combination therapies to treat cancers and autoimmune disorders related to bcma

专利摘要:
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 y-secretase inhibitors, which can be done concomitantly or sequentially to treat or preventing a B cell-related proliferative disease, such as cancer or autoimmune disease, or the like. a bcma specific binding molecule in combination with y-secretase inhibitor can be used, for example, in adoptive immunotherapy.
公开号:BR112019017120A2
申请号:R112019017120
申请日:2018-02-16
公开日:2020-04-14
发明作者:GREEN Damian；R Riddell Stanley；HILL Tyler
申请人:Hutchinson Fred Cancer Res；
IPC主号:

专利说明:

Invention Patent Descriptive Report for: COMBINATION THERAPIES FOR TREATING CANCERS AND AUTOIMMUNE DISORDERS RELATED TO BCMA
GOVERNMENT DECLARATION OF INTEREST [001] This invention was made with government support under CA136551 granted by the National Institutes of Health. The government has certain rights in the invention.
DECLARATION RELATING TO THE SEQUENCE LISTING [002] The Sequence List associated with this application is provided in text format instead of a paper copy and is incorporated by reference in the specification. The name of the text file that contains the String Listing is 360056_445WQ_SEQUENCE_LISTING.txt. The text file is 14.4 KB, was created on February 16, 2018 and is being sent electronically via EFS-Web.
BACKGROUND [003] The human immune system generally protects the body from pathogens and foreign invaders. B lymphocytes, which are also referred to as B cells and a component of the immune system, produce antibodies that bind to, and in some cases mediate the destruction of, the foreign substance or pathogen. In some cases, however, the immune system may be deregulated and result in diseases that involve an uncontrolled proliferation of B cells, such as cancer, autoimmune disease and disease
Petition 870190103473, of 10/14/2019, p. 7/184
2/118 inflammatory.
[004] Mature B cells and their differentiated progeny can be identified by molecules on their cell surface, such as the B cell maturation antigen (BCMA, also known as member 17 of the tumor necrosis factor receptor superfamily (TNFRSF17), TNFRSF13A and CD269), which is expressed in plasma cells and in some mature B cells. BCMA has been shown to specifically bind to B cell activation factor (BAFF, also known as TNFSF13B, TALL-1, and CD257) and a proliferation-inducing ligand (APRIL, also known as TNFSF13, TALL-2 and CD256), which can lead to NF-κΒ activation. Therapies that target BCMA, including adoptive transfer of T cells modified by the BCMA-specific chimeric antigen receptor (CAR), antibodies specific to BCMA naked, or administration of BCMA-specific antibodies conjugated to a therapeutic moiety (antibody-drug conjugate, CAF, such as a radioactive marker) can be used to treat some types of B cell malignancies, such as multiple myeloma, but can be limited in effectiveness by the number of BCMA molecules expressed on the surface of tumor cells and / or the presence of soluble BCMA in the circulation. A low expression of BCMA on the surface of cancer cells or soluble BCMA can limit and prevent
Petition 870190103473, of 10/14/2019, p. 8/184
3/118 the effectiveness of therapeutic agents due to inadequate binding to BCMA present on the surface of tumor cells. Low levels of other target molecules in tumor cells (eg CD19, CD20) that are antibody targets, antibody-drug conjugates or T cells with chimeric antigen receptor have been shown to limit the effectiveness of these therapies and allow cancer cells that express low levels of the target molecule escape elimination. In the case of BCMA, the short extracellular portion of the molecule is cleaved from the cell's surface and discarded through the action of gamma-secretase (γ-secretase), a cell enzyme located in the membrane and involved in protein dividing. This divage decreases the BCMA density in cells, such as myeloma cancer cells that express the molecule, and results in elevated levels of soluble BCMA (sBCMA) in the serum of patients with certain autoimmune diseases (eg, systemic lupus erythematosus) and cancer (for example, multiple myeloma).
[005] Currently, there is still a need in the field of immunotherapy for alternative or improved compositions or methods for the more efficient treatment of autoimmune diseases and cancer.
BRIEF DESCRIPTION OF THE DRAWINGS [007] Figures IA - IP show the design and functional tests of examples of chimeric receptor molecules
Petition 870190103473, of 10/14/2019, p. 9/184
4/118 of antigens (CAR) of this disclosure.
(A) Illustrations of examples of CARs that have an extracellular component consisting of a scFv specific for BCMA derived from antibody A7D12.2 (A7) or antibody C11D5.3 (Cll) and a spacer region (composed of a hinge region of IgG4), a hydrophobic portion (composed of a transmembrane domain of CD28) and an intracellular component composed of an effector domain of CD3 / and a costimulatory domain of 4-1BB. The scFvs were constructed with the C-terminal end of the Vh region linked (linker) of variable regions G4S (SEQ ID NO: 30)) to the N-terminal end of the Vl region (HL orientation) or the C-terminal end of the V1 region linked to the N-terminal end of the Vh region (LH orientation).
(B) Flow cytometry data (staining with succinimidyl ester carboxyfluorescein (CFSE)) showing the proliferation of human T cells that express the CARs shown in Figure IA and control T cells that do not contain a CAR in response to tumor cells that express BCMA .
(C) production of cytokines (IFN-γ) by T cells transduced by CAR shown in Figure 1B when cultured in vitro with the indicated BCMA- (K562) or BCMA ⁺ (K562 BCMA, U266, MM1.S) tumor cell lines .
(D) Specific lysis of target cell lines
Petition 870190103473, of 10/14/2019, p. 10/184
5/118
indicated by the cells T-BCMA CAR. (AND) Illustration of two examples of from CAR this disclosure , where region spacer (Spacer +) can
include a cassette with a tag, a linker module, a hinge, a spacer amino acid and any combination thereof. If the CAR contains one or more markers in the spacer region, it will be referred to as a T-ChARM, as described herein. The upper CAR contains an extracellular component (composed of a scFv specific for BCMA and a spacer region that optionally contains other elements, such as a marker or ligand (ligand)), hydrophobic portion (composed of a transmembrane domain of CD28) and a component intracellular (composed of a CD3 / effector domain and a 4-1BB co-stimulatory domain). The lower CAR contains an extracellular component (composed of a scFv specific for BCMA and a spacer region that optionally contains other elements, such as a marker or ligand (ligand)), hydrophobic portion (composed of a transmembrane domain of CD28) and a component intracellular (composed of a CD3 / effector domain and a co-stimulatory CD28 domain). Both BCMA CAR / T-ChARM specific constructs contain a gene marker for transduction that comprises a truncated human EGFR (EGFRt), which has separated from the BCMA specific constructs
Petition 870190103473, of 10/14/2019, p. 11/184
6/118
CAR / T-ChARM by a self-cleavage peptide sequence of the Thoseaasígna virus 2A (T2A). Other known peptides that self-cleave can also be used, such as porcine teschovirus-1 2A (P2A), equine rhinitis A virus (ERAV) 2A (E2A) and foot-and-mouth disease virus (FMDV) 2A (F2A).
(F) Examples of CAR / T-ChARM constructs that have spacer regions of different 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 = spacer with an intermediate length of 157 amino acids with two strep tag cassettes; and Lo = 228 amino acid long spacer. The Short and Long spacers can optionally contain a cassette with a marker, such as a Strep tag.
(G) Additional example illustrations of CAR / T-ChARM constructs with a scFv Cll or A7 HL and which have different spacer regions, optionally including STII tails.
(Η) The ability that human T cells modified with BCMA-specific CARs have to recognize BCMA and proliferate is measured by labeling T cells with carboxyfluorescein (CFSE), culturing CAR T cells or controlling non-transduced T cells with CFSE with K562 cells transduced with a polynucleotide that
Petition 870190103473, of 10/14/2019, p. 12/184
7/118 encodes full-length BCMA (K562 / BCMA), and using flow cytometry to measure the CFSE dilution at each cell division. CFSE-labeled T cells containing different BCMA-specific CARs with different lengths of spacer, but without control non-transduced (UT) T cells, CFSE diluted after coculture with K562 / BCMA cells. T-CAR cells containing 2 ST spacers or longer proliferated better than T-CAR cells expressing the short spacer.
(I, J) Release of cytokines (ΙΕΝ-γ, I; IL-2, J) by anti-BCMA CAR T cells with different lengths of spacers in response to U266 and 8266 myeloma cells that express BCMA.
(K) Expression of EGFRt and STII on the cell surface in CD8 ⁺ T cells transduced with anti-BCMA Cll TChARM or BCMA-2 CAR constructs after isolation and expansion.
(L, M) release of cytokines (ΙΕΝ-γ, L; IL-2, M) by human CD4 (upper panels) and CD8 (lower panels) T cells designed to express Cll TChARMs of this disclosure that include a 41BB co-stimulatory domain or a co-stimulatory domain CD28, or by a previously disclosed anti-BCMA CAR antigen (BCMA2; see Carpenter et al. Clin. Cancer Res. 15: 2048, 2013).
(N) Proliferation of human T cells designed to express the released Cll T-ChARMs or BCMA-2 CAR
Petition 870190103473, of 10/14/2019, p. 13/184
8/118 when co-cultured with the indicated cell lines that express BCMA.
(O) Lysis of BCMA-negative K562 cells by CD8 T cells designed to express Cll T-ChARMs of this disclosure (circle = 41BB co-stimulatory domain, square = CD28 co-stimulatory domain) or BCMA-2 CAR (triangle) in the indicated E: T ratios.
(P) Lysis of K562 cells transduced to express BCMA, by the projected CD8 T cells indicated in Figure 10, at various E: T ratios (x-axis).
[008] Figures 2A-2Q show the production of BCMA and PD-L1 by cultured multiple myeloma (MM) cells and the effect of soluble BCMA, surface-bound BCMA and surface-bound PD-L1 on the capacity that anti-BCMA T-ChARM-T T cells to recognize tumor cells and produce ΙΕΝ-γ.
(A) U266 myeloma cells were washed and plated in culture media for 1, 3, 5 and 24 hours. The media supernatant was collected and tested for soluble BCMA by ELISA. The data show a time-dependent increase in the levels of soluble BCMA (sBCMA) in the supernatant.
(B) Histograms showing BCMA expression by reference MM cells (RPMI 8226) or by an example of patient's primary MM cells that have expression
Petition 870190103473, of 10/14/2019, p. 14/184
9/118 high (Pt. 1), intermediate (Pt. 2) or low / negative (Pt. 3) measured by ELISA (black = anti-BCMA antibody; gray line = isotype control).
(C) Graph showing percentages of patients with myeloma (n = 19) who have high, intermediate or low / negative BCMA expression in MM cells.
(D) Production of-γ by CD8 ⁺ T-ChARM ⁺ CD8 ⁺ anti-BCMA T cells in response to stimulation with MM cells of patients with high (left, n = 5) or low (right, n = 4) (24h at a 2: 1 E: T (ELISA)). Significance was tested using a two-tailed unpaired T test and the bars represent the average production of ΙΕΝ-γ + SEM.
(E) Histograms showing PD-L1 expression by myeloma cells RPMI 8226 (leftmost panel) and primary MM cells from 3 patients that have high, low / negative or intermediate PD-L1 expression, measured by ELISA ( diagonal shading = staining with anti-PD-Ll antibody; empty histogram = isotype control).
(F) Graph showing percentages of patients (n = 19) who have high, intermediate or low / negative PD-L1 expression in MM cells.
(G) ΙΕΝ-γ production by CD8 ⁺ anti-BCMA Cll cells in response to stimulation with MM cells of patients with high (left, n = 4) or low BCMA expression
Petition 870190103473, of 10/14/2019, p. 15/184
10/118 (right, η = 5) (24h at an E: T of 2: 1 (ELISA)). Significance was tested using a two-tailed unpaired T test and the bars represent the average production of ΙΕΝ-γ + SEM.
(H) Production of ΙΕΝ-γ by BCMA-specific T-ChARM T cells in the presence of exogenous soluble BCMA. SBCMA was added to co-cultures of T cells expressing a BCMA-specific T-ChARM and K562 cells transduced with a full-length BCMA (/ BCMA ⁺ ) encoding polynucleotide. There is a dose-dependent inhibition of the effector function of BCMA-specific T-ChARM T cells as measured by the release of ΙΕΝ-γ in the media supernatant.
(I) Data from another dose titration experiment showing the effect of sBCMA on ΙΕΝ-γ production by BCMA-specific T-ChARM T cells that recognize K562 ⁺ BCM cells, which included the addition of sBCMA to the culture in another and higher concentration (5000 ng / mL).
(J) BCMA release by the indicated MM cells cultured in vitro.
(K) sBCMA measured in bone marrow (MO) sera from patients with less (<2% CD138 ⁺ cells) or greater (> 2% CD138 ⁺ cells) disease burden.
(L) Binding of sBCMA to C113ST-ChARM T cells of this disclosure. T cells were incubated with the levels
Petition 870190103473, of 10/14/2019, p. 16/184
11/118 recombinant sBCMA indicated (right side of the diagram) and then stained with a BCMA-Fc fusion that was conjugated to ABC.
(M) Flow cytometry data showing surface staining (BCMA-Fc conjugated to ABC and antiEGFRt antibody) and CD4 expression of C113ST T-ChARM and FMC63 CAR T cells.
(N) Flow cytometry data showing IFN-γ production (y-axis) and CD4 expression (x-axis) by T cells expressing either a C113ST-ChARM (C113ST) from this disclosure or an anti-CD19 CAR ( FMC632) control, in co-culture with K562 cells that express the target and to which the BCMA-Fc fusion protein was administered (left panels: 0 ng / mL BCMA-Fc; right panels: 1000 ng / mL BCMA- Fc.
(O) Dose titration showing release of IFN-γ by T CAR cells in response to target cell lines, as indicated in the presence or absence of exogenous recombinant BCMA (BCMA-Fc fusion). FMC63 (anti-CD19) vs. K562 CD19 ⁺ (control; triangle facing down); C113ST T-ChARM T cells versus RPMI 8226 (triangle facing up), U266 (square) and K562 BCMA ⁺ cells (circle). The data are representative of 2 independent experiments. The bars represent mean + EPM. P-value = <0.05, as determined by one-way ANOVA with post-test. MFI =
Petition 870190103473, of 10/14/2019, p. 17/184
12/118 medium fluorescence intensity.
(P) Production of IFN-γ (Normalized Mean Fluorescence Intensity, IFM) by the T cells shown in Figure 20 in co-culture with cells that express indicated antigens and in the presence or absence of BCMA-Fc (x-axis).
(Q) Cytolytic activity of CD8 ⁺ C113ST TChARM T cells against K562 BCMA ⁺ target cells and FMC63 T cells
CAR against K562 CD19 ⁺ target cells in varying concentrations of recombinant BCMA, analyzed by a 4h ELC at an E: T ratio of 10: 1. The data are representative of 2 independent experiments. The bars represent mean + EPM. P-value = <0.05, as determined by one-way ANOVA with post-test. IFM = average fluorescence intensity.
[009] Figures 3A-3R show the effect of the γsecretase inhibitor (GSI) RO4929097 on the levels of BCMA on the cell surface and other molecules on the cell surface in myeloma cell lines or primary myeloma cell lines.
(A) BCMA was measured on the cell surface in four myeloma cell lines (8226, U266B1, MM1.R, H929) by flow cytometry with a BCMA monoclonal antibody before, and 5 hours later (see diagrams), from incubation of myeloma cell lines with GSI RO4929097 in a concentration range from 0 μΜ (DMSO control) to 1.0 μΜ.
(B) BCMA expression on the surface by MM.1R cells cultured with the indicated concentrations of RO4929097;
Petition 870190103473, of 10/14/2019, p. 18/184
13/118 staining with anti-BCMA antibody (black lines) compared to isotype control (gray line).
(C) Change in BCMA expression on the surface (number of times) by MM cell lines when cultured with the indicated concentrations of RO4929097; change in the number of times indicated in relation to untreated MM cells of the same lineage.
(D) Kinetics of the change in BCMA expression on the surface (number of times) over time by MM cells indicated in culture with RO4929097 1 μΜ.
(E) U266 myeloma cells were incubated for 1, 3, 5 and 24 hours in the presence of various concentrations of GSI RO429097 (0.01 μΜ, 0.1 μΜ and 1.0 μΜ) and evaluated for BCMA expression in flow cytometry. BCMA expression increased in a dose-dependent manner in the presence of GSI with the peak of increase observed after 5 hours of exposure.
(F) Change in BCMA expression on the surface (number of times) in cell lines (MM1R = triangle; U266 = square; 8226 = circle) grown in GSI 1M over time. The GSI was administered again as a half change of means every 2 days. BCMA change (number of times) is defined as Treated (MFIBCMA-MFIiso) / Control (MFIBCMAMFIiso). The data are representative of at least 2 independent experiments.
(G, H) Concentration of sBCMA in the supernatant of cultures of MM cell lines cultured in the presence of indicated concentrations of RO4929097.
Petition 870190103473, of 10/14/2019, p. 19/184
14/118 (I) U266 myeloma cells were washed and plated in culture media in the presence of various concentrations of GSI RO429097 (0.01 μΜ, 0.1 μΜ and 1.0 μΜ). The media supernatant was collected after 1, 3, 5 and 24 hours and tested for soluble BCMA (sBCMA) by ELISA. The data show that the amount of BCMA released from tumor cells into the supernatant over time decreased when a GSI was present at a concentration of at least about 0.01 μΜ.
(J, K) Change in BCMA expression (number of times) (J) and sBCMA concentrations in the supernatant (K) at various times after GSI 1 μΜ has been removed from cultures of myeloma cell lines (GSI + / - ) compared to cultures with continued presence of GSI (GSI + / +).
(L) Viability of MM cells that have pressure, indicated by staining with propidium iodide from cell lines cultured in 1 mM GSI.
(M) BCMA expression on the surface by the patient's primary MM cells cultured with the indicated concentrations of R04929097. Staining was as described in relation to Figure 3B.
(N) Change in BCMA (number of times) in primary myeloma cells (n = 7) cultured with varying amounts of GSI for 4 h. Primary and cell lines were cultured at 0.5x106 cells / mL. BCMA change (number of times) is defined as Treated (MFIBCMA-MFIiso) / Control (MFIBCMA-MFIiso). The data are representative of 3 independent experiments with T cells derived from different donors.
Petition 870190103473, of 10/14/2019, p. 20/184
15/118 (Ο, Ρ) Co-culture of primary myeloma cells with various concentrations of GSI for 4 hours does not affect the levels of various other cell surface molecules in tumor cells, including CS1, CD86, PD-L1, CD80 and CD38.
(Q) Staining of various surface markers in MM1R cells in the presence (black) or absence (gray) of GSI ΙμΜ in culture medium. The coloring of isotypes is shown as an open trace.
(R) Primary CD138 + myeloma cells were enriched from bone marrow samples from patients, incubated for 3 hours in the presence of various concentrations of GSI RO429097 (0.01 μΜ, to 10 μΜ) and evaluated for BCMA expression in flow cytometry. The average fluorescence intensity of BCMA (IFM) in tumor cells is shown as a change (in the number of times) from that observed in tumor cells incubated without RO429097. BCMA dose-dependent positive regulation was observed.
[010] Figures 4A - 4C show that the release of cytokines after the recognition of primary myeloma cells by BCMA-specific T-CAR cells is increased when myeloma cells are pretreated with a GSI.
(A) The production of IL-2 by BCMA T-CAR cells (BCMA-specific T-ChARM Cll 3ST-CD28 and BCMA-specific T-ChARM Cll 3ST-41BB) or co-cultured control CD19sh T-CAR cells (short spacer) with primary human myeloma tumor cells for 24 hours alone or with varying concentrations of GSI RO429097 (0.003 μΜ to 3.0 μΜ).
Petition 870190103473, of 10/14/2019, p. 21/184
16/118 (B) IFN-γ production by BMCA T-ChARM T cells co-cultured with myeloma cells in various concentrations of RO429097.
(C) Proliferation of CFSE-labeled BCMA-specific T-ChARM T cells increased dose-dependent after co-culture for 3 days with primary human myeloma tumor cells in isolated media or in media containing GSI RO492097 at the indicated concentrations.
[Oil] Figures 5A-5R show the effect of various concentrations of GSI on the viability, growth and functional activity of T-CAR cells.
(A) CD19 staining of K562 CD19 ⁺ cells and Raji cells that were cultured with or without GSI for ± 12-16 h. Isotype control shown as a gray line.
(B) Primary human T cells were cultured in GSI RO4929097 in concentrations ranging from 0.01 μΜ to 100 μΜ and viability was measured by excluding trypan blue dye after 24 hours. There was no effect of GSI, at any concentration, on the viability of T cells.
(C) CD19 T-CAR cells were co-cultured with K562 / CD19 target cells in medium containing various concentrations of RO4929097. RO4929097 inhibits the effector function of CD19 T-CAR cells in concentrations of> 3 μΜ when co-cultured, as determined by measuring the production of IL-2 (upper panel) and ΙΡΝγ (lower panel). The box shows the relevant therapeutic window of the drug that does not inhibit the effector function of T-CAR cells.
(D) IL-2 production by CD19 T-CAR cells cultured with
Petition 870190103473, of 10/14/2019, p. 22/184
17/118 target cells in the presence of increasing concentrations of GSI RO4929097.
(E) Production of IL-2 by CD19 T-CAR cells cultured with target cells (K562 CD19) or control cells (K562 BCMA) in the presence of increasing concentrations of GSI RO4929097. The indicated quantities of GSI were administered to the cells, which were then washed (empty bars) or not (full bars).
(F) Data from another experiment showing the production of IL-2 by CD19 T-CAR cells after coculture overnight with K5 62 BCMA ⁺ or K5 62 CD19 ⁺ cells after pre-incubation with varying concentrations of GSI. After washing, GSI was either added (+ / +) or left outside the coculture (+/-) to assess the reversibility of cytokine production.
(G) Lysis specifies, by CD19 T-CAR cells, of the indicated target cells or control cells in the presence of GSI RO4929097.
(H) Proliferation of CD19 T-CAR cells cultured with target cells that express CD19 in the presence or absence of GSI or with control cells in the absence of GSI. The cells were stained with CFSE and proliferation was measured by flow cytometry.
(I) Graphical representation of the number of cell divisions of CD19 T-CAR cells in the presence of the indicated concentrations of GSI RO4929097. The width of the horizontal bars represents the proportion of T-CAR cells in culture that divided according to the indicated number of generations (that is, 5, 4, 3, 2, 1 or 0 generations) throughout the experiment.
Petition 870190103473, of 10/14/2019, p. 23/184
18/118 (J) Cell counts (CD8 staining) during expansion of CD19-specific T-CAR cells with exogenous LCL TM CD19 ⁺ and IL-2 cells in the absence of GSI (circle) or in the presence of GSI 0, 5μΜ (square) or 5μΜ (triangle).
(K) CD19-specific T-CAR cell counts (CD4: CD8 (1: 1) expanded with TM CD19 ⁺ . LCL cells in the absence or presence of GSI as indicated, but without the addition of exogenous IL-2.
(L) IFN-γ concentrations in the supernatants of GSI-expanded CD8 ⁺ CAR CD8 cells after restimulation with K562 cells (without antigen), K562 CD19 ⁺ cells, or Ra j i cells.
(Μ, N) Production of IFN-γ (M) and IL-2 (N) by mixing CD4 T-CAR: anti-CD19 CD8 cells after restimulation with the indicated cell lines in the absence of GSI or in the presence of GSI 0.5 μΜ or 5 μΜ.
(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 2 patients in the absence (0 μΜ; panels left) or presence (1 μΜ; right panels) of GSI RO4929097.
(P) Production of IFN-y (geometric intensity of average fluorescence (gMFI)) by T-ChARM T cells of the present disclosure, cultured with primary MM cells in the presence of the indicated concentration of RO4929097 (x-axis).
(Q) Production of IFN-γ (normalized IFM; y-axis) by T-ChARM T cells of the present disclosure, cultured with cells
Petition 870190103473, of 10/14/2019, p. 24/184
11/198
primaryRO4929097 of MM in(X axis). presence gives concentration indicated from (R) Proliferation of cells T T-CHARM gives gift disclosure in presence of cells MM primary what were not treated ( shading Gray) OR treated with GSI RO4929097
1.0 μΜ.
[012] Figures 6A-6C show the effects of GSI RO4929097 on BCMA expression in an in vivo preclinical model of multiple myeloma.
(A) Experimental scheme for a model of disseminated murine xenografted myeloma. NSG mice were irradiated (275 rad) to facilitate tumor graft and received human MM tumor cells (5x10 ⁶ MM.1R) and then treated with GSI (30 mg / kg). Subsequently, the mice were euthanized and blood and BM samples were collected to determine whether GSI increased BCMA expression in in vivo myeloma cells.
(B) Change in BCMA expression on the surface (number of times) in myeloma cells in euthanized mice at the indicated times after the second GSI administration.
(C) Levels of sBCMA in sera of mice sacrificed at the indicated times after the administration of RO4929097.
[013] Figures 7A-7E show the effects of GSI RO4929097 on anti-BCMA T cell CAR therapy in the pre-clinical model of MM in mice.
(A) Experimental scheme in which the mice received radiation followed by human MM tumor cells (5x10 ⁶ MM.1R
Petition 870190103473, of 10/14/2019, p. 25/184
20/118 expressing firefly luciferase). Twenty days after that, GSI mice (30 mg / kg) were administered at the indicated times and a single suboptimal dose of T-ChARM antiBCMA T cells (0.33x10 ⁶ cells, CD4: CD8 1: 1) on day 0. Imaging by IBL bioluminescence) and survival were monitored at all times.
(B) IBL images of mice taken on days 2, 17 and 16 after treatment with C113ST T-ChARM T cells (0 mg / kg, 0.33x10 ⁶ cells, CD4: CD8 1: 1, left panels; 30 mg / kg, middle panels) or T cells anti-CD19 FM63 control (0.33 x ^{10 6} cells, CD4: CD8 1: 1, 30 mg / kg, right panels).
(C) Quantified luminescence data from the IBL shown in Figure 8B.
(D) Percent survival of mice shown in Figure 8B after administration of T-ChARM T cells.
(E) (left) Quantified luminescence data from the IBL shown in Figure 8B; (right) percentage survival of mice after administration of T-ChARM T cells.
[014] Figure 8 shows an analysis by binding flow cytometry, by a bispecific fusion molecule with specificity for BCMA, to MM92 cells in the presence or absence of GSI. A bispecific control fusion molecule that did not target BCMA was also tested.
DETAILED DESCRIPTION [015] The present invention provides compositions and methods for the treatment of autoimmune diseases and cancer through the combined use of a binding protein
Petition 870190103473, of 10/14/2019, p. 26/184
21/118 specific to a B cell maturation antigen (BCMA), in soluble form or expressed in a cytotoxic cell or another cell, and an inhibitor of γ-secretase (GSI). Polynucleotides encoding such BCMA-specific binding proteins can be used to generate host-modified immune cells (eg, T cells) for use, for example, in adoptive immunotherapy. In certain respects, the present description addresses the use of such therapy in an individual in need of treatment in combination with a GSI, the latter treatment of which can be administered prior to, concomitantly or subsequently to adoptive immunotherapy (that is, immunotherapy with a modified immune cell that expresses a BCMA specific binding protein on the cell surface). Also provided herein are methods of immunotherapy comprising administering a GSI in combination with a BCMA-specific binding protein comprising an antibody or antigen-binding portion thereof, which may, in certain embodiments, be conjugated or otherwise , coupled to a cytotoxic drug, for example, forming an anticorpopharmaceutical conjugate (CAF). Useful therapeutic applications include the treatment of a proliferative disease or disorder (such as cancer), an autoimmune disease or disease or disorder associated with aging in the individual in which the cells
Petition 870190103473, of 10/14/2019, p. 27/184
11/22
BCMA-positive are pathogenic.
[016] Before presenting this disclosure in more detail, it may be useful for an understanding of it to provide definitions of certain terms to be used here. Additional definitions are established throughout this release.
[017] In this description, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the specified range and, where appropriate, fractions thereof (such as a tenth and one hundredth of an integer), unless otherwise specified. In addition, any range of numbers cited herein, related to any physical characteristic, such as subunits, size or thickness of polymers, should be understood to include any integer within the indicated range, unless otherwise specified. As used herein, the term about means ± 20% of the indicated range, value or structure, unless otherwise stated. It should be understood that the terms one and one as used herein refer to one or more of the listed components. The use of the alternative (for example, or) should be understood as meaning one, both, or any combination of the alternatives. As used herein, the terms include, have
Petition 870190103473, of 10/14/2019, p. 28/184
23/118 and understand are used interchangeably, whose terms and variants are intended to be interpreted as being non-limiting.
[018] Furthermore, it should be understood that the individual compounds, or groups of compounds, derived from the various combinations of the structures and substituents described herein, are disclosed by the present application to the same degree as if each compound or group of compounds were defined individually . Thus, the selection of particular structures or particular substituents is within the scope of this disclosure.
[019] The terms consisting essentially of and consisting (m) essentially of limit the scope of a claim to the specified materials or steps, or to those that do not materially affect the basic features of a claimed invention. For example, a protein domain, region, module or cassette (for example, a binding domain, hinge region, ligand module, marker cassette) or a protein (which can have one or more domains, regions, modules or cassettes) consists essentially of a particular amino acid sequence when the amino acid sequence of a domain, region, module, cassette or protein includes extensions, deletions, mutations or a combination of them (for example, amino acids in the amino- or carboxy-terminal or
Petition 870190103473, of 10/14/2019, p. 29/184
24/118 across domains) which, in combination, contribute a maximum of 20% (for example, a maximum of 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) the length of a domain, region, module, cassette or protein and do not substantially affect (that is, it does not reduce activity by more than 50%, such as not more than 40%, 30%, 25%, 20%, 15% , 10%, 5% or 1%) the activity of the domain (s), region (s), module (s), cassette (s) or protein (for example, the target binding affinity of a protein connector or cassette with marker).
[020] As used herein, proliferative disorder refers to excessive or otherwise abnormal growth or proliferation compared to a normal or non-sick cell. Excessive or abnormal growth includes, for example, unregulated growth or proliferation that can occur quickly (for example, hyperproliferation) or can occur more slowly or progressively over time (for example, multiple myeloma), within a native tissue (for example, example, plasmacytoma growth within the bone marrow), as well as spreading to / growing within a distal tissue or body site that are not native to the diseased cell. Exemplary proliferatives include tumors, cancers, neoplastic tissue, carcinoma, sarcoma, malignant cells, premalignant cells, as well as non-neoplastic or non-malignant proliferative disorders (eg, adenoma, fibroma, lipoma, leiomyoma,
Petition 870190103473, of 10/14/2019, p. 30/184
25/118 hemangioma, fibrosis, restenosis) or autoimmune diseases (such as rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease or the like).
[021] A binding protein (also referred to as a binding domain, binding region or binding portion), as used herein, refers to a molecule, such as a peptide, oligopeptide, polypeptide or protein that it has the ability to associate, join or combine in a specific, non-covalent way with a target molecule (eg BCMA). A binding protein includes any naturally-occurring, synthetic, semi-synthetic or recombinantly produced binding partner for a molecule, compound or other target of biological interest. In some embodiments, a binding protein is an immunoglobulin or an immunoglobulin-like molecule, such as an antibody or T cell receptor (TCR), which includes a functional binding domain or a fragment of antigen binding. Examples of binding proteins include variable regions of single chain antibody (for example, domain antibodies, sFv, scFv, Fab), BCMA ligands (for example, BAFE, ABRIL and binding fragments thereof), binding regions to T cell receptor antigens (TCRs), such as single chain TCR (scTCRs), or selected synthetic polypeptides
Petition 870190103473, of 10/14/2019, p. 31/184
26/118 for the specific ability to bind to a biological molecule.
[022] As used herein, binding specifically refers to an association or union of a binding domain, or a fusion protein thereof, to a target molecule with an affinity or K _a (i.e., an association constant in the balance of a particular bonding interaction with units of 1 / M) equal to or greater than 10 ⁵ M ^-1 , although they do not significantly associate or unite with other molecules or components in a sample. The binding domains (or fusion proteins thereof) can be classified as high affinity binding domains (or fusion proteins) or low affinity binding domains (or fusion proteins). High affinity binding domains refer to binding domains with a Ka of at least 10 ⁷ M ^-1 , at least 10 ⁸ M ^-1, at least 10 ⁹ M ^-1 , at least IO ¹⁰ M ^-1 , at least at least 10 ¹¹ M ^-1 , at least 10 ¹² M ^-1 , or at least 10 ¹³ M ^-1 . Low affinity binding domains refer to binding domains with a Ka of up to 10 ⁷ M ^-1 , up to 10 ⁶ M ^-1 , up to 10 ⁵ M ^-1 . Alternatively, affinity can be defined as an equilibrium dissociation constant (Kd) of a specific binding interaction with units of M (for example, 10 ~ ⁵ M to ICT ¹³ M). In certain embodiments, a liaison domain may have an affinity
Petition 870190103473, of 10/14/2019, p. 32/184
27/118 improved, which refers to a selected or modified binding domain with stronger binding to a target antigen than to a wild type (or parental) binding domain. For example, the increased affinity may be due to a K _a (equilibrium association constant) for the antigen target that is greater than for the wild type binding domain, or due to a Kd (dissociation constant) for the target antigen which is smaller than for the wild type binding domain, or due to an off-rate (K _O ff) ratio for the anti target genome which is less than for the binding domain wild type. Several assays are known to identify binding domains of the present disclosure that specifically bind to a particular target, as well as to determine binding domain affinities or fusion proteins, such as Western blot, ELISA and Biacore analysis (see also by example Scatchard et al. Ann. NY Acad. Sci. 51: 660, 1949; and US Patent Nos. 5,283,173, 5,468,614, or equivalent).
[023] As used herein, heterologous or non-endogenous or exogenous refer to any gene, protein, compound, molecule or activity that is not native to a host cell or an individual, that is to say any gene, protein, compound, molecule or activity native to a host or host cell, but which have been
Petition 870190103473, of 10/14/2019, p. 33/184
28/118 altered or mutated (s) in such a way that the structure, activity or both are different between native molecules and those that have mutated (s). In certain embodiments, heterologous, non-endogenous or exogenous molecules (for example, receptors, ligands) may not be endogenous to a host cell or individual, but, instead, nucleic acids encoding such molecules may have been added to a host cell by conjugation, transformation, transfection, electroporation or the like, where the added nucleic acid molecule can integrate into the genome of a host cell or can exist as extrachromosomal genetic material (for example, as a plasmid or other self-replicating vector). The term homologous refers to a molecule or activity found 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 a molecule or gene encoding the molecule, respectively, of a native host or host cell, but may have a structure, sequence, level of expression or combinations changed. A non-endogenous molecule can be the same species, a different species or a combination of them.
[024] As used herein, the terms endogenous or native refer to a gene, protein, compound, molecule
Petition 870190103473, of 10/14/2019, p. 34/184
29/118 or activity that is normally present in a host or in a host cell.
[025] As used herein, marker cassette refers to a single peptide sequence attached to, fused to, or that is part of a protein of interest, to which a heterologous or non-endogenous cognate binding molecule (eg, receptor, ligand , antibody or other binding partner) has the ability to specifically bind where the binding property can be used to detect, identify, isolate or purify, track, enrich or target a labeled protein or cells that express a labeled protein, particularly when a labeled protein is part of a heterogeneous population of proteins or other material, or when cells expressing a labeled protein are part of a heterogeneous population of cells (for example, from a biological sample such as peripheral blood). In certain embodiments, a cell that expresses a labeled protein can be brought into contact with a heterologous or non-endogenous cognate-binding molecule and induce a biological response in such a way as to promote cell activation, cell proliferation or cell death. In the provided fusion proteins, the ability of the marker cassette (s) to be specifically linked by the cognate-binding molecule (s) is distinct from or in
Petition 870190103473, of 10/14/2019, p. 35/184
30/118 addition to the ability of the binding domain (s) to specifically bind to the target molecule (s). The labeled cassette is generally not an antigen binding molecule, for example, it is not an antibody or TCR or an antigen binding portion thereof.
[026] As used herein, a hinge region or hinge refers to (a) an immunoglobulin hinge sequence (formed, for example, from the upper and central regions) or a functional fragment or variant thereof, (b ) a (stem) region of the Clectin type II interdomain or a functional fragment or variant thereof, or (c) a stem region of the cluster of differentiation (CD) molecule or a functional variant thereof . As used herein, a wild-type immunoglobulin hinge region refers to naturally occurring upper and intermediate hinge amino acid sequences interposed between and connecting the CHI and CH2 domains (for IgG, IgA and IgD) or interposed between and connecting the CHI and CH3 domains (for IgE and IgM) found in the heavy chain of an antibody. In certain embodiments, a hinge region is human and, in particular embodiments, comprises a human IgG hinge region.
[027] As used herein, a spacer region refers to one or more proteins, polypeptides,
Petition 870190103473, of 10/14/2019, p. 36/184
11/31 oligopeptides, peptides, domains, regions, modules, cassettes, motifs or any combination thereof that unite two or more proteins, polypeptides, oligopeptides, peptides, domains, regions, modules, cassettes, motifs or any combination thereof in one fusion protein. For example, a spacer region can provide a separation or spacing function to facilitate the interaction of two single chain fusion proteins, or positioning one or more binding domains, so that the resulting polypeptide structure maintains a specific binding affinity to a target molecule or maintain signaling activity (e.g., effector domain activity) or both. In certain embodiments, a spacer region can comprise a linker module which is a sequence of amino acids that has about two to about 500 amino acids, which can provide flexibility and space for conformational movement between two connected regions, domains, motifs, cassettes or modules by a linker. Examples of ligand modules include those with from one to about ten repetitions of Gly _x Ser _y (SEQ ID NO: 31), where x and y are, independently, an integer from 0 to 10, as long as x and y are not both 0 ( for example, (Gly4Ser) 2 (SEQ ID NO: 32), (Gly ₃ Ser) ₂ (SEQ ID NO: 33), Gly ₂ Ser, or a combination thereof, such as (GlysSer) 2Gly2Ser)) (SEQ ID
Petition 870190103473, of 10/14/2019, p. 37/184
11/28
NO: 34). In certain other embodiments, a spacer region can have a linker module that comprises one or more constant regions of the immunoglobulin heavy chain, such as CH3 alone or CH2CH3. In other embodiments, a spacer region may comprise a hinge region or a marker cassette. Each of these connector components is not mutually exclusive. For example, a spacer region may comprise a hinge and one or more connecting modules, or a spacer region may comprise a hinge, one or more connecting modules, and one or more cassettes with markers. Examples of spacer regions can vary in length, for example, from about five to about 500 amino acids, or from about ten to about 350 amino acids, or from about 15 to about 100 amino acids, or from about 20 to about 75 amino acids, or about 25 to about 35 amino acids. Examples of short spacers range from about five to about 100 amino acids (for example, 12 amino acids, 15 amino acids, 48 amino acids, 50 amino acids, 66 amino acids, 70 amino acids), intermediate spacers range from about 100 to about 200 amino acids ( for example, 110 amino acids, 120 amino acids, 130 amino acids, 140 amino acids, 150 amino acids, 157 amino acids, 175 amino acids) and long spacers range from about 200 to about 500 amino acids (for example, 200 amino acids, 210
Petition 870190103473, of 10/14/2019, p. 38/184
33/118 amino acids, 220 amino acids, 228 amino acids, 230 amino acids, 250 amino acids, 300 amino acids, 350 amino acids, 400 amino acids, 450 amino acids).
[028] A hydrophobic moiety, as used herein, means any sequence of amino acids that has a three-dimensional structure that is thermodynamically stable on a cell membrane and generally varies in length from about 15 amino acids to about 30 amino acids. The structure of a hydrophobic domain can comprise an alpha helix, a beta barrel, a beta leaf, a beta helix or any combination thereof.
[029] 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 in a cell when it receives the appropriate signal. In certain embodiments, an effector domain is part of a protein or protein complex that receives a signal when bound, or that binds directly to a target molecule, which triggers a signal from the effector domain. An effector domain can directly promote a cellular response when it contains one or more domains or signaling motifs, such as a tyrosine immunoreceptor-based activation motif (ITAM), and such a cellular response can be aided or enhanced with a co-stimulatory domain or functional portion thereof. An example of a protein that has an effector domain is CD3 /. In other modalities, an effector domain will indirectly promote an
Petition 870190103473, of 10/14/2019, p. 39/184
34/118 cellular response associating itself with one or more other proteins that directly promote a cellular response.
[030] A co-stimulatory domain, as the term is used here, refers to a signaling fraction that provides T cells with a signal that, in addition to the primary (effector) signal provided by, for example, a chain of CD3 ζ of the TCR / CD3 complex, mediates a T cell response, including activation, proliferation, differentiation, secretion of cytokines or the like. In modality waxes, an intracellular component comprises an effector domain or functional portion thereof, a co-stimulatory domain or functional portion thereof, or any combination thereof.
[031] A variable region linker (variable region linker) refers specifically to a sequence of five to about 35 amino acids that connects a variable region of heavy chain immunoglobulin to a variable region of light chain immunoglobulin or connects the V _a / p and Ch / p chains (for example, V _a -C _a , Vp-Cp, V _a -Vp) of the T cell receptor or connects each V _to -C _a , Vp-Cp, V ^ pair -Vp to a hinge or hydrophobic domain, which provides a spacer function and sufficient flexibility for the interaction of the two binding subdomains so that the resulting single chain polypeptide retains a specific binding affinity to the same target molecule as an antibody or T cell receptor. In certain embodiments, a variable region ligand
Petition 870190103473, of 10/14/2019, p. 40/184
35/118 about ten to about 30 amino acids or about 15 to about 25 amino acids. In particular embodiments, a variable region ligand peptide comprises from one to ten repetitions of Gly _x Ser _y , where x and y are, independently, an integer from 1 to 5 (for example, Gly ₄ Ser (SEQ ID NO: 1 ), Gly ₃ Ser (SEQ ID NO: 2), Gly ₂ Ser, or (Gly ₃ Ser) n (Gly ₄ Ser) i (SEQ ID NO: 3), (Gly ₃ Ser) _n (Gly ₄ Ser) _n (SEQ ID NO: 4) or (Gly ₄ Ser) _n (SEQ ID NO: 5), where n is an integer of 1, 2, 3, 4 or 5) and in which variable regions
connected form a domain binding functional (per example,[032 scFv, scTCR).] Amino acids junction or waste in junction amino acids refer to a or more (per example, 2-20) amino acid residues between two
motifs, regions or adjacent 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 at one or both ends of a linker region ) linking two motifs, regions or domains (for example, between a linker and an adjacent link domain and / or between a linker and an adjacent hinge). The junction amino acids can result from the design of a fusion protein construct (for example, amino acid residues resulting from the use of a
Petition 870190103473, of 10/14/2019, p. 41/184
36/118 restriction enzyme during the construction of a nucleic acid molecule encoding a fusion protein).
[033] The terms understood by those skilled in the art of antibody technology each receive the meaning acquired in the art, unless expressly defined herein to the contrary. The term antibody refers to an intact antibody comprising at least two heavy chains (H) and two light chains (L) interconnected by disulfide bonds, as well as an antigen-binding portion of an intact antibody that has or retains the ability to bind to a target molecule. A monoclonal antibody or antigen-binding portion can be non-human, chimeric, humanized, or human, preferably humanized or human. The structure and function of immunoglobulin are described in the review, for example, by Harlow et al. , Eds., Antibodies: A Laboratory Manual, Chapter 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988).
[034] For example, the terms V1 and Vh refer to the variable binding region of an antibody light and heavy chain, respectively. The variable link regions are made up of discrete and well-defined sub-regions, known as complementarity determining regions (CDRs) and framework regions (framework regions) (FRs). The term CL refers to
Petition 870190103473, of 10/14/2019, p. 42/184
37/118 an immunoglobulin light chain constant region or a light chain constant region, i.e., an antibody light chain constant region. The term CH refers to an immunoglobulin heavy chain constant region or a heavy chain constant region, which can still be divided, depending on the antibody isotype, in the CHI, CH2, and CH3 domains (IgA, IgD, IgG) ,or
CHI, CH2, CH3, and CH4 (IgE, IgM). A Fab (antigen-binding fragment) is the part of an antibody that binds to antigens and includes the variable and CHI region of the heavy chain linked to the light chain via an interchain disulfide bond.
[035] As used herein, portion of the Fc region refers to the segment of the heavy chain constant region of the Fc fragment (the crystallizable region of the Fc fragment or region) of an antibody, which may include one or more constant domains, such as CH2, CH3, CH4 or any combination thereof. In certain embodiments, a portion of the Fc region 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 and any combination thereof. In other embodiments, a CH2CH3 or CH3CH4 structure has sub-region domains of the same antibody isotype and are human, such as IgGl, IgG2, IgG3, IgG4, IgAl, IgA2, IgD, IgE, or human IgM (for example,
Petition 870190103473, of 10/14/2019, p. 43/184
38/118 (human IgGl CH2CH3). As a background, an Fc region is responsible for the effector functions of an immunoglobulin, such as ADCC (antibody-dependent cell-mediated cytotoxicity), CDC (complement-dependent cytotoxicity) and complement fixation, when binding to Fc receptors ( for example, CD16, CD32, FcRn), longer half-life in vivo compared to a polypeptide without an Fc region, binding to protein A and perhaps even placental transfer (see Capon et al., Nature 337: 525, 1989) . In certain embodiments, a portion of the Fc region found in fusion proteins of the present disclosure will be able to mediate one or more of these effector functions, or will not have one or more or none of these activities by means of one or more mutations known in the art.
[036] In addition, the antibodies have a hinge sequence that is typically located between the Fab and Fc region (but a lower section of the hinge may include an amino-terminal portion of the Fc region). As a background, an immunoglobulin hinge acts as a flexible spacer to allow the Fab portion to move freely in space. In contrast to the constant regions, the hinges are structurally diverse, varying both in sequence and in length between classes of immunoglobulins and even between subclasses. Per
Petition 870190103473, of 10/14/2019, p. 44/184
39/118 example, a hinge region of human IgGl is freely flexible, which allows the Fab fragments to rotate around their axes of symmetry and move within a sphere centered on the first of two disulfide bridges between the heavy chains. By comparison, a human IgG2 hinge is relatively short and contains a rigid polyproline double helix stabilized by four disulfide bridges between heavy chains, which restricts flexibility. A human IgG3 hinge differs from other subclasses in its unique hinge region (about four times longer than the IgGl hinge), containing 62 amino acids (including 21 prolines and 11 cysteines), forming an inflexible polyproline double helix and providing greater flexibility because the Fab fragments are relatively distant from the Fc fragment. A hinge of human IgG4 is shorter than that of IgGl, but has the same length as that of IgG2 and its flexibility is intermediate between that of IgGl and IgG2.
[037] T cell receptor (TCR) refers to a molecule found on the surface of T cells (or T lymphocytes) that, in association with CD3, is generally responsible for the recognition of antigens linked to molecules of the major histocompatibility complex ( MHC). The TCR has a highly variable a and β disulfide-linked heterodimer (also known as TCRot and TCRβ, respectively) in most
Petition 870190103473, of 10/14/2019, p. 45/184
40/118 of T cells. In a small subset of T cells, the TCR consists of a heterodimer of γ and δ chains (also known as TCRy and TCR6, respectively). Each TCR chain is a member of the immunoglobulin superfamily and has an immunoglobulin N-terminal variable domain, an immunoglobulin constant domain, a transmembrane region, and a short cytoplasmic tail at the C-terminal end (see Janeway et al. Immunobiology: The Immune System in Health and Disease, ^3rd ed., Current Biology Publications, p. 4:33, 1997). The TCR, as used in the present disclosure, can be of various animal species, including humans, mice, rats, cats, dogs, goats, horses or other mammals. TCRs can be linked to cells (that is, they have a transmembrane region or domain) or they can be in soluble form.
[038] Molecules of the major histocompatibility complex (MHC molecules) refer to glycoproteins that deliver peptide antigens to a cell surface. Class I MHC molecules are heterodimers consisting of a membrane comprising an α chain (with three ot domains) and a non-covalently associated β2 microglobulin. Class II MHC molecules are composed of two transmembrane glycoproteins, α and β, both spanning the membrane. Each chain has two domains. Class I MHC molecules distribute peptides that originate in the cytosol to the cell surface, where the peptide: MHC complex is recognized by CD8 ⁺ T cells. Class II MHC molecules distribute peptides that originate in the system
Petition 870190103473, of 10/14/2019, p. 46/184
41/118 vesicular to the cell surface, where they are recognized by CD4 ⁺ T cells. An MHC molecule can be of several animal species, including humans, mice, rats or other mammals.
[039] A vector is a nucleic acid molecule that has the ability to transport another nucleic acid. The vectors can be, for example, plasmids, cosmids, viruses or phage. An expression vector is a vector that has the ability to direct the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment.
[040] Retroviruses are viruses that have an RNA genome. Gamma-retrovirus refers to a genus of the retroviridae family. Examples of gamma-retroviruses include mouse stem cell viruses, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis virus.
[041] Lentivirus refers to a genus of retroviruses that have the ability to infect cells that are dividing and that are not dividing. Several examples of lentiviruses 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).
[042] T cells or T cell lineage cells refer to cells that exhibit at least one phenotypic characteristic of a T cell or a precursor or progenitor thereof that distinguishes cells from other cells
Petition 870190103473, of 10/14/2019, p. 47/184
42/118 lymphoids and cells of the erythroid or myeloid lineages. Such phenotypic characteristics may include the expression of one or more proteins specific to T cells (for example, CD3 ⁺ , CD4 ⁺ , CD8 ⁺ ), or a specific physiological, morphological, functional or immunological characteristic of a T cell. cells of the T cell lineage can be progenitor or precursor cells committed to the T cell lineage; immature and inactivated CD25 ⁺ T cells; cells that have suffered CD4 or CD8 lineage; thymocyte progenitor cells that are CD4 ⁺ CD8 ⁺ double-positive; positive only for CD4 + and CD8 +; TCRo / β or TCR γδ; or mature and functional or activated T cells.
[043] Nucleic acid molecule, or polynucleotide, can be in the form of RNA or DNA, which include cDNA, genomic DNA and synthetic DNA. A nucleic acid molecule can be double-stranded or single-stranded and, if it is single-stranded, it can be either coding or non-coding (antisense tape). A coding molecule can have a coding sequence identical to a coding sequence known in the art, or it can have a different coding sequence, which, as a result of redundancy or degeneration of the genetic code, or by splicing, can encode the same polypeptide.
[044] Treating or treating or improving refers to the medical control of an individual's disease, disorder or condition (for example, a human or non-human mammal, such as a primate, horse, dog, mouse, rat). For example, a suitable dose or therapeutic regimen comprising protein
Petition 870190103473, of 10/14/2019, p. 48/184
43/118 binding specifies BCMA or a host cell expressing a BCMA specific binding protein used in combination with a γ-secretase inhibitor (GSI) of this disclosure and, optionally, an adjuvant or preconditioning regimen, are administered to induce a therapeutic or prophylactic benefit. Therapeutic or prophylactic / preventive benefit includes improved clinical outcome; decrease or relief of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; decrease in the extent of the disease, stabilization of the disease state; delayed disease progression; remission; survival; prolonged survival; or any combination thereof.
[045] A therapeutically effective amount or effective amount of a BCMA-specific binding protein (which is also referred to as BCMA-specific immunotherapy or targeting BCMA), an γ-secretase inhibitor, a host cell that expresses a BCMA specific binding protein, or a host cell that expresses a γ-secretase inhibitor of this disclosure (eg, BCMA specific CAR, anti-Y-secretase antibody) refer to the amount of compound or cells sufficient to result in improving one or more symptoms of the disease to be treated in a statistically significant way. When referring to an individual active ingredient or to a cell that expresses a single active ingredient, administered alone, a therapeutically effective dose refers to the effects of that ingredient
Petition 870190103473, of 10/14/2019, p. 49/184
44/118 or cell that expresses that ingredient alone. When referring to a combination, a therapeutically effective dose refers to the combined amounts of active ingredients or adjuvant active ingredient combined with a cell that expresses an active ingredient that results in a therapeutic effect, whether administered serially, concurrently or simultaneously. Another combination may be a cell that expresses more than one active ingredient, such as two or more different or similar BCMA specific binding proteins.
[046] Additional definitions are provided throughout this disclosure.
B Cell Maturation Antigen (BCMA) Binding Proteins or Molecules [047] In certain respects, the present disclosure provides methods for treating a proliferative or autoimmune disease or disorder in an individual who has or is suspected to have the disease or disorder, comprising administering to the subject a therapeutically effective amount of a BCMA-specific binding protein (or immunotherapy that targets BCMA) and a therapeutically effective amount of an γ-secretase inhibitor. An example BCMA specific binding protein is a chimeric antigen receptor that comprises an extracellular component and an intracellular component connected by a hydrophobic portion, wherein the extracellular component
Petition 870190103473, of 10/14/2019, p. 50/184
45/118 comprises a BCMA-specific binding domain (e.g., BCMA-specific scFv, BCMA ligand or binding portion thereof, such as BAFF or APRIL) and which optionally comprises a spacer region or hinge, and wherein the intracellular component comprises an effector domain and, optionally, a co-stimulatory domain.
[048] In certain embodiments, the present disclosure provides an immunotherapy that targets BCMA, for use with a γ-secretase inhibitor to treat a proliferative or autoimmune disease or disorder, comprising an antibody specific to BCMA or antigen-binding portion a chimeric antigen receptor (CAR), or a labeled chimeric antigen receptor molecule (T-ChARM). In certain embodiments, an antibody specific to BCMA or antigen-binding portion of it is human or humanized.
[049] Examples of antibodies specific to BCMA include antibodies J22.0-XÍ, J22.9-XÍ, J6M0, J6M1, J6M2, J9M0, J9M1, J9M2, 11D5-3, CA8, A7D12.2, Cll D5.3 , C12A3.2, C13F12.1, 13C2, 17A5, 83A10, 13A4, 13D2, 14B11, 14E1, 29B11, 29F3, 13A7, CA7, SGI, S307118G03, S332121F02, S332126E04, S32211005, S322110D07, S333105 , ET140-24, ET140-37, ET140-40, ET140-54, TBL-CLN1, C4.E2.1, Vicky-1, pSCHLI333, pSCHLI372, and pSCHLI373, and
Petition 870190103473, of 10/14/2019, p. 51/184
46/118 antigen-binding portions thereof. Various modalities of antibodies specific to BCMA and antigen-binding portions thereof, including humanized versions, are disclosed, for example, in PCT Publications 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.
The R. 6 (11): 3009, 2007; and Abbas et al. , Blood 128: 1688,
2016, whose specific antibodies to BCMA, antigen-binding portions of them and humanized versions are all incorporated by reference in their entirety. Variable domains and scFv molecules of these BCMA-specific antibodies can be used as a binding domain in any of the T-ChARM and CAR proteins mentioned herein.
[050] Antigen-binding portions or domains obtained from BCMA-specific antibodies of the present disclosure and useful in the methods disclosed herein include, for example, domain antibodies, sFvs, single chain Fv fragments (scFvs), Fabs , F (ab ') 2), nanobodies, tandem scFv (in series, linked one after the other), scFv-Fcs, scFv dimers, scFv zippers, diabodies, minibodies, tribodies, tetrabs, Fabs, F (ab ) '2s, scFabs, mini antibodies, nanobodies, nanobody
Petition 870190103473, of 10/14/2019, p. 52/184
47/118
HSAs, bispecific T cell engagers (BiTEs), Bispecific T cell Engagers, DARs, scDibodies, scDiacorp-CH3s or scFv-CH3 Knobs-Into-Holes (KIH) clusters (pullers-into-holes).
[051] In certain embodiments, a BCMA-specific binding protein comprises a bispecific or multispecific antibody (or antigen-binding portion thereof) comprising a first binding region (for example, a variable region of the heavy chain, a region light chain variable, or both) that is specific for BCMA and at least one other binding region that is specific for a second target (for example, a BCMA epitope that is different from the epitope of the first binding region, or an epitope of a non-BCMA target, such as, for example, a tumor-associated antigen other than BCMA (for example, CD19 (for example, blinatumomab, MOR-208, SGN-19A, SAR3419, coltuximabravtansina, denituzumabmafodotina, taplitumomabpaptox, XmAb 5574, MDI551, Merck's anti-CD19 patent also known as B4 also known as DI-B4, XmAb 5871, MDX-1342, AEM11), CD20 (for example, rituximab, ofatumumab, ocrelizumab), CD38 (for example, daratumumab or isatuximab (SAR6509 84)), CD45, or a cell surface protein expressed in an effector immune cell, such as a T cell (for example, CD3), an NK cell (for example, CD56) or a T-NK cell
Petition 870190103473, of 10/14/2019, p. 53/184
48/118 (for example, NK1.1), or another antigen or target not associated with a tumor.
[052] In particular embodiments, the present disclosure provides a BCMA-specific binding protein alone or expressed as a T-ChARM in a cell, for use with an γ-secretase inhibitor (GSI). An example of T-ChARM comprises an extracellular component and an intracellular component connected by a hydrophobic portion, wherein the extracellular component comprises a binding domain that specifically binds to BCMA, an optional spacer region, a marker cassette, and a region hinge, and wherein the intracellular component comprises an effector domain and, optionally, a co-stimulatory domain (e.g., a functional domain or portion of 4-1BB, a functional domain or portion of CD28, or both). In certain embodiments, a TChARM binding domain comprises a BCMA-specific scFv, a BCMA-specific scTCR, or a BCMA linker or binding portion thereof (for example, BAFE, APRIL), optionally where the scFv is specific for BCMA. BCMA is human or humanized. Various modalities of T-ChARMs are disclosed in PCT Publication No. WO 2015/095895, whose TChARM frameworks are hereby incorporated by reference in their entirety.
[053] Examples of marker cassettes include Strep
Petition 870190103473, of 10/14/2019, p. 54/184
49/118 tag (which refers to the original Strep® tag, Strep® tag II, or any variant thereof; see, for example, US Patent No. 7,981,632, whose Strep tags are incorporated herein
by reference), His tag, Flag tag, Xpress tag, Avi tag, Calmodulin tag, Polyglutamate tag, HA tag, Myc tag, Nudes tag, S tag, SBP tag, Softag 1, Softag 3, V5 tag, protein
CREB-binding (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), green fluorescent protein (GFP), Thioredoxin tag, or any combination thereof. In some embodiments, a marker cassette may be a genetically engineered affinity site, such as a minimal chelation site (eg, HGGHHG, SEQ ID NO.:6). In certain embodiments, a marker cassette is a Strep tag that has an amino acid sequence of TrpSer-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 0.7) or Trp Arg-HisPro-Gln-Phe- Gly-Gly (SEQ ID NO.:8).
[054] The cassettes with bookmarks may be present in single copies or multiple in proteins of fusion of this disclosure. Per example, a protein from
BCMA specific link of this disclosure can have one, two, three, four or five labeled cassettes (for example, Strep Tag). In certain embodiments, an extracellular component of a BCMA-specific T-ChARM includes a cassette with a marker, two cassettes with a marker, three cassettes with a marker, four cassettes with a marker, or
Petition 870190103473, of 10/14/2019, p. 55/184
50/118 five cassettes with marker. Each of the plurality of marker tapes can be the same or different.
[055] In certain embodiments, a marker cassette comprises from about five to about 500 amino acids, or from about six to about 100 amino acids, or from about seven to about 50 amino acids, or from about eight to about 20 amino acids. In some embodiments, a marker cassette has seven to ten amino acids. Preferably, a marker cassette is non-immunogenic or minimally immunogenic. Essentially, a marker cassette can act as a doorknob or beacon to allow identification, enrichment, isolation, promotion of proliferation, activation, tracking, or elimination of cells that express a BCMA-specific T-ChARM.
[056] In other embodiments, the present disclosure provides a BCMA-specific binding protein for use with a γ-secretase inhibitor, which is a chimeric antigen receptor (CAR), which comprises an extracellular component and an intracellular component linked by a hydrophobic portion, where the extracellular component comprises a binding domain that specifically binds to BCMA and a hinge region, and where the intracellular component comprises an effector domain and, optionally, a co-stimulatory domain. In certain embodiments, a CAR binding domain comprises a BCMA-specific scFv, a BCMA-specific scTCR, a BCMA-specific TCR-binding domain (see, for example,
Petition 870190103473, of 10/14/2019, p. 56/184
51/118
Walseng et al., Scientific Reports 7: 10713 (2017), whose TCR CAR constructs are incorporated herein by reference in their entirety) or a BCMA linker or a linker portion thereof (for example, BAFF, APRIL), optionally where the scFv specific for BCMA is human or humanized. In either of these modalities, a BCMA-specific binding protein in the form of a CAR can be expressed on the surface of a cell, such as a cell in the immune system (e.g., T cell).
[057] A BCMA-specific T-ChARM or CAR can be bound to cells (for example, expressed on the cell surface) or in soluble form. In certain embodiments, polynucleotides that encode T-ChARM or CAR proteins
specific to BCMA may be optimized as the use of codons to improve or maximize the expression in a cell hostess as a cell T (Scholten et al., Clin. Immunol. 119: 135, 2006 (
[058] In certain embodiments, a hinge present in a BCMA-specific T-ChARM or CAR of this disclosure can be an immunoglobulin hinge region, such as a wild type immunoglobulin hinge region or an immunoglobulin hinge region changed from it. In certain embodiments, a hinge is a hinge region of human wild-type immunoglobulin. In certain other embodiments, one or more amino acid residues can be added at the amino- or carboxy-terminal of a hinge region of
Petition 870190103473, of 10/14/2019, p. 57/184
52/118 a wild-type immunoglobulin as part of a fusion protein construct design. For example, one, two or three additional junction amino acid residues may be present at the amino-terminal or carboxiter terminal of the hinge, or a hinge may contain an internal or terminal deletion and have one, two or three additional junction amino acid residues added back.
[059] In certain embodiments, a hinge is an altered immunoglobulin hinge in which one or more cysteine residues in a wild type immunoglobulin hinge region are replaced by one or more other amino acid residues. Examples of altered immunoglobulin hinges include a hinge region of human immunoglobulins IgGl, IgG2 or IgG4 having one, two or three cysteine residues found in a human IgGl, IgG2 or IgG4 hinge replaced by one, two or three human residues different amino acids (for example, serine or alanine). In certain embodiments, a hinge polypeptide comprises or is a sequence that 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
Petition 870190103473, of 10/14/2019, p. 58/184
53/118 a wild-type immunoglobulin hinge region, such as a wild-type human IgG1 hinge, a wild-type human IgG2 hinge, or a wild-type human IgG4 hinge.
[060] In other embodiments, a hinge present in a BCMA-specific T-ChARM or CAR of this disclosure may be a hinge that is not based on or derived from an immunoglobulin hinge (i.e., a wild type immunoglobulin hinge) or an altered immunoglobulin hinge). Examples of such hinges include peptides of about five to about 150 amino acids from the stem region of type II C-lectins or CD molecules, including peptides of about eight to about 25 amino acids or peptides of about seven to about 18 amino acids, or variants thereof.
[061] A region of the C-lectin type II stem or a CD molecule refers to that portion of the extracellular domain of C-lectin type II or the CD molecule that is located between the type C lectin-like domain ( CTLD, from the English C-type lectin-like domain; for example, similar to CTLD of natural killer cell receptors) and the hydrophobic portion (transmembrane domain). For example, the extracellular domain of human CD94 (GenBank Accession No. AAC50291.1) corresponds to amino acid residues 34-179, but CTLD corresponds to residues of
Petition 870190103473, of 10/14/2019, p. 59/184
54/118 amino acids 61-17 6, so that the stem region of the human CD94 molecule comprises amino acid residues 34-60, which are located between the hydrophobic (transmembrane domain) and CTLD (see Boyington et al., Immunity 10:15, 1999; for descriptions of other stem regions, see also Beavil et al., Proc. Nat'1. Acad. Sci. USA 89: 153, 1992; and Figdor et al., Nat. Rev. Immunol 2:11, 2002). These type II or CD C-lectin molecules may also have junction amino acids between the stem region and the transmembrane or CTLD region. In another example, the 233-amino acid human NKG2A protein (GenBank Accession No. P26715.1) has a hydrophobic portion (transmembrane domain) ranging from amino acid 71 to 93 and an extracellular domain ranging from amino acid 94 to 233. CTLD comprises amino acids 119-231 and the stem region comprises amino acids 99-116, which can be flanked by additional joining amino acids. Other C-lectin type II or CD molecules, as well as their extracellular ligand binding domains, stem regions and CTLDs are known in the art (see, for example, GenBank Accession Nos. NP_001993.2; AAH07037.1; NP_001773 .1;
AAL65234.1; CAA04925.1; for the human CD23, CD69, CD72, NKG2A and NKG2D sequences and their descriptions, respectively).
[062] A derivative of a hinge from the region of
Petition 870190103473, of 10/14/2019, p. 60/184
55/118 stem, or a fragment thereof, of a molecule of Clectin type II or CD includes a sequence of about
eight the fence 150 amino acids at which one, two, or three amino acids in the stem region in C-lectin type II type wild or of a molecule in CD have an deletion, insertion, sub constitution or any combination of same. For example, a derivative can understand an or more substitutions of amino acids and / or a deletion in amino acids. In certain modalities , a derivative in an
stem region is more resistant to proteolytic divination compared to the sequence of the wild type stem region, such as those derived from about eight to
about 20 amino acids of NKG2A, NKG2D, CD23, CD64, CD72, or CD94.[063] In certain embodiments, the hinges those of region of the stem can comprise from about seven to fence of 18 amino acids and can form a structure spiral spiral (coiled coil) α-helical. In certain modalities, the hinges of the stem region contain 0, 1,
2, 3, or 4 cysteines. Examples of stem region hinges include fragments of the stem regions, such as portions comprising from about ten to about 150 amino acids of the stem regions of CD69, CD72, CD94, NKG2A and NKG2D.
[064] Alternative hinges that can be used in
Petition 870190103473, of 10/14/2019, p. 61/184
56/118
BCMA-specific T-ChARMs and CARs of this disclosure are portions of cell surface receptors (interdomain regions) that connect immunoglobulin type V or immunoglobulin type C domains. Regions between Ig type V domains where the cell surface receptor contains multiple tandem Ig type V domains and between Ig type C domains where the cell surface receptor contains multiple Ig tandem type C regions they are also contemplated as useful hinges in BCMA-specific T-ChARMs and CARs in this disclosure. In certain embodiments, hinge sequences that comprise cell surface receptor interdomain regions may additionally contain a naturally occurring or added motif, such as a central IgG hinge sequence to provide one or more disulfide bonds to stabilize formation BCMA-specific T-ChARM or CAR dimers. Examples of hinges include interdomain regions between the Ig type V regions and the Ig type C regions of CD2, CD4, CD22, CD33, CD48, CD58, CD66, CD80, CD86, CD150, CD166 or CD244.
[065] In certain embodiments, hinge sequences are 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,
Petition 870190103473, of 10/14/2019, p. 62/184
57/118 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, for example , about 8 to about 20 amino acids, or about 10 to about 15 amino acids. The hinges can be mainly flexible, but they can also provide more rigid characteristics or they can contain mainly a-helical structure with a minimum of β-leaf structure.
[066] In certain embodiments, a hinge sequence is stable in plasma and serum and is resistant to proteolytic degradation. For example, the first lysine in an upper IgG1 hinge region may have been mutated (s) or deleted to minimize proteolytic degradation and the hinges may include joining amino acids. In some embodiments, a hinge sequence may contain a naturally occurring or added motif, such as a central immunoglobulin hinge structure CPPCP (SEQ ID NO .: 9) that provides the ability to form a disulfide or multiple bond disulfide bonds to stabilize the formation of the dimer.
[067] A hydrophobic portion contained in a BCMA-specific binding protein of the present disclosure (eg, BCMA-specific T-ChARM or CAR) will allow
Petition 870190103473, of 10/14/2019, p. 63/184
58/118 that a binding protein specifies the BCMA of this disclosure to associate with a cell membrane, so that a portion of the binding protein is located extracellularly (e.g., labeled cassette, binding domain) and a portion is located intracellularly (for example, effector domain, co-stimulatory domain). A hydrophobic portion will generally be arranged within the phospholipid bilayer of the cell membrane. In certain embodiments, one or more joining amino acids may be arranged between and in order to connect a hydrophobic portion to an effector domain, or arranged between and in order to connect a hydrophobic portion to a spacer region, or arranged between and in order to connect a hydrophobic portion to a marker cassette.
[068] In certain embodiments, a hydrophobic domain is a transmembrane domain, such as a derivative of an integral membrane protein (eg, receptor, differentiation cluster molecule (CD), enzyme, transporter, cell adhesion molecule, or similar). In particular embodiments, a hydrophobic moiety is a transmembrane domain of CD4, CD8, CD27 or CD28.
[069] An intracellular component contained in a BCMA-specific binding protein of the present disclosure (eg, BCMA-specific T-ChARM or CAR) will have the ability to transmit functional signals
Petition 870190103473, of 10/14/2019, p. 64/184
59/118 for a cell. In certain embodiments, a BCMA-specific T-ChARM or CAR will dimerize with a second single-chain T-ChARM or second CAR, respectively, where dimerization allows the intracellular component comprising an effector domain and optionally a co-stimulatory domain is close and promotes signal transduction when exposed to the appropriate signal. In addition to forming such dimeric protein complexes, effector domains and optional co-stimulatory domains can additionally be associated with other signaling factors, such as co-stimulatory factors, to form multiprotein complexes that produce an intracellular signal. In certain embodiments, an effector domain will indirectly promote a cellular response by associating with one or more other proteins that directly promote a cellular response. An intracellular component can include one, two, three or more receptor signaling domains (e.g., effector domains), co-stimulatory domains, or combinations thereof. Any intracellular component comprising an effector domain or a functional portion thereof, a co-stimulatory domain or a functional portion thereof, or any combination thereof of any of a variety of signaling molecules (for example, signal transduction receptors ) can be used in
Petition 870190103473, of 10/14/2019, p. 65/184
60/118 BCMA specific binding proteins of this disclosure.
[070] An intracellular component may have a useful effector or costimulatory domain in the BCMA-specific binding proteins of this disclosure, which may be based on
Or the from an protein from a way signaling Wnt (per example, LRP, Ryk, ROR2), way of NOTCH signaling (per example, NOTCH1, NOTCH2, NOTCH3, NOTCH4), via in
Hedgehog signaling (for example, PTCH, SMO), tyrosine kinase receptors (RTKs) (for example, epidermal growth factor (EGF) family of receptors, family of fibroblast growth factor (FGF) receptors, family of hepatocyte growth factor (HGF) receptors, Insulin receptor family (IR), platelet-derived growth factor receptor (PDGF) family, vascular endothelial growth factor (VEGF) receptor family, receptor family tropomycin kinase (Trk), ephrin receptor family (Eph), AXL receptor family, tyrosine kinase leukocyte receptor family (LTK), tyrosine kinase family with immunoglobulin and EGF 1 (TIE) domains , family of orphan tyrosine kinase receptors (ROR), family of receptors in the discoidin domain (DDR), family of receptors reorganized during transfection (RET), family of tyrosine-protein kinase receptors
Petition 870190103473, of 10/14/2019, p. 66/184
61/118 (PTK7), family of receptors related to the tyrosine kinase receptor (RYK), family of muscle-specific kinase receptors (MuSK); G protein-coupled receptors, GPCRs (curled, smoothed); serine / threonine kinase receptors (BMPR, TGFR); or cytokine receptors (IL-1R, IL-2R, IL-7R, IL-15R).
[071] In certain embodiments, an effector domain comprises a lymphocyte receptor signaling domain or comprises amino acid sequences having one or a plurality of tyrosine immunoreceptor-based activation motifs (ITAMs). Still in additional embodiments, an effector domain comprises a cytoplasmic portion that is associated with a cytoplasmic signaling protein, wherein the cytoplasmic signaling protein is a lymphocyte receptor or signaling domain thereof, a protein comprising a plurality of ITAMs, a co-stimulatory factor, or any combination thereof.
[072] Examples of effector and co-stimulatory domains include those based on or derived from 4-1BB, CD3s,
CD3, CD3 /, CD27, CD28, CD79A , CD79B, CARD11, DAP10, FcRa, FcRp, FcRy, Fyn, HVEM, ICOS, Lek, LAG3, LAT, LRP, NOTCH1, Wnt, NKG2D, 0X40 , ROR2, Ryk , SLAMF1, Slp76, pTa, TCRa,
TCRp, TRIM, Zap70, PTCH2, or any combination thereof.
Petition 870190103473, of 10/14/2019, p. 67/184
62/118 [073] In particular embodiments, BCMA-specific binding proteins of this disclosure comprise (a) a CD3 effector domain / or functional portion thereof and a co-stimulatory domain of CD28 or functional portion thereof, (b) an effector domain of CD3 / or functional portion thereof and a costimulatory domain of 4-1BB or functional portion thereof, or (c) (a) an effector domain of CD3 / or a functional portion thereof and a co-stimulatory domain of CD28 and 4-1BB or functional portions thereof.
Γ-Secretase (GSI) inhibitors [074] As a background, γ-secretase is a complex of integral membrane proteases with multiple subunits, including presenilin (PS), nicastrin (NCT), defective anterior pharynx 1 (APH- 1) and presenilin enhancer 2 (PEN-2) PS is the catalytic subunit that is an aspartate protease that has the ability to form a hydrophilic catalytic pore buried 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), which captures the substrate's amino-terminal as a primary substrate receptor for γ-secretase (Shah et al., Cell 122: 435-41, 2005 ). The γ-secretase complex plays a role in the processing of a variety of substrates, including
Petition 870190103473, of 10/14/2019, p. 68/184
63/118
Notch, CD44, Cadherins, and ephrin B2, as well as it cleaves the amyloid precursor protein in the beta amyloid peptide that is implicated in Alzheimer's disease. The γ-secretase complex is also known to cleave the B cell maturation antigen (BCMA) (Laurent et al., Nature Communications 6, 2015), which is a therapeutic target in several cancers, including multiple myeloma.
[075] Examples of γ-secretase inhibitors (GSIs) include small molecules, peptidomimetic compounds or γ-secretase specific binding proteins. A GSI can target any one or more of the γ-secretase complex proteins, including presenilin 1 (PS1), presenilin 2 (PS2), nicastrin (NCT), defective anterior pharynx 1 (APH-1), and presenilin 2 (PEN2), as long as γ-secretase dividing activity is reduced compared to uninhibited γ-secretase activity. In certain embodiments, γ-secretase activity is reduced by at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or 100%. Assays to measure γ-secretase activity are known in the art (see, for example, Laurent et al., 2015). For example, the level of soluble BCMA can be a surrogate measure for γ-secretase activity. Molecules
Petition 870190103473, of 10/14/2019, p. 69/184
64/118 small that are representative GSIs, for use with an immunotherapy that targets BCMA to treat a proliferative or autoimmune disease or disorder, include avagacestat, DAPT, BMS-906024, BMS-986115, LY411575, MK0752, PF-03084014, RO4929097, semagacestat, YO-01027, and any combination thereof.
[076] Other GSIs are γ-secretase-specific binding proteins, such as antibodies or their antigen-binding portions, than a γ-secretase complex or a γ-secretase complex protein, such as presenilin (PS1), presenilin 2 (PS2), nicastrin (NCT), defective anterior pharynx 1 (APH-1) and presenilin enhancer (PEN-2) An example of a specific binding protein to γsecretase is a specific binding protein to nicastrin, such as antibodies scFvG9, A5226A, 2H6, 10C11 and antigen-binding fragments thereof.
Binding Domains [077] A binding domain can be any peptide that specifically binds to BCMA or that specifically inhibits γ-secretase activity, as described herein. Sources of binding domains include variable antibody regions of various species (which may be in the form of antibodies, sFvs, scFvs, Fabs, scFv-based grababody, or soluble VH domain or antibody domains), including from humans, rodents, birds, or sheep.
Petition 870190103473, of 10/14/2019, p. 70/184
65/118
Additional sources of binding domains include variable regions of antibodies from other species, such as camelids (from camels, dromedaries or llamas; Ghahroudi et al., FEES Lett. 414: 521, 1997; Vincke et al., J. Biol.
Chem. 284: 3213, 2009; Hamers-Casterman et al., Nature
363: 446, 1993 and Nguyen et al. , J. Mol. Biol. 275: 413,
1998), nurse shark (Roux et al., Proc. Nat'1. Acad. Sci. (USA) 95: 11804, 1998), spotted ratfish (Nguyen et al., Immunogen. 54:39, 2002), or lamprey (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 antigen-binding regions using only a variable region of the heavy chain, that is, these functional antibodies are only
homodimers of chains heavy (referred to as antibodies of chain heavy) (Jespers et al., Nat. Biotechnol. 22: 1161, 2004; Cortez-Retamozo et al. , Cancer Res. 64: 2853 , 2004; Baral et al., Nature Med. 12: 580, 200 6; and Barthelemy et al., J. Biol. Chem. 283: 3639, 008).
[078] An alternative source of binding domains for this disclosure includes sequences that encode random peptide libraries or sequences that encode a projected diversity of amino acids in alternative scaffold regions other than antibodies, such as scTCR (see, for example).
Petition 870190103473, of 10/14/2019, p. 71/184
66/118 example Lake et al., Int. Immunol.11: 745, 1999; Maynard et al., J. Immunol. Methods 306: 51, 2005; US Patent No. 8,361,794), fibrinogen domains (see, for example, Weisel et al., Science 230: 1388, 1985), Kunitz domains (see, for example, US Patent No. 6,423,498), proteins Anquirin Repeat Patterns (DARPins) (Binz et al., J. Mol. Biol. 332: 489, 2003 and Binz et al., Nat.
Biotechnol. 22: 515, 2004), fibronectin binding domains (adnectins or antibodies) (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'1. Acad. Sci. (USA) 92 : 6404-6408; Martin et al. (2002) Nat. Biotechnol. 21:11, 2002 and Huang et al. (2005) Structure 13: 155, 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, for example, WO 2006/095164, Beste et al., Proc. Nat '1. Acad. Sci. (USA) 96: 1898, 1999 and Schernfeld et al., Proc. Nat' 1. Acad. Sci. (USA) 106: 8198, 2009), type V domains (see, for example, US Patent Application Publication No. 2007/0065431), type C lectin domains (Zelensky and Gready, FEES J. 272: 6179, 2005 ; Beavil et al., Proc.
Petition 870190103473, of 10/14/2019, p. 72/184
67/118
Nat'1. Acad. Sci. (USA) 89: 153, 1992 and Sato et al., Proc. Nat '1. Acad. Sci. (USA) 100: 1119, 2003), mAb ² or Fcab ™ (see, for example, PCT Patent Application Publications No. WO 2007/098934; WO 2006/072620), armadillo repeat proteins (see, for example, Madhurantakam et al., Protein Sci. 21: 1015, 2012; PCT Patent Application Publication No. WO 2009/040338), afiline (Ebersbach et al., J. Mol. Biol. 372: 172, 2007) , affibody, avimeroses, inhibitor cystine nodes (knottins), finomers (fynomers), atrimers (atrimers), cytotoxic protein-4 associated with T lymphocytes (Weidle et al., Cancer Gen. Proteo. 10: 155, 2013) or similar (Nord et al., Protein Eng. 8: 601, 1995; Nord et al., Nat. Biotechnol. 15: 112, 1997; Nord et al., Euro. J. Biochem. 288: 4269, 2001; Binz et al. ., Nat. Biotechnol. 23: 1251, 2005; Boersma and Plückthun, Curr.
Opin. Biotechnol. 22: 849, 2011).
[079] The binding domains of this disclosure can be generated as described herein or by a variety of methods known in the art (see, for example, US Patent Nos. 6,291,161 and 6,291,158). For example, the binding domains of this disclosure can be identified by screening a Fab phage library in search of Fab fragments that specifically bind to a target of interest (for example, BCMA, component of the γ-secretase complex, such as presenilin or nicastrin) (see Hoet et al., Nat.
Petition 870190103473, of 10/14/2019, p. 73/184
68/118
Biotechnol. 23: 344, 2005). In addition, traditional strategies for the development of hybridomas using a target of interest (eg BCMA, component of the γ-secretase complex, such as presenilin or nicastrin) as an immunogen in convenient systems (eg, mice, HuMAb mouse® , TC mouse ™, Km-mouse®, llamas, chickens, rats, hamsters, rabbits, etc.) can be used to develop binding domains for this disclosure.
[080] In some embodiments, a binding domain is a single-chain Fv fragment (scFv) comprising VH and VL regions specific for a target of interest (e.g. BCMA, component of the γ-secretase complex, such as presenilin or nicastrin). In certain embodiments, the V _H and V _L regions are human. Examples of V _H and V _L regions are the specific anti-BCMA antibody segments J22.0-XÍ, J22.9-XÍ, J6M0, J6M1, J6M2, J9M0, J9M1, J9M2, 11D5-3, CA8, A7D12.2, Cll D5.3, C12A3.2, C13F12.1, 13C2, 17A5, 83A10, 13A4, 13D2, 14B11, 14E1, 29B11, 29F3, 13A7, CA7, SG1, S307118G03, S332121F02, S332126E04, S32261010, S322121010 , ET140-3, ET140-24, ET140-37, ET14040, ET140-54, TBL-CLN1, C4.E2.1, Vicky-1, pSCHLI333, pSCHLI372, or pSCHLI373. Other examples of Vh and Vl regions include specific antinicastrin antibody segments or fragments thereof, which bind to
Petition 870190103473, of 10/14/2019, p. 74/184
69/118 antigens, from scFvG9, A5226A, 2H6 or, 10C11.
[081] In certain embodiments, a binding domain comprises or is a sequence that is 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
minus 98%, at least 99%, at least 99, 5% or 100% identical to a sequence of amino acids of an region variable of light chain (V _L ) (for example, of 22 .0-xi, J22.9-XÍ, J6M0, J6M1, J6M2, J9M0, J9M1, J9M2, 11D5-3 , CA8, A7D12.2, Cll D5.3, C12A3.2, or C13F12.1, 13C2, 17A5, 83A10, 13A4, 13D2, 14B11, 14E1, 29B11, 29F3, 13A7, CA 7,
S307118G03, S332121F02, S332126E04, S322110D07, S336105A07, S335115G01, S335122F05, ET140-3, ET140-24, ET140-37, ET14040, ET140-54, TBL-CLN1, C4.E2.1, C4.E2.1 or pSCHLI373 anti-BCMA; or scFvG9, A5226A, 2H6, or 10C11 anti-nicastrin) or to a variable region of the heavy chain (Vh) (for example, from J22.0-xi, J22.9-xi, J6M0, J6M1, J6M2, J9M0, J9M1, J9M2, 11D5-3, CA8, A7D12.2, Cll D5.3, C12A3.2, C13F12.1, 13C2, 17A5, 83A10, 13A4, 13D2, 14B11, 14E1, 29B11, 29F3, 13A7, CA7, SGI , S307118G03, S332121F02, S332126E04, S322110D07, S336105A07, S335115G01, S335122F05, ET140-3, ET140-24, ET140-37, ET140-40, ET14054, TBL-CLN1, C4E2, CH4 , or anti-BCMA pSCHLI373; or scFvG9, A5226A, 2H6, or anti-nicastrin 10C11), or both, where each CDR comprises zero
Petition 870190103473, of 10/14/2019, p. 75/184
70/118 changes, or at most one, two or three changes, of a monoclonal antibody or fragment or derivative thereof that specifically binds to the target of interest (eg BCMA, component of the γ-secretase complex, such as presenilin or nicastrin).
[082] In certain embodiments, a Vh region of the binding domain of the present disclosure can be derived or based on a Vh from a known monoclonal antibody and contains one or more (for example, 2, 3, 4, 5, 6, 7 , 8, 9, 10) insertions, one or more (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (for example, 2, 3, 4, 5 , 6, 7, 8, 9, 10) amino acid substitutions (for example, conservative amino acid substitutions or non-conservative amino acid substitutions), or a combination of the changes mentioned above, when compared to the Vh of a known monoclonal antibody. An insertion, deletion or substitution can be anywhere in the Vh region, including at the amino- or at the carboxy-terminal or at both ends of this region, provided that each CDR comprises zero changes or at most one, two or three changes and as long as that a binding domain containing the modified Vh region can still specifically bind to its target with an affinity similar to that of the wild type binding domain.
[083] In other modalities, a Vl region in a
Petition 870190103473, of 10/14/2019, p. 76/184
71/118 binding domain of the present disclosure is derived from or based on a Vl of a known monoclonal antibody and contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) inserts , one or more (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (for example, 2, 3, 4, 5, 6, 7, 8, 9 , 10) amino acid substitutions (e.g., conservative amino acid substitutions), or a combination of the aforementioned changes, when compared to the Vl of the known monoclonal antibody. An insertion, deletion or substitution can be anywhere in the V1 region, including at the amino- or carboxy terminal or at both ends of this region, as long as each CDR comprises zero changes or at most one, two or three changes and as long as one binding domain containing the modified V1 region can still specifically bind to its target with an affinity similar to that of the wild type binding domain.
[084] The Vh and Vl domains can be arranged in any orientation (that is, from the amino-terminal to the carboxy-terminal, Vh-Vl or VL-Vh) and can be joined by a sequence of amino acids (for example, having about five to about 35 amino acids in length) that have the ability to provide a spacer function so that the two sublinking domains can interact to form a functional binding domain. In certain
Petition 870190103473, of 10/14/2019, p. 77/184
72/118 modalities, a variable region linker that joins the Vh and Vl domains includes those belonging to the (Gly _n Ser) family, such as (Gly ₃ Ser) _n (Gly4Ser) i (SEQ ID N0: 3) , (Gly ₃ Ser) i (Gly ₄ Ser) n (SEQ ID NO: 10), (Gly ₃ Ser) n (Gly ₄ Ser) n (SEQ ID NO: 4), or (Gly ₄ Ser) _n (SEQ ID NO: 5), where n is an integer from 1 to 5. In certain 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 certain embodiments, these (Gly _n -Ser) based linkers are used to link the V _H and V _L domains in a link domain, and these linkers can also be used to link the link domain to a marker cassette, or to attach a marker cassette to a hydrophobic portion or intracellular component.
[085] In some embodiments, a binding domain is a single-chain T cell receptor (scTCR) comprising ν _α / ρ and Ca / p chains (for example, V _a -C _a , VpCp, Va-Vp) or comprising a V _to -C _a , Vp-Cp, Vp-Vp pair specific for a target of interest (e.g., peptide-MHC complex).
[086] In certain embodiments, a binding domain comprises or is a sequence that is 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%
Petition 870190103473, of 10/14/2019, p. 78/184
73/118 identical to an amino acid sequence of a ν _α , Vp, C _a or Cp of TCR, where each CDR comprises zero changes, or at most one, two or three changes, of a TCR or fragment or derivative of the even if it specifically binds to a target of interest (for example, BCMA, a component of the γ-secretase complex, such as presenilin or nicastrin).
[087] In certain embodiments, a ν _α , Vp, C _{a region} , the Cp of the binding domain of the present disclosure can be derived or based on a ν _α , Vp, C _a , or Cp from a known monoclonal antibody and contains one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) inserts, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (for example, 2, 3, 4, 5, 6, 7, 8, 9, 10) amino acid substitutions (for example, conservative amino acid substitutions or non-conservative amino acid substitutions), or one combination of the aforementioned changes when compared to the ν _α , Vp, C _a , or Cp of a known monoclonal antibody. An insertion, deletion or substitution can be anywhere in the ν _α , Vp, C _a , or Cp region, including at the amino- or at the carboxy-terminal or at both ends of these regions, as long as each CDR comprises zero changes or the maximum one, two or three changes and as long as a binding domain containing a modified ν _α , Vp, C _a , or Cp region can still specifically bind to its target with an affinity similar to that of the
Petition 870190103473, of 10/14/2019, p. 79/184
74/118 wild type.
[088] BCMA, γ-secretase or both can be found in or in association with a cell of interest (target cell). Examples of target cells include a cancer cell, a cell associated with an autoimmune disease or disorder or an inflammatory disease or disorder, and an infectious organism or cell (e.g., bacteria, virus, virus-infected cell).
Cytotoxic Conjugates [089] Antibody-drug conjugates are used to selectively deliver / release a cytotoxic element to a target cell, for example, a tumor or cancer cell. Antibody-drug conjugates and related techniques and chemicals are described in, for example, Nasiri et al., J. Cell. Physiol. (2018), Hedrich et al. , Clin. Pharmacokinet. (2017), Drake and 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), of which the antibody formats, cytotoxic, chemical payloads of ligands (linkers) and conjugation, dosing regimens, treatment methods, pharmacokinetics, and CAF design principles are incorporated herein by reference in their entirety.
[090] In certain embodiments of the methods provided here, a BCMA-specific antibody or antigen-binding portion, a chimeric antigen receptor (CAR), or molecule
Petition 870190103473, of 10/14/2019, p. 80/184
75/118 of labeled chimeric antigen receptor (T-ChARM) is conjugated or, in other circumstances, coupled to a cytotoxic agent, such as chemotherapeutic agent. A chemotherapeutic agent includes, but is not limited to, a chromatin function inhibitor, a topoisomerase inhibitor, a drug that inhibits microtubules, a DNA damaging agent, an antimetabolite (such as folate antagonists, pyrimidine analogs, analogs of purine, and sugar modified analogs), an inhibitor of DNA synthesis, an agent that interacts with DNA (such as an intercalating agent), and a DNA repair inhibitor. Illustrative chemotherapeutic agents include, without limitation, 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 disruptors, such as taxane (paclitaxel, docetaxel), vincristine, vimblastine, nocodazole, epothilones and navelofin, epidophylline, epidophylline, epidolines, epidophylline, epidolines, epidolines, epidolines, epidine, epididinesine , agents that cause DNA damage (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, Cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethyl melamine, hexamethyl melamine, hexamethylamine
Petition 870190103473, of 10/14/2019, p. 81/184
76/118 merchloretamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, temozolamide, teniposide, triethylenothioposforamide and etoposide (VP 16)); antibiotics, such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycin, plicamycin (mitramycin) and mitomycin; enzymes (L-asparaginase that systematically metabolizes L-asparagine and deprives cells that are unable to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents, such as nitrogen mustards (mecloretamine, cyclophosphamide and the like, melphalan, chlorambucil), ethylenimines and methyl melamines (hexamethyl melamine and thiotepa), alkylsulfonates-bulfulfan, nitrosourea (carmustine) and carcinogens (BC) dacarbazine (DTIC); antiproliferative / antimitotic antimetabolites, such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutotimide; hormones, hormone analogues (estrogen, tamoxifen, goserrelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; anti-immigration agents; antisecretory agents
Petition 870190103473, of 10/14/2019, p. 82/184
77/118 (breveldina); immunosuppressants (cyclosporine, tacrolimus (FK506), sirolimus (rapamycin), azathioprine, mofetil mycophenolate); antiangiogenic compounds (TNP470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; 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, cortisone, corticosteroids, irinotecone, cortinone methylprednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers, toxins such as cholera toxin, ricin,
exotoxin of Pseudomonas, toxin in adenylate cyclase in Bordetella pertussis, or toxin from diphtheria, and activators in caspase; and[091] disruptorsAn example chromatin.the CAF that if call specifically to
BCMA is J6M0-mcMMAF (GSK2857 916), described by Tai et al. , Blood 123 (20): 3128-3138 (2014), whose CAF and methods of using them are hereby incorporated by reference in their entirety. Another CAF specific for BCMA (SG1-MMAF) has been described by Ryan et al. (Mol. Cancer Ther., 6 (11): 3009-3018, 2007), whose CAF is here
Petition 870190103473, of 10/14/2019, p. 83/184
78/118 incorporated by reference.
Host Cells and Nucleic Acids [092] In certain respects, the present disclosure provides nucleic acid molecules that encode any one or more of the BCMA specific binding proteins (including multispecific and bispecific binding proteins comprising at least one BCMA binding domain ) or γ-secretase inhibitors described herein. Such nucleic acid molecules can be inserted into an appropriate vector (for example, viral vector or non-viral plasmid vector) for introduction into a host cell of interest (for example, T cell).
[093] As used here, the term recombinant or unnatural refers to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or that has been modified by the introduction of an acid molecule exogenous nucleic acid, in which such changes or modifications are introduced by genetic engineering. Genetic changes include, for example, modifications that introduce nucleic acid molecules that can be expressed and that encode proteins, fusion proteins or enzymes, or other additions, deletions, substitutions or other functional disruptions to the nucleic acid molecules of the genetic material of a cell.
Additional modifications include, for example, non-coding regulatory regions in which the modifications
Petition 870190103473, of 10/14/2019, p. 84/184
79/118 alter the expression of a gene or operon. In certain embodiments, a cell, such as a T cell, obtained from an individual can be converted into an unnatural or recombinant cell (for example, an unnatural or recombinant T cell) by introducing a nucleic acid that encodes a protein from specific binding to BCMA or an γ-secretase inhibitor of this disclosure (for example, BCMA specific T ChARM or CAR; or anti-Y-secretase) as described herein and by which the cell expresses a BCMA specific binding protein located in the cell surface.
[094] Here, it refers to a vector that encodes a central virus as a viral vector. There are a large number of viral vectors available suitable for use with the compositions of the present disclosure, including those identified for human gene therapy applications (see Pfeifer and Verma, Ann. Rev. Genomics Hum. Genet. 2: 111, 2001). Suitable viral vectors include vectors based on RNA viruses, such as vectors derived from retrovirus, for example, vectors derived from the murine leukemia virus Moloney (MLV), and include vectors from more complex retrovirus derivatives, for example, vectors derived from lentivirus. Vectors derived from HIV-1 belong to this category. Other examples include HIV-2-derived lentivirus vectors, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (sheep lentivirus). Methods of using retroviral and lentiviral viral vectors and
Petition 870190103473, of 10/14/2019, p. 85/184
80/118 packaging for the transduction of mammalian host cells with viral particles containing chimeric antigen receptor transgenes are known in the art and have been described previously, for example, in US Patent 8,119,772; Walchli et al. , PLoS One 6: 321930, 2011; Zhao et al., J. Immunol. 274: 4415, 2005; Engels et al., Hum. Gene Ther. 24: 1155, 2003; Frecha et al., Mol. Ther. 28: 1748, 2010; Verhoeyen et al., Methods Mol. Biol. 506: 91, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available.
[095] In certain embodiments, a viral vector is used to introduce a non-endogenous polynucleotide that encodes a BCMA-specific binding protein or a ysecretase inhibitor. A viral vector can be a retroviral vector or a lentiviral vector. A viral vector can also include polynucleotides that encode a marker for transduction. Transduction markers for viral vectors are known in the art and they include selection markers, which can confer resistance to drugs, or detectable markers, such as fluorescent markers or cell surface proteins that can be detected by methods such as flow cytometry. In particular embodiments, a viral vector further comprises a genetic marker for transduction comprising green fluorescent protein, an extracellular domain of human CD2, or a truncated human EGFR (huEGFRt; see Wang et al., Blood 228: 1255, 2011). When a viral vector genome comprises a plurality of nucleic acid sequences to be expressed
Petition 870190103473, of 10/14/2019, p. 86/184
81/118 in a host cell as separate transcripts, the viral vector can also comprise additional sequences between the two (or more) transcripts, allowing for bicistronic or multicistronic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin dividing sites, viral peptide 2A (for example, P2A, T2A, E2A, F2A) or any combination of the same.
[096] Other vectors can also be used for polynucleotide delivery including viral DNA vectors, including, for example, adenovirus-based vectors and adeno-associated virus (AAV) -based vectors (AAV) -based vectors ); vectors derived from the herpes simplex viruses (HSVs, herpes simplex viruses), including amplicon vectors, HSV with replication defects and attenuated HSV (Krisky et al., Gene Ther. 5: 1517, 1998).
[097] Other vectors recently developed for gene therapy uses can also be used with the compositions and methods of this disclosure. Such vectors include those derived from baculovirus and ot virus. (Jolly, D J. 1999. Emerging Viral Vectors, p. 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab) or plasmid vectors (such as Sleeping Beauty) sleeping beauty) or other transposon vectors). In some embodiments, a viral or plasmid vector further comprises a genetic marker for transduction (e.g., green fluorescent protein, huEGFRt).
Petition 870190103473, of 10/14/2019, p. 87/184
82/118 [098] In certain embodiments, hematopoietic progenitor cells or embryonic stem cells are modified to comprise a non-endogenous polynucleotide that encodes a BCMA-specific binding protein (for example, BCMA-specific T-CHARM or CAR) this disclosure. Hematopoietic progenitor cells may comprise thymocyte progenitor cells or induced pluripotent stem cells, which may be derived from or derived from fetal liver tissue, bone marrow, umbilical cord blood, or peripheral blood. Hematopoietic progenitor cells can be from humans, mice, rats, or other mammals. In particular embodiments, CD24 ¹⁰ Lin ~ CD117 ⁺ thymocyte progenitor cells are used.
[099] In certain embodiments, culture conditions involve culturing hematopoietic progenitor cells that express fusion proteins of this disclosure for a time sufficient to induce proliferation or differentiation. The cells are maintained in culture generally for about 3 days to about 5 days, or about 4 to about 10 days, or about 5 to about 20 days. It should be understood that cells can be maintained for an appropriate period of time to achieve the desired result, that is, a desired cell composition or level of proliferation. For example, to generate a cell composition comprising mainly immature and inactivated T cells, the cells can be maintained in culture for about 5 to about 20 days. The cells can be kept in culture for about 20 to about 30 days to generate a
Petition 870190103473, of 10/14/2019, p. 88/184
83/118 cell composition comprising mainly mature T cells. Non-adherent cells can also be collected from the culture at various times, such as between about several days to about 25 days. In certain embodiments, hematopoietic stem cells are co-cultured in stromal cell lines (US Patent No. 7,575,925; Schmitt et al., Nat. Immunol. 5: 410, 2004; Schmitt et al., Immunity 17: 749, 2002).
[100] One or more cytokines that promote impairment or differentiation of hematopoietic progenitor cells can 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; ligand Flt-3, stem cell factor (SCF, stem cell factor), thrombopoietin (TPO) and IL-7. Cytokines can be used in combination with a glycosaminoglycan, such as heparin sulfate.
[101] In some embodiments, host cells that have the ability to express a BCMA-specific binding protein of this disclosure on the cell surface are T cells, including primary cells or cell lines derived from humans, mice, rats, or other mammals. If obtained from a mammal, a T cell can be obtained from numerous sources, including blood, bone marrow, lymph node, thymus, or other tissues or fluids. T cells or subpopulations of them (for example, immature (naive), central memory, effector memory) can be enriched or purified. T cell lines are well known in the
Petition 870190103473, of 10/14/2019, p. 89/184
84/118 technique, some of which are described in Sandberg et al., Leukemia 21: 230, 2000. In certain modalities, T cells that do not have endogenous expression of the TCRot and β chains are used. Such T cells may naturally lack endogenous expression of the TCR ot and β chains or may have been modified to block expression (for example, T cells in a transgenic mouse that does not express ot and β TCR chains or cells that
have been manipulated so The inhibit the expression of chains TCR α and β) or so the if eliminate the TCRot chain, the jail ΤΡΒ, β or both genes. In certain modalities, cells
that have the ability to express a BCMA-specific binding protein of this disclosure on the cell surface are not T cells or cells of a T cell line, but cells that are progenitor cells, stem cells or cells that have been modified to express anti-CD3 on the cell surface.
[102] In any of the modalities provided herein, a host cell can be a universal donor cell that is modified to reduce or eliminate the expression of one or more endogenous genes involved in an immune response. For example, a T cell can be modified to reduce or eliminate the expression of one or more polypeptides selected from PD-1, LAG-3, CTLA4, TIGIT, TIM3, a component of the HLA complex, or a TCR or component of the TCR complex. Without wishing to be bound by theory, certain endogenously expressed immune cell proteins can be recognized as foreign by a
Petition 870190103473, of 10/14/2019, p. 90/184
85/118 allogeneic host that receives the modified immune cells, which can result in the elimination of the modified immune cells (for example, an HLA allele) or down-regulate the immune activity of the modified immune cells (for example, PD1, LAG-3 , CTLA4, TIGIT) or interfere with the binding activity of a heterologically expressed binding protein of the present disclosure (for example, an endogenous TCR that binds to a non-tumor associated antigen and interferes with the specific receptor for modified immune cell antigens that specifically binds to the tumor-associated antigen). Consequently, decreasing or eliminating the expression or activity of such endogenous genes or proteins can improve the activity, tolerance, and persistence of the modified immune cells in an allogeneic environment in the host and may allow for universal administration of the cells (for example, to any receptor, regardless of the type of HLA).
[103] In certain embodiments, a host cell (e.g., modified immune cell) of this disclosure comprises a chromosomal genetic knockout of one or more genes that encode a component of the HLA PD-1, LAG-3, CTLA4 complex, TIM3, TIGIT (for example, a gene encoding an otl macroglobulin, a2 macroglobulin, a u3 macroglobulin, a βΐ microglobulin, or a β2 microglobulin), a component of the TCR (for example, a gene that encodes a variable TCR region or a TCR constant region) (see, for example, Torikai et al., Nature Sci. Rep. 6: 21151 (2016); Torikai et al., Blood 119 (24): 5697 (2012); and Torikai et al. ., Blood 222 (8): 1341 (2013),
Petition 870190103473, of 10/14/2019, p. 91/184
86/118 whose gene editing techniques, compositions and adoptive cell therapies are hereby incorporated by reference in their entirety). For example, in some embodiments, a chromosomal genetic knockout is produced using a CRISPR / Cas9 system and may involve transfection of the immune cell modified with a lentivirus (eg, pLentiCRISPRv2; Torikai et al., Blood (2016 )) that expresses a CRISPR / Cas9 system that targets PD-1, LAG-3, CTLA4, an HLA component, or a TCR component, or any combination thereof. Primers useful for designing a lentivirus that expresses a CRISPR / Cas9 system to inhibit an endogenously expressed immune cell protein include, for example, primer pairs comprising forward and reverse primers having the nucleotide sequences shown in SEQ ID NOS: 22 and 23, 24 and 25, 26 and 27, and 28 and 29.
[104] In certain embodiments, a host T cell transfected to express a BCMA-specific binding protein (eg, T-ChARM, CAR, multispecific binding proteins comprising at least one BCMA binding domain, bispecific binding proteins that comprise at least one BCMA binding domain) of this disclosure is a functional T cell, such as a virus-specific T cell, a tumor antigen-specific cytotoxic T cell, a
immature T cell, an stem cell T of memory, a T cell central memory or effector, or a regulat T cell CD4 + story CD25 +. [105] May if r added to culture one or more
Petition 870190103473, of 10/14/2019, p. 92/184
87/118 growth factor cytokines that promote the proliferation of T cells that express a BCMA-specific binding protein of this disclosure. Cytokines can be human or non-human. Examples of growth factor cytokines that can be used promote T cell proliferation include IL-2, IL-15, IL-21 or the like.
Uses [106] Diseases that can be treated with immunotherapy that targets BCMA or cells that express a BCMA-specific binding protein in combination with an γsecretase inhibitor (GSI), as described in the present disclosure, include cancer (for example , cancers that express BCMA), immune diseases (for example, autoimmune), or diseases related to aging (for example, senescence). Immunotherapy and adoptive 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: el001018, 2010; Luo et al., J. Mol. Med. 89: 903, 2011).
[107] Cancers that are susceptible to the compositions and methods disclosed herein are those that express or have the ability to express BCMA on their cell surface. Examples of cancer that can be treated include myelomas (such as multiple myeloma), leukemias (such as plasma cell leukemia), lymphomas (such as lymphoplasmacytic lymphoma),
Petition 870190103473, of 10/14/2019, p. 93/184
88/118 plasmacytomas, Waldenstrom's macroglobulinemia. Other cancers that can express BCMA include breast adenocarcinoma and bronchogenic lung carcinoma. In certain embodiments, proliferative disorders that respond to a combination therapy of a specific BCMA-binding protein and a GSI are certain types of B cell cancer, including disorders
plasma cells (such as, for example, myeloma multiple).[108] Diseases inflammatory and autoimmune include arthritis, rheumatoid arthritis, arthritis rheumatoid young man,
osteoarthritis, polychondritis, psoriatic arthritis, psoriasis, dermatitis, polymyositis / dermatomyositis, inclusion body myositis, inflammatory myositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, CREST syndrome, inflammatory bowel disease, Crohn's disease, ulcerative colitis, ulcerative colitis respiratory distress, 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, systemic lupus erythematosus (SLE), subacute cutaneous lupus erythematosus, discoid lupus, lupus myelitis, lupus cerebritis, juvenile diabetes, multiple sclerosis, allergic encephalomyelitis, optic neuromyelitis, rheumatic fever, Sydenham's chorea, immune responses associated with late-onset hypertension cytokines and T lymphocytes, tuberculosis, sa rcoidosis, granulomatosis, including
Petition 870190103473, of 10/14/2019, p. 94/184
89/118 Wegener's granulomatosis and Churg-Strauss disease, agranulocytosis, vasculitis (including hypersensitivity / angiitis vasculitis, ANCA and rheumatoid vasculitis), aplastic anemia, Diamond-Blackfan 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 involving leukbocyte diapedesis, inflammatory central nervous system (CNS) disorders, multiple organ injury syndrome, myvisthenia gravis , diseases mediated by the antigen-antibody complex, glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Behçet's syndrome, Castleman's syndrome, Goodpasture's syndrome, Lambert-Eaton myasthenic syndrome, Reynaud's syndrome, Sjorgen's syndrome Stevens-Johnson syndrome, rejection of solid organ transplantation, graft versus host disease (GVHD), penf bullous igoid, pemphigus, autoimmune polyendocrinopathies, seronegative spondyloarthropathies, Reiter's disease, rigid man's syndrome, giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathies, or IgM-mediated neuropathy, ITP thrombotic thrombocytopenic purpura (TTP), Henoch-Schonlein purpura, autoimmune thrombocytopenia, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism; autoimmune endocrine diseases, including autoimmune thyroiditis, chronic thyroiditis (thyroiditis
Petition 870190103473, of 10/14/2019, p. 95/184
90/118 of Hashimoto), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Graves' disease, autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), type I diabetes, which is also referred to as insulin-dependent diabetes mellitus (DMID) and Sheehan's syndrome; autoimmune hepatitis, lymphoid interstitial pneumonitis (HIV), bronchiolitis obliterans (non-transplant), non-specific interstitial pneumonia (PINS), Guillain-Barré syndrome, large vessel vasculitis (including rheumatic polymyalgia and giant cell arteritis (Takayasu)), vasculitis of medium vessels (including Kawasaki disease and polyarteritis nodosa), polyarteritis nodosa (PAN), ankylosing spondylitis, Berger's disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, celiac disease (gluten enteropathy), cryoglobulinemia, cryoglobulinemia, cryoglobulinemia associated with hepatitis, amyotrophic lateral sclerosis (ALS), coronary artery disease, familial Mediterranean fever, microscopic polyangiitis, Cogan's syndrome, Whiskott-Aldrich syndrome and thromboangiitis obliterans.
[109] In particular embodiments, a method for treating an individual with BCMA-binding protein in combination with a secretase-γ inhibitor, as disclosed herein, includes treatment of multiple myeloma, plasmacytoma, plasma cell leukemia, macroglobulinemia Waldenstrom's disease, B-cell lymphoma and lymphoplasmocytic lymphoma.
[110] A BCMA specific binding protein, such as a CAR or T-ChARM, from this disclosure can be administered to a
Petition 870190103473, of 10/14/2019, p. 96/184
91/118 individual in cell-bound form (eg, gene therapy of the target cell population (mature T cells (eg, CD8 ⁺ or CD4 ⁺ T cells) or other cells of the T cell lineage)). In a specific embodiment, cells of the T cell lineage expressing BCMA-specific binding protein (for example, BCMA-specific CAR or T-ChARM) administered to an individual are single, allogeneic or autologous cells.
[111] A method of the present disclosure includes steps for administering a BCMA-specific binding molecule expressed on the surface of an immune cell (for example, a T cell) and administering a γ-secretase inhibitor (GSI). In certain embodiments, the combination can be administered concomitantly, together in the same pharmaceutically acceptable carrier, or in separate formulations (but concurrently). Simultaneous administration means that each component is administered at the same time or 8 to 12 hours between one and the other. The administration of a second component more than 12 hours after the first component will be considered a sequential administration. In other modalities, a BCMA-specific immunotherapy and a GSI can be administered sequentially (for example, one, two, three, four, five, six, seven, eight, or nine days apart; with one, two, three, or four weeks apart; one, two, three, four, five, six, seven, eight, or nine weeks apart; or one, two, three, four, five, six, or more years apart; or similar), in any order and in any
Petition 870190103473, of 10/14/2019, p. 97/184
92/118 any combination. In particular modalities, when administered sequentially, GSI is administered first and immunotherapy that targets BCMA (soluble or cellular form) is administered next. In other modalities, an immunotherapy that targets BCMA (soluble or cellular form) is administered first and GSI is administered next. In particular embodiments, an immunotherapy that targets BCMA comprising a modified immune cell that specifically binds to BCMA (for example, a BCMA specific T cell, such as a bispecific, multispecific T cell for CAR-T, T-CHARM) it is administered first and a GSI is administered next (for example, within hours, days, weeks, months or years from BCMA immunotherapy). In other embodiments, an immunotherapy targeting BCMA comprising a modified immune cell comprising a binding protein that specifically binds to BCMA (for example, a BCMA specific T cell, such as a bispecific, multispecific T cell for CAR-T , T-ChARM) is (a) administered to an individual; (b) after a period of time (for example, about 5 days to about a week, about a week to about two weeks, about two weeks to about three weeks, about a week to about one month, about a month to about six months, about three months to about a year, or as much as necessary), the individual or a tissue of the individual is probed or examined for the presence or persistence of the previously modified immune cell administered comprising a binding protein that binds
Petition 870190103473, of 10/14/2019, p. 98/184
93/118 specifically BCMA and (c) administer a GSI after and after the presence or persistence of immunotherapy involving BCMA has been detected.
[112] In some embodiments, a GSI is administered to an individual at least once before, simultaneously and at least once (for example, at least two, three or four times) after BCMA-targeted immunotherapy. In particular modalities, a combination therapy of this disclosure comprises an immunotherapy that targets BCMA and its
administration from since about 0.01 μΜ of GSI the fence in 5 μΜ of GSI, since fence in 0.03 μΜ from GSI The about 1.5 μΜ in GSI, since about 0.05 μΜ from GSI to fence in 2.5 μΜ of GSI, or since
about 0.1 μΜ of GSI to about 1.0 μΜ of GSI. In certain embodiments, a combination therapy comprising a modified immune cell that specifically binds to BCMA and a GSI comprises a smaller amount of the immune cell that specifically binds to BCMA, GSI, or both, compared to administering these therapies individually.
[113] For example, as a background and without wishing to be bound by theory, during adoptive immunotherapy, the graft of cells administered expressing BCMA-specific binding protein can be facilitated by prior immunosuppressive conditioning with one or more agents or treatments chemotherapy. In any of the modalities described herein, the method further comprises preconditioning an individual concomitantly or prior to administration of an immunotherapy targeting BCMA or a
Petition 870190103473, of 10/14/2019, p. 99/184
94/118 immune cell that expresses a BCMA specific binding protein and a GSI. In particular modalities, a preconditioning regimen is an immunosuppressive conditioning that comprises depleting endogenous lymphocytes (which is also referred to as lymphoblastic depletion, which can be non-myeloablative or myeloablative). As an example, lymphodepletion can be achieved with cyclophosphamide alone, cyclophosphamide in combination with fludarabine, by using other agents or treatments that are cytotoxic to lymphocytes (for example, total irradiation of the body), or any combination thereof.
[114] The binding molecule, inhibitor or combination compositions can be administered orally, topically, transdermally, parenterally, by inhalation spray, vaginally, rectally or by intracranial injection, or any combination thereof. When administered separately, an immunotherapy that targets BCMA and a GSI can be administered by the same or different routes. For example, in certain modalities, immunotherapy directed to BCMA is administered parenterally and GSI is administered orally, which can occur concurrently or sequentially. The term parenteral, as used herein, includes subcutaneous, intravenous, intramuscular injections, intracisternal injection, or infusion techniques. Administration by intravenous, intradermal, intramuscular, intramammary, intraperitoneal, intrathecal, retrobulbar, intrapulmonary and / or surgical implantation in a specific location is also contemplated. Generally, compositions are essentially
Petition 870190103473, of 10/14/2019, p. 100/184
95/118 free of pyrogens, as well as other impurities that can be harmful to the recipient individual. Injection or infusion, especially intravenous, is preferred for administering an immunotherapy that targets BCMA.
[115] In other embodiments, the BCMA-specific binding protein, GSI, or both can be administered to an individual in soluble form (eg, antibody). Soluble TCRs are also known in the art (see, for example, Molloy et al., Curr. Opin. Pharmacol. 5: 438, 2005; U.S. Patent No. 6,759,243).
[116] Pharmaceutical compositions, including
combination in an immunotherapy what targets BCMA it is a inhibitor in γ-secretase of this disclosure, can to be administered in a way that be appropriate to disease Or the
condition to be treated (or avoided), as determined by people skilled in medical technique. An appropriate dose, adequate duration, and frequency of administration of the compositions will be determined by factors such as the patient's condition, size, type and severity of the disease, particular form of the active ingredient, and the method of administration. The present disclosure provides pharmaceutical compositions comprising cells that express a BCMA specific binding protein, such as a CAR or T-ChARM, and a pharmaceutically acceptable carrier, diluents or excipient. Suitable excipients include water, saline, dextrose, glycerol or the like and combinations thereof.
[117] An advantage of the present disclosure is that
Petition 870190103473, of 10/14/2019, p. 101/184
96/118 cells expressing a BCMA-specific binding protein, such as a CAR or T-ChARM, administered to a patient can be depleted using the cognate binding partner to a marker cassette. In certain embodiments, the present disclosure provides a method for depleting a T cell that expresses a BCMA-specific T-ChARM using an antibody specific for the labeled cassette, using a cognate specific binding partner for the labeled cassette , or using a second T cell expressing a CAR and having specificity for the labeled cassette. In certain embodiments, a marker cassette allows the immunodepletion of a T cell that expresses a BCMA-specific T-ChARM of this disclosure. The elimination of projected T cells can be performed using specific depletion agents for a marker cassette. For example, if a Strep marker is used, then an anti-Strep marker antibody, an anti-Strep scFv marker, or Streptactin, each fused or conjugated to a toxic cell reagent (such as a toxin, radiometal), or a bispecific anti-Strep tag / anti-CD3 scFv, or an anti-Strep tag CAR T cell can be used.
[118] In another aspect, the present disclosure provides a method for selectively promoting the proliferation of a recombinant T cell that expresses a
Petition 870190103473, of 10/14/2019, p. 102/184
97/118
BCMA specific T-ChARM of this disclosure. In certain embodiments, the method comprises selective ex vivo proliferation of T cells that express a BCMA-specific T-ChARM using a marker binding partner, such as an antibody. In other embodiments, the method comprises expanding functional T cells (for example, CTL specific for TAA (tumor-associated antigen) and for viruses, or specific subsets of T cells, such as immature T cells (naive), stem T cells of memory, central or effective memory T cells, CD4 + CD25 + regulatory T cells) with a marker binding partner, such as an antibody, which can optionally be performed in the presence of a co-stimulatory molecule binding partner (such as an antibody antiCD27 or antiCD28).
[119] Still in additional modalities, a BCMA-specific T-ChARM allows selective promotion of T cell proliferation in vivo by expressing a BCMA-specific TCHARM in this disclosure. In certain embodiments, a T cell expressing a CAR comprising a labeled cassette allows the expansion of CAR T cells in vivo by contacting cells that express a ligand (for example, including PD-L1 T cell suppressor cells, PD-L2). Such expanded T cells are useful in methods of treating diseases
Petition 870190103473, of 10/14/2019, p. 103/184
98/118 described herein. In certain embodiments, proliferation or expansion of cells expressing BCMA-specific T-ChARM as disclosed herein are induced in vivo, which can be induced with a marker cassette binding partner (such as an anti-marker antibody) and , optionally, a binding partner to co-stimulatory molecules (such as an anti-CD27 or antiCD28 antibody).
[120] In certain modalities additional, cells that express Specific T-ChARM for BCMA, like here disclosed, are activated in vivo, such as in location of a
tumor. For example, a composition (for example, alginate, basement membrane matrix (Matrigel®), biopolymer or other matrix) or a vehicle (for example, microspheres (microbeads), nanoparticles or other solid surface) comprising a cassette binding partner with a marker (such as an anti-marker antibody) and a co-stimulatory molecule binding partner (such as an anti-CD27 or antiCD28 antibody) can be used to activate locally, at the site of a tumor (for example, a solid tumor) , a T cell expressing a BCMA-specific T-ChARM as disclosed herein.
[121] In certain embodiments, recombinant cells that express a BCMA-specific T-ChARM can be detected or screened in vivo using antibodies that bind to
Petition 870190103473, of 10/14/2019, p. 104/184
99/118 specificity to a marker cassette (eg, anti-marker antibodies), or by other cognate binding proteins that specifically bind to the marker cassette sequence (eg, Streptactin that binds to Strep tag), partners of binding to the cassette with a marker, which are conjugated to a fluorescent dye, radiolabel, iron oxide nanoparticle or other imaging agent known in the art for X-ray detection, computed tomography, magnetic resonance, positron emission computed tomography, ultrasound, flow cytometry, near infrared imaging systems, or other imaging modalities (see, for example, Yu et al., Theranostícs 2: 3, 2012).
[122] In other embodiments, cells expressing BCMA-specific TChARM of the present disclosure can be used in diagnostic methods or imaging methods, including methods used in relation to the indications or conditions identified herein.
[123] In some embodiments, a method comprises administering a GSI in combination (for example, concomitantly, separately or sequentially) with a BCMA specific binding protein which may comprise, for example, a BCMA specific antibody or a binding moiety the antigen thereof, such as antibody-drug conjugate, or a bispecific or multispecific binding protein, such as those useful for immunotherapy with pre-targeted radiation (see, for example, Green et al., Blood 131: 611 (2018) ).
Petition 870190103473, of 10/14/2019, p. 105/184
100/118
EXAMPLES
Example 1
Design and Testing of BCMA-Specific Antigen Chimeric Receptors [124] Anti-BCMA CARs have been prepared to examine their usefulness in immunotherapy against multiple myeloma and other disorders. The anti-BCMA CARs were constructed with scFvs composed of the V _H and V _L regions of the C115 D5.3 antibody (Cll) and the A7D12.2 (A7) antibody, which included an IgG4 hinge region (spacer), a domain CD28 transmembrane, a 4-1BB co-stimulatory domain, and a CD3 / effector domain. The scFvs were produced in both the HL and LH orientations (see Figure IA). Human T cells were transduced with expression constructs that encode anti-BCMA CARs and examined for functional characteristics. As shown in Figure IB, T cells expressing Cll CARs and A7 CARs proliferated when co-cultured with target cells that express BCMA, although Cll CARs apparently proliferated somewhat more robustly than T cells. that express A7 CARs. Cll T-CAR cells also produced more cytokines in response to target cell lines (or K562 cells transfected with antigen or MM cell lines that express BCMA; Figure 1C) and
Petition 870190103473, of 10/14/2019, p. 106/184
101/118 had greater specific death activity against target cells compared to LH A7 T-CAR cells (Figure 1D).
[125] Additional A7 and Cll CARs with different intracellular components were generated, including CARs containing a 4-1BB co-stimulatory domain (Figure 1E, top illustration) and a CD28 co-stimulatory domain (Figure 1E, bottom illustration) . The length of the extracellular spacer domain was also varied to improve the interaction between the CAR expressing T cell and the BCMA ⁺ target cell, including short spacers (for example, with 12 amino acids, 48 amino acids and 66 amino acids in length), intermediate (for example, 157 amino acids long), and long (for example, 228 amino acids long). The spacer CARs of 48 amino acids and 157 amino acids included two (2) cassettes with Strep-Tag, while the spacer of 66 amino acids included three (3) cassettes with Strep-Tag (see Figure 1F). It refers here to chimeric antigen-labeled receptors, such as CARs containing cassettes with Strep-Tag, shown in Figures IF and 1G, as T-ChARMs.
[126] Human T cells were transduced to express T-ChARMs (see Example 2) and analyzed for functionality. The length of the spacer affected the T-ChARM constructs comprising an scFv derived from
Petition 870190103473, of 10/14/2019, p. 107/184
102/118 anti-BCMA Cll D5.3 antibody, in which intermediate spacers (with about 65 amino acids; see Cll Cll 3ST, Cll 2STint and CllLo of Figure 1H) in the long run (with about 200 amino acids) worked best in this domain binding. C113ST_4-1BB and C113ST_CD28 were selected as the best performing constructs (Figures 1H-1J) and compared to an anti-BCMA CAR previously disclosed (BCMA-2; Carpenter et al. Clin. Cancer Res. 19: 2048, 2013). The expression of EGFRt in primary T cells was similar for each T-ChARM / CAR construct and the surface expression of T-ChARM was confirmed for those containing STII sequences, as shown by staining with monoclonal anti-STII antibody (Figure IK). T cells that express C113ST T-ChARM produced more cytokines (Figures IL, 1M) and proliferated more robustly (Figure IN) than T cells that express BCMA-2 when co-cultured with target cells. It has also been determined that T cells expressing C113ST T-ChARM can recognize and lyse MM cells from CD138 ⁺ patients (data not shown). T cells that express either C113ST T-ChARM or BCMA-2 CAR did not show cytolytic activity against K562 cells that do not express antigens (Figure 10), but effectively lysed K562 cells transduced to express BCMA antigen (Figure IP), demonstrating that the Projected T cells specifically recognize the antigen.
Petition 870190103473, of 10/14/2019, p. 108/184
103/118
Example 2
Production of Recombinant T Cells and Expression of BCMA-Specific TChARMs [127] CD8 ⁺ and CD4 ⁺ T cells from PBMC were isolated from normal human donors using CD8 ⁺ CD4 ⁺ T cell isolation kit (Miltenyi Biotec), activated with anti-CD3 / CD28 beads (Life Technologies) according to the manufacturer's instructions, and transduced with a lentiviral CAR encoding supernatant (epHIB7) generated by transient transfection of HEK293T cells using PsPAX2 and pMD2G packaging plasmids ( MOI = 3) supplemented with 0.8 pg / mL polybrene (Millipore, Bedford, MA) on day 3 after activation by centrifugation at 2,100 rpm for 45 min at 32 ° C. T cells were expanded 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 each 4 8 hours. After expansion, an aliquot of each transduced T cell line was stained with anti-EGFR antibody conjugated with biotin and streptavidin-PE (Miltenyi, Auburn, CA). TEGFR ⁺ T cells were isolated by sorting on a FACS-Aria cell sorter (Becton Dickinson). The subset of tEGFR ⁺ T cells was then stimulated with irradiated B-LCL CD19 ⁺ (8,000 rad) in a ratio of T: LCL cells of 1: 7 and expanded for 8 days in CTL medium with the addition of 50 U / ml of rh IL-2 every 48 hours or using a rapid expansion protocol for cells expressing T-ChARMs (Riddell and Greenberg, J. Immunol. Methods 128: 189, 1990), where T-ChARM
Petition 870190103473, of 10/14/2019, p. 109/184
104/118 comprises a scFv derived from anti-BCMA antibody of A7 or Cll D5.3.
[128] The following conjugated antibodies were used for phenotyping and flow cytometric analysis: CD4, CD8, CD25, CD137, CD45, Appendix V, CD62L, CD27, CD28 (BD Biosciences), anti-Streptag II antibody (Genscript), EGFR antibody (ImClone Systems Incorporated, Branchburg, NJ); strepTavidin-PE (BD Biosciences, San Jose, CA). Staining with propidium iodide (PI, BD Biosciences) was performed for discrimination of live / dead cells, as indicated by the manufacturer. Flow analyzes were performed in a FACS Canto II, purifications by classification in an FACS Ariall (Becton Dickinson, Franklin Lakes, NJ) and the data were analyzed using the FlowJo software (Treestar, Ashland, OR).
[129] To examine the expression of BCMA-specific T-Charm on the cell surface, transduced T cells were classified for EGFRt expression and evaluated by staining with fluorochrome-labeled anti-Streptag mAb. The mean fluorescence intensity (IMF) of EGFR staining was similar in T cells transduced with each BCMA-specific T-ChARM and the CD19-Short CAR. The introduction of a marker in a CAR to produce a T-ChARM did not interfere with the expression of the transgene (data not shown). An antiStrepTag mAb specifically stained T cells transduced with the various BCMA-specific T-ChARMs, regardless of the position or number of marker sequences in each T-ChARM.
Example 3
Petition 870190103473, of 10/14/2019, p. 110/184
105/118
Soluble BCMA (sBCMA) Inhibits BCMA-Specific T-Cell Activity [130] The potential effectiveness of T-cell therapies targeting BCMA against most myelomas has been examined. As the loss of surface BCMA by myeloma cells can make it difficult to recognize T cells, levels of soluble BCMA were measured in supernatants during the culture of a myeloma cell line. U266 myeloma cells were washed and plated in culture media for 1, 3, 5 and 24 hours. The supernatant from the media was collected and tested for sBCMA by ELISA. The data show a time-dependent increase in sBCMA levels in the supernatant (Figure 2A). Next, the degree of BCMA expression was examined in primary myeloma (MM) cells of the primary patient and in the production of cytokines by BCMA-specific T-ChARM T cells placed in contact with the patient's MM cells. Figure 2B shows the expression of surface BCMA by a reference cell line (RPMI, left panel) and MM cell samples from primary patients with different levels of BCMA expression. The pie-pie pie chart in Figure 2C shows that BCMA expression on the surface of MM cells of nineteen (19) different patients was considered positive for most patients, although
Petition 870190103473, of 10/14/2019, p. 111/184
106/118 there were some patient samples that showed surface BCMA expression between intermediate and low / no surface BCMA expression (~ 25%). In addition, Figure 2D shows that T-ChARM T cells produced more IFN-γ when cultured with MM cells from patients expressing high levels of surface BCMA, compared to culture with MM cells from patients with low BCMA .
[131] Myeloma cells often express PDL1, which is believed to inhibit the function of T cells by binding to PD-1 in T cells (Freeman et al. J. Exp. Med. 192 (1): 1027 (2000)) . The possibility of PD-L1 affecting BCMA-specific T-ChARM T cells was also investigated. Flow cytometry of patient samples showed that PD-L1 was also expressed in 79% of samples with 47% PD-Ll ^high, 32% PD-Ll ^int and 21% PD-Ll ^{low / n} 9 (Figures 2E and 2F). No correlation was observed between BCMA expression and PD-L1. Figure 2G shows that TChARM T cells produced more IFN-γ when cultured with MM cells from patients expressing low levels of PD-L1 versus high levels of PD-L1, although this trend was not statistically significant.
[132] The effect of sBCMA was also examined when adding sBCMA to T cell co-cultures that express a BCMA-specific T-ChARM and transduced K562 cells
Petition 870190103473, of 10/14/2019, p. 112/184
107/118 with a polynucleotide encoding full-length BCMA (K562 / BCMA ⁺ ). A dose-dependent inhibition of the effector function of BCMA-specific T-ChARM T cells was detected as a measure of IFN-γ release in the media supernatant when administering exogenous sBCMA (Figures 2H, 21). In addition, MM cell lines showed substantial increases in sBCMA of the culture supernatant within 24 hours (Figure 2J).
[133] To examine whether high levels of sBCMA can inhibit BCMA-specific T-ChARM T cell activity, bone marrow (MO) sera from patients were examined for sBCMA levels. It was found that the patient's OM had high levels of sBCMA, which correlated approximately with the disease burden, as determined by the percentage of CD138 ⁺ cells present (Figure 2K). To test whether sBCMA would bind to T-ChARM T cells, T cells were incubated with increasing levels of recombinant BCMA and then stained using BCMA-Fc conjugated to APC. As shown in Figure 2L, staining decreased with higher levels of recombinant BCMA, indicating that sBCMA could potentially have a detrimental effect on the function of BCMA-specific T-ChARM cells.
[134] To confirm T-ChARM expression, C113ST T-ChARM T cells and control anti-CD19 CAR T cells
Petition 870190103473, of 10/14/2019, p. 113/184
108/118 (FMC63) were incubated with Fc-BCMA and stained to show EGFRt (CAR / T-ChARM transduction marker) and CD4, showing that T-ChARMs were expressed by cells (Figure 2M). The staining of EGFRt and other T cell surface molecules was unaffected (data not shown).
[135] To determine whether the loss and binding of sBCMA to T-ChARMs inhibits T cell function, BCMA-Fc was added to co-cultures of T cells / target cell lines, resulting in very reduced recognition ( ΙΕΝ-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 that express the CD19 antigen (Figure 2N). in T cells BCMA TChARM was dose-dependent (Figures 20, 2P). The addition of non-fused recombinant BCMA to the Fc also inhibited the production of ΙΕΝ-γ in a dose-dependent manner, but did not inhibit cell lysis by K562 BCMA cells ⁺ by T-ChARM T cells (Figure 2Q), which may be due to the high density of antigens on the surface of the target cells.
Example 4
Effect of γ-Secretase Inhibitor on BCMA levels of MM [136] To examine the effect of γsecretase inhibition on BCMA levels in myeloma cells, used
Petition 870190103473, of 10/14/2019, p. 114/184
109/118 whether the γ-secretase (GSI) inhibitor RO4929097. BCMA is rapidly up-regulated in several myeloma cell lines when incubated with γ-secretase inhibitor (GSI) RO4929097 (the concentration used ranged from 0.001 μΜ to 1.0 μΜ) (Figures 3A-3D).
[137] To examine the effect of GSI on surface BCMA expression kinetics, U266 myeloma cells were incubated for 1, 3, 5 and 24 hours in the presence of various concentrations of GSI RO429097 (0.01 μ, 0 , 1 μΜ and 1.0 μΜ) and evaluated for BCMA expression on the cell surface by flow cytometry. BCMA expression increased in a dose-dependent manner in the presence of a GSI (Figure 3E). Positive regulation persisted for 7 days of culture in GSI 1.0 μΜ (Figure 3F).
[138] For test yourself the effect in GSI on The loss in BCMA in MM, three cell lines many different in myeloma (MM1.R, U266 and 8226) were washed and plated in middle in culture for 24 hours in presence in various concentrations in
GSI RO429097 (0.01 μΜ, 0.1 μΜ and 1.0 μΜ). The supernatant from the media was collected and tested for sBCMA by ELISA. Levels of sBCMA decreased in the supernatant in the presence of a GSI in a dose-dependent manner (Figures 3G and 3H).
[139] To examine the effect on soluble BCMA (sBCMA) levels over time, U266 cells were washed and plated in culture medium for 1, 3, 5 and 24 hours in the presence of various concentrations of GSI RO429097 (0.01 μΜ, 0.1 μΜ and 1.0 μΜ) The media supernatant was collected and tested
Petition 870190103473, of 10/14/2019, p. 115/184
110/118 for soluble BCMA (sBCMA) by ELISA. The data shows a time-dependent increase in sBCMA levels in the supernatant when a GSI is present (Figure 31).
[140] Next, the effect of removing GSI from the MM cell culture was examined. As shown in Figures 3J and 3K, BCMA surface levels decreased after removal of RO429097 1.0 μΜ, while sBCMA in the supernatant increased
When if removed the GSI, but not increased When the GSI remained. These data indicate a ef e inhibited ito reversible river from GSI about the loss in BCMA in surface. Per end, the
Viability of the tested MM cell lines was not affected by the addition of RO429097 1.0 μΜ to the culture (Figure 3L).
[141] The effect of GSI on BCMA expression was then tested on samples of primary myeloma from patients. CD138 + myeloma cells were enriched from bone marrow samples from patients, incubated for 3 hours in the presence of various concentrations of GSI RO429097 (0.01 μΜ to 10 μΜ) and evaluated for surface BCMA expression by flow cytometry . The mean fluorescence intensity (IFM) of BCMA in tumor cells is presented as a change (in the number of times) in relation to tumor cells incubated without RO4929097. Dose-dependent positive regulation of BCMA was observed in tumor cells (Figure 3N), while there was no effect on the levels of several other cell surface molecules, including CS1, CD86, PD-L1, CD80 and CD38 (Figures 303Q) .
Example 5
Petition 870190103473, of 10/14/2019, p. 116/184
111/118
Γ-Secretase Inhibitor Improves Recognition of Bcma + Myeloma Cells by BCMA-specific TChARM Expressions [142] To examine the effect of a GSI on CAR T cell activity against BCMA ⁺ multiple myeloma, IL production -2 by BCMA T-CAR cells (BCMA-specific T-ChARM Cll 3ST-CD28 and BCMA-specific T-ChARM Cll 3ST-41BB) or CD19sh T-CAR cells (short spacer) control co-cultured with primary human myeloma tumor cells for 24 hours in media containing various varying concentrations of GSI RO429097 (0.003 μΜ to 3.0 μΜ) (Figure 4A). Treatment with GSI resulted in a dose-dependent increase in IL-2 production by T-ChARM BCMA T cells. In addition, IFNy production by T-ChARM BMCA T cells co-cultured with myeloma cells in various concentrations of RO429097 was measured and was also increased in the presence of GSI (Figure 4B). These data show that multiple myeloma cells stimulate BCMA-specific TChARM T cells better when pretreated with γ-secretase inhibitor that up-regulates BCMA expression in the tumor cell. Finally, the proliferation of CFSE-labeled BCMA-specific TChARM T cells was increased in a dose-dependent manner after co-culture for 3 days with primary human myeloma tumor cells in the presence of GSI RO492097 (Figure 4C).
[143] Without wishing to be limited by theory, a potential caveat regarding the use of GSI to increase BCMA in myeloma cells to improve recognition of
Petition 870190103473, of 10/14/2019, p. 117/184
112/118 T-CAR cells is that high concentrations of GSI can inhibit T cell signaling and effector functions. See Eagar et al., Immunity 20 (4): 407, 2004. To assess the potential effects of GSI on T CAR cells in an environment where the expression of the target ligand remains stable, CD19 T-CAR cells were co-identified. cultured with Raji or K562 / CD19 ⁺ target cells in the presence of GSI (0.01 μΜ to 100 μΜ) and measured viability and effector function. In this concentration range, there was no effect on the expression of CD19 antigen (Figure 5A) or on the viability of T-CAR cells after 24 hours (Figure 5B). It was found that GSI RO4929097 inhibits the effector function of CD19 T-CAR cells in concentrations> 1 μΜ when co-cultured with K562 CD19 ⁺ cells, as determined when measuring the production of IL-2 (Figure 5C, upper panel) and IFNy (Figure 5C, lower panel) (see also Figures 5D-5F). However, the ability of CD19 T-CAR cells to specifically kill target cells was not affected by the concentration of GSI (Figure 5G). CD19 T-CAR cells also proliferated in response to stimulation by target cells in the presence of 10 μΜ GSI RO4929097, although less effectively than in the absence of GSI RO4929097 (Figure 5H). To further investigate the effect of GSI dosage on T-CAR cell division, a GSI dose titration RO4929097 was added to co-cultures of CD19 T-CAR cells and target cells. The division of T-CAR cells was not essentially affected or only slightly affected by the addition of therapeutic doses of GSI (0.01 μΜ-l μΜ), while GSI at a higher, non-therapeutic dose (10 μΜ), inhibited detectably
Petition 870190103473, of 10/14/2019, p. 118/184
113/118 proliferation (Figure 51). CD19 T-CAR cells cultured in the presence of GSI did not show a decrease in expansion over 8 days in culture with (Figure 5J) or without (Figure 5K) exogenous IL-2. In addition, T cells that were expanded in the presence of 5μΜ, 0.5μΜ GSI or in the absence of GSI did not show a significant difference in IFN-γ production when subsequently restimulated with target cells after expansion in the presence (Figure 5L) or in the absence (Figure 5M) of exogenous IL-2. IL-2 production between groups of T-CAR cells was also not markedly different (Figure 5N). Overall, GSI at therapeutic levels does not affect T cell function, including cytokine production, cell lysis activity and proliferation.
[144] Next, the effect of GSI on the functionality of T cells from T cells containing a BCMA-specific T-ChARM was examined. GSI treatment of primary myeloma tumor samples greatly improved IFN-γ production (Figures 50, 5P and 5Q) and CD8 expression (Figure 50) by co-cultured T-ChARM cells, with effects seen after treatment with GSI 0.1 μΜ or more, and also increased the proliferation of T cells (Figure 5R).
[145] These data indicate the clinical utility of combining BCMA-specific T-CAR cells and a GSI in order to increase BCMA levels in multiple myeloma cells. This can provide an advantage or even have a synergistic effect on the treatment of multiple myeloma by facilitating the elimination of tumor cells that express low levels of BCMA. The data also shows that a range of concentrations
Petition 870190103473, of 10/14/2019, p. 119/184
114/118 γ-secretase inhibitor promoted BCMA expression of tumor cells without affecting T cell activity.
Example 6
In vivo study of GSI Activity in an MM Xenograft Model [146] To investigate whether the sBCMA inhibitory effects of GSI RO4929097 described in Example 4 could be replicated in vivo, a human MM murine xenograft model was tested as illustrated in Figure 6A. Briefly, immunodeficient NOD / SCID gamma (NSG) mice (The Jackson Laboratory) received sublethal irradiation (275 rad) on day -1, followed by 5x10 ⁶ MM.1R cells on the next day (day 0). GSI RO4929097 (30 mg / kg) was administered by oral gavage on day 19 and day 20. The mice were sacrificed at different times after the second GSI gavage administration and blood and bone marrow samples were collected for analysis. The measurement of GSI was followed by a rapid increase (> 3 times for 4 hours after the second gavage) of surface BCMA expression in tumor cells (Figure 6B) and a concomitant decrease in sBCMA (Figure 6C). The observed effects, in general, were reduced within 48 hours after the second gavage treatment.
Example 7
In vivo study of T Cell Combination Therapy
Petition 870190103473, of 10/14/2019, p. 120/184
115/118
GSI + CAR targeting MM [147] The ability of GSI therapy to improve anti-BCMA T-CAR cell therapy in vivo was investigated. In an experiment similar to that described in Example 6, NSG mice were sublethally irradiated on day -21, followed by administration of human MM cells (5x10 ⁶ MM.lR ^ffluc ) on day -20 (see Figure 7A). A first dose of GSI (RO4929097 30 mg / kg) was administered by oral gavage on day -1. On day 0, the mice were injected with a suboptimal dose of T-ChARM Cll 3ST T cells (0.33 x ^{10 6} cells; CD4: CD8 1: 1) and received a second dose of GSI. Additional doses of GSI were administered on day +1, day +8 and day +9. Bioluminescence imaging (BLI, from English bioluminescence imaging) was performed throughout the experiment and the rats were monitored for survival. As shown in Figures 7B, 7C and 7E (left side graph), the group that received T + GSI cells had reduced luminescence and more mice without detectable tumors compared to the group that received only T cells (BLI data from the group only with T cells not shown). Quantified luminescence data (Figures 7C and 7E (left-hand graph)) indicated that the initial effect of T-ChARM T cells began to reverse at approximately day 9, when tumors began to disappear (Simulated + BCMA T cells). The effect
Petition 870190103473, of 10/14/2019, p. 121/184
116/118 antitumor was prolonged by combining with GSI RO4929097 (RO49 + BCMA T cells), but the tumors in that treatment group also ended up disappearing. The imaging data were consistent with the survival of the mice (Figures 7D and 7E (graph on the right side)), with the mice that received combination therapy surviving longer than the group that received T cells just before succumbing. In certain embodiments, treatments with GSI are repeated (from at least 2 to at least about 5 times to at least about 25 times) when used in combination with T cells antiBCMA CAR, treatments with GSI which can be administered concurrently with , before or after administration of anti-BCMA CAR T cells.
Example 8
GSI Improves Binding to MM BCMA + Cells by a Bispecific Anti-BCMA Fusion Protein [148] A bispecific fusion protein having specificity for BCMA, for another antigen and with a YFP marker (yellow fluorescent protein) fluorescent protein). Bispecific fusion protein was added to BC H929 cells expressing BCMA in culture (0.5x10 ⁶ cells) with or without GSI. The binding was assessed by flow cytometry. As shown in Figure 8, the addition of GSI improved the
Petition 870190103473, of 10/14/2019, p. 122/184
117/118 binding by bispecific fusion protein that binds to BCMA, but had no effect on binding by a bispecific control fusion protein that does not target BCMA.
These data show that at immunotherapies involving bispecific molecules that has target BCMA can be increased or improved with GSI.[149] The various modalities described above can be combined to provide other modalities. All
US patents, US patent application publications, US patent applications, foreign patents, foreign patent applications and non-patent publications mentioned in this specification and / or listed in the Order Data Form are hereby incorporated by reference , in its entirety. Aspects of the modalities can be modified, if necessary, to employ concepts from the various patents, orders and publications to provide still additional modalities.
[150] These and other changes can be made to the modalities in the light of the detailed description above. In general, in the following claims, the terms used should not be interpreted in such a way as to limit the claims to the specific modalities disclosed in the specification and in the claims, but should be interpreted to include all possible modalities, together with the
Petition 870190103473, of 10/14/2019, p. 123/184
118/118 full scope of equivalents to which claims are entitled. Consequently, claims are not limited by disclosure.

权利要求:
Claims (13)
[1]
1. Method for treating (i) a proliferative disease or disorder, such as cancer, and / or (ii) an autoimmune disease or disorder, in an individual who has or is suspected to have the disease or disorder associated with BCMA expression, characterized by the fact that it comprises administering to the individual a therapeutically effective amount of a BCMA-specific binding protein and a therapeutically effective amount of a γ-secretase inhibitor.
[2]
2. Method according to claim 1, characterized by the fact that the BCMA specific binding protein is a BCMA specific antibody or antigen binding portion thereof, a chimeric antigen receptor (CAR) or a labeled molecule chimeric antigen receptor (T-ChARM).
[3]
3. Method according to claim 2, characterized by the fact that the BCMA specific binding protein is human or humanized.
[4]
Method according to any one of claims 1 to 3, characterized in that the BCMA-specific binding protein comprises a BCMA-specific scFv, a BCMA-specific scTCR, or a BCMA ligand or binding portion of the same.
Petition 870190079710, of 16/08/2019, p. 44/59
2/13
[5]
5. Method according to claim 4, characterized in that the BCMA-specific binding protein that binds is an scFv comprising variable regions of the heavy chain and the light chain based on the BCMA J22.0-XI antibody, J22.9-XÍ, J6M0, J6M1,
J6M2, J9M0, J9M1, J9M2, 11D5-3, CA8, A7D12.2, Cll D5.3, C12A3.2, C13F12.1, 13C2, 17A5, 83A10, 13A4, 13D2, 14B11, 14E1, 29B11, 29F3, 13A7 , CA7, S307118G03, SGI, S332121F02,
S332126E04, S322110D07, S336105A07, S335115G01, S335122F05, ET140-3, ET140-24, ET140-37, ET140-40, ET140-54, TBL-CLN1, C4.E2.1, Vicky-1, pSCHLI37, pSCHLI37,
[6]
Method according to any one of claims 1 to 5, characterized in that the BCMA-specific binding protein is a chimeric antigen receptor comprising a hydrophobic portion disposed between an extracellular component and a component
intracellular, in which the component extracellular comprises The binding protein specific BCMA, comprising The binding protein specific to BCMA (a) one serving in antigen binding specific BCMA from of
BCMA antibody J22.0-XÍ, J22.9-XÍ, J6M0, 11D5-3, CA8, A7D12.2, Cll D5.3, 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, ET140-24, ET140-37, . 45/59
3/13
40, ET140-54, TBL-CLN1, C4.E2.1, Vicky-1, pSCHLI333, pSCHLI372, or pSCHLI373; or (b) a BCMA binder or binding portion thereof based on or derived from BAFE or APRIL.
[7]
7. Method according to claim 6, characterized by the fact that the hydrophobic portion is a transmembrane domain.
[8]
8. Method according to claim 7, characterized by the fact that the transmembrane domain is a transmembrane domain of CD4, CD8, CD28 or CD27.
[9]
Method according to any one of claims 6 to 8, characterized in that the intracellular component comprises an effector domain or functional portion thereof, a co-stimulatory domain or functional portion thereof, or any combination thereof.
[10]
10. Method according to claim 9, characterized by the fact that the intracellular component comprises 4-1BB (CD137), CD3s, CD3, CD3 /, CD25, CD27, CD28, CD79A, CD79B, CARD11, DAP10, FcRa, ΕοΡβ, FcRy, Fyn, HVEM, ICOS, Lek, LAG3, LAT, LRP, NKG2D, NOTCH1, NOTCH2, NOTCH3, NOTCH4, 0X40 (CD134), ROR2, Ryk, SLAMF1, Slp76, pTa, TCRa, TCRp, TRIM, Z , PTCH2, or a functional part thereof, or in any combination thereof.
[11]
11. Method, according to any of the
Petition 870190079710, of 16/08/2019, p. 46/59
4/13 claims 9 or 10, characterized by the fact that the effector domain comprises CD3 / or the functional portion thereof.
[12]
12. Method according to any of claims 6 to 11, characterized in that the intracellular component comprises a co-stimulatory domain or a functional portion thereof selected from CD27, CD28, 4-1BB (CD137), 0X40 (CD134) , or
any combination of the same. 13. Method, according to any of the claims 6 to 12, characterized by the fact that the
effector domain or effector portion thereof comprises CD3 / or functional portion thereof, and one or more co-stimulatory domain or functional portion thereof 4-1BB (CD137), CD27, CD28, and 0X40 (CD134).
14. Method according to any of claims 6 to 13, characterized in that the intracellular component comprises (a) 4-1BB or a functional portion thereof and CD3 /, (b) CD27 or a functional portion thereof and CD3 /, (c) CD28 or a functional portion thereof and CD3 /, (d) 0X40 or a functional portion thereof and CD3 /, (e) CD28 or a functional portion thereof, 4-1BB or or a portion functional of the same and CD3 /, (f) 0X40 or a functional portion of the same, 4-1BB or or a functional portion of the same and CD3 /, or (g) CD28 or a functional portion of the same,
Petition 870190079710, of 16/08/2019, p. 47/59
5/13
0X40 or a functional portion thereof and CD3 /.
Method according to any one of claims 6 to 14, characterized in that the extracellular component comprises an immunoglobulin hinge region, a CH2 domain and a CH3 domain, a CH3 domain, or any combination thereof arranged between the BCMA specific binding protein and the hydrophobic portion.
16. Method according to claim 15, characterized by the fact that the hinge region is an IgGl hinge region.
17. Method according to claim 15 or claim 16, characterized in that the CH2 domain is an IgG1 CH2 domain and the CH3 domain is an IgG1 CH3 domain.
18. Method, of wake up with any of the claims 1 to 17, characterized by the fact that that specific binding protein BCMA is encoded for one exogenous polynucleotide and is expressed in a cell hostess.19. Method, of wake up with claim 18, characterized by the fact that the host cell is
cell of the human immune system.
20. Method according to claim 19, characterized by the fact that the immune system cell
Petition 870190079710, of 16/08/2019, p. 48/59
The human 6/13 is a CD4 + T cell, a CD8 + T cell, a CD4- CD8- double negative T cell, a γδ T cell, a natural killer cell, a dendritic cell, or any combination thereof.
21. Expression vector according to claim 20, characterized by the fact that the cell of the human immune system is a T cell, the T cell is a naive T cell (inactive or immature), a central memory T cell, an effector memory T cell, mass T cells, or any combination thereof.
22. Method according to any one of claims 1 to 21, characterized in that the γ-secretase inhibitor is avagacestat, DAPT, BMS-906024, BMS-986115, MK-0752, PF-03084014, RO4929097, or YO-01027.
23. The method of claim 22, characterized by the fact that the γ-secretase inhibitor is a nicastrin-specific binding protein.
24. Method according to claim 23, characterized in that the nicastrin-specific binding protein is scFvG9, antibody A5226A, antibody 2H6, or antibody 10C11.
25. Method according to any one of claims 1 to 24, characterized in that the BCMA-specific binding protein comprises a variable domain comprising CDR1, CDR2 and CDR3 of the sequence of
Petition 870190079710, of 16/08/2019, p. 49/59
7/13 amino acids from any of SEQ ID NOS .: 14-16 and a variable domain comprising CDR1, CDR2 and CDR3 from the amino acid sequence of any of SEQ ID NOS .: 17-21.
26. Method according to any one of claims 1 to 25, characterized in that the method additionally comprises preconditioning the individual with an immunosuppressive regimen prior to or concomitant with the BCMA specific binding protein and the γsecretase inhibitor.
27. Method according to claim 26, characterized by the fact that the immunosuppressive regimen is a non-myeloablative treatment or a myeloablative treatment.
28. Method according to claim 27, characterized in that the non-myeloablative treatment comprises cyclophosphamide or cyclophosphamide in combination with fludarabine.
29. Method according to any one of claims 1 to 28, characterized in that the BCMA specific binding protein and γsecretase inhibitor are administered sequentially.
30. Method according to any one of claims 1 to 28, characterized in that the BCMA-specific binding protein and γ inhibitor
Petition 870190079710, of 16/08/2019, p. 50/59
8/13 secretase are administered concomitantly.
31. Method according to claim 30, characterized in that the binding protein specifies BCMA and the γ-secretase inhibitor are formulated together.
32. Method according to any one of claims 1 to 31, characterized in that the BCMA-specific binding protein is administered parenterally and the γ-secretase inhibitor is administered orally.
33. Method according to any one of claims 1 to 32, characterized by the fact that the proliferative disease or disorder is a hematological cancer or a solid cancer.
34. Method according to claim 33, characterized by the fact that hematological cancer is selected from the group consisting of multiple myeloma, plasmacytoma, plasma cell leukemia, Waldenstrom's macroglobulinemia, B-cell lymphoma, and lymphoplasmacytic lymphoma.
35. Method according to claim 33, characterized by the fact that solid cancer is breast adenocarcinoma or bronchogenic lung carcinoma.
36. Method according to any one of claims 1 to 32, characterized by the fact that the
Petition 870190079710, of 16/08/2019, p. 51/59
9/13 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 epidermal necrolysis, toxicological epidermal emulsion 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, systemic lupus erythematosus (SLE), subacute cutaneous lupus erythematosus, discoid lupus, lupus myelitis, lupus cerebritis, juvenile diabetes, multiple sclerosis, encephalitis optic neuromyelitis, rheumatic fever, Sydenham's chorea, immune responses associated with acute and late hypersensitivity mediated by cytokines and T lymphocytes, tuberculosis, sarcoidosis, granulomatosis, including Wegener's granulomatosis and Churg-Strauss disease, agranulocytosis, vasculitis (including hypersensitivity / angiitis vasculitis, ANCA and rheumatoid vasculitis), anemia aplastic, Diamond-Blackfan anemia, immune hemolytic anemia including hemolytic anemia
Petition 870190079710, of 16/08/2019, p. 52/59
10/13 autoimmune (AIHA), pernicious anemia, pure red cell aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, inflammatory central nervous system (CNS) disorders, syndrome multiple organ injury, myasthenia gravis, antigen-antibody complex mediated diseases, glomerular basement membrane disease, antiphospholipid antibody syndrome, allergic neuritis, Behçet's disease, Castleman's syndrome, Goodpasture syndrome, Lambert-Eaton myasthenic syndrome, Reynaud's syndrome, Sjorgen's syndrome, Stevens-Johnson syndrome, rejection of solid organ transplantation, graft versus host disease (GVHD), bullous pemphigoid, pemphigus, autoimmune polyendocrinopathies, seronegative spondyloarthropathies, Reiter's disease, rigid man syndrome giant cell arteritis, immune complex nephritis, IgA nephropathy, IgM polyneuropathies or p-mediated neuropathy or IgM, idiopathic thrombocytopenic purpura (ITP), thrombotic thrombocytopenic purpura (TTP), Henoch-Schonlein purpura, autoimmune thrombocytopenia, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism; autoimmune endocrine diseases, including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's thyroiditis), thyroiditis
Petition 870190079710, of 16/08/2019, p. 53/59
11/13 subacute, idiopathic hypothyroidism, Addison's disease, Graves' disease, autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), type I diabetes, which is also referred to as insulin-dependent diabetes mellitus) and Sheehan's syndrome; autoimmune hepatitis, lymphoid interstitial pneumonitis (HIV), obliterating bronchiolitis (non-transplant), unspecified interstitial pneumonia (PINS), Guillain-Barré syndrome, large vessel vasculitis (including rheumatic polymyalgia and giant cell arteritis (Takayasu)), vasculitis medium median vessels (including Kawasaki disease and polyarteritis nodosa), polyarteritis nodosa (PAN), ankylosing spondylitis, Berger's disease (IgA nephropathy), rapidly progressive glomerulonephritis, primary biliary cirrhosis, celiac disease (gluten enteropathy), cryoglobulinemia, cryoglobulinemia associated with hepatitis, amyotrophic lateral sclerosis (ALS), coronary artery disease, familial Mediterranean fever, microscopic polyangiitis, Cogan's syndrome, WhiskottAldrich's syndrome and thromboangiitis obliterans.
37. Kit to treat a hematological and / or autoimmune disease or disorder, characterized by the fact that it comprises (a) a unit dose of a BCMA specific binding protein, and (b) a unit dose of a y- inhibitor secretase.
Petition 870190079710, of 16/08/2019, p. 54/59
12/13
38. Kit according to claim 37, characterized in that it additionally comprises a CD20-specific binding protein, such as rituximab, ofatumumab, ocrelizumab; a CD19-specific binding protein; a CD45-specific binding protein; a CD38-specific binding protein; a cytokine; a chemokine; a growth factor; a chemotherapeutic agent; or a radiotherapy agent.
39. Method according to any one of claims 6 to 36, characterized in that the chimeric antigen receptor comprises a T-ChARM and in which the extracellular domain of the T-ChARM comprises a Strep Tag.
40. Method according to claim 39, characterized in that the extracellular domain of TCHARM comprises two, three, four, five, six, seven, eight, or nine marker cassettes.
41. Method according to any one of claims 1 to 40, characterized in that the γ-secretase inhibitor is administered to the subject at least once subsequent to a first administration of the BCMA-specific binding protein.
42. Method according to claim 41, characterized in that the γ-secretase inhibitor is administered at least 2, at least 3, at least 4, at least
Petition 870190079710, of 16/08/2019, p. 55/59
[13]
13/13
any less 5, fur any less 6, fur any less 7, fur any less 8, at any less 9, fur any less 10, fur any less 15, fur any less 20, at any less 25, fur any less 30, fur any less 35, fur any less 40, at any less 45, or at least 50 times subsequent to first
administration of the BCMA-specific binding protein.
43. Method, of a deal with any an of claims 1 to 42, featured by the fact that the inhibitor γ-secretase : is administered at a concentration of fence 30 mg / kg. 44. Method, of a deal with any an of claims 1 to 5, featured by the fact that the protein binding specific to BCMA comprises one antibody or an antigen-binding portion what it is coupled to a cytotoxic agent. 45. Method, of a deal with any an of claims 1 to 5 or with claim 44,
characterized by the fact that the BCMA-specific binding protein is multispecific.
46. Method according to claim 45, characterized by the fact that the BCMA specific binding protein is bispecific.

类似技术:

---

公开号 | 公开日 | 专利标题

---

BR112019017120A2|2020-04-14|combination therapies to treat cancers and autoimmune disorders related to bcma

---

Davila et al.2016|Biology and clinical application of CAR T cells for B cell malignancies

---

KR20180130534A|2018-12-07|Chimeric antigen receptor targeting cancer

---

CN107109419A|2017-08-29|Use CD33 Chimeric antigen receptor treating cancers

---

JP2021525509A|2021-09-27|Diverse antigen-binding domains for cell therapy, new platforms and other enhancements

---

JP2022002550A|2022-01-11|Compositions and Methods for CD20 Immunotherapy

---

US20190330302A1|2019-10-31|Chimeric antigen receptors based on alternative signal i domains

---

US20200113940A1|2020-04-16|Chimeric antigen receptor t cells targeting the tumor microenvironment

---

AU2018250336A1|2019-09-26|Engineered cells expressing prostate-specific membrane antigen | or a modified form thereof and related methods

---

BR112020001605A2|2020-08-11|methods for producing genetically modified cell compositions and related compositions

---

KR20200041377A|2020-04-21|STREP-TAG specific binding protein and its use

---

US20210268027A1|2021-09-02|Chimeric antigen receptors targeting cd37 and cd19

---

US20200079870A1|2020-03-12|Chimeric antigen receptors targeting cd37

---

US20190330343A1|2019-10-31|Methods and compositions relating to ex vivo culture and modulation of t cells

---

Hudecek et al.2013|The non-signaling extracellular spacer domain of chimeric antigen receptors is

同族专利:

---

公开号 | 公开日

---

CN110461335A|2019-11-15|

---

PH12019550153A1|2020-03-16|

---

JP2020507605A|2020-03-12|

---

WO2018151836A8|2018-11-15|

---

RU2019128662A|2021-03-17|

---

RU2019128662A3|2021-06-18|

---

KR20190116420A|2019-10-14|

---

EP3582782A1|2019-12-25|

---

SG11201907580SA|2019-09-27|

---

WO2018151836A1|2018-08-23|

---

AU2018222749A1|2019-08-15|

---

US20190359727A1|2019-11-28|

---

MX2019009552A|2019-10-02|

---

CA3052779A1|2018-08-23|

---

IL268349D0|2019-09-26|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US6291158B1|1989-05-16|2001-09-18|Scripps Research Institute|Method for tapping the immunological repertoire|

---

US6291161B1|1989-05-16|2001-09-18|Scripps Research Institute|Method for tapping the immunological repertiore|

---

US5283173A|1990-01-24|1994-02-01|The Research Foundation Of State University Of New York|System to detect protein-protein interactions|

---

JP4137997B2|1994-01-11|2008-08-20|ダイアックスコープ．|Inhibitors of human plasmin derived from the Kunitz domain|

---

US6759243B2|1998-01-20|2004-07-06|Board Of Trustees Of The University Of Illinois|High affinity TCR proteins and methods|

---

AUPP221098A0|1998-03-06|1998-04-02|Diatech Pty Ltd|V-like domain binding molecules|

---

JP2004533997A|2001-02-20|2004-11-11|ザイモジェネティクス，インコーポレイティド|Antibodies that bind both BCMA and TACI|

---

DE10113776B4|2001-03-21|2012-08-09|"Iba Gmbh"|Isolated streptavidin-binding, competitively elutable peptide, this comprehensive fusion peptide, nucleic acid coding therefor, expression vector, methods for producing a recombinant fusion protein and methods for detecting and / or obtaining the fusion protein|

---

US7575925B2|2002-12-10|2009-08-18|Sunnybrook Health Sciences Centre|Cell preparations comprising cells of the T cell lineage and methods of making and using them|

---

DE602005022595D1|2004-06-29|2010-09-09|Immunocore Ltd|A MODIFIED T-CELL RECEPTOR EXPRESSING CELLS|

---

SI1752471T1|2005-01-05|2009-04-30|F Star Biotech Forsch & Entw|Synthetic immunoglobulin domains with binding properties engineered in regions of the molecule different from the complementarity determining regions|

---

GB0504767D0|2005-03-08|2005-04-13|Ares Trading Sa|Lipocalin protein|

---

JP6088723B2|2005-11-23|2017-03-01|ジェネンテック， インコーポレイテッド|Compositions and methods for B cell assays.|

---

EP1829895A1|2006-03-03|2007-09-05|f-star Biotechnologische Forschungs- und Entwicklungsges.m.b.H.|Bispecific molecule binding TLR9 and CD32 and comprising a T cell epitope for treatment of allergies|

---

US8119772B2|2006-09-29|2012-02-21|California Institute Of Technology|MART-1 T cell receptors|

---

CA2700391A1|2007-09-24|2009-04-02|University Of Zuerich|Designed armadillo repeat proteins|

---

SI2406284T1|2009-03-10|2017-01-31|Biogen Ma Inc.|Anti-bcma antibodies|

---

WO2011108008A2|2010-03-04|2011-09-09|Transgene Biotek Ltd.|Antibody for targeted induction of apoptosis, cdc and adcc mediated killing of cancer cells, tbl-cln1|

---

BR112013028779B8|2011-05-27|2021-04-20|Glaxo Group Ltd|antigen-binding protein or immunoconjugate, immunoconjugate, pharmaceutical composition, and, use of a composition|

---

US10189906B2|2012-11-01|2019-01-29|Max-Delrück-Centrum Für Molekulare Medizin|Antibody that binds CD269 suitable for use in the treatment of plasma cell diseases such as multiple myeloma and autoimmune diseases|

---

TW201425336A|2012-12-07|2014-07-01|Amgen Inc|BCMA antigen binding proteins|

---

EP3620468A1|2013-02-05|2020-03-11|EngMab Sàrl|Method for the selection of antibodies against bcma|

---

EP3083671B1|2013-12-20|2020-09-30|Fred Hutchinson Cancer Research Center|Tagged chimeric effector molecules and receptors thereof|

---

US10144782B2|2014-04-30|2018-12-04|Max-Delbrück-Centrum Für Molekulare Medizin In Der Helmholtz-Gemeinschaft|Humanized antibodies against CD269 |

---

EP3023437A1|2014-11-20|2016-05-25|EngMab AG|Bispecific antibodies against CD3epsilon and BCMA|

---

AU2015357533B2|2014-12-05|2021-10-07|Eureka Therapeutics, Inc.|Antibodies targeting B-cell maturation antigen and methods of use|

---

RU2748655C2|2015-07-24|2021-05-28|Онкотрэкер, Инк.|Gamma-secretase inhibitors for treatment of immune system dysfunction|WO2018057776A1|2016-09-22|2018-03-29|Regeneron Pharmaceuticals, Inc.|Methods for treating severe atopic dermatitis by administering an il-4r inhibitor|

---

EP3615068A1|2017-04-28|2020-03-04|Novartis AG|Bcma-targeting agent, and combination therapy with a gamma secretase inhibitor|

---

WO2019028367A1|2017-08-04|2019-02-07|Regeneron Pharmaceuticals, Inc.|Methods for treating active eosinophilic esophagitis|

---

AU2019266150A1|2018-05-06|2021-01-07|Ayala Pharmaceuticals Inc.|Combination compositions comprising bisfluoroalkyl-1,4- benzodiazepinone compounds and methods of use thereof|

---

WO2019215585A1|2018-05-06|2019-11-14|Ayala Pharmaceuticals Inc.|Compositions comprising cd20 inhibitors and bisfluoroalkyl-1,4-benzodiazepinone compounds and methods of use thereof|

---

CN112203661A|2018-05-24|2021-01-08|艾雅拉制药公司|Compositions comprising a difluoroalkyl-1, 4-benzodiazepine compound and an immunotherapeutic agent and methods of using the same|

---

CN109503716B|2018-10-08|2021-04-27|浙江生研生物科技有限公司|Bispecific chimeric antigen receptor molecule and application thereof in tumor treatment|

---

CN112955748A|2018-10-31|2021-06-11|葛兰素史密斯克莱知识产权发展有限公司|Methods of treating cancer|

---

EP3941938A1|2019-03-06|2022-01-26|Dana-Farber Cancer Institute, Inc.|T cell receptors specific to b-cell maturation antigen for treatment of cancer|

---

SG11202109002XA|2019-03-21|2021-09-29|Regeneron Pharma|Combination of il-4/il-13 pathway inhibitors and plasma cell ablation for treating allergy|

---

WO2021051390A1|2019-09-20|2021-03-25|上海吉倍生物技术有限公司|Bcma-targeted antibody and chimeric antigen receptor|

---

WO2021146604A1|2020-01-16|2021-07-22|Allogene Therapeutics, Inc.|Combination therapies of chimeric antigen receptors targeting b-cell maturation antigen and gamma secretase inhibitors|

---

CN111234020B|2020-01-23|2020-10-23|和铂医药有限公司|BCMA binding protein and preparation method and application thereof|

---

WO2021183934A1|2020-03-13|2021-09-16|Springworks Therapeutics, Inc.|A combination therapy with nirogacestat and a bcma-directed therapy and uses thereof|

---

WO2021209498A1|2020-04-14|2021-10-21|Julius-Maximilians-Universität Würzburg|Combination therapy of atra or other retinoids with immunotherapeutic agents binding to bcma|

法律状态:
2021-10-19| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

---

申请号 | 申请日 | 专利标题

---

US201762460612P| true| 2017-02-17|2017-02-17|

---

US201762582270P| true| 2017-11-06|2017-11-06|

---

PCT/US2018/000050|WO2018151836A1|2017-02-17|2018-02-16|Combination therapies for treatment of bcma-related cancers and autoimmune disorders|

---