Combination therapies for treatment of BCMA-related cancers and autoimmune disorders

Abstract

To provide combination therapies for treatment of BCMA-related cancers and autoimmune disorders.SOLUTION: The present disclosure relates to methods for using BCMA-specific binding molecules (such as a BCMA-specific chimeric antigen receptor or antibody) in combination with γ-secretase inhibitors, which can be done concurrently or sequentially, to treat or prevent a B-cell related proliferative disease, such as a cancer or autoimmune disease, or the like. A BCMA-specific binding molecule in combination with γ-secretase inhibitor can be used in, for example, adoptive immunotherapy.SELECTED DRAWING: Figure 7B

Description

[Technical Field]

[0001] [Statement of Government Support] This invention was made with government support under CA136551 awarded by the National Institutes of Health.

[0002] [Sequence Listing Statement] The Sequence Listing accompanying this application is provided in text form in lieu of paper and is incorporated herein by reference. The name of the text file containing the Sequence Listing is 360056_445WO_SEQUENCE_LISTING.txt. The text file is 14.4 KB, was created on February 16, 2018, and is being submitted electronically hereby via EFS-Web. [Background technology]

[0003] The human immune system generally protects the body from invading foreign substances and pathogens. B lymphocytes, also known as B cells, are a component of the immune system that produce antibodies that bind to foreign substances and pathogens and, in some cases, mediate their destruction. However, in some cases, the immune system becomes dysregulated, leading to diseases associated with the uncontrolled proliferation of B cells, such as cancer, autoimmune diseases, and inflammatory disorders.

[0004] Mature B cells and their differentiated progeny can be identified by molecules on their cell surface, such as B cell maturation antigen (BCMA; also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), TNFRSF13A, and CD269), which is expressed on plasma cells and certain mature B cells. BCMA specifically binds to B cell-activating factor (BAFF; also known as TNFSF13B, TALL-1, and CD257) and proliferation-inducing ligand (APRIL; also known as TNFSF13, TALL-2, and CD256), which can lead to NF-κB activation. BCMA-targeted therapies, including adoptive transfer of T cells engineered with BCMA-specific chimeric antigen receptors (CARs), administration of naked BCMA-specific antibodies, or BCMA-specific antibodies conjugated to therapeutic moieties (antibody-drug conjugates (ADCs), such as radiolabeled antibodies), can be used to treat certain types of B-cell malignancies, such as multiple myeloma. However, efficacy can be limited by the number of BCMA molecules expressed on the tumor cell surface and / or the presence of soluble BCMA in the bloodstream. Low BCMA surface expression or the presence of soluble BCMA on cancer cells limits and inhibits the efficacy of therapeutic agents due to inappropriate binding to BCMA present on the surface of tumor cells. Low concentrations of other target molecules (e.g., CD19, CD20) on tumor cells targeted by antibodies, antibody-drug conjugates, or chimeric antigen receptor T cells have been shown to limit the efficacy of these therapies, allowing cancer cells expressing low levels of the target molecule to escape elimination. In the case of BCMA, a short extracellular portion of the molecule is cleaved and shed from the cell surface through the action of gamma-secretase (γ-secretase), a membrane-localized cellular enzyme involved in proteolytic cleavage. This cleavage reduces the density of BCMA on cells that express the molecule, such as myeloma cells, and leads to elevated levels of soluble BCMA (sBCMA) in the serum of patients with certain autoimmune diseases (e.g., systemic lupus erythematosus) and cancers (e.g., multiple myeloma).

[0005] Currently, there continues to be a need in the field of immunotherapy for alternative or improved compositions and methods for more effectively treating autoimmune diseases and cancer. Summary of the Invention

[0006] The present disclosure provides compositions and methods for treating autoimmune diseases and cancer through the combination of a B-cell maturation antigen (BCMA)-specific binding protein, either in soluble form or expressed on cytotoxic or other cells, with a gamma-secretase inhibitor (GSI). Polynucleotides encoding such BCMA-specific binding proteins can be used to create modified host immune cells (e.g., T cells) for use in, for example, adoptive immunotherapy. In certain embodiments, the present disclosure is directed to the use of such treatment in combination with a GSI in a subject in need of such treatment, where the latter treatment can be administered before, simultaneously with, or after adoptive immunotherapy (i.e., immunotherapy using modified immune cells expressing a BCMA-specific binding protein on their cell surface). Also provided are immunotherapies comprising administering a GSI in combination with a BCMA-specific binding protein, including an antibody or antigen-binding portion thereof, where the BCMA-specific binding protein, in certain embodiments, can be conjugated or otherwise linked to a cytotoxic agent, e.g., forming an antibody-drug conjugate (ADC). Useful therapeutic applications include the treatment of a proliferative disease or disorder (such as cancer), an autoimmune disease, or a disease or disorder associated with aging in a subject in which BCMA-positive cells are of pathogenesis. [Brief explanation of the drawings]

[0007] [Figure 1A]1A-1P show the design and functionality testing of exemplary chimeric antigen receptor (CAR) molecules of the present disclosure. (A) Diagram of a representative CAR with a BCMA-specific scFv derived from the A7D12.2 antibody ("A7") or the C11D5.3 antibody ("C11"), an extracellular component consisting of a spacer region (comprising an IgG4 hinge region) and a hydrophobic portion (comprising a CD28 transmembrane domain), and an intracellular component consisting of a CD3ζ effector domain and a 4-1BB costimulatory domain. The scFvs were configured with the C-terminus of the VH region linked to the N-terminus of the VL region (in the "HL" orientation) (via a "G4S" variable region linker (SEQ ID NO: 30)) or the C-terminus of the VL region linked to the N-terminus of the VH region (in the "LH" orientation). [Figure 1B] (B) Flow cytometry data (carboxyfluorescein succinimidyl ester (CFSE) staining) showing proliferation of human T cells expressing the CARs shown in Figure 1A and control T cells without CARs in response to BCMA-expressing tumor cells. [Figure 1C] (C) Cytokine production (IFN-γ) by the CAR-transduced T cells shown in Figure 1B when cultured in vitro with the indicated BCMA- (K562) or BCMA+ (K562BCMA, U266, MM1.S) tumor cell lines. [Figure 1D] (D) Specific lysis of the indicated target cell lines by BCMA CAR-T cells. [Figure 1E](E) Diagram of two exemplary CARs of the present disclosure, where the spacer region (Spacer+) can include a tag cassette, a linker module, a hinge, spacer amino acids, and any combination thereof. If a CAR includes one or more tags in the spacer region, it will be referred to as a T-ChARM, as described herein. The CAR in the upper part of the diagram includes an extracellular component (comprising a BCMA-specific scFv and a spacer region that may optionally include additional elements such as a tag or linker), a hydrophobic portion (comprising a CD28 transmembrane domain), and an intracellular component (comprising a CD3ζ effector domain and a 4-1BB costimulatory domain). The CAR in the lower part of the diagram includes an extracellular component (comprising a BCMA-specific scFv and a spacer region that may optionally include additional elements such as a tag or linker), a hydrophobic portion (comprising a CD28 transmembrane domain), and an intracellular component (comprising a CD3ζ effector domain and a CD28 costimulatory domain). Both of the two BCMA-specific CAR / T-ChARM constructs contain a transduction genetic marker comprising a truncated form of human EGFR (EGFRt), separated from the BCMA-specific CAR / T-ChARM construct by a self-cleaving Thoseaasigna virus 2A (T2A) peptide sequence. Other known self-cleaving peptides can be used as well, such as porcine teschovirus 1 2A (P2A), equine rhinitis A virus (ERAV) 2A (E2A), and foot-and-mouth disease virus (FMDV) 2A (F2A). [Figure 1F] (F) Exemplary CAR / T-ChARM constructs with spacer regions of various lengths. sh = 12 amino acid short spacer; 2ST = 48 amino acid spacer with two Strep tag cassettes; 3ST = 66 amino acid spacer with three Strep tag cassettes; 2ST Int = 157 amino acid intermediate spacer with two Strep tag cassettes; and Lo = 228 amino acid long spacer. The short and long spacers can optionally include a tag cassette, e.g., a Strep tag. [Figure 1G](G) Additional diagrams of exemplary CAR / T-ChARM constructs with C11 or A7 HL scFv and with different spacer regions (optionally including an STII tag). [Figure 1H] (H) The BCMA-recognizing and proliferative capacity of human T cells engineered with BCMA-specific CARs was measured by labeling T cells with carboxyfluorescein (CFSF), co-culture of CAR-T cells or control untransduced CFSE-labeled T cells with K562 cells (K562 / BCMA) transduced with a polynucleotide encoding full-length BCMA (K562 / BCMA), and measuring the dilution of CFSE with each cell division using flow cytometry. CFSE-labeled T cells containing different BCMA-specific CARs with different spacer lengths diluted CFSE after co-culture with K562 / BCMA cells, whereas control untransduced T cells (UT) did not. CAR-T cells containing 2ST or longer spacers proliferated better than CAR-T cells expressing short spacers. [Figure 1I] (I,J) Cytokine release (IFN-γ, I; IL-2, J) by anti-BCMA CAR-T cells with various spacer lengths in response to BCMA-expressing U266 and 8266 myeloma cells. [Figure 1J] (I,J) Cytokine release (IFN-γ, I; IL-2, J) by anti-BCMA CAR-T cells with various spacer lengths in response to BCMA-expressing U266 and 8266 myeloma cells. [Figure 1K] (K) Cell surface EGFRt and STII expression on CD8+ T cells transduced with anti-BCMA C11 T-ChARM or BCMA-2 CAR constructs after isolation and propagation. [Figure 1L](L,M) Cytokine release (IFN-γ, L; IL-2, M) by human CD4 (top panel) and CD8 (bottom panel) T cells engineered to express the disclosed C11 T-ChARM containing a 4-1BB costimulatory domain or a CD28 costimulatory domain, or by a previously disclosed anti-BCMA CAR ("BCMA-2"; see Carpenter et al., Clin. Cancer Res. 19:2048, 2013). [Figure 1M] (L,M) Cytokine release (IFN-γ, L; IL-2, M) by human CD4 (top panel) and CD8 (bottom panel) T cells engineered to express the disclosed C11 T-ChARM containing a 4-1BB costimulatory domain or a CD28 costimulatory domain, or by a previously disclosed anti-BCMA CAR ("BCMA-2"; see Carpenter et al., Clin. Cancer Res. 19:2048, 2013). [Figure 1N] (N) Expansion of human T cells engineered to express the C11 T-ChARM or BCMA-2 CAR of the present disclosure co-cultured with the indicated BCMA-expressing cell lines. [Figure 1O] (O) Lysis of K562 BCMA-negative cells by CD8 T cells engineered to express the disclosed C11 T-ChARM (○=41BB costimulatory domain, □=CD28 costimulatory domain) or BCMA-2 CAR (△) at the indicated E:T ratios. [Figure 1P] (P) Lysis of K562 cells transfected to express BCMA by engineered CD8 T cells shown in Figure 1O at various E:T ratios (x-axis). [Figure 2A]Figures 2A-2Q show the production of BCMA and PD-L1 by multiple myeloma (MM) cells in culture and the effect of soluble BCMA (sBCMA), surface-bound BCMA, and surface-bound PD-L1 on the ability of anti-BCMA T-ChARM-T cells to recognize tumor cells and produce IFN-γ. (A) U266 myeloma cells were washed and plated in medium for 1, 3, 5, and 24 hours. Media supernatants were collected and assayed for soluble BCMA by ELISA. Data show a time-dependent increase in soluble BCMA levels in the supernatants. [Figure 2B] (B) Histogram showing BCMA expression by representative patient primary MM cells or by reference MM cells (RPMI 8226) with high (Pt. 1), moderate (Pt. 2) or low / negative (Pt. 3) expression as measured by ELISA (black = anti-BCMA antibody; gray line = isotype control). [Figure 2C] (C) Chart showing the percentage of myeloma patients (n=19) with high, moderate, or low / negative BCMA expression on MM cells. [Figure 2D] (D) IFN-γ production by anti-BCMA C11 T-ChARM+ CD8+ T cells in response to stimulation with patient MM cells with high (left, n=5) or low (right, n=4) BCMA expression (2:1 E:T ratio for 24 hours (ELISA)). Significance was tested using an unpaired two-tailed T-test, and bars represent mean IFN-γ production + SEM. [Figure 2E] (E) Histogram showing PD-L1 expression by RPMI 8226 myeloma cells (left-most panel) and primary MM cells from three patients with high, low / negative, or moderate PD-L1 expression, as measured by ELISA (diagonal shading = staining with anti-PD-L1 antibody; blank histogram = isotype control). [Figure 2F] (F) Chart showing the percentage of patients (n=19) with high, intermediate, or low / negative PD-L1 expression in MM cells. [Figure 2G](G) IFN-γ production by anti-BCMA C11 T-ChARM+ CD8+ cells of the present disclosure in response to patient MM cells with high (left, n=4) or low (right, n=5) PD-L1 expression (24 h (ELISA) at an E:T ratio of 2:1). Significance was tested using an unpaired two-tailed t-test, and bars represent mean IFN-γ production + SEM. [Figure 2H] (H) IFN-γ production by BCMA-specific T-ChARM T cells in the presence of exogenous soluble BCMA. sBCMA was added to co-cultures of T cells expressing BCMA-specific T-ChARM T cells and K562 cells transduced with a polynucleotide encoding full-length BCMA (K562 / BCMA+). Dose-dependent inhibition of BCMA-specific T-ChARM T cell effector function is observed as measured by IFN-γ release into the culture supernatant. [Figure 2I] (I) Data from another dose titration experiment showing the effect of sBCMA on IFN-γ production by BCMA-specific T-ChARM T cells recognizing K562 BCMA+ cells, which involved adding sBCMA to the cultures at a different, higher concentration (5,000 ng / mL). [Figure 2J] (J) BCMA shedding by the indicated MM cells cultured in vitro. [Figure 2K] (K) sBCMA measured in bone marrow (BM) serum from patients with low (<2% CD138+ cells) or high (>2% CD138+ cells) disease burden. [Figure 2L] (L) sBCMA binding to C113ST-ChARM T cells of the present disclosure. T cells were incubated with the indicated levels of recombinant sBCMA (right side of the figure) and then stained with BCMA-Fc fusion bound to APC. [Figure 2M] (M) Flow cytometry data showing surface staining (BCMA-Fc and anti-EGFRt antibodies conjugated with APC) and CD4 expression by C113ST T-ChARM cells and FMC63 CAR-T cells. [Figure 2N](N) Flow cytometry data showing IFN-γ production (y-axis) and CD4 expression (x-axis) by T cells expressing either the C113ST-ChARM of the present disclosure (“C113ST”) or a control anti-CD19 CAR (“FMC632”) when co-cultured with target-expressing K562 cells and administered BCMA-Fc fusion protein (left panel: 0 ng / mL BCMA-Fc; right panel: 1000 ng / mL BCMA-Fc). [Figure 2O] (O) Dose titration showing IFN-γ release by CAR T cells in response to indicated target cell lines in the presence or absence of exogenous recombinant BCMA (BCMA-Fc fusion). FMC63 (anti-CD19) vs. K562 CD19+ (control; downward-pointing triangle ▽); C113ST T-ChARM T cells vs. RPMI 8226 (upward-pointing triangle △), U266 (square □), and K562 BCMA+ cells (circle ○). Data are representative of two independent experiments. Bars represent mean + SEM. P value (determined by one-way ANOVA with post-hoc test) ≤ 0.05. MFI = mean fluorescence intensity. [Figure 2P] (P) IFN-γ production (normalized mean fluorescence intensity, MFI) by T cells shown in Figure 2O in coculture with the indicated antigen-expressing cells in the presence or absence of BCMA-Fc (x-axis). [Figure 2Q] (Q) Cytolytic activity of CD8+ C113ST T-ChARM T cells against K562 BCMA+ target cells and FMC63 CAR-T cells against K562 CD19+ target cells, analyzed by 4h CRA at various concentrations of recombinant BCMA at an E:T ratio of 10:1. Data are representative of two independent experiments. Bars represent the mean + SEM. P value (determined by one-way ANOVA with post-hoc test) ≤ 0.05. MFI = mean fluorescence intensity. [Figure 3A]Figures 3A-3R show the effect of the γ-secretase inhibitor (GSI) RO4929097 on the levels of cell surface BCMA and other cell surface molecules in myeloma cell lines or primary myeloma cells. (A) Cell surface BCMA was measured by flow cytometry using an anti-BCMA monoclonal antibody in four myeloma cell lines (8226, U266B1, MM1.R, H929) before and 5 hours after incubation with GSI RO4929097 at concentrations ranging from 0 μM (DMSO control) to 1.0 μM (see figure). [Figure 3B] (B) Surface BCMA expression by MM.1R cells cultured with the indicated concentrations of RO4929097; staining with anti-BCMA antibody (black line) compared to isotype control (gray line). [Figure 3C] (C) Fold change in surface BCMA expression by MM cell lines upon culture with the indicated concentrations of RO4929097. Fold change is shown relative to untreated MM cells of the same cell line. [Figure 3D] (D) Kinetics of the change (fold) in surface BCMA expression over time by the indicated MM cells in culture with 1 μM RO4929097. [Figure 3E] (E) U266 myeloma cells were incubated with various concentrations of the GSI RO429097 (0.01 μM, 0.1 μM, 1.0 μM) for 1, 3, 5, and 24 hours, and surface BCMA expression was assessed by flow cytometry. BCMA expression increased in a dose-dependent manner in the presence of the GSI, with the greatest increase observed after 5 hours of exposure. [Figure 3F] (F) Fold change over time in surface BCMA expression on cell lines (MM1R = triangle; U266 = square; 8226 = circle) cultured in 1 μM GSI. GSI was re-administered as a half-volume change of medium every 2 days. BCMA fold change is defined as treatment group (MFIBCMA-MFIiso) / control group (MFIBCMA-MFIiso). Data are representative of at least two independent experiments. [Figure 3G](G,H) Culture supernatant concentrations of sBCMA in MM cell line cells cultured in the presence of the indicated concentrations of RO4929097. [Figure 3H] (G,H) Culture supernatant concentrations of sBCMA in MM cell line cells cultured in the presence of the indicated concentrations of RO4929097. [Figure 3I] (I) U266 myeloma cells were washed and plated in media in the presence of various concentrations of the GSI RO429097 (0.01 μM, 0.1 μM, and 1.0 μM). Media supernatants were collected after 1, 3, 5, and 24 hours and assayed for soluble BCMA (sBCMA) by ELISA. The data show that the presence of GSI at concentrations of at least approximately 0.01 μM reduced the amount of BCMA released from tumor cells into the supernatant over time. [Figure 3J] (J,K) Fold change in BCMA expression (J) and supernatant sBCMA concentration (K) at various time points after removal of 1 μM GSI from myeloma cell line cultures (GSI + / −) compared to cultures in the continued presence of GSI (GSI + / +). [Figure 3K] (J,K) Fold change in BCMA expression (J) and supernatant sBCMA concentration (K) at various time points after removal of 1 μM GSI from myeloma cell line cultures (GSI + / −) compared to cultures in the continued presence of GSI (GSI + / +). [Figure 3L] (L) Viability of the indicated MM-expressing cells as measured by propidium iodide staining of cell lines cultured in 1 μM GSI. [Figure 3M] (M) Surface BCMA expression by primary patient MM cells cultured with the indicated concentrations of RO4929097. Staining was as described for Figure 3B. [Figure 3N] (N) Fold change in BCMA on primary myeloma cells (n=7) cultured for 4 hours with various amounts of GSI. Primary cells and cell lines were cultured at 0.5 x 106 cells / mL. Fold change in BCMA is defined as treated group (MFIBCMA-MFIiso) / control group (MFIBCMA-MFIiso). Data are representative of three independent experiments using T cells from different donors. [Figure 3O] (O,P) Co-culture of primary myeloma cells with various concentrations of GSI for 4 hours does not affect the levels of several other cell surface molecules on tumor cells, including CS1, CD86, PD-L1, CD80, and CD38. [Figure 3P] (O,P) Co-culture of primary myeloma cells with various concentrations of GSI for 4 hours does not affect the levels of several other cell surface molecules on tumor cells, including CS1, CD86, PD-L1, CD80, and CD38. [Figure 3Q] (Q) Staining of various surface markers on MM1R cells in the presence (black) or absence (gray) of 1 μM GSI in the medium. Isotype staining is shown as an open plot. [Figure 3R] (R) CD138+ primary myeloma cells were enriched from patient bone marrow samples, incubated for 3 hours in the presence of various concentrations of the GSI RO4929097 (0.01 μM to 10 μM), and assessed for surface BCMA expression by flow cytometry. BCMA mean fluorescence intensity (MFI) of tumor cells is expressed as the fold increase over that observed in tumor cells incubated in the absence of RO4929097. A dose-dependent upregulation of BCMA is observed. [Figure 4A] Figures 4A-4C show that pretreatment of myeloma cells with GSI increases cytokine release after recognition of primary myeloma cells by BCMA-specific CAR-T cells. (A) IL-2 production by BCMA CAR-T cells (BCMA-specific T-ChARM C11 3ST-CD28 and BCMA-specific T-ChARM C11 3ST-41BB) or control CD19sh CAR (short spacer)-T cells cocultured with primary human myeloma cells for 24 hours alone or in the presence of various concentrations of the GSI RO4929097 (0.003 μM to 3.0 μM). [Figure 4B] (B) IFN-γ production by BMCA T-ChARM T cells cocultured with myeloma cells in the presence of various concentrations of RO4929097. [Figure 4C](C) Proliferation of CFSE-labeled BCMA-specific T-ChARM T cells increased in a dose-dependent manner after 3 days of coculture with primary human myeloma cells in medium alone or medium containing the indicated concentrations of GSI RO492097. [Figure 5A] Figures 5A-5R show the effect of various concentrations of GSI on the viability, proliferation, and functional activity of CAR-T cells. (A) CD19 staining of K562 CD19+ cells and Raji cells cultured with or without GSI for ±12-16 hours. Isotype control is indicated by the gray line. [Figure 5B] (B) Primary human T cells were cultured in concentrations of GSI RO4929097 ranging from 0.01 μM to 100 μM, and viability was measured by trypan blue exclusion 24 hours later. No effect of GSI on T cell viability was observed at any concentration. [Figure 5C] (C) CD19 CAR-T cells were co-cultured with K562 / CD19 target cells in medium containing various concentrations of RO4929097. RO4929097 inhibits CD19 CAR-T cell effector function at concentrations ≥ 3 μM upon co-culture, as determined by measuring IL-2 (upper panel) and IFNγ (lower panel) production. Boxes indicate the relevant therapeutic window of the drug that does not inhibit CAR-T cell effector function. [Figure 5D] (D) IL-2 production by CD19 CAR-T cells cultured with target cells in the presence of increasing concentrations of the GSI RO4929097. [Figure 5E] (E) IL-2 production by CD19 CAR-T cells cultured with target cells ("K562 CD19") or control cells ("K562 BCMA") in the presence of increasing concentrations of the GSI RO4929097. Cells were dosed with the indicated amount of GSI and then washed (open bars) or not (filled bars). [Figure 5F](F) Data from another experiment showing IL-2 production by CD19 CAR-T cells after preincubation with various concentrations of GSI followed by overnight co-culture with K562 BCMA+ or K562 CD19+ cells. After washing, GSI was added back into the co-culture (+ / +) or omitted (+ / -) to assess the reversibility of cytokine production. [Figure 5G] (G) Specific lysis of indicated target or control cells by CD19 CAR-T cells in the presence of GSI RO4929097. [Figure 5H] (H) Proliferation of CD19 CAR-T cells cultured with CD19-expressing target cells in the presence or absence of GSI, or with control cells in the absence of GSI. Cells were stained with CFSE, and proliferation was measured by flow cytometry. [Figure 5I] (I) Graphical representation of the number of CD19 CAR-T cell divisions in the presence of the indicated concentrations of GSI RO4929097. The width of the horizontal bar represents the proportion of CAR-T cells in the culture that underwent the indicated number of generations (i.e., 5, 4, 3, 2, 1, or 0) over the course of the experiment. [Figure 5J] (J) Cell counts (CD8 staining) during expansion of CD19+ TM LCL cells and CD19-specific CAR-T cells with exogenous IL-2 in the absence (circle ○) or presence of 0.5 μM (square □) or 5 μM (triangle ▽) of GSI. [Figure 5K] (K) Cell counts of CD19-specific CAR-T cells (CD4:CD8 (1:1)) grown with CD19+ TM.LCL cells in the absence or presence of GSI as indicated, but without the addition of exogenous IL-2. [Figure 5L] (L) IFN-γ concentrations in the supernatants of anti-CD19 CAR CD8+ T cells grown in the presence of GSI after restimulation with K562 cells (no antigen), K562 CD19+ cells, or Raji cells. [Figure 5M](M,N) IFN-γ (M) and IL-2 (N) production by CD4:CD8 anti-CD19 CAR-T cell mixtures after restimulation with the indicated cell lines in the absence of GSI or in the presence of either 0.5 μM or 5 μM GSI. [Figure 5N] (M,N) IFN-γ (M) and IL-2 (N) production by CD4:CD8 anti-CD19 CAR-T cell mixtures after restimulation with the indicated cell lines in the absence of GSI or in the presence of either 0.5 μM or 5 μM GSI. [Figure 5O] (O) Intracellular staining showing IFN-γ production (y-axis) and CD8 expression (x-axis) by T-ChARM T cells of the present disclosure cultured with primary MM cells from two patients in the absence (0 μM; left panel) or presence (1 μM; right panel) of GSI RO4929097. [Figure 5P] (P) IFN-γ production (geometric mean fluorescence intensity (gMFI)) by T-ChARM T cells of the present disclosure cultured with primary MM cells in the presence of the indicated concentrations of RO4929097 (x-axis). [Figure 5Q] (Q) IFN-γ production (normalized MFI; y-axis) by T-ChARM T cells of the present disclosure cultured with primary MM cells in the presence of the indicated concentrations of RO4929097 (x-axis). [Figure 5R] (R) Proliferation of T-ChARM T cells of the present disclosure in the presence of primary MM cells untreated (gray shading) or treated with 1.0 μM GSI RO4929097. [Figure 6A] Figures 6A-6C show the effect of GSI RO4929097 on BCMA expression in a preclinical in vivo model of multiple myeloma. (A) Experimental scheme for the disseminated xenograft mouse myeloma model. NSG mice were irradiated (275 rad) to promote tumor engraftment and administered human MM tumor cells (5 × 10 MM.1R) followed by GSI treatment (30 mg / kg). Mice were then euthanized, and blood and BM samples were collected to determine whether GSI upregulated BCMA expression on myeloma cells in vivo. [Figure 6B](B) Change (fold) in surface BCMA expression on myeloma cells in mice euthanized at the indicated time points after the second GSI administration. [Figure 6C] (C) sBCMA levels in serum of mice sacrificed at the indicated time points after administration of RO4929097. [Figure 7A] Figures 7A-7E show the effect of GSI RO4929097 on anti-BCMA CAR-T cell therapy in a preclinical mouse MM model. (A) Experimental scheme: Mice were irradiated and then administered human MM tumor cells (5 x 10 MM.1R expressing firefly luciferase). Twenty days later, mice were administered GSI (30 mg / kg) at the indicated time points and a single suboptimal dose of anti-BCMA T-ChARM T cells (0.33 x 10 cells, 1:1 CD4:CD8) on day 0. Bioluminescence imaging (BLI) and viability were monitored throughout the study. [Figure 7B] (B) BLI images of mice taken on days 2, 17, and 16 after treatment with C113ST T-ChARM T cells (0.33 × 10 cells at 0 mg / kg, 1:1 CD4:CD8, left panel; 30 mg / kg, middle panel) or control FM63 anti-CD19 CAR-T cells (0.33 × 10 cells, 1:1 CD4:CD8, 30 mg / kg, right panel). [Figure 7C] (C) Quantitative luminescence data from BLI shown in Figure 8B. [Figure 7D] (D) Survival rate of mice shown in Figure 8B after administration of T-ChARM T cells. [Figure 7E] (E) (Left) Quantitative luminescence data from BLI shown in Figure 8B. (Right) Survival of mice after administration of T-ChARM T cells. [Figure 8] Figure 8 shows flow cytometry analysis of binding to H929 MM cells by bispecific fusion molecules with specificity for BCMA in the presence or absence of GSI. A control bispecific fusion molecule that does not target BCMA was also tested. DETAILED DESCRIPTION OF THE INVENTION

[0008] Before setting forth this disclosure in more detail, it may be helpful to an understanding of the invention to provide definitions of certain terms used herein. Additional definitions are provided throughout this disclosure.

[0009] As used herein, any concentration range, percentage range, ratio range, or integer range should be understood to encompass any integer value within the recited range, and, where appropriate, fractions thereof (e.g., 1 / 10 and 1 / 100 of an integer), unless otherwise specified. Additionally, numerical ranges recited herein relating to any physical characteristic, such as polymer subunits, size, or thickness, are understood to include any integer within the recited range, unless otherwise specified. As used herein, the term "about" means ±20% of the indicated range, value, or structure, unless otherwise specified. The terms "a" and "an," as used herein, should be understood to refer to "one or more" of the indicated component. The use of alternatives (e.g., "or") should be understood to mean any one, two, or any combination of the alternatives. As used herein, the terms "comprise," "have," and "comprise" are used synonymously, and these terms and variations should be construed as open-ended.

[0010] In addition, it should be understood that individual compounds or groups of compounds derived from the various combinations of structures and substituents described herein are disclosed by this application to the same extent as if each compound or group of compounds were individually referenced. Thus, selection of a particular structure or particular substituents is within the scope of this disclosure.

[0011] The term "consisting essentially of" limits the scope of a claim to particular substances or steps or steps that do not materially affect the essential characteristics of the claimed invention. For example, a protein domain, region, module, or cassette (e.g., a binding domain, hinge region, linker module, tag cassette) or protein (which may comprise one or more domains, regions, modules, or cassettes) is said to "consist essentially of" a particular amino acid sequence (e.g., amino- or carboxy-terminal amino acids or inter-domain amino acids) when the amino acid sequence of the domain, region, module, cassette, or protein contains extensions, deletions, mutations, or combinations thereof that, when combined, involve up to 20% of the length of the domain, region, module, cassette, or protein (e.g., up to 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%) and do not substantially affect (i.e., reduce) the activity of the domain, region, module, cassette, or protein (e.g., target binding affinity of a binding protein or tag cassette) (i.e., do not reduce said activity by more than 50%, e.g., reduce said activity by no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%).

[0012] As used herein, "proliferative disorder" refers to excessive or otherwise abnormal growth or proliferation compared to normal or unaffected cells. Excessive or abnormal growth includes, for example, unregulated growth or proliferation that can occur rapidly (e.g., hyperproliferation) or that can progress slowly or incrementally over time within native tissues (e.g., multiple myeloma) (e.g., plasmacytoma growth in the bone marrow), as well as spreading / growing into distant tissues or body sites not native to the affected cells. Exemplary proliferative disorders include tumors, cancers, tumor tissues, carcinomas, sarcomas, malignant cells, precancerous cells, as well as non-neoplastic or non-malignant proliferative disorders (e.g., adenoma, fibroma, lipoma, leiomyoma, hemangioma, fibrosis, restenosis), or autoimmune diseases (e.g., rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, etc.).

[0013] As used herein, a "binding protein" (also referred to as a "binding domain," "binding region," or "binding moiety") refers to a molecule, such as a peptide, oligopeptide, polypeptide, or protein, that has the ability to specifically and non-covalently associate with, integrate with, or bind to a target molecule (e.g., BCMA). Binding proteins include any natural, synthetic, semi-synthetic, or recombinantly produced binding partner for a biological molecule, compound, or other target of interest. In some embodiments, the binding protein is derived from an immunoglobulin or immunoglobulin-like molecule, such as an antibody or T-cell receptor (TCR), that comprises a functional binding domain or an antigen-binding fragment thereof. Exemplary binding proteins include single-chain antibody variable regions (e.g., domain antibodies, sFv, scFv, Fab), BCMA ligands (e.g., BAFF, APRIL, and binding fragments thereof), antigen-binding regions of T-cell receptors (TCRs), such as single-chain TCRs (scTCRs), or synthetic polypeptides selected for their specific ability to bind to a biomolecule.

[0014] As used herein, "specifically binds" means to bind to a target molecule. 5 M -1 or greater affinity or K a (i.e., the equilibrium association constant of a particular binding interaction in units of 1 / M) while not significantly associating with or integrating with any other molecules or components in the sample. A binding domain (or fusion protein thereof) can be classified as a "high affinity" binding domain (or fusion protein thereof) or a "low affinity" binding domain (or fusion protein thereof). A "high affinity" binding domain is one that has at least 10 7 M -1 , at least 10 8 M -1 , at least 10 9 M -1 , at least 10 10 M -1 , at least 10 11 M -1 , at least 10 12M -1 , or at least 10 13 M -1 K a "Low affinity" binding domains refer to those binding domains that have a maximum affinity of 10 7 M -1 , up to 10 6 M -1 , up to 10 5 M -1 K a Alternatively, affinity refers to the equilibrium dissociation constant (K) of a particular binding interaction, in units of M. d ) (e.g., 10 -5 M to 10 -13 In certain embodiments, a binding domain has "enhanced affinity," which refers to a selected or engineered binding domain that binds to a target antigen more strongly than the wild-type (or parent) binding domain. For example, enhanced affinity refers to a K for a target antigen that is higher than that of the wild-type binding domain. a (equilibrium association constant) or a K for the target antigen that is lower than that of the wild-type binding domain d (dissociation constant) or an off-rate (K) for the target antigen that is lower than that of the wild-type binding domain off A variety of assays are known for identifying binding domains of the present disclosure that specifically bind to particular targets, as well as for determining the affinity of binding domains or fusion proteins, such as Western blots, ELISAs, and Biacore® analysis (see, e.g., Scatchard et al., Ann. NY Acad. Sci. 51:660, 1949; and U.S. Pat. Nos. 5,283,173, 5,468,614, or equivalents).

[0015] As used herein, "heterologous" or "non-endogenous" or "exogenous" refers to any gene, protein, compound, molecule, or activity that is not native to a host cell or subject, or any gene, protein, compound, molecule, or activity that is native to a host or host cell but that has been modified or mutated so that its structure, activity, or both differ, such as between the native molecule and a mutant molecule. In certain embodiments, a heterologous, non-endogenous, or exogenous molecule (e.g., receptor, ligand) may not be endogenous to a host cell or subject, but instead, a nucleic acid encoding such a molecule may have been added to the host cell by conjugation, transformation, transfection, electroporation, etc., where the added nucleic acid molecule may be integrated into the host cell genome or exist as extrachromosomal genetic material (e.g., as a plasmid or other self-replicating vector). The term "homologous" or "homolog" refers to a molecule or activity found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous molecule or gene encoding the molecule may be homologous to the native host or host cell or gene encoding the molecule, respectively, but may have an altered structure, sequence, expression level, or a combination thereof. A non-endogenous molecule may be from the same species, a heterologous species, or a combination thereof.

[0016] The terms "endogenous" or "native" as used herein refer to a gene, protein, compound, molecule, or activity that is normally present in a host or host cell.

[0017] As used herein, a "tag cassette" refers to a unique peptide sequence affixed to, fused to, or part of a protein of interest to which a heterologous or non-endogenous cognate binding molecule (e.g., receptor, ligand, antibody, or other binding partner) can specifically bind, where the binding properties are used to detect, identify, isolate, purify, track, enrich, or target the tagged protein or cells expressing the tagged protein, particularly when the tagged protein is a heterogeneous population of proteins or other substances, or when the cells expressing the tagged protein are a heterogeneous population of cells (e.g., a biological sample such as peripheral blood). In certain embodiments, cells expressing the tagged protein can be contacted with a heterologous or non-endogenous cognate binding molecule to elicit a biological response, such as promoting cell activation, cell proliferation, or cell death. The resulting fusion protein is one in which the ability of the tag cassette to be specifically bound by the cognate binding molecule(s) is distinct from or complementary to the ability of the binding domain(s) to specifically bind to the target molecule(s). The tag cassette is generally not an antigen-binding molecule, for example, not an antibody or TCR or antigen-binding portion thereof.

[0018] As used herein, "hinge region" or "hinge" refers to (a) an immunoglobulin hinge sequence (e.g., composed of upper and core regions) or a functional fragment or variant thereof, (b) a type II C-lectin interdomain (stalk) region or a functional fragment or variant thereof, or (c) a stalk region of a clustering (CD) molecule or a functional variant thereof. As used herein, a "wild-type immunoglobulin hinge region" refers to the naturally occurring upper and middle hinge amino acid sequences inserted between and connecting the CH1 and CH2 domains present in the heavy chain of an antibody (in the case of IgG, IgA, and IgD) or between and connecting the CH1 and CH3 domains (in the case of IgE and IgM). In certain embodiments, the hinge region is human, and in certain embodiments, comprises a human IgG hinge region.

[0019] As used herein, a "spacer region" refers to one or more proteins, polypeptides, oligopeptides, peptides, domains, regions, modules, cassettes, motifs, or any combination thereof that link two or more proteins, polypeptides, oligopeptides, peptides, domains, regions, modules, cassettes, motifs, or any combination thereof in a fusion protein. For example, a spacer region can provide a separation or spacing function that promotes interaction between two single-chain fusion proteins, or a function that facilitates positioning of one or more binding domains, such that the resulting polypeptide structure maintains specific binding affinity to a target molecule, maintains signaling activity (e.g., effector domain activity), or both. In certain embodiments, a spacer region can include a "linker module," an amino acid sequence having from about 2 to about 500 amino acids, which can provide flexibility and space for conformational movement between the two regions, domains, motifs, cassettes, or modules connected by the linker. Exemplary linker modules include Gly, Gly(II), ... x Ser yand (SEQ ID NO: 31) having 1 to about 10 repeats of the sequence (SEQ ID NO: 31), where x and y are independently integers between 0 and 10, provided that both x and y are not 0 (e.g., (Gly4Ser)2 (SEQ ID NO: 32), (Gly3Ser)2 (SEQ ID NO: 33), Gly2Ser, or a combination thereof, such as (Gly3Ser)2Gly2Ser (SEQ ID NO: 34)). In certain other embodiments, the spacer region can have a linker module comprising one or more immunoglobulin heavy chain constant regions, such as CH3 alone or CH2CH3. In further embodiments, the spacer region can comprise a hinge region or a tag cassette. Such linking components are not mutually exclusive. For example, the spacer region may comprise one hinge region and one or more linker modules, or the spacer region may comprise one hinge region, one or more linker modules, and one or more tag cassettes. Typical spacer regions can vary in length, for example, from about 5 to about 500 amino acids, or from about 10 to about 350 amino acids, or from about 15 to about 100 amino acids, or from about 20 to about 75 amino acids, or from about 25 to about 35 amino acids. Typical short spacers range from about 5 to about 100 amino acids (e.g., 12, 15, 48, 50, 66, 70 amino acids), medium spacers range from about 100 to about 200 amino acids (e.g., 110, 120, 130, 140, 150, 157, 175 amino acids), and long spacers range from about 200 to about 500 amino acids (e.g., 200, 210, 220, 228, 230, 250, 300, 350, 400, 450 amino acids).

[0020] As used herein, "hydrophobic portion" refers to any amino acid sequence that has a thermodynamically stable three-dimensional structure in a cell membrane and generally ranges in length from about 15 to about 30 amino acids. The structure of the hydrophobic domain can include an α-helix, a β-barrel, a β-sheet, a β-helix, or any combination thereof.

[0021] As used herein, an "effector domain" is an intracellular portion of a fusion protein or receptor that can directly or indirectly promote a biological or physiological response of a cell upon receiving an appropriate signal. In certain embodiments, the effector domain is part of a protein or protein complex that receives a signal when bound thereto, or it directly binds to a target molecule, initiating a signal from the effector domain. An effector domain can directly promote a cellular response if it contains one or more signaling domains or motifs, such as immunoreceptor tyrosine-based activation motifs (ITAMs), and such a cellular response can be assisted or improved by a costimulatory domain or functional portion thereof. An exemplary protein having an effector domain is CD3ζ. In other embodiments, an effector domain will indirectly promote a cellular response by associating with one or more other proteins that directly promote the cellular response.

[0022] The term "costimulatory domain" as used herein refers to a signaling component that provides a signal to a T cell that mediates a T cell response, such as activation, proliferation, differentiation, cytokine secretion, etc., in addition to the primary (effector) signal provided, for example, by the CD3ζ chain of the TCR / CD3 complex. In certain embodiments, the intracellular component comprises an effector domain or a functional portion thereof, a costimulatory domain or a functional portion thereof, or any combination thereof.

[0023] A "variable region linker" refers to a linker that connects a heavy chain immunoglobulin variable region to a light chain immunoglobulin variable region, or a linker that connects a T cell receptor V α / β Chain and C α / β Linking chains (e.g. V α -C α , V β -C β , V α -V β ), or each V α -C α , V β -Cβ , V α -V β The term "variable region linker" refers to a 5 to about 35 amino acid sequence that links the pair to a hinge or hydrophobic domain, providing sufficient spacer function and flexibility for the two sub-binding domains to interact so that the resulting single-chain polypeptide retains specific binding affinity for the same target molecule as an antibody or T cell receptor. In certain embodiments, the variable region linker comprises about 10 to about 30 amino acids or about 15 to about 25 amino acids. In certain embodiments, the variable region linker peptide comprises a nucleotide sequence consisting of Gly. x Ser y wherein x and y are independently integers of 1 to 5, e.g., Gly4Ser (SEQ ID NO: 1), Gly3Ser (SEQ ID NO: 2), Gly2Ser, or (Gly3Ser) n (Gly4Ser)1 (SEQ ID NO: 3), (Gly3Ser) n (Gly4Ser) n (SEQ ID NO: 4), or (Gly4Ser) n (SEQ ID NO: 5), where n is an integer of 1, 2, 3, 4, or 5], and the linked variable regions form a functional binding domain (e.g., scFv, scTCR).

[0024] "Junction amino acids" or "junction amino acid residues" refer to one or more (e.g., about 2-20) amino acid residues that connect two adjacent motifs, regions, or domains of a polypeptide, such as between a binding domain and an adjacent linker region or between a hydrophobic domain and an adjacent effector domain, or that are located on one or both ends of a linker region that connects two motifs, regions, or domains (e.g., between a linker and an adjacent binding domain and / or between a linker and an adjacent hinge region). Junction amino acids can result from the design of a fusion protein construct (e.g., amino acid residues resulting from the use of restriction enzyme sites in the construction of a nucleic acid molecule encoding a fusion protein).

[0025] Terms understood by those skilled in the antibody art will be given their art-acquired meaning unless clearly defined differently herein. The term "antibody" refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as an antigen-binding portion of the intact antibody that has or retains the ability to bind a target molecule. Monoclonal antibodies, or antigen-binding portions thereof, can be non-human, chimeric, humanized, or human, preferably humanized or human. The structure and function of immunoglobulins are reviewed, for example, in Chapter 14 of Antibodies: A Laboratory Manual, edited by Harlow et al. (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).

[0026] For example, the term "V L " and "V H " refers to the variable binding regions from the light and heavy chains of an antibody, respectively. The variable binding regions are composed of separate, well-defined "complementarity-determining regions" (CDRs) and subregions known as "frameworks" (FRs). The term "C L (or CL)" refers to an "immunoglobulin light chain constant region" or "light chain constant region," i.e., the constant region from an antibody light chain. H "(or CH)" refers to "immunoglobulin heavy chain constant region" or "heavy chain constant region," which can be further subdivided into CH1, CH2, and CH3 domains (IgA, IgD, IgG) or CH1, CH2, CH3, and CH4 domains (IgE, IgM) depending on the antibody isotype. "Fab" (antigen-binding fragment) is the portion of an antibody that binds to an antigen, and it contains the variable region and CH1 of the heavy chain linked to a light chain via an interchain disulfide bond.

[0027] As used herein, "Fc region portion" refers to the heavy chain constant region segment of the Fc fragment from an antibody ("fragment crystallizable" or Fc region), which can include one or more constant domains, e.g., CH2, CH3, CH4, or any combination thereof. In certain embodiments, the Fc region portion includes the CH2 and CH3 domains of an IgG, IgA, or IgD antibody, or any combination thereof, or the CH3 and CH4 domains of an IgM or IgE antibody, or any combination thereof. In other embodiments, the CH2CH3 or CH3CH4 structure has subregion domains from the same antibody isotype and is human, such as human IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgD, IgE, or IgM (e.g., CH2CH3 from human IgG1). By way of background, the Fc region is responsible for immunoglobulin effector functions, such as ADCC (antibody-dependent cell-mediated cytotoxicity), CDC (complement-dependent cytotoxicity) and complement fixation, binding to Fc receptors (e.g., CD16, CD32, FcRn), longer in vivo half-life compared to polypeptides lacking the Fc region, protein A binding, and possibly even placental passage (see Capon et al., Nature 337:525, 1989). In some embodiments, the Fc region portion found in the fusion proteins of the present disclosure may be capable of mediating one or more of the foregoing effector functions or may lack one or more or all of these activities, e.g., by one or more mutations known in the art.

[0028] In addition, antibodies typically have a hinge sequence located between the Fab and Fc regions (although the lower portion of the hinge may include the amino-terminal portion of the Fc region). By way of background, immunoglobulin hinges act as flexible spacers that allow the Fab portion to move freely in space. In contrast to constant regions, hinges are structurally diverse, differing in both sequence and length between immunoglobulin classes and even subclasses. For example, the human IgG1 hinge region is freely flexible, allowing the Fab fragments to rotate about their axis of symmetry and move within a sphere centered on the first of the two inter-heavy chain disulfide bridges. In contrast, the human IgG2 hinge is relatively short and contains a rigid polyproline double helix stabilized by four inter-heavy chain disulfide bridges that limit its flexibility. The human IgG3 hinge differs from the other subclasses by a unique extended hinge region (approximately four times the length of the IgG1 hinge) that contains 62 amino acids (including 21 prolines and 11 cysteines), forms a rigid polyproline double helix, and provides greater flexibility because the Fab fragment is relatively far from the Fc fragment. The human IgG4 hinge is shorter than IgG1 but is the same length as IgG2, and its flexibility is intermediate between that of IgG1 and IgG2.

[0029] "T cell receptor" (TCR) refers to a molecule found on the surface of T cells (or T lymphocytes) that typically functions, in association with CD3, to recognize antigens bound to major histocompatibility complex (MHC) molecules. The TCR comprises a disulfide-linked heterodimer of hypervariable α and β chains (also known as TCRα and TCRβ, respectively) in the majority of T cells. In a small subset of T cells, the TCR is composed of a heterodimer of variable γ and δ chains (also known as TCRγ and TCRδ, respectively). Each chain of the TCR is a member of the immunoglobulin superfamily and contains an N-terminal immunoglobulin variable domain, an immunoglobulin constant domain, a transmembrane region, and a short C-terminal cytoplasmic tail (see Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd ed., Current Biology Publications, p. 4:33, 1997). The TCRs used in this disclosure may be derived from a variety of animal species, including human, mouse, rat, cat, dog, goat, horse, or other mammals. The TCRs can be cell-bound (i.e., have a transmembrane region or domain) or in soluble form.

[0030] "Major histocompatibility complex molecules" (MHC molecules) refer to glycoproteins that deliver peptide antigens to the cell surface. MHC class I molecules are heterodimers consisting of a transmembrane α chain (with three α domains) and non-covalently associated β2-microglobulin. MHC class II molecules are composed of two transmembrane glycoproteins, α and β (both spanning the membrane). Each chain has two domains. MHC class I molecules deliver peptides from the cytosol to the cell surface, where they are expressed by CD8 + T cells recognize peptide:MHC complexes. MHC class II molecules deliver peptides from the vesicular system to the cell surface, where they are identified by CD4 + Recognized by T cells. MHC molecules may be derived from a variety of animal species, including human, mouse, rat, or other mammals.

[0031] A "vector" is a nucleic acid molecule capable of transporting another nucleic acid. A vector can be, for example, a plasmid, cosmid, virus, or phage. An "expression vector" is a vector that is capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment.

[0032] A "retrovirus" is a virus with an RNA genome. A "gammaretrovirus" refers to a genus of the Retroviridae family. Exemplary gammaretroviruses include murine stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus.

[0033] "Lentivirus" refers to a genus of retrovirus that can infect dividing and non-dividing cells.Some examples of lentivirus include HIV (human immunodeficiency virus: including HIV type 1 and HIV type 2); equine infectious anemia virus; feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV).

[0034] "T cell" or "cell of T cell lineage" refers to a cell that displays at least one phenotypic characteristic of a T cell or its precursor or progenitor, which distinguishes the cell from other cells of the lymphoid, erythroid, or myeloid lineage. Such phenotypic characteristics include expression of one or more proteins specific for T cells (e.g., CD3 + , CD4 + , CD8 + ), or physiological, morphological, functional, or immunological characteristics specific to T cells. For example, cells of the T cell lineage are precursor or progenitor cells that are committed to the T cell lineage; CD25 + Immature and inactivated T cells; cells that have undergone CD4 or CD8 lineage commitment; CD4 + CD8 + Double positive thymic progenitor cells; single positive CD4 + or CD8+ TCRαβ or TCRγδ; or functional mature or activated T cells.

[0035] A "nucleic acid molecule" or "polynucleotide" can be in the form of RNA or DNA, and includes cDNA, genomic DNA, and synthetic DNA. A nucleic acid molecule can be double-stranded or single-stranded, and if single-stranded, can be the coding strand or the non-coding strand (antisense strand). A coding molecule may have the same coding sequence as a coding sequence known in the art, or may have a different coding sequence that, as a result of redundancy or degeneracy in the genetic code or as a result of splicing, is capable of encoding the same polypeptide.

[0036] "Treating," "treatment," or "ameliorating" refers to the medical management of a disease, disorder, or condition in a subject (e.g., a human or non-human animal, e.g., a primate, horse, dog, mouse, or rat). For example, an appropriate dosing or therapeutic regimen, including the use of a BCMA-specific binding protein or a host cell expressing a BCMA-specific binding protein in combination with a gamma-secretase inhibitor (GSI) of the present disclosure, and optionally an adjuvant or preconditioning regimen, is administered to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic benefit includes improved clinical outcome; alleviation or amelioration of symptoms associated with the disease; reduction in the onset of symptoms; improved quality of life; stabilization of the disease state; delay in disease progression; remission; survival; long-term survival; or any combination thereof.

[0037] A "therapeutically effective amount" or "effective amount" of a BCMA-specific binding protein (also referred to as a BCMA-specific therapy or BCMA-targeted immunotherapy), a γ-secretase inhibitor, a host cell expressing a BCMA-specific binding protein, or a host cell expressing a γ-secretase inhibitor of the present disclosure (e.g., a BCMA-specific CAR, an anti-γ-secretase antibody) refers to an amount of compound or cells sufficient to result in a statistically significant improvement in one or more symptoms of the disease being treated. When referring to an individual active ingredient administered alone or cells expressing a single active ingredient, the therapeutically effective amount refers to the effect of that ingredient or the effect of cells expressing only that ingredient. When referring to a combination, the therapeutically effective amount refers to the combined amount of active ingredients, or the combined amount of supplemental active ingredients in combination with cells expressing active ingredients, whether administered sequentially, simultaneously, or concomitantly, that results in a therapeutic effect. Another combination can be cells expressing more than one active ingredient, such as two or more different BCMA-specific binding proteins.

[0038] Additional definitions are provided throughout this disclosure.

[0039] B-cell maturation antigen (BCMA) binding proteins or molecules In certain aspects, the present disclosure provides methods of treating a proliferative or autoimmune disease or disorder in a subject having or suspected of having the disease or disorder, comprising administering to the subject a therapeutically effective amount of a BCMA-specific binding protein (or BCMA-targeted immunotherapy) and a therapeutically effective amount of a γ-secretase inhibitor. Exemplary BCMA-specific proteins are chimeric antigen receptors comprising an extracellular component and an intracellular component linked by a hydrophobic moiety, wherein the extracellular component comprises a BCMA-specific binding domain (e.g., a BCMA-specific scFv, a BCMA ligand or binding portion thereof, e.g., BAFF or APRIL) and optionally a spacer region or hinge, and the intracellular component comprises an effector domain and optionally a costimulatory domain.

[0040] In certain embodiments, the present disclosure provides BCMA-targeted immunotherapies for use in conjunction with a gamma-secretase inhibitor to treat a proliferative or autoimmune disease or disorder, comprising a BCMA-specific antibody or antigen-binding portion thereof, a chimeric antigen receptor (CAR), or a tagged chimeric antigen receptor molecule (T-ChARM). In certain embodiments, the BCMA-specific antibody or antigen-binding portion thereof is human or humanized.

[0041] Exemplary BCMA-specific antibodies include antibodies J22.0-xi, J22.9-xi, J6M0, J6M1, J6M2, J9M0, J9M1, J9M2, 11D5-3, CA8, A7D12.2, C11D5.3, C12A3.2, C13F12.1, 13C2, 17A5, 83A10, 13A4, 13D2, 14B11, 14E1, 29B11, 29F3, 13A7, CA7, SG1, S307118G03, S332121F02, S332126E04, S322110D07, S336105A07, S335115G01, S335122F05, ET140-3, ET140-24, ET140-37, ET140-40, ET140-54, TBL-CLN1, C4.E2.1, Vicky-1, pSCHLI333, pSCHLI372, and pSCHLI373, and antigen-binding portions thereof. Various embodiments of BCMA-specific antibodies and antigen-binding portions thereof, including humanized variants, are described, for example, in PCT Publication Nos. WO 2002 / 066516, WO 2007 / 062090, WO 2010 / 104949, WO 2011 / 108008, WO 2012 / 163805, WO 2014 / 068079, WO 2015 / 166073, WO 2014 / 122143, WO 2014 / 089335, WO 2016 / 090327, and WO 2016 / 079177; Ryan et al., Mol. Cancer. Ther. 6(11): 3009, 2007; and Abbas et al., Blood 128:1688, and BCMA-specific antibodies, antigen-binding portions thereof, and humanized variants thereof, all of which are incorporated herein by reference. Variable domains and scFv molecules from these BCMA-specific antibodies can be used as binding domains in any of the T-ChARM and CAR proteins referred to herein.

[0042] Antigen-binding portions or domains derived from the BCMA-specific antibodies of the present disclosure and useful in the methods disclosed herein include, for example, domain antibodies, sFv, single-chain Fv fragments (scFv), Fab, F(ab')2, nanobodies, tandem scFv, scFv-Fc, scFv dimers, scFv zippers, diabodies, minibodies, triabodies, tetrabodies, Fab, F(ab)'2, scFab, miniantibodies, nanobodies, nanobody-HSA, bispecific T cell targeting (BiTE), DAR, sc diabody, sc diabody-CH3, or scFv-CH3 Knobs-Into-Holes (KIH) aggregates.

[0043] In certain embodiments, the BCMA-specific binding protein comprises a bispecific or multispecific antibody (or antigen-binding portion thereof), such an antibody comprising a first binding region (e.g., a heavy chain variable region, a light chain variable region, or both) that is specific for BCMA and a second target (e.g., a BCMA epitope that is different from the epitope of the first binding region, or an epitope of a non-BCMA target, e.g., a tumor-associated antigen that is not BCMA, e.g., CD19 (e.g., blinatumomab, MOR-208, SGN-19A, SAR3419, cortuximab ravtansine, denintuzumab mafodotin, taplitumomab paptox, XmAb 5574, MDI-551, Merck proprietary anti-CD19 aka B4 aka DI-B4, XmAb and at least one additional binding region specific for an antigen or target (e.g., CD20 (e.g., rituximab, ofatumumab, ocrelizumab), CD38 (e.g., daratumumab or isatuximab (SAR650984)), CD45, or a cell surface protein expressed on an immune effector cell, e.g., on a T cell (e.g., CD3), on a NK cell (e.g., CD56), on a NK-T cell (e.g., NK1.1), or another non-tumor-associated antigen or target).

[0044] In certain embodiments, the present disclosure provides a BCMA-specific binding protein alone or a BCMA-specific protein expressed intracellularly as a T-ChARM, used in conjunction with a gamma-secretase inhibitor (GSI). A typical T-ChARM comprises an extracellular component and an intracellular component linked by a hydrophobic moiety, wherein the extracellular component comprises a binding domain that specifically binds BCMA, an optional spacer region, a tag cassette, and a hinge region, and the intracellular component comprises an effector domain and optionally a costimulatory domain (e.g., a functional domain or portion from 4-1BB, a functional domain or portion from CD28, or both). In certain embodiments, the T-ChARM binding domain comprises a BCMA-specific scFv, a BCMA-specific scTCR, a BCMA ligand, or a binding portion thereof (e.g., BAFF, APRIL), optionally wherein the BCMA-specific scFv is human or humanized. Various embodiments of T-ChARM are disclosed in PCT Publication No. WO 2015 / 095895, the T-ChARM scaffold of which is incorporated herein by reference in its entirety.

[0045] Exemplary tag cassettes include a Strep tag (which refers to the original Strep® tag, Strep® tag II, or any variant thereof; see, e.g., U.S. Pat. No. 7,981,632; the Strep tag is incorporated herein by reference), His tag, Flag tag, Xpress tag, Avi tag, calmodulin tag, polyglutamic acid tag, HA tag, Myc tag, Nus tag, S tag, SBP tag, Softag 1, Softag 3, V5 tag, CREB binding protein (CBP), glutathione S-transferase (GST), maltose binding protein (MBP), green fluorescent protein (GFP), thioredoxin tag, or any combination thereof. In some embodiments, the tag cassette can be an engineered affinity site, such as a minimal chelating site (e.g., HGGHHG, SEQ ID NO: 6). In certain embodiments, the tag cassette is a Strep tag having the amino acid sequence Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 7) or Trp-Arg-His-Pro-Gln-Phe-Gly-Gly (SEQ ID NO: 8).

[0046] The tag cassette may be present in a single or multiple copies in the fusion protein of the present disclosure. For example, the BCMA-specific binding protein of the present disclosure can have 1, 2, 3, 4, or 5 tag cassettes (e.g., Strep tags). In certain embodiments, the extracellular component of the BCMA-specific T-ChARM comprises 1 tag cassette, 2 tag cassettes, 3 tag cassettes, 4 tag cassettes, or 5 tag cassettes. Each of the multiple tag cassettes may be the same or different.

[0047] In certain embodiments, the tag cassette comprises about 5 to about 500 amino acids, or about 6 to about 100 amino acids, or about 7 to about 50 amino acids, or about 8 to about 20 amino acids. In some embodiments, the tag cassette has 7 to 10 amino acids. Preferably, the tag cassette is non-immunogenic or minimally immunogenic. Essentially, the tag cassette can function as a handle or beacon, allowing for the identification, enrichment, isolation, promotion of proliferation, activation, tracking, or elimination of cells expressing BCMA-specific T-ChARM.

[0048] In further embodiments, the present disclosure provides a BCMA-specific binding protein that is a chimeric antigen receptor (CAR) for use with a gamma-secretase inhibitor, the BCMA-specific binding protein comprising an extracellular component and an intracellular component linked by a hydrophobic moiety, the extracellular component comprising a binding domain that specifically binds BCMA and a hinge region, and the intracellular component comprising an effector domain and optionally a costimulatory domain. In certain embodiments, the CAR binding domain comprises a BCMA-specific scFv, a BCMA-specific scTCR, a BCMA-specific TCR binding domain (see, e.g., Walseng et al., Scientific Reports 7:10713 (2017), the TCR CAR construct of which is incorporated herein by reference), or a BCMA ligand or binding portion thereof (e.g., BAFF, APRIL), optionally wherein the BCMA-specific scFv is human or humanized. In any of these embodiments, the BCMA-specific binding protein in the form of a CAR is expressed on the surface of a cell, such as an immune system cell (e.g., a T cell).

[0049] The BCMA-specific T-ChARM or CAR may be cell-associated (e.g., expressed on the cell surface) or in a soluble form. In certain embodiments, the polynucleotide encoding the BCMA-specific T-ChARM or CAR protein may be codon-optimized to enhance or maximize expression in host cells, such as T cells (Scholten et al., Clin. Immunol. 119:135, 2006).

[0050] In certain embodiments, the hinge present in the BCMA-specific T-ChARM or CAR of the present disclosure can be an immunoglobulin hinge region, such as a wild-type immunoglobulin hinge region or a modified immunoglobulin hinge region thereof. In certain embodiments, the hinge is a wild-type human immunoglobulin hinge region. In certain other embodiments, one or more amino acid residues can be added to the amino or carboxy terminus of the wild-type immunoglobulin hinge region as part of the design of the fusion protein construct. For example, one, two, or three additional junction amino acid residues can be present at the amino or carboxy terminus of the hinge, or the hinge can include a terminal or internal deletion and one, two, or three additional junction amino acid residues can be added behind it.

[0051] In certain embodiments, the hinge is a modified immunoglobulin hinge in which one or more cysteine ​​residues in the wild-type immunoglobulin hinge region have been substituted with one or more different amino acid residues. Exemplary modified immunoglobulin hinges include immunoglobulin human IgG1, IgG2, or IgG4 hinge regions in which one, two, or three cysteine ​​residues found in a wild-type human IgG1, IgG2, or IgG4 hinge have been substituted with one, two, or three different amino acid residues (e.g., serine or alanine). In certain embodiments, the hinge polypeptide is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to a wild-type immunoglobulin hinge region, e.g., a wild-type human IgG1 hinge, a wild-type human IgG2 hinge, or a wild-type human IgG4 hinge.

[0052] In further embodiments, the hinge present in a BCMA-specific T-ChARM or CAR of the present disclosure can be a hinge that is not based on or derived from an immunoglobulin hinge (i.e., not a wild-type or modified immunoglobulin hinge). Examples of such hinges include a peptide of about 5 to about 150 amino acids from the stalk region of a type II C-lectin or CD molecule, such as a peptide of about 8 to about 25 amino acids or a peptide of about 7 to about 18 amino acids, or variants thereof.

[0053] The "stalk region" of a type II C-lectin or CD molecule refers to the portion of the extracellular domain of the type II C-lectin or CD molecule located between the C-type lectin-like domain (CTLD; e.g., similar to the CTLD of the natural killer cell receptor) and the hydrophobic portion (transmembrane domain). For example, the extracellular domain of human CD94 (GenBank accession number AAC50291.1) corresponds to amino acid residues 34-179, while the CTLD corresponds to amino acid residues 61-176. Thus, the stalk region of the human CD94 molecule includes amino acid residues 34-60, located between the hydrophobic portion (transmembrane domain) and the CTLD (see Boyington et al., Immunity 10:75, 1999; for descriptions of other stalk regions, see Beavil et al., Proc. Nat'l. Acad. Sci. USA 89:753, 1992; and Figdor et al., Nat. Rev. Immunol. 2:77, 2002). These type II C-lectin or CD molecules may have a linking amino acid between the stalk region and the transmembrane region or CTLD. In another example, the 233 amino acid human NKG2A protein (GenBank Accession No. P26715.1) has a hydrophobic portion (transmembrane domain) ranging from amino acids 71 ​​to 93 and an extracellular domain ranging from amino acids 94 to 233. The CTLD comprises amino acids 119 to 231, and the stalk region comprises amino acids 99 to 116, optionally flanked by additional linking amino acids. Other type II C-lectin or CD molecules, as well as their extracellular ligand-binding domains, stalk regions, and CTLDs, are known in the art (see, e.g., GenBank Accession Nos. NP_001993.2; AAH07037.1; NP_001773.1; AAL65234.1; CAA04925.1; the sequences and descriptions of human CD23, CD69, CD72, NKG2A, and NKG2D, respectively).

[0054] A "derivative" of the stalk region hinge of a type II C-lectin or CD molecule, or a fragment thereof, comprises a sequence of about 8 to about 150 amino acids, in which there is a deletion, insertion, substitution, or any combination thereof of one, two, or three amino acids from the stalk region of a wild-type type II C-lectin or CD molecule. For example, a derivative can include one or more amino acid substitutions and / or deletions. In certain embodiments, a derivative of the stalk region is more resistant to proteolysis than a wild-type stalk region sequence (e.g., derived from about 8 to about 20 amino acids of NKG2A, NKG2D, CD23, CD64, CD72, or CD94).

[0055] In certain embodiments, the stalk region hinge can comprise about 7 to about 18 amino acids and can form an α-helical coiled-coil structure. In certain embodiments, the stalk region hinge comprises 0, 1, 2, 3, or 4 cysteines. Exemplary stalk region hinges include fragments of the stalk region, such as those portions comprising about 10 to about 150 amino acids from the stalk regions of CD69, CD72, CD94, NKG2A, and NKG2D.

[0056] Alternative hinges that can be used in the BCMA-specific T-ChARMs and CARs of the present disclosure are those from the portion of a cell surface receptor that connects the immunoglobulin V-like or immunoglobulin C-like domains (the interdomain region). Regions between IgV-like domains where the cell surface receptor comprises multiple IgV-like domains in tandem, and regions between IgC-like domains where the cell surface receptor comprises multiple IgC-like domains in tandem, are also considered useful hinges for the BCMA-specific T-ChARMs and CARs of the present disclosure. In certain embodiments, hinge sequences comprised of cell surface receptor interdomain regions may further comprise natural or additional motifs, such as an IgG core hinge sequence, that provide one or more disulfide bonds to stabilize dimerization of the BCMA-specific T ChARM or CAR. Examples of hinges include the interdomain region between the IgV-like and IgC-like regions of CD2, CD4, CD22, CD33, CD48, CD58, CD66, CD80, CD86, CD150, CD166, or CD244.

[0057] In certain embodiments, the hinge sequence has about 5 to about 150 amino acids, about 5 to about 10 amino acids, about 10 to about 20 amino acids, about 20 to about 30 amino acids, about 30 to about 40 amino acids, about 40 to about 50 amino acids, about 50 to about 60 amino acids, about 5 to about 60 amino acids, about 5 to about 40 amino acids, e.g., about 8 to about 20 amino acids or about 10 to about 15 amino acids. The hinge is primarily flexible, but may also have more rigid characteristics, or may comprise primarily an α-helical structure with a small amount of β-sheet.

[0058] In certain embodiments, the hinge sequence is stable in plasma and serum and resistant to proteolytic cleavage. For example, the first lysine in the IgG1 upper hinge region may be mutated or deleted to minimize proteolytic cleavage, and the hinge may include linking amino acids. In some embodiments, the hinge sequence may include a natural or additional motif, such as the immunoglobulin hinge core structure CPPCP (SEQ ID NO: 9), that confers the ability to form one or more disulfide bonds that stabilize dimer formation.

[0059] The hydrophobic moiety contained in a BCMA-specific binding protein of the disclosure (e.g., a BCMA-specific T-ChARM or CAR) will enable the BCMA-specific binding protein of the disclosure to associate with a cell membrane such that a portion of the binding protein is located extracellularly (e.g., tag cassette, binding domain) and a portion is located intracellularly (e.g., effector domain, costimulatory domain). The hydrophobic moiety will generally be located within the cell membrane phospholipid bilayer. In some embodiments, one or more junction amino acids may be located between and connect the hydrophobic moiety and the effector domain, or between and connect the hydrophobic moiety and the spacer region, or between and connect the hydrophobic moiety and the tag cassette.

[0060] In some embodiments, the hydrophobic domain is a transmembrane domain, e.g., derived from an integral membrane protein (e.g., a receptor, a cluster sorting (CD) molecule, an enzyme, a transporter, a cell adhesion molecule, etc.). In specific embodiments, the hydrophobic moiety is a transmembrane domain derived from CD4, CD8, CD27, or CD28.

[0061] An intracellular component contained on a BCMA-specific binding protein (e.g., a BCMA-specific T-ChARM or CAR) of the present disclosure may be capable of transmitting a functional signal to a cell. In certain embodiments, the BCMA-specific T-ChARM or CAR may dimerize with a second single-chain T-ChARM or CAR, respectively. This dimerization brings the intracellular component, including the effector domain and, optionally, the costimulatory domain, into close proximity and allows them to promote signal transduction upon exposure to an appropriate signal. In addition to forming such a dimeric protein complex, the effector domain and optional costimulatory domain may associate with additional signaling factors, e.g., costimulatory factors, to form a multiprotein complex that generates an intracellular signal. In certain embodiments, the effector domain may indirectly promote a cellular response by associating with one or more other proteins that directly promote the cellular response. The intracellular component may comprise one, two, three, or more receptor signaling domains (e.g., effector domains), costimulatory domains, or a combination thereof. Intracellular components including effector domains or functional portions thereof, costimulatory domains or functional portions thereof, or any combination thereof from any of a variety of signaling molecules (e.g., signaling receptors) can be used in the BCMA-specific binding proteins of the disclosure.

[0062] The intracellular component may have an effector or costimulatory domain useful for the BCMA-specific binding proteins of the present disclosure, which may include those involved in the Wnt signaling pathway (e.g., LRP, Ryk, ROR2), the NOTCH signaling pathway (e.g., NOTCH1, NOTCH2, NOTCH3, NOTCH4), the Hedgehog signaling pathway (e.g., PTCH, SMO), receptor tyrosine kinase (RTK) (e.g., epidermal growth factor (EGF) receptor family, fibroblast growth factor (FGF) receptor family, hepatocyte growth factor (HGF) receptor family, insulin receptor (IR) family, platelet-derived growth factor (PDGF) receptor family, vascular endothelial growth factor (VEGF) receptor family, and the like). ) receptor family, tropomycin receptor kinase (Trk) receptor family, ephrin (Eph) receptor family, AXL receptor family, leukocyte tyrosine kinase (LTK) receptor family, tyrosine kinase with immunoglobulin-like and EGF-like domain 1 (TIE) receptor family, receptor tyrosine kinase-like orphan (ROR) receptor family, discoidin domain (DDR) receptor family, rearranged during transfection (RET) receptor family, tyrosine-protein kinase-like (PTK7) receptor family, receptor tyrosine kinase-related (RYK) receptor family, muscle-specific kinase (MuSK) receptor family); G protein-coupled receptors, GPCRs (Frizzled, Smoothened); serine / threonine kinase receptors (BMPR, TGFR); or cytokine receptors (IL-1R, IL-2R, IL-7R, IL-15R).

[0063] In certain embodiments, the effector domain comprises a lymphocyte receptor signaling domain or comprises an amino acid sequence having one or more immunoreceptor tyrosine-based activation motifs (ITAMs). In yet other embodiments, the effector domain comprises a cytoplasmic portion that associates with a cytoplasmic signaling protein, wherein the cytoplasmic signaling protein is a protein comprising a lymphocyte receptor or its signaling domain, multiple ITAMs, a costimulatory factor, or any combination thereof.

[0064] Exemplary effector and costimulatory domains include those based on or derived from 4-1BB, CD3ε, CD3δ, CD3ζ, CD27, CD28, CD79A, CD79B, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, Wnt, NKG2D, OX40, ROR2, Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or any combination thereof.

[0065] In certain embodiments, a BCMA-specific binding protein of the disclosure comprises (a) an effector domain from CD3ζ, or a functional portion thereof, and a costimulatory domain from CD28, or a functional portion thereof; (b) an effector domain from CD3ζ, or a functional portion thereof, and a costimulatory domain from 4-1BB, or a functional portion thereof; or (c) (a) an effector domain from CD3ζ, or a functional portion thereof, and costimulatory domains from CD28 and 4-1BB, or functional portions thereof.

[0066] γ-secretase inhibitors (GSIs) By way of background, γ-secretase is a multisubunit integral membrane protease complex containing presenilin (PS), nicastrin (NCT), anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2). PS is the catalytic subunit, an aspartic protease capable of forming a hydrophilic catalytic pore embedded in the membrane (Takasugi et al., Nature 422:438-41, 2003), which cleaves single-pass transmembrane proteins within the transmembrane domain. NCT is a type I membrane glycoprotein with a large extracellular domain (ECD). The ECD captures the amino terminus of substrates as the primary substrate acceptor for γ-secretase (Shah et al., Cell 122:435-47, 2005). The γ-secretase complex plays a role in the processing of various substrates, such as Notch, CD44, cadherins, and ephrin B2, as well as cleaving the amyloid precursor protein into the amyloid β peptide associated with Alzheimer's disease. The γ-secretase complex is also known to cleave B-cell maturation antigen (BCMA) (Laurent et al., Nature Communications 6, 2015), which is a therapeutic target in various cancers, including multiple myeloma.

[0067] Exemplary gamma-secretase inhibitors (GSIs) include small molecules, peptidomimetic compounds, or gamma-secretase-specific binding proteins. GSIs can target one or more of the gamma-secretase complex proteins, such as presenilin 1 (PS1), presenilin 2 (PS2), nicastrin (NCT), anterior pharynx-defective 1 (APH-1), and presenilin enhancer 2 (PEN-2), provided that gamma-secretase cleavage activity is reduced compared to uninhibited gamma-secretase. In some embodiments, gamma-secretase activity is reduced by at least about 50%, at least about 60%, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or 100%. Assays for measuring γ-secretase activity are known in the art (see, e.g., Laurent et al., 2015). For example, the level of soluble BCMA can be a surrogate measure of γ-secretase activity. Representative small molecule GSIs used in conjunction with BCMA-targeted immunotherapies to treat proliferative or autoimmune diseases or disorders include avagacestat, DAPT, BMS-906024, BMS-986115, LY411575, MK-0752, PF-03084014, RO4929097, semagacestat, YO-01027, and any combination thereof.

[0068] Another GSI is a γ-secretase-specific binding protein, e.g., an antibody or antigen-binding portion thereof, such as a γ-secretase complex or γ-secretase complex protein, e.g., presenilin 1 (PS1), presenilin 2 (PS2), nicastrin (NCT), APH-1 (Anterior pharynx-defective 1), and presenilin enhancer 2 (PEN-2). Exemplary γ-secretase-specific binding proteins are nicastrin-specific binding proteins, e.g., antibodies scFvG9, A5226A, 2H6, 10C11, and antigen-binding fragments thereof.

[0069] Binding domain The binding domain can be any peptide that specifically binds BCMA or specifically inhibits γ-secretase activity as described herein. Sources of binding domains include antibody variable regions (which can be in the form of antibodies, sFv, scFv, Fab, scFv-based "Grababodies", soluble VH domains, or domain antibodies) from a variety of species, including human, rodent, avian, or ovine. Additional sources of binding domains include those from other species, such as camelids (camels, dromedaries, or llamas; Ghahroudi et al., FEBS Lett. 414:521, 1997; Vincke et al., J. Biol. Chem. 284:3273, 2009; Hamers-Casterman et al., Nature 363:446, 1993 and Nguyen et al., J. Mol. Biol. 275:413, 1998), nurse sharks (Roux et al., Proc. Nat'l. Acad. Sci. (USA) 95:11804, 1998), spotted ratfish (Nguyen et al., Immunogen. 54:39, 2002), or lampreys (Herrin et al., Proc. Nat'l. Acad. Sci. (USA) 105:2040, 2008 and Alder et al., Nat. Immunol. 9:319, 2008). These antibodies can form the antigen-binding region using only the heavy chain variable region, i.e., these functional antibodies are homodimers of heavy chains only (also referred to as "heavy chain antibodies") (Jespers et al., Nat. Biotechnol. 22:1161, 2004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008).

[0070] Alternative sources of binding domains of the present disclosure include sequences encoding random peptide libraries, or sequences encoding engineered diversity of amino acids in the loop regions of another non-antibody scaffold, such as scTCRs (see, e.g., Lake et al., Int. Immunol. 11:745, 1999; Maynard et al., J. Immunol. Methods 306:51, 2005; U.S. Pat. No. 8,361,794), fibrinogen domains (see, e.g., Weisel et al., Science 230:1388, 1985), Kunitz domains (see, e.g., U.S. Pat. No. 6,423,498), engineered ankyrin repeat proteins (DARPins) (Binz et al., J. Mol. Biol. 332:489, 2003 and Binz et al., Nat. Biotechnol. 22:575, 2004), fibronectin-binding domains (adnectins or monobodies) (see Richards et al., J. Mol. Biol. 326:1475, 2003; Parker et al., Protein Eng. Des. Selec. 18:435, 2005 and Hackel et al. (2008) J. Mol. Biol. 381:1238-1252), cysteine-knot miniproteins (Vita et al. (1995) Proc. Nat'l. Acad. Sci. (USA) 92:6404-6408; Martin et al. (2002) Nat. Biotechnol. 21:71, 2002 and Huang et al. (2005) Structure 13:755, 2005), tetratricopeptide repeat domains (Main et al., Structure 11:497, 2003 and Cortajarena et al., ACS Chem. Biol. 3:161, 2008), leucine-rich repeat domains (Stumpp et al., J. Mol. Biol. 332:471, 2003), lipocalin domains (see, e.g., WO 2006 / 095164, Beste et al., Proc. Nat'l. Acad. Sci. (USA) 96:1898, 1999 and Schonfeld et al., Proc. Nat'l. Acad. Sci.(USA) 106:8198, 2009), V-like domains (see, e.g., U.S. Patent Application Publication No. 2007 / 0065431), C-type lectin domains (see Zelensky & Gready, FEBS J. 272:6179, 2005; Beavil et al., Proc. Nat'l. Acad. Sci. (USA) 89:753, 1992 and Sato et al., Proc. Nat'l. Acad. Sci. (USA) 100:7779, 2003), mAbs. 2 or Fcab TM (see, e.g., PCT Patent Application Publication Nos. WO 2007 / 098934; WO 2006 / 072620), armadillo repeat proteins (see, e.g., Madhurantakam et al., Protein Sci. 21: 1015, 2012; PCT Patent Application Publication No. WO 2009 / 040338), affilins (see, e.g., Ebersbach et al., J. Mol. Biol. 372: 172, 2007), affibodies, avimers, knottins, fynomers, atrimers, cytotoxic T-lymphocyte-associated protein 4 (Weidle et al., Cancer Gen. Proteo. 10:155, 2013), or the like (Nord et al., Protein Eng. 8:601, 1995; Nord et al., Nat. Biotechnol. 15:772, 1997; Nord et al., Euro. J. Biochem. 268:4269, 2001; Binz et al., Nat. Biotechnol. 23:1257, 2005; Boersma & Pluckthun, Curr. Opin. Biotechnol. 22:849, 2011).

[0071] Binding domains of the present disclosure can be generated as described herein or by various methods known in the art (see, e.g., U.S. Patent Nos. 6,291,161 and 6,291,158). For example, binding domains of the present disclosure can be identified by screening Fab phage libraries for Fab fragments that specifically bind to a target of interest (e.g., BCMA, a γ-secretase complex component, e.g., presenilin or nicastrin) (see Hoet et al., Nat. Biotechnol. 23:344, 2005). Moreover, binding domains can be generated in conventional systems (e.g., mouse, HuMAb Mouse®, TC mouse). TM The binding domains of the present disclosure can be created using traditional techniques for hybridoma development using the target of interest (e.g., BCMA, a γ-secretase complex component such as presenilin or nicastrin) as an immunogen in mammals (e.g., KM-Mouse®, llama, chicken, rat, hamster, rabbit, etc.).

[0072] In some embodiments, the binding domain is a V specific for a target of interest (e.g., BCMA, a γ-secretase complex component, e.g., presenilin or nicastrin). H Area and V L In some embodiments, the V is a single chain Fv fragment comprising a V region. H Area and V L The domain is human. Typical V H and V LThe regions included the following segments of anti-BCMA specific antibodies: J22.0-xi, J22.9-xi, J6M0, J6M1, J6M2, J9M0, J9M1, J9M2, 11D5-3, CA8, A7D12.2, C11D5.3, C12A3.2, C13F12.1, 13C2, 17A5, 83A10, 13A4, 13D2, 14B11, 14E1, 29B11, 29F3, 13A7, CA7, SG1, S307118G03, S332121F02, S332126E04, S322110D07, S336105A07, S335115G01, S335122F05, ET140-3, ET140-24, ET140-37, ET140-40, ET140-54, TBL-CLN1, C4.E2.1, Vicky-1, pSCHLI333, pSCHLI372, or pSCHLI373. Another typical V H and V L The region includes an anti-nicastrin-specific antibody segment or antigen-binding fragment thereof from scFvG9, A5226A, 2H6, or 10C11.

[0073] In certain embodiments, the binding domain comprises a light chain variable region (V L) (e.g., anti-BCMA J22.0-xi, J22.9-xi, J6M0, J6M1, J6M2, J9M0, J9M1, J9M2, 11D5-3, CA8, A7D12.2, C11D5.3, C12A3.2 or C13F12.1, 13C2, 17A5, 83A10, 13A4, 13D2, 14B11, 14E1, 29B11, 29F3, 13A7, CA7, S307118G03, S332121F02, S332126E04, S322110D07, S336105A07, S335115G01, S335122F05, ET140-3, ET140-24, ET140-37, ET140-40, ET140-54, TBL-CLN1, C4.E2.1, Vicky-1, pSCHLI333, pSCHLI372 or pSCHLI373; or anti-nicastrin scFvG9, A5226A, 2H6 or 10C11) or heavy chain variable region (V H) (e.g., anti-BCMA J22.0-xi, J22.9-xi, J6M0, J6M1, J6M2, J9M0, J9M1, J9M2, 11D5-3, CA8, A7D12.2, C11D5.3, C12A3.2, C13F12.1, 13C2, 17A5, 83A10, 13A4, 13D2, 14B11, 14E1, 29B11, 29F3, 13A7, CA7, SG1, S307118G03, S332121F02, S332126E04, S322110D07, S336105A07, S335115G01, S335122F05, ET140-3, ET140-24, ET140-37, ET140-40, ET140-54, TBL-CLN1, C4.E2.1, Vicky-1, derived from pSCHLI333, pSCHLI372, or pSCHLI373; or anti-nicastrin scFvG9, A5226A, 2H6 or 10C11), or both), wherein each CDR contains no, or at most one, two, or three mutations from a monoclonal antibody or fragment or derivative thereof that specifically binds to a target of interest (e.g., BCMA, a γ-secretase complex component, e.g., presenilin or nicastrin).

[0074] In certain embodiments, binding domain V of the present disclosure H The region is the V region of a known monoclonal antibody. H can be derived from or based on the V region of a known monoclonal antibody HThe region can include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions), or a combination of the above changes. H The CDRs may be present anywhere in the region, such as at the amino or carboxy terminus of the region or at both ends, provided that each CDR contains no alterations or at most one, two, or three alterations, and the modified V H A binding domain containing the region will still specifically bind its target with an affinity similar to that of the wild-type binding domain.

[0075] In a further embodiment, the V of the binding domain of the present disclosure L The region is the V region of a known monoclonal antibody. L can be derived from or based on the V region of a known monoclonal antibody L The region can include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions), or a combination of the above changes. The insertions, deletions, or substitutions can be any of the following: L The CDRs may be present anywhere in the region, such as at the amino or carboxy terminus of the region or at both ends, provided that each CDR contains no alterations or at most one, two, or three alterations, and the modified V L A binding domain containing the region will still specifically bind its target with an affinity similar to that of the wild-type binding domain.

[0076] V H and V LThe domains can be oriented in either direction (i.e., from amino to carboxyl termini) as V H -V L or V L -V H and may be connected by an amino acid sequence (e.g., having a length of about 5 to about 35 amino acids) that can provide a spacer function so that the two sub-binding domains can interact to form a functional binding domain. In some embodiments, V H Domains and V L The variable region linker joining the domains is (Gly n Ser) family, e.g. (Gly3Ser) n (Gly4Ser)1 (SEQ ID NO: 3), (Gly3Ser)1(Gly4Ser) n (SEQ ID NO: 10), (Gly3Ser) n (Gly4Ser) n (SEQ ID NO: 4), or (Gly4Ser) n (SEQ ID NO: 5), where n is an integer from 1 to 5. In some embodiments, the linker is (Gly-Gly-Gly-Gly-Ser)3 (SEQ ID NO: 12) or (Gly-Gly-Gly-Ser)4 (SEQ ID NO: 13). In some embodiments, these (Gly n The Ser)-based linker is a V H Domains and V L Used to link domains, these linkers are also used to link binding domains to tag cassettes or to link tag cassettes to hydrophobic moieties or intracellular components.

[0077] In some embodiments, the binding domain is a V that is specific for a target of interest (e.g., a peptide-MHC complex). α / β Chain and C α / β chain (e.g., V α -C α , V β -C β , V α -V β ) or V α -C α , Vβ -C β , V α -V β It is a single-chain T cell receptor (scTCR) that contains a pair.

[0078] In certain embodiments, the binding domain comprises at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or 100% TCR V α , V β , C α or C β and each CDR is a sequence that contains, unaltered or up to 1, 2 or 3 alterations from a TCR or fragment or derivative thereof that specifically binds a target of interest (e.g., BCMA, a γ-secretase complex component, e.g., presenilin or nicastrin),

[0079] In certain embodiments, binding domain V of the present disclosure α , V β , C α or C β The region is the V region of known TCRs. α , V β , C α Or C β and can be derived from or based on the V of a known TCR. α , V β , C α , or C β The insertions, deletions, or substitutions may include one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative or non-conservative amino acid substitutions) compared to V, or a combination of the above changes. α , V β , C α or C βThe CDRs may be unaltered or may have up to one, two, or three alterations, and may be located anywhere, including at the amino terminus, carboxy terminus, or both ends of the region, provided that each CDR is unaltered or has up to one, two, or three alterations, and the V α , V β , C α or C β It is assumed that the binding domain containing the region is still able to specifically bind its target with affinity similar to the wild type.

[0080] BCMA, γ-secretase, or both, are found on or associated with cells of interest ("target cells"). Typical target cells include cancer cells, cells associated with autoimmune or inflammatory diseases or disorders, and infectious organisms or cells (e.g., bacteria, viruses, virus-infected cells).

[0081] cytotoxic complex Antibody-drug conjugates are used to selectively deliver cytotoxic agents to target cells, such as tumor or cancer cells. Antibody-drug conjugates and related technologies and chemistries are described, for example, in Nasiri et al., J. Cell. Physiol. (2018); Hedrich et al., Clin. Pharmacokinet. (2017); Drake & Rabuka, BioDrugs 31(6):521 (2017); Meyer et al., Bioconj. Chem. 27(12):2791 (2016); Moek et al., J. Nucl. Med. 58:83S (2017); Nareshkumar et al., Pharm. Res. 32:3526 (2015); Parslow et al., Biomedicines 4:14 (2016); and Green et al., Blood 131:611. (2018), the entire contents of which are incorporated herein by reference, for the theory of antibody morphology, cytotoxic payloads, linkers and conjugation chemistries, dosing regimens, therapeutic methods, pharmacokinetics, and ADC design.

[0082] In certain embodiments of the methods provided herein, the BCMA-specific antibody or antigen-binding portion thereof, chimeric antigen receptor (CAR), or tagged chimeric antigen receptor molecule (T-ChARM) is conjugated or otherwise linked to a cytotoxic drug, such as a chemotherapeutic drug, including, but not limited to, inhibitors of chromatin function, topoisomerase inhibitors, microtubule inhibitors, DNA damaging agents, antimetabolites (such as antifolates, pyrimidine analogs, purine analogs, sugar-modified analogs), DNA synthesis inhibitors, DNA interacting agents (such as intercalating agents), and DNA repair inhibitors. Exemplary chemotherapeutic agents include, but are not limited to, the following groups: antimetabolites / anticancer agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine, and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin, and 2-chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents, including natural products, such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disrupting agents, such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and nabetaxel. Rubin, epidipodophyllotoxins (etoposide, teniposide), DNA damaging drugs (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethylmelamine, oxaliplatin, ifosfamide, melphalan, merchlorthamine, mitomycin, mitoxantrone, nitrosoureas, plicamycin, procarbazine, taxol, taxotere, temozolamide, teniposide, triethylenethiophosphoramide, and etoposide (VP 16)); antibiotics, such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mithramycin) and mitomycin;enzymes (L-asparaginase, which metabolizes L-asparagine systemically, depriving cells that do not have the ability to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkylsulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazene-dacarbazine (DTIC); folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogens, tamoxifen, goserelin, bicalutamide, nilutamide), and aromatase inhibitors. Anticoagulants (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytic agents (tissue plasminogen activator, streptokinase, urokinase, etc.), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (vleverdin); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); antiangiogenic compounds (TNP470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blockers; nitric oxide donors; antisense oligonucleotides; antibodies (trastuzumab, rituximab); chimeric antigen receptors; cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan (CPT-11) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone and prenisolone); growth factor signaling kinase inhibitors;Mitochondrial dysfunction inducers, toxins such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, or diphtheria toxin, and caspase activators; and chromatin disrupting agents.

[0083] An exemplary ADC that specifically binds to BCMA is J6M0-mcMMAF (GSK2857916), described in Tai et al., Blood 123(20):3128-3138 (2014), the entire contents of which are incorporated herein by reference. Another BCMA-specific ADC (SG1-MMAF) is described by Ryan et al. (Mol. Cancer Ther., 6(11):3009-3018, 2007), the entire contents of which are incorporated herein by reference.

[0084] Host cells and nucleic acids In certain aspects, the present disclosure provides nucleic acid molecules encoding any one or more of the BCMA-specific binding proteins (including multispecific and bispecific binding proteins comprising at least one BCMA-binding domain) or gamma-secretase inhibitors described herein. Such nucleic acid molecules can be inserted into an appropriate vector (e.g., a viral vector or a non-viral plasmid vector) for introduction into a host cell of interest (e.g., a T cell).

[0085] As used herein, the terms "recombinant" or "non-naturally occurring" refer to an organism, microorganism, cell, nucleic acid molecule, or vector that contains at least one genetic change or has been modified by the introduction of an exogenous nucleic acid molecule, where such change or modification is introduced by genetic engineering. Genetic changes include, for example, modifications that introduce expressible nucleic acid molecules encoding proteins, fusion proteins, or enzymes, or the addition, deletion, substitution, or other functional disruption of other nucleic acid molecules in the genetic material of a cell. Additional modifications include, for example, non-coding regulatory regions, where the modification alters the expression of a gene or operon. In certain embodiments, cells, such as T cells obtained from a subject, are converted into non-natural or recombinant cells (e.g., non-natural or recombinant T cells) by introducing a nucleic acid encoding a BCMA-specific binding protein or γ-secretase inhibitor (e.g., a BCMA-specific T-ChARM or CAR; or an anti-γ-secretase) of the present disclosure as described herein, thereby causing the cells to express a BCMA-specific binding protein immobilized on the cell surface.

[0086] Vectors encoding core viruses are referred to herein as "viral vectors." There are numerous viral vectors available that are suitable for use with the compositions of the present disclosure, including those identified for human gene therapy applications (see Pfeifer & Verma, Ann. Rev. Genomics Hum. Genet. 2:177, 2001). Suitable viral vectors include RNA virus-based vectors, such as retrovirus-derived vectors, e.g., Moloney murine leukemia virus (MLV)-derived vectors, as well as more complex retrovirus-derived vectors, e.g., lentivirus-derived vectors. HIV-1-derived vectors fall into this category. Other examples include lentiviral vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus).

[0087] The use of retroviral and lentiviral vectors and packaging cells to transduce mammalian host cells with viral particles containing chimeric antigen receptor transgenes is known in the art and has been described, for example, in U.S. Patent No. 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther. 18:1748, 2010; Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available.

[0088] In certain embodiments, a viral vector is used to introduce a non-endogenous polynucleotide encoding a BCMA-specific binding protein or a γ-secretase inhibitor. The viral vector may be a retroviral or lentiviral vector. The viral vector may also include a polynucleotide encoding a transduction marker. Transduction markers for viral vectors are known in the art and include selectable markers that can confer drug resistance, or detectable markers such as fluorescent markers or cell surface proteins that can be detected by methods such as flow cytometry. In certain embodiments, the viral vector further includes a genetic marker for transduction, including green fluorescent protein, the extracellular domain of human CD2, or a truncated form of human EGFR (huEGFRt; see Wang et al., Blood 118:1255, 2011). When the viral vector genome includes multiple nucleic acid sequences to be expressed in a host cell as separate transcripts, the viral vector may include additional sequences between the two (or more) transcripts to enable bicistronic or polycistronic expression. Examples of such sequences used in viral vectors include an internal ribosome entry site (IRES), a furin cleavage site, a viral 2A peptide (e.g., P2A, T2A, E2A, F2A), or any combination thereof.

[0089] Other vectors can also be used for polynucleotide delivery, including DNA viral vectors such as adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; amplicon vectors, vectors derived from herpes simplex virus (HSV), including replication-defective HSV and attenuated HSV (Krisky et al., Gene Ther. 5:1517, 1998).

[0090] Other vectors recently developed for gene therapy applications can also be used with the compositions and methods of the present disclosure. Such vectors include those derived from baculoviruses and alphaviruses (Jolly, D. J., 1999. Emerging Viral Vectors. P. 209-40, Friedmann, T., ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (e.g., "sleeping beauty" or other transposon-based vectors). In some embodiments, the viral or plasmid vector further comprises a genetic marker for transduction (e.g., green fluorescent protein, huEGFRt).

[0091] In certain embodiments, hematopoietic progenitor cells or embryonic stem cells are modified to contain a non-endogenous polynucleotide encoding a BCMA-specific binding protein (e.g., a BCMA-specific T-ChARM or CAR) of the present disclosure. Hematopoietic progenitor cells may include thymocyte precursor cells or induced pluripotent stem cells derived or derived from fetal liver tissue, bone marrow, umbilical cord blood, or peripheral blood. Hematopoietic progenitor cells may be derived from humans, mice, rats, or other mammals. In certain embodiments, CD24 lo Lin - CD117 + Thymocyte precursor cells are used.

[0092] In certain embodiments, the culture conditions involve culturing hematopoietic progenitor cells expressing the fusion protein of the present disclosure for a time sufficient to induce proliferation or differentiation. Cells are generally maintained in culture for about 3 to about 5 days, or about 4 to about 10 days, or about 5 to about 20 days. It will be understood that cells may be maintained for any suitable amount of time necessary to achieve the desired result, i.e., the desired cell composition or proliferation level. For example, to generate a cell composition comprising primarily immature, inactivated T cells, cells can be maintained in culture for about 5 to about 20 days. Cells can be maintained in culture for about 20 to about 30 days to generate a cell composition comprising primarily mature T cells. Non-adherent cells can also be harvested from the culture at various time points, ranging from about several days to about 25 days. In certain embodiments, hematopoietic stem cells are co-cultured on stromal cell lines (US Pat. No. 7,575,925; Schmit et al., Nat. Immunol. 5:410, 2004; Schmit et al., Immunity 17:749, 2002).

[0093] One or more cytokines that promote the commitment or differentiation of hematopoietic progenitor cells may be added to the culture. Cytokines can be human or non-human. Representative examples of cytokines that can be used include all members of the FGF family, including FGF-4 and FGF-2; Flt-3 ligand, stem cell factor (SCF), thrombopoietin (TPO), and IL-7. Cytokines can be used in combination with glycosaminoglycans, such as heparin sulfate.

[0094] In some embodiments, host cells capable of expressing a BCMA-specific binding protein of the present disclosure on their cell surface are T cells, including primary cells or cell lines derived from humans, mice, rats, or other mammals. When obtained from mammals, T cells can be obtained from a number of sources, including blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells or subpopulations thereof (e.g., naive, central memory, effector memory) can be enriched or purified. T cell lines are well known in the art, some of which are described in Sandberg et al., Leukemia 21:230, 2000. In some embodiments, T cells lacking endogenous expression of the TCR α and β chains are used. Such T cells naturally lack endogenous expression of the TCR α and β chains, or have been modified to prevent expression (e.g., T cells from transgenic mice that do not express the TCR α and β chains, or cells engineered to suppress expression of the TCR α and β chains), or have been modified to knock out the TCR α chain, TCR β chain, or both genes. In certain embodiments, the cell capable of expressing a BCMA-specific binding protein of the disclosure on its cell surface is not a T cell or a cell of T cell lineage, but is a progenitor cell, stem cell, or cell that has been modified not to express cell surface anti-CD3.

[0095] In any of the embodiments provided herein, the host cells may be "universal donor" cells that have been modified to reduce or eliminate expression of one or more endogenous genes involved in the immune response. For example, T cells may be modified to reduce or eliminate expression of one or more polypeptides selected from PD-1, LAG-3, CTLA4, TIGIT, TIM3, an HLA complex component, or a TCR or TCR complex component. Without being bound by theory, it is possible that certain endogenously expressed immune cell proteins are recognized as foreign by the allogeneic host receiving the modified immune cells, causing elimination of the modified immune cells (e.g., HLA alleles), or downregulating the immune activity of the modified immune cells (e.g., PD-1, LAG-3, CTLA4, TIGIT), or interfering with the binding activity of a heterologously expressed binding protein of the present disclosure (e.g., an endogenous TCR that binds to a non-tumor-associated antigen and interferes with the antigen-specific receptor of the modified immune cell that specifically binds to a tumor-associated antigen). Thus, reducing or eliminating the expression or activity of such endogenous genes or proteins can improve the activity, tolerance, and persistence of modified immune cells in allogeneic host preparations and allow for universal administration of those cells (e.g., to any recipient regardless of HLA type).

[0096] In certain embodiments, the host cells (e.g., engineered immune cells) of the disclosure express genes encoding PD-1, LAG-3, CTLA4, TIM3, TIGIT, an HLA complex component (e.g., a gene encoding alpha 1 macroglobulin, alpha 2 macroglobulin, alpha 3 macroglobulin, beta 1 microglobulin, or beta 2 microglobulin), a TCR component (e.g., a gene encoding a TCR variable region or a TCR constant region) (see Torikai et al., Nature Sci. Rep. 6:21757 (2016); Torikai et al., Blood 119 (24): 5697 (2012); and Torikai et al., Blood 122(8): 1341 (2013); the entire contents of which are incorporated by reference herein for their description of gene editing techniques, compositions, and adoptive cell therapy methods). For example, in some embodiments, chromosomal gene knockouts are generated using the CRISPR / Cas9 system and may require transfection of engineered immune cells with a lentivirus expressing a CRISPR / Cas9 system targeting PD-1, LAG-3, CTLA4, an HLA component, a TCR component, or any combination thereof (e.g., pLentiCRISPRv2; Torikai et al., Blood (2016)). Primers useful for designing lentiviruses expressing the CRISPR / Cas9 system to inhibit endogenously expressed immune cell proteins include, for example, primer pairs comprising forward and reverse primers having the nucleotide sequences set forth in SEQ ID NOs: 22 and 23, 24 and 25, 26 and 27, and 28 and 29.

[0097] In certain embodiments, host T cells transfected to express a BCMA-specific binding protein of the disclosure (e.g., a T-ChARM, a CAR, a multispecific binding protein comprising at least one BCMA-binding domain, a bispecific binding protein comprising at least one BCMA-binding domain) are functional T cells, such as virus-specific T cells, tumor antigen-specific cytotoxic T cells, naive T cells, memory stem T cells, central or effector memory T cells, or CD4 + CD25 + Regulatory T cells.

[0098] One or more growth factor cytokines that promote proliferation of T cells expressing the BCMA-specific binding proteins of the present disclosure may be added to the culture. The cytokines may be human or non-human. Exemplary growth factor cytokines that can be used to promote T cell proliferation include IL-2, IL-15, IL-21, etc.

[0099] Purpose As described in this disclosure, diseases that can be treated with BCMA-targeted immunotherapy or cells expressing a BCMA-specific binding protein in combination with a gamma-secretase inhibitor (GSI) include cancer (e.g., a BCMA-expressing cancer), an immune disease (e.g., autoimmunity), or an age-related disease (e.g., senescence). Adoptive immunotherapy and gene therapy are promising treatments for various types of cancer (Morgan et al., Science 314:126, 2006; Schmitt et al., Hum. Gene Ther. 20:1240, 2009; June, J. Clin. Invest. 117:1466, 2007) and infectious diseases (Kitchen et al., PLoS One 4:38208, 2009; Rossi et al., Nat. Biotechnol. 25:1444, 2007; Zhang et al., PLoS Pathog. 6:e1001018, 2010; Luo et al., J. Mol. Med. 89:903, 2011).

[0100] Cancers suitable for the compositions and methods disclosed herein are those that express or are capable of expressing BCMA on their cell surface. Examples of cancer types that can be treated include myeloma (e.g., multiple myeloma), leukemia (e.g., plasma cell leukemia), lymphoma (e.g., lymphoplasmacytic lymphoma), plasmacytoma, and Waldenstrom's macroglobulinemia. Other cancers that may express BCMA include adenocarcinoma of the breast and bronchial carcinoma of the lung. In certain embodiments, proliferative disorders suitable for combination therapy with a BCMA-specific binding protein and a GSI are certain types of B-cell cancers, including plasma cell disorders (e.g., multiple myeloma).

[0101] Inflammatory and autoimmune diseases include arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, polychondritis, psoriatic arthritis, psoriasis, dermatitis, polymyositis / dermatomyositis, inclusion body myositis, inflammatory myositis, toxic epidermal necrolysis, systemic dermatosclerosis and sclerosis, CREST syndrome, inflammatory bowel syndrome, Crohn's disease, ulcerative colitis, respiratory distress syndrome, adult respiratory distress syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, T cell infiltration and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion failure, systemic lupus erythematosus (SLE), subacute cutaneous lupus erythematosus, discoid lupus, lupus myelitis, lupus encephalitis, juvenile-onset diabetes, multiple sclerosis, allergic encephalomyelitis, neuromyelitis optica, rheumatic fever, Sydenham's chorea, immune responses with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, granulomatous diseases, Wegener's granulomatosis and Churg-Strauss disease, agranulocytosis, vasculitis (including hypersensitivity vasculitis / vasculitis, ANCA and rheumatoid vasculitis), aplastic anemia, Diamond-Blackfield's disease Autoimmune hemolytic anemia (AIHA), immune hemolytic anemia (immune hemolytic anemia), pernicious anemia, pure red cell aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte leakage, central nervous system (CNS) inflammatory disorders, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, antiphospholipid syndrome, allergic neuritis, Behçet's disease, Castleman syndrome, Goodpasture syndrome, Lambert-Eaton myasthenic syndrome, Raynaud's syndrome, Jorgen's syndrome, steroids, Evens-Johnson syndrome, solid organ transplant rejection, graft-versus-host disease (GVHD), bullous pemphigoid, pemphigus, autoimmune polyendocrinopathy, seronegative spondyloarthropathy, Reiter's disease, stiff-man syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathy or IgM-mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), Henoch-Schönlein purpura, autoimmune thrombocytopenia, autoimmune diseases of the testes and autoimmune orchitis and oophoritis, primary hypothyroidism, autoimmune thyroiditis,Autoimmune endocrine disorders such as chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Graves' disease, autoimmune polyglandular syndrome (or polyendocrinopathy syndrome), type 1 diabetes mellitus (also known as insulin-dependent diabetes mellitus (IDDM)) and Sheehan's syndrome; autoimmune hepatitis, lymphocytic interstitial pneumonia (HIV), bronchiolitis obliterans (non-transplant), nonspecific interstitial pneumonia (NSIP), Guillain-Barré syndrome, large-vessel vasculitis (polymyalgia rheumatoid and giant cell (Takayasu) arteritis), medium-vessel vasculitis (including Kawasaki disease and polyarteritis nodosa), polyarteritis nodosa (PAN), ankylosing spondylitis, Berger's disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, celiac disease sprue (gluten enteropathy), cryoglobulinemia, cryoglobulinemia with hepatitis, amyotrophic lateral sclerosis (ALS), coronary artery disease, familial Mediterranean fever, microscopic polyangiitis, Cogan's syndrome, Wiskott-Aldrich syndrome, and thromboangiitis obliterans.

[0102] In certain embodiments, methods of treating a subject with a BCMA binding protein in combination with a gamma-secretase inhibitor as disclosed herein include treating multiple myeloma, plasmacytoma, plasma cell leukemia, Waldenstrom's macroglobulinemia, B-cell lymphoma, and lymphoplasmacytic lymphoma.

[0103] A BCMA-specific binding protein, such as a CAR or T-ChARM of the present disclosure, can be administered to a subject in a cell-bound form (e.g., gene therapy of a target cell population (mature T cells (e.g., CD8 + or CD4 + T cells) or other cells of the T cell lineage). In certain embodiments, the cells of the T cell lineage expressing a BCMA-specific binding protein (e.g., a BCMA-specific CAR or T-ChARM) administered to the subject are syngeneic, allogeneic, or autologous cells.

[0104] The disclosed methods include administering a BCMA-specific binding molecule expressed on the surface of immune cells (e.g., T cells) and a gamma-secretase inhibitor (GSI). In certain embodiments, the combination can be administered simultaneously, either together in the same pharmaceutically acceptable carrier or in separate formulations (but simultaneously). Simultaneous administration means that each component is administered simultaneously or within 8-12 hours of each other. Administration of a second component 12 hours or more after the first would be considered sequential administration. In other embodiments, the BCMA-specific immunotherapeutic and GSI can be administered sequentially (e.g., 1, 2, 3, 4, 5, 6, 7, 8, or 9 days apart; 1, 2, 3, or 4 weeks apart; 1, 2, 3, 4, 5, 6, 7, 8, or 9 weeks apart; or 1, 2, 3, 4, 5, 6, or more years apart, etc.) in any order and in any combination. In certain embodiments, when administered sequentially, the GSI is administered first and the BCMA-targeted immunotherapeutic (soluble or cellular form) is administered second. In other embodiments, the BCMA-targeted immunotherapeutic (soluble or cellular form) is administered first and the GSI is administered second. In certain embodiments, the BCMA-targeted immunotherapeutic, including engineered immune cells that specifically bind to BCMA (e.g., BCMA-specific T cells such as CAR-T, T-ChARM, bispecific, multispecific T cells), is administered first, and the GSI is administered second (e.g., within hours, days, weeks, months, or years of the BCMA immunotherapeutic). In further embodiments, a BCMA-targeted immunotherapeutic comprising engineered immune cells (e.g., BCMA-specific T cells such as CAR-T, T-ChARM, bispecific, multispecific T cells) comprising a binding protein that specifically binds BCMA is (a) administered to a subject; (b) after a period of time (e.g., about 5 days to about 1 week, about 1 week to about 2 weeks, about 2 weeks to about 3 weeks, about 1 week to about 1 month, about 1 month to about 6 months, about 3 months to about 1 year, or longer as needed), the subject or the subject's tissues are probed or examined for the presence or persistence of the previously administered engineered immune cells comprising the binding protein that specifically binds BCMA, and (c) a GSI is administered second after the presence or persistence of the BCMA immunotherapy is detected.

[0105] In some embodiments, the GSI is administered to the subject at least once before, simultaneously with, and at least once (e.g., at least 2, 3, or 4 times) after a BCMA-targeted immunotherapy. In certain embodiments, a combination therapy of the present disclosure comprises administration of a BCMA-targeted immunotherapy and about 0.01 μM to about 5 μM GSI, about 0.03 μM to about 1.5 μM GSI, about 0.05 μM to about 2.5 μM GSI, or about 0.1 μM to about 1.0 μM GSI. In certain embodiments, a combination therapy comprising engineered immune cells that specifically bind to BCMA and a GSI results in reduced amounts of immune cells that specifically bind to BCMA, a GSI, or both, compared to administration of these therapies individually.

[0106] For example, by way of background and without wishing to be bound by theory, engraftment of administered cells expressing a BCMA-specific binding protein during adoptive immunotherapy can be promoted by prior immunosuppressive conditioning with one or more chemotherapeutic agents or treatments. In any of the embodiments described herein, the method further includes preconditioning the subject simultaneously with or prior to administering the BCMA-targeted immunotherapy or immune cells expressing a BCMA-specific binding protein and a GSI. In certain embodiments, the preconditioning regimen is immunosuppressive conditioning that includes depletion of endogenous lymphocytes (also referred to as lymphodepletion, which may be non-myeloablative or myeloablative). Exemplary lymphodepletion may be achieved by cyclophosphamide alone, cyclophosphamide in combination with fludarabine, the use of other agents or treatments that are cytotoxic to lymphocytes (e.g., total body irradiation), or any combination thereof.

[0107] The binding molecule, inhibitor, or combination composition may be administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally, or intracranially, or any combination thereof. When administered separately, the BCMA-targeted immunotherapy and the GSI may be administered by the same or different routes. For example, in certain embodiments, the BCMA-targeted immunotherapy is administered parenterally and the GSI is administered orally, which may be simultaneous or sequential. As used herein, the term "parenteral" includes subcutaneous injection, intravenous, intramuscular, intracisternal injection, or infusion techniques. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, or intrapulmonary injection and / or surgical implantation at a specific site is also contemplated. In general, the compositions are essentially free of pyrogens and other impurities that may be harmful to the recipient. Intravenous injection or infusion (infusion) is particularly preferred for administering BCMA-targeted immunotherapy.

[0108] In other embodiments, the BCMA-specific binding protein, the GSI, or both, may be administered to a subject in a soluble form (e.g., an antibody). Soluble TCRs are also known in the art (see, e.g., Molloy et al., Curr. Opin. Pharmacol. 5:438, 2005; U.S. Patent No. 6,759,243).

[0109] The pharmaceutical compositions comprising the combination therapy of the disclosed BCMA-targeted immunotherapy and gamma-secretase inhibitor can be administered in a manner appropriate for the disease or condition to be treated (or prevented), as determined by one skilled in the medical field. The appropriate dose, appropriate duration, and frequency of administration of the composition will be determined by factors such as the patient's condition, the size, type, and severity of the disease, the specific form of the active ingredient, and the method of administration. The present disclosure provides pharmaceutical compositions comprising cells expressing a BCMA-specific binding protein, such as a CAR or T-ChARM, and a pharmaceutically acceptable carrier, diluent, or excipient. Suitable excipients include water, saline, dextrose, glycerol, etc., and combinations thereof.

[0110] An advantage of the present disclosure is that cells expressing a BCMA-specific binding protein (e.g., a CAR or T-ChARM) administered to a patient can be depleted using a cognate binding partner for the tag cassette. In certain embodiments, the present disclosure provides methods for depleting T cells expressing a BCMA-specific T-ChARM by using an antibody specific for the tag cassette, by using a cognate binding partner specific for the tag cassette, or by using a second T cell expressing a CAR and having specificity for the tag cassette. In certain embodiments, the tag cassette allows for immunodepletion of T cells expressing the BCMA-specific T-ChARM of the present disclosure. For example, when a Strep tag is used, an anti-Strep tag antibody, anti-Strep tag scFv, or streptactin, each fused or conjugated to a cytotoxic reagent (e.g., a toxin, radioactive metal), can be used, or an anti-Strep tag / anti-CD3 bispecific scFv, or anti-Strep tag CAR-T cells can be used.

[0111] In another aspect, the present disclosure provides a method for selectively promoting the expansion of recombinant T cells expressing a BCMA-specific T-ChARM of the present disclosure. In certain embodiments, the method comprises selective ex vivo expansion of T cells expressing the BCMA-specific T-ChARM using a tag binding partner, such as an antibody. In further embodiments, the method comprises selectively expanding functional T cells (e.g., virus-specific, TAA (tumor-associated antigen)-specific CTLs, or specific T cell subsets, such as naive T cells, memory stem T cells, central or effector memory T cells, CD4 + CD25 + The method involves expanding the target cells (regulatory T cells) with a tag binding partner, such as an antibody, optionally in the presence of a costimulatory molecule binding partner (such as an anti-CD27 or anti-CD28 antibody).

[0112] In still further embodiments, when the BCMA-specific T-ChARM of the present disclosure is expressed, the BCMA-specific T-ChARM allows for selective promotion of T cell proliferation in vivo. In some embodiments, T cells expressing a CAR comprising a tag cassette, when contacted with cells expressing a ligand (e.g., including T cell inhibitory cell ligands PD-L1 and PD-L2), allow for CAR-T cell proliferation in vivo. Such expanded T cells are useful in the methods of treating disease described herein. In certain embodiments, proliferation or expansion of cells expressing the BCMA-specific T-ChARM disclosed herein can be induced in vivo by a tag cassette binding partner (e.g., an anti-tag antibody) and, optionally, a costimulatory molecule binding partner (e.g., an anti-CD27 or anti-CD28 antibody).

[0113] In certain further embodiments, cells expressing a BCMA-specific T-ChARM disclosed herein are activated in vivo, such as at a tumor site. For example, T cells expressing a BCMA-specific T-ChARM as disclosed herein can be activated locally at the site of a tumor (e.g., a solid tumor) using a composition (e.g., alginate, basement membrane matrix (Matrigel®), biopolymer, or other matrix) or carrier (e.g., microbeads, nanoparticles, or other solid surface) comprising a tag cassette binding partner (e.g., an anti-tag antibody) and a costimulatory molecule binding partner (e.g., an anti-CD27 or anti-CD28 antibody).

[0114] In certain embodiments, recombinant cells expressing BCMA-specific T-ChARM can be detected or tracked in vivo using an antibody that specifically binds to the tag cassette (e.g., an anti-tag antibody) or other cognate binding protein that specifically binds to the tag cassette sequence (e.g., streptactin, which binds to the Strep tag). The binding partner of this tag cassette is conjugated to a fluorescent dye, radioactive tracer, iron oxide nanoparticle, or imaging agent known in the art for detection by X-ray, CT scan, MRI scan, PET scan, ultrasound, flow cytometry, near-infrared imaging system, or other imaging modality (see, e.g., Yu et al., Theranostics 2:3, 2012).

[0115] In further embodiments, cells expressing the BCMA-specific T-ChARM of the present disclosure may be used in diagnostic or imaging methods, including methods used in connection with the indications or conditions identified herein.

[0116] In some embodiments, the method involves administering the GSI in combination (e.g., simultaneously, separately, or sequentially) with a BCMA-specific binding protein, which may include, for example, a BCMA-specific antibody or antigen-binding portion thereof, including administering the GSI with a BCMA-specific binding moiety, such as an antibody-drug conjugate, or a bispecific or multispecific binding protein, e.g., one useful for pre-targeted radioimmunotherapy (see, e.g., Green et al., Blood 131:611 (2018)). [Example]

[0117] Example 1: Creation and testing of BCMA-specific chimeric antigen receptors Anti-BCMA CARs were prepared to investigate their efficacy for immunotherapy of multiple myeloma and other diseases. Anti-BCMA CARs were prepared using V-cells derived from the C115 D5.3 ("C11") and A7D12.2 ("A7") antibodies. H Area and V LThe anti-BCMA CARs were generated using scFvs consisting of an IgG4 hinge region (spacer), a CD28 transmembrane domain, a 4-1BB costimulatory domain, and a CD3ζ effector domain. The scFvs were produced in both "HL" and "LH" orientations (see Figure 1A). Human T cells were transduced with expression constructs encoding the anti-BCMA CARs and functional characteristics were investigated. As shown in Figure 1B, T cells expressing the C11 CAR and the A7 CAR proliferated when co-cultured with BCMA-expressing target cells, although the C11 CAR appeared to proliferate slightly more robustly than T cells expressing the A7 CAR. C11 CAR-T cells produced greater amounts of cytokines in response to target cell lines (either antigen-transfected K562 cells or BCMA-expressing MM cell lines; Fig. 1C ) and had greater specific killing activity against target cells compared with A7 LH CAR-T cells ( Fig. 1D ).

[0118] We generated additional A7 and C11 CARs with different intracellular components, including a CAR containing the 4-1BB costimulatory domain (Figure 1E, top panel) and a CD28 costimulatory domain (Figure 1E, bottom panel). The length of the extracellular spacer domain also influences the binding of CAR-expressing T cells to BCMA. + To improve interaction with target cells, short (e.g., 12, 48, and 66 amino acids in length), intermediate (e.g., 157 amino acids in length), and long (e.g., 228 amino acids in length) spacers were modified. The 48- and 157-amino acid spacer CARs each contained two (2) Strep-Tag cassettes, while the 66-amino acid spacer contained three (3) Strep-Tag cassettes (see Figure 1F). A tagged chimeric antigen receptor (CAR) containing a Strep-Tag cassette is shown in Figures 1F and 1G and is referred to herein as T-ChARM.

[0119] Human T cells were transduced and expressed with T-ChARM (see Example 2) and analyzed for functionality. Spacer length influenced T-ChARM constructs containing scFvs derived from the anti-BCMA antibody C11 D5.3, where intermediate (approximately 65 amino acids; see C11 3ST, C11 2STint, and C11Lo in Figure 1H) to long (approximately 200 amino acids) spacers performed best for this binding domain. C113ST_4-1BB and C113ST_CD28 were selected as the best-performing constructs (Figures 1H-1J) and compared to a previously described anti-BCMA CAR ("BCMA-2"; Carpenter et al., Clin. Cancer Res. 19:2048, 2013). Expression of EGFRt on primary T cells was similar for each T-ChARM / CAR construct, and surface expression of T-ChARMs containing STII sequences was confirmed by staining with anti-STII monoclonal antibodies (Figure 1K). T cells expressing either C113ST T-ChARM produced higher amounts of cytokines (Figures 1L and 1M) and proliferated more robustly than T cells expressing BCMA-2 when cocultured with target cells (Figure 1N). T cells expressing either C113ST T-ChARM expressed CD138 + It was also determined that they were able to recognize and lyse parental MM cells (data not shown). T cells expressing either the C113ST T-ChARM or the BCMA-2 CAR had no cytolytic activity against non-antigen-expressing K562 cells (Figure 1O), but efficiently lysed K562 cells transduced to express the BCMA antigen (Figure 1P), demonstrating that the engineered T cells specifically recognize the antigen.

[0120] Example 2: Production of recombinant T cells and expression of BCMA-specific T-ChARMs CD8 + and CD4 + T cells were isolated from PBMCs of normal human donors and analyzed by CD8 + / CD4 +T cells were isolated using a T cell isolation kit (Miltenyi Biotec), activated with anti-CD3 / CD28 beads (Life Technologies) according to the manufacturer's instructions, and transduced three days after activation by centrifugation at 2,100 rpm for 45 minutes at 32°C with lentivirus (epHIB7) supernatant encoding a CAR generated by transient transfection (MOI = 3) of HEK293T cells with PsPAX2 and pMD2G packaging plasmids supplemented with 0.8 μg / mL polybrene (Millipore, Bedford, MA). T cells were cultured every 48 hours in RPMI, 10% human serum, 2 mM L-glutamine, and 1% penicillin-streptomycin (CTL medium) supplemented with recombinant human (rh) IL-2 to a final concentration of 50 U / mL. After culture, an aliquot of each transduced T cell line was stained with biotin-conjugated anti-EGFR antibody and streptavidin-PE (Miltenyi, Auburn, CA). tEGFR was detected by sorting on a FACS-Aria cell sorter (Becton Dickinson). + T cells were isolated and then tEGFR + T cell subsets were isolated from irradiated (8,000 rad) CD19 + B-LCLs were used to prime T cells at a T cell:LCL ratio of 1:7 and cultured for 8 days in CTL medium with the addition of 50 U / mL rhIL-2 every 48 hours or using the rapid expansion protocol for cells expressing T-ChARMs (Riddell & Greenberg, J. Immunol. Methods 128:189, 1990), where the T-ChARMs contain scFvs derived from the anti-BCMA antibodies A7 or C11 D5.3.

[0121] Flow cytometry phenotyping and analysis used the following conjugated antibodies: CD4, CD8, CD25, CD137, CD45, Annexin V, CD62L, CD27, CD28 (BD Biosciences), anti-Strep-tag II antibody (Genscript), EGFR antibody (ImClone Systems Incorporated, Branchburg, NJ); strepTavidin-PE (BD Biosciences, San Jose, CA). Propidium iodide staining (PI, BD Biosciences) was performed for live / dead cell sorting according to the manufacturer's instructions. Flow analysis was performed on a FACS Canto II, and sorting and purification was performed on a FACS Aria II (Becton Dickinson, Franklin Lakes, NJ), and data analysis was performed using FlowJo software (Treestar, Ashland, OR).

[0122] To examine cell surface expression of BCMA-specific T-ChARMs, transduced T cells were sorted for EGFRt expression and assessed by staining with a fluorochrome-conjugated anti-Streptag mAb. The mean fluorescence intensity (MFI) of EGFR staining was similar for T cells transduced with each of the BCMA-specific T-ChARMs and the CD19-Short CAR. Introduction of a tag into the CAR to generate the T-ChARM did not interfere with transgene expression (data not shown). Anti-Streptag mAb specifically stained T cells transduced with various BCMA-specific T-ChARMs, regardless of the position or number of tag sequences in each T-ChARM.

[0123] Example 3: Soluble BCMA (sBCMA) inhibits BCMA-specific CAR-T cell activity We investigated the potential efficacy of BCMA-targeted T cell therapy for most myelomas. Because shedding of cell surface BCMA by myeloma cells can interfere with T cell recognition, we measured soluble BCMA levels in the supernatants of myeloma cell lines during culture. U266 myeloma cells were washed and plated in culture medium for 1, 3, 5, and 24 hours. Culture supernatants were collected and analyzed for sBCMA by ELISA. The data show a time-dependent increase in sBCMA levels in the supernatants (Figure 2A). Next, we examined the extent of BCMA expression and cytokine production on primary patient multiple myeloma (MM) cells by contacting BCMA-specific T-ChARM T cells with the patient's MM cells. Figure 2B shows surface BCMA expression by a control cell line (RPMI, left panel) and test samples from primary patient MM cells with different levels of BCMA expression. The pie chart in Figure 2C shows that BCMA expression on the surface of MM cells from 19 different patients was found to be positive for the majority of patients, although some patient samples showed intermediate to low / none surface BCMA expression (~25%). Furthermore, Figure 2D shows that T-ChARM T cells produced greater amounts of IFN-γ when cultured with patient MM cells expressing high levels of surface BCMA compared to those cultured with low-BCMA patient MM cells.

[0124] Myeloma cells frequently express PD-L1, which is thought to suppress T cell function by binding to PD-1 on T cells (Freeman et al., J. Exp. Med. 192(7):1027 (2000)). We also investigated the possibility that PD-L1 acts on BCMA-specific T-ChARM T cells. Flow cytometry of patient samples revealed that PD-L1 was expressed on 79% of samples, specifically 47% of which expressed PD-L1. hi , and 32% were PD-L1 int , and 21% had PD-L1 low / negThe results showed that BCMA expression was significantly higher in T-ChARM T cells than in MM cells expressing high levels of PD-L1 (Figures 2E and 2F). No correlation was observed between BCMA and PD-L1 expression. Figure 2G shows that T-ChARM T cells produced greater amounts of IFN-γ when cultured with patient MM cells expressing low levels of PD-L1 than with patient MM cells expressing high levels of PD-L1, although this trend was not statistically significant.

[0125] T cells expressing BCMA-specific T-ChARM and K562 cells transduced with a polynucleotide encoding full-length BCMA (K562 / BCMA + The effect of sBCMA was examined by adding sBCMA to co-cultures with MM cells. Dose-dependent inhibition of BCMA-specific T-ChARM T cell effector function was detected as a measure of IFN-γ release into the culture supernatant upon administration of exogenous sBCMA (Figures 2H, 2I). Furthermore, MM cell lines showed a substantial increase in sBCMA in the culture supernatant within 24 hours (Figure 2J).

[0126] To investigate whether high levels of sBCMA could inhibit the activity of BCMA-specific T-ChARM T cells, the patient's bone marrow (BM) serum was examined for sBCMA levels. The patient's BM was found to have high levels of sBCMA, which was associated with CD138 + This roughly correlated with disease burden as determined by cell abundance (Figure 2K). To test whether sBCMA binds to T-ChARM T cells, T cells were incubated with increasing levels of recombinant BCMA and then stained with BCMA-Fc bound to APC. As shown in Figure 2L, staining decreased with increasing levels of recombinant BCMA, indicating that sBCMA may potentially have a detrimental effect on BCMA-specific T-ChARM T cell function.

[0127] To confirm T-ChARM expression, C113ST T-ChARM T cells and control anti-CD19 CAR T cells ("FMC63") were incubated with Fc-BCMA and stained for EGFRt (a CAR / T-ChARM transduction marker) and CD4, demonstrating that T-ChARMs were expressed by these cells (Figure 2M). Staining for EGFRt and other T cell surface molecules was unaffected (data not shown).

[0128] To examine whether shedding of sBCMA and its binding to T-ChARM suppresses T cell function, we added BCMA-Fc to T cell / target cell line cocultures. This resulted in a significant decrease in cognitive function (IFN-γ production and CD4 expression) by BCMA-targeted T-ChARM T cells, but not by control anti-CD19 CAR-T cells (cultured with K562 cells expressing the CD19 antigen) (Figure 2N). The effect of BCMA on T-ChARM T cells was dose-dependent (Figures 2O, 2P). Addition of recombinant BCMA unfused to Fc also inhibited IFN-γ production in a dose-dependent manner, but not by K562 BCMA T-ChARM T cells. + It did not inhibit cell lysis (Fig. 2Q), a result likely due to the high antigen density on the target cell surface.

[0129] Example 4: Effect of γ-secretase inhibitors on MM BCMA levels To examine the effect of γ-secretase inhibition on BCMA levels in myeloma cells, we used the γ-secretase inhibitor (GSI) RO429097. BCMA was rapidly upregulated in various myeloma cell lines when incubated with the γ-secretase inhibitor (GSI) RO429097 (at concentrations ranging from 0.001 μM to 1.0 μM) (Figures 3A-3D).

[0130] To examine the effect of GSI on surface BCMA expression kinetics, U266 myeloma cells were incubated in the presence of various concentrations of the GSI R0429097 (0.01 μM, 0.1 μM, and 1.0 μM) for 1, 3, 5, and 24 hours and assessed for cell surface BCMA expression by flow cytometry. BCMA expression increased in a dose-dependent manner in the presence of GSI (Figure 3E). At 1.0 μM GSI, upregulation persisted over the 7-day culture period (Figure 3F).

[0131] To examine the effect of GSIs on BCMA shedding in MM, three different myeloma cell lines (MM1.R, U266, and 8226) were washed and plated in the presence of various concentrations of the GSI R0429097 (0.01 μM, 0.1 μM, and 1.0 μM) for 24 hours. Culture supernatants were harvested and assayed for sBCMA by ELISA. sBCMA levels were reduced in a dose-dependent manner in the supernatants in the presence of GSI (Figures 3G & 3H).

[0132] To examine the time course effect on soluble BCMA (sBCMA) levels, U266 cells were washed and plated in media for 1, 3, 5, and 24 hours in the presence of various concentrations of the GSI R0429097 (0.01 μM, 0.1 μM, and 1.0 μM). Media supernatants were collected and assayed for soluble BCMA (sBCMA) by ELISA. The data show that the presence of the GSI reduces soluble BCMA levels in the supernatant in a dose-dependent manner (Figure 3I).

[0133] Next, we examined the effect of removing GSI from MM cell cultures. As shown in Figures 3J and 3K, surface BCMA levels decreased after removal of 1.0 μM R0429097, whereas supernatant BCMA levels increased when GSI was removed but not when GSI was retained. These data demonstrate a reversible inhibitory effect of GSI on surface BCMA shedding. Finally, the viability of the tested MM cell lines was unaffected by the addition of 1.0 μM R0429097 to the cultures (Figure 3L).

[0134] The effect of GSI on BCMA expression was then tested in primary myeloma samples from patients. + Myeloma cells were enriched and incubated for 3 hours in the presence of various concentrations of the GSI RO429097 (0.01 μM to 10 μM) and assessed for surface BCMA expression by flow cytometry (Figures 3M and 3R). BCMA mean fluorescence intensity (MFI) on tumor cells was expressed as the fold increase relative to tumor cells incubated in the absence of R0429097. A dose-dependent upregulation of BCMA on tumor cells was observed (Figure 3N), while the levels of several other cell surface molecules, such as CS1, CD86, PD-L1, CD80, and CD38, were completely unaffected (Figures 3O–3Q).

[0135] Example 5: γ-secretase inhibitors inhibit BCMA uptake by T cells expressing BCMA-specific T-ChARM + Improved recognition of myeloma cells BCMA +To examine the effect of GSIs on CAR T cell activity against multiple myeloma, IL-2 production by BCMA CAR-T cells (BCMA-specific T-ChARM C11 3ST-CD28 and BCMA-specific T-ChARM C11 3ST-41BB) or control CD19sh CAR (short spacer)-T cells was measured after 24 hours of coculture with primary human myeloma cells in medium containing various concentrations of the GSI R0429097 (0.003 μM to 3.0 μM) (Figure 4A). Treatment with GSI caused a dose-dependent increase in IL-2 production by BCMA T-ChARM T cells. IFNγ production by BCMA T-ChARM T cells cocultured with myeloma cells in the presence of various concentrations of R0429097 was also increased in the presence of GSI (Figure 4B). These data indicate that pretreatment of multiple myeloma cells with a γ-secretase inhibitor enhanced BCMA-specific T-ChARM T cell proliferation and upregulated BCMA expression on tumor cells. Finally, proliferation of CFSE-labeled BCMA-specific T-ChARM T cells increased in a dose-dependent manner after 3 days of coculture with primary human myeloma cells in the presence of GSI R0429097 (Figure 4C).

[0136] Without being bound by theory, a possible caveat of using GSIs to increase BCMA on myeloma cells to improve CAR-T cell recognition is that high concentrations of GSIs may inhibit T cell signaling and effector function. See Eagar et al., Immunity 20(4):407, 2004. To evaluate the potential effect of GSIs on CAR-T cells in the setting of stably maintained target ligand expression, CD19 CAR-T cells were transfected with Raji or K562 / CD19 CAR-T cells. +Target cells were co-cultured with GSI (0.01 μM to 100 μM), and viability and effector function were measured. Over this concentration range, no effect on CD19 antigen expression (Figure 5A) or CAR-T cell viability (Figure 5B) was observed after 24 hours. GSI RO4929097 inhibited K562 CD19 expression, as determined by measuring IL-2 production (Figure 5C, upper panel) and IFNγ production (Figure 5C, lower panel). + We found that GSI R04929097 inhibited CD19 CAR-T cell effector function at concentrations of ≥1 μM when cocultured with CD19 CAR-T cells (see also Figures 5D–5F). However, the ability of CD19 CAR-T cells to specifically kill target cells was not affected by GSI concentration (Figure 5G). CD19 CAR-T cells proliferated in response to target cell stimulation in the presence of 10 μM GSI R04929097, but less efficiently than in the absence of GSI R04929097 (Figure 5H). To further investigate the dose effect of GSI on CAR-T cell division, we added increasing doses of GSI R04929097 to cocultures of CD19 CAR-T and target cells. The addition of therapeutic doses of GSI (0.01 μM–1 μM) had virtually no or only a minimal effect on CAR-T cell division, while higher, non-therapeutic doses of GSI (10 μM) detectably inhibited proliferation (Figure 5I). CD19 CAR-T cells cultured in the presence of GSI showed no reduction in proliferation over 8 days in culture medium with (Figure 5J) or without (Figure 5K) exogenous IL-2. Furthermore, T cells cultured in the presence or absence of 5 μM or 0.5 μM GSI did not show significant differences in IFN-γ production when restimulated with target cells after expansion in the presence (Figure 5L) or absence (Figure 5M) of exogenous IL-2. IL-2 production also did not differ significantly between CAR-T cell groups (Figure 5N). Overall, therapeutic levels of GSI do not affect T cell functions such as cytokine production, cytolytic activity, and proliferation.

[0137] Next, we examined the effect of GSI on T cell functionality of T cells containing BCMA-specific T-ChARM. GSI treatment of primary myeloma samples significantly improved IFN-γ production (Figures 5O, 5P, and 5Q) and CD8 expression (Figure 5O) by cocultured T-ChARM T cells, an effect observed after treatment with 0.1 μM GSI or higher, and also increased T cell proliferation (Figure 5R).

[0138] These data demonstrate the clinical utility of combining BCMA-specific CAR-T cells with GSIs to increase BCMA levels on multiple myeloma cells. This may provide benefit in treating multiple myeloma by promoting the elimination of tumor cells expressing low levels of BCMA and may even have a synergistic effect. The data also show that a range of concentrations of γ-secretase inhibitors promoted BCMA expression on tumor cells without affecting T cell activity.

[0139] Example 6: In vivo study of GST activity in MM xenograft models To determine whether the sBCMA inhibitory effects of GSI R04929097 described in Example 4 could be replicated in vivo, a mouse xenograft model of human MM was tested as depicted in Figure 6A. Briefly, immunodeficient NOD / SCIDγ (NSG) mice (The Jackson Laboratory) were sublethally irradiated (275 rad) on day -1, followed by 5x10 rFv the following day (day 0). 6 Mice were administered MM.1R cells. The GSI RO4929097 (30 mg / kg) was administered by oral gavage on days 19 and 20. Mice were sacrificed at various time points after the second GSI gavage, and blood and bone marrow samples were collected for analysis. Following GSI administration, there was a rapid increase in surface BCMA expression on tumor cells (>3-fold increase by 4 hours after the second gavage) (Figure 6B) with a concomitant decrease in sBCMA expression (Figure 6C). The observed effect generally diminished within 48 hours after the second gavage.

[0140] Example 7: In vivo study of GSI + CAR-T cell combination therapy targeting MM The ability of GSI therapy to improve anti-BCMA CAR-T cell therapy in vivo was investigated. In an experiment similar to that described in Example 6, NSG mice were sublethally irradiated on day -21 and subsequently inoculated with human MM cells (5x10 6 MM.1R ffluc ) (see Figure 7A). On day -1, mice received the first dose of GSI (30 mg / kg RO4929097) by oral gavage. On day 0, mice received a suboptimal dose of C11 3ST T-ChARM T cells (0.33 x 10 6 Mice were injected with GST+T cells (CD4:CD8 1:1 ratio) and then given a second dose of GSI. Additional doses of GSI were administered on days +1, +8, and +9. Bioluminescence imaging (BLI) was performed throughout the experiment to monitor survival. As shown in Figures 7B, 7C, and 7E (left graphs), the group receiving GST+T cells exhibited reduced luminescence and more mice lacked detectable tumors compared to the group receiving T cells alone (BLI data from the T cell-only group are not shown). Quantified luminescence data (Figures 7C and 7E (left graphs)) showed that the initial T-ChARM T cell effect began to reverse around day 9 when tumors began to grow ("Mock+BCMA T cells"). The antitumor effect was prolonged by combination with GSI R04929097 ("R049+BCMA T cells"), although tumors in that treatment group eventually grew. The imaging data was consistent with mouse survival (Figures 7D and 7E (right graphs)), with mice receiving the combination therapy surviving longer before death than mice receiving T cells alone. In one embodiment, the GSI treatment is repeated (at least 2 times, at least about 5 times, up to at least 25 times) when used in combination with anti-BCMA CAR T cells, and can be administered simultaneously with, before, or after administration of the anti-BCMA CAR T cells.

[0141] Example 8: GSI inhibits BCMA with bispecific anti-BCMA fusion proteins + Improved binding to MM cells A bispecific fusion protein with specificity for both BCMA and another antigen and a YFP (yellow fluorescent protein) tag was generated. This bispecific fusion protein was injected into BCMA-expressing H929 MM cells (0.5 × 10 cells) in culture with or without GSI. 6 Cells). Binding was assessed by flow cytometry. As shown in Figure 8, the addition of a GSI improved binding by the BCMA-binding bispecific fusion protein, but had no effect on binding by a control bispecific fusion protein that does not target BCMA. These data indicate that immunotherapy using bispecific molecules that target BCMA can be enhanced or improved by a GSI.

[0142] The various embodiments described above can be combined to provide additional embodiments. The U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the Application Data Sheets are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to utilize the concepts of the various patents, applications, and publications to provide further additional embodiments.

[0143] These and other changes can be made to the embodiments in light of the above-detailed description. In general, the terms used in the following claims should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but should be construed to encompass all possible embodiments along with the full range of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims

1. In a subject having or suspected of having a disease or disorder associated with BCMA expression, (i) a proliferative disease or disorder, such as cancer, and / or (ii) an autoimmune disease or disorder 23. A method of treating a BCMA-specific inflammatory bowel disease comprising administering to the subject a therapeutically effective amount of a BCMA-specific binding protein and a therapeutically effective amount of a gamma secretase inhibitor.

2. 2. The method of claim 1, wherein the BCMA-specific binding protein is a BCMA-specific antibody or antigen-binding portion thereof, a chimeric antigen receptor (CAR) or a tagged chimeric antigen receptor molecule (T-ChARM).

3. 3. The method of claim 2, wherein the BCMA-specific binding protein is human or humanized.

4. 4. The method of any one of claims 1 to 3, wherein the BCMA-specific protein comprises a BCMA-specific scFv, a BCMA-specific scTCR, or a BCMA ligand or a binding portion thereof.

5. The BCMA specific binding protein is selected from the group consisting of BCMA antibodies J22.0-xi, J22.9-xi, J6M0, J6M1, J6M2, J9M0, J9M1, J9M2, 11D5-3, CA8, A7D12.2, C11 D5.3, C12A3.2, C13F12.1, 13C2, 17A5, 83A10, 13A4, 13D2, 14B11, 14E1, 29B11, 29F3, 13A7, CA7, S307118G03, SG1, S332121F02, S332126E04, S322110D07, S336105A07, S335115G01, The method of claim 4, wherein the scFv comprises heavy and light chain variable regions based on S335122F05, ET140-3, ET140-24, ET140-37, ET140-40, ET140-54, TBL-CLN1, C4.E2.1, Vicky-1, pSCHLI333, pSCHLI372, or pSCHLI373.

6. the BCMA specific binding protein is a chimeric antigen receptor comprising a hydrophobic portion located between an extracellular component and an intracellular component, wherein the extracellular component comprises a BCMA specific binding protein, the BCMA specific binding protein being (a) the BCMA antibodies J22.0-xi, J22.9-xi, J6M0, 11D5-3, CA8, A7D12.2, C11 D5.3, C12A3.2, C13F12.1, 13C2, 17A5, 83A10, 13A4, 13D2, 14B11, 14E1, 29B11, 29F3, 13A7, CA7, SG1, S307118G03, S332121F02, S332126E04, S322110D07, S336105A07, S335 6. The method of any one of claims 1 to 5, comprising (a) a BCMA-specific antigen binding portion derived from 115G01, S335122F05, ET140-3, ET140-24, ET140-37, ET140-40, ET140-54, TBL-CLN1, C4.E2.1, Vicky-1, pSCHLI333, pSCHLI372 or pSCHLI373; or (b) a BCMA ligand or binding portion thereof based on or derived from BAFF or APRLIL.

7. The method of claim 6 , wherein the hydrophobic portion is a transmembrane domain.

8. The method of claim 7, wherein the transmembrane domain is a CD4, CD8, CD28 or CD27 transmembrane domain.

9. The method of any one of claims 6 to 8, wherein the intracellular component comprises an effector domain or a functional portion thereof, a co-stimulatory domain or a functional portion thereof, or any combination thereof.

10. 10. The method of claim 9, wherein the intracellular component comprises 4-1BB (CD137), CD3ε, CD3δ, CD3ζ, CD25, CD27, CD28, CD79A, CD79B, CARD11, DAP10, FcRα, FcRβ, FcRγ, Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, OX40 (CD134), ROR2, Ryk, SLAMF1, Slp76, pTα, TCRα, TCRβ, TRIM, Zap70, PTCH2, or a functional portion thereof, or any combination thereof.

11. The method of claim 9 or 10, wherein the effector domain comprises CD3ζ or a functional portion thereof.

12. 12. The method of any one of claims 6 to 11, wherein the intracellular component comprises a costimulatory domain or a functional portion thereof, and the costimulatory domain or a functional portion thereof is selected from CD27, CD28, 4-1BB (CD137), OX40 (CD134), or any combination thereof.

13. 13. The method of any one of claims 6 to 12, wherein the effector domain or effector portion thereof comprises CD3zeta or a functional portion thereof and one or more of the costimulatory domains or functional portions thereof of 4-1BB (CD137), CD27, CD28 and OX40 (CD134).

14. The method of any one of claims 6 to 13, wherein the intracellular components comprise: (a) 4-1BB or a functional portion thereof and CD3ζ; (b) CD27 or a functional portion thereof and CD3ζ; (c) CD28 or a functional portion thereof and CD3ζ; (d) OX40 or a functional portion thereof and CD3ζ; (e) CD28 or a functional portion thereof, 4-1BB or a functional portion thereof, and CD3ζ; (f) OX40 or a functional portion thereof, 4-1BB or a functional portion thereof, and CD3ζ; or (g) CD28 or a functional portion thereof, OX40 or a functional portion thereof, and CD3ζ.

15. 15. The method of any one of claims 6 to 14, wherein the extracellular component comprises an immunoglobulin hinge region, a CH2 domain and a CH3 domain, a CH3 domain, or any combination thereof, located between the BCMA-specific binding protein and a hydrophobic moiety.

16. The method of claim 15, wherein the immunoglobulin hinge region is an IgG1 hinge region.

17. The method of claim 15 or 16, wherein the CH2 domain is an IgG1 CH2 domain and the CH3 domain is an IgG1 CH3 domain.

18. 18. The method of any one of claims 1 to 17, wherein the BCMA specific binding protein is encoded by an exogenous polynucleotide and expressed in a host cell.

19. 20. The method of claim 18, wherein the host cell is a human immune system cell.

20. The human immune system cells are CD4 + T cells, CD8 + T cells, CD4 - CD8 - 20. The method of claim 19, wherein the cells are double negative T cells, γδ T cells, natural killer cells, dendritic cells, or any combination thereof.

21. 21. The method of claim 20, wherein the human immune system cells are T cells, and the T cells are naive T cells, central memory T cells, effector memory T cells, bulk T cells, or any combination thereof.

22. 22. The method of any one of claims 1 to 21, wherein the gamma-secretase inhibitor is avagacestat, DAPT, BMS-906024, BMS-986115, MK-0752, PF-03084014, RO4929097 or YO-01027.

23. 23. The method of claim 22, wherein the gamma secretase inhibitor is a nicastrin specific binding protein.

24. 24. The method of claim 23, wherein the nicastrin-specific binding protein is scFvG9, antibody A5226A, antibody 2H6 or antibody 10C11.

25. 25. The method of any one of claims 1 to 24, wherein the BCMA-specific binding protein comprises a variable domain comprising CDR1, CDR2 and CDR3 of the amino acid sequence of any one of SEQ ID NOs: 14 to 16, and a variable domain comprising CDR1, CDR2 and CDR3 of the amino acid sequence of any one of SEQ ID NOs: 17 to 21.

26. 26. The method of any one of claims 1 to 25, wherein the method further comprises pretreating the subject with an immunosuppressive regimen prior to or concurrently with said BCMA-specific binding protein and said gamma-secretase inhibitor.

27. 27. The method of claim 26, wherein the immunosuppressive regimen is a non-myeloablative or myeloablative regimen.

28. 28. The method of claim 27, wherein the non-myeloablative therapy comprises cyclophosphamide or cyclophosphamide in combination with fludarabine.

29. 29. The method of any one of claims 1 to 28, wherein the BCMA-specific binding protein and the gamma-secretase inhibitor are administered sequentially.

30. 29. The method of any one of claims 1 to 28, wherein the BCMA-specific binding protein and the gamma-secretase inhibitor are administered simultaneously.

31. 31. The method of claim 30, wherein the BCMA-specific binding protein and the gamma-secretase inhibitor are formulated together.

32. 32. The method of any one of claims 1 to 31, wherein the BCMA-specific binding protein is administered parenterally and the gamma-secretase inhibitor is administered orally.

33. The method of any one of claims 1 to 32, wherein the proliferative disease or disorder is a hematological or solid cancer.

34. 34. The method of claim 33, wherein the hematological cancer is selected from the group consisting of multiple myeloma, plasmacytoma, plasma cell leukemia, Waldenstrom's macroglobulinemia, B-cell leukemia, and lymphoplasmacytic lymphoma.

35. 34. The method of claim 33, wherein the solid cancer is adenocarcinoma of the breast or bronchial carcinoma of the lung.

36. The autoimmune disease is selected from the group consisting of arthritis, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, polychondritis, psoriatic arthritis, psoriasis, dermatitis, polymyositis / dermatomyositis, inclusion body myositis, inflammatory myositis, toxic epidermal necrolysis, systemic dermatosclerosis and sclerosis, CREST syndrome, inflammatory bowel disease, Crohn's disease, ulcerative colitis, respiratory distress syndrome, adult respiratory distress syndrome (ARDS), meningitis, encephalitis, uveitis, colitis, glomerulonephritis, allergic conditions, eczema, asthma, conditions involving T cell infiltration and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, total inflammatory disease, inflammatory bowel ... Systemic lupus erythematosus (SLE), subacute cutaneous lupus erythematosus, discoid lupus, lupus myelitis, lupus encephalitis, juvenile-onset diabetes mellitus, multiple sclerosis, allergic encephalomyelitis, neuromyelitis optica, rheumatic fever, Sydenham's chorea, immune responses with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, granulomatous diseases such as Wegener's granulomatosis and Churg-Strauss disease, agranulocytosis, vasculitis (including hypersensitivity vasculitis / vasculitis, ANCA and rheumatic vasculitis), aplastic anemia, Diamond Blackfield's disease, Fan's anemia, immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases with leukocyte leakage, central nervous system (CNS) inflammatory diseases, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, antiphospholipid syndrome, allergic neuritis, Behçet's disease, Castleman syndrome, Goodpasture's syndrome, Lambert-Eaton myasthenic syndrome, Raynaud's syndrome, Sjögren's syndrome , Stevens-Johnson syndrome, solid organ transplant rejection, graft-versus-host disease (GVHD), bullous pemphigoid, pemphigus, autoimmune polyendocrinopathy, seronegative spondyloarthropathy, Reiter's disease, stiff man syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathy or IgM-mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), Henoch-Schönlein purpura, autoimmune diseases of the testes and ovaries, including autoimmune orchitis and oophoritis, primary hypothyroidism;Autoimmune endocrine disorders such as autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Graves' disease, autoimmune polyglandular syndrome (or polyendocrinopathy syndrome), type I diabetes mellitus also called insulin-dependent diabetes mellitus (IDDM) and Sheehan's syndrome; autoimmune hepatitis, lymphocytic interstitial pneumonia (HIV), bronchiolitis obliterans (non-transplant), nonspecific interstitial pneumonia (NSIP), Guillain-Barre syndrome, large vessel vasculitis (including polymyalgia rheumatica and giant cell (Takayasu) arteritis), intermediate vasculitis (including Kawasaki disease and polyarteritis nodosa), polyarteritis nodosa (PAN), ankylosing spondylitis, Berger's disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, celiac disease 33. The method of any one of claims 1 to 32, wherein the disease is selected from the group consisting of cerebrospinal fluid (cerebrospinal fluid) sprue (gluten enteropathy), cryoglobulinemia, cryoglobulinemia associated with hepatitis, amyotrophic lateral sclerosis (ALS), coronary artery disease, familial Mediterranean fever, microscopic polyangiitis, Cogan's syndrome, Wiskott-Aldrich syndrome, and thromboangiitis obliterans.

37. 1. A kit for treating a hematological and / or autoimmune disease or disorder, comprising: (a) a unit dose of a BCMA-specific binding protein; and (b) a unit dose of a γ-secretase inhibitor.

38. 38. The kit of claim 37, further comprising a C20-specific binding protein, such as rituximab, ofatumumab, or ocrelizumab; a CD19-specific binding protein; a CD45-specific binding protein; a CD38-specific binding protein; a cytokine; a chemokine; a growth factor; a chemotherapy agent; or a radiotherapy agent.

39. The method of any one of claims 6 to 36, wherein the chimeric antigen receptor comprises T-ChARM and the extracellular domain of the T-ChARM comprises a Strep Tag.

40. The method of claim 39, wherein the extracellular domain of the T-ChARM comprises 2, 3, 4, 5, 6, 7, 8 or 9 tag cassettes.

41. 41. The method of any one of claims 1 to 40, wherein the gamma secretase inhibitor is administered to the subject at least once after an initial administration of a BCMA-specific binding protein.

42. 42. The method of claim 41, wherein the gamma secretase inhibitor is administered at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 times after an initial administration of a BCMA-specific binding protein.

43. 43. The method of any one of claims 1 to 42, wherein the gamma secretase inhibitor is administered at a concentration of about 30 mg / kg.

44. 6. The method of any one of claims 1 to 5, wherein the BCMA-specific binding protein comprises an antibody, or an antigen-binding portion thereof, conjugated to a cytotoxic agent.

45. 45. The method of any one of claims 1 to 5 or 44, wherein the BCMA specific binding protein is multispecific.

46. 46. ​​The method of claim 45, wherein the BCMA-specific binding protein is bispecific.

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