BCMA as target for t cell redirecting antibodies in b cell lymphoma

Abstract

The present disclosure provides a method for treating non-Hodgkin lymphoma (NHL) in a human subject, the method comprising administering to the subject a therapeutically effective amount of a BCMA-specific antibody. Also disclosed is a composition comprising a BCMA-specific antibody and a gamma-secretase inhibitor in an amount that is therapeutically effective for the treatment of non-Hodgkin's lymphoma (NHL) in a human subject.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 194,470 filed on May 28, 2021 and U.S. Provisional Application No. 63 / 209,694 filed on June 11, 2021, the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present disclosure relates to treatments for B lymphoproliferative disorders. Background Art

[0004] In recent years, the treatment of B-cell lymphoproliferative disorders such as B non-Hodgkin lymphoma (B-NHL) has improved significantly. This is mainly due to the rapidly expanding treatment arm, which includes not only chemo-immunotherapy, 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 ). However, 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.

[0005] It is known from stem cell transplantation (SCT) that long-term survival of T cells mediating anticancer responses is feasible 3 . However, this mostly elderly patient population is not suitable for SCT due to the potential for severe graft-versus-host disease (GVHD). Because the use of autologous T-cell-based therapy arguably does not lead to the development of GVHD, it may be a preferred treatment modality. Different autologous-based T cells have 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 patients with follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), and mantle cell lymphoma (MCL). 4 In contrast, CAR T cells have proven to be more promising 4 Currently available CAR T cells are produced in a patient-specific manner, which makes them time-consuming and expensive. CAR-T cells are only given once, and the development of T-cell exhaustion is a common cause of treatment failure. 5 .

[0006] Consistent with the BCMA expression on PB and PC, there have been many reports indicating that BCMA is highly expressed in multiple myeloma (MM) and is therefore a suitable target for BCMA-targeted therapy. 17Similar to plasma cells, BCMA signaling also promotes the survival of MM cells 17,18 However, since BCMA expression has also been observed on tonsillar memory B cells and germinal center B cells 21-23 , it remains unresolved whether other mature B-cell malignancies also express BCMA and can therefore also be targeted using BCMA-directed therapies. Summary of the invention

[0007] Provided herein is a method for treating non-Hodgkin lymphoma (NHL) in a human subject, the method comprising administering to the subject a therapeutically effective amount of a BCMA-specific antibody.

[0008] Also disclosed is a composition comprising a BCMA-specific antibody and a γ-secretase inhibitor in an amount that is therapeutically effective for treating non-Hodgkin lymphoma (NHL) in a human subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figures 1A to 1E Presented are experimental results demonstrating that BCMA is expressed by different B-cell malignancies and can be enhanced by γ-secretase inhibition.

[0010] FIG. 2A to FIG. 2F Provided are evaluation results showing that BCMA is expressed at low levels on primary CLL cells and can be slightly enhanced by γ-secretase inhibition.

[0011] FIG. 3A to FIG. 3B Shown is the expression of BCMA on different B-cell malignancies.

[0012] FIG. 4A to FIG. 4G Demonstrated how BCMA antibodies induce T cell activation, degranulation, cytokine secretion, and cytotoxicity in the presence of B-cell malignancy cell lines.

[0013] FIG. 5A to FIG. 5C Evaluation results are provided showing that healthy donor T cells kill primary CLL cells in the presence of BCMA-specific antibodies that are primarily dependent on CD8+ T cells.

[0014] FIG. 6A to FIG. 6C Shown is how BCMA-specific antibodies induce T cell activation of CLL-derived T cells and lead to CLL killing.

[0015] Figure 7 It was demonstrated that the viability of different cell lines was not affected by γ-secretase inhibition.

[0016] Figure 8Data are presented demonstrating that CLL cell viability is not affected by γ-secretase inhibition.

[0017] 9A to 9F An assessment is provided as to whether anti-BCMA antibody treatment in the presence or absence of a γ-secretase inhibitor results in an increase in markers of T cell activation (CD25), T cell degranulation (CD107a), cytokine expression (IFN-g, IL-2, and TNF-a), and T cell division. DETAILED DESCRIPTION

[0018] The inventive subject matter disclosed by the present invention can be more easily understood by referring to the following detailed description in conjunction with the accompanying drawings and examples forming a part of the present disclosure. It should be understood that these inventions are not limited to the specific products, methods, conditions or parameters described and / or shown herein, and the terms used herein are only used to describe specific embodiments by way of example and are not intended to limit the invention protected by the claims.

[0019] The entire disclosure of each patent, patent application, and publication cited or described in this document is hereby incorporated by reference herein.In this disclosure, superscript numbers refer to the corresponding numbered publications listed below under the "References" heading.

[0020] As used above and throughout this disclosure, the following terms and abbreviations shall be understood to have the following meanings, unless otherwise indicated.

[0021] In this disclosure, unless the context clearly dictates otherwise, the singular forms "a," "an," "the," and "said" include plural references, and references to a specific value include at least that specific value. Thus, for example, reference to "a treatment" refers to one or more such treatments and equivalents thereof known to those skilled in the art, and so forth. Furthermore, when it is indicated that an element "may be" X, Y, or Z, such usage is not intended to exclude other options for the element in all instances.

[0022] When "about" is used above to express a value as an approximation, it should be understood that the specific value constitutes another embodiment. As used herein, "about X" (wherein X is a numerical value) preferably refers to the quoted value ± 10%, including the end value. For example, the phrase "about 8" preferably refers to a value of 7.2 to 8.8, including the end value; as another example, the phrase "about 8%" preferably refers to a value of 7.2% to 8.8%, including the end value. Where present, all ranges include the end value and are combinable. For example, when citing a range of "1 to 5", the quoted range should be understood to optionally include ranges such as "1 to 4", "1 to 3", "1-2", "1-2 and 4-5", "1-3 and 5". In addition, when a list of alternatives is provided affirmatively, such a list may also include embodiments in which any of the alternatives may be excluded. For example, when describing a range of "1 to 5", such description may include situations in which any of 1, 2, 3, 4, or 5 is excluded; thus, a reference to "1 to 5" may include "1 and 3 to 5, but not including 2", or simply "wherein 2 is not included". The phrase "at least about x" is intended to include both "about x" and "at least x". It should also be understood that if a parameter range is provided, the invention provides all integers within the range and their tenths. For example, "2 hours-5 hours" includes 2 hours, 2.1 hours, 2.2 hours, 2.3 hours, etc., up to 5 hours.

[0023] 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 and is a viable target for multiple myeloma (MM). Data on BCMA expression on other B-cell malignancies are sparse, and for CLL, DLBCL, FL, and MCL, conflicting data have been published. 17,24-27 , complicating the question of whether BCMA is a viable target in these diseases. Therefore, it is not clear whether BCMA-specific antibodies (such as BCMA×CD3 Whether terituzumab (terituzumab) can target BCMA in other mature B-cell malignancies besides MM.

[0024] The present inventors evaluated the expression of BCMA on mature B-cell lymphoma cell lines and assessed whether BCMA expression could be enhanced by inhibiting γ-secretase. BCMA expression in primary material of patients with B-NHL (including CLL) was also measured and (semi) quantified. In order to evaluate whether BCMA can be used as a target for B-NHL (including CLL), it was evaluated whether BCMA×CD3BSab (terituzumab, JNJ-7957) can mediate T cell activation and tumor cell killing of lymphoma cell lines. This BsAb has been used by Genmab The technology was developed with enhanced stability compared to the BsAb format. As a proof of concept that BCMA can elicit autologous T cell responses, CLL was used as a target.

[0025] The inventors found that BCMA can be variably detected on all mature B-cell malignancy cell lines tested, with the highest expression in MM, followed by Waldenstrom's macroglobulinemia (WM). In all B-cell lines, γ-sec inhibition increased BCMA expression, even when BCMA basal levels were low. These data were confirmed in primary B-cell lymphomas, where detectable BCMA levels were present in samples from patients with WM, CLL, and diffuse large B-cell lymphoma.

[0026] Co-culturing HD T cells with various B-cell malignancy cell lines in the presence of terituzumab resulted in T-cell activation, proliferation, and cytotoxicity independent of the level of BCMA expression. The efficacy of terituzumab on primary tumor cells was investigated using CLL cells. Despite low BCMA levels, healthy donor T cells lysed CLL cells by up to 40% in the presence of terituzumab. Furthermore, terituzumab induced activation, degranulation, and potent cytotoxicity of CLL-derived T cells when co-cultured with autologous CLL cells.

[0027] Thus, the inventors found that BCMA expression is not limited to MM, but is also present on other mature B-cell malignancies. Targeting BCMA with terituzumab resulted in cytotoxicity of lymphoma cell lines and primary CLL, even in the presence of low BCMA levels.

[0028] Based on these findings, provided herein is a method for treating non-Hodgkin lymphoma (NHL) in a human subject, the method comprising administering to the subject a therapeutically effective amount of a BCMA-specific antibody.

[0029] As used herein, the phrase "therapeutically effective amount" refers to the amount of an active compound that will elicit the biological or medical response sought in a tissue, system, animal, individual or human, including one or more of the following:

[0030] (1) at least partially preventing a disease or condition or its symptoms; for example, preventing a disease, condition or disorder in an individual who may be susceptible to the disease, condition or disorder but does not yet experience or display the pathology or symptomology of the disease;

[0031] (2) inhibiting a disease or condition; for example, inhibiting a disease, condition or disorder in an individual experiencing or displaying pathology or symptomology of the disease, condition or disorder (i.e., including arresting further development of the pathology and / or symptomology); and

[0032] (3) at least partially ameliorating a disease or condition; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying pathology or symptomology of the disease, condition or disorder (i.e., including reversal of pathology and / or symptomology).

[0033] A BCMA-specific antibody can be monospecific or multispecific (eg, bispecific), ie, the antibody can be specific for a target other than BCMA, as long as it is also specific for BCMA.

[0034] In certain embodiments, the BCMA-specific antibody is a BCMA×CD3 bispecific antibody.In light of the present disclosure, any suitable BCMA×CD3 bispecific antibody known to those skilled in the art can be used in the present invention.

[0035] Various bispecific antibody formats include the formats described herein and recombinant IgG-like dual targeting molecules, in which the two sides of the molecule each contain Fab fragments or portions of Fab fragments of at least two different antibodies; IgG fusion molecules, in which a full-length IgG antibody is fused to an additional Fab fragment or a portion of a Fab fragment; Fc fusion molecules, in which a single-chain Fv molecule or a stable diabody is fused to a heavy chain constant domain, an Fc region or a portion thereof; Fab fusion molecules, in which different Fab fragments are fused together; heavy chain antibodies (e.g., domain antibodies, nanobodies) based on ScFv and bivalent antibodies, in which different single-chain Fv molecules or different bivalent antibodies or different heavy chain antibodies (e.g., domain antibodies, nanobodies) are fused to each other or to another protein or carrier molecule or a bispecific antibody generated by arm exchange. Exemplary bispecific formats include dual targeting molecules, including dual targeting (DT)-Ig (GSK / Domantis), 2-in-1 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 (AcademicInstitution), SCORPION (Emergent BioSolutions / Trubion, Zymogenetics / BMS) and dual affinity retargeting technology (Fc-DART) (MacroGenics), F(ab)2 (Medarex / AMGEN), bifunctional or Bis-Fab (Genentech), docking lock (DNL) (ImmunoMedics), bivalent bispecific (Biotecnol) and Fab-Fv (UCB-Celltech), bispecific T cell engager (BITE) (Micromet), tandem bivalent antibody (Tandab) (Affimed), dual affinity targeting technology (DART) (MacroGenics), single chain bivalent antibody (Academic), TCR-like antibody (AIT, ReceptorLogics), human serum albumin ScFv fusion (Merrimack) and COMBODY (Epigen Biotech), dual targeting nanobody (Ablynx), dual targeting heavy chain domain only antibody.Various formats of bispecific antibodies have been described, for example, in Chames and Baty, 2009, Curr Opin Drug Disc Dev, Vol. 12, p. 276 and Nunez-Prado et al., 2015, Drug Discovery Today, Vol. 20, No. 5, pp. 588-594.

[0036] In some embodiments, the BCMA×CD3 bispecific antibody comprises any one of the BCMA binding domains described in WO2017 / 031104, the entire contents of which are incorporated herein by reference in their entirety. In some embodiments, the BCMA×CD3 bispecific antibody comprises any one of the CD3 binding domains described in WO2017 / 031104. In some embodiments, the BCMA×CD3 bispecific antibody comprises any one of the BCMA×CD3 bispecific antibodies or antigen-binding fragments thereof described in WO2017 / 031104.

[0037] In some embodiments, the BCMA×CD3 bispecific antibody comprises a CD3 binding domain comprising a heavy chain complementary determining region 1 (HCDR1) of SEQ ID NO: 11, HCDR2 of SEQ ID NO: 12, HCDR3 of SEQ ID NO: 13, a light chain complementary 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.

[0038] In some embodiments, the BCMA×CD3 bispecific antibody comprises a BCMA binding domain comprising a heavy chain complementarity determining region 1 (HCDR1) of SEQ ID NO:1, HCDR2 of SEQ ID NO:2, HCDR3 of SEQ ID NO:3, LCDR1 of SEQ ID NO:4, LCDR2 of SEQ ID NO:5, and 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.

[0039] In some embodiments, the BCMA×CD3 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.

[0040] In some embodiments, the BCMA×CD3 bispecific antibody is chimeric, humanized, or human.

[0041] In some embodiments, the BCMA×CD3 bispecific antibody is an antigen binding fragment. Exemplary antigen binding fragments are Fab, F(ab')2, Fd, and Fv fragments.

[0042] In some embodiments, the bispecific antibody is of IgG1, IgG2, IgG3 or IgG4 isotype. In a preferred embodiment, the bispecific antibody is of IgG4 isotype. An exemplary wild-type IgG4 comprises the amino acid sequence of SEQ ID NO: 21.

[0043] Bispecific antibodies can have any allotype. It is expected that allotype has no effect on the properties of bispecific antibodies such as binding or Fc-mediated effector functions. The immunogenicity of therapeutic antibodies is associated with increased risk of infusion reactions and reduced duration of therapeutic response (Baert et al., (2003) N Engl J Med, Vol. 348: pp. 602-608). The extent to which therapeutic antibodies induce immune responses in hosts can be determined in part by the allotype of the antibody (Stickler et al., (2011) Genes and Immunity, Vol. 12: pp. 213-221). Antibody allotypes are related to amino acid sequence variations at specific positions in the constant region sequence of antibodies. Table 1 shows selected IgG1, IgG2 and IgG4 allotypes.

[0044] Table 1

[0045]

[0046] In some embodiments, the bispecific antibody comprises one or more Fc substitutions that reduce binding of the bispecific antibody to Fcγ receptors (FcγRs) and / or reduce Fc effector functions, such as C1q binding, complement dependent cytotoxicity (CDC), antibody dependent cell-mediated cytotoxicity (ADCC), or phagocytosis (ADCP). Specific substitutions can be compared to wild-type IgG4 of SEQ ID NO: 21.

[0047] The Fc positions that can be substituted to reduce Fc binding to activating FcγRs and subsequently reduce effector function are the following substitutions: L234A / L235A on IgG1, V234A / G237A / P238S / H268A / V309L / A330S / P331S on IgG2, F234A / L235A on IgG4, S228P / F234A / L235A on IgG4, N297A on all Ig isotypes, V234A / G237A on IgG2, K214T / E233P / L234V / L235A / G236 deletion / A327G / P331S on IgG1. A / D365E / L358M on IgG2, H268Q / V309L / A330S / P331S on IgG2, S267E / L328F on IgG1, L234F / L235E / D265A on IgG1, L234A / L235A / G237A / P238S / H268A / A330S / P331S on IgG1, S228P / F234A / L235A / G237A / P238S on IgG4, and S228P / F234A / L235A / G236 deletion / G237A / P238S on IgG4, where residue numbering is according to the EU index.

[0048] An Fc substitution that may be used to reduce CDC is the K322A substitution.

[0049] The well-known S228P substitution can also be made in IgG4 antibodies to enhance IgG4 stability.

[0050] In some embodiments, the bispecific antibody comprises one or more asymmetric substitutions in the first CH3 domain or in the second CH3 domain or in both the first CH3 domain and the second CH3 domain.

[0051] In some embodiments, the one or more asymmetric substitutions are selected from the group consisting of: 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_Y407 V / T394W, T366I_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.

[0052] In some embodiments, the BCMA×CD3 bispecific antibody is of the IgG4 isotype and comprises a phenylalanine at position 405 and an arginine at position 409 in the first heavy chain (HC1), and a leucine at position 405 and a lysine at position 409 in the second heavy chain (HC2), wherein the residue numbering is according to the EU index.

[0053] In some embodiments, the BCMA×CD3 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.

[0054] In some embodiments, the BCMA×CD3 bispecific antibody comprises HC1 of SEQ ID NO:9, a first light chain (LC1) of SEQ ID NO:10, HC2 of SEQ ID NO:19, and a second light chain (LC2) of SEQ ID NO:20.

[0055] In some embodiments, the BCMA×CD3 bispecific antibody is CC-93269, BI 836909, JNJ-64007957 (terituzumab), or PF-06863135. In a preferred embodiment, the BCMA×CD3 bispecific antibody is terituzumab.

[0056] In some embodiments, the amount of Terituzumab administered to a 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. In certain embodiments, the amount of Terituzumab 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.

[0057] The non-Hodgkin's lymphoma treated according to the inventive method can be, for example, any subtype characterized by the expression of B cell maturation antigen (BCMA). For example, 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 common in adults, so the inventive method may include administering BCMA-specific antibodies to adults (e.g., individuals over 16 years old). However, non-Hodgkin's lymphoma may also occur in children, and the inventive method may also be used to treat minors (16 years old or younger individuals).

[0058] The method of the present invention may also include administering a γ-secretase inhibitor to the subject. As described more fully below, administering a γ-secretase inhibitor to a subject can act synergistically with a BCMA-specific antibody. For example, in order to enhance the efficacy of the BCMA-specific antibody, γ-secretase inhibition can be used for subjects expressing BCMA just below a threshold level. As in the case of administering a BCMA-specific antibody, the γ-secretase inhibitor is administered in a therapeutically effective amount, which means that the amount of the γ-secretase inhibitor should be administered in an amount in which the γ-secretase inhibitor provides a therapeutic effect when the subject is also undergoing treatment with a BCMA-specific antibody. The aforementioned definition of "therapeutically effective amount" applies to the amount of the γ-secretase inhibitor when co-administered with a BCMA-specific antibody.

[0059] According to the method of the present invention, treatment with a BCMA-specific antibody can occur substantially simultaneously with treatment with a γ-secretase inhibitor. Treatment with a BCMA-specific antibody that occurs substantially simultaneously with a γ-secretase inhibitor refers to a situation where there is a time overlap between treatment with a BCMA-specific antibody and treatment with a γ-secretase inhibitor. Therefore, treatment with a BCMA-specific antibody occurs during a time period that at least partially overlaps with the time period during which the administration of the γ-secretase inhibitor occurs, and can be said to be substantially simultaneous. In this case, BCMA-specific antibody treatment may begin before or after the start of treatment with the γ-secretase inhibitor. When there is no overlap between the time period during which treatment with a BCMA-specific antibody occurs and the time period during which the administration of the γ-secretase inhibitor occurs, the treatment can be described as being performed sequentially. Therefore, in certain embodiments, treatment with a BCMA-specific antibody and treatment with a γ-secretase inhibitor may occur sequentially. In this case, BCMA-specific antibody treatment may begin before or after the start of γ-secretase inhibitor therapy.

[0060] In some embodiments, the BCMA-specific antibody and the γ-secretase inhibitor are administered to the subject in a single dosage form. Alternatively, the BCMA-specific antibody can be administered in a first dosage form and the γ-secretase inhibitor in a second dosage form.

[0061] Also disclosed herein is a composition comprising a BCMA-specific antibody and a γ-secretase inhibitor, the amount of which is therapeutically effective for treating non-Hodgkin's lymphoma (NHL) in a human subject. The characteristics and amounts of the BCMA-specific antibody and the γ-secretase inhibitor (including the amount constituting a therapeutically effective amount) can be as described above in conjunction with the method of the present invention for treating non-Hodgkin's lymphoma.

[0062] According to the methods and compositions disclosed herein, BCMA-specific antibodies, γ-secretase inhibitors, or both may be provided in compositions formulated for any type of administration. For example, antibodies and / or inhibitors may be provided in compositions (dosage forms) formulated for oral, topical, parenteral, enteral, or inhaled administration. In certain embodiments, the composition is formulated for oral administration. Antibodies and / or inhibitors may be formulated for pure administration or in combination with conventional pharmaceutical carriers, diluents, or excipients, which may be liquid or solid. Applicable solid carriers, diluents, or excipients may act as binders, disintegrants, fillers, lubricants, glidants, compression aids, processing aids, colorants, sweeteners, preservatives, suspensions / dispersants, tablet disintegrants, encapsulating materials, film formers or coatings, flavoring agents, or printing inks. Any material used to prepare any dosage unit form is preferably pharmaceutically pure and substantially nontoxic at the amount used. In addition, antibodies and / or inhibitors may be incorporated into sustained-release preparations and formulations. Administration in this regard includes administration by, among others, intravenous, intramuscular, subcutaneous, intraocular, intrasynovial, transepithelial including transdermal, ocular, sublingual and buccal; topical including intraocular, epidermal, ocular, rectal and nasal inhalation via insufflation, aerosol and rectal systems.

[0063] In powders, the carrier, diluent or excipient may be a finely divided solid 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 a suitable ratio and compressed into the desired shape and size. For oral therapeutic administration, the antibody and / or inhibitor may be admixed with a carrier, diluent or excipient and used in the form of ingestible tablets, lozenges, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The amount of active compound in such therapeutically useful compositions is preferably such that a suitable dosage will be obtained.

[0064] Liquid carriers, diluents or excipients can be used to prepare solutions, suspensions, emulsions, syrups, elixirs, etc. The antibody and / or inhibitor can be dissolved or suspended in a pharmaceutically acceptable liquid such as water, an organic solvent, a mixture of the two, or a pharmaceutically acceptable oil or fat. The liquid carrier, excipient or diluent may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickeners, colorants, viscosity modifiers, stabilizers or osmotic pressure regulators.

[0065] Suitable solid carriers, diluents and excipients may include, for example, calcium phosphate, silicon dioxide, magnesium stearate, talc, sugar, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, ethyl cellulose, sodium carboxymethyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, a low melting wax, ion exchange resins, cross-linked carboxymethyl cellulose carbon, acacia, pregelatinized starch, crospovidone, HPMC, povidone, titanium dioxide, polycrystalline cellulose, aluminum methahydroxide, agar, tragacanth gum, or mixtures thereof.

[0066] Suitable examples of liquid carriers, diluents and excipients for oral, topical or parenteral administration include water (especially containing the above-mentioned additives, such as cellulose derivatives, preferably sodium carboxymethylcellulose solution), alcohols (including monohydric alcohols and polyhydric alcohols, such as glycols) and their derivatives and oils (such as fractionated coconut oil and peanut oil) or mixtures thereof.

[0067] For parenteral administration, the carrier, diluent or excipient may also be an oily ester, such as ethyl oleate and isopropyl myristate. Also contemplated are sterile liquid carriers, diluents or excipients, which are used in sterile liquid form compositions for parenteral administration. Dispersions may also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under common storage and use conditions, these preparations may contain preservatives to prevent the growth of microorganisms.

[0068] The pharmaceutical form suitable for injection purposes includes, for example, sterile aqueous solutions or dispersions and sterile powders for the immediate preparation of sterile injection solutions or dispersions. In all cases, the form is preferably sterile and fluid, so as to be easily delivered by a syringe. It is preferably stable under manufacturing and storage conditions and preferably can prevent the contamination of microorganisms such as bacteria and fungi. Carriers, diluents or excipients can be solvents or dispersion media containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.), their suitable mixtures and vegetable oils. For example, by utilizing a coating such as lecithin, maintaining the required particle size in the case of dispersions, and utilizing a surfactant, suitable fluidity can be maintained. The effect of preventing microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, etc. In many cases, it is preferred to include isotonic agents, such as sugar or sodium chloride. The extended absorption of the composition for injection can be achieved by using agents such as aluminum monostearate and gelatin that delay absorption.

[0069] Sterile injection solutions can be prepared by incorporating pharmaceutically appropriate amounts of antibodies and / or inhibitors with various other ingredients listed above (as needed) into a suitable solvent, followed by filtration sterilization. Generally speaking, dispersions can be prepared by incorporating antibodies and / or inhibitors into a sterile vehicle containing an alkaline dispersion medium and the other ingredients required in those listed above. With regard to sterile powders for the preparation of sterile injection solutions, preferred preparation methods may include vacuum drying and freeze drying techniques, which obtain powders of active agents or active ingredients and any other desired ingredients from the aforementioned sterile filtered solutions of these ingredients.

[0070] BCMA-specific antibodies (and, when applicable, γ-secretase inhibitors) can be formulated as pharmaceutical compositions comprising from about 1 mg / mL to about 200 mg / mL of the antibody.

[0071] In some embodiments, the pharmaceutical composition further comprises one or more excipients. In some embodiments, the one or more excipients include, but are not limited to, buffers, sugars, surfactants, chelating agents, or any combination thereof.

[0072] In some embodiments, the pharmaceutical composition comprises:

[0073] about 20 mg / mL to about 120 mg / mL of a BCMA-specific antibody, 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;

[0074] About 5 mM to about 20 mM buffer, such as sodium phosphate, KH2PO4, sodium acetate, or sodium citrate at about 5 mM, about 10 mM, about 15 mM, about 20 mM, or any value therebetween;

[0075] about 1% w / v to about 20% w / v sugar, such as 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;

[0076] about 0.01% w / v to about 2% w / v of a surfactant, such as polysorbate 80 (PS-80) or PS-20 at 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

[0077] Ethylenediaminetetraacetic acid (EDTA) or edetate from about 5 mM to about 40 mM, 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 5-6, 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.

[0078] In some embodiments, the pharmaceutical composition further comprises about 0.1 mg / mL to about 5 mg / mL of an amino acid, such as 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 of methionine or arginine.

[0079] In one embodiment, a pharmaceutical composition useful in the present invention comprises a BCMA-specific antibody (such as terituzumab), 20 mM sodium phosphate, 10% weight / volume (w / v) sucrose, 0.06% (w / v) PS80, and 25 μg / mL EDTA at pH 5.4.

[0080] In another embodiment, a pharmaceutical composition useful in the present invention comprises a BCMA-specific antibody (such as terituzumab), 10 mM to 15 mM sodium acetate, 8% (w / v) sucrose, 0.04% (w / v) PS20, and 20 μg / mL EDTA at pH 5.2.

[0081] In another embodiment, a pharmaceutical composition useful in the present invention comprises a BCMA-specific antibody (such as terituzumab), 15 mM KH2PO4, 10% (w / v) cellobiose, 0.05% (w / v) PS20, and 25 μg / mL EDTA at pH 5.1.

[0082] Application

[0083] In some embodiments, the BCMA-specific antibody is administered by intravenous injection.

[0084] In some embodiments, the BCMA-specific antibody is administered by subcutaneous injection.

[0085] The dose of the BCMA-specific antibody administered to a subject with cancer (such as multiple myeloma) is sufficient to alleviate or at least partially arrest the disease being treated (a "therapeutically effective amount"), and includes about 0.1 μg / kg to about 6000 μg / kg, for example, about 0.3 μg / kg to about 5000 μg / kg, about 0.1 μg / kg to about 3000 μg / kg, about 0.2 μg / kg to about 3000 μg / kg, about 0.3 μg / kg to about 3000 μg / kg, about 0.6 μg / kg to about 3000 μg / kg, about 1.2 μg / kg to about 3000 μg / kg, about 19.2 μg / kg to about 15.5 μg / kg, about 20.5 μg / kg to about 25.5 μg / kg, about 25.5 μg / kg to about 25.5 μg / kg, about 20.5 μg / kg to about 25.5 μg / kg about 3000 μg / kg, about 35 μg / kg to about 3000 μg / kg, about 80 μg / kg to about 3000 μg / kg, about 100 μg / kg to about 3000 μg / kg, about 270 μg / kg to about 3000 μg / kg, about 720 μg / kg to about 3000 μg / kg, about 0.1 μg / kg to about 1800 μg / kg, about 0.2 μg / kg to about 1800 μg / kg, about 0.3 μg / kg to about 1800 μg / kg, about 0.6 μg / kg to about 1800 μg / kg, about 1.2 μg / kg to about 1800 μg / kg, about 19.2 μg / kg to about 3000 μg / kg. / kg to about 1800 μg / kg, about 35 μg / kg to about 1800 μg / kg, about 80 μg / kg to about 1800 μg / kg, about 100 μg / kg to about 1800 μg / kg, about 270 μg / kg to about 1800 μg / kg, about 720 μg / kg to about 1800 μg / kg, about 0.1 μg / kg to about 1500 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 1500 μg / kg, about 1.2 μg / kg to about 1500 μg / 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 to 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 19.2 μg / kg to about 850 μg / kg, about 35 μg / kg to about 850 μg / kg, about 80 μg / kg to about 850 μg / kg, about 100 μg / kg to about 850 μg / kg, about 270 μg / kg to about 850 μg / kg, about 720 μg / kg to about 850 μg / kg, about 0.1 μg / kg to about 720 μg / kg, about 0.2 μg / kg to about 720 μg / kg, about 0.3 μg / kg to about 720 μg / kg, about 0.6 μg / kg to about 720 μg / kg g, about 1.2 μg / kg to about 720 μg / kg, about 19.2 μg / kg to about 720 μg / kg, about 35 μg / kg to about 720 μg / kg, about 80 μg / kg to about 720 μg / kg, about 100 μg / kg to about 720 μg / kg, about 270 μg / kg to about 720 μg / kg, about 720 μg / kg to about 720 μg / kg, about 0.1 μg / kg to about 270 μg / kg, about 0.2 μg / kg to about 270 μg / kg, about 0.3 μg / kg to about 270 μg / kg. 0 μg / kg, about 0.6 μg / kg to about 270 μg / kg, about 1.2 μg / kg to about 270 μg / kg, about 19.2 μg / kg to about 270 μg / kg, about 35 μg / kg to about 270 μg / kg, about 80 μg / kg to about 270 μg / kg, about 100 μg / kg to about 270 μg / kg, about 270 μg / kg to about 270 μg / kg, about 720 μg / kg to about 270 μg / kg, about 0.1 μg / kg to about 100 μg / kg, about 0.2 μg / kg g / kg to about 100 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, about 720 μg / kg to about 100 μg / kg of the antibody. 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, about 60μg / kg, about 80μg / kg, about 100μg / kg, about 120μg / kg, about 180μg / kg, about 240μg / kg, about 270μg / kg, about 300μg / kg, about 720μg / kg, about 850μg / kg, about 1000μg / kg, about 1100μg / kg, about 1200μg / kg, about 1300μg / kg, about 1400μg / kg 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, about 5500 μg / kg, about 6000 μg / kg, or any dose therebetween. .

[0086] A fixed unit dose of BCMA-specific antibody may also be administered, such as 50 mg, 100 mg, 200 mg, 500 mg or 1000 mg or any value therebetween, or the dose may be based on the surface area of ​​the patient, such as 500 mg / m 2 , 400mg / m 2 , 300mg / m 2 , 250mg / m 2 , 200mg / m 2 or 100 mg / m 2 Or any value therebetween. Typically 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) doses may be administered to treat cancers such as multiple myeloma, but 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses may also be administered.

[0087] The BCMA-specific antibody can be administered repeatedly after one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, one month, five weeks, six weeks, seven weeks, two months, three months, four months, five months, six months or more. The course of treatment can also be repeated, as in chronic administration. Repeated administration can be the same dose or different doses. For example, the BCMA-specific antibody can be administered at a first dose at weekly intervals for a certain number of weeks, then administered at a second dose once every two weeks for another certain number of weeks, and then administered at a third dose once a week for another certain number of weeks.

[0088] BCMA-specific antibodies can be administered by maintenance therapy, such as, for example, once a week for 6 months or longer. For example, BCMA-specific antibodies can be administered on at least one of days 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 after initiation of treatment, or alternatively, on at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof, in a single dose or every 24, 12, 8, 6, 4, or 2 hours. The invention can be provided as a daily dose 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 to about 850 μg / kg, about 270 μg / kg to about 720 μg / kg of the antibody per day.

[0089] In one embodiment, the BCMA-specific antibody is administered intravenously once a week in a single dose. For example, the BCMA-specific antibody can be 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. / 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, is administered intravenously once a week.

[0090] In one embodiment, the BCMA-specific antibody is administered intravenously twice a week in a single dose. For example, the BCMA-specific antibody can be 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. / 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, administered intravenously twice a week.

[0091] In one embodiment, the BCMA-specific antibody is administered intravenously with an increasing (or "priming") dose, followed by a higher dose once a week. For example, the BCMA-specific antibody can be administered intravenously with an increasing 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, and then administered intravenously once a week at a 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.

[0092] In one embodiment, the BCMA-specific antibody is administered intravenously at an ascending dose, then at a higher ascending dose, followed by administration once weekly at a third higher dose. For example, the BCMA-specific antibody can be administered intravenously at an ascending 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 administration at 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, is administered intravenously in ascending doses, followed by once 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.

[0093] In one embodiment, the BCMA-specific antibody is administered intravenously at an ascending dose, then at a higher ascending dose, then at a third higher ascending dose, and then at a fourth higher dose once a week. For example, the BCMA-specific antibody can be administered intravenously at an ascending 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, and then administered intravenously at an ascending 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, followed by administration once a week. Then, it is intravenously administered at an increasing 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 intravenous administration once a week 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.

[0094] In one embodiment, the BCMA-specific antibody is administered subcutaneously once a week in a single dose. For example, the BCMA-specific antibody can be 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 The amount of about 100 μg / kg, about 200 μg / kg, about 400 μg / kg, about 500 μg / kg, about 600 μg / kg, about 800 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 is administered subcutaneously once a week.

[0095] In one embodiment, the BCMA-specific antibody is administered subcutaneously in ascending doses and then administered once a week at a higher dose. For example, the BCMA-specific antibody can be administered subcutaneously in ascending doses 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, and then administered subcutaneously once a week at a dose of about 80 μg / kg, about 100 μg / kg, about 240 μg / kg, about 300 μg / kg, or any dose therebetween.

[0096] In one embodiment, the BCMA-specific antibody is administered subcutaneously at an ascending dose, then at a higher ascending dose, followed by administration at a third higher dose once a week. For example, the BCMA-specific antibody can be administered subcutaneously at an ascending 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 administration at an ascending dose of about 80 μg / kg, about 100 μg / kg, about 240 μg / kg, about 300 μg / kg, or any dose therebetween, followed by administration at an ascending dose of about 240 μg / kg, about 300 μg / kg, or any dose therebetween, followed by administration at an ascending 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 administration at an ascending dose of about 10 μg / kg, about 20 μg / kg, about 350 μg / kg, about 40 μg / kg, about 50 μg / kg, about 60 μg / kg, or any dose therebetween, followed by administration at an ascending dose of about 100 μg / kg, about 200 μg / kg, about 30 ... about 300 μg / kg, The dosage of about 10 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 is administered subcutaneously once a week.

[0097] In some embodiments, a BCMA-specific antibody is administered for a time sufficient to achieve a complete response, a stringent complete response, a very good partial response, a partial response, a minimal response, or a stable disease state, and can be continued until disease progression or lack of patient benefit. In accordance with the present disclosure, disease status can be determined by any suitable method known to those skilled in the art, including, for example, analysis of serum and urine monoclonal protein concentrations, M protein levels, BCMA levels.

[0098] In some embodiments, a BCMA-specific antibody is administered for a time sufficient to achieve a complete response characterized by a negative minimal residual disease (MRD) state. According to the present disclosure, a negative MRD state can be determined by any suitable method known to those skilled in the art. In some embodiments, a negative MRD state is determined using next generation sequencing (NGS). In some embodiments, a negative MRD state is determined to be at 10 -4 cells, 10 -5 cells or 10 -6 cells.

[0099] BCMA-specific antibodies may also be administered prophylactically to reduce the risk of developing cancer, delay the onset of an event in cancer progression, and / or reduce the risk of recurrence after cancer remission.

[0100] In some embodiments, the method further comprises administering to the subject one or more anti-cancer therapies.

[0101] In some embodiments, the one or more anti-cancer therapies are selected from the group consisting of autologous stem cell transplantation (ASCT), radiation, surgery, chemotherapeutic agents, immunomodulators, and targeted cancer therapies.

[0102] In some embodiments, the one or more anti-cancer therapies are selected from the group consisting of selinexor, venaclat, lenalidomide, thalidomide, pomalidomide, bortezomib, carfilzomib, elotuzumab, ixazomib, melphalan, dexamethasone, vincristine, cyclophosphamide, hydroxydaunorubicin, prednisone, rituximab, imatinib, tinib, dasatinib, nilotinib, bosutinib, ponatinib, bafitinib, sacatinib, selinexor, venaclat, tauzerti or darusertib, 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.

[0103] Example

[0104] The following examples further define the present invention. It should be understood that these examples, although indicating preferred embodiments of the present invention, are given by way of illustration only and should not be construed as limiting the appended claims. Based on the above discussion and these examples, those skilled in the art can ascertain the essential characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications may be made to the present invention to adapt it to various uses and conditions.

[0105] Example 1 - Evaluation of BCMA Expression in B Cell Malignancy Cell Lines and Enhancement by Inhibition of γ-Secretase

[0106] To evaluate whether other malignancies besides multiple myeloma (MM) could potentially be targeted by BCMA-directed immunotherapy, BCMA expression was assessed by flow cytometry on B-cell malignancy cell lines. Figures 1A to 1E Described are the results of an evaluation that revealed that BCMA is expressed by different B-cell malignancies and can be enhanced by γ-secretase inhibition. Figure 1A B cell malignancy cell lines were cultured and basal levels of BCMA were assessed by flow cytometry and compared to isotype controls. (n=3-8). Dashed lines indicate no increase compared to isotype controls. Figure 1B, cell lines were treated with 100 nM γ-secretase inhibitor or with medium 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) Dashed line indicates no increase compared to 0 nM γ-secretase inhibitor. Figure 1C Shown are the results of evaluation of soluble BCMA by ELISA in supernatants of B-cell malignancy cell lines after treatment with 0 nM or 100 nM γ-secretase inhibitor for 24-48 hours (n=2). Figure 1D BCMA mRNA was assessed by qPCR relative to GAPDH control after treatment of B-cell malignancy cell lines for 24 hours without or with 100 nM γ-secretase inhibitor. Figure 1E Figure 3 shows the correlation between BCMA membrane expression and BCMA mRNA expression in B-cell malignancy cell lines without or with 100 nM γ-secretase inhibition for 24 h. Paired t-test with Wilcoxon test ( Figure 1A to Figure 1B ) or simple linear regression ( Figure 1E ) P values ​​were calculated. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001 ****P < 0.0001.

[0107] Thus, the MM cell lines U266 (gMFI 5529) and RPMI-8226 (MM, gMFI 4621) express high levels of BCMA ( Figure 1A ). However, in addition to MM, high levels of BCMA were detected on WM cell lines (MWCL1; gMFI 2762 and BCWM.1; gMFI 2069). Lower but still detectable BCMA levels were found on CLL cell lines (CII; gMFI 2059, PGA; gMFI 2097, Mec-1; gMFI 1376), Burkitt's lymphoma (Daudi; gMFI 1456 and Ramos; gMFI 1300), DLBCL (OCI-Ly7; gMFI 1086 and OCI-Ly3; gMFI 1177), and MCL (JeKo-1; gMFI 675). Figure 1A As expected, BCMA was not detected on Jurkat cells, which are derived from T-cell acute lymphoblastic leukemia ( Figure 1A ).

[0108] Since BCMA is known to be cleaved by γ-secretase, it was evaluated whether inhibition of this enzyme would lead to increased BCMA levels on these B cell lines. To assess this, different B cell malignancy lines were incubated with 100 nM γ-secretase inhibitor (Ly411575) for 24-48 hours and the BCMA fold increase was compared to unstimulated cells. The viability of the different cell lines was not affected by γ-secretase inhibition ( Figure 7 All B-cell malignancy cell lines showed increased BCMA levels following γ-secretase inhibition ( Figure 1B In addition to cell lines that already have high basal BCMA levels (U266, RPMI-8226, MWCL1, and BCWM.1), cell lines that also have low basal BCMA expression, such as JeKo-1 and OCI-Ly7, were also able to upregulate BCMA after γ-secretase inhibition ( Figure 1B ). Again, even after γ-secretase inhibition, Jurkat cells did not show upregulation of BCMA ( Figure 1B ). Upregulated BCMA levels after γ-secretase inhibition suggest that γ-secretase activity sheds BCMA. This was investigated by measuring soluble BCMA (sBCMA) levels in supernatants of B-cell malignancy cell lines cultured for 24 or 48 h in the absence or presence of γ-secretase inhibitors. Indeed, sBCMA levels were detectable when culturing different selected cell lines (except Jurkat) and increased with longer culture times ( Figure 1C However, sBCMA was significantly reduced when γ-secretase inhibitor was added at both time points ( Figure 1C ), suggesting 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, it was observed that the amount of BCMA per cell showed a strong correlation with the mRNA level after γ-secretase inhibition (R 2 =0.92)( Figure 1E This is in contrast to the case without γ-secretase inhibition, which showed only a weak correlation (R 2 =0.36). Taken together, these data indicate that, in addition to MM, cell lines derived from different B-cell malignancies express BCMA and that this expression can be enhanced by γ-secretase inhibition.

[0109] Example 2 - Evaluation of BCMA expression in primary CLL and B cell lymphoma samples

[0110] The results regarding BCMA expression on different B-cell lymphoma cell lines prompted the investigation of whether similar results could be observed in primary material from patients with CLL and B-cell lymphoma. Primary CLL samples were stained for BCMA expression by flow cytometry and compared with isotype controls. FIG. 2A to FIG. 2F As shown in , BCMA expression is low on primary CLL cells and can be slightly enhanced by γ-secretase inhibition. Figure 2A CLL cells were cultured and basal levels of BCMA were assessed by flow cytometry and compared with isotype controls. (n=25). Figure 2B CLL cells were treated with 0 nM or 100 nM γ-secretase inhibitor for 24 h or 48 h and BCMA was assessed by flow cytometry. Values ​​are expressed as fold increase compared to medium control. (n=12-28). Figure 2C Basal levels of BCMA compared to isotype controls in CLL patients with mutated or unmutated IgVH are presented. (n=4-10). Figure 2D Shown are the fold increases in BCMA following treatment with 100 nM γ-secretase inhibitors for 24-48 hours compared to medium controls in CLL patients with mutated or unmutated IgVH (n=5-13). Figure 2E Results are presented for evaluation of BCMA mRNA relative to GAPDH control by qPCR after treatment of primary CLL samples for 24 hours without or with 100 nM γ-secretase inhibitor (n=9). Figure 2F Provided is the assessment of soluble BCMA by ELISA in supernatants of B cell malignancy cell lines after treatment with 100 nM γ-secretase inhibitor or medium control for 24-48 hours. (n=4-12) The BCMA levels were determined by Wilcoxon test ( FIG. 2A to FIG. 2B ), Mann Whitney test ( Figure 2B , Figure 2C , Figure 2D ) or paired t test ( Figure 2E , Figure F) Calculate P value. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0111] Thus, primary CLL cells show low expression of BCMA ( Figure 2A Incubation of CLL with a γ-secretase inhibitor for 24 h did result in a small but significant upregulation of BCMA, which was further enhanced after 48 h ( Figure 2B ). The viability of CLL cells was not affected by the inhibitors ( Figure 8No differences in BCMA levels were observed between CLL samples with mutated versus unmutated immunoglobulin heavy chain variable region gene (IgHV) status before or after γ-secretase inhibitor treatment ( Figure 2C to Figure 2D Low but measurable levels of BCMA mRNA expression were detected on CLL cells ( Figure 2E Although low BCMA was detected by flow cytometry, sBCMA was detectable in the supernatant of CLL cells after 24 h of culture, which increased after 48 h ( Figure 2F Treatment with a γ-secretase inhibitor resulted in a significant decrease in sBCMA levels, suggesting active shedding of BCMA from these CLL cells ( Figure 2F ).

[0112] To evaluate whether BCMA can be detected in LNs of CLL patients, BCMA IHC was performed. BCMA expression by IHC was also evaluated on primary material on bone marrow or lymph nodes of MM, WM, DLBCL, and MCL patients. FIG. 3A to FIG. 3B The expression of BCMA on different B-cell malignancies is described and immunohistochemistry of paraffin-embedded slides of different B-cell malignancies at 400x magnification is shown. Figure 3A In the analysis, tumor cells were identified by pathotype based on Pax-5 staining for CLL (n=4) and DLBCL (n=3), IgM staining for WM (n=3), cyclin D1 staining for MCL (n=3), and CD138 staining for MM (n=4). Figure 3B Examples representing strong, moderate, weak, and no expression of BCMA on the membrane and Golgi apparatus by IHC.

[0113] 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 staining intensity of both membrane and Golgi complex ( Figure 3B ). The results for different B-cell lymphomas and CLL are summarized in Table 2 below.

[0114] Table 2 - BCMA expression by IHC on different B-cell malignancies. The amount of tumor cells was determined for each tumor type. BCMA positivity on the membrane or in the Golgi was determined as a percentage of total tumor cells. The percentages were assessed independently by two pathologists.

[0115]

[0116] Bone marrow biopsy samples from MM patients showed the strongest BCMA expression on tumor cells, as shown by Golgi staining or membrane expression. In addition, BCMA was 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 suggest that BCMA may be expressed on other B-cell malignancies besides MM. However, expression is low and, in some cases, limited to a small proportion of tumor cells.

[0117] Example 3 - Co-culture of healthy donor PBMCs with B cell malignancies in the presence of BCMA-specific antibodies Cell lines

[0118] Since different B-cell malignancy cell lines express BCMA to varying degrees, it was investigated whether co-culture of these cell lines with the BCMA × CD3 BsAb terituzumab in the presence of HD PBMCs would lead to activation of T cells. To assess this, four cell lines were selected based on previously determined BCMA levels: RPMI-8226 (MM, positive control; high BCMA), BCWM.1 (WM, high BCMA), CII (CLL, medium BCMA), and JeKo-1 (MCL, low BCMA). Cell lines and age-matched HD PBMCs were cultured with or without 100 nM γ-secretase inhibitor in the presence of 100 ng / mL terituzumab or control BsAbs (BCMA × blank or blank × CD3). As a positive control, anti-CD3 / CD28 antibodies were added to the co-cultures to induce TCR stimulation.

[0119] FIG. 4A to FIG. 4G Provides an evaluation of BCMA×CD3 in the presence of B-cell malignancy cell lines Induce T cell activation, degranulation, cytokine secretion and cytotoxicity. PBMC from healthy donors were unstimulated or treated with 100ng / mL BCMA×CD3 BCMA×blank, blank×CD3, or anti-CD3 / CD28 antibody stimulation. 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 (Waldenstrom's macroglobulinemia), or CII (chronic lymphocytic leukemia) at a 1:1 E:T ratio. After activation with CD25 for 48 h ( Figure 4A ), degranulation was measured by flow cytometry ( Figure 4B ), IFNγ secretion ( Figure 4D ), IL-2 secretion ( Figure 4E ), TNFα secretion ( Figure 4F ) and cytotoxicity ( Figure 4G ) (n=3-14). Four days after incubation, T cell proliferation was assessed by FACS ( Figure 4C )(n=3-9).

[0120] Therefore, after 2 days of co-culture with different cell lines in the presence of terituzumab or anti-CD3 / CD28 stimulation, CD4 + and CD8 + T cells showed upregulation of the activation marker CD25 (IL-2 receptor) ( Figure 4A and Fig.9A No activation was observed using control BsAbs, and the addition of terituzumab did not result in upregulation of CD25 when PBMCs were cultured in the absence of target cells ( Figure 4A and 9A Similar upregulation was observed for CD107a, IFNγ, IL-2, and TNFα after 24 h ( Figure 4B , Figure 4D To Figure F, Fig. 9B and Fig.9D Activation and proliferation were independent of BCMA expression density, as low BCMA-expressing cells such as JeKo-1 induced activation to levels similar to those of the high BCMA-expressing cell line RPMI-8226 and were not further enhanced by increasing BCMA levels through γ-secretase inhibition ( Figure 4A , Figure 4C and Fig.9A , Fig. 9C In addition to T cell activation, degranulation, and cytokine production, terituzumab also induced cell death of target cells when co-cultured with HD T cells ( Figure 4G Again, since JeKo-1 lysed more efficiently than the higher BCMA-expressing BCWM.1 or CII cell lines and since JeKo-1 did not improve upon addition of a γ-secretase inhibitor, the cytotoxic potential did not appear to be dependent on BCMA levels ( Figure 4G ). Therefore, a specific (low) threshold level of BCMA seems to be necessary for the activity of terituzumab to induce correct T cell activation and cytotoxicity. However, these results also show that tumor-intrinsic factors can also negatively influence the response to terituzumab.

[0121] Example 4 - Despite low BCMA expression in CLL cells, BCMA-specific antibodies induce efficient lysis of CLL cells

[0122] The present invention shows that low amounts of BCMA expression can be sufficient to confer sensitivity to terituzumab on a cell line. Since primary CLL samples express BCMA at even lower levels compared to the JeKo-1 cell line, the inventors explored whether this expression level was still high enough to induce efficient lysis of CLL cells. To assess this, HD T cells were co-cultured with primary CLL cells in the presence of 100 ng / mL terituzumab, with or without 100 nM of a γ-secretase inhibitor for 48-96 hours.

[0123] FIG. 5A to FIG. 5C Figure 3 shows how healthy donor T cells kill primary CLL cells in the presence of BCMA×CD3 DuoBody, which is primarily dependent on CD8 + PBMCs from healthy donors were unstimulated or stimulated with 100 ng / mL BCMA×CD3 in the absence (-) or presence (+) of 100 nM γ-secretase inhibitor. Cytotoxicity measurement after stimulation. PBMCs were co-cultured with primary CLL cells at an E:T ratio of 10:1 for 48 hours ( Figure 5A ) or 96 hours ( Figure 5B ). (n=5) Figure 5C Primary CLL cells were transfected with CD4 + or CD8 + or CD4 + and CD8 + (1:1 ratio) Cytotoxicity measurements of cells co-cultured at an E:T ratio of 5:1 for 96 hours and left untreated (-) or treated with 100 nM γ-secretase inhibitor (+). (n=8) The results were analyzed by Wilcoxon test ( Figure 5A ), paired t test ( Figure 5B ) or repeated measures one-way ANOVA ( Figure 5C ) P values ​​were calculated. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001.

[0124] Thus, after 48 hours, induction of cell death was observed in CLL cells, with an average lysis of 12.9% and 16.4% in the two T cell donors. After treatment with γ-secretase inhibitors, this level increased to an average of 14.9% and 21.6% ( Figure 5A After 96 hours, the amount of cell death in the two T cell donors increased slightly to an average of 15.8% and 20%, and after γ-secretase inhibitor treatment it was 25.8% and 27.4% ( Figure 5B). However, in the cell line data, γ-secretase inhibition did not result in enhanced killing, and although a trend was observed in CLL, it did not reach significance across all T cell donors or time points ( FIG. 5A to FIG. 5B ). CLL cells were isolated from HD CD4 + or CD8 + or CD4 + and CD8 + The contributions of CD4 and / or CD8 were examined by co-culturing them together (1:1 ratio) for 96 hours. + T cells cannot induce CD8 + T cell killing, which is related to CD8 + This is in stark contrast to T cells, which can induce up to 60% lysis ( Figure 5C ). In contrast to HD T cells, T cells derived from CLL patients are known to have dysfunctions in activation, degranulation, synaptogenesis, and cytotoxicity. 28-30 .

[0125] We also evaluated whether terituzumab induces activation and cytotoxicity of CLL-derived T cells.To assess activation and degranulation, whole PBMCs from CLL patients were treated with 100 ng / mL terituzumab or control BsAbs for 96 h in the presence or absence of γ-secretase inhibition. FIG. 6A to FIG. 6C Demonstrated how BCMA×CD3 DuoBody induces T cell activation of CLL-derived T cells and leads to CLL killing. Fig. 6A 6, CLL PBMCs were stimulated with 100 ng / mL BCMA×CD3, BCMA×blank, blank×CD3, or anti-CD3 / CD28 antibodies. Fig. 6A )CD25 and ( Figure 6B ) Flow cytometric analysis of CD107a (n=3-5). Figure 6C T cells were isolated from CLL patients and co-cultured with their autologous CLL at an E:T ratio of 5:1 in the presence or absence of 100 ng / mL BCMA×CD3 DuoBody for 96 h and were either untreated (-) or treated with 100 nM γ-secretase inhibitor (+). (n=6) The data were analyzed by ordinary one-way ANOVA ( FIG. 6A to FIG. 6B ) or paired t test ( Figure 6C ) P values ​​were calculated. Data are presented as mean ± SD. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0126] Therefore, in the presence of terituzumab, CD4+ and CD8 + A trend toward increased CD25 activation was observed in T cells, which was enhanced by the addition of γ-secretase inhibitors ( Fig. 6A ), whereas no upregulation could be detected after addition of control BsAbs. Similar results were obtained when evaluating degranulation (measured by CD107a), although this was not observed in CD8 + Finally, when CLL-derived T cells were co-cultured with their autologous CLL cells in the presence of terituzumab for 96 h, an average lysis of 40% was achieved, which was slightly increased after the addition of γ-secretase inhibition ( Figure 6C These results suggest that despite low BCMA expression on primary CLL cells, these cells can be efficiently lysed by terituzumab after co-culture with CLL-derived T cells.

[0127] Materials and methods

[0128] The following materials, conditions and methods were used in accordance with the experimental work described in Examples 1 to 3 above.

[0129] Patients and Controls: Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood of CLL patients or from buffy coats of (age-matched) healthy donors (HD) from SanquinBlood Supply (Amsterdam, The Netherlands) using Ficoll-Plaque (VWR). All samples were cryopreserved in liquid nitrogen, and the CLL samples used had at least 85% CD5 + CD19 + Purity. Paraffin-embedded bone marrow and lymph node tissue (bone marrow from MM and Waldenstrom's macroglobulinemia (WM) and lymph nodes (LN) from DLBCL, MCL and CLL) were obtained from the Department of Pathology, University Medical Center Amsterdam. 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 Amsterdam UMC (ethical approval number 2013 / 159).

[0130] Bispecific antibodies: whole BCMA×CD3 DuoBody (JNJ-7957, JNJ-64007957) and controls BCMA×blank (BC3B4) and blank×CD3 (CNTO7008) were provided by Janssen Pharmaceuticals.

[0131] 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 RPMI1640 medium (Thermo Fisher Scientific). The medium was supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin.

[0132] Flow cytometry: PBMCs were washed with PBA (PBS, 0.5% BSA and 0.02% sodium azide) and stained with fluorescently labeled antibodies for 20 minutes on ice. The following antibodies were used: BCMA APC, BCMA PE (Biolegend), IgG2aκ isotype PE (BD Biosciences), CD3 V500 (BD Biosciences), CD4 BV605 (BD Biosciences), CD4PerCPefl710 (eBioscience), CD5 PE (eBioscience), CD5 PerCPCy5.5(Biolegend), CD8BV510(Biolegend), CD8 PECy7(eBioscience), CD19 APC(BD Biosciences), CD19 FITC(BDBiosciences), CD20FITC(BD Biosciences), CD25 APC(BD Biosciences), CD25 BV786(BD Biosciences), CD27 PerCPefl710 (eBioscience), CD38 PE (BD Biosciences), CD38 BV421 (Sony), CD45RA 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), TNFα AF700 (BD Biosciences). To exclude dead cells, the fixable viability dye eFluor 780 was used according to the manufacturer's instructions. For staining of intracellular cytokines, cells were fixed and permeabilized using the Fixation / Permeabilization Solution Kit (BD Biosciences). After antibody staining, samples were washed with PBA, acquired on a BD FACS Canto or LSR Fortssa flow cytometer, and analyzed with FlowJo v10. eBeads were counted using flow cytometry, 123 TM Cell numbers were quantified using Counting Beads (Thermo Fisher Scientific) according to the manufacturer's instructions. BCMA molecules per cell were determined using a PE phycoerythrin fluorescence quantification kit (BD Biosciences).

[0133] BCMA was characterized by flow cytometry and quantitative polymerase chain reaction. Cell lines or CLL cells were cultured in culture medium or in the presence of 100nM γ-secretase inhibitor (Ly411575, Sigma). After 24 hours or 48 hours, BCMA was detected by flow cytometry as described above. Relative expression was calculated compared with isotype control. For qPCR, total RNA was isolated using RNeasy micro 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 on Quantstudio 3 (Applied Biosystems). The expression of BCMA was normalized to GAPDH. Linear regression software was used for analysis.

[0134] Cytotoxicity assay: Cell lines or primary CLL samples were labeled with Cell Trace Violet (CTV, ThermoFisher Scientific) or carboxyfluorescein diacetate succinimidyl ester (CFSE, ThermoFisherScientific) according to the manufacturer's instructions and co-cultured with healthy donor PBMC or CLL-derived (autologous) T cells at different effector-target (E:T) ratios. If indicated, CD4 and CD8 T cells were isolated using MACS beads (Miltenyi) according to the manufacturer's instructions before co-culture. Co-culture was performed in the presence of 100 ng / mL BCMA×CD3, BCMA×blank, or blank×CD3. If indicated, 100 nM γ-secretase inhibitor (Ly411575) was added. Flow cytometry was used to assess the viability of target cells using 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 medium control) / (100 - % target cell death in medium control)*100%. Samples were excluded when cell death in medium control exceeded 50%.

[0135] T cell proliferation: PBMCs from HD patients were labeled with CTV and cultured alone or with RPMI-8226, JeKo-1, CII, or BCWM1 at an E:T ratio of 1:1. PBMCs were incubated in the presence of 100 ng / mL BCMA×CD3, BCMA×blank, or blank×CD3 or stimulated with CD3 (clone 1XE) and CD28 (clone 15E8) antibodies. Where indicated, 100 nM γ-secretase inhibitor (Ly411575) was added. After 4 days, proliferation was measured by flow cytometry as described above.

[0136] Activation, cytokine production and degranulation: PBMCs from HD or CLL patients were incubated in the presence of 100 ng / mL BCMA×CD3, BCMA×blank or blank×CD3 or stimulated with CD3 (clone 1XE) and CD28 (clone 15E8) antibodies for 2 days. Where indicated, HD PBMCs were co-cultured with RPMI-8226, JeKo-1, CII or BCWM1 at an E:T ratio of 1:1. Where indicated, 100 nM γ-secretase inhibitor (Ly411575) was added. Brefeldin A (10 ug / mL, Invitrogen), GolgiStop (BD Biosciences) and anti-CD107a PE-Cy7 were added 4-6 hours before evaluating activation, degranulation and cytokine production by flow cytometry as described above.

[0137] sBCMA ELISA: Cell lines or CLL cells were cultured in culture medium or in the presence of 100 nM γ-secretase inhibitor (Ly411575, Sigma). Supernatants were harvested after 24 or 48 hours and stored at -20°C. Soluble BCMA (sBCMA) in the supernatants was measured by ELISA using an antibody pair for BCMA.

[0138] 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 PhenoPath Laboratories (Seattle) on a Dako Autostainer EQ240 system. Results were evaluated by two independent pathologists.

[0139] Statistical analysis: Data were checked for normality by the D'Agostino-Pearson test or by the Shapiro-Wilk test if n < 5. P values ​​were calculated by using two-sided paired or unpaired t-test, Wilcoxon paired signed-rank test, Mann-Whitney test, repeated measures or ordinary one-way ANOVA (followed by Bonferroni post hoc test), or Kruskal-Wallis test (followed by Dunn's test). Correlations were determined by simple linear regression. Statistical analysis was performed using Graphpad PRISM version 8.3.0, and significance was set at P < 0.05.

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Claims

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

2. The method of claim 1, wherein the BCMA-specific antibody is monospecific or bispecific.

3. The method of claim 1 or claim 2, wherein the BCMA-specific antibody is terituzumab.

4. The method of any preceding claim, wherein the non-Hodgkin lymphoma is a subtype characterized by expression of B-cell maturation antigen (BCMA).

5. The method of any preceding claim, wherein the non-Hodgkin lymphoma is chronic lymphocytic leukemia (CLL), lymphoblastic lymphoma, mantle cell lymphoma, diffuse large B-cell lymphoma, or Waldenstrom's macroglobulinemia.

6. The method according to any preceding claim, wherein the subject is an adult.

7. The method of any preceding claim, wherein the amount of terituzumab 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.

8. The method according to any preceding claim, further comprising administering to the subject a γ-secretase inhibitor.

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

10. The method of 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 in an amount that is therapeutically effective for treating non-Hodgkin lymphoma (NHL) in a human subject; and, γ-Secretase inhibitors.

12. The composition of 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.

13. The composition of claim 11 or claim 12, wherein the BCMA-specific antibody is terituzumab.

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