CROSS-REFERENCE TO RELATED APPLICATIONS FOR CAR-T CELL THERAPY TARGETING BCMA FOR MULTIPLE MYELOMA

Abstract

Provided herein are methods of treating a subject suffering from multiple myeloma who has undergone previous therapy and has limited treatment options. The subject is administered an infusion of chimeric antigen receptor (CAR)-T cells comprising an anti-BCMA CAR containing polypeptide. In some embodiments, the dose of CAR-T cells administered to the subject is 1.0 x 10 5 ~5.0 × 10 6 CAR-T cells / kilogram of subject mass. The treatment method effectively obtains and maintains a negative minimal residual disease status and other beneficial clinical outcomes related to efficacy and safety.

Description

[Technical field]

[0001] This application claims priority to U.S. Patent Application No. PCT / CN2021 / 128578, filed November 4, 2021, and U.S. Patent Application No. 63 / 275,471, filed November 4, 2021, the disclosures of each of which are incorporated by reference in their entireties herein.

[0002] Sequence Listing This application contains a computer readable sequence listing that has been submitted with this application in the file format of XML, the contents of which are incorporated herein by reference in their entirety. The sequence listing submitted with this application has an XML filename of "14651-048-228_SEQ_LISTING.xml", was created on October 27, 2022, and is 28,401 bytes in size. [Background technology]

[0003] 1. Background Multiple myeloma is an aggressive plasma cell neoplasm. It is considered to be a B-cell neoplasm that grows uncontrollably in the bone marrow. Symptoms include one or more of hypercalcemia, renal failure, anemia, bone lesions, bacterial infection, hyperviscosity, and amyloidosis. Although available new therapies, including proteasome inhibitors, immunomodulatory agents, and monoclonal antibodies, have significantly improved patient outcomes, multiple myeloma is still considered to be an incurable disease. Since most patients relapse or become refractory, new therapies for multiple myeloma are continually needed. Summary of the Invention

[0004] 2. Overview of this Disclosure In one aspect, a method of treating a subject is provided, the method comprising administering to the subject a composition comprising a therapeutically effective amount of T cells comprising a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising: (a) (1) a first anti-BCMA binding moiety comprising a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 18, a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 19, and a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 20; (2) an extracellular antigen-binding domain comprising a second BCMA-binding portion comprising a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 21, a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 22, and a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 23; (b) a transmembrane domain; (c) an intracellular signaling domain; thereby delivering a dose of CAR-expressing T cells (CAR-T cells) to the subject; wherein the subject has multiple myeloma, has previously undergone one, two or three prior lines of therapy, and is refractory to lenalidomide.

[0005] In some embodiments, the multiple myeloma is refractory to the last line of therapy. In some embodiments, the subject has relapsed after one, two, or three prior lines of therapy. In some embodiments, the subject has received prior treatment with at least one prior line of therapy, the at least one prior line of therapy comprising treatment with lenalidomide and at least one non-lenalidomide drug, the at least one non-lenalidomide drug comprising at least one of a proteasome inhibitor, an immunomodulatory agent, or an anti-CD38 antibody. In some embodiments, the subject has received prior treatment with at least two prior lines of therapy. In some embodiments, the subject has received prior treatment with three prior lines of therapy.

[0006] In some embodiments, the subject has received prior treatment with dexamethasone, an alkylating agent, or daratumumab, hi some embodiments, the multiple myeloma is refractory to three drugs.

[0007] In some embodiments, the method effectively obtains a minimal residual disease (MRD) negative status of the subject as assessed in bone marrow following the administration of the CAR-T cells. In some embodiments, the minimal residual disease (MRD) negative status is obtained at a first follow-up period from about 29 days to about 184 days following the administration of the CAR-T cells. In some embodiments, the method effectively maintains the minimal residual disease (MRD) negative status of the subject as assessed in bone marrow at a second follow-up period from about 57 days to about 191 days following the administration of the CAR-T cells, and further wherein the first follow-up period is earlier than the second follow-up period.

[0008] In some embodiments, the method comprises: -4 , 10 -5 or 10 -6 In some embodiments, the method effectively achieves a minimal residual disease (MRD) negative status at a rate of about 24% to about 61% at a sensitivity threshold level of 10 -4 , 10 -5 or 10 -6 At a sensitivity threshold level of 0.1, the minimal residual disease (MRD) negativity status is effectively achieved in approximately 41% of cases.

[0009] In some embodiments, the method comprises: -5 At a sensitivity threshold level of 100%, the method effectively obtains the minimal residual disease (MRD) negative status in about 64% to about 99% of subjects with evaluable samples. -5 At a sensitivity threshold level of 0.1, approximately a 92% rate of subjects with evaluable samples effectively achieves the minimal residual disease (MRD) negativity status.

[0010] In some embodiments, the method is effective to obtain at least one response in the subject following said administration of the CAR-T cells, wherein the at least one response comprises, in order from best to worst, a stringent complete response, a complete response, a best partial response, a partial response, or a minimal response.

[0011] In some embodiments, the method is effective to obtain an initial response prior to a period of about 21 days to about 99 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response prior to a period of about 21 days to about 55 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response prior to about 36 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response prior to about 30 days after said administration of the CAR-T cells.

[0012] In some embodiments, the method is effective to obtain a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response. In some embodiments, the method is effective to obtain a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response at a rate of about 52% to about 87%. In some embodiments, the method is effective to obtain a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response at a rate of about 72%.

[0013] In some embodiments, the method is effective to obtain a best response of any one of partial response, best partial response, complete response, or stringent complete response. In some embodiments, the method is effective to obtain a best response of any one of partial response, best partial response, complete response, or stringent complete response at a rate of about 49% to about 84%. In some embodiments, the method is effective to obtain a best response of any one of partial response, best partial response, complete response, or stringent complete response at a rate of about 69%.

[0014] In some embodiments, the method is effective to obtain a best response of any one of best partial response, complete response, or stringent complete response. In some embodiments, the method is effective to obtain a best response of any one of best partial response, complete response, or stringent complete response at a rate of about 49% to about 84%. In some embodiments, the method is effective to obtain a best response of any one of best partial response, complete response, or stringent complete response at a rate of about 69%.

[0015] In some embodiments, the method is effective to obtain a best response of complete response or stringent complete response. In some embodiments, the method is effective to obtain a best response of complete response or stringent complete response at a rate of about 39% to about 76%. In some embodiments, the method is effective to obtain a best response of complete response or stringent complete response at a rate of about 58%.

[0016] In some embodiments, the method is effective in obtaining a best response of stringent complete response. In some embodiments, the method is effective in obtaining a best response of stringent complete response at a rate of about 33% to about 70%. In some embodiments, the method is effective in obtaining a best response of stringent complete response at a rate of about 52%.

[0017] In some embodiments, the method effectively obtains progression free survival in the subject. In some embodiments, the method effectively obtains progression free survival in the subject from the administration of the CAR-T cells to about 55 days after the administration of the CAR-T cells. In some embodiments, the method effectively obtains progression free survival in the subject from the administration of the CAR-T cells to about 297 days after the administration of the CAR-T cells.

[0018] In some embodiments, the method is effective to obtain the progression free survival at a rate of about 62% to about 95% at a follow-up period of about 6 months or about 9 months after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the progression free survival at a rate of about 86% at a follow-up period of about 6 months or about 9 months after the administration of the CAR-T cells.

[0019] In some embodiments, the method further comprises treating the cytokine release syndrome in the subject more than about 3 days after the administration of the CAR-T cells. In some embodiments, the method effectively obtains about a 1% to about 90% reversal rate of the cytokine release syndrome in a period of about 7 days after the cytokine release syndrome is first observed.

[0020] In some embodiments, the method effectively achieves a rate of immune effector cell-associated neurotoxicity of about 20% to about 99%.

[0021] In some embodiments, the method is effective to obtain the best effect before a period of about 27 days to about 237 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect before a period of about 46 days to about 172 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect before about 109 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect before about 87 days after the administration of the CAR-T cells.

[0022] In some embodiments, the method effectively maintains a response in a subject for a follow-up period from the time of the initial response to about 270 days after the administration of the CAR-T cells. In some embodiments, the method effectively maintains a response at about 70% to about 99% rate for a follow-up period of about 6 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains a response at about 95% rate for a follow-up period of about 6 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains a response at about 7% to about 92% rate for a follow-up period of about 9 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains a response at about 63% rate for a follow-up period of about 9 months after the administration of the CAR-T cells.

[0023] In some embodiments, the method further comprises administering the CAR-T cells to a patient receiving the CAR-T cells for 10 to about 3 months after the administration of the CAR-T cells. -5 At a sensitivity threshold level of 0.1%, the method effectively obtains a minimal residual disease (MRD) negative status of the subject as assessed in bone marrow. In some embodiments, the method effectively obtains a minimal residual disease (MRD) negative complete response or a minimal residual disease (MRD) negative stringent complete response at a rate of about 18% to about 54% at a follow-up period of about 291 days after the administration of the CAR-T cells. In some embodiments, the method effectively obtains a minimal residual disease (MRD) negative complete response or a minimal residual disease (MRD) negative stringent complete response at a rate of about 35% at a follow-up period of about 291 days after the administration of the CAR-T cells.

[0024] In one aspect, a method of treating a subject is provided, the method comprising administering to the subject a composition comprising a therapeutically effective amount of T cells comprising a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising: (a) (1) a first anti-BCMA binding moiety comprising a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 18, a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 19, and a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 20; (2) an extracellular antigen-binding domain comprising a second BCMA-binding portion comprising a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 21, a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 22, and a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 23; (b) a transmembrane domain; (c) an intracellular signaling domain; thereby delivering a dose of CAR-expressing T cells (CAR-T cells) to the subject; Here, the subject is suffering from multiple myeloma, has previously experienced an early relapse, and has already undergone prior treatment with one prior line of treatment, the one prior line of treatment including treatment with at least two drugs including a proteasome inhibitor and an immunomodulatory agent.

[0025] In some embodiments, the subject is further treated with an anti-CD38 antibody.In some embodiments, the multiple myeloma is refractory to at least one drug.

[0026] In some embodiments, the method effectively obtains a minimal residual disease (MRD) negative status of the subject as assessed in bone marrow following the administration of the CAR-T cells. In some embodiments, the minimal residual disease (MRD) negative status is assessed in bone marrow at a first follow-up period from about 35 days to about 58 days following the administration of the CAR-T cells. In some embodiments, the method effectively maintains the minimal residual disease (MRD) negative status of the subject as assessed in bone marrow at a second follow-up period from about 78 days to about 359 days following the administration of the CAR-T cells, and further wherein the first follow-up period is earlier than the second follow-up period.

[0027] In some embodiments, the method comprises: -4 or 10 -5 At a sensitivity threshold level of about 26% to about 74%, or -6 In some embodiments, the method effectively achieves a minimal residual disease (MRD) negative status at a rate of about 17% to about 64% at a sensitivity threshold level of 10 -4 or 10 -5 At a sensitivity threshold level of about 50% or 10 -6 At a sensitivity threshold level of 0.1, the minimal residual disease (MRD) negativity status is effectively achieved in approximately 39% of cases.

[0028] In some embodiments, the method comprises: -5 At a sensitivity threshold level of 100%, the method effectively achieves the minimal residual disease (MRD) negative status in about 66% to about 100% of subjects with evaluable samples. -5 At a sensitivity threshold level of 0.1, approximately 100% of subjects with evaluable samples will effectively achieve the minimal residual disease (MRD) negativity status.

[0029] In some embodiments, the method is effective to obtain at least one response in the subject following said administration of the CAR-T cells, wherein the at least one response comprises, in order from best to worst, a stringent complete response, a complete response, a best partial response, a partial response, or a minimal response.

[0030] In some embodiments, the method is effective to obtain an initial response prior to a period of about 27 days to about 78 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response prior to a period of about 27 days to about 47 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response prior to about 33 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response prior to about 28 days after said administration of the CAR-T cells.

[0031] In some embodiments, the method is effective to obtain a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response. In some embodiments, the method is effective to obtain a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response at a rate of about 65% to about 99%. In some embodiments, the method is effective to obtain a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response at a rate of about 89%.

[0032] In some embodiments, the method is effective to obtain a best response of any one of partial response, best partial response, complete response, or stringent complete response. In some embodiments, the method is effective to obtain a best response of any one of partial response, best partial response, complete response, or stringent complete response at a rate of about 65% to about 99%. In some embodiments, the method is effective to obtain a best response of any one of partial response, best partial response, complete response, or stringent complete response at a rate of about 89%.

[0033] In some embodiments, the method is effective to obtain a best response of any one of best partial response, complete response, or stringent complete response. In some embodiments, the method is effective to obtain a best response of any one of best partial response, complete response, or stringent complete response at a rate of about 41% to about 87%. In some embodiments, the method is effective to obtain a best response of any one of best partial response, complete response, or stringent complete response at a rate of about 67%.

[0034] In some embodiments, the method is effective to obtain a best response of complete response or stringent complete response. In some embodiments, the method is effective to obtain a best response of complete response or stringent complete response at a rate of about 10% to about 54%. In some embodiments, the method is effective to obtain a best response of complete response or stringent complete response at a rate of about 28%.

[0035] In some embodiments, the method is effective to obtain a best response of stringent complete response. In some embodiments, the method is effective to obtain a best response of stringent complete response at a rate of about 6% to about 48%. In some embodiments, the method is effective to obtain a best response of stringent complete response at a rate of about 22%.

[0036] In some embodiments, the method effectively results in progression-free survival in the subject. In some embodiments, the method effectively results in progression-free survival in the subject from the administration of the CAR-T cells to about 182 days after the administration of the CAR-T cells.

[0037] In some embodiments, the method effectively obtains the progression free survival at about a 100% rate at about a 6 month follow-up period after the administration of the CAR-T cells. In some embodiments, the method effectively obtains the progression free survival at about a 5% to about a 95% rate at about a 9 month or about a 12 month follow-up period after the administration of the CAR-T cells. In some embodiments, the method effectively obtains the progression free survival at about a 67% rate at about a 9 month or about a 12 month follow-up period after the administration of the CAR-T cells.

[0038] In some embodiments, the method further comprises treating the cytokine release syndrome in the subject more than about 3 days after the administration of the CAR-T cells. In some embodiments, the method effectively obtains about a 1% to about 100% reversal rate of the cytokine release syndrome in a period of about 7 days after the cytokine release syndrome is first observed.

[0039] In some embodiments, the method is effective to obtain the best effect before a period of about 27 days to about 354 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect before a period of about 27 days to about 155 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect before about 71 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect before about 42 days after the administration of the CAR-T cells.

[0040] In some embodiments, the method effectively maintains a response in a subject for a follow-up period from the time of the initial response to about 156 days after the administration of the CAR-T cells. In some embodiments, the method effectively maintains a response at a rate of about 5% to about 95% for a follow-up period of about 6 months, about 9 months, or about 12 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains a response at a rate of about 67% for a follow-up period of about 6 months, about 9 months, or about 12 months after the administration of the CAR-T cells.

[0041] In some embodiments, the method further comprises administering the CAR-T cells to a patient receiving the CAR-T cells for 10 to about 3 months after the administration of the CAR-T cells. -5 At a sensitivity threshold level of 0.1%, the method effectively obtains a minimal residual disease (MRD) negative status of the subject as assessed in bone marrow. In some embodiments, the method effectively obtains a minimal residual disease (MRD) negative complete response or a minimal residual disease (MRD) negative stringent complete response at a rate of about 1% to about 35% at a follow-up period of about 141 days after the administration of the CAR-T cells. In some embodiments, the method effectively obtains a minimal residual disease (MRD) negative complete response or a minimal residual disease (MRD) negative stringent complete response at a rate of about 11% at a follow-up period of about 141 days after the administration of the CAR-T cells.

[0042] In one aspect, a method of treating a subject is provided, the method comprising administering to the subject a composition comprising a therapeutically effective amount of T cells comprising a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising: (a) (1) a first anti-BCMA binding moiety comprising a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 18, a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 19, and a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 20; (2) an extracellular antigen-binding domain comprising a second BCMA-binding portion comprising a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 21, a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 22, and a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 23; (b) a transmembrane domain; (c) an intracellular signaling domain; thereby delivering a dose of CAR-expressing T cells (CAR-T cells) to the subject; wherein the subject is afflicted with multiple myeloma and has already received at least one prior line of treatment, the at least one prior line of treatment comprising treatment with at least four drugs, including a non-cellular BCMA targeted drug.

[0043] In some embodiments, the at least four drugs further comprise a proteasome inhibitor, an immunomodulatory agent, and an anti-CD38 antibody.

[0044] In some embodiments, the subject has been previously treated with at least two prior lines of treatment. In some embodiments, the subject has been previously treated with at least four prior lines of treatment. In some embodiments, the subject has been previously treated with at least eight prior lines of treatment. In some embodiments, the subject has been previously treated with at least twelve prior lines of treatment. In some embodiments, the subject relapses after said at least one prior line of treatment.

[0045] In some embodiments, the method effectively obtains a minimal residual disease (MRD) negative status of the subject as assessed in bone marrow following the administration of the CAR-T cells. In some embodiments, the minimal residual disease (MRD) negative status is assessed in bone marrow at a first follow-up period from about 56 days to about 58 days following the administration of the CAR-T cells. In some embodiments, the method effectively maintains the minimal residual disease (MRD) negative status of the subject as assessed in bone marrow at a second follow-up period from about 183 days to about 186 days following the administration of the CAR-T cells, and further wherein the first follow-up period is earlier than the second follow-up period.

[0046] In some embodiments, the method comprises: -4 At the sensitivity threshold level, the ratio is about 9% to about 49%, and -5 At a sensitivity threshold level of about 6% to about 44%, or -6 In some embodiments, the method effectively achieves a minimal residual disease (MRD) negative status at a rate of about 1% to about 31% at a sensitivity threshold level of 10 -4 At the sensitivity threshold level of 10, the ratio is about 25%. -5 At a sensitivity threshold level of about 20%, or 10 -6 At a sensitivity threshold level of 0.1, approximately 10% of patients effectively achieve the minimal residual disease (MRD) negative status.

[0047] In some embodiments, the method comprises: -5At a sensitivity threshold level of 100%, the method effectively obtains the minimal residual disease (MRD) negative status in about 22% to about 96% of subjects with evaluable samples. -5 At a sensitivity threshold level of 0.1, approximately 67% of subjects with evaluable samples effectively achieve the minimal residual disease (MRD) negativity status.

[0048] In some embodiments, the method is effective to obtain at least one response in the subject following said administration of the CAR-T cells, wherein the at least one response comprises, in order from best to worst, a stringent complete response, a complete response, a best partial response, a partial response, or a minimal response.

[0049] In some embodiments, the method is effective to obtain an initial response before a period of about 27 days to about 153 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response before a period of about 27 days to about 88 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response before about 43 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response before about 28 days after said administration of the CAR-T cells.

[0050] In some embodiments, the method is effective to obtain a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response. In some embodiments, the method is effective to obtain a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response at a rate of about 23% to about 69%. In some embodiments, the method is effective to obtain a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response at a rate of about 45%.

[0051] In some embodiments, the method is effective to obtain a best response of any one of partial response, best partial response, complete response, or stringent complete response. In some embodiments, the method is effective to obtain a best response of any one of partial response, best partial response, complete response, or stringent complete response at a rate of about 19% to about 64%. In some embodiments, the method is effective to obtain a best response of any one of partial response, best partial response, complete response, or stringent complete response at a rate of about 40%.

[0052] In some embodiments, the method is effective to obtain a best response of any one of best partial response, complete response, or stringent complete response. In some embodiments, the method is effective to obtain a best response of any one of best partial response, complete response, or stringent complete response at a rate of about 15% to about 59%. In some embodiments, the method is effective to obtain a best response of any one of best partial response, complete response, or stringent complete response at a rate of about 35%.

[0053] In some embodiments, the method is effective to obtain a best response of complete response or stringent complete response. In some embodiments, the method is effective to obtain a best response of complete response or stringent complete response at a rate of about 3% to about 38%.

[0054] In some embodiments, the method is effective to obtain a best response of stringent complete response. In some embodiments, the method is effective to obtain a best response of stringent complete response at a rate of about 1% to about 32%.

[0055] In some embodiments, the method is effective to obtain progression free survival in the subject. In some embodiments, the method is effective to obtain progression free survival in the subject from the administration of the CAR-T cells to about 15 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain progression free survival in the subject from the administration of the CAR-T cells to about 44 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain progression free survival in the subject from the administration of the CAR-T cells to about 159 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain progression free survival at a rate of about 29% to about 75% at a follow-up period of about 6 months or about 9 months after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain progression free survival at a rate of about 55% at a follow-up period of about 6 months or about 9 months after the administration of the CAR-T cells.

[0056] In some embodiments, the method effectively achieves a rate of cytokine release syndrome of about 60% to about 99%. In some embodiments, the method further comprises treating the subject for cytokine release syndrome more than about 3 days after the administration of the CAR-T cells.

[0057] In some embodiments, the method effectively achieves a rate of immune effector cell-associated neurotoxicity of about 20% to about 99%.

[0058] In some embodiments, the method is effective to obtain the best effect before a period of about 27 days to about 171 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect before a period of about 27 days to about 133 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect before about 78 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect before about 56 days after the administration of the CAR-T cells.

[0059] In some embodiments, the method effectively maintains a response in a subject for a follow-up period from the time of the initial response to about 132 days after the administration of the CAR-T cells, and further wherein the initial response is obtained from the time of the administration of the CAR-T cells to about 131 days after the administration of the CAR-T cells. In some embodiments, the method effectively maintains a response at a rate of about 20% to about 96% for a follow-up period of about 6 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains a response at a rate of about 80% for a follow-up period of about 6 months after the administration of the CAR-T cells.

[0060] In some embodiments, the multiple myeloma is refractory to at least two drugs, in some embodiments, the multiple myeloma is refractory to at least three drugs, in some embodiments, the multiple myeloma is refractory to at least four drugs, in some embodiments, the multiple myeloma is refractory to at least five drugs.

[0061] In some embodiments, the subject has about 10% to about 30% bone marrow plasma cells prior to said administration of said CAR-T cells.

[0062] In some embodiments, the dose is 1.0 x 10 per kilogram of the subject's mass. 5 ~5.0 × 10 6 In some embodiments, the dose comprises 5.0 x 10 CAR-T cells per kilogram of the subject's mass. 5 ~1.0 × 10 6 In some embodiments, the dose comprises about 0.75 x 10 CAR-T cells per kilogram of the subject's mass. 6 In some embodiments, the dose is 1.0 x 10 CAR-T cells per subject. 8 The CAR-T cell is preferably a cellular or a cellular component.

[0063] In some embodiments, the administration of the CAR-T cells is by a single intravenous infusion. In some embodiments, the single intravenous infusion is administered using one bag of the CAR-T cells. In some embodiments, the administration of the one bag of the CAR-T cells is completed between the time of thawing the one bag of CAR-T cells and 3 hours after thawing the one bag of CAR-T cells. In some embodiments, the single intravenous administration is administered using two bags of the CAR-T cells. In some embodiments, the administration of each bag of the two bags of the CAR-T cells is completed between the time of thawing the first bag of the two bags of CAR-T cells and 3 hours after thawing the first bag of CAR-T cells.

[0064] In some embodiments, the lymphodepletion regimen is administered about 5 to about 7 days prior to administration of the CAR-T cells. In some embodiments, the lymphodepletion regimen is administered intravenously. In some embodiments, the lymphodepletion regimen includes administration of cyclophosphamide or administration of fludarabine. In some embodiments, the cyclophosphamide is administered at a dose of 300 mg / m 2 In some embodiments, the fludarabine is administered intravenously at a dose of 30 mg / m 2 In some embodiments, cyclophosphamide is administered intravenously at 300 mg / m 2 and fludarabine 30 mg / m 2 and intravenous administration of the CAR-T cells to a lymphodepleting regimen, the lymphodepleting regimen comprising administering the CAR-T cells intravenously to a lymphocyte about 5 to about 7 days prior to administration of the CAR-T cells.

[0065] In some embodiments, the subject further undergoes a bridging therapy, wherein said bridging therapy includes short-term treatment with at least one bridging drug between apheresis and said lymphodepletion regimen, and wherein said at least one bridging drug has previously resulted in the subject having stable disease, minimal response, partial response, best partial response, complete response, or stringent complete response. In some embodiments, the subject experiences an increase in tumor burden despite undergoing said bridging therapy. In some embodiments, the subject experiences an increase in tumor burden of about 25% or more despite undergoing said bridging therapy.

[0066] In some embodiments, the method further comprises treating the subject for cytokine release syndrome more than about 3 days after the administration of the CAR-T cells without significantly reducing in vivo expansion of the CAR-T cells. In some embodiments, the treatment of cytokine release syndrome comprises administering an IL-6R inhibitor to the subject. In some embodiments, the IL-6R inhibitor is an antibody. In some embodiments, the antibody inhibits IL-6R by binding to the extracellular domain of IL-6R. In some embodiments, the IL-6R inhibitor blocks binding of IL-6 to IL-6R. In some embodiments, the IL-6R inhibitor is tocilizumab.

[0067] In some embodiments, the subject is treated at most about 1 hour prior to the administration of the CAR-T cells with an administered prodrug comprising an antipyretic and an antihistamine. In some embodiments, the antipyretic comprises paracetamol or acetaminophen. In some embodiments, the antipyretic is administered orally or intravenously to the subject. In some embodiments, the antipyretic is administered to the subject at a dose between 650 mg and 1000 mg. In some embodiments, the antihistamine comprises diphenhydramine. In some embodiments, the antihistamine is administered orally or intravenously to the subject. In some embodiments, the antihistamine is administered at a dose between 25 mg and 50 mg, or the equivalent thereof. In some embodiments, the antipyretic comprises paracetamol or acetaminophen, and the antipyretic comprises a dose of 650 mg to 1000 mg, and wherein the antihistamine comprises diphenhydramine, and the antihistamine comprises a dose of 25 mg to 50 mg, or an equivalent thereof, and wherein the antipyretic comprises a dose of 25 mg to 50 mg, or an equivalent thereof, and wherein the antipyretic comprises a dose of 25 mg to 50 mg, or an equivalent thereof, and wherein the antihistamine ...

[0068] In some embodiments, the composition comprising CAR-T cells administered to the subject further comprises an excipient selected from dimethylsulfoxide or dextran-40.

[0069] In some embodiments, the first BCMA binding moiety and / or the second BCMA binding moiety is an anti-BCMA VHH. In some embodiments, the first BCMA binding moiety is a first anti-BCMA VHH and the second BCMA binding moiety is a second anti-BCMA VHH. In some embodiments, the first BCMA binding moiety comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the first BCMA binding moiety comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:10. In some embodiments, the second BCMA binding moiety comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the second BCMA binding moiety comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:12.

[0070] In some embodiments, the first BCMA binding moiety and the second BCMA binding moiety are linked to each other via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the peptide linker comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 11.

[0071] In some embodiments, the CAR polypeptide further comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide is derived from CD8α. In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the signal peptide comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 9.

[0072] In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the transmembrane domain comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 14.

[0073] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the intracellular signaling domain is derived from CD3zeta. In some embodiments, the intracellular signaling domain comprises at least one costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:8. In some embodiments, the intracellular signaling domain comprises a polypeptide encoded by a nucleic acid sequence of SEQ ID NO:16. In some embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:7. In some embodiments, the intracellular signaling domain comprises a polypeptide encoded by a nucleic acid sequence of SEQ ID NO:15.

[0074] In some embodiments, the CAR polypeptide further comprises a hinge domain located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the hinge domain comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 13.

[0075] In some embodiments, the CAR comprises the amino acid sequence of SEQ ID NO:17.

[0076] In some embodiments, the T cells are autologous T cells. In some embodiments, the T cells are allogeneic T cells.

[0077] In some embodiments, the subject is a human.

[0078] In some embodiments, the subject has not been previously exposed to a BCMA targeted drug.

[0079] In some embodiments, the multiple myeloma is aggressive. [Brief description of the drawings]

[0080] 3. Brief description of the drawings [Figure 1] Figure 1 shows the expression of BCMA antigen on GC, memory cells and plasmablasts in lymph nodes, on the surface of long-lived plasma cells in bone marrow LN and MALT, and on multiple myeloma cells. BAFF-R antigen is not expressed on plasmablasts, long-lived plasma cells or multiple myeloma cells. TACI is expressed on memory cells and plasmablasts, long-lived plasma cells and multiple myeloma cells. CD138 is expressed only on long-lived plasma cells and multiple myeloma cells.

[0081] [Diagram 2]FIG. 1 shows the design of the ciltacabtagene autoleucel CAR. Ciltacabtagene autoleucel contains two VHH domains, rather than a single VL domain and a single VH domain expressed in various other CARs. Ciltacabtagene autoleucel contains intracellular CD137 and the human CD3 zeta domain.

[0082] [Diagram 3] FIG. 1 is a schematic for producing a virus encoding a siltacabtadine autolucel CAR, transducing the virus into T cells from a patient, and producing CAR T cells expressing siltacabtadine autolucel.

[0083] [Figure 4] Schematic diagram showing the study design of Siltacabtadine Autorcel CAR T cells. The patient population includes patients with relapsed or refractory multiple myeloma, refractory to three prior lines or both PI / IMiD, and previously exposed to PI, IMiD, and anti-CD38. The primary goal is to establish safety and RP2D, e.g., investigating incidence and severity of adverse events (Stage 1b). Another primary goal is efficacy: ORR-PR or better, as defined by the IMWG (Stage 2). Secondary goals are: incidence and severity of adverse events (Stage 2) and any further efficacy characterization.

[0084] [Diagram 5] Figure 1 shows the reduction in disease burden (representing the type of measurable disease, i.e. serum M protein, urinary M protein, or difference between impaired and intact free light chains (dFLC)) for Cohort A responders in the all treatment analysis population. "a" represents Bence-Jones proteinuria at baseline, where a transient response occurred during crosslinking therapy, and output represents dFLC values.

[0085] [Figure 6] FIG. 1 shows the reduction in disease burden (representing the type of measurable disease, i.e., serum M-protein, urinary M-protein, or difference between impaired and intact free light chains (dFLC)) in Cohort B responders in the all treatment analysis population.

[0086] [Figure 7] Figure 1 shows the reduction in disease burden (representing the type of measurable disease, i.e. serum M protein, urinary M protein, or difference between impaired and intact free light chains (dFLC)) for Cohort C responders in the all treatment analysis population. "a" represents Bence Jones protein at baseline, where a transient response occurred during crosslinking therapy, and output represents dFLC value. "b" represents non-estimable (NE) measurable disease type, and output represents dFLC value.

[0087] [Figure 8] FIG. 1 shows response assessment and duration of response for responders in Cohort A in the all treatment analysis population.

[0088] [Figure 9] FIG. 1 shows response assessment and duration of response for responders in Cohort B in the all treatment analysis population.

[0089] [Figure 10] FIG. 1 shows response assessment and duration of response for responders in Cohort C in the all treatment analysis population.

[0090] [Figure 11] Kaplan-Meier plot assessing duration of response among cohort A responders in the all treatment analysis population.

[0091] [Figure 12] Kaplan-Meier plot assessing duration of response among cohort B responders in the all treatment analysis population.

[0092] [Figure 13] Kaplan-Meier plot assessing duration of response among cohort A responders in the all treatment analysis population.

[0093] [Figure 14] Kaplan-Meier plot assessing progression-free survival for responders in Cohort A in the all treatment analysis population.

[0094] [Figure 15] Kaplan-Meier plot assessing progression-free survival for cohort B responders in the all treatment analysis population.

[0095] [Figure 16] Kaplan-Meier plot assessing progression-free survival for Cohort C responders in the all treatment analysis population. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0096] 4. Detailed Description The present disclosure provides methods of treating patients with multiple myeloma with CAR-T cells. In some embodiments, these methods relate to treating patients with multiple myeloma who have undergone previous therapy and have an overall survival rate of about 50% or less. The present disclosure further provides related nucleic acids, recombinant expression vectors, host cells, cell populations, antibodies or antigen-binding portions thereof, and pharmaceutical compositions, which relate to the immune cells and CAR-expressing T cells of the present disclosure. Dosage protocols and dosage forms are also provided.

[0097] In the following, some aspects of the present invention are described with reference to examples for illustrative purposes only. It should be understood that many specific details, relationships and methods are described to provide a thorough understanding of the present disclosure. However, as one skilled in the art can easily recognize, the present disclosure can be practiced without one or more specific details or can be practiced using other methods, regimens, reagents, cell lines and animals. The present disclosure is not limited to the order of acts or events described, since some acts may occur in different orders and / or simultaneously with other acts or events. It should be noted that not all acts, steps or events described are required to implement a method according to the present disclosure.

[0098] Unless otherwise defined, all terms, annotations and other scientific terms or vocabulary used herein are intended to have the meaning commonly understood by those skilled in the art. In some cases, for clarity and / or ease of reference, the present specification defines terms having a commonly understood meaning, and the specification including such definitions should not necessarily be interpreted as indicating a significant difference from the meaning commonly understood in the art. It is further understood that terms (as defined in a general dictionary) should be interpreted as having a meaning consistent with its meaning in the context of the relevant art and / or as otherwise defined in the present specification.

[0099] 4.1. Definition The terms "about" or "approximately" include within a statistically significant range of values. Such ranges may be within the order of a given value or range, preferably within 50%, more preferably within 20%, even more preferably within 10%, and even more preferably within 5%. The acceptable variation encompassed by the terms "about" or "approximately" will depend on the particular system under study and will be readily appreciated by one of ordinary skill in the art.

[0100] The term "protein" or "polypeptide" as used herein encompasses all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including, but not limited to, glycoproteins and all other types of modified proteins (e.g., proteins produced by phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc.).

[0101] Unless otherwise specified, the terms "nucleic acid," "nucleotide," and "polynucleotide" encompass both DNA and RNA. "Nucleic acid sequence" or "nucleotide sequence" refers to a nucleic acid sequence that encodes amino acids, and these terms can also refer to a nucleic acid sequence that includes portions that encode any amino acids (including any amino acids encoded by linkers) that are added as a cloned product.

[0102] The term "antibody" includes monoclonal antibodies (including full-length four-chain antibodies or full-length heavy-chain-only antibodies (having an immunoglobulin Fc region)), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies and single chain molecules), and antibody fragments (e.g., Fab, F(ab')2 and Fv). The terms "immunoglobulin" (Ig) and "antibody" are used interchangeably herein. Antibodies contemplated herein include single domain antibodies, e.g., heavy-chain-only antibodies. The terms "antibody and antibodies" refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fv (scFv), single chain antibodies, Fab fragments, F(ab') fragments, Fvs linked via disulfide bonds (sdFv), intrabodies, miniantibodies, diabodies and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies against antigen-specific TCRs), and epitope-binding fragments of any of the above. The terms "antibody" and "antibodies" further refer to covalent diabodies (e.g., those disclosed in U.S. Patent Application Publication No. 2007 / 0004909) and Ig-DARTS (e.g., those disclosed in U.S. Patent Application Publication No. 2009 / 0060910). Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgM1, IgM2, IgA1, and IgA2), or subclass.

[0103] A "full-length antibody" consists of two heavy chains (HC) and two light chains (LC) linked together via disulfide bonds and their multimers (e.g., IgM). Each heavy chain consists of a heavy chain variable domain (VH) and a heavy chain constant domain, which consists of the subdomains CH1, hinge, CH2, and CH3. Each light chain consists of a light chain variable domain (VL) and a light chain constant domain (CL). VH and VL may be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with framework regions (FWs). Each VH and VL consists of three CDRs and four FW segments, which are arranged from the amino group to the carboxy terminus in the following order: FW1, CDR1, FW2, CDR2, FW3, CDR3, and FW4.

[0104] "Complementarity determining region (CDR)" is an antigen-binding site in an antibody. CDR may be defined in various terms: (i) Complementarity determining region (CDR) (three in VH (HCDR1, HCDR2, HCDR3) and three in VL (LCDR1, LCDR2, LCDR3)) is based on sequence variability (Wu and Kabat, J. Exp. Med. 132:211-50, 1970; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991). (ii) "hypervariable region", "HVR" or "HV" (three in VH (H1, H2, H3) and three in VL (L1, L2, L3)) refers to the regions of an antibody variable domain that are highly variable in structure, as defined by Chothia and Lesk (Chothia and Lesk, Mol. Biol. 196:901-17, 1987). The International ImMunoGeneTics (IMGT) database (http: / / www_imgt_org) provides standard numbering and definitions of antigen-binding sites. The correspondence between CDR, HV and IMGT descriptions is described in Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003. As used herein, the terms "CDR", "HCDR1", "HCDR2", "HCDR3", "LCDR1", "LCDR2" and "LCDR3" include the CDRs as defined by any of the methods described above, Kabat, Chothia or IMGT, unless otherwise expressly indicated herein. The framework regions (FW) flank and are located between any of the CDRs in VL (LFW1, LFW2, LFW3, LFW4) and VH (HFW1, HFW2, HFW3, HFW4).

[0105] The term "heavy chain-only antibody" or "HCAb" includes functional antibodies, which contain heavy chains but lack the light chains normally found in four-chain antibodies. Camelids (e.g., camels, llamas, alpacas) are known to produce HCAbs.

[0106] The term "single domain antibody" or "sdAb" refers to a single antigen-binding polypeptide having three complementarity determining regions (CDRs). A single sdAb can bind to an antigen but does not pair with a corresponding CDR-containing polypeptide. In some cases, single domain antibodies are engineered from camelid HCAbs and the heavy chain variable domain is referred to herein as a "VHH". Some VHHs are also referred to as "nanobodies". Camlidid sdAbs are among the smallest antigen-binding antibody fragments known (see, e.g., Hamers-Casterman et al., Nature 363:446-8 (1993); Greenberg et al., Nature 374:168-73 (1995); Hassanzadeh-Ghassabeh et al., Nanomedicine (Lond), 8:1013-26 (2013)). A basic VHH has the structure, from N-terminus to C-terminus, FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3.

[0107] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The heavy and light chain variable domains may be referred to as "VH" and "VL", respectively. These domains are usually the most variable parts of the antibody (compared to other antibodies of the same class) and contain the antigen binding site. Heavy chain-only antibodies from camelid species have a single heavy chain variable region, referred to as "VHH". Thus, VHH is a special type of VH.

[0108] The term "variable" refers to the fact that the sequences of some segments of the variable domains vary widely between antibodies. The V domains (i.e., variable domains) mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, variability is not uniformly distributed over the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) in the light and heavy chain variable domains. The more conserved parts of the variable domains are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions connected via three HVRs that mainly adopt a β-sheet structure, which connect to form loops that connect the β-sheet structure and in some cases form part of the β-sheet structure. The HVRs in each chain are held tightly by the FR regions and help form the antigen-binding site of the antibody (if the antibody is not an sdAb, an HVR from another chain is used) (see Kabat et al., Sequences of Immunological Interest, 5th ed., National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding the antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

[0109] The terms "fragment of an antibody," "antibody fragment," "functional fragment of an antibody," and "antigen-binding portion" are used interchangeably herein and refer to one or more fragments or portions of an antibody, which fragment or portion retains the ability to specifically bind to an antigen (see generally, Holliger et al., Nat. Biotech., 23(9): 1 126-1129 (2005)). The antigen recognition portion of a CAR encoded by a nucleic acid sequence disclosed herein may contain any BCMA-binding antibody fragment. The antibody fragment desirably includes, for example, one or more CDRs, a variable region (or a portion thereof), a constant region (or a portion thereof), or a combination thereof. Examples of antibody fragments include: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; and (iv) a single-chain Fv (scFv), which is a monovalent molecule consisting of the two domains (i.e., VL and VH) of the Fv fragment linked via a synthetic linker, which allows the synthesis of the two domains into a single polypeptide chain (see, e.g., Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol, 16: 778 (1998)). (v) diabodies that are dimers of polypeptide chains, each polypeptide chain comprising a VH linked to a VL via a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby driving pairing between complementary domains on different VH-VL polypeptide chains to produce a dimeric molecule having two functional antigen binding sites. Antibody fragments are known in the art and are described in more detail, for example, in U.S. Patent Application Publication No. 2009 / 0093024 A1.Antigen-binding fragments may be synthetic, enzymatically obtainable or genetically engineered polypeptides and include parts of immunoglobulins that bind to the antigen, such as VH, VL, VH and VL, Fab, Fab', F(ab')2, Fd and Fv fragments, domain antibodies (dAbs) consisting of one VH domain or one VL domain, shark variable IgNAR domains, camelized VH domains, VHH domains, minimal recognition units consisting of amino acid residues mimicking antibody CDRs (e.g. FR3-CDR3-FR4 portions), HCDR1, HCDR2 and / or HCDR3 and LCDR1, LCDR2 and / or LCDR3, surrogate scaffolds that bind the antigen, and multispecific proteins comprising antigen-binding fragments. Antigen-binding fragments (e.g., VH and VL) can be linked together via synthetic linkers to form various types of single antibody designs, where the VH / VL domains can pair intramolecularly or intermolecularly when the VH and VL domains are expressed as separate single chains, thereby forming monovalent antigen-binding domains, e.g., single chain Fvs (scFvs) or diabodies. Antigen-binding fragments can be further conjugated to other antibodies, proteins, antigen-binding fragments or alternative scaffolds (which may be monospecific or multispecific) to engineer bispecific and multispecific proteins.

[0110] As used herein, the terms "specific binding," "specific recognition," or "having specificity for" refer to a measurable and reproducible interaction, such as the binding of a target to an antigen binding protein (e.g., a CAR or a VHH), that determines the presence of the target in the presence of a heteromolecular population that includes biomolecules.

[0111] The term "specificity" refers to the selective recognition of a particular epitope of an antigen by an antigen-binding protein (e.g., a CAR or a VHH). For example, a natural antibody is monospecific. The term "multispecificity" refers to an antigen-binding protein (e.g., a CAR or an antibody) having two or more antigen-binding sites, where at least two of the antigen-binding sites bind to different antigen-binding specificities. As used herein, "bispecificity" refers to an antigen-binding protein (e.g., a CAR or an antibody) having two different antigen-binding specificities.

[0112] As used herein, the term "operably linked" and similar phrases, when used to refer to a nucleic acid or amino acid, refer to the operably linking of a nucleic acid sequence or an amino acid sequence, respectively, which are placed in a functional relationship with each other. For example, when a promoter, an enhancer element, an open reading frame, a 5' and 3' UTR, and a terminator sequence are operably linked, the correct production of a nucleic acid molecule (e.g., RNA) occurs. In some embodiments, the operably linked nucleic acid elements cause the transcription of the open reading frame, which ultimately causes the production of a polypeptide (i.e., expression of the open reading frame). As another example, an operably linked peptide is one whose functional domains are positioned at an appropriate distance from each other, conferring an intended function to each domain.

[0113] A "chimeric antigen receptor" or "CAR" is an artificially constructed hybrid protein or polypeptide that contains at least one antibody (or antibody fragment) antigen-binding domain linked to a T cell signaling domain. CAR features may include the ability to redirect T cell specificity and reactivity to a selected target in a non-MHC-restricted manner (utilizing the antigen-binding properties of monoclonal antibodies). Non-MHC-restricted antigen recognition endows CAR-expressing T cells with the ability to recognize antigens (not dependent on antigen processing), thus circumventing a major mechanism of tumor escape. Advantageously, when expressed in T cells, CARs do not dimerize with endogenous T cell receptor (TCR) α and β chains. T cells expressing CARs are referred to herein as CAR T cells, CAR-T cells, or CAR-modified T cells, and these terms are used interchangeably herein. The cells can be genetically modified to stably express at least one antigen-antibody binding domain on their surface to confer novel MHC-independent antigen specificity. "BCMA CAR" refers to a CAR with an extracellular binding domain specific for BCMA. "Dual epitope CAR" refers to a CAR with extracellular binding domains specific for two different epitopes of an antigen (e.g., BCMA).

[0114] The terms "T cells" and "T lymphocytes" may be used interchangeably and synonymously herein. As used herein, T cells include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. T cells may be T helper (Th) cells, e.g., T helper 1 (Th1) or T helper 2 (Th2) cells. T cells may be helper T cells (HTL; CD4+ T cells), CD4+ T cells, cytotoxic T cells (CTL; ​​CD8+ T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4+CD8+ T cells, or any other T cell subgroup. Other exemplary T cell populations suitable for use in certain embodiments include naive T cells and memory T cells. Further included is "NKT cells", which refers to a special T cell population that expresses the semi-invariant αβ T cell receptor, but also expresses various molecular markers typically associated with NK cells (e.g., NK1.1). NKT cells include NK1.1+ and NK1.1-, as well as CD4+, CD4-, CD8+ and CD8- cells. The TCR in NKT cells is unique because it recognizes glycolipid antigens presented by the MHC I-like molecule CD1d. NKT cells can produce cytokines that promote inflammation or immune tolerance, and therefore can have protective or harmful effects. Further included is "γ-δ T cells (γδ T cells)", which refers to a special population, a small proportion of T cells that have a unique TCR on their surface, and which are distinct from most T cells, in that the TCR in most T cells consists of two glycoprotein chains (called α- and β-TCR chains), whereas the TCR in γδ T cells consists of one γ-chain and one δ-chain. γδ T cells can play a role in immune surveillance and immune regulation, and are an important source of IL-17, and are found to induce potent CD8+ cytotoxic T cell responses. Further includes "regulatory T cells" or "Tregs", which refer to T cells that suppress aberrant or excessive immune responses and play a role in immune tolerance. Tregs are typically transcription factor Foxp3 positive CD4+ T cells, and may include transcription factor Foxp3 negative regulatory T cells (IL-10 producing CD4+ T cells).

[0115] "Cilta-cel" ("cilta-cel") is a chimeric antigen receptor T cell (CAR-T) therapy that contains two VHH domains targeting B cell maturation antigen (BCMA) designed to confer avidity for BCMA. Cilta-cel may comprise T lymphocytes transduced with cilta-cel CAR (CAR encoded by lentiviral vector). The CAR targets human B cell maturation antigen (anti-BCMA CAR). Figure 2 provided a schematic diagram of the lentiviral vector encoding cilta-cel CAR. The amino acid sequence of cilta-cel CAR is that of SEQ ID NO: 17.

[0116] A "tumor cell" or "cancer cell" is a cancerous, precancerous or transformed cell in vivo, ex vivo or in tissue culture that has spontaneous or induced phenotypic changes. These changes do not necessarily involve the incorporation of new genetic material. Transformation can be caused by infection with a transforming virus and incorporation of new genomic nucleic acid, incorporation of exogenous nucleic acid, but can also occur naturally or after exposure to carcinogens (thereby mutating endogenous genes). Transformation / cancer is exemplified by in vitro, in vivo and ex vivo morphological changes, cell immortalization, aberrant growth control, lesion formation, proliferation, malignancy, modulation of tumor-specific marker levels, invasiveness, tumor growth in suitable animal hosts such as nude mice, and the like.

[0117] The terms "express" and "expression" mean to permit or cause the production of the information in a gene or DNA sequence. For example, expression can take the form of producing a protein by activating cellular functions involved in the transcription and translation of the corresponding gene or DNA sequence. A DNA sequence forms an "expression product," such as a protein, in a cell or by cellular expression. The expression product itself, e.g., the resulting protein, can also be referred to as being "expressed" by the cell. Expression products may be characterized as intracellular, extracellular, or transmembrane.

[0118] The term "treat" refers to a therapeutic treatment, where the objective is to alleviate or relieve an undesirable physiological change or disease, or to provide a beneficial or desired clinical outcome during treatment. Beneficial or desired clinical outcomes include alleviation of symptoms, reduction in the extent of disease, stabilization of the disease state (i.e., cessation of deterioration), delay or slowing of disease progression, improvement or mitigation of the disease state, and / or remission (whether partial or complete remission, and whether detectable or undetectable). "Treatment" may also mean extending survival compared to the expected survival if the subject were not receiving treatment. Subjects in need of treatment include subjects already suffering from an undesirable physiological change or disease, and subjects who are prone to suffering from a physiological change or disease. Treatment may also refer to a therapeutic agent, referred to herein as a "medicament" or "medication," which, by its action, is intended to aid in the realization of a beneficial or desired clinical outcome of interest. The therapeutic agent or drug may be administered to the subject via a number of routes, including at least intravenous and oral routes. The term "intravenous" in relation to administration of a therapeutic agent or drug refers to administering said therapeutic agent or drug into one or more veins. The term "oral" in relation to administration of a therapeutic agent or drug refers to administering said therapeutic agent or drug via an oral passageway, such as the mouth.

[0119] As used herein, the term "subject" refers to an animal. The terms "subject" and "patient" are used interchangeably herein when referring to a subject. Thus, "subject" refers to a person being treated for a disease or prevented as a patient. The methods described herein may be used to treat animal subjects belonging to any class. Examples of such animals include mammals. Mammals include, but are not limited to, rodent (order Rodentia) mammals, such as mice and hamsters, and lagomorph (order Logomorpha) mammals, such as rabbits. Mammals may be of the order Carnivora, including felines (cats) and canines (dogs). Mammals may be of the order Artiodactyla, including bovids (cattle) and porcines (pigs), or of the order Perssodactyla, including equids (horses). The mammal may be of the order Primate, Ceboid, or Simoid (monkeys) or the order Anthropoid (humans and monkeys). In some embodiments, the mammal is a human.

[0120] The term "effective" as applied to a dose or amount refers to the amount of a compound or drug composition sufficient to produce the desired activity after administration to a subject in need thereof. It should be noted that when a combination of active ingredients is administered, the effective amount of the combination may or may not include the amount of each ingredient that is effective when administered individually. The exact amount required will vary from subject to subject and will depend on the species, age and general condition of the subject, the severity of the condition being treated, the particular drug or drugs used, the administration pattern, etc.

[0121] The phrase "pharmacologically acceptable" as used in conjunction with the compositions described herein refers to molecular entities and other components of the composition that are physiologically tolerable and typically do not produce adverse reactions when administered to a mammal (e.g., a human). Preferably, the term "pharmacologically acceptable" means approved by a regulatory agency of the U.S. Federal or state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopoeias for use in animals, and more specifically, in humans.

[0122] The term "line of treatment" as used in conjunction with the method of treatment herein refers to one or more cycles of a planned treatment procedure, which may consist of one or more planned cycles of single agent or combination therapy and the treatment sequence administered in a planned manner. For example, a planned treatment regimen of induction therapy followed by autologous stem cell transplantation followed by maintenance is one line of treatment. A new line of treatment is considered to have been started if the planned course of treatment is amended to include other therapeutic agents or drugs (alone or in combination) due to disease progression, recurrence or toxicity. A new line of treatment is considered to have been started already if the observation period after the planned therapy is interrupted due to the need to administer additional treatment to the disease.

[0123] The term "refractory" as used herein in conjunction with treatment with a particular therapeutic agent or drug or line of therapy refers to a disease or subject with a disease that is not responsive to said therapeutic agent or drug or line of therapy. The phrase "refractory myeloma" refers to multiple myeloma that is not responsive to primary or rescue therapy or that has progressed within 60 days of the last therapy.

[0124] The phrase "refractory disease" refers to a disease in which a minimal response to therapy is not achieved or progresses to disease progression.

[0125] "Enhancement" or "promotion" or "increase" or "expand" or "improvement" generally refers to the ability of the compositions contemplated herein to generate, induce or cause a greater physiological response (i.e., downstream effects) compared to the response from a vehicle or control molecule / composition. Measurable physiological responses include T cell expansion, activation, effector function, persistent expansion and / or increased cancer cell killing capacity, and those apparent from the understanding in the art and the description herein. In some embodiments, an "increased" or "enhanced" amount may be a "statistically significant" amount and may include an increase of 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more (e.g., 500-fold, 1000-fold) (including all integers and decimal points greater than 1 in between, e.g., 1.5, 1.6, 1.7, 1.8, etc.) over the response from a vehicle or control composition.

[0126] "Reduction" or "reduction" or "mitigation" or "decrease" or "attenuation" generally refers to the ability of the compositions contemplated herein to generate, induce or cause a smaller physiological response (i.e., downstream effect) compared to the response from a vehicle or control molecule / composition. In some embodiments, the amount of "reduction" or "reduction" may be a "statistically significant" amount and may include a 1.1-fold, 1.2-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold or more (e.g., 500-fold, 1000-fold) decrease (including all integers and decimal points greater than 1 therebetween, e.g., 1.5, 1.6, 1.7, 1.8, etc.) of the response from the vehicle, the control composition (baseline response) or the response in a particular cell line.

[0127] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used herein, the indefinite articles "a / an" and "the" should be understood to include plural references unless the context clearly dictates otherwise.

[0128] Throughout the disclosure, each aspect of the disclosure may be presented in the form of a range. It should be understood that the description in range format is merely for convenience and brevity, and should not be interpreted as an inflexible limitation on the scope of the disclosure. Thus, the description of a range should be considered to have all possible subranges and individual numerical values ​​within the range precisely disclosed. For example, the description of a range such as 1-6 should be considered to have the precisely disclosed subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, and individual numerical values ​​within the range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range of, for example, 95% to 99% identity includes those with 95%, 96%, 97%, 98% or 99% identity, and includes subranges such as 96% to 99%, 96% to 98%, 96% to 97%, 97% to 99%, 97% to 98% and 98% to 99% identity, regardless of the breadth of the range.

[0129] Vectors Standard recombinant techniques can be used to obtain polynucleotide sequences encoding the CARs described in this application. The desired polynucleotide sequences can be isolated and sequenced from antibody producing cells, such as hybridoma cells. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques.

[0130] The present disclosure further provides a vector comprising a nucleic acid sequence encoding a CAR disclosed herein. The vector may be, for example, a plasmid, a cosmid, a viral vector (e.g., retrovirus or adenovirus) or a phage. Suitable vectors and methods for vector production are well known in the art (see, for example, Sambrook et al. and Ausubel et al.).

[0131] In addition to the nucleic acid sequence encoding the CAR disclosed herein, the vector preferably includes expression control sequences that provide for expression of the nucleic acid sequence in a host cell, such as a promoter, enhancer, polyadenylation signal, transcription terminator, internal ribosome entry site (IRES), etc. Exemplary expression control sequences are known in the art and described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, Vol. 185, Academic Press, San Diego, Calif. (1990).

[0132] In some embodiments, the vector comprises a promoter. Numerous promoters recognized by many potential host cells are well known. The selected promoter can be operably linked to the cistron DNA encoding the CAR disclosed herein by removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present application. Numerous promoters from many different sources are well known in the art, including constitutive, inducible and inhibitable promoters. Representative promoter sources include, for example, viral, mammalian, insect, plant, yeast and bacterial, and suitable promoters from these sources are readily available or can be produced synthetically based on publicly available sequences, for example, from depository centers such as ATCC and other commercial or private sources. Promoters can be unidirectional (i.e., initiate transcription in one direction) or bidirectional (i.e., initiate transcription in the 3' or 5' direction). Non-limiting examples of promoters include, for example, the T7 bacterial expression system, the pBAD(araA) bacterial expression system, the cytomegalovirus (CMV) promoter, the SV40 promoter and the RSV promoter. Inducible promoters include, for example, the Tet system (U.S. Pat. Nos. 5,464,758 and 5,814,618), the ecdysone-inducible system (No et al., Proc. Natl. Acad. Sci., 93: 3346-3351 (1996)), the T-REX™ system (Invitrogen, Carlsbad, Calif.), the LACSWITCH™ system (Stratagene, San Diego, Calif.), and the Cre-ERT tamoxifen-inducible recombinase system (Indra et al., Nuc. Acid. Res., 27: 4324- 4327 (1999), Nuc. Acid. Res., 28: e99 (2000); U.S. Pat. No. 7,112,715; and Kramer and Fussenegger, Methods Mol. Biol, 308: 123-144). (2005)).

[0133] In some embodiments, the vector includes an "enhancer." As used herein, the term "enhancer" refers to a DNA sequence that, for example, increases the transcription of a nucleic acid sequence operably linked to it. Enhancers may be located several kilobases away from the coding region of a nucleic acid sequence and can mediate the binding of regulatory factors, changes in DNA methylation patterns or DNA structure. Numerous enhancers from many different sources are well known in the art and are available as or within cloned polynucleotides (e.g., from depository centers such as ATCC and other commercial or private sources). Many polynucleotides that contain a promoter (e.g., the commonly used CMV promoter) also contain enhancer sequences. Enhancers can be located upstream, internal or downstream of a coding sequence. The term "Ig enhancer" refers to an enhancer element derived from an enhancer region that maps within the immunoglobulin (Ig) locus. Such Ig enhancers include, for example, the heavy chain (μ) 5′ enhancer, the light chain (κ) 5′ enhancer, the κ and μ intronic enhancers, and 3′ enhancers (see generally, Paul WE (ed), Fundamental Immunology, 3rd Edition, Raven Press, New York (1993), pages 353-363, and U.S. Patent No. 5,885,827).

[0134] In some embodiments, a vector contains a "selectable marker gene." As used herein, the term "selectable marker gene" refers to a nucleic acid sequence that allows cells expressing the nucleic acid sequence to be specifically selected or targeted in the presence of a corresponding selection agent. Suitable selectable marker genes are known in the art and are described, for example, in International Patent Application Publication Nos. WO 1992 / 08796 and WO 1994 / 28143, Wigler et al., Proc. Natl. Acad. Sci. USA, 77: 3567 (1980), O'Hare et al., Proc. Natl. Acad. Sci. USA, 78: 1527 (1981), Mulligan and Berg, Proc. Natl. Acad. Sci. USA, 78: 2072 (1981), Colberre-Garapin et al., J. Mol. Biol., 150: 1 (1981), Santerre et al., Gene, 30: 147 (1984), Kent et al., Science, 237: 901-903. (1987), Wigler et al., Cell, IP. 223 (1977), Szybalska and Szybalski, Proc. Natl. Acad. Sci. USA, 48: 2026 (1962), Lowy et al., Cell, 22: 817 (1980), and U.S. Patent Nos. 5,122,464 and 5,770,359.

[0135] In some embodiments, a vector is an "episomal expression vector" or "episome," which is replicable in a host cell and persists as an extrachromosomal segment of DNA in the host cell under appropriate selective pressure (see, e.g., Conese et al., Gene Therapy, 11: 1735-1742 (2004)). Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids that utilize the Epstein-Barr virus nuclear antigen 1 (EBNA1) and the Epstein-Barr virus (EBV) origin of replication (oriP). The vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, CA) and pB-CMV from Stratagene (La Jolla, CA) represent non-limiting examples of episomal vectors that use T antigen and the SV40 origin of replication instead of EBNA1 and oriP.

[0136] In some embodiments, the vector is an "integrative expression vector," which may integrate randomly into the DNA of a host cell, or may contain recombination sites to allow for recombination between the expression vector and specific sites in the chromosomal DNA of the host cell. Such integrative expression vectors can utilize endogenous expression control sequences of the host cell chromosome to achieve expression of a desired protein. Examples of vectors that integrate in a site-specific manner include, for example, components of the flp-in system from Invitrogen (Carlsbad, Calif.) (e.g., pcDNA TM 5 / FRT), or the cre-lox system, which can be found, for example, in the pExchange-6 core vector from Stratagene (La Jolla, Calif.). Examples of vectors that randomly integrate into a host cell chromosome include, for example, pcDNA3.1 (introduced in the absence of T antigen) from Invitrogen (Carlsbad, Calif.), and pCI or pFNI OA (ACT) FLEXI from Promega (Madison, Wis.). TM Examples include:

[0137] In some embodiments, the vector is a viral vector. Exemplary viral expression vectors include, but are not limited to, adenovirus-based vectors (e.g., the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands)), lentivirus-based vectors (e.g., the lentivirus-based pLP1 from Life Technologies (Carlsbad, Calif.)) and retrovirus-based vectors (e.g., pFB-ERV plus pCFB-EGSH from Stratagene (La Jolla, Calif.)). In a preferred embodiment, the vector is a lentivirus vector.

[0138] In some examples, a vector comprising a nucleic acid encoding a CAR disclosed herein is introduced into a host cell that may contain a heterologous nucleic acid. As used herein, the term "host cell" refers to any cell that contains a heterologous nucleic acid. The heterologous nucleic acid may be a vector (e.g., an expression vector). For example, a host cell may be a cell from any organism, selected, modified, transformed, propagated, used or engineered in any manner to produce a substance by the cell, e.g., the cell expresses genes, DNA or RNA sequences, proteins or enzymes. An appropriate host may be determined. For example, a host cell may be selected based on the vector backbone and the desired outcome. For example, a plasmid or cosmid may be introduced into a prokaryotic host cell to replicate some types of vectors. Bacterial cells such as, but not limited to, DH5α, JM109 and KCB, SURE® competent cells and SOLOPACK Gold cells may be used as host cells to replicate and / or express vectors. Bacterial cells such as E. coli LE392 may also be used as host cells for phage viruses. Eukaryotic cells that may be used as host cells include, but are not limited to, yeast (e.g., YPH499, YPH500, and YPH501), insects, and mammals. Examples of mammalian eukaryotic host cells for replicating and / or expressing vectors include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC12. Preferably, the host cell may be a cell that contains an expression vector. In a preferred embodiment, the host cell is one that can be easily and reliably grown, has a very fast growth rate, has a well-characterized expression system, and can be easily and efficiently transformed or transfected. The host cell may be a eukaryotic cell, such as a plant, animal, fungus, or algae, or a prokaryotic cell, such as a bacterium or protozoan. The host cell may be a cultured cell or a primary cell, i.e., one that is directly isolated from a living organism, e.g., a human. The host cell may be an adherent cell or a suspension cell, i.e., a cell that grows in suspension.Suitable host cells are known in the art and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK 293 cells, and the like. In a preferred embodiment, the host cell is a HEK 293 cell. In some embodiments, the HEK 293 cell is from the ATCC SD-3515 line. In some embodiments, the HEK 293 cell is from the IU-VPF MCB line. In some embodiments, the HEK 293 cell is from the IU-VPF MWCB line. In some embodiments, the host cell may be a peripheral blood lymphocyte (PBL), a peripheral blood mononuclear cell (PBMC), or a natural killer cell (NK). Preferably, the host cell is a natural killer (NK) cell. More preferably, the host cell is a T cell.

[0139] To amplify or replicate a recombinant expression vector, the host cell may be a prokaryotic cell, such as a DH5α cell. To produce a virus from a viral expression vector, the host cell may be a eukaryotic cell, such as a HEK 293 cell. To produce a recombinant CAR, the host cell may be a mammalian cell. The mammalian host cell is preferably a human cell. The host cell may be any cell type, from any type of tissue, and at any developmental stage. Methods for selecting suitable mammalian host cells, and for transforming, culturing, amplifying, screening, and purifying the cells are known in the art.

[0140] In some examples, the disclosure provides an isolated host cell that expresses a nucleic acid sequence encoding a CAR described herein.

[0141] In some embodiments, the host cell is a T cell. The T cells of the present disclosure may be any T cell, for example, a cultured T cell, for example, a primary T cell, or a T cell from a cultured T cell line, or a T cell obtained from a mammal. If obtained from a mammal, the T cells may be obtained from many sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. Additionally, the T cells may be enriched or purified. The T cells are preferably human T cells (e.g., isolated from a human). The T cells may be at any stage of development, including, but not limited to, CD4+ / CD8+ double positive T cells, CD4+ helper T cells, for example, Th and Th2 cells, CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, and the like. In one embodiment, the T cells are CD8+ T cells or CD4+ T cells. T cell lines are available, for example, from the American Type Culture Collection (ATCC, Manassas, VA) and the German Collection of Microbial Cell Cultures (DSMZ), and include, for example, Jurkat cells (ATCC TIB-152), Sup-Tl cells (ATCC CRL-1942), RPMI 8402 cells (DSMZ ACC-290), Karpas 45 cells (DSMZ ACC-545), and derivatives thereof.

[0142] In some embodiments, the host cell is a natural killer (NK) cell. NK cells are cytotoxic lymphocytes that function in the innate immune system. NK cells are defined as large granular lymphocytes and constitute a third class of cells differentiated from common lymphoid progenitors, which also give rise to B and T lymphocytes (see, e.g., Immunobiology, 5th ed., Janeway et al., eds., Garland Publishing, New York, NY (2001)). NK cells differentiate and mature in the bone marrow, lymph nodes, spleen, tonsils, and thymus. After maturation, NK cells enter the cycle as large lymphocytes with unique cytotoxic granules. NK cells can recognize and kill some abnormal cells, such as some tumor cells and virus-infected cells, and are believed to be important in the innate immune defense against intracellular pathogens. As discussed above with respect to T cells, the NK cells may be any NK cells, e.g., cultured NK cells, e.g., primary NK cells, or NK cells from a cultured NK cell line, or NK cells obtained from a mammal. If obtained from a mammal, the NK cells may be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. Additionally, the NK cells may be enriched or purified. The NK cells are preferably human NK cells (e.g., isolated from a human). NK cell lines are available, for example, from the American Type Culture Collection (ATCC, Manassas, VA), and include, for example, NK-92 cells (ATCC CRL-2407), NK92MI cells (ATCC CRL-2408), and derivatives thereof.

[0143] A nucleic acid sequence encoding a CAR disclosed herein can be introduced into a cell by "transfection," "transformation," or "transduction." As used herein, "transfection," "transformation," or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell by using physical or chemical methods.

[0144] The term "transformation" refers to the introduction of one or more exogenous polynucleotides into a bacterial cell that already has the ability to be transformed, for example, by using dimethylsulfoxide, divalent cations (e.g., calcium), or polyethylene glycol. Many transformation techniques are known in the art and include heat shock and electric shock.

[0145] The term "transfection" means the introduction of "foreign" (i.e., exogenous or extracellular) nucleic acid into a cell using recombinant DNA techniques. The term "genetic modification" means the introduction of a "foreign" (i.e., exogenous or extracellular) gene, DNA or RNA sequence into a host cell, whereby the host cell expresses the introduced gene or sequence to produce a desired substance, typically a protein or enzyme encoded by the introduced gene or sequence. The introduced gene or sequence may be referred to as a "cloned" or "foreign" gene or sequence and may include regulatory or control sequences, such as initiation sequences, termination sequences, promoter sequences, signal sequences, secretion sequences or other sequences used in the genetic machinery of the cell, that are operably linked to the polynucleotide encoding the chimeric antigen receptor. The gene or sequence may include non-functional sequences or sequences with no known function. A host cell that receives and expresses the introduced DNA or RNA is already "genetically engineered." The DNA or RNA introduced into a host cell may come from any source, including cells that are of the same genus or species as the host cell, or cells from a different genus or species. Many transfection techniques are known in the art and include, for example, calcium phosphate DNA co-precipitation (see, e.g., Murray EJ (ed.), Methods in Molecular Biology, Vol. 7, Gene Transfer and Expression Protocols, Humana Press (1991)), DEAE-dextran, electroporation, cationic liposome-mediated transfection, tungsten particle-facilitated microparticle bombardment (Johnston, Nature, 346: 776-777 (1990)), and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

[0146] The term "transduction" refers to the introduction of foreign nucleic acid into a cell using a viral vector. A phage or viral vector can be introduced into a host cell via the transduction of an infectious viral particle, which can be propagated in a suitable packaging cell, where many packaging cells are commercially available and known in the art.

[0147] The term "regulatory element" refers to any cis-acting genetic element in some embodiments that controls the expression of a nucleic acid sequence. In some embodiments, the term "promoter" includes substantially the minimal sequence required for transcription initiation. In some embodiments, the term "promoter" includes sequences that initiate transcription, and also further includes sequences that can upregulate or downregulate transcription, commonly referred to as "enhancer elements" and "repressor elements," respectively.

[0148] 4.3. Antibodies and their derived proteins Suitable methods for producing antibodies are known in the art, for example standard hybridoma methods described in, e.g., Kohler and Milstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA Janeway et al. (eds.), Immunobiology, 5th Ed., Garland Publishing, New York, NY (2001). Alternatively, other methods are known in the art, such as the EBV-hybridoma method (Haskard and Archer, J. Immunol. Methods, 74(2), 361-67 (1984), and Roder et al., Methods Enzymol., 121, 140-67 (1986)) and phage vector expression systems (see, e.g., Huse et al., Science, 246, 1275-81 (1989)). Methods for producing antibodies in non-human animals are also described, for example, in U.S. Patent Nos. 5,545,806, 5,569,825, and 5,714,352, and U.S. Patent Application Publication No. 2002 / 0197266 A1.

[0149] Phage display may be used to generate antibodies. In this regard, phage libraries encoding antigen-binding variable (V) domains of antibodies can be generated using standard molecular biology and recombinant DNA techniques (see, e.g., Sambrook et al., supra, and Ausubel et al., supra). Phage encoding variable regions with desired specificity are selected to specifically bind to the desired antigen, and a complete or partial antibody comprising the selected variable domain is reconstituted. The nucleic acid sequence encoding the reconstituted antibody is introduced into an appropriate cell line (e.g., myeloma cells to produce hybridomas), whereby antibodies with monoclonal antibody characteristics are secreted from the cells (see, e.g., Janeway et al., supra, Huse et al., supra, and U.S. Pat. No. 6,265,150).

[0150] The antibodies, polypeptides and proteins (including functional portions and functional variants) of the embodiments of the present disclosure may be post-translationally modified. They may be glycosylated, esterified, N-acylated, amidated, carboxylated, phosphorylated, for example, via cyclization by disulfide bridges, or converted to acid addition salts. In some embodiments, they may be dimerized or polymerized, or conjugated.

[0151] The antibodies, polypeptides and / or proteins (including functional portions and functional variants thereof) of the embodiments of the present disclosure can be obtained by methods known in the art. Suitable methods for de novo synthesis of polypeptides and proteins are described in the following references, for example, Chan et al., Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000, Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000, and Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001. Furthermore, the produced polypeptides and proteins can be recombined by standard recombinant methods using the nucleic acids described herein. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001, and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. Also, some antibodies, polypeptides and proteins (including functional portions and functional variants thereof) of the present disclosure may be isolated and / or purified from sources (e.g., plants, bacteria, insects, mammals, etc.). Methods of isolation and purification are known in the art. Alternatively, the antibodies, polypeptides and / or proteins (including functional portions and functional variants thereof) described herein may be commercially synthesized. In this regard, the antibodies, polypeptides and proteins may be synthetic, recombinant, isolated and / or purified.

[0152] 4.4. Chimeric Antigen Receptors International Patent No. WO 2018 / 028647 is incorporated herein by reference in its entirety. U.S. Patent No. 2018 / 0230225 is incorporated herein by reference in its entirety. International Patent Application No. PCT / CN2020 / 133598 is incorporated herein by reference in its entirety.

[0153] The present disclosure provides a method of treating a subject with a cell expressing a chimeric antigen receptor (CAR). The CAR comprises an extracellular antigen binding domain that contains one or more single domain antibodies. In various embodiments, a BCMA-targeting CAR (also referred to herein as a "BCMA CAR") is provided, which comprises a polypeptide, the polypeptide comprising (a) an extracellular antigen binding domain comprising an anti-BCMA binding moiety, (b) a transmembrane domain, and (c) an intracellular signaling domain. In some embodiments, the anti-BCMA binding moiety is camelid, chimeric, human, or humanized. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell (e.g., a T cell). In some embodiments, the primary intracellular signaling domain is derived from CD4. In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain comprises a costimulatory signaling domain. In some embodiments, the costimulatory signaling domain is derived from a costimulatory molecule selected from the group consisting of CD27, CD28, CD137, OX40, CD30, CD40, CD3, LFA-1, ICOS, CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand of CD83, and combinations thereof. In some embodiments, the costimulatory signaling domain is derived from CD137.

[0154] In some embodiments, the BCMA CAR further comprises a hinge domain (e.g., a CD8α hinge domain) located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain. In some embodiments, the BCMA CAR further comprises a signal peptide (e.g., a CD8α signal peptide) located at the N-terminus of the polypeptide. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular antigen binding domain, a CD8α hinge domain, a CD28 transmembrane domain, a first costimulatory signaling domain derived from CD28, a second costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD4. In some embodiments, the polypeptide comprises, from N-terminus to C-terminus, a CD8α signal peptide, an extracellular antigen binding domain, a CD8α hinge domain, a CD8α transmembrane domain, a second costimulatory signaling domain derived from CD137, and a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the BCMA CAR is monospecific. In some embodiments, the BCMA CAR is monovalent.

[0155] The present application further provides a CAR having two or more (including but not limited to any one of 2, 3, 4, 5, 6 or more) binding moieties that specifically bind to an antigen, such as BCMA. In some embodiments, one or more binding moieties are antigen-binding fragments. In some embodiments, one or more binding moieties comprise a single domain antibody. In some embodiments, one or more binding moieties comprise a VHH.

[0156] In some embodiments, the CAR is a multivalent (e.g., bivalent, trivalent or higher) CAR comprising a polypeptide, the polypeptide comprising (a) an extracellular antigen-binding domain comprising a plurality (e.g., at least about any one of 2, 3, 4, 5, 6 or more) binding moieties that specifically bind to an antigen (e.g., a tumor antigen); (b) a transmembrane domain; and (c) an intracellular signaling domain.

[0157] In some embodiments, the binding moieties, e.g., VHHs (including multiple VHHs, or a first VHH and / or a second VHH) are camelid, chimeric, human or humanized. In some embodiments, the binding moieties or VHHs are linked to each other via peptide bonds or peptide linkers. In some embodiments, each peptide linker is no more than about 50 (e.g., no more than about any one of 35, 25, 20, 15, 10 or 5) amino acids in length.

[0158] In some embodiments the first BCMA binding moiety and / or the second BCMA binding moiety is an anti-BCMA VHH. In some embodiments the first BCMA binding moiety is a first anti-BCMA VHH and the second BCMA binding moiety is a second anti-BCMA VHH.

[0159] In some embodiments, the first anti-BCMA binding moiety comprises a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the first anti-BCMA binding moiety comprises a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the first anti-BCMA binding moiety comprises a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the first BCMA binding moiety comprises the amino acid sequence of SEQ ID NO: 2. In some embodiments, the first BCMA binding moiety comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 10. In some embodiments, the first anti-BCMA binding moiety comprises one or more or all of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 2. These sequences correspond to sequences present in cilta-cel.

[0160] In some embodiments, the second BCMA binding moiety comprises a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO:21. In some embodiments, the second BCMA binding moiety comprises a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO:22. In some embodiments, the second BCMA binding moiety comprises a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO:23. In some embodiments, the second BCMA binding moiety comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the second BCMA binding moiety comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:12. In some embodiments, the second anti-BCMA binding moiety comprises one or more or all of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23 and SEQ ID NO:4. These sequences correspond to sequences present in cilta-cel.

[0161] In some embodiments, the first BCMA binding moiety and the second BCMA binding moiety are linked to each other via a peptide linker. In some embodiments, the peptide linker comprises the amino acid sequence of SEQ ID NO: 3. In some embodiments, the peptide linker comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 11.

[0162] In some embodiments, the CAR further comprises a hinge domain (e.g., a CD8α hinge domain) located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain. In some embodiments, the CAR further comprises a signal peptide (e.g., a CD8α signal peptide) located at the N-terminus of the polypeptide.

[0163] Without wishing to be bound by theory, multivalent CARs or those CARs comprising an extracellular antigen binding domain containing a first BCMA binding moiety and a second BCMA binding moiety may be particularly suitable for targeting multimeric antigens through synergistic binding of different antigen binding sites or for enhancing binding affinity or avidity for antigens. Improved avidity may allow for a significant reduction in the dose of CAR-T cells required to achieve a therapeutic effect, for example in the range of 4.0 x 10 4 ~1.0 × 10 6 CAR-T cells / kilogram of subject mass, or a total of 3.0 x 10 6 ~1.0 × 10 8 of CAR-T expressing cells. Monovalent CARs, such as bb2121, may need to be administered at 5-10 times these amounts to achieve comparable efficacy. In various embodiments, the reduced dose ranges can provide a significant reduction in cytokine release syndrome (CRS) and other potentially dangerous side effects of CAR-T therapy.

[0164] The various binding moieties (e.g., extracellular antigen binding domains including a first BCMA binding moiety and a second BCMA binding moiety) in the CAR described herein can be linked to each other via a peptide linker. The peptide linkers linking the different binding moieties (e.g., VHHs) can be the same or different. The different domains of the CAR can also be linked to each other via a peptide linker. In some examples, the binding moieties (e.g., VHHs) are directly linked to each other without any peptide linker.

[0165] The peptide linker in the CAR described herein may have any suitable length. In some embodiments, the length of the peptide linker is at least about any one of the following: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 75, 100 or more amino acids. In some embodiments, the length of the peptide linker is less than or equal to about any one of the following: 100, 75, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5 or fewer amino acids. In some embodiments, the length of the peptide linker is any one of about 1 amino acid to about 10 amino acids, about 1 amino acid to about 20 amino acids, about 1 amino acid to about 30 amino acids, about 5 amino acids to about 15 amino acids, about 10 amino acids to about 25 amino acids, about 5 amino acids to about 30 amino acids, about 10 amino acids to about 30 amino acids, about 30 amino acids to about 50 amino acids, about 50 amino acids to about 100 amino acids, or about 1 amino acid to about 100 amino acids.

[0166] The CAR of the present application comprises a transmembrane domain that can be directly or indirectly linked to an extracellular antigen-binding domain.

[0167] The CAR may comprise a T cell activation moiety. The T cell activation moiety may be any suitable moiety derived or derived from any suitable molecule. In one embodiment, for example, the T cell activation moiety comprises a transmembrane domain. The transmembrane domain may be any transmembrane domain derived or derived from any molecule known in the art. For example, the transmembrane domain may be derived or derived from a CD8α molecule or a CD28 molecule. Without wishing to be bound by theory, CD8 is a transmembrane glycoprotein that functions as a co-receptor for the T cell receptor (TCR) and is primarily expressed on the surface of cytotoxic T cells. The most common form of CD8 exists as a dimer composed of CD8α and CD8β chains. CD28 is expressed on T cells and provides a costimulatory signal necessary for T cell activation. CD28 is a receptor for CD80 (B7.1) and CD86 (B7.2). In a preferred embodiment, CD8α and CD28 are human.

[0168] In addition to the transmembrane domain, the T cell activation moiety may further comprise an intracellular (i.e., cytoplasmic) T cell signaling domain. The intracellular T cell signaling domain may be obtained or derived from a CD28 molecule, a CD3ζ molecule or modified forms thereof, a human Fc receptor gamma (FcRγ) chain, a CD27 molecule, an OX40 molecule, a 4-1BB molecule, or other intracellular signaling molecules known in the art. Without wishing to be bound by theory, (1) CD28 is a T cell marker important for T cell costimulation, (2) CD3ζ associates with the TCR to produce a signal and contains an immunoreceptor tyrosine-based activation motif (ITAM), and (3) 4-1BB, also called CD137, delivers a strong costimulatory signal to T cells, promotes differentiation of T lymphocytes, and improves long-term survival of T lymphocytes. In a preferred embodiment, CD28, CD3ζ, 4-IBB, OX40, and CD27 are human.

[0169] The T cell activation domain of a CAR encoded by a nucleic acid sequence disclosed herein may comprise any one of the above transmembrane domains in any combination with any one or more of the above intercellular T cell signaling domains. For example, a nucleic acid sequence disclosed herein can encode a CAR comprising a CD28 transmembrane domain and the intracellular T cell signaling domains of CD28 and CD3ζ. Alternatively, for example, a nucleic acid sequence disclosed herein can encode a CAR comprising a CD8α transmembrane domain and the intracellular T cell signaling domains of CD28, CD3ζ, Fc receptor gamma (FcRy) chain, and / or 4-1 BB.

[0170] In some embodiments, the CAR polypeptide further comprises a signal peptide located at the N-terminus of the polypeptide. In some embodiments, the signal peptide is derived from CD8α (CD8α SP). In some embodiments, the signal peptide comprises the amino acid sequence of SEQ ID NO: 1. In some embodiments, the signal peptide comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 9.

[0171] In some embodiments, the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the transmembrane domain comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 14.

[0172] In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. In some embodiments, the intracellular signaling domain is derived from CD3zeta. In some embodiments, the intracellular signaling domain comprises at least one costimulatory signaling domain. In some embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:8. In some embodiments, the intracellular signaling domain comprises a polypeptide encoded by a nucleic acid sequence of SEQ ID NO:16. In some embodiments, the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:7. In some embodiments, the intracellular signaling domain comprises a polypeptide encoded by a nucleic acid sequence of SEQ ID NO:15.

[0173] In some embodiments, the CAR polypeptide further comprises a hinge domain located between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain comprises the amino acid sequence of SEQ ID NO: 5. In some embodiments, the hinge domain comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 13.

[0174] In some embodiments, the CAR comprises one or more or all of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23. In one embodiment, the CAR comprises SEQ ID NO:17. In some embodiments, the CAR comprises a polypeptide encoded by one or more or all of the nucleic acid sequences of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, and SEQ ID NO:16.

[0175] 4.5. Immune Effector Cell Compositions "Immune effector cell" refers to an immune cell capable of exerting an immune effector function. In some embodiments, the immune effector cell expresses at least FcγRIII and exerts ADCC effector function. Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils. In some embodiments, the immune effector cell is a T cell. In some embodiments, the T cell is an autologous T cell. In some embodiments, the T cell is an allogeneic T cell. In some embodiments, the T cell is CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8-, or a combination thereof. In some embodiments, the T cell expresses a CAR and produces IL-2, TNF, and / or TNF after binding to a target cell (e.g., a CD20+ or ​​CD19+ tumor cell). In some examples, CD8+ T cells express the CAR and lyse the antigen-specific target cells after binding to the target cells.

[0176] Biological methods of introducing vectors into immune effector cells include using DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Chemical methods of introducing vectors into immune effector cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0177] This specification is 3.0 × 10 7 ~1.0 × 10 8The present invention provides a dosage form comprising 3.0×10 CAR-T cells, the CAR-T cells comprising a CAR containing a polypeptide, the polypeptide comprising (a) an extracellular antigen binding domain comprising a first BCMA binding moiety that specifically binds to a first epitope of BCMA and a second BCMA binding moiety that specifically binds to a second epitope of BCMA, (b) a transmembrane domain, and (c) an intracellular signaling domain, wherein the first epitope and the second epitope are different. In some embodiments, the CAR-T cells are 3.0×10 7 ~1.0 × 10 8 In some embodiments, the dosage form comprises 3.0×10 engineered immune effector cells (e.g., T cells), the engineered immune effector cells comprising a CAR containing a polypeptide, the polypeptide comprising (a) an extracellular antigen binding domain comprising a first anti-BCMA VHH that specifically binds to a first epitope of BCMA and a second anti-BCMA VHH that specifically binds to a second epitope of BCMA, (b) a transmembrane domain, and (c) an intracellular signaling domain, where the first epitope and the second epitope are different. In some embodiments, the dosage form comprises 3.0×10 7 ~4.0 × 10 7 In some embodiments, the dosage form contains 3.5 × 10 CAR-T cells. 7 ~4.5 × 10 7 In some embodiments, the dosage form contains 4.0 × 10 CAR-T cells. 7 ~5.0 × 10 7 In some embodiments, the dosage form contains 4.5 × 10 CAR-T cells. 7 ~5.5 × 10 7 In some embodiments, the dosage form contains 5.0 × 10 CAR-T cells. 7 ~6.0 × 10 7 In some embodiments, the dosage form contains 5.5 x 10 CAR-T cells. 7 ~6.5 × 10 7 In some embodiments, the dosage form contains 6.0 × 10 CAR-T cells. 7~7.0 × 10 7 In some embodiments, the dosage form contains 6.5 × 10 CAR-T cells. 7 ~7.5 × 10 7 In some embodiments, the dosage form contains 7.0 × 10 CAR-T cells. 7 ~8.0 × 10 7 In some embodiments, the dosage form contains 7.5 × 10 CAR-T cells. 7 ~8.5 × 10 7 In some embodiments, the dosage form contains 8.0 × 10 CAR-T cells. 7 ~9.0 × 10 7 In some embodiments, the dosage form contains 8.5 × 10 CAR-T cells. 7 ~9.5 × 10 7 In some embodiments, the dosage form contains 9.0 x 10 CAR-T cells. 7 ~1.0 × 10 8 Contains CAR-T cells.

[0178] In some embodiments, the cell population of the CAR-T formulation described herein includes, for example, different stages of differentiation, T cells or T cell populations. Stages of T cell differentiation include naive T cells, stem central memory T cells, central memory T cells, effector memory T cells, and final effector T cells, from the least differentiated to the most differentiated. After antigen exposure, naive T cells proliferate and differentiate into memory T cells, for example, stem central memory T cells and central memory T cells, and then into effector memory T cells. After receiving the appropriate T cell receptor, costimulatory signals, and inflammatory signals, memory T cells further differentiate into final effector T cells. See, for example, Restifo. Blood. 124.4(2014):476-77, and Joshi et al. J. Immunol. 180.3(2008):1309-15.

[0179] Naive T cells may have the following expression pattern of cell surface markers: CCR7+, CD62L+, CD45RO-, CD95-. Stem central memory T cells (Tscm) may have the following expression pattern of cell surface markers: CCR7+, CD62L+, CD45RO-, CD95+. Central memory T cells (Tcm) may have the following expression pattern of cell surface markers: CCR7+, CD62L+, CD45RO+, CD95+. Effector memory T cells (Tem) may have the following expression pattern of cell surface markers: CCR7-, CD62L-, CD45RO+, CD95+. Final effector T cells (Teff) may have the following expression pattern of cell surface markers: CCR7-, CD62L-, CD45RO-, CD95+. See, e.g., Gattinoni et al. Nat. Med. 17(2011):1290-7, and Flynn et al. Clin. Translat. Immunol. 3(2014):e20.

[0180] 4.6. Pharmaceutical Compositions and Formulations The present application further provides a pharmaceutical composition, which comprises any one of the engineered immune effector cells comprising any one of the anti-BCMA antibodies disclosed herein or any one of the CARs (e.g., BCMA CARs) described herein, and a pharmaceutically acceptable carrier agent. The pharmaceutical composition can be prepared by mixing any of the immune effector cells having a desired purity described herein with an optional pharmaceutically acceptable carrier agent, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or an aqueous solution. In some embodiments, the CAR-T cell drug composition further comprises an excipient selected from dimethyl sulfoxide or dextran-40.

[0181] The compositions described herein can be administered as part of a pharmaceutical composition that includes one or more carrier agents. The choice of carrier agent is determined in part by the particular nucleic acid sequence, vector, or host cell expressing the CAR disclosed herein, and the particular method for administering the nucleic acid sequence, vector, or host cell expressing the CAR disclosed herein. As such, there are multiple suitable formulations of the pharmaceutical compositions of the present disclosure.

[0182] For example, the pharmaceutical composition may contain a preservative. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate and benzalkonium chloride. Optionally, a mixture of two or more preservatives may be used. The preservative or mixture thereof is typically present in an amount of about 0.0001% to about 2% by weight of the total composition.

[0183] A buffering agent may also be used in the composition. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. Optionally, a mixture of two or more buffering agents may be used. The buffering agent or mixture thereof is typically present in an amount of about 0.001% to about 4% by weight of the total composition.

[0184] A composition comprising a nucleic acid sequence encoding a CAR disclosed herein or a host cell expressing a CAR disclosed herein may be formulated as an inclusion complex, such as a cyclodextrin inclusion complex, or a liposome. Liposomes may be used to target host cells (e.g., T cells or NK cells) or the nucleic acid sequences disclosed herein to a specific tissue. Liposomes may also be used to increase the half-life of the nucleic acid sequences disclosed herein. Many methods may be used to prepare liposomes, such as those described in, for example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9: 467 (1980), and U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369. The compositions may use timed, delayed and sustained release delivery systems so that delivery of the compositions disclosed herein occurs before sensitization of the site to be treated and has sufficient time to cause sensitization of the site to be treated.Many types of release delivery systems are available and known to those skilled in the art.Such systems increase convenience for subjects and physicians by avoiding repeated administration of the composition, and may be particularly suitable for some embodiments of the compositions disclosed herein.

[0185] In some embodiments, the CAR-T cells are about 1.0 × 10 5 ~2.0 × 10 5 Cells / kg, 1.5 × 10 5 ~2.5 × 10 5 Cells / kg, 2.0 x 10 5 ~3.0 × 10 5 Cells / kg, 2.5 × 10 5 ~3.5 × 10 5 Cells / kg, 3.0 x 10 5 ~4.0 × 10 5 Cells / kg, 3.5 × 10 5 ~4.5 × 10 5 Cells / kg, 4.0 x 10 5 ~5.0 × 10 5Cells / kg, 4.5 × 10 5 ~5.5 × 10 5 Cells / kg, 5.0 x 10 5 ~6.0 × 10 5 Cells / kg, 5.5 × 10 5 ~6.5 × 10 5 Cells / kg, 6.0 × 10 5 ~7.0 × 10 5 Cells / kg, 6.5 × 10 5 ~7.5 × 10 5 Cells / kg, 7.0 × 10 5 ~8.0 × 10 5 Cells / kg, 7.5 × 10 5 ~8.5 × 10 5 Cells / kg, 8.0 × 10 5 ~9.0 × 10 5 Cells / kg, 8.5 × 10 5 ~9.5 × 10 5 Cells / kg, 9.0 × 10 5 ~1.0 × 10 6 In a preferred embodiment, the dose is about 0.75 x 10 cells / kg. 6 In some embodiments, the CAR-T cells are formulated at 1.0 x 10 cells / kg. 8 It is formulated in doses of less than 1 cell / subject.

[0186] 4.7. Methods of Treating Subjects The present application further relates to methods and compositions for use in cellular immunotherapy. In some embodiments, the cellular immunotherapy is for treating cancer in a subject, including but not limited to hematological malignancies and solid tumors. In some embodiments, the subject is a human. In some embodiments, the methods are suitable for treating adult and pediatric populations, including all age subgroups, and may be used as any line of treatment, including first or subsequent lines.

[0187] Any of the anti-BCMA VHHs, CARs and engineered immune effector cells (e.g., CAR-T cells) described herein can be used in methods to treat cancer. In some embodiments, the immune effector cells are autologous. In some embodiments, the immune effector cells are allogeneic.

[0188] In some embodiments, the CAR-T cells are about 1.0 × 10 5 ~2.0 × 10 5 Cells / kg, 1.5 × 10 5 ~2.5 × 10 5 Cells / kg, 2.0 x 10 5 ~3.0 × 10 5 Cells / kg, 2.5 × 10 5 ~3.5 × 10 5 Cells / kg, 3.0 x 10 5 ~4.0 × 10 5 Cells / kg, 3.5 × 10 5 ~4.5 × 10 5 Cells / kg, 4.0 x 10 5 ~5.0 × 10 5 Cells / kg, 4.5 × 10 5 ~5.5 × 10 5 Cells / kg, 5.0 x 10 5 ~6.0 × 10 5 Cells / kg, 5.5 × 10 5 ~6.5 × 10 5 Cells / kg, 6.0 × 10 5 ~7.0 × 10 5 Cells / kg, 6.5 × 10 5 ~7.5 × 10 5 Cells / kg, 7.0 × 10 5 ~8.0 × 10 5 Cells / kg, 7.5 × 10 5 ~8.5 × 10 5 Cells / kg, 8.0 × 10 5 ~9.0 × 10 5 Cells / kg, 8.5 × 10 5~9.5 × 10 5 Cells / kg, 9.0 × 10 5 ~1.0 × 10 6 Cells / kg, 1.0 x 10 6 ~2.0 × 10 6 Cells / kg, 1.5 × 10 6 ~2.5 × 10 6 Cells / kg, 2.0 x 10 6 ~3.0 × 10 6 Cells / kg, 2.5 × 10 6 ~3.5 × 10 6 Cells / kg, 3.0 x 10 6 ~4.0 × 10 6 Cells / kg, 3.5 × 10 6 ~4.5 × 10 6 Cells / kg, 4.0 x 10 6 ~5.0 × 10 6 Cells / kg, 4.5 × 10 6 ~5.5 × 10 6 cells / kg or 5.0 x 10 6 ~6.0 × 10 6 In a preferred embodiment, the dose is about 0.75 x 10 cells / kg. 6 In some embodiments, the CAR-T cells are about 1.0 x 10 8 The antibody will be administered at a dose of cells / subject.

[0189] In some embodiments, the CAR-T cells are 1.0 × 10 8 In some embodiments, the CAR-T cells are administered at a dose of about 3.0-4.0 x 10 cells / subject. 7 In some embodiments, the CAR-T cells are administered at a dose of about 3.5-4.5 x 10 cells. 7 In some embodiments, the CAR-T cells are administered at a dose of about 4.0-5.0 × 10 7 In some embodiments, the CAR-T cells are administered at a dose of about 4.5-5.5 x 10 cells. 7In some embodiments, the CAR-T cells are administered at a dose of about 5.0-6.0 × 10 7 In some embodiments, the CAR-T cells are administered at a dose of about 5.5-6.5 x 10 cells. 7 In some embodiments, the CAR-T cells are administered at a dose of about 6.0-7.0 × 10 7 In some embodiments, the CAR-T cells are administered at a dose of about 6.5-7.5 x 10 cells. 7 In some embodiments, the CAR-T cells are administered at a dose of about 7.0-8.0 × 10 cells. 7 In some embodiments, the CAR-T cells are administered at a dose of about 7.5-8.5 x 10 cells. 7 In some embodiments, the CAR-T cells are administered at a dose of about 8.0-9.0 × 10 cells. 7 In some embodiments, the CAR-T cells are administered at a dose of about 8.5-9.5 x 10 cells. 7 In some embodiments, the CAR-T cells are administered at a dose of about 9.0 x 10 cells. 7 ~1.0 × 10 8 The cells are administered in doses of 100 cells.

[0190] In some embodiments, the CAR-T cells are about 0.693 × 10 6 In some embodiments, the CAR-T cells are administered at a dose of about 0.52 × 10 live CAR-positive T cells / kg. 6 In some embodiments, the CAR-T cells are administered at a dose of about 0.94 × 10 live CAR-positive T cells / kg. 6 In some embodiments, the CAR-T cells are administered at a dose of about 0.709 × 10 live CAR-positive T cells / kg. 6 In some embodiments, the CAR-T cells are administered at a dose of about 0.51 × 10 live CAR-positive T cells / kg. 6In some embodiments, the CAR-T cells are administered at a dose of about 0.95 × 10 live CAR-positive T cells / kg. 6 The CAR-T cells are administered at a dose of 100 mg / kg of live CAR-positive T cells. In some embodiments, the CAR-T cells are administered in an outpatient setting.

[0191] In some embodiments, the CAR-T cells are administered (e.g., at any of the doses described above) in one or more intravenous infusions. In some embodiments, the administration of the CAR-T cells is by a single intravenous infusion. In some embodiments, the single intravenous infusion is administered using one bag of the CAR-T cells. In some embodiments, the administration of the one bag of the CAR-T cells is completed between the time of thawing the one bag of CAR-T cells and 3 hours after thawing the one bag of CAR-T cells. In some embodiments, a single intravenous administration is administered using two bags of the CAR-T cells. In some embodiments, the administration of each bag of the two bags of the CAR-T cells is completed between the time of thawing the first bag of the two bags of CAR-T cells and 3 hours after thawing the first bag of CAR-T cells.

[0192] In some embodiments, the time from initial apheresis to administration of CAR-T cells is less than 41, 47, 54, 61, 68, 75, 82, 89, 96, 103, 110, 117, 124, 131, 138, 145, 152, 159, 166, or 167 days. In some embodiments, the time from initial apheresis to administration of CAR-T cells is more than 41, 47, 54, 61, 68, 75, 82, 89, 96, 103, 110, 117, 124, 131, 138, 145, 152, 159, 166, or 167 days.

[0193] The composition may be administered to the mammal using standard administration techniques, including host cells expressing a CAR-encoding nucleic acid sequence as disclosed herein, or a vector comprising a CAR-encoding nucleic acid sequence as disclosed herein, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository administration. The composition is preferably suitable for parenteral administration. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the composition is administered to the mammal by intravenous, intraperitoneal, or subcutaneous injection using peripheral systemic delivery. Most preferably, the composition is administered by intravenous infusion.

[0194] A composition comprising a host cell expressing a CAR-encoding nucleic acid sequence disclosed herein or a vector comprising a CAR-encoding nucleic acid sequence disclosed herein can be administered with one or more additional therapeutic agents, and these therapeutic agents can be co-administered to a mammal. "Co-administration" refers to administration of one or more additional therapeutic agents and a composition comprising a host cell disclosed herein or a vector disclosed herein sufficiently close in time that a CAR disclosed herein can potentiate the effect of the one or more other therapeutic agents, or vice versa. In this embodiment, a composition comprising a host cell disclosed herein or a vector disclosed herein can be administered first, and then one or more additional therapeutic agents can be administered, or vice versa.

[0195] The CAR-expressing cells described herein and at least one additional therapeutic agent may be administered simultaneously (in the same or separate compositions) or sequentially. In the case of sequential administration, the CAR-expressing cells described herein may be administered first and the additional agent may be administered second, or the order of administration may be reversed.

[0196] In some embodiments, the lymphodepletion regimen is administered about 5 to about 7 days prior to administration of the CAR-T cells. In some embodiments, the lymphodepletion regimen is administered intravenously. In some embodiments, the lymphodepletion regimen includes administration of cyclophosphamide or administration of fludarabine. In some embodiments, the cyclophosphamide is administered at a dose of 300 mg / m 2 In some embodiments, the fludarabine is administered intravenously at a dose of 30 mg / m 2 In some embodiments, cyclophosphamide is administered intravenously at 300 mg / m 2 and fludarabine 30 mg / m 2 and intravenously administering the lymphodepleting regimen to a subject about 5 to about 7 days prior to administration of the CAR-T cells.

[0197] In some embodiments, the subject further undergoes bridging therapy, wherein said bridging therapy includes short-term treatment with at least one bridging drug between apheresis and said lymphodepletion regimen, and wherein said at least one bridging drug has previously resulted in the subject having stable disease, minimal response, partial response, best partial response, complete response, or stringent complete response. In some embodiments, the subject's tumor burden increases despite said bridging therapy. In some embodiments, the subject's tumor burden increases by about 25% or more despite receiving said bridging therapy.

[0198] In some embodiments, the subject is treated at most about 1 hour prior to said administration of said CAR-T cells with an administered prodrug comprising an antipyretic agent and an antihistamine. In some embodiments, the antipyretic agent comprises paracetamol or acetaminophen. In some embodiments, the antipyretic agent is administered orally or intravenously to the subject. In some embodiments, the antipyretic agent is administered to the subject at a dose between 650 mg and 1000 mg. In some embodiments, the antihistamine comprises diphenhydramine. In some embodiments, the antihistamine is administered to the subject at least or intravenously. In some embodiments, the antihistamine is administered at a dose between 25 mg and 50 mg, or the equivalent thereof. In some embodiments, the antipyretic comprises paracetamol or acetaminophen, and the antipyretic comprises a dose of 650 mg to 1000 mg, and wherein the antihistamine comprises diphenhydramine, and the antihistamine comprises a dose of 25 mg to 50 mg, or an equivalent thereof, and wherein the antipyretic comprises a dose of 25 mg to 50 mg, or an equivalent thereof, and wherein the antipyretic comprises a dose of 25 mg to 50 mg, or an equivalent thereof, and wherein the antihistamine ...

[0199] In some embodiments, the method further comprises diagnosing the subject for cytokine release syndrome (CRS). In a preferred embodiment, the diagnosis is based on the American Society for Transplantation and Cellular Therapy (ASTCT) consensus classification, formerly the American Society for Blood and Marrow Transplantation (ASBMT). Table 30 provides a non-limiting summary of the ASTCT consensus classification for CRS diagnosis. In some embodiments, CRS is assessed by evaluating the levels of one or more or all of IL-6, IL-10, IFN-γ, C-reactive protein (CRP) and ferritin.

[0200] In some embodiments, the method further comprises treating the subject for cytokine release syndrome (CRS). In some embodiments, the CRS is treated with an antipyretic. In some embodiments, the CRS is treated with an anticytokine therapy. In some embodiments, the treatment of CRS occurs more than about 3 days after infusion. In some embodiments, the treatment of CRS occurs without significantly reducing in vivo expansion of the CAR-T cells. In some embodiments, the method further comprises treating the subject for cytokine release syndrome more than about 3 days after the administration of the CAR-T cells without significantly reducing in vivo expansion of the CAR-T cells. In some embodiments, the treatment of CRS comprises administering to the subject an IL-6R inhibitor. In some embodiments, the IL-6R inhibitor is an antibody. In some embodiments, the antibody inhibits IL-6R by binding to the extracellular domain of IL-6R. In some embodiments, the IL-6R inhibitor blocks the binding of IL-6 to IL-6R. In some embodiments, the IL-6R inhibitor is tocilizumab. In some embodiments, the anti-cytokine therapy includes administration of tocilizumab. In some embodiments, the anti-cytokine therapy includes administration of a steroid. In some embodiments, the treatment of CRS includes treatment with a monoclonal antibody other than tocilizumab. In some embodiments, the antibody other than tocilizumab targets a cytokine. In some embodiments, the cytokine targeted by the antibody other than tocilizumab is IL-1. In some embodiments, the antibody targeting IL-1 is anakinra. In some embodiments, the cytokine targeted by the antibody other than tocilizumab is TNFα. In some embodiments, the treatment of CRS includes administering a corticosteroid to the subject. In some embodiments, the treatment of CRS includes the use of a vasoconstrictor. In some embodiments, the treatment of CRS includes intubation or mechanical ventilation. In some embodiments, the treatment of CRS includes administering cyclophosphamide to the subject. In some embodiments, the treatment of CRS includes administering etanercept to the subject.In some embodiments, treating CRS comprises administering levetiracetam to the subject. In some embodiments, treating CRS comprises supportive care.

[0201] In some embodiments, the method further comprises diagnosing the subject for immune cell effector-associated neurotoxicity (ICANS). In some embodiments, the diagnosis is made based on the National Cancer Institute Common Terminology Criteria for Adverse Events (NCI CTCAE) criteria. In some embodiments, the diagnosis is made based on the NCI CTCAE criteria version 5.0. In some embodiments, the diagnosis is made based on the American Society for Transplant and Cellular Therapy (ASTCT) consensus classification system. In some embodiments, the subject has neurotoxicity consistent with ICAN. Table 31 provides a non-limiting summary of the ASTCT consensus classification system for ICANS diagnosis. In some embodiments, treating ICANS comprises administering an IL-6R inhibitor to the subject. In some embodiments, the IL-6R inhibitor is an antibody. In some embodiments, the antibody inhibits IL-6R by binding to the extracellular domain of IL-6R. In some embodiments, the IL-6R inhibitor blocks the binding of IL-6 to IL-6R. In some embodiments, the IL-6R inhibitor is tocilizumab. In some embodiments, the treatment of ICANS comprises administering to the subject an IL-1 inhibitor. In some embodiments, the IL-1 inhibitor is an antibody. In a preferred embodiment, the IL-1 inhibitory antibody is anakinra. In some embodiments, the treatment of ICANS comprises administering to the subject a corticosteroid. In some embodiments, the treatment of ICANS comprises administering to the subject levetiracetam. In some embodiments, the treatment of ICANS comprises administering to the subject dexamethasone. In some embodiments, the treatment of ICANS comprises administering to the subject methylprednisolone sodium succinate. In some embodiments, the treatment of ICANS comprises administering to the subject pethidine. In some embodiments, the treatment of ICANS comprises administering to the subject one or more or all of tocilizumab, anakinra, a corticosteroid, levetiracetam, dexamethasone, methylprednisolone sodium succinate, or pethidine.

[0202] In some embodiments, the method further comprises diagnosing the subject for cytopenia. In some embodiments, the cytopenia comprises one or more or all of lymphopenia, neutropenia, and thrombocytopenia. Without being bound by theory, grade 3 or grade 4 lymphopenia, but not below grade 2, is defined as a lymphocyte count below 0.5×10 9Grade 3 or 4 neutropenia, but not grade 2 or less, is characterized by a neutrophil count of less than 1000 cells / microliter of the subject's blood sample, and Grade 3 or 4 thrombocytopenia, but not grade 2 or less, is characterized by a platelet count of less than 50,000 cells / microliter of the subject's blood sample. In some examples, more than 75% of subjects who suffered from Grade 3 or Grade 4 lymphopenia after administration of the CAR-T cells recovered to Grade 2 or less lymphopenia at day 60 after administration of the CAR-T cells. In some examples, more than 80% of subjects who suffered from Grade 3 or Grade 4 lymphopenia after administration of the CAR-T cells recovered to Grade 2 or less lymphopenia at day 60 after administration of the CAR-T cells. In some examples, greater than 85% of subjects who suffered from grade 3 or grade 4 lymphopenia after administration of the CAR-T cells recovered to grade 2 or less lymphopenia at day 60 after administration of the CAR-T cells. In some examples, greater than 90% of subjects who suffered from grade 3 or grade 4 lymphopenia after administration of the CAR-T cells recovered to grade 2 or less lymphopenia at day 60 after administration of the CAR-T cells. In some examples, greater than 70% of subjects who suffered from grade 3 or grade 4 neutropenia after administration of the CAR-T cells recovered to grade 2 or less neutropenia at day 60 after administration of the CAR-T cells. In some examples, greater than 75% of subjects who suffered from grade 3 or grade 4 neutropenia after administration of the CAR-T cells recovered to grade 2 or less neutropenia at day 60 after administration of the CAR-T cells. In some examples, greater than 80% of subjects who experienced grade 3 or grade 4 neutropenia after administration of the CAR-T cells recovered to grade 2 or less neutropenia at day 60 after administration of the CAR-T cells. In some examples, greater than 85% of subjects who experienced grade 3 or grade 4 neutropenia after administration of the CAR-T cells recovered to grade 2 or less neutropenia at day 60 after administration of the CAR-T cells.In some embodiments, more than 30% of subjects who suffered grade 3 or grade 4 thrombocytopenia after administration of the CAR-T cells recovered to grade 2 or less thrombocytopenia at day 60 after administration of the CAR-T cells. In some embodiments, more than 34% of subjects who suffered grade 3 or grade 4 thrombocytopenia after administration of the CAR-T cells recovered to grade 2 or less thrombocytopenia at day 60 after administration of the CAR-T cells. In some embodiments, more than 38% of subjects who suffered grade 3 or grade 4 thrombocytopenia after administration of the CAR-T cells recovered to grade 2 or less thrombocytopenia at day 60 after administration of the CAR-T cells. In some embodiments, more than 42% of subjects who suffered grade 3 or grade 4 thrombocytopenia after administration of the CAR-T cells recovered to grade 2 or less thrombocytopenia at day 60 after administration of the CAR-T cells.

[0203] When a host cell expressing a CAR-encoding nucleic acid sequence disclosed herein or a composition comprising a vector comprising a CAR-encoding nucleic acid sequence disclosed herein is administered to a mammal (e.g., a human), the biological activity of the CAR can be measured by any suitable method known in the art. According to the methods disclosed herein, the CAR binds to BCMA in multiple myeloma cells, and these multiple myeloma cells are destroyed. The binding of the CAR to BCMA on the surface of multiple myeloma cells can be measured by any suitable method known in the art, including, for example, ELISA and flow cytometry. The ability of the CAR to destroy multiple myeloma cells can be measured by any suitable method known in the art, such as, for example, the cytotoxicity assays described in Kochenderfer et al., J. Immunotherapy, 32(7):689-702 (2009) and Herman et al., J. Immunological Methods, 285(1):25-40 (2004). The biological activity of the CAR can also be measured by assaying the expression of several cytokines, such as CD 107a, IFNγ, IL-2, and TNF.

[0204] The methods described herein may be used to treat a variety of cancers, including solid and liquid cancers. In some embodiments, these methods are for treating multiple myeloma. The methods described herein may be used in adjunctive or novel adjunctive treatments, as a combination therapy with first, second, third, or other types of cancer therapy known in the art (e.g., chemotherapy, surgery, radiation, gene therapy, immunotherapy, bone marrow transplantation, stem cell transplantation, targeted therapy, cryotherapy, ultrasound therapy, photodynamic therapy, radiofrequency ablation, etc.).

[0205] In some embodiments, the cancer is multiple myeloma. In some embodiments, the cancer is stage I, stage II or stage III, and / or stage A or stage B multiple myeloma according to the Durie-Salmon classification system. In some embodiments, the cancer is stage I, stage II or stage III multiple myeloma according to the International Classification System published by the International Myeloma Working Group (IMWG). In some embodiments, the multiple myeloma is aggressive.

[0206] In some embodiments, the subject has received prior treatment with at least one prior line of therapy. In some embodiments, the at least one prior line of therapy includes a drug treatment, and the drug is a proteasome inhibitor (PI). Non-limiting examples of PIs include bortezomib, carbenzimib, and izomib. In some embodiments, the at least one prior line of therapy includes a drug treatment, and the drug is an immunomodulatory drug (IMiD). Non-limiting examples of IMiDs include lenalidomide, pomalidomide, and thalidomide. In some embodiments, the at least one prior line of therapy includes a drug treatment, and the drug is a corticosteroid. Non-limiting examples of corticosteroids include dexamethasone and prednisone. In some embodiments, the at least one prior line of therapy includes a drug treatment, and the drug is an alkylating agent. In some embodiments, the at least one prior line of therapy includes a drug treatment, and the drug is an anthracycline. In some embodiments, at least one prior line of treatment includes a drug treatment, and the drug is an anti-CD38 antibody. Non-limiting examples of anti-CD38 antibodies include daratumumab, isatuximab, and the research antibody TAK-079. In some embodiments, at least one prior line of treatment includes a drug treatment, and the drug is elotuzumab. In some embodiments, at least one prior line of treatment includes a drug treatment, and the drug is panobinostat. In some embodiments, the subject relapses after the at least one prior line of treatment. In some embodiments, the cancer is refractory to one or more or all of bortezomib, carbenzimib, isomib, lenalidomide, pomalidomide, thalidomide, dexamethasone, prednisone, alkylating agents, daratumumab, isatuximab, TAK-079, elotuzumab, and panobinostat. In some embodiments, the previous line of treatment includes surgery, radiation therapy, or autologous or allogeneic transplantation, or any combination of such treatments.

[0207] In some embodiments, the multiple myeloma is refractory to at least two drugs, in some embodiments, the multiple myeloma is refractory to at least three drugs, in some embodiments, the multiple myeloma is refractory to at least four drugs, in some embodiments, the multiple myeloma is refractory to at least five drugs.

[0208] In some embodiments, the subject has about 10% to about 30% bone marrow plasma cells prior to said administration of said CAR-T cells.

[0209] In some embodiments, a bone marrow aspirate or biopsy may be performed for clinical evaluation, or the bone marrow aspirate may be performed for biomarker evaluation. In some embodiments, clinical classification (morphology, cytogenetics, immunohistochemistry or immunofluorescence or flow cytometry) may be performed. In some embodiments, immunophenotyping may be performed on a portion of the bone marrow aspirate to monitor checkpoint ligand expression in BCMA, CD138 positive multiple myeloma cells, and checkpoint expression in T cells. In some embodiments, next generation sequencing (NGS) of bone marrow aspirate DNA may be used to monitor minimal residual disease (MRD) in subjects. NGS of bone marrow aspirate DNA is known to those of skill in the art. In some embodiments, NGS is performed via clonoSEQ. In some embodiments, a baseline bone marrow aspirate may be used to define a myeloma clone, and a post-treatment sample may be used to assess MRD negativity. In some embodiments, the MRD negativity status may be based on an evaluable sample. In some embodiments, an evaluable sample is one or more or all of correction, quality control, and evaluable cell sufficiency under a particular sensitivity level. In some embodiments, the sensitivity level is 10 -6 In some embodiments, the sensitivity level is 10 -6 and the sensitivity level is 10 -5 In some embodiments, the sensitivity level is 10-4 In some embodiments, the sensitivity level is 10 -3 It is.

[0210] In some embodiments, the International Myeloma Working Group (IMWG) response criteria are used to assess the subject's response to the treatment method, and the response criteria are summarized in Table 2. In some embodiments, the response may be classified as stringent complete response (sCR). In some embodiments, the response may be classified as complete response (CR), which is worse than stringent complete response (sCR). In some embodiments, the response may be classified as very good partial response (VGPR), which is worse than complete response (CR). In some embodiments, the response may be classified as partial response (PR), which is worse than very good partial response (VGPR). In some embodiments, the response may be classified as minimal response (MR), which is worse than partial response (PR). In some embodiments, the response may be classified as stable disease (SD), which is worse than minimal response (MR). In some embodiments, the response may be classified as progressive disease (PD), which is worse than stable disease.

[0211] In some embodiments, tests to assess response criteria based on the International Myeloma Working Group (IMWG) are measurement of myeloma protein (M-protein) in serum and urine, serum calcium corrected for albumin, bone marrow examination, bone survey, and documentation of extramedullary plasmacytoma.

[0212] Non-limiting examples of tests for measuring M-protein in blood and urine are known to those of skill in the art and include serum quantitative Ig, serum protein electrophoresis (SPEP), serum immunofixation electrophoresis, serum FLC assay, 24-hour urinary M-protein electrophoresis quantitative (UPEP), urine immunofixation electrophoresis and serum β2-microglobulin.

[0213] It is known to those skilled in the art to calculate serum calcium corrected for albumin in a blood sample to detect hypercalcemia. Without wishing to be bound by theory, calcium is bound to albumin, and only unbound (free) calcium has biological activity, therefore serum calcium levels must be adjusted for abnormal albumin levels ("corrected serum calcium").

[0214] In some embodiments, a bone survey of any or all of the skull, entire spine, pelvis, thorax, humerus, femur, and any other bones may be performed and assessed by radiography ("X-ray") or low-dose computed tomography (CT) diagnostic quality scans without the use of IV contrast, both of which are known to those of skill in the art. In some embodiments, X-ray or CT scans may be performed locally after T cell administration and before disease progression is confirmed to document response or progression with or without clinical indication based on symptoms. In some embodiments, magnetic resonance imaging (MRI) may be used to assess bone disease, but does not replace a bone survey. MRI is known to those of skill in the art. In some embodiments, a radionuclide bone scan may be used at screening in addition to a complete bone survey to document disease status in two ways. Radionuclide bone scans are known to those of skill in the art. In some embodiments, a radionuclide bone scan and a complete bone survey may be performed simultaneously. In some embodiments, a radionuclide bone scan may not replace a complete bone survey. In some embodiments, if a subject shows disease progression manifested by painful symptoms in bone changes, disease progression can be documented by bone surveys or other radiographs depending on the symptoms experienced by the subject.

[0215] In some embodiments, extramedullary plasmacytoma can be documented by clinical examination or MRI. In some embodiments, extramedullary plasmacytoma can be documented by CT scan if there are no contraindications to the use of IV contrast. In some embodiments, extramedullary plasmacytoma can be documented by fusion of a positron emission tomography (PET) scan with a CT scan if the CT portion is of sufficient diagnostic quality. In some embodiments, assessments of measurable sites of extramedullary disease in subjects can be performed and measured or assessed locally every 4 weeks until a confirmed CR or confirmed disease progression occurs. In some embodiments, assessments of extramedullary plasmacytoma can be performed every 12 weeks.

[0216] In some embodiments, the sum of the products of perpendicular diameters of previous extramedullary plasmacytomas may be decreased by more than 90% or at least 50% to qualify for VGPR or PR or MR, respectively. In some embodiments, the sum of the products of perpendicular diameters of previous extramedullary plasmacytomas must increase by at least 50% or the longest diameter of a previous lesion that is > 1 cm (in the short axis) must increase by at least 50% or a new plasmacytoma must develop to qualify for disease progression. In some embodiments, if not all extramedullary plasmacytomas present are reported, the sum of the products of perpendicular diameters of reported plasmacytomas is increased by at least 50% to qualify for disease progression. In some embodiments, if the study treatment interferes with the immunofixation assay, CR may be defined as the disappearance of the original M protein associated with multiple myeloma upon immunofixation.

[0217] In some embodiments, the subject's response to the treatment method is assessed based on the change in disease burden or tumor burden. Disease burden or tumor burden refers to the type of measurable disease in the subject. In some embodiments, the change in tumor burden can be assessed based on the change in paraprotein levels after treatment. In some embodiments, the paraprotein is M-protein in serum. In some embodiments, the paraprotein is M-protein in serum. In some embodiments, the change in tumor burden can be assessed based on the difference between impaired and intact free light chains (dFLC). In some embodiments, the change in tumor burden is assessed based on the maximum paraprotein reduction relative to baseline (i.e., relative to before administration of the CAR-T cells). In some embodiments, the change in tumor burden is assessed at a median follow-up period of 28 days or more after administration of the CAR-T cells. In some embodiments, the change in tumor burden is assessed at a median follow-up period of 1 month or more after administration of the CAR-T cells. In some embodiments, the change in tumor burden is assessed at a median follow-up period of 3 months or more after administration of the CAR-T cells. In some embodiments, the change in tumor burden is assessed at a median follow-up period of 6 months or more after administration of the CAR-T cells. In some embodiments, the change in tumor burden is assessed at a median follow-up period of 9 months or more after administration of the CAR-T cells. In some embodiments, the change in tumor burden is assessed at a median follow-up period of 12 months or more after administration of the CAR-T cells.

[0218] In some embodiments, the subject is re-treated by administration of a second dose of CAR-T cells via a second intravenous infusion. In some embodiments, the re-treatment dose is 1.0×10 per kilogram of subject mass. 5 ~5.0 × 10 6 In some embodiments, the retreatment dose comprises about 0.75 x 10 CAR-T cells per kilogram of subject mass. 5In some embodiments, the subject is retreated if the subject exhibits disease progression after a best response of minimal response or better response after the initial infusion of CAR-T cells. In some embodiments, the time from the initial infusion of CAR-T cells to detection of disease progression comprises at least six months.

[0219] 4.8. Methods of Treating Subjects Who Are Refractory to Lenalidomide In one aspect, a method of treating a subject is provided, the method comprising administering to the subject a composition comprising a therapeutically effective amount of T cells comprising a chimeric antigen receptor (CAR), wherein the subject has multiple myeloma and is refractory to lenalidomide. In some embodiments, the subject has received prior treatment with one, two, or three prior lines of therapy.

[0220] In some embodiments, the multiple myeloma is refractory to the last line of therapy. In some embodiments, the subject has relapsed after one, two, or three prior lines of therapy. In some embodiments, the subject has received prior treatment with at least one prior line of therapy, the at least one prior line of therapy comprising treatment with lenalidomide and at least one non-lenalidomide drug, the at least one non-lenalidomide drug comprising at least one of a proteasome inhibitor, an immunomodulatory agent, or an anti-CD38 antibody. In some embodiments, the subject has not been previously exposed to a BCMA-targeted drug. In some embodiments, the subject has received prior treatment with at least two prior lines of therapy. In some embodiments, the subject has received prior treatment with three prior lines of therapy.

[0221] In some embodiments, the subject has received prior treatment with dexamethasone, an alkylating agent, or daratumumab, hi some embodiments, the multiple myeloma is refractory to three drugs.

[0222] In some embodiments, the method of treatment effectively obtains a reduced tumor burden in the subject. In some embodiments, the method of treatment effectively obtains a tumor burden reduction of about 1% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 92% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100% in the subject. In some embodiments, the method of treatment effectively obtains a tumor burden reduction of about 100% in the subject. In some embodiments, the treatment method effectively obtains about 1% to about 100% tumor burden reduction in the subject at a rate of about 1% to about 100%. In some embodiments, the treatment method effectively obtains about 60% to about 100% tumor burden reduction in the subject at a rate of about 1% to about 100%. In some embodiments, the treatment method effectively obtains about 65% to about 100% tumor burden reduction in the subject at a rate of about 1% to about 92%. In some embodiments, the treatment method effectively obtains about 70% to about 100% tumor burden reduction in the subject at a rate of about 1% to about 88%. In some embodiments, the treatment method effectively obtains about 90% to about 100% tumor burden reduction in the subject at a rate of about 1% to about 88%. In some embodiments, the treatment method effectively obtains about 95% to about 100% tumor burden reduction in the subject at a rate of about 1% to about 88%. In some embodiments, the treatment method is effective in obtaining about a 99% to about 100% tumor burden reduction in a proportion of subjects between about 1% to about 88%. In some embodiments, the treatment method is effective in obtaining about a 100% tumor burden reduction in a proportion of subjects between about 1% to about 83%.

[0223] In some embodiments, the method of treatment effectively achieves a minimal residual disease (MRD) negative status or effectively maintains said minimal residual disease (MRD) status in the subject.

[0224] In some embodiments, the method of treatment is effective to obtain a negative minimal residual disease (MRD) status in the subject. -6 In some embodiments, the method of treatment effectively achieves minimal residual disease (MRD) negativity in a subject at a sensitivity level of 10 -5 In some embodiments, the method of treatment effectively achieves minimal residual disease (MRD) negativity in a subject at a sensitivity level of 10 -4 In some embodiments, the method of treatment effectively achieves minimal residual disease (MRD) negativity in a subject at a sensitivity level of 10 -3 In some embodiments, the method of treatment effectively obtains a minimal residual disease (MRD) negative status at a sensitivity level of 0.1 to 1.0. In some embodiments, the method of treatment effectively obtains a MRD negative status when assessed in bone marrow. In some embodiments, the method of treatment effectively maintains a MRD negative status when assessed in an evaluable bone marrow sample. In some embodiments, the method of treatment effectively obtains a MRD negative status when assessed in bone marrow DNA. In some embodiments, the method effectively obtains a minimal residual disease (MRD) negative status of the subject as assessed in bone marrow at a follow-up period of about 29 days or more after the administration of the CAR-T cells, about 2 months or more after the administration of the CAR-T cells, about 3 months or more after the administration of the CAR-T cells, about 6 months or more after the administration of the CAR-T cells, about 9 months or more after the administration of the CAR-T cells, or about 12 months or more after the administration of the CAR-T cells. In some embodiments, the minimal residual disease (MRD) negative status is obtained at a first follow-up period of about 29 days to about 184 days after the administration of the CAR-T cells.

[0225] In some embodiments, the treatment method effectively maintains the minimal residual disease (MRD) negativity initially achieved in the subject. -5 In some embodiments, the method of treatment effectively maintains MRD negativity at a sensitivity level of 10 -6In some embodiments, the method of treatment effectively achieves minimal residual disease (MRD) negativity at a sensitivity level of 10 -4 In some embodiments, the treatment method effectively maintains MRD negativity at a sensitivity level of 10 -3 In some embodiments, the treatment method effectively maintains the MRD-negative status at a sensitivity level of 0.1 to 1.0 days after administration of the CAR-T cells. In some embodiments, the treatment method effectively maintains the MRD-negative status when assessed with a bone marrow sample. In some embodiments, the treatment method effectively maintains the MRD-negative status when assessed with an evaluable bone marrow sample. In some embodiments, the treatment method effectively maintains the MRD-negative status when assessed with bone marrow DNA. In some embodiments, the method effectively maintains the minimal residual disease (MRD)-negative status of the subject as assessed in bone marrow at a follow-up period of about 29 days to about 291 days after administration of the CAR-T cells, about 29 days to about 9 months after administration of the CAR-T cells, about 29 days to about 6 months after administration of the CAR-T cells, about 29 days to about 3 months after administration of the CAR-T cells, or about 29 days to about 2 months after administration of the CAR-T cells. In some examples, the method effectively maintains the minimal residual disease (MRD) negative status of the subject as assessed in bone marrow at a second follow-up period from about 57 days to about 191 days after the administration of the CAR-T cells, and further wherein the first follow-up period is earlier than the second follow-up period.

[0226] In some embodiments, the efficacy of the treatment method is assessed by evaluating the proportion of subjects with MRD-negative status. -6 The efficacy of the treatment method is assessed by assessing the proportion of subjects with MRD-negative status at a sensitivity level of 10 -5 The efficacy of the treatment method is assessed by assessing the proportion of subjects with MRD-negative status at a sensitivity level of 10 -4 The efficacy of the treatment method is assessed by assessing the proportion of subjects with MRD-negative status at a sensitivity level of 10 -3The efficacy of the treatment method is assessed by assessing the proportion of subjects with MRD-negative status at a median follow-up period from administration of the CAR-T cells to about 291 days after administration of the CAR-T cells, from administration of the CAR-T cells to about 9 months after administration of the CAR-T cells, from administration of the CAR-T cells to about 6 months after administration of the CAR-T cells, from administration of the CAR-T cells to about 3 months after administration of the CAR-T cells, from administration of the CAR-T cells to about 2 months after administration of the CAR-T cells, or from administration of the CAR-T cells to about 29 days after administration of the CAR-T cells. In some embodiments, the method assesses the efficacy of the treatment method ... days after administration of the CAR-T cells. -4 , 10 -5 or 10 -6 In some embodiments, the method effectively achieves a minimal residual disease (MRD) negative status at a rate of about 24% or less at a sensitivity threshold level of 10 -4 , 10 -5 or 10 -6 In some embodiments, the method effectively achieves a minimal residual disease (MRD) negative status at a rate of about 41% or less at a sensitivity threshold level of 10 -4 , 10 -5 or 10 -6 In some embodiments, the method effectively achieves a minimal residual disease (MRD) negative status at a rate of about 61% or less at a sensitivity threshold level of 10 -4 , 10 -5 or 10 -6 In some embodiments, the method effectively achieves a minimal residual disease (MRD) negative status at a rate of about 24% to about 61% at a sensitivity threshold level of 10 -4 , 10 -5 or 10 -6 At a sensitivity threshold level of 0.1, the minimal residual disease (MRD) negativity status is effectively achieved in approximately 41% of cases.

[0227] In some embodiments, the efficacy of the treatment method is assessed by evaluating the proportion of subjects with evaluable bone marrow and MRD-negative status. -6The efficacy of the treatment method is assessed by assessing the proportion of subjects with evaluable bone marrow and MRD-negative status at a sensitivity level of 10. -5 The efficacy of the treatment method is assessed by assessing the proportion of subjects with evaluable bone marrow and MRD-negative status at a sensitivity level of 10. -4 The efficacy of the treatment method is assessed by assessing the proportion of subjects with evaluable bone marrow and MRD-negative status at a sensitivity level of 10. -3 The efficacy of the treatment method is assessed by assessing the proportion of subjects with evaluable bone marrow and MRD negative status at a median follow-up time from CAR-T cell administration to about 291 days post-CAR-T cell administration, from CAR-T cell administration to about 9 months post-CAR-T cell administration, from CAR-T cell administration to about 6 months post-CAR-T cell administration, from CAR-T cell administration to about 3 months post-CAR-T cell administration, from CAR-T cell administration to about 2 months post-CAR-T cell administration, or from CAR-T cell administration to about 29 days post-CAR-T cell administration. In some embodiments, the method assesses the efficacy of the treatment method ... days post-CAR-T cell administration. -5 At a sensitivity threshold level of 100%, the method effectively achieves minimal residual disease (MRD) negativity in about 64% or less of subjects with evaluable samples. -5 At a sensitivity threshold level of 100%, the method effectively achieves minimal residual disease (MRD) negativity in about 92% or less of subjects with evaluable samples. -5 At a sensitivity threshold level of 100%, the method effectively obtains minimal residual disease (MRD) negativity in about 99% or less of subjects with evaluable samples. -5 At a sensitivity threshold level of 100%, the method effectively obtains the minimal residual disease (MRD) negative status in about 64% to about 99% of subjects with evaluable samples.-5 At a sensitivity threshold level of 0.1, approximately a 92% rate of subjects with evaluable samples effectively achieves the minimal residual disease (MRD) negativity status.

[0228] In some embodiments, the method is effective to obtain at least one response in the subject following said administration of the CAR-T cells, wherein the at least one response comprises, in order from best to worst, a stringent complete response, a complete response, a best partial response, a partial response, or a minimal response.

[0229] In some embodiments, the method is effective to obtain an initial response within about 21 days or more after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response within about 30 days or more after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response within about 36 days or more after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response within about 56 days or more after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response within about 99 days or more after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response before about 21 days to about 99 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response before about 21 days to about 55 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response no earlier than about 36 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response no earlier than about 30 days after said administration of the CAR-T cells.

[0230] In some embodiments, the effectiveness of the treatment method is assessed by assessing the proportion of subjects having a stringent complete response. In some embodiments, the effectiveness of the treatment method is assessed by assessing the proportion of subjects having a complete response or better. In some embodiments, the effectiveness of the treatment method is assessed by assessing the proportion of subjects having a best partial response or better. In some embodiments, the effectiveness of the treatment method is assessed by assessing the proportion of subjects having a partial response or better. In some embodiments, the effectiveness of the treatment method is assessed by assessing the proportion of subjects having a minimal response or better.

[0231] In some embodiments, the method effectively obtains a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response, i.e., a best response of minimal response or better. In some embodiments, the rate at which the method effectively obtains a best response of minimal response or better is referred to as a clinical benefit rate. In some embodiments, the method effectively obtains a best response of minimal response or better at a rate of about 52% or less. In some embodiments, the method effectively obtains a best response of minimal response or better at a rate of about 72% or less. In some embodiments, the method effectively obtains a best response of minimal response or better at a rate of about 87% or less. In some embodiments, the method effectively obtains a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response at a rate of about 52% to about 87%. In some embodiments, the method effectively achieves a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response at a rate of about 72%.

[0232] In some embodiments, the method effectively obtains a best response of any one of partial response, best partial response, complete response, or stringent complete response, i.e., a best response of partial response or better. In some embodiments, the rate at which the method effectively obtains a best response of partial response or better is referred to as the overall survival rate or overall response rate. In some embodiments, the method effectively obtains the best response of partial response or better at a rate of about 49% or less. In some embodiments, the method effectively obtains the best response of partial response or better at a rate of about 69% or less. In some embodiments, the method effectively obtains the best response of partial response or better at a rate of about 84% or less. In some embodiments, the method effectively obtains the best response of any one of partial response, best partial response, complete response, or stringent complete response at a rate of about 49% to about 84%. In some embodiments, the method is effective to obtain a best response of any one of the partial response, best partial response, complete response, or stringent complete response at a rate of about 69%.

[0233] In some embodiments, the method effectively obtains a best response of any one of best partial response, complete response, or stringent complete response, i.e., a best response of best partial response or better. In some embodiments, the method effectively obtains the best response of best partial response or better at a rate of about 49% or less. In some embodiments, the method effectively obtains the best response of best partial response or better at a rate of about 69% or less. In some embodiments, the method effectively obtains the best response of best partial response or better at a rate of about 84% or less. In some embodiments, the method effectively obtains the best response of any one of best partial response, complete response, or stringent complete response at a rate of about 49% to about 84%. In some embodiments, the method effectively obtains the best response of any one of best partial response, complete response, or stringent complete response at a rate of about 69%.

[0234] In some embodiments, the method effectively obtains a best effect of complete response or stringent complete response, i.e., a best effect of complete response or better. In some embodiments, the method effectively obtains the best effect of complete response or better at a rate of about 39% or less. In some embodiments, the method effectively obtains the best effect of complete response or better at a rate of about 58% or less. In some embodiments, the method effectively obtains the best effect of complete response or better at a rate of about 76% or less. In some embodiments, the method effectively obtains the best effect of complete response or stringent complete response at a rate of about 39% to about 76%. In some embodiments, the method effectively obtains the best effect of complete response or stringent complete response at a rate of about 58%.

[0235] In some embodiments, the method is effective in obtaining a best response of stringent complete response. In some embodiments, the method is effective in obtaining a best response of stringent complete response at a rate of about 33% or less. In some embodiments, the method is effective in obtaining a best response of stringent complete response at a rate of about 52% or less. In some embodiments, the method is effective in obtaining a best response of stringent complete response at a rate of about 70% or less. In some embodiments, the method is effective in obtaining a best response of stringent complete response at a rate of about 33% to about 70%. In some embodiments, the method is effective in obtaining a best response of stringent complete response at a rate of about 52%.

[0236] In some embodiments, the method is effective to obtain the best effect about 27 days or more after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect about 87 days or more after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect about 109 days or more after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect about 172 days or more after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect about 237 days or more after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect before about 27 days to about 237 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect before about 46 days to about 172 days after the administration of the CAR-T cells. In some embodiments, the method is effective to achieve the best effect prior to about 109 days after the administration of the CAR-T cells. In some embodiments, the method is effective to achieve the best effect prior to about 87 days after the administration of the CAR-T cells.

[0237] In some embodiments, the method effectively maintains a response in a subject for a follow-up period from the time of the initial response to about 270 days after the administration of the CAR-T cells. In some embodiments, the method effectively maintains a response at about 70% to about 99% rate for a follow-up period of about 6 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains a response at about 95% rate for a follow-up period of about 6 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains a response at about 7% to about 92% rate for a follow-up period of about 9 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains a response at about 63% rate for a follow-up period of about 9 months after the administration of the CAR-T cells.

[0238] In some embodiments, wherein the method effectively obtains the initial response within about 269 days or less after the administration of the CAR-T cells, and the method effectively maintains the response in the subject for a follow-up period from the time of the initial response to about 270 days after the administration of the CAR-T cells. In some embodiments, the method effectively maintains the response at about 70% or less for a follow-up period of 6 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains the response at about 95% or less for a follow-up period of 6 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains the response at about 99% or less for a follow-up period of 6 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains the response at about 63% or less for a follow-up period of 9 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains a response rate of about 92% or less over a 9-month follow-up period following the administration of the CAR-T cells.

[0239] In some embodiments, the method further comprises administering the CAR-T cells to a patient receiving the CAR-T cells for 10 to about 3 months after the administration of the CAR-T cells. -5At a sensitivity threshold level of 0.1%, the method effectively obtains a minimal residual disease (MRD) negative status of the subject as assessed in bone marrow. In some embodiments, the method effectively obtains a minimal residual disease (MRD) negative complete response or a minimal residual disease (MRD) negative stringent complete response at a rate of about 18% or less at a follow-up period of about 291 days after the administration of the CAR-T cells. In some embodiments, the method effectively obtains a minimal residual disease (MRD) negative complete response or a minimal residual disease (MRD) negative stringent complete response at a rate of about 35% or less at a follow-up period of about 291 days after the administration of the CAR-T cells. In some embodiments, the method effectively obtains a minimal residual disease (MRD) negative complete response or a minimal residual disease (MRD) negative stringent complete response at a rate of about 54% or less at a follow-up period of about 291 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain about an 18% to about a 54% rate of minimal residual disease (MRD) negative complete responses or minimal residual disease (MRD) negative stringent complete responses at a follow-up period of about 291 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain about a 35% rate of minimal residual disease (MRD) negative complete responses or minimal residual disease (MRD) negative stringent complete responses at a follow-up period of about 291 days after the administration of the CAR-T cells.

[0240] In some embodiments, the method is effective to obtain progression free survival in the subject. In some embodiments, the method is effective to obtain progression free survival in the subject from the administration of the CAR-T cells to about 55 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain progression free survival in the subject from the administration of the CAR-T cells to about 297 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain progression free survival at about 62% or less at about 6 months or about 9 months follow-up after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain progression free survival at about 86% or less at about 6 months or about 9 months follow-up after the administration of the CAR-T cells. In some embodiments, the method effectively obtains the progression free survival at about 95% or less at about 6 months or about 9 months follow-up period after the administration of the CAR-T cells. In some embodiments, the method effectively obtains the progression free survival at about 62% to about 95% at about 6 months or about 9 months follow-up period after the administration of the CAR-T cells. In some embodiments, the method effectively obtains the progression free survival at about 86% at about 6 months or about 9 months follow-up period after the administration of the CAR-T cells.

[0241] In some embodiments, the method further comprises treating the subject for cytokine release syndrome more than about 3 days after the administration of the CAR-T cells. In some embodiments, the cytokine release syndrome has a reversal rate of about 90% or less 7 days after the cytokine release syndrome is first observed. In some embodiments, the method effectively obtains a reversal rate of about 1% to about 90% of the cytokine release syndrome in a period of about 7 days after the cytokine release syndrome is first observed.

[0242] In some embodiments, the rate of immune effector cell-associated neurotoxicity is about 20% or greater. In some embodiments, the method is effective to obtain a rate of immune effector cell-associated neurotoxicity of about 20% to about 99%. 4.9. Methods for Treating Subjects Who Have Previously Had Early Relapse

[0243] In one embodiment, a method of treating a subject is provided, comprising administering to the subject a composition comprising a therapeutically effective amount of T cells comprising a chimeric antigen receptor (CAR), wherein the subject is afflicted with multiple myeloma and has had a prior early relapse. The term "prior early relapse" refers to disease progression based on International Myeloma Working Group (IMWG) response criteria from the time of autologous hematopoietic stem cell transplant (ASCT) treatment to about 12 months after the autologous hematopoietic stem cell transplant (ASCT) treatment for participants who have already undergone an autologous hematopoietic stem cell transplant (ASCT), or from the time of initiation of anti-myeloma therapy to about 12 months after the initiation of anti-myeloma therapy for participants who have not undergone an autologous hematopoietic stem cell transplant (ASCT).

[0244] In some embodiments, the subject has already received prior treatment with one prior line of therapy. In some embodiments, the one prior line of therapy includes treatment with at least two drugs. In some embodiments, the at least two drugs include a proteasome inhibitor and an immunomodulatory agent. In some embodiments, the subject is further treated with an anti-CD38 antibody. In some embodiments, the subject has not been previously exposed to a BCMA-targeted drug. In some embodiments, the multiple myeloma is refractory to at least one drug.

[0245] In some embodiments, the method of treatment effectively obtains a reduced tumor burden in the subject. In some embodiments, the method of treatment effectively obtains a tumor burden reduction of about 1% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 92% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100% in the subject. In some embodiments, the method of treatment effectively obtains a tumor burden reduction of about 100% in the subject. In some embodiments, the method of treatment effectively obtains about 1% to about 100% tumor burden reduction in the subject at a rate of about 1% to about 100%. In some embodiments, the method of treatment effectively obtains about 80% to about 100% tumor burden reduction in the subject at a rate of about 1% to about 100%. In some embodiments, the method of treatment effectively obtains about 85% to about 100% tumor burden reduction in the subject at a rate of about 1% to about 100%. In some embodiments, the method of treatment effectively obtains about 90% to about 100% tumor burden reduction in the subject at a rate of about 1% to about 88%. In some embodiments, the method of treatment effectively obtains about 95% to about 100% tumor burden reduction in the subject at a rate of about 1% to about 88%. In some embodiments, the method of treatment effectively obtains about 96% to about 100% tumor burden reduction in the subject at a rate of about 1% to about 82%. In some embodiments, the treatment method is effective in obtaining about a 99% to about 100% tumor burden reduction in a proportion of subjects between about 1% to about 82%. In some embodiments, the treatment method is effective in obtaining about a 100% tumor burden reduction in a proportion of subjects between about 1% to about 76%.

[0246] In some embodiments, the method of treatment effectively achieves a minimal residual disease (MRD) negative status or effectively maintains said minimal residual disease (MRD) status in the subject.

[0247] In some embodiments, the method of treatment is effective to obtain a negative minimal residual disease (MRD) status in the subject. -6 In some embodiments, the method of treatment effectively achieves minimal residual disease (MRD) negativity in a subject at a sensitivity level of 10 -5 In some embodiments, the method of treatment effectively achieves minimal residual disease (MRD) negativity in a subject at a sensitivity level of 10 -4 In some embodiments, the method of treatment effectively achieves minimal residual disease (MRD) negativity in a subject at a sensitivity level of 10 -3 In some embodiments, the method of treatment effectively obtains a minimal residual disease (MRD) negative status at a sensitivity level of 0.1 to 1.0. In some embodiments, the method of treatment effectively obtains a MRD negative status when assessed in bone marrow. In some embodiments, the method of treatment effectively maintains a MRD negative status when assessed in an evaluable bone marrow sample. In some embodiments, the method of treatment effectively obtains a MRD negative status when assessed in bone marrow DNA. In some embodiments, the method effectively obtains a minimal residual disease (MRD) negative status of the subject as assessed in bone marrow at a follow-up period of about 35 days or more after the administration of the CAR-T cells, about 2 months or more after the administration of the CAR-T cells, about 3 months or more after the administration of the CAR-T cells, about 6 months or more after the administration of the CAR-T cells, about 9 months or more after the administration of the CAR-T cells, or about 12 months or more after the administration of the CAR-T cells. In some embodiments, the minimal residual disease (MRD) negative status is assessed in bone marrow at a first follow-up period of about 35 days to about 58 days after the administration of the CAR-T cells.

[0248] In some embodiments, the treatment method effectively maintains the minimal residual disease (MRD) negativity initially achieved in the subject. -5 In some embodiments, the method of treatment effectively maintains MRD negativity at a sensitivity level of 10 -6In some embodiments, the method of treatment effectively achieves minimal residual disease (MRD) negativity at a sensitivity level of 10 -4 In some embodiments, the treatment method effectively maintains MRD negativity at a sensitivity level of 10 -3 In some embodiments, the treatment method effectively maintains the MRD-negative status at a sensitivity level of 0.1 to 1.0 days after administration of the CAR-T cells. In some embodiments, the treatment method effectively maintains the MRD-negative status when assessed with a bone marrow sample. In some embodiments, the treatment method effectively maintains the MRD-negative status when assessed with an evaluable bone marrow sample. In some embodiments, the treatment method effectively maintains the MRD-negative status when assessed with bone marrow DNA. In some embodiments, the method effectively maintains the minimal residual disease (MRD)-negative status of the subject as assessed in bone marrow at a follow-up period of about 35 days to about 359 days after administration of the CAR-T cells, about 35 days to about 9 months after administration of the CAR-T cells, about 35 days to about 6 months after administration of the CAR-T cells, about 35 days to about 3 months after administration of the CAR-T cells, or about 35 days to about 2 months after administration of the CAR-T cells. In some examples, the method effectively maintains the minimal residual disease (MRD) negative status of the subject as assessed in bone marrow at a second follow-up period from about 78 days to about 359 days after the administration of the CAR-T cells, and further wherein the first follow-up period is earlier than the second follow-up period.

[0249] In some embodiments, the efficacy of the treatment method is assessed by evaluating the proportion of subjects with MRD-negative status. -6 The efficacy of the treatment method is assessed by assessing the proportion of subjects with MRD-negative status at a sensitivity level of 10 -5 The efficacy of the treatment method is assessed by assessing the proportion of subjects with MRD-negative status at a sensitivity level of 10 -4 The efficacy of the treatment method is assessed by assessing the proportion of subjects with MRD-negative status at a sensitivity level of 10 -3The efficacy of the treatment regimen is assessed by assessing the proportion of subjects with MRD-negative status at a sensitivity level of .

[0250] In some embodiments, the efficacy of the treatment method is assessed by evaluating the proportion of subjects with MRD-negative status at a median follow-up period from administration of the CAR-T cells to about 141 days after administration of the CAR-T cells, from administration of the CAR-T cells to about 4 months after administration of the CAR-T cells, from administration of the CAR-T cells to about 3 months after administration of the CAR-T cells, from administration of the CAR-T cells to about 2 months after administration of the CAR-T cells, or from administration of the CAR-T cells to about 35 days after administration of the CAR-T cells. -4 or 10 -5 At a sensitivity threshold level of about 26% or less, or at a sensitivity threshold level of about 10 -6 In some embodiments, the method effectively achieves a minimal residual disease (MRD) negative status at a rate of about 17% or less at a sensitivity threshold level of 10 -4 or 10 -5 At a sensitivity threshold level of about 50% or less, or at a sensitivity threshold level of about 10 -6 In some embodiments, the method effectively achieves a minimal residual disease (MRD) negative status at a rate of about 39% or less at a sensitivity threshold level of 10 -4 or 10 -5 At a sensitivity threshold level of about 74% or less, or at a sensitivity threshold level of about 10 -6 In some embodiments, the method effectively achieves a minimal residual disease (MRD) negative status at a rate of about 64% or less at a sensitivity threshold level of 10 -4 or 10 -5 At a sensitivity threshold level of about 26% to about 74%, or -6 In some embodiments, the method effectively achieves a minimal residual disease (MRD) negative status at a rate of about 17% to about 64% at a sensitivity threshold level of 10 -4 or 10 -5 At a sensitivity threshold level of about 50% or 10 -6At a sensitivity threshold level of 0.1, the minimal residual disease (MRD) negativity status is effectively achieved in approximately 39% of cases.

[0251] In some embodiments, the efficacy of the treatment method is assessed by evaluating the proportion of subjects with evaluable bone marrow and MRD-negative status. -6 The efficacy of the treatment method is assessed by assessing the proportion of subjects with evaluable bone marrow and MRD-negative status at a sensitivity level of 10. -5 The efficacy of the treatment method is assessed by assessing the proportion of subjects with evaluable bone marrow and MRD-negative status at a sensitivity level of 10. -4 The efficacy of the treatment method is assessed by assessing the proportion of subjects with evaluable bone marrow and MRD-negative status at a sensitivity level of 10. -3 The efficacy of the treatment method is assessed by assessing the proportion of subjects with evaluable bone marrow and MRD-negative status at a median follow-up time from administration of the CAR-T cells to about 141 days after administration of the CAR-T cells, from administration of the CAR-T cells to about 4 months after administration of the CAR-T cells, from administration of the CAR-T cells to about 3 months after administration of the CAR-T cells, from administration of the CAR-T cells to about 2 months after administration of the CAR-T cells, or from administration of the CAR-T cells to about 35 days after administration of the CAR-T cells. In some embodiments, the method assesses the efficacy of the treatment method by assessing the proportion of subjects with evaluable bone marrow and MRD-negative status at a median follow-up time from administration of the CAR-T cells to about 141 days after administration of the CAR-T cells, from administration of the CAR-T cells to about 4 months after administration of the CAR-T cells, from administration of the CAR-T cells to about 3 months after administration of the CAR-T cells, from administration of the CAR-T cells to about 2 months after administration of the CAR-T cells, or from administration of the CAR-T cells to about 35 days after administration of the CAR-T cells. -5 At a sensitivity threshold level of 100%, the method effectively achieves minimal residual disease (MRD) negativity in about 66% or less of subjects with evaluable samples. -5 At a sensitivity threshold level of 100%, the method effectively achieves minimal residual disease (MRD) negativity in about 100% or less of subjects with evaluable samples. -5At a sensitivity threshold level of 100%, the method effectively achieves the minimal residual disease (MRD) negative status in about 66% to about 100% of subjects with evaluable samples. -5 At a sensitivity threshold level of 0.1, approximately 100% of subjects with evaluable samples will effectively achieve the minimal residual disease (MRD) negativity status.

[0252] In some embodiments, the method is effective to obtain at least one response in the subject following said administration of the CAR-T cells, wherein the at least one response comprises, in order from best to worst, a stringent complete response, a complete response, a best partial response, a partial response, or a minimal response.

[0253] In some embodiments, the method is effective to obtain an initial response about 27 days or more after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response about 28 days or more after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response about 33 days or more after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response about 46 days or more after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response about 78 days or more after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response before about 27 days to about 78 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response before about 27 days to about 47 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response no earlier than about 33 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response no earlier than about 28 days after said administration of the CAR-T cells.

[0254] In some embodiments, the effectiveness of the treatment method is assessed by assessing the proportion of subjects having a stringent complete response. In some embodiments, the effectiveness of the treatment method is assessed by assessing the proportion of subjects having a complete response or better. In some embodiments, the effectiveness of the treatment method is assessed by assessing the proportion of subjects having a best partial response or better. In some embodiments, the effectiveness of the treatment method is assessed by assessing the proportion of subjects having a partial response or better. In some embodiments, the effectiveness of the treatment method is assessed by assessing the proportion of subjects having a minimal response or better.

[0255] In some embodiments, the method effectively obtains a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response, i.e., a best response of minimal response or better. In some embodiments, the rate at which the method effectively obtains a best response of minimal response or better is referred to as a clinical benefit rate. In some embodiments, the method effectively obtains a best response of the minimal response or better at a rate of about 65% or less. In some embodiments, the method effectively obtains a best response of the minimal response or better at a rate of about 89% or less. In some embodiments, the method effectively obtains a best response of the minimal response or better at a rate of about 99% or less. In some embodiments, the method effectively obtains a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response at a rate of about 65% to about 99%. In some embodiments, the method is effective to obtain a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response at a rate of about 89%.

[0256] In some embodiments, the method effectively obtains a best response of any one of partial response, best partial response, complete response, or stringent complete response, i.e., a best response of partial response or better. In some embodiments, the rate at which the method effectively obtains a best response of partial response or better is referred to as the overall survival rate or overall response rate. In some embodiments, the method effectively obtains a best response of the partial response or better at a rate of about 65% or less. In some embodiments, the method effectively obtains a best response of the partial response or better at a rate of about 89% or less. In some embodiments, the method effectively obtains a best response of the partial response or better at a rate of about 99% or less. In some embodiments, the method effectively obtains a best response of any one of partial response, best partial response, complete response, or stringent complete response at a rate of about 65% to about 99%. In some embodiments, the method is effective to obtain a best response of any one of the partial response, best partial response, complete response, or stringent complete response at a rate of about 89%.

[0257] In some embodiments, the method effectively obtains a best response of any one of best partial response, complete response, or stringent complete response, i.e., a best response of best partial response or better. In some embodiments, the method effectively obtains the best response of best partial response or better at a rate of about 41% or less. In some embodiments, the method effectively obtains the best response of best partial response or better at a rate of about 67% or less. In some embodiments, the method effectively obtains the best response of best partial response or better at a rate of about 87% or less. In some embodiments, the method effectively obtains the best response of any one of best partial response, complete response, or stringent complete response at a rate of about 41% to about 87%. In some embodiments, the method effectively obtains the best response of any one of best partial response, complete response, or stringent complete response at a rate of about 67%.

[0258] In some embodiments, the method effectively obtains a best effect of complete response or stringent complete response, i.e., a best effect of complete response or better. In some embodiments, the method effectively obtains the best effect of complete response or better at a rate of about 10% or less. In some embodiments, the method effectively obtains the best effect of complete response or better at a rate of about 28% or less. In some embodiments, the method effectively obtains the best effect of complete response or better at a rate of about 54% or less. In some embodiments, the method effectively obtains the best effect of complete response or stringent complete response at a rate of about 10% to about 54%. In some embodiments, the method effectively obtains the best effect of complete response or stringent complete response at a rate of about 28%.

[0259] In some embodiments, the method is effective to obtain a best effect of stringent complete response. In some embodiments, the method is effective to obtain a best effect of stringent complete response at a rate of about 6% or less. In some embodiments, the method is effective to obtain a best effect of stringent complete response at a rate of about 22% or less. In some embodiments, the method is effective to obtain a best effect of stringent complete response at a rate of about 48% or less. In some embodiments, the method is effective to obtain a best effect of stringent complete response at a rate of about 6% to about 48%. In some embodiments, the method is effective to obtain a best effect of stringent complete response at a rate of about 22%.

[0260] In some embodiments, the method is effective to obtain the best effect about 27 days or more after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect about 42 days or more after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect about 71 days or more after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect about 154 days or more after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect about 354 days or more after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect before about 27 days to about 354 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect before about 27 days to about 155 days after the administration of the CAR-T cells. In some embodiments, the method is effective to achieve the best effect no earlier than about 71 days after the administration of the CAR-T cells. In some embodiments, the method is effective to achieve the best effect no earlier than about 42 days after the administration of the CAR-T cells.

[0261] In some embodiments, the method effectively maintains a response in a subject for a follow-up period from the time of the initial response to about 156 days after the administration of the CAR-T cells. In some embodiments, the method effectively maintains a response at a rate of about 5% to about 95% for a follow-up period of about 6 months, about 9 months, or about 12 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains a response at a rate of about 67% for a follow-up period of about 6 months, about 9 months, or about 12 months after the administration of the CAR-T cells.

[0262] In some embodiments, the method effectively obtains the initial response within about 269 days or less after the administration of the CAR-T cells, and the method effectively maintains the response in the subject for a follow-up period from the time of the initial response to about 156 days after the administration of the CAR-T cells. In some embodiments, the method effectively maintains the response at about 5% or less for a follow-up period of 6 months, 9 months, or 12 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains the response at about 67% or less for a follow-up period of 6 months, 9 months, or 12 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains the response at about 95% or less for a follow-up period of 6 months, 9 months, or 12 months after the administration of the CAR-T cells.

[0263] In some embodiments, the method further comprises administering the CAR-T cells to a patient receiving the CAR-T cells for 10 to about 3 months after the administration of the CAR-T cells. -5At a sensitivity threshold level of 0.1%, the method effectively obtains a minimal residual disease (MRD) negative status of the subject as assessed in bone marrow. In some embodiments, the method effectively obtains a minimal residual disease (MRD) negative complete response or a minimal residual disease (MRD) negative stringent complete response at a rate of about 1% or less at a follow-up period of about 141 days after the administration of the CAR-T cells. In some embodiments, the method effectively obtains a minimal residual disease (MRD) negative complete response or a minimal residual disease (MRD) negative stringent complete response at a rate of about 11% or less at a follow-up period of about 141 days after the administration of the CAR-T cells. In some embodiments, the method effectively obtains a minimal residual disease (MRD) negative complete response or a minimal residual disease (MRD) negative stringent complete response at a rate of about 35% or less at a follow-up period of about 141 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain a rate of about 1% to about 35% of minimal residual disease (MRD) negative complete responses or MRD negative stringent complete responses at a follow-up period of about 141 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain a rate of about 11% of minimal residual disease (MRD) negative complete responses or MRD negative stringent complete responses at a follow-up period of about 141 days after the administration of the CAR-T cells.

[0264] In some embodiments, the method is effective to obtain progression-free survival in the subject. In some embodiments, the method is effective to obtain progression-free survival in the subject from the administration of the CAR-T cells to about 182 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain progression-free survival at about 100% or less at a follow-up period of 6 months after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain progression-free survival at about 5% or less at a follow-up period of about 9 months or about 12 months after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain progression-free survival at about 67% or less at a follow-up period of about 9 months or about 12 months after the administration of the CAR-T cells. In some embodiments, the method effectively obtains the progression free survival at about 95% or less at about 9 months or about 12 months follow-up after the administration of the CAR-T cells. In some embodiments, the method effectively obtains the progression free survival at about 100% at about 6 months follow-up after the administration of the CAR-T cells. In some embodiments, the method effectively obtains the progression free survival at about 5% to about 95% at about 9 months or about 12 months follow-up after the administration of the CAR-T cells. In some embodiments, the method effectively obtains the progression free survival at about 67% at about 9 months or about 12 months follow-up after the administration of the CAR-T cells.

[0265] In some embodiments, the cytokine release syndrome has a reversal rate of about 100% or less 7 days after the cytokine release syndrome is first observed. In some embodiments, the method effectively achieves a reversal rate of about 1% to about 100% of the cytokine release syndrome in a period of about 7 days after the cytokine release syndrome is first observed.

[0266] 4.10. Methods of Treating Subjects Who Have Previously Received Non-Cellular BCMA-Targeted Therapy In one embodiment, a method of treating a subject is provided, the method comprising administering to the subject a composition comprising a therapeutically effective amount of T cells comprising a chimeric antigen receptor (CAR), wherein the subject has multiple myeloma and has already received at least one prior line of therapy comprising a treatment with a non-cellular BCMA targeted drug. In some embodiments, the at least one prior line of therapy comprises treatment with at least four drugs including a non-cellular BCMA targeted drug. In some embodiments, the at least four drugs further comprise a proteasome inhibitor, an immunomodulatory drug, and an anti-CD38 antibody.

[0267] In some embodiments, the subject has received prior treatment with at least two prior lines of therapy, at least three prior lines of therapy, at least four prior lines of therapy, at least five prior lines of therapy, at least six prior lines of therapy, at least seven prior lines of therapy, at least eight prior lines of therapy, at least nine prior lines of therapy, at least ten prior lines of therapy, at least eleven prior lines of therapy, or at least twelve prior lines of therapy. In some embodiments, the subject relapses after the at least one prior line of therapy, at least two prior lines of therapy, at least three prior lines of therapy, at least four prior lines of therapy, at least five prior lines of therapy, at least six prior lines of therapy, at least seven prior lines of therapy, at least eight prior lines of therapy, at least nine prior lines of therapy, at least ten prior lines of therapy, at least eleven prior lines of therapy, or at least twelve prior lines of therapy.

[0268] In some embodiments, the method of treatment effectively obtains a reduced tumor burden in the subject. In some embodiments, the method of treatment effectively obtains a tumor burden reduction of about 1% to about 100%, about 20% to about 100%, about 60% to about 100%, about 65% to about 100%, about 70% to about 100%, about 75% to about 100%, about 80% to about 100%, about 85% to about 100%, about 90% to about 100%, about 92% to about 100%, about 95% to about 100%, about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100% in the subject. In some embodiments, the method of treatment effectively obtains a tumor burden reduction of about 100% in the subject. In some embodiments, the treatment method effectively obtains a tumor burden reduction of about 1% to about 100% in the subject at a rate of about 1% to about 83%. In some embodiments, the treatment method effectively obtains a tumor burden reduction of about 5% to about 100% in the subject at a rate of about 1% to about 77%. In some embodiments, the treatment method effectively obtains a tumor burden reduction of about 20% to about 100% in the subject at a rate of about 1% to about 77%. In some embodiments, the treatment method effectively obtains a tumor burden reduction of about 30% to about 100% in the subject at a rate of about 1% to about 77%. In some embodiments, the treatment method effectively obtains a tumor burden reduction of about 40% to about 100% in the subject at a rate of about 1% to about 77%. In some embodiments, the treatment method effectively obtains a tumor burden reduction of about 50% to about 100% in the subject at a rate of about 1% to about 77%. In some embodiments, the treatment method effectively obtains about a 60% to about 100% tumor burden reduction in the subject at a rate of about 1% to about 72%. In some embodiments, the treatment method effectively obtains about a 70% to about 100% tumor burden reduction in the subject at a rate of about 1% to about 72%. In some embodiments, the treatment method effectively obtains about an 80% to about 100% tumor burden reduction in the subject at a rate of about 1% to about 67%. In some embodiments, the treatment method effectively obtains about a 90% to about 100% tumor burden reduction in the subject at a rate of about 1% to about 67%.In some embodiments, the treatment method is effective in obtaining about a 95% to about 100% tumor burden reduction in subjects at rates between about 1% and about 61%. In some embodiments, the treatment method is effective in obtaining about a 100% tumor burden reduction in subjects at rates between about 1% and about 50%.

[0269] In some embodiments, the method of treatment effectively achieves a minimal residual disease (MRD) negative status or effectively maintains said minimal residual disease (MRD) status in the subject.

[0270] In some embodiments, the method of treatment is effective to obtain a negative minimal residual disease (MRD) status in the subject. -6 In some embodiments, the method of treatment effectively achieves minimal residual disease (MRD) negativity in a subject at a sensitivity level of 10 -5 In some embodiments, the method of treatment effectively achieves minimal residual disease (MRD) negativity in a subject at a sensitivity level of 10 -4 In some embodiments, the method of treatment effectively achieves minimal residual disease (MRD) negativity in a subject at a sensitivity level of 10 -3In some embodiments, the method of treatment effectively obtains a minimal residual disease (MRD) negative status at a sensitivity level of 0.1 to 1.0. In some embodiments, the method of treatment effectively obtains a MRD negative status when assessed in bone marrow. In some embodiments, the method of treatment effectively maintains a MRD negative status when assessed in an evaluable bone marrow sample. In some embodiments, the method of treatment effectively obtains a MRD negative status when assessed in bone marrow DNA. In some embodiments, the method effectively obtains a minimal residual disease (MRD) negative status of the subject as assessed in bone marrow at a follow-up period of about 27 days or more after the administration of the CAR-T cells, about 2 months or more after the administration of the CAR-T cells, about 3 months or more after the administration of the CAR-T cells, about 6 months or more after the administration of the CAR-T cells, about 9 months or more after the administration of the CAR-T cells, or about 12 months or more after the administration of the CAR-T cells. In some embodiments, the minimal residual disease (MRD) negative status is assessed in bone marrow at a first follow-up period of about 56 days to about 58 days after the administration of the CAR-T cells.

[0271] In some embodiments, the treatment method effectively maintains the minimal residual disease (MRD) negativity initially achieved in the subject. -5 In some embodiments, the method of treatment effectively maintains MRD negativity at a sensitivity level of 10 -6 In some embodiments, the method of treatment effectively achieves minimal residual disease (MRD) negativity at a sensitivity level of 10 -4 In some embodiments, the treatment method effectively maintains MRD negativity at a sensitivity level of 10 -3In some embodiments, the treatment method effectively maintains the MRD-negative status at a sensitivity level of 0.1 to 1.0 days after administration of the CAR-T cells. In some embodiments, the treatment method effectively maintains the MRD-negative status when assessed in a bone marrow sample. In some embodiments, the treatment method effectively maintains the MRD-negative status when assessed in an evaluable bone marrow sample. In some embodiments, the treatment method effectively maintains the MRD-negative status when assessed in bone marrow DNA. In some embodiments, the method effectively maintains the minimal residual disease (MRD)-negative status of the subject as assessed in bone marrow at a follow-up period of about 27 days to about 186 days after administration of the CAR-T cells, about 29 days to about 6 months after administration of the CAR-T cells, about 29 days to about 3 months after administration of the CAR-T cells, or about 29 days to about 2 months after administration of the CAR-T cells. In some examples, the method effectively maintains the minimal residual disease (MRD) negative status of the subject as assessed in bone marrow at a second follow-up period from about 183 days to about 186 days after the administration of the CAR-T cells, and further wherein the first follow-up period is earlier than the second follow-up period.

[0272] In some embodiments, the efficacy of the treatment method is assessed by evaluating the proportion of subjects with MRD-negative status. -6 The efficacy of the treatment method is assessed by assessing the proportion of subjects with MRD-negative status at a sensitivity level of 10 -5 The efficacy of the treatment method is assessed by assessing the proportion of subjects with MRD-negative status at a sensitivity level of 10 -4 The efficacy of the treatment method is assessed by assessing the proportion of subjects with MRD-negative status at a sensitivity level of 10 -3The efficacy of the treatment method is assessed by assessing the proportion of subjects with MRD-negative status at a median follow-up period from administration of the CAR-T cells to about 186 days after administration of the CAR-T cells, from administration of the CAR-T cells to about 6 months after administration of the CAR-T cells, from administration of the CAR-T cells to about 3 months after administration of the CAR-T cells, from administration of the CAR-T cells to about 2 months after administration of the CAR-T cells, or from administration of the CAR-T cells to about 27 days after administration of the CAR-T cells. In some embodiments, the method assesses the efficacy of the treatment method by assessing the proportion of subjects with MRD-negative status at a median follow-up period from administration of the CAR-T cells to about 186 days after administration of the CAR-T cells, from administration of the CAR-T cells to about 6 months after administration of the CAR-T cells, from administration of the CAR-T cells to about 3 months after administration of the CAR-T cells, from administration of the CAR-T cells to about 2 months after administration of the CAR-T cells, or from administration of the CAR-T cells to about 27 days after administration of the CAR-T cells. -4 At a sensitivity threshold level of about 9% or less, 10 -5 At a rate of about 6% or less at the sensitivity threshold level of 10 -6 In some embodiments, the method effectively achieves a minimal residual disease (MRD) negative status at a rate of about 1% or less at a sensitivity threshold level of 10 -4 At a sensitivity threshold level of about 25% or less, 10 -5 At a sensitivity threshold level of about 20% or less, or at a sensitivity threshold level of about 10 -6 In some embodiments, the method effectively achieves a minimal residual disease (MRD) negative status at a rate of about 10% or less at a sensitivity threshold level of 10. -4 At a sensitivity threshold level of about 49% or less, 10 -5 At a sensitivity threshold level of about 44% or less, or at a sensitivity threshold level of about 10 -6 In some embodiments, the method effectively achieves a minimal residual disease (MRD) negative status at a rate of about 31% or less at a sensitivity threshold level of 10 -4 At the sensitivity threshold level, the ratio is about 9% to about 49%, and -5 At a sensitivity threshold level of about 6% to about 44%, or -6 In some embodiments, the method effectively achieves a minimal residual disease (MRD) negative status at a rate of about 1% to about 31% at a sensitivity threshold level of 10 -4 At the sensitivity threshold level of 10, the ratio is about 25%. -5At a sensitivity threshold level of about 20%, or 10 -6 At a sensitivity threshold level of 0.1, approximately 10% of patients effectively achieve the minimal residual disease (MRD) negative status.

[0273] In some embodiments, the efficacy of the treatment method is assessed by evaluating the proportion of subjects with evaluable bone marrow and MRD-negative status. -6 The efficacy of the treatment method is assessed by assessing the proportion of subjects with evaluable bone marrow and MRD-negative status at a sensitivity level of 10. -5 The efficacy of the treatment method is assessed by assessing the proportion of subjects with evaluable bone marrow and MRD-negative status at a sensitivity level of 10. -4 The efficacy of the treatment method is assessed by assessing the proportion of subjects with evaluable bone marrow and MRD-negative status at a sensitivity level of 10. -3 The efficacy of the treatment method is assessed by assessing the proportion of subjects with evaluable bone marrow and MRD-negative status at a median follow-up time from administration of the CAR-T cells to about 186 days post-CAR-T cell administration, from administration of the CAR-T cells to about 6 months post-CAR-T cell administration, from administration of the CAR-T cells to about 3 months post-CAR-T cell administration, from administration of the CAR-T cells to about 2 months post-CAR-T cell administration, or from administration of the CAR-T cells to about 27 days post-CAR-T cell administration. In some embodiments, the method assesses the efficacy of the treatment method by assessing the proportion of subjects with evaluable bone marrow and MRD-negative status at a median follow-up time from administration of the CAR-T cells to about 186 days post-CAR-T cell administration, from administration of the CAR-T cells to about 6 months post-CAR-T cell administration, from administration of the CAR-T cells to about 3 months post-CAR-T cell administration, from administration of the CAR-T cells to about 2 months post-CAR-T cell administration, or from administration of the CAR-T cells to about 27 days post-CAR-T cell administration. -5 At a sensitivity threshold level of 100%, the method effectively achieves minimal residual disease (MRD) negativity in about 22% or less of subjects with evaluable samples. -5 At a sensitivity threshold level of 100%, the method effectively achieves minimal residual disease (MRD) negativity in about 67% or less of subjects with evaluable samples. -5At a sensitivity threshold level of 100%, the method effectively achieves minimal residual disease (MRD) negativity in about 96% or less of subjects with evaluable samples. -5 At a sensitivity threshold level of 100%, the method effectively obtains the minimal residual disease (MRD) negative status in about 22% to about 96% of subjects with evaluable samples. -5 At a sensitivity threshold level of 0.1, approximately 67% of subjects with evaluable samples effectively achieve the minimal residual disease (MRD) negativity status.

[0274] In some embodiments, the method is effective to obtain at least one response in the subject following said administration of the CAR-T cells, wherein the at least one response comprises, in order from best to worst, a stringent complete response, a complete response, a best partial response, a partial response, or a minimal response.

[0275] In some embodiments, the method is effective to obtain an initial response about 27 days or more after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response about 28 days or more after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response about 43 days or more after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response about 87 days or more after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response about 153 days or more after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response before about 27 days to about 153 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response before about 27 days to about 88 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response no earlier than about 43 days after said administration of the CAR-T cells. In some embodiments, the method is effective to obtain an initial response no earlier than about 28 days after said administration of the CAR-T cells.

[0276] In some embodiments, the effectiveness of the treatment method is assessed by assessing the proportion of subjects having a stringent complete response. In some embodiments, the effectiveness of the treatment method is assessed by assessing the proportion of subjects having a complete response or better. In some embodiments, the effectiveness of the treatment method is assessed by assessing the proportion of subjects having a best partial response or better. In some embodiments, the effectiveness of the treatment method is assessed by assessing the proportion of subjects having a partial response or better. In some embodiments, the effectiveness of the treatment method is assessed by assessing the proportion of subjects having a minimal response or better.

[0277] In some embodiments, the method effectively obtains a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response, i.e., a best response of minimal response or better. In some embodiments, the rate at which the method effectively obtains a best response of minimal response or better is referred to as a clinical benefit rate. In some embodiments, the method effectively obtains a best response of minimal response or better at a rate of about 23% or less. In some embodiments, the method effectively obtains a best response of minimal response or better at a rate of about 45% or less. In some embodiments, the method effectively obtains a best response of minimal response or better at a rate of about 68% or less. In some embodiments, the method effectively obtains a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response at a rate of about 23% to about 69%. In some embodiments, the method effectively achieves a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response at a rate of about 45%.

[0278] In some embodiments, the method effectively obtains a best response of any one of partial response, best partial response, complete response, or stringent complete response, i.e., a best response of partial response or better. In some embodiments, the rate at which the method effectively obtains a best response of partial response or better is referred to as the overall survival rate or overall response rate. In some embodiments, the method effectively obtains the best response of partial response or better at a rate of about 19% or less. In some embodiments, the method effectively obtains the best response of partial response or better at a rate of about 40% or less. In some embodiments, the method effectively obtains the best response of partial response or better at a rate of about 63% or less. In some embodiments, the method effectively obtains the best response of any one of partial response, best partial response, complete response, or stringent complete response at a rate of about 19% to about 64%. In some embodiments, the method is effective to obtain a best response of any one of the partial response, best partial response, complete response, or stringent complete response at a rate of about 40%.

[0279] In some embodiments, the method effectively obtains a best response of any one of best partial response, complete response, or stringent complete response, i.e., a best response of best partial response or better. In some embodiments, the method effectively obtains the best response of best partial response or better at a rate of about 15% or less. In some embodiments, the method effectively obtains the best response of best partial response or better at a rate of about 35% or less. In some embodiments, the method effectively obtains the best response of best partial response or better at a rate of about 59% or less. In some embodiments, the method effectively obtains the best response of any one of best partial response, complete response, or stringent complete response at a rate of about 15% to about 59%. In some embodiments, the method effectively obtains the best response of any one of best partial response, complete response, or stringent complete response at a rate of about 35%.

[0280] In some embodiments, the method effectively obtains a best effect of complete response or stringent complete response, i.e., a best effect of complete response or better. In some embodiments, the method effectively obtains the best effect of complete response or better at a rate of about 3% or less. In some embodiments, the method effectively obtains the best effect of complete response or better at a rate of about 15% or less. In some embodiments, the method effectively obtains the best effect of complete response or better at a rate of about 38% or less. In some embodiments, the method effectively obtains the best effect of complete response or stringent complete response at a rate of about 3% to about 38%. In some embodiments, the method effectively obtains the best effect of complete response or stringent complete response at a rate of about 15%.

[0281] In some embodiments, the method is effective to obtain a best effect of stringent complete response. In some embodiments, the method is effective to obtain a best effect of stringent complete response at a rate of about 1% or less. In some embodiments, the method is effective to obtain a best effect of stringent complete response at a rate of about 10% or less. In some embodiments, the method is effective to obtain a best effect of stringent complete response at a rate of about 32% or less. In some embodiments, the method is effective to obtain a best effect of stringent complete response at a rate of about 1% to about 32%. In some embodiments, the method is effective to obtain a best effect of stringent complete response at a rate of about 10%.

[0282] In some embodiments, the method is effective to obtain the best effect about 27 days or more after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect about 56 days or more after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect about 77 days or more after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect about 132 days or more after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect about 171 days or more after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect before about 27 days to about 171 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain the best effect before about 27 days to about 133 days after the administration of the CAR-T cells. In some embodiments, the method is effective to achieve the best effect no earlier than about 78 days after the administration of the CAR-T cells. In some embodiments, the method is effective to achieve the best effect no earlier than about 56 days after the administration of the CAR-T cells.

[0283] In some embodiments, the method effectively maintains a response in a subject for a follow-up period from the time of the initial response to about 132 days after the administration of the CAR-T cells, and further wherein the initial response is obtained from the time of the administration of the CAR-T cells to about 131 days after the administration of the CAR-T cells. In some embodiments, the method effectively maintains a response at a rate of about 20% to about 96% for a follow-up period of about 6 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains a response at a rate of about 80% for a follow-up period of about 6 months after the administration of the CAR-T cells.

[0284] In some embodiments, wherein the method effectively obtains the initial response within about 131 days or less after the administration of the CAR-T cells, and the method effectively maintains the response in the subject for a follow-up period from the time of the initial response to about 132 days after the administration of the CAR-T cells. In some embodiments, the method effectively maintains the response at about 20% or less for a follow-up period of 6 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains the response at about 80% or less for a follow-up period of 6 months after the administration of the CAR-T cells. In some embodiments, the method effectively maintains the response at about 96% or less for a follow-up period of 6 months after the administration of the CAR-T cells.

[0285] In some embodiments, the method is effective to obtain progression free survival in the subject. In some embodiments, the method is effective to obtain progression free survival in the subject from the administration of the CAR-T cells to about 15 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain progression free survival in the subject from the administration of the CAR-T cells to about 44 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain progression free survival in the subject from the administration of the CAR-T cells to about 159 days after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain progression free survival at about 29% or less at a follow-up period of 6 or 9 months after the administration of the CAR-T cells. In some embodiments, the method is effective to obtain progression free survival at about 55% or less at a follow-up period of 6 or 9 months after the administration of the CAR-T cells. In some embodiments, the method effectively obtains the progression free survival at about 75% or less at about 6 months or about 9 months follow-up period after the administration of the CAR-T cells. In some embodiments, the method effectively obtains the progression free survival at about 29% to about 75% at about 6 months or about 9 months follow-up period after the administration of the CAR-T cells. In some embodiments, the method effectively obtains the progression free survival at about 55% at about 6 months or about 9 months follow-up period after the administration of the CAR-T cells.

[0286] In some embodiments, the rate of cytokine release syndrome is about 60% or greater. In some embodiments, the method effectively achieves a rate of cytokine release syndrome of about 60% to about 99%. In some embodiments, the method further comprises treating the subject for cytokine release syndrome more than about 3 days after the administration of the CAR-T cells.

[0287] In some embodiments, the rate of immune effector cell-associated neurotoxicity is about 20% or greater. In some embodiments, the method is effective to obtain a rate of immune effector cell-associated neurotoxicity of about 20% to about 99%.

[0288] 4.11. Kits and Products Any of the compositions described herein may be included in a kit. In some examples, the engineered immortalized CAR-T cells are provided in a kit, which may further include reagents suitable for expanding the cells, such as culture medium.

[0289] In non-limiting examples, the components include a chimeric receptor expression construct, one or more reagents for producing the chimeric receptor expression construct, cells for transfecting the expression construct, and / or one or more instruments for obtaining immortalized T cells for transfecting the expression construct (such instruments may be a syringe, pipette, forceps, and / or any such medically approved device).

[0290] In some aspects, the kit comprises reagents or equipment for cell electroporation.

[0291] In some embodiments, the kit comprises an artificial antigen presenting cell.

[0292] The kit may include one or more of the compositions of the present disclosure, suitably aliquoted, or reagents for producing the compositions of the present disclosure. The components of the kit may be packaged in aqueous media or lyophilized form. The container device of the kit may include at least one vial, test tube, flask, bottle, syringe, or other container device, into which the components may be placed, and preferably suitably aliquoted. If there is more than one component in the kit, the kit will typically further include a second, third, or other additional container, into which the additional components may be placed separately. However, various combinations of components may be included in a vial. The kit of the present disclosure typically further includes a device for containing the chimeric receptor construct, and any other sealed reagent container for commercial sale. Such containers may include, for example, injection-molded or blow-molded plastic containers into which the desired vials are held.

[0293] 4.12. Specific Examples Particular embodiments of the present disclosure are described in the following numbered paragraphs: 1. A method of treating a subject, the method comprising administering to the subject a composition comprising a therapeutically effective amount of T cells comprising a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising: (a) (1) a first anti-BCMA binding moiety comprising a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 18, a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 19, and a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 20; (2) an extracellular antigen-binding domain comprising a second BCMA-binding portion comprising a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 21, a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 22, and a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 23; (b) a transmembrane domain; (c) an intracellular signaling domain; thereby delivering a dose of T cells expressing a CAR (CAR-T cells) to the subject; Here, the subject is (i) suffers from multiple myeloma; (ii) have already received prior treatment with one, two or three previous lines of therapy; (iii) The method is refractory to lenalidomide. 2. A method of treating a subject, the method comprising administering to the subject a composition comprising a therapeutically effective amount of T cells comprising a chimeric antigen receptor (CAR), the chimeric antigen receptor comprising: (a) (1) a first anti-BCMA binding moiety comprising a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 18, a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 19, and a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 20; (2) an extracellular antigen-binding domain comprising a second BCMA-binding portion comprising a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 21, a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 22, and a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 23; (b) a transmembrane domain; (c) an intracellular signaling domain; thereby delivering a dose of T cells expressing a CAR (CAR-T cells) to the subject; Here, the subject is (i) suffers from multiple myeloma; (ii) has already received prior treatment with one prior line of treatment, said prior line of treatment including treatment with at least two drugs, including a proteasome inhibitor and an immunomodulatory drug; (iii) previous early recurrence, method; 3. A method of treating a subject, the method comprising administering to the subject a composition comprising a therapeutically effective amount of T cells comprising a chimeric antigen receptor (CAR), the chimeric antigen receptor being: (a) (1) a first anti-BCMA binding moiety comprising a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 18, a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 19, and a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 20; (2) an extracellular antigen-binding domain comprising a second BCMA-binding portion comprising a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 21, a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 22, and a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 23; (b) a transmembrane domain; (c) an intracellular signaling domain; thereby delivering a dose of T cells expressing a CAR (CAR-T cells) to the subject; Here, the subject is (i) suffers from multiple myeloma; (ii) having already received at least one prior line of treatment, said at least one prior line of treatment comprising treatment with at least four drugs including a non-cellular BCMA targeted drug. 4. The subject has received prior treatment with at least one prior line of treatment, the at least one prior line of treatment comprising treatment with lenalidomide and at least one non-lenalidomide drug, the at least one non-lenalidomide drug being (a) proteasome inhibitors, (b) an immunomodulatory agent; or (c) the method of paragraph 1, comprising at least one anti-CD38 antibody. 5. The method of paragraph 1 or paragraph 4, wherein the subject has received prior treatment with at least two prior lines of therapy. 6. The method of paragraph 1, paragraph 4 or paragraph 5, wherein the subject has received prior treatment with three prior lines of therapy. 7. The method of any one of paragraphs 1 or 4-6, wherein the subject has received prior treatment with dexamethasone, an alkylating agent, or daratumumab. 8. The method of any one of paragraphs 1 or 4-7, wherein the multiple myeloma is refractory to a last line of therapy. 9. The method of any one of paragraphs 1 or 4-8, wherein the subject has relapsed after the one, two, or three prior lines of therapy. 10. The method of any one of paragraphs 1 or 4-9, wherein the multiple myeloma is refractory to three drugs. 11. The method of any one of paragraphs 1 or 4-10, wherein the method effectively obtains a minimal residual disease (MRD) negative status in the subject as assessed in bone marrow following the administration of the CAR-T cells. 12. The method of paragraph 11, wherein the minimal residual disease (MRD)-negative status is obtained at a first follow-up period from about 29 days to about 184 days after the administration of the CAR-T cells. 13. The method of paragraph 12, wherein the method effectively maintains the minimal residual disease (MRD)-negative status of the subject as assessed in the bone marrow at a second follow-up period from about 57 days to about 191 days after the administration of the CAR-T cells, and further wherein the first follow-up period is earlier than the second follow-up period. 14. The method comprises the steps of: -4 , 10 -5 or 10 -6 12. The method of paragraph 11, wherein the method effectively obtains the minimal residual disease (MRD) negative status at a rate of about 24% to about 61% at a sensitivity threshold level of 1. 15. The method comprises the steps of: -4 , 10 -5 or 10 -6 12. The method of paragraph 11, effectively obtaining said minimal residual disease (MRD) negative status at a rate of about 41% at a sensitivity threshold level of 0.1%. 16. The method comprises the steps of: -512. The method of paragraph 11, wherein at a sensitivity threshold level of 0.1%, the method effectively obtains the minimal residual disease (MRD) negative status in about 64% to about 99% of subjects with evaluable samples. 17. The method comprises the steps of: -5 12. The method of paragraph 11, wherein at a sensitivity threshold level of 0.1, 0.2 or 0.3, effectively obtains said minimal residual disease (MRD) negative status in about a 92% proportion of subjects with evaluable samples. 18. The method effectively obtains at least one response in the subject following said administration of the CAR-T cells, wherein the at least one response is, in order from best to worst: (i) stringent complete response; (ii) complete response; (iii) best partial response, (iv) partial response, or (v) The method of paragraphs 1 or 4-17, comprising a minimal response. 19. The method of paragraph 18, wherein the method effectively obtains an initial response prior to a period of about 21 to about 99 days after the administration of the CAR-T cells. 20. The method of paragraph 18, wherein the method effectively obtains an initial response prior to a period of about 21 to about 55 days after the administration of the CAR-T cells. 21. The method of paragraph 18, wherein the method effectively obtains an initial response prior to about 36 days after the administration of the CAR-T cells. 22. The method of paragraph 18, wherein the method effectively obtains an initial response no sooner than about 30 days after the administration of the CAR-T cells. 23. The method of any one of paragraphs 18 to 22, wherein the method effectively achieves a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response. 24. The method of paragraph 23, wherein the method effectively achieves a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response in a rate of about 52% to about 87%. 25. The method of paragraph 23, wherein the method effectively achieves a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response in about a 72% rate. 26. The method of any one of paragraphs 18 to 25, wherein the method effectively achieves a best response of any one of partial response, best partial response, complete response, or stringent complete response. 27. The method of paragraph 26, wherein the method effectively achieves a best response of any one of the partial response, best partial response, complete response, or stringent complete response at a rate of about 49% to about 84%. 28. The method of paragraph 26, wherein the method effectively achieves a best response of any one of the partial response, best partial response, complete response, or stringent complete response at a rate of about 69%. 29. The method of any one of paragraphs 18 to 28, wherein the method effectively achieves a best response of any one of best partial response, complete response, or stringent complete response. 30. The method of paragraph 29, wherein the method effectively achieves a best response of any one of the best partial response, complete response, or stringent complete response at a rate of about 49% to about 84%. 31. The method of paragraph 29, wherein the method effectively achieves a best response of any one of the best partial response, complete response, or stringent complete response at a rate of about 69%. 32. The method of any one of paragraphs 18 to 31, wherein the method effectively achieves an optimal response of complete response or stringent complete response. 33. The method of paragraph 32, wherein the method effectively achieves a best outcome of complete response or stringent complete response at a rate of about 39% to about 76%. 34. The method of paragraph 32, wherein the method effectively achieves a best outcome of complete response or stringent complete response at a rate of about 58%. 35. The method of any one of paragraphs 18 to 34, wherein the method effectively achieves an optimal response of stringent complete response. 36. The method of paragraph 35, wherein the method effectively achieves the optimal stringent complete response at a rate of about 33% to about 70%. 37. The method of paragraph 35, wherein the method effectively achieves the optimal response of stringent complete response at a rate of about 52%. 38. The method of any one of paragraphs 1 or 4 to 37, wherein the method effectively results in progression-free survival in the subject. 39. The method of paragraph 38, wherein the method effectively achieves the progression-free survival in the subject from the administration of the CAR-T cells to about 55 days after the administration of the CAR-T cells. 40. The method of paragraph 38, wherein the method effectively achieves the progression-free survival in the subject from the administration of the CAR-T cells to about 297 days after the administration of the CAR-T cells. 41. The method of paragraph 38, wherein the method effectively achieves a progression-free survival rate of about 62% to about 95% at a follow-up period of about 6 months or about 9 months after the administration of the CAR-T cells. 42. The method of paragraph 38, wherein the method effectively achieves a progression-free survival rate of about 86% at a follow-up period of about 6 months or about 9 months after the administration of the CAR-T cells. 43. The method of any one of paragraphs 1 or 4-42, wherein the method further comprises treating cytokine release syndrome in the subject more than about 3 days after the administration of the CAR-T cells. 44. The method of paragraph 43, wherein the method effectively results in about a 1% to about a 90% reversal rate of the cytokine release syndrome within about a 7 day period after the cytokine release syndrome is first observed. 45. The method of any one of paragraphs 1 or 4 to 44, wherein the method effectively obtains a rate of about 20% to about 99% of neurotoxicity associated with immune effector cells. 46. ​​The method of any one of paragraphs 23 to 45, wherein the method effectively achieves the optimal effect prior to a period of from about 27 days to about 237 days after the administration of the CAR-T cells. 47. The method of any one of paragraphs 23 to 45, wherein the method effectively achieves the optimal effect prior to a period of from about 46 days to about 172 days after the administration of the CAR-T cells. 48. The method of any one of paragraphs 23 to 45, wherein the method effectively achieves the optimal effect prior to about 109 days after the administration of the CAR-T cells. 49. The method of any one of paragraphs 23 to 45, wherein the method effectively achieves the optimal effect prior to about 87 days after the administration of the CAR-T cells. 50. The method of any one of paragraphs 19 to 49, wherein the method effectively maintains the response in the subject for a follow-up period from the time of the initial response to about 270 days after the administration of the CAR-T cells. 51. The method of any one of paragraphs 18 to 50, wherein the method effectively maintains response at a rate of about 70% to about 99% over a follow-up period of about 6 months after the administration of the CAR-T cells. 52. The method of any one of paragraphs 18 to 50, wherein the method effectively maintains the response at a rate of about 95% over a follow-up period of about 6 months after the administration of the CAR-T cells. 53. The method of any one of paragraphs 18 to 50, wherein the method effectively maintains response at a rate of about 7% to about 92% over a follow-up period of about 9 months after the administration of the CAR-T cells. 54. The method of any one of paragraphs 18 to 50, wherein the method effectively maintains response at a rate of about 63% over a follow-up period of about 9 months after the administration of the CAR-T cells. 55. Further, the method includes administering 10 to about 3 months after administration of the CAR-T cells. -5 55. The method of any one of paragraphs 32 to 54, effectively obtaining a minimal residual disease (MRD) negative status of the subject as assessed in the bone marrow at a sensitivity threshold level of 0.1 to 0.5 μg / mL. 56. The method of paragraph 55, wherein the method effectively obtains a minimal residual disease (MRD)-negative complete response or a minimal residual disease (MRD)-negative stringent complete response at a rate of about 18% to about 54% during a follow-up period of about 291 days after the administration of the CAR-T cells. 57. The method of paragraph 55, wherein the method effectively achieves a minimal residual disease (MRD)-negative complete response or a minimal residual disease (MRD)-negative stringent complete response rate of about 35% during a follow-up period of about 291 days after the administration of the CAR-T cells. 58. The method of paragraph 2, wherein the subject is further treated with an anti-CD38 antibody. 59. The method of paragraph 2 or paragraph 58, wherein the multiple myeloma is refractory to at least one drug. 60. The method of any one of paragraphs 2 or 58-59, wherein the method effectively obtains a minimal residual disease (MRD) negative status in the subject as assessed in bone marrow following administration of the CAR-T cells. 61. The method of paragraph 60, wherein the minimal residual disease (MRD)-negative status is assessed in the bone marrow at a first follow-up period from about 35 days to about 58 days after the administration of the CAR-T cells. 62. The method of paragraph 61, wherein the method effectively maintains the minimal residual disease (MRD)-negative status of the subject as assessed in the bone marrow at a second follow-up period from about 78 days to about 359 days after the administration of the CAR-T cells, and further wherein the first follow-up period is earlier than the second follow-up period. 63. The method comprises: -4 or 10 -5 At a sensitivity threshold level of about 26% to about 74%, or -6 61. The method of paragraph 60, wherein the method effectively obtains the minimal residual disease (MRD) negative status at a rate of about 17% to about 64% at a sensitivity threshold level of 61. 64. The method comprises: -4 or 10 -5 At a sensitivity threshold level of about 50% or 10 -6 61. The method of paragraph 60, effectively obtaining said minimal residual disease (MRD) negative status at a rate of about 39% at a sensitivity threshold level of 0.1. 65. The method comprises: -561. The method of paragraph 60, wherein at a sensitivity threshold level of 0.1, 0.1 or 0.2, 0.2 or 0.3, effectively obtains the minimal residual disease (MRD) negative status in a proportion of subjects having an evaluable sample of about 66% to about 100%. 66. The method comprises: -5 61. The method of paragraph 60, wherein at a sensitivity threshold level of 0.1, 0.2 or 1.5 effectively obtains said minimal residual disease (MRD) negative status in about a 100% proportion of subjects with evaluable samples. 67. The method effectively obtains at least one response in the subject following said administration of the CAR-T cells, wherein the at least one response is, in order from best to worst: (i) stringent complete response; (ii) complete response; (iii) best partial response, (iv) partial response, or (v) The method of any one of paragraphs 2 or 58-66, comprising a minimal response. 68. The method of paragraph 67, wherein the method effectively obtains an initial response prior to a period of about 27 to about 78 days after the administration of the CAR-T cells. 69. The method of paragraph 67, wherein the method effectively obtains an initial response prior to a period of about 27 to about 47 days after the administration of the CAR-T cells. 70. The method of paragraph 67, wherein the method effectively obtains an initial response prior to about 33 days after the administration of the CAR-T cells. 71. The method of paragraph 67, wherein the method effectively obtains an initial response prior to about 28 days after the administration of the CAR-T cells. 72. The method of any one of paragraphs 67 to 71, wherein the method effectively achieves a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response. 73. The method of paragraph 72, wherein the method effectively achieves a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response in a rate of about 65% to about 99%. 74. The method of paragraph 72, wherein said method effectively achieves a best response of any one of said minimal response, partial response, best partial response, complete response, or stringent complete response in about an 89% rate. 75. The method of any one of paragraphs 67 to 74, wherein the method effectively achieves a best response of any one of a partial response, a best partial response, a complete response, or a stringent complete response. 76. The method of paragraph 75, wherein the method effectively achieves a best response of any one of the partial response, best partial response, complete response, or stringent complete response in a rate of about 65% to about 99%. 77. The method of paragraph 75, wherein the method effectively achieves a best response of any one of the partial response, best partial response, complete response, or stringent complete response at a rate of about 89%. 78. The method of any one of paragraphs 67 to 77, wherein the method effectively achieves a best response of any one of best partial response, complete response, or stringent complete response. 79. The method of paragraph 78, wherein the method effectively achieves a best response of any one of the best partial response, complete response, or stringent complete response at a rate of about 41% to about 87%. 80. The method of paragraph 78, wherein the method effectively achieves a best response of any one of the best partial response, complete response, or stringent complete response at a rate of about 67%. 81. The method of any one of paragraphs 67 to 80, wherein the method effectively achieves an optimal response of complete response or stringent complete response. 82. The method of paragraph 81, wherein the method effectively achieves a best outcome of complete response or stringent complete response at a rate of about 10% to about 54%. 83. The method of paragraph 81, wherein said method effectively achieves a best outcome of said complete response or stringent complete response at a rate of about 28%. 84. The method of any one of paragraphs 67 to 83, wherein the method effectively achieves an optimal response of stringent complete response. 85. The method of paragraph 84, wherein the method effectively achieves the optimal stringent complete response at a rate of about 6% to about 48%. 86. The method of paragraph 84, wherein the method effectively achieves the best outcome of stringent complete response at a rate of about 22%. 87. The method of any one of paragraphs 2 or 58 to 86, wherein the method effectively results in progression-free survival in the subject. 88. The method of paragraph 87, wherein the method effectively achieves said progression-free survival in said subject from said administration of said CAR-T cells to about 182 days after said administration of said CAR-T cells. 89. The method of paragraph 87, wherein the method effectively achieves a progression-free survival rate of about 100% at a follow-up period of about 6 months after the administration of the CAR-T cells. 90. The method of paragraph 87, wherein the method effectively achieves a progression-free survival rate of about 5% to about 95% over a follow-up period of about 9 months or about 12 months after the administration of the CAR-T cells. 91. The method of paragraph 87, wherein the method effectively achieves a progression-free survival rate of about 67% at a follow-up period of about 9 months or about 12 months after the administration of the CAR-T cells. 92. The method of any one of paragraphs 2 or 58 to 91, wherein the method further comprises treating cytokine release syndrome in the subject more than about 3 days after the administration of the CAR-T cells. 93. The method of paragraph 92, wherein the method effectively obtains about a 1% to about 100% reversal rate of the cytokine release syndrome within about a 7 day period after the cytokine release syndrome is first observed. 94. The method of any one of paragraphs 72 to 93, wherein the method effectively achieves the optimal effect prior to a period of from about 27 days to about 354 days after the administration of the CAR-T cells. 95. The method of any one of paragraphs 72 to 93, wherein the method effectively achieves the optimal effect prior to a period of from about 27 days to about 155 days after the administration of the CAR-T cells. 96. The method of any one of paragraphs 72 to 93, wherein the method effectively achieves the optimal effect prior to about 71 days after administration of the CAR-T cells. 97. The method of any one of paragraphs 72 to 93, wherein the method effectively achieves the optimal effect prior to about 42 days after administration of the CAR-T cells. 98. The method of any one of paragraphs 68 to 97, wherein the method effectively maintains the response in the subject for a follow-up period from the time of the initial response to about 156 days after the administration of the CAR-T cells. 99. The method of any one of paragraphs 68 to 98, wherein the method effectively maintains response at a rate of about 5% to about 95% at a follow-up period of about 6 months, about 9 months, or about 12 months after the administration of the CAR-T cells. 100. The method of any one of paragraphs 68 to 98, wherein the method effectively maintains response at a rate of about 67% at a follow-up period of about 6 months, about 9 months, or about 12 months after the administration of the CAR-T cells. 101. Further, the method includes administering the CAR-T cells to a patient in need of treatment with 10 -5 101. The method of any one of paragraphs 81 to 100, effectively obtaining a minimal residual disease (MRD) negative status of the subject as assessed in the bone marrow at a sensitivity threshold level of 0.1 to 0.5%. 102. The method of paragraph 101, wherein the method effectively obtains a minimal residual disease (MRD)-negative complete response or a minimal residual disease (MRD)-negative stringent complete response at a rate of about 1% to about 35% during a follow-up period of about 141 days after the administration of the CAR-T cells. 103. The method of paragraph 101, wherein the method effectively achieves a minimal residual disease (MRD)-negative complete response or a minimal residual disease (MRD)-negative stringent complete response rate of about 11% during a follow-up period of about 141 days after the administration of the CAR-T cells. 104. The method of paragraph 3, wherein the subject has received prior treatment with at least two prior lines of therapy. 105. The method of paragraph 3 or paragraph 104, wherein the subject has received prior treatment with at least four prior lines of therapy. 106. The method of any one of paragraphs 3 or 104-105, wherein the subject has received prior treatment with at least eight prior lines of therapy. 107. The method of any one of paragraphs 3 or 104-106, wherein the subject has received prior treatment with at least 12 prior lines of therapy. 108. The method of any one of paragraphs 3 or 104-107, wherein the subject has relapsed after at least one prior line of treatment. 109. The method of any one of paragraphs 3 or 104 to 108, wherein the method effectively obtains a minimal residual disease (MRD) negative status in the subject as assessed in bone marrow following the administration of the CAR-T cells. 110. The method of paragraph 109, wherein the minimal residual disease (MRD)-negative status is assessed in the bone marrow at a first follow-up period from about 56 days to about 58 days after the administration of the CAR-T cells. 111. The method of paragraph 110, wherein the method effectively maintains the minimal residual disease (MRD)-negative status of the subject as assessed in the bone marrow at a second follow-up period from about 183 days to about 186 days after the administration of the CAR-T cells, and further wherein the first follow-up period is earlier than the second follow-up period. 112. The method comprises: -4 At the sensitivity threshold level, the ratio is about 9% to about 49%, and -5 At a sensitivity threshold level of about 6% to about 44%, or -6 109. The method of paragraph 109, wherein the method effectively obtains the minimal residual disease (MRD) negative status at a rate of about 1% to about 31% at a sensitivity threshold level of 10. 113. The method comprises: -4 At the sensitivity threshold level of 10, the ratio is about 25%. -5 At a sensitivity threshold level of about 20%, or 10 -6 110. The method of paragraph 109, wherein the method effectively obtains said minimal residual disease (MRD) negative status at a rate of about 10% at a sensitivity threshold level of 0.1%. 114. The method comprises: -5109. The method of paragraph 109, wherein at a sensitivity threshold level of 0.1, 0.2 or 0.3, 0.4 or 0.5, effectively obtains the minimal residual disease (MRD) negative status in about 22% to about 96% of subjects with evaluable samples. 115. The method comprises: -5 110. The method of paragraph 109, wherein at a sensitivity threshold level of 0.1, 0.2 or 0.3 effectively obtains said minimal residual disease (MRD) negative status in about a 67% rate in subjects with evaluable samples. 116. The method effectively obtains at least one response in the subject after said administration of the CAR-T cells, wherein the at least one response is, in order from best to worst: (i) stringent complete response; (ii) complete response; (iii) best partial response, (iv) partial response, or (v) The method of any one of paragraphs 3 or 104-115, comprising a minimal response. 117. The method of paragraph 116, wherein the method effectively obtains an initial response prior to a period of about 27 days to about 153 days after the administration of the CAR-T cells. 118. The method of paragraph 116, wherein the method effectively obtains an initial response prior to a period of about 27 to about 88 days after the administration of the CAR-T cells. 119. The method of paragraph 116, wherein the method effectively obtains an initial response prior to about 43 days after the administration of the CAR-T cells. 120. The method of paragraph 116, wherein the method effectively obtains an initial response prior to about 28 days after the administration of the CAR-T cells. 121. The method of any one of paragraphs 116 to 120, wherein the method effectively achieves a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response. 122. The method of paragraph 121, wherein the method effectively achieves a best response of any one of minimal response, partial response, best partial response, complete response, or stringent complete response in a rate of about 23% to about 69%. 123. The method of paragraph 121, wherein said method effectively achieves a best response of any one of said minimal response, partial response, best partial response, complete response, or stringent complete response at a rate of about 45%. 124. The method of any one of paragraphs 116 to 123, wherein the method effectively achieves a best response of any one of partial response, best partial response, complete response, or stringent complete response. 125. The method of paragraph 124, wherein the method effectively achieves a best response of any one of the partial response, best partial response, complete response, or stringent complete response in a rate of about 19% to about 64%. 126. The method of paragraph 124, wherein said method effectively achieves a best response of any one of said partial response, best partial response, complete response, or stringent complete response at a rate of about 40%. 127. The method of any one of paragraphs 116 to 126, wherein the method effectively achieves a best response of any one of best partial response, complete response, or stringent complete response. 128. The method of paragraph 127, wherein the method effectively achieves a best response of any one of the best partial response, complete response, or stringent complete response at a rate of about 15% to about 59%. 129. The method of paragraph 127, wherein the method effectively achieves a best response of any one of the best partial response, complete response, or stringent complete response at a rate of about 35%. 130. A method according to any one of paragraphs 116 to 129, wherein the method effectively achieves an optimal response of complete response or stringent complete response. 131. The method of paragraph 130, wherein the method effectively achieves a best outcome of complete response or stringent complete response at a rate of about 3% to about 38%. 132. The method of any one of paragraphs 116 to 131, wherein the method effectively achieves an optimal response of stringent complete response. 133. The method of paragraph 132, wherein the method effectively achieves the optimal stringent complete response at a rate of about 1% to about 32%. 134. The method of any one of paragraphs 3 or 104 to 133, wherein the method effectively results in progression-free survival in the subject. 135. The method of paragraph 134, wherein the method effectively achieves the progression-free survival in the subject from the administration of the CAR-T cells to about 15 days after the administration of the CAR-T cells. 136. The method of paragraph 134, wherein the method effectively achieves the progression-free survival in the subject from the administration of the CAR-T cells to about 44 days after the administration of the CAR-T cells. 137. The method of paragraph 134, wherein the method effectively achieves said progression-free survival in said subject from said administration of said CAR-T cells to about 159 days after said administration of said CAR-T cells. 138. The method of paragraph 134, wherein the method effectively achieves a progression-free survival rate of about 29% to about 75% at a follow-up period of about 6 months or about 9 months after the administration of the CAR-T cells. 139. The method of paragraph 134, wherein the method effectively achieves a progression-free survival rate of about 55% at a follow-up period of about 6 months or about 9 months after the administration of the CAR-T cells. 140. The method of any one of paragraphs 3 or 104 to 139, wherein the method effectively achieves a rate of cytokine release syndrome of about 60% to about 99%. 141. The method of paragraph 140, wherein the method further comprises treating cytokine release syndrome in the subject more than about 3 days after the administration of the CAR-T cells. 142. The method of any one of paragraphs 3 or 104 to 141, wherein the method effectively obtains a rate of about 20% to about 99% of neurotoxicity associated with immune effector cells. 143. The method of any one of paragraphs 3 or 104-142, wherein the at least four drugs further comprise a proteasome inhibitor, an immunomodulatory agent, and an anti-CD38 antibody. 144. The method of any one of paragraphs 121 to 143, wherein the method effectively achieves the optimal effect prior to a period of from about 27 days to about 171 days after the administration of the CAR-T cells. 145. The method of any one of paragraphs 121 to 143, wherein the method effectively achieves the optimal effect prior to a period of from about 27 days to about 133 days after the administration of the CAR-T cells. 146. The method of any one of paragraphs 121 to 143, wherein the method effectively achieves the optimal effect prior to about 78 days after the administration of the CAR-T cells. 147. The method of any one of paragraphs 121 to 143, wherein the method effectively achieves the optimal effect prior to about 56 days after the administration of the CAR-T cells. 148. The method of any one of paragraphs 117 to 147, wherein the method effectively maintains a response in the subject for a follow-up period from the time of the initial response to about 132 days after the administration of the CAR-T cells, and further wherein the initial response is obtained from the time of the administration of the CAR-T cells to about 131 days after the administration of the CAR-T cells. 149. The method of any one of paragraphs 117 to 147, wherein the method effectively maintains response at a rate of about 20% to about 96% over a follow-up period of about 6 months after the administration of the CAR-T cells. 150. The method of any one of paragraphs 117 to 147, wherein the method effectively maintains response at a rate of about 80% over a follow-up period of about 6 months after the administration of the CAR-T cells. 151. The method of any one of paragraphs 1 to 150, wherein the multiple myeloma is refractory to at least two drugs. 152. The method of any one of paragraphs 1-151, wherein the multiple myeloma is refractory to at least three drugs. 153. The method of any one of paragraphs 1-152, wherein the multiple myeloma is refractory to at least four drugs. 154. The method of any one of paragraphs 1 to 153, wherein the multiple myeloma is refractory to at least five drugs. 155. The method of any one of paragraphs 1 to 154, wherein the subject has about 10% to about 30% bone marrow plasma cells prior to said administration of the CAR-T cells. 156. The dose is 1.0 × 10 per kilogram of the subject's mass. 5 ~5.0 × 10 6 156. The method of any one of paragraphs 1 to 155, comprising said CAR-T cells. 157. The dose is 5.0 × 10 per kilogram of the subject's mass. 5 ~1.0 × 10 6 157. The method of any one of paragraphs 1 to 156, comprising said CAR-T cells. 158. The dose is about 0.75 × 10 per kilogram of the subject's mass. 6 158. The method of any one of paragraphs 1 to 157, comprising said CAR-T cells. 159. The dose is 1.0 × 10 8 The method of any one of paragraphs 1 to 158, comprising less than one of said CAR-T cells. 160. The method of any one of paragraphs 1 to 159, wherein the administration of the CAR-T cells is by single intravenous infusion. 161. The method of paragraph 160, wherein the single intravenous infusion is administered using one bag of the CAR-T cells. 162. The method of paragraph 161, wherein the administering of the bag of CAR-T cells is completed between the time of thawing the bag of CAR-T cells and 3 hours after thawing the bag of CAR-T cells. 163. The method of paragraph 160, wherein the single intravenous administration is administered using two bags of the CAR-T cells. 164. The method of paragraph 163, wherein the administering of each bag of the two bags of CAR-T cells is completed between the time of thawing of a first bag of the two bags of CAR-T cells and 3 hours after thawing of the first bag of CAR-T cells. 165. The method of any one of paragraphs 1 to 164, wherein the lymphodepletion regimen is performed about 5 days to about 7 days prior to administration of the CAR-T cells. 166. The method of paragraph 165, wherein the lymphodepleting regimen is administered intravenously. 167. The lymphodepleting regimen comprises: (a) administration of cyclophosphamide; or (b) The method of paragraph 166, comprising administration of fludarabine. 168. The cyclophosphamide is 300 mg / m 2 168. The method of paragraph 167, wherein the compound is administered intravenously at 100 mg / kg. 169. The fludarabine is administered at a dose of 30 mg / m 2 168. The method of paragraph 167, wherein the compound is administered intravenously at 100 mg / kg. 170. Cyclophosphamide 300 mg / m 2 and fludarabine 30 mg / m 2 and intravenous administration of said CAR-T cells to said patient, said lymphodepletion regimen comprising administering to said patient an effective amount of said CAR-T cells intravenously to said patient, said lymphodepletion regimen comprising administering to said patient an effective amount of said CAR-T cells ... 171. The method of any one of paragraphs 165 to 170, wherein the subject further undergoes bridging therapy, wherein the bridging therapy comprises short-term treatment with at least one bridging drug between apheresis and the lymphodepletion regimen, and wherein the at least one bridging drug has previously resulted in the subject achieving stable disease, minimal response, partial response, best partial response, complete response, or stringent complete response. 172. The method of paragraph 171, wherein the subject has increased tumor burden despite receiving the bridging therapy. 173. The method of paragraph 171, wherein the subject has an increase in tumor burden of about 25% or more despite receiving the bridging therapy. 174. The method of any one of paragraphs 43-44, 92-93, or 141, wherein the method further comprises treating cytokine release syndrome in the subject more than about 3 days after administration of the CAR-T cells without significantly reducing in vivo expansion of the CAR-T cells. 175. The method of any one of paragraphs 43-44, 92-93, 141 or 174, wherein the treating of cytokine release syndrome comprises administering to the subject an IL-6R inhibitor. 176. The method of paragraph 175, wherein the IL-6R inhibitor is an antibody. 177. The method of paragraph 176, wherein the antibody inhibits IL-6R by binding to the extracellular domain of IL-6R. 178. The method of any one of paragraphs 175 to 177, wherein the IL-6R inhibitor blocks the binding of IL-6 to IL-6R. 179. The method of any one of paragraphs 175 to 178, wherein the IL-6R inhibitor is tocilizumab. 180. The method of any one of paragraphs 1 to 179, wherein the subject is treated with an administered prodrug comprising an antipyretic and an antihistamine at most about 1 hour prior to the administration of the CAR-T cells. 181. The method of paragraph 180, wherein the antipyretic comprises paracetamol or acetaminophen. 182. The method of paragraph 180, wherein the antipyretic is administered orally or intravenously to the subject. 183. The method of paragraph 180, wherein the antipyretic is administered to the subject at a dose of 650 mg to 1000 mg. 184. The method of paragraph 180, wherein the antihistamine comprises diphenhydramine. 185. The method of paragraph 180, wherein the antihistamine is administered to the subject orally or intravenously. 186. The method of paragraph 180, wherein the antihistamine is administered in a dose of 25 mg to 50 mg, or an equivalent dose thereof. 187. The method of paragraph 180, wherein the antipyretic comprises paracetamol or acetaminophen, and the antipyretic is administered orally or intravenously to the subject at a dose of 650 mg to 1000 mg, and wherein the antihistamine comprises diphenhydramine, and the antihistamine is administered orally or intravenously to the subject at a dose of 25 mg to 50 mg, or an equivalent dose. 188. The method of any one of paragraphs 1 to 187, wherein the composition comprising CAR-T cells administered to the subject further comprises an excipient selected from dimethyl sulfoxide or dextran-40. 189. The method of any one of paragraphs 1 to 188, wherein the first BCMA binding moiety and / or the second BCMA binding moiety is an anti-BCMA VHH. 190. The method of paragraph 189, wherein the first BCMA binding moiety is a first anti-BCMA VHH and the second BCMA binding moiety is a second anti-BCMA VHH. 191. The method of any one of paragraphs 1 to 190, wherein the first BCMA binding portion comprises the amino acid sequence of SEQ ID NO:2. 192. The method of any one of paragraphs 1 to 191, wherein the first BCMA binding portion comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:10. 193. The method of any one of paragraphs 1 to 192, wherein the second BCMA binding portion comprises the amino acid sequence of SEQ ID NO:4. 194. The method of any one of paragraphs 1 to 193, wherein the second BCMA binding portion comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:12. 195. The method of any one of paragraphs 1 to 194, wherein the first BCMA binding moiety and the second BCMA binding moiety are linked to each other via a peptide linker. 196. The method of paragraph 195, wherein the peptide linker comprises the amino acid sequence of SEQ ID NO:3. 197. The method of paragraph 195, wherein the peptide linker comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:11. 198. The method of any one of paragraphs 1 to 197, wherein the CAR polypeptide further comprises a signal peptide located at the N-terminus of the polypeptide. 199. The method of paragraph 198, wherein the signal peptide is derived from CD8α. 200. The method of paragraph 198, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO:1. 201. The method of paragraph 198, wherein the signal peptide comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:9. 202. The method of any one of paragraphs 1 to 201, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO:6. 203. The method of any one of paragraphs 1 to 202, wherein the transmembrane domain comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:14. 204. The method of any one of paragraphs 1 to 203, wherein the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell. 205. The method of any one of paragraphs 1 to 204, wherein the intracellular signaling domain is derived from CD3ζ. 206. The method of any one of paragraphs 1 to 205, wherein the intracellular signaling domain comprises at least one costimulatory signaling domain. 207. A method according to any one of paragraphs 1 to 206, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:8. 208. The method of any one of paragraphs 1 to 207, wherein the intracellular signaling domain comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:16. 209. A method according to any one of paragraphs 1 to 208, wherein the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO:7. 210. A method according to any one of paragraphs 1 to 209, wherein the intracellular signaling domain comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:15. 211. The method of any one of paragraphs 1 to 210, wherein the CAR polypeptide further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain. 212. The method of paragraph 211, wherein the hinge domain comprises the amino acid sequence of SEQ ID NO:5. 213. The method of paragraph 211, wherein the hinge domain comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:13. 214. The method of any one of paragraphs 1 to 213, wherein the CAR comprises the amino acid sequence of SEQ ID NO:17. 215. The method of any one of paragraphs 1 to 214, wherein the T cells are autologous T cells. 216. The method of any one of paragraphs 1 to 215, wherein the T cells are allogeneic T cells. 217. The method of any one of paragraphs 1 to 216, wherein the subject is a human. 218. The method of any one of paragraphs 1 to 103, wherein the subject has not been previously exposed to a BCMA targeted drug. 219. The method of any one of paragraphs 1 to 218, wherein the multiple myeloma is progressive.

[0294] 5. Examples The following examples are provided to further illustrate some of the embodiments disclosed herein. The examples are intended to illustrate, but not to limit, the disclosed embodiments.

[0295] 5.1. Example 1: Siltacavtadine Autorusel B-cell maturation antigen (BCMA, also called CD269 and TNFRSF17) is a 20 kilodalton type III membrane protein that is part of the tumor necrosis receptor superfamily. BCMA is a cell surface antigen that is expressed at high levels mainly on B-lineage cells. Figure 1 shows the expression of BCMA on various immune-derived cells. Comparative studies have shown that BCMA is deficient in most normal tissues and is not expressed on CD34-positive hematopoietic stem cells. BCMA binds to two ligands that induce B-cell proliferation and plays a crucial role in B-cell maturation and subsequent differentiation into plasma cells. BCMA has become a promising target for CAR-T-based immunotherapy (siltacabtadine autreucel) due to its selective expression and biological importance to myeloma cell proliferation and survival.

[0296] Siltacabtadine Autorcel is an autologous chimeric antigen receptor T cell (CAR-T) therapy that targets BCMA. The Siltacabtadine Autorcel chimeric antigen receptor (CAR) is designed to confer avidity and contains two VHH domains that target the B cell maturation antigen (BCMA). Figure 2 shows a chart of the construct.

[0297] 5.2. Example 2: Treatment of Cohort A, Cohort B, and Cohort C with Siltacabtadine Autolucel In this multi-cohort, open-label, phase 2 study, we evaluated the safety and efficacy of cilta-cel in patients with multiple myeloma who need urgent treatment and have an overall survival rate of approximately 50% or less in various clinical settings. In Figure 4, a schematic summary of the study flow chart is described, which consists of a lymphodepleting regimen before cilta-cel infusion.

[0298] Apheresis was performed on eligible subjects to collect peripheral blood mononuclear cells (PBMCs). Study entry was defined as the day of apheresis. Siltacabtadine Autolucel Drug Product (DP) was produced by selected T cells from the apheresis. Subjects who failed apheresis or production were allowed to reattempt apheresis.

[0299] Bridging therapy (anti-plasma cell-directed therapy between apheresis and the first dose conditioning regimen) was permitted if clinically indicated (i.e., to maintain disease stability while waiting for siltacabtadine auto-repair). Additional cycles of bridging therapy were considered based on the subject's clinical status and the time of availability of CAR-T product. Bridging therapy is defined as a short-term treatment that previously caused at least a stable disease response in the subject.

[0300] After meeting the treatment safety criteria, subjects were administered a conditioning regimen to help them achieve lymphodepletion and promote CAR-T cell expansion. The lymphodepletion regimen consisted of cyclophosphamide 300 mg / m 2 and fludarabine 30 mg / m 2 cyclophosphamide 300 mg / m before the cilta-cel infusion 2 and fludarabine 30 mg / m 2 This was consistent with the lymphodepleting regimen used in the commercially available CAR-T products Kymriah and Yescarta.

[0301] Five to seven days after the start of the conditioning regimen, cilta-cel produced from apheresis material via viral transduction as shown in Figure 3 was administered on a day defined as day 1. Approximately 1 hour prior to cilta-cel infusion, subjects received premedication. Corticosteroids were not used during preinfusion. Infused prodrugs are listed in Table 1. After treatment with infused prodrugs, subjects received 0.75 × 10 6CAR-positive live T cells / kg (range: 0.5–1.0 × 10 6 Administer a single infusion of cilta-cel at a total target dose of 1.0 × 10 live CAR-positive T cells / kg, with a maximum total dose of 1.0 × 10 8 were live CAR-positive T cells.

[0302] One or two patient-specified infusion bags stored frozen contained a fixed dose of cilta-cel. Thawing time for the cilta-cel was matched to the infusion time. Infusion time was confirmed in advance and the start of thawing was timed to ensure that cilta-cel was available for infusion when the patient was ready. When receiving more than one bag to perform a therapeutic infusion, one bag was thawed at a time. The next bag was thawed / infused once it was determined that the previous bag was safe to administer.

[0303] 5.3. Example 3: Evaluation of the efficacy of treating cohort A, cohort B, and cohort C with siltacabtadine autolucel Using the IMWG-based response criteria summarized in Table 2, the study classified responses from best to worst as stringent complete response (sCR), complete response (CR), best partial response (VGPR), partial response (PR), minimal response (MR), stable disease, or progressive disease. Progression of disease was recorded consistently across clinical study sites. The studies performed to evaluate the IMWG-based response criteria were as follows: · Measurement of myeloma protein in serum and urine: Myeloma protein (M protein) was measured using the following tests from blood and 24-hour urine samples: serum quantitative Ig, serum protein electrophoresis (SPEP), serum immunofixation electrophoresis, serum FLC assay (per disease assessment for subjects with suspected CR / sCR and subjects with serum FLC disease only), 24-hour urine M protein electrophoresis quantitative (UPEP), urine immunofixation electrophoresis, serum β2-microglobulin. At least one repeat study demonstrated disease progression based on only one laboratory test. After relapse from CR, disease evaluation continued until disease progression was confirmed. Serum and urine immunofixation and serum free light chain (FLC) assays were performed at screening and thereafter when CR was suspected (if serum or 24-hour urine M protein electrophoresis [by SPEP or UPEP] was zero or not quantifiable). Serum and urine immunofixation tests were typically performed for subjects with light chain multiple myeloma. Serum calcium corrected for albumin: Collect and analyze blood samples to calculate serum calcium corrected for albumin until proven disease progression develops; development of hypercalcemia (corrected serum calcium > 11.5 mg / dL [> 2.9 mmol / L]) in the absence of any other cause may indicate disease progression or recurrence. Calcium is bound to albumin, and only unbound (free) calcium has biological activity; therefore, serum calcium levels must be adjusted for abnormal albumin levels ("corrected serum calcium"). Bone marrow examination: Bone marrow aspirate or biopsy was performed for clinical evaluation. Bone marrow aspirate was performed for biomarker evaluation. Clinical classification (morphology, cytogenetics, immunohistochemistry or immunofluorescence or flow cytometry) was performed. Immunophenotyping was performed on a portion of the bone marrow aspirate to monitor checkpoint ligand expression in BCMA, CD138 positive multiple myeloma cells, and checkpoint expression in T cells. When possible, bone marrow aspirate was performed to confirm CR and sCR and disease progression. Since minimal residual disease (MRD) negativity is a potential surrogate for PFS and OS in multiple myeloma treatment, next generation sequencing (NGS) was used on bone marrow aspirate DNA to monitor MRD in subjects. Baseline bone marrow aspirates were used to define myeloma clones, and post-treatment samples were used to evaluate MRD negativity. Fresh bone marrow aspirates were collected prior to the first-agent conditioning regimen (≤ 7 days). · Bone Survey: A bone survey (including skull, entire spine, pelvis, thorax, humerus, femur, and any other bones suspected by the investigator to be disease-related) was performed during the screening phase and was assessed by radiography ("X-ray") or low-dose computed tomography (CT) scan without the use of IV contrast. If a CT scan was used, it was of diagnostic quality. After cilta-cel injection and before disease progression was confirmed, X-rays or CT scans were performed locally to document response or progression, with or without clinical indication based on symptoms. Magnetic resonance imaging (MRI) was an acceptable method to assess bone disease and was incorporated as appropriate, however, it did not replace a bone survey. If a radionuclide bone scan was used at screening in addition to a complete bone survey, disease status was documented in two ways. These studies were performed simultaneously. A radionuclide bone scan cannot replace a complete bone survey. If a subject showed disease progression as manifested by pain symptoms with bone changes, disease progression was documented by bone survey or other radiography depending on the symptoms experienced by the subject. If radiological studies clearly indicated disease progression, repeat confirmatory x-rays were not considered necessary. If no changes were evident, repeat x-rays were performed within 1 to 3 weeks. Documentation of extramedullary plasmacytoma: Known sites of extramedullary plasmacytoma were documented ≤14 days prior to the first conditioning regimen. Clinical examination or MRI was used to document extramedullary site of disease. If there was no contraindication to IV contrast use, CT scan evaluation was considered an acceptable alternative regimen. Positron emission tomography scan or ultrasound examination cannot document the size of extramedullary plasmacytoma. However, if the CT portion of the PET / CT fusion scan was of sufficient diagnostic quality, the PET / CT fusion scan was optionally to document extramedullary plasmacytoma. Extramedullary plasmacytoma was assessed by clinical examination or radiological imaging for all subjects with a history of plasmacytoma or clinical indication ≤14 days prior to the first conditioning regimen. For subjects with a history of plasmacytoma or other subjects with clinical indications during treatment, local assessment, measurement or evaluation of measurable sites of extramedullary disease was performed (for physical examination) every 4 weeks until a confirmed CR or confirmed disease progression occurred. If only radiological assessment was available, evaluation of extramedullary plasmacytoma was performed every 12 weeks. Irradiated or resected lesions were considered non-measurable and were monitored for disease progression only. To qualify for VGPR or PR / minimal response (MR), the sum of the products of the perpendicular diameters of previous extramedullary plasmacytomas had to decrease by more than 90% or at least 50%, respectively, and no new plasmacytomas had to develop. To qualify for disease progression, the sum of the products of the perpendicular diameters of previous extramedullary plasmacytomas had to increase by at least 50%, or the longest diameter of a previous lesion that was > 1 cm had to increase by at least 50% (short axis), or new plasmacytomas had to develop. Although not all extramedullary plasmacytomas present were reported, disease progression criteria were met if the sum of the products of the perpendicular diameters of the reported plasmacytomas increased by at least 50%.

[0304] If the study treatment was identified as interfering with the immunofixation assay, CR was defined as disappearance of the original M protein associated with multiple myeloma at the time of immunofixation, and confirmation of CR was not influenced by unrelated M protein secondary to the study treatment.

[0305] The study endpoints (as assessed by an Independent Review Committee (IRC)) were: Overall response rate (ORR) was defined as the proportion of subjects who achieved a PR or better response based on IMWG criteria. VGPR or better response rate (sCR+CR+VGPR) was defined as the proportion of subjects who achieved VGPR or better response based on IMWG criteria. Duration of response (DOR) was calculated from the time from the first documented date of response (PR or better response) to the first documented date of evidence of progressive disease (as defined by IMWG criteria) in responders (those with PR or better response). Relapse from CR with positive immunofixation or trace M protein was not considered to be disease progression. After relapse from CR, disease evaluation continued until disease progression was confirmed. Duration of response (TTR) was defined as the time from the date of initial infusion of cilta-cel to the first efficacy assessment at which a subject met all criteria for PR or better response. Progression-free survival (PFS) was defined as the time from the date of initial cilta-cel infusion to the date of first documented disease progression (as defined by IMWG criteria) or death from any cause (whichever occurred first). Overall survival (OS) was measured from the date of initial cilta-cel infusion to the date of patient death.

[0306] For ORR, response rates and their 95% exact confidence intervals (CIs) were calculated based on a binomial distribution, and the...

Claims

1. 1. A pharmaceutical composition comprising T cells comprising a chimeric antigen receptor (CAR) for use in a method of treating multiple myeloma in a subject, wherein the chimeric antigen receptor is (a) an extracellular antigen-binding domain, (1) a first anti-BCMA binding moiety comprising a first complementarity determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 18, a second complementarity determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 19, and a third complementarity determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 20; (2) the extracellular antigen-binding domain comprising a second anti-BCMA binding portion comprising a first complementarity-determining region (CDR1) comprising the amino acid sequence of SEQ ID NO: 21, a second complementarity-determining region (CDR2) comprising the amino acid sequence of SEQ ID NO: 22, and a third complementarity-determining region (CDR3) comprising the amino acid sequence of SEQ ID NO: 23; (b) a transmembrane domain; and (c) an intracellular signaling domain; The method includes administering to the subject a dose of T cells expressing a CAR (CAR-T cells); Here, the subject: (i) have already received prior treatment with one, two, or three previous lines of treatment; and (ii) The pharmaceutical composition, wherein the patient is refractory to lenalidomide.

2. The subject has received prior treatment with at least one prior line of treatment, the at least one prior line of treatment comprising treatment with lenalidomide and at least one non-lenalidomide drug, the at least one non-lenalidomide drug being (a) a proteasome inhibitor, (b) an immunomodulatory agent; or (c) an anti-CD38 antibody.

3. 3. The pharmaceutical composition of claim 2, wherein the subject has received prior treatment with at least two prior lines of therapy or at least three prior lines of therapy.

4. The subject has received prior treatment with dexamethasone, an alkylating agent, or daratumumab. the multiple myeloma is refractory to a last line of therapy; the subject has relapsed after one, two, or three prior lines of therapy; or 2. The pharmaceutical composition of claim 1, wherein the multiple myeloma is refractory to three drugs.

5. 10. The pharmaceutical composition of claim 1, wherein the method effectively obtains a minimal residual disease (MRD) negative status in the subject as assessed in bone marrow after the administration of the CAR-T cells.

6. 6. The pharmaceutical composition of claim 5, wherein the minimal residual disease (MRD)-negative status is obtained in a first follow-up period from 29 to 184 days after the administration of the CAR-T cells.

7. 7. The pharmaceutical composition of claim 6, wherein the method effectively maintains the minimal residual disease (MRD)-negative status of the subject as assessed in the bone marrow at a second follow-up period from 57 to 191 days after the administration of the CAR-T cells, and further wherein the first follow-up period is earlier than the second follow-up period.

8. The method comprises: －４ , 10 －５ or 10 －６ At a sensitivity threshold level of 24% to 61% 10 －４ , 10 －５ or 10 －６ At a sensitivity threshold level of 41%, 10 －５ in 64% to 99% of subjects with evaluable samples at a sensitivity threshold level of 10 －５ 6. The pharmaceutical composition of claim 5, which effectively achieves the minimal residual disease (MRD) negative status in a 92% rate in subjects with evaluable samples at a sensitivity threshold level of 0.1%.

9. The method effectively obtains at least one response in the subject after said administration of the CAR-T cells, wherein said at least one response is, in order from best to worst: (i) stringent complete response, (ii) complete response, (iii) best partial response, (iv) partial response, or (v) The pharmaceutical composition of claim 1, comprising a minimal response.

10. The method includes: receiving an initial response prior to a time period of 21 to 99 days after administration of the CAR-T cells; an initial response prior to a period of 21 to 55 days after said administration of said CAR-T cells; an initial response prior to 36 days after said administration of said CAR-T cells; or The pharmaceutical composition of claim 9, which effectively obtains an initial response prior to 30 days after said administration of said CAR-T cells.

11. The method may include determining a best response as any one of minimal response, partial response, best partial response, complete response, or severe complete response. The best response was defined as partial response, best partial response, complete response, or severe complete response. The best response was defined as either best partial response, complete response, or severe complete response. The best response was complete response or severe complete response, or The pharmaceutical composition according to claim 9, which effectively achieves the best effect of severe complete response.

12. The method comprises achieving a best response of any one of minimal response, partial response, best partial response, complete response, or severe complete response at a rate of 52% to 87%. A 72% rate of patients achieved the best response of any one of minimal response, partial response, best partial response, complete response, or severe complete response. A best response of any one of the partial response, best partial response, complete response, or severe complete response was achieved in a rate of 49% to 84%. The best response of any one of the partial response, best partial response, complete response, or severe complete response was 69%. The best response was either the best partial response, complete response, or severe complete response at a rate of 49% to 84%. The best response of any one of the best partial response, complete response, or severe complete response was 69%. The best response was a complete response or severe complete response in a rate of 39% to 76%. The best response was a complete response or severe complete response in 58% of cases. A best response of 33% to 70% of patients with a stringent complete response; or The pharmaceutical composition of claim 11, which effectively achieves the best effect of stringent complete response at a rate of 52%.

13. The pharmaceutical composition of claim 1 , wherein the method effectively results in progression-free survival in the subject.

14. The method includes measuring the progression-free survival of the subject from the administration of the CAR-T cells to 55 days after the administration of the CAR-T cells; and measuring the progression-free survival of the subject from the administration of the CAR-T cells to 297 days after the administration of the CAR-T cells. a progression-free survival rate of 62% to 95% at a follow-up period of 6 months or 9 months after said administration of said CAR-T cells; or The pharmaceutical composition of claim 13, which effectively achieves the progression-free survival rate of 86% during a follow-up period of 6 months or 9 months after the administration of the CAR-T cells.

15. 10. The pharmaceutical composition of claim 1, wherein the method further comprises treating cytokine release syndrome in the subject more than 3 days after the administration of the CAR-T cells.

16. 16. The pharmaceutical composition of claim 15, wherein the method effectively achieves a 1% to 90% reversal rate of the cytokine release syndrome within a 7 day period from when the cytokine release syndrome is first observed.

17. The pharmaceutical composition of claim 1, wherein the method effectively achieves a 20% to 99% rate of immune effector cell-associated neurotoxicity.

18. The method includes determining the best response before a period of 27 to 237 days after administration of the CAR-T cells; the best response is observed before a period of 46 to 172 days after administration of the CAR-T cells; the best response occurs prior to 109 days after administration of the CAR-T cells; or The pharmaceutical composition of claim 11 or 12, wherein the best effect is effectively achieved before 87 days after the administration of the CAR-T cells.

19. 11. The pharmaceutical composition of claim 10, wherein the method effectively maintains the response in the subject for a follow-up period from the time of the initial response to 270 days after the administration of the CAR-T cells.

20. The method comprises achieving a response rate of 70% to 99% during a follow-up period of 6 months after the administration of the CAR-T cells; A 95% response rate was observed during the 6-month follow-up period after the administration of the CAR-T cells. a response rate of 7% to 92% during a 9-month follow-up period after said administration of said CAR-T cells; or The pharmaceutical composition of claim 9 or 12, which effectively maintains a response rate of 63% during a follow-up period of 9 months after the administration of the CAR-T cells.

21. Further, the method includes administering 10 or more CAR-T cells to a patient within 3 months after the administration of the CAR-T cells. －５ 13. The pharmaceutical composition of claim 11 or 12, which effectively obtains a minimal residual disease (MRD) negative status in the subject as assessed in the bone marrow at a sensitivity threshold level of 0.1% or more.

22. The method comprises achieving a minimal residual disease (MRD)-negative complete response or a minimal residual disease (MRD)-negative stringent complete response at a rate of 18% to 54% during a follow-up period of 291 days after the administration of the CAR-T cells; or The pharmaceutical composition of claim 21, which effectively achieves a minimal residual disease (MRD)-negative complete response or a minimal residual disease (MRD)-negative stringent complete response at a rate of 35% over a follow-up period of 291 days after the administration of the CAR-T cells.

23. 18. The pharmaceutical composition of any one of claims 1 to 17, wherein the multiple myeloma is refractory to at least two drugs, at least three drugs, at least four drugs, or at least five drugs.

24. The pharmaceutical composition of any one of claims 1 to 17, wherein the subject has 10% to 30% bone marrow plasma cells prior to the administration of the CAR-T cells.

25. The dose is 1.0 x 10 per kilogram of the subject's mass. ５ ~5.0 x 10 ６ the CAR-T cells, The dose is 5.0 x 10 per kilogram of the subject's mass. ５ ~1.0 x 10 ６ the CAR-T cells, 0.75 x 10 per kilogram of the subject's mass ６ the CAR-T cells, or 1.0 × 10 per subject ８ The pharmaceutical composition of any one of claims 1 to 17, comprising less than one of the CAR-T cells.

26. The pharmaceutical composition of any one of claims 1 to 17, wherein the administration of the CAR-T cells is by single intravenous infusion.

27. 27. The pharmaceutical composition of claim 26, wherein the single intravenous infusion is administered with one bag of the CAR-T cells or two bags of the CAR-T cells.

28. the administration of the one bag of CAR-T cells is completed between the time of thawing the one bag of CAR-T cells and 3 hours after thawing the one bag of CAR-T cells; or 28. The pharmaceutical composition of claim 27, wherein the administration of each bag of the two bags of CAR-T cells is completed between the time of thawing a first bag of the two bags of CAR-T cells and 3 hours after thawing the first bag of CAR-T cells.

29. The pharmaceutical composition of any one of claims 1 to 17, wherein the lymphodepletion regimen is carried out 5 to 7 days prior to administration of the CAR-T cells.

30. 30. The pharmaceutical composition of claim 29, wherein the lymphodepleting regimen is by intravenous administration.

31. The lymphodepleting regimen comprises: (a) administration of cyclophosphamide; or 31. The pharmaceutical composition of claim 30, comprising (b) administration of fludarabine.

32. The cyclophosphamide is 300 mg / m ２ administered intravenously with The fludarabine is 30 mg / m ２ 32. The pharmaceutical composition of claim 31 , wherein the composition is administered intravenously at

33. Cyclophosphamide 300 mg / m ２ and fludarabine at 30 mg / m ２ and administering to a subject a lymphodepleting regimen comprising administering to a subject a lymphoma cell line comprising the CAR-T cells intravenously, the ...

34. 30. The pharmaceutical composition of claim 29, wherein the subject further undergoes bridging therapy, wherein the bridging therapy comprises short-term treatment with at least one bridging drug between apheresis and the lymphodepletion regimen, and wherein the at least one bridging drug has previously resulted in stable disease, minimal response, partial response, best partial response, complete response, or stringent complete response in the subject.

35. the subject has an increased tumor burden despite receiving the cross-linking therapy, and / or 35. The pharmaceutical composition of claim 34, wherein the subject has a tumor burden that increases by 25% or more despite receiving the crosslinking therapy.

36. 16. The pharmaceutical composition of claim 15, wherein the method further comprises treating cytokine release syndrome in the subject more than 3 days after the administration of the CAR-T cells without significantly reducing in vivo expansion of the CAR-T cells.

37. The pharmaceutical composition according to claim 15, wherein the treatment of cytokine release syndrome comprises administering to the subject an IL-6R inhibitor.

38. The IL-6R inhibitor is an antibody. The IL-6R inhibitor blocks the binding of IL-6 to IL-6R, and / or The pharmaceutical composition of claim 37, wherein the IL-6R inhibitor is tocilizumab.

39. The pharmaceutical composition of claim 38, wherein the antibody inhibits IL-6R by binding to the extracellular domain of IL-6R.

40. 18. The pharmaceutical composition of any one of claims 1-17, wherein the subject is treated with an administration prodrug comprising an antipyretic and an antihistamine at most 1 hour prior to the administration of the CAR-T cells.

41. The antipyretic includes paracetamol or acetaminophen. The antipyretic is administered orally or intravenously to the subject. the antipyretic is administered to the subject at a dose of 650 mg to 1000 mg; The antihistamine includes diphenhydramine. The antihistamine is administered orally or intravenously to the subject. the antihistamine is administered in a dose of 25 mg to 50 mg or its equivalent; or 41. The pharmaceutical composition of claim 40, wherein the antipyretic comprises paracetamol or acetaminophen, and is administered orally or intravenously to the subject at a dose of 650 mg to 1000 mg, and wherein the antihistamine comprises diphenhydramine, and is administered orally or intravenously to the subject at a dose of 25 mg to 50 mg, or an equivalent dose thereof.

42. The pharmaceutical composition of any one of claims 1 to 17, wherein the composition comprising CAR-T cells to be administered to the subject further comprises an excipient selected from dimethyl sulfoxide or dextran-40.

43. The pharmaceutical composition of any one of claims 1 to 17, wherein the first anti-BCMA binding moiety and / or the second anti-BCMA binding moiety is an anti-BCMA VHH.

44. 44. The pharmaceutical composition of claim 43, wherein the first anti-BCMA binding moiety is a first anti-BCMA VHH and the second anti-BCMA binding moiety is a second anti-BCMA VHH.

45. the first anti-BCMA binding portion comprises the amino acid sequence of SEQ ID NO:2; the first anti-BCMA binding moiety comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO: 10; the second anti-BCMA binding moiety comprises the amino acid sequence of SEQ ID NO:4; or 18. The pharmaceutical composition of any one of claims 1 to 17, wherein the second anti-BCMA binding moiety comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:

12.

46. 18. The pharmaceutical composition of any one of claims 1 to 17, wherein the first anti-BCMA binding moiety and the second anti-BCMA binding moiety are linked to each other via a peptide linker.

47. the peptide linker comprises the amino acid sequence of SEQ ID NO: 3; or 47. The pharmaceutical composition of claim 46, wherein the peptide linker comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:

11.

48. The pharmaceutical composition of any one of claims 1 to 17, wherein the CAR polypeptide further comprises a signal peptide located at the N-terminus of the polypeptide.

49. the signal peptide is derived from CD8α; the signal peptide comprises the amino acid sequence of SEQ ID NO: 1; or 49. The pharmaceutical composition of claim 48, wherein the signal peptide comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:

9.

50. the transmembrane domain comprises the amino acid sequence of SEQ ID NO: 6; or The pharmaceutical composition of any one of claims 1 to 17, wherein the transmembrane domain comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:

14.

51. the intracellular signaling domain comprises a primary intracellular signaling domain of an immune effector cell; the intracellular signaling domain is derived from CD3ζ; the intracellular signaling domain comprises at least one costimulatory signaling domain; the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 8; The intracellular signaling domain comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:

16. the intracellular signaling domain comprises the amino acid sequence of SEQ ID NO: 7; or 18. The pharmaceutical composition of any one of claims 1 to 17, wherein the intracellular signaling domain comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:

15.

52. The pharmaceutical composition of any one of claims 1 to 17, wherein the CAR polypeptide further comprises a hinge domain located between the C-terminus of the extracellular antigen-binding domain and the N-terminus of the transmembrane domain.

53. the hinge domain comprises the amino acid sequence of SEQ ID NO: 5; or 53. The pharmaceutical composition of claim 52, wherein the hinge domain comprises a polypeptide encoded by the nucleic acid sequence of SEQ ID NO:

13.

54. The pharmaceutical composition according to any one of claims 1 to 17, wherein the CAR comprises the amino acid sequence of SEQ ID NO:

17.

55. the T cells are autologous T cells, or The pharmaceutical composition of any one of claims 1 to 17, wherein the T cells are allogeneic T cells.

56. The pharmaceutical composition according to any one of claims 1 to 17, wherein the subject is a human.

57. The pharmaceutical composition of any one of claims 1 to 17, wherein the subject has not been previously exposed to a BCMA-targeted drug.

58. The pharmaceutical composition of any one of claims 1 to 17, wherein the multiple myeloma is aggressive.

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