Use of chimeric antigen receptor (CAR) T-cell therapy in combination with inhibitors of inflammation-related soluble factors

Abstract

Provided herein is the use of chimeric antigen receptors (CARs) to treat cancer (e.g., B-cell related cancers, e.g., multiple myeloma).

Description

[Technical Field]

[0001] Cross-references to related applications This application claims the benefits of U.S. Application No. 63 / 121,716, filed on 4 December 2020. This disclosure is incorporated herein by reference in its entirety.

[0002] Reference to electronically submitted sequence listings This application incorporates by reference an ASCII text file titled 14247-617-228_SEQ_LISTING.txt, created on November 23, 2021, with a size of 33,941 bytes, which was submitted with this application.

[0003] 1. Field The disclosures presented herein relate to methods for treating cancer (e.g., B-cell-associated cancers, e.g., multiple myeloma). More specifically, the disclosure relates to improved methods for treating cancer (e.g., B-cell-associated cancers, e.g., multiple myeloma) using chimeric antigen receptors (CARs), comprising the steps of: determining the level of one or more inflammation-related soluble factors; then, based on the determination, administering a CAR comprising an antibody or an antigen-binding fragment thereof (e.g., an anti-BCMA antibody or an antigen-binding fragment thereof); and to immune effector cells (e.g., T cells) genetically modified to express these CARs. [Background technology]

[0004] 2.Background 2.1. Background Many options are currently available to approach cancer treatment, including conventional chemotherapy approaches and immunotherapies, such as chimeric antigen receptor (CAR) T-cell therapy. In certain cases, the level of certain factors (e.g., proteins or biomarkers) in the patient's serum may be relevant to the decision of whether or not to administer CAR-T therapy. Therefore, there is a need for information on certain factors (e.g., proteins or biomarkers) in the patient's serum that may be relevant to the decision of whether or not to administer CAR-T therapy. [Overview of the Initiative]

[0005] 3. Brief Overview This disclosure provides, in general terms, an improved method for treating cancer (e.g., B-cell related cancer, e.g., multiple myeloma) using chimeric antigen receptors (CARs), comprising the steps of: determining the level of one or more inflammation-related soluble factors; and, based on the determination, administering a CAR comprising an antibody or an antigen-binding fragment thereof (e.g., an anti-BCMA antibody or an antigen-binding fragment thereof), as well as immunoeffector cells (e.g., T cells) genetically modified to express these CARs.

[0006] In one embodiment, a method for predicting whether a cancer in a human is responsive to chimeric antigen receptor (CAR) T cells is provided herein, comprising the steps of: (i) determining the level of one or more inflammation-related soluble factors in the serum of a human; and (ii) administering a therapeutically effective dose of CAR T cells to a human if the level of the one or more soluble factors in (i) is similar to that in the serum of a patient who is responsive to chimeric antigen receptor (CAR) T cells.

[0007] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0008] In certain embodiments, the cancer is multiple myeloma.

[0009] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0010] In certain embodiments, the levels of one or more inflammation-related soluble factors are determined by the O-link IO analyte assay.

[0011] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0012] In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, 4, 5, 6, or 7 days after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in the serum of a patient who is responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, or 4 weeks after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in the serum of a patient who is responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells approximately one, two, three, four, five, or six months after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in the serum of a patient who is responsive to chimeric antigen receptor (CAR) T cells.

[0013] In one embodiment, a method for treating cancer in a person in need is provided herein, comprising the step of determining the level of one or more inflammation-related soluble factors in a serum sample from the person in need, wherein if the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells, the person in need is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells.

[0014] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0015] In certain embodiments, the cancer is multiple myeloma.

[0016] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0017] In certain embodiments, the levels of one or more inflammation-related soluble factors are determined by the O-link IO analyte assay.

[0018] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0019] In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, 4, 5, 6, or 7 days after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, or 4 weeks after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells approximately one, two, three, four, five, or six months after it is determined that the levels of one or more inflammation-related soluble factors are similar to the levels of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells.

[0020] In one embodiment, a method for treating cancer in a person in need is provided herein, comprising: a) determining that the level of one or more inflammation-related soluble factors in a serum sample from the person in need is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells; and b) subsequently providing the person in need with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells based on the determination in step a.

[0021] In certain embodiments, the one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, the one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, the one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, the one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0022] In certain embodiments, the cancer is multiple myeloma.

[0023] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are idecabtagene vicleucel (ide-cel) cells.

[0024] In certain embodiments, the level of the one or more inflammation-related soluble factors is determined by the O-link IO analyte assay.

[0025] In certain embodiments, the CAR T cells are administered at a dose ranging from 150×10 6 cells to 450×10 6 cells. In certain embodiments, the BCMA CAR T cells are administered at a dose ranging from 150×10 6 cells to 450×10 6 cells.

[0026] In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, 4, 5, 6, or 7 days after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, or 4 weeks after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells approximately one, two, three, four, five, or six months after it is determined that the levels of one or more inflammation-related soluble factors are similar to the levels of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells.

[0027] In one embodiment, a method for treating cancer is provided herein, comprising the step of administering a therapeutically effective dose of chimeric antigen receptor (CAR) T cells to a human patient diagnosed with cancer, wherein the level of one or more inflammation-related soluble factors in a serum sample from the human patient prior to the administration is determined to be similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells.

[0028] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0029] In certain embodiments, the cancer is multiple myeloma.

[0030] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0031] In certain embodiments, the levels of one or more inflammation-related soluble factors are determined by the O-link IO analyte assay.

[0032] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0033] In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, 4, 5, 6, or 7 days after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, or 4 weeks after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells approximately one, two, three, four, five, or six months after it is determined that the levels of one or more inflammation-related soluble factors are similar to the levels of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells.

[0034] In one embodiment, a method is provided herein for determining whether a patient diagnosed with cancer should be administered chimeric antigen receptor (CAR) T cells, comprising the step of determining the level of one or more inflammation-related soluble factors in a serum sample from the patient, wherein the patient is a candidate for CAR T cells if the level of one or more inflammation-related soluble factors in the serum sample from the patient is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient who is responsive to chimeric antigen receptor (CAR) T cells.

[0035] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0036] In certain embodiments, the cancer is multiple myeloma.

[0037] In certain embodiments, the levels of one or more inflammation-related soluble factors are determined by the O-link IO analyte assay.

[0038] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0039] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0040] In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, 4, 5, 6, or 7 days after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, or 4 weeks after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells approximately one, two, three, four, five, or six months after it is determined that the levels of one or more inflammation-related soluble factors are similar to the levels of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells.

[0041] In one embodiment, a method for treating cancer in a person in need thereof is provided herein, comprising the step of administering to a person chimeric antigen receptor (CAR) T cells and an antagonist of an inflammation-related soluble factor selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1.

[0042] In certain embodiments, the inflammation-related soluble factor is selected from the group consisting of PGF, CD70, TNFRSF9, and CD83.

[0043] In certain embodiments, an inflammation-related soluble factor antagonist is administered to humans in a therapeutically effective dose to reduce the level of inflammation-related soluble factor in humans to the level of inflammation-related soluble factor in patients responsive to chimeric antigen receptor (CAR) T cells.

[0044] In certain embodiments, the cancer is multiple myeloma.

[0045] In certain embodiments, the CAR T cells are CAR T cells that target BCMA.

[0046] In certain embodiments, BCMA-targeting CAR T cells are ide-cel cells.

[0047] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0048] In certain embodiments, the inflammation-related soluble factor antagonist is administered prior to the administration of human chimeric antigen receptor (CAR) T cells. In certain embodiments, the inflammation-related soluble factor antagonist is administered about 1, 2, 3, 4, 5, 6, or 7 days before the administration of human chimeric antigen receptor (CAR) T cells. In certain embodiments, the inflammation-related soluble factor antagonist is administered about 1, 2, 3, or 4 weeks before the administration of human chimeric antigen receptor (CAR) T cells. In certain embodiments, the inflammation-related soluble factor antagonist is administered about 1, 2, 3, 4, 5, or 6 months before the administration of human chimeric antigen receptor (CAR) T cells.

[0049] 4. Brief explanation of the drawing [Brief explanation of the drawing]

[0050] [Figure 1] Figure 1 shows the correlation of non-responder at 9 months in the O-link IO analyte assay. AUC, area under the curve; DCN, decorin; FDR, false detection rate; IL-10, interleukin-10; IL-12, interleukin-12; IO, immuno-oncology; M, month; NCR1, natural cytotoxicity triggering receptor 1; NR, non-responder; PD, progressive disease; PDCD1, programmed cell death 1; PGF, placental growth factor; R, responder; TNFRSF, tumor necrosis factor receptor superfamily member. AUC > 0.5 indicates a higher assay in NR; O-link IO panel analytes with FDR < 0.1 are shown. bAUC > 0.5 indicates a higher assay in NR; O-link IO panel analytes with FDR < 0.1 are shown. cM9 PD indicates a responder that progressed before M9. dM9 R indicates responders that still responded at ≥M9. eFDR correction was determined by the Benjamini-Hochberg method. [Figure 2] Figure 2 shows that inflammation-related soluble factors that can negatively modulate T cell effector function were associated with suboptimal responses. The upper left graph in Figure 2 shows the change in CD83 log multiple at 3 months after ide-cel injection in non-responders (M3 NR) and responders (M3 R). The upper right graph in Figure 2 shows the change in TNFRSF9 (sCD137) log multiple at 3 months after ide-cel injection in non-responders (M3 NR) and responders (M3 R). The lower left graph in Figure 2 shows the change in PGF log multiple at 9 months after ide-cel injection in non-responders (M9 NR) and responders (M9 R). The lower right graph in Figure 2 shows the change in CD70 log multiple at 9 months after ide-cel injection in non-responders (M9 NR) and responders (M9 R). [Modes for carrying out the invention]

[0051] 5. A brief explanation of the sequence number. Sequence IDs 1-3 describe the amino acid sequences of exemplary light chain CDR sequences for BCMA CARs as intended herein.

[0052] Sequence IDs 4-6 describe the amino acid sequences of exemplary heavy chain CDR sequences for BCMA CARs as intended herein.

[0053] Sequence ID 7 describes the amino acid sequence of an exemplary light chain sequence for the BCMA CAR intended herein.

[0054] Sequence ID 8 describes the amino acid sequence of an exemplary heavy chain sequence for the BCMA CAR intended herein.

[0055] Sequence ID 9 describes the amino acid sequence of an exemplary BCMA CAR as intended herein, having a signal peptide (amino acids 1-22). The amino acid sequence of the mature form of BCMA02 is described in Sequence ID 37.

[0056] Sequence ID 10 describes a polynucleotide sequence encoding an exemplary BCMA CAR as intended herein.

[0057] Sequence ID 11 describes the amino acid sequence of human BCMA.

[0058] Sequence IDs 12-22 describe the amino acid sequences of various linkers.

[0059] Sequence IDs 23-35 describe the amino acid sequences of the protease cleavage site and the self-cleaving polypeptide cleavage site.

[0060] Sequence ID 36 describes the polynucleotide sequence of an exemplary BCMA CAR encoding vector. See Table 1.

[0061] Sequence ID 37 describes the amino acid sequence of an exemplary mature BCMA CAR (i.e., without a signal sequence) as intended herein.

[0062] Sequence ID 38 describes the amino acid sequence of BCMA02 scFv.

[0063] Table 1: List of arrays: [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0064] 6. Detailed explanation 6.1. Methods for treating cancer using chimeric antigen receptor (CAR) T cells The disclosures presented herein relate, in general, to improved methods for treating cancer (e.g., B-cell related cancers, e.g., multiple myeloma) using chimeric antigen receptors (CARs), comprising the steps of: determining the level of one or more inflammation-related soluble factors; and, based on the determination, administering a CAR comprising an antibody or its antigen-binding fragment (e.g., an anti-BCMA antibody or its antigen-binding fragment); and to immune effector cells (e.g., T cells) genetically modified to express these CARs.

[0065] In one embodiment, a method for predicting whether a cancer in a human is responsive to chimeric antigen receptor (CAR) T cells is provided herein, comprising the steps of: (i) determining the level of one or more inflammation-related soluble factors in the serum of a human; and (ii) administering a therapeutically effective dose of CAR T cells to a human if the level of the one or more soluble factors in (i) is similar to that in the serum of a patient who is responsive to chimeric antigen receptor (CAR) T cells.

[0066] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0067] In certain embodiments, the cancer is multiple myeloma.

[0068] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0069] In certain embodiments, the levels of one or more inflammation-related soluble factors are determined by the O-link IO analyte assay.

[0070] In a particular embodiment, CAR T cells are 150 × 106 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0071] In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, 4, 5, 6, or 7 days after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, or 4 weeks after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells approximately one, two, three, four, five, or six months after it is determined that the levels of one or more inflammation-related soluble factors are similar to the levels of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells.

[0072] In any particular embodiment of the above embodiments, the level of CD83 in a human serum sample is a log-multiple change of approximately 3.25 compared to the control. In any particular embodiment of the above embodiments, the level of CD83 in a human serum sample is a log-multiple change of approximately 3.25 compared to the control or less. In any particular embodiment of the above embodiments, the level of TNFRSF9 (sCD137) in a human serum sample is a log-multiple change of approximately 6.3 compared to the control. In any particular embodiment of the above embodiments, the level of TNFRSF9 (sCD137) in a human serum sample is a log-multiple change of approximately 6.3 compared to the control or less. In any particular embodiment of the above embodiments, the level of PGF in a human serum sample is a log-multiple change of approximately 8.6 compared to the control. In any particular embodiment of the above embodiments, the level of PGF in a human serum sample is a log-multiple change of approximately 8.6 compared to the control or less. In any particular embodiment of the above embodiments, the level of CD70 in a human serum sample is a log-multiple change of approximately 5.0 compared to the control. In any particular embodiment of the above embodiments, the level of CD70 in a human serum sample is approximately a log-multiple change of 5.0 or less compared to a control.

[0073] In one embodiment, a method for treating cancer in a person in need is provided herein, comprising the step of determining the level of one or more inflammation-related soluble factors in a serum sample from the person in need, wherein if the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells, the person in need is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells.

[0074] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0075] In certain embodiments, the cancer is multiple myeloma.

[0076] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0077] In certain embodiments, the levels of one or more inflammation-related soluble factors are determined by the O-link IO analyte assay.

[0078] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0079] In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, 4, 5, 6, or 7 days after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, or 4 weeks after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells approximately one, two, three, four, five, or six months after it is determined that the levels of one or more inflammation-related soluble factors are similar to the levels of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells.

[0080] In any particular embodiment of the above embodiments, the level of CD83 in a human serum sample is a log-multiple change of approximately 3.25 compared to the control. In any particular embodiment of the above embodiments, the level of CD83 in a human serum sample is a log-multiple change of approximately 3.25 compared to the control or less. In any particular embodiment of the above embodiments, the level of TNFRSF9 (sCD137) in a human serum sample is a log-multiple change of approximately 6.3 compared to the control. In any particular embodiment of the above embodiments, the level of TNFRSF9 (sCD137) in a human serum sample is a log-multiple change of approximately 6.3 compared to the control or less. In any particular embodiment of the above embodiments, the level of PGF in a human serum sample is a log-multiple change of approximately 8.6 compared to the control. In any particular embodiment of the above embodiments, the level of PGF in a human serum sample is a log-multiple change of approximately 8.6 compared to the control or less. In any particular embodiment of the above embodiments, the level of CD70 in a human serum sample is a log-multiple change of approximately 5.0 compared to the control. In any particular embodiment of the above embodiments, the level of CD70 in a human serum sample is approximately a log-multiple change of 5.0 or less compared to a control.

[0081] In one embodiment, a method for treating cancer in a person in need is provided herein, comprising: a) determining that the level of one or more inflammation-related soluble factors in a serum sample from the person in need is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells; and b) subsequently providing the person in need with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells based on the determination in step a.

[0082] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0083] In certain embodiments, the cancer is multiple myeloma.

[0084] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0085] In certain embodiments, the levels of one or more inflammation-related soluble factors are determined by the O-link IO analyte assay.

[0086] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0087] In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, 4, 5, 6, or 7 days after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, or 4 weeks after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells approximately one, two, three, four, five, or six months after it is determined that the levels of one or more inflammation-related soluble factors are similar to the levels of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells.

[0088] In any particular embodiment of the above embodiments, the level of CD83 in a human serum sample is a log-multiple change of approximately 3.25 compared to the control. In any particular embodiment of the above embodiments, the level of CD83 in a human serum sample is a log-multiple change of approximately 3.25 compared to the control or less. In any particular embodiment of the above embodiments, the level of TNFRSF9 (sCD137) in a human serum sample is a log-multiple change of approximately 6.3 compared to the control. In any particular embodiment of the above embodiments, the level of TNFRSF9 (sCD137) in a human serum sample is a log-multiple change of approximately 6.3 compared to the control or less. In any particular embodiment of the above embodiments, the level of PGF in a human serum sample is a log-multiple change of approximately 8.6 compared to the control. In any particular embodiment of the above embodiments, the level of PGF in a human serum sample is a log-multiple change of approximately 8.6 compared to the control or less. In any particular embodiment of the above embodiments, the level of CD70 in a human serum sample is a log-multiple change of approximately 5.0 compared to the control. In any particular embodiment of the above embodiments, the level of CD70 in a human serum sample is approximately a log-multiple change of 5.0 or less compared to a control.

[0089] In one embodiment, a method for treating cancer is provided herein, comprising the step of administering a therapeutically effective dose of chimeric antigen receptor (CAR) T cells to a human patient diagnosed with cancer, wherein the level of one or more inflammation-related soluble factors in a serum sample from the human patient prior to the administration is determined to be similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells.

[0090] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0091] In certain embodiments, the cancer is multiple myeloma.

[0092] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0093] In certain embodiments, the levels of one or more inflammation-related soluble factors are determined by the O-link IO analyte assay.

[0094] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0095] In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, 4, 5, 6, or 7 days after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, or 4 weeks after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells approximately one, two, three, four, five, or six months after it is determined that the levels of one or more inflammation-related soluble factors are similar to the levels of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells.

[0096] In any particular embodiment of the above embodiments, the level of CD83 in serum samples from human patients is a log-multiple change of approximately 3.25 compared to the control. In any particular embodiment of the above embodiments, the level of CD83 in serum samples from human patients is a log-multiple change of approximately 3.25 compared to the control or less. In any particular embodiment of the above embodiments, the level of TNFRSF9(sCD137) in serum samples from human patients is a log-multiple change of approximately 6.3 compared to the control. In any particular embodiment of the above embodiments, the level of TNFRSF9(sCD137) in serum samples from human patients is a log-multiple change of approximately 6.3 compared to the control or less. In any particular embodiment of the above embodiments, the level of PGF in serum samples from human patients is a log-multiple change of approximately 8.6 compared to the control. In any particular embodiment of the above embodiments, the level of PGF in serum samples from human patients is a log-multiple change of approximately 8.6 compared to the control or less. In any particular embodiment of the above embodiments, the level of CD70 in a serum sample from a human patient is a change of approximately 5.0 log-multiples compared to the control. In any particular embodiment of the above embodiments, the level of CD70 in a serum sample from a human patient is a change of approximately 5.0 log-multiples compared to the control or less.

[0097] In one embodiment, a method is provided herein for determining whether a patient diagnosed with cancer should be administered chimeric antigen receptor (CAR) T cells, comprising the step of determining the level of one or more inflammation-related soluble factors in a serum sample from the patient, wherein the patient is a candidate for CAR T cells if the level of one or more inflammation-related soluble factors in the serum sample from the patient is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient who is responsive to chimeric antigen receptor (CAR) T cells.

[0098] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0099] In certain embodiments, the cancer is multiple myeloma.

[0100] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0101] In certain embodiments, the levels of one or more inflammation-related soluble factors are determined by the O-link IO analyte assay.

[0102] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0103] In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, 4, 5, 6, or 7 days after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, or 4 weeks after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells approximately one, two, three, four, five, or six months after it is determined that the levels of one or more inflammation-related soluble factors are similar to the levels of one or more inflammation-related soluble factors in a serum sample from a patient responsive to chimeric antigen receptor (CAR) T cells.

[0104] In any particular embodiment of the above embodiments, the level of CD83 in the serum sample from the patient is a log-multiple change of about 3.25 compared to the control. In any particular embodiment of the above embodiments, the level of CD83 in the serum sample from the patient is a log-multiple change of about 3.25 compared to the control or less. In any particular embodiment of the above embodiments, the level of TNFRSF9(sCD137) in the serum sample from the patient is a log-multiple change of about 6.3 compared to the control. In any particular embodiment of the above embodiments, the level of TNFRSF9(sCD137) in the serum sample from the patient is a log-multiple change of about 6.3 compared to the control or less. In any particular embodiment of the above embodiments, the level of PGF in the serum sample from the patient is a log-multiple change of about 8.6 compared to the control. In any particular embodiment of the above embodiments, the level of PGF in the serum sample from the patient is a log-multiple change of about 8.6 compared to the control or less. In any particular embodiment of the above embodiments, the level of CD70 in the serum sample from the patient is a log-multiple change of about 5.0 compared to the control. In any particular embodiment of the above embodiments, the level of CD70 in the serum sample from the patient is about a log multiple of 5.0 or less compared to the control.

[0105] In one embodiment, a method for treating cancer in a person in need is provided herein, comprising the step of determining the level of one or more inflammation-related soluble factors in a serum sample from the person in need, and if the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy person, the person in need is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells.

[0106] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0107] In certain embodiments, the cancer is multiple myeloma.

[0108] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0109] In certain embodiments, the levels of one or more inflammation-related soluble factors are determined by the O-link IO analyte assay.

[0110] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0111] In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, 4, 5, 6, or 7 days after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy person. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, or 4 weeks after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy person. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, 4, 5, or 6 months after it is determined that the level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy person.

[0112] In one embodiment, a method for treating cancer in a person in need is provided herein, comprising: a) determining that the level of one or more inflammation-related soluble factors in a serum sample from the person in need is similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy person; and b) subsequently providing the person in need with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells based on the determination in step a.

[0113] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0114] In certain embodiments, the cancer is multiple myeloma.

[0115] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0116] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0117] In certain embodiments, the levels of one or more inflammation-related soluble factors are determined by the O-link IO analyte assay.

[0118] In certain embodiments, the step of subsequently providing a therapeutically effective dose of chimeric antigen receptor (CAR) T cells to a person in need is carried out approximately 1, 2, 3, 4, 5, 6, or 7 days after the decision in step a.

[0119] In certain embodiments, the step of subsequently providing a therapeutically effective dose of chimeric antigen receptor (CAR) T cells to a person in need is carried out approximately one, two, three, or four weeks after the decision in step a. In certain embodiments, the step of subsequently providing a therapeutically effective dose of chimeric antigen receptor (CAR) T cells to a person in need is carried out approximately one, two, three, four, five, or six months after the decision in step a.

[0120] In one embodiment, a method for treating cancer is provided herein, comprising the step of administering a therapeutically effective dose of chimeric antigen receptor (CAR) T cells to a human patient diagnosed with cancer, wherein the level of one or more inflammation-related soluble factors in a serum sample from the human patient prior to the administration is determined to be similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy human.

[0121] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0122] In certain embodiments, the cancer is multiple myeloma.

[0123] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0124] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0125] In certain embodiments, the level of one or more inflammation-related soluble factors in a serum sample from a human patient is determined to be similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy human about 1, 2, 3, 4, 5, 6, or 7 days prior to the administration. In certain embodiments, the level of one or more inflammation-related soluble factors in a serum sample from a human patient is determined to be similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy human about 1, 2, 3, or 4 weeks prior to the administration. In certain embodiments, the level of one or more inflammation-related soluble factors in a serum sample from a human patient is determined to be similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy human about 1, 2, 3, 4, 5, or 6 months prior to the administration.

[0126] In certain embodiments, the levels of one or more inflammation-related soluble factors are determined by the O-link IO analyte assay.

[0127] In one embodiment, a method is provided herein for determining whether a patient diagnosed with cancer should be administered chimeric antigen receptor (CAR) T cells, comprising the step of determining the level of one or more inflammation-related soluble factors in a serum sample from the patient, wherein the patient is a candidate for CAR T cells if the level of one or more inflammation-related soluble factors in the serum sample from the patient is similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy person.

[0128] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0129] In certain embodiments, the cancer is multiple myeloma.

[0130] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0131] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0132] In one embodiment, the present invention provides a method for predicting whether a cancer in a human is responsive to chimeric antigen receptor (CAR) T cells, comprising the steps of (i) determining the level of one or more inflammation-related soluble factors in a serum sample; and (ii) administering a therapeutically effective dose of CAR T cells to a human if the level of one or more soluble factors in (i) is similar to that in a healthy human.

[0133] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0134] In certain embodiments, the cancer is multiple myeloma.

[0135] In certain embodiments, the CAR T cells are CAR T cells that target BCMA.

[0136] In certain embodiments, BCMA-targeting CAR T cells are ide-cel cells.

[0137] In a particular embodiment, CAR T cells are 150 × 10 6cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0138] In certain embodiments, the levels of one or more inflammation-related soluble factors are determined by the O-link IO analyte assay.

[0139] In one embodiment, a method for treating cancer in a person in need thereof is provided herein, comprising the step of administering to a person chimeric antigen receptor (CAR) T cells and an antagonist of an inflammation-related soluble factor selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1.

[0140] In certain embodiments, the inflammation-related soluble factor is selected from the group consisting of PGF, CD70, TNFRSF9, and CD83.

[0141] In certain embodiments, an antagonist of inflammation-related soluble factors is administered to humans in a therapeutically effective dose to reduce the level of inflammation-related soluble factors in humans to the level of inflammation-related soluble factors in healthy humans.

[0142] In certain embodiments, an inflammation-related soluble factor antagonist is administered to humans in a therapeutically effective dose to reduce the level of inflammation-related soluble factor in humans to the level of inflammation-related soluble factor in patients responsive to chimeric antigen receptor (CAR) T cells.

[0143] In certain embodiments, the cancer is multiple myeloma.

[0144] In certain embodiments, the CAR T cells are CAR T cells that target BCMA.

[0145] In certain embodiments, BCMA-targeting CAR T cells are ide-cel cells.

[0146] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0147] In certain embodiments, the inflammation-related soluble factor antagonist is administered prior to the administration of human chimeric antigen receptor (CAR) T cells. In certain embodiments, the inflammation-related soluble factor antagonist is administered about 1, 2, 3, 4, 5, 6, or 7 days before the administration of human chimeric antigen receptor (CAR) T cells. In certain embodiments, the inflammation-related soluble factor antagonist is administered about 1, 2, 3, or 4 weeks before the administration of human chimeric antigen receptor (CAR) T cells. In certain embodiments, the inflammation-related soluble factor antagonist is administered about 1, 2, 3, 4, 5, or 6 months before the administration of human chimeric antigen receptor (CAR) T cells.

[0148] In one embodiment, a method for treating cancer in a person in need is provided herein, comprising the step of administering to a person an inhibitor of a chimeric antigen receptor (CAR) T cell and an inflammation-related soluble factor selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1.

[0149] In certain embodiments, the inflammation-related soluble factor is selected from the group consisting of PGF, CD70, TNFRSF9, and CD83.

[0150] In certain embodiments, an inhibitor of inflammation-related soluble factors is administered to humans in a therapeutically effective dose to reduce the level of inflammation-related soluble factors in humans to the level of inflammation-related soluble factors in healthy humans.

[0151] In certain embodiments, the cancer is multiple myeloma.

[0152] In certain embodiments, the CAR T cells are CAR T cells that target BCMA.

[0153] In certain embodiments, BCMA-targeting CAR T cells are ide-cel cells.

[0154] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0155] In certain embodiments, the inhibitor of inflammation-related soluble factors is administered prior to the administration of human chimeric antigen receptor (CAR) T cells. In certain embodiments, the inhibitor of inflammation-related soluble factors is administered about 1, 2, 3, 4, 5, 6, or 7 days before the administration of human chimeric antigen receptor (CAR) T cells. In certain embodiments, the inhibitor of inflammation-related soluble factors is administered about 1, 2, 3, or 4 weeks before the administration of human chimeric antigen receptor (CAR) T cells. In certain embodiments, the inhibitor of inflammation-related soluble factors is administered about 1, 2, 3, 4, 5, or 6 months before the administration of human chimeric antigen receptor (CAR) T cells.

[0156] In one aspect, provided herein is a method of treating cancer in a human in need thereof, comprising: a. determining that a first level of one or more inflammation-related soluble factors in a serum sample from the human in need thereof is higher than the level of the one or more inflammation-related soluble factors in a serum sample from a healthy human; b. subsequently administering to the human a therapeutically effective dose of an antagonist of the inflammation-related soluble factor based on the determination in step a; c. after step b, determining a second level of the one or more inflammation-related soluble factors in a serum sample from the human in need thereof; wherein when the second level of the one or more inflammation-related soluble factors is similar to the level of the one or more inflammation-related soluble factors in a serum sample from a healthy human, the human in need thereof is subsequently administered a therapeutically effective dose of chimeric antigen receptor (CAR) T cells.

[0157] In certain embodiments, the one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, the one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, the one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, the one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0158] In certain embodiments, the cancer is multiple myeloma.

[0159] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are idecabtagene vicleucel (ide-cel) cells.

[0160] In certain embodiments, the CAR T cells are administered at a dose ranging from 150×10 6 cells to 450×10 6 cells. In certain embodiments, the BCMA CAR T cells are administered at a dose ranging from 150×10 6 cells to 450×10 6 cells.

[0161] In certain embodiments, the CAR T cells are administered at a dose ranging from 150×10 6 cells to 450×10 6 cells. In certain embodiments, the BCMA CAR T cells are administered at a dose ranging from 150×10 6 cells to 450×10 6 cells.

[0162] In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, 4, 5, 6, or 7 days after it is determined that a second level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy person. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, or 4 weeks after it is determined that a second level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy person. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells approximately one, two, three, four, five, or six months after it is determined that a second level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy person.

[0163] In one embodiment, a method for treating cancer in a person in need is provided herein, comprising: a) determining that the level of one or more inflammation-related soluble factors in a serum sample from the person in need is higher than the level of one or more inflammation-related soluble factors in a serum sample from a healthy person; b) subsequently providing the person with a therapeutically effective dose of an inflammation-related soluble factor antagonist based on the determination in step a; c) determining, after step b, that the level of one or more inflammation-related soluble factors in a serum sample from the person in need is similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy person; and d) subsequently providing the person in need with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells based on the determination in step c.

[0164] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0165] In certain embodiments, the cancer is multiple myeloma.

[0166] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0167] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0168] In certain embodiments, the step of subsequently providing a therapeutically effective dose of chimeric antigen receptor (CAR) T cells to a person in need is carried out approximately one, two, three, or four weeks after the decision in step c. In certain embodiments, the step of subsequently providing a therapeutically effective dose of chimeric antigen receptor (CAR) T cells to a person in need is carried out approximately one, two, three, four, five, or six months after the decision in step c.

[0169] In one embodiment, a method for treating cancer is provided herein, comprising the step of administering a therapeutically effective dose of chimeric antigen receptor (CAR) T cells to a human patient diagnosed with cancer, wherein the patient has been previously administered a therapeutically effective dose of an inflammation-related soluble factor antagonist, and a serum sample from the patient prior to the administration of the chimeric antigen receptor (CAR) T cells is determined to be similar in level to one or more inflammation-related soluble factors in a serum sample from a healthy human.

[0170] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0171] In certain embodiments, the cancer is multiple myeloma.

[0172] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0173] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0174] In certain embodiments, the level of one or more inflammation-related soluble factors in a serum sample from a human patient is determined to be similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy human about 1, 2, 3, 4, 5, 6, or 7 days before the administration of a therapeutically effective dose of chimeric antigen receptor (CAR) T cells. In certain embodiments, the level of one or more inflammation-related soluble factors in a serum sample from a human patient is determined to be similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy human about 1, 2, 3, or 4 weeks before the administration of a therapeutically effective dose of chimeric antigen receptor (CAR) T cells. In certain embodiments, the level of one or more inflammation-related soluble factors in a serum sample from a human patient is determined to be similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy human about 1, 2, 3, 4, 5, or 6 months before the administration of a therapeutically effective dose of chimeric antigen receptor (CAR) T cells.

[0175] In one aspect, provided herein is a method of determining whether a patient diagnosed with cancer should be administered chimeric antigen receptor (CAR) T cells, comprising the step of determining the level of one or more inflammation-related soluble factors in a serum sample from the patient, wherein the patient is a candidate for CAR T cells if the patient has been previously administered an antagonist of the one or more inflammation-related soluble factors, and the level of the one or more inflammation-related soluble factors in the serum sample from the patient is similar to the level of the one or more inflammation-related soluble factors in a serum sample from a healthy human.

[0176] In certain embodiments, the one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, the one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, the one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, the one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0177] In certain embodiments, the cancer is multiple myeloma.

[0178] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are idecabtagene vicleucel (ide-cel) cells.

[0179] In certain embodiments, the CAR T cells are 150×10 6 cells to 450×10 6administered at a dose in the range of cells. In certain embodiments, the BCMA CAR T cells are 150×10 6 cells to 450×10 6 cells.

[0180] In one aspect, provided herein is a method of treating cancer in a human in need thereof, comprising: a. determining that a first level of one or more inflammation-related soluble factors in a serum sample from the human in need thereof is higher than the level of the one or more inflammation-related soluble factors in a serum sample from a healthy human; b. subsequent to step a, providing the human with a therapeutically effective dose of an inhibitor of the inflammation-related soluble factor based on the determination in step a; c. after step b, determining a second level of the one or more inflammation-related soluble factors in a serum sample from the human in need thereof; wherein if the second level of the one or more inflammation-related soluble factors is similar to the level of the one or more inflammation-related soluble factors in a serum sample from a healthy human, the human in need thereof is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells.

[0181] In certain embodiments, the one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, the one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, the one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, the one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0182] In certain embodiments, the cancer is multiple myeloma.

[0183] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are idecabtagene vicleucel (ide-cel) cells.

[0184] In certain embodiments, the CAR T cells are administered at a dose in the range of 150×10 6 cells to 450×10 6 cells. In certain embodiments, the BCMA CAR T cells are administered at a dose in the range of 150×10 6 cells to 450×10 6 cells.

[0185] In certain embodiments, the CAR T cells are administered at a dose in the range of 150×10 6 cells to 450×10 6 cells. In certain embodiments, the BCMA CAR T cells are administered at a dose in the range of 150×10 6 cells to 450×10 6 cells.

[0186] In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, 4, 5, 6, or 7 days after it is determined that a second level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy person. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells about 1, 2, 3, or 4 weeks after it is determined that a second level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy person. In certain embodiments, a person requiring it is subsequently provided with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells approximately one, two, three, four, five, or six months after it is determined that a second level of one or more inflammation-related soluble factors is similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy person.

[0187] In one embodiment, a method for treating cancer in a person in need is provided herein, comprising: a) determining that the level of one or more inflammation-related soluble factors in a serum sample from the person in need is higher than the level of one or more inflammation-related soluble factors in a serum sample from a healthy person; b) subsequently providing the person with a therapeutically effective dose of an inhibitor of the inflammation-related soluble factors based on the determination in step a; c) determining, after step b, that the second level of one or more inflammation-related soluble factors in a serum sample from the person in need is similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy person; and d) subsequently providing the person in need with a therapeutically effective dose of chimeric antigen receptor (CAR) T cells based on the determination in step c.

[0188] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0189] In certain embodiments, the cancer is multiple myeloma.

[0190] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0191] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0192] In certain embodiments, the step of subsequently providing a therapeutically effective dose of chimeric antigen receptor (CAR) T cells to a person in need is carried out approximately one, two, three, or four weeks after the decision in step c. In certain embodiments, the step of subsequently providing a therapeutically effective dose of chimeric antigen receptor (CAR) T cells to a person in need is carried out approximately one, two, three, four, five, or six months after the decision in step c.

[0193] In one embodiment, a method for treating cancer is provided herein, comprising the step of administering a therapeutically effective dose of chimeric antigen receptor (CAR) T cells to a human patient diagnosed with cancer, wherein the patient has been previously administered a therapeutically effective dose of an inhibitor of inflammation-related soluble factors, and a serum sample from the patient prior to the administration of the therapeutically effective dose of chimeric antigen receptor (CAR) T cells is determined to be similar in level to one or more inflammation-related soluble factors in a serum sample from a healthy human.

[0194] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0195] In certain embodiments, the cancer is multiple myeloma.

[0196] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0197] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6 The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0198] In certain embodiments, the level of one or more inflammation-related soluble factors in a serum sample from a human patient is determined to be similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy human about 1, 2, 3, 4, 5, 6, or 7 days before the administration of a therapeutically effective dose of chimeric antigen receptor (CAR) T cells. In certain embodiments, the level of one or more inflammation-related soluble factors in a serum sample from a human patient is determined to be similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy human about 1, 2, 3, or 4 weeks before the administration of a therapeutically effective dose of chimeric antigen receptor (CAR) T cells. In certain embodiments, the level of one or more inflammation-related soluble factors in a serum sample from a human patient is determined to be similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy human about 1, 2, 3, 4, 5, or 6 months before the administration of a therapeutically effective dose of chimeric antigen receptor (CAR) T cells.

[0199] In one embodiment, a method is provided herein for determining whether a patient diagnosed with cancer should be administered chimeric antigen receptor (CAR) T cells, comprising the step of determining the level of one or more inflammation-related soluble factors in a serum sample from the patient, wherein the patient is a candidate for CAR T cells if the patient has been previously administered one or more inhibitors of inflammation-related soluble factors, and the level of one or more inflammation-related soluble factors in a serum sample from the patient is similar to the level of one or more inflammation-related soluble factors in a serum sample from a healthy person.

[0200] In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, one or more inflammation-related soluble factors are one or more of PGF, CD70, TNFRSF9, and CD83.

[0201] In certain embodiments, the cancer is multiple myeloma.

[0202] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells. In certain embodiments, the BCMA-directed CAR T cells are ide-cel cells.

[0203] In a particular embodiment, CAR T cells are 150 × 10 6 cells ~450×10 6The cells are administered in doses within a range. In certain embodiments, BCMA CAR T cells are administered in doses of 150 × 10⁶. 6 cells ~450×10 6 It is administered in doses corresponding to the cell range.

[0204] In the methods presented herein, the determination may be carried out using standard techniques well known to those skilled in the art. Additionally, the determination step may be carried out by utilizing standard techniques for determining the levels of inflammation-related soluble factors (e.g., PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1), such as serological, plasma, or hematological tests. For example, the level of one or more inflammation-related soluble factors in a blood sample (e.g., peripheral blood sample) from a patient or healthy person responsive to chimeric antigen receptor (CAR) T cells (e.g., BCMA CAR T cells, e.g., ide-cel) may be determined using standard techniques well known to those skilled in the art, such as serological, hematological, or plasma tests, such as the assays used in the examples. A healthy person may be, for example, a person not diagnosed with a disease (e.g., a person without cancer).

[0205] In certain embodiments, tumors or cancers include lymphoma, lung cancer, breast cancer, prostate cancer, adrenocortical carcinoma, thyroid cancer, nasopharyngeal cancer, melanoma, skin cancer, colorectal cancer, tendonoid, adhesive small round cell tumor, endocrine tumor, Ewing's sarcoma, peripheral primitive neuroectodermal tumor, solid germ cell tumor, hepatoblastoma, neuroblastoma, non-rhabdomyosarcoma, soft tissue sarcoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, Wilms' tumor, glioblastoma, myxoma, fibroma, lipomatous chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prelymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenstrom macroglobulinemia, splenic marginal zone lymphoma, plasmacytic myeloma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, nodal marginal zone B-cell lymphoma, filtration Bolsterol lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, Burkitt lymphoma, prelympho These include intestinal T lymphocyte lymphoma, hepatosplenic T lymphocyte lymphoma, blastocyte NK cell lymphoma, mycosis fungoides, Sézary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papuloma, angioimmunoblastic T lymphocyte lymphoma, peripheral T lymphocyte lymphoma (unspecified), anaplastic large cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, or multiple myeloma.

[0206] In certain embodiments, cancer is multiple myeloma, chronic lymphocytic leukemia, or non-Hodgkin lymphoma. In certain embodiments, cancer is non-Hodgkin lymphoma, and non-Hodgkin lymphoma is Burkitt lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large cell lymphoma, progenitor cell B lymphoblastic lymphoma, or mantle cell lymphoma. In certain embodiments, cancer is multiple myeloma. In certain embodiments, multiple myeloma is high-risk multiple myeloma or relapsed and refractory multiple myeloma. In certain embodiments, multiple myeloma is high-risk multiple myeloma, and high-risk multiple myeloma is a disease characterized by R-ISS stage III disease and / or early relapse. In certain embodiments, multiple myeloma is not a disease of R-ISS stage III.

[0207] In any particular embodiment of the above embodiments, cancer is brain cancer, glioblastoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma, lung cancer, uterine cancer, ovarian cancer, colorectal cancer, anal cancer, liver cancer, hepatocellular carcinoma, stomach cancer, testicular cancer, endometrial cancer, cervical cancer, Hodgkin's disease, non-Hodgkin lymphoma, esophageal cancer, intestinal cancer, thyroid cancer, adrenal cancer, bladder cancer, kidney cancer, breast cancer, multiple myeloma, sarcoma, anal cancer, or squamous cell carcinoma.

[0208] In a particular embodiment of any of the embodiments described above, the inflammation-related soluble factor is one or more of the following: placental growth factor (PGF), CD70, tumor necrosis factor receptor superfamily member 4 (TNFRSF4), tumor necrosis factor receptor superfamily member 9 (TNFRSF9 or sCD137 or s4-1BB), decorin (DCN), differentiation cluster 83 (CD83), interleukin 10 (IL-10), programmed cell death 1 (PDCD1), interleukin 12 (IL-12), and native cytotoxic trigger receptor 1 (NCR1).

[0209] In a particular embodiment of any of the above embodiments, the inhibitor or antagonist of inflammation-related soluble factors is an inhibitor or antagonist of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, or NCR1. In a particular embodiment of any of the above embodiments, the inhibitor or antagonist of inflammation-related soluble factors is an inhibitor or antagonist of PGF. In a particular embodiment of any of the above embodiments, the inhibitor or antagonist of inflammation-related soluble factors is an inhibitor or antagonist of CD70. In a particular embodiment of any of the above embodiments, the inhibitor or antagonist of inflammation-related soluble factors is an inhibitor or antagonist of TNFRSF4. In a particular embodiment of any of the above embodiments, the inhibitor or antagonist of inflammation-related soluble factors is an inhibitor or antagonist of TNFRSF9 (sCD137 or s4-1BB). In a particular embodiment of any of the above embodiments, the inhibitor or antagonist of inflammation-related soluble factors is an inhibitor or antagonist of DCN. In a particular embodiment of any of the above embodiments, the inhibitor or antagonist of inflammation-related soluble factors is an inhibitor or antagonist of CD83. In a particular embodiment of any of the above embodiments, the inhibitor or antagonist of inflammation-related soluble factors is an inhibitor or antagonist of IL10. In a particular embodiment of any of the above embodiments, the inhibitor or antagonist of inflammation-related soluble factors is an inhibitor or antagonist of PDCD1. In a particular embodiment of any of the above embodiments, the inhibitor or antagonist of inflammation-related soluble factors is an inhibitor or antagonist of IL12. In a particular embodiment of any of the above embodiments, the inhibitor or antagonist of inflammation-related soluble factors is an inhibitor or antagonist of NCR1.

[0210] In a particular embodiment of any of the embodiments described above, the inhibitor of inflammation-related soluble factors is an inhibitor of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, or NCR1. In a particular embodiment of any of the embodiments described above, the inhibitor of inflammation-related soluble factors is an inhibitor of inflammation-related soluble factors selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In a particular embodiment of any of the embodiments described above, the inhibitor of inflammation-related soluble factors is an inhibitor of PGF. In a particular embodiment of any of the embodiments described above, the inhibitor of inflammation-related soluble factors is an inhibitor of CD70. In a particular embodiment of any of the embodiments described above, the inhibitor of inflammation-related soluble factors is an inhibitor of TNFRSF4. In any particular embodiment of the above embodiments, the inhibitor of inflammation-related soluble factors is an inhibitor of TNFRSF9 (sCD137 or s4-1BB). In any particular embodiment of the above embodiments, the inhibitor of inflammation-related soluble factors is an inhibitor of DCN. In any particular embodiment of the above embodiments, the inhibitor of inflammation-related soluble factors is an inhibitor of CD83. In any particular embodiment of the above embodiments, the inhibitor of inflammation-related soluble factors is an inhibitor of IL10. In any particular embodiment of the above embodiments, the inhibitor of inflammation-related soluble factors is an inhibitor of PDCD1. In any particular embodiment of the above embodiments, the inhibitor of inflammation-related soluble factors is an inhibitor of IL12. In any particular embodiment of the above embodiments, the inhibitor of inflammation-related soluble factors is an inhibitor of NCR1.

[0211] In a particular embodiment of any of the embodiments described above, the inflammation-related soluble factor antagonist is an antagonist of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, or NCR1. In a particular embodiment of any of the embodiments described above, the inflammation-related soluble factor antagonist is an antagonist of an inflammation-related soluble factor selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In a particular embodiment of any of the embodiments described above, the inflammation-related soluble factor antagonist is an antagonist of PGF. In a particular embodiment of any of the embodiments described above, the inflammation-related soluble factor antagonist is an antagonist of CD70. In any particular embodiment of the above embodiments, the antagonist of inflammation-related soluble factors is the antagonist of TNFRSF4. In any particular embodiment of the above embodiments, the antagonist of inflammation-related soluble factors is the antagonist of TNFRSF9 (sCD137 or s4-1BB). In any particular embodiment of the above embodiments, the antagonist of inflammation-related soluble factors is the antagonist of DCN. In any particular embodiment of the above embodiments, the antagonist of inflammation-related soluble factors is the antagonist of CD83. In any particular embodiment of the above embodiments, the antagonist of inflammation-related soluble factors is the antagonist of IL10. In any particular embodiment of the above embodiments, the antagonist of inflammation-related soluble factors is the antagonist of PDCD1. In any particular embodiment of the above embodiments, the antagonist of inflammation-related soluble factors is the antagonist of IL12. In any particular embodiment of the above embodiments, the antagonist of inflammation-related soluble factors is an antagonist of NCR1.

[0212] In a particular embodiment of any of the embodiments described above, the inhibitor or antagonist of an inflammation-related soluble factor is an antibody. In a particular embodiment, the antagonist of an inflammation-related soluble factor is an antibody against PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, or NCR1. In a particular embodiment, the antagonist of an inflammation-related soluble factor is an antibody against an inflammation-related soluble factor selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In a particular embodiment, the antagonist of an inflammation-related soluble factor is an antibody against PGF. In a particular embodiment, the antagonist of an inflammation-related soluble factor is an antibody against CD70. In certain embodiments, the antagonist of inflammation-related soluble factors is an antibody against TNFRSF4. In certain embodiments, the antagonist of inflammation-related soluble factors is an antibody against TNFRSF9 (sCD137 or s4-1BB). In certain embodiments, the antagonist of inflammation-related soluble factors is an antibody against DCN. In certain embodiments, the antagonist of inflammation-related soluble factors is an antibody against CD83. In certain embodiments, the antagonist of inflammation-related soluble factors is an antibody against IL10. In certain embodiments, the antagonist of inflammation-related soluble factors is an antibody against PDCD1. In certain embodiments, the antagonist of inflammation-related soluble factors is an antibody against IL12. In certain embodiments, the antagonist of inflammation-related soluble factors is an antibody against NCR1.

[0213] In certain embodiments of any of the embodiments described above, the IL-12 antagonist is STELARA® (ustekinumab). In certain embodiments, STELARA® (ustekinumab) is administered to adult humans weighing 100 kg or less in a dose of approximately 45 mg initially and after 4 weeks subcutaneously, followed by a dose of 45 mg every 12 weeks subcutaneously. In certain embodiments, STELARA® (ustekinumab) is administered to adult humans weighing more than 100 kg in a dose of approximately 90 mg initially and after 4 weeks subcutaneously, followed by a dose of 90 mg every 12 weeks subcutaneously. In certain embodiments, STELARA® (ustekinumab) is administered to pediatric human patients weighing less than 60 kg in a dose of approximately 0.75 mg / kg. In certain embodiments, STELARA® (ustekinumab) is administered to pediatric human patients weighing 60 kg to 100 kg at a dose of approximately 45 mg. In certain embodiments, STELARA® (ustekinumab) is administered to pediatric human patients weighing more than 100 kg at a dose of approximately 90 mg. In certain embodiments, STELARA® (ustekinumab) is administered to adult humans weighing up to 55 kg at a dose of approximately 260 mg. In certain embodiments, STELARA® (ustekinumab) is administered to adult humans weighing 55 kg to 85 kg at a dose of approximately 390 mg. In certain embodiments, STELARA® (ustekinumab) is administered to adult humans weighing more than 85 kg at a dose of approximately 520 mg.

[0214] Inhibitors or antagonists of a factor (e.g., a protein) may be protein traps. Protein traps can be constructed, for example, by fusing the first three Ig domains of a receptor (i.e., ligand-binding elements) to the constant region (Fc) of human IgG1. See Holash et al., Proc Natl Acad Sci US A. 2002 Aug 20; 99(17):11393-11398. In any particular embodiment of the above embodiments, the antagonist of the inflammation-related soluble factor is a protein trap. In a particular embodiment, the protein trap binds to an inflammation-related soluble factor selected from the group consisting of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1. In certain embodiments, the protein trap binds to PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, or NCR1. In certain embodiments, the protein trap binds to an inflammation-related soluble factor selected from the group consisting of PGF, CD70, TNFRSF9, and CD83. In certain embodiments, the protein trap binds to PGF, CD70, TNFRSF9, or CD83.

[0215] Exemplary soluble factor antagonists, which may be used in combination with CAR T therapy, for example, and which may be administered prior to CAR T therapy to reduce the level or activity of one or more inflammation-related soluble factors described herein, include: ustekinumab for IL-12 and urerumab and utomirumab for CD137.

[0216] In certain embodiments, the subject undergoes a leukocyte apheresis procedure to collect PBMCs for the production of CAR T cells prior to administration of CAR T cells to the subject.

[0217] In certain embodiments, CAR T cells are administered by intravenous infusion.

[0218] In any particular embodiment of the above aspects or embodiments, the subject is a human (e.g., a human patient).

[0219] In certain embodiments of any of the above aspects or embodiments, the CAR T cells include BCMA02, ABECMA (copyright), JCARH125, JNJ-68284528 (LCAR-B38M; containing SEQ ID NO: 265 of 10,934,363 or SEQ ID NO: 300 of WO2018 / 028647, and having or not having a signal peptide) (Janssen / Legend), P-BCMA-101 (Poseida), PBCAR269A (Poseida), P-BCMA-Allo1 (Poseida), Allo-715 (Pfizer / Allogene), CT053 (Carsgen), Descartes-08 (Cartesian), PHE885 (Novartis), CTX120 (CRISPR Therapeutics); CD19 CAR T therapy, such as CAR T therapy targeting Yescarta, Kymriah, Tecartus, lysocabtadiene malareucel (liso-cel), or any other cell surface marker.

[0220] In a particular embodiment of any of the above aspects or embodiments, the CAR T cell therapy includes BCMA02, ABECMA (copyright), JCARH125, JNJ-68284528 (LCAR-B38M; containing SEQ ID NO: 265 of 10,934,363 or SEQ ID NO: 300 of WO2018 / 028647, a protein with or without a signal peptide) (Janssen / Legend), P-BCMA-101 (Poseida), PBCAR269A (Poseida), P-BCMA-Allo1 (Poseida), Allo-715 (Pfizer / Allogene), CT053 (Carsgen), Descartes-08 (Cartesian), PHE885 (Novartis), CTX120 (CRISPR Therapeutics); CD19 CAR T therapy, such as CAR T therapy targeting Yescarta, Kymriah, Tecartus, lysocabtadiene malareucel (liso-cel), or any other cell surface marker.

[0221] In certain embodiments, the CAR T cells are BCMA-directed CAR T cells (BCMA CAR T cells). BCMA is a member of the tumor necrosis factor receptor superfamily (see, e.g., Thompson et al., J. Exp. Medicine, 192(1): 129-135, 2000 and Mackay et al., Annu. Rev. Immunol, 21: 231-264, 2003). BCMA binds to B cell activator (BAFF) and proliferation-inducing ligand (APRIL) (see, e.g., Mackay et al., 2003 and Kalled et al., Immunological Reviews, 204: 43-54, 2005). Among benign cells, BCMA has been reported to be expressed primarily in a subset of plasma cells and mature B cells (see, for example, Laabi et al., EMBO J., 77(1): 3897-3904, 1992; Laabi et al., Nucleic Acids Res., 22(7): 1147-1154,, 1994; Kalled et al., 2005; O'Connor et al., J. Exp. Medicine, 199(1): 91-97, 2004; and Ng et al., J. Immunol., 73(2): 807-817, 2004). Mice deficient in BCMA are healthy and have a normal number of B cells, but the survival of long-lived plasma cells is impaired (see, e.g., O'Connor et al., 2004; Xu et al., Mol. Cell. Biol., 21(12): 4067-4074, 2001; and Schiemann et al., Science, 293(5537): 2 111-21 14, 2001).BCMA RNA is universally detected in cells of multiple myeloma and other lymphomas, and BCMA protein has been detected by several researchers on the surface of plasma cells derived from multiple myeloma patients (see, e.g., Novak et al., Blood, 103(2): 689-694, 2004; Neri et al., Clinical Cancer Research, 73(19): 5903-5909, 2007; Bellucci et al., Blood, 105(10): 3945-3950, 2005; and Moreaux et al., Blood, 703(8): 3148-3157, 2004).

[0222] In certain embodiments, BCMA CAR T cells include a BCMA-targeting CAR, and the BCMA-targeting CAR includes an antibody or antibody fragment that targets BCMA. In certain embodiments, BCMA CAR T cells include a BCMA-targeting CAR, and the BCMA-targeting CAR includes a single-chain Fv antibody or antibody fragment (scFv). In certain embodiments, BCMA CAR T cells include a BCMA-targeting CAR, and the BCMA-targeting CAR includes BCMA02 scFv, for example, SEQ ID NO: 38.

[0223] In certain embodiments of any of the above aspects or embodiments, BCMA CAR T cells include a BCMA-targeting CAR. In certain embodiments, the BCMA-targeting CAR includes an antibody or antibody fragment that targets BCMA. In certain embodiments of any of the above aspects or embodiments, BCMA CAR T cells include a BCMA-targeting CAR, and the BCMA-targeting CAR includes a single-chain Fv antibody or antibody fragment (scFv). In certain embodiments of any of the above aspects or embodiments, BCMA CAR T cells include a BCMA-targeting CAR, and the BCMA-targeting CAR includes SEQ ID NO: 37. In certain embodiments of any of the above aspects or embodiments, BCMA CAR T cells include a BCMA-targeting CAR, and the BCMA-targeting CAR includes BCMA02 scFv, for example, SEQ ID NO: 38. In certain embodiments, the BCMA-targeting CAR is coded by SEQ ID NO: 10. In certain embodiments, BCMA CAR T cells include a nucleic acid encoding BCMA CAR T, such as a vector, containing amino acids 22-493 or 1-493 of SEQ ID NO: 9, SEQ ID NO: 37, or SEQ ID NO: 38, or a nucleic acid including SEQ ID NO: 10, such as a vector. In certain embodiments of any of the above aspects or embodiments, BCMA CAR T cells are idekabuta jene bicleucell cells.

[0224] In a particular embodiment of any of the above aspects or embodiments, the CAR T cell therapy (e.g., immune cells expressing a BCMA-directed chimeric antigen receptor (CAR) (BCMA CAR T cells), e.g., ide-cel cells) includes a population of cells comprising 10%, 5%, 3%, 2%, or 1% of senescent CAR T cell populations, e.g., CD4 CAR T cells (CD3+ / CD4+ / CAR+ / CD57+). In a particular embodiment of any of the above aspects or embodiments, the tissue sample is blood, plasma, or serum. In another specific embodiment of any of the above aspects or embodiments, the disease caused by cells expressing BCMA is multiple myeloma, chronic lymphocytic leukemia, or non-Hodgkin lymphoma (e.g., Burkitt lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large B-cell lymphoma, precursor B-lymphoblastic lymphoma, and mantle cell lymphoma). In a particular embodiment, the disease is multiple myeloma, e.g., high-risk multiple myeloma or relapsed and refractory multiple myeloma. In another specific embodiment, high-risk multiple myeloma is a disease characterized by R-ISS stage III disease and / or early relapse (e.g., progressive disease within 12 months from the date of the last treatment regimen, e.g., a last treatment regimen using proteasome inhibitors, immunomodulators, and / or dexamethasone). In certain embodiments, multiple myeloma is not a R-ISS stage III disease. In certain embodiments, the disease caused by cells expressing BCMA is a non-Hodgkin lymphoma, which is selected from the group consisting of Burkitt lymphoma, chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL / SLL), diffuse large B-cell lymphoma, follicular lymphoma, immunoblastic large B-cell lymphoma, precursor B-lymphoblastic lymphoma, and mantle cell lymphoma.

[0225] In one embodiment, prior to the administration of T cells expressing a chimeric antigen receptor (CAR) that targets B cell maturation antigen (BCMA) (BCMA CAR T cells), subjects with tumors are evaluated for BCMA expression by the tumor.

[0226] In certain embodiments of any of the above aspects or embodiments, the immune cells are T cells, for example, CD4+ T cells, CD8+ T cells, or cytotoxic T lymphocytes (CTLs), T killer cells, or natural killer (NK) cells. In another particular embodiment, the immune cells are 150 × 10⁻¹⁰ 6 cells ~450×10 6 It is administered in the form of a single cell dose.

[0227] In any particular embodiment of the above aspects or embodiments, immune cells (e.g., CAR T cells, e.g., BCMA CAR T cells) are 150 × 10 6 cells ~450×10 6 Cells, 300×10 6 cells ~600×10 6 cells, 350×10 6 cells ~600×10 6 cells, 350×10 6 cells ~550×10 6 cells, 400×10 6 cells ~600×10 6 cells, 150 x 10 6 cells ~300×10 6 Cells or 400 × 10 6 cells ~500×10 6 They are administered in doses ranging from cells to cells. In some embodiments, immune cells are approximately 150 × 10⁶ 6 cells, approximately 200 x 10 6 cells, approximately 250 x 10 6 Cells, approximately 300 x 10 6 Cells, approximately 350 x 10 6 Cells, approximately 400 x 10 6 Cells, approximately 450 x 10 6 cells, approximately 500 x 10 6 Cells or approximately 550 × 10 6 It is administered in doses of cells. In one embodiment, immune cells are approximately 450 × 10⁶6 The immune cells are administered in cellular doses. In some embodiments, the subject receives a single infusion of immune cells expressing a chimeric antigen receptor (CAR) (e.g., CAR T cells). In some embodiments, the administration of CAR-expressing immune cells is repeated (e.g., a second dose of immune cells is administered to the subject). In some embodiments, the subject receives a single infusion of immune cells expressing a chimeric antigen receptor (CAR) that targets B cell maturation antigen (BCMA). In some embodiments, the administration of immune cells expressing a BCMA-targeting CAR is repeated (e.g., a second dose of immune cells is administered to the subject).

[0228] In any particular embodiment of the embodiments described herein, immune cells expressing CAR (e.g., CAR T cells) are approximately 150 × 10⁻⁶ 6 Cells ~ approx. 300×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 350 × 10⁶ 6 Cells ~ approx. 550×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 400 × 10⁶ 6 Cells ~ approx. 500×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, CAR-expressing immune cells are approximately 150 × 10⁶ 6 Cells ~ approx. 250×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 300 × 10⁶ 6 Cells ~ approx. 500×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 350 × 10⁶ 6 Cells ~ approx. 450×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 300 × 10⁶ 6Cells ~ approx. 450×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 250 × 10⁶ 6 Cells ~ approx. 450×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 300 × 10⁶ 6 Cells ~ approx. 600×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 250 × 10⁶ 6 Cells ~ approx. 500×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 350 × 10⁶ 6 Cells ~ approx. 500×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 400 × 10⁶ 6 Cells ~ approx. 600×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 400 × 10⁶ 6 Cells ~ approx. 450×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 200 × 10⁶ 6 Cells ~ approx. 400×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 200 × 10⁶ 6 Cells ~ approx. 350×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 200 × 10⁶ 6 Cells ~ approx. 300×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, CAR-expressing immune cells are approximately 450 × 10⁶ 6Cells ~ approx. 500×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 250 × 10⁶ 6 Cells ~ approx. 400×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 250 × 10⁶ 6 Cells ~ approx. 350×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, CAR-expressing immune cells are approximately 450 × 10⁶ 6 The immune cells are administered in cellular doses. In certain embodiments of any of the embodiments described herein, the immune cells are T cells (e.g., autologous T cells). In certain embodiments of any of the embodiments described herein, the subject being treated undergoes a leukocyte apheresis procedure to collect autoimmune cells to produce immune cells that express CAR prior to its administration to the subject. In certain embodiments of any of the embodiments described herein, the immune cells (e.g., T cells) are administered by intravenous infusion.

[0229] In any particular embodiment of the embodiments described herein, immune cells expressing CAR (e.g., CAR T cells) are approximately 150 × 10⁻⁶ 6 Cells ~ approx. 300×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 350 × 10⁶ 6 Cells ~ approx. 550×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 400 × 10⁶ 6 Cells ~ approx. 500×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, CAR-expressing immune cells are approximately 150 × 10⁶ 6 Cells ~ approx. 250×10 6It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 300 × 10⁶ 6 Cells ~ approx. 500×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 350 × 10⁶ 6 Cells ~ approx. 450×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 300 × 10⁶ 6 Cells ~ approx. 450×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 250 × 10⁶ 6 Cells ~ approx. 450×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 300 × 10⁶ 6 Cells ~ approx. 600×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 250 × 10⁶ 6 Cells ~ approx. 500×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 350 × 10⁶ 6 Cells ~ approx. 500×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 400 × 10⁶ 6 Cells ~ approx. 600×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 400 × 10⁶ 6 Cells ~ approx. 450×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 200 × 10⁶ 6 Cells ~ approx. 400×10 6It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 200 × 10⁶ 6 Cells ~ approx. 350×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 200 × 10⁶ 6 Cells ~ approx. 300×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, CAR-expressing immune cells are approximately 450 × 10⁶ 6 Cells ~ approx. 500×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 250 × 10⁶ 6 Cells ~ approx. 400×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing CAR are approximately 250 × 10⁶ 6 Cells ~ approx. 350×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, CAR-expressing immune cells are approximately 450 × 10⁶ 6 The immune cells are administered in cellular doses. In certain embodiments of any of the embodiments described herein, the immune cells are T cells (e.g., autologous T cells). In certain embodiments of any of the embodiments described herein, the subject being treated undergoes a leukocyte apheresis procedure to collect autoimmune cells to produce immune cells that express CAR prior to its administration to the subject. In certain embodiments of any of the embodiments described herein, the immune cells (e.g., T cells) are administered by intravenous infusion.

[0230] In any particular embodiment of the embodiments described herein, immune cells expressing BCMA-directed CARs (BCMA CAR T cells) are approximately 150 × 10⁻⁶ 6 Cells ~ approx. 300×10 6It is administered in a cellular dose. In a particular embodiment of any of the embodiments described herein, immune cells expressing BCMA-directed CARs are approximately 350 × 10⁶ 6 Cells ~ approx. 550×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing BCMA-directed CARs are administered in approximately 400 × 10⁶ doses. 6 Cells ~ approx. 500×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing BCMA-directed CARs are approximately 150 × 10⁶ 6 Cells ~ approx. 250×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing BCMA-directed CARs are approximately 300 × 10⁶ 6 Cells ~ approx. 500×10 6 It is administered in a cellular dose. In a particular embodiment of any of the embodiments described herein, immune cells expressing BCMA-directed CARs are approximately 350 × 10⁶ 6 Cells ~ approx. 450×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing BCMA-directed CARs are approximately 300 × 10⁶ 6 Cells ~ approx. 450×10 6 It is administered in a cellular dose. In a particular embodiment of any of the embodiments described herein, immune cells expressing BCMA-directed CARs are approximately 250 × 10⁶ 6 Cells ~ approx. 450×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing BCMA-directed CARs are approximately 300 × 10⁶ 6 Cells ~ approx. 600×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing BCMA-directed CARs range from approximately 250 × 10⁶ cells to approximately 500 × 10⁶ cells. 6It is administered in a cellular dose. In a particular embodiment of any of the embodiments described herein, immune cells expressing BCMA-directed CARs are approximately 350 × 10⁶ 6 Cells ~ approx. 500×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing BCMA-directed CARs are administered in approximately 400 × 10⁶ doses. 6 Cells ~ approx. 600×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing BCMA-directed CARs are administered in approximately 400 × 10⁶ doses. 6 Cells ~ approx. 450×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing BCMA-directed CARs are approximately 200 × 10⁶ 6 Cells ~ approx. 400×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing BCMA-directed CARs are approximately 200 × 10⁶ 6 Cells ~ approx. 350×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing BCMA-directed CARs are approximately 200 × 10⁶ 6 Cells ~ approx. 300×10 6 It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing BCMA-directed CARs are approximately 450 × 10⁶ 6 Cells ~ approx. 500×10 6 It is administered in cellular doses. In certain embodiments of any of the embodiments described herein, immune cells expressing BCMA-directed CARs range from approximately 250 × 10⁶ cells to approximately 400 × 10⁶ cells. 6 It is administered in a cellular dose. In a particular embodiment of any of the embodiments described herein, immune cells expressing BCMA-directed CARs are approximately 250 × 10⁶ 6 Cells ~ approx. 350×10 6It is administered in cellular doses. In any particular embodiment of the embodiments described herein, immune cells expressing BCMA-directed CARs are approximately 450 × 10⁶ 6 The immune cells are administered in cellular doses. In certain embodiments of any of the embodiments described herein, the immune cells are T cells (e.g., autologous T cells). In certain embodiments of any of the embodiments described herein, the subject being treated undergoes a leukocyte apheresis procedure to collect autoimmune cells to produce immune cells expressing BCMA-directed CARs prior to their administration to the subject. In certain embodiments of any of the embodiments described herein, the immune cells (e.g., T cells) are administered by intravenous infusion.

[0231] In certain embodiments of any of the embodiments or models disclosed herein, the subject being treated is administered lymphocyte depletion (LD) chemotherapy prior to the administration of CAR-expressing immune cells (e.g., CAR T cells). In certain embodiments, the LD chemotherapy comprises fludarabine and / or cyclophosphamide. In certain embodiments, the LD chemotherapy comprises fludarabine (e.g., about 30 mg / m2 intravenously) and cyclophosphamide (e.g., about 300 mg / m2 intravenously) for a period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 3 days). In other specific embodiments, the LD chemotherapy comprises any of the chemotherapeutic agents described in Section 5.9. In certain embodiments, the subject is administered CAR-expressing immune cells 1, 2, 3, 4, 5, 6, or 7 days after the administration of the LD chemotherapy (e.g., 2 or 3 days after the administration of the LD chemotherapy). In certain embodiments, the subject had not received any therapy for at least one week or longer, at least two weeks or longer (at least 14 days or longer), at least three weeks or longer, at least four weeks or longer, at least five weeks or longer, or at least six weeks or longer prior to the initiation of LD chemotherapy. In certain embodiments of any of the embodiments disclosed herein, the subject being treated had received only a single prior treatment regimen prior to the administration of immune cells expressing a chimeric antigen receptor (CAR).

[0232] In certain embodiments of any of the embodiments or models disclosed herein, the subject being treated is administered lymphocyte depletion (LD) chemotherapy prior to the administration of immune cells expressing BCMA-directed CARs (e.g., BCMA CAR T cells). In certain embodiments, the LD chemotherapy comprises fludarabine and / or cyclophosphamide. In certain embodiments, the LD chemotherapy comprises fludarabine (e.g., about 30 mg / m2 intravenously) and cyclophosphamide (e.g., about 300 mg / m2 intravenously) for a period of 1, 2, 3, 4, 5, 6, or 7 days (e.g., 3 days). In other specific embodiments, the LD chemotherapy comprises any of the chemotherapeutic agents described in Section 5.9. In certain embodiments, the subject is administered immune cells expressing B-cell maturation antigen (BCMA)-directed chimeric antigen receptors (CARs) 1, 2, 3, 4, 5, 6, or 7 days after the administration of the LD chemotherapy (e.g., 2 or 3 days after the administration of the LD chemotherapy). In certain embodiments, the subject had not received any therapy for at least one week or longer, at least two weeks or longer (at least 14 days or longer), at least three weeks or longer, at least four weeks or longer, at least five weeks or longer, or at least six weeks or longer prior to the initiation of LD chemotherapy. In certain embodiments of any of the embodiments disclosed herein, the subject being treated had received only a single prior treatment regimen prior to the administration of immune cells expressing a chimeric antigen receptor (CAR) directed towards B cell maturation antigen (BCMA).

[0233] For any of the embodiments described above, the subject undergoes apheresis to collect and isolate the immune cells, e.g., T cells. In a particular embodiment of any of the embodiments described above, the subject exhibits the following at the time of apheresis: M-protein (serum protein electrophoresis [sPEP] or urine protein electrophoresis [uPEP]): sPEP ≥ 0.5 g / dL or uPEP ≥ 200 mg / 24 hours; light chain multiple myeloma with measurable disease in serum or urine, serum immunoglobulin free light chain ≥ 10 mg / dL and an abnormal serum immunoglobulin kappa-lambda free light chain ratio; and / or Eastern Cooperative Oncology Group (ECOG) activity status ≤ 1. In more specific embodiments, the subject had further received at least three lines of the aforementioned prior treatments, including prior treatments using proteasome inhibitors, immunomodulators (lenalidomide or pomalidomide), and anti-CD38 antibodies, at the time of apheresis; had received at least two consecutive cycles of treatment for each of the aforementioned at least three lines of prior treatments until progressive disease was the best response to the line of treatment; had evidence of progressive disease with the most recent line of prior treatment, or within 60 days thereof; and / or had achieved a response (minimal or better) to at least one of the aforementioned prior lines of treatment. In any particular embodiment of the above embodiments, the subject exhibits the following at the time of administration: M-protein (serum protein electrophoresis [sPEP] or urine protein electrophoresis [uPEP]): sPEP ≥ 0.5 g / dL or uPEP ≥ 200 mg / 24 hours; light chain multiple myeloma with measurable disease in serum or urine, serum immunoglobulin free light chain ≥ 10 mg / dL and abnormal serum immunoglobulin kappa-lambda free light chain ratio; and / or East Coast Cancer Group (ECOG) activity status ≤ 1.In another, more specific embodiment, the subject further: has received only one previous antimyeloma treatment regimen; has the following high-risk factors: R-ISS stage III and early relapse as defined as (i) progressive disease (PD) less than 12 months from the date of the first transplant if the subject has received induction and stem cell transplantation; or (ii) PD less than 12 months from the date of the last treatment regimen which must contain at least a proteasome inhibitor, immunomodulator and dexamethasone if the subject has received induction only.

[0234] In certain embodiments of any of the above aspects or embodiments, the CAR comprises an antibody or antibody fragment that targets BCMA. In more specific embodiments, the CAR comprises a single-chain Fv antibody fragment (scFv). In more specific embodiments, the CAR comprises BCMA02 scFv, e.g., SEQ ID NO: 38. In certain embodiments of any of the above aspects or embodiments, the immune cells are idekabuta genbicleucell cells. In one embodiment, the chimeric antigen receptor comprises BCMA, e.g., a mouse single-chain Fv antibody fragment that targets BCMA. In one embodiment, the chimeric antigen receptor comprises a mouse anti-BCMA scFv that binds to a human BCMA polypeptide hinge domain comprising a CD8α polypeptide, e.g., a CD8α transmembrane domain, a CD137(4-1BB) intracellular costimulatory signaling domain, and a CD3ζ primary signaling domain. In one embodiment, the chimeric antigen receptor comprises a mouse scFv that targets BCMA, e.g., the scFV is the anti-BCMA02 CAR of SEQ ID NO: 9. In one embodiment, the chimeric antigen receptor is or comprises SEQ ID NO: 9 or SEQ ID NO: 37. In one embodiment, the chimeric antigen receptor is or comprises SEQ ID NO: 9. In one embodiment, the chimeric antigen receptor is or comprises SEQ ID NO: 37. In a more specific embodiment of any embodiment herein, the immune cell is an ide-cel cell. In one embodiment, the immune cell comprises a chimeric antigen receptor comprising BCMA, for example, a mouse single-stranded Fv antibody fragment targeting BCMA. In one embodiment, the immune cell comprises a chimeric antigen receptor comprising a BCMA polypeptide, for example, a BCMA comprising a CD8α polypeptide, a CD8α transmembrane domain, a CD137(4-1BB) intracellular costimulatory signaling domain, and a CD3ζ primary signaling domain, and a mouse anti-BCMA scFv that binds to the hinge domain. In one embodiment, the immune cell comprises a chimeric antigen receptor comprising SEQ ID NO: 9 or SEQ ID NO: 37. In one embodiment, the immune cell comprises a chimeric antigen receptor comprising SEQ ID NO: 9.In one embodiment, the immune cell includes a chimeric antigen receptor, which is or contains SEQ ID NO: 37.

[0235] In other embodiments, the genetically modified immune effector cells intended herein are administered to patients with B-cell-associated conditions, such as B-cell malignancies.

[0236] The amount of soluble (i.e., non-membrane-bound) BCMA (sBCMA) after CAR T-cell therapy, such as anti-BCMA CAR T-cell therapy, can be used to determine whether a subject is likely to respond well to CAR T-cell therapy or whether a different anticancer therapy should be administered. A greater decrease in sBCMA levels in tissue samples (e.g., serum, plasma, lymph, or blood) after CAR T-cell therapy correlates with more clinically beneficial outcomes (e.g., very good partial response, complete response, or stringent complete response). In one embodiment, a method is provided herein for treating a disease induced by cells expressing a B-cell maturation agent (BCMA) in a subject requiring treatment, comprising, for example, the steps of: determining a first level of soluble BCMA (sBCMA) in a tissue sample obtained from the subject; performing a first BCMA-based therapeutic modality on the subject, comprising the steps of: then determining a second level of soluble BCMA (sBCMA) in a tissue sample obtained from the subject, wherein if the second level of sBCMA is greater than about 30% of the first level of sBCMA, the subject is provided with a second BCMA-based therapeutic modality for further treatment of the disease, wherein the first BCMA-based therapeutic modality and the second BCMA-based therapeutic modality are different BCMA-based therapeutic modalities. In certain embodiments, the immune cells are idekabutagenbicleucell cells. In certain embodiments, the second BCMA-based therapeutic modality is not idekabutagenbicleucel cells.

[0237] In any particular embodiment of the above aspects or embodiments, the CAR T cell therapy (e.g., immune cells expressing a BCMA-directed chimeric antigen receptor (CAR) (BCMA CAR T cells, e.g., ide-cel cells)) comprises a population of cells including about 10%, 5%, 3%, 2%, or 1% activated CAR T cells, e.g., activated CD8 CAR T cells (CD3+ / CD8+ / CAR+ / CD25+).

[0238] In a particular embodiment of any of the above aspects or embodiments, the CAR comprises an antibody or antibody fragment that targets the antigen of interest. The antigen of interest may be any antigen of interest, for example, an antigen on tumor cells. Tumor cells may be, for example, cells in a solid tumor or cells of a hematological cancer. The antigen may be any antigen expressed on cells of any tumor or cancer type, for example, lymphoma, lung cancer, breast cancer, prostate cancer, adrenocortical carcinoma, thyroid cancer, nasopharyngeal cancer, melanoma, for example, malignant melanoma, skin cancer, colorectal cancer, tendonoid, adhesive round cell tumor, endocrine tumor, Ewing's sarcoma, peripheral primitive neuroectodermal tumor, solid germ cell tumor, hepatoblastoma, neuroblastoma, non-rhabdomyosarcoma, soft tissue sarcoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, Wilms' tumor, glioblastoma, myxoma, fibroma, or lipoma. In more specific embodiments, the lymphoma is chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prelymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, splenic marginal zone lymphoma, plasmacytoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, Burkitt lymphoma, T-cell lymphoma It may be pre-lymphocytic leukemia, macrogranular lymphocytic T lymphocyte leukemia, invasive NK cell leukemia, adult T lymphocyte leukemia / lymphoma, extranodal NK / T lymphocyte leukemia, nasal / intestinal T lymphocyte lymphoma, hepatosplenic T lymphocyte lymphoma, blastogenic NK cell lymphoma, mycosis fungoides, Sézary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T lymphocyte lymphoma, peripheral T lymphocyte lymphoma (unspecified), anaplastic large cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, or multiple myeloma.

[0239] In certain embodiments, the antigen is a tumor-associated antigen (TAA) or tumor-specific antigen (TSA). In various specific embodiments, without limitation, tumor-associated antigens or tumor-specific antigens include Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD19, CD20, CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein (GFAP), and globulin. These include cystic disease fluid protein (GCDFP-15), HMB-45 antigen, high molecular weight melanoma-associated antigen (HMW-MAA), protein melan-A (MART-1), myo-D1, muscle-specific actin (MSA), neurofilaments, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, dimeric pyruvate kinase isoenzyme M2 (tumor M2-PK), abnormal ras protein, or abnormal p53 protein.

[0240] In certain embodiments, TAA or TSA is a cancer / testicular (CT) antigen, such as BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ESO-1, NY-SAR-35, OY-TES-1, SPANXB1, SPA17, SSX, SYCP1, or TPTE.

[0241] In certain other embodiments, TAA or TSA is a carbohydrate or ganglioside, such as fuc-GM1, GM2 (carcinoembryonic antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2), GM3, and GD3.

[0242] In certain other embodiments, TAA or TSA may be alpha-actinin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, beta-catenin, CA125, CA15-3 (CA27, 29\BCAA), CA195, CA242, CA50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-1, dek-can fusion protein, EBNA, EF2, Epstein-Barr virus antigen, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triose phosphate isomerase, Gage 3, 4, 5, 6, 7, GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, TRP2-Int2, gp100(Pmel 17) Tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, p15(58), RAGE, SCP-1, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA19-9, CA72-4, CAM17.1, NuMa, K-ras, 13-catenin, Mum-1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KP1, CO-029, FGF-5, G250, Ga733(EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, These include TLP, TPS, CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostain, TARP (T cell receptor gamma surrogate leading frame protein), Trp-p8, STEAP1 (six-transmembrane prostatic epithelial antigen 1), abnormal ras protein, or abnormal p53 protein. In another specific embodiment, the tumor-associated antigen or tumor-specific antigen is integrin αvβ3 (CD61), galectin (galactin), K-Ras (V-Ki-ras2 Kirsten rat sarcoma virus oncogene), or Ral-B.

[0243] In certain embodiments, the TAA or TSA is CD20, CD123, CLL-1, CD38, CS-1, CD138, ROR1, FAP, MUC1, PSCA, EGFRvIII, EPHA2, or GD2. In further specific embodiments, the TAA or TSA is CD123, CLL-1, CD38, or CS-1. In certain embodiments, the extracellular domain of the CAR binds to CS-1. In further specific embodiments, the extracellular domain includes a single-stranded version of elotuzumab and / or an antigen-binding fragment of elotuzumab. In certain embodiments, the extracellular domain of the CAR binds to CD20. In even more specific embodiments, the extracellular domain of the CAR is scFv, or its antigen-binding fragment binds to CD20.

[0244] Other tumor-related and tumor-specific antigens are known to those skilled in the art.

[0245] Antibodies that bind to TSA and TAA, as well as scFv, are known in the art, as are the nucleotide sequences that encode them.

[0246] In certain embodiments, the antigen is not considered to be TSA or TAA, but is nevertheless an antigen associated with tumor cells or damage induced by the tumor. In certain embodiments, the antigen is a tumor microenvironment-associated antigen (TMAA). In certain embodiments, for example, the TMAA is a growth factor, cytokine, or interleukin, for example, a growth factor, cytokine, or interleukin associated with angiogenesis or vascularization. Such growth factors, cytokines, or interleukins may include, for example, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-8). Tumors can also create a localized hypoxic environment for the tumor. Therefore, in other certain embodiments, the TMAA is a hypoxia-associated factor, for example, HIF-1α, HIF-1β, HIF-2α, HIF-2β, HIF-3α, or HIF-3β. Tumors can also induce localized damage to normal tissue, triggering the release of molecules known as damage-associated molecular pattern molecules (DAMPs; also known as alarmins). Thus, in certain other specific embodiments, TMAA may be DAMPs, e.g., heat shock proteins, chromatin-associated protein high-mobility group box 1 (HMGB1), S100A8 (MRP8, cargranulin A), S100A9 (MRP14, cargranulin B), serum amyloid A (SAA), or deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate. In certain embodiments, TMAA may be VEGF-A, EGF, PDGF, IGF, or bFGF. In a particular embodiment of any of the above aspects or embodiments, the CAR comprises an antibody or antibody fragment that targets the antigen of interest. In a more particular embodiment, the CAR comprises a single-chain Fv antibody fragment (scFv). In one embodiment, the chimeric antigen receptor comprises an scFv that binds to the antigen of interest, e.g., an antigen on tumor cells, a hinge domain containing a CD8α polypeptide, a CD8α transmembrane domain, a CD137(4-1BB) intracellular costimulatory signaling domain, and a CD3ζ primary signaling domain. The tumor cells may be, for example, cells in a solid tumor or cells of a hematological malignancy. The antigen may be any antigen expressed on cells of any tumor or cancer type. In one embodiment, the immune cell comprises a chimeric antigen receptor comprising a single-chain Fv antibody fragment that targets the antigen of interest. In one embodiment, the immune cell comprises a chimeric antigen receptor comprising an scFv that binds to the antigen of interest, a hinge domain containing a CD8α polypeptide, a CD8α transmembrane domain, a CD137(4-1BB) intracellular costimulatory signaling domain, and a CD3ζ primary signaling domain.

[0247] In certain embodiments of any of the above aspects or embodiments, the CAR comprises an antibody or antibody fragment that targets BCMA. In more specific embodiments, the CAR comprises a single-chain Fv antibody fragment (scFv). In more specific embodiments, the CAR comprises BCMA02 scFv, e.g., SEQ ID NO: 38. In certain embodiments of any of the above aspects or embodiments, the immune cells are idekabuta genbicleucell cells. In one embodiment, the chimeric antigen receptor comprises BCMA, e.g., a mouse single-chain Fv antibody fragment that targets BCMA. In one embodiment, the chimeric antigen receptor comprises a BCMA polypeptide, e.g., a mouse anti-BCMA scFv that binds to human BCMA polypeptide, a hinge domain containing a CD8α polypeptide, a CD8α transmembrane domain, a CD137(4-1BB) intracellular costimulatory signaling domain, and a CD3ζ primary signaling domain. In one embodiment, the chimeric antigen receptor comprises a BCMA-targeting mouse scFv, e.g., the scFV is the anti-BCMA02 CAR of SEQ ID NO: 9 or SEQ ID NO: 37. In one embodiment, the chimeric antigen receptor is SEQ ID NO: 9 or includes SEQ ID NO: 9. In one embodiment, the chimeric antigen receptor is SEQ ID NO: 37 or includes SEQ ID NO: 37. In more specific embodiments of any embodiment herein, the immune cells are ide-cel cells. In one embodiment, the immune cells include a chimeric antigen receptor comprising BCMA, e.g., a mouse single-stranded Fv antibody fragment targeting BCMA. In one embodiment, the immune cells include a chimeric antigen receptor comprising a BCMA polypeptide, e.g., mouse anti-BCMA scFv that binds to BCMA, a hinge domain containing a CD8α polypeptide, a CD8α transmembrane domain, a CD137(4-1BB) intracellular costimulatory signaling domain, and a CD3ζ primary signaling domain. In one embodiment, the immune cells include a chimeric antigen receptor comprising SEQ ID NO: 9 or includes SEQ ID NO: 9. In one embodiment, the immune cells include a chimeric antigen receptor comprising SEQ ID NO: 37 or includes SEQ ID NO: 37.

[0248] In other embodiments, the genetically modified immune effector cells intended herein are administered to patients with B-cell-related conditions, such as autoimmune diseases associated with B-cell or B-cell malignancies.

[0249] The implementation of the subject matter presented herein will, unless otherwise specifically indicated, utilize conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology, and cell biology, which are within the technical scope of this art, many of which are described below for illustrative purposes. Such techniques are well described in the literature. For example, Sambrook, et al., Molecular Cloning: A Laboratory Manual (3rd Edition, 2001);Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989);Maniatis et al., Molecular Cloning: A Laboratory Manual (1982);Ausubel et al., Current Protocols in Molecular Biology (John Wiley and Sons, updated July 2008);Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience;Glover, DNA Cloning: A Practical Approach, vol. I & II (IRL Press, Oxford, 1985);Anand, Techniques for the Analysis of Complex Genomes, (Academic Press, New York, 1992);Transcription and Translation (B. Hames & S. Higgins, Eds.See also monographs in academic journals such as *Current Protocols in Immunology* (QE Coligan, AM Kruisbeek, DH Margulies, EM Shevach and W. Strober, eds., 1991), *Annual Review of Immunology*, and *Advances in Immunology* (1984); Perbal, *A Practical Guide to Molecular Cloning* (1984); Harlow and Lane, *Antibodies* (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1998); *Current Protocols in Immunology* (QE Coligan, AM Kruisbeek, DH Margulies, EM Shevach and W. Strober, eds., 1991); *Annual Review of Immunology*; and *Advances in Immunology*.

[0250] 6.2.Definition Unless otherwise defined, all scientific and technical terms used herein have the same meanings as those commonly understood by those skilled in the art to which this disclosure belongs. Any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this disclosure, but preferred embodiments of compositions, methods and materials are described herein. For the purposes of this disclosure, the following terms are defined below:

[0251] The articles “a,” “an,” and “the” are used herein to refer to one or more (i.e., at least one, or one or more) grammatical objects of the articles. For example, “an element” means one or more elements.

[0252] The use of options (e.g., "or") should be understood to mean one of the options, both, or any combination thereof.

[0253] The terms "and / or" should be understood to mean either one or both of the options.

[0254] As used herein, the terms “about” or “approximately” refer to a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length that varies by approximately 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% relative to the reference quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length. In one embodiment, the terms “about” or “approximately” refer to a range of a quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length of ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% relative to the reference quantity, level, value, number, frequency, percentage, dimension, size, volume, weight, or length.

[0255] Throughout this specification, unless the context otherwise requires, the words “comprise,” “comprises,” and “comprising” are understood to imply that they include the process or element or group of processes or elements described, but do not exclude any other process or element or group of processes or elements. “Consists of” means to include and be limited to whatever follows the phrase “consists of.” Thus, the phrase “consists of” indicates that the listed elements are required or mandatory, and that no other elements should be present. “Essentially consists of” means to include any elements listed after the phrase, and be limited to other elements that do not interfere with or contribute to the activity or action of the listed elements as specified in this disclosure. Thus, the phrase “essentially consists of” indicates that the listed elements are required or mandatory, but no other elements substantially affect the activity or action of the listed elements.

[0256] Any reference throughout this specification to “one embodiment,” “a specific embodiment,” “a related embodiment,” “a particular embodiment,” “an additional embodiment,” or “a further embodiment,” or any combination thereof, means that any particular feature, structure, or characteristic described in connection with such embodiment is included in at least one embodiment of the disclosure presented herein. Therefore, the appearance of these terms in various places throughout this specification does not necessarily all refer to the same embodiment. Furthermore, any particular feature, structure, or characteristic may be combined in any preferred manner in one or more embodiments. It is also understood that a positive description of a feature in one embodiment may serve as a basis for excluding such feature in a particular embodiment.

[0257] 6.3. Mode of administration In certain embodiments of any of the above aspects or embodiments, CAR T cells and antagonists of inflammation-related soluble factors (e.g., antagonists of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1) may be administered to the subject simultaneously (i.e., concurrently) and / or sequentially (i.e., sequentially) to appropriate levels of inflammation-related soluble factors in the subject's serum to be determined to be similar to levels of inflammation-related soluble factors in the serum of patients responsive to chimeric antigen receptor (CAR) T cells.

[0258] In certain embodiments of any of the above aspects or embodiments, CAR T cells and antagonists of inflammation-related soluble factors (e.g., antagonists of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1) are administered simultaneously. The term “simultaneous administration,” as used herein, means that the CAR T cells and the antagonists of inflammation-related soluble factors are administered with a time separation of no more than about 15 minutes, for example, no more than about 10, 5, or 1 minute. When CAR T cells and antagonists of inflammation-related soluble factors are administered simultaneously, the CAR T cells and antagonists of inflammation-related soluble factors may be contained in the same composition (e.g., a composition containing both CAR T cells and antagonists of inflammation-related soluble factors) or in separate compositions (e.g., the CAR T cells and antagonists of inflammation-related soluble factors are contained in separate compositions).

[0259] In certain embodiments of any of the above aspects or embodiments, CAR T cells and antagonists of inflammation-related soluble factors (e.g., antagonists of PGF, CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1) are administered sequentially. The term “sequential administration,” as used herein, means that the CAR T cells and the antagonists of inflammation-related soluble factors are administered with time intervals greater than about 15 minutes, e.g., about 20, 30, 40, 50, 60 minutes, or more minutes. Either the CAR T cells or the antagonists of inflammation-related soluble factors may be administered first. The CAR T cells and the antagonists of inflammation-related soluble factors are contained in separate compositions, which may be contained in the same or different packages.

[0260] In any particular embodiment of the above aspects or embodiments, the administration of CAR T cells and inflammation-related soluble factor antagonists is synchronous, i.e., the administration periods of CAR T cells and inflammation-related soluble factor antagonists overlap with each other.

[0261] In some embodiments, an antagonist of inflammation-related soluble factors is administered for at least one cycle (e.g., at least two, three, or four cycles) prior to the administration of CAR T cells. In certain embodiments, the CAR T cell administration period begins following the administration of this antagonist and approximately one, two, three, four, five, six, or seven days after it is determined that the levels of inflammation-related soluble factors are similar to those in serum from patients responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, the CAR T cell administration period begins following the administration of this antagonist and approximately one, two, three, or four weeks after it is determined that the levels of inflammation-related soluble factors are similar to those in serum from patients responsive to chimeric antigen receptor (CAR) T cells. In certain embodiments, the administration period of CAR T cells is initiated following the administration of this antagonist and approximately one, two, three, four, five, or six months after it is determined that the levels of inflammation-related soluble factors are similar to those of inflammation-related soluble factors in the serum of patients responsive to chimeric antigen receptor (CAR) T cells.

[0262] In some embodiments, administration of CAR T cells and antagonists of inflammation-related soluble factors is initiated at approximately the same time (e.g., within one of 1, 2, 3, 4, 5, 6, or 7 days). In some embodiments, administration of CAR T cells and antagonists of inflammation-related soluble factors is terminated at approximately the same time (e.g., within one of 1, 2, 3, 4, 5, 6, or 7 days). In some embodiments, administration of CAR T cells is continued after the termination of administration of antagonists of inflammation-related soluble factors (e.g., over one of approximately 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, or 12 months). In some embodiments, administration of CAR T cells is initiated after the start of administration of antagonists of inflammation-related soluble factors (e.g., after one of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months). In some embodiments, the administration of CAR T cells and antagonists of inflammation-related soluble factors is initiated and terminated at approximately the same time. In some embodiments, the administration of CAR T cells and antagonists of inflammation-related soluble factors is terminated at approximately the same time, and the administration of CAR T cells is initiated after the initiation of the administration of antagonists of inflammation-related soluble factors (e.g., after one of approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months).

[0263] In certain embodiments of any of the above aspects or embodiments, the administration of CAR T cells and antagonists of inflammation-related soluble factors is asynchronous. For example, in some embodiments, the administration of antagonists of inflammation-related soluble factors is terminated before the administration of CAR T cells. In some embodiments, the administration of CAR T cells is terminated before the administration of antagonists of inflammation-related soluble factors. The period between these two asynchronous administrations may range from about 2 to 8 weeks, for example, about 4 weeks.

[0264] Antagonists of CAR T cells and inflammation-related soluble factors described herein may be administered to an individual (e.g., a human, e.g., a human patient) via a variety of routes, including, for example, intravenous, intra-arterial, intraperitoneal, intrapulmonary, oral, inhaled, intracapsular, intramuscular, intratracheal, subcutaneous, intraocular, intrathecal, transmucosal, and transdermal. In some embodiments, a sustained-release formulation of the composition may be used. In one embodiment, an antagonist of inflammation-related soluble factors may be administered by any acceptable route, including, but not limited to, oral, intramuscular, transdermal, intravenous, via an inhaler or other airborne delivery system.

[0265] 6.4. Chimeric Antigen Receptors In various embodiments, genetically engineered receptors are provided that redirect the cytotoxicity of immune effector cells to B cells. These genetically engineered receptors are referred to herein as chimeric antigen receptors (CARs). A CAR is a molecule that combines antibody-based specificity for a desired antigen (e.g., BCMA) with a T cell receptor-activating intracellular domain to produce a chimeric protein that exhibits specific anti-BCMA cell immune activity. As used herein, the term “chimeric” describes a molecule composed of different protein or DNA portions from different origins.

[0266] The CAR T cell therapies to which the embodiments described herein are applied include any CAR T therapies, e.g., BCMA CAR T cell therapies, e.g., BCMA02, ABECMA (copyright), JCARH125, JNJ-68284528 (LCAR-B38M; comprising SEQ ID NO: 265 of 10,934,363 or SEQ ID NO: 300 of WO2018 / 028647, with or without a signal peptide) (Janssen / Legend), P-BCMA-101 (Poseida), PBCAR269A (Poseida), P-BCMA-Allo1 (Poseida), Allo-715 (Pfizer / Allogene), CT053 (Carsgen), Descartes-08 (Cartesian), PHE885 (Novartis), CTX120 (CRISPR Therapeutics); CD19 CAR T therapies, including CAR T therapies targeting Yescarta, Kymriah, Tecartus, lysocabtadiene malareucel (liso-cel), and any other cell surface markers.

[0267] The extracellular domain of a polypeptide (also referred to as a binding domain or antigen-specific binding domain) binds to the antigen of interest. In certain embodiments, the extracellular domain includes a receptor or a portion of a receptor that binds to the antigen. The extracellular domain may, for example, be a receptor or a portion of a receptor that binds to the antigen. In certain embodiments, the extracellular domain includes or is an antibody or its antigen-binding portion. In certain embodiments, the extracellular domain includes or is a single-stranded Fv domain. The single-stranded Fv domain may, for example, be linked by a mobile linker. H V connected to L and the V L and V H This may include cases where the antigen is derived from an antibody that binds to the antigen.

[0268] The antigen to which the extracellular domain of the polypeptide binds can be any antigen of interest, for example, an antigen on tumor cells. Tumor cells may be, for example, cells in a solid tumor or cells of a hematological cancer. The antigen may be any antigen expressed on cells of any tumor or cancer type, such as lymphoma, lung cancer, breast cancer, prostate cancer, adrenocortical carcinoma, thyroid cancer, nasopharyngeal cancer, melanoma, such as malignant melanoma, skin cancer, colorectal cancer, tendonoid, adhesive round cell tumor, endocrine tumor, Ewing's sarcoma, peripheral primitive neuroectodermal tumor, solid germ cell tumor, hepatoblastoma, neuroblastoma, non-rhabdomyosarcoma, soft tissue sarcoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, Wilms' tumor, glioblastoma, myxoma, fibroma, or lipoma. In more specific embodiments, the lymphoma is chronic lymphocytic leukemia (small lymphocytic lymphoma), B-cell prelymphocytic leukemia, lymphoplasmacytic lymphoma, Waldenström macroglobulinemia, splenic marginal zone lymphoma, plasmacytoma, plasmacytoma, extranodal marginal zone B-cell lymphoma, MALT lymphoma, nodal marginal zone B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, mediastinal (thymic) large B-cell lymphoma, intravascular large B-cell lymphoma, primary exudative lymphoma, Burkitt lymphoma, T-cell lymphoma It may be pre-lymphocytic leukemia, macrogranular lymphocytic T lymphocyte leukemia, invasive NK cell leukemia, adult T lymphocyte leukemia / lymphoma, extranodal NK / T lymphocyte leukemia, nasal / intestinal T lymphocyte lymphoma, hepatosplenic T lymphocyte lymphoma, blastogenic NK cell lymphoma, mycosis fungoides, Sézary syndrome, primary cutaneous anaplastic large cell lymphoma, lymphomatoid papulosis, angioimmunoblastic T lymphocyte lymphoma, peripheral T lymphocyte lymphoma (unspecified), anaplastic large cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, or multiple myeloma.

[0269] In certain embodiments, the antigen is a tumor-associated antigen (TAA) or tumor-specific antigen (TSA). In various specific embodiments, without limitation, the tumor-associated antigen or tumor-specific antigen may be Her2, prostate stem cell antigen (PSCA), alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), cancer antigen-125 (CA-125), CA19-9, calretinin, MUC-1, epithelial membrane protein (EMA), epithelial tumor antigen (ETA), tyrosinase, melanoma-associated antigen (MAGE), CD19, CD20, CD34, CD45, CD99, CD117, chromogranin, cytokeratin, desmin, glial fibrillary acidic protein ( These include GFAP, Gross cystic disease fluid protein (GCDFP-15), HMB-45 antigen, high molecular weight melanoma-associated antigen (HMW-MAA), protein melan-A (MART-1), myo-D1, muscle-specific actin (MSA), neurofilaments, neuron-specific enolase (NSE), placental alkaline phosphatase, synaptophysis, thyroglobulin, thyroid transcription factor-1, dimeric pyruvate kinase isoenzyme M2 (tumor M2-PK), abnormal ras protein, or abnormal p53 protein.

[0270] In certain embodiments, TAA or TSA is a cancer / testicular (CT) antigen, such as BAGE, CAGE, CTAGE, FATE, GAGE, HCA661, HOM-TES-85, MAGEA, MAGEB, MAGEC, NA88, NY-ESO-1, NY-SAR-35, OY-TES-1, SPANXB1, SPA17, SSX, SYCP1, or TPTE.

[0271] In certain other embodiments, TAA or TSA is a carbohydrate or ganglioside, such as fuc-GM1, GM2 (carcinoembryonic antigen-immunogenic-1; OFA-I-1); GD2 (OFA-I-2), GM3, and GD3.

[0272] In certain other embodiments, TAA or TSA may be alpha-actinin-4, Bage-1, BCR-ABL, Bcr-Abl fusion protein, beta-catenin, CA125, CA15-3 (CA27, 29\BCAA), CA195, CA242, CA-50, CAM43, Casp-8, cdc27, cdk4, cdkn2a, CEA, coa-1, dek-can fusion protein, EBNA, EF2, Epstein-Barr virus antigen, ETV6-AML1 fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, triose phosphate isomerase, Gage 3, 4, 5, 6, 7, GnTV, Herv-K-mel, Lage-1, NA-88, NY-Eso-1 / Lage-2, SP17, SSX-2, TRP2-Int2, gp100(Pmel 17) Tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, RAGE, GAGE-1, GAGE-2, p15(58), RAGE, SCP-1, Hom / Mel-40, PRAME, p53, H-Ras, HER-2 / neu, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, 13-catenin, Mum-1, p16, TAGE, PSMA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, 13HCG, BCA225, BTAA, CD68\KP1, CO-029, FGF-5, G250, Ga733(EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90, TAAL6, TAG72, These include TLP, TPS, CD19, CD22, CD27, CD30, CD70, GD2 (ganglioside G2), EGFRvIII (epidermal growth factor variant III), sperm protein 17 (Sp17), mesothelin, PAP (prostatic acid phosphatase), prostain, TARP (T cell receptor gamma surrogate leading frame protein), Trp-p8, STEAP1 (six-transmembrane prostatic epithelial antigen 1), abnormal ras protein, or abnormal p53 protein. In another specific embodiment, the tumor-associated antigen or tumor-specific antigen is integrin αvβ3 (CD61), galectin (galactin), K-Ras (V-Ki-ras2 Kirsten rat sarcoma virus oncogene), or Ral-B.

[0273] In certain embodiments, the TAA or TSA is CD20, CD123, CLL-1, CD38, CS-1, CD138, ROR1, FAP, MUC1, PSCA, EGFRvIII, EPHA2, or GD2. In further specific embodiments, the TAA or TSA is CD123, CLL-1, CD38, or CS-1. In certain embodiments, the extracellular domain of the CAR binds to CS-1. In further specific embodiments, the extracellular domain includes a single-stranded version of elotuzumab and / or an antigen-binding fragment of elotuzumab. In certain embodiments, the extracellular domain of the CAR binds to CD20. In even more specific embodiments, the extracellular domain of the CAR is scFv, or its antigen-binding fragment binds to CD20.

[0274] Other tumor-related and tumor-specific antigens are known to those skilled in the art.

[0275] Antibodies that bind to TSA and TAA, as well as scFv, are known in the art, as are the nucleotide sequences that encode them.

[0276] In certain embodiments, the antigen is not considered to be TSA or TAA, but is nevertheless an antigen associated with tumor cells or damage induced by the tumor. In certain embodiments, the antigen is a tumor microenvironment-associated antigen (TMAA). In certain embodiments, for example, the TMAA is a growth factor, cytokine, or interleukin, for example, a growth factor, cytokine, or interleukin associated with angiogenesis or vascularization. Such growth factors, cytokines, or interleukins may include, for example, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), or interleukin-8 (IL-8). Tumors can also create a localized hypoxic environment for the tumor. Therefore, in other certain embodiments, the TMAA is a hypoxia-associated factor, for example, HIF-1α, HIF-1β, HIF-2α, HIF-2β, HIF-3α, or HIF-3β. Tumors can also induce localized damage to normal tissue, triggering the release of molecules known as damage-associated molecular pattern molecules (DAMPs; also known as alarmins). Thus, in certain other specific embodiments, TMAA may be DAMPs, e.g., heat shock proteins, chromatin-associated protein high-mobility group box 1 (HMGB1), S100A8 (MRP8, cargranulin A), S100A9 (MRP14, cargranulin B), serum amyloid A (SAA), or deoxyribonucleic acid, adenosine triphosphate, uric acid, or heparin sulfate. In certain embodiments, TMAA may be VEGF-A, EGF, PDGF, IGF, or bFGF.

[0277] In certain embodiments, the extracellular domain is joined to the transmembrane domain by a linker, spacer, or hinge polypeptide sequence, such as a sequence from CD28.

[0278] In certain embodiments, the CARs contemplated herein include an extracellular domain that binds to BCMA, a transmembrane domain, and an intracellular signaling domain. Engagement of the anti-BCMA antigen-binding domain of the CAR with BCMA on the surface of target cells results in clustering of the CARs, delivering activating stimuli to CAR-containing cells. A key characteristic of CARs is their ability to redirect immunoeffector cell specificity by leveraging the cell-specific targeting capabilities of monoclonal antibodies, soluble ligands, or cell-specific co-receptors, thereby triggering the production of molecules that can mediate cell death of target antigen-expressing cells in a manner independent of proliferation, cytokine production, phagocytosis, or major histocompatibility (MHC).

[0279] In various embodiments, the CAR comprises an extracellular binding domain including a mouse anti-BCMA (e.g., human BCMA) specific binding domain; a transmembrane domain; one or more intracellular costimulatory signaling domains; and a primary signaling domain.

[0280] In certain embodiments, the CAR comprises an extracellular binding domain containing a mouse anti-BCMA (e.g., human BCMA) antibody or its antigen-binding fragment; a transmembrane domain containing one or more hinge domains or spacer domains; one or more intracellular costimulatory signaling domains; and a primary signaling domain.

[0281] 6.4.1. Joining Domains In certain embodiments, the CAR contemplated herein comprises an extracellular binding domain containing a mouse anti-BCMA antibody or its antigen-binding fragment that specifically binds to a human BCMA polypeptide expressed on B cells. As used herein, the terms “binding domain,” “extracellular domain,” “extracellular binding domain,” “antigen-specific binding domain,” and “extracellular antigen-specific binding domain” are used synonymously to provide a CAR having the ability to specifically bind to a target antigen of interest, e.g., BCMA. The binding domain may be derived from natural, synthetic, semi-synthetic, or recombinant sources.

[0282] The terms “specific binding affinity,” “specifically binding,” “specifically bound,” “specifically conjugated,” “specific binding,” or “specifically targeted,” as used herein, describe the binding of an anti-BCMA antibody or its antigen-binding fragment (or a CAR containing it) to BCMA with a binding affinity greater than background binding. The binding domain (or a CAR containing the binding domain or a fusion protein containing the binding domain) is, for example, about 10 5 M -1 The above affinity or K a "Specifically binds" to BCMA when it binds to or associates with BCMA at a specific equilibrium association constant of a particular binding interaction having a unit of 1 / M (i.e., a specific binding interaction equilibrium association constant having a unit of 1 / M). In a particular embodiment, the binding domain (or its fusion protein) is approximately 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 , 10 11 M -1 , 10 12 M -1 , or 10 13 M -1 The above K a It binds to the target. The "high affinity" binding domain (or its single-strand fusion protein) is at least 10 7 M -1 , at least 108 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12 M -1 , at least 10 13 M -1 , or a larger K a This refers to those binding domains that possess [the characteristic].

[0283] Alternatively, affinity can be expressed in units of M (for example, 10 -5 M~10 -13 The equilibrium dissociation constant (K) of a specific bond interaction having M or less d ) may be defined as. The affinity of binding domain polypeptides and CAR proteins according to this disclosure can be readily determined using prior art, for example, by competitive ELISA (enzyme-linked immunosorbent assay), by binding association, by displacement assay using labeled ligands, or by surface plasmon resonance devices, e.g., the Biacore T100 available from Biacore, Inc., Piscataway, New Jersey, or optical biosensor technologies, e.g., the EPIC system or EnSpire available from Corning and Perkin Elmer, respectively (see also, e.g., Scatchard et al. (1949) Ann. NY Acad. Sci. 51:660; and U.S. Patents 5,283,173; 5,468,614, or equivalents).

[0284] In one embodiment, the affinity of the specific binding is approximately twice as great as the background binding, approximately five times greater than the background binding, approximately ten times greater than the background binding, approximately twenty times greater than the background binding, approximately fifty times greater than the background binding, approximately one hundred times greater than the background binding, or approximately one thousand times greater than the background binding, or greater than that.

[0285] In certain embodiments, the extracellular binding domain of the CAR contains an antibody or its antigen-binding fragment. "Antibody" refers to a binder which is a polypeptide containing at least a light-chain or heavy-chain immunoglobulin variable region that specifically recognizes and binds to an epitope of an antigen, such as a peptide, lipid, polysaccharide, or nucleic acid containing an antigenic determinant, for example, one recognized by immune cells.

[0286] "Antigen (Ag)" refers to a compound, composition, or substance that can stimulate antibody production or a T-cell response in an animal, and includes compositions (e.g., those containing cancer-specific proteins) that are injected or absorbed into the animal. Antigens react with products of specific humoral or cellular immunity, including heterologous antigens, e.g., those induced by the disclosed antigen. In certain embodiments, the target antigen is an epitope of a BCMA polypeptide.

[0287] An "epitope" or "antigenic determinant" refers to a region of an antigen to which a binder binds. Epitopes can be formed from both continuous amino acids or discontinuous amino acids juxtaposed by the tertiary folding of a protein. Epitopes formed from continuous amino acids are usually retained upon exposure to denaturing solvents, while epitopes formed by tertiary folding are usually lost during processing with denaturing solvents. Epitopes typically contain at least three, more commonly at least five, about nine, or about eight to ten amino acids in a distinctive spatial conformation.

[0288] Antibodies include their antigen-binding fragments, e.g., camel Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv protein ("scFv"), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide-stabilized Fv protein ("dsFv"), and single-domain antibodies (sdAb, nanobody) and the portion of a full-length antibody responsible for antigen binding. This term also includes genetically modified forms, e.g., chimeric antibodies (e.g., humanized mouse antibodies), heteroconjugate antibodies (e.g., bispecific antibodies) and their antigen-binding fragments. See also Pierce Catalog and Handbook, 1994–1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3rd Ed., WH Freeman & Co., New York, 1997.

[0289] As will be understood by those skilled in the art, and as will be described elsewhere herein, a complete antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region and first, second, and third constant regions, while each light chain consists of a variable region and a constant region. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as λ or κ. Immunoglobulins containing α, δ, ε, γ, and μ heavy chains are classified as immunoglobulin (Ig) A, IgD, IgE, IgG, and IgM. A complete antibody forms a "Y" shape. The stem of the Y consists of second and third constant regions of two heavy chains joined together (a fourth constant region for IgE and IgM), and a disulfide bond (interchain) is formed at the hinge. The heavy chains γ, α, and δ have a constant region composed of three tandem (single-row) Ig domains and a hinge region for added mobility; the heavy chains μ and ε have a constant region composed of four immunoglobulin domains. The second and third constant regions are called the "CH2 domain" and the "CH3 domain," respectively. Each arm of Y contains a variable region of a single heavy chain and a first constant region, which are coupled to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding.

[0290] The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also known as "complementarity determination regions" or "CDRs." CDRs can be defined or identified by conventional methods, for example, by sequences following Kabat et al (Wu, TT and Kabat, EA, J Exp Med. 132(2):211-50, (1970); Borden, P. and Kabat EA, PNAS, 84: 2440-2443 (1987); (see also Kabat et al., Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, 1991, incorporated herein by reference), or by structures following Chothia et al (Chothia, C. and Lesk, AM, J Mol. Biol., 196(4): 901-917 (1987), Chothia, C. et al, Nature, 342: 877-883 (1989)).

[0291] The sequences of different light chain or heavy chain framework regions are relatively conserved within a species, such as humans. The framework region of an antibody, which is the combined framework region of the light and heavy chain components, works to position and align the CDRs in three-dimensional space. CDRs are primarily responsible for binding to the epitope of the antigen. The CDRs of each chain are usually numbered sequentially starting from the N-terminus and called CDR1, CDR2, and CDR3, and are usually identified by the chain to which a particular CDR is located. Thus, CDRs located in the variable domain of the antibody's heavy chain are called CDRH1, CDRH2, and CDRH3, while CDRs located in the variable domain of the antibody's light chain are called CDRL1, CDRL2, and CDRL3. Antibodies with different specificities (i.e., different combined sites for different antigens) have different CDRs. While it is the CDRs that differ from antibody to antibody, a limited number of amino acid positions within the CDR are directly involved in antigen binding. These positions within the CDR are called specificity-determining residues (SDRs). Examples of light chain CDRs suitable for constructing the humanized BCMA CAR intended in this specification include, but are not limited to, the CDR sequences shown in SEQ ID NOs: 1-3. Examples of heavy chain CDRs suitable for constructing the humanized BCMA CAR intended in this specification include, but are not limited to, the CDR sequences shown in SEQ ID NOs: 4-6.

[0292] "V H References to "VH" or "VH" refer to the variable region of an immunoglobulin heavy chain, including those of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragments as disclosed herein. L References to "VL" or "VL" refer to the variable region of an immunoglobulin light chain, including those of an antibody, Fv, scFv, dsFv, Fab, or other antibody fragments as disclosed herein.

[0293] A "monoclonal antibody" is an antibody produced by a single clone of a B lymphocyte or by cells transfected with the light and heavy chain genes of a single antibody. Monoclonal antibodies are produced by methods known to those skilled in the art, for example, by creating hybrid antibody-forming cells from the fusion of myeloma cells with immunosplenic cells. Monoclonal antibodies include humanized monoclonal antibodies.

[0294] A "chimeric antibody" has framework residues derived from one species, for example, humans, and CDRs (generally antigen-binding residues) derived from another species, for example, mice. In certain embodiments, the CAR contemplated herein includes an antigen-specific binding domain which is the chimeric antibody or its antigen-binding fragment.

[0295] A "humanized" antibody is an immunoglobulin that contains a human framework region and one or more CDRs derived from a non-human (e.g., mouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulin that provides the CDRs is called the "donor," and the human immunoglobulin that provides the framework is called the "acceptor."

[0296] In certain embodiments, the mouse anti-BCMA (e.g., human BCMA) antibody or its antigen-binding fragment may include, but is not limited to, camel Ig (camel antibody (VHH)), Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, single-chain Fv antibody ("scFv"), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide-stabilized Fv protein ("dsFv"), and single-domain antibody (sdAb, nanobody).

[0297] As used herein, "Camel Ig" or "Camel VHH" refers to the smallest known antigen-binding unit of a heavy-chain antibody (Koch-Nolte, et al, FASEB J., 21: 3490-3498 (2007)). "Heavy-chain antibody" or "Camel antibody" refers to an antibody containing two VH domains and no light chain (Riechmann L. et al, J. Immunol. Methods 231:25 38 (1999); WO94 / 04678; WO94 / 25591; U.S. Patent No. 6,005,079).

[0298] "IgNAR," in the context of "novel immunoglobulin antigen receptors," refers to a class of antibodies derived from the shark immune repertoire consisting of homodimers of one variable novel antigen receptor (VNAR) domain and five constant novel antigen receptor (CNAR) domains. IgNARs represent the smallest known immunoglobulin-based protein scaffolds, are highly stable, and possess efficient binding characteristics. Their inherent stability may stem from both (i) the underlying Ig scaffold, which represents a significant number of charged hydrophilic surface-exposed residues compared to conventional antibody VH and VL domains found in mouse antibodies; and (ii) stabilizing structural features in the complementarity-determining region (CDR) loop, including patterns of inter-loop disulfide bridges and intra-loop hydrogen bonding.

[0299] Papain digestion of the antibody yields two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and the remaining "Fc" fragment, whose name reflects its ability to easily crystallize. Pepsin treatment yields an F(ab')2 fragment with two antigen-binding sites, which is still capable of crosslinking antigens.

[0300] "Fv" is the smallest antibody fragment containing a complete antigen-binding site. In one embodiment, a double-stranded Fv species consists of a dimer of one heavy-chain variable domain and one light-chain variable domain that are closely, non-covalently associated. In a single-stranded Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a mobile peptide linker so that the light and heavy chains can associate in a "dimer" structure similar to that in a double-stranded Fv species. It is in this configuration that the three hypervariable regions (HVRs) of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. In total, six HVRs give the antibody antigen-binding specificity. However, even a single variable domain (or half of an Fv containing only three antigen-specific HVRs) has the ability to recognize and bind to an antigen, albeit with lower affinity than the complete binding site.

[0301] The Fab fragment contains heavy and light chain variable domains, as well as the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the herein designation for Fab' fragments in which the cysteine ​​residue(s) of the constant domain have a free thiol group. The F(ab')2 antibody fragment was originally generated as a pair of Fab' fragments having a hinge cysteine ​​between them. Other chemical couplings of antibody fragments are also known.

[0302] The term "diabody" refers to an antibody fragment having two antigen-binding sites, where the fragment contains a heavy chain variable domain (VH) (VH-VL) connected to a light chain variable domain (VL) within the same polypeptide chain. By using a linker that is too short to allow pairing between two domains on the same chain, the domains are forced to pair with a complementary domain on another chain, creating two antigen-binding sites. Diabodies can be bivalent or bispecific. Diabodies are well described, for example, by EP404,097;WO1993 / 01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., PNAS USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described by Hudson et al., Nat. Med. 9:129-134 (2003).

[0303] A "single-domain antibody," "sdAb," or "nanobody" refers to an antibody fragment consisting of the variable region (VH domain) of the antibody heavy chain or the variable region (VL domain) of the antibody light chain (Holt, L., et al, 2003, Trends in Biotechnology, 21(11): 484-490).

[0304] A "single-chain Fv" or "scFv" antibody fragment contains the VH and VL domains of the antibody, which are present in either orientation (e.g., VL-VH or VH-VL) within a single polypeptide chain. Generally, scFv polypeptides further contain a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. For an overview of scFv, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp. 269-315.

[0305] In certain embodiments, the CAR contemplated herein includes an antigen-specific binding domain that is a mouse scFv. Single-chain antibodies can be cloned from the V-region gene of a hybridoma specific to the desired target. The generation of such hybrid vectors has become routine. Techniques that can be used to clone variable region heavy chains (VH) and variable region light chains (VL) are described, for example, in Orlandi et al., PNAS, 1989; 86: 3833-3837.

[0306] In certain embodiments, the antigen-specific binding domain is a mouse scFv that binds to a human BCMA polypeptide. Examples of heavy chain variable sequences suitable for constructing the BCMA CAR intended herein include, but are not limited to, the amino acid sequence shown in SEQ ID NO: 8. Examples of light chain variable sequences suitable for constructing the BCMA CAR intended herein include, but are not limited to, the amino acid sequence shown in SEQ ID NO: 7.

[0307] The BCMA-specific binding domains provided herein also comprise one, two, three, four, five, or six CDRs. Such CDRs may be non-human CDRs selected from light chain CDRL1, CDRL2, and CDRL3 and heavy chain CDRH1, CDRH2, and CDRH3, or modified non-human CDRs. In certain embodiments, the BCMA-specific binding domain comprises (a) a light chain variable region comprising light chain CDRL1, light chain CDRL2, and light chain CDRL3, and (b) a heavy chain variable region comprising heavy chain CDRH1, heavy chain CDRH2, and heavy chain CDRH3.

[0308] 6.4.2. Linker In certain embodiments, the CAR contemplated herein may include linker residues between various domains, for example, added for proper spacing and conformation of the molecule. In certain embodiments, the linker is a variable region linking sequence. The "variable region linking sequence" is configured such that the resulting polypeptide maintains specific binding affinity to the same target molecule as an antibody containing the same light and heavy chain variable regions. H and V L This is an amino acid sequence that connects domains and provides a spacer function adapted to the interaction of two subbinding domains. The CARs contemplated herein may comprise one, two, three, four, or five linkers. In certain embodiments, the linker length is about 1 to about 25 amino acids, about 5 to about 20 amino acids, or about 10 to about 20 amino acids, or any intervening length of amino acids. In some embodiments, the linker has an amino acid length of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids.

[0309] As an example of a linker, glycine polymer (G) n ;Glycine-serine polymer (G 1~5 S 1~5 ) nExamples include (wherein n is an integer of at least 1, 2, 3, 4, or 5); glycine-alanine polymers; alanine-serine polymers; and other mobile linkers known in the art. Glycine and glycine-serine polymers are relatively amorphous and therefore can potentially act as neutral tethers between domains in fusion proteins such as CARs described herein. Glycine accesses considerably more fi-psy space than even alanine and is far less restricted than residues with longer side chains (see Scheraga, Rev. Computational Chem. 11173-142 (1992)). Those skilled in the art will recognize that the design of a CAR may, in certain embodiments, include all or part of a mobile linker, and consequently the linker may include one or more parts that give the desired CAR structure a less mobile structure as well as a mobile linker.

[0310] Other exemplary linkers, though not limited to these, include the following amino acid sequences: GGG;DGGGS (SEQ ID NO: 12);TGEKP (SEQ ID NO: 13) (see, for example, Liu et al., PNAS 5525-5530 (1997));GGRR (SEQ ID NO: 14) (Pomerantz et al. 1995, op. cit.);(GGGGS) n(In the formula, n=1, 2, 3, 4 or 5, GGGGS is identified as SEQ ID NO: 15 (Kim et al., PNAS 93, 1156-1160 (1996); EGKSSGSGSESKVD (SEQ ID NO: 16) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87:1066-1070); KESGSVSSEQLAQFRSLD (SEQ ID NO: 17) (Bird et al., 1988, Science 242:423-426), GGRRGGGS (SEQ ID NO: 18); LRQRDGERP (SEQ ID NO: 19); LRQKDGGGSERP (SEQ ID NO: 20); LRQKd(GGGS)2ERP (SEQ ID NO: 21). Alternatively, the mobile linker is identified using a computer program capable of modeling both the DNA binding site and the peptide itself (Desjarlais & Berg, PNAS) It can be rationally designed by 90:2256-2260 (1993), PNAS 91:11099-11103 (1994), or by phage display. In one embodiment, the linker contains the following amino acid sequence: GSTSGSGKPGSGEGSTKG (SEQ ID NO: 22) (Cooper et al., Blood, 101(4): 1637-1644 (2003)).

[0311] 6.4.3. Spacer Domain In certain embodiments, the CAR binding domain is followed by one or more “spacer domains,” which refer to regions that detach the antigen-binding domain from the effector cell surface, enabling proper cell / cell contact, antigen binding, and activation. (Patel et al., Gene Therapy, 1999; 6: 412-419). The spacer domains may originate from natural, synthetic, semi-synthetic, or recombinant sources. In certain embodiments, the spacer domains may include, but are not limited to, one or more heavy chain constant regions, e.g., portions of immunoglobulin containing CH2 and CH3. The spacer domains may include amino acid sequences of naturally occurring or modified immunoglobulin hinge regions.

[0312] In one embodiment, the spacer domain includes the CH2 and CH3 domains of IgG1 or IgG4.

[0313] 6.4.4. Hinged Domain The binding domain of a CAR is generally followed by one or more "hinge domains," which play a role in positioning the antigen-binding domain away from the effector cell surface to enable proper cell / cell contact, antigen binding, and activation. CARs generally contain one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domains may originate from natural, synthetic, semi-synthetic, or recombinant sources. The hinge domains may contain amino acid sequences from naturally occurring immunoglobulin hinge regions or modified immunoglobulin hinge regions.

[0314] "Modified hinge region" means (a) a naturally occurring hinge region having up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions), (b) a portion of a naturally occurring hinge region having up to 30% amino acid changes (e.g., up to 25%, 20%, 15%, 10%, or 5% amino acid substitutions or deletions) and being at least 10 amino acids long (e.g., at least 12, 13, 14, or 15 amino acids), or (c) a portion of a naturally occurring hinge region containing a core hinge region (which may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids long). In certain embodiments, one or more cysteine ​​residues in the naturally occurring immunoglobulin hinge region may be replaced by one or more other amino acid residues (e.g., one or more serine residues). The modified immunoglobulin hinge region may, or further, have a proline residue in the wild-type immunoglobulin hinge region that has been replaced by another amino acid residue (e.g., a serine residue).

[0315] Other exemplary hinge domains suitable for use in the CARs described herein include hinge domains derived from the extracellular regions of type 1 membrane proteins, such as CD8α, CD4, CD28, and CD7, which may be wild-type hinge domains from these molecules or may be modified. In another embodiment, the hinge domain includes the CD8α hinge domain.

[0316] 6.4.5. Transmembrane domains The transmembrane (TM) domain is the portion of the CAR that fuses the extracellular binding portion with the intracellular signaling domain, tethering the CAR to the cell membrane of immunoeffector cells. The TM domain may originate from a native, synthetic, semi-synthetic, or recombinant source. The TM domain may originate from the alpha, beta, or zeta chains of T cell receptors, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and PD-1 (i.e., it may include at least its transmembrane region). In certain embodiments, the TM domain is synthetic and predominantly contains hydrophobic residues, such as leucine and valine.

[0317] In one embodiment, the CAR contemplated herein comprises a TM domain derived from CD8α. In another embodiment, the CAR contemplated herein comprises a TM domain derived from CD8α and a short oligo or polypeptide linker, preferably having an amino acid length between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, that links the TM domain of the CAR to an intracellular signaling domain. Glycine-serine-based linkers provide particularly suitable linkers.

[0318] 6.4.6. Intracellular signaling domains In certain embodiments, the CARs contemplated herein include an intracellular signaling domain. The “intracellular signaling domain” refers to the portion of the CAR that is involved in inducing effector cell functions, such as activation, cytokine production, proliferation, and cytotoxic activity, including the transmission of a message from an effective BCMA CAR that binds to a human BCMA polypeptide into the interior of an immune effector cell, and the release of cytotoxic factors to the CAR-bound target cell or other cellular responses induced by antigen binding to the extracellular CAR domain.

[0319] The term "effector function" refers to the specialized function of immune effector cells. The effector function of T cells may include, for example, cytolytic activity or helper activity, including cytokine secretion. Therefore, the term "intracellular signaling domain" refers to the portion of a protein that transmits effector function signals, instructing cells to perform specialized functions. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire domain. To the extent that a cleaved portion of an intracellular signaling domain is used, such a cleaved portion can be used in place of the entire domain, as long as it transmits effector function signals. The term "intracellular signaling domain" shall include any cleaved portion of an intracellular signaling domain sufficient to transmit effector function signals.

[0320] It is known that signals generated by TCRs alone are insufficient for complete T cell activation, and that secondary or co-stimulatory signals are also required. Therefore, T cell activation is sometimes said to be mediated by two distinct classes of intracellular signaling domains: a primary signaling domain that initiates antigen-dependent primary activation by a TCR (e.g., the TCR / CD3 complex) and a co-stimulatory signaling domain that acts antigen-independently to provide secondary or co-stimulatory signals. In certain embodiments, the CAR contemplated herein comprises an intracellular signaling domain including one or more "co-stimulatory signaling domains" and "primary signaling domains."

[0321] Primary signaling domains modulate the primary activation of the TCR complex either stimulatingly or inhibitorily. Primary signaling domains acting stimulatingly may contain an immunoreceptor tyrosine-based activation motif or a signaling motif known as ITAM.

[0322] Examples of ITAMs containing primary signaling domains particularly used in the subject matter presented herein include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d. In certain embodiments, the CAR comprises the CD3ζ primary signaling domain and one or more co-stimulatory signaling domains. The intracellular primary signaling and co-stimulatory signaling domains may be tandem-linked to the carboxyl terminus of the transmembrane domain in any order.

[0323] The CARs contemplated herein include one or more costimulatory signaling domains that enhance the efficacy and proliferation of T cells expressing CAR receptors. As used herein, the terms “costimulatory signaling domain” or “costimulatory domain” refer to the intracellular signaling domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or Fc receptor that provides efficient activation upon binding to an antigen and a secondary signal necessary for T lymphocyte function. Examples of such co-stimulatory molecules include CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In one embodiment, CAR includes one or more co-stimulatory signaling domains selected from the group consisting of CD28, CD137, and CD134, and a CD3ζ primary signaling domain.

[0324] In another embodiment, the CAR includes CD28 and CD137 co-stimulatory signaling domains and a CD3ζ primary signaling domain.

[0325] In yet another embodiment, the CAR includes CD28 and CD134 co-stimulatory signaling domains and a CD3ζ primary signaling domain.

[0326] In one embodiment, the CAR includes CD137 and CD134 co-stimulatory signaling domains and a CD3ζ primary signaling domain.

[0327] In certain embodiments, the CAR contemplated herein comprises a BCMA polypeptide expressed on B cells, for example, a human anti-BCMA antibody or its antigen-binding fragment that specifically binds to human BCMA expressed on human B cells.

[0328] In certain embodiments, the CAR contemplated herein comprises a BCMA polypeptide expressed on B cells, for example, a mouse anti-BCMA antibody or its antigen-binding fragment that specifically binds to human BCMA expressed on human B cells.

[0329] In one embodiment, CAR is a transmembrane domain derived from a polypeptide selected from the group consisting of BCMA polypeptides, e.g., mouse anti-BCMA scFv that binds to human BCMA polypeptide; T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and the alpha, beta, or zeta chains of PD1; and CARD1 1. One or more intracellular co-stimulatory signaling domains from co-stimulatory molecules selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70; and primary signaling domains from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0330] In one embodiment, CAR is a transmembrane domain derived from a polypeptide selected from the group consisting of BCMA polypeptides, e.g., mouse anti-BCMA scFv that binds to human BCMA polypeptide; T cell receptor, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and the alpha, beta, or zeta chains of PD1; and CARD1 1. A costimulatory signaling domain from one or more intracellular costimulatory signaling domains selected from the group consisting of CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70; and one or more primary signaling domains from polypeptides selected from the group consisting of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0331] In one embodiment, the CAR is a hinge domain selected from the group consisting of BCMA polypeptides, e.g., mouse anti-BCMA scFv that binds to human BCMA polypeptide, IgG1 hinge / CH2 / CH3, IgG4 hinge / CH2 / CH3, and CD8α hinge; T cell receptors, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD Transmembrane domains derived from polypeptides selected from the group consisting of 134, CD137, CD152, CD154, and the alpha, beta, or zeta chains of PD1; as well as CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C One or more intracellular costimulatory signaling domains from costimulatory molecules selected from the group consisting of SLP76, TRIM, and ZAP70; and primary signaling domains from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0332] In one embodiment, the CAR is selected from the group consisting of BCMA polypeptides, e.g., mouse anti-BCMA scFv that binds to human BCMA polypeptide; hinge domains selected from the group consisting of IgG1 hinge / CH2 / CH3, IgG4 hinge / CH2 / CH3, and CD8α hinge; and T cell receptors, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and alpha, beta, or zeta chains of PD1. Transmembrane domains derived from polypeptides; as well as CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C The system comprises one or more intracellular costimulatory signaling domains from costimulatory molecules selected from the group consisting of SLP76, TRIM, and ZAP70; and one or more primary signaling domains from polypeptides selected from the group consisting of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0333] In one embodiment, CAR comprises a hinge domain selected from the group consisting of BCMA polypeptides, e.g., mouse anti-BCMA scFv that binds to human BCMA polypeptide; IgG1 hinge / CH2 / CH3, IgG4 hinge / CH2 / CH3, and CD8α hinge; a transmembrane domain derived from a polypeptide selected from the group consisting of T cell receptors, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and the alpha, beta, or zeta chains of PD1; and the TM domain of CAR and intracellular signaling domain. Short oligo or polypeptide linkers, preferably with an amino acid length between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, link the amino acids; and CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C One or more intracellular costimulatory signaling domains from costimulatory molecules selected from the group consisting of SLP76, TRIM, and ZAP70; and primary signaling domains from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0334] In one embodiment, CAR comprises a hinge domain selected from the group consisting of BCMA polypeptides, e.g., mouse anti-BCMA scFv that binds to human BCMA polypeptide; IgG1 hinge / CH2 / CH3, IgG4 hinge / CH2 / CH3, and CD8α hinge; a transmembrane domain derived from a polypeptide selected from the group consisting of T cell receptors, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and the alpha, beta, or zeta chains of PD1; and the TM domain of CAR and intracellular signaling domain. Short oligo or polypeptide linkers, preferably with an amino acid length between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, link the amino acids; and CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), CD223 (LAG3), CD270 (HVEM), CD273 (PD-L2), CD274 (PD-L1), CD278 (ICOS), DAP10, LAT, NKD2C The system comprises one or more intracellular costimulatory signaling domains from costimulatory molecules selected from the group consisting of SLP76, TRIM, and ZAP70; and one or more primary signaling domains from polypeptides selected from the group consisting of TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d.

[0335] In certain embodiments, the CAR comprises a BCMA polypeptide, e.g., mouse anti-BCMA scFv that binds to human BCMA polypeptide; a hinge domain containing an IgG1 hinge / CH2 / CH3 polypeptide and a CD8α polypeptide; a CD8α transmembrane domain containing a polypeptide linker of about 3 to about 10 amino acids; a CD137 intracellular costimulatory signaling domain; and a CD3ζ primary signaling domain.

[0336] In certain embodiments, the CAR comprises a hinge domain containing a BCMA polypeptide, e.g., mouse anti-BCMA scFv that binds to a human BCMA polypeptide; a CD8α polypeptide; a CD8α transmembrane domain containing a polypeptide linker of about 3 to about 10 amino acids; a CD134 intracellular costimulatory signaling domain; and a CD3ζ primary signaling domain.

[0337] In certain embodiments, the CAR comprises a hinge domain containing a BCMA polypeptide, e.g., mouse anti-BCMA scFv that binds to a human BCMA polypeptide; a CD8α polypeptide; a CD8α transmembrane domain containing a polypeptide linker of about 3 to about 10 amino acids; a CD28 intracellular costimulatory signaling domain; and a CD3ζ primary signaling domain.

[0338] In certain embodiments, the CAR comprises a hinge domain containing a BCMA polypeptide, e.g., a mouse anti-BCMA scFv that binds to a human BCMA polypeptide; a CD8α polypeptide; a CD8α transmembrane domain; a CD137(4-1BB) intracellular costimulatory signaling domain; and a CD3ζ primary signaling domain.

[0339] Furthermore, the CAR designs envisioned herein enable improved proliferation, long-term persistence, and acceptable cytotoxic properties in CAR-expressing T cells compared to unmodified T cells or T cells modified to express other CARs.

[0340] 6.5. Polypeptides This disclosure intends, to some extent, to CAR polypeptides and their fragments, cells and compositions comprising them, and vectors expressing polypeptides. In certain embodiments, polypeptides comprising one or more CARs, such as those shown in SEQ ID NO: 9, are provided.

[0341] "Polypeptide," "polypeptide fragment," "peptide," and "protein" are used synonymously according to their conventional meanings, i.e., as sequences of amino acids, unless otherwise specified. Polypeptides are not limited to a specific length; for example, they may include full-length protein sequences or fragments of full-length proteins, and may include post-translational modifications of polypeptides, e.g., glycosylation, acetylation, phosphorylation, etc., as well as other modifications known in the art, both naturally occurring and non-naturally occurring. In various embodiments, the CAR polypeptides contemplated herein include a signal (or leader) sequence at the N-terminus of the protein, which directs the movement of the protein, either concurrently or post-translationally. Examples of suitable signal sequences useful in the CARs disclosed herein, but not limited to, include the IgG1 heavy chain signal sequence and the CD8α signal sequence. Provided herein are proteins that are mature proteins, i.e., they do not contain signal peptides. For example, a CAR may be a mature CAR. Polypeptides can be prepared using any of a variety of well-known recombinant and / or synthetic techniques. Polypeptides contemplated herein specifically include CARs or sequences of this disclosure having deletions, additions, and / or substitutions of one or more amino acids of a CAR as disclosed herein.

[0342] When used herein, "isolated peptide" or "isolated polypeptide," etc., refers to the in vitro isolation and / or purification of a peptide or polypeptide molecule from the cellular environment and in relation to other components of the cell, i.e., not substantially related to the in vivo substance. Similarly, "isolated cell" refers to a cell obtained from an in vivo tissue or organ that is substantially free of extracellular matrix.

[0343] Polypeptides include "polypeptide variants." Polypeptide variants may differ from naturally occurring polypeptides by one or more substitutions, deletions, additions, and / or insertions. Such variants may occur naturally or may be produced synthetically, for example, by modifying one or more of the polypeptide sequences described above. For example, in certain embodiments, it may be desirable to improve the binding affinity and / or other biological properties of a CAR polypeptide by introducing one or more substitutions, deletions, additions, and / or insertions into the binding domain, hinge, TM domain, co-stimulatory signaling domain, or primary signaling domain of the CAR polypeptide. In certain embodiments, such polypeptides include polypeptides having at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% amino acid identity to it.

[0344] Polypeptides include "polypeptide fragments." Polypeptide fragments refer to polypeptides that may be monomers or polymers having amino-terminal deletions, carboxyl-terminal deletions, and / or internal deletions or substitutions of naturally occurring or recombinant polypeptides. In certain embodiments, a polypeptide fragment may contain amino acid chains of at least 5 to about 500 amino acids in length. In certain embodiments, a fragment may contain at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, It can be observed that the amino acid lengths are 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 150, 200, 250, 300, 350, 400, or 450. Particularly useful polypeptide fragments include functional domains containing the antigen-binding domain or fragment of the antibody. In the case of mouse anti-BCMA (e.g., human BCMA) antibodies, useful fragments include, but are not limited to, the CDR region of the heavy or light chain, the CDR3 region; the variable region of the heavy or light chain; and a portion of the antibody chain or variable region containing two CDRs.

[0345] Polypeptides can also be fused or conjugated in-frame to linkers, or to other sequences (e.g., polyHis) to facilitate the synthesis, purification, or identification of polypeptides, or to enhance the binding of polypeptides to solid supports.

[0346] As described above, the polypeptides of this disclosure can be modified in various ways, including amino acid substitution, deletion, terminal cleavage, and insertion. Methods of such operations are generally known in the art. For example, amino acid sequence variants of a reference polypeptide can be prepared by mutation in DNA. Methods of mutagenicity and nucleotide sequence modification are well known in the art. See, for example, Kunkel (1985, Proc. Natl. Acad. Sci. USA. 82: 488-492), Kunkel et al., (1987, Methods in Enzymol, 154: 367-382), U.S. Patent No. 4,873,192, Watson, JD et al., (Molecular Biology of the Gene, Fourth Edition, Benjamin / Cummings, Menlo Park, Calif., 1987) and the references cited therein. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the target protein can be found in the model in Dayhoff et al., (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC).

[0347] In certain embodiments, the variant contains a conservative substitution. A “conservative substitution” is one in which an amino acid is substituted with another amino acid having similar properties, such that a person skilled in the art of peptide chemistry would predict that the secondary structure and hydropathic properties of the polypeptide would not be substantially altered. Modifications can be made in the structures of the polynucleotides and polypeptides of this disclosure, and functional molecules encoding variant or derivative polypeptides with desired characteristics can still be obtained. If it is desirable to modify the amino acid sequence of the polypeptide to produce an equivalent, or even an improved variant polypeptide, a person skilled in the art can, for example, change one or more codons encoding the DNA sequences according to Table 2.

[0348] Table 2 - Amino Acid Codons [Table 2]

[0349] Guidance in determining which amino acid residues can be substituted, inserted, or deleted without loss of biological activity can be found using computer programs well known in the art, such as DNASTAR® software. Preferably, the amino acid changes in the protein variants disclosed herein are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of those of a related family of amino acids in their side chains. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In peptides or proteins, suitable conservative amino acid substitutions are known to those skilled in the art and can generally be made without altering the biological activity of the resulting molecule. Those skilled in the art generally recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially alter its biological activity (see, for example, Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, The Benjamin / Cummings Pub. Co., p.224). An example of a conservative substitution is described in U.S. Provisional Patent Application No. 61 / 241,647, the disclosure of which is incorporated herein by reference.

[0350] In making such changes, the hydropathic index of amino acids may be considered. The importance of hydropathic amino acid indices in conferring interactive biological functions to proteins is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). Each amino acid is assigned a hydropathic index based on its hydrophobic and charged characteristics (Kyte and Doolittle, 1982). These values ​​are as follows: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamic acid (-3.5); glutamine (-3.5); aspartic acid (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

[0351] It is known in the art that certain amino acids may be substituted with other amino acids having a similar hydropathic index or score, and that this still results in a protein with similar biological activity, i.e., a biologically functionally equivalent protein is still obtained. In making such changes, substitutions of amino acids with a hydropathic index of ±2 are preferred, those within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred. It is also understood in the art that similar amino acid substitutions can be effectively carried out based on hydrophilicity.

[0352] As detailed in U.S. Patent No. 4,554,101, the following hydrophilic values ​​are assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid (+3.0±1); glutamic acid (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). It is understood that amino acids may be substituted with others having similar hydrophilic values, and that biologically equivalent, and in particular immunologically equivalent, proteins may still be obtained. In such changes, substitutions of amino acids with a hydrophilicity value of ±2 or less are preferred, those with a value of ±1 or less are particularly preferred, and those with a value of ±0.5 or less are even more particularly preferred.

[0353] As outlined above, amino acid substitutions may be based on the relative similarities of amino acid side chain substituents, such as their hydrophobicity, hydrophilicity, charge, and size.

[0354] Polypeptide variants further include glycosylated forms, aggregated conjugates with other molecules, and covalent conjugates with unrelated chemical moieties (e.g., pegylated molecules). As is well known in the art, covalent variants can be prepared by linking functional groups to groups found in amino acid chains or at N- or C-terminal residues. Variants also include allelic variants, species variants, and mutains. Terminal breaks or deletions of regions that do not affect the functional activity of a protein are also variants.

[0355] In one embodiment where the expression of two or more polypeptides is desired, the encoding polynucleotide sequence can be separated by an IRES sequence, as discussed elsewhere herein. In another embodiment, the two or more polypeptides may be expressed as a fusion protein containing one or more self-cleaving polypeptide sequences.

[0356] Polypeptides disclosed herein include fusion polypeptides. In certain embodiments, fusion polypeptides and polynucleotides encoding fusion polypeptides, such as CARs, are provided. Fusion polypeptides and fusion proteins refer to polypeptides having at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more polypeptide segments. Fusion polypeptides are typically linked C-terminus to N-terminus, but may be linked C-terminus to C-terminus, N-terminus to N-terminus, or N-terminus to C-terminus. The polypeptides of a fusion protein may be in any order or a specific order. Fusion polypeptides or fusion proteins may also include conservatively modified variants, polymorphic variants, alleles, mutants, subsequences, and interspecies homologs, as long as the desired transcriptional activity of the fusion polypeptide is conserved. Fusion polypeptides may be produced by chemical synthesis or by chemical linkage between two parts, or may be prepared using other standard techniques. As discussed elsewhere herein, a ligated DNA sequence containing a fusion polypeptide is operably linked to a suitable transcriptional or translational regulatory element.

[0357] In one embodiment, the fusion partner includes a sequence (expression enhancer) that assists in the expression of the protein in a higher yield than the naturally occurring recombinant protein. Other fusion partners may be selected to increase the solubility of the protein, to enable the protein to be targeted to a desired intracellular compartment, or to facilitate the transport of the fusion protein across the cell membrane.

[0358] The fusion polypeptide may further contain polypeptide cleavage signals between each of the polypeptide domains described herein. Furthermore, polypeptide moieties can be placed within any linker peptide sequence. Examples of polypeptide cleavage signals include polypeptide cleavage recognition sites, e.g., protease cleavage sites, nuclease cleavage sites (e.g., rare restriction enzyme recognition sites, autocleavable ribozyme recognition sites), and autocleavable viral oligopeptides (see deFelipe and Ryan, 2004. Traffic, 5(8); 616-26).

[0359] Suitable protease cleavage sites and self-cleaving peptides are known to those skilled in the art (see, for example, Ryan et al., 1997. J. Gener. Virol. 78, 699-722; Scymczak et al. (2004) Nature Biotech. 5, 589-594). Examples of protease cleavage sites include, but are not limited to, those of potivirus NIa protease (e.g., tobacco etch virus protease), potivirus HC protease, potivirus P1 (P35) protease, biovirus NIa protease, proteases encoded by biovirus RNA-2, aftovirus L protease, enterovirus 2A protease, rhinovirus 2A protease, picorna 3C protease, comovirus 24K protease, nepovirus 24K protease, RTSV (rice tungro spherical virus) 3C-like protease, PYVF (parsnip macular virus) 3C-like protease, heparin, thrombin, factor X, and enterokinase. Due to its high cleavage stringency, the TEV (tobacco etch virus) protease cleavage site is preferred. In one embodiment, for example, EXXYXQ(G / S) (SEQ ID NO: 23), for example, ENLYFQG (SEQ ID NO: 24) and ENLYFQS (SEQ ID NO: 25) (wherein X represents any amino acid) (cleavage by TEV occurs between Q and G or between Q and S).

[0360] In certain embodiments, the self-cleaving peptides include polypeptide sequences obtained from potivirus and cardiovirus 2A peptide, FMDV (foot-and-mouth disease virus), equine rhinitis A virus, Thosea asigna virus, and porcine teeshou virus.

[0361] In certain embodiments, the self-cleaving polypeptide site includes a 2A or 2A-like site, sequence, or domain (Donnelly et al., 2001. J. Gen. Virol. 82:1027-1041).

[0362] Table 3: Exemplary site 2A includes the following sequence: [Table 3]

[0363] In certain embodiments, the polypeptides intended herein include CAR polypeptides.

[0364] 6.6. Polynucleotides In certain embodiments, polynucleotides encoding one or more CAR polypeptides, e.g., Sequence ID No. 10, are provided. As used herein, the terms “polynucleotide” or “nucleic acid” refer to messenger RNA (mRNA), RNA, genomic RNA (gRNA), positive RNA (RNA(+)), negative RNA (RNA(-)), genomic DNA (gDNA), complementary DNA (cDNA), or recombinant DNA. Polynucleotides include single-stranded and double-stranded polynucleotides. Preferably, the polynucleotides disclosed herein include polynucleotides or variants having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein (e.g., see Sequence Listings), the variant typically retaining at least one biological activity of the reference sequence. In various exemplary embodiments, this disclosure partially intends to describe polynucleotides comprising expression vectors, viral vectors, and transfer plasmids, as well as compositions and cells comprising them.

[0365] In certain embodiments, polynucleotides encoding all intermediate-length consecutive amino acid residues are provided by this disclosure, in addition to at least about 5, 10, 25, 50, 100, 150, 200, 250, 300, 350, 400, 500, 1000, 1250, 1500, 1750, or 2000 or more consecutive amino acid residues of a polypeptide. It will be readily understood in this context that “intermediate length” means any length between the values ​​described, e.g., 6, 7, 8, 9, etc.; 101, 102, 103, etc.; 151, 152, 153, etc.; 201, 202, 203, etc.

[0366] As used herein, terms such as “polynucleotide variant” and “variant” refer to a polynucleotide that exhibits substantial sequence identity with a reference polynucleotide sequence or a polynucleotide that hybridizes with the reference sequence under stringent conditions as defined below. These terms include polynucleotides in which one or more nucleotides are added or deleted or replaced with different nucleotides compared to the reference polynucleotide. In this regard, it is well understood in the art that certain modifications, including mutations, additions, deletions, and substitutions, can be made to a reference polynucleotide so that the modified polynucleotide retains the biological function or activity of the reference polynucleotide.

[0367] When used herein, “sequence identity” or, for example, “sequences that are 50% identical to ~” refers to the degree to which sequences are identical across a comparison window, either nucleotide-wise or amino acid-wise. Thus, “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions in both sequences where identical nucleic acid bases (e.g., A, T, C, G, I) or identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) are present, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to obtain the percentage of sequence identity. The nucleotides and polypeptides described herein have at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any of the reference sequences described herein, and typically the polypeptide variant maintains at least one biological activity of the reference polypeptide.

[0368] Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence,” “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity.” A “reference sequence” is the length of a monomer unit containing at least 12, but frequently 15-18, and often at least 25, nucleotides and amino acid residues. Since each of two polynucleotides may contain (1) similar sequences between the two polynucleotides (i.e., only portions of the complete polynucleotide sequence) and (2) different sequences between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing the sequences of the two polynucleotides across a “comparison window” to identify and compare local regions of sequence similarity. The “comparison window” refers to a conceptual segment of at least six, typically about 50 to 100, and more typically about 100 to 150, consecutive positions within which the sequences are compared after the two sequences have been optimally aligned with a reference sequence at the same number of consecutive positions. The comparison window may contain approximately 20% or less of additions or deletions (i.e., gaps) compared to the reference sequence (which does not include additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning the comparison window may be performed by computer implementation of the algorithm (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by the best alignment (i.e., the one that yields the highest percentage of homology across the comparison window) generated by any of the various methods of inspection and selection. For example, the program of the BLAST family disclosed by Altschul et al., 1997, Nucl. Acids Res. 25:3389 may also be referenced.A detailed discussion of sequence analysis can be found in Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc, 1994-1998, Chapter 15, Unit 19.3.

[0369] As used herein, “isolated polynucleotide” refers to a polynucleotide that has been purified from sequences adjacent to it in its naturally occurring state, for example, a DNA fragment that has been removed from sequences normally adjacent to it. “Isolated polynucleotide” also refers to complementary DNA (cDNA), recombinant DNA, or other polynucleotides that do not exist in nature but are created by humans.

[0370] The terminology used to describe the orientation of polynucleotides includes the 5' (usually the end of the polynucleotide with a free phosphate group) and the 3' (usually the end of the polynucleotide with a free hydroxyl (OH) group). Polynucleotide sequences can be annotated in either the 5'-to-3' or 3'-to-5' orientation. For DNA and mRNA, the 5'-to-3' strand is referred to as the “sense,” “plus,” or “coding” strand because its sequence is identical to the pre-messenger (premRNA) sequence [except for uracil (U) in RNA instead of thymine (T) in DNA]. For DNA and mRNA, the complementary 3'-to-5' strand, which is the strand transcribed by RNA polymerase, is referred to as the “template,” “antisense,” “minus,” or “non-coding” strand. As used herein, the term “reverse orientation” refers to a 5'-to-3' sequence written in the 3'-to-5' orientation or a 3'-to-5' sequence written in the 5'-to-3' orientation.

[0371] The terms "complementary" and "complementarity" refer to polynucleotides (i.e., sequences of nucleotides) that are related by base pairing rules. For example, the complementary strand of the DNA sequence 5' AGTCATG 3' is 3' TCAGTAC 5'. The latter sequence is often written as a reverse complementary strand, 5' CATGACT 3', with a 5' end on the left and a 3' end on the right. A sequence equal to that reverse complementary strand is called a palindrome sequence. Complementarity can be "partial," in which case only some of the bases of the nucleic acid match according to base pairing rules. Alternatively, there can be "complete" or "total" complementarity between nucleic acids.

[0372] Furthermore, as a result of the degeneracy of the genetic code, it will be understood by those skilled in the art that there are many nucleotide sequences that encode polypeptides, or fragments or variants thereof, as described herein. Some of these polynucleotides have minimal homology to the nucleotide sequences of any native gene. Nevertheless, polynucleotides that vary due to differences in codon usage are particularly intended by this disclosure, which are, for example, polynucleotides optimized for human and / or primate codon selection. Furthermore, alleles of genes containing the polynucleotide sequences provided herein may also be used. An allele is an endogenous gene that has been altered as a result of one or more mutations, such as nucleotide deletions, additions, and / or substitutions.

[0373] The term “nucleic acid cassette,” as used herein, refers to a gene sequence in a vector capable of expressing RNA and subsequently a protein. A nucleic acid cassette contains the gene of interest, e.g., a CAR. The nucleic acid cassette is locologically and sequencely oriented within the vector so that the nucleic acid in the cassette is transcribed into RNA, and, if necessary, translated into a protein or polypeptide, undergoes appropriate post-translational modifications required for activity in transformed cells, and can be moved to a suitable compartment for biological activity by targeting to a suitable intracellular compartment or secretion to an extracellular compartment. Preferably, the cassette has its 3' and 5' ends adapted for ready insertion into the vector, for example, having restriction endonuclease sites at each end. In one embodiment, the nucleic acid cassette contains a sequence of a chimeric antigen receptor used to treat a tumor or cancer. In one embodiment, the nucleic acid cassette contains a sequence of a chimeric antigen receptor used to treat a B-cell malignancy. The cassette can be removed and inserted as a single unit into a plasmid or viral vector.

[0374] In certain embodiments, the polynucleotide comprises at least one polynucleotide of interest. As used herein, the term “polynucleotide of interest” refers to a polynucleotide encoding a polypeptide (i.e., the polypeptide of interest) inserted into an expression vector in which expression is desired. The vector may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 polynucleotides of interest. In certain embodiments, the polynucleotide of interest encodes a polypeptide that provides a therapeutic effect in the treatment or prevention of a disease or disorder. The polynucleotide of interest, and the polypeptide encoded therein, comprises both a polynucleotide encoding a wild-type polypeptide and a polynucleotide encoding its functional variant and fragment. In certain embodiments, the functional variant has at least 80%, at least 90%, at least 95%, or at least 99% identity to the corresponding wild-type reference polynucleotide or polypeptide sequence. In certain embodiments, the functional variant or fragment has at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the biological activity of the corresponding wild-type polypeptide.

[0375] In one embodiment, the polynucleotide of interest does not encode a polypeptide but serves as a template for transcribing miRNA, siRNA, or shRNA, ribozyme, or other inhibitory RNA. In various other embodiments, the polynucleotide of interest comprises one or more additional polynucleotides of interest that encode inhibitory nucleic acid sequences, including but not limited to CARs, siRNA, miRNA, shRNA, and ribozyme.

[0376] As used herein, the terms “siRNA” or “short interfering RNA” refer to short polynucleotide sequences that mediate sequence-specific post-transcriptional gene silencing, translation inhibition, transcription inhibition, or epigenetic RNAi processes in animals (Zamore et al., 2000, Cell, 101, 25-33; Fire et al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286, 950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, Genes & Dev., 13, 139-141; and Strauss, 1999, Science, 286, 886). In certain embodiments, the siRNA comprises a first and second strand having the same number of nucleosides; however, the first and second strands are offset so that two terminal nucleosides on the first and second strands do not pair with residues on the complementary strand. In certain cases, the two unpaired nucleosides are thymidine residues. The siRNA should contain a region of sufficient homology to the target gene and be of sufficient length in terms of nucleotides, so that the siRNA, or a fragment thereof, can mediate downregulation of the target gene. Therefore, the siRNA contains a region that is at least partially complementary to the target RNA. Perfect complementarity between the siRNA and the target is not required, but the correspondence must be sufficient to allow the siRNA, or its cleavage product, to be directed toward sequence-specific silencing, such as by RNAi cleavage of the target RNA. The degree of complementarity, or homology to the target strand, is most essential in the antisense strand. Perfect complementarity is often desired, particularly in the antisense strand, but some embodiments include one or more, preferably 10, 8, 6, 5, 4, 3, 2, or fewer, mismatches with respect to the target RNA. Mismatches are most acceptable in terminal regions, and if present, are preferably within one or more terminal regions, for example, within 6, 5, 4, or 3 nucleotides of the 5' and / or 3' ends.The sense strand only needs to be sufficiently complementary to the antisense strand to maintain the overall double-stranded characteristics of the molecule.

[0377] Additionally, the siRNA may be modified or contain a nucleoside analog. The single-stranded region of the siRNA may be modified or contain a nucleoside analog; for example, one or more unpaired regions of a hairpin structure, such as a region linking two complementary regions, may have modifications or a nucleoside analog. Modifications are also useful, for example, to stabilize one or more 3' or 5' ends of the siRNA against an exonuclease, or to facilitate the entry of an antisense siRNA agent into the RISC. Modifications may include C3 (or C6, C7, C12) aminolinkers, thiol linkers, carboxyl linkers, non-nucleotide spacers (C3, C6, C9, C12, debase, triethylene glycol, hexaethylene glycol), phosphoramidites, and special biotin or fluorescein reagents having another DMT-protected hydroxyl group that allows for multiple couplings during RNA synthesis. Each strand of siRNA may have a length equal to or less than 30, 25, 24, 23, 22, 21, or 20 nucleotides. The strands are preferably at least 19 nucleotides long. For example, each strand may be 21 to 25 nucleotides long. Preferred siRNAs have a double-stranded region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more overhangs of 2 to 3 nucleotides, preferably one or two 3' overhangs of 2 to 3 nucleotides.

[0378] As used herein, the terms “miRNA” or “microRNA” refer to small non-coding RNAs of 20–22 nucleotides, typically excised from a folded RNA precursor structure of approximately 70 nucleotides known as pre-miRNA. miRNAs negatively modulate their targets in one of two ways, depending on the degree of complementarity between the miRNA and the target. Firstly, miRNAs that bind to protein-coding mRNA sequences with perfect or near-perfect complementarity induce the RNA-mediated interference (RNAi) pathway. miRNAs that exert their regulatory effect by binding to an imperfectly complementary site within the 3' untranslated region (UTR) of their mRNA targets repress target gene expression post-transcriptionally, apparently at the translational level, through a RISC complex similar to, or sometimes identical to, that used for the RNAi pathway. Consistent with translational regulation, miRNAs using this mechanism reduce the protein levels of their target genes, but the mRNA levels of these genes are minimally affected. miRNAs encompass both naturally occurring miRNAs and artificially designed miRNAs that can specifically target any mRNA sequence. For example, in one embodiment, those skilled in the art can design a short hairpin RNA construct expressed as a primary transcript of a human miRNA (e.g., miR-30 or miR-21). This design has been shown to significantly increase knockdown efficiency by adding a Drosha processing site to the hairpin construct (Pusch et al., 2004). The hairpin stem consists of a 22-nt dsRNA (e.g., an antisense that has perfect complementarity to the desired target) and a 15-19-nt loop from a human miR. Addition of the miR loop and miR30 flanking sequence on either or both sides of the hairpin results in a more than 10-fold increase in Drosha and Dicer processing of the expressed hairpin compared to a conventional shRNA design without microRNA. Increased Drosha and Dicer processing translates to higher siRNA / miRNA production and greater potency for expressed hairpins.

[0379] As used herein, the terms “shRNA” or “short hairpin RNA” refer to a double-stranded structure formed by a single self-complementary RNA strand. ShRNA constructs containing a nucleotide sequence identical to a portion of the target gene, either a coding or non-coding sequence, are preferred for inhibition. RNA sequences having insertions, deletions, and single point mutations compared to the target sequence have also been found to be effective for inhibition. Sequence identity higher than 90%, or even 100%, between the inhibitory RNA and the portion of the target gene is preferred. In certain preferred embodiments, the length of the double-stranded portion of the shRNA is at least 20, 21, or 22 nucleotides, corresponding, for example, to the size of the RNA product produced by Dicer-dependent cleavage. In certain embodiments, the shRNA construct is at least 25, 50, 100, 200, 300, or 400 bases long. In certain embodiments, the shRNA construct is 400–800 bases long. The shRNA construct is highly tolerant of morphological variations in loop sequence and loop size.

[0380] As used herein, the term “ribozyme” refers to a catalytically active RNA molecule that has the ability to site-specifically cleave target mRNA. Several subtypes have been described, including, for example, hammerhead and hairpin ribozymes. The catalytic activity and stability of ribozymes can be improved by substituting ribonucleotides in non-catalyzative bases with deoxyribonucleotides. While specific mRNAs can be disrupted using ribozymes that cleave mRNA at site-specific recognition sequences, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNA at a site designated by an adjacent region that forms a complementary base pair with the target mRNA. The only requirement is that the target mRNA has the following two-base sequence: 5'-UG-3'. The construction and manufacture of hammerhead ribozymes are well known in the art.

[0381] In certain embodiments, the soluble factor antagonist is an siRNA, miRNA, shRNA, or ribozyme.

[0382] In certain embodiments, a method for delivering a polynucleotide of interest, including siRNA, miRNA, shRNA, or ribozyme, comprises one or more regulatory sequences, such as a strongly constitutive pol III, e.g., the human U6 snRNA promoter, the mouse U6 snRNA promoter, the human and mouse H1 RNA promoter, and the human tRNA-val promoter, or the strongly constitutive pol II promoter, as described elsewhere in this specification.

[0383] The polynucleotides disclosed herein, regardless of the length of the coding sequence itself, may be combined with other DNA sequences, such as promoters and / or enhancers, untranslated regions (UTRs), signal sequences, Kozak sequences, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, internal ribosome entry sites (IRESs), recombinase recognition sites (e.g., LoxP, FRT, and Att sites), stop codons, transcription termination signals, and self-cleaving polypeptides and epitope tags, as disclosed elsewhere herein or as known in the art, and as a result, their overall lengths may vary considerably. Therefore, it is intended that polynucleotide fragments of almost any length may be used, and the full length is preferably limited by the ease of preparation and use in the intended recombinant DNA protocol.

[0384] Polynucleotides can be prepared, manipulated, and / or expressed using any of the various well-established techniques known and available in the art. To express a desired polypeptide, the nucleotide sequence encoding the polypeptide can be inserted into a suitable vector. Examples of vectors include plasmids, autonomous replication sequences, and transposing elements. Additional exemplary vectors include, but are not limited to, plasmids, phagemids, cosmids, artificial chromosomes, e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), bacteriophages, e.g., lambda phages or M13 phages, and animal viruses. Examples of categories of animal viruses useful as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (e.g., SV40). Examples of expression vectors include the pClneo vector (Promega) for expression in mammalian cells; and pLenti4 / V5-DEST®, pLenti6 / V5-DEST®, and pLenti6.2 / V5-GW / lacZ (Invitrogen) for lentiviral-mediated gene transfer and expression in mammalian cells. In certain embodiments, the coding sequences of chimeric proteins disclosed herein may be ligated into such expression vectors for the expression of chimeric proteins in mammalian cells.

[0385] In one embodiment, the vector encoding the CAR intended herein comprises the polynucleotide sequence described in SEQ ID NO: 36.

[0386] In certain embodiments, the vector is an episomal vector or a vector maintained outside of chromosomes. As used herein, the term “episome” means a vector that can replicate without integration into the host’s chromosomal DNA and without stepwise loss from a dividing host cell, and also means that the vector replicates outside of chromosomes or episomally. The vector is engineered to harbor a sequence encoding a DNA replication origin or “ori” from a lymphocytotropic herpesvirus or gamma herpesvirus, adenovirus, SV40, bovine papillomavirus, or yeast, particularly a replication origin of a lymphocytotropic herpesvirus or gamma herpesvirus corresponding to oriP of EBV. In certain embodiments, the lymphocytotropic herpesvirus may be Epstein-Barr virus (EBV), Kaposi’s sarcoma herpesvirus (KSHV), herpesvirus thymili (HS), or Marek’s disease virus (MDV). Epstein-Barr virus (EBV) and Kaposi's sarcoma herpesvirus (KSHV) are also examples of gamma herpesviruses. Typically, host cells contain viral replication transactivator proteins that activate replication.

[0387] The “regulatory elements” or “regulatory sequences” present in an expression vector are the untranslated regions of the vector that interact with host cell proteins that perform transcription and translation (origin of replication, selection cassette, promoter, enhancer, translation initiation signal (Shine Dalgarno sequence or Kozak sequence), intron, polyadenylated sequence, 5' and 3' untranslated regions). Such elements can vary in strength and specificity. Depending on the vector system and host used, any number of suitable transcription and translation elements, including ubiquitous and inducible promoters, may be used.

[0388] In certain embodiments, vectors for use herein include, but are not limited to, expression vectors and viral vectors, and include exogenous, endogenous, or heterologous regulatory sequences, such as promoters and / or enhancers. An “endogenous” regulatory sequence is a regulatory sequence that is naturally linked to a given gene in the genome. An “exogenous” regulatory sequence is a regulatory sequence that is placed adjacent to a gene by means of genetic engineering (i.e., molecular biology techniques) such that the transcription of that gene is directed by the linked enhancer / promoter. A “heterologous” regulatory sequence is an exogenous sequence from a different species than the cell being genetically engineered.

[0389] As used herein, the term “promoter” refers to a recognition site of a polynucleotide (DNA or RNA) to which RNA polymerase binds. RNA polymerase initiates and transcribes the polynucleotide operably linked to the promoter. In certain embodiments, the operable promoter in mammalian cells includes an AT-rich region located about 25–30 bases upstream from the site where transcription begins and / or another sequence, the CNCAAT region (where N may be any nucleotide), found 70–80 bases upstream from the start of transcription.

[0390] The term "enhancer" refers to a segment of DNA containing a sequence capable of providing enhanced transcription, which in some cases can function independently of the orientation of other regulatory sequences. Enhancers can function cooperatively or additively with promoters and / or other enhancer elements. The term "promoter / enhancer" refers to a segment of DNA containing a sequence capable of providing both promoter and enhancer functions.

[0391] The term “operably linked” refers to a juxtaposition of the described components in a relationship that allows them to function in their intended manner. In one embodiment, the term refers to a functional linkage between a nucleic acid expression regulatory sequence (e.g., a promoter and / or enhancer) and a second polynucleotide sequence, e.g., the polynucleotide of interest, such that the expression regulatory sequence directs the transcription of the nucleic acid corresponding to the second sequence.

[0392] As used herein, the term “constitutive regulatory sequence” refers to a promoter, enhancer, or promoter / enhancer that enables continuous or sequential transcription of an operablely linked sequence. A constitutive regulatory sequence may be a “ubiquitous” promoter, enhancer, or promoter / enhancer that enables expression in a diverse range of cell and tissue types, or a “cell-specific,” “cell-type-specific,” “cell-lineage-specific,” or “tissue-specific” promoter, enhancer, or promoter / enhancer that enables expression in a limited range of cell and tissue types, respectively.

[0393] Examples of ubiquitous expression regulatory sequences suitable for use in the specific embodiments presented herein include the cytomegalovirus (CMV) early promoter, the Simianvirus 40 (SV40) (e.g., early or late), the Moloney's mouse leukemia virus (MoMLV) LTR promoter, the Roussarcoma virus (RSV) LTR, the herpes simplex virus (HSV) (thymidine kinase) promoter, the H5, P7.5, and P11 promoters from vaccinia virus, the elongation factor 1-alpha (EF1a) promoter, and the early growth response 1. 1;EGR1), ferritin H (FerH), ferritin L (FerL), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), eukaryotic translation initiation factor 4A1 (EIF4A1), heat shock 70kDa protein 5 (HSPA5), heat shock protein 90kDa beta, member 1 (HSP90B1), heat shock protein 70kDa (HSP70), β-kinesin (β-KIN), human ROSA 26 loci (Irions et al., Nature Biotechnology 25, 1477 - 1482) This includes, but is not limited to, the ubiquitin C promoter (UBC), phosphoglycerate kinase-1 (PGK) promoter, cytomegalovirus enhancer / chicken β-actin (CAG) promoter, β-actin promoter and myeloproliferative sarcoma virus enhancer, negative regulatory region deletion, and dl587rev primer-binding site substitution (MND) promoter (Challita et al., J Virol. 69(2):748-55 (1995)).

[0394] In one embodiment, the vector of this disclosure includes an MND promoter.

[0395] In one embodiment, the vector of this disclosure includes an EF1a promoter containing a first intron of the human EF1a gene.

[0396] In one embodiment, the vector of this disclosure comprises an EF1a promoter lacking the first intron of the human EF1a gene.

[0397] In certain embodiments, it may be desirable to express polynucleotides containing CARs from a T cell-specific promoter.

[0398] As used herein, “conditional expression” can refer to any type of conditional expression, including but not limited to inducible expression; repressive expression; and expression in cells or tissues having a particular physiological, biological, or disease condition. This definition is not intended to exclude cell-type or tissue-specific expression. Certain embodiments provide conditional expression of a polynucleotide of interest, for example, where expression is controlled by subjecting cells, tissues, organisms, etc., to a treatment or condition that induces the expression of the polynucleotide or to an increase or decrease in the expression of the polynucleotide encoded by the polynucleotide of interest.

[0399] Examples of inductive promoters / systems include, but are not limited to, steroid-inductive promoters, such as promoters for genes encoding glucocorticoid or estrogen receptors (inducible by treatment with the corresponding hormone), metallothionine promoters (inducible by treatment with various heavy metals), MX-1 promoters (inducible by interferon), the "GeneSwitch" mifepristone-controlled system (Sirin et al., 2003, Gene, 323:67), cumate-inducible gene switches (International Publication No. 2002 / 088346), and tetracycline-dependent regulatory systems.

[0400] Conditional expression can also be achieved by using site-specific DNA recombinases. According to certain embodiments, the vector contains at least one (typically two) sites for recombination mediated by site-specific recombinases. As used herein, the terms “recombinase” or “site-specific recombinase” include excised or incorporated proteins, enzymes, cofactors or related proteins involved in a recombination reaction with one or more recombination sites (e.g., 2, 3, 4, 5, 7, 10, 12, 15, 20, 30, 50, etc.), which may be wild-type proteins (see Landy, Current Opinion in Biotechnology 3:699-707 (1993)) or mutants, derivatives (e.g., fusion proteins containing recombinant protein sequences or fragments thereof), fragments, and variants thereof. Examples of recombinases suitable for use herein include, but are not limited to, Cre, Int, IHF, Xis, Flop, Fis, Hin, Gin, ΦC31, Cin, Tn3 resolverase, TndX, XerC, XerD, TnpX, Hjc, Gin, SpCCE1, and ParA.

[0401] A vector may contain one or more recombination sites for any of a variety of site-specific recombinases. It should be understood that the target sites for site-specific recombinases are additional to any site required for the incorporation of the vector, e.g., a retroviral vector or a lentiviral vector. As used herein, the terms “recombinant sequence,” “recombinant site,” or “site-specific recombinant site” refer to a specific nucleic acid sequence that a recombinase recognizes and binds to.

[0402] For example, one recombination site for Cre recombinase is loxP, which is a 34-base pair sequence containing two 13-base pair inverted repeats (serving as recombinase-binding sites) adjacent to an 8-base pair core sequence (see FIG. 1 in Sauer, B., Current Opinion in Biotechnology 5:521-527 (1994)). Other exemplary loxP sites include, but are not limited to, lox511 (Hoess et al., 1996; Bethke and Sauer, 1997), lox5171 (Lee and Saito, 1998), lox2272 (Lee and Saito, 1998), m2 (Langer et al., 2002), lox71 (Albert et al., 1995), and lox66 (Albert et al., 1995).

[0403] Preferred recognition sites for FLP recombinase include, but are not limited to, FRT (McLeod, et al., 1996), F1, F2, F3 (Schlake and Bode, 1994), F4, F5 (Schlake and Bode, 1994), FRT(LE) (Senecoff et al., 1988), and FRT(RE) (Senecoff et al., 1988).

[0404] Other examples of recognition sequences are the attB, attP, attL, and attR sequences, which are recognized by the recombinase enzyme λ-integrase, e.g., phi-c31. The φC31 SSR mediates recombination only between the heterotyped sites attB (34 bp long) and attP (39 bp long) (Groth et al., 2000). Both attB and attP, named for attachment sites for phage integrases on bacterial and phage genomes, respectively, contain incomplete inverted repeats that appear to be linked by the φC31 homodimer (Groth et al., 2000). The generated sites, attL and attR, are effectively inactive to further φC31-mediated recombination (Belteki et al., 2003), making the reaction irreversible. To catalyze insertion, attB-containing DNA has been found to be more readily inserted into genomic attP sites than attP sites are inserted into genomic attB sites (Thyagarajan et al., 2001; Belteki et al., 2003). Therefore, a typical strategy is to position an attP-containing "docking site" at a defined locus by homologous recombination, and then partner this site with an incoming attB-containing sequence for insertion.

[0405] As used herein, “internal ribosome entry site” or “IRES” refers to an element that facilitates direct internal ribosome entry into the start codon of a cistron (protein-coding region), e.g., ATG, thereby leading to cap-independent translation of the gene. See, for example, Jackson et al., 1990. Trends Biochem Sci 15(12):477-83) and Jackson and Kaminski. 1995. RNA 1(10):985-1000. In certain embodiments, the vector contemplated herein comprises one or more polynucleotides of interest encoding one or more polypeptides. In certain embodiments, to achieve efficient translation of each of the multiple polypeptides, the polynucleotide sequences may be separated by one or more IRES sequences or polynucleotide sequences encoding self-cleaving polypeptides.

[0406] As used herein, the term “Kozak sequence” refers to a short nucleotide sequence that greatly promotes the initial binding of mRNA to the small subunit of the ribosome, thereby increasing translation. The consensus Kozak sequence is (GCC)RCCATGG, where R is purine (A or G) (Kozak, 1986. Cell. 44(2):283-92, and Kozak, 1987. Nucleic Acids Res. 15(20):8125-48). In certain embodiments, the vectors contemplated herein contain a polynucleotide having the consensus Kozak sequence and encoding a desired polypeptide, e.g., CAR.

[0407] In some embodiments, polynucleotides or cells harboring polynucleotides utilize suicide genes, including inducible suicide genes, to reduce the risk of direct toxicity and / or uncontrolled proliferation. In certain embodiments, the suicide genes are not immunogenic to the host harboring the polynucleotides or cells. Certain examples of suicide genes that may be used are caspase-9 or caspase-8 or cytosine deaminase. Caspase-9 may be activated using a specific chemical inducer of dimerization (CID).

[0408] In certain embodiments, the vector includes a gene segment that induces immunoeffector cells of the present disclosure, e.g., T cells, to become sensitive to negative selection in vivo. "Negative selection" means that the injected cells can be eliminated as a result of changes in the individual's in vivo conditions. Negatively selectable phenotypes may result from the insertion of genes that confer sensitivity to administered agents, e.g., compounds. Negatively selectable genes are known in the art and include, in particular, the herpes simplex virus type I thymidine kinase (HSV-I TK) gene that confers ganciclovir sensitivity (Wigler et al., Cell 11:223, 1977); the cellular hypoxanthine phosphoribosyltransferase (HPRT) gene, the cellular adenine phosphoribosyltransferase (APRT) gene, and the bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[0409] In some embodiments, genetically modified immune effector cells, such as T cells, further comprise a polynucleotide containing a positive marker that enables the selection of cells with a negatively selectable phenotype in vitro. The positively selectable marker may be a gene that, when introduced into a host cell, expresses a dominant phenotype and enables the positive selection of cells possessing that gene. This type of gene is known in the art and includes, in particular, the hygromycin-B phosphotransferase gene (hph) that confers resistance to hygromycin B, the aminoglycoside phosphotransferase gene (neo or aph) from Tn5 encoding resistance to the antibiotic G418, the dihydrofolate reductase (DHFR) gene, the adenosine deaminase gene (ADA), and the multidrug resistance (MDR) gene.

[0410] Preferably, a positively selectable marker and a negatively selectable element are fused such that the loss of the negatively selectable element necessarily accompanies the loss of the positively selectable marker. More preferably, the positively and negatively selectable markers are fused such that the loss of one obligately leads to the loss of the other. An example of a fused polynucleotide that yields a polypeptide as an expression product conferring both the desired positively and negatively selective features described above is the hygromycin phosphotransferase thymidine kinase fusion gene (HyTK). Expression of this gene yields a polypeptide that confers hygromycin B resistance for positive selection in vitro and ganciclovir sensitivity for negative selection in vivo. See Lupton SD, et al, Mol. and Cell. Biology 1 1:3374-3378, 1991. Additionally, in certain embodiments, the polynucleotide encoding the chimeric receptor is found in a retroviral vector containing a fused gene, particularly one conferring hygromycin B resistance for positive selection in vitro and ganciclovir sensitivity for negative selection in vivo, e.g., Lupton, SD et al. (1991), in the HyTK retroviral vector described above. See also the publications PCT US91 / 08442 and PCT / US94 / 05601 by SD Lupton, which describe the use of a bifunctional selectable fusion gene derived from fusing a dominant positive selectable marker with a negative selectable marker.

[0411] Positive selectable markers may be derived from genes selected from the group consisting of hph, nco, and gpt, for example, while negative selectable markers may be derived from genes selected from the group consisting of cytosine deaminase, HSV-I TK, VZV TK, HPRT, APRT, and gpt. In certain embodiments, the marker is a bifunctional selectable fusion gene, where the positive selectable marker is derived from hph or neo, and the negative selectable marker is derived from a cytosine deaminase or TK gene or a selectable marker.

[0412] Viral vector In certain embodiments, cells (e.g., immune effector cells) are transduced using retroviral vectors encoding CARs, such as lentiviral vectors. For example, immune effector cells are transduced using a vector encoding a CAR containing a BCMA polypeptide, such as a mouse anti-BCMA antibody that binds to a human BCMA polypeptide, or an antigen-binding fragment thereof, along with intracellular signaling domains of CD3ζ, CD28, 4-1BB, Ox40, or any combination thereof. Alternatively, immune effector cells are transduced using a vector encoding a CAR containing an extracellular antigen, such as an antibody that binds to a tumor antigen, or an antigen-binding fragment thereof, along with intracellular signaling domains of CD3ζ, CD28, 4-1BB, Ox40, or any combination thereof. Thus, these transduced cells can induce a CAR-mediated cytotoxic response.

[0413] Retroviruses are a common tool for gene delivery (Miller, 2000, Nature. 357: 455-460). In certain embodiments, retroviruses are used to deliver polynucleotides encoding chimeric antigen receptors (CARs) to cells. As used herein, the term “retrovirus” refers to an RNA virus whose genomic RNA is reverse transcribed into a linear double-stranded DNA copy, and subsequently its genomic DNA is covalently incorporated into the host genome. Once incorporated into the host genome, the virus is referred to as a “provirus.” The provirus serves as a template for RNA polymerase II, directing the expression of RNA molecules encoding structural proteins and enzymes required to produce new viral particles.

[0414] Exemplary retroviruses suitable for use in specific embodiments include, but are not limited to, Moloney's mouse leukemia virus (MMuLV), Moloney's mouse sarcoma virus (MoMSV), Harvey's mouse sarcoma virus (HaMuSV), mouse mammary tumor virus (MuMTV), gibbon leukemia virus (GaLV), feline leukemia virus (FLV), spumavirus, friend mouse leukemia virus, mouse stem cell virus (MSCV), and Rous sarcoma virus (RSV) and lentivirus.

[0415] As used herein, the term “lentivirus” refers to a group (or genus) of compound retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV types 1 and HIV type 2); Visna-Maedi virus (VMV) virus; Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Ape immunodeficiency virus (SIV). In one embodiment, an HIV-based vector skeleton (i.e., an HIV cis-acting sequence element) is utilized. In certain embodiments, the lentivirus is used to deliver polynucleotides containing CARs to cells.

[0416] Retroviral vectors, more particularly lentiviral vectors, may be used in the implementation of certain embodiments disclosed herein. Thus, as used herein, the terms “retrovirus” and “retroviral vector” are to include “lentivirus” and “lentiviral vector,” respectively.

[0417] The term "vector" is used herein to refer to a nucleic acid molecule that has the ability to transfer or transport another nucleic acid molecule. The nucleic acid to be transferred is generally ligated, for example, inserted, into the vector nucleic acid molecule. The vector may contain a sequence directed toward autonomous replication in a cell, or it may contain a sequence sufficient to enable integration into host cell DNA. Useful vectors include, for example, plasmids (e.g., DNA plasmids or RNA plasmids), transposons, cosmids, bacterial artificial chromosomes, and viral vectors. Useful viral vectors include, for example, replication-deficient retroviruses and lentiviruses.

[0418] As will be apparent to those skilled in the art, the term “viral vector” is widely used to refer to either a nucleic acid molecule (e.g., an import plasmid) containing a virus-derived nucleic acid element that typically facilitates the transfer or integration of nucleic acid molecules into the cell’s genome, or a viral particle that mediates nucleic acid transfer. Viral particles typically contain various viral components and, in some cases, host cell components in addition to nucleic acids.

[0419] The term "viral vector" can refer to either a virus or a viral particle that has the ability to transfer nucleic acids into cells or into the nucleic acids themselves. Viral vectors and transfer plasmids contain structural and / or functional genetic elements primarily derived from viruses. The term "retroviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or parts thereof, primarily derived from retroviruses. The term "lentiviral vector" refers to a viral vector or plasmid containing structural and functional genetic elements, or parts thereof, primarily derived from lentiviruses, including LTRs. The term "hybrid vector" refers to a vector, LTR, or other nucleic acid containing both retroviral, e.g., lentiviral, sequence and non-lentiviral viral sequence. In one embodiment, a hybrid vector refers to a vector or transfer plasmid containing a retroviral, e.g., lentiviral, sequence for reverse transcription, replication, integration, and / or packaging.

[0420] In certain embodiments, the terms “lentiviral vector” and “lentiviral expression vector” may be used to refer to lentiviral transfer plasmids and / or infectious lentiviral particles. Where references are made herein to elements, such as cloning sites, promoters, regulatory elements, heterologous nucleic acids, etc., it should be understood that the sequences of these elements are present in RNA form in the lentiviral particles of this disclosure and in DNA form in the DNA plasmids of this disclosure.

[0421] At each end of a provirus, there is a structure called a "long-terminal repeat" or "LTR." The term "long-terminal repeat (LTR)" refers to a domain of base pairs located at the end of retroviral DNA, which is a direct repeat in the context of their native sequence, as well as containing the U3, R, and U5 regions. LTRs generally provide fundamental functions for retroviral gene expression (e.g., promotion, initiation, and polyadenylation of gene transcripts) as well as for viral replication. LTRs contain numerous regulatory signals, including transcriptional regulatory elements, polyadenylation signals, and sequences required for replication and integration of the viral genome. Viral LTRs are divided into three regions called U3, R, and U5. The U3 region contains enhancer and promoter elements. The U5 region is the sequence between the primer-binding site and the R region, and contains the polyadenylation sequence. The R (repeat) region is adjacent to the U3 and U5 regions. LTRs consist of the U3, R, and U5 regions and are present at both the 5' and 3' ends of the viral genome. Adjacent to the 5' LTR are sequences necessary for reverse transcription of the genome (tRNA primer binding sites) and sequences necessary for efficient packaging of viral RNA into particles (psi sites).

[0422] As used herein, the terms “packaging signal” or “packaging sequence” refer to a sequence located within the retroviral genome required for the insertion of viral RNA into a viral capsid or particle. See, for example, Clever et al., 1995. J. of Virology, Vol. 69, No. 4; pp. 2101–2109. Some retroviral vectors use the smallest packaging signal (also referred to as the psi [Ψ] sequence) required for capsid encapsulation of the viral genome. Therefore, as used herein, the terms “packaging sequence,” “packaging signal,” “psi,” and the symbol “Ψ” are used in reference to non-coding sequences required for capsid encapsulation of the retroviral RNA strand during viral particle formation.

[0423] In various embodiments, the vector includes modified 5' LTR and / or 3' LTR. Either or both LTRs may include one or more modifications, including but not limited to one or more deletions, insertions, or substitutions. Modifications to the 3' LTR are often made to improve the security of a lentiviral or retroviral system by making the virus replication-defective. As used herein, the term “replication-defective” refers to a virus that lacks the ability to replicate completely and effectively, and as a result does not produce infectious virions (e.g., a replication-defective lentiviral offspring). The term “replication-competent” refers to a wild-type virus or mutant virus that has the ability to replicate, and as a result the viral replication of the virus has the ability to produce infectious virions (e.g., a replication-competent lentiviral offspring).

[0424] A “self-inactivating” (SIN) vector refers to a replication-defective vector, such as a retroviral or lentiviral vector, in which the right (3') LTR enhancer-promoter region, known as the U3 region, is modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication. This is because the right (3') LTR U3 region is used as a template for the left (5') LTR U3 region during viral replication, and therefore viral transcripts cannot be produced without the U3 enhancer-promoter. In further embodiments, the 3' LTR is modified so that the U5 region is replaced, for example, with an ideal poly(A) sequence. It should be noted that modifications to the LTR, e.g., to the 3' LTR, 5' LTR, or both the 3' LTR and 5' LTR are also included herein.

[0425] Additional safety enhancements are provided by replacing the U3 region of the 5' LTR with a heterologous promoter to drive transcription of the viral genome during the production of viral particles. Examples of heterologous promoters that may be used include, for example, the Simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., very early), Moloney's mouse leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) (thymidine kinase) promoters. Typical promoters can drive high levels of transcription in a Tat-independent manner. This substitution reduces the likelihood that recombination will produce a replicant virus because the complete U3 sequence is not present in the virus production system. In certain embodiments, heterologous promoters have additional advantages in controlling the manner in which the viral genome is transcribed. For example, heterologous promoters can be inducible, and as a result, transcription of all or part of the viral genome occurs only in the presence of an inducing factor. Inducing factors include, but are not limited to, one or more chemical compounds or physiological conditions, such as the temperature or pH under which host cells are cultured.

[0426] In some embodiments, the viral vector includes a TAR element. The term "TAR" refers to a "transactivation response" gene element located in the R region of the lentiviral (e.g., HIV) LTR. This element interacts with the lentiviral transactivator (tat) gene element to enhance viral replication. However, this element is not required in embodiments where the U3 region of the 5' LTR is replaced by a heterologous promoter.

[0427] The "R region" refers to a region within the retroviral LTR that begins at the start of the capping group (i.e., the start of transcription) and ends immediately before the start of the polyA tract. The R region is also defined as being adjacent to the U3 and U5 regions. The R region plays a role in allowing the movement of nascent DNA from one end of the genome to the other during reverse transcription.

[0428] As used herein, the term “FLAP element” refers to a nucleic acid whose sequence includes the central polyprint lactate and central termination sequences (cPPT and CTS) of a retrovirus, e.g., HIV-1 or HIV-2. Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and Zennou, et al., 2000, Cell, 101:173. During HIV-1 reverse transcription, the central start of the positive-strand DNA in the central polyprint lactate (cPPT) and the central termination in the central termination sequence (CTS) lead to the formation of a three-strand DNA structure: the HIV-1 central DNA flap. While we do not wish to be bound by any theory, the DNA flap may act as a cis-activity determinant of lentiviral genome nuclear transport and / or may increase viral titer. In certain embodiments, the retroviral or lentiviral vector backbone includes one or more FLAP elements upstream or downstream of the heterologous gene of interest in the vector. For example, in certain embodiments, the transfer plasmid includes a FLAP element. In one embodiment, the vector includes a FLAP element isolated from HIV-1.

[0429] In one embodiment, a retroviral or lentiviral transfer vector contains one or more export elements. The term “export element” refers to a cis-acting post-transcriptional regulatory element that regulates the transport of RNA transcripts from the nucleus to the cytoplasm of a cell. Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) rev response element (RRE) (see, e.g., Cullen et al., 1991. J. Virol. 65: 1053; and Cullen et al., 1991. Cell 58: 423), as well as the hepatitis B virus post-transcriptional regulatory element (HPRE). Generally, RNA export elements are located within the 3' UTR of a gene and may be inserted as one or more copies.

[0430] In certain embodiments, the expression of heterologous sequences in a viral vector is increased by incorporating post-transcriptional regulatory elements, efficient polyadenylation sites, and, as appropriate, transcription termination signals into the vector. Various post-transcriptional regulatory elements can increase the expression of heterologous nucleic acids in proteins, such as the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE; Zufferey et al., 1999, J. Virol., 73:2886); the post-transcriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell. Biol., 5:3864); and others (Liu et al., 1995, Genes Dev., 9:1766). In certain embodiments, the vector may contain post-transcriptional regulatory elements, such as WPRE or HPRE.

[0431] In certain embodiments, the vector lacks or does not contain post-transcriptional regulatory elements (PTEs), such as WPRE or HPRE, because, in some cases, these elements increase the risk of cell transformation and / or substantially or significantly increase the amount of mRNA transcript or mRNA stability. Therefore, in some embodiments, the vector lacks or does not contain PTEs. In other embodiments, the vector lacks or does not contain WPRE or HPRE as an additional safety measure.

[0432] Elements that direct the efficient termination and polyadenylation of heterologous nucleic acid transcripts increase heterologous gene expression. Transcription termination signals are generally found downstream of polyadenylation signals. In certain embodiments, the vector contains a polyadenylation sequence at the 3' end of the polynucleotide encoding the polypeptide to be expressed. The terms “polyA site” or “polyA sequence,” as used herein, refer to a DNA sequence that directs both termination and polyadenylation of the nascent RNA transcript by RNA polymerase II. Polyadenylation sequences can promote mRNA stability by adding a polyA tail to the 3' end of the coding sequence, and thus can contribute to increased translation efficiency. Since transcripts lacking a polyA tail are unstable and rapidly degraded, efficient polyadenylation of recombinant transcripts is desirable. Examples of polyA signals that may be used in vectors herein include ideal polyA sequences (e.g., AATAAA, ATTAAA, AGTAAA), bovine growth hormone polyA sequences (BGHpA), rabbit β-globin polyA sequences (rβgpA), or other suitable heterologous or endogenous polyA sequences known in the art.

[0433] In certain embodiments, the retroviral or lentiviral vector further comprises one or more insulator elements. The insulator elements may contribute to the protection of lentiviral expression sequences, such as therapeutic polypeptides, from integration site effects (i.e., positional effects; see, e.g., Burgess-Beusse et al., 2002, Proc. Natl. Acad. Sci., USA, 99:16433; and Zhan et al., 2001, Hum. Genet., 109:471) that may be mediated by cis-acting elements present in the genomic DNA, leading to the unregulated expression of the imported sequence. In some embodiments, the import vector comprises one or more insulator elements in the 3' LTR, and in the integration of the provirus into the host genome, the provirus comprises one or more insulators in both the 5' LTR or 3' LTR due to replication in the 3' LTR. Suitable insulators for use herein include, but are not limited to, chicken β-globin insulators (see Chung et al., 1993. Cell 74:505; Chung et al., 1997. PNAS 94:575; and Bell et al., 1999. Cell 98:387; these are incorporated herein by reference). Examples of insulator elements include, but are not limited to, insulators derived from β-globin loci, such as chicken HS4.

[0434] According to certain embodiments, most or all of the viral vector backbone sequence is derived from a lentivirus, e.g., HIV-1. However, it should be understood that many different sources of retroviral and / or lentiviral sequences are available or can be combined, and that numerous substitutions and modifications in certain lentiviral sequences can be adapted without impairing the ability of the transfer vector to perform the functions described herein. Furthermore, various lentiviral vectors are known in the art; see Naldini et al., (1996a, 1996b, and 1998); Zufferey et al., (1997); Dull et al., 1998, U.S. Patent No. 6,013,516; and U.S. Patent No. 5,994,136. Many of these may be adapted for the production of the viral vectors or transfer plasmids of this disclosure.

[0435] In various embodiments, the vectors described herein may include a promoter operably ligated to a polynucleotide encoding a CAR polypeptide. The vector may have one or more LTRs, any of which may include one or more modifications, e.g., one or more nucleotide substitutions, additions, or deletions. The vector may further include one or more accessory elements for increasing transduction efficiency (e.g., cPPT / FLAP), viral packaging (e.g., psi(Ψ) packaging signals, RREs), and / or other elements for increasing therapeutic gene expression (e.g., poly(A) sequences), and may optionally include WPREs or HPREs.

[0436] In certain embodiments, the transfer vector comprises a left (5') retroviral LTR; a central polyprint lact / DNA flap (cPPT / FLAP); a retroviral export element; a promoter operably linked to a polynucleotide encoding the CAR polypeptide intended herein and active in T cells; and a right (3') retroviral LTR; and optionally a WPRE or HPRE.

[0437] In certain embodiments, the transfer vector comprises a left (5') retroviral LTR; a retroviral export element; a promoter operably linked to a polynucleotide encoding the CAR polypeptide intended herein, which is active in T cells; a right (3') retroviral LTR; and a poly(A) sequence; and optionally a WPRE or HPRE. In another particular embodiment, provided herein is a lentiviral vector comprising a left (5') LTR; cPPT / FLAP; RRE; a promoter operably linked to a polynucleotide encoding the CAR polypeptide intended herein, which is active in T cells; a right (3') LTR; and a polyadenylated sequence; and optionally a WPRE or HPRE.

[0438] In certain embodiments, the lentiviral vector provided herein comprises a left (5') HIV-1 LTR; a psi(Ψ) packaging signal; cPPT / FLAP; RRE; a promoter operably linked to a polynucleotide encoding the CAR polypeptide intended herein, which is active in T cells; a right (3') autoinactivating (SIN) HIV-1 LTR; and a rabbit β-globin polyadenylated sequence; and optionally a WPRE or HPRE.

[0439] In another embodiment, the vector provided herein comprises at least one LTR; a central polyprint lactate / DNA flap (cPPT / FLAP); a retroviral export element; and a promoter operably linked to a polynucleotide encoding a CAR polypeptide intended herein, which is active in T cells; and optionally a WPRE or HPRE.

[0440] In certain embodiments, the vector provided herein comprises at least one LTR; cPPT / FLAP; RRE; a promoter operably linked to a polynucleotide encoding a CAR polypeptide intended herein, which is active in T cells; and a polyadenylation sequence; and optionally a WPRE or HPRE.

[0441] In certain embodiments, provided herein are at least one SIN HIV-1 LTR; a psi(Ψ) packaging signal; cPPT / FLAP; a T cell-active promoter operably linked to a polynucleotide encoding the CAR polypeptide intended herein; and a rabbit β-globin polyadenylation sequence; and optionally a WPRE or HPRE.

[0442] In various embodiments, the vector is an embedded viral vector.

[0443] In various other embodiments, the vector is an episomal or non-embedded viral vector.

[0444] In various embodiments, the vectors contemplated herein include non-integrating or integration-deficient retroviruses. In one embodiment, an "integrating-deficient" retrovirus or lentivirus refers to a retrovirus or lentivirus having an integrase that lacks the ability to integrate the viral genome into the host cell's genome. In various embodiments, the integrase protein is mutated to specifically reduce its integrase activity. An integration-incompetent lentivirus vector is obtained by modifying the pol gene encoding the integrase protein to obtain a mutant pol gene encoding an integration-deficient integrase. Such an integration-incompetent virus vector is described in Patent Application International Publication No. 2006 / 010834 (which is incorporated herein by reference in its entirety).

[0445] Exemplary mutations in the HIV-1 pol gene suitable for reducing integrase activity include H12N, H12C, H16C, H16V, S81R, D41A, K42A, H51A, Q53C, D55V, D64E, D64V, E69A, K71A, E85A, E87A, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, K This includes, but is not limited to, 160A, R166A, D167A, E170A, H171A, K173A, K186Q, K186T, K188T, E198A, R199c, R199T, R199A, D202A, K211A, Q214L, Q216L, Q221L, W235F, W235E, K236S, K236A, K246A, G247W, D253A, R262A, R263A, and K264H.

[0446] Exemplary mutations in the HIV-1 pol gene suitable for reducing integrase activity include, but are not limited to, D64E, D64V, E92K, D116N, D1161, D116A, N120G, N1201, N120E, E152G, E152A, D35E, K156E, K156A, E157A, K159E, K159A, W235F, and W235E.

[0447] In certain embodiments, the integrase includes a mutation in one or more of the amino acids D64, D116, or E152. In one embodiment, the integrase includes mutations in the amino acids D64, D116, and E152. In certain embodiments, the defective HIV-1 integrase includes a D64V mutation.

[0448] "Host cells" include cells electroporated, transfected, infected, or transduced in vivo, ex vivo, or in vitro using recombinant vectors or polynucleotides disclosed herein. Host cells may include packaging cells, producer cells, and cells infected with viral vectors. In certain embodiments, host cells infected with viral vectors disclosed herein are administered to subjects in need of treatment. In certain embodiments, the term "target cells" is used interchangeably with host cells and refers to cells that have been transfected, infected, or transduced to a desired cell type. In certain embodiments, the target cells are T cells.

[0449] Large-scale production of viral particles is often necessary to achieve reasonable viral titers. Viral particles are produced by transfecting a packaging cell system containing a viral structure and / or accessory genes, such as gag, pol, env, tat, rev, vif, vpr, vpu, vpx, or nef genes or other retroviral genes, with an import vector.

[0450] As used herein, the term “packaging vector” refers to an expression vector or viral vector that lacks a packaging signal and contains polynucleotides encoding one, two, three, four or more viral structures and / or accessory genes. Typically, a packaging vector is contained within a packaging cell and introduced into the cell via transfection, transduction, or infection. Methods of transfection, transduction, or infection are well known to those skilled in the art. The retroviral / lentiviral transfer vectors disclosed herein may be introduced into a packaging cell system via transfection, transduction, or infection to generate producer cells or cell lines. The packaging vectors disclosed herein may be introduced into human cells or cell lines by standard methods, including, for example, calcium phosphate transfection, lipofection, or electroporation. In some embodiments, the packaging vector is introduced into cells with a dominantly selectable marker, such as neomycin, hygromycin, puromycin, blastosidine, zeosin, thymidine kinase, DHFR, Gln synthetase, or ADA, followed by selection and isolation of clones in the presence of the appropriate drug. Selectable marker genes can be physically ligated to the gene encoded by the packaging vector, for example, by IRES or self-cleaving viral peptides.

[0451] The viral envelope protein (env) determines the range of host cells that can ultimately be infected and transformed by recombinant retroviruses produced from the cell line. In the case of lentiviruses, such as HIV-1, HIV-2, SIV, FIV, and EIV, the env proteins include gp41 and gp120. Preferably, the viral env proteins expressed by the packaging cells disclosed herein are encoded on a vector separate from the viral gag and pol genes, as previously described.

[0452] Examples of retrovirus-derived env genes that may be used herein include, but are not limited to, the MLV envelope, 10A1 envelope, BAEV, FeLV-B, RD114, SSAV, Ebola, Sendai, FPV (avian plague virus), and influenza virus envelopes. Similarly, in addition to RNA viruses (e.g., the RNA virus families of Picornaviridae, Caliciviridae, Astroviridae, Togaviridae, Flaviviridae, Coronaviridae, Paramyxoviridae, Rhabdoviridae, Filoviridae, Orthomyxoviridae, Bunyaviridae, Arenaviridae, Reoviridae, Birnaviridae, and Retroviridae), genes encoding envelopes from DNA viruses (families of Hepadnaviridae, Circoviridae, Parvoviridae, Papovaviridae, Adenoviridae, Herpesviridae, Poxviridae, and Iridoviridae) may be used. Typical examples include FeLV, VEE, HFVW, WDSV, SFV, rabies, ALV, BIV, BLV, EBV, CAEV, SNV, ChTLV, STLV, MPMV, SMRV, RAV, FuSV, MH2, AEV, AMV, CT10, and EIAV.

[0453] In other embodiments, the envelope protein for pseudotyping a virus in connection with the present disclosure is the following viruses: influenza A, e.g., H1N1, H1N2, H3N2 and H5N1 (avian influenza), influenza B, influenza C virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, hepatitis E virus, rotavirus, any virus of the Norwalk virus group, enteric adenovirus, parvovirus, dengue virus, sarpox, mononegative Viral orders, lyssaviruses, e.g., rabies virus, Lagos bat virus, Mocola virus, Dubenhage virus, European bat virus 1 & 2 and Australian bat virus, ephemerovirus, becyclovirus, vesicular stomatitis virus (VSV), herpesviruses, e.g., herpes simplex virus types 1 and 2, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus (EBV), human herpesvirus (HHV), human herpesvirus types 6 and 8, human immunodeficiency virus Viruses (HIV), papillomaviruses, mouse gamma herpesviruses, arenaviruses such as Argentine hemorrhagic fever virus, Bolivian hemorrhagic fever virus, Sabia-associated hemorrhagic fever virus, Venezuelan hemorrhagic fever virus, Lassa fever virus, Machupo virus, lymphocytic choriomeningitis virus (LCMV), Bunyaviridae, such as Crimean-Congo hemorrhagic fever virus, hantaviruses, viruses that cause hemorrhagic fever with renal syndrome, Rift Valley fever virus, and filoviruses including Ebola hemorrhagic fever and Marburg hemorrhagic fever. This includes, but is not limited to, filoviruses, including the Flaviviridae family (including Kasanul Forest disease virus, Omsk hemorrhagic fever virus, tick-borne encephalitis-causing viruses) and Paramyxoviridae families, such as Hendra virus and Nipah virus, smallpox and varicella, alphaviruses, such as Venezuelan horse encephalitis virus, Eastern equine encephalitis virus, Western equine encephalitis virus, SARS-related coronavirus (SARS-CoV), West Nile virus, and any encephalitis-causing viruses.

[0454] In one embodiment, the herein provides packaging cells that produce a recombinant retrovirus, such as a lentivirus, pseudotyped with VSV-G glycoprotein.

[0455] The terms “pseudotyped” or “pseudotyped,” as used herein, refer to a virus in which its viral envelope protein is replaced with the viral envelope protein of another virus having desirable characteristics. For example, HIV can be pseudotyped with the vesicular stomatitis virus G-protein (VSV-G) envelope protein, which allows HIV to infect a wider range of cells because the HIV envelope protein (encoded by the env gene) typically targets the virus to CD4+ presenting cells. In one embodiment, the lentiviral envelope protein is pseudotyped with VSV-G. In one embodiment, provided herein are packaging cells that produce a recombinant retrovirus, e.g., a lentivirus, pseudotyped with the VSV-G envelope glycoprotein.

[0456] As used herein, the term “packaging cell system” refers to a cell system that does not contain a packaging signal but stably or transiently expresses viral structural proteins and replication enzymes (e.g., gag, pol, and env) necessary for the precise packaging of viral particles. Any suitable cell system may be used to prepare packaging cells in connection with this disclosure. Generally, the cells are mammalian cells. In certain embodiments, the cells used to produce the packaging cell system are human cells. Suitable cell lines that may be used include, for example, CHO cells, BHK cells, MDCK cells, C3H 10T1 / 2 cells, FLY cells, Psi-2 cells, BOSC 23 cells, PA317 cells, WEHI cells, COS cells, BSC 1 cells, BSC 40 cells, BMT 10 cells, VERO cells, W138 cells, MRC5 cells, A549 cells, HT1080 cells, 293 cells, 293T cells, B-50 cells, 3T3 cells, NIH3T3 cells, HepG2 cells, Saos-2 cells, Huh7 cells, HeLa cells, W163 cells, 211 cells, and 211A cells. In certain embodiments, the packaging cells are 293 cells, 293T cells, or A549 cells. In other specific embodiments, the cells are A549 cells.

[0457] As used herein, the term “producer cell system” refers to a cell system capable of producing recombinant retroviral particles, comprising a packaging cell system and an import vector construct containing a packaging signal. The preparation of infectious viral particles and viral stock solutions may be carried out using prior art. Methods for preparing viral stock solutions are known in the art and are shown, for example, in Soneoka et al. (1995) Nucl. Acids Res. 23:628-633 and NR Landau et al. (1992) J. Virol. 66:5110-5113. Infectious viral particles may be collected from packaging cells using prior art. For example, infectious particles can be collected by cell lysis or collection of the supernatant of a cell culture, as is known in the art. The collected viral particles may be purified as desired. Suitable purification techniques are well known to those skilled in the art.

[0458] The delivery of genes or other polynucleotide sequences using retroviral or lentiviral vectors by means of viral infection rather than transfection is referred to as “transduction.” In one embodiment, a retroviral vector is transduced into a cell through infection and proviral incorporation. In certain embodiments, a target cell, e.g., a T cell, is “transduced” if it contains genes or other polynucleotide sequences delivered to the cell by infection using a virus or retroviral vector. In certain embodiments, a transduced cell contains one or more genes or other polynucleotide sequences delivered by a retroviral or lentiviral vector in its cellular genome.

[0459] In certain embodiments, host cells transduced using a viral vector disclosed herein expressing one or more polypeptides are administered to a subject to treat and / or prevent B-cell malignancies. Other methods relating to the use of viral vectors in gene therapy, which may be utilized according to certain embodiments herein, include, for example, Kay, MA (1997) Chest 111(6 Supp.):138S-142S; Ferry, N. and Heard, JM (1998) Hum. Gene Ther. 9:1975-81; Shiratory, Y. et al. (1999) Liver 19:265-74; Oka, K. et al. (2000) Curr. Opin. Lipidol. 11:179-86; Thule, PM and Liu, JM (2000) Gene Ther. 7:1744-52; Yang, NS (1992) Crit. Rev. Biotechnol. 12:335-56; Alt, M. (1995) J. Hepatol. 23:746-58; Brody, This information can be found in SL and Crystal, RG (1994) Ann. NY Acad. Sci. 716:90-101; Strayer, DS (1999) Expert Opin. Investig. Drugs 8:2159-2172; Smith-Arica, JR and Bartlett, JS (2001) Curr. Cardiol. Rep. 3:43-49; and Lee, HC et al. (2000) Nature 408:483-8.

[0460] 6.7 Genetically modified cells In certain embodiments, disclosed herein are genetically modified cells to express the CARs intended herein for use in the treatment of tumors or cancer. In certain embodiments, disclosed herein are genetically modified cells to express the CARs intended herein for use in the treatment of B-cell-associated conditions. As used herein, the terms “genetically engineered” or “genetically modified” refer to the addition of additional genetic material in the form of DNA or RNA to the total genetic material in a cell. The terms “genetically modified cell,” “modified cell,” and “redirected cell” are used interchangeably. As used herein, the term “gene therapy” refers to the introduction of additional genetic material in the form of DNA or RNA to the total genetic material in a cell for the purpose of restoring, correcting, or modifying gene expression, or for the purpose of expressing therapeutic polypeptides, such as CARs.

[0461] In certain embodiments, the CARs contemplated herein are introduced into and expressed in immune effector cells to redirect the specificity of the immune effector cells to a target antigen of interest, e.g., a BCMA polypeptide. "Immune effector cells" are any cells of the immune system that have one or more effector functions (e.g., cytotoxic cell-killing activity, cytokine secretion, induction of ADCC and / or CDC).

[0462] The immune effector cells of this disclosure may be autologous ("self") or non-self ("non-self," e.g., allogeneic, syngeneic, or heterogeneic).

[0463] As used herein, "autologous" refers to cells from the same subject.

[0464] Where used herein, "homogeneous" refers to cells of the same species that are genetically distinct from the cells being compared.

[0465] As used herein, "syngeneic" refers to a different subject cell that is genetically identical to the cell being compared.

[0466] When used herein, "heterogeneous" refers to cells of a different species than the cells being compared. In certain embodiments, the cells of this disclosure are homogeneous.

[0467] Exemplary immune effector cells used with CARs as intended herein include T lymphocytes. The terms “T cell” or “T lymphocyte” are recognized in the art and are intended to include thymocytes, immature T lymphocytes, mature T lymphocytes, quiescent T lymphocytes, or activated T lymphocytes. T cells may be helper T(Th) cells, such as helper T1(Th1) or helper T2(Th2) cells. T cells are helper T cells (HTL;CD4 + T cells)CD4 + T cells, cytotoxic T cells (CTL; ​​CD8 + T cells), CD4 + CD8 + T cells, CD4 - CD8 - This may be T cells, or any other subset of T cells. Other exemplary populations of T cells suitable for use in a particular embodiment include naive T cells and memory T cells.

[0468] As those skilled in the art will understand, other cells may also be used as immune effector cells in conjunction with the CARs described herein. In particular, immune effector cells also include NK cells, NKT cells, neutrophils, and macrophages. Immune effector cells also include effector cell progenitor cells, such progenitor cells may be induced to differentiate into immune effector cells in vivo or in vitro. Thus, in certain embodiments, immune effector cells include immune effector cell progenitor cells, such as hematopoietic stem cells (HSCs) contained within a CD34+ population of cells derived from umbilical cord blood, bone marrow, or recruited peripheral blood, which differentiate into mature immune effector cells upon administration to a subject or may be induced to differentiate into mature immune effector cells in vitro.

[0469] As used herein, immune effector cells genetically engineered to contain BCMA-specific CARs may be referred to as “BCMA-specific redirected immune effector cells.”

[0470] "CD34 + The term “cell” as used herein refers to a cell that expresses the CD34 protein on its cell surface. “CD34” as used herein refers to a cell surface glycoprotein (e.g., sialomucin protein) that often acts as a cell-cell adhesion molecule and is involved in T cell entry into lymph nodes. + The cell population contains hematopoietic stem cells (HSCs), which differentiate upon administration to the patient and contribute to all hematopoietic lineages, including T cells, NK cells, NKT cells, neutrophils, and monocyte / macrophage lineages.

[0471] In certain embodiments, what is provided herein is a method for producing immunoeffector cells expressing CARs as intended herein. In one embodiment, the method includes the step of transfecting or transfecting immunoeffector cells isolated from an organism so that the immunoeffector cells express one or more CARs as described herein. In certain embodiments, immunoeffector cells are isolated from an organism and genetically modified without further in vitro manipulation. Such cells can then be directly re-administered to an organism. In further embodiments, immunoeffector cells are first activated in vitro and stimulated to proliferate before being genetically modified to express CARs. In this regard, immunoeffector cells may be cultured before and / or after being genetically modified (i.e., transfected or transfected to express CARs as intended herein).

[0472] In certain embodiments, the cell source is obtained from the subject prior to...

Claims

1. A method for determining whether cancer in humans is responsive to chimeric antigen receptor (CAR) T cells, (i) the step of determining the level of one or more inflammation-related soluble factors in human serum; then A method comprising the step of demonstrating responsiveness to CAR T cells if the level of one or more inflammation-related soluble factors in (i) is similar to that in serum from another human who is responsive to CAR T cells, wherein the one or more inflammation-related soluble factors are selected from the group consisting of placental growth factor (PGF), CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1.

2. A pharmaceutical composition comprising chimeric antigen receptor (CAR) T cells for the treatment of cancer in a person requiring treatment, characterized in that the pharmaceutical composition is used to be administered to the person when the level of one or more inflammation-related soluble factors in a serum sample from the person is similar to the level of one or more inflammation-related soluble factors in a serum sample from another person responsive to CAR T cells, wherein the one or more inflammation-related soluble factors are selected from the group consisting of placental growth factor (PGF), CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1.

3. A method for selecting a human patient diagnosed with cancer as a candidate for administration of chimeric antigen receptor (CAR) T cells, comprising the step of determining the level of one or more inflammation-related soluble factors in a serum sample from the patient, wherein the patient is indicated as a candidate for the CAR T cells if the level of one or more inflammation-related soluble factors in the serum sample from the patient is similar to the level of one or more inflammation-related soluble factors in a serum sample from a patient responsive to CAR T cells, and the one or more inflammation-related soluble factors are selected from the group consisting of placental growth factor (PGF), CD70, TNFRSF4, TNFRSF9 (sCD137 or s4-1BB), DCN, CD83, IL10, PDCD1, IL12, and NCR1.

4. The method according to claim 1 or 3, wherein one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83.

5. The method according to any one of claims 1, 3, and 4, wherein the cancer is multiple myeloma.

6. CAR T cells have a heavy chain variable region (V) containing the amino acid sequence shown in SEQ ID NO:

8. Ｈ ) and the light chain variable region (V) containing the amino acid sequence shown in Sequence ID No. 7 Ｌ The method according to any one of claims 1 and 3 to 5, comprising a CAR including an extracellular antigen-binding domain including ).

7. The method according to any one of claims 1 and 3 to 6, wherein the CAR T cell contains CAR, and the CAR contains the amino acid sequence shown in SEQ ID NO: 9 or 37.

8. The method according to any one of claims 1 and 3 to 7, wherein the CAR T cells are BCMA-targeting CAR T cells.

9. BCMA-targeting CAR T cells include BCMA02, idekabuta genbiculocell cells (ide-cel), JCARH125, JNJ-68284528 (LCAR-B38M; containing SEQ ID NO: 265 of 10,934,363 or SEQ ID NO: 300 of WO2018 / 028647, and having or not having a signal peptide) (Janssen / Legend), The method according to claim 8, wherein a selected material is obtained from the group comprising P-BCMA-101 (Poseida), PBCAR269A (Poseida), P-BCMA-Allo1 (Poseida), Allo-715 (Pfizer / Allogene), CT053 (Carsgen), Descartes-08 (Cartesian), PHE885 (Novartis), and CTX120 (CRISPR Therapeutics).

10. The method according to claim 9, wherein the BCMA-targeting CAR T cells are idekabuta genbiculocel cells (ide-cell).

11. The method according to any one of claims 1 and 3 to 10, wherein the level of one or more inflammation-related soluble factors is determined by an O-link IO analyte assay.

12. The method according to any one of claims 1 and 3 to 11, wherein CAR T cells are for treating cancer in a person in need of treatment.

13. The pharmaceutical composition according to claim 2, wherein the one or more inflammation-related soluble factors are selected from the group consisting of PGF, CD70, TNFRSF9, and CD83.

14. The pharmaceutical composition according to claim 2 or 13, wherein the cancer is multiple myeloma.

15. CAR T cells have a heavy chain variable region (V) containing the amino acid sequence shown in SEQ ID NO:

8. Ｈ ) and the light chain variable region (V) containing the amino acid sequence shown in Sequence ID No. 7 Ｌ A pharmaceutical composition according to any one of claims 2, 13, and 14, comprising a CAR containing an extracellular antigen-binding domain containing ).

16. The pharmaceutical composition according to any one of claims 2 and 13 to 15, wherein the CAR T cell comprises a CAR, and the CAR comprises an amino acid sequence shown in SEQ ID NO: 9 or 37.

17. The pharmaceutical composition according to any one of claims 2 and 13 to 16, wherein the CAR T cells are CAR T cells that target BCMA.

18. BCMA-targeting CAR T cells include BCMA02, idekabuta genbiculocell cells (ide-cel), JCARH125, JNJ-68284528 (LCAR-B38M; containing SEQ ID NO: 265 of 10,934,363 or SEQ ID NO: 300 of WO2018 / 028647, and having or not having a signal peptide) (Janssen / Legend), The pharmaceutical composition according to claim 17, selected from the group comprising P-BCMA-101 (Poseida), PBCAR269A (Poseida), P-BCMA-Allo1 (Poseida), Allo-715 (Pfizer / Allogene), CT053 (Carsgen), Descartes-08 (Cartesian), PHE885 (Novartis), and CTX120 (CRISPR Therapeutics).

19. The pharmaceutical composition according to claim 18, wherein the BCMA-targeting CAR T cells are idekabuta genbiculocel cells (ide-cel).

20. The pharmaceutical composition according to any one of claims 2 and 13 to 19, wherein the level of one or more inflammation-related soluble factors is determined by an O-link IO analyte assay.

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