BCMA monoclonal antibody drug conjugate

Abstract

To provide methods for treating multiple myeloma, and compositions that can be used in the methods.SOLUTION: The disclosure provides an antibody-drug conjugate (ADC) comprising a monoclonal antibody, or an antigen-binding fragment thereof, specific to B-cell maturation antigen (BCMA) conjugated to a cytotoxin. The disclosure also provides compositions comprising the antibody-drug conjugate and methods of killing multiple myeloma cells (including multiple myeloma stems cells) that express BCMA, by contact with the ADC.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Incorporation by Reference of Electronically Submitted Materials The computer readable nucleotide / amino acid sequence listing submitted and set forth herein as a single 16,498 byte ASCII (text) file entitled "BCMA-100-WO-PCT-SeqListing.TXT," created July 31, 2018, is incorporated herein by reference in its entirety. [Background technology]

[0002] Multiple myeloma (MM) is a malignant tumor characterized by the accumulation of clonal plasma cells (see, for example, Non-Patent Document 1 and Non-Patent Document 2). Current treatments for MM include chemotherapy, radiation, surgery, biophosphonates, and autologous stem cell transplantation (ASCT). While these treatments often result in remission, nearly all patients eventually relapse and die (see, for example, Non-Patent Document 2 and Non-Patent Document 3).

[0003] B-cell maturation antigen (BCMA) is a tumor necrosis family receptor (TNFR) member expressed on cells of the B-cell lineage (NPL 4). BCMA expression is most abundant on well-differentiated B cells. BCMA is involved in mediating plasma cell survival to maintain long-term humoral immunity. BCMA expression is associated with several cancers, autoimmune disorders, and infectious diseases. Several researchers have detected BCMA RNA widely within multiple myeloma cells and BCMA protein on the surface of plasma cells from multiple myeloma patients (see, e.g., NPL 5; NPL 6; NPL 7; and NPL 8). Therefore, BCMA is being investigated as a promising therapeutic target in multiple myeloma. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Palumbo et al., New England J.Med.,364(11):1046-1060(2011) [Non-patent document 2] Lonial et al.,Clinical Cancer Res.,77(6):1264-1277(2011) [Non-patent document 3] Rajkumar, Nature Rev. Clinical Oncol, 5(8):479-491(2011) [Non-patent document 4] Laabi et al., Nucleic Acids Research, 22(7):1147-1154(1994) [Non-Patent Document 5] Novak et al, Blood, 103(2):689-694(2004) [Non-patent document 6] Neri et al.,Clinical Cancer Research,73(19):5903-5909(2007) [Non-Patent Document 7] Bellucci et al.,Blood,105(10):3945-3950(2005) [Non-patent document 8] Moreaux et al.,Blood,703(8):3148-3157(2004) Summary of the Invention [Problem to be solved by the invention]

[0005] There remains a need for compositions that can be used in methods for treating multiple myeloma. The present invention provides such compositions and methods. [Means for solving the problem]

[0006] The present disclosure provides an antibody drug conjugate (ADC) comprising a monoclonal antibody, or antigen-binding fragment thereof, specific for B-cell maturation antigen (BCMA) conjugated to a cytotoxin. The monoclonal antibody comprises (a) a heavy chain variable region comprising the complementarity determining region 1 (HCDR1) amino acid sequence of SEQ ID NO: 1, the HCDR2 amino acid sequence of SEQ ID NO: 2, and the HCDR3 amino acid sequence of SEQ ID NO: 3, and (b) a light chain variable region comprising the complementarity determining region 1 (LCDR1) amino acid sequence of SEQ ID NO: 4, the LCDR2 amino acid sequence of SEQ ID NO: 5, and the LCDR3 amino acid sequence of SEQ ID NO: 6.

[0007] Additionally, the present disclosure provides compositions comprising the aforementioned antibody drug conjugates and methods of killing BCMA-expressing multiple myeloma cells (including multiple myeloma stem cells) by contacting the multiple myeloma cells with the ADC.

[0008] The present disclosure also provides a monoclonal antibody, or antigen-binding fragment thereof, specific for BCMA, comprising: (a) a heavy chain variable region comprising the complementarity determining region 1 (HCDR1) amino acid sequence of SEQ ID NO: 1, the HCDR2 amino acid sequence of SEQ ID NO: 2, and the HCDR3 amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable region comprising the complementarity determining region 1 (LCDR1) amino acid sequence of SEQ ID NO: 4, the LCDR2 amino acid sequence of SEQ ID NO: 5, and the LCDR3 amino acid sequence of SEQ ID NO: 6. [Brief explanation of the drawings]

[0009] [Figure 1] Figure 1 is a series of graphs (Figures 1A-1C) depicting FACS binding of purified antibodies to adherent 293 (Ad293) cells expressing huBCMA, cynoBCMA, BAFF-R, and TACI, as described in Example 1. The 15B2GL monoclonal antibody was the only cynomolgus monkey cross-reactive antibody tested that did not bind to BAFF-R and / or TACI. [Figure 2]Figure 2 is a graph illustrating the ability of BCMA antibody drug conjugates to kill multiple myeloma (MM) and plasma cell leukemia (PCL) cells in vitro, as described in Example 4. Figures 2A-2H show the viability of specific BCMA-expressing multiple myeloma and plasma cell leukemia cell lines treated with the indicated ADCs, while Figures 21 and 2J show the viability of cell lines that do not express BCMA. [Figure 3] Figure 3 includes graphs depicting the killing of multiple myeloma cell lines in the presence of soluble BCMA by antibody drug conjugate 15B2GL-SG3249 compared to I09-SG3249 ADC in conditioned medium collected from Ad293 cells expressing human BCMA, as described in Example 4 (Figure 3A), or compared to J6M0-mc-MMAF and J6M0-SG3249 ADC in conditioned medium collected from Ad293 cells expressing human BCMA (Figure 3B). [Figure 4] Figure 4 is a graph illustrating the change in tumor volume in an H929 xenograft mouse model of multiple myeloma in response to treatment with the BCMA-targeting ADCs 15B2GL-SG3249, I09-SG3249, L15-SG3249, and J6M0-mc-MMAF compared to untreated mice. [Figure 5] Figure 5 is a graph illustrating the change in tumor volume in the JJN3 xenograft mouse model of multiple myeloma in response to treatment with ADCs 15B2GL-SG3249, I09-SG3249, L15-SG3249, J6M0-mc-MMAF, and isotype control IgG1-SG3249 compared to untreated mice. [Figure 6] FIG. 6 is a graph illustrating the change in tumor volume in the MM.1S xenograft mouse model of plasma cell leukemia in response to treatment with the ADCs 15B2GL-SG3249, I09-SG3249, L15-SG3249, and J6M0-mc-MMAF compared to untreated mice. [Figure 7]FIG. 7 is a graph illustrating the change in tumor volume in the MM.1R xenograft mouse model of plasma cell leukemia in response to treatment with the ADCs 15B2GL-SG3249 and J6M0-mc-MMAF compared to untreated mice. [Figure 8] Figure 8 includes a series of graphs and flow cytometry plots illustrating BCMA expression on MM stem cells. BCMA expression was detected on both MM plasma cells (CD19-CD138+, gray trace) and MM stem cells (CD19+CD138-, black trace). [Figure 9] Figure 9 includes a series of graphs illustrating the sensitivity of MM stem cells from patient samples MM263 (Figure 9A), MM276 (Figure 9B), MM277 (Figure 9C), and MM284 (Figure 9D) to the ADC 15B2GL-SG3249 compared to the ADC J6M0-mc-MMAF in a clonogenic assay. Controls included untreated cells and a nonspecific IgG1-SG3249 conjugate at the highest dose of 400 ng / mL. The number of colonies formed was normalized to the number formed in untreated cultures, which was set at 100%. [Figure 10] Figure 10 is a graph illustrating the ability of BCMA antibody drug conjugates to kill multiple myeloma (MM) and plasma cell leukemia (PCL) cells in vitro, as described in Example 7. Figures 10A-10H show the viability of specific BCMA-expressing multiple myeloma and plasma cell leukemia cell lines treated with the indicated ADCs, while Figures 101 and 10J show the viability of cell lines that do not express BCMA. [Figure 11] Figure 11 is a graph illustrating the change in tumor volume in an H929 xenograft mouse model of multiple myeloma in response to treatment with BCMA-targeting ADCs 15B2GL-SG3400, J6M0-SG3400, and isotype control IgG1-SG3400 compared to untreated mice. [Figure 12]Figure 12 is a graph illustrating the change in tumor volume in the MM.1S xenograft mouse model of plasma cell leukemia in response to treatment with the BCMA-targeting ADCs 15B2GL-SG3400, J6M0-SG3400, and the isotype control IgG1-SG3400 compared to untreated mice. [Figure 13] FIG. 13 is a graph illustrating the measurement of the affinity and kinetics of 15B2GL, I09, P10, and L15 antibodies binding to human BCMA using the SPR-based ProteOn system. [Figure 14] FIG. 14 is a graph illustrating the measurement of the affinity and kinetics of N22, M02, and J6M0 antibodies binding to human BCMA using the SPR-based ProteOn system. [Figure 15] FIG. 15 is a graph depicting the binding of 15B2GL, L15, I09, or J6M0 antibodies to NCI-H929, MM.1S, and Ad293+huBCMA cell lines as measured by flow cytometry. DETAILED DESCRIPTION OF THE INVENTION

[0010] The present disclosure provides antibody-drug conjugates (ADCs) comprising a monoclonal antibody, or antigen-binding fragment thereof, specific for B-cell maturation antigen (BCMA) conjugated to a cytotoxin. The term "antibody-drug conjugate," as used herein, refers to a compound comprising a monoclonal antibody (mAb) linked to a cytotoxin (generally a small molecule drug with high systemic toxicity) via a chemical linker. In some embodiments, the ADC may comprise a small molecule cytotoxin that has been chemically modified to include a linker. The linker is then used to conjugate the cytotoxin to the antibody or antigen-binding fragment thereof. Upon binding to a target antigen on the surface of a cell, the ADC is internalized and transported to the lysosome, where the cytotoxin is released either by proteolysis of the cleavable linker (e.g., by cathepsin B found within lysosomes) or by proteolysis of the antibody if linked to the cytotoxin via a non-cleavable linker. The cytotoxin then travels out of the lysosome and into the cytosol or into the nucleus, where it can then bind to its target, depending on its mechanism of action.

[0011] The term "monoclonal antibody," as used herein, refers to an antibody produced by a single clone of B cells and binding to the same epitope. In contrast, the term "polyclonal antibody" refers to a population of antibodies produced by different B cells and binding to different epitopes of the same antigen. The antibody-drug conjugates described herein may comprise whole antibodies or antibody fragments. A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain has an N-terminal variable (VH) region and three C-terminal constant (CH1, CH2, and CH3) regions, and each light chain has an N-terminal variable (VL) region and a C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen-binding site of an antibody. The VH and VL regions have the same general structure, and each region contains four framework regions whose sequences are relatively conserved. The framework regions are connected by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of the antibody, which is responsible for antigen binding.

[0012] ADCs may comprise antigen-binding fragments of antibodies. The terms "antibody fragment," "antigen-binding fragment," "functional fragment of an antibody," and "antigen-binding portion" are used interchangeably herein and refer to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (see generally Holliger et al., Nat. Biotech., 23(9):1 126-1129 (2005)). An antibody fragment may include, for example, one or more CDRs, a variable region (or portion thereof), a constant region (or portion thereof), or a combination thereof. Examples of antibody fragments include, but are not limited to, (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fv fragment, which consists of the VL and VH domains of a single arm of an antibody; and (iv) a single-chain Fv (scFv), which is a monovalent molecule consisting of two domains (i.e., VL and VH) of an Fv fragment linked by a synthetic linker that allows synthesis of the two domains as a single polypeptide chain (see, e.g., Bird et al., Science, 242:423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988); and Osbourn et al., J. Immunol. 1999, 2000). (See, e.g., U.S. Pat. No. 6,111,161 (1998)), and (v) bispecific antibodies that are dimers of polypeptide chains, each comprising a VH connected to a VL by a peptide linker that is too short to allow pairing between the VH and VL on the same polypeptide chain, thereby driving pairing between complementary domains on different VH-VL polypeptide chains to create a dimeric molecule having two functional antigen-binding sites. Antibody fragments are known in the art and are described in more detail, for example, in U.S. Patent Application Publication No. 2009 / 0093024 A1.

[0013] In one embodiment, the antibody-drug conjugate described herein comprises a monoclonal antibody, or antigen-binding fragment thereof, specific for B-cell maturation antigen (BCMA, also known as CD269). BCMA is a member of the tumor necrosis factor receptor superfamily (see, e.g., Thompson et al., J. Exp. Medicine, 192(1):129-135 (2000), and Mackay et al., Annu. Rev. Immunol., 21:231-264 (2003)). BCMA binds to B-cell activating factor (BAFF) and proliferation-inducing ligand (APRIL) (see, e.g., Mackay et al., supra, and Kalled et al., Immunological Reviews, 204:43-54 (2005)). Among non-malignant cells, BCMA has been reported to be expressed predominantly in plasma cells and a subset of mature B cells (see, e.g., Laabi et al., EMBO J., 77(11):3897-3904 (1992); Laabi et al., Nucleic Acids Res., 22(7):1147-1154 (1994); Kalled et al., supra; O'Connor et al., J. Exp. Medicine, 199(1):91-97 (2004); and Ng et al., J. Immunol., 173(2):807-817 (2004)). Mice deficient in BCMA are healthy and have normal numbers of B cells, but have impaired survival of long-lived plasma cells (see, e.g., O'Connor et al., supra; Xu et al., Mol. Cell. Biol., 21(12):4067-4074 (2001); and Schiemann et al., Science, 293(5537):2111-2114 (2001)).BCMA RNA has been widely detected within multiple myeloma cells and BCMA protein has been detected on the surface of plasma cells from multiple myeloma patients by several investigators (see, e.g., Novak et al., Blood, 103(2):689-694 (2004); Neri et al., Clinical Cancer Research, 73(19):5903-5909 (2007); Bellucci et al., Blood, 105(10):3945-3950 (2005); and Moreaux et al., Blood, 703(8):3148-3157 (2004)).

[0014] In some embodiments, the present disclosure provides a monoclonal antibody, or antigen-binding fragment thereof, specific for BCMA as described above, independent of the antibody-drug conjugate. The monoclonal antibody, or antigen-binding fragment thereof, may comprise (a) a heavy chain variable region comprising the complementarity-determining region 1 (HCDR1) amino acid sequence of SEQ ID NO: 1, the HCDR2 amino acid sequence of SEQ ID NO: 2, and the HCDR3 amino acid sequence of SEQ ID NO: 3, and (b) a light chain variable region comprising the complementarity-determining region 1 (LCDR1) amino acid sequence of SEQ ID NO: 4, the LCDR2 amino acid sequence of SEQ ID NO: 5, and the LCDR3 amino acid sequence of SEQ ID NO: 6. In another embodiment, the monoclonal antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and / or a light chain variable region of the amino acid sequence of SEQ ID NO: 8.

[0015] A monoclonal antibody, or antigen-binding fragment thereof, specific for BCMA may comprise any suitable binding affinity to BCMA or an epitope thereof. The term "affinity" refers to the equilibrium constant for the reversible binding of two agents, and is also referred to as the dissociation constant (K D) The affinity of an antibody or antigen-binding fragment thereof for a target antigen or epitope can be measured using any method known in the art. Such methods include, for example, fluorescence-activated cell sorting (FACS), surface plasmon resonance (e.g., Biacore, ProteOn), biolayer interferometry (BLI, e.g., Octet), kinetic exclusion assays (e.g., KinExA), separable beads (e.g., magnetic beads), antigen panning, and / or ELISA (see, e.g., Janeway et al. (eds.), Immunobiology, 5th ed., Garland Publishing, New York, NY, 2001). It is known in the art that the binding affinity of a particular antibody will vary depending on the method used to analyze the binding affinity.

[0016] A soluble form of BCMA (sBCMA) has been detected in the serum of multiple myeloma patients, with reported values ​​ranging from 3.8 to 1062 ng / mL (Lee et al. Br J Haematol 2016, Sanchez et al. Br J Haematol 2012) and consists of the entire extracellular domain of the molecule (Laurent et al. Nat Commun 2015). Therefore, sBCMA may reduce the efficacy of antibody-based therapies. The functional characteristics of BCMA and recombinant monomeric human BCMA are similar (Laurent et al. Nat Commun 2015). Therefore, to mitigate the potential effect of sBCMA on the efficacy of BCMA antibody-drug conjugates, it is desirable to select an antibody component with weak binding to recombinant monomeric human BCMA and strong binding to membrane-bound BCMA.

[0017] The affinity of a binding agent for a ligand, e.g., the affinity of an antibody for an epitope, can be, for example, about 1 picomolar (pM) to about 1 micromolar (μM) (e.g., about 1 picomolar (pM) to about 1 nanomolar (nM), or about 1 nM to about 1 micromolar (μM)). In one embodiment, the monoclonal antibody or antigen-binding fragment thereof has a K of 100 nanomolar or less (e.g., 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, or about 10 nM, or a range defined by any two of the foregoing values). D In another embodiment, the monoclonal antibody may bind to BCMA with a K of 10 nanomolar or less (e.g., about 9 nM, about 8.5 nM, about 8 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5.5 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM, about 0.05 nM, about 0.025 nM, about 0.01 nM, about 0.001 nM, or a range defined by any two of the foregoing values). D In another embodiment, the monoclonal antibody may bind to BCMA with a K of 200 pM or less (e.g., about 190 pM, about 175 pM, about 150 pM, about 125 pM, about 110 pM, about 100 pM, about 90 pM, about 80 pM, about 75 pM, about 60 pM, about 50 pM, about 40 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, about 10 pM, about 5 pM, about 1 pM, or a range defined by any two of the foregoing values). D may bind to BCMA.

[0018] In one embodiment, the affinity of the BCMA antibody, or antigen-binding fragment thereof, for monomeric BCMA, as measured by surface plasmon resonance (SPR), is about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, or a range defined by any two of the above values, e.g., about 50 nM to about 70 nM, about 55 nM to about 65 nM, or about 58 nM to about 62 nM.

[0019] In one embodiment, the affinity of the BCMA antibody or antigen-binding fragment thereof for membrane-bound BCMA as measured by FACS is 10 nanomolar or less (e.g., about 9 nM, about 8.5 nM, about 8 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5.5 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, about 0.3 nM, about 0.2 nM, about 0.1 nM, about 0.05 nM, about 0.025 nM, about 0.01 nM, about 0.001 nM, or a range defined by any two of the foregoing values).

[0020] Antigen-binding portions or fragments of monoclonal antibodies can be of any size, so long as the portion binds to BCMA. In this regard, antigen-binding portions or fragments of monoclonal antibodies specific for BCMA (also referred to herein as "anti-BCMA monoclonal antibodies") desirably contain between about 5 and 18 amino acids (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or a range defined by any two of the foregoing values).

[0021] In one embodiment, the antibody-drug conjugate comprises the variable region of an anti-BCMA monoclonal antibody. In this regard, the ADC may comprise the light chain variable region, the heavy chain variable region, or both the light chain variable region and the heavy chain variable region of an anti-BCMA monoclonal antibody. Preferably, the ADC comprises the light chain variable region and the heavy chain variable region of an anti-BCMA monoclonal antibody. Monoclonal antibodies that bind to BCMA are disclosed, for example, in International Patent Application Publication No. WO 2010 / 104949. In one embodiment, the monoclonal antibodies of the ADCs described herein comprise (a) a heavy chain variable region comprising the complementarity determining region 1 (HCDR1) amino acid sequence of SYSMN (SEQ ID NO: 1), the HCDR2 amino acid sequence of SISGSSNYIYYADSVKG (SEQ ID NO: 2), and the HCDR3 amino acid sequence of GGNYYVEYFQY (SEQ ID NO: 3), and (b) a light chain variable region comprising the complementarity determining region 1 (LCDR1) amino acid sequence of RASQYISSNYLA (SEQ ID NO: 4), the LCDR2 amino acid sequence of GASNRAT (SEQ ID NO: 5), and the LCDR3 amino acid sequence of QQYGSSPIT (SEQ ID NO: 6). In another embodiment, the monoclonal antibodies of the ADCs described herein may comprise a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8.

[0022] The terms "cytotoxin" and "cytotoxic drug" refer to any molecule that inhibits or prevents the function of a cell and / or causes destruction of the cell (cell death) and / or exerts an anti-proliferative effect. It will be understood that the cytotoxin or cytotoxic drug of an ADC is also referred to in the art as the "payload" of the ADC. Several classes of cytotoxic drugs are known in the art to have potential utility in ADC molecules and can be used in the ADCs described herein. Such classes of cytotoxic drugs include, for example, anti-microtubule agents (e.g., auristatins and maytansinoids), pyrrolobenzodiazepines (PBDs), RNA polymerase II inhibitors (e.g., amatoxins), and DNA alkylating agents (e.g., indolinobenzodiazepine pseudodimers). Examples of specific cytotoxic drugs that may be used in the ADCs described herein include, but are not limited to, amanitin, auristatin, calicheamicin, daunomycin, doxorubicin, duocarmycin, dolastatin, enediyne, lexitropsin, taxane, puromycin, maytansinoid, vinca alkaloid, tubulisin, and pyrrolobenzodiazepines (PBDs). More specifically, the cytotoxic drug may be, for example, AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatstatin, calicheamicin, maytansine, DM1, DM4, vinblastine, methotrexate, netropsin, or a derivative or analog thereof. Cytotoxins suitable for use in ADCs are also described, for example, in International Patent Application Publication Nos. WO 2015 / 155345 and WO 2015 / 157592.

[0023] In one embodiment, the cytotoxic drug may be an anti-microtubule agent, such as tubulysin, maytansinoid, auristatin, or a derivative thereof. The terms "anti-microtubule agent" and "microtubule-targeting agent" are synonymous and refer to agents that inhibit cell division by interfering with microtubules. Tubulysin is a member of a class of natural products isolated from Myxobacterium species (Sasse et al., J. Antibiot., 53:879-885 (2000)), which inhibit tubulin polymerization and act as a mitotic toxin, leading to cell cycle arrest and apoptosis (Steinmetz et al., Chem. Int. Ed., 43:4888-4892 (2004); Khalil et al., Chem. Biochem., 7:678-683 (2006); Kaur et al., Biochem. J., 396:235-242 (2006)). Examples of tubulysins are disclosed in, for example, International Patent Application Publication Nos. WO 2015 / 157594, WO 2004 / 005326, WO 2012 / 019123, WO 2009 / 134279, WO 2009 / 055562, WO 2004 / 005327; U.S. Pat. Nos. 7,776,841, 7,754,885, and 7,816,377; and U.S. Pat. Appl. Nos. 2010 / 0240701, 2011 / 0021568, and 2011 / 0263650.

[0024] In certain embodiments, tubulysin is a compound described in WO 2015 / 157594 (hereby incorporated by reference), such as a compound having the following structure: [ka] or a compound having the following structure: [ka] and compounds having the formula:

[0025] Maytansinoids inhibit the polymerization of the microtubule protein tubulin, thereby preventing the formation of microtubules (see, e.g., U.S. Pat. No. 6,441,163 and Remillard et al., Science, 189:1002-1005 (1975)). Maytansinoids have been shown to inhibit tumor cell growth in vitro using cell culture models and in vivo using experimental animal systems. Furthermore, the cytotoxicity of maytansinoids is 1,000-fold greater than that of conventional chemotherapeutic agents, such as methotrexate, daunorubicin, and vincristine (see, e.g., U.S. Pat. No. 5,208,020). Maytansinoids include maytansine, maytansinol, C-3 esters of maytansinol, and other maytansinol analogs and derivatives (see, e.g., U.S. Pat. Nos. 5,208,020 and 6,441,163). C-3 esters of maytansinol can be naturally occurring or synthetically derived. Furthermore, natural and synthetic C-3 maytansinol esters can be classified as C-3 esters with simple carboxylic acids or C-3 esters with derivatives of N-methyl-L-alanine, the latter being more cytotoxic than the former. Synthetic maytansinoid analogs are also known in the art and are described, for example, in Kupchan et al., J. Med. Chem., 21:31-37 (1978). Methods for making maytansinol and its analogs and derivatives are described, for example, in U.S. Pat. No. 4,151,042. Examples of maytansinoids that may be used in connection with the ADCs described herein include, but are not limited to, N2'-deacetylase-N2'-(3-mercapto-1-oxopropyl)-maytansine (DM1) and N2'-deacetylase-N2'-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4).

[0026] Auristatins represent a class of highly potent antimitotic agents that have shown substantial preclinical activity at well-tolerated doses (Law et al., Cancer Res., 66:2328-2337 (2006); Ma et al., Clin. Cancer Res., 12:2591-2596 (2006); Tse et al., Cancer Res., 12:1373-1382 (2006); and Oflazoglu et al., Br. J. Haematol., 142:69-73 (2008); and Oflazoglu et al., Clin. Cancer Res., 14:6171-6180 (2008)). Auristatin ADCs are currently being evaluated in preclinical and clinical trials. Examples of auristatins that may be used in connection with the ADCs described herein include, but are not limited to, monomethyl auristatin E (MMAE) and the related molecule monomethyl auristatin F (MMAF) (see, e.g., Doronina et al., Nat. Biotechnol., 21:778-784 (2003); and Doronina et al., Bioconjug. Chem., 17:114-124 (2006)).

[0027] In one embodiment, the cytotoxic drug may be a pyrrolobenzodiazepine (PBD) or a PBD derivative. PBDs translocate to the nucleus where they crosslink to DNA, block replication during mitosis, and damage DNA by inducing single-strand breaks, ultimately leading to apoptosis. Some PBDs also have the ability to recognize and bind to specific sequences in DNA. PBDs have the general structure [ka] is.

[0028] PBDs vary in the number, type, and position of substituents on both their aromatic A-ring and pyrrolo C-ring, as well as the degree of saturation of the C-ring. The B-ring contains either an imine (N=C), a carbinolamine (NH-CH(OH)), or a carbinolamine methyl ether (NH-CH(OMe)) at the N10-C11 position, the electrophilic center responsible for alkylating DNA. All known natural products possess the (S)-configuration at the chiral C11a position, which results in a right-handed twist from the C-ring toward the A-ring. This feature also gives the PBD the proper three-dimensional shape to be isohelically aligned with the minor groove of B-form DNA, resulting in an ordered fit at the binding site (Kohn, In: Antibiotics III. Springer-Verlag, New York, pp. 3-11 (1975); and Hurley and Needham-VanDevanter, Acc. Chem. Res., 19:230-237 (1986)). PBDs can form adducts within the minor groove, resulting in interference with DNA processing.

[0029] The first PBD antitumor antibiotic, anthramycin, was discovered in 1965 (Leimgruber et al., J. Am. Chem. Soc., 87:5793-5795 (1965); Leimgruber et al., J. Am. Chem. Soc., 87:5791-5793 (1965)). Since then, several naturally occurring PBDs have been reported, and more than 10 synthetic routes to various analogs have been developed (Thurston et al., Chem. Rev., 433-465 (1994); and Antonow, D. and Thurston, DE, Chem. Rev., 111:2815-2864 (2011)).Family members include abeimicin (Hochlowski et al., J. Antibiotics, 40:145-148 (1987)), ticamycin (Konishi et al., J. Antibiotics, 37:200-206 (1984)), DC-81 (Japanese Patent No. 58180487; Thurston et al., Chem. Brit., 26:767-772 (1990); and Bose et al., Tetrahedron, 48:751-758 (1992)), mazetramycin (Kuminoto et al., J. Antibiotics, 33:665-667 (1980)), neothramycin A and B (Takeuchi et al., al., J. Antibiotics, 29:93-96 (1976)), polothramycin (Tsunakawa et al., J. Antibiotics, 41:1366-1373 (1988)), prothracarcin (Shimizu et al., J. Antibiotics, 29:2492-2503 (1982); and Langley and Thurston, J. Org. Chem., 52:91-97 (1987)), sivanomycin (DC-102) (Hara et al., J. Antibiotics, 41:702-704 (1988); and Itoh et al., J. Antibiotics, 41:1281-1284 (1988)), sibiromycin (Leber et al. al., J. Am. Chem. Soc., 110:2992-2993 (1988)), and tomamycin (Arima et al., J. Antibiotics, 25:437-444 (1972)). PBDs, as well as ADCs containing PBDs, are also described in International Patent Application Publication Nos. WO 2015 / 155345 and WO 2015 / 157592.

[0030] In one embodiment, the PBD is PBD3249 (also referred to herein as “SG3249”), which is described in detail in WO 2014 / 057074 and has the following structure: [ka] It has.

[0031] In another embodiment, the PBD is PBD3315 (also referred to herein as “SG3315”), which is described in detail in WO 2015 / 052322 and has the following structure: [ka] It has.

[0032] In another embodiment, the PBD is SG3400 (also referred to as compound 23), which is described in detail in International Application PCT / EP2017 / 052988, filed February 10, 2017, and has the following structure: [ka] It has.

[0033] A BCMA monoclonal antibody, or antigen-binding fragment thereof, may be conjugated to a cytotoxin using any suitable method known in the art, including site-specific or non-site-specific conjugation methods. Conventional conjugation methods for antibodies typically rely on random (i.e., non-specific) conjugation of a payload to an antibody or antigen-binding fragment thereof through lysines or cysteines. Thus, in some embodiments, an antibody or antigen-binding fragment thereof is randomly conjugated to a cytotoxic drug, e.g., by partial reduction of the antibody or antibody fragment, followed by reaction with the desired drug in the presence or absence of an attached linker moiety. For example, the antibody or antigen-binding fragment thereof may be reduced using dithiothreitol (DTT) or a similar reducing agent. The cytotoxic drug may then be added in molar excess to the reduced antibody or antibody fragment in the presence of dimethyl sulfoxide (DMSO), with or without an attached linker moiety. After conjugation, an excess of free cysteine ​​may be added to quench any unreacted drug. The reaction mixture may then be purified and buffer exchanged into phosphate buffered saline (PBS).

[0034] In other embodiments, cytotoxic agents may be conjugated to BCMA monoclonal antibodies using site-specific conjugation methods at specific reactive amino acid residues to yield homogeneous ADC formulations with uniform stoichiometry. Site-specific conjugation may be through a cysteine ​​residue or a non-natural amino acid. In one embodiment, a cytotoxic agent may be conjugated to an antibody, or antigen-binding fragment thereof, through at least one cysteine ​​residue. In particular, for example, a cytotoxic agent may be chemically conjugated to the side chain of an amino acid at a specific Kabat position within the Fc region of a BCMA monoclonal antibody. In this regard, the cytotoxic agent may be conjugated to the BCMA monoclonal antibody via a cysteine ​​residue at any suitable position within the Fc region of the antibody, including but not limited to, a cysteine ​​at at least one of positions 239, 248, 254, 273, 279, 282, 284, 286, 287, 289, 297, 298, 312, 324, 326, 330, 335, 337, 339, 350, 355, 356, 359, 360, 361, 375, 383, 384, 389, 398, 400, 413, 415, 418, 422, 440, 441, 442, 443 and 446, where numbering corresponds to the EU index in Kabat. In one embodiment, a cytotoxic agent may be conjugated to a BCMA monoclonal antibody through cysteine ​​residues at specific Kabat positions 239 and / or 442 of the BCMA monoclonal antibody, and / or through amino acid residues inserted between Kabat positions 239 and 240 of the BCMA antibody (Dimasi et al., Mol Pharm, 14(5):1501-1516 (2017)). Alternatively, a cytotoxic agent may be conjugated to a BCMA monoclonal antibody or antigen-binding fragment thereof through a thiol-maleimide bond, for example, via sulfhydryl-reactive groups in the hinge and heavy-light chains.

[0035] The BCMA monoclonal antibodies described herein comprise at least one cytotoxic molecule conjugated thereto, although the BCMA monoclonal antibodies may comprise any suitable number of cytotoxic molecules conjugated thereto (e.g., 1, 2, 3, 4, or more cytotoxic molecules) to achieve the desired therapeutic effect. Desirably, the ADCs described herein comprise two cytotoxic molecules conjugated to the BCMA monoclonal antibody.

[0036] The BCMA antibodies described herein are useful for any therapy where targeting of BCMA is desirable, for example, adoptive cell transfer (ACT), bispecific T cell targeting (BiTE), and nanoparticles. In one embodiment, the present disclosure provides a chimeric antigen receptor (CAR) comprising the antigen-binding domain of a BCMA monoclonal antibody described herein linked to a T cell activation moiety. A "chimeric antigen receptor (CAR)" is an artificially constructed hybrid protein or polypeptide comprising the antigen-binding domain of an antibody (e.g., a single-chain variable fragment (scFv)) linked to a T cell signaling or T cell activation moiety. CAR structures have evolved over the last two decades, most commonly incorporating signaling motifs from single-chain variable fragments (scFv) derived from monoclonal antibodies (mAbs) and the TCR ζ chain (referred to as "first-generation" CARs (see, e.g., Okur, FV, Brenner, MK, Methods Mol. Biol., 651:319-45 (2010); and Lee et al., Clin. Cancer. Res., 18(10):2780-2790 (2012)). More recently, second- and third-generation CARs have been developed that incorporate one ("second-generation") or two ("third-generation") costimulatory activation motifs, e.g., from CD28, 4-1BB (CD137), and / or CD134 (OX-40), which enhance proliferation, cytotoxicity, and persistence in vivo (see, e.g., Finney et al., al., J. Immunol., 172:104-13 (2004); Imai et al., Leukemia, 18:676-84 (2004); Maher et al., Nat Biotechnol., 20:70-5 (2002); Milone et al., Mol Ther., 17:1453-64 (2009); and Lee et al., supra).

[0037] The antigen-binding domain of the CAR may comprise a whole monoclonal antibody or a monoclonal antibody fragment, as described herein. In one embodiment, the antigen-binding domain of the CAR may comprise a single-chain Fv (scFv) fragment of an anti-BCMA monoclonal antibody. Methods for generating chimeric antigen receptors and CARs are further described, for example, in Riviere, I. and M. Sadelain, Mol. Ther., 25(5):1117-1124 (2017); Davila, M. L. and M. Sadelain, Int. J. Hematol., 104(1):6-17 (2016); and U.S. Patent Application Publication No. 2015 / 0051266 A1.

[0038] The present disclosure also provides a composition comprising the above-described antibody or antibody-drug conjugate and a pharmaceutically acceptable (e.g., physiologically acceptable) carrier. Any suitable carrier known in the art can be used within the context of the present invention. The choice of carrier will be determined, in part, by the particular site to which the composition may be administered and the particular method used to administer the composition. The composition may optionally be sterile. The composition may be prepared according to conventional techniques, for example, as described in Remington: The Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

[0039] The composition desirably contains the antibody or antibody-drug conjugate in an amount effective to treat or prevent multiple myeloma. Accordingly, the present disclosure provides a method of killing multiple myeloma cells, comprising contacting BCMA-expressing multiple myeloma cells with an antibody or antibody-drug conjugate described herein, or a composition comprising the antibody or ADC described herein, whereby the antibody or antibody-drug conjugate binds to BCMA on the multiple myeloma cells and kills the multiple myeloma cells. The present disclosure also provides the use of an antibody or ADC described herein, or a composition comprising the antibody or ADC, in the manufacture of a medicament for treating multiple myeloma. As discussed herein, multiple myeloma, also known as plasma cell myeloma or Kahler's disease, is a cancer of plasma cells, a type of white blood cell normally involved in the production of antibodies (Raab et al., Lancet, 374:324-329 (2009)). Multiple myeloma affects 1 to 4 people per 100,000 per year. The disease is more common in men and, for reasons still unknown, is twice as common in African Americans as in Caucasian Americans. Multiple myeloma is an extremely rare hematologic malignancy (14%), accounting for 1% of all cancers (Raab et al., supra). Treatment for multiple myeloma typically involves high-dose chemotherapy followed by hematopoietic stem cell transplantation (allogeneic or autologous), but high rates of relapse are common in multiple myeloma patients undergoing such treatment. As discussed above, BCMA is highly expressed by multiple myeloma cells (see, e.g., Novak et al., supra; Neri et al., supra; Bellucci et al., supra; and Moreaux et al., supra).

[0040] As shown herein, BCMA is also expressed on multiple myeloma stem cells. Thus, the present disclosure provides a method for killing multiple myeloma stem cells, comprising contacting BCMA-expressing multiple myeloma stem cells with a composition comprising an antibody-drug conjugate described herein or an ADC described herein, whereby the antibody-drug conjugate binds to BCMA on the multiple myeloma stem cells and kills the multiple myeloma stem cells. Multiple myeloma stem cells can be identified in the bone marrow of multiple myeloma patients by their surface expression of CD19 and lack of CD138 surface expression (see, e.g., Matsui et al., Blood, 103:2332-6 (2004)). These cells are clonogenic in nature and engraft in immunodeficient mice, whereas myeloma plasma cells, defined as CD138+CD19-, do not. Multiple myeloma stem cells are also resistant to current therapies (Matsui et al., Cancer Res., 68:190-7 (2008)).

[0041] An antibody drug conjugate described herein, or a composition comprising an antibody drug conjugate, may be contacted with a population of BCMA-expressing multiple myeloma cells ex vivo, in vivo, or in vitro. "Ex vivo" refers to a method performed inside or on a cell or tissue in an artificial environment outside an organism that minimizes alteration of the natural state. In contrast, the term "in vivo" refers to a method performed within a living organism in its normal, intact state, while an "in vitro" method is performed using components of an organism that have been isolated from their usual biological context. In one embodiment, the multiple myeloma cells are human multiple myeloma cells that are contacted in vivo with an ADC described herein, or a composition comprising an ADC.

[0042] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. Preferably, the effect is therapeutic, i.e., the effect partially or completely cures the disease and / or adverse symptoms that may result from the disease. To this end, the methods of the invention involve administering a "therapeutically effective amount" of an antibody or ADC, or a composition comprising an antibody or ADC, and a pharmaceutically acceptable carrier. A "therapeutically effective amount" refers to an amount effective, at the dosage and for the period necessary, to achieve the desired therapeutic result. A therapeutically effective amount may vary depending on factors such as the individual's condition, age, sex, and weight, as well as the ability of the antibody or ADC to elicit a desired response in the individual. For example, a therapeutically effective amount of an ADC of the invention is an amount that binds to and destroys BCMA on multiple myeloma cells.

[0043] Alternatively, the pharmacological and / or physiological effect may be prophylactic, i.e., the effect completely or partially prevents a disease or its symptoms. In this regard, the methods of the invention involve administering a "prophylactically effective amount" of an ADC or a composition comprising an ADC to a mammal susceptible to multiple myeloma. A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result (e.g., prevention of disease onset).

[0044] Therapeutic or prophylactic effectiveness may be monitored by periodic evaluation of treated patients. In one embodiment, the ADCs described herein inhibit or suppress the proliferation of BCMA-expressing myeloma cells by at least about 10% (e.g., at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%). Cell proliferation can be measured using any suitable method known in the art, including, for example, measuring the incorporation of labeled nucleosides (e.g., 3H-thymidine or bromodeoxyuridine Brd(U)) into genomic DNA (see, e.g., Madhavan, HN, J. Stem Cells Regen. Med., 3(1):12-14 (2007)).

[0045] The antibodies or ADCs described herein, or compositions comprising the antibodies or ADCs, can be administered to a mammal (e.g., a human) using standard administration techniques, including, for example, intravenous, intraperitoneal, or subcutaneous. More preferably, the antibodies or ADCs, or compositions containing them, are administered to a mammal by intravenous injection.

[0046] The antibodies or ADCs described herein, or compositions comprising the antibodies or ADCs, can be administered to a mammal with one or more additional therapeutic agents, which can be co-administered. As used herein, the term "co-administer" refers to administering one or more additional therapeutic agents and an antibody or ADC described herein, or an antibody- or ADC-containing composition, within a sufficiently short period of time that the antibody or ADC can enhance the effect of the one or more additional therapeutic agents, or vice versa. In this regard, the antibody or ADC or composition containing it can be administered first, and the one or more additional therapeutic agents can be administered second, or vice versa. For example, the antibody or ADC or composition containing it can be administered in combination with other agents (e.g., as an adjuvant) to treat or prevent multiple myeloma. In this regard, the antibody or ADC, or antibody- or ADC-containing composition, can be used in combination with at least one other anti-cancer agent, such as any suitable chemotherapeutic agent known in the art, ionizing radiation, small molecule anti-cancer agents, cancer vaccines, biotherapeutic agents (e.g., other monoclonal antibodies, cancer-killing viruses, gene therapy agents, and adoptive T-cell transfer), and / or surgery. Further aspects of the present invention are described below: [Section 1] An antibody drug conjugate (ADC) comprising a monoclonal antibody, or antigen-binding fragment thereof, specific for B-cell maturation antigen (BCMA) conjugated to a cytotoxin, wherein the monoclonal antibody comprises (a) a heavy chain variable region comprising the complementarity determining region 1 (HCDR1) amino acid sequence of SEQ ID NO: 1, the HCDR2 amino acid sequence of SEQ ID NO: 2, and the HCDR3 amino acid sequence of SEQ ID NO: 3, and (b) a light chain variable region comprising the complementarity determining region 1 (LCDR1) amino acid sequence of SEQ ID NO: 4, the LCDR2 amino acid sequence of SEQ ID NO: 5, and the LCDR3 amino acid sequence of SEQ ID NO: 6. [Section 2] The antibody-drug conjugate of item 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:7. [Section 3] Item 3. The antibody-drug conjugate of Item 1 or 2, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:8. [Section 4] Item 4. The antibody-drug conjugate according to any one of Items 1 to 3, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8. [Section 5] Item 5. The antibody-drug conjugate according to any one of Items 1 to 4, wherein the cytotoxin is an anti-microtubule agent, a pyrrolobenzodiazepine (PBD), an RNA polymerase II inhibitor, or a DNA alkylating agent. [Section 6] 6. The antibody-drug conjugate of paragraph 5, wherein the cytotoxin is an anti-microtubule agent selected from the group consisting of maytansinoids, auristatins, and tubulisins. [Section 7] Item 6. The antibody-drug conjugate of item 5, wherein the cytotoxin is a pyrrolobenzodiazepine (PBD). [Section 8] The pyrrolobenzodiazepine has the following formula:

change

[0047] The following examples further illustrate the invention but, of course, should not be construed as in any way limiting its scope. [Example]

[0048] Example 1 This example describes the production of monoclonal antibodies specific for B cell maturation antigen (BCMA).

[0049] Following the RIMMS immunization regimen described by Kilpatrick et al., Hybridoma, 16(4):381-389 (1997), 6-week-old female Ablexis transgenic mice (Ablexis, LLC, San Francisco, CA) received six subcutaneous injections of purified recombinant human (rHu) BCMA-Fc alone (Campaign 1) or alternating immunizations with both rHu BCMA-Fc and cynomolgus monkey BCMA-Fc (Cyno BCMA-Fc) or adherent 293 cells (Ad293) expressing either Hu BCMA or Cyno BCMA (Campaign 2) at multiple sites. Mice were immunized at 2-3 day intervals over a 13-day period. For each immunization, mice were first anesthetized with isoflurane. Immunogens were emulsified in complete or incomplete Freund's adjuvant and TITERMAX® Gold adjuvant (Sigma-Aldrich, St. Louis, MO) and injected bilaterally at multiple sites. Test bleeds were collected on day 13 and assayed by antigen ELISA and FACS binding to adherent 293 cells expressing human and cynomolgus BCMA. Mice with good serum titers received a prefusion boost intraperitoneally and were sacrificed on day 17. Lymph node cells were collected and fused to the myeloma cell line P3-X63-Ag8.653 using the polyethylene glycol fusion method (Roche Diagnostics, Indianapolis, IN) to generate stable hybridomas.

[0050] Anti-BCMA-specific hybridomas were identified by screening hybridoma supernatants in a direct-binding ELISA against BCMA-expressing Ad293 cells followed by FACS. Positive hybridomas were further tested for their ability to bind, internalize, and kill NCI-H929 multiple myeloma cells in vitro using a secondary saporin-conjugated Fab-ZAP (Advanced Targeting Systems, San Diego, CA, IT-48) and by FACS binding to endogenous BCMA expressed in the cell line. Based on internalization and cell-killing potency, hybridomas were then cloned and expanded by limiting dilution for antibody purification and variable region gene rescue.

[0051] The first campaign yielded a panel of 44 human-only binders and four human and cynomolgus cross-reactive binders. These 48 antibodies were then further tested by FACS on adherent 293 cells expressing BCMA and the NCI-H929 cell line expressing endogenous huBCMA. A lead panel of 25 hybridoma lines was identified by ranking antibodies by best binding to endogenous human BCMA. A total of 11 hybridomas were progressed to subcloning, scale-up, sequencing, and purification. Clones were also evaluated for Fab Zap-based killing. One antibody (clone 4679) was recombinantly cloned as a human IgG1 for further evaluation.

[0052] The second campaign yielded a panel of 98 binders for immunization with rHu / Cyno BCMA-Fc, 9 binders for immunization with Ad293 cells expressing rHu / Cyno BCMA-Fc and Cyno BCMA, and 0 binders for immunization with Ad293 cells expressing both Hu and Cyno BCMA. These hybridomas were further tested by FACS for binding to TACI and BAFF-R, and antibodies showing any detectable binding to TACI and BAFF-R were removed. These secondary screens, coupled with Fab Zap assays, resulted in the identification of eight hybridomas that advanced in limiting dilution cloning (LDC). Based on their activity, two clones (clones 756 and 15B2) were then converted to human IgG1 for further evaluation.

[0053] Antibodies 4679, 756, and 15B2GL (as described below) were analyzed by FACS to assess antibody binding to human BCMA, cynomolgus BCMA, TACI, and BAFF-R using recombinant forms of the receptors stably expressed on Ad293 cells. Binding assays were performed by incubating antibodies 4679, 756, and 15B2GL with 200,000 cells for 45 minutes at 4°C and then washing twice with PBS + 2% FBS. Cells were then incubated with Alexa-Flour 647-conjugated secondary antibodies at 4°C and then washed twice with PBS + 2% FBS. Anti-BAFF-R, anti-TACI, and anti-human BCMA-APC-conjugated antibodies were added according to the manufacturer's recommended dilutions in control wells. Cells were resuspended in 200 μL of PBS + 2% FBS + DAPI, and antibody binding to live cells was analyzed using a Becton Dickinson Biosciences LSRII hemocytometer. Antibody 15B2GL was the only cynomolgus cross-reactive antibody tested that did not bind to BAFF-R and / or TACI, as shown in FIG.

[0054] The 15B2 monoclonal antibody was mutated to its germlined form (15B2GL) using primers designed to mutate four non-germlined residues in 15B2. 15B2 wild-type DNA was used as template DNA for QuikChange Lightning mutagenesis (Agilent Genomics, Santa Clara, CA). STBIII cells (Invitrogen / Thermofisher Scientific, Waltham, MA) were used for transformation. After sequence verification, BCMA binding and kinetic assays were performed to compare the binding of 15B2GL and 15B2WT and generate a population of lead-optimized (LO) clones of 15B2GL. Briefly, 15B2 was cloned into a FAb expression vector designed for bacterial expression. 27 residues in the heavy chain and 19 residues in the light chain were parsimoniously mutated using primers designed to allow cysteine ​​removal from 19 different amino acids, respectively. Mutagenesis was performed using the QuikChange Lightning Mutagenesis kit (Agilent Genomics, Santa Clara, CA).

[0055] Approximately 100 colonies per site were screened by binding ELISA for a total of 6,000 clones. ELISA binding was measured by capturing low-density human BCMA and using bacterial supernatants 48 hours after bacterial expression. Hits were defined as those that exceeded the 15B2GL control by more than twofold. Individual amino acid hits were confirmed by ELISA against cynomolgus monkey BCMA, and no binding to nonspecific proteins was observed. Identified hits from the simple screen were then combined for primer design to generate a combinatorial library. The FAb screening procedure described above by ELISA binding was repeated as a combinatorial library using 15B2GL as the parental template and ELISA control. Hits identified after combinatorial screening were then cloned into an IgG mammalian expression vector ("Maia") designed for ADC conjugation. For further testing, proteins were expressed in 293HEK cells and purified by Protein A affinity chromatography.

[0056] The ability of the 15B2GL antibody and its LO clone to bind, internalize, and kill H929 multiple myeloma cells in vitro was assessed using Fab-ZAP and FACS binding to endogenous BCMA expressed in the cell lines described above. Briefly, antibodies were incubated with 200,000 cells at 4°C for 30 minutes and then washed twice with PBS + 2% FBS. Cells were resuspended in 100 μL of cold PBS + 2% FBS and maintained at 4°C. At specified time points, cells were washed, resuspended in warm RPMI + 10% FBS, and placed in a 37°C incubator (5% CO). At the end of the experiment, cells were washed and then incubated with Alexa-Fluor 647-conjugated secondary antibody at 4°C, followed by two washes with PBS + 2% FBS. Cells were resuspended in 200 μL of PBS + 2% FBS + DAPI and antibody binding to live cells was analyzed using a Becton Dickinson Biosciences LSRII hemocytometer. The 15B2GL antibody showed inherently rapid internalization by this method when compared to the anti-BCMA antibody J6M0 (described in U.S. Patent No. 9,273,141) and the LO antibody.

[0057] Monoclonal antibody 15B2GL was selected based on BCMA binding, kinetic screens (discussed above), and internalization, and five LO clones (i.e., I09, L15, P10, N22, and M02) were selected for purification and conjugation with 15B2GL.

[0058] The amino acid sequences of the heavy and light chain variable regions of monoclonal antibody 15B2 (wild-type and germlined) and LO clones I09, L15, P10, N22, and M02 are shown in Table 1.

[0059] [Table 1]

[0060] [Table 2]

[0061] The results of this example demonstrate the production of monoclonal antibodies specific for BCMA.

[0062] Example 2 This example demonstrates a method of producing an antibody drug conjugate (ADC) comprising a BCMA monoclonal antibody conjugated to a cytotoxin in accordance with the present disclosure.

[0063] The 15B2GL monoclonal antibody and optimized clones described in Example 1 were conjugated to the PBD payload SG3249 using site-specific conjugation (Thompson et al., J. Control Release, 236:100-116 (2016); Dimasi et al. Mol Pharm. 2017 May 1;14(5):1501-1516). Specifically, the purified antibody was incubated with a 40 molar excess of the reducing agent TCEP (tris(2-carboxyethyl)phosphine) in PBS (pH 7.2), 1 mM EDTA (ethylenediaminetetraacetic acid) for 3 hours at 37°C. After incubation, the reducing agent was removed by dialysis twice against PBS (pH 7.2), 1 mM EDTA at 4°C using a 10,000 MWCO dialysis cassette, followed by incubation with 20 molar equivalents of dehydroascorbic acid at 25°C for 4 hours. Eight equivalents of the PBD payload SG3249 from a stock solution in 10% (v / v) DMSO were then added sequentially, followed by incubation at room temperature for 1 hour with gentle rotation. The conjugation reaction was quenched by the addition of 4 molar equivalents (over SG3249) of N-acetylcysteine.

[0064] The conjugation process resulted in 8-10% aggregate formation. Polymeric aggregates, including conjugation reagents such as cysteine-quenched SG3249, were removed using ceramic hydroxyapatite type II chromatography (CHT) as previously described (Thompson et al., J. Control Release, 236:100-116 (2016)). The site-specific ADC was formulated in 25 mM histidine-HCl, 7% sucrose, and 0.02% polysorbate-80 (pH 6).

[0065] To measure monomer content, aggregates, and fragments, analytical size-exclusion chromatography (SEC-HPLC) was performed using 100 μg (100 μL volume) of antibody or ADC loaded onto a TSKgel G3000WXL column (Tosoh Bioscience, Tokyo, Japan). The mobile phase consisted of 0.1 M sodium sulfate, 0.1 M sodium phosphate, and 10% isopropanol (pH 6.8). The flow rate was 1 mL / min, and each analysis was performed for 20 min at room temperature. Hydrophobic interaction chromatography (HIC-HPLC) was used to assess conjugation and drug loading distribution and was performed using a butyl-nonporous resin (NPR) column (4.6 μm ID × 3.5 cm, 2.5 μm, Tosoh Bioscience). Mobile phase A consisted of 25 mM Tris-HCl, 1.5 M (NH4)2SO4 (pH 8.0); and mobile phase B consisted of 25 mM Tris-HCl and 5% isopropanol (pH 8.0). 100 μL of antibody or ADC at a concentration of 1 mg / mL was loaded and eluted at a flow rate of 1 mL / min using a gradient from 5% B to 100% B over 13 min. Reducing reversed-phase chromatography (rRP-HPLC) was used to confirm chain-specific conjugation. Antibodies and ADCs were reduced with 42 mM dithiothreitol (DTT) in PBS (pH 7.2) for 20 min at 37 °C. Ten micrograms of reduced antibody or ADC was loaded onto a polymeric reversed-phase (PLRP-S) 1000A column (2.1 x 50 mm) (Agilent Technologies, Santa Clara, CA) and eluted with a gradient of 5% B to 100% B over 25 minutes at a flow rate of 1 mL / min at 80°C (mobile phase A: 0.1% trifluoroacetic acid in water; mobile phase B: 0.1% trifluoroacetic acid in acetonitrile).

[0066] Conjugation of heavy and light chains and drug:antibody ratios (DAR) were determined by reduced liquid chromatography-mass spectrometry (rLCMS) performed on an Agilent 1290 Series uHPLC coupled to an Agilent 6230TOF (Agilent Technologies, Santa Clara, CA). 2 μg of reduced antibody or ADC was loaded onto a ZORBAX rapid resolution high definition (RRHD) 300-diphenyl column (2.1 × 50 mm, 1.8 μm) (Agilent Technologies, Santa Clara, CA) and eluted at a flow rate of 0.5 mL / min with a step gradient of 80% B after 2.1 min (mobile phase A: 0.1% formic acid in water, and mobile phase B: 0.1% formic acid in acetonitrile). Forward time-of-flight mass spectrometry scans were acquired, and data collection and processing were performed using MassHunter software (Agilent Technologies, Santa Clara, CA). The rLCMS data were used to calculate the DAR as described in Thompson et al., supra.

[0067] Conjugation efficiency was determined using the following equation (with a theoretical DAR of 2):

number

[0068] [Table 3]

[0069] The results of this example demonstrate the production of an ADC comprising a BCMA monoclonal antibody conjugated to a pyrrolobenzodiazepine in accordance with the present disclosure.

[0070] Example 3 This example demonstrates the binding affinity of the monoclonal BCMA antibodies described herein to monomeric (soluble) and membrane-bound BCMA.

[0071] The binding of the 15B2GL monoclonal antibody and the optimized clones I09, L15, P10, N22, and M02 (described in Example 1) was assessed using a ProteOn XPR36 instrument (Bio-Rad, Hercules, CA) with monomeric human sBCMA (GenScript, Piscataway, NJ). For comparison, binding of J6M0 was also assessed. Using standard amine coupling, 25 μg / ml of anti-Fc polyclonal antibody (Jackson ImmunoResearch, West Grove, PA) prepared in 10 mM sodium acetate buffer (pH 4.5) was immobilized onto the surface of a ProteOn GLC biosensor chip (Bio-Rad, Hercules, CA) preactivated with 20 mM EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 5 mM sulfo-NHS (N-hydroxysulfosuccinimide) at a density of approximately 200–600 resonance units (RU). Subsequently, 15B2GL, I09, L15, P10, N22, M02, and J6M0 were injected at a concentration of 1 μg / ml for capture by the immobilized anti-Fc polyclonal antibody. Two-fold serial dilutions of sBCMA, ranging from 100 to 6.25 nM, prepared in PBS (pH 7.4) with 0.005% (v / v) Tween-20, were flowed over the captured surface at 75 μL / min for 150 seconds (with a 600-second dissociation time). Sensorgrams were recorded. Data were analyzed using ProteOn data analysis software.

[0072] The results of this experiment are shown in Figures 13 and 14 and Table 3.

[0073] [Table 4]

[0074] The binding of 15B2GL, I09, L15, and J6M0 to membrane-bound human BCMA was assessed using flow cytometry in multiple myeloma and plasma cell leukemia cell lines (NCI-H929 and MM.1S, respectively) that endogenously express BCMA. The binding of 15B2GL, I09, and L15 to membrane-bound human BCMA was also assessed in Ad293 cells that express human BCMA. Binding assays were performed by incubating anti-BCMA antibodies with 200,000 cells for 30 minutes at 4°C and then washing twice with PBS + 2% FBS (FACS buffer). Various antibody concentrations were assessed using a 12-point, 3-fold dilution series. Next, cells were incubated with 5 μg / mL goat anti-human IgG-AF647 secondary antibody (Thermo Fisher Scientific, Waltham, MA) at 4°C and then washed twice with PBS + 2% FBS. Cells were resuspended in 200 uL of PBS + 2% FBS + DAPI.

[0075] Fluorescence of live single cells was measured using a BD Biosciences LSRII hemocytometer and BD FACSDiva software (BD Biosciences, San Jose, CA). Data was analyzed using FlowJo software (FlowJo, LLC, Ashland, OR). Mean fluorescence intensity values ​​were used to determine the percentage of binding, and EC50 was determined using Prism software (GraphPad Software Inc, La Jolla, CA). The results of this experiment are shown in Figure 15. A summary of the SPR and flow cytometry "apparent affinity" data is shown in Table 4.

[0076] [Table 5]

[0077] The results of this example demonstrate that the monoclonal anti-BCMA antibody 15B2GL binds strongly to membrane-bound BCMA and weakly to monomeric (soluble) BCMA, which is unique compared to other monoclonal antibodies analyzed in these assays.

[0078] Example 4 This example demonstrates the use of the antibody drug conjugates described herein to kill multiple myeloma and plasma cell leukemia cells in vitro.

[0079] The killing of multiple myeloma and plasma cell leukemia cell lines by antibody-drug conjugates containing 15B2GL, or its affinity-optimized clone, conjugated to SG3249 was evaluated in vitro using the protocol recommended in the CELLTITER-GLO® kit (Promega, Madison, WI). The killing of multiple myeloma and plasma cell leukemia cell lines by free warhead SG3199 was also evaluated using the protocol recommended in the CELLTITER-GLO® kit (Promega, Madison, WI). Briefly, 5 x 10 cells were cultured in 80 μL of RPMI + 10% FBS. 3 Cells were added to the inner wells of a white-walled 96-well plate (Corning® Costar®, Fisher Scientific, Waltham, MA). The following BCMA-expressing cell lines were tested: NCI-H929, EJM, MM.1R.JJN3, OPM-2, MM.1S, U266.B1, and L363. The BMCA-negative cell lines Raji and Jurkat were also tested. Antibody-drug conjugates were diluted to a 5x stock (2.5 μg / mL) in RPMI + 10% FBS. Treatments were then serially diluted 1:3 in RPMI + 10% FBS. 20 μL of this series was added to cells in duplicate, ranging from a highest concentration of 0.5 μg / mL to a lowest concentration of 3 x 10 -6A 12-point dose curve of the antibody-drug conjugates was obtained in the μg / mL range. Isotype antibody-drug conjugates (IgG1-SG3249 and IgG1-mc-MMAF) and media-only controls were also included. Plates were incubated at 37°C and 5% CO for 96 hours. At the end of the incubation period, 100 μL of substrate solution (Promega, Madison, WI) was added to each well. Luminescence was measured using an EnVision Multilabel plate reader (Perkin Elmer, Waltham, MA). Data were analyzed and graphed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA), and the 50% inhibitory concentration (IC50) was determined.

[0080] Chromosomal translocation information for each cell line was obtained from Moreaux et al., 2011 and Boersma-Vreugdenhil et al., 2004. BCMA receptor counts were measured using AF647-labeled 15B2 (Alexa Fluor 647 Protein Labeling Kit, Thermo Fisher Scientific, Waltham, MA) and the Quantum™ MESF Kit for Alexa Fluor 647 (Bangs Laboratories, Fishers, IN).

[0081] The results of this experiment are shown in Table 5 and Figures 2A-2J.

[0082] [Table 6]

[0083] The ability of the 15B2GL-SG3249 ADC to kill multiple myeloma cells in vitro in the presence of soluble BCMA (sBCMA), compared to the I09-SG3249 ADC, was evaluated in MM.1S cells using the protocol described above, except that the cell lines tested were also treated with BCMA-containing conditioned media collected from Ad293 cells, which express human BCMA (Figures 3A and 3B). 15B2GL-SG3249 ADC cell killing in the presence of sBCMA was compared to an ADC containing the anti-BCMA antibody J6M0 (described in U.S. Patent No. 9,273,141). The results of this experiment are shown in Figure 3, which demonstrate that 15B2GL-SG3249 ADC activity is maintained in the presence of clinically relevant levels of sBCMA to a greater extent than the I09-SG3249 (Figure 3A), J6M0-mc-MMAF, and J6M0-SG3249 ADCs (Figure 3B and Table 6).

[0084] [Table 7]

[0085] The results of this example demonstrate that the 15B2GL-SG3249 ADC kills multiple myeloma and plasma cell leukemia cells in vitro, and that cell-killing activity is maintained even in the presence of soluble BCMA. Notably, the 15B2GL-SG3249 ADC was cytotoxic against both MM.1S and NCI-H929 in vitro, killing an average of 95% of tumor cells in the presence of sBCMA levels up to 720 ng / mL, with little effect on IC50. ADCs developed from antibodies with similar affinity for monomeric and membrane-bound BCMA showed a sBCMA dose-dependent decrease in potency, with an IC50 change of 20-fold in the presence of 720 ng / mL sBCMA.

[0086] Example 5 This example demonstrates the in vivo killing of multiple myeloma and plasma cell leukemia cells using the antibody drug conjugates described herein.

[0087] Subcutaneous xenograft mouse models of multiple myeloma and plasma cell leukemia were established by transfecting BCMA-expressing multiple myeloma or plasma cell leukemia cell lines (i.e., NCI-H929, JJN-3, MM.1S, and MM.1R) into female CB-17 SCID (CB-17 / IcrHsd-Prkdc-scid) or athymic nude (Foxn1) mice using MATRIGEL™ (BD Biosciences, San Jose, CA). nu The tumor was generated by transplanting it into a mouse. Once the tumor grew to approximately 180 mm 3 (NCI-H929 cells), 190mm 3 (JJN3 cells), 160mm 3 (MM.1S cells), or 175 mm 3 Once tumor size reached 100% (MM.1R cells), mice were randomized based on tumor size, placed into treatment groups, and treated with BCMA-targeted ADCs (as described below).

[0088] NCI-H929 xenograft model Mice were treated with a single intravenous dose of 0.3 mg / kg of either the 15B2GL-SG3249, I09-SG3249, or L15-SG3249 ADC, or J6M0-mc-MMAF was administered intravenously at a dose of 0.3 mg / kg per week for 2 weeks. Control mice were left untreated. Mice treated with 15B2GL-SG3249, I09-SG3249, and L15-SG3249 were observed for 99 days after tumor implantation, and no evidence of tumor regrowth was observed, as shown in Figure 4. No weight loss was observed in any of the treatment groups.

[0089] JJN3 xenograft model Mice were treated with a single intravenous dose of 1 mg / kg of either the 15B2GL-SG3249, I09-SG3249, or L15-SG3249 ADC, or J6M0-mc-MMAF ADC was administered intravenously at a dose of 1 mg / kg per week for 3 weeks. Control mice were left untreated. Mice treated with 15B2GL-SG3249, I09-SG3249, and L15-SG3249 were observed for 104 days after tumor implantation, and no evidence of tumor regrowth was observed, as shown in Figure 5. No weight loss was observed in any of the treatment groups.

[0090] MM.1S xenograft model Mice were treated with a single intravenous dose of 1 mg / kg of either the 15B2GL-SG3249, I09-SG3249, or L15-SG3249 ADC, or J6M0-mc-MMAF was administered intravenously at a dose of 1 mg / kg twice weekly for 4 weeks. Control mice were left untreated. Mice treated with 15B2GL-SG3249, I09-SG3249, and L15-SG3249 were observed for 99 days after tumor implantation, and no evidence of tumor regrowth was observed, as shown in Figure 6. No weight loss was observed in any of the treatment groups.

[0091] MM.1R xenograft model Mice were treated with a single intravenous dose of 1 mg / kg of 15B2GL-SG3249 or J6M0-mc-MMAF administered intravenously at a weekly dose of 3 mg / kg for 4 weeks. Control mice were left untreated. Mice treated with 15B2GL-SG3249 were observed for 109 days after tumor implantation, and no evidence of tumor regrowth was observed, as shown in Figure 7. No weight loss was observed in any of the treatment groups.

[0092] The results of this example demonstrate that the 15B2GL-SG3249 ADC exhibits enhanced anti-tumor efficacy in vivo compared to other ADCs that target BCMA-expressing cells.

[0093] Example 6 This example demonstrates that multiple myeloma stem cells express BCMA.

[0094] The bone marrow of patients with multiple myeloma (MM) contains a small population of cancer stem cells (CSCs) that can be identified in the patient's bone marrow by their surface expression of CD19 and the lack of CD138 surface expression (Matsui et al., Blood, 103:2332-6 (2004)).

[0095] BCMA expression was assessed by flow cytometry on stem cell populations from four multiple myeloma patient samples. Samples were obtained from Proteogenex, Inc. (Culver City, CA) (see Table 7), and individual multiple myeloma (MM) samples were thawed in a 37°C water bath.

[0096] [Table 8]

[0097] Thawed cells were added to 10 mL of PBS and counted using a ViCELL™ counter (Beckmann-Coulter Life Sciences, Indianapolis, IN). An aliquot of the cell suspension was prepared for the colony formation assay, while the remainder of the cell suspension was centrifuged at low speed to pellet the cells. Cells were Fc-blocked according to the manufacturer's instructions and then plated at 200,000 cells / well in 96-well plates. The plates were centrifuged, the cells pelleted, the Fc-blocking solution decanted, and the cell samples resuspended in BV staining buffer and then stained with an appropriate antibody panel consisting of commercially available directly conjugated antibodies, as shown in Tables 8 and 9.

[0098] [Table 9]

[0099] [Table 10]

[0100] Additionally, compensation beads were individually stained with the test antibodies. The plate was incubated at 4°C in the dark for 30 minutes. The plate was centrifuged, and the cells were washed with DPBS + 2% FBS and then resuspended in 200 μL of DPBS + 2% FBS + DAPI. Cells from each well were evaluated on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA), and FCS files were generated. Cell identification was performed using the following gating method: compensation was performed using the autocomp matrix in FlowJo® 10 (FlowJo LLC, Ashland, OR), utilizing compensation beads and single staining data. Plasma cells were then gated on an FSC-A vs. SSC-A plot, and live single cells were selected on a DAPI vs. SSC-W plot. Next, an exclusion gate was used to remove cells that stained positive for CD3, CD14, CD34, and CD193 on a BV-510 vs. SSC-A plot. This population was then analyzed in a CD138-PE vs. CD19-APC plot. Histograms for BCMA expression were generated for the MM CSC population, defined as CD19+ / CD138-, and the MM plasma cell population, defined as CD19- / CD138+. Analysis gates were set based on appropriate fluorescence minus one (FMO) controls. As shown in Figure 7, all samples exhibited a small percentage of CD138+CD19- cells positive for BCMA expression. The levels of BCMA expression on stem cell populations were generally comparable to those observed on plasma cells, with the exception of MM277, where levels were reduced but still positive for BCMA expression.

[0101] The results of this example demonstrate that BCMA is expressed on multiple myeloma cancer stem cells.

[0102] Example 7 This example demonstrates that the 15B2GL-SG3249 antibody drug conjugate kills multiple myeloma stem cells.

[0103] Given that MM stem cells are capable of forming colonies in vitro (Matsui et al., Blood, 103:2332-6 (2004)), the ability of ADC15B2GL-SG3249 to kill clonogenic cells in MM bone marrow biopsies characterized in Example 2 was tested. Specifically, cells were counted using a ViCELL™ counter (Beckmann-Coulter Life Sciences, Indianapolis, IN) and resuspended in IMDM + 2% FBS at a density 10-fold higher than that required for plating. METHOCULT™ H4434 Classic (StemCell Technologies, Inc., Vancouver, BC, Canada) was mixed according to the manufacturer's instructions at 2000 cells / mL for MM263, MM284, and MM276, and 4000 cells / mL for MM277. Next, 25–400 ng / mL of test 15B2GL-SG3249 and J6M0-mc-MMAF ADCs were added to the appropriate tubes. The control IgG1-SG3249 antibody was added only at the high dose of 400 ng / mL. All tubes were thoroughly vortexed and then allowed to settle, allowing air bubbles to rise to the top. Once the bubbles had risen, 400 μL was removed with a 16-gauge blunt-end needle and carefully plated into one well of a 24-well ultra-low attachment plate (VWR, Radnor, PA). Each treatment was plated in duplicate in the inner wells of the plate. PBS was added to the outer wells, and the plate was incubated at 37°C for 7–10 days. Colonies were visually counted, with colony formation recorded by scanning plates on a Celigo® Image Cytometer (Nexcelom Biosciences, Lawrence, MA). In all four cases, 15B2GL-SG3249 was able to kill clonogenic cells, whereas J6M0-mc-MMAF was not, as shown in Figure 8. At the highest dose tested (400 ng / mL), 15B2GL-SG3249 was able to kill 100% of colonies for MM263 and MM284, and 87.5% and 91% of colonies for MM276 and MM277, respectively.In contrast, 400 ng / mL J6M0-mc-MMAF did not reduce the number of colonies formed for MM263 and only resulted in a 12.5%, 40%, and 50% decrease in colony formation for MM276, MM277, and MM284, respectively.

[0104] The results of this example demonstrate that the antibody-drug conjugate 15B2GL-SG3249 targets and kills BCMA-expressing multiple myeloma cancer stem cells.

[0105] Example 8 This example demonstrates the in vitro killing of multiple myeloma and plasma cell leukemia cells using the antibody drug conjugates described herein.

[0106] Killing of multiple myeloma and plasma cell leukemia cell lines by antibody-drug conjugates, including 15B2GL conjugated to SG3400, was evaluated in vitro using the CELLTITER-GLO® kit (Promega, Madison, WI), as described in Example 4. The following BCMA-expressing cell lines were tested: NCI-H929, EJM, MM.1R.JJN3, OPM-2, MM.1S, U266.B1, and L363. The BMCA-negative cell lines Raji and Jurkat were also tested. The antibody-drug conjugates were diluted to a 5x stock (25 μg / mL) in RPMI + 10% FBS. The treatments were then serially diluted 1:3 in RPMI + 10% FBS. 20 μL of this series was added to cells in duplicate, ranging from a highest concentration of 5 μg / mL to a lowest concentration of 2.8 x 10 -5A 12-point dose curve of the antibody-drug conjugate was obtained in the μg / mL range. An isotype antibody-drug conjugate (IgG1-SG3400) and media-only controls were also included. Plates were incubated at 37°C and 5% CO for 96 hours. At the end of the incubation period, 100 μL of substrate solution (Promega, Madison, WI) was added to each well. Luminescence was measured using an EnVision Multilabel plate reader (Perkin Elmer, Waltham, MA). Data were analyzed and graphed using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA). The results of this experiment are shown in Figures 10A-10J.

[0107] The results of this example demonstrate that the 15B2GL-SG3400 ADC kills multiple myeloma and plasma cell leukemia cells in vitro.

[0108] Example 9 This example demonstrates the use of the antibody drug conjugates described herein to kill multiple myeloma and plasma cell leukemia cells in vivo.

[0109] Subcutaneous xenograft mouse models of multiple myeloma and plasma cell leukemia were generated by implanting BCMA-expressing multiple myeloma or plasma cell leukemia cell lines (i.e., NCI-H929 and MM.1S) into female CB-17 SCID (CB-17 / IcrHsd-Prkdc-scid) mice using MATRIGEL™ (BD Biosciences, San Jose, CA). Once tumors reached approximately 200 mm 3 (NCI-H929 cells) or 180mm 3 Once tumor size reached 100% (MM.1S cells), mice were randomized based on tumor size, placed into treatment groups, and treated with BCMA-targeting ADCs (as described below).

[0110] NCI-H929 xenograft model Mice were treated with a single intravenous dose of either 1 mg / kg IgG1-SG3400 or 0.3 mg / kg 15B2GL-SG3400 ADC, or J6M0-SG3400 was administered intravenously at a single dose of 0.3 mg / kg or 1 mg / kg. Control mice were left untreated. Mice treated with 15B2GL-SG3400 were observed for 74 days after tumor implantation, and no evidence of tumor regrowth was observed, as shown in Figure 11. No weight loss was observed in any of the treatment groups.

[0111] MM.1S xenograft model Mice were treated with a single intravenous dose of either IgG1-SG3400 or 15B2GL-SG3400 ADC at 1 mg / kg or 3 mg / kg, or J6M0-SG3400 was administered intravenously at a dose of 1 mg / kg or 3 mg / kg. Control mice were left untreated. Mice treated with 3 mg / kg J6M0-SG3400 were observed for 85 days after tumor implantation, and no evidence of tumor regrowth was observed, as shown in Figure 12. No weight loss was observed in any of the treatment groups.

[0112] The results of this example demonstrate that the 15B2GL-SG3400 ADC exhibits antitumor efficacy in vivo.

[0113] The data described in this example demonstrate that an ADC containing the monoclonal antibody 15B2GL exhibits potent antitumor activity in preclinical models of MM. Importantly, in vitro experiments suggest that this activity is maintained in the presence of sBCMA. These data further demonstrate that the 15B2GL-SG3249 ADC, which carries a potent PBD payload, effectively targets both the majority of myeloma plasma cells and the more quiescent CD19+ / CD138- clonogenic cells, which may offer an opportunity for more durable clinical responses in this genetically heterogeneous disease.

[0114] All references, including publications, patent applications, and patents, cited in this specification are herein incorporated by reference to the same extent as if each individual reference was individually and specifically indicated to be incorporated by reference.

[0115] Use of the terms "a," "an," "the," "at least one," and similar referents in the context of describing the invention (particularly in the context of the claims that follow) should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The term "at least one" followed by a list of one or more items (e.g., "at least one of A and B") should be construed to mean one item selected from the listed item (A or B) or any combination of two or more of the listed items (A and B), unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise noted. The recitation of ranges of values ​​herein is intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is encompassed herein as if it were individually recited herein, unless otherwise indicated herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language provided herein (e.g., "etc."), is intended merely to more fully clarify the invention and not to pose limitations on the scope of the invention, unless specifically claimed. No language herein should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0116] Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of these preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors contemplate that those of ordinary skill in the art will utilize such variations as appropriate, and the inventors intend that the invention may be practiced differently from as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

1. A pharmaceutical composition for treating multiple myeloma in an animal, comprising a therapeutically effective amount of a composition comprising an antibody drug conjugate (ADC), the therapeutically effective amount is 0.3 mg / kg, 1 mg / kg, or 3 mg / kg; The ADC comprises a monoclonal antibody or antigen-binding fragment thereof that binds to membrane-bound B-cell maturation antigen (BCMA) conjugated to a cytotoxin; and The monoclonal antibody comprises: (a) a heavy chain variable region comprising a complementarity determining region 1 (HCDR1) amino acid sequence of SEQ ID NO: 1, a HCDR2 amino acid sequence of SEQ ID NO: 2, and a HCDR3 amino acid sequence of SEQ ID NO: 3; and (b) a light chain variable region comprising a complementarity determining region 1 (LCDR1) amino acid sequence of SEQ ID NO: 4, a LCDR2 amino acid sequence of SEQ ID NO: 5, and a LCDR3 amino acid sequence of SEQ ID NO:

6. Pharmaceutical compositions.

2. The pharmaceutical composition of claim 1 , wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:

7.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO:

8.

4. 4. The pharmaceutical composition of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 7 and the light chain variable region comprises the amino acid sequence of SEQ ID NO:

8.

5. 5. The pharmaceutical composition of claim 1, wherein the cytotoxin is an anti-microtubule agent, a pyrrolobenzodiazepine (PBD), an RNA polymerase II inhibitor, or a DNA alkylating agent.

6. 6. The pharmaceutical composition of claim 5, wherein the cytotoxin is an anti-microtubule agent selected from the group consisting of maytansinoids, auristatins, and tubulisins.

7. 6. The pharmaceutical composition of claim 5, wherein the cytotoxin is a pyrrolobenzodiazepine (PBD).

8. The pyrrolobenzodiazepine has the following formula: 【Chemical 1】 8. The pharmaceutical composition of claim 7, wherein the compound is SG3249 having the formula:

9. A pharmaceutical composition according to any one of claims 1 to 8, further comprising a pharmaceutically acceptable carrier.

10. 10. The pharmaceutical composition of claim 1, wherein tumor volume is reduced in the presence of soluble BCMA.

11. 11. The pharmaceutical composition of any one of claims 1 to 10, wherein the antibody preferentially binds to membrane-bound BCMA.

12. A pharmaceutical composition described in any one of claims 1 to 11, which is administered using any administration means selected from the group consisting of intravenous administration, intraperitoneal administration, and subcutaneous administration.

13. The pharmaceutical composition of claim 12, which is administered by intravenous injection.

14. A pharmaceutical composition according to any one of claims 1 to 13, administered in combination with one or more additional therapeutic agents.

15. 15. The pharmaceutical composition of any one of claims 1 to 14, wherein the animal is a human.

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