BCMA as a target for T cell redirecting antibodies in B cell lymphoma

Abstract

Methods are provided for treating non-Hodgkin's lymphoma (NHL) in a human subject, comprising administering to the subject a therapeutically effective amount of a BCMA-specific antibody. Also disclosed are compositions comprising a BCMA-specific antibody and a gamma-secretase inhibitor in amounts that are therapeutically effective to treat non-Hodgkin's lymphoma (NHL) in a human subject.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 194,470, filed May 28, 2021, and U.S. Provisional Patent Application No. 63 / 209,694, filed June 11, 2021, both of which are incorporated by reference in their entireties herein.

[0002] FIELD OF THEINVENTION The present disclosure relates to the treatment of B-lymphoproliferative disorders. [Background technology]

[0003] The treatment of B-cell lymphoproliferative disorders, such as B non-Hodgkin lymphoma (B-NHL), has progressed significantly in recent years, primarily due to a rapidly expanding therapeutic armamentarium that includes not only chemoimmunotherapy but also targeted agents such as inhibitors of key B-cell receptor kinases, such as BTK and PI3K, and inhibitors of key apoptosis regulators, such as venetoclax. 1、2 Nevertheless, for many B-NHL subtypes, these treatments are not curative and ultimately lead to disease recurrence in patients, highlighting the need for new therapies for these malignancies.

[0004] Stem cell transplantation (SCT) reveals that long-lived T cell-mediated anti-cancer responses are feasible 3However, this large proportion of elderly patients are not candidates for SCT because severe graft-versus-host disease (GVHD) may occur. The application of autologous-based T-cell therapy may be a preferred treatment since it will arguably not result in the development of GVHD. Different autologous-based T-cells have already been developed, including immune checkpoint blockade (ICB) and chimeric antigen receptor (CAR) T-cells. ICB has been shown to be effective only in a small number of B-NHL patients, including those with follicular lymphoma (FL), diffuse large B cell lymphoma (DLBCL), and mantle cell lymphoma (MCL). 4 In contrast, CAR T cells have proven more promising. 4 Currently available CAR T cells are produced in a patient-specific manner, which is time-consuming and costly. CAR-T cells are given once, and the development of T cell exhaustion is a common reason for treatment failure. 5 .

[0005] Consistent with the expression of BCMA in PBS and PC, it is well documented that BCMA is highly expressed in multiple myeloma (MM) and is therefore a suitable target for BCMA-targeted therapy. 17 Comparable to plasma cells, BCMA signaling also promotes survival of MM cells 17、18 However, BCMA expression has also been observed in tonsillar memory B cells and germinal center B cells. 21~23 However, it remains to be determined whether other mature B cell malignancies also express BCMA and therefore could be targeted using BCMA-directed therapies. Summary of the Invention [Means for solving the problem]

[0006] Provided herein are methods for treating non-Hodgkin lymphoma (NHL) in a human subject, comprising administering to the subject a therapeutically effective amount of a BCMA-specific antibody.

[0007] Also disclosed is a composition comprising a BCMA-specific antibody and a gamma-secretase inhibitor in an amount that is therapeutically effective to treat non-Hodgkin's lymphoma (NHL) in a human subject. [Brief description of the drawings]

[0008] [Figure 1A] We present the results of experiments demonstrating that BCMA is expressed by different B cell malignancies and can be enhanced by γ-secretase inhibition. [Figure 1B] We present the results of experiments demonstrating that BCMA is expressed by different B cell malignancies and can be enhanced by γ-secretase inhibition. [Figure 1C] We present the results of experiments demonstrating that BCMA is expressed by different B cell malignancies and can be enhanced by γ-secretase inhibition. [Figure 1D] We present the results of experiments demonstrating that BCMA is expressed by different B cell malignancies and can be enhanced by γ-secretase inhibition. [Figure 1E] We present the results of experiments demonstrating that BCMA is expressed by different B cell malignancies and can be enhanced by γ-secretase inhibition. [Figure 2A] We provide the results of an evaluation showing that BCMA is expressed at low levels in primary CLL cells and can be slightly enhanced by γ-secretase inhibition. [Figure 2B] We provide the results of an evaluation showing that BCMA is expressed at low levels in primary CLL cells and can be slightly enhanced by γ-secretase inhibition. [Figure 2C]We provide the results of an evaluation showing that BCMA is expressed at low levels in primary CLL cells and can be slightly enhanced by γ-secretase inhibition. [Figure 2D] We provide the results of an evaluation showing that BCMA is expressed at low levels in primary CLL cells and can be slightly enhanced by γ-secretase inhibition. [Figure 2E] We provide the results of an evaluation showing that BCMA is expressed at low levels in primary CLL cells and can be slightly enhanced by γ-secretase inhibition. [Figure 2F] We provide the results of an evaluation showing that BCMA is expressed at low levels in primary CLL cells and can be slightly enhanced by γ-secretase inhibition. [Figure 3A] BCMA expression in different B cell malignancies. [Figure 3B] BCMA expression in different B cell malignancies. [Figure 4A] We demonstrate how BCMA antibodies induce activation, degranulation, cytokine secretion, and cytotoxicity by T cells in the presence of B cell malignant cell lines. [Figure 4B] We demonstrate how BCMA antibodies induce activation, degranulation, cytokine secretion, and cytotoxicity by T cells in the presence of B cell malignant cell lines. [Figure 4C] We demonstrate how BCMA antibodies induce activation, degranulation, cytokine secretion, and cytotoxicity by T cells in the presence of B cell malignant cell lines. [Figure 4D] We demonstrate how BCMA antibodies induce activation, degranulation, cytokine secretion, and cytotoxicity by T cells in the presence of B cell malignant cell lines. [Figure 4E] We demonstrate how BCMA antibodies induce activation, degranulation, cytokine secretion, and cytotoxicity by T cells in the presence of B cell malignant cell lines. [Figure 4F]We demonstrate how BCMA antibodies induce activation, degranulation, cytokine secretion, and cytotoxicity by T cells in the presence of B cell malignant cell lines. [Figure 4G-1] We demonstrate how BCMA antibodies induce activation, degranulation, cytokine secretion, and cytotoxicity by T cells in the presence of B cell malignant cell lines. [Figure 4G-2] We demonstrate how BCMA antibodies induce activation, degranulation, cytokine secretion, and cytotoxicity by T cells in the presence of B cell malignant cell lines. [Diagram 5] AC provide the results of an evaluation showing that healthy donor T cells kill primary CLL cells in the presence of BCMA-specific antibodies that is highly dependent on CD8+ T cells. [Figure 6A] We show how BCMA-specific antibodies induce T cell activation of CLL-derived T cells, leading to CLL killing. [Figure 6B] We show how BCMA-specific antibodies induce T cell activation of CLL-derived T cells, leading to CLL killing. [Figure 6C] We show how BCMA-specific antibodies induce T cell activation of CLL-derived T cells, leading to CLL killing. [Figure 7] We demonstrate that the viability of various cell lines was not affected by γ-secretase inhibition. [Figure 8] Data demonstrating that CLL cell viability was unaffected by γ-secretase inhibition. [Figure 9A-1] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9A-2]We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9A-3] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9A-4] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9A-5] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9B-1] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9B-2] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9B-3]We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9B-4] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9B-5] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9C-1] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9C-2] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9C-3] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9C-4]We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9C-5] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9D-1] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9D-2] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9D-3] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9D-4] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9D-5]We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9E-1] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9E-2] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9E-3] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9E-4] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9E-5] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9F-1]We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9F-2] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9F-3] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9F-4] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. [Figure 9F-5] We provide the results of an evaluation of whether anti-BCMA antibody treatment, with or without a γ-secretase inhibitor, results in increases in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-α), and T cell division. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The inventive subject matter disclosed in this invention may be more readily understood by reference to the following detailed description taken in conjunction with the accompanying figures and examples, which form a part of this disclosure. It is to be understood that these inventions are not limited to the specific products, methods, conditions, or parameters described and / or illustrated herein, and that the terminology used herein is for the purpose of describing specific embodiments by way of example only, and is not intended to limit the invention as claimed.

[0010] The entire disclosure of each patent, patent application, and publication cited or described in this document is incorporated herein by reference. In this disclosure, superscript numbers refer to correspondingly numbered publications listed under the heading "References" below.

[0011] As used above, and throughout this disclosure, the following terms and abbreviations shall be understood to have the following meanings, unless otherwise indicated:

[0012] In this disclosure, the singular forms "a," "an," and "the" include plural references and a reference to a particular numerical value includes at least that particular value unless the context clearly dictates otherwise. Thus, for example, a reference to "a treatment" is a reference to one or more of such treatments and equivalents thereof known to those skilled in the art, and so forth. Furthermore, when indicating that a particular element "may be" X, Y, or Z, such use is not intended to exclude other options for that element in all instances.

[0013] When values ​​are expressed as approximations by use of the antecedent "about," it is understood that the particular value forms another embodiment. As used herein, "about X," where X is a numerical value, preferably refers to ±10% of the stated value, inclusive. For example, the phrase "about 8" preferably refers to values ​​between 7.2 and 8.8, inclusive, and as another example, the phrase "about 8%" preferably refers to values ​​between 7.2% and 8.8%, inclusive. Where present, all ranges are inclusive and combinable. For example, when reciting a range of "1-5," the recited range should be construed to optionally include ranges such as "1-4," "1-3," "1-2," "1-2 and 4-5," "1-3 and 5," etc. Additionally, when a list of alternatives is expressly provided, such list may also include embodiments in which any of the alternatives may be excluded. For example, when a range of "1 to 5" is described, such description can support the situation where any of 1, 2, 3, 4, or 5 is excluded, and thus a recitation of "1 to 5" can support "1 and 3 to 5, but not 2" or simply "2 is not included." The phrase "at least about x" is intended to encompass both "about x" and "at least x." Also, when a parameter range is provided, it is understood that all integers and tenths thereof within that range are also provided by the present invention. For example, "2 to 5 hours" includes 2 hours, 2.1 hours, 2.2 hours, 2.3 hours, etc., up to 5 hours.

[0014] B cell maturation antigen (BCMA) is highly expressed on normal and malignant mature B cells, which can be enhanced by inhibition of γ-secretase (γ-sec) inhibition, making it a viable target for multiple myeloma (MM). Data on BCMA expression on other B cell malignancies are scarce, with conflicting data published for CLL, DLBCL, FL, and MCL. 17、24~27 This complicates the question of whether BCMA is a viable target in these diseases. It is currently not known whether other mature B cell malignancies besides MM can be targeted by BCMA-specific antibodies such as BCMAxCD3 DuoBody® teclistamab.

[0015] We evaluated BCMA expression in mature B-cell lymphoma cell lines to assess whether BCMA expression could be enhanced by inhibiting γ-secretase. BCMA expression was also measured and (semi)quantified on primary material from B-NHL (including CLL) patients. To assess whether BCMA could be used as a target in B-NHL (including CLL), we evaluated whether BCMAxCD3 BsAb (teclistamab, JNJ-7957) could mediate T-cell activation as well as tumor cell killing of lymphoma cell lines. This BsAb was developed using Genmab DuoBody® technology, resulting in increased stability compared to the BsAb format. CLL was used as a target as a proof of concept that BCMA can trigger an autologous T-cell response.

[0016] We found that BCMA was variably detectable on all mature B cell malignant cell lines tested, with highest expression in MM, followed by Waldenstrom's macroglobulinemia (WM). In all B cell lines, γ-sec inhibition increased BCMA expression, even when basal levels of BCMA were low. These data were corroborated in primary B cell lymphomas, with detectable levels of BCMA in samples from WM, CLL, and diffuse large B cell lymphoma patients.

[0017] Co-culture of HD T cells with various B cell malignant cell lines in the presence of teclistamab resulted in T cell activation, proliferation, and cytotoxicity, independent of the level of BCMA expression. The efficacy of teclistamab against primary tumor cells was tested using CLL cells. Despite low BCMA levels, healthy donor T cells lysed up to 40% of CLL cells in the presence of teclistamab. Furthermore, teclistamab induced activation, degranulation, and efficient cytotoxicity by CLL-derived T cells when co-cultured with autologous CLL cells.

[0018] Thus, we discovered that BCMA expression is not restricted to MM but is also present on other mature B cell malignancies. Targeting BCMA with teclistamab resulted in cytotoxicity of lymphoma cell lines and primary CLL, even when BCMA levels were low.

[0019] In accordance with these discoveries, provided herein are methods for treating non-Hodgkin's lymphoma (NHL) in a human subject comprising administering to the subject a therapeutically effective amount of a BCMA-specific antibody.

[0020] As used herein, the phrase "therapeutically effective amount" refers to that amount of an active compound that elicits the biological or medical response desired in a tissue, system, animal, individual, or human by a researcher, physician, or other clinician, including, but not limited to, the following: (1) At least partially preventing a disease or condition or a symptom thereof, e.g., preventing a disease, condition, or disorder in an individual who may be predisposed to the disease, condition, or disorder but who has not yet experienced or manifested symptoms of the disease; (2) inhibiting a disease or condition, e.g., inhibiting a disease, condition, or disorder in an individual experiencing or displaying a pathology or symptom of the disease, condition, or disorder (i.e., including arresting further progression of the pathology and / or symptom); and (3) At least partially ameliorating a disease or condition, e.g., ameliorating a disease, condition, or disorder in an individual experiencing or displaying the pathology or symptoms of the disease, condition, or disorder (i.e., including reversal of the pathology and / or symptoms).

[0021] BCMA-specific antibodies may be monospecific or multispecific (e.g. bispecific), i.e. the antibody may be specific for a target other than BCMA, as long as it is also specific for BCMA.

[0022] In certain embodiments, the BCMA-specific antibody is a BCMAxCD3 bispecific antibody. In light of the present disclosure, any suitable BCMAxCD3 bispecific antibody known to the skilled artisan can be used in the present invention.

[0023] Various bispecific antibody formats include those described herein, as well as recombinant IgG-like dual targeting molecules in which the two sides of the molecule each contain a Fab fragment or a portion of a Fab fragment of at least two different antibodies, IgG fusion molecules in which a full length IgG antibody is fused to an extra Fab fragment or a portion of a Fab fragment, Fc fusion molecules in which a single chain Fv molecule or a stabilized diabody is fused to a heavy chain constant domain, Fc region, or a portion thereof, Fab fusion molecules in which different Fab fragments are fused together, ScFv and diabody-based heavy chain antibodies (e.g., domain antibodies, nanobodies) in which different single chain Fv molecules or different diabodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule, or to bispecific antibodies generated by arm swapping.Exemplary bispecific antibody formats include dual targeting molecules including Dual Targeting (DT)-Ig (GSK / Domantis), Two-in-one antibodies (Genentech), and mAb2 (F-Star), Dual Variable Domain (DVD)-Ig (Abbott), DuoBody (Genmab), Ts2Ab (MedImmune / AZ), and BsAb (Zymogenetics), HERCULES (Biogen Idec), and TvAb (Roche), ScFv / Fc fusions (Academic Institution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS), and Dual Affinity Retargeting Technology (DAT). Technology, Fc-DART (MacroGenics), F(ab)2 (Medarex / AMGEN), dual activity or Bis-Fab (Genentech), Dock-and-Lock (DNL) (ImmunoMedics), bivalent bispecific (Biotecnol), and Fab-Fv (UCB-Celltech), Bispecific T Cell Engager (BITE) (Micromet), Tandem Diabody (Tandab) (Affimed), Dual Affinity Retargeting Technology (DART) (MacroGenics), single chain diabodies (Academic), TCR-like antibodies (AIT, ReceptorLogics), human serum albumin ScFv fusions (Merrimack), and COMBODY (Epigen Biotech), dual targeting nanobodies (Ablynx), dual targeting heavy chain only domain antibodies. Various formats of bispecific antibodies are described, for example, in Chames and Baty (2009) Curr Opin Drug Disc Dev 12:276 and Nunez-Prado et al., (2015) Drug Discovery Today 20(5):588-594.

[0024] In some embodiments, the BCMAxCD3 bispecific antibody comprises any one of the BCMA binding domains described in WO 2017 / 031104, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the BCMAxCD3 bispecific antibody comprises any one of the CD3 binding domains described in WO 2017 / 031104. In some embodiments, the BCMAxCD3 bispecific antibody comprises any one of the BCMAxCD3 bispecific antibodies or antigen-binding fragments thereof described in WO 2017 / 031104.

[0025] In some embodiments, the BCMAxCD3 bispecific antibody comprises a CD3 binding domain comprising heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 11, HCDR2 of SEQ ID NO: 12, HCDR3 of SEQ ID NO: 13, light chain complementarity determining region 1 (LCDR1) of SEQ ID NO: 14, LCDR2 of SEQ ID NO: 15, and LCDR3 of SEQ ID NO: 16, or a heavy chain variable region (VH) of SEQ ID NO: 17, and a light chain variable region (VL) of SEQ ID NO: 18.

[0026] In some embodiments the BCMAxCD3 bispecific antibody comprises a BCMA binding domain comprising a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO: 1, an HCDR2 of SEQ ID NO: 2, an HCDR3 of SEQ ID NO: 3, an LCDR1 of SEQ ID NO: 4, an LCDR2 of SEQ ID NO: 5, and an LCDR3 of SEQ ID NO: 6, or a heavy chain variable region (VH) of SEQ ID NO: 7, and a light chain variable region (VL) of SEQ ID NO: 8.

[0027] In some embodiments the BCMAxCD3 bispecific antibody comprises a first heavy chain (HC1) of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10, a second heavy chain (HC2) of SEQ ID NO: 19, and a second light chain (LC2) of SEQ ID NO: 20.

[0028] In some embodiments, the BCMAxCD3 bispecific antibody is chimeric, humanized, or human.

[0029] In some embodiments, the BCMAxCD3 bispecific antibody is an antigen-binding fragment. Exemplary antigen-binding fragments are Fab, F(ab')2, Fd, and Fv fragments.

[0030] In some embodiments, the bispecific antibody is an IgG1, IgG2, IgG3, or IgG4 isotype. In a preferred embodiment, the bispecific antibody is an IgG4 isotype. An exemplary wild-type IgG4 comprises the amino acid sequence of SEQ ID NO:21.

[0031] Bispecific antibodies can be of any allotype. Allotype is not expected to affect the properties of bispecific antibodies, such as binding or Fc-mediated effector functions. Immunogenicity of therapeutic antibodies is associated with a high risk of infusion reactions and a short duration of therapeutic response (Baert et al., (2003) N Engl J Med 348:602-08). The extent to which a therapeutic antibody induces an immune response in a host may be determined in part by the antibody allotype (Stickler et al., (2011) Genes and Immunity 12:213-21). Antibody allotypes are related to amino acid sequence variations at specific positions in the antibody constant region sequence. Table 1 shows selected IgG1, IgG2, and IgG4 allotypes.

[0032] [Table 1]

[0033] In some embodiments, the bispecific antibody comprises one or more Fc substitutions that reduce binding of the bispecific antibody to Fcγ receptors (FcγR) and / or reduce an Fc effector function such as C1q binding, complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), or antibody-dependent cell-mediated phagocytosis (ADCP). Particular substitutions can be made compared to the wild type IgG4 of SEQ ID NO: 21.

[0034] Fc positions that may be substituted to reduce binding of the Fc to activating FcγRs and in turn reduce effector function are L234A / L235A in IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S in IgG2, F234A / L235A in IgG4, S228P / F234A / L235A in IgG4, N297A in all Ig isotypes, V234A / G237A in IgG2, K214T / E233P / L234V / L235A / G236 deletion / A327G / P33 IgG1; H268Q / V309L / A330S / P331S in IgG2; S267E / L328F in IgG1; L234F / L235E / D265A in IgG1; L234A / L235A / G237A / P238S / H268A / A330S / P331S in IgG1; S228P / F234A / L235A / G237A / P238S in IgG4; and S228P / F234A / L235A / G236 deletion / G237A / P238S substitutions in IgG4, where residue numbering is according to the EU index.

[0035] An Fc substitution that can be used to reduce CDC is the K322A substitution.

[0036] To increase the stability of IgG4, the well-known S228P substitution can further be made in the IgG4 antibody.

[0037] In some embodiments, the bispecific antibody comprises one or more asymmetric substitutions in the first CH3 domain or the second CH3 domain, or in both the first CH3 domain and the second CH3 domain.

[0038] In some embodiments, the one or more asymmetric substitutions are F405L / K409R, wild type / F405L_R409K, T366Y / F405A, T366W / F405W, F405W / Y407A, T394W / Y407T, T394S / Y407A, T366W / T394S, F405W / T394S, and T366W / T366S_L368A_Y407V, L351Y_F405A_Y407V / T394W, T3 66I_K392M_T394W / F405A_Y407V, T366L_K392M_T394W / F405A_Y407V, L351Y_Y407A / T366A_K409F, L351Y_Y407A / T366V_K409F, Y407A / T366A_K409F, and T350V_L351Y_F405A_Y407V / T350V_T366L_K392L_T394W.

[0039] In some embodiments the BCMAxCD3 bispecific antibody is of IgG4 isotype and comprises a phenylalanine at position 405 and an arginine at position 409 of the first heavy chain (HC1), and a leucine at position 405 and a lysine at position 409 of the second heavy chain (HC2), where the numbering of the residues is according to the EU index.

[0040] In some embodiments, the BCMAxCD3 bispecific antibody further comprises a proline at position 228, an alanine at position 234, and an alanine at position 235 in both HC1 and HC2.

[0041] In some embodiments the BCMAxCD3 bispecific antibody comprises an HC1 of SEQ ID NO: 9, a first light chain (LC1) of SEQ ID NO: 10, an HC2 of SEQ ID NO: 19, and a second light chain (LC2) of SEQ ID NO: 20.

[0042] In some embodiments, the BCMAxCD3 bispecific antibody is CC-93269, BI836909, JNJ-64007957 (teclistamab), or PF-06863135. In a preferred embodiment, the BCMAxCD3 bispecific antibody is teclistamab.

[0043] In some embodiments, the amount of teclistamab administered to a subject is effective to activate T cells in the subject, induce neutrophil degranulation in the subject, and induce cytokine production in the subject, or any combination thereof. In certain embodiments, the amount of teclistamab administered to a subject is effective to activate T cells in the subject, induce neutrophil degranulation in the subject, and induce cytokine production in the subject.

[0044] The non-Hodgkin's lymphoma treated according to the method can be, for example, any subtype characterized by the expression of B cell maturation antigen (BCMA). For example, the non-Hodgkin's lymphoma can be chronic lymphocytic leukemia (CLL), lymphoblastic lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, or Waldenstrom's macroglobulinemia. Non-Hodgkin's lymphoma is more frequently observed in adults, and therefore the method can include administration of BCMA-specific antibodies to adults (e.g., individuals over 16 years old). However, non-Hodgkin's lymphoma can also occur in children, and the method of the present invention can also be used to treat immature humans (individuals under 16 years old).

[0045] The method may further comprise administering a gamma-secretase inhibitor to the subject. As described more fully below, administration of a gamma-secretase inhibitor to the subject may obtain a synergistic effect with a BCMA-specific antibody. For example, gamma-secretase inhibition may be useful for subjects who express BCMA just below a threshold level, to enhance the effectiveness of the BCMA-specific antibody. As in the case of administration of a BCMA-specific antibody, the gamma-secretase inhibitor is administered in a therapeutically effective amount, and by therapeutically effective amount, it is meant that the amount of the gamma-secretase inhibitor should be administered in an amount that provides a therapeutic effect when the subject is also treated with a BCMA-specific antibody. The above definition of "therapeutically effective amount" applies with respect to the amount of the gamma-secretase inhibitor when co-administered with a BCMA-specific antibody.

[0046] According to the method, the BCMA-specific antibody treatment may be performed substantially simultaneously with the gamma-secretase inhibitor treatment. The BCMA-specific antibody treatment performed substantially simultaneously with the gamma-secretase inhibitor refers to a situation where there is a time overlap between the BCMA-specific antibody treatment and the gamma-secretase inhibitor treatment. Thus, the BCMA-specific antibody treatment performed during a period that at least partially overlaps with the period during which the gamma-secretase inhibitor administration is performed can be said to be substantially simultaneous. In such cases, the BCMA-specific antibody treatment may be started before or after the initiation of the gamma-secretase inhibitor treatment. When there is no overlap between the period during which the BCMA-specific antibody treatment is performed and the period during which the gamma-secretase inhibitor administration is performed, the treatment may be described as sequential. Thus, in certain embodiments, the BCMA-specific antibody treatment and the gamma-secretase inhibitor treatment may be performed sequentially. In such cases, the BCMA-specific antibody treatment may be started before or after the initiation of the gamma-secretase inhibitor therapy.

[0047] In some embodiments, the BCMA-specific antibody and the gamma-secretase inhibitor are administered to the subject in a single dosage form. Alternatively, the BCMA-specific antibody may be administered in a first dosage form and the gamma-secretase inhibitor is administered in a second dosage form.

[0048] Also disclosed herein is a composition comprising a BCMA-specific antibody and a gamma-secretase inhibitor in amounts that are therapeutically effective to treat non-Hodgkin's lymphoma (NHL) in a human subject. The characteristics and amounts (including what constitutes a therapeutically effective amount) of the BCMA-specific antibody and gamma-secretase inhibitor may be as each described above in connection with the present methods for treating non-Hodgkin's lymphoma.

[0049] In accordance with the methods and compositions of the present disclosure, the BCMA-specific antibody, the gamma-secretase inhibitor, or both, may be provided in a composition formulated for any type of administration. For example, the antibody and / or inhibitor may be provided in a composition (dosage form) formulated for administration orally, topically, parenterally, enterally, or by inhalation. In certain embodiments, the composition is formulated for oral administration. The antibody and / or inhibitor may be formulated for neat administration alone or in combination with conventional pharmaceutical carriers, diluents, or excipients, which may be liquid or solid. Applicable solid carriers, diluents, or excipients may function as, among others, binders, disintegrants, fillers, lubricants, glidants, compression aids, processing aids, colorants, sweeteners, preservatives, suspending / dispersing agents, tablet disintegrants, encapsulating materials, film formers or coatings, flavoring agents, or printing inks. Any material used in preparing any unit dosage form is preferably pharma- ceutically pure and substantially non-toxic in the amounts employed. In addition, the antibodies and / or inhibitors may be incorporated into sustained release preparations and formulations. Administration in this regard includes administration by topical routes including, inter alia, the following routes: intravenous, intramuscular, subcutaneous, intraocular, intrasynovial, transepithelial including transdermal, ocular, sublingual, and buccal, ophthalmic, dermal, ocular, rectal, and insufflation, aerosol nasal inhalation, and rectal systemic.

[0050] In powders, the carrier, diluent, or excipient may be a finely divided solid that is mixed with the finely divided active ingredient. In tablets, the antibody and / or inhibitor is mixed with a carrier, diluent, or excipient having the necessary compression properties in suitable proportions and compressed into the desired shape and size. For oral therapeutic administration, the antibody and / or inhibitor may be incorporated with a carrier, diluent, or excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active compound(s) in such therapeutically useful compositions is preferably such that a suitable dosage will be obtained.

[0051] Liquid carriers, diluents, or excipients may be used in preparing solutions, suspensions, emulsions, syrups, elixirs, and the like. The antibody and / or inhibitor may be dissolved or suspended in a pharma- ceutically acceptable liquid, such as water, an organic solvent, a mixture of both, or a pharma- ceutically acceptable oil or fat. The liquid carriers, excipients, or diluents may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, coloring agents, viscosity adjusting agents, stabilizers, or osmolality adjusting agents.

[0052] Suitable solid carriers, diluents, and excipients may include, for example, calcium phosphate, silicon dioxide, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, polyvinylpyrrolidine, low melting waxes, ion exchange resins, croscarmellose carbon, acacia, pregelatinized starch, crospovidone, HPMC, povidone, titanium dioxide, polycrystalline cellulose, aluminum methahydroxide, agar, tragacanth, or mixtures thereof.

[0053] For example, suitable examples of liquid carriers, diluents, and excipients for oral, topical, or parenteral administration include water (specifically containing the additives described above, such as cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric and polyhydric alcohols, such as glycols), and derivatives thereof, and oils (e.g., fractionated coconut oil and peanut oil), or mixtures thereof.

[0054] For parenteral administration, carrier, diluent or excipient may also be oily ester such as ethyl oleate and isopropyl myristate.Sterile liquid carrier, diluent or excipient used in sterile liquid form composition for parenteral administration is also contemplated.Dispersion can also be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oil.Under normal storage and use conditions, these preparations may contain preservatives to prevent the growth of microorganisms.

[0055] Pharmaceutical forms suitable for injection use include, for example, sterile aqueous solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form is preferably sterile and fluid to provide easy delivery by syringe. The form is preferably stable under the conditions of manufacture and storage, and is preferably preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier, diluent, or excipient may be, for example, a solvent or dispersion medium containing water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial activity can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of the injectable compositions can be achieved by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0056] Sterile injectable solutions can be prepared by incorporating a pharma- ceutically appropriate amount of the antibody and / or inhibitor in an appropriate solvent, along with various other ingredients as enumerated above, as needed, followed by filtered sterilization. In general, dispersions can be prepared by incorporating the antibody and / or inhibitor into a sterile vehicle containing the basic dispersion medium and the other required ingredients selected from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation can include vacuum drying and freeze-drying techniques, which yield a powder of the active agent or active ingredient, plus any additional desired ingredients, from a previously sterile-filtered solution thereof.

[0057] The BCMA-specific antibody (and, where applicable, the γ-secretase inhibitor) may be formulated as a pharmaceutical composition containing from about 1 mg / mL to about 200 mg / mL of antibody.

[0058] In some embodiments, the pharmaceutical composition further comprises one or more excipients, including, but not limited to, a buffer, a sugar, a surfactant, a chelating agent, or any combination thereof.

[0059] In some embodiments, the pharmaceutical composition comprises: and a BCMA-specific antibody at about 20 mg / mL to about 120 mg / mL, such as about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL, about 120 mg / mL, or any value therebetween; A buffering agent, such as sodium phosphate, KH2PO4, sodium acetate, or sodium citrate, at about 5 mM to about 20 mM, about 5 mM, about 10 mM, about 15 mM, about 20 mM, or any value therebetween; about 1% w / v to about 20% w / v of a sugar such as glucose, sucrose, or cellobiose, about 1% w / v, about 2% w / v, about 3% w / v, about 4% w / v, about 5% w / v, about 6% w / v, about 7% w / v, about 8% w / v, about 9% w / v, about 10% w / v, about 15% w / v, about 20% w / v, or any value therebetween; about 0.01% w / v to about 2% w / v of a surfactant such as polysorbate 80 (PS-80) or PS-20, about 0.01% w / v, about 0.02% w / v, about 0.03% w / v, about 0.04% w / v, about 0.05% w / v, about 0.06% w / v, about 0.07% w / v, about 0.08% w / v, about 0.09% w / v, about 0.1% w / v, about 0.5% w / v, about 1% w / v, about 1.5% w / v, about 2% w / v, or any value therebetween; and about 5 mM to about 40 mM ethylenediaminetetraacetic acid (EDTA) or edetate, such as about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, or any value therebetween, and a pH of about 5 to 6, such as about 5, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6, or any value therebetween.

[0060] In some embodiments, the pharmaceutical composition further comprises about 0.1 mg / mL to about 5 mg / mL of an amino acid, such as methionine or arginine, at about 0.1 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, or any value therebetween.

[0061] In one embodiment, a pharmaceutical composition useful in the invention comprises a BCMA-specific antibody such as teclistamab, 20 mM sodium phosphate, 10% weight / volume (w / v) sucrose, 0.06% (w / v) PS80, and 25 μg / mL EDTA, pH 5.4.

[0062] In another embodiment, a pharmaceutical composition useful in the invention comprises a BCMA-specific antibody such as teclistamab, 10-15 mM sodium acetate, 8% (w / v) sucrose, 0.04% (w / v) PS20, and 20 μg / mL EDTA, pH 5.2.

[0063] In another embodiment, a pharmaceutical composition useful in the invention comprises a BCMA-specific antibody such as teclistamab, 15 mM KH2PO4, 10% (w / v) cellobiose, 0.05% (w / v) PS20, and 25 μg / mL EDTA, pH 5.1.

[0064] Administration In some embodiments, the BCMA-specific antibody is administered by intravenous injection.

[0065] In some embodiments, the BCMA-specific antibody is administered by subcutaneous injection.

[0066] The dose of a BCMA-specific antibody given to a subject with cancer, such as multiple myeloma, is an amount sufficient to ameliorate or at least partially prevent the disease being treated (a "therapeutically effective amount"), and may be from about 0.1 μg / kg to about 6000 μg / kg, for example, from about 0.3 μg / kg to about 5000 μg / kg, from about 0.1 μg / kg to about 3000 μg / kg, from about 0.2 μg / kg to about 3000 μg / kg, from about 0.3 μg / kg to about 3000 μg / kg, from about 0.6 μg / kg to about 3000 μg / kg, from about 1.2 μg / kg to about 3000 μg / kg, μg / kg, approximately 19.2μg / kg to approximately 3000μg / kg, approximately 35μg / kg to approximately 3000μg / kg, approximately 80μg / kg to approximately 3000μg / kg, approximately 100μg / kg to approximately 3000μg / kg, approximately 270μg / kg to approximately 3000μg / kg, approximately 720μg / kg~about 3000μg / kg, about 0.1μg / kg~about 1800μg / kg, about 0.2μg / kg~about 1800μg / kg, about 0.3μg / kg~about 1800μg / kg, about 0.6μg / kg~about 1800μg / kg, about 1.2μg / kg~about 1800μg / kg, about 19.2μg / kg to about 1800μg / kg, about 35μg / kg to about 1800μg / kg, about 80μg / kg to about 1800μg / kg, about 100μg / kg to about 1800μg / kg, about 270μg / kg to about 1800μg / kg, about 720μg / kg kg~about 1800μg / kg, about 0.1μg / kg~about 1500μg / kg, about 0.2μg / kg~about 1500μg / kg, about 0.3μg / kg~about 1500μg / kg, about 0.6μg / kg~about 1500μg / kg, about 1.2μg / kg~about 1500μg / kg kg, about 19.2μg / kg to about 1500μg / kg, about 35μg / kg to about 1500μg / kg, about 80μg / kg to about 1500μg / kg, about 100μg / kg to about 1500μg / kg, about 270μg / kg to about 1500μg / kg, about 720μg / kg ~about 1500μg / kg, about 0.1μg / kg to about 850μg / kg, about 0.2μg / kg to about 850μg / kg, about 0.3μg / kg to about 850μg / kg, about 0.6μg / kg to about 850μg / kg, about 1.2μg / kg to about 850μg / kg, about 19.2μg / kg to about 850μg / kg, about 35μg / kg to about 850μg / kg, about 80μg / kg to about 850μg / kg, about 100μg / kg to about 850μg / kg, about 270μg / kg to about 850μg / kg, about 7 20μg / kg to about 850μg / kg, about 0.1μg / kg to about 720μg / kg, about 0.2μg / kg to about 720μg / kg, about 0.3μg / kg to about 720μg / kg, about 0.6μg / kg to about 720μg / kg, Approximately 1.2μg / kg to approximately 720μg / kg, approximately 19.2μg / kg to approximately 720μg / kg, approximately 35μg / kg to approximately 720μg / kg, approximately 80μg / kg to approximately 720μg / kg, approximately 100μg / kg to approximately 720μg / k g, about 270μg / kg to about 720μg / kg, about 720μg / kg to about 720μg / kg, about 0.1μg / kg to about 270μg / kg, about 0.2μg / kg to about 270μg / kg, about 0.3μg / kg to about 270μg / kg, about 0.6μg / kg to about 270μg / kg, about 1.2μg / kg to about 270μg / kg, about 19.2μg / kg to about 270μg / kg, about 35μg / kg to about 270μg / kg, about 80μg / kg to about 270 μg / kg, approximately 100 μg / kg to approximately 270 μg / kg, approximately 270 μg / kg to approximately 270 μg / kg, approximately 720 μg / kg to approximately 270 μg / kg, approximately 0.1 μg / kg to approximately 100 μg / kg, approximately 0.2 μg / kg to approximately 1 00 μg / kg, about 0.3 μg / kg to about 100 μg / kg, about 0.6 μg / kg to about 100 μg / kg, about 1.2 μg / kg to about 100 μg / kg, about 19.2 μg / kg to about 100 μg / kg, about 35 μg / kg to about 100 μg / kg, about 80 μg / kg to about 100 μg / kg, about 100 μg / kg to about 100 μg / kg, about 270 μg / kg to about 100 μg / kg, and about 720 μg / kg to about 100 μg / kg of antibodies. Suitable doses include, for example, about 0.1 μg / kg, about 0.2 μg / kg, about 0.3 μg / kg, about 0.6 μg / kg, about 1.2 μg / kg, about 2.4 μg / kg, about 4.8 μg / kg, about 9.6 μg / kg, about 19.2 μg / kg, about 20 μg / kg, about 35 μg / kg, about 38.4 μg / kg, about 40 μg / kg, about 50 μg / kg, about 57.6μg / kg, approximately 60μg / kg, approximately 80μg / kg, approximately 100μg / kg, approximately 120μg / kg, approximately 180μg / kg, approximately 240μg / kg, approximately 270μg / kg, approximately 300μ g / kg, approximately 720μg / kg, approximately 850μg / kg, approximately 1000μg / kg, approximately 1100μg / kg, approximately 1200μg / kg, approximately 1300μg / kg, approximately 1400μg / kg, approximately 1500 μg / kg, about 1600 μg / kg, about 1700 μg / kg, about 1800 μg / kg, about 2000 μg / kg, about 2500 μg / kg, about 3000 μg / kg, about 3500 μg / kg, about 4000 μg / kg, about 4500 μg / kg, about 5000 μg / kg, about 5500 μg / kg, about 6000 μg / kg, or any dose therebetween.

[0067] A fixed unit dose of BCMA-specific antibody can also be provided, for example, 50, 100, 200, 500, or 1000 mg, or any value therebetween, or the dose can be based on the patient's body surface area, for example, 500, 400, 300, 250, 200, or 100 mg / m 2 Typically, 1 to 8 doses (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) can be administered to treat a cancer such as multiple myeloma, although 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more doses can be given.

[0068] Administration of the BCMA-specific antibody can be repeated after 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 1 month, 5 weeks, 6 weeks, 7 weeks, 2 months, 3 months, 4 months, 5 months, 6 months, or more. Repeated courses of treatment are also possible as long-term administration. Repeated administration can be at the same dose or at different doses. For example, the BCMA-specific antibody can be administered at a first dose at weekly intervals for a certain number of weeks, followed by a second dose every 2 weeks for a further certain number of weeks, followed by a third dose every week for a further certain number of weeks.

[0069] The BCMA-specific antibody can be administered by maintenance therapy, such as, for example, once a week for a period of six months or more. For example, the BCMA-specific antibody can be administered as a daily dosage in an amount of about 0.1 μg / kg to about 6000 μg / kg, for example, about 0.2 μg / kg to about 3000 μg / kg, about 0.2 μg / kg to about 2000 μg / kg, about 0.2 μg / kg to about 1500 μg / kg, about 0.3 μg / kg to about 1500 μg / kg, about 0.6 μg / kg to about 720 μg / kg, about 1.2 μg / kg to about 270 μg / kg, about 19.2 μg / kg to about 720 μg / kg, about 35 μg / kg per day, using single or divided doses every 24, 12, 8, 6, 4, or 2 hours, or using a combination thereof. about 850 μg / kg, about 270 μg / kg to about 720 μg / kg of antibody can be provided on at least one of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or at least one of the 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 weeks, or a combination thereof, after initiation of treatment.

[0070] In one embodiment, the BCMA specific antibody is administered intravenously in a single dose once a week. For example, the BCMA specific antibody is administered at a dose of about 0.1 μg / kg, about 0.2 μg / kg, about 0.3 μg / kg, about 0.6 μg / kg, about 1.2 μg / kg, about 2.4 μg / kg, about 4.8 μg / kg, about 9.6 μg / kg, about 19.2 μg / kg, about 20 μg / kg, about 35 μg / kg, about 38.4 μg / kg, about 40 μg / kg, about 50 μg / kg, about 57.6 μg / kg, about 60 μg / kg, about 80 μg / kg, about 100 μg / kg, about 120 μg / kg , about 180 μg / kg, about 240 μg / kg, about 270 μg / kg, about 300 μg / kg, about 720 μg / kg, about 850 μg / kg, about 1000 μg / kg, about 1100 μg / kg, about 1200 μg / kg, about 1300 μg / kg, about 1400 μg / kg, about 1500 μg / kg, about 1500 μg / kg, about 1600 μg / kg, about 1700 μg / kg, about 1800 μg / kg, or any dose therebetween.

[0071] In one embodiment, the BCMA specific antibody is administered intravenously in a single dose twice weekly. For example, the BCMA specific antibody is administered at a dose of about 0.1 μg / kg, about 0.2 μg / kg, about 0.3 μg / kg, about 0.6 μg / kg, about 1.2 μg / kg, about 2.4 μg / kg, about 4.8 μg / kg, about 9.6 μg / kg, about 19.2 μg / kg, about 20 μg / kg, about 35 μg / kg, about 38.4 μg / kg, about 40 μg / kg, about 50 μg / kg, about 57.6 μg / kg, about 60 μg / kg, about 80 μg / kg, about 100 μg / kg, about 120 μg / kg, It may be administered intravenously twice weekly in an amount of about 180 μg / kg, about 240 μg / kg, about 270 μg / kg, about 300 μg / kg, about 720 μg / kg, about 850 μg / kg, about 1000 μg / kg, about 1100 μg / kg, about 1200 μg / kg, about 1300 μg / kg, about 1400 μg / kg, about 1500 μg / kg, about 1500 μg / kg, about 1600 μg / kg, about 1700 μg / kg, about 1800 μg / kg, or any dose therebetween.

[0072] In one embodiment, the BCMA-specific antibody is administered intravenously at a step-up (or "priming") dose, followed by a higher dose administered once weekly. For example, the BCMA-specific antibody may be administered intravenously at a step-up dose of about 0.1 μg / kg, about 0.2 μg / kg, about 0.3 μg / kg, about 0.6 μg / kg, about 1.2 μg / kg, about 2.4 μg / kg, about 4.8 μg / kg, about 9.6 μg / kg, about 10 μg / kg, about 19.2 μg / kg, about 20 μg / kg, or any dose therebetween, followed by a weekly intravenous dose of about 35 μg / kg, about 38.4 μg / kg, about 40 μg / kg, about 50 μg / kg, about 57.6 μg / kg, about 60 μg / kg, about 80 μg / kg, or any dose therebetween.

[0073] In one embodiment, the BCMA specific antibody is administered intravenously at a step-up dose, followed by a higher step-up dose, followed by a third higher dose once weekly. For example, the BCMA specific antibody is administered intravenously at a step-up dose of about 0.1 μg / kg, about 0.2 μg / kg, about 0.3 μg / kg, about 0.6 μg / kg, about 1.2 μg / kg, about 2.4 μg / kg, about 4.8 μg / kg, about 9.6 μg / kg, about 10 μg / kg, about 19.2 μg / kg, about 20 μg / kg, or any dose therebetween, followed by a step-up dose of about 35 μg / kg, about 38.4 μg / kg, about 40 μg / kg, about 45 μg / kg, about 50 μg / kg, about 55 μg / kg, about 60 μg / kg, about 65 μg / kg, about 70 μg / kg, about 75 μg / kg, about 80 μg / kg, about 85 μg / kg, about 90 μg / kg, about 100 μg / kg, about 120 μg / kg, about 140 μg / kg, about 160 μg / kg, about 180 μg / kg, about 190 μg / kg, about 200 μg / kg, or any dose therebetween. The compound may be administered intravenously at a step-up dose of about 40 μg / kg, about 50 μg / kg, about 57.6 μg / kg, about 60 μg / kg, about 80 μg / kg, or any dose therebetween, followed by weekly intravenous administration at a dose of about 80 μg / kg, about 100 μg / kg, about 120 μg / kg, about 180 μg / kg, about 240 μg / kg, about 270 μg / kg, or any dose therebetween.

[0074] In one embodiment, the BCMA-specific antibody is administered intravenously in a step-up dose, followed by a higher step-up dose, followed by a third higher step-up dose, followed by a fourth higher dose once weekly. For example, a BCMA specific antibody is administered intravenously at a step-up dose of about 0.1 μg / kg, about 0.2 μg / kg, about 0.3 μg / kg, about 0.6 μg / kg, about 1.2 μg / kg, about 2.4 μg / kg, about 4.8 μg / kg, about 9.6 μg / kg, about 10 μg / kg, about 19.2 μg / kg, about 20 μg / kg, or any dose therebetween, followed by a step-up dose of about 35 μg / kg, about 38.4 μg / kg, about 40 μg / kg, about 50 μg / kg, about 57.6 μg / kg, about 60 μg / kg, about 80 μg / kg, or any dose therebetween; This may then be followed intravenously at a step-up dose of about 80 μg / kg, about 100 μg / kg, about 120 μg / kg, about 180 μg / kg, about 240 μg / kg, about 270 μg / kg, or any dose therebetween, followed by weekly intravenous administration at a dose of about 300 μg / kg, about 720 μg / kg, about 850 μg / kg, about 1000 μg / kg, about 1100 μg / kg, about 1200 μg / kg, about 1300 μg / kg, about 1400 μg / kg, about 1500 μg / kg, about 1600 μg / kg, about 1700 μg / kg, about 1800 μg / kg, or any dose therebetween.

[0075] In one embodiment, the BCMA specific antibody is administered subcutaneously in a single dose once weekly. For example, the BCMA specific antibody is administered at a dose of about 0.1 μg / kg, about 0.2 μg / kg, about 0.3 μg / kg, about 0.6 μg / kg, about 1.2 μg / kg, about 2.4 μg / kg, about 4.8 μg / kg, about 9.6 μg / kg, about 19.2 μg / kg, about 20 μg / kg, about 35 μg / kg, about 38.4 μg / kg, about 40 μg / kg, about 50 μg / kg, about 57.6 μg / kg, about 60 μg / kg, about 80 μg / kg, about 100 μg / kg, about 120 μg / kg, about 180 μg / kg, about 240 μg / kg, about 270 μg / kg, about 300 μg / kg, about 4 ...

[0043] The present invention may be administered subcutaneously once weekly in an amount of about 720 μg / kg, about 850 μg / kg, about 1000 μg / kg, about 1100 μg / kg, about 1200 μg / kg, about 1300 μg / kg, about 1400 μg / kg, about 1500 μg / kg, about 1500 μg / kg, about 1600 μg / kg, about 1700 μg / kg, about 1800 μg / kg, about 2000 μg / kg, about 2500 μg / kg, about 3000 μg / kg, about 3500 μg / kg, about 4000 μg / kg, about 4500 μg / kg, about 5000 μg / kg, or any dose therebetween.

[0076] In one embodiment, the BCMA specific antibody is administered subcutaneously in a step-up dose followed by weekly administration of a higher dose. For example, the BCMA specific antibody may be administered subcutaneously at a step-up dose of about 10 μg / kg, about 20 μg / kg, about 35 μg / kg, about 40 μg / kg, about 50 μg / kg, about 60 μg / kg, or any dose therebetween, followed by weekly administration of a dose of about 80 μg / kg, about 100 μg / kg, about 240 μg / kg, about 300 μg / kg, or any dose therebetween.

[0077] In one embodiment, the BCMA specific antibody is administered subcutaneously at a step-up dose, followed by a higher step-up dose, followed by a third higher dose once weekly. For example, the BCMA specific antibody is administered subcutaneously at a step-up dose of about 10 μg / kg, about 20 μg / kg, about 35 μg / kg, about 40 μg / kg, about 50 μg / kg, about 60 μg / kg, or any dose therebetween, followed by a step-up dose of about 80 μg / kg, about 100 μg / kg, about 240 μg / kg, about 300 μg / kg, or any dose therebetween; It may then be administered subcutaneously once weekly at a dose of about 240 μg / kg, about 720 μg / kg, about 1100 μg / kg, about 1200 μg / kg, about 1300 μg / kg, about 1400 μg / kg, about 1500 μg / kg, about 1600 μg / kg, about 1700 μg / kg, about 1800 μg / kg, about 2000 μg / kg, about 2500 μg / kg, about 3000 μg / kg, or any dose therebetween.

[0078] In some embodiments, the BCMA specific antibody is administered for a sufficient time to achieve a complete response, stringent complete response, very good partial response, partial response, minimal response, or stable disease state and may continue until disease progression or lack of patient benefit. Disease state may be determined by any suitable method known to those of skill in the art in light of the present disclosure, such as, for example, analysis of serum and urinary monoclonal protein concentrations, M protein levels, BCMA levels, etc.

[0079] In some embodiments, the BCMA-specific antibody is administered for a sufficient time to achieve a complete response characterized by a negative minimal residual disease (MRD) status. Negative MRD status can be determined by any suitable method known to one of skill in the art in view of the present disclosure. In some embodiments, negative MRD status is determined using next generation sequencing (NGS). In some embodiments, negative MRD status is determined within 10 -4 cells, 10 -5cells, or 10 -6 It is determined by cells.

[0080] BCMA-specific antibodies can also be administered prophylactically to reduce the risk of cancer progression, delay the onset of occurrence of events in the progression of cancer, and / or reduce the risk of recurrence when the cancer goes into remission.

[0081] In some embodiments, the method further comprises administering to the subject one or more anti-cancer therapies.

[0082] In some embodiments, the one or more anti-cancer therapies are selected from the group consisting of autologous stem cell transplant (ASCT), radiation, surgery, chemotherapeutic agents, immunomodulatory agents, and targeted cancer therapies.

[0083] In some embodiments, the one or more anticancer therapies are selinexor, venetoclax, lenalidomide, thalidomide, pomalidomide, bortezomib, carfilzomib, elotozumab, ixazomib, melphalan, dexamethasone, vincristine, cyclophosphamide, hydroxydaunorubicin, prednisone, rituximab, imatinib, dasatinib, nilotinib, bosutinib, ponatinib, bafetinib, saracatinib , selinexor, venetoclax, tozasertib or danusertib, cytarabine, daunorubicin, idarubicin, mitoxantrone, hydroxyurea, decitabine, cladribine, fludarabine, topotecan, etoposide 6-thioguanine, corticosteroids, methotrexate, 6-mercaptopurine, azacitidine, arsenic trioxide and all-trans retinoic acid, or any combination thereof. EXAMPLES

[0084] The present invention is further defined in the following examples. It should be understood that these examples, while showing preferred embodiments of the present invention, are provided as examples only and should not be interpreted as limiting the scope of the appended claims. From the above discussion and these examples, those skilled in the art can confirm that the essential features of the present invention can be variously changed and modified to suit various uses and conditions without departing from the spirit and scope of the present invention.

[0085] Example 1 - Evaluation of BCMA expression by B-cell malignant cell lines and enhancement by inhibition of γ-secretase To assess whether other malignancies besides multiple myeloma (MM) could potentially be targeted by BCMA-directed immunotherapy, B-cell malignant tumor cell lines were assessed for BCMA expression by flow cytometry. Figure 1A-Figure 1E show the results of the assessment in which BCMA was found to be expressed by different B-cell malignancies and could be enhanced by γ-secretase inhibition. According to Figure 1A, B-cell malignant tumor cell lines were cultured and basal levels of BCMA were assessed by flow cytometry and compared to isotype control. (n=3-8). The dotted line indicates no increase compared to isotype control. According to Figure 1B, cell lines were treated with 100 nM γ-secretase inhibitor or media control for 24-48 hours and BCMA was assessed by flow cytometry. Values ​​are expressed as fold increase compared to 0 nM γ-secretase inhibitor. (n=3-12) The dotted line indicates no increase compared to 0 nM γ-secretase inhibitor. Figure 1C represents the results of evaluation of soluble BCMA by ELISA in supernatants of B cell malignant cell lines after treatment for 24-48 hours with 0 nM or 100 nM γ-secretase inhibitors (n=2). Figure 1D represents evaluation of BCMA mRNA relative to GAPDH control by qPCR after treatment of B cell malignant cell lines with or without 100 nM γ-secretase inhibitors for 24 hours. Figure 1E shows the correlation between BCMA membrane expression and BCMA mRNA expression in B cell malignant cell lines without or with 100 nM γ-secretase inhibition for 24 hours. P values ​​were calculated by Wilcoxon paired t-test (Figure 1A-B) or simple linear regression (Figure 1E). Data are presented as mean ± SD. * P < .05; ** P < .01; *** P<0.001 **** P<.0001.

[0086] Thus, MM cell lines U266 (gMFI5529) and RPMI-8226 (MM, gMFI4621) expressed high levels of BCMA (Figure 1A). However, in addition to MM, high levels of BCMA could be detected on WM cell lines (MWCL1; gMFI2762 and BCWM.1; gMFI2069). Lower, but still detectable levels of BCMA were found on cell lines of CLL (CII; gMFI2059, PGA; gMFI2097, Mec-1; gMFI1376), Burkitt's lymphoma (Daudi; gMFI1456 and Ramos; gMFI1300), DLBCL (OCI-Ly7; gMFI1086 and OCI-Ly3; gMFI1177) and MCL (JeKo-1; gMFI675) (Figure 1A). As expected, BCMA was not detected on Jurkat cells derived from T-cell acute lymphoblastic leukemia ( Fig. 1A ).

[0087] As BCMA is known to be cleaved by γ-secretase, we assessed whether inhibition of this enzyme would result in enhanced BCMA levels in these B cell lines. To assess this, the different B cell malignant tumor lines were incubated with 100 nM of a γ-secretase inhibitor (Ly411575) for 24–48 h and the fold increase in BCMA was compared to unstimulated cells. The viability of the various cell lines was not affected by γ-secretase inhibition (Figure 7A). All B cell malignant tumor cell lines showed increased BCMA levels after γ-secretase inhibition (Figure 1B). In addition to cell lines that already had high basal levels of BCMA (U266, RPMI-8226, MWCL1, and BCWM.1), cell lines that had low basal expression of BCMA, such as JeKo-1 and OCI-Ly7, were also able to upregulate BCMA after γ-secretase inhibition (Figure 1B). Again, Jurkat cells did not show upregulation of BCMA even after γ-secretase inhibition (Figure 1B). The upregulated levels of BCMA after γ-secretase inhibition suggest an active shedding of BCMA by γ-secretase. This was tested by determination of soluble BCMA (sBCMA) levels in the supernatants of B-cell malignant cell lines cultured for 24 or 48 h in the absence or presence of γ-secretase inhibitors. Indeed, sBCMA levels were detectable upon culture of selected different cell lines (except Jurkat) and increased with longer culture times (Figure 1C). Nevertheless, sBCMA was strongly reduced upon addition of γ-secretase inhibitors at both time points (Figure 1C), indicating that the observed increase was due to prevention of cleavage. This was further confirmed when evaluating mRNA levels, which remained equal before and after γ-secretase inhibition (Figure 1D). Consistent with this, when BCMA molecules per cell were quantified, the amount of BCMA per cell correlated strongly with mRNA levels after γ-secretase inhibition (R 2 = 0.92) (Figure 1E). This is a weak correlation (R 2= 0.36) Collectively, these data indicate that in addition to MM, cell lines derived from different B cell malignancies express BCMA and that such expression can be enhanced by γ-secretase inhibition.

[0088] Example 2 - Assessment of BCMA expression in primary CLL and B cell lymphoma samples The results regarding BCMA expression in different B-cell lymphoma cell lines prompted us to investigate whether similar results could be observed in primary material from CLL and B-cell lymphoma patients. Primary CLL samples were stained for BCMA expression by flow cytometry and compared to isotype controls. As shown in Figure 2A-F, BCMA is lowly expressed on primary CLL cells and can be slightly enhanced by γ-secretase inhibition. As provided in Figure 2A, CLL cells were cultured and basal levels of BCMA were assessed by flow cytometry and compared to isotype controls. (n=25). For Figure 2B, CLL cells were treated with 0 nM or 100 nM of γ-secretase inhibitor for 24 h or 48 h and BCMA was assessed by flow cytometry. Values ​​are expressed as fold increase compared to media control. (n=12-28). Figure 2C provides basal levels of BCMA compared to isotype controls among CLL patients with mutated or non-mutated IgVH. (n=4-10). Figure 2D shows the fold increase in BCMA after 24-48 hours of treatment with 100 nM γ-secretase inhibitor compared to media control among CLL patients with mutated or non-mutated IgVH. (n=5-13). Figure 2E provides the results of assessment of BCMA mRNA by qPCR relative to GAPDH control after 24 hours of treatment of primary CLL samples without or with 100 nM γ-secretase inhibitor. (n=9). Figure 2F provides assessment of soluble BCMA by ELISA in supernatants of B cell malignant cell lines after 24-48 hours of treatment with 100 nM γ-secretase inhibitor or media control. (n = 4–12) P values ​​were calculated by Wilcoxon test (Figures 2A–B), Mann-Whitney test (Figures 2B, 2C, and 2D), or paired t test (Figures 2E and 2F). Data are presented as mean ± SD. * P < .05; ** P < .01; *** P < 0.001; **** P<.0001.

[0089] Thus, primary CLL cells showed slight expression of BCMA (Figure 2A). Incubation of CLL with γ-secretase inhibitors for 24 hours resulted in a small but significant upregulation of BCMA, which was further enhanced after 48 hours (Figure 2B). Viability of CLL cells was not affected by the inhibitors (Figure 8A). No difference in BCMA levels was observed before or after γ-secretase inhibitor treatment between CLL samples with mutated and non-mutated immunoglobulin heavy variable region (IgHV) status (Figures 2C-D). Low but measurable mRNA levels of BCMA expression could be detected on CLL cells (Figure 2E). Despite low BCMA detection by flow cytometry, sBCMA could be detected in the supernatants of CLL cells already after 24 hours of culture, which increased after 48 hours (Figure 2F). Treatment with a γ-secretase inhibitor led to a marked decrease in sBCMA levels, indicating active shedding of BCMA from these CLL cells ( Fig. 2F ).

[0090] To evaluate whether BCMA could be detected in LNs of CLL patients, BCMA IHC was performed. BCMA expression by IHC was also evaluated on primary material in bone marrow or lymph nodes of MM, WM, DLBCL, and MCL patients. Figures 3A-B show BCMA expression in different B-cell malignancies, showing immunohistochemistry of paraffin-embedded slides of different B-cell malignancies at 400x magnification. In Figure 3A, tumor cells were identified by disease type based on staining for Pax-5 for CLL (n=4) and DLBCL (n=3), IgM for WM (n=3), Cyclin D1 for MCL (n=3), and CD138 for MM (n=4). Figure 3B represents examples of strong, moderate, weak, and no expression of BCMA by IHC both in the membrane and Golgi.

[0091] Therefore, to determine the amount of tumor cells expressing BCMA, tissues were also stained for CD138 (MM), IgM (WM), Cyclin D1 (MCL), and Pax-5 (CLL and DLBCL) (Figure 3A). BCMA expression was classified based on the intensity of both membrane and Golgi complex staining (Figure 3B). The results for different B cell lymphomas and CLL are summarized below in Table 2.

[0092] Table 2 - BCMA expression by IHC in different B cell malignancies. For each tumor type, the amount of tumor cells was determined. BCMA positivity, either on the membrane or within the Golgi, was determined as a percentage of the total tumor cells. Percentages were assessed independently by two pathologists.

[0093] [Table 2]

[0094] Bone marrow biopsy samples from MM patients showed the strongest BCMA expression in tumor cells, either as Golgi staining or membrane expression. BCMA could also be easily detected in bone marrow samples from patients with WM. BCMA expression was weak in LN biopsy specimens from CLL and DLBCL patients, and BCMA could not be detected in LN samples obtained from MCL patients. These results indicate that BCMA can be expressed in other B-cell malignancies besides MM. However, expression was low and in some cases restricted to only a small proportion of tumor cells.

[0095] Example 3 - Co-culture of healthy donor PBMCs with B cell malignant cell lines in the presence of BCMA-specific antibodies Since different B cell malignant cell lines express BCMA to varying degrees, we investigated whether co-culture of these cell lines with the BCMAxCD3 BsAb teclistamab in the presence of HD PBMCs would result in T cell activation. To assess this, four cell lines were selected based on previously determined BCMA levels. These were RPMI-8226 (MM, positive control; high BCMA), BCWM.1 (WM, high BCMA), CII (CLL, medium BCMA), and JeKo-1 (MCL, low BCMA). Cell line and age-matched HD PBMCs were cultured in the presence of 100 ng / mL teclistamab or control BsAbs (BCMAxnull or nullxCD3) with or without 100 nM γ-secretase inhibitor. As a positive control, anti-CD3 / CD28 antibodies were added to the co-cultures to induce TCR stimulation.

[0096] Figures 4A-G provide the results of an evaluation of BCMAxCD3 DuoBody® inducing activation, degranulation, cytokine secretion, and cytotoxicity by T cells in the presence of B cell malignant cell lines. PBMCs from healthy donors were left unstimulated or stimulated with 100 ng / mL BCMAxCD3 DuoBody®, BCMAxnull, nullxCD3, or anti-CD3 / CD28 antibodies. Cells were left untreated (-) or treated with 100 nM γ-secretase inhibitor (+). T cells were co-cultured with cell lines RPMI-8226 (multiple myeloma), JeKo-1 (mantle cell lymphoma), BCWM.1 (Waldenström's macroglobulinemia), or CII (chronic lymphocytic leukemia) at a 1:1 E:T ratio. After 48 h, activation by CD25 (Figure 4A), degranulation (Figure 4B), secretion of IFNγ (Figure 4D), IL-2 (Figure 4E), TNFα (Figure 4F), and cytotoxicity (Figure 4G) were measured by flow cytometry (n = 3-14). After 4 days of incubation, T cell proliferation was assessed by FACS (Figure 4C) (n = 3-9).

[0097] Therefore, CD4 + T cells and CD8 +Both T cells showed upregulation of the activation marker CD25 (IL-2 receptor) after 2 days of coculture with various cell lines in the presence of either teclistamab or anti-CD3 / CD28 stimulation (Figures 4A and 9A). No activation was observed using a control BsAb, and addition of teclistamab did not result in upregulation of CD25 when PBMCs were cultured without target cells (Figures 4A and 9A). Similar upregulation was observed after 24 hours for CD107a, IFNγ, IL-2, and TNFα (Figures 4B, 4D-F, 9B, and 9D-F). Activation and proliferation were not dependent on BCMA expression density, as low BCMA expressing cells such as JeKo-1 induced activation to similar levels as the high BCMA expressing cell line RPMI-8226, which was not further enhanced by increasing BCMA levels by γ-secretase inhibition (Figure 4A, Figure 4C, and Figure 9A, Figure 9C). In addition to T cell activation, degranulation, and cytokine production, teclistamab also induced cell death of target cells when co-cultured with HD T cells (Figure 4G). Again, cytotoxicity did not appear to be dependent on BCMA levels, as JeKo-1 was lysed more efficiently than the high BCMA expressing BCWM.1 or CII cell lines, and did not improve with the addition of γ-secretase inhibitors (Figure 4G). Thus, for teclistamab activity, a certain (low) threshold level of BCMA appears to be required to induce adequate T cell activation and cytotoxicity. However, these results also indicate that intratumoral factors may also negatively affect the response to teclistamab.

[0098] Example 4 - Despite low expression of BCMA by CLL cells, BCMA-specific antibodies induce potent lysis of CLL cells We now show that low levels of BCMA expression may be sufficient to confer sensitivity of cell lines to teclistamab. As primary CLL samples express BCMA at even lower levels compared to the JeKo-1 cell line, we investigated whether this expression level was still high enough to induce effective lysis of CLL cells. To assess this, HD T cells were co-cultured with primary CLL cells for 48-96 hours in the presence of 100 ng / mL teclistamab with or without 100 nM γ-secretase inhibitor.

[0099] Figures 5A to 5C show that healthy donor T cells express CD8 + Figure 5 shows how primary CLL cells are killed in the presence of BCMAxCD3 DuoBody, which is highly dependent on T cells. Cytotoxicity was measured after PBMCs from healthy donors were left unstimulated or stimulated with 100ng / mL BCMAxCD3 DuoBody® in the absence (-) or presence (+) of 100nM γ-secretase inhibitor. PBMCs, T cells, were co-cultured with primary CLL cells at an E:T ratio of 10:1 for (Figure 5A) 48 hours or (Figure 5B) 96 hours (n=5). Figure 5C shows the CD4 + , or CD8 + , or CD4 + and CD8 + Figure 5 shows the results of cytotoxicity measurements of primary CLL cells co-cultured for 96 h at a 5:1 E:T ratio with 1:1 IgG (1:1 ratio) and either left untreated (-) or treated with 100 nM γ-secretase inhibitor (+). (n=8) P values ​​were calculated by Wilcoxon test (Figure 5A), paired t test (Figure 5B), or repeated measures one-way ANOVA (Figure 5C). Data are presented as mean ± SD. * P < .05; ** P < .01; *** P<0.001.

[0100] Thus, after 48 hours, induction of cell death could be observed in CLL cells, with an average lysis of 12.9% and 16.4% in both T cell donors. This level increased to an average of 14.9% and 21.6% upon treatment with γ-secretase inhibitors (Figure 5A). After 96 hours, the amount of cell death increased slightly to an average of 15.8% and 20% for both T cell donors, and 25.8% and 27.4% with γ-secretase inhibitor treatment (Figure 5B). In the cell line data, inhibition of γ-secretase did not result in enhanced killing, whereas in CLL this trend could be observed, although it did not reach significance in all T cell donors or at all time points (Figures 5A-B). The contribution of CD4 and / or CD8 could be seen to be related to the increased lysis of CLL cells compared to HD CD4 + , or CD8 + , or CD4 + and CD8 + The effect of CD4+ / CD5+ cells on the expression of CD4+ / CD5+ cells was examined by co-culturing the cells with either CD4+ / CD5+ cells (1:1 ratio) in the presence of teclistamab for 96 h. + T cells are CD8 + It was unable to induce the death of T cells, which was able to induce lysis up to 60% of CD8 + This was in stark contrast to T cells (Figure 5C). In contrast to HD T cells, T cells from CLL patients are known to be dysfunctional, particularly with respect to activation, degranulation, synapse formation, and cytotoxicity. 28~30 .

[0101] We also evaluated whether teclistamab induces activation and cytotoxicity of CLL-derived T cells. To assess activation and degranulation, whole PBMCs from CLL patients were treated with 100ng / mL teclistamab or control BsAb for 96 hours in the presence or absence of γ-secretase inhibition. Figures 6A-C demonstrate how BCMAxCD3 DuoBody induces T cell activation of CLL-derived T cells, resulting in CLL killing. In Figures 6A-C, CLL PBMCs were stimulated with 100ng / mL BCMAxCD3, BCMAxnull, nullxCD3, or anti-CD3 / CD28 antibodies. Flow cytometry analysis of (Figure 6A) CD25 and (Figure 6B) CD107a was performed 4 days later (n=3-5). In Figure 6C, T cells from a CLL patient were isolated and co-cultured with autologous CLL at a 5:1 E:T ratio for 96 h in the presence or absence of 100 ng / mL BCMAxCD3 DuoBody and were left untreated (-) or treated with 100 nM γ-secretase inhibitor (+). (n=6) P values ​​were calculated by regular one-way ANOVA (Figures 6A-B) or paired t-test (Figure 6C). Data are presented as mean ± SD. * P < .05; ** P < .01; *** P < 0.001; **** P<.0001.

[0102] Thus, in the presence of teclistamab, CD4 + T cells and CD8 + A trend towards increased CD25 activation in both CD8 T cells could be observed, which was enhanced by the addition of a γ-secretase inhibitor (Figure 6A), whereas no upregulation could be detected by the addition of a control BsAb. Similar results were obtained when assessing degranulation (measured by CD107a), which was consistent with the CD8 +The BCMA expression was more pronounced in T cells. Finally, co-culture of CLL-derived T cells with their autologous CLL cells for 96 h in the presence of teclistamab resulted in a mean lysis of 40%, which was slightly increased upon addition of γ-secretase inhibition (Figure 6C). These results imply that despite the low BCMA expression in primary CLL cells, these cells can be efficiently lysed by teclistamab when co-cultured with CLL-derived T cells.

[0103] Materials and Methods The following materials, conditions and methods were used in accordance with the experimental work described in Examples 1-3 above.

[0104] Patients and controls. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of CLL patients or buffy coats of (age-matched) healthy donors (HD) from Sanquin Blood Supply (Amsterdam, The Netherlands) using Ficoll-Plaque (VWR). All samples were stored frozen in liquid nitrogen and the CLL samples used had at least 85% CD5 + CD19 + Paraffin-embedded bone marrow and lymph node tissues (bone marrow from MM and Waldenström's Macroglobulinemia (WM) and lymph node (LN) from DLBCL, MCL, and CLL) were obtained from the Department of Pathology at the Amsterdam University Medical Centers, location AMC. Written informed consent was obtained from all subjects in accordance with the Declaration of Helsinki and the study was approved by the Medical Ethics Committee of the Amsterdam UMC (ethical approval number 2013 / 159).

[0105] Bispecific antibodies. Full BCMAxCD3 DuoBody (JNJ-7957, JNJ-64007957) and controls BCMAxnull (BC3B4) and nullxCD3 (CNTO7008) were provided by Janssen Pharmaceuticals.

[0106] Culture conditions. CLL cells, RPMI-8226, MWCL1, BCWM1, Mec-1, Ramos, OCI-Ly7, and Jurkat cells were cultured in Iscove's Modified Dulbecco's Medium (IMDM, Thermo Fisher Scientific). HD or tonsil-derived PBMC, U266, CII, PGA-1, Daudi, OCI-Ly3, and JeKo-1 cells were cultured in RPMI 1640 medium (Thermo Fisher Scientific). The medium was supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin.

[0107] Flow cytometry. PBMCs were washed with PBA (PBS, 0.5% BSA, and 0.02% sodium azide) and stained with fluorescently labeled antibodies for 20 min on ice. BCMA APC, BCMA PE(Biolegend), IgG2a kappa isotype PE(BD Biosciences), CD3 V500(BD Biosciences), CD4 BV605(BD Biosciences), CD4 PerCPefl710(eBioscience), CD5 PE(eBioscience), CD5 PerCPCy5.5(Biolegend), CD8 BV510(Biolegend), CD8 PECy7(eBioscience), CD19 APC(BD Biosciences), CD19 FITC(BD Biosciences), CD20 FITC(BD Biosciences), CD25 APC(BD Biosciences), CD25 BV786(BD Biosciences), CD27 PerCPefl710(eBioscience), CD38 PE(BD Biosciences), CD38 BV421(Sony), CD45RA The following antibodies were used: BV650 (Biolegend), CD107a PECy7 (BD Biosciences), CD138 FITC (Molecular Probes), CCR7 BUV395 (BD Biosciences), IgD PE-CF594 (BD Biosciences), IFNγ BV421 (BD Biosciences), IL-2 PE-Dazzle594 (Biolegend), and TNFα AF700 (BD Biosciences). To exclude dead cells, Fixable Viability Dye eFluor780 was used according to the manufacturer's instructions. For staining of intracellular cytokines, cells were fixed and permeabilized using Fixation / Permeabilization Solution Kit (BD Biosciences). After antibody staining, samples were washed using PBA and acquired on a BD FACS Canto or LSR Fortessa flow cytometer and analyzed with FlowJo v10.To quantify cell numbers using flow cytometry, 123count eBeads™ Counting Beads (Thermo Fisher Scientific) were used according to the manufacturer's instructions. BCMA molecules per cell were determined by using the PE Phycoerythrin Fluorescence Quantification Kit (BD Biosciences).

[0108] BCMA was characterized by flow cytometry and quantitative polymerase chain reaction. Cell lines or CLL cells were cultured either in medium or in the presence of 100 nM γ-secretase inhibitor (Ly411575, Sigma). After 24 or 48 h, BCMA was detected by flow cytometry as described above. Relative expression was calculated compared to isotype control. For qPCR, total RNA was isolated using RNeasy mini kit (Qiagen) and cDNA was transcribed by RevertAid (Fermentas) using random hexamer primers (Promega). qPCR was performed using SYBR Green master mix (Applied Biosystems) and measured with Quantstudio 3 (Applied Biosystems). BCMA expression was normalized to GAPDH. Linear regression software was used for analysis.

[0109] Cytotoxicity assays. Cell lines or primary CLL samples were labeled with Cell Trace Violet (CTV, ThermoFisher Scientific) or carboxyfluorescein diacetate succinimidyl ester (CFSE, ThermoFisher Scientific) according to the manufacturer's instructions and co-cultured with healthy donor PBMCs or CLL-derived (autologous) T cells at different effector to target (E:T) ratios. Where indicated prior to co-culture, CD4 and CD8 T cells were isolated using MACS beads (Miltenyi) according to the manufacturer's instructions. Co-cultures were performed in the presence of 100 ng / mL BCMAxCD3, BCMAxnull, or nullxCD3. Where indicated, 100 nM γ-secretase inhibitor (Ly411575) was added. Target cell viability was assessed using flow cytometry with TO-PRO-3 (Invitrogen) and MitoTracker Orange (Invitrogen). Specific lysis of target cells was calculated as (% target cell death in treated samples - % target cell death in media control) / (100 - % target cell death in media control). * Calculated as 100%. Samples were excluded when cell death in the medium control exceeded 50%.

[0110] T cell proliferation. PBMCs from HD patients were labeled with CTV and cultured alone or with RPMI-8226, JeKo-1, CII, or BCWM1 at a 1:1 E:T ratio. PBMCs were incubated in the presence of 100 ng / mL BCMAxCD3, BCMAxnull, or nullxCD3 or stimulated with CD3 (clone 1XE) and CD28 (clone 15E8) antibodies. Where indicated, 100 nM of γ-secretase inhibitor (Ly411575) was added. After 4 days, proliferation was measured by flow cytometry as described above.

[0111] Activation, cytokine production, and degranulation. PBMCs from HD or CLL patients were incubated in the presence of 100 ng / mL BCMAxCD3, BCMAxnull, or nullxCD3 or stimulated with CD3 (clone 1XE) and CD28 (clone 15E8) antibodies for 2 days. Where indicated, HD PBMCs were cocultured with RPMI-8226, JeKo-1, CII, or BCWM1 at a 1:1 E:T ratio. Where indicated, 100 nM γ-secretase inhibitor (Ly411575) was added. Brefeldin A (10 μg / mL, Invitrogen), GolgiStop (BD Biosciences), and anti-CD107a PE-Cy7 were added for 4–6 h, after which activation, degranulation, and cytokine production were assessed by flow cytometry as described above.

[0112] sBCMA ELISA. Cell lines or CLL cells were cultured either in media or in the presence of 100 nM γ-secretase inhibitor (Ly411575, Sigma). After 24 or 48 hours, supernatants were collected and stored at -20°C. Soluble BCMA (sBCMA) in the supernatants was measured by ELISA using an antibody pair against BCMA.

[0113] BCMA Immunohistochemistry. IHC staining for BCMA (clone E6D7B, Cell Signaling), CD138, Pax-5, Cyclin D1, and IgM was performed on paraffin-embedded bone marrow and LN tissues. Staining was performed by PhenPath Laboratories (Seattle, WA) on a Dako Autostainer EQ240 system. Results were evaluated by two independent pathologists.

[0114] Statistical analysis. Data were checked for normality by D'Agostino-Pearson test or by Shapiro-Wilk test if n<5. P values ​​were calculated by using paired or unpaired two-tailed t-test, Wilcoxon paired-pairs signed-rank test, Mann-Whitney test, repeated measures or ordinary one-way ANOVA (after Bonferroni post-hoc test), or Kruskal-Wallis test (after Dunn's post-hoc test). Correlations were determined by simple linear regression. Statistical analysis was performed using Graphpad PRISM version 8.3.0 with significance set at P<0.05.

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Claims

1. A pharmaceutical composition for use in a method for treating non-Hodgkin lymphoma (NHL) in a human subject, comprising a BCMA-specific antibody, wherein the method comprises administering to the subject a therapeutically effective amount of the BCMA-specific antibody, a pharmaceutical composition.

2. The pharmaceutical composition according to claim 1, wherein the BCMA-specific antibody is bispecific or monospecific.

3. The pharmaceutical composition according to claim 1 or 2, wherein the BCMA-specific antibody is teclistamab.

4. The pharmaceutical composition according to claim 1 or 2, wherein the non-Hodgkin lymphoma is a subtype characterized by the expression of B cell maturation antigen (BCMA).

5. The pharmaceutical composition according to claim 1 or 2, wherein the non-Hodgkin lymphoma is chronic lymphocytic leukemia (CLL), lymphoblastic lymphoma, mantle cell lymphoma, diffuse large B cell lymphoma, or Waldenström macroglobulinemia.

6. The pharmaceutical composition according to claim 1 or 2, wherein the subject is an adult.

7. The amount of teclistamab administered to the subject is effective to activate T cells in the subject, induce neutrophil degranulation in the subject, induce cytokine production in the subject, or any combination thereof, the pharmaceutical composition according to claim 1 or 2.

8. The pharmaceutical composition according to claim 1 or 2, wherein the method further comprises administering to the subject a γ-secretase inhibitor.

9. The pharmaceutical composition according to claim 8, wherein the BCMA-specific antibody and the γ-secretase inhibitor are administered to the subject in a single dosage form.

10. The pharmaceutical composition according to claim 8, wherein the BCMA-specific antibody is administered in a first dosage form and the γ-secretase inhibitor is administered in a second dosage form.

11. A composition comprising a BCMA-specific antibody and a γ-secretase inhibitor in an amount therapeutically effective to treat non-Hodgkin lymphoma (NHL) in a human subject, a composition.

12. A composition for use in a method for treating non-Hodgkin lymphoma (NHL) in a human subject, comprising a BCMA-specific antibody, wherein the composition is used in combination with a γ-secretase inhibitor, a composition. A composition for use in a method for treating non-Hodgkin lymphoma (NHL) in a human subject, the composition comprising a γ-secretase inhibitor, wherein the composition is used in combination with a BCMA-specific antibody. **Claim 14** The composition according to claim 11, wherein the amount of the BCMA-specific antibody is effective to activate T cells in the subject, induce neutrophil degranulation in the subject, induce cytokine production in the subject, or any combination thereof. **Claim 15** The composition according to any one of claims 11 to 13, wherein the BCMA-specific antibody is teclistamab.

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