not limited.

[0311] In some embodiments, the amount and re-injection time are determined by the subject's initial tumor burden. For example, in some situations, the number of cells in the initial amount generally administered to the subject is relatively small, and when the tumor burden is relatively low, for example, solid tumors can be detected via tumor markers to assess the tumor burden size and / or microscopic residual lesions, and the initial amount may be relatively large. In other situations, for subjects with a relatively high tumor burden, the initial amount may be administered in successive doses, divided into 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses, preferably divided into 1 to 5 doses, and more preferably divided into 2 to 3 doses. The interval between doses is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, or consecutive. In some cases, the subsequent dose may be equal to, greater than, or less than the initial dose.

[0312] As used herein, the term "treatment" means to completely or partially alleviate or reduce a tumor or associated symptoms. Desired therapeutic effects include, but are not limited to, prevention of tumor development or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the tumor, prevention of metastasis, reduction of the rate of tumor progression, improvement or alleviation of the tumor status, and alleviation or improvement of the prognosis. As used herein, "delay of tumor development" means delaying, inhibiting, alleviating, slowing down, stabilizing, suppressing, and / or limiting the development of said tumor. Such delay may have different durations, which are determined by the disease history and / or the subject awaiting treatment. A person skilled in the art should understand that the delay may include prevention (in subjects that have not developed said tumor). For example, terminal cancer, such as the development of metastasis, may be delayed.

[0313] In some embodiments, the provided cells and compositions delay tumor development or alleviate tumor progression.

[0314] The term "inhibition" of function or activity as used in the text refers to a decrease in function or activity compared to the same condition in other cases or compared to another condition.

[0315] For reagents such as drug preparations, cells, or compositions, the "effective dose" in administration refers to the amount effective in obtaining a desired result, such as a therapeutic or preventive effect, when used for the required duration at a given dosage.

[0316] The “therapeutic effective dose” of a drug formulation or reagent, such as a cell, refers to an amount effective in obtaining a desired therapeutic outcome, such as the treatment of a tumor and / or the pharmacokinetic or pharmacokinetic effects of said treatment, in terms of usage and required time course. The therapeutic effective dose may vary depending on changes in various factors, such as disease state, age, gender, and subject body weight, and the cell population administered. In some embodiments, the provided method relates to the administration of said cells and / or composition in an effective amount (e.g., therapeutic effective dose).

[0317] "Prophylactic effective dose" refers to an amount that is advantageous for obtaining a desired result over a given usage amount and required time course. Generally, since the prophylactic dose is administered to the subject early or prior to the onset of disease, the prophylactic effective dose is lower than the therapeutic effective dose. In some modalities, where the tumor burden is relatively low, the prophylactic effective dose may be higher than the therapeutic effective dose.

[0318] A method for administering cells used in immune effector cell therapy is known and can be used in combination with the method and composition provided in the text.

[0319] In some embodiments, the cell therapy, for example, adoptive T cell or immune effector cell therapy, is carried out via a self-re-injection method. Accordingly, in some embodiments, the cells are derived from the subject requiring the treatment and said cells, and after being isolated and processed, are administered to the same subject.

[0320] In some embodiments, the cell therapy, for example, adoptive T cell or immune effector cell therapy, is performed via allogeneic re-infusion, wherein the cells are isolated and / or extracted and prepared from a donor under other circumstances, and the donor and the subject receiving the cells are different. In these embodiments, the cells are administered to a subject with high genetic histocompatibility. In some embodiments, the donor and the subject are genetically identical. In some embodiments, the donor and the subject are genetically similar. In some embodiments, the subject and the donor belong to the same HLA classification or supertype.

[0321] The cells may be administered in any suitable manner, for example, by injection such as intravenous or subcutaneous injection, intraocular injection, fundus injection, subretinal injection, intravitreal injection, counter-interval injection, subscleral injection, intrachoroidal injection, anterior chamber injection, subconjectval injection, subconjunctival injection, episcleral injection, retropondermal injection, periorbital injection, or periorbital delivery. In some embodiments, they are administered parenterally, intrapulmonaryly, and intranasally, and if local treatment is required, intralesional administration is performed. Extraperitoneal administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In some embodiments, a specified amount of cells is administered in a single intravenous injection. In some embodiments, cells are administered via multiple intravenous injections, for example, multiple administrations not exceeding 20 days, or cells are administered via continuous infusion.

[0322] In preventing or treating a disease, the appropriate dosage may be determined by the type of disease to be treated, the type of chimeric antigen receptor or cell, the severity and course of the disease, whether the cell is administered for preventive or therapeutic purposes, previous treatment, the subject's clinical history and response to the cell, and the judgment of the attending physician. In some embodiments, the composition and cell are suitable for administration to the subject in a single time period or a series of treatments.

[0323] In some embodiments, the immune effector cells are administered as part of a combination therapy, for example, in combination with other therapeutic agents or other treatment methods, such as other interventional therapies, such as antibodies, engineered immune effector cells, receptors or reagents, cytotoxic drugs, etc., either simultaneously or sequentially in any order. In some embodiments, the immune effector cells are administered in combination with one or more other treatment methods or in combination with other interventional therapies, either simultaneously or sequentially in any order. In some situations, the immune effector cells are administered with other therapies in sufficiently close proximity to produce a greater therapeutic effect than the 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, etc., to enhance persistence.

[0324] The other therapeutic agents mentioned above may be administered in any suitable manner, for example, by injection such as intravenous or subcutaneous injection, intraocular injection, fundus injection, subretinal injection, intravitreal injection, counter-interval injection, subscleral injection, intrachoroidal injection, anterior chamber injection, subconjectival injection, subconjunctival injection, episcleral injection, retropondermal injection, periorbital injection, or periorbital delivery. In some embodiments, they are delivered parenterally, intrapulmonaryly, and intranasally, and if local treatment is required, intralesional administration may be performed. Extraperitoneal administration includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. In some embodiments, a specified amount of the therapeutic agent is administered via a single intravenous injection. In some embodiments, it is administered via multiple intravenous injections, for example, multiple administrations not exceeding 20 days, or via continuous infusion.

[0325] In some embodiments, the method of the present invention comprises the step of administering a tubulin inhibitor and the adoptive cell or immune effector cell to a subject. The method of the present invention comprises the step of administering a tubulin inhibitor and an immune effector cell to a subject in one course of treatment. In some embodiments, the method of the present invention comprises the step of administering a tubulin inhibitor to a subject and then administering an immune effector cell. In some embodiments, the method of the present invention comprises the step of administering an immune effector cell to the subject first and then administering a tubulin inhibitor. In some embodiments, the method of the present invention comprises the step of administering a tubulin inhibitor and an immune effector cell to the subject almost simultaneously. In some embodiments, the method of the present invention comprises the step of administering a tubulin inhibitor and an immune effector cell alternately to the subject. In a multiple course of treatment, the tubulin inhibitor and the immune effector cell may be the same or different.

[0326] Tubulin inhibitors according to the text include tubulin polymerization promoters and tubulin polymerization inhibitors. Tubulin polymerization promoters include, for example, taxanes, epithiolones, sponge lactones, and laulimalide, but are not limited thereto. Tubulin polymerization inhibitors include, but are not limited to, colchicine, Combretastatin A-4, BPR0L075, Plinabulin (NPI-2358), Nakiterpiosin, Vincristine, Nocodazole, Podophyllotoxin, Dolastatins10, Indibulin (D-24851), and Eribulin.

[0327] Taxane compounds include paclitaxel (trade name: taxol), albumin-bound paclitaxel (nab-paclitaxel, Abraxane), docetaxel, etc., and their derivatives. Taxane tubulin inhibitors promote the assembly of tubulin into microtubules and inhibit the depolymerization of microtubules, thereby causing the abnormal arrangement of microtubule bundles to form astrocytes, which ultimately leads to the loss of normal spindle function and cell death.

[0328] In some embodiments, the method of the present invention comprises the step of administering a taxane-based drug and immune effector cells to a subject in one treatment course. In some embodiments, the method of the present invention comprises the step of administering a taxane-based drug to a subject and then administering immune effector cells to the subject in one treatment course. In some embodiments, the method of the present invention comprises the step of administering immune effector cells to the subject first and then administering a taxane-based drug to the subject in one treatment course. In some embodiments, the method of the present invention comprises the step of administering a taxane-based drug and immune effector cells to the subject almost simultaneously in one treatment course. In some embodiments, the method of the present invention comprises the step of administering a taxane-based drug and immune effector cells alternately to the subject in one treatment course.

[0329] In some embodiments, the method of the present invention comprises the step of administering a taxane-based drug and immune effector cells to a subject during a multiple treatment course. In some embodiments, the method of the present invention comprises the step of administering a taxane-based drug to a subject and then administering immune effector cells during a multiple treatment course. In some embodiments, the method of the present invention comprises the step of administering immune effector cells to the subject first and then administering a taxane-based drug during a multiple treatment course. In some embodiments, the method of the present invention comprises the step of administering a taxane-based drug and immune effector cells to the subject almost simultaneously during a multiple treatment course. In some embodiments, the method of the present invention comprises the step of administering a taxane-based drug and immune effector cells alternately to the subject during a multiple treatment course. In some embodiments, the method of the present invention comprises the step of administering the same amount of a taxane-based drug and / or immune effector cells to the subject during different treatment courses. In some embodiments, the method of the present invention comprises the step of administering different amounts of a taxane-based drug and / or immune effector cells to the subject during different treatment courses. The term "taxane drug" refers to a drug comprising a taxane compound as a major component, wherein the taxane compound has a bridge methylene benzocyclodecene core structure similar to that of a taxane. In some embodiments, the bridge methylene benzocyclodecene core structure of the taxane compound contains unsaturated bonds. In some embodiments, the bridge methylene benzocyclodecene core structure of the taxane compound does not contain unsaturated bonds. In some embodiments, carbon atoms in the bridge methylene benzocyclodecene core structure of the taxane are substituted by heteroatoms selected from N, O, S, and P. In some embodiments, the taxane compound is administered by injection.In some embodiments, the method of the present invention may increase, enhance, or prolong the activity and / or number of immune cells during the course of cancer treatment, or induce a medically effective response. The term “increase” or “enhance” the activity of immune cells means allowing a subject or tumor cell to have an improved ability to respond to the treatment disclosed herein. For example, an enhanced response may include a responsiveness enhanced by 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, “enhance” may also mean an increase in the number of subjects of a response treatment, such as immune effector cell therapy. For example, an enhanced response may mean a total percentage of subjects receiving response 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 some embodiments, the dosage of a taxane compound, such as Abraxane, is 300 mg / m². 2 No more than / dose, or about 200 mg / m² 2 Not more than / times, preferably 100 mg / m² 2 / dose ~ 200 mg / m² 2 It is per dose. In some embodiments, the dosage of a taxane-based compound such as Abraxane is about 200 mg / m² 2 / dose, 195 mg / m² 2 / dose, 190 mg / m² 2 / dose, 185 mg / m² 2 / dose, 180 mg / m² 2 / dose, 175 mg / m² 2 / dose, 170 mg / m² 2 / dose, 165 mg / m² 2 / dose, 160 mg / m² 2 / dose, 155 mg / m² 2 / dose, 150 mg / m² 2 / dose, 145 mg / m² 2 / dose, 140 mg / m² 2 / dose, 135 mg / m² 2 / dose, 130 mg / m² 2 / dose, 125 mg / m² 2 / dose, 120 mg / m² 2 / dose, 115 mg / m² 2 / dose, 110 mg / m² 2 / dose, 105 mg / m² 2 / dose, 100 mg / m² 2 / dose, 95 mg / m² 2 / dose, 90 mg / m² 2 / dose, 85 mg / m² 2 / dose, 80 mg / m² 2 / dose, 75 mg / m² 2 / dose, 70 mg / m² 2 / dose, 65 mg / m² 2 / dose, 60 mg / m² 2 / dose, 55 mg / m² 2 / dose, 50 mg / m² 2 / dose, 45 mg / m² 2 / dose, 40 mg / m² 2 / dose, 35 mg / m² 2 / dose, 30 mg / m² 2 / dose, 25 mg / m² 2 / dose, 20 mg / m² 2 / dose, 15 mg / m² 2 / dose, 10 mg / m² 2 / dose, 8 mg / m² 2 / dose, 6 mg / m² 2 / dose, 4 mg / m² 2 / dose, 3 mg / m² 2 / dose, 2 mg / m² 2 / dose or 1 mg / m² 2 / times, preferably, 100 mg / m² 2 / dose ~ 200 mg / m² 2 / times. In some embodiments, a taxane compound such as Abraxane is intravenously administered 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, a taxane compound such as Abraxane is intravenously administered once during each course of treatment. In some embodiments, immune effector cell therapy is performed 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 1 month, or any combination thereof before administering a taxane compound. In some embodiments, immune effector cell therapy is performed after 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 administering a taxane compound.

[0330] In some embodiments, the method of the present invention comprises the step of performing a pretreatment, such as a chemotherapy drug (chemotherapy agent), whole-body radiation, local radiation therapy, or a combination thereof, before administering an initial dose or a subsequent dose. In some embodiments, the method comprises the step of performing a pretreatment on a subject using one or more chemotherapy agents. In some embodiments, the method comprises the step of performing a pretreatment on a subject using a tubulin inhibitor and one or more other chemotherapy agents. Unless theoretically limited, it is understood that the action of the pretreatment includes, but is not limited to, lymphocyte removal, reduction of tumor burden, etc. In some embodiments, the method according to the text comprises the step of not performing the pretreatment before administering an initial dose or a subsequent dose. In some embodiments, not performing the pretreatment includes not administering a lymphocyte remover, whole-body radiation therapy or a combination thereof, or performing the pretreatment by other means, and the lymphocyte removal rate in the subject after the pretreatment is still lower than 50%, 55%, 60%, 65%, or 70%.

[0331] In some embodiments, the chemotherapy agent according to the text refers to a drug used in chemotherapy, and refers to a chemical drug having a prophylactic therapeutic effect against microbial infections, parasitic diseases, and malignant tumors. The chemotherapy agent includes, but is not limited to, synthetic antimicrobial agents, antibiotics, antiparasitic agents, antifungal agents, antiviral agents, alkylating agents, metabolic antagonists, antituberculosis agents, and antitumor agents. Examples include diterpene alkaloid compounds (e.g., taxane), cyclophosphamide, fludarabine, cyclosporine, rapamycin, mycophenolic acid, steroids, melphalan, bendamustine, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, hydroxyurea, methotrexate, rituximab, vinblastine, and / or vincristine. In some embodiments, the metabolic antagonist is, for example, carmofur, tegafur, pentostatin, deoxyfluridine, trimexate, fludarabine, capecitabine, gallopitabine, cytarabine stearyl sodium phosphate, fosteabine sodium hydrate, raltitrexed, paltitrexid, dipyriteflu, tiazofurin, norlatrexed, pemetrexed, nelzarabine,2'-deoxy-2'-methylenecytidine, 2'-fluoromethylene-2'-deoxycytidine, N-[5-(2,3-dihydrogen-benzofuranyl)sulfonyl]-N'-(3,4-dichlorophenyl)urea, N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycerol-BL-mannose-heptanosylpyranosyl]adenine(N6-[4-deoxy-4-[N2-[2(E),4(E)-tetradecadienoyl]glycylamino]-L-glycerol-BL-mannose-heptanosylpyranosyl]adenine), aplidine, ascidin, 4-[2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimido[5,4-b]thiazin-6-yl-(S)-ethyl]-2,5-thienoyl-L-glutamic acid), aminopterin, 5-fluorouracil, alanosine, 11-acetyl-8-(carbamoyloxymethyl)-4-formyl-6-methoxy-14-oxa-1,11-diazatetracycline(7.4.1.0.0)-tetradecano-2,4,6-triene-9-yl acetate), swainsonine, lometrisol, dexrazoxane, methioninase,Metabolic antagonists include, but are not limited to, 2'-cyano-2'-deoxy-N4-palmitoyl-1-BD-arabinofuranosyl cytosine and 3-aminopyridine-2-aldehyde thiosemicarbazone. In some embodiments, the alkylating agent includes, but is not limited to, examples such as dacarbazine, phenylalanine mustard, cyclophosphamide, temozolomide, chlorambucil, busulfan, mechlorethamine, and nitrosourea.

[0332] The effect of immuno-effector cell therapy may be improved by pretreating the subject before administering the initial dose or subsequent doses. The first infusion day in which immuno-effector cells (e.g., CAR T cells) are administered during each treatment course is designated as Day 0. Pretreatment is performed before performing the immuno-effector cell infusion. In some embodiments, pretreatment is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days prior to administering adoptive cells or immuno-effector cells. In some embodiments, pretreatment is performed using a single chemotherapy agent. In some embodiments, pretreatment is performed using two or more chemotherapy agents. In some embodiments, pretreatment is performed using one microtubule inhibitor and one or more other chemotherapy agents. In some embodiments, pretreatment is performed using two microtubule inhibitors and one or more other chemotherapy agents. In some embodiments, pretreatment is performed using one microtubule inhibitor, such as a tubulin polymerization promoter, one or more alkylating agents, and one or more metabolic antagonists. In some embodiments, pretreatment is performed using one or more paclitaxel derivatives, one or more alkylating agents, and one or more metabolic antagonists. In some embodiments, pretreatment is performed using one microtubule inhibitor (e.g., paclitaxel, particularly albumin-bound paclitaxel) and two other chemotherapy agents (e.g., fludarabine and cyclophosphamide). For the sake of convenience of explanation, specific embodiments are described for pretreatment using one microtubule inhibitor and two other chemotherapy agents, with fludarabine and cyclophosphamide as representative chemotherapy agents (albumin-bound) and paclitaxel as a representative microtubule inhibitor.For example, in some embodiments, fludarabine, or cyclophosphamide and albumin-conjugated paclitaxel are used alone, in combination of two, or in combination of three and administered to a subject as a 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 prior to injecting CAR-T cells, and preferably, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, It is administered to the subject 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days in advance, and more preferably, at least 2, 3, 4, 5, 6, 7, or 8 days in advance. In some embodiments, pretreatment comprises administering fludarabine and cyclophosphamide 6 days before injecting CAR-T cells. In some embodiments, pretreatment comprises administering fludarabine and cyclophosphamide 7 days before injecting CAR-T cells. In some embodiments, pretreatment comprises administering fludarabine and cyclophosphamide 5 days before injecting CAR-T cells. In some embodiments, pretreatment includes administering fludarabine, cyclophosphamide, and albumin-bound paclitaxel 6 days before injecting CAR-T cells. In some embodiments, pretreatment includes administering albumin-bound paclitaxel 4 days before injecting CAR-T cells.

[0333] As described above, the day on which CAR T cell therapy is performed during each course of treatment for the patient is designated as Day 0. In some embodiments, fludarabine, cyclophosphamide, and albumin-bound paclitaxel may be administered at any time prior to performing CAR T cell therapy. In some embodiments, fludarabine, cyclophosphamide, or albumin-bound paclitaxel is started at least 7, 6, 5, 4, 3, 2, or 1 day prior to infusing CAR T cells. In some embodiments, fludarabine, cyclophosphamide, or albumin-bound paclitaxel is started at least 12, 11, 10, 9, or 8 days prior to infusing CAR T cells. In some embodiments, fludarabine, cyclophosphamide, or albumin-bound paclitaxel is started 7 days prior to infusing CAR T cells. In some embodiments, fludarabine, cyclophosphamide, or albumin-bound paclitaxel is started 6 days prior to infusion of CAR T cells. In some embodiments, fludarabine, cyclophosphamide, or albumin-bound paclitaxel is started 12 days prior to infusion of CAR T cells. In some embodiments, fludarabine, cyclophosphamide, or albumin-bound paclitaxel is started 5 days prior to infusion of CAR T cells. In some embodiments, fludarabine, cyclophosphamide, or albumin-bound paclitaxel is started 4 days prior to infusion of CAR T cells.

[0334] In some embodiments, fludarabine is started 7 days before injecting CAR T cells, and cyclophosphamide is started 7 days before injecting CAR T cells. In some embodiments, fludarabine is started 6 days before injecting CAR T cells, and cyclophosphamide is started 6 days before injecting CAR T cells. In some embodiments, fludarabine is started 5 days before injecting CAR T cells, and cyclophosphamide is started 5 days before injecting CAR T cells. In some embodiments, fludarabine is started 12 days before injecting CAR T cells, and cyclophosphamide is started 12 days before injecting CAR T cells. In some embodiments, fludarabine is started 5 days before injecting CAR T cells, cyclophosphamide is started 5 days before injecting CAR T cells, and albumin-bound paclitaxel is started 4 days before injecting CAR T cells. In some embodiments, fludarabine is started 6 days before injecting CAR T cells, cyclophosphamide is started 6 days before injecting CAR T cells, and albumin-bound paclitaxel is started 5 days before injecting CAR T cells. In some embodiments, fludarabine is started 12 days before injecting CAR T cells, cyclophosphamide is started 12 days before injecting CAR T cells, and albumin-bound paclitaxel is administered 11 days before injecting CAR T cells.

[0335] The time when the pretreatment component can be adjusted is when the therapeutic effect of CAR T is greatest. Generally, fludarabine, cyclophosphamide, and / or albumin-bound paclitaxel may be administered daily. In some embodiments, fludarabine, cyclophosphamide, and albumin-bound paclitaxel are administered daily for about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days. In some embodiments, fludarabine is administered daily for 4 days and cyclophosphamide is administered daily for 2 days. In some embodiments, fludarabine is administered daily for 2 days and cyclophosphamide is administered daily for 3 days. In some embodiments, fludarabine is administered daily for 2 days and cyclophosphamide is administered daily for 4 days. In some embodiments, fludarabine is administered daily for 1 day 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 albumin-bound 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 albumin-bound paclitaxel is administered daily for 1 day.

[0336] As described above, the day on which CAR T-cell therapy is performed on each patient is designated as Day 0. In some embodiments, fludarabine is administered to the patient on Day 4 prior to Day 0 (i.e., Day-4). In some embodiments, fludarabine is administered to the patient on Day-5. In some embodiments, fludarabine is administered to the patient on Day-6. In some embodiments, fludarabine is administered to the patient on Day-12. In some embodiments, fludarabine is administered to the patient on Day-6 and Day-5. In some embodiments, fludarabine is administered to the patient on Day-5 and Day-4. In some embodiments, fludarabine is administered to the patient on Day-6, Day-5, Day-4, and Day-3. In some embodiments, fludarabine is administered to the patient on Day-7, Day-6, Day-5, and Day-4. In some embodiments, cyclophosphamide is administered to the patient on days 6, 5, 4, and 3. In some embodiments, cyclophosphamide is administered to the patient on days 6, 5, 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, albumin-bound paclitaxel is administered to the patient on day 4. In some embodiments, albumin-bound paclitaxel is administered to the patient on day 5. In some embodiments, albumin-bound paclitaxel is administered to the patient on day 11. In some embodiments, albumin-bound paclitaxel is administered to the patient on days 5 and 4.

[0337] Fludarabine, cyclophosphamide, and albumin-bound paclitaxel may be administered on the same or different days. If fludarabine, cyclophosphamide, and albumin-bound paclitaxel are administered on the same day, cyclophosphamide and / or albumin-bound paclitaxel may be administered before or after fludarabine; or fludarabine and / or albumin-bound paclitaxel may be administered before or after cyclophosphamide; or cyclophosphamide and / or fludarabine may be administered before or after albumin-bound 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 albumin-bound 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 albumin-bound 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 albumin-bound 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 albumin-bound paclitaxel is administered on day 5.

[0338] In some embodiments, fludarabine, cyclophosphamide, and albumin-bound paclitaxel may be administered simultaneously or sequentially. In some embodiments, cyclophosphamide is administered to the patient before administering fludarabine. In some embodiments, cyclophosphamide is administered to the patient after administering fludarabine. In some embodiments, albumin-bound paclitaxel is administered to the patient before administering fludarabine. In some embodiments, albumin-bound paclitaxel is administered to the patient before or after administering fludarabine. In some embodiments, albumin-bound paclitaxel is administered to the patient before administering cyclophosphamide. In some embodiments, albumin-bound paclitaxel is administered after administering cyclophosphamide.

[0339] Fludarabine, cyclophosphamide, and albumin-bound paclitaxel may be administered via any route (including intravenous injection (IV)). In some embodiments, fludarabine is administered via IV within 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, and about 120 minutes. In some embodiments, cyclophosphamide is administered via IV within about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 90 minutes, and about 120 minutes. In some embodiments, albumin-bound paclitaxel is administered via IV within 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, and about 120 minutes.

[0340] In some embodiments, T cell therapy is performed on the patient by administering fludarabine and cyclophosphamide, or by administering fludarabine, cyclophosphamide, and albumin-conjugated paclitaxel. In some embodiments, the T cell therapy includes 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 includes the administration of engineered antigen-specific chimeric antigen receptor (CAR T) positive T cells. In some embodiments, eACT includes the administration of engineered antigen-specific T cell receptor (TCR) positive T cells. In some embodiments, the engineered T cells treat the patient's tumor.

[0341] In some embodiments, the pretreatment is about 500 mg / m² 2 / day, 490 mg / m² 2 / day, 480 mg / m²2 / day, 470 mg / m² 2 / day, 460 mg / m² 2 / day, 450 mg / m² 2 / day, 440 mg / m² 2 / day, 430 mg / m² 2 / day, 420 mg / m² 2 / day, 410 mg / m² 2 / day, 400 mg / m² 2 / day, 390 mg / m² 2 / day, 380 mg / m² 2 / day, 370 mg / m² 2 / day, 360 mg / m² 2 / day, 350 mg / m² 2 / day, 340 mg / m² 2 / day, 330 mg / m² 2 / day, 320 mg / m² 2 / day, 310 mg / m² 2 / day, 300 mg / m² 2 / day, 290 mg / m² 2 / day, 280 mg / m² 2 / day, 270 mg / m² 2 / day, 260 mg / m² 2 / day, 250 mg / m² 2 / day, 240 mg / m² 2 / day, 230 mg / m² 2 / day, 220 mg / m² 2 / day, 210 mg / m² 2 / day, 200 mg / m² 2 / day, 190 mg / m² 2 / day, 180 mg / m² 2 / day, 170 mg / m² 2 / day, 160 mg / m² 2 / day, 150 mg / m² 2 / day, 140 mg / m² 2 / day, 130 mg / m² 2 / day, 120 mg / m² 2 / day, 110 mg / m² 2 / day, 100 mg / m² 2 / day, 90 mg / m² 2 / day, 85 mg / m²2 / day, 70 mg / m² 2 / day, 65 mg / m² 2 / day, 60 mg / m² 2 / day, 55 mg / m² 2 / day, 50 mg / m² 2 / day, 49 mg / m² 2 / day, 48 mg / m² 2 / day, 47 mg / m² 2 / day, 46 mg / m² 2 / day, 45 mg / m² 2 / day, 44 mg / m² 2 / day, 43 mg / m² 2 / day, 42 mg / m² 2 / day, 41 mg / m² 2 / day, 40 mg / m² 2 / day, 39 mg / m² 2 / day, 38 mg / m² 2 / day, 37 mg / m² 2 / day, 36 mg / m² 2 / day, 35 mg / m² 2 / day, 34 mg / m² 2 / day, 33 mg / m² 2 / day, 32 mg / m² 2 / day, 31 mg / m² 2 / day, 30 mg / m² 2 / day, 29 mg / m² 2 / day, 28 mg / m² 2 / day, 27 mg / m² 2 / day, 26 mg / m² 2 / day, 25 mg / m² 2 / day, 24 mg / m² 2 / day, 23 mg / m² 2 / day, 22 mg / m² 2 / day, 21 mg / m² 2 / day, 20 mg / m² 2 / day, 19 mg / m² 2 / day, 18 mg / m² 2 / day, 17 mg / m² 2 / day, 16 mg / m² 2 / day, 15 mg / m² 2 / day, 14 mg / m² 2 / day, 13 mg / m² 2 / day, 12 mg / m² 2 / day, 11 mg / m² 2 / day, 10 mg / m² 2 / day, 9 mg / m² 2 / day, 8 mg / m² 2 / day, 7 mg / m² 2 / day, 6 mg / m² 2 / day, 5 mg / m² 2 / day, 4 mg / m² 2 / day, 3 mg / m² 2 / day, 2 mg / m² 2 / day, 1 mg / m² 2 Administration of fludarabine not exceeding / day; and / or about 1000 mg / m² 2 / day, 950 mg / m² 2 / day, 900 mg / m² 2 / day, 850 mg / m² 2 / day, 800 mg / m² 2 / day, 750 mg / m² 2 / day, 700 mg / m² 2 / day, 690 mg / m² 2 / day, 680 mg / m² 2 / day, 670 mg / m² 2 / day, 660 mg / m² 2 / day, 650 mg / m² 2 / day, 640 mg / m² 2 / day, 630 mg / m² 2 / day, 620 mg / m² 2 / day, 610 mg / m² 2 / day, 600 mg / m² 2 / day, 595 mg / m² 2 / day, 590 mg / m² 2 / day, 585 mg / m² 2 / day, 580 mg / m² 2 / day, 579 mg / m² 2 / day, 578 mg / m² 2 / day, 576 mg / m² 2 / day, 575 mg / m² 2 / day, 574 mg / m² 2 / day, 573 mg / m²2 / day, 572 mg / m² 2 / day, 571 mg / m² 2 / day, 570 mg / m² 2 / day, 569 mg / m² 2 / day, 568 mg / m² 2 / day, 567 mg / m² 2 / day, 566 mg / m² 2 / day, 565 mg / m² 2 / day, 564 mg / m² 2 / day, 563 mg / m² 2 / day, 562 mg / m² 2 / day, 561 mg / m² 2 / day, 560 mg / m² 2 / day, 559 mg / m² 2 / day, 558 mg / m² 2 / day, 557 mg / m² 2 / day, 556 mg / m² 2 / day, 555 mg / m² 2 / day, 554 mg / m² 2 / day, 553 mg / m² 2 / day, 552 mg / m² 2 / day, 551 mg / m² 2 / day, 550 mg / m² 2 / day, 549 mg / m² 2 / day, 548 mg / m² 2 / day, 547 mg / m² 2 / day, 546 mg / m² 2 / day, 545 mg / m² 2 / day, 544 mg / m² 2 / day, 543 mg / m² 2 / day, 542 mg / m² 2 / day, 541 mg / m² 2 / day, 540 mg / m² 2 / day, 539 mg / m² 2 / day, 538 mg / m² 2 / day, 537 mg / m² 2 / day, 536 mg / m² 2 / day, 535 mg / m² 2 / day, 534 mg / m² 2 / day, 533 mg / m²2 / day, 532 mg / m² 2 / day, 531 mg / m² 2 / day, 530 mg / m² 2 / day, 529 mg / m² 2 / day, 528 mg / m² 2 / day, 527 mg / m² 2 / day, 526 mg / m² 2 / day, 525 mg / m² 2 / day, 524 mg / m² 2 / day, 523 mg / m² 2 / day, 522 mg / m² 2 / day, 521 mg / m² 2 / day, 520 mg / m² 2 / day, 519 mg / m² 2 / day, 518 mg / m² 2 / day, 517 mg / m² 2 / day, 516 mg / m² 2 / day, 515 mg / m² 2 / day, 514 mg / m² 2 / day, 513 mg / m² 2 / day, 512 mg / m² 2 / day, 511 mg / m² 2 / day, 510 mg / m² 2 / day, 509 mg / m² 2 / day, 508 mg / m² 2 / day, 507 mg / m² 2 / day, 506 mg / m² 2 / day, 505 mg / m² 2 / day, 504 mg / m² 2 / day, 503 mg / m² 2 / day, 502 mg / m² 2 / day, 501 mg / m² 2 / day, 500 mg / m² 2 / day, 490 mg / m² 2 / day, 480 mg / m² 2 / day, 470 mg / m² 2 / day, 460 mg / m² 2 / day, 450 mg / m² 2 / day, 440 mg / m² 2 / day, 430 mg / m²2 / day, 420 mg / m² 2 / day, 410 mg / m² 2 / day, 400 mg / m² 2 / day, 390 mg / m² 2 / day, 380 mg / m² 2 / day, 370 mg / m² 2 / day, 360 mg / m² 2 / day, 350 mg / m² 2 / day, 340 mg / m² 2 / day, 330 mg / m² 2 / day, 320 mg / m² 2 / day, 310 mg / m² 2 / day, 300 mg / m² 2 / day, 290 mg / m² 2 / day, 280 mg / m² 2 / day, 270 mg / m² 2 / day, 260 mg / m² 2 / day, 250 mg / m² 2 / day, 240 mg / m² 2 / day, 230 mg / m² 2 / day, 220 mg / m² 2 / day, 210 mg / m² 2 / day, 200 mg / m² 2 / day, 190 mg / m² 2 / day, 180 mg / m² 2 / day, 170 mg / m² 2 / day, 160 mg / m² 2 / day, 150 mg / m² 2 / day, 140 mg / m² 2 / day, 130 mg / m² 2 / day, 120 mg / m² 2 / day, 110 mg / m² 2 / day, 100 mg / m² 2 / day, 90 mg / m² 2 / day, 80 mg / m² 2 / day, 70 mg / m² 2 / day, 60 mg / m² 2 / day, 50 mg / m² 2 / day, 40 mg / m² 2 / day, 30 mg / m²2 / day, 20 mg / m² 2 / day or 10 mg / m² 2 Administration of cyclophosphamide no more than / day; and / or about 500 mg / m² 2 / day, 450 mg / m² 2 / day, 400 mg / m² 2 / day, 350 mg / m² 2 / day, 300 mg / m² 2 / day, 290 mg / m² 2 / day, 280 mg / m² 2 / day, 270 mg / m² 2 / day, 265 mg / m² 2 / day, 260 mg / m² 2 / day, 255 mg / m² 2 / day, 250 mg / m² 2 / day, 245 mg / m² 2 / day, 240 mg / m² 2 / day, 235 mg / m² 2 / day, 230 mg / m² 2 / day, 225 mg / m² 2 / day, 220 mg / m² 2 / day, 215 mg / m² 2 / day, 210 mg / m² 2 / day, 205 mg / m² 2 / day, 200 mg / m² 2 / day, 195 mg / m² 2 / day, 190 mg / m² 2 / day, 185 mg / m² 2 / day, 180 mg / m² 2 / day, 175 mg / m² 2 / day, 170 mg / m² 2 / day, 165 mg / m² 2 / day, 160 mg / m² 2 / day, 155 mg / m² 2 / day, 150 mg / m² 2 / day, 145 mg / m² 2 / day, 140 mg / m² 2 / day, 135 mg / m² 2 / day, 130 mg / m² 2 / day, 125 mg / m²2 / day, 120 mg / m² 2 / day, 115 mg / m² 2 / day, 110 mg / m² 2 / day, 105 mg / m² 2 / day, 100 mg / m² 2 / day, 99 mg / m² 2 / day, 98 mg / m² 2 / day, 97 mg / m² 2 / day, 96 mg / m² 2 / day, 95 mg / m² 2 / day, 94 mg / m² 2 / day, 93 mg / m² 2 / day, 92 mg / m² 2 / day, 91 mg / m² 2 / day, 90 mg / m² 2 / day, 89 mg / m² 2 / day, 88 mg / m² 2 / day, 87 mg / m² 2 / day, 86 mg / m² 2 / day, 85 mg / m² 2 / day, 84 mg / m² 2 / day, 83 mg / m² 2 / day, 82 mg / m² 2 / day, 81 mg / m² 2 / day, 80 mg / m² 2 / day, 79 mg / m² 2 / day, 78 mg / m² 2 / day, 77 mg / m² 2 / day, 76 mg / m² 2 / day, 75 mg / m² 2 / day, 74 mg / m² 2 / day, 73 mg / m² 2 / day, 72 mg / m² 2 / day, 71 mg / m² 2 / day, 70 mg / m² 2 / day, 69 mg / m² 2 / day, 68 mg / m² 2 / day, 67 mg / m² 2 / day, 66 mg / m² 2 / day, 65 mg / m² 2 / day, 64 mg / m² 2 / day, 63 mg / m² 2 / day, 62 mg / m² 2 / day, 61 mg / m² 2 / day, 60 mg / m² 2 / day, 59 mg / m² 2 / day, 58 mg / m² 2 / day, 57 mg / m² 2 / day, 56 mg / m² 2 / day, 55 mg / m² 2 / day, 54 mg / m² 2 / day, 53 mg / m² 2 / day, 52 mg / m² 2 / day, 51 mg / m² 2 / day, 49 mg / m² 2 / day, 48 mg / m² 2 / day, 47 mg / m² 2 / day, 46 mg / m² 2 / day, 45 mg / m² 2 / day, 44 mg / m² 2 / day, 43 mg / m² 2 / day, 42 mg / m² 2 / day, 41 mg / m² 2 / day, 40 mg / m² 2 / day, 35 mg / m² 2 / day, 30 mg / m² 2 / day, 25 mg / m² 2 / day, 20 mg / m² 2 / day, 15 mg / m² 2 / day, 10 mg / m² 2 / day, 5 mg / m² 2 / day or 1 mg / m² 2 It includes the administration of albumin-bound paclitaxel no more than / day.

[0342] In some embodiments, the pretreatment is about 10 mg / m² 2 / day to 50 mg / m² 2 Administer fludarabine at a daily dosage of approximately 300 mg / m² 2 / day to 700 mg / m² 2 It includes administering cyclophosphamide at a daily usage amount.

[0343] In some embodiments, the pretreatment is about 10 mg / m² 2 / day to 50 mg / m² 2 Administer fludarabine at a daily dosage of approximately 300 mg / m² 2 / day to 700 mg / m² 2 Administer cyclophosphamide at a daily dosage of approximately 300 mg / m² 2 Not more than / day, about 200 mg / m² 2 Not more than / day, approximately 150 mg / m² 2 Not more than / day, 100 mg / m² 2 Not more than / day, 80 mg / m² 2 Not more than / day or 70 mg / m² 2 It includes administering albumin-bound paclitaxel at a dosage not exceeding one day.

[0344] In some embodiments, the pretreatment is about 15 mg / m² 2 / day to 40 mg / m² 2 Administer fludarabine at a daily dosage of approximately 400 mg / m² 2 / day to 650 mg / m² 2 It includes administering cyclophosphamide at a daily usage amount.

[0345] In some embodiments, the pretreatment is about 15 mg / m² 2 / day to 40 mg / m² 2 Administer fludarabine at a daily dosage of approximately 400 mg / m² 2 / day to 650 mg / m² 2 Administer cyclophosphamide at a daily dosage of approximately 300 mg / m² 2 Not more than / day, about 200 mg / m² 2 Not more than / day, approximately 150 mg / m² 2 Not more than / day, 100 mg / m² 2 Not more than / day, 80 mg / m² 2 Not more than / day or 70 mg / m² 2It includes administering albumin-bound paclitaxel at a dosage not exceeding one day.

[0346] In some embodiments, the pretreatment is about 15 mg / m² 2 / day to 30 mg / m² 2 Administer fludarabine at a daily dosage of approximately 450 mg / m² 2 / day to 600 mg / m² 2 / day, 450 mg / m² 2 / day to 550 mg / m² 2 / day or 490 mg / m² 2 / day to 550 mg / m² 2 It includes administering cyclophosphamide at a daily usage amount.

[0347] In some embodiments, the pretreatment is about 15 mg / m² 2 / day to 30 mg / m² 2 Administer fludarabine at a daily dosage of approximately 450 mg / m² 2 / day to 600 mg / m² 2 / day, 450 mg / m² 2 / day to 550 mg / m² 2 / day or 490 mg / m² 2 / day to 550 mg / m² 2 Administer cyclophosphamide at a daily dosage of approximately 300 mg / m² 2 Not more than / day, about 200 mg / m² 2 Not more than / day, approximately 150 mg / m² 2 Not more than / day, 100 mg / m² 2 Not more than / day, 80 mg / m² 2 Not more than / day or 70 mg / m² 2 It includes administering albumin-bound paclitaxel at a dosage not exceeding one day.

[0348] In some embodiments, the pretreatment is about 20 mg / m² 2 / day to 30 mg / m² 2 Administer fludarabine at a daily dosage of approximately 450 mg / m²2 / day to 600 mg / m² 2 / day, 450 mg / m² 2 / day to 550 mg / m² 2 / day or 490 mg / m² 2 / day to 550 mg / m² 2 It includes administering cyclophosphamide at a daily usage amount.

[0349] In some embodiments, the pretreatment is about 20 mg / m² 2 / day to 30 mg / m² 2 Administer fludarabine at a daily dosage of approximately 450 mg / m² 2 / day to 600 mg / m² 2 / day, 450 mg / m² 2 / day to 550 mg / m² 2 / day or 490 mg / m² 2 / day to 550 mg / m² 2 Administer cyclophosphamide at a daily dosage of approximately 300 mg / m² 2 Not more than / day, about 200 mg / m² 2 Not more than / day, approximately 150 mg / m² 2 Not more than / day, 100 mg / m² 2 Not more than / day, 80 mg / m² 2 Not more than / day or 70 mg / m² 2 It includes administering albumin-bound paclitaxel at a dosage not exceeding one day.

[0350] In some embodiments, the pretreatment is about 20 mg / m² 2 / day to 25 mg / m² 2 Administer fludarabine at a daily dosage of approximately 450 mg / m² 2 / day to 600 mg / m² 2 / day, 450 mg / m² 2 / day to 550 mg / m² 2 / day or 490 mg / m² 2 / day to 550 mg / m² 2It includes administering cyclophosphamide at a daily usage amount.

[0351] In some embodiments, the pretreatment is about 15 mg / m² 2 / day to 25 mg / m² 2 Administer fludarabine at a daily dosage of approximately 450 mg / m² 2 / day to 600 mg / m² 2 / day, 450 mg / m² 2 / day to 550 mg / m² 2 / day or 490 mg / m² 2 / day to 550 mg / m² 2 Administer cyclophosphamide at a daily dosage of approximately 300 mg / m² 2 Not more than / day, about 200 mg / m² 2 Not more than / day, approximately 150 mg / m² 2 Not more than / day, 100 mg / m² 2 Not more than / day, 80 mg / m² 2 Not more than / day or 70 mg / m² 2 It includes administering albumin-bound paclitaxel at a dosage not exceeding one day.

[0352] In some embodiments, the present invention comprises a method for pre-treating immune effector cells before injecting them, wherein the method has a dosage of about 20 mg / m² to the patient. 2 Fludarabine per person and a usage of approximately 500 mg / m² 2 The method comprises the step of administering cyclophosphamide on a daily basis, 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 comprises a method of pretreatment before injecting immune effector cells, wherein the method has a dosage of approximately 20 mg / m² to the patient. 2 Fludarabine per person and a usage of approximately 514 mg / m² 2The method comprises the step of administering cyclophosphamide on a daily basis, wherein fludarabine is administered on days 7, 6, 5, and 4, and cyclophosphamide is administered on days 7 and 6. In some embodiments, the present invention comprises a method of pretreatment before injecting immune effector cells, wherein the method has a dosage of approximately 20 mg / m² to the patient. 2 Fludarabine per person and a usage of approximately 514 mg / m² 2 The method comprises the step of administering cyclophosphamide on a daily basis, 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 comprises a method of pretreatment before injecting immune effector cells, wherein the method has a dosage of approximately 20 mg / m² to the patient. 2 Fludarabine per person and a usage of approximately 514 mg / m² 2 The method comprises the step of administering cyclophosphamide on a given day, 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 comprises a method of pretreatment before injecting immune effector cells, wherein the method has a dosage of approximately 20 mg / m² to the patient. 2 Fludarabine per person and a usage of approximately 510 mg / m² 2 The method comprises the step of administering cyclophosphamide on a given day, 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 comprises a method of pretreatment before injecting immune effector cells, wherein the method has a dosage of approximately 20 mg / m² to the patient. 2 Fludarabine per person and a usage of approximately 500 mg / m² 2The method comprises the step of administering cyclophosphamide on a daily basis, wherein fludarabine is administered on day 5, and cyclophosphamide is administered on days 5, 4, and 3. In some embodiments, the present invention comprises a method of pretreatment before injecting immune effector cells, wherein the method has a dosage of approximately 20 mg / m² to the patient. 2 Fludarabine per person and a usage of approximately 540 mg / m² 2 The method comprises the step of administering cyclophosphamide on a daily basis, wherein fludarabine is administered on day 6, and cyclophosphamide is administered on days 6, 5, and 4. In some embodiments, the invention comprises a method of pretreatment before injecting immune effector cells, wherein the method has a dosage of approximately 20 mg / m² to the patient. 2 Fludarabine per person and a usage of approximately 500 mg / m² 2 The method comprises the step of administering cyclophosphamide on a daily basis, wherein fludarabine is administered on day 6, and cyclophosphamide is administered on days 6, 5, and 4. In some embodiments, the invention comprises a method of pretreatment before injecting immune effector cells, wherein the method has a dosage of approximately 20 mg / m² to the patient. 2 Fludarabine per person and a usage of approximately 500 mg / m² 2 The method comprises the step of administering cyclophosphamide on a daily basis, wherein fludarabine is administered on day 6, and cyclophosphamide is administered on days 6, 5, 4, and 3. In some embodiments, the present invention comprises a method of pretreatment before injecting immune effector cells, wherein the method has a dosage of approximately 20 mg / m² to the patient. 2 Fludarabine per person and a usage of approximately 500 mg / m² 2The method comprises the step of administering cyclophosphamide on a daily basis, wherein fludarabine is administered on day 12, and cyclophosphamide is administered on days 12, 11, and 10. In some embodiments, the present invention comprises a method of pretreatment before injecting immune effector cells, wherein the method has a dosage of approximately 20 mg / m² to the patient. 2 Fludarabine per person, dosage approximately 500 mg / m² 2 / cyclophosphamide per person and usage amount is approximately 100 mg / m² 2 The method comprises the step of administering albumin-bound paclitaxel on day 1, wherein fludarabine is administered on day 5, cyclophosphamide is administered on days 5, 4, and 3, and albumin-bound paclitaxel is administered on day 4. In some embodiments, the present invention comprises a method of pretreatment before injecting immune effector cells, wherein the method has a dosage of about 20 mg / m² to the patient. 2 Fludarabine per person, dosage is approximately 540 mg / m² 2 Cyclophosphamide per person and usage amount is approximately 71 mg / m² 2 The method comprises the step of administering albumin-bound paclitaxel on day 1, wherein fludarabine is administered on day 6, cyclophosphamide is administered on days 6, 5, and 4, and albumin-bound paclitaxel is administered on day 5. In some embodiments, the present invention comprises a method of pretreatment before injecting immune effector cells, wherein the method has a dosage of about 20 mg / m² to the patient. 2 Fludarabine per person, dosage approximately 500 mg / m² 2 Cyclophosphamide per person and usage amount is approximately 71 mg / m² 2The method comprises the step of administering albumin-bound paclitaxel on day 12, wherein fludarabine is administered on day 12, cyclophosphamide is administered on days 12, 11, and 10, and albumin-bound paclitaxel is administered on day 11. In some embodiments, the present invention comprises a method of pretreatment before injecting immune effector cells, wherein the method has a dosage of about 20 mg / m² to the patient. 2 Fludarabine per person, dosage approximately 500 mg / m² 2 / cyclophosphamide per person and usage amount is approximately 100 mg / m² 2 The method comprises the step of administering albumin-bound paclitaxel on day 1, wherein fludarabine is administered on day 6, cyclophosphamide is administered on days 6, 5, and 4, and albumin-bound paclitaxel is administered on day 5. In some embodiments, the present invention comprises a method of pretreatment before injecting immune effector cells, wherein the method has a dosage of about 20 mg / m² to the patient. 2 Fludarabine per person, usage is approximately 507 mg / m² 2 Cyclophosphamide per person and a usage of approximately 68 mg / m² 2 The method comprises the step of administering albumin-bound paclitaxel on day 1, wherein fludarabine is administered on day 6, cyclophosphamide is administered on days 6, 5, 4, and 3, and albumin-bound paclitaxel is administered on day 5. In some embodiments, the invention comprises a method of pretreatment before injecting immune effector cells, wherein the method has a dosage of approximately 137 mg / m² to the patient. 2 The step of administering albumin-bound paclitaxel on day 1 is included, wherein albumin-bound paclitaxel is administered on day 4.

[0353] The above method may include various other interventions. For example, cyclophosphamide and fludarabine may cause adverse effects in the patient after administration. The scope of the invention includes administering a composition to the patient to reduce some of these adverse effects. In some embodiments, the method includes the step of administering physiological saline to the patient. Physiological saline may be administered to the patient before or after administering cyclophosphamide and / or fludarabine, or before and after administering cyclophosphamide and / or fludarabine. In some embodiments, physiological saline is administered to the patient before administering cyclophosphamide and / or fludarabine on each infusion day, and after administering cyclophosphamide and / or fludarabine. In addition, an adjuvant and an excipient may be administered to the patient. For example, Mesna (sodium 2-mercaptoethane sulfonate) may be administered. In addition, exogenous cytokines may be administered to the patient.

[0354] In some embodiments, treatment outcomes are improved by pretreatment before infusion of the initial or subsequent doses. For example, in some embodiments, pretreatment improves the effect of treatment with the initial 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 the disease stable period.

[0355] Once immune effector cells are administered to a subject (e.g., human), in some embodiments, the biological activity of the engineered immune effector cell population is measured through one of various known methods. Parameters used for evaluation include the specific binding of antigens to engineered or natural T cells or other immune cells, in vivo (e.g., via imaging) or in vitro (e.g., via ELISA or flow cytometry). In some embodiments, the ability of the engineered immune effector cells to destroy target cells can be detected by any suitable method known in the art. For example, it can be detected through cytotoxicity tests in the literature, such as 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 may be measured by measuring the expression and / or secretion of certain cytokines, such as CD107a, IFNγ, IL-2, and TNF. In some embodiments, biological activity is measured by evaluating clinical outcomes, such as tumor burden or reduction of burden. In some embodiments, a reduction in tumor markers is evaluated. In some embodiments, the results of cell toxicity, persistence and / or proliferation, and / or the presence or absence of a host immune response are evaluated.

[0356] administration

[0357] In the present invention, the timing and size of administration of multiple doses of immune effector cells are generally designed to reduce risk, minimize toxicity consequences, and / or enhance effect, for example, by providing increased subject exposure to immune effector cells over time. The method generally comprises the step of administering an initial dose within a specific time range between different doses prior to one or more subsequent doses.

[0358] In the case of immuno-effector cell therapy, administration in a specified "dose" includes administering a single composition and / or administering it sequentially in a single dose, for example, administering a specified amount or number of immuno-effector cells in a single injection or sequential injection, and also includes administering a specified amount or number of immuno-effector cells by providing a plurality of single compositions or injectables in divided amounts within a specific time period not exceeding 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days. Accordingly, the initial dose or subsequent dose is a single or sequential dose of a specified number of immuno-effector cells administered or initiated within a single time period. However, in some cases, the initial dose or subsequent dose is injected or infused multiple times within a time period not exceeding 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days, for example, once daily for 3 or 2 days, or infused multiple times a day. In some embodiments, the immune effector cells of the initial dose are administered as a single drug composition. In some embodiments, the immune effector cells of the subsequent dose are administered as a single drug composition. In some embodiments, the immune effector cells of the initial dose are administered as a plurality of compositions containing the immune effector cells of the initial dose in total. In some embodiments, the immune effector cells of the subsequent dose are administered as a plurality of compositions containing the immune effector cells of the subsequent dose in total. In some embodiments, additional subsequent amounts of a plurality of compositions may be administered within a time not exceeding 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 days.

[0359] The term "divided dose" refers to a portion of a single dose that is divided into multiple doses and administered to the subject within a period exceeding one day. In some embodiments, the initial dose and / or each subsequent dose may all be administered as two or more divided doses. For example, in some embodiments, the dose (which is the total dose of the initial dose or the total dose of the subsequent dose) may be administered to the subject within 3, 5, 14, or 15 days. An exemplary method of divided administration includes the step of administering a divided dose of 50% of the total dose on day 0 (designating the day on which each CAR T-cell therapy is performed on the patient as day 0) and administering a divided dose of 50% of the total dose on day 2. In other embodiments, 10% of the total dose may 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, on day 0, a divided amount of 10% of the total usage is administered, on day 1, a divided amount of 30% of the total usage is administered, and on day 2, a divided amount of 60% of the total usage is administered. In some embodiments, on day 0, a divided amount of 1 / 3 of the total usage is administered, on day 11, a divided amount of 1 / 3 of the total usage is administered, and on day 14, a divided amount of 1 / 3 of the total usage is administered. In some embodiments, on day 0, a divided amount of 46% of the total usage is administered, on day 1, a divided amount of 18% of the total usage is administered, and on day 2, a divided amount of 36% of the total usage is administered. In some embodiments, on day 0, a divided amount of 25% of the total usage is administered, on day 2, a divided amount of 25% of the total usage is administered, on day 9, a divided amount of 25% of the total usage is administered, and on day 13, a divided amount of 25% of the total usage is administered. In some embodiments, the total time spread of administering a single amount divided into multiple doses does not exceed 15 days.

[0360] As used in the text, "initial dose" is intended to describe the total dose administered during the first course of treatment according to the method described in the text. The said dose is equal to the total dose administered during the course of treatment in a single course of treatment, or the total dose administered during the first course of treatment in a multiple course of treatment. The said term does not mean a dose for which the subject has not received immuno-effector cell therapy prior to treatment according to the method described in the text, or for which the subject has not previously been administered the same immuno-effector cells expressing the same chimeric antigen receptor or targeting the same antigen.

[0361] Compared to the initial dose, the term "subsequent dose" refers to a single dose or the total dose administered in each treatment course after the initial dose has been administered in the performance of multiple treatment courses. In some embodiments, the method comprises the step of performing multiple treatment courses, i.e., administering one or more subsequent doses, wherein the first subsequent dose is referred to as the second treatment course dose and the second subsequent dose is referred to as the third treatment course dose, and so is inferred. Furthermore, in a series of doses, the previous dose is the dose immediately preceding the subsequent dose administered thereafter. In some embodiments, the subsequent dose is administered by applying a time and method of administration similar to that of the initial dose. In some embodiments, the interval between the initial dose and the first subsequent dose or the second treatment course dose, and the interval between multiple subsequent doses, may be the same or different, as described above.

[0362] Amount or size of usage

[0363] Generally, the dosage size of the initial dose and / or one or more subsequent doses is designed to provide improved efficacy and / or reduced toxicity risk. In some embodiments, the number of immune effector cells in the initial dose or a single subsequent dose is approximately 1 x 10⁻⁶ 6 Cells / kg subject body weight to about 3×10 7 cells / kg is greater than, less than, or equal to the subject's body weight, for example, about 1 x 10⁻⁶ 5 , 1.5Х10 5 , 2Х10 5 , 2.5Х10 5 , 3X10 5 , 3.5Х10 5 , 4X10 5 , 4.5Х10 5 , 5Х10 5 , 5.5Х10 5 , 6Х10 5 , 6.5Х10 5 , 7Х10 5 , 7.5Х10 5 , 8Х10 5 , 8.5Х10 5 , 9Х10 5 , 9.5Х10 5 , 1X10 6 , 1.5Х10 6 , 2Х10 6 , 2.5Х10 6 , 3X10 6 , 3.5Х10 6 , 4X10 6 , 4.5Х10 6 , 5Х10 6 , 5.5Х10 6 , 6Х10 6 , 6.5Х10 6 , 7Х10 6 , 7.5Х10 6 , 8Х10 6 , 8.5Х10 6 , 9Х10 6 , 9.5Х10 6 , 1X10 7 , 1.5Х10 7 , 2Х10 7, 2.5X10 7 , 3X10 7 , 3.5X10 7 , 4X10 7 , 4.5X10 7 , 5X10 7 , 6X10 7 , 6.5X10 7 , 7X10 7 , 7.5X10 7 , 8X10 7 , 8.5X10 7 , 9X10 7 , 9.5X10 7 , 1X10 8 , 1.5X10 8 , 2X10 8 , 2.5X10 8 , 3X10 8 , 3.5X10 8 , 4X10 8 , 4.5X10 8 , 5X10 8 , 5.5X10 8 , 6X10 8 , 6.5X10 8 , 7X10 8 , 7.5X10 8 , 8X10 8 , 8.5X10 8 , 9X10 8 , 9.5X10 8 , 1X10 9 , 1.5X10 9 , 2X10 9 , 2.5X10 9 , 3X10 9 , 3.5X10 9 , 4X10 9 , 4.5X10 9 , 5X10 9 , 5.5X10 9 , 6X10 9 , 6.5X10 9 , 7X10 9 , 7.5X10 9 , 8X10 9 , 8.5X10 9 , 9X10 9 , 9.5X10 9, 1X10 10 , 1.5Х10 10 , 2Х10 10 , 2.5Х10 10 , 3X10 10 , 3.5Х10 10 , 4X10 10 , 4.5Х10 10 , 5Х10 10 , 5.5Х10 10 , 6Х10 10 , 6.5Х10 10 , 7Х10 10 , 7.5Х10 10 , 8Х10 10 , 8.5Х10 10 , 9Х10 10 , 9.5Х10 10 , 1X10 11 Cells / kg is greater than or less than the subject's body weight.

[0364] In a specific embodiment, the number and / or concentration of immune effector cells refers to the number of chimeric antigen receptor (e.g., CAR)-expressing immune effector cells. In another embodiment, the number and / or concentration of immune effector cells refers to the number or concentration of all administered cells, T cells, or peripheral blood mononuclear cells (PBMCs).

[0365] 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) at the initial use is approximately 1 x 10⁻⁶ 6 These cells / kg are greater than the subject's body weight, for example, about or at least about 1X10 6 , 1.1X10 6 , 2Х10 6 , 2.9Х10 6 , 3X10 6 , 3.3Х10 6 , 5Х10 6 , 1X10 7 , 1.3X10 7 , 1.6Х10 7 , 2.6Х10 7, 2.8X10 7 , 3X10 7 , 5Х10 7 , 2.5Х10 8 , 1X10 9 , 1X10 10 , 5Х10 10 The number of such cells / kg subject body weight, or a range between any two of the aforementioned values.

[0366] In some embodiments, the number of immune effector cells administered in a subsequent amount is equal to, similar to, or greater than the number of immune effector cells administered in the first amount according to any embodiment of the present invention, for example, 1X10 6 Equal to or about 1X10 6 , 1X10 7 Equal to or about 1X10 7 , 1.3X10 7 Equal to or about 1.3X10 7 , 1.6Х10 7 Equal to or about 1.6 x 10 7 , 1.9Х10 7 Equal to or about 1.9 x 10 7 , 2Х10 7 Equal to or about 2×10 7 , 2.2Х10 7 Equal to or about 2.2X10 7 , 3.7Х10 7 Equal to or about 3.7 x 10 7 , 5Х10 7 Equal to or about 5×10 7 , or 5.1Х10 7 Equal to or about 5.1X10 7 , or 1X10 8 Equal to or about 1X10 8 , or 2.5X10 8 Equal to or about 2.5 x 10 8 , or 1X10 9 Equal to or about 1X10 9 , or 1X10 10 Equal to or about 1X10 10 , or 5Х10 10Equal to or about 5×10 10 These are values ​​within the range of 1 cell / kg subject body weight, or any 2 of the above values.

[0367] Regarding 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.

[0368] In some embodiments, the subsequent usage is greater than, equal to, or less than the initial usage. For example, in some embodiments, the subsequent usage is approximately 1 x 10 6 It comprises chimeric antigen receptor (e.g., CAR)-expressing cells, T cells, and / or PBMCs at a rate of 1 cell / kg subject body weight, e.g., about or at least about 1 x 10⁶ 6 , 1X10 7 , 1.3X10 7 , 1.6Х10 7 , 1.9Х10 7 , 2Х10 7 , 2.2Х10 7 , 3.7Х10 7 , 5Х10 7 , 5.1X10 7 , 1X10 8 , 2.5Х10 8 , 1X10 9 , 1X10 10 , 5Х10 10 These cells / kg subject body weight, or values ​​within the range between any two of the above values, each including an end value.

[0369] In some embodiments, the amount or size of the subsequent use sufficiently reduces the tumor burden or an index thereof and / or one or more symptoms of the disease or pathology. In some embodiments, the use is of a size that effectively improves the survival of the subject, for example, by inducing the survival of the subject and improving the survival of the subject such as recurrence-free survival or event-free survival for at least one month or at least one, two, three, four, or five years.

[0370] In some embodiments, compared to the time before administration of the first dose or subsequent dose, after administration of the subsequent dose, the tumor burden, including tumor size, tumor volume and / or tumor mass, is reduced to at least 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 90% or more.

[0371] In another embodiment, the number of immune effector cells administered in a subsequent dose is less than the number of immune effector cells administered in an initial dose.

[0372] In some embodiments, the size of the initial and / or subsequent dose is determined based on one or more standards, for example, the subject's response to prior treatment such as chemotherapy, tumor burden such as tumor volume, size, and extent, or the type and stage of metastasis and / or complications such as cancerous pleuroplasty and ascites common in late-stage cancer, and / or the likelihood or incidence of toxic outcomes such as CRS, macrophage activation syndrome, tumor lysis syndrome, and neurotoxicity in the subject, and / or the host immune response to the administered cells and / or chimeric antigen receptors.

[0373] Based on the implications of the present invention, a person skilled in the art should understand that the usage amount specifically disclosed in the present invention is a safe and effective usage amount obtained by the inventor through research, but a person skilled in the art should understand that, for example, a clinician may determine the specific initial usage amount according to various actual situations, such as factors such as the patient's tumor burden and the patient's own physical condition; and if a subsequent usage amount needs to be administered additionally, a person skilled in the art may determine the subsequent usage amount according to, for example, changes in tumor burden after administering immune effector cells.

[0374] A person skilled in the art should also understand that the immune effector cells of the present invention, such as CLD18A2-CAR-T cells, may be used in combination with other CAR-Ts. For example, since a patient's tumor may express several tumor antigens, the patient may receive CLD18A2-CAR-T cell therapy of the present invention after having previously received CAR-T cell therapy targeting a different tumor antigen; or the patient may switch to CLD18A2-CAR-T cell therapy of the present invention after having previously received other treatment different from the CLD18A2-CAR-T cell therapy of the present invention if antibody specificity is weak or if the antibody causes mouse myogenic rejection. Accordingly, in such cases, a person skilled in the art should understand that, for example, a clinician may determine the number of administrations and dosage of the CLD18A2-CAR-T cells of the present invention based on the circumstances of the previous treatment. For example, one may refer to the safe and effective dosage and number of administrations disclosed in the text, namely, about 2 x 10 10 Cells / kg not exceeding the subject's body weight or a total amount of approximately 1 x 10⁻⁶ 12 Without exceeding the cell; preferably, about 2×10 9 Cells / kg not exceeding the subject's body weight or a total amount of approximately 2×10 11Not exceeding the cell size; more preferably, about 2.5 x 10 8 Cells / kg subject body weight, or 5×10 7 Cells / kg subject body weight, or 3×10 7 Cells / kg must not exceed the subject's body weight, or the total amount must be 5×10 9 or about 1 x 10 10 It does not exceed the cell.

[0375] In some embodiments, the size of the initial dose and / or subsequent doses is determined by the burden of the subject's disease or pathology. For example, in some embodiments, the number of immune effector cells administered in the initial dose is determined based on the tumor burden present in the subject at the time prior to administration of the initial dose. In some embodiments, the size of the initial dose and / or subsequent doses is inversely proportional to the tumor burden.

[0376] In some embodiments, the number of immune effector cells administered in subsequent doses is determined based on the tumor burden present in the subject prior to administration of the initial dose. In some embodiments, for example, where the initial dose reduces or lowers the tumor burden, or where the tumor burden is already lower than a certain threshold or level, for example, where the risk of a higher toxic outcome than the toxic outcome increases, the subsequent dose is increased, for example, about 1 x 10 7 Exceeding 10 cells (e.g., total cells, receptor-expressing cells, T cells, or PBMCs) / kg body weight, e.g., about 2.0 x 10 7 , 2.5Х10 7 , 3.0Х10 7 , 5.0Х10 7 , 1.0Х10 8 , 2.5Х10 8 More than 1 cell / kg, and / or more than the initial usage.

[0377] In some situations, even if the dosage burden on a subject administered the initial dose is reduced, subsequent doses may be increased, for example, about 1 x 10 7 Exceeding 10 cells (e.g., total cells, receptor-expressing cells, T cells, or PBMCs) / kg body weight, e.g., about 2.0 x 10 7 , 2.5Х10 7 , 3.0Х10 7 , 5.0Х10 7 , 1X10 8 , 2.5Х10 8 Exceeds the number of cells / kg and / or is greater than the initial usage.

[0378] T cells (e.g., CAR T cells) may be administered via any route (including intravenous injection (IV)). In some embodiments, CAR T cells are administered via IV within 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, and about 60 minutes.

[0379] In some embodiments, the initial dose comprises an amount of immune effector cells capable of not causing or reducing toxic or toxic consequences, such as cytokine release syndrome (CRS), severe CRS (sCRS), macrophage activation syndrome, tumor lysis syndrome, fever of at least 38°C or about 38°C lasting for 3 days or more, CRP plasma levels of at least about 20 mg / dL, and / or neurotoxicity. In some embodiments, the number of cells administered as the initial dose is determined based on the likelihood that toxic or toxic consequences (e.g., CRS, sCRS, and / or CRS-related consequences) will occur in the subject after administration of the cells. For example, in some embodiments, the likelihood of toxic consequences progressing in the subject is predicted based on the tumor burden. In some embodiments, the method includes the step of detecting or evaluating toxic consequences and / or tumor burden before administering the dose.

[0380] In some embodiments, when a biochemical marker such as the CRS marker or other marker is administered after the first dose of toxicity results, the serum level is not increased to a level higher than a specified level, such as an acceptable level, e.g., about 10, 15, 20, 25, 50, 75, or 100 times higher than or equal to the serum level of the marker at the time before the first dose is administered, or is already increased above an acceptable level but is reduced to a level lower than or equal to the already acceptable level, a subsequent dose is administered to the subject. In some embodiments, if the level of the label is reduced to 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 after the initial dose of the label is administered, it is administered in a subsequent dose, but if the level of the label is not reduced to an acceptable level within the above time period, it is not administered in a subsequent dose.

[0381] In some embodiments, if there is no clinical risk of cytokine release syndrome (CRS), macrophage activation syndrome or tumor lysis syndrome or neurotoxicity occurring, or if it has already passed or has already been cleared after administration of the initial dose, subsequent doses are administered, for example, after a critical time when such conditions have generally already been cleared and / or are unlikely to occur, for example, in about 60%, 70%, 80%, 90%, or 95% of subjects with a specific disease or pathology.

[0382] In some embodiments, whether to administer a subsequent dose, the time of administration of the subsequent dose, and / or the number of cells administered in the subsequent dose are determined by the presence, absence, or degree of an immune response or detectable immune response to the cells administered to the subject in the first dose or the chimeric antigen receptors expressed by them. In some embodiments, a subsequent dose containing cells expressing the receptor of the cells of the first dose is not administered to a subject having a detectable host-adaptive immune response, or an immune response that has already been established or reached a certain level, stage, or degree.

[0383] Administration time

[0384] In some embodiments, the time for administering subsequent amounts begins to be calculated from the day after the administration of the first commercial amount is completed (the day the total amount of the first amount is administered is set as day 0).

[0385] In some embodiments, if the serum level of the factor indicating CRS in a subject exceeds about 10, 25, 50, or 100 times the serum level of the marker in the subject at the time before the first dose is administered, a subsequent dose is administered.

[0386] In some embodiments, a subsequent dose is administered after the result associated with CRS (e.g., serum factors associated with or indicating CRS) or its clinical signs or symptoms such as fever, hypoxia, hypotension, or neurological disorders in the subject have already reached a peak level and have begun to decrease after the initial dose. In some embodiments, a subsequent dose is administered when it is observed that the result has decreased below its peak level after administration, or when the level has decreased after reaching a maximum value or level of the result after administration.

[0387] In some embodiments, a subsequent dose is administered when the level of a toxicity result marker (e.g., a serum marker of CRS) decreases to about 25 times the level of the marker at the time prior to the first dose. In some embodiments, a subsequent dose is administered when no CRS occurs in the subject or when severe CRS does not occur.

[0388] In some embodiments, the subsequent dose is administered at a time when the tumor burden prior to administration of the first dose is reduced compared to the patient's tumor burden. In some embodiments, the subsequent dose is administered when, after administration of the first dose, the tumor burden or its markers, e.g., 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 already decreased by about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or more.

[0389] In some embodiments, a subsequent dose is administered if the subject's disease or condition does not recur after a decrease in response to the initial dose or previous dose. In some embodiments, the reduction of tumor burden is indicated by a decrease in one or more factors, said factors being, for example, the expression level of tumor markers in the subject or in their body fluids or organs or tissues, the burden or number of tumor cells, the mass or volume of the tumor, or the degree of metastasis. In some embodiments, recurrence is general, or one or more factors, or tumor burden. A subsequent dose is administered if the subject, disease burden, or factor thereof recurs compared to the lowest point measured or reached after the initial or previous administration, but is still less than the amount administered at the start before the initial dose. In some embodiments, if the tumor burden or its indicating factor does not change, for example, if the increase in tumor burden stops, a subsequent dose is administered to the subject.

[0390] In some embodiments, a subsequent dose is administered when a host adaptive immune response is not detected, has not been established, or has not reached a certain level, degree, or stage. In some embodiments, a subsequent dose is administered before the subject's memory immune response proceeds.

[0391] In some embodiments, the time between the administration of the first dose and the administration of the subsequent dose is about 21 to about 80 days, about 25 to about 60 days, or 25 to 55 days. The time for the administration of the subsequent dose does not exceed 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 first dose. In some embodiments, the time for administration of the subsequent amount does not exceed about 44, 45, 46, 47, or 48 days after administration of the first amount.

[0392] In some embodiments, after administering a subsequent dose (i.e., referred to as the first subsequent dose or the second course of treatment dose), additional or more subsequent doses are 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 embodiments, an additional subsequent dose is administered at least about 21 days to about 80 days after administering a previous subsequent dose (e.g., the first subsequent dose). In some embodiments, the exemplary dose plan includes a timetable for administering chimeric antigen receptor-expressing cells (e.g., CAR-expressing cells, e.g., CAR T cells) at or approximately at what time. In some embodiments, an additional dose (second subsequent dose) is administered within about 49 days after administering the first subsequent dose, and so on. In some embodiments, the time of administration of a subsequent dose administered later does not exceed about 21 to about 80 days, about 25 to about 60 days, or 25 to 55 days after administration of the immediately preceding subsequent dose. In some embodiments, the time of administration of a subsequent dose administered later does not exceed about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 days after administration of the immediately preceding subsequent dose.

[0393] In any embodiment, in some circumstances, the method comprises the step of administering an initial dose or a previous dose and a subsequent dose, and in other circumstances, comprises the step of administering a subsequent dose to a subject who has previously been administered an initial dose or a previous dose, said subject having previously been administered an initial dose or a previous dose that does not include the initial dose or the previous dose itself. Accordingly, in some cases, the method relates to an intensifying treatment, such as administering an intensifying subsequent dose to a subject who has been administered a previous dose (e.g., a reduced dose) of chimeric antigen receptor-expressing (e.g., CAR-expressing) cells. In some aspects, the previous dose of cells expressing a receptor such as CAR sufficiently reduces the disease or pathological burden of the subject so that the efficacy and / or safety of administering the subsequent dose of cells is improved compared to the dose administered to a subject who has not been administered an initial dose.

[0394] In some embodiments, compared to the time before administration of the first dose or subsequent dose, after administration of the subsequent dose, the tumor burden, including tumor markers, tumor size, tumor volume and / or tumor mass, is reduced to at least 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 90% or more.

[0395] Host immune response to reinjected cells

[0396] In some embodiments, one or more doses, e.g., subsequent doses, are administered when the subject's immune response (e.g., adaptive or specific immune response to the genetically modified receptor or cell) is not present, is not detected, or is not detected above a certain level. The presence or extent of a specific immune response to the genetic modification is generally related to the immunogenicity of the receptor (e.g., CAR or genetically modified TCR expressed by the cell) and / or the time 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 28, 35, or 42 days prior to the subject first contact with the cell expressing the receptor. Accordingly, in some embodiments, subsequent doses are administered before an immune response to the chimeric antigen receptor or cell, an adaptive or specific immune response, a detectable immune response, and / or a memory response develops in the subject. In this embodiment, compared to other methods in which a subsequent dose is administered at a relatively later time than the previous or initial dose, the ability of the subsequent dose to proliferate cells and / or remain continuously present in the subject is improved. The method comprises, for example, the step of detecting the presence or absence or level of such immune response or its markers after administration of the initial or subsequent dose and before administration of the subsequent or next subsequent dose.

[0397] In some embodiments, when and / or whether to administer a subsequent dose is determined by whether the subject exhibits such an immune response or a detectable reading, e.g., a detectable specific or adaptive host immune response specific to the cell or chimeric antigen receptor, e.g., a CAR expressed by the cell of the initial dose, and / or at what level such a response is detected. In some embodiments, if such a response is detected, the subject is not administered a subsequent dose. If the subject does not exhibit a specific or adaptive (e.g., humoral or cell-mediated) immune response to the receptor (e.g., CAR expressed by the cell of the initial dose), or if such a response or marker is not exhibited at a detectable level or higher than an acceptable level, the subject is administered a subsequent dose. In some embodiments, when administered a subsequent dose, the subject exhibits a reduced humoral or cell-mediated immune response to the CAR expressed by the cell of the initial dose compared to when the initial dose was relatively high.

[0398] In any one of the above embodiments, a detectable immune response refers to any detectable amount among numerous resonant methods for evaluating a specific immune response to a specific antigen and cell. For example, in some embodiments, a specific type of immune response is detected by performing ELISPOTT, ELISA on the subject's serum or by detecting the presence of an epitope, e.g., a CAR antigen, that binds to a chimeric antigen receptor and specifically binds to and / or neutralizes an antigen present in the cell based on a method for detecting antibodies in the cell (e.g., a method via flow cytometry). In some of these measurements, the isotype of the detected antibody, and the type indicating the response and / or whether the response is a memory response are determined.

[0399] In some embodiments, for example, after administration of an initial dose or a subsequent dose, the presence or absence of such host immune response and / or the number or degree thereof is detected or measured.

[0400] In some embodiments, a reduction is observed in comparison methods using alternative administration methods, for example, wherein the subject is administered a single dose (e.g., a single large dose of cells), for example, by replacing the single dose with the total number of cells administered uniformly in the first and subsequent doses by the provided method, or compared to the administration of multiple large doses or multiple doses that are less than about 21 days or more than about 80 days apart, the method reduces the burden of disease or pathology, such as, for example, the number of tumor cells, tumor size, duration of patient survival or event-free survival, and a longer time period. In some embodiments, the subject's survival time, survival at a given time period, survival range, presence or duration of event-free or symptom-free survival, or recurrence-free survival is evaluated.

[0401] In some embodiments, the disease burden is measured or detected before administering the first dose, after administering the first dose but before administering the subsequent dose, and / or after administering the subsequent dose. For multiple subsequent doses, the disease burden in some embodiments may be measured before or after administering any subsequent dose, or between administering the subsequent doses.

[0402] In some embodiments, after administration of the initial amount, the burden is reduced by at least or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 100%. In some aspects, the administration of a subsequent dose may further reduce the disease burden or tumor burden, for example, compared to the time before administration of the subsequent dose or compared to the time before administration of the first dose, the burden is reduced to 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% or approximately this amount.

[0403] In some embodiments, the event-free survival rate or overall survival rate of a subject is improved through the method compared to other methods. For example, in some embodiments, the progression-free survival rate or probability of a subject treated with the method within one month of administration of the initial dose is higher than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. In some embodiments, the overall survival rate is higher than about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%. In some embodiments, progression-free survival, recurrence-free survival, or survival of at least about 1 month, or survival of at least about 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or survival of 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years was achieved in subjects treated by the above method. In some embodiments, the time to progression is improved, for example, to be longer than or equal to about 1 month, or at least about 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or 10 years.

[0404] In some embodiments, treatment performed through the above method has a reduced probability of recurrence compared to other methods. For example, in some embodiments, the probability of recurrence or progression within one month after the first use is lower than about 80%, about 70%, about 60%, about 50%, about 40%, about 30%, about 20%, about 10%, about 9%, about 8%, about 7%, and about 6%.

[0405] Cell exposure and persistence

[0406] In some embodiments, the amount and / or duration thereof are designed to facilitate the subject's exposure to the cell, for example, to promote its proliferation and / or persistence with increasing time.

[0407] In some embodiments, the provided method increases the subject's exposure to the cells used (e.g., the number of cells or duration increased over time) and / or the efficacy and treatment outcomes in immunotherapy. For example, an advantage of the method is that, compared to other methods, the degree of exposure to cells expressing a MERA antigen receptor (e.g., CAR-expressing cells) is greater and / or treatment outcomes are improved to a greater extent. These results may include patient survival and remission, and are even observed in individuals with a severe tumor burden.

[0408] In some embodiments, the presence and / or amount of cells expressing a chimeric antigen receptor (e.g., CAR-expressing cells) in a subject is detected after administration of an initial dose and / or subsequent doses. In some embodiments, quantitative PCR (qPCR) is used to evaluate the amount of cells expressing a chimeric antigen receptor (e.g., CAR-T) in the subject's blood or serum or in an organ or tissue (e.g., a disease site). In some embodiments, persistence is quantified as the number of copies of DNA or plasmid encoding a CAR-like receptor in DNA per μg, or the number of receptor-expressing cells, such as CAR-expressing cells, in a sample such as blood or serum per μL, or the total number of peripheral blood mononuclear cells (PBMCs) or leukocytes or T cells in the sample per μL.

[0409] In some embodiments, cells are detected in a subject after administering the first dose or at least on day 1, day 2, day 3, day 4, day 5, day 6, day 7, day 8, day 9, day 10, day 11, day 12, day 13, day 14, day 15, day 16, day 17, day 18, day 19, day 20, day 21, day 22, day 23, day 24, day 25, day 26, day 27, day 28, day 29 or day 30. In some embodiments, or at least after administration of the first or subsequent dose, cells are detected at 2 weeks, 4 weeks, or 6 weeks, or 3 months, 6 months, or 12 months, 18 months, or 24 months, or 30 months, or 36 months, or 1 year, 2 years, 3 years, 4 years, 5 years, or 5 years or more.

[0410] In some embodiments, the total amount of cells is used as a single dose through an alternative method, such as a method of administering a single dose, for example, a method containing more cells than the first dose, and subsequent doses of cells are administered to subjects who were not administered the first dose, and / or subsequent doses are administered at a time other than a specified time, for example, at a prescribed time later than or after the time when the subject’s immune response to the receptor (e.g., CAR) appears.

[0411] Contact indicating cell number, proliferation, and / or persistence, for example, can be indicated based on the maximum number of cells exposed to the subject, the duration of detectable cells above a specific number or percentage, the area under the cell number curve over time, and / or combinations thereof and their exponents. These results are evaluated using known methods, such as qPCR, relative to the total amount of nucleic acid or DNA in a specific sample, such as blood or serum, to detect the number of copies of nucleic acid encoding a chimeric antigen receptor, and / or flow cytometry measurements are used to detect cells expressing the receptor with antibodies for receptor specificity. Based on cell measurements, the number or percentage of functional cells may also be detected, for example, to induce disease or pathology, or to detect reactions (e.g., cytotoxic reactions) that bind to and / or neutralize cells expressing antigens identified by the receptor.

[0412] In some embodiments, increased cell exposure of the subject includes increased cell proliferation. In some embodiments, receptor-(e.g., CAR-) expressing cells proliferate in the subject after administration of an initial dose and / or subsequent doses. In some embodiments, compared to other methods, for example, administering cells in a single dose, administering a relatively large initial dose, administering subsequent doses but not the initial dose, and / or administering subsequent doses at a designated time or before or after a time to induce an immune response, for example, before administration of subsequent doses, the method increases cell proliferation more.

[0413] In some embodiments, the method significantly increases the proliferation of administered cells in vivo, for example, measured by flow cytometry. In some embodiments, a high peak rate of cells is detected. For example, in some embodiments, after administration of the first or subsequent dose, when the subject's blood or disease site or its leukocyte portion (e.g., PBMC portion or T cell portion) reaches a peak level or maximum level, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% of the cells express a chimeric antigen receptor such as a CAR.

[0414] In some embodiments, the method results in at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000, 1500, 2000, 5000, 10000, or 15000 replicated or maximum concentrations of receptor-encoding nucleic acids in the subject's blood or serum or other body fluids or organs or tissues, e.g., CAR of DNA per μg, or at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 CAR of receptors, total number of cells / peripheral blood mononuclear cells (PBMCs), total number of mononuclear cells, total number of T cells, or total number of μL of cells / peripheral blood mononuclear cells (PBMCs) expressing receptors such as CAR, e.g., CAR of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9 μL. In some embodiments, cells expressing the receptor were detected to be at least 10%, 20%, 30%, 40%, 50%, or 60% of the total PBMCs in the subject's blood, and / or persist for at least 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 24 weeks, 36 weeks, 48 ​​weeks, or 52 weeks after administration at the initial or subsequent dose at the said level, or persist for 1 year, 2 years, 3 years, 4 years, or 5 years or more after administration in this manner.

[0415] In some embodiments, the replication of the nucleic acid encoding a chimeric antigen receptor, such as a CAR, in the above method causes the DNA per μg to increase by at least 2 times, at least 4 times, at least 10 times, or at least 20 times.

[0416] In some embodiments, cells expressing the receptor are detected in the subject's blood or serum, for example, by a specified method such as qPCR or a flow cytometry-based detection method, for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 days after administration of the first dose or after administration of a subsequent dose, It lasts for 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 days or 60 days or more, or for at least or about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 weeks or 24 or more weeks after administration of the initial dose or subsequent dose.

[0417] In some modes, at least about 1 χ 10 2 , at least about 1x10 3 , at least about 1x10 4 , at least about 1x10 5 , or at least about 1X10 6 or at least about 5x10 6 or at least about 1 x 10 7 or at least about 5x10 7or at least about 1 x 10 8 or at least about 2.5 x 10 8 Candidates of chimeric antigen receptor-expressing cells, e.g., CAR-expressing cells, and / or at least 10, 25, 50, 100, 200, 300, 400, or 500 or 1000 receptor-expressing cells per μL, e.g., at least 10 per μL, may be detected or present in a subject or their fluid, tissue, or compartment, e.g., blood, e.g., peripheral blood, or the disease site thereof. In some embodiments, after administration of the first dose or after administration of a subsequent dose, the number or concentration of cells may be detected to persist in the subject for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, at least about 20 days, at least about 40 days, or at least about 60 days, or at least about 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months, or at least 2 or 3 years. These cell numbers can be detected through flow cytometry-based or quantitative PCR-based methods, and the total number of cells can be estimated using known methods.

[0418] In some embodiments, the number of copies of nucleic acid encoding a chimeric antigen receptor in cells per 100, e.g., the number of carrier copies, is measured by immunohistochemistry, PCR and / or flow cytometry, e.g., in peripheral blood or bone marrow or other compartments, at least 0.01, at least 0.1, at least 1, or at least 10, e.g., about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, or at least about 6 weeks after administering the first dose or subsequent dose of cells, or at least about 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 or 12 months, or at least 2 or 3 years. In some embodiments, the number of copies of a carrier expressing a CAR-like receptor in genomic DNA per μg is at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 90, 100, at least 1000, at least 5000, or at least 10000, or at least 15000, or at least 20000 when measured at about 1 week, about 2 weeks, about 3 weeks, or at least about 4 weeks after administering the first or subsequent dose of cells expressing a CAR-like receptor, or at least 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 9 months, or at least 2 years after administering in this manner.

[0419] In some embodiments, receptors such as CARs expressed by cells are detected in the subject's blood and / or the disease site thereof by quantitative PCR (qPCR) or flow cytometry, and are detected after administration of the cells, for example, after administration of the first dose or subsequent dose, or after administration of the subsequent dose, 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 3 years.

[0420] Chimeric antigen receptors expressed by cells

[0421] Cells generally express chimeric antigen receptors, and the chimeric antigen receptors include functional non-TCR antigen receptors such as receptors to which other antigens are bound, such as genetically modified T cell receptors (TCR), T cell fusion protein (TFP), and T cell antigen coupler (TAC).

[0422] An exemplary antigen receptor comprises a CAR, and a method for manipulating and introducing the receptor into a cell comprises, for example, a Chinese patent application with publication numbers CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN106397593A, CN106467573A, and an international patent application with publication numbers WO2018006882A1 and WO2015172339A8.

[0423] The above chimeric receptor includes a chimeric antigen receptor (CAR). Chimeric receptors such as CARs generally include an extracellular antigen binding domain, for example, a portion of the antibody molecule is typically the heavy chain variable (VH) region and / or light chain variable (VL) region of the antibody, such as the scFv antibody fragment. In some embodiments, the chimeric receptor may include an amino acid sequence represented by, for example, SEQ ID NO: 34, of a splice variant 1 (CLD18A1, CLD18.1) of Claudin 18 (CLD18) (Registration No. NP_057453, NM016369) 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 99% or more of sequence homology with SEQ ID NO: 34, and splice variant 2 (CLD18A2, CLD18.2) It may be possible to identify an amino acid sequence comprising (registration number NM_001002026, NP_001002026) or a variant thereof, for example, SEQ ID NO: 33, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% or more of sequence homology with SEQ ID NO: 33. In some embodiments, the chimeric receptor specifically identifies CLD18A2 but does not identify CLD18A1.

[0424] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) comprises, for example, an amino acid sequence comprising an HCDR or LCDR represented by SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or a variant thereof, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% or more sequence homology with SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7, etc., scFv antibody Includes short stories.

[0425] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) comprises an amino acid sequence or variant thereof comprising an HCDR or LCDR represented, for example, by SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13, or an scFv antibody fragment such as an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99% or more of sequence homology with SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13.

[0426] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) comprises an amino acid sequence or variant thereof comprising an HCDR or LCDR represented, for example, by SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13, or an scFv antibody fragment such as an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99% or more of sequence homology with SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13.

[0427] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) comprises an amino acid sequence or variant thereof comprising a heavy or light chain variable region represented, for example, SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18 or SEQ ID NO: 19, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99% or more of sequence homology with SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18 or SEQ ID NO: 19.

[0428] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) comprises an amino acid sequence or a variant thereof comprising a heavy or light chain variable region represented, for example, SEQ ID NO: 20 or SEQ ID NO: 21 or SEQ ID NO: 22 or SEQ ID NO: 23, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99% or more of sequence homology with SEQ ID NO: 20 or SEQ ID NO: 21 or SEQ ID NO: 22 or SEQ ID NO: 23, etc. scFv antibody fragment.

[0429] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) is an amino acid sequence comprising a heavy or light chain variable region represented, for example, by SEQ ID NO: 20 or SEQ ID NO: 21 or SEQ ID NO: 22 or SEQ ID NO: 23, or a variant thereof, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% or more of sequence homology with SEQ ID NO: 20 or SEQ ID NO: 21 or SEQ ID NO: 22 or SEQ ID NO: 23.

[0430] In some embodiments, the chimeric antigen receptor (e.g., CAR) is an amino acid sequence represented, for example, by SEQ ID NO: 24 or SEQ ID NO: 25 or SEQ ID NO: 26 or SEQ ID NO: 27 or SEQ ID NO: 28 or SEQ ID NO: 29 or SEQ ID NO: 30 or SEQ ID NO: 31 or SEQ ID NO: 32 or a variant thereof, or SEQ ID NO: 24 or SEQ ID NO: 25 or SEQ ID NO: 26 or SEQ ID NO: 27 or SEQ ID NO: 28 or SEQ ID NO: 29 or SEQ ID NO: 30 or SEQ ID NO: 31 or SEQ ID NO: 32 and at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 It includes amino acid sequences exhibiting %, 96%, 97%, 98%, 99%, or 99% or more of sequence homology.

[0431] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) may be at least a portion of an immunoglobulin constant region, such as a hinge region, such as an IgG4 hinge region and / or a CH1 / CL and / or Fc region, or a variant or modified form thereof, or further comprises a linking sequence containing these. In some embodiments, the constant region or portion is of human IgG, such as IgG4 or IgG1. In some aspects, the portion of the constant region is an antigen-identification portion, such as a linking sequence between a scFv and a transmembrane domain. The linking sequence may have such a length, and compared to the case where the linking sequence is absent, said length increases cellular reactivity after binding to the antigen. In some embodiments, the length of the linking sequence is about 12 amino acids or a length not exceeding 12 amino acids. An exemplary linking sequence comprises a length of at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 amino acids, and comprises an integer between any endpoint of the range. In some embodiments, the linking sequence region has a length of about 12 amino acids or shorter, about 119 amino acids or shorter, or about 229 amino acids or shorter. An exemplary linking sequence comprises a single IgG4 hinge, an IgG4 hinge connected to a CH2 and CH3 domain, or an IgG4 hinge connected to a CH3 domain.

[0432] The antigen generally identifies a domain that connects to one or more intracellular signaling regions; for example, in the case of a CAR, it identifies a simulated activated signaling region through an antigen-receptor complex (e.g., a TCR complex) and / or a signal through other cell surface receptors. Thus, the antigen-binding component (e.g., an antibody) is connected to one or more transmembrane and intracellular signaling domains. In some embodiments, the transmembrane domain is fused to the extracellular domain. In one embodiment, a transmembrane domain that is one of the domains in a natural related receptor (e.g., a CAR) is used. In some situations, the transmembrane domain is selected or modified through amino acid substitution to prevent the domain from binding to the transmembrane domain of the same or different surface membrane protein, thereby minimizing interaction with other members of the receptor complex.

[0433] In some embodiments, the transmembrane domain is natural or synthetic. In the case of natural, in some embodiments, the domain is derived from any membrane-bound or transmembrane protein. The transmembrane domain comprises α-chains, β-chains, or ζ-chains derived from T-cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, and / or the transmembrane domain comprises functional variants thereof (e.g., structural parts thereof (e.g., transmembrane structural parts), those that essentially retain the properties), etc. (i.e., a transmembrane domain comprising at least these). In some embodiments, the synthetic transmembrane domain comprises hydrophobic residues such as leucine and valine. In some embodiments, a trimer of phenylalanine, tryptophan, and valine appears at each end of the synthesized transmembrane domain. In some embodiments, the connection is implemented through a linker, a spacer, and / or a transmembrane domain.

[0434] The intracellular signaling domain described above includes a simulated or approximate signal through a natural antigen receptor, a signal that binds to a co-stimulatory receptor through such receptor, and / or a signal through a single co-stimulatory receptor, etc. In some embodiments, a short oligopeptide or polypeptide linker, for example, a linker having a length of 2 to 10 amino acids including glycine and serine, for example, a linker of a glycine-serine doublet, is present and forms a link between the cytoplasmic signaling domain and the transmembrane domain of the CAR.

[0435] The receptor, such as a CAR, generally comprises at least one or more intracellular signaling portions. In some embodiments, the receptor comprises an intracellular component of a TCR complex, for example, a TCRCD3+ chain such as a CD3ζ chain that mediates T-cell activation and cytotoxicity. Thus, in some embodiments, the antigen-binding portion is connected to one or more cell signaling modules. In some embodiments, the cell signaling module comprises a CD3 transmembrane domain, a CD3 intracellular signaling domain, and / or other CD transmembrane domains. In some embodiments, the receptor, such as a CAR, further comprises a portion of one or more other molecules such as Fc receptor γ, CD8, CD4, CD25, or CD16. For example, in some embodiments, the CAR or other chimeric receptor comprises a chimeric molecule between CD3ζ (CD3-ζ) or Fc receptor γ and CD8, CD4, CD25, or CD16.

[0436] In some embodiments, when bound to a CAR or other chimeric receptor, the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of immune cells, for example, the manipulation of T cells to express the CAR. For example, in some situations, the CAR induces T cell functions, for example, cytolytic activity or helper T cell activity, for example, the secretion of cytokines or other factors. In some embodiments, for example, when transducing an effector function signal, the complete immunostimulatory chain is replaced by the antigen receptor portion or the cleavage portion of the intracellular signaling domain of the co-stimulator. In some embodiments, the intracellular signaling domain comprises the cytoplasmic sequence of a T cell receptor (TCR), and in some aspects, further comprises a receptor that acts in conjunction with a naturally occurring co-receptor to initiate signaling after being conjugated to the antigen receptor.

[0437] In the case of natural TCRs, full activation generally requires not only signaling through the TCR but also a co-stimulatory signal. In some embodiments, to promote full activation, a component for generating a second or co-stimulatory signal is also included in the CAR. In other embodiments, the CAR does not include a component for generating a co-stimulatory signal. In some aspects, another CAR is expressed in the same cell and provides a component for generating a second or co-stimulatory signal.

[0438] In some embodiments, T cell activation is described as being mediated through two cytoplasmic signaling sequences: those that are initially activated dependently by the TCR initiator antigen (first cytoplasmic signaling sequence), and those that provide a second or co-stimulatory signal through the antigen acting in a non-dependent manner (second cytoplasmic signaling sequence). In some embodiments, the CAR comprises one or both of these signaling components.

[0439] In some embodiments, the antibody portion of the chimeric antigen receptor (e.g., CAR) further comprises a signal peptide, such as a signal peptide comprising CD8 or a variant thereof, for example, an amino acid sequence represented by SEQ ID NO: 35, or a signal peptide comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% or more of sequence homology with SEQ ID NO: 35.

[0440] In some embodiments, the CAR comprises a major cytoplasmic signaling sequence that regulates the initial activation of the TCR complex. The initial cytoplasmic signaling sequence acting in a stimulating manner may comprise a tyrosine-based activated motif of the immune receptor or a signaling motif known as ITAM. Embodiments of ITAM include TCRζ, FcRγ, FcR It includes an initial cytoplasmic signaling sequence derived from CD3γ, CD3δ, CD3ε, CDS, CD22, CD79a, CD79b, and CD66d. In some embodiments, the cytoplasmic signaling molecule in the CAR includes a cytoplasmic signaling domain, a portion thereof, or a sequence derived from CD3ζ.

[0441] In some embodiments, the CAR comprises a transmembrane portion and / or signaling domain of a co-stimulatory receptor such as CD28, CD137, OX40, DAP10, and ICOS. S. In some embodiments, the same CAR comprises an activating portion and a co-stimulatory portion simultaneously.

[0442] In some embodiments, the activating domain is contained within a single CAR, but the co-stimulating component is provided by a different CAR that identifies a different antigen. In some embodiments, the CARs include an activating or stimulating CAR and a co-stimulating CAR, all of which are expressed in the same cell (see WO2014 / 055668). In some embodiments, the cell includes one or more stimulating or activating CARs and / or co-stimulating CARs. In some embodiments, the cell further includes an inhibitory CAR (see Fedorov et al., Sci. Transl. Medicine, 5 (215) (December 2013)), and by identifying, for example, a disease or pathology-related and / or specific CAR, the stimulating signal delivered through the disease-targeting CAR is reduced or inhibited through the binding of the inhibitory CAR and its ligand, for example, by reducing off-target effects.

[0443] In some embodiments, the intracellular signaling portion of the chimeric antigen receptor, such as a CAR, comprises a CD3ζ intracellular domain and a co-stimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises a CD28 transmembrane and signaling domain connected to a CD3 (e.g., CD3-ζ) intracellular domain. In some embodiments, the intracellular signaling domain comprises a chimeric CD28 and / or CD137 (4-1BB, TNFRSF9) co-stimulatory domain connected to a CD3ζ intracellular domain.

[0444] In some embodiments, the CAR comprises one or more activation domains, such as two or more co-stimulatory domains and an initial activation domain in the cytoplasmic portion. An exemplary CAR comprises the intracellular portions of CD3-ζ, CD28, and CD137.

[0445] In some cases, the CAR is referred to as a first-generation, second-generation, and / or third-generation CAR. In some embodiments, the first-generation CAR provides only a CAR of CD3 chain-induced signaling upon antigen binding; in some embodiments, the second-generation CAR provides a CAR such as a CAR containing an intracellular signaling domain derived from a co-stimulatory receptor (e.g., CD28 or CD137) of such signaling and co-stimulatory signaling; and in some embodiments, the third-generation CAR is a CAR containing multiple co-stimulatory domains of different co-stimulatory receptors.

[0446] In some embodiments, the chimeric antigen receptor comprises an extracellular portion containing an antibody or antibody fragment. In some embodiments, the chimeric antigen receptor comprises an extracellular portion containing an antibody or fragment and an intracellular signaling domain. In some embodiments, the antibody or fragment comprises scFv, and the intracellular domain comprises ITAM. In some embodiments, the intracellular signaling domain comprises the signaling domain of the ζ chain of the CD3-ζ chain. In some embodiments, the chimeric antigen receptor comprises an extracellular domain and a transmembrane domain connected to the intracellular signaling domain. In some embodiments, the transmembrane domain comprises the transmembrane portion of CD28. In some embodiments, the chimeric antigen receptor comprises the intracellular domain of a T cell co-stimulatory molecule. The extracellular domain and the transmembrane domain may be connected directly or indirectly. In some embodiments, the extracellular domain and the transmembrane domain are connected via a linking sequence. In some embodiments, the receptor comprises an extracellular portion such as the CD28 extracellular portion, which is a molecule derived from the transmembrane domain. In some embodiments, the chimeric antigen receptor comprises an intracellular domain, such as between a transmembrane domain and an intracellular signaling domain, derived from a T cell co-stimulatory molecule or a functional variant thereof. In some embodiments, the T cell co-stimulatory molecule is CD28 or 41BB. For example, in some embodiments, the CAR comprises an antibody such as an antibody fragment, or comprises a transmembrane portion of CD28 or a transmembrane domain of a functional variant thereof, a signaling portion of CD28 or an intracellular signaling domain of a functional variant thereof, and a signaling portion of CD3ζ or a functional variant thereof.In some embodiments, the CAR comprises an antibody such as an antibody fragment, comprises a transmembrane portion of CD28 or a transmembrane domain of a functional variant thereof, comprises a signaling portion of CD137 or an intracellular signaling domain of a functional variant thereof, and comprises a signaling portion of CD3ζ or a functional variant thereof. In some of the above embodiments, the receptor further comprises a linking sequence comprising a portion of an Ig molecule (e.g., human Ig molecule), e.g., an Ig hinge, e.g., an IgG4 hinge, e.g., merely a hinge linking sequence. In some embodiments, the chimeric antigen receptor comprises: (i) an antibody that specifically identifies a tumor antigen, a transmembrane domain of CD28 or CD8, a co-stimulatory signaling domain of CD28, and CD3ζ; or (ii) an antibody that specifically identifies a tumor antigen, a transmembrane domain of CD28 or CD8, a co-stimulatory signaling domain of CD137, and CD3ζ; or (iii) an antibody that specifically identifies a tumor antigen, CD28 or a transmembrane domain of CD8, a co-stimulation signal domain of CD28, a co-stimulation signal domain of CD137 and CD3ζ.

[0447] In some embodiments, the transmembrane domain of the chimeric antigen receptor (e.g., CAR) is or comprises a transmembrane domain of human CD28 (e.g., registration number: P01747.1) or a variant thereof, for example, a transmembrane domain comprising an amino acid sequence represented by SEQ ID NO: 39, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% or more of sequence homology with SEQ ID NO: 39.

[0448] In some embodiments, the transmembrane domain of the chimeric antigen receptor (e.g., CAR) is or comprises a transmembrane domain of human CD8 or a variant thereof, for example, a transmembrane domain comprising an amino acid sequence represented by SEQ ID NO: 45, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% or more of sequence homology with SEQ ID NO: 45.

[0449] In some embodiments, the intracellular signaling portion of the chimeric antigen receptor (e.g., CAR) comprises an intracellular co-stimulatory signaling domain of human CD28 or a functional variant thereof or a portion thereof. For example, the intracellular signaling domain may comprise an amino acid sequence represented by SEQ ID NO: 41, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% or more of sequence homology with SEQ ID NO: 41.

[0450] In some embodiments, the intracellular domain comprises an intracellular co-stimulatory signaling domain of CD137 (e.g., registration number: Q07011.1) or a functional variant or a portion thereof, for example, an amino acid sequence represented by SEQ ID NO: 47, or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% or more sequence homology with SEQ ID NO: 47.

[0451] In some embodiments, the intracellular signaling domain of the chimeric antigen receptor (e.g., CAR) comprises a human CD3ζ-stimulated signaling domain or a functional variant thereof. For example, in some embodiments, the intracellular signaling domain comprises an amino acid sequence represented by SEQ ID NO: 43 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99% or more of sequence homology with SEQ ID NO: 43.

[0452] In some embodiments, 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 represented by SEQ ID NO: 37 or an amino acid sequence exhibiting at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence homology with SEQ ID NO: 37.

[0453] For example, in some embodiments, the CAR comprises an antibody such as an antibody fragment, a scFv, a linkage sequence, for example, a linkage sequence comprising a portion of an immunoglobulin molecule, for example, a hinge region and / or one or more heavy chain molecular constant regions, for example, an Ig-hinge, a linkage sequence comprising a linkage sequence comprising a hinge region and / or one or more heavy chain molecular constant regions, for example, an Ig-hinge, a transmembrane domain comprising a transmembrane domain induced wholly or partially by CD28, an intracellular signaling domain induced by CD28, and a CD3ζ signaling domain. In some embodiments, the CAR comprises an antibody or fragment, for example, a scFv, a linkage sequence, for example, any linkage sequence comprising an Ig-hinge, a transmembrane domain induced by CD28, an intracellular signaling domain induced by CD137, and a signaling domain induced by CD3ζ.

[0454] The terms “polypeptide” and “protein” may be used interchangeably and refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides comprising the provided receptor and other polypeptides (e.g., linkers or peptides) may comprise amino acid residues including natural and / or non-natural amino acid residues. The terms further include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, and phosphorylation. In some embodiments, the polypeptide may further comprise modifications to the raw or natural sequences, provided that the protein retains the required activity. Such modifications may be significant through site-specific mutagenesis or unexpected through the occurrence of host mutations of the protein or errors resulting from PCR amplification.

[0455] In some embodiments, the method according to the text comprises the step of administering one adoptive cell or immune effector cell to the subject. In some embodiments, the method according to the text comprises the step of administering two or more adoptive cells or immune effector cells for the same tumor antigen to the subject during different treatment courses. In some embodiments, the method according to the text comprises the step of administering two or more adoptive cells or immune effector cells for different tumor antigens to the subject during different treatment courses. In some embodiments, the method according to the text comprises the step of administering two or more adoptive cells or immune effector cells for the same epitope of the same tumor antigen to the subject during different treatment courses. In some embodiments, the method according to the text comprises the step of administering two or more adoptive cells or immune effector cells for different epitopes of the same tumor antigen to the subject during different treatment courses. In some embodiments, the method according to the text comprises the step of treating a tumor at the same site by administering two or more adoptive cells or immune effector cells to the subject during different treatment courses. In some embodiments, the method according to the text comprises the step of treating tumors in different sites by administering two or more adoptive cells or immune effector cells to the subject during different treatment courses. In some embodiments, at least one of the adoptive cells or immune effector cells used in the method according to the text is a CAR-T cell that targets CLD18A2. In some embodiments, at least one of the adoptive cells or immune effector cells used in the method according to the text is a CAR-T cell that targets CLD18A2 according to the text.

[0456] A chimeric antigen receptor (e.g., CAR) expressed by cells administered to a subject in varying doses generally identifies or specifically binds to a related and / or specific molecule expressed by the disease or pathology being treated or by the cells. When specifically binding to an antigen-like molecule, the receptor generally promotes an immune response targeting the disease or pathology by delivering an immune-stimulating signal (e.g., a signal delivered by ITAM) to the cells. For example, in some embodiments, the cells in the first dose express a CAR that specifically binds to an antigen expressed by the cells or tissues of the disease or pathology or to an antigen related to the disease or pathology.

[0457] The receptor (e.g., CAR) expressed by the cells of the subsequent use is generally specifically bound to the same antigen as the CAR of the first use and is generally the same receptor or extremely similar to the receptor of the cells of the first use. In some embodiments, the receptor in the cells of the subsequent use is the same as or very similar to the receptor in the cells of the first use.

[0458] In some embodiments, the CAR expressed by the cells of the subsequent use comprises the same scFv, the same signaling domain, and / or the same linkage as the CAR expressed by the cells of the first use. In some embodiments, it further comprises the same co-stimulation, stimulation, transmembrane, and / or other domains as the first use. In some embodiments, one or more components of the CAR of the subsequent use differ from the CAR of the first use.

[0459] immune effector cells

[0460] In the present invention, the cell expressing the chimeric receptor and provided through the method according to the text is an immune effector cell. The cell is generally a eukaryotic cell, such as a mammalian cell, which is generally a human cell. In some embodiments, the cell is a cell of the immune system, such as a bone marrow or lymphoid cell, which is derived from blood, bone marrow, lymph, or lymphoid organs, and includes lymphocytes, and is generally a T cell and / or NK cell, and is a cell of innate or adaptive immunity. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, but include induced pluripotent stem cells (iPSCs). The cell is generally a primary cell, such as a cell isolated directly from a subject and / or a cell isolated from a subject and frozen. In some embodiments, the cells comprise one or more subgroups of T cells or other cell types, for example, pre-T cell populations, CD4+ cells, CD8+ cells and subgroups thereof, for example, those defined by function, activation status, maturation, differentiation potential, amplification, recycling, localization, and / or persistence ability, antigen specificity, antigen receptor type, presence in a specific organ or compartment, status of marker or cytokine secretion, and / or degree of differentiation. With respect to a subject awaiting treatment, the cells may be allogeneic and / or self. The method comprises conventional methods. In some embodiments, in the prior art, the cells are pluripotent and / or multipotent stem cells, such as induced pluripotent stem cells (iPSCs). In some embodiments, the method comprises the step of preparing, processing, culturing, and / or manipulating cells isolated from a subject and introducing them into the same patient before or after cryopreservation.

[0461] T cells and / or CD4+ and / or CD8+ T cell subtypes and subgroups include primitive T (TN) cells, effector T cells (TEFF), memory T cells and their subtypes, for example, stem cell memory T (TSCM), centrome memory T (TCM), effector memory T (TEM), or finally 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, for example, TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells, and δ / γ T cells.

[0462] In some embodiments, the cell is a natural killer (NK) cell. In some embodiments, the cell is a monocyte or granulocyte, for example, a bone marrow cell, macrophage, neutrophil, dendritic cell, mast cell, eosinophil, and / or basophil.

[0463] In some embodiments, the cell comprises one or more nucleic acids introduced by genetic engineering, thereby expressing a recombinant of said nucleic acid or a genetically engineered product. In some embodiments, the nucleic acid is heterologous, that is, generally not present in the cell or a sample obtained from said cell, for example, in another organism or a sample obtained from said cell, for example, generally not found in the engineered cell and / or an organism derived from said cell. In some embodiments, the nucleic acid is not naturally occurring, for example, the nucleic acid is found in nature and comprises a chimeric combination including nucleic acids encoding domains derived from various different cell types.

[0464] Method and carrier for genetic engineering manipulation

[0465] The present invention further provides a method, composition, and kit for generating genetically engineered cells expressing a chimeric antigen receptor. The genetic engineering operation generally relates to an operation that introduces a nucleic acid encoding the recombinant or engineered portion into a cell, e.g., through retroviral transduction, transfusion, or transformation.

[0466] In some embodiments, first, cells are stimulated, for example, by combining them with a stimulant to induce a response such as proliferation, survival, and / or activation with said stimulant, for example, by detecting through the expression of a cytokine or an activation marker, and then gene transfer is carried out by transducing said activated cells and amplifying them in a culture to a number sufficient for clinical application.

[0467] In some situations, the overexpression of stimulating factors (e.g., lymphokines or cytokines) may be toxic to the subject. Therefore, in some situations, engineered cells are designed to negatively select gene segments that are sensitive to the body, for example, after administration in an immunotherapy. For example, in some embodiments, the cells are engineered so that they may be eliminated due to changes in the body conditions of the subject to whom they are administered. A negatively selectable phenotype can be generated through a gene that provides sensitivity to an administered reagent (e.g., a compound). A negatively selectable gene is the herpes simplex virus type I thymidine kinase (HSV-I TK) gene that provides sensitivity to ganciclovir (Wigler et al., Cell II:223, I977); It includes the cellular hypoxanthine phosphoribosyl transferase (HPRT) gene, the cellular adenine phosphoribosyl transferase (APRT) gene, and bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89: 33 (1992)).

[0468] In some embodiments, cells are also engineered to promote the expression of cytokines or other factors. Various methods for introducing genetically engineered components, such as antigen receptors (e.g., CARs), are known, and the methods and compositions provided herein may be used. Exemplary methods include a method for transferring nucleic acids encoding a receptor, including a virus such as a retrovirus or lentivirus, a transduction factor, and a method via electroporation.

[0469] In some embodiments, recombinant nucleic acid is transferred to a cell using recombinant infectious virus granules, for example, using a carrier derived from simian virus 40 (SV40), adenovirus, or adeno-associated virus (AAV). In some embodiments, recombinant nucleic acids are transferred to T cells using a retroviral carrier, such as a recombinant lentivirus carrier or a γ-retrovirus carrier (see, e.g., Koste et al., (2014) Gene Therapy Apr 3, 2014. doi:10.1038 / gt.2014.25; Carlens et al., (2000) Exp Hematol 28(10): 1137-46; Alonso-Camino et al., (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. Nov 2011; 29 (11): 550-557).

[0470] In some embodiments, the retrovirus carrier comprises a long terminal duplicate sequence (LTR) and is, for example, a retrovirus carrier derived from Molony murine leukemia virus (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), spleen fociforming virus (SFFV), or adeno-associated virus (AAV). Most retrovirus carriers are derived from murine retroviruses. In some embodiments, the retrovirus includes those derived from any avian or mammalian cell. Retroviruses are generally amphiphilic, which means they can infect numerous types of host cells (including humans). Lentiviral transduction methods are known. Exemplary methods are described, for example, in Wang et al., (2012) J. Immunother. 35 (9): 689-701; Cooper et al., (2003) Blood. 101: 1637-1644; Verhoeyen et al., (2009) Methods Mol Biol. 506:97-114; and Cavalieri et al., (2003) Blood. 102 (2): 497-505.

[0471] In some embodiments, recombinant nucleic acid is transferred to T cells via electroporation (see, e.g., Chicaybam et al., (2013) PLoS ONE 8(3): e60298 and Van Tedeloo et al., (2000) Gene Therapy 7(16): 1431-1437). In some embodiments, recombinant nucleic acid is transferred to T cells via translocation (see, e.g., Manuri et al., (2010) Hum Gene Ther 21(4): 427-437; Sharma et al., (2013) Molec Ther Nucl Acids 2, e74; and Huang et al., (2009) Methods Mol Biol 506: 115-126). Other methods for introducing and expressing genetic material in immune cells include calcium phosphate transduction (e.g., described in “Current Protocols in Molecular Biology”, John Wiley & Sons, New York), protoplast fusion, cationic liposome-mediated transduction; particle impaction facilitated by tungsten particles (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

[0472] Other methods and carriers for transferring nucleic acids encoding recombinant products are described, for example, in the international patent application with publication number WO2014055668 and the U.S. patent with number 7,446,190.

[0473] Other nucleic acids for introduction (e.g., genes) include those intended to improve therapeutic effects by promoting the activity and / or function of the translocated cells, for example; genes for selecting and / or evaluating cells by providing genetic markers, for example, genes for evaluating in vivo survival or localization; safety genes, for example, genes that cause cells to negatively select gene segments sensitive to the body, as described, for example, in Lupton S.D. et al., Mol. and Cell Biol., 11: 6 (1991); and Riddell et al., Human Gene Therapy 3: 319-338 (1992); and also refer to Lupton’s publication numbers PCT / US91 / 08442 and PCT / US94 / 05601, etc., in which the application of dual-function selectable fusion genes by inducing the fusion of a dominant positive selectable marker with a negative selectable marker is described. For example, refer to columns 14 through 17 of U.S. Patent No. 6,040,177 by Riddell et al.

[0474] Manufacturing of immune effector cells

[0475] In some embodiments, the preparation of the engineered cells comprises one or more culture and / or one or more preparation steps. Cells for introducing nucleic acids encoding a genetically modified receptor (e.g., CAR) may be isolated from a sample (e.g., a biological sample, e.g., a sample obtained from or derived from a subject). In some embodiments, the subject from whom the cells are isolated is a subject who suffers from a certain disease or pathology, or who requires cell therapy, or who is to undergo cell therapy. In some embodiments, the subject is a specific therapeutically mediated human, e.g., a human requiring adoptive cell therapy or effector cell therapy, and the cells used in said therapy have been isolated, processed, and / or engineered. In some embodiments, the cells are primary cells, such as primary human cells. The sample comprises samples obtained directly from the subject's tissues, body fluids, and other samples, and samples obtained from one or more processing steps, such as isolation, centrifugation, genetic engineering (e.g., transduction using a viral carrier), washing, and / or culture. The above biological samples may be obtained directly from samples derived from or processed from living organisms. Biological samples include, but are not limited to, body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, as well as tissue and organ samples, and include processed samples derived from these.

[0476] In some embodiments, the sample from which cells are derived or isolated is blood or a blood-derived sample, or a product of apheresis or leukocyte removal. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsies, tumors, leukemia, lymphoma, lymph nodes, intestinal lymphoid tissue, mucosal lymphoid tissue, spleen, other lymphoid tissue, liver, lungs, stomach, intestines, colon, kidneys, pancreas, breasts, bones, prostates, cervix, testes, ovaries, tonsils, 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 of their own and allogeneic origin.

[0477] In some embodiments, the cells are derived from a cell line such as a T cell line. In some embodiments, the cells are derived from xenogeneic sources such as mice, rats, non-human primates, and pigs.

[0478] In some embodiments, cell separation comprises one or more cell separation steps not based on manufacturing and / or evolution. In some embodiments, cells undergo washing, centrifugation, and / or culture in the presence of one or more substances, for example, to remove unnecessary components, to enrich necessary components, and to degrade or remove cells sensitive to specific substances. In some embodiments, cells are separated based on one or more properties, such as density, adhesion properties, size, sensitivity to specific components, and / or resistance. In some embodiments, cells derived from a subject's circulating blood are obtained, for example, through component collection or leukocyte ablation. In some embodiments, the sample comprises lymphocytes, but includes T cells, monocytes, granulocytes, B cells, other nucleated blood leukocytes, erythrocytes, and / or platelets, and in some embodiments, cells different from erythrocytes and platelets.

[0479] In some embodiments, blood cells collected from the subject are washed, for example, by removing the plasma portion and placing the cells in a suitable buffer or medium for use in a subsequent processing step. In some embodiments, the cells are washed with phosphate-buffered saline (PBS). In some embodiments, the washing solution is deficient in calcium and / or magnesium and / or numerous or all divalent cations. In some embodiments, the washing step is completed via semi-automated "circulating" centrifugation (e.g., COBE 2991 cell processor, Baxter) as described by the manufacturer. In some embodiments, the washing step is completed via tangential flow filtration (TFF) as described by the manufacturer. In some embodiments, after washing, the cells are resuspended in various biocompatible buffers, such as Ca++ / Mg++-free PBS. In some embodiments, blood cell sample components are removed, and the cells are resuspended directly in a medium.

[0480] In some embodiments, the method comprises a density-based cell separation method, for example, centrifuging peripheral blood through a Percoll or Ficoll gradient with or without breaking down red blood cells, or obtaining peripheral blood mononuclear cells (PBMCs) by performing apheresis or leukocyte removal on the sample.

[0481] In some embodiments, the separation method comprises the step of separating different cell types based on the expression or presence of one or more specific molecules among the cells, e.g., surface markers, e.g., surface proteins, intracellular markers, or nucleic acids. In some embodiments, any known method of separation based on such markers may be used. In some embodiments, the separation is based on affinity or immuno-affinity. For example, in some embodiments, the separation comprises separating cells and cell populations based on the expression or expression level of one or more markers (typically cell surface markers) of the cells, e.g., incubating with an antibody or binding partner that specifically binds to such markers, then generally performing a washing step, and separating cells already bound to said antibody or binding partner from cells that are not yet bound to said antibody or binding partner.

[0482] This separation step may be based on positive selection and / or negative selection, whereby in positive selection, cells already bound to the reagent are retained and further applied, and in negative selection, cells not yet bound to the antibody or binding partner are retained. In some embodiments, both parts are retained and further applied. In some embodiments, where there is no antibody capable of specifically verifying the cell type in the heterogeneous group, negative selection can be very useful, so it is preferable to proceed with separation based on markers expressed through cells different from the required group.

[0483] The above separation does not require enriching or removing 100% of a specific cell population or cells expressing a specific marker. For example, positively selecting or enriching a specific type of cell, for example, expressing a marker, increases the number or percentage of said cells, but does not mean that there is no need to completely eliminate cells that do not express said marker. Likewise, negatively selecting, removing, or consuming a specific type of cell, for example, expressing a marker, decreases the number or percentage of said cells, but does not mean that there is no need to completely remove all such cells.

[0484] In some embodiments, multiple separation steps are performed, wherein a portion of the positive or negative selection from one step is performed in another separation step, for example, to perform subsequent positive or negative selection. In some embodiments, a single separation step consumes cells expressing multiple markers simultaneously, for example, by culturing the cells with various antibodies or binding partners specific to the markers that target each for negative selection. Similarly, cells can be cultured with various antibodies or binding partners expressed in various cell types to allow for simultaneous positive selection against multiple cell types.

[0485] For example, in some embodiments, specific subgroups of T cells, e.g., one or more surface marker-positive cells, or cells expressing high levels of one or more surface markers, e.g., CD3+, CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+ and / or CD45RO+ T cells are separated through positive selection or negative selection techniques.

[0486] For example, CD3+, CD28+ T cells can undergo positive selection using magnetic beads connected to CD3 / CD28 (e.g., DYNA Bead M-450 CD3 / CD28 T cell amplifier).

[0487] In some embodiments, separation is performed by enriching a specific cell population through positive selection or by depleting a specific cell population through negative selection. In some embodiments, positive selection or negative selection is completed by culturing cells with one or more antibodies or other binding reagents that specifically bind to one or more surface markers, and said one or more surface markers are expressed (marker+) or expressed at a relatively high level (marker high) in the positively selected or negatively selected cells, respectively.

[0488] In some embodiments, T cells are isolated from PBMC samples by performing negative selection on a marker (e.g., CD14) expressed on non-T cells (e.g., B cells, monocytes, or other blood leukocytes). In some embodiments, helper CD4+ and CD8+ cytotoxic T cells are isolated using a CD4+ or CD8+ selection step. These CD4+ and CD8+ groups may be further isolated into subgroups by performing positive or negative selection on a marker expressed or expressed to a relatively high degree on one or more primary, memory, and / or effector T cell subgroups.

[0489] In some embodiments, CD8+ cells are additionally enriched with or consumed primary, central memory, effector memory, and / or central memory stem cells, for example, by performing positive or negative selection based on surface antigens associated with the corresponding subgroups. In some embodiments, central memory T (TCM) cells are enriched to increase the effect, for example, to improve long-term survival, amplification, and / or transplantation after administration, and in some embodiments, this is particularly robust in these subgroups. See Terakura et al., (2012) Blood. 1: 72-82; Wang et al., (2012) J Immunother. 35(9): 689-701. In some embodiments, CD8+ T cells enriched with TCM are combined with CD4+ T cells to further enhance the effect.

[0490] In an embodiment, memory T cells are present in the CD62L+ and CD62L- subgroups of CD8+ peripheral blood lymphocytes. PBMCs enrich or consume the CD62L-CD8+ and / or CD62L+CD8+ portions, for example, using anti-CD8 antibodies and anti-CD62L antibodies.

[0491] In some embodiments, enrichment of central memory T (TCM) cells is performed by positive or surface overexpression of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD127; in some embodiments, this is negatively selected based on cells expressing or overexpressing CD45RA and / or granzyme B. In some embodiments, separation of the CD8+ group enriching TCM cells is performed by consuming cells expressing CD4, CD14, and CD45RA, and then positively selecting or enriching cells expressing CD62L. Meanwhile, enrichment of central memory T (TCM) cells is performed by starting with a negative portion of cells selected based on CD4 expression, negatively selecting based on the expression of CD14 and CD45RA, and positively selecting based on CD62L. In some embodiments, these selections are performed simultaneously, and in other embodiments, these selections are performed sequentially according to a specified order. The same selection step based on CD4 expression is intended to produce a CD8+ cell population or subgroup and also to generate a CD4+ cell population or subgroup; therefore, the positive and negative portions from the CD4-based isolation are retained, and after selectively performing one or more additional positive selection or negative selection steps, they are used in a subsequent step of the method.

[0492] In a specific embodiment, CD4+ cell selection is performed on a PBMC sample or other blood leukocyte sample, wherein the negative and positive portions are retained. Next, negative selection is performed on the negative portion based on the expression of CD14 and CD45RA or CD19, and positive selection is performed based on central memory T cell characteristic markers such as CD62L or CCR7, wherein the positive and negative selections are performed in a specified order.

[0493] Helper CD4+ T cells are separated into primary, central memory, and effector cells by verifying a cell population equipped with cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, the primary CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, and CD4+ T cells. In some embodiments, the central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, the effector CD4+ cells are CD62L- and CD45RO-.

[0494] In one embodiment, to enrich CD4+ cells through negative selection, a monoclonal antibody mixture generally comprises antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, said antibodies or binding partners are bound to a solid support or substrate, such as magnetic beads or paramagnetic beads, to allow the separation of cells for positive and / or negative selection.

[0495] In some embodiments, the manufacturing method includes a freezing step, such as freezing the cells before or after isolation, culture, and / or manipulation. In some embodiments, granulocytes are removed during the freezing and subsequent thawing steps, and, to some extent, monocytes in the cell population are also removed. In some embodiments, for example, a washing step is performed to suspend the cells in a freezing solution after removing plasma and platelets. In some aspects, any various known freezing solutions and parameters may be used. In one embodiment, PBS or other suitable cell freezing medium containing 20% ​​DMSO and 8% human serum albumin (HAS) is used. Next, the medium is diluted 1:1 so that the final concentrations of DMSO and HAS are 10% and 4%, respectively. Next, the cells are frozen to -80°C or -90°C according to a program already set on a generally programmable cooling device or a principle such as a speed of 1° / min, and stored in the gas phase of a liquid nitrogen storage tank.

[0496] In some embodiments, the provided method includes a culture and / or genetic engineering manipulation step. For example, in some embodiments, a method for culturing and / or manipulating a consumed cell population and a culture starter composition is provided.

[0497] In some embodiments, the cell population is cultured in a culture start composition. It is cultured and / or manipulated in an incubator, such as a shell, chamber, well, column, tube, tube set, valve, vial, petri dish, bag, or other container for culturing cells.

[0498] In some embodiments, cells are cultured before or together with genetic engineering operations. The culture step may include culture, stimulation, activation, and / or proliferation. In some embodiments, cells or compositions are cultured under stimulation conditions or in the presence of stimulating reagents. These conditions include conditions designed to induce cell proliferation, reproduction, activation, and / or survival in a group to simulate contact with the House, and / or to induce cells to be used in genetic engineering operations such as introduction into recombinant antigen receptors.

[0499] The above conditions may include specific media, temperature, oxygen content, carbon dioxide content, time, reagents such as nutrients, amino acids, antibiotics, and ions, and / or stimulating factors such as cytokines, chemokines, antigens, binding partners, fusion proteins, and recombinant soluble receptors, and any other substances capable of maintaining the activation of the cell state through design.

[0500] In some embodiments, the stimulating condition or reagent comprises one or more substances, such as ligands, capable of activating the intracellular signaling domain of the TCR complex. In some embodiments, said substance initiates or causes a TCR / CD3 intracellular signaling cascade response in T cells. Such substances may comprise, for example, antibodies specific to the TCR component and / or co-stimulating receptor, such as anti-CD3 antibodies, anti-CD28 antibodies, and / or one or more cytokines that bind to a solid support, such as beads. Optionally, the amplification method may further comprise the step of adding anti-CD3 antibodies and / or anti-CD28 antibodies to a medium (e.g., at a concentration of at least about 0.5 ng / ml). In some embodiments, the stimulator comprises IL-2 and / or IL-15 and / or IL-7 and / or IL-21, such as IL-2, at a concentration of at least about 10 units / ml.

[0501] In some embodiments, the culture is carried out according to some of the techniques described in, for example, U.S. patent no. 6,040,177, 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.

[0502] In some embodiments, the T cell population is increased to culture starter cells, such as non-dividing peripheral blood mononuclear cells (PBMCs) (e.g., the cell population obtained for each T lymph in the initial population to be amplified comprises at least about 5, 10, 20, or 40 or more PBMC culture starter cells); and said culture is amplified by culturing said culture (e.g., for a sufficient amount of time to amplify said number of T cells). In some embodiments, said non-dividing culture starter cells may comprise γ-ray irradiated PBMC culture starter cells. In some embodiments, said PBMCs are prevented from dividing by γ-ray irradiation in the range of about 3000 to 3600 rads. In some embodiments, said culture starter cells are added to the medium before adding the T cells.

[0503] In some embodiments, the stimulation conditions include a temperature suitable for human T lymphocyte growth, e.g., about 25°C, generally at least about 30°C, and also generally or about 37°C. Optionally, the culture may further include the step of adding non-dividing EBV-transformed lymphoblasts (LCL) as culture aids. LCL may be irradiated with γ rays in the range of about 6,000 to 10,000 rads. In some embodiments, LCL culture aids are provided in any suitable amount, e.g., the ratio of LCL culture aids to initial T lymphocytes is at least about 10:1.

[0504] In some embodiments, 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 is generated against a cytomegalovirus antigen, and the process is carried out by isolating T cells from an infected subject and extrastimulating the cells using the same antigen.

[0505] Composition and preparation

[0506] The present invention further provides drug compositions and formulations for the method of the present invention. In some embodiments, the drug compositions and formulations of the present invention comprise a composition of cells for administration, for example, a unit formulation composition of a number of cells for administration comprising a specified amount or a portion thereof. The drug compositions and formulations generally comprise one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition comprises at least one other therapeutic agent.

[0507] The term "drug preparation" refers to a preparation of this form, wherein the preparation allows for effective biological activity containing an active ingredient and does not contain additional ingredients having toxicity that is unacceptable to the subject to whom the preparation is to be administered.

[0508] "Pharmaceuticalally acceptable carrier" means one component of a drug formulation that is not an active ingredient and is not toxic to the subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, sealants, or preservatives.

[0509] In some embodiments, the selection of the carrier is determined by the specific cell and / or method of administration. Accordingly, various suitable preparation methods exist. For example, the drug composition may contain a preservative. Suitable preservatives may include, for example, methyl paraben, propyl paraben, sodium benzoate, and benzalkonium chloride. In some embodiments, a mixture of two or more preservatives is used. The amount of the preservative or a mixture thereof is generally about 0.0001% to about 2% (calculated by the total weight of the composition). Carriers are described, for example, in *Remington's Pharmaceutical Sciences*, 16th edition, Osol, A. Ed. (1980). At the dosage and concentration used, pharmaceutically acceptable carriers are generally non-toxic to subjects and are buffers such as phosphate, citrate, and other organic acid buffers; Antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride); hexaalkyl quaternary ammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; p-hydroxyalkyl benzoates such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate; catechol; resorcinol;Cyclohexanol; 3-pentanol; and m-cresol; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; 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 (e.g., Zn-protein complexes); and / or nonionic surfactants such as polyethylene glycol (PEG), but not limited thereto.;

[0510] In some embodiments, the composition comprises a buffer. Suitable buffers include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some embodiments, a mixture of two or more buffers is used. The amount of the buffer or a mixture thereof is generally about 0.001% to about 4% (calculated by the total weight of the composition). Methods for preparing an administerable drug composition are known. Exemplary methods are specifically described, for example, in *Remington: The Science and Practice of Pharmacy*, Lippincott Williams & Wilkins, 21st edition (May 1, 2005).

[0511] The above formulation may include an aqueous solution. The above formulation or composition may further include one or more active ingredients, said active ingredients may be used for specific indications, diseases, or pathologies to which the cell therapy is to be applied, and preferably, may be used for those having supplementary activity against said cells, wherein the corresponding active ingredients do not have a negative effect on each other. These active ingredients are appropriately present in combination at effective doses for the required purpose. Accordingly, in some embodiments, the drug composition further includes other pharmaceutically active substances or drugs, for example, chemotherapy agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, and / or vincristine.

[0512] In some embodiments, the drug composition comprises an amount of cells that effectively treat or prevent a disease or pathology, such as a therapeutically effective amount or a preventively effective amount. In some embodiments, the therapeutic or preventive effect is monitored by regularly evaluating the subject being treated. The required amount may be delivered by administering the cells in a single bolus, administering the cells in multiple boluses, or by injecting the cells in succession.

[0513] In some embodiments, the composition comprises an amount of cells that effectively reduces the burden of disease or pathology and / or does not cause CRS or severe CRS in a subject and / or produces any other result of the method according to the text.

[0514] The above cells and compositions may be administered using standard administration techniques, formulations, and / or devices. The administration of the cells may be autologous or heterologous. For example, immunosuppressive cells or progenitor cells may be obtained from the same subject and administered to the same subject or different compatible subjects. Immunosuppressive cells derived from peripheral blood or their progenitors (e.g., derived in vivo, ex vivo, or in vitro) may be administered via catheter administration, systemic injection, local injection, intravenous injection, or local injection including parenteral administration. When administering a therapeutic composition (e.g., a drug composition containing genetically modified immunosuppressive cells), it is generally prepared in an injectable form (solution, suspension, fluid) of a unit formulation.

[0515] The formulations include formulations used for oral administration, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, mucosal, sublingual, or suppository administration. In some embodiments, the cell group is administered parenterally. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some embodiments, the cells are administered to a subject using peripheral systemic delivery via intravenous, intraperitoneal, or subcutaneous injection.

[0516] In some embodiments, the composition is provided in the form of a sterile liquid formulation, for example, as an isotonic aqueous solution, suspension, emulsion, dispersion, or viscous composition, and in some embodiments thereof, may be buffered to a selected pH. Liquid formulations are generally easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are somewhat easier to administer, particularly by injection. In other embodiments, viscous compositions may be prepared within a suitable viscosity range to provide a longer contact time than that of a specific tissue. The liquid or viscous composition may comprise a carrier that may be a solvent or dispersion medium containing, for example, water, saline solution, phosphate-buffered physiological saline, a polyhydroxy compound (e.g., glycerol, propylene glycol, liquid polyethylene glycol), and a suitable mixture thereof. For example, the cells may be placed in a solvent such as a suitable carrier, diluent, or excipient such as sterile water, physiological saline, or glucose dextrose to prepare a sterile injectable solution. The composition may include auxiliary substances such as wetting, dispersing, or emulsifying agents (e.g., methyl cellulose), pH buffers, gelling agents or thickening additives, preservatives, flavoring agents, and / or coloring agents, as determined by the required administration and preparation routes. In some embodiments, suitable preparations may be prepared by referring to standard textbooks.

[0517] Various additives, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, may be added to enhance the stability and sterility of the composition. Additionally, the action of microorganisms may be prevented through antibacterial and antifungal agents such as various parabens, chlorobutanol, phenol, and sorbic acid. Absorption of the injectable drug form may be extended by using substances that delay absorption, such as aluminum monostearate and gelatin.

[0518] Preparations for administration into the body are generally sterile. Sterility can be easily achieved, for example, by filtration using a sterile filter membrane.

[0519] product

[0520] The present invention further provides products, such as kits and devices, for administering cells to a subject according to a provided method of adoptive cell therapy or immune effector cell therapy and for storing said cells and compositions.

[0521] The product comprises one or more containers, but generally includes multiple containers, packaging materials, and a label combined with or attached to one or more containers and / or packaging, or a packaging insert page containing instructions for administering cells to a subject.

[0522] A container generally contains, for example, one or more units of cells to be administered. A product generally comprises multiple containers containing a single unit of cells. A unit of cells may be the amount or number of cells to be administered to a subject as an initial dose, or twice (or more than twice) the number of cells to be administered as an initial or subsequent dose. This may be the smallest amount or the smallest possible amount of cells administered to a subject in relation to the method of administration. In some embodiments, a unit of cells is administered to any subject suffering from a specific disease or pathology as a unit of cells according to the method of the present invention, or is the number of cells or minimum number of cells for any subject. For example, in some embodiments, a unit of cells may include a minimum amount of cells administered to a subject with a relatively small body weight and / or a relatively low disease burden, for example, administered to a subject designated as an initial dose in one or more circumstances in a unit of cells, and also administered to a subject designated as one or more subsequent doses in one or more subsequent doses in a unit of cells, for example, according to the provided method. In some embodiments, the number of cells in a unit dose, such as the number of chimeric antigen receptor-expressions or CAR-expressions of a subject derived from cells or the number of cells, must be administered to a specific subject as an initial dose. In some embodiments, cells are derived from a subject who is to be treated or who requires treatment by the method provided in the text.

[0523] In some embodiments, each container independently contains a unit amount of cells, for example, containing the same or essentially the same number of cells. Thus, in some embodiments, each container contains the same, nearly, or essentially the same number of cells or chimeric antigen receptor-expressing cells. In some embodiments, the unit amount is approximately 1 x 10⁻⁶ 10, approximately 1x10 9 , approximately 1x10 8 or about 1 x 10 7 It includes subjects derived from fewer than 10 engineered cells, total cells, T cells, or PBMCs / kg to be treated and / or derived from cells.

[0524] Suitable containers include, for example, bottles, vials, syringes, and flexible bags such as freeze bags. In certain embodiments, the container is a bag, for example, a flexible bag suitable for injecting cells into a subject, for example, a flexible plastic or PVC bag or an EVA or ULPDE bag, and / or an IV solution bag. In some embodiments, the bag is a sealable or / or sterile bag for delivering sterile solutions and cells and compositions. In some embodiments, the volume of the container, e.g., bag is equal to or about 10 ml, 20 ml, 30 ml, 40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, 100 ml, 200 ml, 300 ml, 400 ml, 500 ml, or 1000 ml, or at least this amount, e.g., about 10 ml to 100 ml or about 100 ml, or about 10 ml to 500 ml or about 500 ml. In some embodiments, a container such as a bag is manufactured from a material that is stable at one or more different temperatures and / or provides and / or maintains stable storage of cells, for example, so as to be frozen and thawed immediately before treatment, at a low temperature, for example, lower than or equal to about -20°C, -80°C, -120°C, 135°C, -196°C and / or a temperature suitable for freezing storage and / or other temperatures, for example, temperatures suitable for freezing and thawing cells and body temperature, for example, about 37°C or -38°C, or -39°C, or -40°C, and is carried out, for example, at a subject's point or treatment point, for example, at a bed.

[0525] The above-mentioned container may be manufactured from various materials, such as glass or plastic. In some embodiments, the container comprises one or more ports, such as a sterile infiltration port, which are connected to one or more tubes, for example, via a tube or catheter, and are connected for the purpose of transferring from another container, for example, intravenous or other infusion and / or from another container, such as a cell culture and / or storage bag or another container. Exemplary containers include freezing bags, intravenous solution bags, and vials, and are equipped with a membrane that can be penetrated through an injection needle.

[0526] The product may further include one or more packaging insert pages or labels displaying usage information and / or descriptions. In some embodiments, the information or descriptions indicate that a specific disease or condition may or should be treated, and / or provide such descriptions. The label or packaging insert page may indicate the contents of the product to treat the disease or condition. In some embodiments, the label or packaging insert page provides instructions for treating a subject, for example, to administer an initial and one or more subsequent doses of cells to a subject derived from cells, for example, by any one of the embodiments of the provided method. In some embodiments, the instructions specify administering one or more subsequent doses after administering a unit dose, for example, the contents of a single container of the product, at the initial dose, and within a specified time period or / or after detecting the presence or absence or amount or degree of one or more factors or results in the subject.

[0527] In some embodiments, the description specifies an initial administration and subsequent administration to a subject in multiple unit doses. In some embodiments, the initial administration involves delivering one of the unit doses to the subject, and subsequent administration involves administering one or more of the unit doses to the subject.

[0528] In some embodiments, the description specifies a subsequent administration that takes place after the first administration, for example, about 21 to about 80 days or about 25 to about 60 days, for example, equal to or equal to 50 days or about 50 days after the first administration or the start of the previous administration. In some embodiments, the description specifies administering a subsequent dose after determining that the serum level of the factor indicating cytokine release syndrome (CRS) in the subject is about 10 times, about 25 times, and / or about 50 times lower than the serum level of the subject's marker immediately before the first administration, and / or that the index of CRS has already reached a peak level and is decreasing, and / or that no detectable adaptive host immune response specific to the receptor (e.g., CAR) expressed by the cell in the subject has appeared.

[0529] In some embodiments, the label or packaging insert page or packaging includes a mark to indicate the identity of the subject from whom the cells are derived and / or the subject to whom the cells are to be administered. In the case of autologous transplantation, the identity of the subject from whom the cells are derived is the same as the identity of the subject to whom the cells are to be administered. Thus, the identification information may specify the administration of cells, such as the cells of the original cell origin, to a specific patient. This information may be present on the packaging material and / or label in the form of a barcode or other coding mark, or it may indicate the subject's name and / or other identifying features.

[0530] In some embodiments, the product comprises a plurality of containers containing one or more compositions generally containing cells, for example, in a single unit use form, and further comprises one or more other containers containing the composition, said composition comprising a cytotoxic or other therapeutic agent combined with other reagents, for example, cells, and administered simultaneously or in any order as a single dose. Alternatively, the product may further comprise other or the same containers containing a pharmaceutically acceptable buffer. It may further comprise other materials such as other buffers, diluents, filters, tubes, needles, and / or syringes.

[0531] The term "package insert page" refers to an instruction manual commonly included in the product packaging of a therapeutic product, and said instruction manual includes information such as instructions for using the therapeutic product, method of use, dosage, administration, concomitant treatment, contraindications, and / or warnings.

[0532] Treatment of cancer patients using CAR-expressing autologous T cells for initial and subsequent doses

[0533] The method of the present invention can be summarized as follows.

[0534] Peripheral blood mononuclear cells (PBMCs) or T cells are isolated from human subjects with cancer based on "monocyte isolation," and the cells are cultured and transduced using a viral carrier encoding a chimeric antigen receptor (CAR), said chimeric antigen receptor (CAR) specifically binds to an antigen that is a tumor-associated or tumor-specific antigen expressed by the cancer in the subject. The cells are cryopreserved in an infusion medium in a single flexible cryo-bag, and each is approximately 1 x 10⁶ 6 Dog cells to 5X10 7 It contains a single unit dose of cells, consisting of 10 cells. The initial dose injected into each subject was approximately 1 x 10⁶ 12No more than 1 x 10 cells, preferably about 1 x 10 11 No more than 1 x 10 cells, more preferably about 1 x 10 10 10 cells or about 5×10 9 or about 2x10 9 No more than one cell. Before injection, keep the cells at a temperature lower than approximately -130°C or -175°C.

[0535] Before initiating cell therapy, blood is obtained from the subject, and one or more serum factors indicating cytokine release syndrome (CRS) in the serum, such as tumor necrosis factor α (TNFα), interferon γ (IFNγ), IL-10, and IL-6, are evaluated via methods of ELISA and / or MSD and / or CBA. Before initiating treatment, the tumor burden may be selectively assessed by measuring the size or mass of the solid tumor, for example, via PET or CT scans.

[0536] Resuscitate by raising the temperature to approximately 38°C, and administer the initial amount of cells to the subject through multiple infusions. Each infusion is performed as a continuous intravenous (IV) administration within a time interval of approximately 3 to 30 minutes.

[0537] After administering the initial dose, the subject undergoes a physical examination and is monitored for any symptoms of toxicity or toxic consequences, e.g., fever, hypotension, hypoxia, neurological disorders, or elevated serum levels of inflammatory cytokines or C-reactive protein (CRP). Optionally, when administering one or multiple doses after the initial dose, blood is obtained from the patient, and levels of serum factors indicating CRS are evaluated using ELISA and / or MSD and / or CBA methods. The serum factor levels are compared to those obtained before administering the final initial dose. If necessary, anti-IL6 or other CRS treatments are administered to reduce the symptoms of CRS.

[0538] After administering the initial dose, for example, at 1, 2, 3 and / or 4 weeks after the start of administration, the presence or absence of an anti-CAR immune response in the subject is selectively detected, for example, through qPCR, ELISA, ELISPOT, cell-based antibody assay and / or mixed lymphocyte reaction.

[0539] The percentage reduction in tumor burden achieved through the initial dose may be measured once or multiple times after administration of the initial dose to patients with solid tumors via optional scans (e.g., PET and CT scans), and / or may be measured in blood or disease-positive cells at the tumor site through quantification.

[0540] Subsequent doses are administered. In some subjects, a subsequent dose is administered within approximately 80 days of the start of administration of the initial dose, preferably within approximately 60 days, more preferably within approximately 50 days. In some cases, a subsequent dose is administered only if the tested CRS-related results or serum factor levels are lower than acceptable levels and an anti-CAR immune response is not detected in the subject approximately 60 days after administration of the initial dose. In other subjects, a subsequent dose is administered if more than 30 days after administration of the initial dose is deemed not to have CRS or severe CRS, or if 20% of CRS is observed when the levels of all tested serum factors indicating CRS are lower than the peak levels after administration of the initial dose and an anti-CAR immune response is not detected in the subject. In some embodiments, one or more subsequent doses of cells are administered. The time interval for administering each dose is 21 to 80 days, preferably 25 to 60 days, and more preferably 25 to 55 days.

[0541] The size of the subsequent dose is patient-specific, based on tumor burden, the presence of an anti-CAR immune response, and the level of conclusions regarding CRS. Some subjects are administered subsequent doses of 1, 2, 3, or even more units of cells. The subsequent dose infused to each subject is approximately 1 x 10⁻⁶ 12 No more than 1 x 10 cells, or about 1 x 10 11 No more than 1 x 10 cells, or about 1 x 10 10 No more than 10 cells, or about 5 x 10 9 No more than 2 x 10 cells, or about 2 x 10 9 It is not more than the number of cells. It is administered as a subsequent dose within about 3 to 30 minutes via continuous intravenous injection.

[0542] Starting from the initial dose, subjects are regularly monitored over several years. During patient visits, tumor burden is measured, and / or the proliferation and persistence of administered cells in vivo are measured by detecting CAR-expressing cells via flow cytometry and quantitative polymerase chain reaction (qPCR), and / or the progression of the anti-CAR immune response is evaluated.

[0543] The advantages of the present invention are as follows.

[0544] 1. The present invention significantly enhances the therapeutic effect of tumor treatment using immune effector cells expressing chimeric antigen receptors, namely by reducing the number or volume of tumors, and / or reducing the number and / or extent of metastases, and / or reducing tumor markers, and / or eliminating or weakening complications commonly seen in advanced cancer.

[0545] 2. The antitumor effect against solid tumors is significant, safe, and effective, and provides a treatment method that can stabilize or even cure CLA18A2-positive adenocarcinoma of the middle to late stage of the digestive tract.

[0546] 3. Provides a pretreatment composition that enhances anti-tumor therapy of immune effector cells, and by administering a composition of fludarabine, cyclophosphamide, and albumin-bound paclitaxel before administering immune effector cells, the anti-tumor therapeutic effect of immune effector cells can be significantly promoted, the tumor burden on the patient can be greatly reduced, the patient's quality of life can be improved, and the survival period can be extended.

[0547] The present invention is further explained in conjunction with the specific examples below. It should be understood that these examples are intended only to illustrate the invention and do not limit the scope of the invention. Experimental methods for which specific conditions are not specified in the examples below are generally carried out according to conditions, for example, those specified in J. Sambrook et al., *The Experiment Guide on Molecular Cloning*, 3rd edition, Science Publishers, 2002, or conditions suggested by the manufacturer.

[0548] Materials and Methods:

[0549] ingredient:

[0550] All of the various materials, including the reagents used in this invention, are commercially available.

[0551] An exemplary antigen receptor of the present invention comprises a CAR, and methods for engineering and introducing the receptor into a cell refer to all contents disclosed, for example, in Chinese patent applications with publication numbers CN107058354A, CN107460201A, CN105194661A, CN105315375A, CN105713881A, CN106146666A, CN106519037A, CN106554414A, CN105331585A, CN106397593A, CN106467573A, and international patent applications with publication numbers WO2018006882A1 and WO2015172339A8.

[0552] In the following examples of the present invention, the scFv portion of the chimeric antigen receptor comprises a heavy chain variable region represented by SEQ ID NO: 14 and a light chain variable region represented by SEQ ID NO: 18, the chimeric antigen receptor comprises a sequence represented by SEQ ID NO: 24, an amino acid sequence of the scFv represented by SEQ ID NO: 54, and a nucleic acid sequence represented by SEQ ID NO: 55, and the provided CDR regions are HCDR1 represented by SEQ ID NO: 1, HCDR2 represented by SEQ ID NO: 7, HCDR3 represented by SEQ ID NO: 3, LCDR1 represented by SEQ ID NO: 4, LCDR2 represented by SEQ ID NO: 5, and LCDR3 represented by SEQ ID NO: 6.

[0553] The method for constructing CAR-T cells is as follows. First, construct the CAR gene; for example, the construction of hu8E5-2I-BBZ involves sequentially, from the 5' end to the 3' end, the CD8α signaling peptide (nucleotide sequence indicated by SEQ ID NO: 36), hu8E5-2I scFV (nucleotide sequence indicated by SEQ ID NO: 55), CD8 hinge (nucleotide sequence indicated by SEQ ID NO: 38), CD8 transmembrane domain (nucleotide sequence indicated by SEQ ID NO: 46), and the intracellular signaling domain of CD137 (nucleotide sequence indicated by SEQ ID NO: 48), and CD3 intracellular signaling domain CD3 It contains (the nucleotide sequence indicated by SEQ ID NO: 44). Next, the CAR gene is cloned into a shuttle plasmid to obtain a target plasmid containing the CAR gene. The constructed target plasmid and packaging plasmid are co-transfected into 293T cells to prepare a lentivirus carrier, and the lentivirus carrier is transfected into T cells obtained from the patient's peripheral blood to obtain CAR-T cells. For example, hu8E5-2I-BBZ is used in the following examples.

[0554] Example 1: Treatment of cancer patients using CAR-expressing autologous T cells at initial and subsequent doses

[0555] Autologous T cells expressing the anti-CLD18A2 chimeric antigen receptor (CAR) are administered to patients with CLD18A2-positive gastrointestinal tumors (e.g., gastric adenocarcinoma, pancreatic cancer). Prior to cell administration, the patient undergoes pretreatment using the component aliquot separation technique of "monocyte aliquot." To obtain autologous CAR-expressing T cells, PBMCs are obtained by isolating them from component aliquot samples from individual subjects, transfected via a viral carrier encoding the anti-CLD18A2 CAR, and then amplified in large quantities. The prepared autologous CAR-expressing T cells are then frozen and stored in an infusion medium within a single flexible cryo-bag. The initial dose administered to each subject is approximately 1 x 10⁶ 12 No more than 1 x 10 cells, preferably about 1 x 10 11 No more than 1 x 10 cells, preferably about 1 x 10 10 No more than 10 cells, or about 5 x 10 9 No more than 2 x 10 cells 9 No more than 10 cells. Before injection, keep the cells at a temperature lower than -175°C.

[0556] Before initiating cell therapy, blood is obtained from the subject, and one or more factors indicating cytokine release syndrome (CRS) in the serum, such as tumor necrosis factor α (TNFα), interferon γ (IFNγ), IL-10, and IL-6, are evaluated through methods of ELISA and / or MSD and / or CBA. Before initiating treatment, the tumor burden may be evaluated selectively by measuring the size or characteristics of the solid tumor, for example, via PET or CT scans, and may also be evaluated by detecting tumor markers and / or observing the occurrence and severity of tumor complications.

[0557] The subject is revived by raising the temperature to approximately 38°C, and the initial amount of cells is administered to the subject through multiple infusions. The cells are administered in multiple doses within a period not exceeding 20 days, for example, in 1 to 6 doses, preferably 1 to 5 doses, preferably 1 to 4 doses, preferably 1 to 3 doses, more preferably 2 to 3 doses, to administer the initial amount. Each infusion is administered via continuous intravenous (IV) infusion within a time of approximately 3 to 30 minutes, preferably 5 to 25 minutes.

[0558] After administering the initial dose, the subject undergoes a physical examination and is monitored for any symptoms of toxicity or toxic consequences, e.g., fever, hypotension, hypoxia, neurological disorders, or elevated serum levels of inflammatory cytokines or C-reactive protein (CRP). Optionally, when administering one or multiple doses after the initial dose, blood is obtained from the patient, and levels of serum factors indicating CRS are evaluated using ELISA and / or MSD and / or CBA methods. The levels of serum factors are compared to the levels obtained before administering the final initial dose. If necessary, anti-IL6 or other CRS treatments are administered to reduce the symptoms of CRS.

[0559] After administering the initial dose, for example, at 1, 2, 3 and / or 4 weeks after the start of administration, the presence or absence of an anti-CAR immune response in the subject is selectively detected, for example, through qPCR, ELISA, ELISPOT, cell-based antibody assay and / or mixed lymphocyte reaction.

[0560] The percentage reduction in tumor burden achieved through the initial dose may be measured once or multiple times after administration of the initial dose to patients with solid tumors via optional scans (e.g., PET and CT scans), and / or may be measured in blood or disease-positive cells at the tumor site through quantification.

[0561] Subsequent doses are administered. In some subjects, a subsequent dose is administered within approximately 80 days of the start of administration of the initial dose, preferably within approximately 60 days, more preferably within approximately 50 days. In some cases, a subsequent dose is administered only if the tested CRS-related results or serum factor levels are lower than acceptable levels and an anti-CAR immune response is not detected in the subject approximately 60 days after administration of the initial dose. In other subjects, a subsequent dose is administered if more than 30 days after administration of the initial dose is deemed not to have CRS or severe CRS, or if 20% of CRS is observed when the levels of all tested serum factors indicating CRS are lower than the peak levels after administration of the initial dose and an anti-CAR immune response is not detected in the subject. In some embodiments, one or more subsequent doses of cells are administered. The time interval for administering each dose is 21 to 80 days, preferably 25 to 60 days, and more preferably 25 to 55 days.

[0562] The magnitude of the subsequent dose is patient-specific, based on tumor burden, the presence of an anti-CAR immune response, and the level of CRS-related outcomes. Some subjects are administered 1, 2, 3, or even more subsequent doses. The subsequent dose infused to each subject is approximately 1 x 10⁻¹⁰ 12 No more than 1 x 10 cells, preferably about 1 x 10 11 No more than 1 x 10 cells, more preferably about 1 x 10 10 No more than 10 cells, more preferably about 5×10 9 No...

Claims

Claim 1 A pharmaceutical composition for treating a CLD18A2-positive solid tumor comprising an amount of immune effector cells, wherein the immune effector cells express a chimeric antigen receptor (CAR) that specifically identifies CLD18A2, and the composition further comprises a chemotherapy agent comprising cyclophosphamide, fludarabine, and albumin-bound paclitaxel, wherein the chemotherapy agent is administered before the amount of immune effector cells is administered to a subject. Claim 2 A pharmaceutical composition according to claim 1, characterized in that a chemotherapy agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days prior to administering the amount of immune effector cells. Claim 3 In paragraph 1, the above usage amount is 2X10 9 A pharmaceutical composition characterized by containing a total amount of immune effector cells that does not exceed the cell count. Claim 4 A pharmaceutical composition according to claim 1, characterized by administering a subsequent amount of immune effector cells expressing a chimeric antigen receptor (CAR) that specifically identifies CLD18A2 at least once after administering an amount of immune effector cells. Claim 5 In paragraph 4, at least one subsequent use is 2X10 9 A pharmaceutical composition characterized by containing a total amount of immune effector cells that does not exceed the cell count. Claim 6 In paragraph 1, the dosage of fludarabine is 10 to 50 mg / m² 2 / day, or 15 ~ 40 mg / m² 2 / day, or 15 ~ 30 mg / m² 2 / day, or 20 ~ 30 mg / m² 2 / day, or 25 mg / m² 2 The dosage of cyclophosphamide is 300 to 700 mg / m² 2 / day, or 400 ~ 650 mg / m² 2 / day, or 450 ~ 600 mg / m² 2 / day, or 450 ~ 550 mg / m² 2 / day, or 490 ~ 550 mg / m² 2 / day, or 250 mg / m² 2 / iligo; the dosage of albumin-bound paclitaxel is 300 mg / m² 2 Not more than / day, or 200 mg / m² 2 Not more than / day, or 150 mg / m² 2 Not more than / day, or 100 mg / m² 2 Not more than / day, or 80 mg / m² 2 Not more than / day, or 70, 69, 68, 67, 66, 65, 64, 63, 62, or 61 mg / m² 2 No more than / day, or the dose of albumin-bound paclitaxel is 100 mg / m² 2 A pharmaceutical composition characterized by being a single unit. Claim 7 In paragraph 6, fludarabine is 25 mg / m² for 2 days. 2 Administered per day, cyclophosphamide 250 mg / m² for 3 days 2 It is administered per day, and albumin-bound paclitaxel is 100 mg / m² per day. 2 A pharmaceutical composition characterized by being administered daily. Claim 8 A pharmaceutical composition according to claim 1, characterized in that the chemotherapy agent is administered daily for 2, 3, 4, 5, 6, or 7 days. Claim 9 A pharmaceutical composition according to claim 8, characterized in that cyclophosphamide and fludarabine are administered daily for 2, 3, or 4 days and albumin-bound paclitaxel is administered once; or cyclophosphamide, fludarabine, and albumin-bound paclitaxel are administered daily for 2, 3, or 4 days. Claim 10 A pharmaceutical composition according to claim 1, wherein the CLD18A2 positive solid tumor is breast cancer, colon cancer, rectal cancer, kidney cancer, liver cancer, lung cancer, stomach cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, ovarian cancer, anal cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, Hodgkin lymphoma, non-Hodgkin lymphoma, endocrine cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, bladder cancer, kidney cancer, ureteral cancer, renal pelvis cancer, central nervous system (CNS) cancer, primary CNS lymphoma, spinal tumor, brainstem glioma, pituitary adenoma, Kaposi sarcoma, epidermal carcinoma, or squamous cell carcinoma. Claim 11 A pharmaceutical composition according to claim 1, characterized in that the CLD18A2 positive solid tumor is a gastrointestinal tumor. Claim 12 A pharmaceutical composition according to claim 11, characterized in that the above-mentioned digestive tract tumor is an adenocarcinoma. Claim 13 A pharmaceutical composition according to claim 1, characterized in that the CLD18A2 positive solid tumor is pancreatic cancer, gastric cancer, gastric adenocarcinoma, esophageal cancer, rectal cancer, anal cancer, or small intestine cancer. Claim 14 In claim 1, the chimeric antigen receptor comprises a scFv antibody fragment specifically binding to CLD18A2, a transmembrane domain, and an intracellular domain, wherein the scFv antibody fragment is HCDR1 represented by SEQ ID NO: 1, HCDR2 represented by SEQ ID NO: 2, HCDR3 represented by SEQ ID NO: 3, LCDR1 represented by SEQ ID NO: 4, LCDR2 represented by SEQ ID NO: 5, and LCDR3 represented by SEQ ID NO: 6; or HCDR1 represented by SEQ ID NO: 1, HCDR2 represented by SEQ ID NO: 7, HCDR3 represented by SEQ ID NO: 3, LCDR1 represented by SEQ ID NO: 4, LCDR2 represented by SEQ ID NO: 5, and LCDR3 represented by SEQ ID NO: 6; A pharmaceutical composition characterized by comprising HCDR1 indicated by SEQ ID NO: 8, HCDR2 indicated by SEQ ID NO: 9 or SEQ ID NO: 68, HCDR3 indicated by SEQ ID NO: 10, LCDR1 indicated by SEQ ID NO: 11, LCDR2 indicated by SEQ ID NO: 12, and LCDR3 indicated by SEQ ID NO:

13. Claim 15 A pharmaceutical composition according to claim 14, wherein the scFv antibody fragment comprises: a heavy chain variable region represented by SEQ ID NO: 14 and a light chain variable region represented by SEQ ID NO: 16; or a heavy chain variable region represented by SEQ ID NO: 18 and a light chain variable region represented by SEQ ID NO: 16; or a heavy chain variable region represented by SEQ ID NO: 22 and a light chain variable region represented by SEQ ID NO: 20; or a heavy chain variable region represented by SEQ ID NO: 53 and a light chain variable region represented by SEQ ID NO:

52. Claim 16 A pharmaceutical composition according to claim 14, wherein the scFv antibody fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 57, and SEQ ID NO:

58. Claim 17 A pharmaceutical composition according to claim 1, wherein the chimeric antigen receptor comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO:

32. Claim 18 A pharmaceutical composition according to claim 1, characterized in that the immune effector cell is a T lymphocyte, NK cell, or NKT lymphocyte. Claim 19 A pharmaceutical composition according to claim 1, characterized in that the chimeric antigen receptor comprises at least two or three cytoplasmic signaling domains. Claim 20 A pharmaceutical composition according to claim 19, wherein the at least two or three cytoplasmic signaling domains are selected from the group consisting of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CDS, CD22, CD79a, CD79b, CD66d, CD28, CD137, OX40, DAP10, and ICOS. Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete Claim 48 delete Claim 49 delete Claim 50 delete Claim 51 delete Claim 52 delete Claim 53 delete Claim 54 delete Claim 55 delete Claim 56 delete Claim 57 delete Claim 58 delete Claim 59 delete Claim 60 delete Claim 61 delete Claim 62 delete

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