Combination therapy

Abstract

The present disclosure relates to methods and compositions for treating B-cell malignancies. Specifically, the present disclosure relates to a B-cell malignancy drug 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 cross-linker; and (b) a proteasome inhibitor.

Description

[0001] Reference to a sequence listing submitted electronically

[0002] The contents of the electronically submitted sequence listing as an ASCII text file submitted with this application (name: BCMA-150-US-PSP-SequenceListing.txt; size: 11,279 bytes; and creation date: May 28, 2019) are incorporated herein by reference in their entirety. Background Art

[0003] Blood cancer is a term used to describe many different types of cancer that affect blood cells, bone marrow, or the lymphatic system. It reportedly accounts for almost 10% of new cancer cases in the United States each year, with over 1.2 million people living with or in remission from a blood cancer in the US alone. It is the fifth most common cancer in the UK, with over 240,000 people diagnosed with blood cancer each year and 40,000 diagnosed. The three main types of blood cancer are leukemia, lymphoma, and myeloma, each representing a B-cell malignancy.

[0004] For example, the B-cell malignancy myeloma (e.g., multiple myeloma (MM)) is a malignancy of clonal plasma cells (e.g., B cells) in which persistent DNA damage is associated with progression from malignant promonoclonal gammopathy of undetermined significance (MGUS) to active MM. Current treatment options for myeloma include conventional corticosteroids, alkylating agents, proteasome inhibitors (PIs), and immunomodulatory drugs (IMiDs), all of which contribute to improving the overall survival of myeloma patients. A particularly interesting therapeutic approach that has been explored in recent years is immunotherapy (e.g., using monoclonal antibodies). For example, WO 2010 / 104949 and WO 2019 / 025983 describe antibodies that bind to an antigen called B-cell maturation antigen (BCMA), which has been shown to have good selectivity for B-cell malignancies (particularly myeloma cells), wherein the antibodies described exhibit anti-B-cell malignancy activity.

[0005] Despite the increasing availability of different treatments, the development of drug resistance underlies disease relapse (particularly in myeloma), and the effectiveness of any treatment is limited by the maximal efficacy achievable at a tolerable dose.

[0006] Therefore, there is a need for improved drugs having activity against B-cell malignancies.The present invention addresses one or more of the above problems. Summary of the Invention

[0007] The present invention relates to combination therapies for B-cell malignancies.

[0008] The present invention is based on the surprising discovery that proteasome inhibitors (e.g., bortezomib) can be used to synergize with anti-BCMA antibody-drug conjugates (and vice versa), which increases the cytotoxicity of cells of B-cell malignancies following contact with the combination of these agents. Thus, the seminal discovery of the present invention is that proteasome inhibitors can be used to enhance the anti-B-cell malignancy activity of antibody-drug conjugates (and vice versa), more particularly, when the drug (or the antibody-drug conjugate) is a nucleic acid cross-linking agent.

[0009] In one aspect, the present invention provides a B-cell malignancy drug comprising:

[0010] 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 cross-linker; and

[0011] b. Proteasome inhibitors;

[0012] wherein the medicament provides enhanced inhibition of B-cell malignancies when compared to an otherwise identical medicament except for the absence of the proteasome inhibitor; or

[0013] wherein the agent provides enhanced inhibition of B cell malignancies when compared to an otherwise identical agent except for the absence of the ADC.

[0014] In another aspect, provided herein are therapeutic combinations for the treatment of B-cell malignancies, comprising:

[0015] a. an ADC comprising an antibody or antigen-binding fragment thereof that binds to BCMA conjugated to a nucleic acid cross-linker; and

[0016] b. Proteasome inhibitors;

[0017] wherein the therapeutic combination provides enhanced inhibition of B-cell malignancies when compared to an otherwise identical composition except for the absence of the proteasome inhibitor; or

[0018] wherein the therapeutic combination provides enhanced inhibition of B cell malignancies when compared to an otherwise identical composition except for the absence of the ADC.

[0019] In another aspect, provided herein is a method for treating a B-cell malignancy, the method comprising administering to a subject a therapeutic combination comprising:

[0020] a. an ADC comprising an antibody or antigen-binding fragment thereof that binds to BCMA conjugated to a nucleic acid cross-linker; and

[0021] b. Proteasome inhibitors;

[0022] wherein the therapeutic combination provides enhanced inhibition of B-cell malignancies when compared to an otherwise identical composition except for the absence of the proteasome inhibitor; or

[0023] wherein the therapeutic combination provides enhanced inhibition of B cell malignancies when compared to an otherwise identical composition except for the absence of the ADC.

[0024] In another aspect, provided herein is an in vitro method for enhancing ADC inhibition of malignant B cells, the method comprising contacting the 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-linker, in combination with (b) a proteasome inhibitor.

[0025] In another aspect, provided herein is an in vitro method for enhancing inhibition by a proteasome inhibitor, the method comprising contacting a malignant B cell 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-linker. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 M2 was shown to be more cytotoxic to MM cells than its MMAF ADC homologue (M3). A Serial dilutions of M2 or M3 were added to MM cell cultures for 3 days, followed by CCK8 cell viability assay. Shown are the ED values ​​of M2 (black circles) and M3 (open circles). 50 Values ​​are determined from one representative experiment with three replicates, with each dose performed in triplicate. MM1S(R) and H929(R) cells, derived from MM1S and H929, respectively, are resistant to lenalidomide and pomalidomide. RPMI-BCMA, RPMI8226 cells overexpressing BCMA; RPMI, RPMI8226 cells. B M2 (black circles) or M3 (open circles) were added for 3 days, followed by [H 3 ] Cell proliferation assay based on thymidine incorporation (left), and viability assay based on CCK8 in RPMI8226 and luminescence-based cell titer growth (CTG) of paired ANBL6 and ANBL6-BR (bortezomib (btz)-resistant) cells. C Dexamethasone (Dex) and btz-resistant MM cells were treated with M2 or M3 for 2 days, followed by flow cytometry (FCM) analysis to determine the percentage of apoptotic cells (Annexin V+ / Aqua- and Annexin V+ / Aqua+). ***, p < 0.0005; **, p < 0.005.

[0027] Figure 2 Shown that M2 blocks the MM cell viability of BMSC induction more effectively than M3 and has cytotoxicity to primary patient-derived MM cells. A MM1Sluc cells were processed 4d with M2 or M3 alone or together with BMSC, and cell viability was determined by BLI. B CFSE-labeled IMiD-resistant MM1S (R) or H929 (R) cells were incubated 2d with specified drugs with or without BMSC, and then FCM analysis was performed using Annexin V and live / dead Aqua staining. Shown is the percentage of Annexin V- / Aqua- (live) CFSE+ MM cells from one of three experiments, each dose in triplicate. C H929 cells were processed 2d with M2 alone or together with IL-6 (5ng / ml), and the percentage of Annexin V- / Aqua- (live) cells was measured. D CD138+ cells from representative RRMM patients were incubated 3d with M2 or M3, and the live / dead cell fraction was measured. E CD138+ cells from RRMM patients (n=3) were incubated with M2 for 3 days, followed by CTG assay. F BMMCs from MM patients (NDMM=4, RRMM=2) were treated with M2 (10 μg / ml) for 5 days. The percentage of viable CD38 high CD138+ cells was determined by FCM analysis.

[0028] Figure 3 M2 was shown to be more effective than M3 in blocking cell proliferation and inducing apoptosis in MM cell lines, regardless of drug sensitivity. A M1 (isotype-PBD), M2 (anti-BCMA-PBD), M3 (anti-BCMA-MMAF homolog), and M4 (isotype-MMAF) were added to MM cells in triplicate for 3 days, followed by CCK8 activity assay. B M2 or M3 were added for 3 days, followed by [H 3 [Illegible text:]-thymidine incorporation was used for proliferation assays. C After 2 days of treatment with btz, ANBL6 (btz-sensitive) and ANBL6-BR (btz-resistant) cells were confirmed using a CTG-based survival assay (left) and an FCM-based apoptosis assay using Annexin V and Live / Dead Aqua staining (right). D MM1S (upper panel) and MM1R (lower panel) cells were treated with the indicated drugs. The percentage of Annexin V+ MM cells is shown.

[0029] Figure 4M2 was shown to induce specific cytotoxicity against MM cells protected by BMSCs and IL-6, and further depleted CD38 high CD138+ patient MM cells. A Various drug-sensitive and resistant MM cell lines (n=6) were treated with M2 for 3 days, alone or together with BMSCs, followed by CTG assay. B BCMA-negative BMSCs, PBMCs, and NK cells were treated with M2 for 5 days. CH929 cells were treated with M2 for 3 days, alone or together with IL-6 (5ng / ml). D BMMCs from a representative NDMM patient were incubated with M2 for 5 days. M2 reduced CD38 high CD138+ MM cells in a dose-dependent manner.

[0030] Figure 5 M2 was shown to synergistically induce MM cell death with bortezomib. AB MM cells were treated with the specified drugs for 2d, followed by FCM analysis using PI and Annexin V staining. Results for one representative sample of each cell line (A) and a summary of the percentage of Annexin V+ cells from three replicates (B) are shown. *, p < 0.01; **, p < 0.005, ***, p < 0.002, C Data from a CTG-based cell viability assay were used to determine the combination index (CI). "Effect" refers to the degree of reduction in cell viability by M2 and bortezomib. A CI < 1 indicates that the two drugs have a synergistic effect. Similar results were obtained from three additional replicates.

[0031] Figure 6 The combination of low-dose M2 and btz was shown to trigger synergistic MM cell death. The designated MM cell lines were incubated alone or with M2 and btz for 3 days, followed by a CTG-based viability assay. "Effect" represents the fraction of cells showing reduced viability under the combined treatment of M2 plus btz. A combination index (CI) of <1 indicates synergy. All experiments were performed in triplicate, and the average values ​​are shown.

[0032] Figure 7Shown, when compared to the individual drugs alone, the combination of M2 and bortezomib induced more effective in vivo anti-MM activity and prolonged survival in mice. A CB17 SCID mice (n = 7 per group) with palpable MM1S tumor implants were randomized and treated for 2 weeks with control vehicle, a single dose of M2 (0.4 mg / kg), six doses of btz (0.4 mg / kg), or a combination of M2 and btz (M2+btz). Compared to the control group (cnt), tumor growth in the combination treatment group was significantly inhibited. (M2 vs. M2+btz, p = 0.035; btz vs. M2+btz, p < 0.005; cnt vs. M2+btz; p = 0.0012; btz vs. cnt, p < 0.005; M2 vs. cnt, p < 0.005). *, p < 0.04, **, p < 0.005, B tracked the body weight of the animals. C Using Kaplan-Meier and log-rank analysis, the median overall survival of animals treated with the combination therapy was significantly prolonged (cnt, 22 days; M2, 40.5 days; btz, 35 days; M2+btz, 57 days) (M2 vs. M2+btz, p < 0.045; btz vs. M2+btz, p < 0.023; cnt vs. M2+btz, p < 0.002). D Immunohistochemistry analysis of tumor tissue sections from each group for Ki-67 (original magnification, x400).

[0033] Figure 8 It is shown that combined treatment with M2 and btz significantly reduced the in vivo growth of MM1S xenografts, demonstrating the in vivo synergistic effect of M2 and btz in the treatment of myeloma. Tumors were removed from representative mice on the same treatment day.

[0034] Figure 9 Figure 2 shows that M2 significantly induces phosphorylation of the DNA damage response (DDR) signaling pathway in MM cells, regardless of p53 status and drug resistance. MM cells with wild-type (MM1S, MM1R, H929) and mutant p53 (*) were treated with the indicated doses of M2 (ad) for the indicated time periods (a), overnight (b, d), or 2 days (c). Cell lysates were prepared and analyzed by immunoblotting using specific antibodies against the indicated molecules. cPARP, cleaved PARP; cCas3, cleaved caspase 3. The experiment was repeated three times.

[0035] Figure 10M2 was shown to significantly induce the DDR signaling cascade in a BCMA-dependent manner, followed by apoptosis. The indicated MM cells were treated with the indicated doses of M2 (AB, DF) or M3 (A, F, G) overnight (A, D, G) or 2d (B, EF). Cell lysates were prepared and analyzed by immunoblotting using specific antibodies against the indicated molecules. M2, but not M3, induces the DDR signaling pathway. cPARP, cleaved PARP; cCas3, cleaved caspase 3. (C) BCMA levels were measured using qRT-PCR. BCMA 中 , BCMA 低 and BCMA 高 Derived from the parent RPMI8226.

[0036] Figure 11 M2 treatment induced the expression of DDR-related genes, including RAD51. Human DNA repair pathway array analysis of RNA from live H929 MM cells treated with M2 (a). Transcripts were normalized by the geometric mean of the internal control, and the fold change of M2 treatment relative to the control (cnt) group is shown. Cell lysates were prepared from various M2-treated MM cell lines and used for immunoblotting to determine the protein level of RAD51 (bc). DETAILED DESCRIPTION

[0037] In one aspect, the present invention provides a B-cell malignancy drug comprising:

[0038] 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 cross-linker; and

[0039] b. Proteasome inhibitors;

[0040] wherein the medicament provides enhanced inhibition of B-cell malignancies when compared to an otherwise identical medicament except for the absence of the proteasome inhibitor; or

[0041] wherein the agent provides enhanced inhibition of B cell malignancies when compared to an otherwise identical agent except for the absence of the ADC.

[0042] Another aspect provides a therapeutic combination for treating a B-cell malignancy, the therapeutic combination comprising:

[0043] a. an ADC comprising an antibody or antigen-binding fragment thereof that binds to BCMA conjugated to a nucleic acid cross-linker; and

[0044] b. Proteasome inhibitors;

[0045] wherein the therapeutic combination provides enhanced inhibition of B-cell malignancies when compared to an otherwise identical composition except for the absence of the proteasome inhibitor; or

[0046] wherein the therapeutic combination provides enhanced inhibition of B cell malignancies when compared to an otherwise identical composition except for the absence of the ADC.

[0047] In a related aspect, a method for treating a B-cell malignancy is provided, the method comprising administering to a subject a therapeutic combination comprising:

[0048] a. an ADC comprising an antibody or antigen-binding fragment thereof that binds to BCMA conjugated to a nucleic acid cross-linker; and

[0049] b. Proteasome inhibitors;

[0050] wherein the therapeutic combination provides enhanced inhibition of B-cell malignancies when compared to an otherwise identical composition except for the absence of the proteasome inhibitor; or

[0051] wherein the therapeutic combination provides enhanced inhibition of B cell malignancies when compared to an otherwise identical composition except for the absence of the ADC.

[0052] The enhanced inhibition of a B cell malignancy may comprise one or more selected from the group consisting of increased tumor growth delay, increased tumor size reduction, increased tumor metastasis reduction, improved survival of a subject harboring a B cell malignancy, or a combination thereof.

[0053] The term "B cell malignancy" includes any disease in which B cells become cancerous and divide uncontrollably (e.g., in the bone marrow and blood) and can invade other sites (e.g., tissues and lymphatic system). In one embodiment, the B cell malignancy is one or more selected from the following: B cell lymphoma, B cell leukemia, myeloma (e.g., multiple myeloma and / or myeloma progenitor cells), or a combination thereof. The term "B cell" includes both mature (differentiated) B cells and their precursors (e.g., stem cells). For example, it includes myeloma stem cells and myeloma progenitor cells.

[0054] In one embodiment, the B cell malignancy is characterized by comprising malignant B cells that express BCMA. In one embodiment, the malignant B cells express high levels of BCMA antigen (relative to a reference non-malignant B cell). Malignant B cells are considered to express "high levels of BCMA" when the level of BCMA antigen expression in the malignant B cells is increased to a statistically significant level compared to the level of BCMA expression in non-malignant (e.g., healthy) B cells.

[0055] Examples of B cell lymphomas 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 leukemias include B cell chronic lymphocytic leukemia / small lymphocytic lymphoma, acute lymphocytic leukemia, B cell prolymphocytic leukemia, precursor B lymphocytic leukemia, and hairy cell leukemia.

[0056] In one embodiment, the B cell malignancy is a myeloma (eg, multiple myeloma).

[0057] Multiple myeloma (MM), also known as plasma cell myeloma or Kahler's disease, is a cancer of B cells (plasma cells), which are a type of white blood cell that is usually responsible for producing antibodies. Current therapies for MM include chemotherapy, radiotherapy, surgery, biophosphonates, and autologous stem cell transplantation (ASCT). Although these therapies often lead to remission, almost all patients eventually relapse and die. Multiple myeloma affects 1-4 people per 100,000 people each year. The disease is more common in men, and due to unknown reasons, its incidence in African Americans is twice that of Caucasians.

[0058] B cell maturation antigen (BCMA), also known as tumor necrosis factor receptor superfamily member 17 (TNFRSF17), is a member of the tumor necrosis family receptor (TNFR) expressed on cells of the B cell lineage. BCMA expression is highest on terminally differentiated B cells. BCMA is involved in mediating the survival of plasma cells and serves to maintain long-term humoral immunity. BCMA expression is associated with a variety of cancers, autoimmune disorders, and infectious diseases. BCMA RNA has been commonly detected in multiple myeloma cells, and some researchers have detected BCMA protein on the surface of plasma cells from multiple myeloma patients. Therefore, BCMA represents a therapeutic target for B cell malignancies, particularly multiple myeloma.

[0059] The nucleotide sequence of human BCMA (TNFRSF17) is described in Ensembl (see accession number ENSG 00000048462), 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 provided in SEQ ID NO: 13.

[0060] BCMA is also expressed on multiple myeloma stem cells. Therefore, the term "myeloma" includes multiple myeloma "stem" cells and myeloma "progenitor" cells. In addition, the methods and uses of the present invention include the treatment of malignant tumors comprising 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 expression. These cells are uniquely cloned and transplanted into immunodeficient mice, while myeloma plasma cells, defined as CD138+CD19-, are not.

[0061] The term "antibody-drug conjugate" means an antibody (or its antigen-binding fragment), which is attached to a cytotoxic agent (typically a small molecule drug with high systemic toxicity) via a chemical linker. The term is used herein to describe an antibody or its antigen-binding fragment that is conjugated to a nucleic acid cross-linking agent. In one embodiment, ADC can include a nucleic acid cross-linking agent (e.g., a small molecule cytotoxin) that has been chemically modified to contain a linker. This linker can then be used to conjugate the nucleic acid cross-linking agent (cytotoxin) to an antibody or its antigen-binding fragment. After being combined with the target antigen (BCMA) on the cell surface, ADC is internalized and transported to the lysosome, where the nucleic acid cross-linking agent (cytotoxin) is released by proteolysis of a cleavable linker (e.g., by cathepsin B found in the lysosome) or by protein degradation of the antibody (e.g., if attached to the cytotoxin via a non-cleavable linker). The cytotoxin is then transferred out of the lysosome and transferred to the cytosol or nucleus, where it can then be bound to its target according to its mechanism of action.

[0062] In one embodiment, the nucleic acid cross-linking agent is a cytotoxic nucleic acid cross-linking agent.

[0063] Proteasome inhibitors have been found to be useful in a variety of cancer therapies. The inventors were surprised to find that proteasome inhibitors can be used to enhance (e.g., synergistically enhance) the anti-B cell malignancy activity of the ADCs of the present invention, and conversely, the ADCs of the present invention can be used to enhance (e.g., synergistically enhance) the anti-B cell malignancy activity of proteasome inhibitors. Without wishing to be bound by theory, the inventors believe that the activity of proteasome inhibitors can enhance the activity of the ADCs of the present invention by causing downstream (molecular) effects leading to inhibition of one or more molecules, for example, it can generally inhibit the activity of the ADC's nucleic acid crosslinker (e.g., PBD cytotoxin) and may even induce resistance to nucleic acid crosslinkers. This activity may be related to or separate from its "normal" activity as a direct proteasome inhibitor. This theory is supported by the observation that the therapeutic combination of the present invention even shows enhanced activity against malignant B cells that are resistant to proteasome inhibitors (e.g., bortezomib) as monotherapy.

[0064] Rather, without wishing to be bound by theory, the activity of the ADCs of the invention may enhance the activity of the proteasome inhibitor by causing downstream (molecular) effects leading to the inhibition of one or more molecules, e.g., which may generally inhibit the activity of the proteasome inhibitor and may even lead to resistance to the proteasome inhibitor.

[0065] Proteasome inhibitors have several consequences. For example, they can lead to increased levels of biologically active proteins such as IκB, an inhibitor of nuclear factor-κB (a protein involved in cell survival). In addition, misfolded and other discarded proteins can accumulate and trigger the unfolded protein response (UPR), which can lead to endoplasmic reticulum (ER) stress.

[0066] In one embodiment, the proteasome inhibitor is a boronic acid-based proteasome inhibitor (eg, bortezomib).

[0067] In one embodiment, the proteasome inhibitor is one or more selected from the group consisting of bortezomib, carfilzomib, ixazomib, marizomib, oprozomib, delanzomib, or a combination thereof. In one embodiment, the proteasome inhibitor is bortezomib.

[0068] Bortezomib (e.g., Velcade), formerly known as PS-341 (Millennium Pharmaceuticals, Cambridge, MA, USA), acts 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:

[0069]

[0070] Carfilzomib It is a epoxyketone proteasome inhibitor that binds irreversibly to the β5 subunit (PSMB5). Its formula is:

[0071]

[0072] Ishazomi Selectively and reversibly inhibits protein proteasome subunit type 5 (PSMB5). Its formula is:

[0073]

[0074] Marizomib (salinosporin A) inhibits proteasome activity by covalently modifying the active site threonine residue of the 20S proteasome. It irreversibly binds to the three main catalytic sites on the protein proteasome subunits β5, β1, and β2. Its formula is:

[0075]

[0076] Oprazomib (known as ONX 0912) has the following formula:

[0077]

[0078] Delanzomib (known as CEP-18770) has the following formula:

[0079]

[0080] In one embodiment, medicine and / or therapeutic composition is included in pharmaceutical composition.Term " pharmaceutical composition " refers to following preparation, and this preparation is in the form that allows the biological activity of active ingredient to be effective, and does not contain other, the experimenter that composition will be used has unacceptable toxicity component.Such composition can be sterile.Medicine and / or therapeutic composition can comprise pharmaceutically acceptable carrier.Exemplary carrier is normal saline.Suitable pharmaceutical composition can comprise one or more buffers (for example, acetate, phosphate or citrate buffer), surfactant (for example, polysorbate), stabilizer (for example, human albumin), preservative (for example, benzyl alcohol) and the absorption promoter and / or other conventional solubilizing agent or dispersant that enhance bioavailability.

[0081] In one embodiment, the medicament, therapeutic combination or pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable non-toxic sterile carrier, such as physiological saline, a non-toxic buffer, a preservative, and the like. Suitable formulations for use in the methods of treatment disclosed herein are described in Remington's Pharmaceutical Sciences, 22nd edition, edited by Lloyd V. Allen, Jr. (2012). In one embodiment, the medicament, therapeutic combination or pharmaceutical composition of the present invention may be contained in one or more formulations selected from capsules, tablets, aqueous suspensions, solutions, nasal aerosols, or combinations thereof. In one embodiment, the pharmaceutical composition may comprise a buffer (e.g., acetate, phosphate or citrate buffer), a surfactant (e.g., polysorbate), an optional stabilizer agent (e.g., human albumin), and the like.

[0082] An agent with "cytotoxicity" (referred to herein as a "cytotoxin" or "cytotoxic agent") is an agent that inhibits or prevents the function of a cell and / or causes cell destruction (cell death), and / or exerts an anti-proliferative effect. It should be understood that the cytotoxin or cytotoxic agent of an ADC is also referred to in the art as the "payload" of the ADC.

[0083] The term "nucleic acid crosslinker" refers to a molecule that reacts with two nucleotides of a nucleic acid to form a covalent bond between them. This crosslinking can occur within the same strand (intrastrand) or between opposite strands of double-stranded DNA (interstrand). These links (adducts) interfere with cellular metabolism, such as DNA replication and transcription, and typically induce cell death. In one embodiment, the nucleic acid is DNA.

[0084] 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 generally subsequently leads to apoptosis. In one embodiment, the nucleic acid cross-linking agent is a cytotoxic nucleic acid cross-linking agent.

[0085] In one embodiment, the nucleic acid cross-linking agent is one or more selected from the group consisting of: pyrrolobenzodiazepines; (PBD), nitrogen mustard, cisplatin, chloroethylnitrosourea (CENU), psoralen, mitomycin C (MMC) antibiotics, or combinations thereof. The nitrogen mustard can be selected from one or more of the following: cyclophosphamide, nitrogen mustard (chlormethine, mechlorethamine or mustine), uracil mustard, melphalan, chlorambucil, ifosfamide, bendamustine or combinations thereof. In one embodiment, the CENU is carmustine.

[0086] In one embodiment, the nucleic acid cross-linking agent is a pyrrolobenzodiazepine (PBD).

[0087] The term "pyrrolobenzodiazepine ” including pyrrolobenzodiazepines and their functional derivatives.

[0088] PBD is a class of cytotoxic agents that transfer to the nucleus before cross-linking DNA, prevent replication during mitosis, damage DNA by inducing DNA chain breaks (single-strand breaks and / or double-strand breaks), and subsequently cause apoptosis. Some PBDs also have the ability to recognize and bind to specific sequences of DNA. In one embodiment, a PBD comprises the general structure:

[0089]

[0090] PBDs differ in the number, type and position of substituents, in both their aromatic A ring and pyrrolo C ring, and in the degree of saturation of the C ring. In the B ring, at the N10-C11 position, there is an imine (N=C), carbinolamine (NH-CH(OH)), or carbinolamine methyl ether (NH-CH(OMe)), which is the electrophilic center responsible for alkylating DNA. All known natural products have an (S)-configuration at the chiral C11a position, which provides them with a right-handed twist when viewed from the C ring towards the A ring. This feature also gives the PBD a suitable three-dimensional shape to have isohelicity with the minor groove of B-form DNA, thereby fitting tightly at the binding site. PBDs can form adducts in the minor groove, thereby interfering with DNA processing.

[0091] The first PBD antitumor antibiotic, anthramycin, was discovered in 1965. Since then, many naturally occurring PBDs have been reported, and more than 10 synthetic routes have been developed for a variety of analogs. Family members include abinomycin (abbeymycin), chicamycin (chicamycin), DC-81, methyl anthramycin (mazethramycin), new anthramycin (neothramycin) A and B, porothramycin (porothramycin), prothracarcin, sibanomicin (DC-102), siberian mycin and tomamycin. PBDs and ADCs comprising them are also described in WO 2015 / 155345 and WO 2015 / 157592, which are incorporated herein by reference in their entirety.

[0092] In one embodiment, the PBD is PBD 3249, also referred to herein as "SG3249" (described in more detail, for example, in WO 2014 / 057074, which is incorporated herein by reference). PBD 3249 (SG3249) comprises the following structure:

[0093]

[0094] In one embodiment, the PBD is PBD 3315, also referred to herein as "SG3315" (described in more detail, for example, in WO 2015 / 052322, which is incorporated herein by reference). PBD 3315 (SG3315) comprises the following structure:

[0095]

[0096] In one embodiment, the PBD (e.g., described in detail in WO 2017 / 137553, which is incorporated herein by reference) comprises the formula:

[0097]

[0098] in:

[0099] (a)R 10 and R 11 Form a nitrogen-carbon double bond between the nitrogen and carbon atoms to which they are attached; or

[0100] (b)R 10 is OH and R 11 yes:

[0101]

[0102] In another embodiment, the PBD is SG3400, also known as compound 23 (described in detail, for example, in WO2017 / 137553, which is incorporated herein by reference), and has the following structure:

[0103]

[0104] In one embodiment, the PBD is a PBD dimer comprising at least two PBD monomers, for example, the at least two PBD monomers are linked via a flexible propylenedioxy tether at the C8 position of their aromatic A-ring phenols.

[0105] The antibodies or antigen-binding fragments thereof of the present invention can be conjugated to cytotoxins (heterologous agents, such as PBDs) 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 PBD moieties (conjugated to the antibody or antigen-binding fragment thereof) comprise the same structure.

[0106] The nucleic acid cross-linking agent (cytotoxin) of the present invention can be connected (e.g., conjugated) to the antibody or antigen-binding fragment thereof through 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.

[0107] Conventional conjugation strategies for antibodies or their Fabs rely on the random conjugation of payload (cytotoxin) to antibodies or Fabs by lysine or cysteine. In one embodiment, the antibody or its Fab is randomly conjugated to an agent (such as a cytotoxin), for example, by partially reducing the antibody or fragment, subsequently reacting with the desired agent, wherein a linker moiety is attached or not attached. The antibody or Fab can be reduced using DTT or similar reducing agents. The linker moiety can then be attached or unattached to the reduced antibody or fragment in a molar excess in the presence of DMSO. After conjugation, excessive free cysteine ​​can be added to quench unreacted agent. The reaction mixture can then be purified, and buffer exchanged with PBS.

[0108] In one embodiment, the nucleic acid cross-linker (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-linker (e.g., cytotoxin) to the antibody or antigen-binding fragment thereof using reactive amino acid residues at specific positions produces a homogeneous ADC formulation with consistent stoichiometry.

[0109] The site-specific conjugation can be through cysteine, residues or unnatural amino acids. In one embodiment, the nucleic acid cross-linking agent (eg, cytotoxin) is conjugated to the antibody or antigen-binding fragment thereof through at least one cysteine ​​residue.

[0110] In one embodiment, nucleic acid cross-linking agents (eg, cytotoxins) are chemically conjugated to amino acid side chains (eg, at specific Kabat positions in 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 the antibody or antigen-binding fragment thereof through a cysteine ​​at at least one of positions 239, 248, 254, 273, 279, 282, 284, 286, 287, 289, 297, 298, 312, 324, 326, 330, 335, 337, 339, 350, 355, 356, 359, 360, 361, 375, 383, 384, 389, 398, 400, 413, 415, 418, 422, 440, 441, 442, 443, and 446, wherein the numbering corresponds to the EU index as in Kabat. In one embodiment, the specific position is 239, 442, or both, wherein the numbering corresponds to the EU index as in Kabat. In one embodiment, the specific position is position 442, an amino acid (cysteine) insertion between positions 239 and 240, or both, where numbering corresponds to the EU index in Kabat. In one embodiment, the nucleic acid cross-linking agent (cytotoxin) is conjugated to the antibody or its antigen-binding fragment via a thiol-maleimide linkage. In some aspects, the amino acid side chain is a sulfhydryl side chain, such as a sulfhydryl reactive group located at the hinge and heavy and light chains of the antibody or its antigen-binding fragment.

[0111] In one embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:11.

[0112] 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.

[0113] In one embodiment, the antibody or antigen-binding fragment thereof comprises:

[0114] i. HCDR1 comprising the amino acid sequence of SEQ ID NO: 1, or a functional variant thereof;

[0115] ii. a HCDR2 comprising the amino acid sequence of SEQ ID NO: 2, or a functional variant thereof;

[0116] iii. HCDR3 comprising the amino acid sequence of SEQ ID NO: 3, or a functional variant thereof;

[0117] iv. LCDR1 comprising the amino acid sequence of SEQ ID NO: 4, or a functional variant thereof;

[0118] v. LCDR2 comprising the amino acid sequence of SEQ ID NO: 5, or a functional variant thereof; and

[0119] vi. LCDR3 comprising the amino acid sequence of SEQ ID NO: 6, or a functional variant thereof.

[0120] In one embodiment, the antibody or antigen-binding fragment thereof comprises:

[0121] 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

[0122] 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;

[0123] 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.

[0124] In one embodiment, the antibody or antigen-binding fragment thereof comprises:

[0125] i. a variable heavy chain comprising the amino acid sequence of SEQ ID NO: 7, or a functional variant thereof; and

[0126] ii. a variable light chain comprising the amino acid sequence of SEQ ID NO: 8, or a functional variant thereof.

[0127] SEQ ID NO: 7 and SEQ ID NO: 8 are germlined versions of the VH and VL. Alternatively, the antibody or antigen-binding fragment thereof may comprise a non-germlined VH and / or VL (e.g., the VH of SEQ ID NO: 9 and the VL of SEQ ID NO: 10).

[0128] The present invention encompasses antibodies (reference antibodies) (e.g., antibodies or antigen-binding fragments) having the CDR sequences or variable heavy and variable light chain sequences defined herein and functional variants thereof. Functional variants bind to the same target antigen as the reference antibody (e.g., BCMA) and may exhibit the same antigen cross-reactivity (or lack thereof) as the reference antibody. These functional variants may have different affinities for the target antigen when compared to the reference antibody. In one embodiment, the functional variants have substantially the same affinity.

[0129] In one embodiment, when compared to the corresponding reference CDR sequence, the functional variant of the reference antibody shows sequence variation at one or more CDRs. Therefore, the functional antibody variant may comprise a functional variant of the CDR. When the term "functional variant" is used in the context of a CDR sequence, this means that when compared to the corresponding reference CDR sequence, the CDR has a maximum of 2 or a maximum of 1 amino acid difference, and when combined with the remaining 5 CDRs (or variants thereof), the variant antibody is able to bind 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 lacks antigen cross-reactivity) as the reference antibody.

[0130] In one embodiment, the functional variant antibody or antigen-binding fragment thereof comprises:

[0131] light chain CDR1 having a maximum of 2 amino acid differences when compared to the corresponding reference CDR sequence;

[0132] a light chain CDR2 having a maximum of 2 amino acid differences when compared to the corresponding reference CDR sequence;

[0133] a light chain CDR3 having a maximum of 2 amino acid differences when compared to the corresponding reference CDR sequence;

[0134] a heavy chain CDR1 having at most 2 amino acid differences when compared to the corresponding reference CDR sequence;

[0135] a heavy chain CDR2 having at most 2 amino acid differences when compared to the corresponding reference CDR sequence; and

[0136] a heavy chain CDR3 having a maximum of 2 amino acid differences when compared to the corresponding reference CDR sequence;

[0137] Wherein 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 of antigen cross-reactivity) as the reference antibody.

[0138] In one embodiment, the functional variant antibody or antigen-binding fragment thereof comprises:

[0139] light chain CDR1 having at most 1 amino acid difference when compared to the corresponding reference CDR sequence;

[0140] a light chain CDR2 having at most 1 amino acid difference when compared to the corresponding reference CDR sequence;

[0141] a light chain CDR3 having at most 1 amino acid difference when compared to the corresponding reference CDR sequence;

[0142] a heavy chain CDR1 having at most 1 amino acid difference when compared to the corresponding reference CDR sequence;

[0143] a heavy chain CDR2 that has at most 1 amino acid difference when compared to the corresponding reference CDR sequence; and

[0144] a heavy chain CDR3 having at most 1 amino acid difference when compared to the corresponding reference CDR sequence;

[0145] Wherein 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 of antigen cross-reactivity) as the reference antibody.

[0146] For example, a functional variant of the antibody or antigen-binding fragment may comprise:

[0147] a heavy chain CDR1 having a maximum of 2 amino acid differences when compared to SEQ ID NO: 1;

[0148] a heavy chain CDR2 having at most 2 amino acid differences when compared to SEQ ID NO: 2; and

[0149] a heavy chain CDR3 having a maximum of 2 amino acid differences when compared to SEQ ID NO: 3;

[0150] a light chain CDR1 having up to 2 amino acid differences when compared to SEQ ID NO: 4;

[0151] a light chain CDR2 having up to 2 amino acid differences when compared to SEQ ID NO: 5;

[0152] a light chain CDR3 having up to 2 amino acid differences when compared to SEQ ID NO: 6;

[0153] wherein the variant antibody binds to BCMA (e.g., a BCMA polypeptide epitope), and / or wherein the variant antibody may exhibit the same antigenic cross-reactivity (or lack of antigenic cross-reactivity) as a reference antibody or antigen-binding fragment.

[0154] In one embodiment, the functional variant of the antibody or antigen-binding fragment may comprise:

[0155] a heavy chain CDR1 having at most 1 amino acid difference when compared to SEQ ID NO: 1;

[0156] a heavy chain CDR2 having at most 1 amino acid difference when compared to SEQ ID NO: 2; and

[0157] a heavy chain CDR3 having at most 1 amino acid difference when compared to SEQ ID NO: 3;

[0158] a light chain CDR1 having at most 1 amino acid difference when compared to SEQ ID NO: 4;

[0159] a light chain CDR2 having at most 1 amino acid difference when compared to SEQ ID NO: 5;

[0160] a light chain CDR3 having at most 1 amino acid difference when compared to SEQ ID NO: 6;

[0161] wherein the variant antibody binds to BCMA (e.g., a BCMA polypeptide epitope), and / or wherein the variant antibody may exhibit the same antigenic cross-reactivity (or lack of antigenic cross-reactivity) as a reference antibody or antigen-binding fragment.

[0162] The foregoing can be similarly applied to variants of the other antibodies described herein, wherein the amino acid differences are defined relative to their CDR sequences, and wherein the variant antibodies bind to the same target antigen as the antibody, and / or wherein the variant antibodies can exhibit the same antigen cross-reactivity (or lack of antigen cross-reactivity).

[0163] In one embodiment, a functional variant antibody may have a total of up to 5, 4 or 3 amino acid differences in its CDRs when compared to a corresponding reference antibody, provided that there are at most 2 (e.g., at most 1) amino acid differences per CDR. In one embodiment, a functional variant antibody may have a total of up to 2 (e.g., at most 1) amino acid differences in its CDRs when compared to a corresponding reference antibody, provided that there are at most 2 amino acid differences per CDR. In one embodiment, a functional variant antibody may have a total of up to 2 (e.g., at most 1) amino acid differences in its CDRs when compared to a corresponding reference antibody, provided that there are at most 1 amino acid difference per CDR.

[0164] The amino acid difference can be an amino acid substitution, insertion or deletion. In one embodiment, the amino acid difference is a conservative amino acid substitution as described herein.

[0165] 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 comprise a framework region having at most 2, or at most 1, amino acid differences (when compared to the corresponding reference framework sequence). Thus, each framework region may have at most 2, or at most 1, amino acid differences (when compared to the corresponding reference framework sequence).

[0166] In one embodiment, the functional variant antibody may have a total of up to 5, 4 or 3 amino acid differences in its framework regions when compared to the corresponding reference antibody, provided that there are at most 2 (e.g., at most 1) amino acid differences per framework region. In one embodiment, the functional variant antibody has a total of up to 2 (e.g., at most 1) amino acid differences in its framework regions when compared to the corresponding reference antibody, provided that there are at most 2 amino acid differences per framework region. In one embodiment, the functional variant antibody has a total of up to 2 (e.g., at most 1) amino acid differences in its framework regions when compared to the corresponding reference antibody, provided that there are at most 1 amino acid difference per framework region.

[0167] Thus, a functional variant antibody may comprise a variable heavy chain and a variable light chain as described herein, wherein:

[0168] The heavy chain has a maximum of 14 amino acid differences (a maximum of 2 amino acid differences in each CDR and a maximum of 2 amino acid differences in each framework region) when compared to the heavy chain sequence described herein (e.g., SEQ ID NO.: 7); and

[0169] When compared to the light chain sequence described herein (e.g., SEQ ID NO.: 8), the light chain has a maximum of 14 amino acid differences (a maximum of 2 amino acid differences in each CDR and a maximum of 2 amino acid differences in each framework region);

[0170] wherein the functional variant antibody binds to the same target antigen as the reference antibody, and / or wherein the functional variant antibody exhibits the same antigen cross-reactivity (or lack of antigen cross-reactivity) as the reference antibody.

[0171] Such variant heavy or light chains can be referred to as "functional equivalents" of the reference heavy or light chain.

[0172] In one embodiment, a functional variant antibody may comprise a variable heavy chain and a variable light chain as described herein, wherein:

[0173] The heavy chain has a maximum of 7 amino acid differences (a maximum of 1 amino acid difference in each CDR and a maximum of 1 amino acid difference in each framework region) when compared to the heavy chain sequence herein (e.g., SEQ ID NO.: 7); and

[0174] When compared to the light chain sequence herein (e.g., SEQ ID NO.: 8), the light chain has a maximum of 7 amino acid differences (a maximum of 1 amino acid difference in each CDR and a maximum of 1 amino acid difference in each framework region);

[0175] wherein the functional variant antibody binds to the same target antigen as the reference antibody, and / or wherein the functional variant antibody exhibits the same antigen cross-reactivity (or lack of antigen cross-reactivity) as the reference antibody.

[0176] 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 between about 0.1 nM to about 40 nM, between about 0.5 nM to about 30 nM, between 1 nM to about 20 nM, or between about 1.5 nM to about 20 nM. In one embodiment, the antibody or antigen-binding fragment thereof binds to BCMA (e.g., human BCMA) with a KD of between about 23 nM to about 27 nM. In conjunction with BCMA (for example, human BMCA). In one embodiment, the antibody or its Fab binds to BCMA (for example, human BCMA) with a KD between about 1nM and about 1.5nM. KD measurement (binding affinity) can be carried out by any suitable assay known in the art. Such methods include, for example, fluorescence activated cell sorting (FACS), surface plasmon resonance (for example, Biacore, ProteOn), biofilm interferometry (BLI, such as Octet), kinetic exclusion assay (for example, KinExA), separable beads (for example, magnetic beads), antigen panning, ELISA, and / or ForteBio Octet system. Suitable kinetic exclusion assays include KinExA systems (for example, KinExA 3100, KinExA 3200 or KinExA 4000) (Sapidyne Instruments, Idaho).

[0177] Advantageously, this therapeutic combination provides for the purposeful use of such proteasome inhibitors to target cells that would normally be unresponsive (e.g., resistant) to such proteasome inhibitors. For example, the present inventors have demonstrated that proteasome inhibitors (e.g., bortezomib) can enhance the anti-B cell malignancy activity of ADCs even when the B cell malignancies are resistant to proteasome inhibitors (see Example 3).

[0178] Thus, the present invention encompasses administering an ADC in combination with a proteasome inhibitor (which otherwise provides only low / poor inhibition of B-cell malignancies) at a dosage of the proteasome inhibitor such that, following said addition, the combination is now able to demonstrate improved inhibition of B-cell malignancies. Thus, the present invention provides for the "repurposing" of proteasome inhibitors (e.g., bortezomib) as potentiators for ADCs, rather than using them as (stand-alone) monotherapy (which would otherwise be ineffective against drug-resistant malignancies).

[0179] Similarly, the present invention encompasses administering a proteasome inhibitor in combination with an ADC (which otherwise provides only low / poor inhibition of B-cell malignancies) at doses of the ADC such that, following said addition, the combination is now able to demonstrate improved inhibition of B-cell malignancies. Thus, the present invention provides for the "repurposing" of ADCs as potentiators of proteasome inhibitors, rather than using (stand-alone) monotherapy (which would otherwise be ineffective against B-cell malignancies that express low levels of the BCMA antigen).

[0180] Thus, in one embodiment, the B cell malignancy is resistant to a proteasome inhibitor (e.g., a drug comprising a proteasome inhibitor in the absence of an ADC of the invention). In one embodiment, the proteasome inhibitor is bortezomib.

[0181] In one embodiment, the B cell malignancy is resistant to an ADC of the invention (e.g., a medicament comprising an ADC in the absence of a proteasome inhibitor of the invention). In one embodiment, the B cell malignancy resistant to an ADC of the invention is characterized by comprising malignant B cells that do not have increased or decreased expression levels of the BCMA antigen relative to a reference non-malignant B cell.

[0182] The therapeutic combination has also been shown to have enhanced activity against B-cell malignancies that are resistant to many other common anticancer drugs (see Example 3). Thus, in one embodiment, the B-cell malignancy is resistant to one or more drugs selected from the group consisting of dexamethasone, lenalidomide, pomalidomide, bortezomib, or a combination thereof.

[0183] Furthermore, due to the synergistic nature of the combination, lower doses of the components can be used, thereby reducing the risk of developing resistance to any one component due to overuse (a fundamental public health threat). In fact, the present invention reduces the need for prescription of chronic treatment regimens. For example, the present inventors have shown that the in vivo efficacy of suboptimal doses of ADCs is still enhanced when administered in combination with a proteasome inhibitor (e.g., bortezomib).

[0184] Thus, in one embodiment, the ADC and / or proteasome inhibitor is administered at a suboptimal dose.

[0185] The order of application / administration of the components of the therapeutic combination can vary. The ADC and the proteasome inhibitor can be administered simultaneously as part of a single composition or in separate compositions (e.g., both at their own specific optimal doses for achieving a synergistic effect). For example, the ADC can be present in a first composition (e.g., suitable for intravenous administration to a subject) and the proteasome inhibitor can be present in a second composition (e.g., suitable for intravenous, subcutaneous, or oral administration to a subject). For example, when the proteasome inhibitor is bortezomib, the second composition can be suitable for intravenous or subcutaneous injection. In embodiments where the proteasome inhibitor is ixazomib, the second composition can additionally or alternatively be suitable for oral administration.

[0186] In addition, the ADC and the proteasome inhibitor can be administered at different times (e.g., the proteasome inhibitor can be pre-administered to sensitize the malignant B cells to the ADC). Thus, in another embodiment, the ADC and the proteasome inhibitor are administered to the subject at different times in separate compositions.

[0187] 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 after the ADC.

[0188] As used herein, the terms "treat" or "treating" include preventative treatment (e.g., preventing the onset of a B-cell malignancy), as well as corrective treatment (treating a subject who already has a B-cell malignancy). In one embodiment, the terms "treat" or "treating" as used herein refer to corrective treatment. The terms "treat" or "treating" include treating both a B-cell malignancy and its symptoms. In some embodiments, "treat" or "treating" refers to a symptom of a B-cell malignancy.

[0189] Thus, a therapeutically effective amount or a prophylactically effective amount of the drug and / or therapeutic combination may be administered to a subject.

[0190] A "therapeutically effective amount" is any amount of a drug and / or therapeutic combination that, when administered alone or in combination to a subject to treat a B-cell malignancy (or a symptom thereof), is sufficient to effect such treatment of the B-cell malignancy, or a symptom thereof.

[0191] A "prophylactically effective amount" is any amount of a drug and / or therapeutic combination that, when administered alone or in combination to a subject, inhibits or delays the onset or recurrence of a B-cell malignancy (or its symptoms). In some embodiments, a prophylactically effective amount completely prevents the onset or recurrence of a B-cell malignancy. "Inhibiting" the onset means reducing the likelihood of an onset of a B-cell malignancy (or its symptoms), or completely preventing an onset.

[0192] Additional aspects of the invention provide an in vitro method for enhancing ADC inhibition of malignant B cells, the method comprising contacting the 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-linker, in combination with (b) a proteasome inhibitor.

[0193] In another aspect, an in vitro method for enhancing inhibition by a proteasome inhibitor is provided, the method comprising contacting a malignant B cell 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-linker.

[0194] The terms "suppresses" or "suppressing" in this context or in any medicament, method, or use described herein include "inhibiting the growth of," "inhibiting the proliferation of," or "killing" malignant B cells. Reference to "malignant B cells" includes reference to "tumors comprising malignant B cells."

[0195] The terms "inhibits" or "inhibiting" are synonymous with the terms "slowing the growth of" or "stopping the proliferation of" malignant B cells, or "slowing the growth of" a tumor comprising malignant B cells. In one embodiment, the therapeutic combination of the present invention can "kill" or "be used to kill" a malignant B cell, or a tumor comprising a malignant B cell. The term "inhibits" also includes preventing the growth (e.g., proliferation) of a malignant B cell, or a tumor comprising a malignant B cell.

[0196] In one embodiment, the enhanced inhibition of B cell malignancies may comprise one or more selected from the group consisting of: increased tumor growth delay, increased tumor size reduction, increased tumor metastasis reduction, improved survival of subjects harboring B cell malignancies, or a combination thereof. In one embodiment, the enhanced inhibition of B cell malignancies may comprise one or more selected from the group consisting of: increased tumor growth delay, increased tumor size reduction, or a combination thereof.

[0197] In one embodiment, the medicament and / or therapeutic combination of the present invention enhances inhibition of 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% compared to an otherwise identical medicament and / or composition except for the lack of a proteasome inhibitor. In one embodiment, the medicament and / or therapeutic combination of the present invention enhances inhibition of 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% compared to an otherwise identical medicament and / or composition except for the lack of an ADC of the present invention.

[0198] Inhibition can be measured by measuring cell proliferation, which can be assayed using art-recognized techniques that measure the rate of cell division, and / or the fraction of cells in a cell population that undergo cell division, and / or the rate of cell loss from a cell population due to terminal differentiation or cell death (e.g., thymidine incorporation).

[0199] The assessment of "enhanced inhibition of B-cell malignancies" is demonstrated by reference to the accompanying examples and can be assessed using the methods described in the examples (e.g., Example 3). For example, Example 3 describes a method comprising an Annexin V / PI-based FMC assay that measures the "% apoptotic cells observed" value in an in vitro culture of malignant B cells after contact with a test sample. This allows for direct comparison of B-cell malignancy inhibition between the agents of the present invention and an otherwise identical agent except for the lack of a proteasome inhibitor or ADC.

[0200] In one embodiment, inhibition of B-cell malignancies is considered enhanced when the "% apoptotic cells observed" value obtained for the combination of two primarily active compounds (e.g. an ADC of the invention, and a proteasome inhibitor) is greater than the "% apoptotic cells observed" value obtained in the absence of the ADC of the invention, or the proteasome inhibitor, suitably a proteasome inhibitor, but under otherwise identical conditions.

[0201] Thus, in one embodiment, enhanced inhibition of B-cell malignancies can be determined by comparing the "% apoptotic cells observed" value obtained for a combination of an ADC of the invention and a protease inhibitor to the "% apoptotic cells observed" value obtained for the same formulation (e.g., drug) in the absence of the ADC of the invention or the proteasome inhibitor (suitably in the absence of the proteasome inhibitor) under the same conditions.

[0202] In one embodiment, the present invention provides an enhancement of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% (observed % apoptotic cells) against B cell malignancies compared to the observed % apoptotic cells provided by the same formulation (e.g., drug) under the same conditions in the absence of an ADC or a proteasome inhibitor of the invention (suitably lacking a proteasome inhibitor). In one embodiment, the present invention provides an enhancement of at least about 65% (observed % apoptotic cells) against B cell malignancies compared to the observed % apoptotic cells provided by the same formulation (e.g., drug) under the same conditions in the absence of an ADC or a proteasome inhibitor of the invention (suitably lacking a proteasome inhibitor).

[0203] In one embodiment, the combination of an ADC of the present invention and a proteasome inhibitor can exhibit synergistic inhibition of B-cell malignancies.

[0204] As used herein, the term "synergistic" means that the inhibition of B-cell malignancies exhibited is greater than the sum of its parts. In other words, the inhibition of B-cell malignancies is greater than simple addition.

[0205] Synergy can be measured by determining a "combination index" (CI) using an analytical tool (e.g., CompuSyn (ComboSyn, Inc.)), where a CI < 1 indicates synergism between the combination of the main active ingredients of the invention, a CI > 1 indicates antagonism, and a CI of 1 indicates additive effects. The CI can be measured by comparing the performance of a composition comprising a drug / therapeutic combination of the invention with a composition that is otherwise identical except for the absence of an ADC or proteasome inhibitor of the invention (suitably the absence of a proteasome inhibitor) in any cell viability assay known in the art (e.g., alternative or additional methods relative to those described in Examples 1-3, such as the "CellTiter-Glo based cell viability" assay described in Example 3).

[0206] With reference to the examples (e.g., Example 3), synergistic inhibition of B cell malignancies can be considered to be present when the CI is less than about 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, or 0.05. In one embodiment, the CI can be less than about 0.7. In one embodiment, the CI can be less than about 0.6.

[0207] In one embodiment, a "cell viability assay" comprises: incubating a test sample comprising malignant B cells in the presence of an amount of a composition comprising a therapeutic combination (an ADC of the invention and a proteasome inhibitor); and comparing the number of non-viable cells (e.g., apoptotic cells) in the test sample after incubation (e.g., after at least 0.5, 1, 1.5, or 2 days of incubation) to the number of non-viable cells in a control sample incubated in the presence of an otherwise identical composition except for the absence of: (a) the proteasome inhibitor, or (b) the ADC (suitably lacking the proteasome inhibitor).

[0208] 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, cat, and / or rabbit), livestock (e.g., pigs, sheep, cattle, and / or goats), and / or a horse. In one embodiment, the subject is a human.

[0209] In the methods of the present invention, the subject may not have been previously diagnosed with a B-cell malignancy. Alternatively, the subject may have been previously diagnosed with a B-cell malignancy. The subject may also be a person who exhibits risk factors for the disease, or a person who is asymptomatic for a B-cell malignancy. The subject may also be a person who has a B-cell malignancy, or a person who is at risk of developing a B-cell malignancy. In one embodiment, the subject has previously been administered a therapy for a B-cell malignancy.

[0210] 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 a combination thereof. In one embodiment, administration is selected from one or more of the following: intravenous, intraarterial (e.g., by injection or infusion), subcutaneous, or a combination thereof.

[0211] Antibody preparation

[0212] The antibodies of the present invention can be obtained using conventional techniques known to those skilled in the art, and their utility can be demonstrated by conventional binding studies - exemplary methods are described in Example 2. For example, a simple binding assay is to incubate cells expressing an antigen with the antibody. If the antibody is labeled with a fluorophore, binding of the antibody to the antigen can be detected by FACS analysis.

[0213] Methods for producing BCMA antibodies and antibody fragments thereof of the present invention are described in WO 2010 / 104949 and WO 2019 / 025983 (particularly WO 2019 / 025983), both of which are incorporated herein by reference.

[0214] The antibodies of the present invention can be produced in a variety of animals, including mice, rats, rabbits, goats, sheep, monkeys, or horses. Antibodies can be produced following immunization with a single involucral polysaccharide or with multiple involucral polysaccharides. Blood isolated from these animals contains polyclonal antibodies—multiple antibodies that bind to the same antigen. Antigens can also be injected into chickens to produce polyclonal antibodies in egg yolk. To obtain monoclonal antibodies specific for a single epitope of an antigen, antibody-secreting lymphocytes are isolated from the animal and immortalized by fusing them with a cancer cell line. The fused cells are called hybridomas and continue to grow and secrete antibodies in culture. Individual 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 producing 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 typically purified using protein A / G or antigen affinity chromatography.

[0215] The antibodies or antigen-binding fragments thereof of the present invention can be prepared as monoclonal antibodies, which can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature 256:495 (1975). Using the hybridoma method, mice, hamsters, or other appropriate host animals are immunized as described above to elicit lymphocytes that produce antibodies that will specifically bind to the immunizing antigen. Lymphocytes can also be immunized in vitro. Following immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line using, for example, polyethylene glycol to form hybridoma cells that can then be selected from the unfused lymphocytes and myeloma cells. Hybridomas that produce monoclonal antibodies specific for the selected antigen (as determined by immunoprecipitation, immunoblotting, or by in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)) can then be propagated in vitro using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as ascites tumors in animals. Monoclonal antibodies can then be purified from the culture medium or ascites using known methods.

[0216] Alternatively, the antibody or its antigen-binding fragment (for example, monoclonal antibody) can also be prepared using the recombinant DNA method as described in U.S. Patent number 4,816,567. From mature B cells or hybridoma cells, for example, by using oligonucleotide primers (the gene of the heavy chain and light chain of its specific amplification encoding antibody) RT-PCR separation polynucleotide encoding monoclonal antibody, and use conventional procedures to measure their sequence.Then the polynucleotide encoding the separation of heavy chain and light chain is cloned into a suitable expression vector, and these expression vectors, when being transfected into a host cell (such as Escherichia coli cells, ape COS cells, Chinese hamster ovary (CHO) cells or myeloma cells) that does not produce immunoglobulin, are produced by these host cells. In addition, 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).

[0217] One or more polynucleotides encoding the antibodies or antigen-binding fragments thereof of the present invention can be further modified in a variety of ways using recombinant DNA technology to produce alternative antibodies. In some embodiments, for example, the constant domains of the light and heavy chains of a mouse monoclonal antibody can be replaced by (1) those of a human antibody, for example, to produce a chimeric antibody, or by (2) a non-immunoglobulin polypeptide, for example, to produce a fusion antibody. In some embodiments, these constant regions are truncated or removed to produce antibody fragments of the desired monoclonal antibody. Site-directed mutagenesis or high-density mutagenesis of the variable region can be used to optimize the specificity, affinity, etc. of the monoclonal antibody.

[0218] In one embodiment, the antibody or antigen-binding fragment thereof is a human antibody or antigen-binding fragment thereof. Human antibodies can be directly prepared using various techniques known in the art. Immortalized human B lymphocytes immunized in vitro or isolated from an immune individual that produces antibodies against a target antigen can be produced. 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); U.S. Pat. No. 5,750,373.

[0219] In one embodiment, the antibody or antigen-binding fragment thereof can be selected from a phage library, wherein the phage library expresses human antibodies as described in, for example, 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 producing and using antibody phage libraries are also described in U.S. Pat. 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.

[0220] 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), which is incorporated by reference in its entirety.

[0221] In one embodiment, the antibody or its antigen-binding fragment (e.g., monoclonal antibody) can be a humanized antibody. Methods for engineering, humanizing or resurfacing non-human antibodies or human antibodies can also be used and are well known in the art. Humanized, resurfacing or similarly engineered antibodies can have one or more amino acid residues from non-human sources, such as, but not limited to, mice, rats, rabbits, non-human primates or other mammals. These non-human amino acid residues are replaced by residues commonly referred to as "input" residues, which are typically taken from the "input" variable domains, constant domains or other domains of known human sequences. Such input sequences can be used to reduce immunogenicity or reduce, enhance or change binding, affinity, association rate, dissociation rate, avidity, specificity, half-life or any other suitable features as known in the art. Suitably, CDR residues can directly and most substantially participate in affecting antigen (e.g., BCMA) binding. Therefore, some or all of the non-human or human CDR sequences can be maintained, while the non-human sequences of the variable and constant regions can be replaced by human amino acids or other amino acids.

[0222] The antibody can also be optionally humanized, resurfaced, engineered or human antibody engineered, wherein high affinity for antigens (e.g., BCMA) and other favorable biological properties are retained. To achieve this goal, humanized (or human) or engineered antibodies and resurfaced antibodies can be optionally prepared by analyzing parental sequences and various conceptual humanized and engineered products using three-dimensional models of parental, engineered and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those of ordinary skill in the art. Computer programs that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences are available. Examining these displays allows analysis of the possible effects of residues in the function of candidate immunoglobulin sequences, i.e., analyzing the residues that affect the ability of candidate immunoglobulins to bind to their antigens (e.g., BCMA). In this way, FW residues can be selected and combined from the consensus sequence and the input sequence so that the desired antibody characteristics (e.g., increasing affinity to one or more target antigens) are achieved.

[0223] Humanization, resurfacing or engineering of the antibodies of the present invention or antigen-binding fragments thereof can be performed using any known method, such as, but not limited to, those described in 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 239:1534 (1988); Proceedings of the National Academy of Sciences of the United States of America 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 No. PCT / US 98 / 16280, PCT / US96 / 18978, PCT / US91 / 09630, PCT / US91 / 05939, PCT / US94 / 01234, PCT / GB89 / 01334, PCT / GB91 / 01134, PCT / GB92 / 01755; International Patent Application Publication Nos. WO90 / 14443, WO90 / 14424, WO90 / 14430; and European Patent Publication No. EP229246; each of which is incorporated herein by reference in its entirety, including the references cited therein.

[0224] Antibodies or antigen-binding fragments thereof can also be made in transgenic mice containing human immunoglobulin loci that, upon immunization, are capable of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. 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.

[0225] In one embodiment, fragments of the antibodies of the present invention (e.g., antibody fragments) are provided. Various techniques for producing 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., an "intact" or "parent" antibody) binds. Thus, reference to an "antigen-binding fragment thereof" refers to an antigen-binding fragment that binds to BCMA (e.g., a BCMA-antigen-binding fragment of an antibody).

[0226] Traditionally, these fragments are derived by proteolysis 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, anti-BCMA antibody fragments are produced recombinantly. All Fab, Fv, and scFv antibody fragments can be expressed in and secreted from E. coli or other host cells, thereby allowing the production of large quantities of these fragments. Such anti-BCMA antibody fragments can also be isolated from the antibody phage libraries discussed above. These anti-BCMA antibody fragments can also be linear antibodies as described in U.S. Patent No. 5,641,870. Other techniques for producing antibody fragments will be clear to the skilled practitioner.

[0227] As provided herein, the modified antibodies or antigen-binding fragments thereof may comprise any type of variable region that provides for the association of the antibody or polypeptide with BCMA. In this regard, the variable region may be composed of or derived from any type of mammal that can induce an increased humoral response and generate immunoglobulins against the desired antigen. For this reason, the variable region of the anti-BCMA antibody or its antigen-binding fragment may be, for example, human, mouse, non-human primate (e.g., cynomolgus monkey, macaque, etc.) or wolf. In one embodiment, both the variable region and the constant region of the modified antibody or its antigen-binding fragment are human. In one embodiment, the variable region of a compatible antibody (usually derived from a non-human source) may be engineered or specially tailored to improve binding properties or reduce the immunogenicity of the molecule. In this regard, the variable region useful in the present invention may be humanized or otherwise altered by incorporating an imported amino acid sequence.

[0228] In one embodiment, the variable domains in the heavy chain and light chain of antibody or its Fab are changed by at least partially replacing one or more CDRs and / or by replacing and sequence alteration by part of the framework region. Although CDR can be derived from the same category or even subclass of the antibody derived from the framework region, it is envisioned that CDR will be derived from the antibody of different classes and in certain embodiments derived from the antibody of different species. It is not necessary to replace all CDRs with the complete CDR from the donor variable region so that the antigen binding ability of a variable domain is transferred to another. Instead, it is only necessary to transfer those residues necessary for the activity of the antigen binding site. In view of the explanation illustrated in U.S. Patent number 5,585,089, 5,693,761 and 5,693,762, those skilled in the art are fully capable of obtaining the immunogenic functional antibody with reduction by carrying out routine experiments.

[0229] Despite the changes to the variable region, it will be understood by those skilled in the art that the modified antibodies or antigen-binding fragments thereof of the present invention will comprise antibodies (e.g., full-length antibodies or antigen-binding fragments thereof) in which at least a portion of one or more constant region domains has been deleted or otherwise altered to provide desired biochemical characteristics, such as increased tumor localization or decreased serum half-life when compared to an antibody having approximately the same immunogenicity comprising a native or unaltered constant region. In one embodiment, the constant region of the modified antibody will comprise a human constant region. Modifications to the constant region compatible with the present invention include additions, deletions, or substitutions of one or more amino acids in one or more domains. That is, the modified antibodies disclosed herein may comprise changes or modifications to one or more of the three heavy chain constant domains (CH1, CH2, or CH3) and / or to 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 will comprise a domain-deleted construct or variant in which the entire CH2 domain is removed (ΔCH2 construct). In one embodiment, the omitted constant region domain may be replaced by a short amino acid spacer (e.g., 10 residues) that provides some molecular flexibility normally conferred by the absent constant region.

[0230] In addition to deleting the entire constant region domain, the antibodies or antigen-binding fragments thereof provided herein can be modified by partial deletion or substitution of several or even single amino acids in the constant region. For example, the mutation of a single amino acid in a selected region in the CH2 domain can be sufficient to substantially reduce Fc binding, and thereby increase tumor localization. Similarly, one or more constant region domains that control effector functions (e.g., complement C1Q binding) can be completely or partially deleted. This type of partial deletion of the constant region can improve the selected features (e.g., serum half-life) of the antibody or its antigen-binding fragment, while keeping other desired functions related to the subject's constant region domain intact. In addition, the constant region of the antibody and its antigen-binding fragment can be modified by mutation or substitution of one or more amino acids that enhance the resulting construct properties. In this respect, the activity (e.g., Fc binding) provided by the conserved binding site can be interfered with while substantially maintaining the configuration and immunogenicity of the modified antibody or its antigen-binding fragment. In one embodiment, one or more amino acids can be added to the constant region to enhance desired features, such as reducing or increasing effector functions, or providing more cytotoxins or carbohydrate attachments. In one embodiment, it may be desirable to insert or replicate specific sequences derived from selected constant region domains.

[0231] The present invention further includes variants and equivalents that are substantially homologous to the antibodies or antigen-binding fragments of the present invention (e.g., murine, chimeric, humanized or human antibodies or antigen-binding fragments thereof). These can contain, for example, conservative substitution mutations, i.e., substitutions of one or more amino acids by similar amino acids. For example, conservative substitutions refer to replacing one amino acid with another within the same general class, such as, for example, replacing an acidic amino acid with another acidic amino acid, replacing a basic amino acid with another basic amino acid, or replacing a neutral amino acid with another neutral amino acid. Conservative amino acid substitutions are well known in the art.

[0232] In one embodiment, the antibody or antigen-binding fragment thereof can be further modified to contain additional chemical moieties that are not normally part of the protein. Those derived moieties can improve the solubility, biological half-life, or absorption of the protein. These moieties can also reduce or eliminate any desired side effects of the protein, etc. A review of those moieties can be found in Remington's Pharmaceutical Sciences, 22nd edition, Lloyd V. Allen, Jr. ed. (2012).

[0233] Sequence homology

[0234] Percent identity can be determined using any of a variety of sequence alignment methods, including but not limited to global methods, local methods, and hybrid methods such as, for example, segment methods. Protocols for determining percent identity are routine procedures within the skill of the art. Global methods align sequences from the beginning to the end of the molecule and determine the optimal alignment by adding the scores for individual residue pairs and applying gap penalties. Non-limiting methods include, for example, CLUSTAL W, see, for example, 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, see, for example, Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments [The accuracy of multiple protein sequence alignments is significantly improved by iterative refinement evaluated using reference structure alignments], 264(4) J. MoI. Biol. [Journal of Molecular Biology] 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-MA New Algorithm for Multiple Alignment of Highly Divergent Sequences [Align-M - a new algorithm for multiple alignment of highly divergent sequences], 20(9) Bioinformatics [Bioinformatics]: 1428-1435 (2004).

[0235] Thus, 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 using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum62" scoring matrix of Henikoff and Henikoff (described above) as shown below to optimize the alignment score (amino acids are represented by the standard single-letter code).

[0236] "Percentage 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, % identity can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids, multiplied by 100. The calculation of % sequence identity can also take into account the number of rooms, and the length of each room that needs to be introduced to optimize the comparison of two or more sequences. Sequence comparisons and percent identity determinations between two or more sequences can be performed using a specific mathematical algorithm familiar to the skilled person (such as BLAST).

[0237] Substantially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions, or additions. These changes are preferably minor in nature, 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 1 to about 30 amino acids; and small amino or carboxyl terminal extensions, such as an amino terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.

[0238] Conservative amino acid substitutions

[0239] Alkaline: Arginine

[0240] Lysine

[0241] Histidine

[0242] Acidic: Glutamic acid

[0243] Aspartic acid

[0244] Polarity: Glutamine

[0245] Asparagine

[0246] Hydrophobicity: Leucine

[0247] Isoleucine

[0248] Valine

[0249] Aromatic: Phenylalanine

[0250] Tryptophan

[0251] Tyrosine

[0252] Small: Glycine

[0253] Alanine

[0254] Serine

[0255] Threonine

[0256] Methionine

[0257] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) can replace the 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 unnatural amino acids can replace polypeptide amino acid residues. The polypeptides of the present invention may also contain non-naturally occurring amino acid residues.

[0258] A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids can be substituted for amino acid residues in the polypeptides of the invention.

[0259] Essential amino acids in the polypeptides of the invention can be identified by methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, Science 244:1081-5, 1989). Biological interaction sites can also be determined by physical analysis of the structure, such as by nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined with mutating putative contact site amino acids. 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 homology analysis with related components of the polypeptides of the invention, such as the translocation component or the protease component.

[0260] 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 methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptides, and then sequencing the mutagenized polypeptides to determine the range of substitutions allowed 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, WIPO Publication No. WO 92 / 06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0261] Unless defined otherwise, 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, 20th ed., John Wiley and Sons, New York (1994), and Hale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, New York (1991) provide one of ordinary skill in the art with a general dictionary of many of the terms used in this disclosure.

[0262] The present disclosure is not limited by 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 the embodiments of the present disclosure. Numerical ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequence is written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right in an amino to carboxyl orientation, respectively.

[0263] The headings provided herein are not limitations of the various aspects or embodiments of this disclosure.

[0264] The name, three-letter abbreviation or single-letter abbreviation of amino acid is used to refer to amino acid in this article. As used herein, the term "protein" includes proteins, polypeptides and peptides. As used herein, the term "amino acid sequence" is synonymous with the term "polypeptide" and / or the term "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 in this article. In this disclosure and the claims, conventional one-letter and three-letter codes for amino acid residues can be used. The 3-letter code for amino acids follows the definition of the Joint Commission on Biochemical Nomenclature (JCBN) of IUPAC IUB. It should also be understood that due to the degeneracy of the genetic code, a polypeptide can be encoded by more than one nucleotide sequence.

[0265] Other definitions of terms may appear throughout this specification. Before describing exemplary embodiments in more detail, it should be understood that the present disclosure is not limited to the specific embodiments described, and thus these embodiments may vary. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the present disclosure is limited only by the appended claims.

[0266] Where a range of values ​​is provided, it is understood that each intervening value (to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise) between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any specified value or intervening value in a stated range and any other specified value or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in that range, and each range in which either, neither, or both of the limits are included in the smaller range is also encompassed in this disclosure, subject to any specifically excluded limits in the stated range. Where a stated range includes one or both of the limits, ranges excluding one or both of those included limits are also encompassed in this disclosure.

[0267] It must be noted that, as used herein and in 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 skilled in the art, and so forth.

[0268] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art for the appended claims.

[0269] Examples

[0270] The invention will now be described, by way of example only, with reference to the following examples.

[0271] Materials and Methods

[0272] Anti-BCMA antibody production

[0273] Antibodies were generated as described in WO 2010 / 104949 and WO 2019 / 025983, both of which are incorporated herein by reference. Suitable antibodies were generated as described in Kinneer et al. (2018), Leukemia 33, 766-771 and WO 2019 / 025983.

[0274] Generation of anti-BCMA ADCs

[0275] Anti-BCMA ADCs (anti-BCMA antibodies conjugated to PBD are referred to herein as "M2") were prepared by site-specific conjugation of the PBD dimer ticarlin (SG3249) to the BCMA-Abl antibody described in the following literature: Kinneer et al. (2018), Leukemia 33, 766-771 ("Kinneer et al. (2018)") using a protease-cleavable linker as previously described (see, e.g., Kinneer et al. (2018); incorporated herein by reference). An example of a BCMA antibody is BCMA-Ab2, also described in Kinneer et al. (2018). The antibodies are further described in WO 2019 / 025983 (incorporated herein by reference) as 15B2GL and J6M0-mc, respectively. ADC "M3" was similarly generated by attaching a monomethylreocetin F (MMAF) payload to the antibody BCMA-Abl. As previously described, both payloads were site-specifically conjugated to an engineered cysteine ​​inserted after position 239 (C239i) in the CH2 domain of the BCMA antibody. Briefly, BCMA-Ab1 was reduced with a 40 molar excess of TCEP at 37°C for three hours, followed by three consecutive dialysis cycles to remove TCEP. The antibody was then oxidized with a 20 molar excess of DHAA at room temperature for four hours, and conjugated using eight molar equivalents of payload. After conjugation, free payload and protein aggregates were removed by purification over ceramic hydroxyapatite.

[0276] Mouse xenograft model of human MM

[0277] All animal experiments were approved by the Institutional Animal Care and Use Committee of the Dana-Farber Cancer Institute and met relevant regulatory standards. CB-17SCID mice were subcutaneously inoculated with 5.0×106MM.1S cells in 100 μl of serum-free RPMI 1640 medium. When tumors were measurable approximately 3 weeks after MM cell injection, mice (8 mice / group) were randomized and treated with vehicle alone, M2, btz, or M2 and btz. Tumor 2-dimensional size was measured every three days using a caliper, and tumor volume was calculated using the following formula: V=0.5a×b2, where "a" and "b" are the long and short diameters of the tumor, respectively. When the animal's tumor reached 2 cm 3 Kill them when they are killed.

[0278] Analysis of tumors harvested from mice using immunoblotting and immunostaining

[0279] After 3d treatment, the tumor from each group was harvested and cell lysates were prepared for immunoblotting. Tumor sections collected from mice were immunostained for proliferation by Ki67 (BCR CRM325). Immunohistochemical images were taken for Ki67 on a Zeiss inverted fluorescence microscope. Plan-Apochromat 63X / 1.40 Oil DIC M27 objective lens was used.

[0280] Cells and cell culture

[0281] MM cell lines were cultured in RPMI containing 10% fetal bovine serum (GIBCO, 10437028), 2mM / L L-glutamine, 100U / mL penicillin, and 100mg / mL streptomycin (GIBCO, 15140122). Their authenticity and mycoplasma contamination were regularly checked by human STR profiling cell authentication. Patient MM and normal donor samples were obtained after providing informed consent in accordance with the Declaration of Helsinki and under the support of a protocol approved by the Dana-Farber Cancer Institute Institutional Review Board. Primary CD138+ plasma cells (>95% purity) were purified from bone marrow mononuclear cells (BMMCs) from MM patient BM aspirates using anti-CD138 microbeads (Miltenyi Biotech, Auburn, California). The remaining CD138-negative BMMCs were further cultured to derive BMSCs. Peripheral blood mononuclear cells (PBMCs) were isolated from PB samples using a Ficoll-diatrizoate density gradient. Bortezomib was purchased from Selleckchem (Selleck Chemicals).

[0282] Cell viability and apoptosis assays

[0283] Cell viability was analyzed by CCK8 (Abcam, Cambridge, MA), CellTiter-Glo (CTG) (Promega), and BLI measurements. Cells were expressed in the presence of FITC Annexin-V (BD Bioscience), PE-Annexin-V (BioLegend), and / or LIVE / DEAD ELISA kits according to the manufacturer's instructions. TM Apoptosis can be assessed by flow cytometry analysis after fixation and staining with Aqua (Invitrogen, L34957). MM cells were labeled with CFSE (Invitrogen) and then cultured for 2 days alone or with BMSCs, followed by Annexin V / Aqua staining and flow cytometry analysis.

[0284] Luciferase proliferation assay

[0285] BMSCs were seeded in 96-well plates and incubated for 24 hours to allow cell adhesion. MM1Sluc cells were cultured in RPMI culture medium at a ratio of 100:1 on a confluent layer of BMSCs for 4 days. Proliferation was measured using luciferase assay according to the manufacturer's protocol (Promega, Madison, Wisconsin).

[0286] Statistical analysis

[0287] Each experiment was performed at least three times, and the data are presented as mean ± SD. Data were analyzed using a Student's t-test for comparisons of two groups or a one-way analysis of variance (ANOVA) with multiple comparisons using GraphPad software (GraphPad Software, Inc., La Jolla, CA, USA). P values ​​< 0.05 were considered statistically significant. Drug interactions were evaluated using CompuSyn software to determine the combination index (CI). A CI < 1 indicates synergy, while a CI > 1 indicates antagonism and a CI = 1 indicates additive effect.

[0288] Example 1

[0289] The results of the experiments reflected in this example demonstrate that the anti-BCMA antibody-PBD conjugate (M2) induces more potent cytotoxicity against drug-resistant MM cells compared to its MMAF ADC homolog (M3).

[0290] The cytotoxicity of an ADC consisting of an anti-BCMA antibody conjugated to a PBD (M2) was compared to that of its MMAF ADC homologue (M3) against a panel of MM cell lines with varying levels of BCMA expression and response to current anti-MM drugs. Both ADCs consisted of the same anti-BCMA mAb (BCMA-Ab1 / 15B2GL, described above), but were conjugated to different payloads: a DNA-crosslinking PBD for M2 (e.g., ticarcillin) and a microtubule-binding MMAF for M3. Using a CCK8-based viability assay lasting 3 days, the ED50 values ​​of M2 were lower than those of M3 in all MM cell lines tested (n=10), regardless of sensitivity to anti-MM therapies including dexamethasone and IMiDs ( Figure 1 A, Figure 3 A) ED of 8MM cell lines, excluding RPMI8226 (RPMI) and its BCMA-overexpressing derivative RPMI-BCMA, M2, and M3 50The values ​​ranged from 11.85 to 3499 ng / ml and from 21.28 to 271431 ng / ml, respectively. All MM cells carried various p53 mutations, except for MM1S and H929 cells, from which two IMiD-resistant MM1S(R) and H929(R) cells were derived, respectively. M2, but not M3, was cytotoxic to RPMI8226 cells expressing the lowest BCMA levels and resistant to IMiDs ( Figure 1 A-1B). Using a DNA synthesis assay, M2 showed greater potency (>1-2-log) than M3 in blocking proliferation of all MM cells ( Figure 1 B, Figure 3 B) For example, in RPMI8226 cells, the ED of M2 versus M3 50 In addition, M2, but not M3, reduced the viability of both ANBL6 and its derivative bortezomib (btz)-resistant ANBL6-BR cells cultured with IL-6 ( Figure 3 C). These paired IL-6-dependent ANBL6 cells are insensitive to M3 and express cell membrane BCMA protein comparable to RPMI8226 cells (data not shown). Therefore, MM cells with relatively lower BCMA expression are also significantly more susceptible to the effects of M2 and M3.

[0291] Flow cytometry (FCM) analysis after staining with Annexin V and Live / Dead Aqua revealed that M2 induced earlier and increased apoptosis in paired MM cell lines sensitive or resistant to dexamethasone (dex) or bortezomib (btz) in a dose- and time-dependent manner when compared with M3 ( Figure 1 C, Figure 3 D) These in vitro results indicate that, regardless of BCMA levels and p53 status, M2 overcomes resistance to current anti-MM drugs (dexamethasone, lenalidomide, pomalidomide, bortezomib) to a greater extent than M3 in MM cells.

[0292] Example 2

[0293] The results of the experiments reflected in this example demonstrate that M2 is more effective than M3 in inducing cytotoxicity against MM cells in the bone marrow microenvironment and in patient MM cells.

[0294] Next, we evaluated the effects of M2 and M3 on MM cells co-cultured with bone marrow stromal cells (BMSCs) and IL-6, both of which promote the growth, survival, and drug resistance of MM cells. Using BLI measurements, BMSCs showed a significant increase in the growth and survival of MM1Sluc cells ( Figure 4A). M2 inhibited the viability of MM1Sluc and all other tested MM cell lines (n=6) co-cultured with BMSCs more effectively than M3 in BLI and CTG-based assays ( Figure 2 A, Figure 4 A), and minimally affects BCMA-negative non-MM cell subsets, including BMSCs, PBMCs, and NK cells ( Figure 4 B). Using FCM analysis to identify viable MM cells, M2 reduced the survival of IMiD-resistant MM1S(R) and H929(R) cells more effectively than M3, even in the presence of BMSCs ( Figure 2 B). In quantitative FCM-based ( Figure 2 C) and CTG( Figure 4 C), M2 reduced the growth and survival of H929 MM cells in the presence or absence of IL-6.

[0295] After 3 days of treatment, the live and dead BM CD138+ cell fractions from RRMM patients were quantified by FCM analysis. Importantly, M2 increased (>2-fold) the apoptotic CD138+ patient MM cells in a dose-dependent manner compared to M3 ( Figure 4 D). In a CTG-based assay, M2 also showed dose-dependent toxicity in CD138-purified BM cells from three additional RRMM patients ( Figure 2 E), and a significant depletion of data from 4 newly diagnosed MM (NDMM) patients ( Figure 2 F, left, Figure 4 D) and 2 RRMM patients ( Figure 2 F, right) Viable CD38 high CD138+ BM cells. 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.

[0296] Example 3

[0297] The results of the experiments reflected in this example demonstrate that M2, used in combination with bortezomib, induces synergistic cytotoxicity against MM cells in vitro and in vivo.

[0298] Bortezomib (Btz) was selected as a candidate co-therapy for M2 because Btz is an existing myeloma therapy. Based on the FMC analysis of Annexin V / PI, the low-dose combination of M2 and Btz further enhanced apoptosis in JJN3 and RPMI8226 cells compared with either agent alone ( Figure 5AB, p < 0.01). In btz-resistant ANBL6-BR cells cultured in IL-6, a significant increase in cell death was also seen after combination treatment (supporting the observation that the addition of btz provided more than an additive effect). Next, the results from the CTG-based viability assay were analyzed to calculate the combination index (CI). A CI < 1 was obtained in more than 6 representative MM cell lines, indicating that M2 plus btz had a synergistic effect ( Figure 5 C, Figure 6 ).

[0299] Next, the in vivo efficacy of suboptimal doses of M2 and btz was evaluated in the MM1S xenograft mouse model. Mice with palpable MM1S tumors were randomly divided into 4 groups and received vehicle control, single treatment with M2, or 6 btz (0.4 mg / kg) (alone or with M2) treatments. On the 24th day after treatment, a single dose of M2 or a total of 6 doses of btz significantly delayed MM1S tumor growth in mice compared to vehicle control ( Figure 7 A, p < 0.005). Combination treatment significantly reduced tumor volume compared to either agent alone (p < 0.04). M2 plus btz treatment was well tolerated as body weights of all animals were unaffected ( Figure 7 B). Follow-up of 177 days showed that the median overall survival of the combination treatment group was significantly prolonged compared with the cohort treated with either agent alone (cnt, 22 days; M2, 40.5 days; btz, 35 days; M2+btz, 57 days) (p < 0.045) ( Figure 7 C) On day 177, 15% of the mice in the combination treatment group were still alive without any tumor growth.

[0300] Immunohistochemistry (IHC) of Ki67 (a proliferative cell marker) further confirmed that combination therapy inhibited proliferation more effectively than single-agent therapy ( Figure 7 D) - Note the reduced number of stained cells (dark color) in M2+btz treatment.

[0301] Treatment with the combination of M2 and btz significantly reduced the in vivo growth of MM1S xenografts ( Figure 8 ), demonstrating that M2 and btz have an in vivo synergistic effect in the treatment of myeloma.

[0302] In conclusion, the synergistic activity of M2 and btz observed at the cellular level in vitro was translated into superior in vivo efficacy in a plasmacytoma model of MM.

[0303] Discussion of Examples 1-3

[0304] Disease relapse due to drug resistance remains a major obstacle to longer MM survival. Therefore, in RRMM, novel therapies are needed to overcome drug resistance and address unmet medical needs. Here, the inventors of the present invention first showed that ADC (M2; an anti-BCMA antibody conjugated to PBD) had better cytotoxicity than its MMAF ADC homologue for all tested MM cell lines and patient MM cells. PBD dimers cause cell death in both rapidly dividing cells and more dormant cells, which is different from MMAF, which mainly binds to tubulin to target proliferating tumor cells. M2 has a more effective effect on MM cell proliferation than its MMAF ADC homologue, including cells with low levels of BCMA expression and resistance to current therapies, even in the presence of BMSCs and IL-6. These data suggest that M2 may be more effective than its MMAF ADC homologue in treating aggressive MM.

[0305] Importantly, in all MM cells tested, combined treatment with M2 and btz induced synergistic cell death in vitro, as evidenced by a CI < 1. Notably, M2 synergized with btz even in btz-resistant ANBL6-BR cells, suggesting that other, undefined molecules are also responsible for the enhanced cytotoxicity.

[0306] In mice bearing MM1S tumors, M2 was significantly more effective than btz as a single-agent therapy. Importantly, when M2 was combined with btz, the inhibitory effect on tumor growth in vivo was further enhanced. In mice receiving both drugs, significant tumor necrosis was observed earlier than with either agent alone, and on day 177, 15% of the mice in the combination treatment group were still alive and tumor-free. Importantly, no weight loss was observed in any group, indicating that M2 has a favorable safety profile in vivo, suggesting that the combination treatment of M2 and btz can be safely administered in vivo.

[0307] In conclusion, M2 specifically triggers potent growth inhibition and cell 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 combining M2 with btz further enhances efficacy and prolongs host survival.

[0308] Example 4

[0309] The results of the experiments reflected in this example demonstrate that M2 significantly activates DNA damage response and repair signaling cascades, followed by apoptosis, in both drug-sensitive and drug-resistant MM cells.

[0310] In MM cell lines, immunoblot analysis was used to determine the induction of the DNA damage response (DDR) signaling cascade triggered by M2 in a time- and dose-dependent manner. M2, but not M3, activated phosphorylation of ATM, cell cycle checkpoint kinases 1 (CHK1) and CHK2 (CHK1 / 2), and histone 2AX (H2AX), an early event in the response to DNA double-strand breaks (DSBs). Figure 9 A and 10A). M2-stimulated phosphorylation of ATM and CHK1 / 2 was detected at 4 h and persisted for >1 d after treatment. Earlier and more pronounced activation of ATM and CHK1 / 2 triggered by M2 was observed in H929 cells (which express significantly higher levels of BCMA than MM1S cells). Figure 10 A, 10C). The intensity of M2-induced ATM and CHK1 / 2 phosphorylation also correlated with BCMA levels in parental RPMI8226MM cells ( Figure 10 D). In the MM cells tested, M2 induced phosphorylation of ATM and CHK1 / 2 to a significantly greater extent than ATR phosphorylation. After 2 days of treatment with M2, PARP cleavage (cPARP) and caspase 3 cleavage (cCas3) were induced in a BCMA-dependent manner ( Figure 10 B, 10E-10F), which is associated with increased phosphorylated H2AX (γH2AX) ( Figure 10 B), indicating that M2 induces DNA damage in MM cells, followed by apoptosis. Importantly, in all MM cells (including 6 cell lines with p53 mutations), M2 induced the phosphorylation of ATM and CHK1 / 2 in a dose-dependent manner ( Figure 9 b-9c). Under the same treatment conditions as M2, M3 neither induced ATM / ATR nor CHK1 / 2 ( Figure 10 A). M2 is more effective than M3 in inducing cPARP and cCas3 ( Figure 10 F), consistent with its higher potency than M3 in triggering apoptosis in MM cells. Significantly, M2 triggered ATM / ATR and downstream CHK1 / 2 signaling pathways, γH2AX, and PARP cleavage in ANBL6 and paired btz-resistant ANBL6-BR cells ( Figure 9 c). Prominent activation of ATM and CHK1 / 2 by M2 was also observed in IMiD-resistant H929(R) cells to a similar extent as in parental H929MM cells ( Figure 9 d) Therefore, in btz- and len-resistant MM cells, M2 still induces BCMA-dependent DDR signaling pathways via activation of the ATR / ATM-CHK1 / 2 signaling cascade, followed by apoptosis.

[0311] DNA repair mechanisms Array analysis showed that M2 altered the expression of 51 of 72 DNA damage repair-related genes in H929 MM cells ( Figure 11 a). In various drug-sensitive and drug-resistant MM cells (n>6), M2 induces RAD51 in a dose-dependent manner, which binds to DNA ICLs before generating DSBs ( Figure 11 b-11c), but not M3 ( Figure 10 G). Thus, in MM cells, M2 specifically activates the ATM / ATR-CHK1 / 2-mediated DDR signaling cascade and induces downstream DDR-related molecules associated with increased γH2AX and RAD51, followed by apoptosis.

[0312] All publications mentioned in the above specification are incorporated herein by reference. 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 invention. Although the present invention has been described in conjunction with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described embodiments for carrying out the present invention that are apparent to those skilled in the art of biochemistry and biotechnology or related fields are intended to fall within the scope of the following claims.

[0313] sequence

[0314]

Claims

1. A drug for treating B-cell malignancies, comprising: a. Antibody-drug conjugates (ADCs) comprising a pyrrolobenzodiazepine an antibody or antigen-binding fragment thereof that binds to the B cell maturation antigen (BCMA) of a human leukemia cell line (PBD); and b. proteasome inhibitor bortezomib, The antibody or antigen-binding fragment thereof comprises the following six CDRs: a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2; a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3; a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4; a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5; and A light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6; and wherein the PBD has the formula wherein in the B ring, at the N10-C11 position is an imine (N═C), methanolamine (NH—CH(OH)), or methanolamine methyl ether (NH—CH(OMe)), which is the electrophilic center responsible for alkylating DNA, and wherein it has an (S)-configuration at the chiral C11a position; wherein the medicament provides enhanced inhibition of B-cell malignancies when compared to an otherwise identical medicament except for the absence of the proteasome inhibitor; or wherein the agent provides enhanced inhibition of B cell malignancies when compared to an otherwise identical agent except for the absence of the ADC.

2. A therapeutic combination for treating a B-cell malignancy, comprising: a. ADC comprising a pyrrolobenzodiazepine a BCMA-binding antibody or antigen-binding fragment thereof (PBD); and b. proteasome inhibitor bortezomib, The antibody or antigen-binding fragment thereof comprises the following six CDRs: a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2; a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3; a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4; a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5; and A light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6; and wherein the PBD has the formula wherein in the B ring, at the N10-C11 position is an imine (N═C), methanolamine (NH—CH(OH)), or methanolamine methyl ether (NH—CH(OMe)), which is the electrophilic center responsible for alkylating DNA, and wherein it has an (S)-configuration at the chiral C11a position; wherein the therapeutic combination provides enhanced inhibition of B-cell malignancies when compared to an otherwise identical composition except for the absence of the proteasome inhibitor; or wherein the therapeutic combination provides enhanced inhibition of B cell malignancies when compared to an otherwise identical composition except for the absence of the ADC.

3. Use of a combination of an ADC and a proteasome inhibitor in the preparation of a medicament for treating a B-cell malignancy, wherein the ADC comprises a proteasome inhibitor conjugated to a pyrrolobenzodiazepine (PBD) or an antigen-binding fragment thereof, wherein the proteasome inhibitor is bortezomib, The antibody or antigen-binding fragment thereof comprises the following six CDRs: a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2; a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3; a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4; a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5; and A light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6; and wherein the PBD has the formula wherein in the B ring, at the N10-C11 position is an imine (N═C), methanolamine (NH—CH(OH)), or methanolamine methyl ether (NH—CH(OMe)), which is the electrophilic center responsible for alkylating DNA, and wherein it has an (S)-configuration at the chiral C11a position; wherein the combination provides enhanced inhibition of B-cell malignancies when compared to an otherwise identical composition except for the absence of the proteasome inhibitor; or wherein the combination provides enhanced inhibition of B cell malignancies when compared to an otherwise identical composition except for the absence of the ADC.

4. An in vitro method for enhancing ADC inhibition of B cell malignancies, the method comprising contacting a malignant B cell with: (a) an ADC comprising a pyrrolobenzodiazepine (PBD) a BCMA-binding antibody or antigen-binding fragment thereof, in combination with (b) bortezomib, The antibody or antigen-binding fragment thereof comprises the following six CDRs: a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2; a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3; a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4; a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5; and A light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6; and wherein the PBD has the formula Among them, in the B ring, at the N10-C11 position is an imine (N=C), methanolamine (NH—CH(OH)), or methanolamine methyl ether (NH—CH(OMe)), which is the electrophilic center responsible for the alkylation of DNA, and wherein it has the (S)-configuration at the chiral C11a position.

5. An in vitro method for enhancing proteasome inhibitor inhibition of B cell malignancies, the method comprising contacting a malignant B cell with: (a) bortezomib in combination with (b) an ADC comprising a pyrrolobenzodiazepine (PBD) or a BCMA-binding antibody or antigen-binding fragment thereof, The antibody or antigen-binding fragment thereof comprises the following six CDRs: a heavy chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 1; a heavy chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 2; a heavy chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 3; a light chain CDR1 consisting of the amino acid sequence of SEQ ID NO: 4; a light chain CDR2 consisting of the amino acid sequence of SEQ ID NO: 5; and A light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6; and wherein the PBD has the formula Among them, in the B ring, at the N10-C11 position is an imine (N=C), methanolamine (NH—CH(OH)), or methanolamine methyl ether (NH—CH(OMe)), which is the electrophilic center responsible for the alkylation of DNA, and wherein it has the (S)-configuration at the chiral C11a position.

6. The medicament according to claim 1, the therapeutic combination according to claim 2, the use according to claim 3 or the in vitro method according to claim 4 or 5: a. wherein the proteasome inhibitor is administered before, simultaneously with, or after the ADC; or b. wherein the ADC is administered before, simultaneously with or after the proteasome inhibitor.

7. The medicament according to claim 1 , the therapeutic combination according to claim 2, the use according to claim 3 or the in vitro method according to claim 4 or 5, wherein the B-cell malignancy is characterized by malignant B cells comprising increased expression levels of the BCMA antigen relative to reference non-malignant B cells.

8. The medicament according to claim 1, the therapeutic combination according to claim 2, the use according to claim 3 or the in vitro method according to claim 4 or 5, wherein the B-cell malignancy is one or more selected from the group consisting of: B-cell lymphoma, B-cell leukemia, myeloma or a combination thereof.

9. The medicament according to claim 1, the therapeutic combination according to claim 2, the use according to claim 3 or the in vitro method according to claim 4 or 5, wherein the B-cell malignancy is multiple myeloma.

10. The medicament according to claim 1, the therapeutic combination according to claim 2, the use according to claim 3 or the in vitro method according to claim 4 or 5, wherein the antibody or antigen-binding fragment thereof comprises: a. a heavy chain variable region comprising the amino acid sequence of SEQ ID: NO 7; and / or b. a light chain variable region comprising the amino acid sequence of SEQ ID: NO 8.

11. The medicament according to claim 1, the therapeutic combination according to claim 2, the use according to claim 3 or the in vitro method according to claim 4 or 5, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising a cysteine ​​(C) insertion between the serine (S) at position 239 and the valine (V) at position 240, wherein the numbering corresponds to the EU index as in Kabat.

12. The medicament according to claim 1, the therapeutic combination according to claim 2, the use according to claim 3 or the in vitro method according to claim 4 or 5, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:

11.

13. The medicament according to claim 1, the therapeutic combination according to claim 2, the use according to claim 3 or the in vitro method according to claim 4 or 5, wherein the antibody or antigen-binding fragment thereof comprises a human kappa constant region comprising the amino acid sequence of SEQ ID NO:

12.

14. The medicament according to claim 1, the therapeutic combination according to claim 2, the use according to claim 3 or the in vitro method according to claim 4 or 5, wherein the PBD is selected from one or more of the following: (a) SG3249, (b) SG3315 or (c) SG3400, each comprising the following formula: (a) (b) as well as (c) or a combination thereof.

15. The medicament according to claim 1, the therapeutic combination according to claim 2, the use according to claim 3 or the in vitro method according to claim 4 or 5, wherein the PBD is SG3249 comprising the formula:

16. The medicament according to claim 1, the therapeutic combination according to claim 2, the use according to claim 3, or the in vitro method according to claim 4 or 5, wherein the antibody or antigen-binding fragment thereof is a monoclonal antibody.

17. The medicament according to claim 1, the therapeutic combination according to claim 2 or the use according to claim 3, wherein the medicament or therapeutic combination comprises a pharmaceutically acceptable carrier.

18. The medicament according to claim 1, the therapeutic combination according to claim 2, the use according to claim 3 or the in vitro method according to claim 4 or 5, wherein the B-cell malignancy is resistant to one or more selected from the group consisting of dexamethasone, lenalidomide, pomalidomide, bortezomib, or a combination thereof.

19. The medicament according to claim 1, the therapeutic combination according to claim 2, the use according to claim 3 or the in vitro method according to claim 4 or 5, wherein the B-cell malignancy is resistant to bortezomib.

20. The medicament of claim 1, the therapeutic combination of claim 2, the use of claim 3, or the in vitro method of claim 4 or 5, wherein the enhanced inhibition of B-cell malignancies comprises one or more selected from the group consisting of increased tumor growth delay, increased tumor size reduction, increased tumor metastasis reduction, improved survival of subjects harboring B-cell malignancies, or a combination thereof.

21. The medicament according to claim 1, the therapeutic combination according to claim 2 or the use according to claim 3, wherein the medicament or therapeutic combination is administered by intravenous infusion.

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