Combination therapy

Abstract

The present disclosure relates to methods and compositions for the treatment of B-cell malignancies. Specifically, the disclosure relates to a B-cell malignancy agent or composition comprising: (a) an antibody-drug conjugate (ADC) comprising an antibody or antigen-binding fragment thereof that binds to B-cell maturation antigen (BCMA), conjugated to a nucleic acid crosslinking agent; and (b) a proteasome inhibitor.

Description

[Technical field]

[0001] REFERENCE TO ELECTRONICALLY SUBMITTED SEQUENCE LISTING The contents of the Sequence Listing submitted electronically in an ASCII text file with this application (Name: BCMA-150-US-PSP-SequenceListing.txt, Size: 11,279 bytes, Created: May 28, 2019) are incorporated herein by reference in their entirety. [Background technology]

[0002] Hematological cancer is a term used to describe many different types of cancer that affect blood cells, bone marrow, or the lymphatic system. Hematological cancer accounts for nearly 10% of new cancer cases each year in the United States, with over 1.2 million people in the United States alone reported to have or be in remission from blood cancer. Hematological cancer is the fifth most common cancer in the UK, with over 240,000 cases and 40,000 cases diagnosed with blood cancer each year in the UK. The three main groups are leukemia, lymphoma, and myeloma, each representing a B-cell malignancy.

[0003] For example, B-cell malignant myeloma (e.g., multiple myeloma (MM)) is a malignant tumor of clonal plasma cells (e.g., B cells) with ongoing DNA damage associated with the progression from premalignant monoclonal gammopathy of undetermined significance (MGUS) to active MM. Current treatment regimens for myeloma include conventional corticosteroids, alkylating agents, proteasome inhibitors (PIs), and immunomodulatory drugs (IMiDs), which have helped to increase the overall survival rate of myeloma patients. A particularly interesting therapeutic approach that has been explored in recent years is immunotherapy (e.g., using monoclonal antibodies). For example, US Pat. No. 5,399,433 and US Pat. No. 5,499,433 describe antibodies that bind to an antigen known as B-cell maturation antigen (BCMA), which has been shown to have good selectivity for B-cell malignancies (especially myeloma cells), and the described antibodies exhibit anti-B-cell malignancy activity.

[0004] Despite the increasing availability of various treatments, the development of drug resistance underlies the recurrence of diseases, particularly multiple myeloma, and the effectiveness of any treatment is limited by the maximum effectiveness achievable with tolerated doses.

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0006] Therefore, there is a need for improved agents having anti-B cell malignancy activity. The present invention addresses one or more of the above problems.

Means for Solving the Problems

[0007] The present invention relates to a combination therapy for B cell malignancies.

[0008] The present invention is based on the surprising discovery that a proteasome inhibitor (e.g., bortezomib) can be used (and vice versa) to act synergistically with an anti-BCMA antibody-drug conjugate to increase the cytotoxicity of cells of B cell malignancies after contact with a combination of these agents. Thus, the promising finding of the present invention is that a proteasome inhibitor can be used (and vice versa) to enhance the anti-B cell malignancy activity of an antibody-drug conjugate, and more particularly when the drug (or the above antibody-drug conjugate) is a nucleic acid cross-linking agent.

[0009] In one aspect, provided herein is an agent for B cell malignancies, comprising a. An antibody-drug conjugate (ADC) comprising an antibody or an antigen-binding fragment thereof that binds to B cell maturation antigen (BCMA) conjugated to a nucleic acid cross-linking agent, and b. A proteasome inhibitor, and when compared to a drug that is identical except for lacking the proteasome inhibitor, results in enhanced suppression of B cell malignancies, or when compared to a drug that is identical except for lacking the ADC, results in enhanced suppression of B cell malignancies, a drug for B cell malignancies.

[0010] In another aspect, provided herein is a therapeutic combination for use in treating B cell malignancies, comprising a. An ADC comprising an antibody or an antigen-binding fragment thereof that binds to BCMA conjugated to a nucleic acid cross-linking agent, and b. A proteasome inhibitor, and when compared to a composition that is identical except for lacking the proteasome inhibitor, results in enhanced suppression of B cell malignancies, or when compared to a composition that is identical except for lacking the ADC, results in enhanced suppression of B cell malignancies, a therapeutic combination for use in treating B cell malignancies.

[0011] In another aspect, provided herein is a method for treating B cell malignancies, comprising a. Administering to a subject a therapeutic combination comprising an ADC comprising an antibody or an antigen-binding fragment thereof that binds to BCMA conjugated to a nucleic acid cross-linking agent, and b. A proteasome inhibitor, wherein the therapeutic combination results in enhanced suppression of B cell malignancies when compared to a composition that is identical except for lacking the proteasome inhibitor, or the therapeutic combination results in enhanced suppression of B cell malignancies when compared to a composition that is identical except for lacking the ADC, a method for treating B cell malignancies.

[0012] In another aspect, provided herein is an in vitro method for enhancing the inhibition of malignant B cells by an ADC, the method comprising contacting malignant B cells with (a) an ADC comprising an antibody or antigen-binding fragment thereof that binds to BCMA conjugated to a nucleic acid cross-linking agent, in combination with (b) a proteasome inhibitor.

[0013] In another aspect, provided herein is an in vitro method for enhancing the inhibition of malignant B cells by a proteasome inhibitor, the method comprising contacting malignant B cells with (a) a proteasome inhibitor in combination with (b) an ADC comprising an antibody or antigen-binding fragment thereof that binds to BCMA conjugated to a nucleic acid cross-linking agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0014]

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Mode for Carrying Out the Invention

[0015] In one aspect, provided herein is an agent for B-cell malignancies, comprising: a. an antibody-drug conjugate (ADC) comprising an antibody or an antigen-binding fragment thereof that binds to B-cell maturation antigen (BCMA) conjugated to a nucleic acid cross-linking agent, and b. a proteasome inhibitor, and which results in enhanced suppression of B-cell malignancies when compared to an agent that is identical except lacking the proteasome inhibitor, or which results in enhanced suppression of B-cell malignancies when compared to an agent that is identical except lacking the ADC, an agent for B-cell malignancies.

[0016] Another aspect is a therapeutic combination for use in the treatment of B-cell malignancies, comprising: a. an ADC comprising an antibody or antigen-binding fragment thereof that binds to BCMA conjugated to a nucleic acid cross-linking agent, and b. a proteasome inhibitor, and which, when compared to a composition that is identical except for lacking the proteasome inhibitor, results in enhanced suppression of B-cell malignancies, or which, when compared to a composition that is identical except for lacking the ADC, results in enhanced suppression of B-cell malignancies, and provides a therapeutic combination for use in the treatment of B-cell malignancies.

[0017] In a related aspect, a method for treating B-cell malignancies is provided, the method comprising administering to a subject a therapeutic combination comprising: a. an ADC comprising an antibody or antigen-binding fragment thereof that binds to BCMA conjugated to a nucleic acid cross-linking agent, and b. a proteasome inhibitor, wherein the therapeutic combination, when compared to a composition that is identical except for lacking the proteasome inhibitor, results in enhanced suppression of B-cell malignancies, or wherein the therapeutic combination, when compared to a composition that is identical except for lacking the ADC, results in enhanced suppression of B-cell malignancies.

[0018] The enhanced suppression of B-cell malignancies can include one or more selected from increased delay of tumor growth, enhanced reduction of tumor size, enhanced reduction of tumor metastasis, increased survival rate, or combinations thereof, in a subject with B-cell malignancies.

[0019] The term "B-cell malignancy" encompasses any disease in which B cells become cancerous, divide without control (e.g., in the bone marrow and blood), and have the potential to infiltrate other sites (e.g., tissues and the lymphatic system). In one embodiment, the B-cell malignancy is one or more selected from B-cell lymphoma, B-cell leukemia, myeloma (e.g., multiple myeloma and myeloma precursor cells), or combinations thereof. The term "B cell" encompasses both mature (differentiated) B cells and their precursors (e.g., stem cells). For example, myeloma stem cells and myeloma precursor cells are included.

[0020] In one embodiment, the B-cell malignancy is characterized by including malignant B cells that express BCMA. In one embodiment, the malignant B cells express a high level of BCMA antigen (compared to reference non-malignant B cells). When the expression level of BCMA antigen in malignant B cells increases to a statistically significant level compared to the BCMA expression level in non-malignant (e.g., healthy) B cells, the malignant B cells are considered to express "high levels of BCMA".

[0021] Examples of B-cell lymphoma include diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, primary central nervous system (CNS) lymphoma, and primary intraocular lymphoma. Examples of B-cell leukemia include B-cell chronic lymphocytic leukemia / small lymphocytic lymphoma, acute lymphoblastic leukemia, B-cell prolymphocytic leukemia, precursor B lymphoblastic leukemia, and hairy cell leukemia.

[0022] In one embodiment, the B-cell malignancy is myeloma (e.g., multiple myeloma).

[0023] Multiple myeloma (MM), also known as plasma cell myeloma or Kahler's disease, is a cancer of B cells (plasma cells), a type of white blood cell that normally produces antibodies. Current treatments for MM include chemotherapy, radiation, surgery, biophosphonates, and autologous stem cell transplantation (ASCT). These treatments often result in remission, but almost all patients eventually relapse and die. Multiple myeloma affects 1 to 4 people per 100,000 per year. The disease is more common in men and the cause is still unknown, but it is seen more than twice as often in African Americans as in Caucasians.

[0024] B-cell maturation antigen (BCMA), also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), is a member of the tumor necrosis factor family receptor (TNFR) that is expressed on cells of the B-cell lineage. BCMA expression is highest in terminally differentiated B cells. BCMA is involved in mediating the survival of plasma cells for maintaining humoral immunity over the long term. BCMA expression is associated with a number of cancers, autoimmune disorders, and infectious diseases. BCMA RNA has been ubiquitously detected in multiple myeloma cells, and BCMA protein has been detected on the surface of plasma cells from multiple myeloma patients by multiple researchers. Thus, BCMA represents a therapeutic target for B-cell malignancies, particularly multiple myeloma.

[0025] The nucleotide sequence of human BCMA (TNFRSF17) is described in Ensembl (see accession number ENSG00000048462, which is incorporated herein by reference). The amino acid sequence of BCMA (TNFRSF17) is described in UniProt (see accession number Q02223, which is incorporated herein by reference). The amino acid sequence of human BCMA is shown in SEQ ID NO: 13.

[0026] BCMA is also expressed in multiple myeloma stem cells. Thus, the term "myeloma" encompasses multiple myeloma "stem" cells, and myeloma "progenitor" cells. Further, the methods and uses of the present invention encompass the treatment of malignancies including multiple myeloma stem cells (e.g., those expressing BCMA). Multiple myeloma stem cells (and / or myeloma progenitor cells) can be identified in the bone marrow of multiple myeloma patients by their surface expression of CD19 and lack of CD138 surface expression. These cells are uniquely clonogenic and engraft in immunodeficient mice, while myeloma plasma cells defined as CD138+CD19− do not engraft.

[0027] The term "antibody-drug conjugate" means an antibody (or antigen-binding fragment thereof) conjugated to a cytotoxic agent (generally a small molecule drug with high systemic toxicity) via a chemical linker. This term is used herein to describe an antibody or antigen-binding fragment thereof conjugated to a nucleic acid cross-linking agent. In one embodiment, the ADC can comprise a nucleic acid cross-linking agent (e.g., a small molecule cytotoxin) chemically modified to contain a linker. The linker can then be used to conjugate the nucleic acid cross-linking agent (cytotoxin) to an antibody or antigen-binding fragment thereof. Upon binding to a target antigen (e.g., BCMA) on the surface of a cell, the ADC is internalized and transported to lysosomes where the nucleic acid cross-linking agent (cytotoxin) is released either by proteolytic cleavage of a cleavable linker (e.g., by cathepsin B found within lysosomes) or proteolysis of the antibody when conjugated to the cytotoxin via, for example, a non-cleavable linker. The cytotoxin then migrates from the lysosome to the exterior and into the cytosol or nuclear interior where it can then bind to its target depending on its mechanism of action.

[0028] In one embodiment, the nucleic acid cross-linking agent is a cytotoxic nucleic acid cross-linking agent.

[0029] Proteasome inhibitors have been found to be useful in many cancer therapies. Surprisingly, the inventors have found that the anti-B cell malignancy activity of the ADCs of the present invention can be enhanced (e.g., synergistically enhanced) using proteasome inhibitors, and conversely, the anti-B cell malignancy activity of proteasome inhibitors can be enhanced (e.g., synergistically enhanced) using the ADCs of the present invention. Without wishing to be bound by theory, the inventors believe that the activity of the proteasome inhibitor can enhance the activity of the ADCs of the present invention by causing a downstream (molecular) effect (e.g., suppression of molecules that can normally inhibit the activity of the nucleic acid cross-linking agent (e.g., PBD cytotoxin) of the ADC and can even confer resistance to the nucleic acid cross-linking agent). This activity may or may not be associated with its "normal" activity as a direct proteasome inhibitor. This theory is supported by the observation that the therapeutic combinations of the present invention exhibit enhanced activity even against malignant B cells that are resistant to proteasome inhibitors (e.g., bortezomib) as monotherapies.

[0030] Conversely, without wishing to be bound by theory, the activity of the ADCs of the present invention can enhance the activity of the proteasome inhibitor by causing a downstream (molecular) effect (e.g., suppression of molecules that can normally inhibit the activity of the proteasome inhibitor and can even confer resistance to the proteasome inhibitor).

[0031] Proteasome inhibitors produce various results. For example, proteasome inhibitors can cause an increase in the levels of biologically active proteins such as IκB, an inhibitor of nuclear factor kappa B (a protein involved in cell survival). Furthermore, misfolded proteins and other aged proteins are similarly accumulated, which induce the unfolded protein response (UPR) associated with endoplasmic reticulum (ER) stress.

[0032] In one embodiment, the proteasome inhibitor is a boronic acid-based proteasome inhibitor (e.g., bortezomib).

[0033] In one embodiment, the proteasome inhibitor is one or more selected from bortezomib, carfilzomib, ixazomib, marizomib, oprozomib, delanzomib, or combinations thereof. In one embodiment, the proteasome inhibitor is bortezomib.

[0034] Bortezomib (e.g., Velcade) (Millennium Pharmaceuticals, Cambridge, MA, USA), formerly known as PS-341, functions as an inhibitor of the 26S proteasome, a multi-subunit protein complex responsible for the degradation of ubiquitinated proteins. Bortezomib is a peptide boronate with the molecular formula C 19 H 25 BN4O:

Chemical formula

[0035] Carfilzomib (Kyprolis®) is an epoxyketone proteasome inhibitor that irreversibly binds to the β5 subunit (PSMB5). Its formula is as follows:

Chemical formula

[0036] Ixazomib (Ninlaro®) selectively and reversibly inhibits the protein proteasome subunit type β5 (PSMB5). Its formula is as follows:

Chemical formula

[0037] Marizomib (Salinosporamide A) inhibits proteasome activity by covalently modifying the threonine residue, which is the active site of the 20S proteasome. Marizomib irreversibly binds to three major catalytic sites on the protein proteasome subunits β5, β1, and β2. Its formula is as follows:

Chem.

[0038] Oprozomib (known as ONX 0912) has the following formula:

Chem.

[0039] Delanzomib (known as CEP-18770) has the following formula:

Chem.

[0040] In one embodiment, the agent and / or therapeutic composition is included within a pharmaceutical composition. The term "pharmaceutical composition" refers to a preparation in a form in which the biological activity of the active ingredient can be effective and which does not contain additional ingredients that are highly toxic to the subject to which the composition is administered. Such a composition can be sterile. The agent and / or therapeutic composition can include a pharmaceutically acceptable carrier. An example of a carrier is physiological saline. Suitable pharmaceutical compositions can include one or more of a buffer (e.g., acetate, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), a stabilizer (e.g., human albumin), a preservative (e.g., benzyl alcohol), and an absorption enhancer that enhances bioavailability, and / or other conventional solubilizing or dispersing agents.

[0041] In one embodiment, the agent, therapeutic combination, or pharmaceutical composition of the present invention may include a pharmaceutically acceptable non-toxic sterile carrier, such as physiological saline, non-toxic buffer, preservative, etc. Formulations suitable for use in the treatment methods disclosed herein are described in Remington’s Pharmaceutical Sciences, 22nd ed., Ed. Lloyd V. Allen, Jr. (2012). In one embodiment, the agent, therapeutic combination, or pharmaceutical composition of the present invention may be contained within one or more formulations selected from capsules, tablets, aqueous suspensions, solutions, nasal aerosols, or combinations thereof. In one embodiment, the pharmaceutical composition may include a buffer (e.g., acetic acid, phosphate, or citrate buffer), a surfactant (e.g., polysorbate), and optionally a stabilizer (e.g., human albumin), etc.

[0042] A “cytotoxic” agent (referred to herein as “cytotoxin” or “cytotoxic agent”) is an agent that inhibits or prevents the function of cells, and / or causes the destruction of cells (cell death), and / or exerts an anti-proliferative effect. It will be understood that the cytotoxin or cytotoxic agent of an ADC is also referred to as the “payload” of the ADC in the art.

[0043] The term “nucleic acid cross-linking agent” means a molecule that reacts with two nucleotides of a nucleic acid and forms a covalent bond between them. This cross-linking can occur within the same strand (intrastrand) or between opposite strands (interstrand) of double-stranded DNA. These bonds (adducts) interfere with cellular metabolism such as DNA replication and transcription and typically cause cell death. In one embodiment, the nucleic acid is DNA.

[0044] In one embodiment, the nucleic acid cross-linking agent is an agent that damages DNA by inducing DNA strand breaks (single-strand breaks and / or double-strand breaks) and typically subsequently results in apoptosis. In one embodiment, the nucleic acid cross-linking agent is a cytotoxic nucleic acid cross-linking agent.

[0045] In one embodiment, the nucleic acid crosslinking agent is one or more selected from pyrrolobenzodiazepine (PBD), nitrogen mustard, cisplatin, chloroethylnitrosourea (CENU), psoralen, mitomycin C (MMC) antibiotics, or combinations thereof. The nitrogen mustard can be one or more selected from cyclophosphamide, chloromethine (e.g., mechlorethamine or mustine), uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine, or combinations thereof. In one embodiment, the CENU is carmustine.

[0046] In one embodiment, the nucleic acid crosslinking agent is pyrrolobenzodiazepine (PBD).

[0047] The term "pyrrolobenzodiazepine" encompasses both pyrrolobenzodiazepine and its functional derivatives.

[0048] PBD is a class of cytotoxic agents that translocate to the nucleus before crosslinking DNA, prevent replication during mitosis, damage DNA by inducing DNA strand breaks (single-strand breaks and / or double-strand breaks), and subsequently lead to apoptosis. Some PBDs also have the ability to recognize and bind to specific sequences of DNA. In one embodiment, PBD includes the following general structure:

Chemical formula

[0049] PBDs differ in the number, type, and position of their substituents in both the aromatic A-ring and the pyrrolo C-ring, and also in the degree of saturation of the C-ring. In the B-ring, either an imine (N=C), a carbinolamine (NH-CH(OH)), or a carbinolamine methyl ether (NH-CH(OMe)) is present at the N10–C11 positions, which are the electrophilic centers responsible for the alkylation of DNA. All known natural products have an (S) configuration at the chiral C11a position, which confers a right-handed twist on the natural product when viewed from the C-ring towards the A-ring. This feature also gives PBDs the appropriate three-dimensional shape for isohelicity with respect to the minor groove of B-form DNA, fitting snugly into the binding site. PBDs can form adducts within the minor groove and interfere with DNA processing.

[0050] The first PBD antitumor antibiotic, anthramycin, was discovered in 1965. Since then, numerous naturally occurring PBDs have been reported, and more than 10 synthetic routes leading to various analogs have been developed. Family members include abeymycin, chicamycin, DC-81, mazetramycin, neothramycins A and B, porothramycin, protracarcin, sibanomycin (DC-102), sibiromycin, and tomamycin. PBDs and ADCs containing them are also described in WO 2015 / 155345 and WO 2015 / 157592, which are hereby incorporated by reference in their entirety.

[0051] In one embodiment, the PBD is PBD3249, also referred to herein as “SG3249” (for example, as described in more detail in WO 2014 / 057074, which is hereby incorporated by reference). PBD3249 (SG3249) includes the following structure:

Chemical formula

[0052] In one embodiment, the PBD is PBD3315, also referred to herein as "SG3315" (for example, as described in more detail in WO 2015 / 052322 pamphlet incorporated herein by reference). PBD3315 (SG3315) includes the following structure:

Chemical formula

[0053] In one embodiment, the PBD (for example, as described in detail in WO 2017 / 137553 pamphlet incorporated herein by reference) includes the following formula:

Chemical formula

Chemical formula

[0054] In another embodiment, the PBD is SG3400, also referred to as Compound 23 (for example, as described in detail in WO 2017 / 137553 pamphlet incorporated herein by reference), and has the following structure:

Chemical formula

[0055] In one embodiment, the PBD is a PBD dimer including at least two PBD monomers. For example, the at least two PBD monomers are linked through a flexible propyldioxy tether through the C8 position of their aromatic A-ring phenols.

[0056] The antibody or antigen-binding fragment thereof of the present invention can be conjugated to a cytotoxin (heterologous agent) such as a PBD using site-specific or non-site-specific conjugation methods. In one embodiment, the antibody or antigen-binding fragment thereof comprises one, two, three, four or more PBD moieties. In one embodiment, all of the PBD moieties (conjugated to the antibody or antigen fragment thereof) contain the same structure.

[0057] The nucleic acid cross-linking agent (cytotoxin) of the present invention can be linked (e.g., conjugated) to an antibody or antigen-binding fragment thereof by a spacer (e.g., at least one spacer). In one embodiment, the spacer is a peptide spacer. In one embodiment, the spacer is a non-peptide (e.g., chemical) spacer.

[0058] Conventional conjugation strategies for antibodies or antigen-binding fragments thereof rely on randomly conjugating a payload (cytotoxin) to the antibody or antigen-binding fragment via lysine or cysteine. In one embodiment, the antibody or antigen-binding fragment thereof is randomly conjugated to a drug (e.g., a cytotoxin) by, for example, partial reduction of the antibody or fragment and subsequent reaction with the desired drug, with or without an added linker moiety. The antibody or antigen-binding fragment can be reduced using DTT or a similar reducing agent. Next, the drug, with or without an added linker moiety, can be added in molar excess to the reduced antibody or fragment in the presence of DMSO. After conjugation, excess free cysteine may be added to quench unreacted drug. The reaction mixture can then be purified and buffer-exchanged into PBS.

[0059] In one embodiment, the nucleic acid cross-linking agent (e.g., cytotoxin) is conjugated to the antibody or antigen-binding fragment thereof by site-specific conjugation. In one embodiment, site-specific conjugation of the nucleic acid cross-linking agent (e.g., cytotoxin) to the antibody or antigen-binding fragment thereof using reactive amino acid residues at specific positions results in a homogeneous ADC preparation of a defined stoichiometry.

[0060] Site-specific conjugation can occur via cysteine residues or unnatural amino acids. In one embodiment, a nucleic acid cross-linking agent (e.g., a cytotoxin) is conjugated to an antibody or an antigen-binding fragment thereof via at least one cysteine residue.

[0061] In one embodiment, a nucleic acid cross-linking agent (e.g., a cytotoxin) is chemically conjugated to an amino acid side chain (e.g., at a specific Kabat position within the Fc region of an antibody or antigen-binding fragment). In one embodiment, a nucleic acid cross-linking agent (e.g., a cytotoxin) is conjugated to an antibody or an antigen-binding fragment thereof via a cysteine substitution at any suitable position in the Fc region via at least one cysteine among 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 (numbering corresponding to the Kabat EU index). In one embodiment, the specific position is 239, 442, or both (numbering corresponding to the Kabat EU index). In one embodiment, the specific position is position 442, an insertion of an amino acid (cysteine) between positions 239 and 240, or both (numbering corresponding to the Kabat EU index). In one embodiment, the nucleic acid cross-linking agent (cytotoxin) is conjugated to an antibody or an antigen-binding fragment thereof via a thiol-maleimide bond. In some aspects, the amino acid side chain is a sulfhydryl side chain, e.g., a sulfhydryl-reactive group located in the hinge and heavy chain-light chain of an antibody or an antigen-binding fragment thereof.

[0062] In one embodiment, the antibody or an antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 11.

[0063] In one embodiment, the antibody or antigen-binding fragment thereof comprises a human kappa constant region comprising the amino acid sequence of SEQ ID NO: 12.

[0064] In one embodiment, the antibody or antigen-binding fragment thereof i. an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a functional variant thereof; ii. an HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a functional variant thereof; iii. an HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a functional variant thereof; iv. an LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a functional variant thereof; v. an LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, or a functional variant thereof; and vi. an LCDR3 comprising the amino acid sequence of SEQ ID NO: 6, or a functional variant thereof.

[0065] In one embodiment, the antibody or antigen-binding fragment thereof i. a variable heavy chain (VH) comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, or 95% sequence identity to the reference amino acid sequence of SEQ ID NO: 7, or a functional variant thereof; and / or ii. a variable light chain (VL) comprising an amino acid sequence having at least 70%, 75%, 80%, 90%, or 95% sequence identity to the reference amino acid sequence of SEQ ID NO: 8, or a functional variant thereof.

[0066] In one embodiment, the antibody or antigen-binding fragment thereof comprises a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 7, or a functional variant thereof. In one embodiment, the antibody or antigen-binding fragment thereof comprises a variable light chain comprising the amino acid sequence of SEQ ID NO: 8, or a functional variant thereof.

[0067] In one embodiment, the antibody or antigen-binding fragment thereof i. a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 7, or a functional variant thereof; and ii. a variable light chain comprising the amino acid sequence of SEQ ID NO: 8, or a functional variant thereof.

[0068] SEQ ID NO: 7 and SEQ ID NO: 8 are the germline versions of VH and VL. Alternatively, the antibody or antigen-binding fragment thereof may comprise non-germline VH and / or VL (e.g., VH of SEQ ID NO: 9 and VL of SEQ ID NO: 10).

[0069] The present invention encompasses antibodies (e.g., antibody or antigen-binding fragment) as defined herein having the recited CDR sequences or variable heavy and variable light chain sequences (reference antibody), and functional variants thereof. The functional variants may bind to the same target antigen (e.g., BCMA) as the reference antibody and may exhibit the same antigen cross-reactivity (or lack thereof) as the reference antibody. The functional variants may have a different affinity for the target antigen when compared to the reference antibody. In one embodiment, the functional variants have substantially the same affinity.

[0070] In one embodiment, a functional variant of a reference antibody exhibits sequence variation in one or more CDRs when compared to the corresponding reference CDR sequences. Thus, a functional antibody variant may comprise functional variants of the CDRs. When the term "functional variant" is used with respect to a CDR sequence, this term means that the CDR has at most two, or at most one amino acid difference when compared to the corresponding reference CDR sequence, and when combined with the remaining five CDRs (or variants thereof), enables binding of the variant antibody to the same target antigen (e.g., BCMA) as the reference antibody. In one embodiment, the functional variant exhibits the same antigen cross-reactivity (or lack thereof) as the reference antibody.

[0071] In one embodiment, the functional variant antibody or antigen-binding fragment thereof a light chain CDR1 having at most two amino acid differences when compared to the corresponding reference CDR sequence; a light chain CDR2 having at most two amino acid differences when compared to the corresponding reference CDR sequence; a light chain CDR3 having at most two amino acid differences when compared to the corresponding reference CDR sequence; a heavy chain CDR1 having at most two amino acid differences when compared to the corresponding reference CDR sequence; a heavy chain CDR2 having at most two amino acid differences when compared to the corresponding reference CDR sequence; and a heavy chain CDR3 having at most two amino acid differences when compared to the corresponding reference CDR sequence; The functional variant binds to the same target antigen as the reference antibody. In one embodiment, the functional variant exhibits the same antigen cross-reactivity (or lack thereof) as the reference antibody.

[0072] In one embodiment, the functional variant antibody or antigen-binding fragment thereof a light chain CDR1 having at most one amino acid difference when compared to the corresponding reference CDR sequence; a light chain CDR2 having at most one amino acid difference when compared to the corresponding reference CDR sequence; a light chain CDR3 having at most one amino acid difference when compared to the corresponding reference CDR sequence; a heavy chain CDR1 having at most one amino acid difference when compared to the corresponding reference CDR sequence; a heavy chain CDR2 having at most one amino acid difference when compared to the corresponding reference CDR sequence; and a heavy chain CDR3 having at most one amino acid difference when compared to the corresponding reference CDR sequence; The functional variant binds to the same target antigen as the reference antibody. In one embodiment, the functional variant exhibits the same antigen cross-reactivity (or lack thereof) as the reference antibody.

[0073] For example, a functional variant of an antibody or antigen-binding fragment a heavy chain CDR1 having at most two amino acid differences when compared to SEQ ID NO: 1; a heavy chain CDR2 having at most two amino acid differences when compared to SEQ ID NO: 2; and a heavy chain CDR3 having at most two amino acid differences when compared to SEQ ID NO: 3; a light chain CDR1 having at most two amino acid differences when compared to SEQ ID NO: 4; a light chain CDR2 having at most two amino acid differences when compared to SEQ ID NO: 5; When compared to SEQ ID NO: 6, it may contain a light chain CDR3 with a maximum of two amino acid differences; The variant antibody binds to BCMA (e.g., a BCMA polypeptide epitope), and / or the variant antibody may exhibit the same antigen cross-reactivity (or lack thereof) as the reference antibody or antigen-binding fragment.

[0074] In one embodiment, a functional variant of the antibody or antigen-binding fragment is a heavy chain CDR1 having a maximum of one amino acid difference when compared to SEQ ID NO: 1; a heavy chain CDR2 having a maximum of one amino acid difference when compared to SEQ ID NO: 2; and a heavy chain CDR3 having a maximum of one amino acid difference when compared to SEQ ID NO: 3; a light chain CDR1 having a maximum of one amino acid difference when compared to SEQ ID NO: 4; a light chain CDR2 having a maximum of one amino acid difference when compared to SEQ ID NO: 5; may contain a light chain CDR3 having a maximum of one amino acid difference when compared to SEQ ID NO: 6; The variant antibody binds to BCMA (e.g., a BCMA polypeptide epitope), and / or the variant antibody may exhibit the same antigen cross-reactivity (or lack thereof) as the reference antibody or antigen-binding fragment.

[0075] The above can be similarly applied to variants of other antibodies described herein, where the amino acid differences are defined with respect to their CDR sequences, the variant antibody binds to the same target antigen as the above antibody, and / or the variant antibody may exhibit the same antigen cross-reactivity (or lack thereof).

[0076] In one embodiment, the functional variant antibody may have up to 5, 4, or 3 amino acid differences in its entirety of CDRs, provided that there are up to 2 (e.g., up to 1) amino acid differences per CDR when compared to the corresponding reference antibody. In one embodiment, the functional variant antibody has up to 2 (e.g., up to 1) amino acid differences in its entirety of CDRs, provided that there are up to 2 amino acid differences per CDR when compared to the corresponding reference antibody. In one embodiment, the functional variant antibody has up to 2 (e.g., up to 1) amino acid differences in its entirety of CDRs, provided that there is up to 1 amino acid difference per CDR when compared to the corresponding reference antibody.

[0077] The amino acid differences can be amino acid substitutions, insertions, or deletions. In one embodiment, the amino acid differences are conservative amino acid substitutions as described herein.

[0078] In one embodiment, the functional variant antibody has the same framework sequence as the exemplary antibodies described herein. In another embodiment, the functional variant antibody may include a framework region having up to 2, or up to 1 amino acid difference (when compared to the corresponding framework sequence). Thus, each framework region may have up to 2, or up to 1 amino acid difference (when compared to the corresponding reference framework sequence).

[0079] In one embodiment, the functional variant antibody may have up to 5, 4, or 3 amino acid differences in the entire framework region, provided that there are up to 2 (e.g., up to 1) amino acid differences per framework region when compared to the corresponding reference antibody. In one embodiment, the functional variant antibody may have up to 2 (e.g., up to 1) amino acid differences in the entire framework region, provided that there are up to 2 amino acid differences per framework region when compared to the corresponding reference antibody. In one embodiment, the functional variant antibody may have up to 2 (e.g., up to 1) amino acid differences in the entire framework region, provided that there is up to 1 amino acid difference per framework region when compared to the corresponding reference antibody.

[0080] Accordingly, the functional variant antibody may comprise the variable heavy and variable light chains described herein, the heavy chain having up to 14 amino acid differences (up to 2 amino acid differences in each CDR and up to 2 amino acid differences in each framework region) when compared to the heavy chain sequence described herein (e.g., SEQ ID NO: 7); the light chain having up to 14 amino acid differences (up to 2 amino acid differences in each CDR and up to 2 amino acid differences in each framework region) when compared to the light chain sequence described herein (e.g., SEQ ID NO: 8); the functional variant antibody binds to the same target antigen as the reference antibody and / or the functional variant antibody exhibits the same antigen cross-reactivity (or lack thereof) as the reference antibody.

[0081] The variant heavy or light chain may be referred to as a "functional equivalent" of the reference heavy or light chain.

[0082] In one embodiment, the functional variant antibody may comprise the variable heavy and variable light chains described herein, The heavy chain has at most 7 amino acid differences (at most 1 amino acid difference in each CDR and at most 1 amino acid difference in each framework region) when compared to the heavy chain sequence herein (e.g., SEQ ID NO: 7); The light chain has at most 7 amino acid differences (at most 1 amino acid difference in each CDR and at most 1 amino acid difference in each framework region) when compared to the light chain sequence herein (e.g., SEQ ID NO: 8); The functional variant antibody binds to the same target antigen as the reference antibody and / or the functional variant antibody exhibits the same antigen cross-reactivity (or lack thereof) as the reference antibody.

[0083] In one embodiment, the antibody or antigen-binding fragment thereof binds to BCMA (e.g., human BCMA) with a dissociation constant (KD) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤10 pM, ≤1 pM, or ≤0.1 pM. In one embodiment, the antibody or antigen-binding fragment thereof binds to BCMA (e.g., human BCMA) with a KD of about 0.1 nM to about 40 nM, about 0.5 nM to about 30 nM, about 1 nM to about 20 nM, or about 1.5 nM to about 20 nM. In one embodiment, the antibody or antigen-binding fragment thereof binds to BCMA (e.g., human BMCA) with a KD of about 23 nM to about 27 nM. In one embodiment, the antibody or antigen-binding fragment thereof binds to BCMA (e.g., human BCMA) with a KD between about 1 nM and about 1.5 nM. KD measurements (binding affinity) can be performed by any suitable assay known in the art. Such methods include, for example, fluorescence-activated cell sorting (FACS), surface plasmon resonance (e.g., Biacore, ProteOn), biolayer interferometry (BLI, e.g., Octet), binding equilibrium exclusion (e.g., KinExA), separable beads (e.g., magnetic beads), antigen panning, ELISA, and / or the ForteBio Octet system. Suitable binding equilibrium exclusion methods include the KinExA system (e.g., KinExA3100, KinExA3200, or KinExA4000) (Sapidyne Instruments, Idaho).

[0084] Advantageously, this therapeutic combination provides for the meaningful use of such proteasome inhibitors to target cells that are normally non-responsive (e.g., resistant) to such proteasome inhibitors. For example, the inventors have demonstrated that proteasome inhibitors (e.g., bortezomib) enhance the anti-B cell malignancy activity of the ADC even when B cell malignancies are resistant to the proteasome inhibitor (see Example 3).

[0085] Accordingly, the present invention encompasses administering an ADC in combination with a proteasome inhibitor at a dose of the proteasome inhibitor that would normally result in only low / inadequate suppression of B cell malignancies, such that, following said addition, improved suppression of B cell malignancies can now be demonstrated by this combination. Accordingly, the present invention provides for the “off-label use” of a proteasome inhibitor (e.g., bortezomib) for use as an enhancer of an ADC, as contrasted with the use of (stand-alone) monotherapy, which is not normally effective against resistant malignancies.

[0086] Similarly, the present invention encompasses administering a proteasome inhibitor in combination with an ADC at a dose of the ADC that would normally result in only low / inadequate suppression of B cell malignancies, such that, following said addition, improved suppression of B cell malignancies can now be demonstrated by this combination. Accordingly, the present invention provides for the “off-label use” of an ADC for use as an enhancer of a proteasome inhibitor, as contrasted with the use of (stand-alone) monotherapy, which is not normally effective against B cell malignancies expressing low levels of the BCMA antigen.

[0087] Accordingly, in one embodiment, the B cell malignancy is resistant to a proteasome inhibitor (e.g., an agent comprising a proteasome inhibitor in the absence of the ADC of the present invention). In one embodiment, the proteasome inhibitor is bortezomib.

[0088] In one embodiment, the B cell malignancy is resistant to the ADCs of the present invention (e.g., an agent comprising an ADC in the absence of a proteasome inhibitor of the present invention). In one embodiment, the B cell malignancy resistant to the ADC of the present invention is characterized by comprising malignant B cells with no increase or decrease in the expression level of the BCMA antigen compared to reference non-malignant B cells.

[0089] This therapeutic combination has also been shown to be more active against B cell malignancies that are resistant to many other common anti-cancer agents (see Example 3). Thus, in one embodiment, the B cell malignancy is resistant to one or more drugs selected from dexamethasone, lenalidomide, pomalidomide, bortezomib, or combinations thereof.

[0090] Furthermore, due to the synergistic nature of the combination, lower doses of some of the components can be used, thereby reducing the risk of the development of resistance (a major public health threat) to any of some of the components due to overuse. Indeed, the present invention reduces the need for chronic treatment regimens. For example, the inventors have shown that the in vivo efficacy of an ADC below the optimal dose is still enhanced when administered in combination with a proteasome inhibitor (e.g., bortezomib).

[0091] Thus, in one embodiment, the ADC and / or the proteasome inhibitor is administered below the optimal dose.

[0092] The order of administration / application of a portion of the components of the therapeutic combination can be changed. The ADC and the proteasome inhibitor can be administered simultaneously (e.g., each at its own specific optimal dose to achieve synergy), either as part of a single composition or within separate compositions. For example, the ADC may be present in a first composition (e.g., one adapted for intravenous administration to a subject), and the proteasome inhibitor may be present in a second composition (e.g., one adapted for intravenous, subcutaneous, or oral administration to a subject). For example, if the proteasome inhibitor is bortezomib, the second composition may be adapted for intravenous or subcutaneous injection. In embodiments where the proteasome inhibitor is ixazomib, the second composition may additionally or alternatively be adapted for oral administration.

[0093] Furthermore, the ADC and the proteasome inhibitor can be administered at different times (e.g., the proteasome inhibitor can be administered beforehand to sensitize malignant B cells to the ADC). Thus, in further embodiments, the ADC and the proteasome inhibitor are administered to a subject at different times within separate compositions.

[0094] In one embodiment, the proteasome inhibitor is administered before the ADC. In one embodiment, the proteasome inhibitor is administered simultaneously with the ADC. In one embodiment, the proteasome inhibitor is administered subsequent to the ADC.

[0095] The term "treating" or "treatment" as used herein encompasses prophylactic treatment (e.g., to prevent the onset of B cell malignancies) and corrective treatment (treatment of a subject already suffering from B cell malignancies). In one embodiment, the term "treating" or "treatment" as used herein refers to corrective treatment. The term "treating" or "treatment" encompasses treating both B cell malignancies and their symptoms. In some embodiments, "treating" or "treatment" refers to the symptoms of B cell malignancies.

[0096] Thus, the agent and / or therapeutic combination can be administered to a subject in a therapeutically effective amount or a prophylactically effective amount.

[0097] A "therapeutically effective amount" is any amount of an agent and / or therapeutic combination that, when administered to a subject alone or in combination, is sufficient to effect such treatment of a B-cell malignancy or its symptoms.

[0098] A "prophylactically effective amount" is any amount of an agent and / or therapeutic combination that, when administered to a subject alone or in combination, inhibits or delays the onset or recurrence of a B-cell malignancy (or its symptoms). In some embodiments, the prophylactically effective amount completely prevents the onset or recurrence of a B-cell malignancy. "Inhibition" of onset means either reducing the likelihood of onset (or its symptoms) of a B-cell malignancy or completely preventing onset.

[0099] A further aspect of the invention provides an in vitro method for enhancing the inhibition of malignant B cells by an ADC, the method comprising contacting malignant B cells with (a) an ADC comprising an antibody or antigen-binding fragment thereof that binds to BCMA conjugated to a nucleic acid crosslinking agent, in combination with (b) a proteasome inhibitor.

[0100] In another aspect, an in vitro method for enhancing the inhibition of malignant B cells by a proteasome inhibitor is provided, the method comprising contacting malignant B cells with (a) a proteasome inhibitor in combination with (b) an ADC comprising an antibody or antigen-binding fragment thereof that binds to BCMA conjugated to a nucleic acid crosslinking agent.

[0101] The terms "inhibit" or "inhibition" include, in the context described herein or in any agent, method, or use, "inhibition of growth" of malignant B cells, "inhibition of proliferation" of malignant B cells, or "death" of malignant B cells. References to "malignant B cells" include "tumors containing malignant B cells".

[0102] The terms "inhibit" or "inhibition" are synonymous with the terms "slow the growth" or "stop the proliferation" of malignant B cells, or "slow the growth" of tumors containing malignant B cells. In one embodiment, the therapeutic combination of the present invention can "kill" malignant B cells, or can be "used to kill" tumors containing malignant B cells, or tumors containing malignant B cells. The term "suppress" also encompasses preventing the growth (e.g., proliferation) of malignant B cells, or tumors containing malignant B cells.

[0103] In one embodiment, the enhanced suppression of B cell malignancies can include one or more selected from an increase in the delay of tumor growth, an enhanced reduction in tumor size, an enhanced reduction in tumor metastasis, an increase in survival rate, or a combination thereof, in a subject with a B cell malignancy. In one embodiment, the enhanced suppression of B cell malignancies includes one or more selected from an increase in the delay of tumor growth, an enhanced reduction in tumor size, or a combination thereof.

[0104] In one embodiment, the agent and / or therapeutic combination of the present invention suppresses B cell malignancies by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or about 100% more than an agent and / or composition that is identical except for lacking a proteasome inhibitor. In one embodiment, the agent and / or therapeutic combination of the present invention suppresses B cell malignancies by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or about 100% more than an agent and / or composition that is identical except for lacking the ADC of the present invention.

[0105] Inhibition can be measured by measuring cell proliferation, which can be assayed using techniques recognized in the art for measuring the rate of cell division and / or the proportion of cells in a cell population in which cell division is occurring and / or the rate of cell decrease from a cell population due to terminal differentiation or cell death (e.g., thymidine incorporation).

[0106] The above evaluation of "enhanced suppression of B cell malignancies" is demonstrated by reference to the appended examples and can be evaluated using the methods described in the examples (e.g., Example 3). For example, Example 3 describes a method involving Annexin V / PI-based FMC analysis that measures the "% of apoptotic cells observed" value in an in vitro culture of malignant B cells after contact with a test sample. This allows for a direct comparison of the suppression of B cell malignancies between the agent of the present invention and an agent that is identical except for the absence of a proteasome inhibitor or ADC.

[0107] In one embodiment, the suppression of B cell malignancies is considered enhanced when the "% of apoptotic cells observed" value obtained for a combination of two major active compounds (e.g., the ADC of the present invention and a proteasome inhibitor) is greater than the "% of apoptotic cells observed" value obtained in the absence of either the ADC or the proteasome inhibitor of the present invention (preferably the proteasome inhibitor), provided that the conditions are otherwise the same.

[0108] Accordingly, in one embodiment, enhanced suppression of B cell malignancies can be determined by comparing the "% of apoptotic cells observed" value obtained for a combination of the ADC of the present invention and a proteasome inhibitor with the "% of apoptotic cells observed" value obtained for the same formulation (e.g., agent) lacking the ADC or the proteasome inhibitor of the present invention (preferably lacking the proteasome inhibitor) under the same conditions.

[0109] In one embodiment, the present invention provides an enhancement of the suppression of B-cell malignancies that is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% greater (in % of observed apoptotic cells) than the % of observed apoptotic cells provided by the same formulation (e.g., agent) lacking the ADC or proteasome inhibitor of the present invention under the same conditions (preferably lacking the proteasome inhibitor). In one embodiment, the present invention provides an enhancement of the suppression of B-cell malignancies that is at least about 65% greater (in % of observed apoptotic cells) than the % of observed apoptotic cells provided by the same formulation (e.g., agent) lacking the ADC or proteasome inhibitor of the present invention under the same conditions (preferably lacking the proteasome inhibitor).

[0110] In one embodiment, the combination of the ADC and the proteasome inhibitor of the present invention can exhibit synergistic suppression of B-cell malignancies.

[0111] As used herein, the term "synergistic" means that the suppression of the indicated B-cell malignancies is greater than the sum of its parts. In other words, the suppression of B-cell malignancies exceeds additive suppression.

[0112] Synergy can be measured by determining the "combination index" (CI) using an analysis tool such as CompuSyn (ComboSyn, Inc.), where a CI less than 1 indicates synergy between the combination of the major active ingredients of the present invention, a CI greater than 1 indicates antagonism, and a CI of 1 indicates that the effects are additive. The above CI can be measured by comparing the performance of a composition comprising the pharmaceutical / treatment combination of the present invention with the performance of a composition that is otherwise identical except for lacking the ADC or proteasome inhibitor of the present invention (preferably lacking the proteasome inhibitor) in any cell viability assay known in the art (e.g., instead of or in addition to the methods of Examples 1-3 such as the "CellTiter-Glo based cell viability" assay described in Example 3).

[0113] Referring to the examples (e.g., Example 3), when the CI is less than about 0.95, less than 0.9, less than 0.85, less than 0.8, less than 0.75, less than 0.7, less than 0.65, less than 0.6, less than 0.55, less than 0.5, less than 0.45, less than 0.4, less than 0.35, less than 0.3, less than 0.25, less than 0.2, less than 0.15, less than 0.1, or less than 0.05, it can be considered that there is a synergistic inhibition of B cell malignancies. In one embodiment, the CI can be less than about 0.7. In one embodiment, the CI can be less than about 0.6.

[0114] In one embodiment, the "cell viability assay" comprises incubating a test sample containing malignant B cells in the presence of an amount of a composition comprising a therapeutic combination (the ADC and proteasome inhibitor of the present invention); and following the incubation (e.g., after at least 0.5 days, 1.5 days, or 2 days of incubation), determining the number of non-viable cells (e.g., apoptotic cells) in the test sample, and comparing it to the number of non-viable cells in a control sample incubated in the presence of a composition that is identical except for lacking (a) the proteasome inhibitor, or (b) the ADC (preferably lacking the proteasome inhibitor).

[0115] In one embodiment, the therapeutic combination is administered to a subject. The terms "subject", "individual", and "patient" are used interchangeably herein to refer to a mammalian subject. In one embodiment, the "subject" is a human, a companion animal (e.g., a pet such as a dog, a cat, and / or a rabbit), a livestock animal (e.g., a pig, a sheep, a cow, and / or a goat), and / or a horse. In one embodiment, the subject is a human.

[0116] In the method of the present invention, the subject may not have been previously diagnosed as having a B-cell malignancy. Alternatively, the subject may have been previously diagnosed as having a B-cell malignancy. The subject may also be a person showing a disease risk factor or a person asymptomatic for B-cell malignancy. The subject may also be a person suffering from or at risk of developing B-cell malignancy. In one embodiment, the subject has previously been treated for B-cell malignancy.

[0117] In one embodiment, the methods and uses of the present invention include one or more administration steps selected from oral, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal, inhalation, topical, or combinations thereof. In one embodiment, the administration is one or more selected from intravenous, intraarterial (e.g., by injection or infusion), subcutaneous, or combinations thereof.

[0118] Antibody preparation The antibodies of the present invention can be obtained using conventional techniques known to those skilled in the art, and their usefulness can be confirmed by conventional binding tests (exemplary methods are described in Example 2). As an example, a simple binding assay incubates cells expressing the antigen with the antibody. If the antibody is tagged with a fluorophore, the binding of the antibody to the antigen can be detected by FACS analysis.

[0119] Methods for making the BCMA antibodies and antibody fragments of the present invention are described in International Publication Nos. WO 2010 / 104949 and WO 2019 / 025983 (especially WO 2019 / 025983), both of which are incorporated herein by reference.

[0120] The antibodies of the present invention can be produced in various animals including mice, rats, rabbits, goats, sheep, monkeys, or horses. Antibodies can be produced after immunization with individual capsular polysaccharides, or multiple capsular polysaccharides. Blood isolated from these animals contains polyclonal antibodies (multiple antibodies that bind to the same antigen). The antigen can also be injected into chickens to produce polyclonal antibodies in egg yolk. To obtain monoclonal antibodies specific for a single epitope of the antigen, antibody-secreting lymphocytes are isolated from the animal and immortalized by fusing them with cancer cell lines. The fused cells are called hybridomas, which grow continuously in culture and secrete antibodies. Single hybridoma cells are isolated by dilution cloning to generate cell clones that all produce the same antibody. These antibodies are called monoclonal antibodies. Methods for generating monoclonal antibodies are conventional techniques known to those skilled in the art (see, for example, Making and Using Antibodies: A Practical Handbook. GC Howard. CRC Books. 2006. ISBN 0849335280). Polyclonal and monoclonal antibodies are often purified using protein A / G or antigen affinity chromatography.

[0121] The antibody or antigen-binding fragment thereof of the present invention can be prepared as a monoclonal antibody, which can be prepared using the hybridoma method (for example, the method described by Kohler and Milstein, Nature 256:495 (1975)). Using the hybridoma method, a mouse, hamster, or other suitable host animal is immunized as described above, and production of antibodies capable of specifically binding to the immunizing antigen by lymphocytes is induced. Lymphocytes can also be immunized in vitro. After immunization, the lymphocytes are isolated and hybridoma cells are formed by fusing them with a suitable myeloma cell line using, for example, polyethylene glycol, and then unfused lymphocytes and myeloma cells can be selectively removed therefrom. Hybridomas that produce monoclonal antibodies specifically targeting the selected antigen, as determined by immunoprecipitation, immunoblotting, or in vitro binding assays (for example, radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)), can then be grown either in in vitro culture using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as ascites tumors in animals. The monoclonal antibodies can then be purified from the culture medium or ascites using known methods.

[0122] Alternatively, an antibody or an antigen-binding fragment thereof (e.g., as a monoclonal antibody) can also be produced using recombinant DNA methods as described in U.S. Patent No. 4,816,567. Polynucleotides encoding monoclonal antibodies are isolated from mature B cells or hybridoma cells by, for example, RT-PCR using oligonucleotide primers that specifically amplify the genes encoding the heavy and light chains of the antibody, and their sequences are determined using conventional procedures. Next, the isolated polynucleotides encoding the heavy and light chains are cloned into a suitable expression vector, which is then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not normally produce immunoglobulin proteins, and monoclonal antibodies are produced by the host cells. Also, recombinant monoclonal antibodies or antigen-binding fragments thereof of a desired species can be isolated from phage display libraries expressing the CDRs of the desired species, as described in McCafferty et al., Nature 348:552-554 (1990), Clackson et al., Nature 352:624-628 (1991), and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0123] The polynucleotides encoding the antibodies or antigen-binding fragments thereof of the present invention can be further modified in many different ways using recombinant DNA techniques to produce alternative antibodies. In some embodiments, for example, the constant domains of the light and heavy chains of a murine monoclonal antibody can be (1) replaced with those regions of, for example, a human antibody to produce a chimeric antibody, or (2) replaced with a non-immunoglobulin polypeptide to produce a fusion antibody. In some embodiments, the constant regions are truncated or removed to produce the desired antibody fragments of the monoclonal antibody. Site-directed mutagenesis or high-density mutagenesis of the variable regions can be used to optimize the specificity, affinity, etc. of the monoclonal antibody.

[0124] In one embodiment, the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof. Human antibodies can be prepared directly using a variety of techniques known in the art. Immortalized human B lymphocytes can be generated that are either immunized in vitro or isolated from immunized individuals that produce antibodies against the target antigen. See, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985), Boemer et al., J. Immunol. 147(1):86-95 (1991), and U.S. Patent No. 5,750,373.

[0125] In one embodiment, the antibody or antigen-binding fragment thereof can be selected from a phage library that expresses human antibodies, such as those described in Vaughan et al., Nat. Biotech. 14:309-314 (1996), Sheets et al., Proc. Natl. Acad. Sci. USA, 95:6157-6162 (1998), Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991), and Marks et al., J. Mol. Biol. 222:581 (1991). Techniques for the production and use of antibody phage libraries are also described in U.S. Patent Nos. 5,969,108; 6,172,197; 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915; 6,593,081; 6,300,064; 6,653,068; 6,706,484; and 7,264,963; and Rothe et al., J. Molec. Biol. 376:1182-1200 (2008) (each of which is incorporated by reference in its entirety).

[0126] Affinity maturation strategies and chain shuffling strategies are known in the art and can be used to generate high-affinity human antibodies or antigen-binding fragments thereof. See Marks et al., BioTechnology 10:779-783 (1992) (incorporated herein by reference in its entirety).

[0127] In one embodiment, the antibody or antigen-binding fragment thereof (e.g., monoclonal antibody) can be a humanized antibody. Methods for engineering, humanizing, or resurfacing non-human or human antibodies can also be used, which are well known in the art. A humanized antibody, resurfaced antibody, or similarly engineered antibody can have one or more amino acid residues from a non-human source, e.g., but not limited to, mouse, rat, rabbit, non-human primate, or other mammalian source. These non-human amino acid residues are often replaced by residues referred to as "import" residues, which are typically obtained from the "import" variable domain, constant domain, or other domain of a known human sequence. Such imported sequences can be used to reduce immunogenicity or to reduce, enhance, or alter binding, affinity, on-rate, off-rate, binding activity, specificity, half-life, or any other suitable property known in the art. Preferably, CDR residues can be most directly and substantially involved in affecting antigen (e.g., BCMA) binding. Thus, non-human CDR sequences or some or all of the human CDR sequences can be maintained while simultaneously replacing non-human sequences of the variable and constant regions with human or other amino acids.

[0128] The antibody can also optionally be a humanized antibody, resurfaced antibody, engineered antibody, or human antibody that has been engineered while retaining high affinity for an antigen (e.g., BCMA) and other advantageous biological properties. To achieve this goal, three-dimensional models of the parent sequence, engineered sequence, and humanized sequence are used, and the humanized (or human) antibody or engineered antibody and resurfaced antibody can optionally be prepared by a process that analyzes the parent sequence as well as various conceptual humanized and engineered products. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display the predicted three-dimensional conformational structure of a selected candidate immunoglobulin sequence. By examining these displays, it is possible to analyze the likely role of residues when the candidate immunoglobulin sequence functions, i.e., to analyze the residues that affect the ability of the candidate immunoglobulin to bind its antigen (e.g., BCMA). In this way, FW residues can be selected and combined from the consensus sequence and the import sequence, thereby achieving the desired antibody properties, such as an increase in affinity for the target antigen.

[0129] Humanization, resurfacing, or engineering of the antibody or antigen-binding fragment thereof of the present invention can be carried out by any known method, such as, but not limited to, Jones et al., Nature 321:522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239:1534 (1988); Sims et al., J. Immunol. 151:2296 (1993); Chothia and Lesk, J. Mol. Biol. 196:901 (1987); Carter et al., Proc. Natl. Acad. Sci. USA 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993); U.S. Patent Nos. 5,639,641, 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; 4,816,567, 7,557,189; 7,538,195; and 7,342,110; International Application Nos. PCT / US98 / 16280, PCT / US96 / 18978, PCT / US91 / 09630, PCT / US91 / 05939, PCT / US94 / 01234, PCT / GB89 / 01334, PCT / GB91 / 01134, PCT / GB92 / 01755; International Publication Nos. 90 / 14443, 90 / 14424, 90 / 14430, and European Patent No. 229246 (each of which is hereby incorporated by reference in its entirety, including the references cited therein).

[0130] An antibody or antigen-binding fragment thereof can also be produced in transgenic mice containing the human immunoglobulin locus that can produce the complete repertoire of human antibodies without producing endogenous immunoglobulins upon immunization. This approach is described in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016.

[0131] In one embodiment, a fragment of an antibody of the invention (e.g., an antibody fragment) is provided. Various techniques for generating antibody fragments are known. The terms "antibody fragment", "antigen-binding fragment", "functional fragment of an antibody", and "antigen-binding portion" are used interchangeably herein and refer to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen (e.g., BCMA) to which the antibody (e.g., a "whole" antibody or "parent" antibody) binds. Thus, reference to "its antigen-binding fragment" means an antigen-binding fragment that binds to BCMA (e.g., the BCMA antigen-binding fragment of an antibody).

[0132] Conventionally, these fragments have been obtained via proteolytic digestion of intact antibodies, as described, for example, by Morimoto et al., J. Biochem. Biophys. Meth. 24:107-117 (1993) and Brennan et al., Science 229:81 (1985). In one embodiment, the anti-BCMA antibody fragment is produced recombinantly. Fab, Fv, and scFv antibody fragments can all be expressed and secreted from Escherichia coli (E. coli) or other host cells, thereby allowing these fragments to be produced in large quantities. Such anti-BCMA antibody fragments can also be isolated from the antibody phage libraries described above. The anti-BCMA antibody fragment may also be a linear antibody as described in U.S. Patent No. 5,641,870. Other techniques for generating antibody fragments will be apparent to those of skill in the art.

[0133] The modified antibodies or antigen-binding fragments thereof provided herein may include any type of variable region that results in the association of an antibody or polypeptide with BCMA. In this regard, the variable region may include or be derived from any type of mammalian variable region that can be induced to initiate a humoral response against a desired antigen and produce immunoglobulins. Thus, the variable region of an anti-BCMA antibody or antigen-binding fragment thereof can be, for example, of human, mouse, non-human primate (e.g., cynomolgus monkey, macaque, etc.) or lupine origin. In one embodiment, both the variable and constant regions of the modified antibody or antigen-binding fragment thereof are human. In one embodiment, the variable regions of an adapted antibody (usually derived from a non-human source) can be engineered or specifically tailored to improve the binding properties of the molecule or reduce its immunogenicity. In this regard, variable regions useful in the present invention can be humanized by including imported amino acid sequences or modified in other ways.

[0134] In one embodiment, the variable domains of both the heavy and light chains of the antibody or antigen-binding fragment thereof are modified by at least partial substitution of one or more CDRs and / or by substitution and sequence alteration of partial framework regions. The CDRs can be derived from an antibody of the same class or even the same subclass as the antibody from which the framework region is derived, but it is contemplated that the CDRs can be derived from antibodies of different classes and, in certain embodiments, from antibodies of different species. To transfer the antigen-binding ability of one variable domain to another, it is not necessary to replace all the CDRs with the complete CDRs of the donor variable region. Rather, it is sufficient to transfer only the residues necessary to maintain the activity of the antigen-binding site. Given the teachings described in U.S. Patent Nos. 5,585,089, 5,693,761, and 5,693,762, performing routine experiments to obtain a functionally active antibody with reduced immunogenicity is well within the ability of one of ordinary skill in the art.

[0135] Notwithstanding the modification of the variable regions, one of ordinary skill in the art will understand that the modified antibody or antigen-binding fragment thereof of the present invention, when compared to an antibody of substantially the same immunogenicity that includes a native or unmodified constant region, will result in desired biochemical properties such as increased tumor localization or reduced serum half-life, such that at least a portion of one or more of the constant region domains is deleted or otherwise modified. In one embodiment, the constant region of the modified antibody may include a human constant region. Modifications to the constant region that are compatible with the present invention include addition, deletion, or substitution of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein may include modifications or alterations to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and / or the light chain constant domain (CL). In one embodiment, modified constant regions are contemplated in which one or more domains are partially or completely deleted. In one embodiment, the modified antibody may include a domain deletion construct or variant (ΔCH2 construct) in which the entire CH2 domain has been removed. In one embodiment, the deleted constant region domain can be replaced with a short amino acid spacer (e.g., 10 residues) that typically provides some degree of molecular flexibility imparted by the missing constant region.

[0136] In addition to deletions of the full constant region domain, the antibodies or antigen-binding fragments thereof provided herein can be modified by partial deletions or substitutions of several or even a single amino acid in the constant region. For example, a single amino acid mutation in a selected region of the CH2 domain may be sufficient to substantially reduce Fc binding, thereby increasing tumor localization. Similarly, one or more constant region domains that control effector functions (e.g., complement C1Q binding) can be completely or partially deleted. Such partial deletions of the constant region can improve selected properties (e.g., serum half-life) of the antibody or antigen-binding fragment thereof while leaving other desirable functions associated with the constant region domain of interest intact. Furthermore, the constant regions of antibodies and antigen-binding fragments thereof can be modified by one or more amino acid mutations or substitutions that enhance the profile of the resulting construct. In this regard, it is possible to interfere with the activity provided by conserved binding sites (e.g., Fc binding) while substantially maintaining the disposition and immunogenic profile of the modified antibody or antigen-binding fragment thereof. In one embodiment, there may be an addition of one or more amino acids to the constant region to enhance desirable properties such as a decrease or increase in effector function, or to provide more binding of cytotoxins or carbohydrates. In one embodiment, it may also be desirable to insert or duplicate a specific sequence derived from a selected constant region domain.

[0137] The invention further includes variants and equivalents that are substantially homologous to the antibodies or antigen-binding fragments of the invention (e.g., murine, chimeric, humanized or human antibodies, or antigen-binding fragments thereof). These can include, for example, conservative substitution mutations, i.e., substitutions of one or more amino acids with similar amino acids. For example, conservative substitutions include substituting an amino acid with another amino acid within the same general class, e.g., substituting one acidic amino acid with another acidic amino acid, substituting one basic amino acid with another basic amino acid, or substituting one neutral amino acid with another neutral amino acid. What is intended by conservative amino acid substitutions is well known in the art.

[0138] In one embodiment, the antibody or antigen-binding fragment thereof can be further modified to include additional chemical moieties that are not normally part of the protein. These derivatized moieties can improve the solubility, biological half-life, or absorption of the protein. These moieties can also reduce or eliminate any undesirable side effects of the protein, etc. An overview of these moieties can be found in Remington’s Pharmaceutical Sciences, 22nd ed. Lloyd V. Allen, Jr. (2012).

[0139] Sequence homology Any of a variety of sequence alignment methods can be used to determine the percent identity, including, but not limited to, global methods, local methods, and hybrid methods such as, for example, the segment approach method. Protocols for determining the percent identity are routine procedures within the scope of those skilled in the art. Global methods align sequences from the beginning to the end of the molecule, sum the scores of individual residue pairs, and assign gap penalties to determine the best alignment. Non-limiting methods include, for example, CLUSTAL W (see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994)), and iterative refinement methods (see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein.Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996)). Local methods align sequences by identifying one or more conserved motifs shared by all input sequences.Non-limiting methods include, for example, Match-box (see, for example, Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992)), Gibbs sampling (see, for example, C.E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993)), Align-M (see, for example, Ivo Van WaIIe et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004)).

[0140] Thus, the percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment score using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum62" scoring matrix of Henikoff and Henikoff (supra) as shown below (amino acids are represented by the standard one-letter code).

[0141] The "percent sequence identity" between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, the percent identity can be calculated by dividing the number of identical nucleotides / amino acids by the total number of nucleotides / amino acids and multiplying by 100. Calculation of the percent sequence identity may also take into account the number of gaps introduced to optimize the alignment of two or more sequences and the length of each gap. Comparison of sequences and determination of percent identity between two or more sequences can be performed using specific mathematical algorithms well known to those of skill in the art, such as BLAST.

[0142] Substantially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions, or additions. These changes are preferably of a nature that is not critical, i.e., conservative amino acid substitutions (see below) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically on the order of 1 to about 30 amino acids; and minor extensions at the amino or carboxyl terminus, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

[0143] Conservative amino acid substitutions Basic: arginine lysine histidine Acidic: glutamic acid aspartic acid Polar: glutamine asparagine Hydrophobic: leucine isoleucine valine Aromatic: phenylalanine tryptophan tyrosine Small: glycine alanine serine threonine methionine

[0144] In addition to the 20 standard amino acids, non-standard amino acids (e.g., 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) can be substituted for amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and non-natural amino acids can be substituted for polypeptide amino acid residues. The polypeptides of the present invention can also contain amino acid residues that do not occur naturally.

[0145] A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, amino acids that do not occur naturally, and non-natural amino acids can be substituted for amino acid residues of the polypeptides of the present invention.

[0146] Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244:1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of the structure as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling in combination with mutagenesis of the amino acids at the putative contact sites. See, for example, de Vos et al., Science 255:306-12, 1992, Smith et al., J. Mol. Biol. 224:899-904, 1992, Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identity of essential amino acids can also be inferred from analysis of homology with related components of the polypeptides of the present invention (e.g., translocation components or protease components).

[0147] Multiple amino acid substitutions can be made and tested using known mutagenesis and screening methods such as those disclosed by Reidhaar - Olson and Sauer (Science 241:53 - 7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152 - 6, 1989). Briefly, these authors disclosed a method of simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptides, and then determining the sequence of the mutagenized polypeptides to determine the range of acceptable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832 - 7, 1991, Ladner et al., U.S. Patent No. 5,223,409, Huse, International Publication No. 92 / 06204 pamphlet) and site - directed mutagenesis (Derbyshire et al., Gene 46:145, 1986, Ner et al., DNA 7:127, 1988).

[0148] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide many common dictionaries of terms used in this disclosure to one of ordinary skill in the art.

[0149] The present disclosure is not limited to the exemplary methods and materials disclosed herein, and any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present disclosure. Numerical ranges include the numbers defining the range. Unless otherwise indicated, each nucleic acid sequence is written left to right in the 5' to 3' direction, and amino acid sequences are written left to right in the amino to carboxy direction.

[0150] The headings provided herein do not limit the various aspects or embodiments of the present disclosure.

[0151] In this specification, amino acids are referred to using the name of the amino acid, the three-letter abbreviation, or the one-letter abbreviation. The term "protein" as used herein includes proteins, polypeptides, and peptides. As used herein, the term "amino acid sequence" is synonymous with the terms "polypeptide" and / or "protein". In some cases, the term "amino acid sequence" is synonymous with the term "peptide". In some cases, the term "amino acid sequence" is synonymous with the term "enzyme". The terms "protein" and "polypeptide" are used interchangeably herein. Conventional one-letter and three-letter codes for amino acid residues may be used in the present disclosure and claims. The three-letter codes for amino acids defined in accordance with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It should also be understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by multiple nucleotide sequences.

[0152] Other definitions of terms may appear throughout this specification. Before discussing exemplary embodiments in more detail, it is to be understood that the present disclosure is not limited to the particular embodiments described, and thus may vary. Since the scope of the present disclosure is defined only by the appended claims, it is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0153] Where a range of values is provided, each intervening value, to the tenth of the unit of the lower limit value, between the upper and lower limit values of that range is also specifically disclosed, unless the context clearly dictates otherwise. Each smaller range between any of the stated values or intervening values within the stated range and any other stated value or intervening value within the stated range is included within the present disclosure. The upper and lower limits of these smaller ranges may independently be included in or excluded from the range, and each range that includes any one of the excluded limit values, or neither or both of the excluded limit values, in accordance with any specifically excluded limit values in the stated range, is also included within the present disclosure. Where the stated range includes one or both of the limit values, ranges excluding one or both of the included limit values are also included in the present disclosure.

[0154] It should be noted that, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cytotoxin" includes a plurality of such cytotoxins, and reference to "the cytotoxin" includes reference to one or more cytotoxins and equivalents thereof known to those of ordinary skill in the art.

[0155] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing in this specification should be construed as an admission that such publications constitute prior art to the claims appended hereto. The present disclosure provides, for example, the following embodiments. [1] An agent for B-cell malignancies, a. An antibody-drug conjugate (ADC) comprising an antibody or an antigen-binding fragment thereof that binds to B-cell maturation antigen (BCMA) conjugated to a nucleic acid cross-linking agent, and b. A proteasome inhibitor, which, when compared with an agent that is identical except for lacking the proteasome inhibitor, results in enhanced suppression of B-cell malignancies, or which, when compared with an agent that is identical except for lacking the ADC, results in enhanced suppression of B-cell malignancies. An agent for B-cell malignancies. [2] A therapeutic combination for use in the treatment of B-cell malignancies, a. An ADC comprising an antibody or an antigen-binding fragment thereof that binds to BCMA conjugated to a nucleic acid cross-linking agent, and b. A proteasome inhibitor, which, when compared with a composition that is identical except for lacking the proteasome inhibitor, results in enhanced suppression of B-cell malignancies, or which, when compared with a composition that is identical except for lacking the ADC, results in enhanced suppression of B-cell malignancies. A therapeutic combination for use in the treatment of B-cell malignancies. [3] A method for treating B-cell malignancies, a. Administering to a subject a therapeutic combination comprising an ADC comprising an antibody or an antigen-binding fragment thereof that binds to BCMA conjugated to a nucleic acid cross-linking agent, and b. A proteasome inhibitor, wherein the therapeutic combination, when compared with a composition that is identical except for lacking the proteasome inhibitor, results in enhanced suppression of B-cell malignancies, or wherein the therapeutic combination, when compared with a composition that is identical except for lacking the ADC, results in enhanced suppression of B-cell malignancies. A method for treating B-cell malignancies. [4] (a) An ADC comprising an antibody or an antigen-binding fragment thereof that binds to BCMA conjugated to a nucleic acid cross-linking agent, and (b) contacting a malignant B cell with a proteasome inhibitor in combination, an in vitro method for enhancing the suppression of malignant B cells by the ADC. [5] (a) A proteasome inhibitor, and (b) contacting a malignant B cell with an ADC comprising an antibody or an antigen-binding fragment thereof that binds to BCMA conjugated to a nucleic acid cross-linking agent in combination, an in vitro method for enhancing the suppression of malignant B cells by the proteasome inhibitor. [6] a. The proteasome inhibitor is administered before, simultaneously with, or subsequent to the ADC, or b. The ADC is administered before, simultaneously with, or subsequent to the proteasome inhibitor, the pharmaceutical agent, therapeutic combination, method, or in vitro method for use according to any one of the above 1 to 5. [7] The B cell malignancy comprises malignant B cells with increased expression levels of the BCMA antigen compared to reference non-malignant B cells, the pharmaceutical agent, therapeutic combination, method, or in vitro method for use according to any one of the above 1 to 6. [8] The B cell malignancy is one or more selected from B cell lymphoma, B cell leukemia, myeloma, multiple myeloma, or combinations thereof, the pharmaceutical agent, therapeutic combination, method, or use for use according to any one of the above 1 to 7. [9] The B cell malignancy is multiple myeloma, the pharmaceutical agent, therapeutic combination, method, or use for use according to any one of the above 1 to 8.

[10] The antibody or its antigen-binding fragment has the following six CDRs: a. Heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a functional variant thereof; b. Heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a functional variant thereof; c. A heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a functional variant thereof; d. A light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a functional variant thereof; e. A light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, or a functional variant thereof; and f. The agent, therapeutic combination, method, or use for use according to any one of the above items 1 to 9, comprising a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6, or a functional variant thereof.

[11] The antibody or antigen-binding fragment thereof is a. A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, or a functional equivalent thereof; and / or b. The agent, therapeutic combination, method, or use for use according to any one of the above items 1 to 10, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, or a functional equivalent thereof.

[12] The antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising an insertion of cysteine (C) between serine (S) at position 239 and valine (V) at position 240, and the numbering corresponds to the EU index of Kabat. The agent, therapeutic combination, method, or use for use according to any one of the above items 1 to 11.

[13] The antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO: 11. The agent, therapeutic combination, method, or use for use according to any one of the above items 1 to 12.

[14] The antibody or antigen-binding fragment thereof comprises a human kappa constant region comprising the amino acid sequence of SEQ ID NO: 12. The agent, therapeutic combination, method, or use for use according to any one of the above items 1 to 13.

[15] The proteasome inhibitor is one or more selected from bortezomib, carfilzomib, ixazomib, marizomib, oprozomib, delanzomib, or combinations thereof. The agent, therapeutic combination, method, or use for use according to any one of the above items 1 to 14.

[16] The proteasome inhibitor is bortezomib, the agent, therapeutic combination, method, or use for use according to any one of items 1 to 15 above.

[17] The nucleic acid crosslinking agent is pyrrolobenzodiazepine (PBD), the agent, therapeutic combination, method, or use for use according to any one of items 1 to 16 above.

[18] The nucleic acid crosslinking agent is of the formula:

Chemical formula

[19] The nucleic acid crosslinking agent is the PBD SG3249 containing the following formula, the agent, therapeutic combination, method, or use for use according to any one of items 1 to 18 above.

Chemical formula

[20] The antibody or antigen-binding fragment thereof is a monoclonal antibody, the agent, therapeutic combination, method, or use for use according to any one of items 1 to 19 above.

[21] The agent or the therapeutic combination contains a pharmaceutically acceptable carrier, the agent, therapeutic combination, method, or use for use according to any one of items 1 to 20 above.

[22] The B-cell malignancy is resistant to one or more selected from dexamethasone, lenalidomide, pomalidomide, bortezomib, or a combination thereof, the agent, therapeutic combination, method, or use for use according to any one of items 1 to 21 above.

[23] The agent, therapeutic combination, method, or use for use according to any one of the above items 1 to 22, wherein the B-cell malignancy is resistant to bortezomib.

[24] The agent, therapeutic combination, method, or use for use according to any one of the above items 1 to 23, wherein the enhancement of the suppression of the B-cell malignancy includes one or more selected from an increase in the delay of tumor growth, an enhancement of the reduction of tumor size, an enhancement of the reduction of tumor metastasis, an increase in survival rate, or a combination thereof, in a subject with a B-cell malignancy.

[25] The agent, therapeutic combination, method, or use for use according to any one of the above items 1 to 24, wherein the agent or the therapeutic combination is administered by intravenous injection.

Examples

[0156] The present invention will be described merely by way of illustration with reference to the following examples hereinafter.

[0157] Materials and Methods Preparation of anti-BCMA antibody Antibodies were prepared as described in International Publication No. WO 2010 / 104949 pamphlet and International Publication No. WO 2019 / 025983 pamphlet (both incorporated herein by reference). Suitable antibodies were prepared as described in Kinneer et al (2018), Leukemia 33, 766-771 and International Publication No. WO 2019 / 025983 pamphlet.

[0158] Preparation of anti-BCMA ADC An anti-BCMA ADC (the anti-BCMA antibody conjugated to PBD is referred to herein as "M2") was prepared via site-specific conjugation of the PBD dimer tesirine (SG3249) to the BCMA-Ab1 antibody described in Kinneer et al (2018), Leukemia 33,766-771 ("Kinneer et al (2018)") using a protease-cleavable linker as described above (see, e.g., Kinneer et al (2018), which is incorporated herein by reference). An example of the BCMA antibody is BCMA-Ab2, which is also described in Kinneer et al (2018). The above antibodies are further described as 15B2GL and J6M0-mc, respectively, in International Publication No. WO 2019 / 025983 (which is incorporated herein by reference). An ADC "M3" was similarly prepared by conjugating the monomethyl auristatin F (MMAF) payload to the antibody BCMA-Ab1. Both payloads were site-specifically conjugated to a engineered cysteine (C239i) inserted after position 239 in the CH2 domain of the BCMA antibody as described above. Briefly, BCMA-Ab1 was reduced with 40 molar excess of TCEP at 37 °C for 3 hours, followed by dialysis three times in succession to remove TCEP. The antibody was then oxidized with 20 molar excess of DHAA at room temperature for 4 hours and conjugated using 8 molar equivalents of the payload. After conjugation, free payload and protein aggregates were removed by ceramic hydroxyapatite purification.

[0159] Mouse xenograft model of human MM All animal experiments were approved and conducted in accordance with the relevant regulatory standards of the Dana-Farber Cancer Institute's Animal Care and Use Committee. 5.0 × 106 MM.1S cells in 100 μl of serum-free RPMI 1640 medium were subcutaneously inoculated into CB-17 SCID mice. When tumors were measurable approximately 3 weeks after injection of MM cells, the mice (8 per group) were randomized and treated with vehicle only, M2, btz, or M2 and btz. Tumor size was measured two-dimensionally every 3 days using calipers, and tumor volume was calculated using the following formula: V = 0.5a × b2 (where "a" and "b" are the major and minor diameters of the tumor, respectively). Animals were sacrificed when the tumor reached 2 cm 3 in diameter.

[0160] Analysis of tumors collected from mice using immunoblotting and immunohistochemistry After 3 days of treatment, tumors from each group were collected and cell lysates for immunoblotting were prepared. Tumor sections recovered from mice were subjected to immunohistochemical staining for proliferation with Ki67 (BCR CRM325). Immunohistochemical images were taken for Ki67 with a Zeiss Inverted Fluorescence Microscope. A Plan-Apochromat 63X / 1.40 Oil DIC M27 objective lens was used.

[0161] Cells and cell culture MM cell lines were cultured in RPMI containing 10% fetal bovine serum (GIBCO, 10437028), 2 mM / L L-glutamine, 100 U / mL penicillin, and 100 mg / mL streptomycin (GIBCO, 15140122). For the authenticity and mycoplasma contamination of MM cell lines, MM cell lines were routinely confirmed by human STR profiling cell authentication. In accordance with the Helsinki Declaration, patient MM and normal donor samples were obtained after informed consent under the support of a protocol approved by the Institutional Review Board of Dana-Farber Cancer Institute. Primary CD138+ plasma cells (purity >95%) were purified from bone marrow mononuclear cells (BMMCs) derived from BM aspirates of MM patients using anti-CD138 microbeads (Miltenyi Biotech, Auburn, CA). The remaining CD138-negative BMMCs were further cultured to induce BMSCs. Peripheral blood mononuclear cells (PBMCs) were isolated from PB samples using Ficoll-Hypaque density gradient. Bortezomib was purchased from Selleckchem (Selleck Chemicals).

[0162] Cell viability assay and apoptosis assay Cell viability was analyzed by CCK8 (Abcam, Cambridge, MA), CellTiter-Glo (CTG) (Promega), and BLI measurement. Apoptosis was evaluated by flow cytometry analysis after staining with FITC Annexin-V (BD Biosciences), PE-Annexin-V (BioLegend), and / or LIVE / DEAD™ Fixable Aqua (Invitrogen, L34957) according to the manufacturer's instructions. MM cells were labeled with CFSE (Invitrogen) and then cultured alone or with BMSCs for 2 days, followed by Annexin V / Aqua staining and flow cytometry analysis.

[0163] Luciferase proliferation assay BMSCs were seeded in 96-well plates and incubated for 24 hours to allow the cells to adhere. MM1Sluc cells were cultured for 4 days at a ratio of 100:1 on a confluent layer of BMSCs in RPMI medium. Proliferation was measured using a luciferase assay according to the manufacturer's (Promega, Madison, WI) protocol.

[0164] Statistical analysis Each experiment was performed at least three times, and the data were presented as mean ± SD. The data were analyzed using Student's t-test for comparison of two groups or one-way analysis of variance (ANOVA) for multiple comparisons using Graphpad software (GraphPad Software, La Jolla, CA, USA). A P-value of less than 0.05 was considered statistically significant. Drug interactions were evaluated by CompuSyn software to determine the combination index (CI). A CI less than 1 indicates synergy, a CI greater than 1 indicates antagonism, and CI = 1 indicates additivity.

[0165] Example 1 The experimental results reflected in this example demonstrate that the anti-BCMA antibody-PBD conjugate (M2) induces more potent cytotoxicity against drug-resistant MM cells than its MMAF ADC homolog (M3).

[0166] The cytotoxicity of an ADC composed of an anti-BCMA antibody (M2) conjugated to PBD was compared to that of its MMAF ADC homolog (M3) against a panel of MM cell lines with various levels of BCMA expression and responses to current anti-MM drugs. Both ADCs are composed of the same anti-BCMA mAb (BCMA-Ab1 / 15B2GL as described above), but conjugated to different payloads (i.e., M2 to PBD (e.g., tesirine) that crosslinks DNA and M3 to MMAF that binds to microtubules). Using a CCK8-based viability assay for 3 days, regardless of sensitivity to anti-MM therapies including dexamethasone and IMiD, the ED of M2 50The values were lower than those of M3 in all MM cell lines tested (n = 10) (Figure 1A, Figure 3A). In eight MM cell lines (excluding RPMI8226 (RPMI) and RPMI-BCMA that overexpresses BCMA derived from it), ED 50 The values for M2 and M3 were in the ranges of 11.85 - 3499 ng / ml and 21.28 - 271431 ng / ml, respectively. Except for MM1S cells and H929 cells from which MM1S(R) cells and H929(R) cells, which are resistant to IMiD, are respectively derived, all MM cells carry various p53 mutations. M2 is cytotoxic to RPMI8226 cells that express the lowest BCMA levels and are resistant to IMiD, while M3 is not (Figure 1A - B). Using a DNA synthesis assay, M2 shows a greater (>1 - 2 log) potency than M3 against the blockade of the proliferation of all MM cells (Figure 1B, Figure 3B). For example, the ED 50 of M2 to M3 is 189.7 vs 21427 ng / ml in RPMI8226 cells. Furthermore, M2 decreased the survival rates of both ANBL6 and its bortezomib (btz)-resistant ANBL6-BR cells cultured with IL-6, while M3 did not (Figure 3C). These paired IL-6-dependent ANBL6 cells are insensitive to M3 and express a comparable level of cell membrane BCMA protein to RPMI8226 cells (data not shown). Therefore, MM cells with relatively low BCMA expression are also significantly more sensitive to M2 compared to M3.

[0167] Flow cytometry (FCM) analysis after staining with Annexin V and live / dead Aqua showed that M2 induced earlier and increased apoptosis in a dose- and time-dependent manner compared to M3 in paired MM cell lines sensitive or resistant to dexamethasone (dex) or bortezomib (btz) (Figure 1C, Figure 3D). These in vitro results indicate that M2 significantly overcomes resistance to current anti-MM drugs (dexamethasone, lenalidomide, pomalidomide, bortezomib) in MM cells compared to M3, regardless of BCMA levels and p53 status.

[0168] Example 2 The experimental results reflected in this example demonstrate that M2 is more effective than M3 in inducing cytotoxicity against MM cells in the bone marrow microenvironment and patient MM cells.

[0169] Next, the effects of M2 and M3 on MM cells co-cultured with bone marrow stromal cells (BMSC) and IL-6, which promote the proliferation, survival, and drug resistance of MM cells, were evaluated. Using BLI measurement, BMSC was shown to significantly increase the proliferation and survival of MM1Sluc cells (Figure 4A). In BLI-based and CTG-based assays, M2 potently inhibits the viability of MM1Sluc and all other tested MM cell lines (n = 6) co-cultured with BMSC compared to M3, while minimizing the impact on BCMA-negative non-MM cell subsets such as BMSC, PBMC, and NK cells (Figure 4B) (Figure 2A, Figure 4A). Using FCM analysis to identify viable MM cells, M2 more effectively reduces the survival of IMiD-resistant MM1S(R) and H929(R) cells even in the presence of BMSC compared to M3 (Figure 2B). In quantitative FCM-based analysis (Figure 2C) and quantitative CTG-based analysis (Figure 4C), M2 reduced the proliferation and survival of H929 MM cells in the presence or absence of IL-6.

[0170] After 3 days of treatment, the viable and dead cell fractions of BM CD138+ from patients with RRMM were quantified by FCM analysis. Importantly, M2 increased apoptotic CD138+ patient MM cells in a dose-dependent manner (more than 2-fold) compared to M3 (Figure 4D). In the CTG-based assay, M2 also showed dose-dependent toxicity in CD138-purified BM cells from three additional patients with RRMM (Figure 2E), and additionally, significantly depleted viable CD38highCD138+ BM cells from four patients with newly diagnosed MM (NDMM) (Figure 2F, left, Figure 4D) and two patients with RRMM (Figure 2F, right). These data indicate that M2 depletes patient MM cells regardless of disease state and is significantly more cytotoxic to MM cells in the BM microenvironment than M3.

[0171] Example 3 The experimental results reflected in this example demonstrate that M2 in combination with bortezomib induces synergistic cytotoxicity against MM cells in vitro and in vivo.

[0172] Bortezomib (btz) was selected as a candidate co-therapy for M2 because btz is an existing myeloma therapy. Annexin V / PI-based FMC analysis with a 2-day co-treatment of M2 and btz at low doses further enhanced apoptosis in JJN3 cells and RPMI8226 cells compared to either drug alone (Figure 5A - B, p < 0.01). The significantly increased cell death after co-treatment was also seen in btz-resistant ANBL6-BR cells cultured in IL-6 (confirming the observation that the addition of btz results in an effect that exceeds an additive effect). Next, the results from the CTG-based survival assay were analyzed to calculate the combination index (CI). A CI of less than 1 was obtained in more than six representative MM cells, indicating a synergistic effect of M2 + btz (Figure 5C, Figure 6).

[0173] Next, in the MM1S xenograft mouse model, the in vivo efficacy of M2 and btz below the optimal dose was evaluated. Mice with palpable MM1S tumors were randomized into four groups that received either a vehicle control, single treatment with M2, or six single treatments with btz (0.4 mg / kg) or a combination treatment with M2. Twenty-four days after treatment, a single dose of M2 or a total of six doses of btz significantly delayed MM1S tumor growth in mice compared to the vehicle control (Figure 7A, p < 0.005). The combination treatment significantly reduced tumor volume compared to either single agent alone (p < 0.04). Since the body weights of all animals were not affected, the treatment with M2 + btz was well tolerated (Figure 7B). A 177-day follow-up showed a significant extension of the median overall survival period in the combination treatment group compared to the cohorts treated with either agent alone (cnt, 22 days; M2, 40.5 days; btz, 35 days; M2 + btz, 57 days) (p < 0.045) (Figure 7C). In the combination treatment group, 15% of the mice were still alive without any tumor growth on day 177.

[0174] Immunohistochemistry (IHC) for Ki67 (a cell marker of proliferation) further demonstrated that proliferation was more potently inhibited after combination treatment compared to single-agent treatment (Figure 7D) (note the decrease in the number of stained cells (dark color) in the M2 + btz treatment).

[0175] The combination treatment of M2 and btz significantly decreased the in vivo growth of MM1S xenografts (Figure 8), demonstrating the in vivo synergistic effect of M2 and btz in the treatment of multiple myeloma.

[0176] In conclusion, the synergistic activity of M2 with btz observed in vitro at the cellular level was translated into excellent in vivo efficacy in the multiple myeloma plasma cell tumor model.

[0177] Discussion of Examples 1-3 Disease recurrence due to drug resistance remains a major obstacle to further long-term survival in MM. Therefore, new therapies are needed to overcome drug resistance and address the unmet medical needs in RRMM. In this regard, the inventors first show that ADC (anti-BCMA antibody conjugated to PBD) has superior cytotoxicity to its MMAF ADC homolog against all MM cell lines and patient MM cells tested. Unlike MMAF, which mainly targets proliferating tumor cells through binding to tubulin, the PBD dimer causes cell death in both rapidly dividing cells and more quiescent cells. M2 induces a more potent effect on MM cell proliferation than its MMAF ADC homolog, including cells with low BCMA expression levels and resistance to current therapies even in the presence of BMSC and IL-6. These data suggest that M2 may be more effective than its MMAF ADC homolog in the treatment of high-grade MM.

[0178] Importantly, the combination treatment of M2 and btz in vitro induces synergistic death, demonstrated by CI < 1, in all MM cells tested. In particular, M2 synergizes with btz even in btz-resistant ANBL6-BR cells, indicating that other undefined molecules are also involved in enhancing cytotoxicity.

[0179] In MM1S tumor-bearing mice, M2 is extremely effective as a single agent compared to btz. Importantly, the inhibition of in vivo tumor growth is further enhanced when M2 is combined with btz. In mice administered both drugs, significant tumor necrosis is observed earlier than with either drug alone, and on day 177, 15% of the mice in the combination treatment group are still alive without tumors. Importantly, weight loss is not observed in any of the groups, indicating a favorable safety profile of M2 in vivo and suggesting that the combination treatment of M2 and btz can be safely administered in vivo.

[0180] In summary, M2 specifically induces potent growth inhibition and death even in MM cells that are resistant to current MM therapies and protected by the BM microenvironment. In vivo, M2 is more effective than btz, and the efficacy is further enhanced and host survival is prolonged by combining M2 with btz.

[0181] Example 4 The experimental results reflected in this example demonstrate that M2 significantly activates the DNA damage response and repair signaling cascade in drug-sensitive and drug-resistant MM cells, subsequently activating apoptosis.

[0182] Using immunoblot analysis, we demonstrated the induction of DNA damage response (DDR) signaling cascades induced by M2 in MM cell lines in a time- and dose-dependent manner. M2 induced the phosphorylation of ATM, cell cycle checkpoint kinase 1 (CHK1), and CHK2 (CHK1 / 2), and histone 2AX (H2AX), which are early events in the DNA double-strand break (DSB) response, while M3 did not (Figures 9A and 10A). Phosphorylation of ATM and CHK1 / 2 by M2 stimulation was detected at 4 hours and persisted for more than 1 day after treatment. In H929 cells, which express significantly higher levels of BCMA than MM1S cells, earlier and more prominent activation of ATM and CHK1 / 2 induced by M2 was observed (Figures 10A, C). The intensity of M2-induced phosphorylation of ATM and CHK1 / 2 also correlated with the BCMA levels in parental RPMI8226 MM cells (Figure 10D). In the MM cells tested, M2-induced phosphorylation of ATM and CHK1 / 2 occurred much more extensively than phosphorylation of ATR. After 2 days of treatment with M2, cleavage of PARP (cPARP) and caspase 3 (cCas3) was induced in a BCMA-dependent manner, accompanied by an increase in phosphorylated H2AX (γH2AX) (Figures 10B, E–F), indicating that M2 induces DNA damage and subsequently apoptosis in MM cells. Importantly, M2 induced phosphorylation of ATM and CHK1 / 2 in a dose-dependent manner in all MM cells, including six cell lines with p53 mutations (Figures 9B–C). M3 did not induce ATM / ATR or CHK1 / 2 under the same treatment conditions as M2 (Figure 10A). M2 was more effective than M3 in inducing cPARP and cCas3 (Figure 10F), consistent with the higher efficacy of M2 in inducing apoptosis in MM cells. Significantly, M2 induced ATM / ATR and downstream CHK1 / 2 signaling pathways, cleavage of γH2AX and PARP in ANBL6 cells and paired btz-resistant ANBL6-BR cells (Figure 9C). Marked activation of ATM and CHK1 / 2 by M2 was also observed in IMiD-resistant H929 (R) cells to a similar extent as in parental H929 MM cells (Figure 9D).Therefore, in btz- and len-resistant MM cells, M2 still induces the BCMA-dependent DDR signaling pathway via the activation of the ATR / ATM-CHK1 / 2 signaling cascade, subsequently inducing apoptosis.

[0183] Analysis of the DNA repair mechanism TagMan (registered trademark) array shows that M2 changes the expression of DNA damage repair-related genes in H929 MM cells (51 out of 72) (Figure 11a). In various drug-sensitive MM cells and drug-resistant MM cells (n>6), M2 dose-dependently induces RAD51 (which binds to DNA ICLs before the occurrence of DSBs) (Figures 11b - c), while M3 does not (Figure 10G). Thus, in MM cells, M2 specifically activates the DDR signaling cascade via ATM / ATR-CHK1 / 2, induces downstream DDR-related molecules with an increase in γH2AX and RAD51, and subsequently induces apoptosis.

[0184] All publications mentioned in the above specification are hereby incorporated by reference into this specification. Various modifications and variations of the described methods and systems of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been described in connection with specific embodiments, it should be understood that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications in the manner described for carrying out the invention, which are obvious to those skilled in the art in the fields of biochemistry and biotechnology or related fields, are intended to be within the scope of the following claims.

[0185]

Table 1

Claims

1. A medicament for B-cell malignancies, comprising: a. An antibody-drug conjugate (ADC) comprising an antibody or an antigen-binding fragment thereof that binds to B-cell maturation antigen (BCMA) conjugated to pyrrolobenzodiazepine (PBD); and b. Bortezomib, a proteasome inhibitor, which, when compared with a medicament identical except for lacking the proteasome inhibitor or the ADC, enhances the suppression of B-cell malignancies. A medicament for B-cell malignancies.

2. a. The proteasome inhibitor is administered before, simultaneously with, or after the ADC, or b. The ADC is administered before, simultaneously with, or after the proteasome inhibitor. The medicament according to Claim 1.

3. The medicament according to Claim 1 or 2, wherein the B-cell malignancy comprises malignant B cells with an increased expression level of BCMA antigen compared to reference non-malignant B cells.

4. The medicament according to any one of Claims 1 to 3, wherein the B-cell malignancy is one or more selected from B-cell lymphoma, B-cell leukemia, myeloma, multiple myeloma, or combinations thereof.

5. The medicament according to any one of Claims 1 to 4, wherein the B-cell malignancy is multiple myeloma.

6. The antibody or its antigen-binding fragment comprises the following six CDRs: a. Heavy-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1; b. Heavy-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2; c. Heavy-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3; d. Light-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4; e. Light-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and f. Light-chain CDR3 comprising the amino acid sequence of SEQ ID NO:

6. The medicament according to any one of Claims 1 to 5.

7. The antibody or its antigen-binding fragment comprises a. A heavy-chain variable region comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7; and / or b. A light-chain variable region comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

8. The medicament according to any one of Claims 1 to 6.

8. The agent according to any one of claims 1 to 7, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region having at least 95% amino acid sequence identity to SEQ ID NO: 7 and comprising an insertion of cysteine (C) between serine (S) at position 239 and valine (V) at position 240.

9. The agent according to any one of claims 1 to 8, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:

11.

10. The agent according to any one of claims 1 to 9, wherein the antibody or antigen-binding fragment thereof comprises a human kappa constant region comprising the amino acid sequence of SEQ ID NO:

12.

11. The PBD is of the formula: 【Chemical Formula 1】 One or more PBDs selected from (a) SG3249, (b) SG3315, or (c) SG3400, each containing The agent according to any one of claims 1 to 10, which is one or more PBDs selected from these combinations.

12. The agent according to any one of claims 1 to 11, wherein the PBD is SG3249 containing the following formula. [Chemical Formula 2]

13. The agent according to any one of claims 1 to 12, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

14. The agent according to any one of claims 1 to 13, wherein the agent comprises a pharmaceutically acceptable carrier.

15. The agent according to any one of claims 1 to 14, wherein the B cell malignancy is resistant to one or more selected from dexamethasone, lenalidomide, pomalidomide, bortezomib, or combinations thereof.

16. The agent according to any one of claims 1 to 15, wherein the B cell malignancy is resistant to bortezomib.

17. The enhancement of the suppression of the B cell malignancy comprises one or more selected from an increase in the delay of tumor growth, an enhancement of the reduction of tumor size, an enhancement of the reduction of tumor metastasis, an increase in survival rate, or combinations thereof, in a subject with B cell malignancy. The agent according to any one of claims 1 to 16.

18. The agent according to any one of claims 1 to 17, wherein the agent is administered by intravenous infusion.

19. A pharmaceutical composition for treating B cell malignancy, comprising a. An antibody-drug conjugate (ADC) comprising an antibody or an antigen-binding fragment thereof that binds to B-cell maturation antigen (BCMA) conjugated to a pyrrolobenzodiazepine (PBD), wherein the pharmaceutical composition is used in combination with a proteasome inhibitor, and the proteasome inhibitor is bortezomib; or b. Bortezomib, which is a proteasome inhibitor, wherein the pharmaceutical composition is used in combination with an antibody-drug conjugate (ADC) comprising an antibody or an antigen-binding fragment thereof that binds to B-cell maturation antigen (BCMA) conjugated to a pyrrolobenzodiazepine (PBD), A pharmaceutical composition that provides enhanced suppression of B-cell malignancies when compared to a composition that is identical except for not being used in combination with the proteasome inhibitor or the ADC.

20. a. The proteasome inhibitor is administered before, simultaneously with, or subsequent to the ADC, or b. The ADC is administered before, simultaneously with, or subsequent to the proteasome inhibitor. The pharmaceutical composition according to claim 19.

21. The pharmaceutical composition according to claim 19 or 20, wherein the B-cell malignancy comprises malignant B cells with increased expression levels of BCMA antigen compared to reference non-malignant B cells.

22. The pharmaceutical composition according to any one of claims 19 to 21, wherein the B-cell malignancy is one or more selected from B-cell lymphoma, B-cell leukemia, myeloma, multiple myeloma, or combinations thereof.

23. The pharmaceutical composition according to any one of claims 19 to 22, wherein the B-cell malignancy is multiple myeloma.

24. The antibody or its antigen-binding fragment comprises the following six CDRs: a. Heavy-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1; b. Heavy-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2; c. Heavy-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3; d. Light-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4; e. Light-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and f. Light-chain CDR3 comprising the amino acid sequence of SEQ ID NO:

6. The pharmaceutical composition according to any one of claims 19 to 23.

25. The antibody or its antigen-binding fragment is a. A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7; and / or b. A light chain variable region comprising the amino acid sequence of SEQ ID NO: 8 or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8 The pharmaceutical composition according to any one of claims 19 to 24, comprising the same.

26. The pharmaceutical composition according to any one of claims 19 to 25, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region having at least 95% amino acid sequence identity to SEQ ID NO: 7 and comprising an insertion of cysteine (C) between serine (S) at position 239 and valine (V) at position 240.

27. The pharmaceutical composition according to any one of claims 19 to 26, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:

11.

28. The pharmaceutical composition according to any one of claims 19 to 27, wherein the antibody or antigen-binding fragment thereof comprises a human kappa constant region comprising the amino acid sequence of SEQ ID NO:

12.

29. The PBD is of the formula: [Chemical Formula 3] Comprising respectively (a) SG3249, (b) SG3315, or (c) SG3400 The pharmaceutical composition according to any one of claims 19 to 28, which is one or more PBDs selected from these combinations.

30. The pharmaceutical composition according to any one of claims 19 to 29, wherein the PBD is SG3249 comprising the following formula. [Chemical Formula 4]

31. The pharmaceutical composition according to any one of claims 19 to 30, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

32. The pharmaceutical composition according to any one of claims 19 to 31, further comprising a pharmaceutically acceptable carrier.

33. The pharmaceutical composition according to any one of claims 19 to 32, wherein the B cell malignancy is resistant to one or more selected from dexamethasone, lenalidomide, pomalidomide, bortezomib, or combinations thereof.

34. The pharmaceutical composition according to any one of claims 19 to 33, wherein the B cell malignancy is resistant to bortezomib.

35. Enhanced suppression of the B-cell malignancy includes one or more selected from increased delay of tumor growth, enhanced reduction of tumor size, enhanced reduction of tumor metastasis, increased survival rate, or combinations thereof, in a subject with a B-cell malignancy, the pharmaceutical composition according to any one of claims 19 to 34.

36. The pharmaceutical composition according to any one of claims 19 to 35, wherein the pharmaceutical composition is administered by intravenous injection.

37. An in vitro method for enhancing the suppression of malignant B cells by an ADC, comprising contacting malignant B cells with (a) an ADC comprising an antibody or an antigen-binding fragment thereof that binds to BCMA conjugated to a pyrrolobenzodiazepine (PBD), and (b) bortezomib, a proteasome inhibitor.

38. An in vitro method for enhancing the suppression of malignant B cells by a proteasome inhibitor, comprising contacting malignant B cells with (a) bortezomib, a proteasome inhibitor, and (b) an ADC comprising an antibody or an antigen-binding fragment thereof that binds to BCMA conjugated to a pyrrolobenzodiazepine (PBD).

39. a. contacting the proteasome inhibitor with malignant B cells before, simultaneously with, or subsequent to the ADC, or b. contacting the ADC with malignant B cells before, simultaneously with, or subsequent to the proteasome inhibitor, the in vitro method according to claim 37 or 38.

40. The in vitro method according to any one of claims 37 to 39, wherein the B-cell malignancy comprises malignant B cells with an increased expression level of BCMA antigen compared to reference non-malignant B cells.

41. The in vitro method according to any one of claims 37 to 40, wherein the B-cell malignancy is one or more selected from B-cell lymphoma, B-cell leukemia, myeloma, multiple myeloma, or combinations thereof.

42. The in vitro method according to any one of claims 37 to 41, wherein the B-cell malignancy is multiple myeloma.

43. The antibody or its antigen-binding fragment has the following six CDRs: a. Heavy-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 1; b. Heavy-chain CDR2 comprising the amino acid sequence of SEQ ID NO: 2; c. Heavy-chain CDR3 comprising the amino acid sequence of SEQ ID NO: 3; d. Light-chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4; e. A light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5; and f. The in vitro method according to any one of claims 37 to 42, comprising a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

6.

44. The antibody or antigen-binding fragment thereof is a. A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 7, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 7; and / or b. The in vitro method according to any one of claims 37 to 43, comprising a light chain variable region comprising the amino acid sequence of SEQ ID NO: 8, or an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

8.

45. The antibody or antigen-binding fragment thereof has at least 95% amino acid sequence identity to SEQ ID NO: 7 and comprises a heavy chain constant region comprising an insertion of cysteine (C) between serine (S) at position 239 and valine (V) at position 240. The in vitro method according to any one of claims 37 to 44.

46. The in vitro method according to any one of claims 37 to 45, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:

11.

47. The in vitro method according to any one of claims 37 to 46, wherein the antibody or antigen-binding fragment thereof comprises a human kappa constant region comprising the amino acid sequence of SEQ ID NO:

12.

48. The PBD is Each containing the formula: 【Chemical Formula 5】 One or more PBDs selected from (a) SG3249, (b) SG3315, or (c) SG3400 Or a combination thereof. The in vitro method according to any one of claims 37 to 47.

49. The in vitro method according to any one of claims 37 to 48, wherein the PBD is SG3249 containing the following formula. 【Chemical Formula 6】

50. The in vitro method according to any one of claims 37 to 49, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

51. The in vitro method according to any one of claims 37 to 50, wherein the ADC and / or bortezomib further comprises a pharmaceutically acceptable carrier.

52. The in vitro method according to any one of claims 37 to 51, wherein the B cell malignancy is resistant to one or more selected from dexamethasone, lenalidomide, pomalidomide, bortezomib, or a combination thereof.

53. The in vitro method according to any one of claims 37 to 52, wherein the B cell malignancy is resistant to bortezomib. **Claim 54** The enhancement of the suppression of the B cell malignancy includes one or more selected from an increase in the delay of tumor growth, an enhancement of the reduction in tumor size, an enhancement of the reduction in tumor metastasis, an increase in survival rate, or a combination thereof in a subject including the B cell malignancy. The in vitro method according to any one of claims 37 to 53.

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