EA046403B1 - MONOCLONAL ANTIBODY TO BCMA AND DRUG CONJUGATE - Google Patents

Description

Incorporated herein by reference in its entirety is the machine-readable nucleotide/amino acid sequence listing filed contemporaneously with this application and identified as follows: a 16,498 byte ASCII (text) file entitled BCMA-100-WO-PCT-SeqListing.TXT, created by July 31, 2018

State of the art

Multiple myeloma (MM) is a malignancy characterized by the accumulation of clonal plasma cells (see, for example, Palumbo et al., New England J. Med., 364(11): 1046-1060 (2011), and Lonial et al. , Clinical Cancer Res., 77(6): 1264-1277 (2011). Current therapies for MM include chemotherapy, radiation, surgery, biosphosphonates, and autologous stem cell transplantation (ASCT). Although these therapies often induce remission, almost all patients eventually relapse and die (see, e.g., Lonial et al., supra, and Rajkumar, Nature Rev. Clinical Oncol, 5(8): 479-491 (2011 )).

B-cell maturation antigen (BCMA) is a member of the tumor necrosis factor receptor (TNFR) family expressed on B-cell lineage cells (Laabi et al., Nucleic Acids Research, 22(7): 1147-1154 (1994)). BCMA expression is highest on terminally differentiated B cells. BCMA is involved in mediating plasma cell survival to maintain long-term humoral immunity. BCMA expression has been associated with a number of cancers, autoimmune disorders, and infectious diseases. BCMA RNA has been found ubiquitously in multiple myeloma cells, and BCMA protein has been detected on the surface of plasma cells from patients with multiple myeloma by several investigators (see, for example, 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)). Therefore, BCMA has been investigated as a possible therapeutic target in multiple myeloma.

There continues to be a need for compositions that can be used in methods of treating multiple myeloma. The present invention provides such compositions and methods.

Brief description of the invention

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

The present invention further provides compositions containing the above antibody-drug conjugate and methods for killing multiple myeloma cells (including multiple myeloma stem cells) that express BCMA by contacting the multiple myeloma cells with an ADC.

The present invention also provides a monoclonal antibody or antigen binding fragment thereof directed against BCMA which comprises (a) a heavy chain variable region comprising the amino acid sequence of complementarity determining region 1 (HCDR1) of SEQ ID NO: 1, the amino acid sequence of HCDR2 of SEQ ID NO : 2 and the amino acid sequence of HCDR3 of SEQ ID NO: 3, and (b) a light chain variable region comprising the amino acid sequence of complementarity determining region 1 (LCDR1) of SEQ ID NO: 4, the amino acid sequence of LCDR2 of SEQ ID NO: 5 and the amino acid sequence LCDR3 sequence under SEQ ID NO: 6.

Brief description of several images of the graphic(s)

In fig. 1 is a series of graphs (FIGS. 1A-1C) illustrating FACS analysis of the binding of purified antibodies to adherent 293 (Ad293) cells expressing huBCMA, cynoBCMA, BAFF-R and TACI as described in Example 1. Monoclonal antibody 15B2GL was the only tested cross-reacting antibody against cynomolgus monkey that did not bind to BAFF-R and/or TACI.

In fig. 2 are graphs illustrating the ability of anti-BCMA antibody-drug conjugates to kill multiple myeloma (MM) and plasma cell leukemia (PCL) cells in vitro as described in Example 4. FIG. 2A-2H show the viability of specific BCMA-expressing multiple myeloma and plasma cell leukemia cell lines treated with the indicated ADCs, while FIG. 2I and 2J depict the viability of cell lines that do not express BCMA.

Fig. 3 includes graphs illustrating the killing of multiple mye cell lines

- 1 046403 crowbars in the presence of soluble BCMA using the antibody-drug conjugate 15B2GL-SG3249 compared to ADC I09-SG3249 in conditioned media collected from Ad293 cells expressing human BCMA (Fig. 3A) or compared to ADC J6M0- mc-MMAF and J6M0SG3249 in conditioned media collected from Ad293 cells expressing human BCMA (Fig. 3B) as described in Example 4.

In fig. 4 is a graph illustrating the change in tumor volume in the 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.

In fig. 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, I09SG3249, L15-SG3249, J6M0-mc-MMAF and IgG1 isotype control-SG3249 compared to untreated mice.

In fig. 6 is a graph illustrating the change in tumor volume in a xenograft mouse model of plasma cell leukemia MM.1S in response to treatment with ADCs 15B2GL-SG3249, I09-SG3249, L15-SG3249 and J6M0-mc-MMAF compared to untreated mice.

In fig. 7 is a graph illustrating the change in tumor volume in the MM.1R plasma cell leukemia xenograft mouse model in response to treatment with ADCs 15B2GL-SG3249 and J6M0-mc-MMAF compared to untreated mice.

In fig. Figure 8 presents a series of flow cytometry graphs and graphics illustrating the expression of BCMA on MM stem cells. BCMA expression was detected on both MM plasma cells (CD19-CD138+, gray waveform) and MM stem cells (CD19+CD138-, black waveform).

Fig. 9 includes a series of graphs illustrating the sensitivity of MM stem cells in patient samples MM263 (FIG. 9A), MM276 (FIG. 9B), MM277 (FIG. 9C), and MM284 (FIG. 9D) to ADC 15B2GL-SG3249 compared to ADC J6M0-mc-MMAF in clonogenicity assay. Controls include untreated cells and nonspecific IgG1-SG3249 conjugate at the highest dose of 400 ng per ml. The number of colonies formed was normalized to the number formed in the untreated culture, which was set as 100%.

In fig. 10 are graphs illustrating the ability of the anti-BCMA antibody-drug conjugate to kill multiple myeloma (MM) and plasma cell leukemia (PCL) cells in vitro as described in Example 7. FIG. 10A-10H show the viability of specific multiple myeloma and plasma cell leukemia cell lines expressing BCMA treated with the indicated ADC, while FIG. 10I and 10J show the viability of cell lines that do not express BCMA.

In fig. 11 is a graph illustrating changes in tumor volume in the H929 xenograft mouse model of multiple myeloma in response to treatment with the BCMA-targeting ADCs 15B2GL-SG3400, J6M0-SG3400 and IgG1 isotype control-SG3400 compared to untreated mice.

In fig. 12 is a graph illustrating changes in tumor volume in a xenograft mouse model of plasma cell leukemia MM. 1S in response to treatment with the BCMA-targeting ADCs 15B2GL-SG3400, J6M0-SG3400, and IgG1 isotype control-SG3400 compared to untreated mice.

In fig. 13 are graphs illustrating the binding affinity and kinetics measurements of antibodies 15B2GL, I09, P10 and L15 to human BCMA using the SPR ProteOn system.

In fig. 14 are graphs illustrating the affinity and binding kinetics measurements of antibodies N22, M02 and J6M0 to human BCMA using the ProteOn SPR-based system.

In fig. 15 are graphs illustrating the binding of antibodies 15B2GL, L15, I09 or J6M0 to the NCI-H929, MM.1S and Ad293+huBCMA cell lines measured by flow cytometry.

Detailed Description of the Invention

The present invention provides an antibody-drug conjugate (ADC) comprising a monoclonal antibody or antigen-binding fragment thereof directed against B-cell maturation antigen (BCMA) conjugated to a cytotoxin. The term antibody-drug conjugate as used herein refers to a compound containing a monoclonal antibody (mAb) attached to a cytotoxic agent (typically a small molecule drug with high systemic toxicity) via chemical linkers. In some embodiments, the ADC may contain a small molecule cytotoxin that has been chemically modified to contain a linker. The linker is then used to conjugate the cytotoxin to the antibody or antigen-binding fragment thereof. After

- 2 046403 binding to a target antigen on the cell surface The ADC is internalized and transferred to the lysosome, where the cytotoxin is released either by proteolysis of the cleavable linker (for example, by cathepsin B located in the lysosome) or by proteolytic degradation of the antibody, if attached to the cytotoxin via a non-cleavable linker. The cytotoxin then moves from the lysosome to the cytosol or nucleus, where it can then bind to its target depending on its mechanism of action.

The term monoclonal antibodies as used herein refers to antibodies that are produced by a single clone of B cells and bind to the same epitope. In contrast, the term polyclonal antibodies refers to a population of antibodies that are produced by different B cells and bind to different epitopes of the same antigen. The antibody-drug conjugate described herein may contain a whole antibody or an antibody fragment. 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 of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CH1, CH2 and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant ( CL) area. The variable regions of each pair of light and heavy chains form the antigen-binding site of the antibody. The VH and VL regions have the same overall structure, with each region containing four framework regions whose sequences are relatively conserved. The framework regions are connected by three complementarity determining regions (CDRs). Three CDRs, known as CDR1, CDR2 and CDR3, form the hypervariable region of the antibody, which is responsible for antigen binding.

The ADC may contain an antigen binding fragment of an antibody. The terms antibody fragment, antigen-binding fragment, functional antibody fragment, and antigen-binding moiety are used interchangeably herein and refer to one or more fragments or portions of an antibody that retain the ability to specifically bind an antigen (see generally, Holliger et al., Nat. Biotech., 23(9): 1 126-1129 (2005)). An antibody fragment may contain, for example, one or more CDRs, a variable region (or portions thereof), a constant region (or portions thereof), or combinations thereof. Examples of antibody fragments include, but are not limited to (i) a Fab fragment, which is a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) F(ab')2 fragment, which is a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fv fragment consisting of the VL and VH domains of one arm of the antibody; (iv) single chain Fv (scFv), which is a monovalent molecule consisting of two Fv fragment domains (i.e. VL and VH) connected by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain (see, for example, Bird et al., Science, 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879-5883 (1988); and Osbourn et al., Nat. Biotechnol., 16: 778 (1998)); and (v) a diabody that is a dimer of polypeptide chains, where each polypeptide chain contains a VH attached to a VL by a peptide linker that is too short to allow pairing between VH and VL in the same polypeptide chain, thereby driving pairing between complementary domains in various VH-VL polypeptide chains to form 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 US patent application publication 2009/0093024 A1.

In one embodiment, the antibody-drug conjugate described herein comprises a monoclonal antibody or antigen-binding fragment thereof directed against B-cell maturation antigen (BCMA, also known as CD269). BCMA is a member of the tumor necrosis factor receptor superfamily (see, for example, 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, for example, 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 primarily in plasma cells and subclasses of mature B cells (see, for example, 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)). BCMA-deficient mice are healthy and have normal B cell numbers, but the survival of long-lived plasma cells is impaired (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 found ubiquitously in multiple myeloma cells, and BCMA protein has been detected on the surface of plasma cells from patients with multiple myeloma by several investigators (see, for example, Novak et al, Blood, 103(2): 689-694 (2004); Neri et al., Clinical Cancer Research, 73(19): 5903-5909 (2007); Bellucci et al., Blood, 705(10): 3945-3950 (2005); and Moreaux et al., Blood, 703( 8): 3148-3157 (2004)).

In some embodiments, the present invention provides a monoclonal antibody or antigen binding fragment thereof directed against BCMA described above, without

- 3 046403 depending on the antibody-drug conjugate. The monoclonal antibody or antigen binding fragment thereof may comprise (a) a heavy chain variable region comprising the amino acid sequence of complementarity determining region 1 (HCDR1) of SEQ ID NO: 1, the amino acid sequence of HCDR2 of SEQ ID NO: 2, and the amino acid sequence of HCDR3 of SEQ ID NO: : 3, and (b) a light chain variable region comprising the amino acid sequence of complementarity determining region 1 (LCDR1) of SEQ ID NO: 4, the amino acid sequence of LCDR2 of SEQ ID NO: 5, and the amino acid sequence of LCDR3 of SEQ ID NO: 6. B 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 comprising the amino acid sequence of SEQ ID NO: 8.

A monoclonal antibody or antigen binding fragment thereof directed against BCMA may have any suitable binding affinity for BCMA or an epitope thereof. The term affinity refers to the equilibrium constant for the reversible binding of two agents and is expressed as the dissociation constant (KD). The affinity of an antibody or antigen-binding fragment thereof for an antigen or epitope of interest 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), interferometric technique (BLI, e.g., Octet), kinetic exclusion assay (e.g., KinExA), separable beads (e.g., magnetic beads), panning antigen and/or ELISA (see, for example, Janeway et al. (eds.), Immunobiology, 5th ed., Garland Publishing, New York, N.Y., 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.

The soluble form of BCMA (sBCMA) has been detected in the serum of patients with multiple myeloma, 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 consisted of the complete extracellular domain of the molecule (Laurent et al. Nat Commun 2015). Therefore, sBCMA may attenuate the effects of antibody-based therapies. The functional features of sBCMA and recombinant monomeric human BCMA are identical (Laurent et al. Nat Sottype 2015). Therefore, to reduce the potential effects of sBCMA on the effectiveness of the anti-BCMA antibody-drug conjugate, it is desirable to select an antibody component that has weak binding to recombinant monomeric human BCMA and strong binding to membrane-bound BCMA.

The affinity of the binding agent for a ligand, e.g., the affinity of an antibody for an epitope, can be, for example, from about 1 picomol/L (pM) to about 1 micromol/L (μM) (for example, from about 1 picomol/L (pM) to about 1 nanomol/L (nM) or from about 1 nmol/L to about 1 micromol/L (μM)). In one embodiment, the monoclonal antibody or antigen binding fragment thereof may bind to BCMA with a K _D of less than or equal to 100 nanomol/L (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 above). In another embodiment, the monoclonal antibody may bind to BCMA with a KD of less than or equal to 10 nanomol/L (e.g., about 9 nM, about 8.5 nM, about 8 nM, about 7.5 nM, about 7 nM, about 6 .5 nM, approximately 6 nM, approximately 5.5 nM, approximately 5 nM, approximately 4.5 nM, approximately 4 nM, approximately 3.5 nM, approximately 3 nM, approximately 2.5 nM, approximately 2 nM, approximately 1 .5 nM, approximately 1 nM, approximately 0.9 nM, approximately 0.8 nM, approximately 0.7 nM, approximately 0.6 nM, approximately 0.5 nM, approximately 0.4 nM, approximately 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 above values). In another embodiment, the monoclonal antibody may bind to BCMA with a KD of less than or equal to 200 pM (e.g., about 190 pM, about 175 pM, about 150 pM, about 125 pM, about 110 pM, about 100 pM, about 90 pM, approximately 80 pM, approximately 75 pM, approximately 60 pM, approximately 50 pM, approximately 40 pM, approximately 30 pM, approximately 25 pM, approximately 20 pM, approximately 15 pM, approximately 10 pM, approximately 5 pM, approximately 1 pM or range, defined by any two of the above values).

In one embodiment, the affinity of the anti-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, for example, from about 50 nM to about 70

- 4,046,403 nM, from about 55 nM to about 65 nM, or from about 58 nM to about 62 nM.

In one embodiment, the affinity of the anti-BCMA antibody or antigen-binding fragment thereof for membrane-bound BCMA, as measured by FACS, is less than or equal to 10 nanomol/L (e.g., about 9 nM, about 8.5 nM, about 8 nM, about 7.5 nM, approximately 7 nM, approximately 6.5 nM, approximately 6 nM, approximately 5.5 nM, approximately 5 nM, approximately 4.5 nM, approximately 4 nM, approximately 3.5 nM, approximately 3 nM, approximately 2.5 nM, approximately 2 nM, approximately 1.5 nM, approximately 1 nM, approximately 0.9 nM, approximately 0.8 nM, approximately 0.7 nM, approximately 0.6 nM, approximately 0.5 nM, approximately 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 above values).

The antigen-binding portion or fragment of a monoclonal antibody can be of any size as long as the portion binds to BCMA. In this regard, the antigen binding portion or fragment of an anti-BCMA monoclonal antibody (also referred to herein as an anti-BCMA monoclonal antibody) desirably contains from about 5 to 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 above values).

In one embodiment, the antibody-drug conjugate comprises the variable region of an anti-BCMA monoclonal antibody. In this regard, the ADC may comprise a light chain variable region, a heavy chain variable region, or both a light chain variable region and a heavy chain variable region of an anti-BCMA monoclonal antibody. Preferably, the ADC contains a light chain variable region and a 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 WO 2010/104949. In one embodiment, the ADC monoclonal antibody described herein comprises (a) a heavy chain variable region comprising the amino acid sequence of complementarity determining region 1 (HCDR1) SYSMN (SEQ ID NO: 1), the amino acid sequence HCDR2 SISGSSNYIYYADSVKG (SEQ ID NO: 2) and the amino acid sequence of HCDR3 GGNYYVEYFQY (SEQ ID NO: 3), and (b) a light chain variable region containing the amino acid sequence of complementarity determining region 1 (LCDR1) RASQYISSNYLA (SEQ ID NO: 4), the amino acid sequence of LCDR2 GASNRAT (SEQ ID NO: 5) and the amino acid sequence of LCDR3 QQYGSSPIT (SEQ ID NO: 6). In another embodiment, an ADC monoclonal antibody 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.

The terms cytotoxin and cytotoxic agent refer to any molecule that inhibits or interferes with cellular function and/or causes cell destruction (cell death) and/or has antiproliferative effects. It will be understood that a cytotoxin or cytotoxic ADC is also referred to in the art as an ADC payload. It is known from the prior art that a number of classes of cytotoxic agents are potentially useful in ADC molecules and may be used in the ADC described herein. Such classes of cytotoxic agents include, for example, microtubule-targeting agents (eg, auristatins and maytansinoids), pyrrolobenzodiazepines (PBDs), RNA polymerase II inhibitors (eg, amatoxins), and DNA alkylating agents (eg, indolinobenzodiazepine pseudodimers). Examples of specific cytotoxic agents that may be used in the ADC described herein include, but are not limited to, amanitines, auristatins, calicheamycins, daunomycins, doxorubicins, duocarmycins, dolastatins, enediynes, lexitropsins, taxanes, puromycins, maytansinoids, vinca alkaloids, tubulisins, and pyrrolobenzodiazepines ( PBD). More specifically, the cytotoxic agent may be, for example, AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, CC-1065, SN-38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholinodoxorubicin, dolastatin-10, echinomycin , combretastatin, calicheacin, maytansine, DM1, DM4, vinblastine, vinblastine, methotrexate, netropsin or their derivatives or analogues. Cytotoxins suitable for use in ADCs are also described, for example, in International Patent Application Publications Nos. WO 2015/155345 and WO 2015/157592.

In one embodiment, the cytotoxic agent may be a microtubule-targeting agent such as tubulisin, maytansinoid, auristatin, or derivatives thereof. The terms microtubule-acting agent and microtubule-targeting agent are synonymous and refer to an agent that inhibits cell division by acting on microtubules. Tubulisins are members of a class of natural substances isolated from myxobacterial species (Sasse et al., J. Antibiot., 53: 879-885 (2000)) that act as

- 5 046403 mitotic poisons that inhibit tubulin polymerization and lead 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 tubulisins are disclosed, for example, in the publications of International patent applications No. WO 2015/157594, WO 2004/005326, WO 2012/019123, WO 2009/134279, WO 2009/055562, WO 2004/005327; in US patents 7776841, 7754885 and 7816377; and in published US patent applications 2010/0240701, 2011/0021568 and 2011/0263650.

In certain aspects, tubulisin is a compound described in WO 2015/157594, which is incorporated herein by reference, such as, for example, a compound having the following structure

N

or a compound having the following structure

Maytansinoids inhibit the polymerization of the microtubule protein tubulin, thereby preventing the formation of microtubules (see, for example, US 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 laboratory animal systems. Moreover, the cytotoxicity of maytansinoids is 1000 times higher than that of traditional chemotherapeutic agents, such as, for example, methotrexate, daunorubicin and vincristine (see, for example, US patent 5208020). Maytansinoids include maytansine, maytansinol, maytansinol C-3 esters, and other maytansinol analogs and derivatives (see, for example, US Pat. Nos. 5,208,020 and 6,441,163). C-3 esters of maytansinol can be naturally occurring or produced synthetically. Moreover, both naturally occurring and synthetic C-3-esters of maytansinol can be classified as C-3-ester with carboxylic acids or C-3-ester with N-methyl-alanine derivatives, the latter being more cytotoxic than the first. 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 preparing maytansinol and its analogs and derivatives are described, for example, in US Pat. No. 4,151,042. Examples of maytansinoids that can be used in the ADCs described herein include, but are not limited to, N2'-deacetyl-N2'-(3-mercapto1-oxopropyl) maytansine (DM1) and N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)maytansine (DM4).

Auristatins are a class of highly effective antimitotic agents that have demonstrated significant 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 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: 778784 (2003); and Doronina et al., Bioconjug. Chem., 17: 114-124 (2006)).

In one embodiment, the cytotoxic agent may be a pyrrolobenzodiazepine (PBD) or a PBD derivative. The PBD translocates to the nucleus where it cross-links DNA, preventing replication during mitosis, damaging DNA through single-strand breaks and subsequently leading to apoptosis. Some PBDs are also capable of recognizing and binding to specific DNA sequences. General structure for PBD

- 6 046403

Il AI B ^11a /~n ¹ 6 )\^ ²

3

PBDs differ in the number, type, and position of substituents on both their aromatic A-rings and pyrrole C-rings, as well as the degree of saturation of the C-rings. The B-ring contains either an imine (N=C), a carbinolamine (NH-CH(OH)), or a carbinolamine methyl ester (NH-CH(OMe)) at the N10-C11 position, which is the electrophilic center responsible for alkylation DNA. All known natural products have a ^-configuration at the C11a chiral position, which gives them a dextrorotatory rotation when viewed from the C-ring towards the A-ring. This feature also gives the PBD the appropriate three-dimensional shape for isomeric minor groove helicity of B-form DNA, resulting in a tight 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: 230237 (1986)). PBDs can form adducts in the minor groove, resulting in interference with DNA processing.

The first PBD-based 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, a number of naturally occurring PBDs have been discovered, and more than ten synthetic routes have been developed to produce various analogues (Thurston et al., Chem. Rev., 433-465 (1994); and Antonow, D. and Thurston, D.E. , Chem. Rev., 111: 2815-2864 (2011). Members of this family include abbemycin (Hochlowski et al., J. Antibiotics, 40: 145-148 (1987)), chikamycin (Konishi et al., J. Antibiotics, 37: 200-206 (1984)), DC-81 ( Japanese patent 58180487; Thurston et al., Chem. Brit., 26: 767-772 (1990); and Bose et al., Tempahedron, 48: 751-758 (1992)), masethramycin (Kuminoto et al., J. Antibiotics, 33: 665-667 (1980)), neotramycins A and B (Takeuchi et al., J. Antibiotics, 29: 93-96 (1976)), porotramycin (Tsunakawa et al., J. Antibiotics, 41: 1366 -1373 (1988)), protracarcin (Shimizu et al., J. Antibiotics, 29: 2492-2503 (1982); and Langley and Thurston, J. Org. Chem., 52: 91-97 (1987)), sibanomycin (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. , J. Am. Chem. Soc., 110: 2992-2993 (1988)) and tomycin (Arima et al., J. Antibiotics, 25: 437-444 (1972)). PBDs, as well as ADCs containing PBDs, are also described in International Patent Application Publications Nos. WO 2015/155345 and WO 2015/157592.

In one embodiment, the PBD is PBD 3249, also referred to herein as SG3249 and described in detail in WO 2014/057074, which has the following structure

In another embodiment, the PBD is PBD 3315, also referred to herein as SG3315 and described in detail in WO 2015/052322, which has the following structure

In another embodiment, the PBD is SG3400, also called compound 23, which is described in detail in PCT/EP2017/052988, filed February 10, 2017, and has the following structure

Anti-BCMA monoclonal antibody or antigen-binding fragment thereof may be conjugated

- 7 046403 are conjugated with the cytotoxin using any suitable method known in the art, including site-specific or site-non-specific conjugation methods. Conventional antibody conjugation strategies typically rely on random conjugation (i.e., nonspecific) of the payload to the antibody or its antigen-binding fragment at lysine or cysteine residues. Accordingly, in some aspects, an antibody or antigen-binding fragment thereof is randomly conjugated to a cytotoxic agent, for example, by partially reducing the antibody or antibody fragment and then reacting with the desired agent with or without the addition of a linker moiety. For example, the antibody or antigen binding fragment thereof can be reduced using dithiothreitol (DTT) or a similar reducing agent. The cytotoxic agent, with or without a linker moiety attached thereto, can then be added in molar excess to the reduced antibody or antibody fragment in the presence of dimethyl sulfoxide (DMSO). After conjugation, excess free cysteine can be added to quench the unreacted agent. The reaction mixture can then be purified and buffer exchanged with phosphate buffered saline (PBS).

In other embodiments, a cytotoxic agent can be conjugated to an anti-BCMA monoclonal antibody using site-specific conjugation techniques at specific reactive amino acid residues to produce homogeneous ADC preparations with identical stoichiometry. Site-specific conjugation can occur at a cysteine residue or at an unnatural amino acid. In one embodiment, the cytotoxic agent can be conjugated to an antibody or an antigen-binding fragment thereof at at least one cysteine residue. In particular, for example, a cytotoxic agent may be chemically conjugated to an amino acid side chain at a specific position according to Kabat in the Fc region of an anti-BCMA monoclonal antibody. In this regard, the cytotoxic agent can be conjugated to an anti-BCMA monoclonal antibody at a cysteine residue at any suitable position in the Fc region of the antibody, including, without limitation, 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 the numbering corresponds to the Kabat EU index. In one embodiment, the cytotoxic agent can be conjugated to the anti-BCMA monoclonal antibody at a cysteine residue at specific Kabat positions 239 and/or 442 of the anti-BCMA monoclonal antibody and/or at an amino acid residue inserted between Kabat positions 239 and 240 of the anti-BCMA antibody ( Dimasi et al., Mol Pharm, 14(5): 1501-1516 (2017) Alternatively, the cytotoxic agent may be conjugated to an anti-BCMA monoclonal antibody or antigen-binding fragment thereof at a thiol-maleimide linkage, such as, for example, at a sulfhydryl reactivity group in the hinge area and heavy-light chains.

The anti-BCMA monoclonal antibody described herein contains at least one cytotoxin molecule conjugated thereto; however, the anti-BCMA monoclonal antibody may contain any suitable number of cytotoxin molecules conjugated thereto (eg, 1, 2, 3, 4 or more cytotoxin molecules) to achieve the desired therapeutic effect. Desirably, the ADC described herein contains two cytotoxin molecules conjugated to an anti-BCMA monoclonal antibody.

The anti-BCMA antibody described herein is applicable to any therapeutic agent that needs to target BCMA, such as adoptive cell transfer (ACT), bispecific T-cell activators (BiTE) and nanoparticles. In one embodiment, the present invention provides a chimeric antigen receptor (CAR) comprising the antigen binding domain of an anti-BCMA monoclonal antibody described herein linked to a T cell activation moiety. A chimeric antigen receptor (CAR) is an artificially engineered fusion protein or polypeptide containing the antigen binding domain of an antibody (eg, single chain variable fragment (scFv)) linked to T cell signaling or T cell activation moieties. CAR structures have evolved over the past twenty years and most often include a single chain variable fragment (scFv) derived from a monoclonal antibody (mAb) and a signaling motif from the TCR ζ chain (referred to as first-generation CARs (see, e.g., Okur, F.V., Brenner, M.K., Methods Mol. Biol., 651: 319-45 (2010); and Lee et al., Clin. Cancer. Res., 75(10): 2780-2790 (2012). More recently, a second and third generation CARs that include one (second generation) or two (third generation) co-stimulatory activating motifs, for example from CD28, 4-1BB (CD137) and/or CD134 (OX-40), which enhance proliferation, cytotoxicity and persistence in vivo (see, for example, Finney et 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).

The antigen binding domain of a CAR may comprise a whole monoclonal antibody or a fragment of a monoclonal antibody described herein. In one embodiment, the CAR antigen binding domain may comprise a single chain Fv fragment (scFv) of a monoclonal anti

- 8 046403 body to BCMA. Chimeric antigen receptors and methods for producing 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 US Patent Application Publication 2015/0051266 A1.

The present invention also provides a composition comprising the above-described antibody or antibody-drug conjugate and a pharmaceutically acceptable (eg, physiologically acceptable) carrier. Any suitable carrier known in the art may be used in the context of the present invention. The choice of carrier will be determined in part by the particular site at which the composition may be administered and the particular method used to administer the composition. The composition may not necessarily be sterile. The compositions can be prepared in accordance with traditional techniques described, for example, in Remington: the Science and Practice of Pharmacy, 21st Edition, Lippincott Williams & Wilkins, Philadelphia, PA (2001).

Desirably, the composition contains an antibody or antibody-drug conjugate in an amount that is effective for treating or preventing multiple myeloma. Thus, the present invention provides a method of killing multiple myeloma cells, which includes contacting multiple myeloma cells that express BCMA with an antibody or antibody-drug conjugate described herein, or a composition containing an antibody or ADC described in herein, whereby the antibody or antibody-drug conjugate binds to BCMA on multiple myeloma cells and causes the killing of multiple myeloma cells. The present invention also provides for the use of an antibody or ADC described herein, or a composition containing an antibody or ADC, in the manufacture of a medicament for the treatment of multiple myeloma. As discussed herein, multiple myeloma, also known as plasma cell myeloma or Kahler disease, is a cancer of plasma cells, which are a type of white blood cell typically responsible for producing antibodies (Raab et al., Lancet, 374: 324-329 (2009) ). Multiple myeloma affects 1-4 people per 100,000 people per year. The disease is more common in men and, for unknown reasons, is twice as common in African Americans as in Caucasian Americans. Multiple myeloma is the least common hematologic malignancy (14%) and accounts for 1% of all cancers (Raab et al., supra). Treatment of multiple myeloma usually involves high-dose chemotherapy followed by hematopoietic stem cell transplantation (allogeneic or autologous); however, high relapse rates are common in patients with multiple myeloma who have undergone such treatment. As discussed above, BCMA is expressed at high levels in multiple myeloma cells (see, for example, Novak et al., supra; Neri et al., supra; Bellucci et al., supra; and Moreaux et al., supra).

As demonstrated herein, BCMA is also expressed on multiple myeloma stem cells. Thus, the present invention provides a method of killing multiple myeloma stem cells, which includes contacting multiple myeloma stem cells that express BCMA with an antibody-drug conjugate described herein or a composition containing an ADC described herein , whereby the antibody-drug conjugate binds to BCMA on multiple myeloma stem cells and causes the destruction of multiple myeloma stem cells. Multiple myeloma stem cells can be identified in the bone marrow of patients with multiple myeloma by their surface expression of CD19 and lack of surface expression of CD138 (see, for example, Matsui et al., Blood, 103: 2332-6 (2004)). These cells are uniquely capable of colony formation and engraftment in immunodeficient mice, while myeloma plasma cells, defined as CD138+CD19-, are not. Multiple myeloma stem cells are also resistant to current therapies (Matsui et al., Cancer Res., 68: 190-7 (2008)).

The antibody-drug conjugate described herein, or a composition containing the antibody-drug conjugate, can be contacted with a population of multiple myeloma cells that express BCMA ex vivo, in vivo or in vitro. Ex vivo refers to methods carried out in or on cells or tissue in an artificial environment outside the body with minimal modification of natural conditions. In contrast, the term in vivo refers to a method that is carried out inside living organisms in their normal, intact state, while an in vitro method is carried out using components of the organism that have been isolated from their normal biological environment. In one embodiment, the multiple myeloma cells are human multiple myeloma cells that are contacted with an ADC described herein, or a composition containing an ADC, in vivo.

The terms treatment, implementation of treatment, etc. used in this document. refer to obtaining the desired pharmacological and/or physiological effect. Preferably, the effect is therapeutic, ie, the effect provides partial or complete cure of the disease and/or an adverse symptom related to the disease. For this purpose, the method in accordance with

- 9046403 The present invention involves administering a therapeutically effective amount of an antibody or ADC, or a composition containing an antibody or ADC, and a pharmaceutically acceptable carrier. A therapeutically effective amount refers to an amount effective in doses and for periods of time necessary to achieve the desired therapeutic result. The therapeutically effective amount may vary according to factors such as the disease state, age, sex and weight of the individual and the ability of the antibody or ADC to elicit the desired response in the individual. For example, a therapeutically effective amount of the ADC of the present invention is an amount that binds to and destroys BCMA on multiple myeloma cells.

Alternatively, the pharmacological and/or physiological effect may be prophylactic, i.e. the effect provides complete or partial prevention of the disease or its symptom. In this regard, the method in accordance with the present invention includes administering a prophylactically effective amount of an ADC or a composition containing an ADC to a mammal that is predisposed to multiple myeloma. A prophylactically effective amount refers to an amount effective at doses and for periods of time necessary to achieve the desired prophylactic result (eg, preventing the onset of a disease).

Therapeutic or prophylactic effectiveness can be monitored by periodic evaluation of patients receiving treatment. In one embodiment, the ADC described herein inhibits or suppresses the proliferation of BCMA-expressing myeloma cells by at least about 10% (e.g., at least about 20%, at least about 30%, at least 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 90% approximately 100%). Cell proliferation can be measured using any suitable method known in the art, such as measuring the incorporation of labeled nucleosides (e.g., 3-tomidi or bromodeoxyuridine Brd(U)) into genomic DNA (see, e.g., Madhavan, H.N., J. Stem Cells Regen Med., 3(1): 12-14 (2007).

The antibody or ADC described herein, or a composition containing the antibody, ADC, can be administered to a mammal (eg, human) using standard administration techniques, including, for example, intravenous, intraperitoneal, subcutaneous. More preferably, the antibody or ADC, or a composition containing them, is administered to the mammal by intravenous injection.

An antibody or ADC described herein, or a composition containing an antibody or ADC, can be administered with one or more additional therapeutic agents that can be co-administered to a mammal. The term coadministration as used herein refers to the administration of one or more additional therapeutic agents and an antibody or ADC described herein, or a composition containing an antibody or ADC, sufficiently close in time so that the antibody or ADC can enhance the effect of one or more several additional therapeutic agents, or vice versa. In this regard, the antibody or ADC, or a composition containing them, can be administered first, and one or more additional therapeutic agents can be administered second, or vice versa. For example, an antibody or ADC, or a composition containing them, can be administered in combination with other agents (eg, as an adjuvant) for the treatment or prevention of multiple myeloma. In this regard, the antibody or ADC, or a composition containing the antibody or ADC, can be used in combination with at least one other anticancer agent including, for example, any suitable chemotherapeutic agent known in the art, ionizing radiation, small molecule anticancer agents, vaccines against cancer, biological therapies (eg, other monoclonal antibodies, cancer-killing viruses, gene therapy, and T-cell adoptive transfer), and/or surgery.

The following examples further illustrate the present invention, but, of course, they should not be construed as limiting its scope in any way.

Example 1.

This example describes the production of a monoclonal antibody directed against B-cell maturation antigen (BCMA).

According to the RIMMS immunization schedule described in Kilpatrick et al., Hybridoma, 16(4): 381-389 (1997), six-week-old female Ablexis transgenic mice (Ablexis, LLC, San Francisco, Calif.) received six courses of subcutaneous injections of purified recombinant human (rHu) BCMA-Fc alone (cycle 1) or alternate immunization with both rHu BCMA-Fc and cynomolgus macaque BCMA-Fc (Cyno BCMA-Fc) or adherent 293 cells (Ad293) expressing either Hu BCMA , or Cyno BCMA (cycle 2) to various areas. Mice were immunized over a course of 13 days at intervals of 2-3 days. For each immunization cycle, mice were first anesthetized with isoflurane. The immunogen was emulsified in Freund's complete or incomplete adjuvant and TITERMAX® Gold adjuvant (Sigma-Aldrich, St. Louis, MO) and injected bilaterally into different areas. Blood samples

- 10 046403 was harvested on day 13 and analyzed for antigen binding by ELISA and FACS on human and cynomolgus BCMA-expressing adherent 293 cells. Mice with good serum titers were given a prefusion booster injection and sacrificed on day 17. Lymph node cells were collected and fused with the myeloma cell line P3-X63-Ag8.653 according to the polyethylene glycol fusion method (Roche Diagnostics, Indianapolis, IN) to generate stable hybridomas.

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

The first round resulted in a panel of 44 human-only binding antibodies and 4 binding antibodies with cross-reactivity to humans and cynomolgus monkeys. These 48 antibodies were then further tested by FACS on adherent 293 cells expressing BCMA and endogenous NCI-H929 cell lines expressing huBCMA. A core panel of 25 hybridoma lines was identified by ranking the antibodies for best binding to endogenous human BCMA. A total of 11 hybridomas were nominated for subcloning, scale-up, sequencing, and purification. Clones were also assessed for Fab-Zap-mediated killing. One antibody (clone 4679) was recombinantly cloned as human IgG1 for further evaluation.

The second round resulted in a panel of 98 binding antibodies for immunization with BCMA-Fc rHu/Cyno, nine binding antibodies for immunization with BCMA-Fc rHu/Cyno and Ad293 cells expressing BCMA Cyno, and zero binding antibodies for immunization with Ad293 cells expressing both Hu and Cyno BCMA. These hybridomas were further tested for binding to TACI and BAFF-R by FACS, and antibodies exhibiting any detectable binding to TACI and BAFF-R were removed. These secondary screens, along with the Fab-Zap assay, led to the identification of eight hybridomas that were nominated for limiting dilution cloning (LDC). Based on their activity, two clones (clones 756 and 15B2) were then developed into human IgG1 for further evaluation.

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 at 4°C for 45 min, followed by two washes with PBS+2% FBS. Cells were then incubated with Alexa-Fluor 647-labeled secondary antibodies at 4°C, followed by two washes in PBS+2%FBS. APC-labeled antibodies to human BAFF-R, TACI, and BCMA were added at the manufacturer's recommended dilution to control wells. Cells were resuspended in 200 μl PBS+2%FBS+DAPI and antibody binding to live cells was analyzed using a Becton Dickinson Biosciences LSRII cytometer. Antibody 15B2GL was the only cross-reacting anti-cynomolgus antibody tested that does not bind to BAFF-R and/or TACI, as shown in FIG. 1.

Monoclonal antibody 15B2 was mutated to a germline form (15B2GL) using primers designed to mutate four residues in 15B2 without undergoing germline conversion. Wild-type 15B2 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 analyzes were performed to compare the binding of 15B2GL and 15B2WT and a collection of major optimized (LO) 15B2GL clones was obtained. Briefly, 15B2 was cloned into a FAb expression vector designed for bacterial expression. Twenty-seven residues in the heavy chain and nineteen residues in the light chain, each of which was mutated in a simple manner using primers designed to accommodate nineteen different amino acids, excluding cysteine. Mutagenesis was performed using the QuikChange Lightning Mutagenesis kit (Agilent Genomics, Santa Clara, CA).

Approximately one hundred colonies per position were screened by ELISA for binding for a total of 6000 clones. ELISA binding was measured by low-density capture of human BCMA and bacterial supernatant after 48 h of bacterial expression. Hits were defined as being more than twice that of the 15B2GL control. Individual amino acid hits were confirmed by cynomolgus monkey BCMA ELISA

- 11 046403 without binding to a nonspecific protein. The identified hits from the simple model screen were then combined to design a primer to generate a combinatorial library. The above FAb screening approach by ELISA binding assay was repeated as for the combinatorial library using 15B2GL as the starting template and ELISA control. The hits identified from the combinatorial screen were then cloned into a mammalian IgG expression vector (Maia) designed for conjugation to produce ADCs. Proteins were expressed in 293HEK cells and purified by Protein A affinity chromatography for additional testing.

The ability of antibody 15B2GL and LO clones 15B2GL to bind, internalize and kill multiple myeloma H929 cells in vitro was assessed using Fab-ZAP and FACS binding assay to endogenous BCMA expressed in cell lines as described above. Briefly, antibodies were incubated with 200,000 cells for 30 min at 4°C, followed by two washes with PBS+2% FBS. Cells were resuspended in 100 μl of cold PBS+2% FBS and maintained at 4°C. At certain time points, cells were washed and resuspended in warm RPMI+10% FBS and placed in a 37°C, 5% CO ₂ incubator. At the end of the experiment, cells were washed and then incubated with Alexa-Fluor 647-labeled secondary antibodies at 4°C, followed by two washes in PBS+2%FBS. Cells were resuspended in 200 μl PBS+2%FBS+DAPI and antibody binding to live cells was analyzed using a Becton Dickinson Biosciences LSRII cytometer. Antibody 15B2GL exhibited unique and rapid internalization by this method compared to anti-BCMA J6M0 antibody (described in US Pat. No. 9,273,141) and LO antibodies.

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

The amino acid sequences of the variable regions of the heavy and light chains of monoclonal antibodies 15B2 (wild type and germline) and LO clones I09, L15, P10, N22 and M02 are shown in table. 1.

Table 1

Antibody Amino acid sequence SEQ ID NO: VH 15B2GL EVQLVESGGGLVKPGGSLRLSCAASGFTFRSYSMNW VRQAPGKGLEWVSSISGSSNYIYYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCARGGNYYVEYFQ YWGQGTLVTVSS 7 VL 15B2GL EIVLTQSPGTLLSLSPGERATLSCRASQYISSNYLAWYQ QKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTI SRLEPEDF AVYYCQQ YGS SPITFGQGTKLEIK 8 Heavy chain variable region (VH) 15B2WT EIQLVESGGGLVKPGGSLRLSCAASGFFRSYSMNW VRQAPGKGLEWVSSISGSSNYIYYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTALYYCARGGNYYVEYFQ YWGQGTLVTVSS 9 Light chain variable region (VL) 15B2WT EIVLTQSPGTLLSLSPGERATLSCRASQYISSNYLAWYQ QKPGQAPRLLIYGASNRATGIPDRFSGSGSGTGGFTLTI SRLEPEDF A VFYCQQ YGS SPITFGQGTKLEIK 10 VHI09 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVSSISGSSNYIYYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCARGGNYFVEYFQ QWGQGTLVTVSS eleven VLI09 EIVLTQSPGTLLSLSPGERATLSCRASQYISSNYLAWYQ QKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTI SRLEPEDF AVYYCQQ YS SDPITFGQGTKLEIK 12 VHL15 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVSSISGQSNYIYYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCARGGNYFVEYFQ YWGQGTLVTVSS 13 VLL15 EIVLTQSPGTLLSLSPGERATLSCRASQYISSNNLAWYQ QKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTI SRLEPEDF A VYYCQQYADSPITFGQGTKLEIK 14

- 12 046403

VHM02 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVSSISGQSNYIYYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCARGGNYYVEYFQ YWGQGTLVTVSS 15 VLM02 EIVLTQSPGTLLSLSPGERATLSCRASQYISSNNLAWYQ QKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTI SRLEPEDF AVYYCQQ YS SDPITFGQGTKLEIK 16 VHN22 EVQLVESGGGLVKPGGSLRLSCAASGFTFSSYSMNW VRQAPGKGLEWVSSISGSSNYIYYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCARGGNYFVEYFQ YWGQGTLVTVSS 17 VLN22 EIVLTQSPGTLLSLSPGERATLSCRASQYISSNYLAWYQ QKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTI SRLEPEDF AVYYCQQ YS S SPITFGQGTKLEIK 18 VHP10 EVQLVESGGGLVKPGGSLRLSCAASGFTFRSYSMNW VRQAPGKGLEWVSSISGQSNYIYYADSVKGRFTISRD NAKNSLYLQMNSLRAEDTAVYYCARGGNYYVEYFQ YWGQGTLVTVSS 19 VLP10 EIVLTQSPGTLLSLSPGERATLSCRASQYISSNYLAWYQ QKPGQAPRLLIYGASNRATGIPDRFSGSGSGTDFTLTI SRLEPEDF A VYYCQQYTD SPITFGQGTKLEIK 20

The results of this example demonstrate the production of monoclonal antibodies directed against BCMA.

Example 2.

This example demonstrates a method for preparing an antibody-drug conjugate (ADC) containing an anti-BCMA monoclonal antibody conjugated to a cytotoxin in accordance with the present invention.

Monoclonal antibody 15B2GL and the optimized clones described in Example 1 were conjugated to the PBD SG3249 payload 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(2carboxyethyl)phosphine) in PBS, pH 7.2, 1 mM EDTA (ethylenediaminetetraacetic acid) for 3 h at 37°C. After incubation, the reducing agent was removed by 2X dialysis in PBS, pH 7.2, 1 mM EDTA at 4°C using 10,000 MWCO dialysis cassettes, followed by incubation with 20 molar equivalents of dehydroascorbic acid for 4 h at 25°C. Eight equivalents of PBD SG3249 payload were then added sequentially from a stock solution in 10% (v/v) DMSO, followed by incubation at room temperature for 1 h with gentle rotation. The conjugation reaction was quenched by adding four molar equivalents (relative to SG3249) of N-acetylcysteine.

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

To determine the content of monomers, aggregates and fragments, analytical size exclusion chromatography (BTOP-HPLC) was performed using 100 μg (volume 100 μl) of antibodies or ADCs, which were 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 assay was performed for 20 min at room temperature. Hydrophobic interaction chromatography (HIC-HPLC) was used to evaluate conjugation and drug loading distribution and was performed using a non-porous butyl resin (NPR) column (4.6 μm IDx3.5 cm, 2.5 μm, Tosoh Bioscience). Mobile phase A consisted of 25 mM TrisHCl, 1.5 M (NH ₄ ) ₂ SO ₄ , 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, with a gradient from 5% B to 100% B over 13 min. Reductive reverse phase chromatography (rRP-HPLC) was used to confirm chain-specific conjugation. Antibodies and ADCs were reconstituted at 37°C for 20 min using 42 mM dithiothreitol (DTT) in PBS (pH 7.2). 10 μg of reconstituted antibody or ADC was loaded onto a 1000 A (2.1 x 50 mm) reverse phase polymer media (PLRP-S) column (Agilent Technologies, Santa Clara, CA) and eluted at 80°C with a flow rate of 1 ml/mL. min, with a gradient from 5% B to 100% B in

- 13 046403 for 25 minutes (mobile phase A: 0.1% trifluoroacetic acid in water, mobile phase B: 0.1% trifluoroacetic acid in acetonitrile).

Heavy and light chain conjugation and drug:antibody ratios (DAR) were determined using reduction liquid chromatography mass spectrometry (rLCMS) assays performed on an Agilent 1290 series uHPLC coupled to an Agilent 6230 TOF (Agilent Technologies, Santa Clara, California). 2 μg of reduced antibodies or ADCs were loaded onto a 300-diphenyl ZORBAX rapid high resolution separation (RRHD) column (2.1 x 50 mm, 1.8 μm) (Agilent Technologies, Santa Clara, CA) and eluted at a flow rate of 0.5 ml/min, using 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). A positive time-of-flight MS scan image was acquired, and data acquisition and processing was performed using MassHunter software (Agilent Technologies, Santa Clara, CA). DAR was calculated using rLCMS data as described in Thompson et al., supra.

Conjugation efficiency was determined using the following equation, where the theoretical DAR 2 was used:

Conjugation efficiency = (determined DAR x theoretical DAR) x

The conjugation efficiency and DAR values of the tested antibodies are presented in table. 2.

table 2

Name designs Useful load Conjugation efficiency Ratio drug: antibody (DAR) 15B2GL SG3249 91 1.82 109 SG3249 91 1.82 L15 SG3249 90 1.80 M02 SG3249 92 1.84 N22 SG3249 91 1.82 RU SG3249 93 1.86

The results of this example demonstrate the preparation of ADCs containing a pyrrolobenzodiazepine-conjugated anti-BCMA monoclonal antibody in accordance with the present invention.

Example 3.

This example demonstrates the binding affinity of the anti-BCMA monoclonal antibodies described herein to monomeric (soluble) and membrane-bound BCMA.

Binding of monoclonal antibody 15B2GL and optimized clones I09, L15, P10, N22 and M02 (described in Example 1) was assessed using monomeric human sBCMA (GenScript, Piscataway, NJ) using a ProteOn XPR36 device (Bio-Rad, Hercules, CA ). J6M0 binding was also assessed for comparison. Standard amine immobilization was used to immobilize 25 μg/mL polyclonal anti-Fc antibody (Jackson ImmunoResearch, West Grove, PA) prepared in 10 mM sodium acetate buffer (pH 4.5) 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 (Nhydroxysulfosuccinimide), with a density of ~200-600 resonance units (RU). 15B2GL, I09, L15, P10, N22, M02, and J6M0 were sequentially injected at a concentration of 1 μg/ml for capture by immobilized polyclonal anti-Fc antibody. The sensorgram was recorded by flowing twofold dilutions of sBCMA prepared in PBS (pH 7.4) with 0.005% (v/v) Tween-20, in the range of 100–6.25 nM, over the captured surface for 150 s at 75 µl/min with a dissociation time of 600 s. ProteOn data analysis software was used for data analysis.

The results of this experiment are shown in Fig. 13 and 14 and in table. 3.

- 14 046403

Table 3

Kinetics measurements using BioRad ProteOn

Antibody Cop Koff Kd nM 15B2GL 3.73E+05 2.27E-02 6.07E-08 60.7 N22 5.36E+05 8.73E-03 1.63E-08 16 109 6.09E+05 5.99E-03 9.82E-09 9.8 M02 4.99E+05 4.97E-03 9.95E-09 9.9 L15 8.54E+05 3.86E-03 4.51E-09 4.5 P10 7.01E+05 3.73E-03 5.32E-09 5.3 J6M0 4.21E+05 4.69E-04 1D1E-09 1

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 that endogenously express BCMA (NCI-H929 and MM.1S, respectively). The binding of 15B2GL, I09, L15 to membrane-bound human BCMA was also assessed in Ad293 cells expressing human BCMA. Binding assays were performed by incubating anti-BCMA antibodies with 200,000 cells for 30 min at 4°C, followed by two washes with PBS+2% FBS (FACS buffer). The range of antibody concentrations was assessed using a 12-digit series of 3-fold dilutions. Cells were then incubated with 5 μg/ml goat anti-human IgG secondary antibody AF647 (Thermo Fisher Scientific, Waltham, MA) at 4°C, followed by two washes with PBS+2%FBS. Cells were resuspended in 200 μl PBS+2%FBS+DAPI.

Fluorescence from live single cells was measured using a BD Biosciences LSRII cytometer and BD FACSDiva software (BD Biosciences, San Jose, CA). Data were analyzed using FlowJo software (FlowJo, LLC, Ashland, OR). Average fluorescence intensities were used to determine percent binding, and EC50 was determined using Prism software (GraphPad Software Inc, La Jolla, CA). The results of this experiment are shown in Fig. 15. A summary of the SPR and apparent affinity data measured by flow cytometry are shown in Table. 4.

Table 4

Binding affinity Apparent affinity (nM) bound to BCMA cells Antibody (nM), monomeric NCI-H929 MM.1S Ad293+huBCMA WSMA

15B2GL 60.7 3.14 2.56 3.87 N22 16 But. But. But. 109 9.8 5.4 4.8 5.3 M02 9.9 But. But. But. L15 4.5 4.2 4.2 4.48 RU 5.3 But. But. But. J6M0 1 6.02 6.65 But.

But. = not defined.

The results of this example demonstrate that the anti-BCMA monoclonal antibody 15B2GL exhibits strong binding to membrane-bound BCMA and weak binding to monomeric (soluble) BCMA, which in this regard is unique among the monoclonal antibodies that were analyzed in these assays.

Example 4.

This example demonstrates methods for killing multiple myeloma and plasma cell leukemia cells in vitro using the antibody-drug conjugates described herein.

Killing of multiple myeloma and plasma cell leukemia cell lines by antibody-drug conjugates containing 15B2GL or its affinity optimized clones conjugated to SG3249 was assessed in vitro using the protocol recommended in the CELLTITER-GLO® kit (Promega, Madison, WI) . Killing Multiple Myeloma and Plasma Cell Leukemia Cell Lines Using a Free Attack Element

- 15 046403

SG3199 was also evaluated using the protocol recommended in the CELLTITER-GLO® kit (Promega, Madison, WI). Briefly, 5 x 10 ³ cells in 80 μl RPMI+10% FBS were added to the inner wells of a 96-well white-walled 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. BMCA-negative cell lines Raji and Jurkat were also tested. Antibody-drug conjugates were diluted to 5x stock solution (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 the cells in duplicate, resulting in a 12-point dose curve of the antibody-drug conjugate ranging from 0.5 μg/ml at the highest concentration to 3x10 ^-6 μg/ml at the lowest. Isotype antibody-drug conjugate controls (IgG1-SG3249 and IgG1-mc-MMAF) and media alone were also included. The plates were incubated at 37°C, 5% CO2 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 plotted using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA) and the half-maximal inhibition concentration (IC50) was determined.

Chromosomal translocation information for each cell line was taken from Moreaux et al, 2011 and Boersma-Vreugdenhil et al, 2004. BCMA receptor abundance was determined 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).

The results of this experiment are shown in table. 5 and fig. 2A-2J.

Table 5

In vitro cytotoxicity in multiple myeloma and plasma cell leukemia cell lines

Cell line Origin of the disease Chromosomal translocation Quantity BCMA receptors 15B2GLSG3249 IC50 (ng/ml) SG3199 IC50 (pM) NCI-H929 MM t(4;14) 18931 5.99 5 E.J.M. MM t(14;20) 14325 153 22 MM.1R PCL t(14;16) 9449 8.64 3 JJN-3 MM t(14;16) 3221 36.28 13 ORM-2 mm t(4;14) 2873 201 19 MM.1S PCL t(14;16) 2698 26.8 3 U266B1 MM t(ll;14) 2340 39.7 155 L-363 PCL t(20;22) 930 31.4 H. and. Raji Burkitt's lymphoma H.n. 0 >500 N. And. Jurkat T lymphocyte H.n. 0 >500 3

BCMA = B-cell maturation antigen; n. n. = not applicable; n. And. = not investigated.

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 assessed in MM.1S cells using the protocol described above, except that the cell lines tested were also treated with conditioned media. containing BCMA, collected from Ad293 cells expressing human BCMA (Fig. 3A and 3B). Cell killing by the ADC 15B2GL-SG3249 in the presence of sBCMA was also compared with an ADC containing the anti-BCMA antibody J6M0, which is described in US Pat. No. 9,273,141. The results of this experiment are shown in FIG. 3, which demonstrated that the activity of ADC 15B2GL-SG3249 was maintained in the presence of clinically relevant levels of sBCMA to a greater extent than ADC I09-SG3249 (Fig. 3A), J6M0-mc-MMAF, and J6M0-SG3249 (Fig. 3B and Table 3B). 6).

- 16 046403

Table 6

sBCMA (ng/ml) Test sample IC50 (ng/ml) Efficiency loss ratio 720 15B2GL-SG3249 29.12 2.01 270 15B2GL-SG3249 22.86 1.58 75 15B2GL-SG3249 16.09 1,I 0 15B2GL-SG3249 14.47 1.00 720 J6M0-SG3249 92.92 19.29 270 J6M0-SG3249 32.26 6.70 75 J6M0-SG3249 12.83 2.66 0 J6M0-SG3249 4.816 1.00 720 I09-SG3249 16.16 5.75 270 I09-SG3249 5,548 1.98 75 I09-SG3249 3,791 1.35 0 I09-SG3249 2,809 1.00 720 J bMO-shs-MMAB 1458 49.80 270 J bMO-shs-MMAB 159.7 5.45 75 J bMO-shs-MMAB 55.55 1.90 0 J bMO-shs-MMAB 29.28 1.00

The results of this Example demonstrate that ADC 15B2GL-SG3249 kills multiple myeloma and plasma cell leukemia cells in vitro, and that cell killing activity was maintained even in the presence of soluble BCMA. Specifically, ADC 15B2GL-SG3249 was cytotoxic to both MM.1S and NCI-H929 in vitro, killing an average of 95% of tumor cells in the presence of sBCMA at levels as low as 720 ng/mL with little effect on IC50. ADCs developed from antibodies that have similar affinities to monomeric BCMA and membrane-bound BCMA exhibited a dose-dependent decrease in efficacy of sBCMA, with a 20-fold shift in IC50 in the presence of 720 ng/mL sBCMA.

Example 5.

This example demonstrates methods for killing multiple myeloma and plasma cell leukemia cells in vivo using the antibody-drug conjugates described herein.

Subcutaneous xenograft mouse models of multiple myeloma and plasma cell leukemia were generated by implanting multiple myeloma or plasma cell leukemia cell lines expressing BCMA (i.e., NCI-H929, JJN-3, MM.1S, and MM.1R) into CB-17 SCID females (C .B17/IcrHsd-Prkdc-scid) or nude (Foxn1 ^nu ) mice using MATRIGEL™ (BD Biosciences, San Jose, CA). When tumors reached approximately 180 mm ³ (NCI-H929 cells), 190 mm ³ (JJN3 cells), 160 mm ³ (MM.1S cells), or 175 mm ³ (MM.1R cells), mice were randomized based on tumor size and placed into dosing groups and were treated with ADCs targeting BCMA as described below.

NCI-H929 xenograft model.

Mice were treated with either a single intravenous dose of ADC 15B2GL-SG3249, I09-SG3249, L15-SG3249 at 0.3 mg/kg or weekly intravenous dosing of J6M0-mc-MMAF at 0.3 mg/kg 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 with no evidence of tumor regrowth, as shown in FIG. 4. No weight loss was observed in any of the dosing groups.

JJN3 xenograft model.

Mice were treated with either a single intravenous dose of ADCs 15B2GL-SG3249, I09-SG3249, and L15-SG3249 at 1 mg/kg or weekly intravenous doses of ADC J6M0-mc-MMAF at 1 mg/kg 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 with no evidence of tumor regrowth, as shown in FIG. 5. No weight loss was observed in any of the dosing groups.

Xenograft model MM.1S.

Mice were treated with either a single intravenous dose of ADC 15B2GL-SG3249, I09-SG3249,

- 17 046403

L15-SG3249 at 1 mg/kg, or IV dosing of J6M0-mc-MMAF twice weekly at 1 mg/kg 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 with no evidence of tumor regrowth, as shown in FIG. 6. No weight loss was observed in any of the dosing groups.

Xenograft model MM.1R.

Mice were treated with either a single intravenous dose of 15B2GL-SG3249 at 1 mg/kg or weekly intravenous doses of J6M0-mc-MMAF at 3 mg/kg for 4 weeks. Control mice were left untreated. Mice treated with 15B2GL-SG3249 were observed for 109 days after tumor implantation with no evidence of tumor regrowth as shown in FIG. 7. No weight loss was observed in any of the dosing groups.

The results of this example demonstrate that the ADC 15B2GL-SG3249 exhibits increased antitumor efficacy in vivo compared to other ADCs that target BCMA-expressing cells.

Example 6.

This example demonstrates that multiple myeloma stem cells express BCMA.

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

BCMA expression was assessed in the stem cell population of samples from four multiple myeloma patients by flow cytometry. Samples were obtained from Proteogenex, Inc. (Culver City, CA) (see Table 7) and individual multiple myeloma (MM) samples were thawed in a water bath at 37°C.

Table 7 MM Patient Information

ID sample Floor Age Ethnicity Clinical diagnosis Disease status MM263V m M 63 Caucasian MM Diagnosis established MM277V M F 66 Caucasian MM Diagnosis established MM276V M F 81 Caucasian mm Diagnosis established MM284V M M 84 Caucasian mm Resistant to therapy

Thawed cells were added to 10 ml PBS and counted using a ViCELL™ counter (Beckmann-Coulter Life Sciences, Indianapolis, IN). An aliquot of the cell suspension was obtained for colony formation assay, while the rest of the cell suspension was centrifuged at low speed to pellet the cells. Fc cells were blocked according to the manufacturer's instructions, then seeded at 200,000 cells per well in a 96-well plate. The plate was centrifuged to pellet the cells, the Fc blocking solution was discarded, and cell samples were resuspended in BV staining buffer followed by staining with an appropriate panel of antibodies consisting of directly conjugated antibodies supplied from commercial sources, which are shown in Table. 8 and 9.

- 18 046403

Table 8 Antibody staining panel

Sample Description Antibody panel 1 Separate staining B V510 CD3-BV510, CD34-BV510, CD14-BV510, CD193-BV510 2 Separate PE staining CD 13 8-PE 3 Separate APC staining CD19-APC 4 Separate PE/Cy7 staining WSMA-RE/Su7 5 No coloring 6 Separate DAPI staining DAPI 7 FMO(BV510) CD 13 8-PE, CD19-APC, BCMA-PE/Cy7, DAPI 8 FMO (PE) CD3-BV510, CD34-BV510, CD14-BV510, CD193-BV510, CD19-APC, BCMA-PE/Cy7, DAPI 9 FMO (ARS) CD3-BV510, CD34-BV510, CD14-BV510, CD193-BV510, CD 13 8-PE, WSMA-PE/Су7, DAPI 10 FMO (PE/Su7) CD3-BV510, CD34-BV510, CD14-BV510, CD193-BV510, CD 13 8-PE, CD19-APC, DAPI AND All stains CD3-BV510, CD34-BV510, CD14-BV510, CD193-BV510, CD138-PE, CD19-APC, VSMARE/Cy7, DAPI 12 FMO (DAPI) CD3-BV510, CD34-BV510, CD14-BV510, CD193-BV510, CD138-PE, CD19-APC, VSMARE/Cy7 13 Compensation granules BV510 CD193-BV510 14 Compensation granules PE CD 13 8-PE 15 Compensatory granules APC CD19-APC 16 Compensatory granules PE/Sy7 WSMA-RE/Su7

- 19 046403

Table 9

Antibodies used for flow cytometry analysis

Antibody/reagent Clone Provider Cat. No. CD3-BV510 oktz Biolegend 317332 CD34-BV510 581 Biolegend 343528 CD14-BV510 M5E2 Biolegend 301842 CD193-BV510 5E8 Biolegend 310722 CD19-APC SJ25C1 BD Biosciences 340437 CD 13 8-PE V-AZ 8 Beckman Coulter A40316 WSMA-RE/Su7 19F2 Biolegend 357508 DAPI ThermoFisher Scientific 62248 FC Block BD Pharmingen 564220 Buffer for Horizon Brilliant staining BD Biosciences 563794 Ultra Comp granules eBiosciences 01-2222-42

In addition, compensatory granules were individually stained with the antibodies tested. The plate was incubated at 4°C in the dark for 30 min. The plate was centrifuged and the cells were washed in DPBS+2% FBS followed by resuspension in 200 μl DPBS+2% FBS+DAPI. Cells from each well were assessed on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA) and FCS files were obtained. Cell identification was performed using the following gating strategy: compensation was performed using an automated computer matrix in FlowJo®10 (FlowJo LLC, Ashland, OR) using data for compensatory beads and separate staining. Plasma cells were gated on a FSC-A versus SSC-A plot, then screened for live single cells on a DAPI versus SSC-W plot. Size exclusion gating was then used to remove cells that stained positive for CD3, CD14, CD34, and CD193 in the BV-510 versus SSC-A plot. This population was then analyzed by plotting CD138-PE versus CD19-APC. Histograms for BCMA expression were obtained for the MM CSC population defined as CD19+/CD138− and the MM plasma cell population defined as CD19−/CD138+. The location of the gates in the assay was established based on the corresponding sample controls with a combination of fluorescence-detected labels without one (FMO). All samples showed a small percentage of CD138+CD19− cells that were positive for BCMA expression, as shown in FIG. 7. The level of BCMA expression in the stem cell population was generally equal to that observed in plasma cells, with the exception of MM277, where the level was lower but still positive for BCMA expression.

Using the results of this example, it is demonstrated that BCMA is expressed on multiple myeloma cancer stem cells.

Example 7.

This example demonstrates that the antibody-drug conjugate 15B2GL-SG3249 kills multiple myeloma stem cells.

Because MM stem cells are capable of colony formation in vitro (Matsui et al., Blood, 103: 2332-6 (2004)), the ability of ADC 15B2GL-SG3249 to kill clonogenic cells in bone marrow biopsies from MM patients that were 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 times higher than that required for plating. METHOCULT™ H4434 Classic (StemCell Technologies, Inc., Vancouver, BC, Canada) was mixed with 2000 cells per ml for MM263, MM284, and MM276 and 4000 cells per ml for MM277, following the manufacturer's instructions. 25-400 ng per ml of test ADCs 15B2GL-SG3249 and J6M0-mc-MMAF were then added to the appropriate tubes. Control antibody IgG1-SG3249 was added only at the high dose of 400 ng per ml. All tubes were shaken thoroughly to mix and then left to rest, allowing air bubbles to rise to the top. Once the bubbles had risen, 400 μL was removed using a 16-gauge blunt-tipped needle and carefully placed into one well of a 24-well ultra-low adhesion plate (VWR, Radnor, PA). Each treatment was placed in duplicate in the inner wells of the plate.

- 20 046403

PBS was added to the outer wells and the plate was incubated at 37°C for 7-10 days. Colonies were counted by eye and colony formation was confirmed by scanning the plates with a Celigo® Image Cytometer (Nexcelom Biosciences, Lawrence, MA). As shown in FIG. 8, in all 4 cases, 15B2GL-SG3249 was able to kill clonogenic cells, while J6M0-mc-MMAF was not. 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, a concentration of 400 ng/ml J6M0-mc-MMAF did not reduce the number of colonies formed in the case of MM263, but only resulted in a decrease in colony formation by 12.5%, 40% and 50% in the case of MM276, MM277 and MM284, respectively.

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

Example 8.

This example demonstrates methods for killing multiple myeloma and plasma cell leukemia cells in vitro using the antibody-drug conjugates described herein.

Killing of multiple myeloma and plasma cell leukemia cell lines by antibody-drug conjugates containing 15B2GL conjugated to SG3400 was assessed in vitro using the CELLTITER-GLO® kit (Promega, Madison, WI) as described in Example 4. The following cells were tested lines expressing BCMA: NCI-H929, EJM, MM.1R. JJN3, OPM-2, MM.1S, U266.B1 and L363. BMCA-negative cell lines Raji and Jurkat were also tested. Antibody-drug conjugates were diluted to 5x stock solution (25 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 the cells in duplicate, resulting in a 12-point dose curve of the antibody-drug conjugate ranging from 5 μg/ml at the highest concentration to 2.8 x 10 ^-5 μg/ml at the lowest. Isotype antibody-drug conjugate (IgG1-SG3400) and media alone controls were also included. The plates were incubated at 37°C, 5% CO ₂ for 96 hours. At the end of the incubation period, 100 μl of substrate solution (Promega, Madison, Wis.) was added to each well. Luminescence was measured using an EnVision Multilabel plate reader (Perkin Elmer, Waltham, MA). Data were analyzed and graphs were generated using GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA). The results of this experiment are shown in Fig. 10A-10J.

The results of this Example demonstrate that ADC 15B2GL-SG3400 kills multiple myeloma and plasma cell leukemia cells in vitro.

Example 9.

This example demonstrates methods for killing multiple myeloma and plasma cell leukemia cells in vivo using the antibody-drug conjugates described herein.

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 mice (CB-17/IcrHsdPrkdc-scid ) using MATRIGEL™ (BD Biosciences, San Jose, CA). When tumors reached approximately 200 mm ³ (NCI-H929 cells) or 180 mm ³ (MM.1S cells), mice were randomized based on tumor size and placed into dosing groups and treated with ADCs targeting BCMA as described below.

NCI-H929 xenograft model.

Mice were treated with either a single intravenous dose of ADC IgG1-SG3400 at 1 mg/kg, 15B2GL-SG3400 at 0.3 mg/kg or 1 mg/kg, or an intravenous dose of J6M0-SG3400 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 with no evidence of tumor regrowth as shown in FIG. 11. No weight loss was observed in any of the dosing groups.

Xenograft model MM.1S.

Mice were treated with either a single intravenous dose of ADC IgG1-SG3400 or 15B2GL-SG3400 at 1 mg/kg or 3 mg/kg, or an intravenous dose of J6M0-SG3400 at 1 mg/kg or 3 mg/kg. Control mice were left untreated. Mice treated with 3 mg/kg J6M0SG3400 were observed for 85 days after tumor implantation with no evidence of tumor regrowth as shown in FIG. 12. No weight loss was observed in any of the dosing groups.

The results of this example demonstrate that the ADC 15B2GL-SG3400 exhibits

- 21 046403 has antitumor efficacy in vivo.

Using the data described in the Examples, it is demonstrated that an ADC containing the monoclonal antibody 15B2GL exhibits potent antitumor activity in preclinical models of MM. Importantly, in vitro experiments indicate that this activity is maintained in the presence of sBCMA. These data further demonstrate that the ADC 15B2GLSG3249, carrying a potent PBD payload, effectively targets both bulk myeloma plasma cells and the more passive clonogenic CD19+/CD138- cells, which may enable longer-lasting clinical responses while genetically heterogeneous disease.

All references, including publications, patent applications and patents cited herein, are incorporated herein by reference to the same extent as if each document had been specifically and separately identified as being incorporated by reference and had been presented in its entirety in this document.

The use of the singular terms and the term at least one and similar definitions in the context of the description of the present invention (especially in the context of the following claims) are to be construed to include both the singular and the plural, unless otherwise expressly stated herein. does not contradict the context. Use of the term at least one followed by a list of one or more elements (for example, at least one of A and B) implies one element selected from a list of elements (A or B), or any combination of two or more of listed elements (A and B), unless otherwise stated herein or otherwise clearly inconsistent with the context. The terms consisting of, having, including and containing are to be understood as open terms (i.e., meaning including without limitation) unless otherwise noted. It is intended that the statements of ranges of values herein serve solely as a means of abbreviating the individual indication of each individual value included within a given range unless otherwise indicated herein, and each individual value is included herein as if it were individually mentioned. in this document. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise clearly inconsistent with the context. The use of any kind of examples or illustrative phrases (eg, such) provided herein is intended solely to better illuminate the present invention and not to state a limitation on the scope of the present invention, unless otherwise stated. Nothing in this specification should be construed as indicating that any unstated element is essential to the practice of the present invention.

Preferred embodiments of the present invention are described herein, including the best embodiment known to the inventors of the present invention for carrying out the present invention. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The present inventors expect that those skilled in the art will use such variations as appropriate, and the present inventors expect that the present invention will be practiced differently than specifically described herein. Accordingly, the present invention includes all modifications and equivalents to the subject matter mentioned in the appended claims as permitted by applicable law. Moreover, any combination of the above-described elements, in all possible variations thereof, is encompassed by the present invention unless otherwise indicated herein or otherwise clearly contrary to the context.

Sequence Listing <110> MEDIMMUNE, LLC <120> BCMA MONOCLONAL ANTIBODY AND DRUG CONJUGATE <130> BCMA-100-US-NP <150> 62/596194 <151> 2017-12-08 <150> 62/539825 < 151> 2017-08-01 <160>20 <170> PatentIn version 3.5 <210>1 <211>5

- 22 046403 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400>1

Ser Tyr Ser Met Asn <210>2 <211>17 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400>2

Ser Ile Ser Gly Ser Ser Asn Tyr Ile Tyr

510

Tyr Ala Asp Ser Val Lys

Gly <210>3 <211>11 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400>3

Gly Gly Asn Tyr Tyr Val Glu Tyr Phe Gln Tyr

510 <210>4 <211>12 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400>4

Arg Ala Ser Gln Tyr Ile Ser Ser Asn Tyr Leu Ala

510 <210>5 <211>7 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400> 5

Gly Ala Ser Asn Arg Ala Thr

5

- 23 046403 <210> 6 <211> 9 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400>6

Gln Gln Tyr Gly Ser Ser Pro Ile Thr <210>7 <211>120 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400>7

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Arg Ser Tyr 20 2530

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045

Ser Ser Ile Ser Gly Ser Ser Asn Tyr Ile Tyr Tyr Ala Asp Ser Val 50 5560

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 7580

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 9095

Ala Arg Gly Gly Asn Tyr Tyr Val Glu Tyr Phe Gln Tyr Trp Gly Gln

100 105110

Gly Thr Leu Val Thr Val Ser Ser

115120 <210>8 <211>108 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400> 8

Glu Ile Val Leu Thr Gln Ser Pro

5

Gly Thr Leu Ser Leu Ser Pro Gly

15

- 24 046403

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

25

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly 35 40

Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly

55

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 65 70

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln

90

Ile Thr Phe Gly Gln Gly Thr Lys Leu Glu

100 105 <210> 9 <211> 120 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400> 9

Glu Ile Gln Leu Val Glu Ser Gly Gly Gly

5 10

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

25

Ser Met Asn Trp Val Arg Gln Ala Pro Gly 35 40

Ser Ser Ile Ser Gly Ser Ser Asn Tyr Ile 50 55

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 65 70

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

90

Ala Arg Gly Gly Asn Tyr Tyr Val Glu Tyr

100 105

Tyr Ile Ser Ser Asn 30

Ala Pro Arg Leu Leu 45

Pro Asp Arg Phe Ser 60

Ile Ser Arg Leu Glu

Tyr Gly Ser Ser Pro 95

Lys

Val Lys Pro Gly Gly 15

Thr Phe Arg Ser Tyr 30

Gly Leu Glu Trp Val 45

Tyr Ala Asp Ser Val 60

Lys Asn Ser Leu Tyr

Ala Leu Tyr Tyr Cys 95

Gln Tyr Trp Gly Gln 110

Gly Thr Leu Val Thr Val Ser Ser

115 120 <210> 10 <211> 108

- 25 046403 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400> 10

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr

5 10

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

25

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly 35 40

Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly

55

Gly Ser Gly Ser Gly Thr Gly Phe Thr Leu 65 70

Pro Glu Asp Phe Ala Val Phe Tyr Cys Gln

90

Ile Thr Phe Gly Gln Gly Thr Lys Leu Glu

100 105 <210> 11 <211> 120 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400> 11

Glu Val Gln Leu Val Glu Ser Gly Gly Gly

5 10

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

25

Ser Met Asn Trp Val Arg Gln Ala Pro Gly 35 40

Ser Ser Ile Ser Gly Ser Ser Asn Tyr Ile 50 55

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 65 70

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

90

Ser Leu Ser Pro Gly 15

Tyr Ile Ser Ser Asn 30

Ala Pro Arg Leu Leu 45

Pro Asp Arg Phe Ser 60

Ile Ser Arg Leu Glu

Tyr Gly Ser Ser Pro 95

Lys

Val Lys Pro Gly Gly 15

Thr Phe Ser Ser Tyr 30

Gly Leu Glu Trp Val 45

Tyr Ala Asp Ser Val 60

Lys Asn Ser Leu Tyr

Ala Val Tyr Tyr Cys 95

- 26 046403

Ala Arg Gly Gly Asn Tyr Phe Val Glu Tyr Phe Gln 100105

Gln Trp Gly Gln

110

Gly Thr Leu Val Thr Val Ser Ser

115120 <210>12 <211>108 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400>12

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly

5 1015

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Tyr Ile Ser Ser Asn 20 2530

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 4045

Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 5560

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 7580

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Ser Ser Asp Pro 85 9095

Ile Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100105 <210>13 <211>120 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400>13

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

5 1015

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 2530

Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 4045

Ser Ser Ile Ser Gly Gln Ser Asn Tyr Ile Tyr Tyr Ala Asp Ser Val 50 5560

- 27 046403

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 65 70

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

90

Ala Arg Gly Gly Asn Tyr Phe Val Glu Tyr

100 105

Gly Thr Leu Val Thr Val Ser Ser

115 120 <210> 14 <211> 108 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400> 14

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr

5 10

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

25

Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly 35 40

Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly 50 55

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 65 70

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln

90

Ile Thr Phe Gly Gln Gly Thr Lys Leu Glu

100 105 <210> 15 <211> 120 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400> 15

Glu Val Gln Leu Val Glu Ser Gly Gly Gly

5 10

Lys Asn Ser Leu Tyr

Ala Val Tyr Tyr Cys 95

Gln Tyr Trp Gly Gln 110

Ser Leu Ser Pro Gly 15

Tyr Ile Ser Ser Asn 30

Ala Pro Arg Leu Leu 45

Pro Asp Arg Phe Ser 60

Ile Ser Arg Leu Glu

Tyr Ala Asp Ser Pro 95

Lys

Val Lys Pro Gly Gly 15

- 28 046403

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

25

Ser Met Asn Trp Val Arg Gln Ala Pro Gly 35 40

Ser Ser Ile Ser Gly Gln Ser Asn Tyr Ile 50 55

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 65 70

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

90

Ala Arg Gly Gly Asn Tyr Tyr Val Glu Tyr

100 105

Gly Thr Leu Val Thr Val Ser Ser

115 120 <210> 16 <211> 108 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400> 16

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr

5 10

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

25

Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly 35 40

Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly 50 55

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 65 70

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln

90

Ile Thr Phe Gly Gln Gly Thr Lys Leu Glu

100 105

Thr Phe Ser Ser Tyr 30

Gly Leu Glu Trp Val 45

Tyr Ala Asp Ser Val 60

Lys Asn Ser Leu Tyr

Ala Val Tyr Tyr Cys 95

Gln Tyr Trp Gly Gln 110

Ser Leu Ser Pro Gly 15

Tyr Ile Ser Ser Asn 30

Ala Pro Arg Leu Leu 45

Pro Asp Arg Phe Ser 60

Ile Ser Arg Leu Glu

Tyr Ser Ser Asp Pro 95

Lys <210> 17 <211> 120 <212> PROTEIN <213> Artificial sequence

- 29 046403 <220>

<223> synthetic <400> 17

Glu Val Gln Leu Val Glu Ser Gly Gly Gly

5 10

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly

25

Ser Met Asn Trp Val Arg Gln Ala Pro Gly 35 40

Ser Ser Ile Ser Gly Ser Ser Asn Tyr Ile 50 55

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 65 70

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp

90

Ala Arg Gly Gly Asn Tyr Phe Val Glu Tyr

100 105

Gly Thr Leu Val Thr Val Ser Ser

115 120 <210> 18 <211> 108 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400> 18

Glu Ile Val Leu Thr Gln Ser Pro Gly Thr

5 10

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser

25

Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly 35 40

Ile Tyr Gly Ala Ser Asn Arg Ala Thr Gly

55

Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 65 70

Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln

90

Val Lys Pro Gly Gly 15

Thr Phe Ser Ser Tyr 30

Gly Leu Glu Trp Val 45

Tyr Ala Asp Ser Val 60

Lys Asn Ser Leu Tyr

Ala Val Tyr Tyr Cys 95

Gln Tyr Trp Gly Gln 110

Ser Leu Ser Pro Gly 15

Tyr Ile Ser Ser Asn 30

Ala Pro Arg Leu Leu 45

Pro Asp Arg Phe Ser 60

Ile Ser Arg Leu Glu

Tyr Ser Ser Ser Pro

- 30 046403

Ile Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100105 <210>19 <211>120 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400> 19

Glu 1

Val

Gln

Leu

Val 5

Glu

Ser

Gly

Gly

Gly 10

Leu

Val

Lys

Pro

Gly 15

Gly

Ser

Leu

Arg

Leu

Ser

Cys

Ala

Ala

Ser 25

Gly

Phe

Thr

Phe

Arg 30

Ser

Tyr

Ser

Met

Asn 35

Trp

Val

Arg

Gln

Ala 40

Pro

Gly

Lys

Gly

Leu

Glu

Trp

Val

Ser

Ser 50

Ile

Ser

Gly

Gln

Ser 55

Asn

Tyr

Ile

Tyr

Tyr 60

Ala

Asp

Ser

Val

Lys 65

Gly

Arg

Phe

Thr

Ile

Ser

Arg

Asp

Asn

Ala 75

Lys

Asn

Ser

Leu

Tyr 80

Leu

Gln

Met

Asn

Ser 85

Leu

Arg

Ala

Glu

Asp 90

Thr

Ala

Val

Tyr

Tyr 95

Cys

Ala

Arg

Gly

Gly 100

Asn

Tyr

Tyr

Val

Glu

105

Tyr

Phe

Gln

Tyr

Trp

110

Gly

Gln

Gly

Thr

Leu

115

Val

Thr

Val

Ser

Ser

120 <210> 20 <211> 108 <212> PROTEIN <213> Artificial sequence <220>

<223> synthetic <400> 20

Glu Ile Val Leu Thr Gln Ser Pro 1 5

Gly Thr Leu Ser Leu Ser Pro Gly

15

Glu Arg Ala Thr Leu Ser Cys Arg 20

Ala Ser Gln Tyr Ile Ser Ser Asn 25 30

Tyr Leu Ala Trp Tyr Gln Gln Lys

40

Pro Gly Gln Ala Pro Arg Leu Leu 45

-

Claims (11)

Ile Tyr 50 Gly Ala Ser Asn Gly Ser 65 Gly Ser Gly Thr 70 Pro Glu Asp Phe Ala Val 85 Ile Thr Phe Gly 100 Gln Gly Arg Ala 55 Asp Phe Tyr Tyr Thr Thr Gly Thr Leu Cys Gln 90 Lys Leu Glu 105 Ile Pro Thr Ile 75 Gln Tyr Ile Lys Asp Arg Ser Arg Thr Asp Phe Ser Leu Glu Ser Pro 95 CLAIM 1. An antibody-drug conjugate (ADC) comprising a monoclonal antibody or antigen-binding fragment thereof directed against B-cell maturation antigen (BCMA) conjugated to a cytotoxin, wherein the monoclonal antibody contains (a) a heavy chain variable region containing the amino acid sequence defining complementarity region 1 (HCDR1) of SEQ ID NO: 1, the amino acid sequence of HCDR2 of SEQ ID NO: 2 and the amino acid sequence of HCDR3 of SEQ ID NO: 3, and (b) a light chain variable region containing the amino acid sequence of complementarity determining region 1 ( LCDR1) under SEQ ID NO: 4, the amino acid sequence of LCDR2 under SEQ ID NO: 5 and the amino acid sequence of LCDR3 under SEQ ID NO: 6. 2. The antibody-drug conjugate of claim 1, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 7. 3. The antibody-drug conjugate of claim 1 or 2, wherein the light chain variable region comprises the amino acid sequence of SEQ ID NO: 8. 4. The antibody-drug conjugate according to any one of claims 1 to 3, wherein the heavy chain variable region contains the amino acid sequence of SEQ ID NO: 7, and the light chain variable region contains the amino acid sequence of SEQ ID NO: 8. 5. The antibody-drug conjugate of any one of claims 1 to 4, wherein the cytotoxin is a microtubule-targeting agent, a pyrrolobenzodiazepine (PBD), an RNA polymerase II inhibitor, or a DNA alkylating agent. 6. The antibody-drug conjugate of claim 5, wherein the cytotoxin is a microtubule-targeting agent selected from the group consisting of a maytansinoid, auristatin and tubulisin. 7. The antibody-drug conjugate of claim 5, wherein the cytotoxin is a pyrrolobenzodiazepine (PBD). 8. The antibody-drug conjugate of claim 7, wherein the pyrrolobenzodiazepine is SG3249, characterized by the following formula 9. A composition for the treatment of multiple myeloma, comprising an antibody-drug conjugate according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier. 10. A method of killing multiple myeloma cells, comprising contacting multiple myeloma cells that express BCMA with an antibody-drug conjugate according to any one of claims. 1-8, whereby the antibody-drug conjugate binds to BCMA on multiple myeloma cells and causes the killing of multiple myeloma cells. 11. A method for killing multiple myeloma cells, comprising contacting multiple myeloma cells that express BCMA with the composition of claim 9, whereby -