BCMA (cd269 / tnfrsf17) binding proteins

Abstract

To provide an antigen-binding protein which binds to a membrane-bound target and can be internalized. Also provided is an immunoconjugate comprising the antigen binding protein and a cytotoxic agent.SOLUTION: Provided is an antigen binding protein that specifically binds to BCMA and inhibits the binding of BAFF and / or APRIL to BCMA, wherein the antigen binding protein is capable of binding to Fc γ RIIIA or is capable of Fc γ RIIIA-mediated effector function and is capable of internalization.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to antigen-binding proteins and fragments thereof that specifically bind to B-cell maturation antigen (BCMA), in particular human BCMA (hBCMA).

[0002] The invention also relates to methods of treating diseases or disorders using said antigen-binding fragments, pharmaceutical compositions comprising said antigen-binding fragments, and methods of manufacture. Other embodiments of the invention will become apparent from the details below. [Background technology]

[0003] BCMA (CD269 or TNFRSF17) is a member of the TNF receptor superfamily. It is a non-glycosylated integral membrane receptor for the ligands BAFF and APRIL. BCMA's ligand can also bind to an additional receptor: TACI (transmembrane activator and calcium regulator and cyclophilin ligand interactor), which binds APRIL and BAFF, as well as BAFF-R (BAFF receptor or BR3), which exhibits limited but high affinity for BAFF. Collectively, these receptors and their corresponding ligands regulate various aspects of humoral immunity, B cell development, and homeostasis.

[0004] BCMA expression is typically restricted to the B cell lineage and has been reported to increase in response to terminal B cell differentiation. BCMA is expressed by human plasma blasts, plasma cells from tonsils, spleen, and bone marrow, but also by tonsillar memory B cells and germinal center B cells with a TACI-BAFFR-low phenotype (Darce et al., 2007). BCMA is largely absent on naive and memory B cells (Novak et al., 2004a and b). The BCMA antigen is expressed on the cell surface, making it accessible to antibodies, but it is also expressed in the Golgi apparatus. As suggested by its expression profile, BCMA signaling is typically associated with B cell survival and proliferation and is important in the later stages of B cell differentiation, but also for the survival of long-lived bone marrow plasma cells (O'Connor et al., 2004) and plasmablasts (Avery et al., 2003). Furthermore, because BCMA binds to APRIL with high affinity, it has been suggested that the BCMA-APRIL signaling axis is dominant at later stages of B cell differentiation and is perhaps the most physiologically important interaction.

[0005] Multiple myeloma (MM) is a clonal B-cell malignancy that arises at multiple sites within the bone marrow, either de novo or as a progression from monoclonal gammopathy of undetermined significance (MGUS), before spreading to the circulation. It is generally characterized by increased paraproteins and osteoclast activity, as well as hypercalcemia, cytopenias, renal dysfunction, hyperviscosity, and peripheral neuropathy. Decreases in both normal antibody levels and neutrophil numbers are also common, leading to life-threatening susceptibility to infection. BCMA is involved in the proliferation and survival of myeloma cell lines in vitro (Novak et al., 2004a and b; Moreaux et al., 2004).

[0006] BCMA expression (both transcript and protein) has been reported to correlate with disease progression in MM. Using Affymetrix microarrays, it was demonstrated that the TACI and BCMA genes were overexpressed in multiple myeloma cells (MMC) compared with their normal counterparts (Moreaux et al., 2004). Gene expression analysis has been used to compare human myeloma cells with purified plasma cells from patients with MGUS and from normal bone marrow, as well as primary tumor cells from B-cell lineage leukemia (Bellucci et al., 2005). The BCMA gene was highly expressed in all myeloma samples. Purified plasma cells from patients with MGUS expressed BCMA at a lower level, but there was no significant difference compared with the expression found in normal plasma cells or myeloma cells. In contrast, BCMA expression was significantly reduced in B-cell chronic lymphocytic leukemia (CLL), pre-B acute lymphocytic leukemia (ALL), and T-cell ALL (T-ALL). Mouse models transgenic for overexpressing BAFF or APRIL significantly increase the incidence of B-cell lymphomas (Batten et al., 2004-BAFF, Planelles et al., 2004-APRIL). In humans, excess BAFF and APRIL have been detected in the serum and microenvironment of patients with multiple B-cell malignancies, as well as other B-cell disorders.

[0007] All patent and literature references disclosed within this specification are expressly and entirely incorporated herein by reference. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention provides antigen-binding proteins that bind to membrane-bound targets and are capable of internalization. In a further embodiment, there is provided an immunoconjugate comprising the antigen-binding protein of the present invention and a cytotoxic agent. In a further embodiment, the antigen-binding protein has ADCC effector function. For example, the antigen-binding protein has enhanced ADCC effector function. [Means for solving the problem]

[0009] The present invention provides antigen binding proteins that specifically bind to BCMA, for example antibodies that specifically bind to BCMA and inhibit binding of BAFF and / or APRIL to the BCMA receptor. The present invention also provides antigen binding proteins that specifically bind to BCMA and inhibit binding of BAFF and / or APRIL to BCMA, which antigen binding proteins may bind to FcγRIIIA or have FcγRIIIA-mediated effector function.

[0010] The antigen binding proteins of the present invention specifically bind to BCMA and inhibit the binding of BAFF and / or APRIL to BCMA, and the antigen binding proteins have enhanced binding to FcγRIIIA or have enhanced FcγRIIIA-mediated effector function. In one embodiment, the antigen binding proteins are capable of internalization.

[0011] In one aspect of the invention there is provided an antigen binding protein that binds to non-membrane bound BCMA, such as serum BCMA.

[0012] In one embodiment of the present invention, there is provided an immunoconjugate comprising an antigen binding protein of the invention and a cytotoxic agent.

[0013] In a further embodiment, the antigen binding protein is conjugated to a toxin, such as an auristatin.

[0014] In yet a further embodiment, the drug conjugate is vcMMAE or mcMMAF. In one embodiment, the immunoconjugate also has enhanced ADCC.

[0015] The antigen binding protein may be related to or derived from the murine monoclonal antibody CA8. The CA8 murine heavy chain variable region amino acid sequence is provided as SEQ ID NO:7, and the CA8 murine light chain variable region amino acid sequence is provided as SEQ ID NO:9.

[0016] The antigen binding protein may be related to or derived from the murine monoclonal antibody S336105A07. The S336105A07 murine heavy chain variable region amino acid sequence is provided as SEQ ID NO: 140, and the S336105A07 murine light chain variable region amino acid sequence is provided as SEQ ID NO: 144.

[0017] Other murine monoclonal antibodies from which the antigen binding proteins of the present invention can also be derived are included in Table C.

[0018] The heavy chain variable region (VH) of the antigen binding protein may comprise the following CDRs or variants of those CDRs (as defined by Kabat (Kabat et al., Sequences of proteins of Immunological Interest NIH, 1987)): CDRH1 is provided as SEQ ID NO: 1 or SEQ ID NO: 182; CDRH2 is provided as SEQ ID NO:2 or SEQ ID NO:183; CDRH3 is provided as SEQ ID NO:3 or SEQ ID NO:184.

[0019] The light chain variable region (VL) of the antigen binding protein may comprise the following CDRs or variants of those CDRs (as defined by Kabat (Kabat et al., Sequences of proteins of Immunological Interest NIH, 1987)): CDRL1 is provided as SEQ ID NO:4 or SEQ ID NO:185; CDRL2 is provided as SEQ ID NO:5 or SEQ ID NO:186; CDRL3 is provided as SEQ ID NO:6 or SEQ ID NO:187.

[0020] The present invention also provides polynucleotide sequences encoding the heavy chain variable region of any of the antigen binding proteins described herein, and polynucleotides encoding the light chain variable region of any of the antigen binding proteins described herein.

[0021] The present invention also provides polynucleotide sequences encoding the heavy chain of any of the antigen binding proteins described herein, and polynucleotides encoding the light chain of any of the antigen binding proteins described herein.

[0022] While such polynucleotides represent coding sequences corresponding to the equivalent polypeptide sequence, it will be understood that such polynucleotide sequences may be cloned into an expression vector with an initiation codon, an appropriate signal sequence, and a termination codon.

[0023] The present invention also provides host cells transformed or transfected with a recombinant comprising one or more polynucleotides encoding the heavy and / or light chains of any of the antigen binding proteins described herein.

[0024] The present invention further provides a method for producing any of the antigen binding proteins described herein, the method comprising the step of culturing a host cell comprising a first and a second vector in a suitable medium (e.g. a serum-free medium), wherein said first vector comprises a polynucleotide encoding the heavy chain of any of the antigen binding proteins described herein and said second vector comprises a polynucleotide encoding the light chain of any of the antigen binding proteins described herein.

[0025] The present invention further provides a pharmaceutical composition comprising an antigen binding protein described herein and a pharmaceutically acceptable carrier.

[0026] In a further aspect, the present invention provides a method of treating or preventing a disease or disorder that responds to the inhibition or blockage of BCMA, such as modulation of the interaction between BCMA and its ligands, BAFF or APRIL, comprising the step of administering to said patient a therapeutically effective amount of an antigen binding protein thereof as described herein.

[0027] It is therefore an object of the present invention to provide a therapeutic approach to the treatment of B-cell related disorders or diseases, such as antibody-mediated or plasma cell-mediated diseases or plasma cell malignancies, such as multiple myeloma (MM). In particular, it is an object of the present invention to provide antibodies that specifically bind to antigen binding proteins, in particular BCMA (e.g., hBCMA), and modulate (i.e., inhibit or block) the interaction between BCMA and its ligands, such as BAFF and / or APRIL, in the treatment of diseases and disorders responsive to modulation of that interaction.

[0028] In another aspect of the invention, there is provided a method of treating a human patient suffering from a B-cell related disorder or disease, such as an antibody-mediated or plasma cell-mediated disease, or a plasma cell malignancy, such as multiple myeloma (MM), comprising administering to said patient a therapeutically effective amount of an antigen binding protein as described herein.

[0029] In another aspect of the invention, there is provided a method of treating a human patient suffering from rheumatoid arthritis, psoriasis, type 1 diabetes or multiple sclerosis, the method comprising administering to said patient a therapeutically effective amount of an antigen binding protein as described herein. [Brief explanation of the drawings]

[0030] [Figure 1] FMAT Binding Assay—Figure showing the results of an FMAT assay for CA8 antibody binding to HEK293 cells expressing human and cyno BCMA. Human chimeric CA8 binds well to cells expressing human and cyno BCMA. [Figure 2]ELISA binding assay—Figure 1 shows the results of an ELISA for CA8 antibody binding to human and cyno BCMA recombinant proteins, clearly demonstrating that the human chimeric CA8 antibody binds equally to human and cyno BCMA proteins. [Figure 3] BiaCore Binding Assay—Figure showing binding of CA8 to BCMA-Fc, TACI-Fc and BAFF-R-Fc proteins in a Biacore experiment. The CA8 chimeric antibody does not bind to TACI or BAFF-R proteins. [Figure 4] Cell Binding Assay—Binding of murine S307118G03, S3222110D07, S332121F02, and S332126E04 to H929 multiple myeloma cells and S3322110D07, S332121F02, and S332126E04 to BCMA-transfected ARH77 cells as determined by FACS. Multiple myeloma cell line H929 or ARH77-hBCMA 10B5 BCMA-expressing transfectant cells were stained with either a murine anti-BCMA antibody (solid histogram) or a murine IgG2a isotype control (open histogram). Cells were analyzed by FACS to detect cell-bound antibody. [Figure 5] Cell Binding Assay—Figure 1 shows the binding of chimeric CA8 to a panel of multiple myeloma cell lines as determined by FACS. Binding to H929, OPM-2, JJN-3, and U266 was tested by flow cytometry, and binding was determined by measuring mean fluorescence intensity (MFI) values. Synagis was used as an irrelevant isotype control. [Figure 6] Cell Binding Assay—Figure 1 shows binding curves of humanized CA8 variants to BCMA-transfected ARH77 cells (A) and multiple myeloma H929 cells (B) as determined by FACS. Humanized variants J6M0, J6M1, J6M2, J9M0, J9M1, and J9M2 were tested by flow cytometry and mean fluorescence intensity (MFI) values ​​were measured to determine binding compared to the CA8 chimera. [Figure 7-1]Ligand Neutralization Assay—(A and B) Ability of CA8 and J6M0 to neutralize binding of recombinant BAFF or APRIL to recombinant BCMA coated on ELISA plates. OD values ​​were used to calculate antibody-mediated inhibition of the maximal signal achieved by the relevant ligand alone bound to recombinant BCMA. Data are reported as percent inhibition of maximal signal. Antibodies tested were chimeric CA8 and humanized CA8 J6M0 in both wild-type and afucosylated (Potelligent) forms. (A) Neutralization of BAFF ligand binding. (B) Neutralization of APRIL ligand binding. (C) Ability of J6M0 BCMA antibody to inhibit BAFF- or APRIL-induced phosphorylation of NF-kappaB in H929 cells. H929 cells were washed three times to remove any sBCMA and resuspended in serum-free medium. J6M0 Potelligent® antibody was added to a 96-well plate to give a final well concentration of up to 100 μg / ml, along with BAFF or APRIL ligand, giving final well concentrations of 0.6 or 0.2 μg / ml, respectively. H929 cells were then seeded at 7.5 x 10 cells / well in serum-free medium. After 30 minutes, cells were lysed and phosphorylated NF-kappa B levels were measured using the MSD pNF-kappa B assay. MSD Reader 502819. This is data from one independent experiment. Each data point is the mean / standard deviation of two replicates. [Figure 7-2] This is a continuation of Figure 7-1. [Figure 8] ADCC assay—Figure showing ADCC activity of chimeric CA8 and defucosylated (Fc-enhanced) CA8 with BCMA-expressing target cells. Human NK cells were incubated with europium-labeled ARH77 10B5 BCMA-transfected target cells in the presence of various concentrations of antibody. Europium release from target cells was measured and specific lysis was calculated. (A) ADCC dose-response curve for chimeric CA8 compared to isotype control. (B) ADCC dose-response curve for chimeric CA8 and defucosylated chimeric CA8 (Fc-enhanced) against the BCMA-expressing cell line ARH77 10B5. [Figure 9]ADCC assay - ADCC assay with CA8 humanized antibody using ARH77 BCMA-expressing target cells. Human PBMCs were incubated with europium-labeled ARH77 BCMA-transfected target cells in the presence of various concentrations of the J5, J6, J7, J8, or J9 series of humanized CA8 antibodies. Europium release from the target cells was measured and specific lysis was calculated. EC50 values ​​are shown in μg / ml. [Figure 10] ADCC assay—Figure showing ADCC activity of chimeric, S332121F02 (A), S3322110D07 (B), S307118G03 (C), and humanized S307118G03 H3L0 (D) antibodies against ARH77 10B5 target cells with purified NK cells as effector cells. Human NK target cells were incubated with europium-labeled ARH77 10B5 BCMA-transfected target cells in the presence of various concentrations of antibody. Europium release from target cells was measured and specific lysis was calculated. [Figure 11] Viability assay dose-response curves—Figure 1 shows dose-response curves in cell viability assays for chimeric CA8 antibody, chimeric CA8-vcMMAE, and chimeric CA8-mcMMAF antibody-drug conjugates in human multiple myeloma cell lines (A) NCI-H929, (B) U266-B1, (C) JJN3, and (D) OPM2. Antibodies were added to cells, and the number of viable cells after 96 hours was determined using CelltiterGlo. Data points represent the average of triplicate CellTiterGlo measurements. Error bars represent standard error. [Figure 12]Figure 1 shows the effect of CA8 chimeric antibodies on the cell cycle. (A) Cell cycle histograms of NCI-H929 cells treated with unconjugated chimeric CA8, chimeric CA8-vcMMAE ADC, or chimeric CA8-mcMMAF ADC at 50 ng / mL for the indicated time points. Pactitaxel (100 nM) was used as a positive control for G2 / M cell cycle arrest and cell death. Control human IgG1 was used as a negative control. Cell cycle analysis was performed at the time points indicated on the graph. (B) Quantification of 4N DNA cell populations, indicative of G2 / M arrest, and (C) sub-2N DNA cell populations, indicative of cell death for each of the indicated treatments. Cells were seeded in 12-well plates (2 x 10 cells / well in 1 mL of RPMI + 10% FBS). Antibodies or ADCs were added 6 hours after cell seeding. [Figure 13] Figure 1 shows the effect of chimeric CA8 on phospho-histone H3. Chimeric CA8 ADC treatment results in increased phospho-histone H3 staining in NCI-H929 cells. (A, B) Dot plots of cells stained with propidium iodide to measure DNA content (FL3-H) on the x-axis and anti-phospho-histone H3 (Thr11) antibody (FL1-H) on the y-axis after treatment with either control IgG (A) or chimeric CA8-mcMMAF (B). (C) Quantification of phospho-histone H3-positive NCI-H929 cells after 48 hours of treatment with the indicated concentrations of chimeric CA8 ADC. Pactitaxel (100 nM) was used as a positive control for mitotic arrest, and control chimeric IgG1 was used as a negative control. Cells were seeded in 12-well plates (2 x 10 cells / well in 1 mL of RPMI + 10% FBS). Antibodies or ADCs were added 6 hours after cell seeding. [Figure 14]Figure 1 shows the effect of chimeric CA8 on Annexin-V. Chimeric CA8 ADC treatment results in increased Annexin-V staining of NCI-H929 cells. (A) Histograms of Annexin-V-FITC (FL1-H, upper panel) and live cell propidium iodide staining (FL3-H, lower panel) after treatment with increasing concentrations of chimeric CA8 ADC. (B) Quantification of Annexin-V-positive NCI-H929 cells after 96 hours of treatment with the indicated concentrations of chimeric CA8 ADC. Pactitaxel (100 nM) was used as a positive control for apoptosis, and control chimeric IgG1 was used as a negative control. Cells were seeded in 12-well plates (2 x 10 cells / well in 1 mL of RPMI + 10% FBS). Antibodies or ADCs were added 6 hours after cell seeding. [Figure 15] Viability assay dose-response curves—Figure 1 shows dose-response curves for unconjugated (naked) chimeric CA8 or humanized J6M0 antibodies and vcMMAE and mcMMAF antibody-drug conjugates. Antibody drug conjugates were tested against human multiple myeloma cell lines NCI-H929 and OPM2. [Figure 16] FIG. 14 shows dose response curves for unconjugated, vcMMAE, and mcMMAF antibody-drug conjugates of murine anti-BCMA antibodies S332121F02, S322110D07, S332126E04, and S307118G03 in human multiple myeloma cell lines NCI-H929 and U266-B1. [Figure 17] Figure 1 shows ADCC activity of ADC J6M0 molecule - ADCC assay with J6M0 antibody using ARH77 BCMA-expressing target cells. Human PBMCs were incubated with europium-labeled ARH77 BCMA-transfected target cells in the presence of various concentrations of J6M0 WT and Potelligent BCMA antibody conjugated to MMAE, MMAF, or unconjugated Potelligent BCMA antibody, and europium release was monitored using a Victor 2 1420 multilabel reader. [Figure 18]Figure 1 shows ADCC dose-response curves for CA8 J6M0 Potelligent against a panel of five multiple myeloma lines. Human PBMCs were incubated with multiple myeloma target cells in the presence of various concentrations of CA8 J6M0 Potelligent antibody at an E:T ratio of 50:1 for 18 hours. The percentage of target cells remaining in the effector and target mixture was then measured by FACS using a fluorescently labeled anti-CD138 antibody to detect target cells, and the percentage of cytotoxicity was calculated. A) Example dose-response curves for CA8 J6M0 Potelligent against the five multiple myeloma cell lines tested. Each data point is from a single value. [Figure 19] Figure 1 shows the effect of increasing doses of J6M0 and drug-conjugated J6M0 on the growth and establishment of NCI-H929 cells in CB.17 SCID mice. Calculated tumor volumes of NCI-H929 tumors in CB17 SCID mice after twice-weekly intraperitoneal administration of 50 or 100 μg of either unconjugated or MMAE- or MMAF-conjugated J6M0 anti-BCMA or an IgG1 isotype control for two weeks. Data points represent the mean tumor volume of n=5 per group. [Figure 20] Figure 1 shows the determination of soluble BCMA levels in serum from healthy volunteers and myeloma patients. Serum samples were collected from MM patients, and the samples were from various stages (progressive, remission, relapse, newly diagnosed, and others). Samples shown in the figure are from serum diluted 1 / 500 before assay. BCMA was detected using a human BCMA / TNFRSF17 sandwich ELISA kit from R&D Systems, which measures soluble human BCMA levels, following the standard protocol provided with the kit. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention provides antigen-binding proteins that bind to membrane-bound targets and are capable of internalization. In a further embodiment, an immunoconjugate is provided comprising the antigen-binding protein of the present invention and a cytotoxic agent. In a further embodiment, the antigen-binding protein has ADCC effector function. For example, the antigen-binding protein has enhanced ADCC effector function.

[0032] In one such embodiment, there is provided an antigen binding protein or fragment thereof that specifically binds to BCMA, for example that specifically binds to human BCMA (hBCMA) and inhibits binding of BAFF and / or APRIL to the BCMA receptor.

[0033] In a further embodiment, the antigen binding protein or fragment specifically binds to BCMA and inhibits binding of BAFF and / or APRIL to BCMA, and the antigen binding protein or fragment thereof has the ability to bind to FcγRIIIA and mediate FcγRIIIA-mediated effector function, or has enhanced FcγRIIIA-mediated effector function. In one embodiment of the invention provided herein, the antigen binding protein is capable of internalization.

[0034] In one aspect of the invention there is provided an antigen binding protein according to the invention as described herein which binds to non-membrane bound BCMA, such as serum BCMA.

[0035] In one aspect of the invention there is provided an antigen binding protein as described herein, wherein the antigen binding protein comprises a CDRH3 of SEQ ID NO: 3 or a variant of SEQ ID NO: 3.

[0036] In a further aspect of the present invention there is provided an antigen binding protein as described herein further comprising one or more of CDRH1 of SEQ ID NO: 1, CDRH2: SEQ ID NO: 2: CDRL1: SEQ ID NO: 4, CDRL2: SEQ ID NO: 5 and / or CDRL3: SEQ ID NO: 6 and or variants thereof.

[0037] In one aspect of the invention there is provided an antigen binding protein as described herein, wherein the antigen binding protein comprises a CDRH3 of SEQ ID NO: 184 or a variant of SEQ ID NO: 184.

[0038] In a further aspect of the present invention there is provided an antigen binding protein as herein described, further comprising one or more of CDRH1 of SEQ ID NO: 182, CDRH2: SEQ ID NO: 183: CDRL1: SEQ ID NO: 185, CDRL2: SEQ ID NO: 186 and / or CDRL3: SEQ ID NO: 187 and or variants thereof.

[0039] In a still further embodiment the antigen binding protein comprises CDRH3 of SEQ ID NO:3:CDRH2:SEQ ID NO:2:CDRH1 of SEQ ID NO:1:CDRL1:SEQ ID NO:4:CDRL2:SEQ ID NO:5 and CDRL3:SEQ ID NO:6.

[0040] In a still further embodiment the antigen binding protein comprises CDRH3 of SEQ ID NO:184: CDRH2: SEQ ID NO:183: CDRH1 of SEQ ID NO:182: CDRL1: SEQ ID NO:185: CDRL2: SEQ ID NO:186 and CDRL3: SEQ ID NO:187.

[0041] In one aspect of the invention, the antigen binding protein has enhanced effector function. In another aspect, the antigen binding protein is conjugated to a cytotoxic agent. In yet a further embodiment, the antigen binding protein both has enhanced effector function and is conjugated to a cytotoxic agent.

[0042] Antigen-binding proteins of the present invention may comprise heavy and light chain variable regions of the present invention, which may be formatted into the structure of a native antibody or a functional fragment or equivalent thereof. Thus, antigen-binding proteins of the present invention may comprise VH regions of the present invention formatted into a full-length antibody, a (Fab')2 fragment, a Fab fragment, or equivalents thereof (e.g., scFV, bibody, tribody, or tetrabody, tandab, etc.) when paired with an appropriate light chain. The antibody may be IgG1, IgG2, IgG3, or IgG4, or IgM, IgA, IgE, or IgD, or modified variants thereof. The constant domain of the antibody heavy chain can be selected accordingly. The light chain constant domain may be a kappa or lambda constant domain. Furthermore, antigen-binding proteins may include modifications of all classes, e.g., IgG dimers, Fc variants that no longer bind Fc receptors or no longer mediate C1q binding. The antigen binding protein may also be a chimeric antibody of the type described in WO86 / 01533 which comprises an antigen binding region and a non-immunoglobulin region.

[0043] The constant region is selected according to any functionality required, for example IgG1 can demonstrate lytic capacity by binding complement and / or mediate ADCC (antibody dependent cellular cytotoxicity).

[0044] The antigen binding proteins of the present invention are derived from murine antibodies having the variable regions set out in SEQ ID NO:7 and SEQ ID NO:9 or their non-murine equivalents, such as rat, human, chimeric or humanized variants thereof. For example they are derived from antibodies having the variable heavy chain sequences set out in SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:29 and / or the variable light chain sequences set out in SEQ ID NO:31, SEQ ID NO:33 and / or SEQ ID NO:35.

[0045] In another embodiment, the antigen binding protein of the present invention is derived from an antibody having the variable heavy chain sequence set forth in SEQ ID NO: 116 or SEQ ID NO: 118 and / or the variable light chain sequence set forth in SEQ ID NO: 120 or SEQ ID NO: 122.

[0046] In another embodiment, the antigen binding protein of the present invention is derived from an antibody having the variable heavy chain sequence set forth in SEQ ID NO:140 and / or the variable light chain sequence set forth in SEQ ID NO:144.

[0047] In one aspect of the invention there is provided an antigen binding protein comprising an isolated heavy chain variable domain selected from any one of the following: SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:116 or SEQ ID NO:118.

[0048] In another aspect of the invention there is provided an antigen binding protein comprising an isolated light chain variable domain selected from any one of the following: SEQ ID NO:31, SEQ ID NO:33 or SEQ ID NO:35, SEQ ID NO:120 or SEQ ID NO:122.

[0049] In a further aspect of the invention there is provided an antigen binding protein comprising an isolated heavy chain variable domain selected from any one of the following: SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27 and SEQ ID NO:29 and an isolated light chain variable domain selected from any one of the following: SEQ ID NO:31, SEQ ID NO:33 and / or SEQ ID NO:35.

[0050] In one aspect, the antigen binding protein of the present invention comprises a heavy chain variable region encoded by SEQ ID NO: 23 and a light chain variable region encoded by SEQ ID NO: 31. In one aspect, the antigen binding protein of the present invention comprises a heavy chain variable region encoded by SEQ ID NO: 27 and a light chain variable region encoded by SEQ ID NO: 31. In one aspect, the antigen binding protein of the present invention comprises a heavy chain variable region encoded by SEQ ID NO: 29 and a light chain variable region encoded by SEQ ID NO: 31.

[0051] In one aspect, the antigen binding protein of the present invention comprises a heavy chain variable region encoded by SEQ ID NO:116 and a light chain variable region encoded by SEQ ID NO:120.

[0052] In one aspect, the antigen binding protein of the present invention comprises a heavy chain variable region encoded by SEQ ID NO:118 and a light chain variable region encoded by SEQ ID NO:122.

[0053] In one aspect there is provided a polynucleotide encoding an isolated variable heavy chain, wherein the polynucleotide comprises SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16 or SEQ ID NO:18 or SEQ ID NO:20 or SEQ ID NO:22 or SEQ ID NO:24 or SEQ ID NO:26 or SEQ ID NO:28 or SEQ ID NO:30 or SEQ ID NO:117 or SEQ ID NO:119 or SEQ ID NO:141.

[0054] In one aspect there is provided a polynucleotide encoding an isolated variable light chain, wherein the polynucleotide comprises SEQ ID NO:32 or SEQ ID NO:34 or SEQ ID NO:36 or SEQ ID NO:121 or SEQ ID NO:123 or SEQ ID NO:145.

[0055] In a further aspect, there is provided a polynucleotide encoding an isolated variable heavy chain, wherein the polynucleotide comprises SEQ ID NO:24 or SEQ ID NO:28 or SEQ ID NO:30, and a polynucleotide encoding an isolated variable light chain, wherein the polynucleotide comprises SEQ ID NO:32 or SEQ ID NO:34.

[0056] In a still further aspect, there is provided a polynucleotide encoding an isolated variable heavy chain, said polynucleotide comprising SEQ ID NO: 24, and a polynucleotide encoding an isolated variable light chain, said polynucleotide comprising SEQ ID NO: 32.

[0057] In a still further aspect, there is provided a polynucleotide encoding an isolated variable heavy chain, said polynucleotide comprising SEQ ID NO: 117, and a polynucleotide encoding an isolated variable light chain, said polynucleotide comprising SEQ ID NO: 121.

[0058] In a still further aspect, there is provided a polynucleotide encoding an isolated variable heavy chain, said polynucleotide comprising SEQ ID NO: 119, and a polynucleotide encoding an isolated variable light chain, said polynucleotide comprising SEQ ID NO: 123.

[0059] In a still further aspect, there is provided a polynucleotide encoding an isolated variable heavy chain, said polynucleotide comprising SEQ ID NO: 141, and a polynucleotide encoding an isolated variable light chain, said polynucleotide comprising SEQ ID NO: 145.

[0060] In a further embodiment, the antigen binding protein may comprise any one of the variable heavy chains described herein in combination with any one of the light chains described herein.

[0061] In one aspect the antigen binding protein is an antibody or antigen binding fragment thereof comprising one or more CDRs according to the invention as described herein or one or both of the heavy or light chain variable domains according to the invention as described herein. In one embodiment the antigen binding protein binds to primate BCMA. In one such embodiment the antigen binding protein further binds to non-human primate BCMA, for example cynomolgus macaque BCMA.

[0062] In another embodiment, the antigen binding protein is selected from the group consisting of a dAb, Fab, Fab', F(ab')2, Fv, diabody, triabody, tetrabody, miniantibody and minibody.

[0063] In one aspect of the invention the antigen binding protein is a humanized or chimeric antibody, in a further aspect the antibody is humanized.

[0064] In one embodiment, the antibody is a monoclonal antibody.

[0065] In one aspect of the invention there is provided an antibody having a heavy chain sequence as set forth in SEQ ID NO: 55 or SEQ ID NO: 59 or SEQ ID NO: 61.

[0066] In one aspect of the invention there is provided an antibody having a light chain sequence as set forth in SEQ ID NO: 63 or SEQ ID NO: 65.

[0067] In a further aspect of the invention there is provided an antibody having a heavy chain sequence of SEQ ID NO: 55 and a light chain sequence as set forth in SEQ ID NO: 63.

[0068] In one embodiment there is provided an antigen binding protein which competes with the antigen binding proteins of the invention described herein. In one such embodiment there is therefore provided an antigen binding protein which competes with an antigen binding protein comprising the heavy chain variable sequence of SEQ ID NO: 23 and the light chain variable region of SEQ ID NO: 31.

[0069] In a further embodiment there is therefore provided an antigen binding protein which competes with an antigen binding protein comprising a heavy chain variable sequence selected from one of SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:116, SEQ ID NO:118 and SEQ ID NO:140 and a light chain variable region selected from one of SEQ ID NO:31, SEQ ID NO:120, SEQ ID NO:122 and SEQ ID NO:144.

[0070] In another aspect, the antigen binding protein binds to human BCMA with high affinity, for example when measured by Biacore, the antigen binding protein binds to human BCMA with an affinity of 20 nM or less, or an affinity of 15 nM or less, or an affinity of 5 nM or less, or an affinity of 1000 pM or less, or an affinity of 500 pM or less, or an affinity of 400 pM or less, or 300 pM or less, or for example an affinity of about 120 pM. In a further embodiment, the antigen binding protein binds to human BCMA as measured by Biacore, between about 100 pM and about 500 pM, or between about 100 pM and about 400 pM, or between about 100 pM and about 300 pM. In one embodiment of the invention, the antigen binding protein binds to BCMA with an affinity of less than 150 pM. In one such embodiment, this is measured by Biacore, for example as described in Example 4.

[0071] In another aspect, the antigen binding protein binds to human BCMA and neutralises the binding of the ligands BAFF and / or APRIL to the BCMA receptor in a cell neutralisation assay, wherein the antigen binding protein has an IC50 of between about 1 nM and about 500 nM, or between about 1 nM and about 100 nM, or between about 1 nM and about 50 nM, or between about 1 nM and about 25 nM, or between about 5 nM and about 15 nM. In a further embodiment of the invention, the antigen binding protein binds to BCMA and neutralises BCMA in a cell neutralisation assay, wherein the antigen binding protein has an IC50 of about 10 nM.

[0072] In one such embodiment, this is measured by a cell neutralization assay, eg, as described in Example 4.6.

[0073] Antigen binding proteins, such as antibodies of the present invention, can be produced by transfecting host cells with an expression vector comprising a coding sequence for the antigen binding protein of the present invention. Expression vectors or recombinant plasmids are produced by incorporating these coding sequences for the antigen binding protein operably linked to conventional regulatory control sequences capable of controlling replication and expression in the host cell and / or secretion from the host cell. Regulatory sequences include promoter sequences, such as the CMV promoter, and signal sequences, which may be derived from other known antibodies. Similarly, a second expression vector can be produced carrying DNA sequences encoding complementary antigen binding protein light or heavy chains. In certain embodiments, this second expression vector is identical to the first, except that, to the extent possible, the coding sequences and selectable markers are associated to ensure that each polypeptide chain is functionally expressed. Alternatively, the heavy and light chain coding sequences for the antigen binding protein may be present on a single vector.

[0074] The selected host cells are co-transfected by conventional techniques with both the first and second vectors (or simply transfected with a single vector) to generate transfected host cells of the invention containing both recombinant or synthetic light and heavy chains. The transfected cells are then cultured by conventional techniques to produce the engineered antigen binding proteins of the invention. Antigen binding proteins containing both recombinant heavy and / or light chains are screened from the culture by a suitable assay, such as ELISA or RIA. Similar conventional techniques can be used to construct other antigen binding proteins.

[0075] Those skilled in the art will be able to select appropriate vectors for the cloning and subcloning steps utilized in the methods and construction of the compositions of the present invention. For example, the conventional pUC series of cloning vectors may be used. One vector, pUC19, is commercially available from suppliers such as Amersham (Buckinghamshire, UK) or Pharmacia (Uppsala, Sweden). Furthermore, any vector that replicates easily, has multiple cloning sites and selectable genes (e.g., antibiotic resistance), and is easily manipulated may be used for cloning. Thus, the choice of cloning vector is not a limiting factor in the present invention.

[0076] Expression vectors may also be characterized by genes suitable for amplifying expression of heterologous DNA sequences, such as the mammalian dihydrofolate reductase gene (DHFR). Other vector sequences include poly(A) signal sequences such as those derived from bovine growth hormone (BGH) and the beta globin promoter sequence (betaglobin). Expression vectors useful herein can be synthesized by techniques well known to those skilled in the art.

[0077] The components of such vectors, such as replicons, selection genes, enhancers, promoters, signal sequences, etc., may be obtained from commercial or natural sources, or can be synthesized by known procedures for use in driving the expression and / or secretion of recombinant DNA products in a selected host. Other suitable expression vectors, many of which are known in the art for mammalian, bacterial, insect, yeast, and fungal expression, can also be selected for this purpose.

[0078] The present invention also encompasses cell lines transfected with recombinant plasmids containing the coding sequences of the antigen binding proteins of the present invention. Host cells useful for cloning and other manipulations of these cloning vectors are also conventional. However, cells from various strains of E. coli may also be used for replication of the cloning vectors and other steps in the construction of the antigen binding proteins of the present invention.

[0079] Suitable host cells or cell lines for expression of the antigen binding proteins of the invention include mammalian cells such as NS0, Sp2 / 0, CHO (e.g., DG44), COS, HEK, fibroblasts (e.g., 3T3), and myeloma cells, for example, it can be expressed in CHO or myeloma cells. Human cells may also be used, allowing the molecule to be modified with a human glycosylation pattern.

[0080] Alternatively, other eukaryotic cell lines may be utilized. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening, and production and purification of the products are known in the art. See, e.g., Sambrook et al., cited above.

[0081] Bacterial cells can be useful as suitable host cells for the expression of recombinant Fabs or other embodiments of the present invention (see, e.g., Pluckthun, A., Immunol. Rev., 130:151-188 (1992)). However, due to the tendency of proteins expressed in bacterial cells to be in an unfolded or improperly folded or non-glycosylated form, any recombinant Fabs produced in bacterial cells must be screened for retention of antigen-binding ability. If the molecule expressed by the bacterial cell is produced in a properly folded form, the bacterial cell is a desirable host, or in an alternative embodiment, the molecule can be expressed in a bacterial host and then subsequently refolded. For example, various strains of E. coli used for expression are well known as host cells in the field of biotechnology. Various strains of B. subtilis, Streptomyces, other bacilli, etc., can also be used in the present invention.

[0082] If desired, strains of yeast cells known to those skilled in the art are also available as host cells, and insect cells, such as Drosophila and Lepidoptera, and viral expression systems. See, e.g., Miller et al., Genetic Engineering, 8:277-298, Plenum Press (1986) and references cited therein.

[0083] The general methods by which vectors may be constructed, the transfection methods required to produce the host cells of the present invention, and the culture methods required to produce the antigen-binding proteins of the present invention from such host cells may all be conventional. Typically, the culture methods of the present invention are serum-free culture methods, usually by culturing cells in serum-free suspension. Similarly, once produced, the antigen-binding proteins of the present invention can be purified from the cell culture contents according to standard procedures in the art, including ammonia-16 eroxidi precipitation, affinity columns, column chromatography, gel electrophoresis, and the like. Such techniques are within the skill of the art and are not intended to limit the present invention. For example, the preparation of modified antibodies is described in WO99 / 58679 and WO96 / 16990.

[0084] Yet another method of expressing antigen binding proteins may utilize expression in transgenic animals as described in U.S. Patent No. 4,873,316, which relates to an expression system using an animal casein promoter that, when transgenically incorporated into a mammal, enables the female to produce the desired recombinant protein in her milk.

[0085] In a further embodiment of the invention, there is provided a method of producing an antibody of the invention, the method comprising culturing a host cell transformed or transfected with a vector encoding the light chain and / or the heavy chain of an antibody of the invention, and recovering the antibody produced thereby.

[0086] According to the present invention, there is provided a method for producing an anti-BCMA antibody of the present invention that binds to and neutralizes the activity of human BCMA, comprising the steps of: providing a first vector encoding a heavy chain of an antibody; providing a second vector encoding the light chain of the antibody; transforming a mammalian host cell (e.g., CHO) with the first and second vectors; culturing the host cells of step (c) under conditions conducive to secretion of antibody from the host cells into the medium; recovering the secreted antibodies of step (d); A method is provided, comprising:

[0087] Once expressed by the desired method, the antibody is then tested for in vitro activity using an appropriate assay. Currently, traditional ELISA assay formats are utilized to assess the qualitative and quantitative binding of antibodies to BCMA. Additionally, other in vitro activity assays can be used to verify neutralizing efficacy prior to subsequent human clinical studies, which may be performed to assess the persistence of antibodies in the body despite normal clearance mechanisms.

[0088] Dosage and duration of treatment relate to the relevant duration of the molecules of the invention in the human circulation and may be adjusted by one skilled in the art depending on the condition being treated and the patient's overall health. It is also contemplated that repeated administration (e.g., once per week, or once every two weeks, or once every three weeks) over an extended period of time (e.g., 4-6 months) may be required to achieve maximal therapeutic efficacy.

[0089] In one embodiment of the present invention there is provided a recombinantly transformed, transfected or transduced host cell comprising at least one expression cassette, for example where the expression cassette comprises a polynucleotide encoding the heavy chain of an antigen binding protein according to the invention as described herein and further comprises a polynucleotide encoding the light chain of an antigen binding protein according to the invention as described herein, or where there are two expression cassettes, the first encoding the light chain and the second encoding the heavy chain. For example in one embodiment a first expression cassette comprises a polynucleotide encoding the heavy chain of an antigen binding protein comprising a constant region or an antigen binding fragment thereof linked to a constant region according to the invention as described herein, and further comprises a second cassette comprising a polynucleotide encoding the light chain of an antigen binding protein comprising a constant region or an antigen binding fragment thereof linked to a constant region according to the invention as described herein, for example the first expression cassette comprises a polynucleotide encoding a heavy chain selected from SEQ ID NO: 56 or SEQ ID NO: 60 or SEQ ID NO: 62, and the second expression cassette comprises a polynucleotide encoding a light chain selected from SEQ ID NO: 64 or SEQ ID NO: 66.

[0090] In another embodiment of the invention, a stably transformed host cell is provided comprising a vector comprising one or more expression cassettes encoding the heavy and / or light chain of an antibody comprising a constant region or antigen-binding fragment thereof linked to a constant region described herein. For example, such a host cell may comprise a first vector encoding a light chain and a second vector encoding a heavy chain, e.g., the first vector encoding a heavy chain selected from SEQ ID NO:55, or SEQ ID NO:59, or SEQ ID NO:61, and the second vector encoding a light chain, e.g., the light chain of SEQ ID NO:63 or SEQ ID NO:65. In one such example, the first vector encodes a heavy chain selected from SEQ ID NO:55, and the second vector encodes a light chain, e.g., the light chain of SEQ ID NO:63.

[0091] In another embodiment of the present invention there is provided a host cell according to the invention as described herein, wherein the cell is eukaryotic, for example where the cell is mammalian. Examples of such cell lines include CHO or NS0.

[0092] In another embodiment of the present invention, there is provided a method for producing an antibody comprising a constant region or an antigen-binding fragment thereof linked to a constant region according to the invention described herein, the method comprising culturing a host cell in a medium, e.g., a serum-free medium.

[0093] In another embodiment of the invention, there is provided a method according to the invention described herein, wherein the antibody is further purified to at least 95% or more (e.g., 98% or more) relative to serum-free medium containing the antibody.

[0094] In yet another embodiment, a pharmaceutical composition is provided comprising the antigen binding protein and a pharmaceutically acceptable carrier.

[0095] In another embodiment of the present invention, there is provided a kit of parts comprising a composition according to the invention as described herein together with written instructions for use.

[0096] The mode of administration of the therapeutic agents of the present invention may be any suitable route that delivers the agent to a host. The antigen-binding proteins and pharmaceutical compositions of the present invention are particularly useful for parenteral administration, i.e., subcutaneously (sc), intrathecally, intraperitoneally, intramuscularly (im) or intravenously (iv). In one such embodiment, the antigen-binding proteins of the present invention are administered intravenously or subcutaneously.

[0097] Therapeutic agents of the present invention can be prepared as pharmaceutical compositions containing an effective amount of an antigen-binding protein of the present invention as an active ingredient in a pharmaceutically acceptable carrier. In one embodiment, a prophylactic agent of the present invention is an aqueous suspension or solution containing the antigen-binding protein in a form ready for injection. In one embodiment, the suspension or solution is buffered at physiological pH. In one embodiment, a composition for parenteral administration comprises a solution of an antigen-binding protein of the present invention, or a cocktail thereof, dissolved in a pharmaceutically acceptable carrier. In one embodiment, the carrier is an aqueous carrier. A variety of aqueous carriers may be utilized, such as 0.9% saline, 0.3% glycine, etc. These solutions may be sterile and generally free of particulate matter. These solutions can be sterilized by conventional, well-known sterilization techniques (e.g., filtration). The composition may contain pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, as needed to achieve appropriate physiological conditions. The concentration of the antigen-binding protein of the present invention in such pharmaceutical formulations may vary widely, i.e., from less than about 0.5% by weight, typically or at least about 1% by weight, up to about 15 or 20% by weight, and will be selected primarily based on fluid volume, viscosity, etc., depending on the particular mode of administration selected.

[0098] Thus, a pharmaceutical composition of the invention for intravenous injection can be constructed to contain about 250 ml of sterile Ringer's solution and about 1 to about 30 or 5 mg to about 25 mg of an antigen-binding protein of the invention per ml of Ringer's solution. Actual methods for preparing parenterally administrable compositions will be known or will become apparent to those skilled in the art and are described, for example, in Remington's Pharmaceutical Science, 15 th ed., Mack Publishing Company, Easton, Pennsylvania. The preparation of intravenously administrable antigen-binding protein formulations of the present invention is described in detail in Lasmar U and Parkins D, "The formulation of Biopharmaceutical products," Pharma.Sci.Tech.Today, pages 129-137, Vol. 3(3 rdAkers, MJ "Excipient-Drug interactions in Parenteral Formulations" J.Pharm Sci 91(2002) 2283-2300, Imamura, K et al "Effects of types of sugar on stabilization of Protein in the dried state", J Pharm Sci 92(2003)266-274, Izuts, Kkojima, S. "Excipient crystalinity and its protein-structure-stabilizing effect during freeze-drying", J Pharm. Pharmacol, 54(2002) 1033-1039, Johnson, R, "Mannitol-sucrose mixtures - versatile formulations for protein peroxidization 19g19n", J. Pharm. Sci, 91 (2002) 914-922, and Ha, E, Wang W, Wang Yj, "Peroxide formation in polysorbate 80 and protein stability", J. Pharm. Sci, 91, 2252-2264, (2002) (the entire contents of which are incorporated herein by reference and the reader is expressly referred to).

[0099] In one embodiment, the therapeutic agent of the present invention, when in a pharmaceutical formulation, is present in a unit dosage form. A suitable therapeutically effective dose can be easily determined by one skilled in the art. An appropriate dose can be calculated for a patient based on the patient's weight. For example, an appropriate dose may be in the range of about 0.1 to about 20 mg / kg, e.g., about 1 to about 20 mg / kg, e.g., about 10 to about 20 mg / kg, or about 1 to about 15 mg / kg, e.g., about 10 to about 15 mg / kg, or e.g., 1 to 5 mg / kg. In one embodiment, the antibody is administered at 1 to 5 mg / kg every three weeks. For effective treatment of conditions such as multiple myeloma, SLE, or IPT in humans, an appropriate dose may be about 0.1 to about 1000 mg, for example, about 0.1 to about 500 mg, for example, about 500 mg, for example, about 0.1 to about 100 mg, or about 0.1 to about 80 mg, or about 0.1 to about 60 mg, or about 0.1 to about 40 mg, or for example, about 1 to about 100 mg, or about 1 to about 50 mg of the antigen-binding protein of the present invention, which can be administered parenterally, for example, subcutaneously, intravenously, or intramuscularly. Such doses can be repeated, if necessary, at appropriate time intervals selected as necessary by a physician.

[0100] The antigen binding proteins described herein may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immunoglobulins and peroxides known in the art and are available for reconstitution.

[0101] In another aspect of the present invention there is provided an antigen binding protein as described herein for use in medicine.

[0102] In one aspect of the invention there is provided an antigen binding protein according to the invention as described herein for use in the treatment of rheumatoid arthitis, type 1 diabetes, multiple sclerosis or psoriasis, said method comprising the step of administering to said patient a therapeutically effective amount of the antigen binding protein as described herein.

[0103] In one embodiment of the invention, there is provided a method for treating cancer in a human, comprising administering to said human an antigen binding protein that specifically binds to BCMA. In some instances, the antigen binding protein is part of an immunoconjugate.

[0104] In another embodiment of the present invention, the patient develops neutralizing antibodies to recombinant protein replacement therapy, and the patient is treated with multiple myeloma (MM), chronic lymphocytic leukemia (CLL), non-secretory multiple myeloma, smoldering multiple myeloma, or other leukemia-associated ... There is provided an antigen binding protein according to the invention as described herein for use in the treatment of a B cell mediated or plasma cell mediated disease or antibody mediated disease or disorder selected from: myeloma, monoclonal gammopathy of undetermined significance (MGUS), solitary plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenström's macroglobulinemia, plasma cell leukemia, primary amyloidosis (AL), heavy chain disease, systemic lupus erythematosus (SLE), POEMS syndrome / osteosclerotic myeloma, Type I and II cryoglobulinemia, light chain deposition disease, Goodpasture's syndrome, idiopathic thrombocytopenic purpura (ITP), acute glomerulonephritis, pemphigus and pemphigoid disorders, and epidermolysis bullosa acquisita, or any non-Hodgkin's lymphoma B cell leukemia or Hodgkin's lymphoma (HL) or any disease with BCMA expression, said method comprising the step of administering to said patient a therapeutically effective amount of the antigen binding protein as described herein.

[0105] B cell disorders can be divided into defects in B cell development / immunoglobulin production (immunodeficiencies) and excessive / uncontrolled proliferation (lymphomas, leukemias). As used herein, B cell disorders refer to both types of disease, and methods for treating B cell disorders using antigen binding proteins are provided.

[0106] In certain embodiments, the disease or disorder is selected from the group consisting of multiple myeloma (MM), chronic lymphocytic leukemia (CLL), solitary plasmacytoma (bone, extramedullary), Waldenstrom's macroglobulinemia.

[0107] In one aspect of the invention, the disease is multiple myeloma, smoldering multiple myeloma (SMM) or solitary plasmacytoma (bone, extramedullary).

[0108] In one aspect of the invention, the disease is multiple myeloma.

[0109] In one embodiment of the invention, the disease is systemic lupus erythematosus (SLE).

[0110] In one embodiment of the invention, the disease is idiopathic thrombocytopenic purpura (ITP).

[0111] Also provided is the use of the antigen binding proteins described herein in the manufacture of a medicament for treating the diseases and disorders described herein.

[0112] For example, in one aspect of the invention there is provided the use of an antigen binding protein as described herein for use in the treatment or prevention of diseases and disorders that respond to modulation (such as inhibition or blocking) of the interaction between BCMA and the ligands BAFF and APRIL.

[0113] In one aspect of the invention there is provided use of an antigen binding protein as described herein for use in the treatment or prevention of an antibody-mediated or plasma cell-mediated disease or disorder selected from rheumatoid arthritis, Type 1 Diabetes Mellitus, multiple sclerosis or psoriasis.

[0114] In another embodiment of the present invention, patients develop neutralizing antibodies to recombinant protein replacement therapy, including multiple myeloma (MM), chronic lymphocytic leukemia (CLL), monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), solitary plasmacytoma (bone, extramedullary), Waldenstrom's macroglobulinemia, primary amyloidosis (AL), heavy chain disease, systemic lupus erythematosus (SLE), POEMS syndrome / osteosclerotic myeloma, type I and type II cryoglobulinemia, There is provided a use of an antigen binding protein as described herein for use in the treatment or prophylaxis of an antibody mediated or plasma cell mediated disease or disorder selected from light chain deposition disease, Goodpasture's syndrome, idiopathic thrombocytopenic purpura (ITP), acute glomerulonephritis, pemphigus and pemphigoid disorders and epidermolysis bullosa acquisita, any non-Hodgkin's lymphoma and leukemia or any disease with BCMA expression, said method comprising the step of administering to said patient a therapeutically effective amount of an antigen binding protein as described herein.

[0115] In one aspect, the present invention relates to a method for treating rheumatoid arthritis, type 1 diabetes, multiple sclerosis or psoriasis, or multiple myeloma (MM), chronic lymphocytic leukemia (CLL), monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), solitary plasmacytoma (bone, extramedullary), Waldenstrom's macroglobulinemia, primary amyloidosis (AL), heavy chain disease, systemic lupus erythematosus (SLE), POEMS syndrome / osteosclerotic myeloma, type I and type II cryoglobulinemia, in which patients develop neutralizing antibodies to recombinant protein replacement therapy. In accordance with the present invention, there is provided a pharmaceutical composition comprising an antigen binding protein of the present invention or a functional fragment thereof and a pharmaceutically acceptable carrier for the treatment or prevention of an antibody mediated or plasma cell mediated disease or disorder selected from idiopathic thrombocytopenic purpura (ITP), light chain deposition disease, Goodpasture's syndrome, idiopathic thrombocytopenic purpura (ITP), acute glomerulonephritis, pemphigus and pemphigoid disorders and epidermolysis bullosa acquisita, any non-Hodgkin's lymphoma and leukemia or any disease with BCMA expression, said method comprising the step of administering to said patient a therapeutically effective amount of the antigen binding protein as described herein.

[0116] In another embodiment of the present invention there is provided a method of treating a human patient suffering from rheumatoid arthritis, type 1 diabetes, multiple sclerosis or psoriasis or an antibody-mediated or plasma cell-mediated disorder or disease in which the patient develops neutralising antibodies to recombinant protein replacement therapy, the method comprising the step of administering a therapeutically effective amount of an antigen binding protein according to the invention as described herein, e.g. a method of treating a human patient suffering from a selected antibody-mediated or plasma cell-mediated disease or disorder. In another embodiment of the present invention, patients develop neutralizing antibodies to recombinant protein replacement therapy, including multiple myeloma (MM), chronic lymphocytic leukemia (CLL), monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), solitary plasmacytoma (bone, extramedullary), Waldenstrom's macroglobulinemia, primary amyloidosis (AL), heavy chain disease, systemic lupus erythematosus (SLE), POEMS syndrome / osteosclerotic myeloma, type I and type II cryoglobulinemia, light chain deposition disease, Goodman syndrome, and others. There is provided an antigen binding protein according to the invention as described herein for use in the treatment of an antibody mediated or plasma cell mediated disease or disorder selected from Depasture's syndrome, idiopathic thrombocytopenic purpura (ITP), acute glomerulonephritis, pemphigus and pemphigoid disorders and epidermolysis bullosa acquisita, any non-Hodgkin's lymphoma and leukemia or any disease with BCMA expression, said method comprising the step of administering a pharmaceutical composition comprising an antigen binding protein according to the invention herein in combination with a pharmaceutically acceptable carrier.

[0117] In a further embodiment, a method of treating a human patient suffering from multiple myeloma (MM) is provided.

[0118] definition As used herein, the terms "cancer," "neoplasm," and "tumor" are used interchangeably and, in either the singular or plural, refer to cells that have undergone malignant transformation that renders them pathological to the host organism. Primary cancer cells can be readily distinguished from noncancerous cells by well-established techniques, particularly histological examination. As used herein, the definition of cancer cells includes not only primary cancer cells but also any cells derived from a cancer cell ancestor. This includes metastatic cancer cells, as well as in vitro cultures and cell lines derived from cancer cells. When referring to types of cancer that typically manifest as solid tumors, a "clinically detectable" tumor is one that is detectable based on the tumor mass and / or the expression of one or more cancer-specific antigens in a sample obtained from a patient, for example, by procedures such as computed tomography (CT) scan, magnetic resonance imaging (MRI), X-ray, ultrasound, or palpation during a physical exam. The tumor may also be a hematopoietic (or hematologic or hematological or blood-related) cancer, such as a cancer derived from blood cells or immune cells, which may be referred to as a "liquid tumor." Specific examples of clinical conditions based on hematological tumors include leukemias such as chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia and acute lymphocytic leukemia, plasma cell malignancies such as multiple myeloma, MGUS and Waldenstrom's macroglobulinemia, and lymphomas such as non-Hodgkin's lymphoma and Hodgkin's lymphoma.

[0119] The cancer may be any cancer in which an abnormal number of blast cells or unwanted cell proliferation is present or diagnosed as a hematological cancer, including both lymphoid and myeloid malignancies. Myeloid malignancies include, but are not limited to, acute myeloid (or myelocytic or myeloid or myeloblastic) leukemia (undifferentiated or differentiated), acute promyelocytic (or promyelogenous or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia, and megakaryocytic (or megakaryoblastic) leukemia. These leukemias are sometimes collectively referred to as acute myeloid (or myelocytic or myeloid) leukemia (AML). Myeloid malignancies also include myeloproliferative disorders (MPDs), including, but not limited to, chronic myelogenous (or myeloid) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and polycythemia vera (PCV). Myeloid malignancies also include myelodysplasia (or myelodysplastic syndromes or MDS), which may also be referred to as refractory anemia (RA), refractory anemia with excess blasts (RAEB), and refractory anemia with excess blasts in transformation (RAEBT), and myelofibrosis with or without primary myelofibrosis (MFS).

[0120] Hematopoietic cancers also include lymphoid malignancies, which can affect lymph nodes, spleen, bone marrow, peripheral blood, and / or extra-lymphatic sites. Lymphoid cancers include B-cell malignancies, including, but not limited to, B-cell non-Hodgkin's lymphoma (B-NHL). B-NHL may be indolent (or low-grade), intermediate-grade (or aggressive), or high-grade (highly aggressive). Indolent B-cell lymphomas include follicular lymphoma (FL), small lymphocytic lymphoma (SLL), marginal zone lymphoma (MZL), including nodal MZL, extranodal MZL, splenic MZL, and splenic MZL with villous lymphocytes, lymphoplasmacytic lymphoma (LPL), and mucosa-associated lymphoid tissue (MALT or extranodal marginal zone) lymphoma. Intermediate-grade B-NHL includes leukemia-related or non-related mantle cell lymphoma (MCL), diffuse large cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade 3B) lymphoma, and primary mediastinal lymphoma (PML). Aggressive B-NHL includes Burkitt lymphoma (BL), Burkitt-like lymphoma, small noncleaved cell lymphoma (SNCCL), and lymphoblastic lymphoma. Other B-NHLs include immunoblastic lymphoma (or immunocytoma), primary effusion, HIV-associated (or AIDS-associated) lymphoma, and post-transplant lymphoproliferative disorder (PTLD) or lymphoma. B-cell malignancies also include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenstrom's macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocyte (LGL) leukemia, acute lymphocytic (or lymphocytic or lymphoblastic) leukemia, and Castremann's disease. NHLs can also include T-cell non-Hodgkin's lymphoma (T-NHL), including, but not limited to, T-cell non-Hodgkin's lymphoma, not otherwise specified (NOS), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphoma (AILD), nasal natural killer (NK) cell / T-cell lymphoma, gamma / delta lymphoma, cutaneous T-cell lymphoma, mycosis fungoides, and Sézary syndrome.

[0121] Hematopoietic cancers also include Hodgkin lymphomas (or diseases), including classical Hodgkin lymphoma, nodular sclerosing Hodgkin lymphoma, mixed cytology Hodgkin lymphoma, lymphocyte-predominant (LP) Hodgkin lymphoma, nodular LP Hodgkin lymphoma, and lymphocytopenic Hodgkin lymphoma. Hematopoietic cancers also include plasma cell disorders or cancers, such as multiple myeloma (MM), including smoldering MM, monoclonal gammopathy of undetermined (or unknown or unclear) significance (MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenstrom's macroglobulinemia, plasma cell leukemia, and primary amyloidosis (AL). Hematopoietic cancers can also include other cancers of additional hematopoietic cells, including polymorphonuclear leukocytes (or neutrophils), eosinophils, dendritic cells, platelets, erythrocytes, and natural killer cells. Tissues containing hematopoietic cells, referred to herein as "hematopoietic cell tissues," include peripheral lymphoid tissues such as bone marrow, peripheral blood, thymus and spleen, lymph nodes, lymphoid tissues associated with mucous membranes (such as gut-associated lymphoid tissue), tonsils, Peyer's patches and appendages, and lymphoid tissues associated with other mucous membranes, e.g., the bronchial lining.

[0122] As used herein, the term "antigen binding protein" refers to antibodies, antibody fragments and other protein constructs capable of binding to and neutralizing human BCMA.

[0123] The terms Fv, Fc, Fd, Fab, or F(ab)2 are used with their standard meaning (see, e.g., Harlow et al., Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, (1988)).

[0124] The term "antibody" is used herein in a broad sense and specifically encompasses monoclonal antibodies (full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (eg, bispecific antibodies).

[0125] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising a population that are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen-binding site. Furthermore, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0126] "Chimeric antibody" refers to a type of engineered antibody in which portions of the heavy and / or light chain are identical to or homologous to corresponding sequences in antibodies from a particular donor antibody class or subclass, while the remainder of the chain(s) are identical to or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and in fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567 and Morrison et al. Proc. Natl. Acad. Sci. USA 81:6851-6855) (1984)).

[0127] "Humanized antibody" refers to a type of engineered antibody that has its CDRs derived from a non-human donor immunoglobulin, with the remaining immunoglobulin-derived portions of the molecule being derived from one (or more) human immunoglobulins. In addition, framework support residues can be modified to preserve binding affinity (see, e.g., Queen et al., Proc. Natl Acad Sci USA, 86:10029-10032 (1989); Hodgson et al., Bio / Technology, 9:421 (1991)). Suitable human acceptor antibodies may be selected from conventional databases, such as the KABAT® database, the Los Alamos database, and the Swiss Protein Database, based on their homology with the nucleotide and amino acid sequences of the donor antibody. Human antibodies characterized by homology (on an amino acid basis) with the framework regions of the donor antibody may be suitable to provide heavy chain constant and / or heavy chain variable framework regions for insertion of donor CDRs. A suitable acceptor antibody that can donate light chain constant or variable framework regions can be selected as well. It should be noted that the acceptor antibody heavy and light chains do not have to originate from the same acceptor antibody. The prior art describes several methods for producing such humanized antibodies (see, for example, EP-A-0239400 and EP-A-054951).

[0128] With respect to nucleic acids, the term "substantially identical" refers to two nucleic acids, or designated sequences thereof, that, when optimally aligned and compared, are identical in at least about 80% of the nucleotides, about 90% to about 95% of the nucleotides, or at least about 98% to about 99.5% of the nucleotides, with appropriate nucleotide insertions or deletions. Alternatively, substantial identity exists when the segments hybridize under selective hybridization conditions to the complement of the strand. "Identical," with respect to polynucleotides and polypeptides, as the case may be, refers to a comparison calculated using the algorithms provided in (1) and (2) below: (1) Identity to a polynucleotide is determined by multiplying the total number of nucleotides in a given sequence by an integer (divided by 100) that defines the percent identity, and then subtracting that product from the total number of nucleotides in the sequence; or: nn ≦ xn-(xn y) where nn is the number of nucleotide alterations, xn is the total number of nucleotides in a given sequence, y is 0.95 for 95%, 0.97 for 97%, or 1.00 for 100%, · is the multiplication operator symbol, and any non-integer product of xn and y is rounded down to the nearest integer before being subtracted from xn. Modifications of a polynucleotide sequence that encodes a polypeptide may result in nonsense, missense, or frameshift mutations in the coding sequence, thereby altering the polypeptide encoded by the polynucleotide after such modification.

[0129] (2) Identity for a polypeptide is determined by multiplying the total number of amino acids by an integer (divided by 100) that defines the percent identity, and then subtracting that product from said total number of amino acids; or: na ≦ xa-(xa·y) where na is the number of amino acid alterations, xa is the total number of amino acids in the sequence, y is 0.95 for 95%, 0.97 for 97%, or 1.00 for 100%, and · is the multiplication operator symbol; and any non-integer product of xa and y is rounded down to the nearest integer before subtracting it from xa. It is calculated by:

[0130] With respect to nucleotide and amino acid sequences, the term "identical" refers to the degree of identity between two nucleic acid or amino acid sequences when optimally aligned and compared for appropriate insertions or deletions.

[0131] "Isolated" means altered "by the hand of man" from its natural state, being changed or removed from its original environment, or both. For example, a polynucleotide or polypeptide that is naturally present in an organism is not "isolated," but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated," including, but not limited to, when such a polynucleotide or polypeptide is introduced back into a cell, even if the cell is of the same species or type (the cell from which the polynucleotide or polypeptide was isolated).

[0132] Throughout this specification and the appended claims, the terms "comprising" and "comprises" encompass "consisting of" and "consisting of," that is, these terms are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context permits.

[0133] The term "specifically binds", as used throughout the specification in reference to antigen binding proteins of the invention, means that the antigen binding protein binds to human BCMA (hBCMA) without binding or significantly binding to other human proteins. However, this term does not exclude the fact that antigen binding proteins of the invention may also be cross-reactive with other forms of BCMA, for example primate BCMA. For example, in one embodiment the antigen binding protein does not bind to TACI or BAFF-R.

[0134] As used throughout the specification with respect to antigen binding proteins of the invention, the term "inhibit" means that the biological activity of BCMA is reduced in the presence of an antigen binding protein of the invention compared to the activity of BCMA in the absence of such antigen binding protein. Inhibition may be due to, but is not limited to, one or more of blocking ligand binding, preventing the ligand from activating the receptor, and / or downregulating BCMA. Inhibition may also refer to binding of the antigen binding protein to BCMA and causing cell apoptosis or ADCC. Antibodies of the invention can neutralize the activity of the BCMA ligands BAFF and / or APRIL binding to BCMA. The level of neutralization can be measured in several ways, for example, by using the assay described in the Examples below, for example, in 4.4 in the H929 cell NFkB signaling assay. The BCMA ligands BAFF and APRIL can induce NFkB signaling and downstream events after binding to BCMA. Neutralization of BCMA in this assay is measured by assessing the ability of an anti-BCMA monoclonal antibody to inhibit NFkB induction driven by BAFF or APRIL.

[0135] If the antibody or antigen-binding fragment thereof is capable of neutralizing, this indicates inhibition of the interaction between human BAFF or APRIL and BCMA. Antibodies considered to have neutralizing activity against human BCMA have an IC50 of less than 30 micrograms / ml, or less than 20 micrograms / ml, or less than 10 micrograms / ml, or less than 5 micrograms / ml, or less than 1 microgram / ml, or less than 0.1 micrograms / ml in the H929 stimulation assay described in Example 4.4.

[0136] "CDR" is defined as the complementarity determining region amino acid sequences of an antibody, which are the hypervariable domains of the immunoglobulin heavy and light chains. There are three heavy chain and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, as used herein, "CDR" can refer to all three heavy chain CDRs, or all three light chain CDRs (or both all heavy chain and all light chain CDRs, as appropriate).

[0137] CDRs provide the majority of contact residues for antibody binding to an antigen or epitope. CDRs of interest in the present invention are derived from donor antibody variable heavy and light chain sequences and include analogs of naturally occurring CDRs that also share or retain the same antigen-binding specificity and / or neutralizing ability as the donor antibody from which they are derived.

[0138] The CDR sequences of antibodies can be determined by the Kabat numbering system (Kabat et al., (Sequences of proteins of Immunological Interest NIH, 1987) or they can be determined using the Chothia numbering system (Al-Lazikani et al., (1997) JMB 273, 927-948), the contact point definition method (MacCallum RM and Martin ACR and Thornton JM, (1996), Journal of Molecular Biology, 262(5), 732-745), or any other established method for numbering residues in antibodies and determining CDRs known to those skilled in the art.

[0139] Other numbering conventions for CDR sequences available to those skilled in the art include the "AbM" (University of Bath) and "contact" (University College London) methods. The minimum overlap region using at least two of the Kabat, Chothia, AbM, and contact methods can be determined to provide a "minimum binding unit." The minimum binding unit may be a subportion of a CDR.

[0140] Table A below presents one definition using each numbering convention for each CDR or binding unit. The Kabat numbering scheme is used in Table X to number the variable domain amino acid sequences. It should be noted that some of the CDR definitions may vary depending on the particular publication used.

[0141] TIFF2026010000000002.tif53161Throughout this specification, amino acid residues in antibody sequences are numbered according to the Kabat scheme. Similarly, the terms "CDR," "CDRL1," "CDRL2," "CDRL3," "CDRH1," "CDRH2," and "CDRH3" follow the Kabat numbering system as set forth in Kabat et al., Sequences of proteins of Immunological Interest, NIH, 1987.

[0142] The term "variant" refers to at least one, two, or three amino acid changes in a sequence. These amino acid changes may be deletions, substitutions, or additions, but are preferably substitutions. In one such embodiment, the substitutions are conservative substitutions.

[0143] In an alternative embodiment, the variant sequence contains at least one substitution while retaining the canonical identity of the antigen binding protein.

[0144] Complementarity-determining regions (CDRs) L1, L2, L3, H1, and H2 tend to structurally exhibit one of a finite number of main-chain conformations. A CDR of a particular canonical structural class is defined by both the length of the CDR and the loop packing (structurally determining residues or SDRs) determined by residues at key positions in both the CDRs and framework regions. Martin and Thornton (1996, J Mol Biol 263:800-815) developed an automated method for defining canonical templates of "critical residues." Cluster analysis is used to define canonical classes for sets of CDRs, and canonical templates are then identified by analyzing buried hydrophobic hydrogen-bonding residues and, for example, conserved glycines. CDRs of an antibody sequence can be assigned to a canonical class by comparing the sequence to the critical residue templates and scoring each template using an identity or similarity matrix.

[0145] The terms "VH" and "VL" are used herein to refer to the heavy and light chain variable domains, respectively, of an antibody.

[0146] As used herein, the term "domain" refers to a folded protein structure that has a tertiary structure independent of the rest of the protein. Generally, domains are responsible for discrete functional properties of a protein and can often be added, removed, or transferred to other proteins without losing function of the remainder of the protein and / or domain. A "single antibody variable domain" is a folded polypeptide domain that comprises sequences characteristic of antibody variable domains. Thus, it includes complete antibody variable domains and modified variable domains (e.g., in which one or more loops have been replaced by sequences not characteristic of antibody variable domains), or antibody variable domains that have been truncated or contain N- or C-terminal extensions, as well as folded fragments of variable domains that retain at least the binding activity and specificity of the full-length domain.

[0147] The term "immunoglobulin single variable domain" refers to an antibody variable domain (VH, VHH, VL) that specifically binds to an antigen or epitope independently of a different V region or domain. Immunoglobulin single variable domains can exist in formats (e.g., homo- or heteromultimers) with other, different variable regions or domains, where the other regions or domains are not required for antigen binding by the single immunoglobulin variable domain (i.e., the immunoglobulin single variable domain binds to the antigen independently of the new variable domain). A "domain antibody" or "dAb," as the term is used herein, is the same as an "immunoglobulin single variable domain" that is capable of binding to an antigen. Immunoglobulin single variable domains may be human antibody variable domains, but may also include single antibody variable domains from other species, such as rodent (e.g., as disclosed in WO 00 / 29004), nurse shark, and camel VHH dAbs. Camelid VHHs are immunoglobulin single variable domain polypeptides derived from species including camels, llamas, alpacas, dromedaries, and guanacos, which naturally produce heavy chain antibodies devoid of light chains. Such VHH domains can be humanized using standard methods available to those skilled in the art, and such domains are also considered "domain antibodies" according to the present invention. As used herein, "VH" includes camelid VHH domains. NARVs are another type of immunoglobulin single variable domain identified in cartilaginous fish, including nurse sharks. These domains are also known as novel antigen receptor variable regions (commonly abbreviated as V(NAR) or NARV). For further details, see Mol. Immunol. 44, 656-665 (2006) and U.S. Patent Application No. 20050043519A.

[0148] The term "epitope binding domain" refers to a domain that specifically binds to an antigen or epitope independently from a different V region or domain, which may be a domain antibody (dAb), e.g. a human, camel or shark immunoglobulin single variable domain, or a protein A-derived molecule such as CTLA-4 (Evibody), lipocalin, the Z-domain of protein A (Affibody, SpA), the A-domain (Avimer / Maxibody), heat shock proteins such as GroEI and GroES, 29eroxidise 29g (trans-body), ankyrin repeat proteins (DARPins), peptide aptamers, C-type lectin domains (tetranectin), human gamma-crystallin and human ubiquitin (affilin), PDZ domains, the human protease inhibitor scorpion toxin Kunitz The domain may be a derivative of a scaffold selected from the group consisting of a toxinkunitz-type domain, and fibronectin (adnectin), which has been subjected to protein engineering to obtain binding to a ligand other than the natural ligand.

[0149] CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) is a CD28-family receptor expressed primarily on CD4+ T cells. Its extracellular domain has a variable domain-like Ig fold. The loops corresponding to the CDRs of antibodies can be replaced with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as Ebibodies. For further details, see Journal of Immunological Methods 248(1-2), 31-45 (2001).

[0150] Lipocalins are a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bilins, retinoids, and lipids. They have a rigid β-sheet secondary structure with many loops at the open end of a cone-shaped structure, which can be engineered to bind to various target antigens. Anticalins are between 160 and 180 amino acids in size and are derived from lipocalins. For further details, see Biochim Biophys Acta 1482:337-350 (2000), U.S. Patent No. 7,250,297 B1, and U.S. Patent Application Publication No. 20070224633.

[0151] Affibodies are scaffolds derived from Staphylococcus aureus protein A that can be engineered to bind antigens. This domain consists of a three-helical bundle of approximately 58 amino acids. Libraries have been generated by randomization of surface residues. For further details, see Protein Eng. Des. Sel. 17, 455-462 (2004) and EP 1641818 A1.

[0152] Avimers are multidomain proteins derived from the A-domain scaffold family. Native domains of approximately 35 amino acids adopt specific disulfide-bonded structures. Diversity is generated by the natural mixture of mutations exhibited by the A-domain family. For further details, see Nature Biotechnology 23(12), 1556-1561 (2005) and Expert Opinion on Investigational Drugs 16(6), 909-917 (June 2007).

[0153] Transferrin is a monomeric serum transport glycoprotein. Transferrin can be engineered to bind to various target antigens by inserting peptide sequences into permissive surface loops. Examples of engineered transferrin scaffolds include transbodies. For further details, see J. Biol. Chem 274, 24066-24073 (1999).

[0154] Designed ankyrin repeat proteins (DARPins) are derived from ankyrins, a family of proteins that mediate the linkage of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif composed of two α-helices and a β-turn. They can be engineered to bind to various target antigens by randomizing residues in the first α-helix and β-turn of each repeat. Their binding surface can be increased by increasing the number of molecules (a method of affinity maturation). For further details, see J. Mol. Biol. 332, 489-503 (2003), PNAS 100(4), 1700-1705 (2003), and J. Mol. Biol. 369, 1015-1028 (2007), and U.S. Patent Application Publication No. 20040132028A1.

[0155] Fibronectin is a scaffold that can be engineered to bind antigens. Adnectins consist of a backbone that replicates the natural amino acid sequence of the tenth domain of the 15 repeating units of human type III fibronectin (FN3). Three loops at one end of the β-sandwich can be engineered to enable Adnectins to specifically recognize therapeutic targets of interest. For further details, see Protein Eng. Des. Sel. 18, 435-444 (2005), U.S. Patent Application Publication No. 20080139791, WO2005056764, and U.S. Patent No. 6,818,418 B1.

[0156] Peptide aptamers are combinatorial recognition molecules composed of a constant scaffold protein, typically thioredoxin (TrxA), containing a constrained variable peptide loop inserted into the active site. For further details, see Expert Opin. Biol. Ther. 5, 783-797 (2005).

[0157] Microbodies are derived from naturally occurring microproteins that are 25-50 amino acids in length and contain 3-4 cysteine ​​bridges; examples of microproteins include KalataB1, conotoxins, and knottins. Microproteins have loops that can be engineered to contain up to 25 amino acids without affecting the overall folding of the microprotein. For further details on engineered knottin domains, see WO2008098796.

[0158] Other epitope-binding domains include proteins that have been used as scaffolds to engineer various target antigen-binding properties, including human gamma-crystallin and human ubiquitin (affilin), Kunitz-type domains of human protease inhibitors, the PDZ-domain of the Ras-binding protein AF-6, scorpion toxin (charybdotoxin), and the C-type lectin domain (tetranectin), which are reviewed in Chapter 7—Non-Antibody Scaffolds from the Handbook of Therapeutic Antibodies (2007, edited by Stefan Dubel) and Protein Science 15:14-27 (2006). The epitope-binding domains of the present invention may be derived from any of these alternative protein domains.

[0159] As used herein, the term "antigen-binding site" refers to a site on a protein capable of specifically binding to an antigen, which may be a single domain, such as an epitope-binding domain, or a paired VH / VL domain such as may be found in a standard antibody. In some embodiments of the invention, a single-chain Fv (ScFv) domain may provide the antigen-binding site.

[0160] The terms "mAbdAb" and "dAbmAb" are used herein to refer to the antigen binding proteins of the present invention. The two terms can be used interchangeably and are intended to have the same meaning when used herein.

[0161] As used herein, the term "antigen-binding protein" refers to antibodies, antibody fragments, such as domain antibodies (dAbs), ScFvs, Fabs, Fabs, and other protein constructs. An antigen-binding molecule may comprise at least one Ig variable domain, such as an antibody, domain antibody (dAb), Fab, Fab', F(ab')2, Fv, ScFv, diabody, mAbdAb, affibody, heteroconjugate antibody, or bispecific antibody. In one embodiment, the antigen-binding molecule is an antibody. In another embodiment, the antigen-binding molecule is a dAb, i.e., an immunoglobulin single variable domain, such as a VH, VHH, or VL, that specifically binds an antigen or epitope independently from different V regions or domains. Antigen-binding molecules can bind to two targets; i.e., they may be dual-targeting proteins. Antigen-binding molecules may also be a combination of an antibody and an antigen-binding fragment, such as one or more domain antibodies and / or one or more ScFvs linked to a monoclonal antibody. Antigen-binding molecules may also comprise non-Ig domains, for example domains that are derivatives of scaffolds selected from the group consisting of CTLA-4 (e.g., Epibibodies), lipocalins, Protein A-derived molecules such as the Z-domain of Protein A (affibodies, SpA), A-domains (avimers / maxibodies), heat shock proteins such as GroEI and GroES, 31eroxidized 31g (transbodies), ankyrin repeat proteins (DARPins), peptide aptamers, C-type lectin domains (tetranectins), human gamma-crystallin and human ubiquitin (affilins), PDZ domains, scorpion toxin Kunitz-type domains of human protease inhibitors, and fibronectin (adnectins), which have been subjected to protein engineering to obtain binding to OSM. As used herein, an "antigen-binding protein" is capable of antagonizing and / or neutralizing human OSM. In addition, antigen binding proteins can inhibit and block OSM activity by binding to OSM and preventing the natural ligand from binding to and / or activating the gp130 receptor.

[0162] As used herein, the term "effector function" refers to one or more of antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC)-mediated reactions, Fc-mediated phagocytosis, and antibody recycling via the FcRn receptor. For IgG antibodies, effector functions, including ADCC and ADCP, are mediated by the interaction of the heavy chain constant region with a family of Fcγ receptors present on the surface of immune cells. In humans, these include FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). The interaction between antigen-bound antigen-binding proteins and the formation of Fc / Fcγ complexes induces a wide range of effects, including cytotoxicity, immune cell activation, phagocytosis, and the release of inflammatory cytokines.

[0163] The interaction between the constant region of an antigen-binding protein and various Fc receptors (FcRs) is thought to mediate the effector functions of the antigen-binding protein. Significant biological effects can be the result of effector functions, particularly antibody-dependent cellular cytotoxicity (ADCC), complement fixation (complement-dependent cytotoxicity or CDC), and the half-life / clearance of the antigen-binding protein. Typically, the ability to mediate an effector function requires binding of the antigen-binding protein to an antigen, and not all antigen-binding proteins mediate all effector functions.

[0164] Effector function can be measured in a number of ways, including, for example, measuring ADCC effector function via FcγRIII binding to natural killer cells or via FcγRI binding to monocytes / macrophages. For example, antigen binding proteins of the invention can be assessed for ADCC effector function in a natural killer cell assay. Examples of such assays can be found in Shields et al., 2001 The Journal of Biological Chemistry, Vol. 276, pp. 6591-6604; Chappel et al., 1993 The Journal of Biological Chemistry, Vol. 268, pp. 25124-25131; and Lazar et al., 2006 PNAS, 103; 4005-4010.

[0165] Examples of assays for measuring CDC function include those described in 1995 J Imm Meth 184:29-38.

[0166] Some isotypes of human constant regions, particularly the IgG4 and IgG2 isotypes, essentially lack the functions of a) classical pathway complement activation and b) antibody-dependent cellular cytotoxicity. Depending on the desired effector properties, various modifications to the heavy chain constant region of the antigen-binding protein may be made. It has been reported separately that IgG1 constant regions containing specific mutations reduce binding to Fc receptors and therefore reduce ADCC and CDC (Duncan et al. Nature 1988, 332; 563-564; Lund et al. J. Immunol. 1991, 147; 2657-2662; Chappel et al. PNAS 1991, 88; 9036-9040; Burton and Woof, Adv. Immunol. 1992, 51, 1-84; Morgan et al., Immunology 1995, 86; 319-324; Hezareh et al., J. Virol. 2001, 75(24); 12161-12168).

[0167] In one embodiment of the present invention, there is provided an antigen binding protein comprising a constant region such that the antigen binding protein has reduced ADCC and / or complement activity or effector function. In one such embodiment, the heavy chain constant region may comprise a naturally occurring neutral constant region of the IgG2 or IgG4 isotype, or a mutated IgG1 constant region. Examples of suitable modifications are described in EP 0 307 434. One example includes the substitution of alanine residues at positions 235 and 237 (EU index numbering).

[0168] Human IgG1 constant regions containing specific mutations or altered glycosylation at residue Asn297 have also been described to enhance binding to Fc receptors. In some cases, these mutations have also been shown to enhance ADCC and CDC (Lazar et al. PNAS 2006, 103;4005-4010; Shields et al. J Biol Chem 2001, 276;6591-6604; Nechansky et al. Mol Immunol, 2007, 44;1815-1817).

[0169] In one embodiment of the invention, such mutations are at one or more of positions selected from 239, 332 and 330 (IgG1), or equivalent positions in other IgG isotypes. Examples of suitable mutations are S239D and 1332E and A330L. In one embodiment, the antigen binding protein of the invention described herein is mutated at positions 239 and 332, for example S239D and 1332E, or in a further embodiment, it is mutated at three or more positions selected from 239 and 332 and 330, for example S239D and 1332E and A330L (EU index numbering).

[0170] In an alternative embodiment of the present invention, there is provided an antigen-binding protein comprising a heavy chain constant region with an altered glycosylation profile such that the antigen-binding protein has enhanced effector function. For example, the antigen-binding protein has enhanced ADCC or enhanced CDC, or it has both enhanced ADCC and CDC effector function. Examples of suitable methods for producing antigen-binding proteins with altered glycosylation profiles are described in WO2003011878, WO2006014679 and EP1229125, all of which are applicable to the antigen-binding proteins of the present invention.

[0171] The present invention also provides a method for producing an antigen-binding protein according to the invention, comprising the steps of: a) culturing a recombinant host cell containing an expression vector comprising an isolated nucleic acid described herein, wherein the FUT8 gene encoding alpha-1,6-fucosyltransferase is inactivated in the recombinant host cell; b) recovering the antigen-binding protein; The present invention provides a method comprising:

[0172] Such methods for producing antigen-binding proteins can be carried out, for example, using the Potelligent™ technology system available from BioWa, Inc. (Princeton, NJ), in which CHOK1SV cells lacking a functional copy of the FUT8 gene produce monoclonal antibodies with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity that is increased relative to the same monoclonal antibodies produced in cells with a functional FUT8 gene. Aspects of the Potelligent™ technology system are described in U.S. Patent No. 7,214,775, U.S. Patent No. 6,946,292, WO0061739, and WO0231240, all of which are incorporated herein by reference. Those skilled in the art will also recognize other suitable systems.

[0173] In one embodiment of the present invention, there is provided an antigen binding protein comprising a chimeric heavy chain constant region, for example an antigen binding protein comprising a chimeric heavy chain constant region with at least one CH2 domain derived from IgG3, such that the antigen binding protein has enhanced effector function, for example it has enhanced ADCC or enhanced CDC, or enhanced ADCC and CDC functions. In one such embodiment, the antigen binding protein may comprise one CH2 domain derived from IgG3, or both CH2 domains may be derived from IgG3.

[0174] 1. A method of producing an antigen binding protein according to the invention, comprising: a) culturing a recombinant host cell comprising an expression vector comprising an isolated nucleic acid described herein, wherein the expression vector comprises a nucleic acid sequence encoding an Fc domain having IgG1 and IgG3 Fc domain amino acid residues; b) recovering the antigen-binding protein; A method is also provided, which includes:

[0175] Such methods for producing antigen-binding proteins can be carried out, for example, using the Complegent™ Technology system available from BioWa, Inc. (Princeton, NJ) and Kyowa Hakko Kogyo (now Kyowa Hakko Kirin Co., Ltd.). In that system, recombinant host cells containing an expression vector whose nucleic acid sequence encodes a chimeric Fc domain having both IgG1 and IgG3 Fc domain amino acid residues are expressed to produce an antigen-binding protein with enhanced complement-dependent cytotoxicity (CDC) activity that is increased relative to an otherwise identical antigen-binding protein lacking such chimeric Fc domain. Aspects of the Complegent™ Technology system are described in WO2007011041 and U.S. Patent Application Publication No. 20070148165, each of which is incorporated herein by reference. In an alternative embodiment, CDC activity can be increased by introducing sequence-specific mutations into the Fc region of the IgG chain. Those skilled in the art will also recognize other suitable systems.

[0176] Those skilled in the art will appreciate that such modifications can be used alone as well as in combination with each other to further enhance effector function.

[0177] In one such embodiment of the invention there is provided an antigen binding protein comprising a heavy chain constant region, including variant and chimeric heavy chain constant regions, for example the antigen binding protein comprises at least one CH2 domain from IgG3 and one CH2 domain from IgG1, and wherein the IgG1 CH2 domain has one or more mutations at positions selected from 239, 332 and 330 (for example the mutations may be selected from S239D, I332E and A330L), such that the antigen binding protein has enhanced effector function, for example it has one or more of the following functions: enhanced ADCC or enhanced CDC, for example it has enhanced ADCC and enhanced CDC. In one embodiment the IgG1 CH2 domain has the mutations S239D and I332E.

[0178] In an alternative embodiment of the present invention there is provided an antigen binding protein comprising a chimeric heavy chain constant region and having an altered glycosylation profile. In one such embodiment, the heavy chain constant region comprises at least one CH2 domain from IgG3 and one CH2 domain from IgG1, and has an altered glycosylation profile such that the ratio of fucose to mannose is 0.8:3 or less, e.g. the antigen binding protein is defucosylated, whereby said antigen binding protein has enhanced effector function compared to an equivalent antigen binding protein having an immunoglobulin heavy chain constant region lacking said mutations and altered glycosylation profile, e.g. it has one or more of the following functions: enhanced ADCC or enhanced CDC, e.g. it has enhanced ADCC and enhanced CDC.

[0179] In an alternative embodiment, the antigen binding protein has at least one IgG3 CH2 domain and at least one heavy chain constant domain derived from IgG1, and both IgG CH2 domains are mutated according to the restrictions described herein.

[0180] In one aspect of the invention there is provided a method of producing an antigen binding protein according to the invention described herein, comprising the steps of: a) culturing a recombinant host cell containing an expression vector containing an isolated nucleic acid described herein, wherein the expression vector further comprises an Fc nucleic acid sequence encoding a chimeric Fc domain having both IgG1 and IgG3 Fc domain amino acid residues, and wherein the FUT8 gene encoding alpha-1,6-fucosyltransferase is inactivated in the recombinant host cell; b) recovering the antigen-binding protein; A method is provided, comprising:

[0181] Such methods for producing antigen binding proteins can, for example, use the Aclitamab™ technology system available from BioWa, Inc. (Princeton, NJ), which combines the Potelligent™ and Complegent™ technology systems to produce antigen binding proteins with enhanced activity of both ADCC and CDC, which is increased compared to an otherwise identical monoclonal antibody lacking a chimeric Fc domain and having fucose on the oligosaccharides.

[0182] In yet another embodiment of the present invention there is provided an antigen binding protein comprising a mutated and chimeric heavy chain constant region, wherein said antigen binding protein has an altered glycosylation profile such that the antigen binding protein has enhanced effector function, for example it has one or more of the following functions: enhanced ADCC function or enhanced CDC function. In one embodiment the mutations are selected from positions 239, 332 and 330, for example the mutations are selected from S239D, I332E and A330L. In a further embodiment the heavy chain constant region comprises at least one CH2 domain from IgG3 and one CH2 domain from IgG1. In one embodiment the heavy chain constant region has an altered glycosylation profile such that the ratio of fucose to mannose is 0.8:3 or less, for example the antigen binding protein is defucosylated, whereby said antigen binding protein has enhanced effector function compared to an equivalent non-chimeric antigen binding protein or an immunoglobulin heavy chain constant region lacking said mutations and altered glycosylation profile.

[0183] Immunoconjugates Also provided are immunoconjugates (interchangeably referred to as "antibody-drug conjugates" or "ADCs") comprising the antigen binding proteins according to the invention as described herein, including antibodies conjugated to one or more cytotoxic agents such as, but not limited to, a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (e.g., a protein toxin, an enzymatically active toxin of bacterial, fungal, plant, or animal origin or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[0184] Immunoconjugates have been used for the local delivery of cytotoxic agents, i.e., drugs that kill or inhibit cell growth or proliferation, in the treatment of cancer (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549; Wu et al. (2005) Nature Biotechnology 23(9):1137-1146; Payne, G. (2003) i 3:207-212; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Deliv. Rev. 26:151-172; U.S. Pat. No. 4,975,278). Immunoconjugates allow for targeted delivery of drug moieties to tumors, where their intracellular accumulation can occur, when systemic administration of unconjugated drugs can result in unacceptable levels of toxicity to normal cells as well as the tumor cells being eliminated (Baldwin et al., Lancet (Mar. 15, 1986) pp. 603-05; Thorpe (1985) "Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review," in Monoclonal Antibodies '84: Biological And Clinical Applications (A. Pinchera et al., eds) pp. 475-506. Both polyclonal and monoclonal antibodies have been reported to be useful in these strategies (Rowland et al., (1986) Cancer Immunol. Immunother. 21:183-87). Drugs used in these methods include daunomycin, doxorubicin, methotrexate, and vindesine (Rowland et al., (1986) supra).Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as ricin, geldanamycin (Mandler et al (2000) J. Nat. Cancer Inst. 92(19):1573-1581, Mandler et al (2000) Bioorganic & Med. Chem. Letters 10:1025-1028, Mandler et al (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213, Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998) Cancer Res. 58:2928, Hinman et al (1993) Cancer Res. 53:3336-3342).

[0185] In one embodiment, the present invention provides compounds having the following general structure: ABP-((linker) n -Ctx) m wherein ABP is an antigen-binding protein; the linker is either absent or is a cleavable or non-cleavable linker as described herein; Ctx is any cytotoxic agent described herein; n is 0, 1, 2, or 3; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 and an immunoconjugate having the formula:

[0186] Examples of antibodies linked by an MC linker to auristatins such as MMAE and MMAF have the following structures: [ka]

[0187] As shown in.

[0188] In certain embodiments, the immunoconjugate comprises an antigen-binding protein, including, but not limited to, an antibody and a chemotherapeutic agent or other toxin. Chemotherapeutic agents useful for generating immunoconjugates are described herein. Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, and the like. oficinalis inhibitors, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes. See, for example, WO 93 / 21232, published October 28, 1993. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include: 211 At, 212 Bi, 131 I, 131 In, 90 Y, and 186 Re is an example.

[0189] The antigen binding proteins of the invention can also be conjugated to one or more toxins, including, but not limited to, calicheamicin, maytansinoids, dolastatins, aurostatins, trichothecenes, and CC1065, and derivatives of these toxins that have toxin activity. Suitable cytotoxic agents include, but are not limited to, auristatins, including dovaline-valine-dolaisoleunine-dolaproine-phenylalanine (MMAF) and monomethyl auristatin E (MMAE) and ester forms of MMAE, DNA minor groove binders, DNA minor groove alkylating agents, enediynes, lexitropsins, duocarmycins, taxanes, including paclitaxel and docetaxel, puromycins, dolastatins, maytansinoids, and vinca alkaloids. Specific cytotoxic agents include topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, dolastatin-10, echinomycin, combretatostatin, calicheamicin, maytansine, DM-1, DM-4, ​​and netropsin. Other suitable cytotoxic agents include antitubulin agents such as auristatins, vinca alkaloids, podophyllotoxins, taxanes, baccatin derivatives, cryptophysins, maytansinoids, combretastatins, or dolastatins. Antitubulin agents include dimethylvaline-valine-dolaisoleuin-dolaproine-phenylalanine-p-phenylenediamine, MMAF, MMAE, auristatin E, vincristatine, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epothilone A, epothilone B, nocodazole, cohilutin, colcimid, estramustine, cemadotin, discodermolide, maytansine, DM-1, DM-4, ​​or eleutherobin.

[0190] Antibody-drug conjugates were produced by conjugating the small molecule antitubulin agents monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF) to antibodies. In the case of MMAE, the linker consists of a thiol-reactive maleimide, a caproyl spacer, the dipeptide valine-citrulline, and a p-aminobenzyloxycarbonyl, self-immolative fragmenting group. In the case of MMAF, a protease-resistant maleimide caproyl linker is used. The conjugation process leads to heterogeneity in drug-antibody binding, varying both the number of drugs attached to each antibody molecule (molar ratio [MR]) and the binding site. The most common species are those with an MR of 4, while less common are those with MRs of 0, 2, 6, and 8. The overall average drug-antibody MR is approximately 4.

[0191] Production of immunoconjugates The attachment point is a cysteine ​​produced by mild reduction of the antibody's interchain disulfides (which allows the use of a large excess of reagents without intermediate purification) while the antibody is immobilized on the Protein G affinity resin. During immobilization, a large excess of TCEP completely reduces the interchain disulfides but does not affect antibody binding to the resin.

[0192] The number of thiols per antibody generated by this procedure depends on the source and isotype of the antibody. For example, human (and mouse-human chimeric) IgG1 has four reducible disulfides, generating eight thiols upon complete reduction, while mouse IgG1 has five reducible disulfides, generating ten thiols. If an ADC with maximum drug loading (e.g., 10 drugs per antibody for mouse IgG1) is desired, the maleimide-drug-linker may simply be added to the immobilized antibody in sufficient excess to ensure complete conjugation. However, ADCs with fewer drugs per antibody can also be prepared from fully reduced antibodies by including a biologically inert capping agent, such as N-ethylmaleimide (NEM), which occupies a portion of the available thiols on the antibody. When the maleimide-drug-linker and capping agent are added simultaneously in large excess (at least threefold) to the fully reduced antibody, the two maleimide electrophiles compete for the limited number of available thiols. In this format, drug loading can be considered kinetically controlled, as it is determined by the relative thiol reaction rates of the drug-linker and capping agent. Because the relative reaction rates of maleimide-drug-linkers vary significantly, the molar ratio of drug-linker to NEM present in the reaction mixture must be experimentally determined to arrive at a panel of ADCs with the desired level of drug loading. The molar fractions of the drug linkers SGD-1006 (vcMMAE) and SGD-1269 (mcMMAF) in NEM mixtures that yield ADCs with approximately four drugs per antibody are summarized in Table 2 for common human and mouse IgG isotypes.

[0193] Auristatins and dolastatins In some embodiments, the immunoconjugate comprises an antigen-binding protein or antibody conjugated to auristatins (U.S. Patent Nos. 5,635,483, 5,780,588), which are dolastatins or peptide analogs and derivatives of dolastatins. Dolastatins and auristatins have been shown to interfere with microtubule dynamics, GTP hydrolysis, and nuclear and cell division (Woyke et al. (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584), and have anticancer (U.S. Patent No. 5,663,149) and antifungal (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965) activities. Dolastatin or auristatin (which are pentapeptide derivatives of dolastatins) drug moieties can be attached to antibodies via either the N (amino) terminus or the C (carboxyl) terminus of the peptide drug moiety (WO 02 / 088172).

[0194] Exemplary auristatin embodiments include the N-terminally attached monomethyl auristatin drug moieties DE and DF, as disclosed in "Monomethylvaline Compounds Capable of Conjugation to Ligands," U.S. Pat. No. 7,498,298, the disclosure of which is expressly incorporated by reference in its entirety. As used herein, the abbreviation "MMAE" refers to monomethyl auristatin E. As used herein, the abbreviation "MMAF" refers to dovaline-valine-dolaisoloin-dolaproine-phenylalanine.

[0195] Typically, peptide-based drug moieties can be prepared by forming a peptide bond between two or more amino acids and / or peptide fragments, for example, by liquid phase synthesis, which is well known in the field of peptide chemistry (see E. Schroder and K. Lubke, "The Peptides", volume 1, pp. 76-136, 1965, Academic Press). Auristatin / dolastatin drug moieties can be prepared according to the methods of U.S. Pat. No. 5,635,483, U.S. Pat. No. 5,780,588, Pettit et al. (1989) J. Am. Chem. Soc. 111:5463-5465, Pettit et al. (1998) Anti-Cancer Drug Design 13:243-277, Pettit, GR, et al. Synthesis, 1996, 719-725, and Pettit et al. (1996) J. Chem. Soc. Perkin Trans. 15:859-863. See also Doronina (2003) Nat Biotechnol 21(7):778-784; "Monomethylvaline Compounds Capable of Conjugation to Ligands," U.S. Patent No. 7,498,298, filed November 5, 2004, incorporated herein by reference in its entirety (disclosing, for example, linkers and methods for preparing monomethylvaline compounds such as MMAE and MMAF conjugated to linkers). Biologically active organic compounds that act as cytotoxic agents, particularly pentapeptides, are disclosed in U.S. Patent Nos. 6,884,869, 7,498,298, 7,098,308, 7,256,257, and 7,423,116. Monoclonal antibodies conjugated to various derivatives of MMAE and MMAF and auristatins and methods for making them are described in US Pat. No. 7,964,566.

[0196] Examples of auristatins include MMAE and MMAF, the structures of which are shown below: [ka]

[0197] Maytansine and maytansinoids Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata (U.S. Pat. No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters (U.S. Pat. No. 4,151,042). Highly cytotoxic maytansinoid drugs can be prepared from ansamitocin precursors produced by fermentation of microorganisms such as Actinosynnema. A method for isolating ansamitocins is described in U.S. Pat. No. 6,573,074. Synthetic maytansinol and its derivatives and analogs are described, for example, in U.S. Patent Nos. 4,137,230, 4,248,870, 4,256,746, 4,260,608, 4,265,814, 4,294,757, 4,307,016, 4,308,268, 4,308,269, and 4,309, 428, 4,313,946, 4,315,929, 4,317,821, 4,322,348, 4,331,598, 4,361,650, 4,364,866, 4,424,219, 4,450,254, 4,362,663, and 4,371,533.

[0198] Antibody-maytansinoid conjugates are prepared by chemically linking an antibody to a maytansinoid molecule without significantly reducing the biological activity of either the antibody or the maytansinoid molecule. See, e.g., U.S. Patent No. 5,208,020. An average of 3-4 maytansinoid molecules conjugated per antibody molecule have been shown to be effective in enhancing target cell cytotoxicity without adversely affecting antibody function or solubility, although even a single molecule of toxin per antibody is expected to enhance cytotoxicity over the use of naked antibodies.

[0199] Maytansinoids are well known in the art and can be synthesized by known techniques or isolated from natural sources. Suitable maytansinoids are disclosed, for example, in U.S. Patent No. 5,208,020 and the other documents and non-patent literature referenced hereinabove. Maytansinoids are maytansinol and maytansinol analogs modified within the aromatic ring or at other positions of the maytansinol molecule, such as various maytansinol esters. Methods for preparing maytansinoids for conjugation with antibodies are disclosed in U.S. Patent Nos. 6,570,024 and 6,884,874.

[0200] Calicheamicin The calicheamicin family of antibiotics is capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of calicheamicin family conjugates, see U.S. Patent Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296 (all to American Cyanamid Company). Structural analogs of calicheamicin that can be used include, but are not limited to, gamma 1I, alpha 2I, alpha 3I, N-acetyl-gamma 1I, PSAG, and theta 1I (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the aforementioned U.S. patent by American Cyanamid). Another antitumor agent to which an antibody can be conjugated is the antifolate QFA. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents via antibody-mediated internalization enhances their cytotoxic effects.

[0201] Other cytotoxic agents Other antitumor agents that can be conjugated to the antibodies include BCNU, streptozoicin, vincristine, and 5-fluorouracil, a family of drugs described in U.S. Pat. Nos. 5,053,394 and 5,770,710 and collectively known as LL-E33288 conjugates, and esperamicine (U.S. Pat. No. 5,877,296).

[0202] Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogelin, restrictocin, phenomycin, enomycin, and trichothecenes. See, e.g., WO 93 / 21232, published October 28, 1993.

[0203] The present invention further contemplates immunoconjugates formed between an antibody and a compound with nucleolytic activity (eg, a ribonuclease, such as a deoxyribonuclease, DNase, or a DNA endonuclease).

[0204] To selectively destroy tumors, antibodies may contain highly radioactive atoms. A variety of radioisotopes are available for producing radioconjugated antibodies. Examples include radioactive isotopes of At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and Lu. When the conjugate is used for detection, it may contain radioactive atoms for scintigraphy studies, such as tc99m or I123, or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0205] Radioactive or other labels can be introduced into the conjugate by known methods. For example, peptides can be biosynthesized or synthesized by chemical amino acid synthesis using appropriate amino acid precursors containing, for example, fluorine-19 instead of hydrogen. Labels such as tc99m or I123, Re186, Re188, and In111 can be attached via cysteine ​​residues in the peptide. Yttrium-90 can be attached via lysine residues. To introduce iodine-123, the IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57) can be used. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.

[0206] Preparation of ADCs In antibody-drug conjugates, the antibody can be conjugated to the cytotoxic agent directly or via a linker. Suitable linkers include, for example, cleavable and non-cleavable linkers. Cleavable linkers are typically susceptible to cleavage under intracellular conditions. Suitable cleavable linkers include, for example, peptide linkers cleavable by intracellular proteases, such as lysosomal or endosomal proteases. In exemplary embodiments, the linker may be a dipeptide linker, such as a valine-citrulline (val-cit) or phenylalanine-lysine (phe-lys) linker. Other suitable linkers include linkers hydrolyzable at a pH below 5.5, such as hydrazone linkers. Further suitable cleavable linkers include disulfide linkers.

[0207] Bristol-Myers Squibb has described certain lysosomal enzyme-cleavable antitumor drug conjugates. See, for example, U.S. Patent No. 6,214,345. Seattle Genetics has published U.S. Patent Application Publication No. 2003 / 0096743 and U.S. Patent Application Publication No. 2003 / 0130189, which describe p-aminobenzyl ethers in drug delivery agents. The linkers described in these applications are limited to aminobenzyl ether compositions.

[0208] Conjugates of antigen-binding proteins and cytotoxic agents can be produced using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-activated fluorine compounds (1,5-difluoro-2,4-dinitrobenzene).

[0209] Furthermore, the linker may be comprised of one or more linker moieties. Exemplary linker moieties include 6-maleimidocaproyl ("MC"), maleimidopropanoyl ("MP"), valine-citrulline ("val-cit"), alanine-phenylalanine ("ala-phe"), p-aminobenzyloxycarbonyl ("PAB"), N-succinimidyl 4-(2-pyridylthio)pentanoate ("SPP"), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate ("SMCC"), and N-succinimidyl (4-iodo-acetyl)aminobenzoate ("SIAB"). Additional linker moieties are known in the art, and some are described herein. See also, "Monomethylvaline Compounds Capable of Conjugation to Ligands," U.S. Patent No. 7,498,298, filed November 5, 2004, the contents of which are incorporated herein by reference in their entirety.

[0210] Linkers may also contain amino acids and / or amino acid analogs. Amino acid linker components include dipeptides, tripeptides, tetrapeptides, or pentapeptides. Exemplary dipeptides include valine-citrulline (vc or val-cit) and alanine-phenylalanine (af or ala-phe). Exemplary tripeptides include glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acid residues comprising amino acid linker components include naturally occurring amino acids as well as minor amino acids and non-naturally occurring amino acid analogs, such as citrulline. Amino acid linker components can be designed and optimized for their selectivity for enzymatic cleavage by specific enzymes, such as tumor-associated proteases, cathepsins B, C, and D, or plasmin proteases.

[0211] Antigen-binding proteins and antibodies can be reactive for conjugation with linker reagents. Nucleophilic groups on antibodies include, but are not limited to, (i) N-terminal amine groups, (ii) side-chain amine groups, such as lysine, (iii) side-chain thiol groups, such as cysteine, and (iv) sugar hydroxyl or amino groups on which antibodies are glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and can react to form covalent bonds with electrophilic groups on linker moieties and linker reagents, including (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; and (iii) aldehyde, ketone, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e., cysteine ​​bridges. Antibodies can be reactive for conjugation with linker reagents by treatment with a reducing agent such as DTT (dithiothreitol). Therefore, each cysteine ​​bridge theoretically forms two reactive thiol nucleophiles. Additional nucleophilic groups can be introduced into antibodies by reaction of lysines with 2-iminothiolane (Traut's reagent), resulting in conversion of amines to thiols. Reactive thiol groups can be introduced into antibodies (or fragments thereof) by introducing one, two, three, four, or more cysteine ​​residues (e.g., by preparing a mutant antibody containing one or more non-naturally occurring cysteine ​​amino acid residues).

[0212] Antigen-binding proteins and antibodies can also be modified to introduce electrophilic moieties that can react with nucleophilic substituents on linker reagents or drugs. The sugars of glycosylated antibodies can be oxidized, for example, with periodate oxidizing agents, to form aldehyde or ketone groups that can react with amine groups on linker reagents or drug moieties. The resulting imine Schiff bases can form stable bonds or can be reduced, for example, with borohydride, to form stable amine linkages. In one embodiment, reaction of the carbohydrate moiety of a glycosylated antibody with either galactose oxidase or sodium metaperiodate can generate carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on a drug (Hermanson, Bioconjugate Techniques). In another embodiment, proteins containing N-terminal serine or threonine residues can be reacted with sodium metaperiodate, resulting in the generation of an aldehyde in place of the initial amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146; U.S. Pat. No. 5,362,852). Such aldehydes can react with drug moieties or linker nucleophiles.

[0213] Nucleophilic groups on a drug moiety include, but are not limited to, (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; and (iii) amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups that can react to form covalent bonds with electrophilic groups on linker moieties and linker reagents, including aldehyde, ketone, carboxyl, and maleimide groups.

[0214] In some embodiments, the linker is cleavable by a cleaving agent present in the intracellular environment (e.g., in a lysosome, endosome, or caveolae). The linker may be, for example, a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including, but not limited to, a lysosomal or endosomal protease. Typically, the peptidyl linker is at least two amino acids long or at least three amino acids long. Cleaving agents include cathepsin B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release the active drug within target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). The peptidyl linker may be cleavable by an enzyme present in the cell. For example, a peptidyl linker cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancer tissue, may be used (e.g., a Phe-Leu or Gly-Phe-Leu-Gly (SEQ ID NO: 50) linker). Other such linkers are described, for example, in U.S. Pat. No. 6,214,345. In certain embodiments, the peptidyl linker cleavable by an intracellular protease is a Val-Cit linker or a Phe-Lys linker (see, for example, U.S. Pat. No. 6,214,345, which describes the synthesis of doxorubicin with a val-cit linker). One advantage of using intracellular proteolytic release of a therapeutic agent is that the agent is typically attenuated when conjugated, and the serum stability of the conjugate is typically high.

[0215] In other embodiments, the cleavable linker is pH-sensitive, i.e., sensitive to hydrolysis at a specific pH value. Typically, pH-sensitive linkers are hydrolyzed under acidic conditions. For example, acid-labile linkers (e.g., hydrazones, semicarbazones, thiosemicarbazones, cis-aconitic amides, orthoesters, acetals, ketals, etc.) that are hydrolyzable in lysosomes may be used. (See, e.g., U.S. Patent Nos. 5,122,368, 5,824,805, 5,622,929; Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem. 264:14653-14661). Such linkers are relatively stable under neutral pH conditions, such as those found in blood, but are unstable below pH 5.5 or 5.0, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (such as a thioether attached to the therapeutic agent via an acylhydrazone bond (see, eg, US Pat. No. 5,622,929)).

[0216] In yet other embodiments, the linker is cleavable under reducing conditions (eg, a disulfide linker). A variety of disulfide linkers are known in the art, including, for example, those that can be formed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP (N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB (N-succinimidyl-3-(2-pyridyldithio)butyrate), and SMPT (N-succinimidyl-oxycarbonyl-α-methyl-α-(2-pyridyl-dithio)toluene)-, SPDB, and SMPT (e.g., Thorpe et al., 1987, Cancer Res. 47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody Conjugates in Radioimagery and Therapy of Cancer (C.W. Vogel ed., Oxford U. Press, 1987; see also U.S. Pat. No. 4,880,935).

[0217] In yet other specific embodiments, the linker is a malonic acid linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3'-N-amide analog (Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

[0218] Typically, the linker is substantially insensitive to the extracellular environment. As used herein, "substantially insensitive to the extracellular environment" with respect to a linker means that when the ADC or ADC derivative is present in an extracellular environment (e.g., in plasma), about 20% or less, typically about 15% or less, more typically about 10% or less, and even more typically about 5%, about 3%, or about 1% or less of the linkers are cleaved in a sample of the ADC or ADC derivative. Whether a linker is substantially insensitive to the extracellular environment can be determined, for example, by independently incubating both (a) the ADC or ADC derivative ("ADC sample") and (b) an equimolar amount of unconjugated antibody or therapeutic agent ("control sample") with plasma for a predetermined period of time (e.g., 2, 4, 8, 16, or 24 hours), and then comparing the amount of unconjugated antibody or therapeutic agent present in the ADC sample with the amount present in the control sample, as measured, for example, by high performance liquid chromatography.

[0219] In other, non-mutually exclusive embodiments, the linker promotes cellular internalization. In certain embodiments, when conjugated to a therapeutic agent (i.e., in the context of the linker-therapeutic agent moiety of an ADC or ADC derivative described herein), the linker promotes cellular internalization. In yet other embodiments, when conjugated to both a therapeutic agent and an antigen binding protein, or antibody or derivative thereof (i.e., in the context of an ADC or ADC derivative described herein), the linker promotes cellular internalization.

[0220] Various linkers that can be used with the present compositions and methods are described in WO2004010957, filed July 31, 2003, entitled "Drug Conjugates and Their Use for Treating Cancer, An Autoimmune Disease or an Infectious Disease," and U.S. Provisional Patent Application No. 60 / 400,403, filed July 31, 2002, entitled "Drug Conjugates and Their Use for Treating Cancer, an Autoimmune Disease or an Infectious Disease," the disclosures of which are incorporated herein by reference.

[0221] Alternatively, a fusion protein comprising the antigen-binding protein and cytotoxic agent can be made, for example, by recombinant techniques or peptide synthesis. The length of DNA may include regions encoding the two parts of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide that does not destroy the desired properties of the conjugate.

[0222] In yet another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for use in tumor pretargeting, and the antibody-receptor conjugate is administered to the patient, after which unbound conjugate is removed from the circulation using a detergent, followed by administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionucleotide).

[0223] As used herein, the term "non-human antibody or antibody fragment thereof" is meant to refer to an antibody or fragment thereof that originates from any species other than human, where human includes chimeric antibodies.

[0224] The term "donor antibody" refers to an antibody (monoclonal and / or recombinant) that contributes the amino acid sequences of its variable domains, CDRs, or other functional fragments or analogs thereof to a first immunoglobulin partner to provide an altered immunoglobulin coding region and thereby express an altered antibody having the antigen specificity and neutralizing activity characteristic of the donor antibody.

[0225] The term "acceptor antibody" refers to an antibody (monoclonal and / or recombinant) that is heterologous to the donor antibody and that contributes to a first immunoglobulin partner all (or any portion, but preferably all) of the amino acid sequence encoding its heavy and / or light chain framework regions and / or its heavy and / or light chain constant regions. A human antibody is an acceptor antibody.

[0226] As used herein, the term "human acceptor sequence" is meant to refer to the framework of an antibody or antibody fragment thereof that comprises the amino acid sequence of a VH or VL framework derived from a human antibody or antibody fragment thereof, or a human consensus sequence framework into which CDRs from a non-human species can be incorporated.

[0227] As used herein, the term "incorporation" of a CDR or hypervariable region encompasses any means by which a non-human CDR is placed with a human acceptor framework. It will be understood that this can be achieved in a variety of ways. For example, a nucleic acid encoding the desired amino acid sequence can be generated by mutating a nucleic acid encoding a non-human variable domain sequence so that its framework residues are changed to human acceptor framework residues, or can be generated by mutating a nucleic acid encoding a human variable domain sequence so that the CDRs are changed to non-human residues, or can be generated by synthesizing a nucleic acid encoding the desired sequence. In one embodiment, the final sequence is generated in silico.

[0228] The present invention will now be described by way of example only. The appended claims may include generalizations of one or more of the following examples. [Example]

[0229] Example 1: Monoclonal antibody generation and selection 1.1 Immunization strategies The anti-human BCMA mAb, murine parent CA8, was identified from a hybridoma derived from a mouse immunized with full-length BCMA. BALB / c mice were immunized i.p. with 25 μg of recombinant (rBCMA) protein combined with CFA. Mice were boosted three times at monthly intervals with 25 μg of full-length rBCMA protein plus 10 μg of monophosphoryl lipid A-stable emulsion (MPL-SE) (Corixa Corporation, Seattle, WA), and given a prefusion boost of 30 μg of rBCMA protein i.v. three days before fusion. Hybridomas were generated and cloned either using the ClonaCell-HY Hybridoma Cloning Kit (StemCell Technologies, Vancouver, BC) or conventional methods. In the conventional method, B cells from the spleens of immunized animals were fused with Sp2 / 0 myeloma cells in the presence of PEG (Sigma-Aldrich, St. Louis, MO). After overnight recovery, fused cells were seeded at limiting dilution in 96-well plates and subjected to hypoxanthine-aminopterin-thymidine selection. Hybridoma culture supernatants were tested for the presence of anti-BCMA antibodies by ELISA and flow cytometry.

[0230] The anti-human BCMA mAb murine parent S307118G03 was identified from a hybridoma derived from SJL mice immunized with recombinant human BCMA / TNFRSF17-Fc chimera (R&D 193-Fc) using the RIMMS (Rapid Immunization Multiple Site) method. On day 0, 5 μg of protein per mouse was emulsified in AS02a adjuvant at two sites on the back (over the hip and over the shoulder) and below the major lymph nodes at four sites on the front. On days 6 and 11, 2.5 μg of protein per mouse in RIBI adjuvant was injected below the major lymph nodes at four sites on the front. Animals were sacrificed on day 14. Lymph nodes and spleens were removed and disrupted, and PEG1500-induced somatic cell fusions were performed using mouse myeloma cells X63 AG8 653.GFP.Bcl-2.11 (BioCat 112754; R17209 / 58) at a 3:1 ratio. Fusions were plated into 10 x 96-well plates and screened directly from them.

[0231] The murine parental anti-human BCMA mAb S336105A07 was identified from a hybridoma derived from the same immunization. Lymph nodes and spleens were removed and disrupted on day 14, and Cytopulse electrofusion was performed using a 1:1 ratio with mouse myeloma cells X63 AG8 653.GFP.Bcl-2.11 (BioCat 112754; R17209 / 58). Fusions were plated into omnitrays containing semi-solid medium before being harvested into 10 x 96-well plates and screened directly after 5 days.

[0232] The anti-human BCMA murine parental mAbs S332121F02 and S332126E04 were identified from hybridomas derived from SJL mice immunized with a recombinant Fc fusion of the extracellular domain of human BCMA (4-53) BCMA using the RIMMS (Rapid Immunization Method) method. On day 0, 5 μg of protein per mouse was emulsified in AS02a adjuvant at two sites on the dorsum (over the hip and over the shoulder) and below the major lymph nodes at four sites on the front. On day 6, 5 μg of recombinant cyno BCMA-Fc protein per mouse in RIBI adjuvant was injected below the major lymph nodes at four sites on the front. On day 11, 2.5 μg of recombinant human BCMA-Fc and 2.5 μg of recombinant cyno BCMA-Fc per mouse in RIBI adjuvant were injected below the major lymph nodes at four sites on the front. On day 14, the animals were sacrificed and the cells were treated as for S307118G03.

[0233] The anti-human BCMA murine parental mAb S322110D07 was identified from a hybridoma derived from SJL mice immunized with a recombinant Fc fusion of the extracellular domain of human BCMA (4-53) complexed with recombinant human April (R&D 5860-AP / CF) premixed at a 1:1 molar ratio. Mice were immunized i.p. with 5 μg of April / Cyno BCMA-Fc complex in PBS suspended in RIBI adjuvant (100 μl dose) per mouse and boosted three times at 3-4 week intervals with 2.5 μg of April / Cyno BCMA-Fc complex in PBS suspended in RIBI adjuvant (100 μl dose) per mouse injected via the intraperitoneal route. A pre-fusion boost of the same immunogen was given one day prior to the fusion, and treated as for S307118G03.

[0234] The anti-human BCMA murine parental mAbs S335115G01 and S335122F05 were identified from hybridomas derived from SJL mice immunized with a mixture of recombinant Fc fusions of the extracellular domains of human BCMA (4-53) and cyno BCMA (4-52) using the RIMMS (Rapid Immunization Multiple Site) method. On day 0, 2.5 μg of each protein per mouse was emulsified in AS02a adjuvant and injected at two sites on the dorsal side (over the hip and over the shoulder) and four sites on the front, below the major lymph nodes. On days 6 and 11, 2.5 μg of each protein per mouse in RIBI adjuvant was injected at four sites on the front, below the major lymph nodes. Animals were sacrificed on day 14. Lymph nodes and spleens were removed, disrupted, and subjected to cytopulse electrofusion with mouse myeloma cells X63 AG8 653.GFP.Bcl-2.11 (BioCat 112754; R17209 / 58) at a 1:1 ratio. Fusions were plated into Omnitrays containing semi-solid medium before harvesting into 32 x 96-well plates and screened directly after 5 days.

[0235] [Example 2] Humanization 2.1 Cloning of CA8 hybridoma variable regions Total RNA was extracted from CA8 hybridoma cells, and then heavy and light chain variable domain cDNA sequences were generated by reverse transcription and polymerase chain reaction (RT-PCR). The forward primer for RT-PCR was a mixture of degenerate primers specific to mouse immunoglobulin gene leader sequences, and the reverse primer was specific to the antibody constant region. Since the isotype was unknown, reverse primers for IgG1, IgG2a, and IgG2b were used in this case. To design the primers, mouse V H and V k A DNA multiple sequence alignment of the gene leader sequences was generated.

[0236] 2.2 Cloning of chimeric CA8 DNA expression constructs encoding chimeric antibodies were prepared de novo by assembling overlapping oligonucleotides containing restriction enzyme recognition sites and a human signal sequence for cloning into mammalian expression vectors. HindIII and SpeI restriction enzyme recognition sites were introduced to construct the VH domain containing a signal sequence for cloning into a mammalian expression vector containing the human γ1 constant region. HindIII and BsiWI restriction enzyme recognition sites were introduced to construct the VL domain containing a signal sequence for cloning into a mammalian expression vector containing the human kappa constant region.

[0237] 2.3 Cloning of humanized CA8 variants DNA expression constructs encoding humanized antibody variants were prepared de novo by assembling overlapping oligonucleotides containing restriction enzyme recognition sites and a human signal sequence for cloning into mammalian expression vectors. HindIII and SpeI restriction enzyme recognition sites were introduced to construct the VH domain containing a signal sequence for cloning into a mammalian expression vector containing the human γ1 constant region. HindIII and BsiWI restriction enzyme recognition sites were introduced to construct the VL domain containing a signal sequence for cloning into a mammalian expression vector containing the human kappa constant region.

[0238] 2.4 Expression of Recombinant CA8 Antibody (including Antibody Quantification) Expression plasmids encoding the heavy and light chains were transiently cotransfected into HEK293 6E cells and expressed at a small scale to produce antibody. Antibodies were quantified by ELISA. ELISA plates were coated with anti-human IgG (Sigma I3382) at 1 mg / ml and blocked with blocking solution (4% BSA in Tris-buffered saline). Various dilutions of tissue culture supernatants were added, and the plates were incubated for 1 hour at room temperature. Dilutions of a known standard antibody were also added to the plates. The plates were washed in TBST, and binding was detected by the addition of peroxide-labeled anti-human kappa light chain antibody (Sigma A7164) at a 1 / 1000 dilution in blocking solution. The plates were incubated for 1 hour at room temperature before washing in TBST. The plates were developed by the addition of OPD substrate (Sigma P9187), and color development was stopped by the addition of 2 M H2SO4. Absorbance was measured at 490 nm, and a standard curve was plotted using data from the known standard dilutions. A standard curve was used to estimate the concentration of antibody in tissue culture supernatants. Large-scale antibody preparations were purified using Protein A and concentrations were measured using a Nanodrop (Thermo Scientific). [Table 1]

[0239] 2.5 Defucosylated antibody production To generate defucosylated antibodies, the heavy and light chains were co-transfected into CHO DG44 MS705 BioWa cells and expressed at antibody production scale. Briefly, 30 μg of DNA was linearized overnight with Not1, and the DNA was ethanol precipitated and redissolved in TE buffer. 2.4 × 10 BioWa DG44 cells were obtained from the culture and washed in 14 ml of warm PBS-sucrose. The cells were spun, and the pellet was resuspended in 1.6 ml of PBS-sucrose. Half of the cells suspended in PBS-sucrose (0.8 ml) was added to a BioRad cuvette along with 30 μg of linearized DNA (in 50 μl of TE buffer). A BioRad GenePulser was programmed to 380 V using a capacitance of 25 μF, and the cuvette was loaded for electroporation. The resulting 850 μl of electroporated cells and DNA were added to (80 ml) warm SFM512 medium (containing phenol red, 2XHT (nucleosides), glutamax, and Gibco supplement 4). Finally, the resulting 80 ml cell suspension was transferred to each well of one of four 96-well plates (150 μl / well). After 48 hours, the medium was changed to nucleoside-free by removing approximately 130 μl of conditioned medium and replacing it with 150 μl of fresh selective medium, SFM512 medium (containing phenol red and glutamax). Every 3–4 days, 130–150 μl of conditioned medium was removed and replaced with fresh selective medium. Wells were monitored for color change and assayed for IgG concentration as previously described.

[0240] 2.6 Further Antibodies - Cloning of Hybridoma Variable Regions Total RNA was extracted from S307118G03, S332121F02, S332126E04, S322110D07, S336105A07, S335115G01, and S335122F05 hybridoma cells. Heavy and light chain variable domain cDNA sequences were then generated by reverse transcription and polymerase chain reaction (RT-PCR). The forward primer for RT-PCR was a mixture of degenerate primers specific to mouse immunoglobulin gene leader sequences, and the reverse primer was specific to the antibody constant region, in this case, isotype IgG2a. Primers were designed based on the strategy described by Jones and Bendig (Bio / Technology 9:88, 1991). RT-PCR was performed on both V-region sequences to allow subsequent verification of the correct V-region sequences. DNA sequence data was obtained for the V-region products generated by RT-PCR.

[0241] 2.7 Further antibodies - cloning of chimeras DNA expression constructs encoding chimeric antibodies were prepared de novo by Infusion Advantage PCR cloning (Clonetech) of V gene PCR products into mammalian expression vectors. This cloning method allowed for the fusion of mouse variable regions with human IgG1 heavy and kappa light chain constant regions.

[0242] 2.8 S307118G03 - Cloning of humanized variant Cloning was carried out as for paragraph 2.3.

[0243] 2.9 S307118G03 Expression of Recombinant Antibody Expression plasmids encoding the relevant heavy and light chains (listed in Table 8 below) were transiently co-transfected into HEK293 6E cells and expressed at small scale to produce antibodies, which were Protein A purified from the supernatant and quantified using a Nanodrop spectrophotometer.

[0244] The following 8) were transiently co-transfected into HEK293 6E cells and expressed at small scale to produce antibodies. Antibodies were purified from the supernatant using Protein A and quantified using a Nanodrop spectrophotometer.

[0245] Example 3: Conjugation of antibodies to vcMMAE and mcMMAF to generate antibody drug conjugates (ADCs) Table B. Chemical structures of drug-linkers [ka]

[0246] Gammabind Plus Protein G Sepharose (GE Healthcare) resin slurry (75 μL) was added to each well of a deep-well (2 mL volume) filter plate. The conjugated antibodies were classified by species and isotype, and 0.5 mg or less of each antibody was transferred to each well of the plate. Each antibody was transferred to two separate wells to facilitate the preparation of two conjugates with the drug-linkers SGD-1006 and SGD-1269. The filter plate was then shaken at 1200 RPM for 2 hours at 5°C to allow binding of the antibodies to the resin. The filter plate was then centrifuged at 500 × g for 3 minutes to ensure complete withdrawal of all fluid and resin to the bottom of each well.

[0247] The bound antibody was then reduced by adding 500 μL of 10 mM TCEP, 150 mM NaCl, pH 7, 1 mM EDTA in 100 mM KPO4 and shaking at 22°C for 30 minutes. After reduction, the plate was centrifuged again to remove the TCEP solution and then washed with PBS + 1 mM EDTA, 1 mL / well. The wash solution was removed by centrifugation, and the process was repeated three times for a total of four washes. The bound and reduced antibody was then conjugated using a mixture of NEM and drug linker prepared according to the molar fractions shown in Table 2. [Table 2]

[0248] Thus, separate mixtures of NEM and drug linker were prepared for each antibody species / isotype using 10 mM DMSO stock solutions of SGD-1006, SGD-1269 (see Table B), and NEM. When mixed in the appropriate ratio, the total maleimide concentration was still 10 mM, and this value was used to calculate the volume of maleimide solution added to each well. For example, for a mouse IgG1 with five reducible disulfides (10 available thiols when reduced), 0.5 mg of antibody at 150 kDa is 3.33 nmol, corresponding to 33.3 nmol of thiols. Thus, a 3-fold excess is 100 nmol total maleimide or 10 μl of 10 mM drug linker / NEM mixture. For the SGD-1269 conjugate, this mixture is then prepared with 5.86 μL of SGD-1269 and 4.14 μL of NEM. The maleimide mixture was then diluted in 500 μL of PBS before being added to the immobilized reduced antibody. In practice, multiple antibodies of each isotype were conjugated simultaneously with a single SGD-1269 / NEM, so a mixed solution for each isotype was prepared by multiplying 10 μL / well by the number of wells containing that isotype and then diluted in a volume of PBS equal to 500 μL times the number of wells. Similarly, a total of eight drug-linker / NEM mixtures were prepared (four with SGD-1006 and four with SGD-1269) and diluted in PBS. Three mixtures were then added to the reduced antibody (500 μL / well), and the plate was shaken at 22 °C for 30 minutes. The plate was then centrifuged as above to remove excess reaction solution, followed by four washes with PBS as before. The bound ADC was then eluted by adding 200 μL of 50 mM glycine pH 2.5 to each well and shaking the plate at 1200 RPM for 3 minutes. While shaking, 20 uL of neutralization buffer (1 M potassium phosphate, pH 7.4, 500 mM NaCl, 0.2% Tween®-20) was added to each well of 1 mL of the collection plate. The ADCs were then eluted in the collection plate by spinning at 1500×g for 6 minutes. The collection plate was then briefly shaken to ensure complete mixing of the neutralization buffer.

[0249] The solutions were then transferred into UV assay plates (Costar model 3635, Corning) and the concentration of each ADC was determined using an absorbance plate reader by measuring the optical density at 280 nm. An average IgG extinction coefficient of 1.45 mL mg cm was used to provide a reasonable estimate of ADC concentration across the panel. To confirm successful conjugation, the drug loading of the isotype controls was estimated using a reverse-phase protein HPLC method (described below). For plates containing humanized variants of CA8, this method was used to directly estimate the loading of all ADCs.

[0250] The reversed-phase protein chromatography method for determining drug loading utilizes a PLRP-S polymeric stationary phase (Agilent Technologies). Because the antibody was fully reduced during the conjugation process, all of the antibody subunits eluted from the column as single polypeptide chains, allowing for separate evaluation of subpopulations of light and heavy chain species with varying levels of drug loading. Analysis of these data therefore allowed for the calculation of the average light chain drug loading and the average heavy chain drug loading as independent factors, which were then combined to determine the average antibody drug loading, based on the basic knowledge that each antibody consists of two light chains and two heavy chains. Chromatographic conditions were as follows: a PRLP-S column, 1000 Å, 50 x 2.1 mm, 8 μm particle size (Agilent Technologies), with water + 0.05% TFA as mobile phase A and acetonitrile + 0.01% TFA as mobile phase B, elution with a linear gradient from 27% B to 42% B in 12.5 minutes.

[0251] Anti-BCMA antibodies were conjugated with SGD-1006 and SGD-1269 in three separate batches over a period of several months. In the first batch, a total of 29 antibodies were conjugated (resulting in 58 ADCs). The drug loading of each isotype control as determined by PLRP chromatography and the data are summarized in Table 3. [Table 3]

[0252] For the second batch, an additional 25 antibodies were conjugated (resulting in 50 ADCs). The drug loading of each isotype control was again determined by PLRP chromatography and the data are summarized in Table 4. [Table 4]

[0253] In the third batch, 30 antibodies were conjugated (resulting in 60 ADCs), including 13 humanized variants of CA8. The drug loading of all ADCs in this final batch was determined and is summarized in the following two plate maps (Tables 5 and 6). [Table 5] [Table 6]

[0254] The mean drug loading and %CV are shown at the bottom for each isotype species. Uncharacteristically large variability in drug loading was observed for the SGD-1269 ADC prepared with the mIgG2b antibody, the reason for this is unclear. Also, the Fc-enhanced CA8 antibody produced somewhat lower drug loading levels than the other CA8 human variants; to address this, additional Fc-enhanced CA8 was conjugated in a solution-phase reaction to better match the drug loading achieved for the other antibodies.

[0255] Example 4: Binding data 4.1 FMAT Binding Assay Showing Binding of Chimeric CA8 to Cells Expressing Human or Cyno BCMA Cryopreserved transfected human, cyno BCMA, and mock-transfected HEK293 cells were collected from LN2 storage. Assay wells were prepared with human chimeric CA8 antibody at a range of different concentrations and mixed with human BCMA HEK293, cyno BCMA HEK293, and mock-transfected cells, respectively. An anti-human IgG FMAT Blue secondary conjugate was added to detect human chimeric CA8. Assay plates were left undisturbed for a minimum of 90 minutes before results were read on an ABI8200 (FMAT) plate reader.

[0256] This demonstrated that the chimeric form of the CA8 antibody binds well to both human and cyno BCMA proteins expressed in HEK293 cells.

[0257] The results are shown in Figure 1.

[0258] 4.2 ELISA experiments demonstrating binding of chimeric CA8 to recombinant BCMA protein The chimeric CA8 antibody was tested for binding to human BCMA and cyno BCMA expressed as Fc fusions. Human BCMA-Fc and cyno BCMA-Fc were coated onto ELISA plates, and the plates were blocked with BSA to reduce nonspecific binding. CA8 chimeric antibody was added to the human and cyno BCMA-coated ELISA plates at concentrations ranging from 5 μg / ml to 0.1 μg / ml. Where necessary, an anti-human IgG HRP-conjugated secondary antibody was used to detect any bound human chimeric CA8 antibody. The ELISA was developed with the addition of HRP substrate (TMB). This demonstrated that the CA8 antibody bound to recombinant human and cyno BCMA in the ELISA assay.

[0259] The results are shown in Figure 2.

[0260] 4.3 Biacore Experiments Showing CA8 Antibody Binding to BCMA and TACI Proteins to Determine Cross-Reactivity with TACI Protein CA8 chimeric antibody was injected and captured on Protein A (a Protein A-inducible sensor chip was used). Residual Protein A binding was blocked by injecting a high concentration of human IgG solution. BCMA-Fc, TACI-Fc, or BAFF-R-Fc solutions were then tested for binding to the antibody. The three proteins were injected sequentially, and binding events were measured. The surface was regenerated between each protein injection.

[0261] The sensorgrams were analyzed in the Biaevaluation program. Double reference subtraction was performed to remove instrument noise and any nonspecific binding from the sensorgram curves.

[0262] This demonstrated that CA8 is specific for binding to BCMA and not TACI or BAFFR.

[0263] The binding of CA8 antibody to BCMA-Fc, TACI-Fc, and BAFF-R-Fc was plotted as shown in Figure 3.

[0264] 4.4 Cell Binding and Neutralization Data 4.4.1 Binding of Murine Anti-BCMA Antibodies to Multiple Myeloma Cells and BCMA-Expressing Cells Multiple myeloma cell line H929 and transfected cells expressing ARH77-hBCMA 10B5 BCMA were stained with murine S332211D07, S3332121F02, or S332126E04 at 5 μg / mL or murine isotype control. Multiple myeloma cell line H929 was stained with murine S307118G03. Cells were incubated for 20 minutes at room temperature (RT) and then washed with FACS buffer (PBS + 0.5% BSA + 0.1% sodium azide) to remove unbound antibody. Cells were incubated with a secondary PE-labeled anti-mouse IgG antibody for 15 minutes at RT and then washed with FACS buffer to remove unbound antibody. Cells were analyzed by FACS to detect cell-bound antibody.

[0265] The results (Figure 4) showed that all four murine antibodies bound to the H929 multiple myeloma cell line, and the three antibodies tested on ARH77 BCMA-transfected cells.

[0266] 4.4.2 Binding curves of chimeric CA8 to multiple myeloma cells as determined by FACS Chimeric CA8 binding was determined using a panel of multiple myeloma cell lines. Cell lines H929, OPM-2, JJN-3, and U266 were stained with either chimeric CA8 or an irrelevant antibody (Synagis) at various concentrations for 20 minutes at room temperature. Cells were then washed with FACS buffer (PBS + 0.5% BSA + 0.1% sodium azide) to remove unbound antibody. Cells were incubated with a secondary PE-labeled anti-human IgG antibody for 15 minutes at room temperature and then washed with FACS buffer to remove unbound antibody. Cells were analyzed by FACS, and binding was determined by measuring mean fluorescence intensity (MFI) values.

[0267] The results showed that chimeric CA8 bound to the multiple myeloma cell lines H929, OPM-2, JJN-3, and U266 in a dose-dependent manner (Fig. 5).

[0268] 4.4.3 Binding of Humanized CA8 to BCMA-Transfected Cells as Determined by FACS ARH77-hBCMA 10B5 BCMA-expressing transfected cells or H929 cells were stained with either chimeric CA8 or humanized variants of CA8 designated J6M0, J6M1, J6M2, J9M0, J9M1, or J9M2 at various concentrations for 20 minutes at room temperature. Cells were then washed with FACS buffer (PBS + 0.5% BSA + 0.1% sodium azide) to remove unbound antibody. Cells were incubated with a secondary PE-labeled anti-human IgG antibody for 15 minutes at room temperature and then washed with FACS buffer to remove unbound antibody. Cells were analyzed by FACS, and binding was determined by measuring mean fluorescence intensity (MFI) values.

[0269] The results showed that all antibodies tested except for chimeric CA8 and J9M2 bound to ARH77-hBCMA 10B5 BCMA-expressing transfected cells and H929 cells in a dose-dependent manner (Figure 6).

[0270] 4.5 Demonstration of the ability of CA8 and humanized J6M0 to neutralize binding of BAFF or APRIL to recombinant BCMA The purpose of this assay was to evaluate the ability of antibodies CA8, in both wild-type and afucosylated (Potelligent) forms, and humanized version J6M0, at various concentrations to neutralize the binding capacity of either BCMA ligand, BAFF or APRIL.

[0271] 96-well flat-bottom plates were coated overnight with a 1 μg / mL solution of recombinant human BCMA Fc4-53 in PBS. After a washing step with 0.05% TWEEN® 20, the plates were blocked with a 2% bovine serum albumin solution in PBS for 1 hour at room temperature. The plates were washed as above, and 40 μL of each antibody (mouse IgG, mouse CA8, and chimeric CA8), starting at 10 μg / mL and titrated 1 in 2 in duplicate, was added to the relevant wells and incubated for 1 hour at room temperature. 40 μL of 2% BSA was added to the relevant control wells. 10 μL of either recombinant human BAFF (2149-BF / CF, R&D Systems) or recombinant human APRIL (5860-AP / CF, R&D Systems) was added at 30 ng / mL and 750 ng / mL, respectively, to give final concentrations of 6 ng / mL and 150 ng / mL in each well, respectively. An equivalent volume of 2% BSA was added to the relevant control wells. The plates were incubated at room temperature for 2 hours, after which they were washed as described above. Biotinylated anti-human ligand (BAFF BAF124 or APRIL BAF884, R&D Systems) was added to the relevant wells at 50 ng / mL and incubated for 1 hour. After a washing step, 50 μL of a 1:4000 dilution of streptavidin-HRP (Amersham RPN4401) was added to each well and incubated for 30 minutes at room temperature. The washing process was repeated again, after which 100 μL of tetramethylbenzidine substrate solution (T8665, Sigma) was added to each well. The plates were incubated at room temperature for 20-25 minutes and wrapped in foil. The reaction was stopped by adding 100 μL of 1 M H2SO4. The optical density was determined at 450 nm using a Spectromax reader. See Figures 7A and B.

[0272] In plate-based assays to neutralize BAFF or APRIL binding to BCMA, calculated EC50 values ​​for chimeric CA8 were 0.695 μg / mL and 0.773 μg / mL, respectively. Values ​​for humanized J6M0 were 0.776 ng / mL and 0.630 ng / mL. Values ​​for J6M0 Potelligent™ were 0.748 and 0.616 ng / mL, respectively.

[0273] 4.6 Effect of chimeric CA8 and humanized J6M0 BCMA antibodies on BAFF- or APRIL-induced phosphorylation of NFkB in H929 cells In one set of experiments, H-929 cells were seeded at 75,000 cells / well in serum-free medium in 96-well plates. Chimeric CA8 antibody was added 24 hours later to give a final well concentration of 200 μg / ml. After 10 minutes, BAFF or APRIL ligand was added to the cells to give a final well concentration of 0.6 or 0.3 μg / ml, respectively. After 30 minutes, cells were lysed and phosphorylated NFkappaB levels were measured using the MSD pNFkappaB assay.

[0274] The chimeric BCMA antibody CA8 neutralized both BAFF and APRIL-induced Nfkappa B cell signaling in H-929 cells. It was particularly effective at neutralizing BAFF-induced Nfkappa B cell signaling in this cell type with a mean IC50 of 10 nM compared to 257 nM for APRIL-induced Nfkappa B cell signaling.

[0275] Meaned data from two experiments The IC50 was 10 nM for BAFF-induced NfkappaB neutralization and 257 nM for APRIL-induced NfkappaB neutralization (average of two independent experiments) and are shown in Table 7. [Table 7]

[0276] A further set of experiments was conducted to understand why there was such a discrepancy between the potencies of APRIL and BAFF in neutralizing BCMA in a cell-based system. After discovering the soluble form of BCMA, the experimental design was modified to include a step of washing H929 cells prior to the assay to reduce interference with BCMA lysis by antibody binding. H-929 cells were washed three times to remove some sBCMA and resuspended in serum-free medium. J6M0 Potelligent antibody was added to 96-well plates to obtain a final concentration of 100 μg / ml, along with BAFF or APRIL ligand to obtain final concentrations of 0.6 or 0.2 μg / ml, respectively. H-929 cells were then seeded at 7.5 x 10 cells / well in serum-free medium. After 30 minutes, cells were lysed and phosphorylated NF-kappa B levels were measured using the MSD pNF-kappa B assay. This is data from a single experiment. Each data point is the mean / sd of two replicates. Data from this experiment are shown in Figure 7c. The IC50 for inhibiting BAFF and APRIL signaling was determined to be 0.91 ug / ml and 2.43 ug / ml, respectively.

[0277] 4.7 ProteOn Analysis of Anti-BCMA CA8 Chimeric and Humanized Constructs Initial screening of CA8 chimeric and humanized variants was performed on a ProteON XPR36 (Biorad). The method was as follows: Protein A was immobilized on a GLC chip (Biorad, catalog number: 176-5011) by primary amine coupling; CA8 variants were then captured on this surface; recombinant human BCMA (in-house or commercially available US Biological, B0410) material (only two runs) was passed over at 256, 64, 16, 4, and 1 nM, using a 0 nM injection (i.e., buffer only). The buffer used was HBS-EP buffer. The capture surface was regenerated using 50 mM NaOH. Data were fitted to a 1:1 model using the analysis software built into the ProteOn XPR36. Run 1 corresponds to the first screening of humanized CA8 variants (J0-J5 series), and Run 2 corresponds to the second screening of humanized CA8 variants (J5-J9 series). Both runs were carried out at 25°C.

[0278] Data from Run 1 is shown in Table 8 and data from Run 2 is shown in Table 9. Some molecules in Run 2 (Table 09) failed to obtain measurable affinity values ​​by ProteOn due to off-rates that were beyond the sensitivity of the instrument in this assay, indicating that all these molecules bound tightly to recombinant human BCMA. From Run 1, the data show that some constructs showed no binding to recombinant cyno BCMA at all. [Table 8] [Table 9]

[0279] 4.8 BIAcore analysis of anti-BCMA CA8 chimeric and humanized constructs (J7-J9 series) Protein A was immobilized onto a CM5 chip (GE Healthcare, Catalog No. BR-1005-30) by primary amine coupling, and then this surface was used to capture antibody molecules. Recombinant human BCMA (US Biological, B0410) was used as the analyte at 256 nM, 64 nM, 16 nM, 4 nM, and 1 nM. The capture surface was regenerated using 50 mM NaOH. All binding curves were double-referenced with a buffer injection (i.e., 0 nM), and the data were fitted using the 1:1 model inherent in the T100 evaluation software. Runs were performed at 37°C using HBS-EP as the running buffer.

[0280] The results showed that the molecules tested, with the exception of J9M2, bound to recombinant human BCMA with similar affinity as the chimeric molecule. The data generated from this experiment are presented in Table 10. [Table 10]

[0281] 4.9 BIAcore analysis of anti-BCMA CA8 chimeric and humanized constructs J6M0 and J9M0 Protein A was immobilized onto a CM5 chip (GE Healthcare, Catalog No. BR-1005-30) by primary amine coupling, and then this surface was used to capture antibody molecules. Recombinant human BCMA (US Biological, B0410) was used as the analyte at 256 nM, 64 nM, 16 nM, 4 nM, and 1 nM. The capture surface was regenerated using 50 mM NaOH. All binding curves were double-referenced with a buffer injection (i.e., 0 nM), and the data were fitted using the 1:1 model inherent in the T100 evaluation software. Experiments were run at 25°C and 37°C for Experiment 1 and 37°C only for Experiment 2, using HBS-EP as the running buffer.

[0282] Both runs identified J9M0 as the best molecule in terms of overall affinity to human BCMA. The data generated from this experiment is presented in Table 11. [Table 11]

[0283] 4.10. ProteOn Analysis of Novel Anti-BCMA Chimeric Constructs Initial screening of novel chimeric variants from the second batch of hybridomas was performed on a ProteOn XPR36 (Biorad). The method was as follows: Protein A was immobilized on a GLM chip (Biorad, catalog number: 176-5012) by primary amine coupling, and anti-BCMA variants were then captured on this surface. Recombinant human BCMA (in-house material) was passed over at 256, 64, 16, 4, and 1 nM, with a 0 nM injection (i.e., buffer only) used to double-reference the binding curves. The buffer used was HBS-EP buffer. Regeneration of the capture surface was performed using 50 mM NaOH. Data were fitted to a 1:1 model using the analysis software native to the ProteOn XPR36. Runs were performed at 25°C.

[0284] The data generated from this experiment is presented in Table 12. [Table 12]

[0285] [Example 5] Cell death assay 5.1 ADCC Potency of Chimeric CA8 and Defucosylated Chimeric CA8 Forms in BCMA-Expressing ARH77 Cells Human natural killer (NK) cells were incubated with europium-labeled ARH77 BCMA-transfected target cells (10B5) in the presence of various concentrations of antibody at an E:T ratio of 5:1 for 2 hours. Europium release from target cells was measured and specific lysis was calculated.

[0286] Results: Chimeric CA8 and defucosylated chimeric CA8 killed BCMA-expressing target cells by ADCC. The defucosylated chimeric antibody demonstrated fully potent ADCC activity, as measured by a higher percentage of lysis achieved in all target cells tested, and a 10-fold lower EC50 in the highly BCMA-expressing target cell line 10B5 compared to the parent chimeric antibody. See Figures 8A and 8B.

[0287] 5.2 ADCC Activity of CA8 Humanized Antibody Using ARH77 BCMA-Expressing Target Cells and PBMCs as Effectors Human PBMCs were incubated with europium-labeled ARH77 BCMA-transfected target cells (10B5) in the presence of various concentrations of the humanized form of CA8 antibody (5 μg / ml to 0.005 μg / ml) at an E:T ratio of 5:1 for 2 hours. Europium release from the target cells was measured and specific lysis was calculated.

[0288] result: Results: All of the humanized versions of CA8, J5, J6, J7, J8, and J9 series, demonstrated ADCC activity against the ARH77-high BCMA-expressing cell line 10B5 in a dose-dependent manner. ADCC was at levels similar to those found in experiments using chimeric CA8 molecules. See Figure 9.

[0289] 5.3 ADCC Potency of Chimeric S322110F02, S322110D07, and S307118G03 and Humanized S307118G03 H3L0 Against BCMA-Expressing ARH77 10B5 Cells Using Purified NK Cells as Effector Cells Human natural killer (NK) target cells were incubated with europium-labeled ARH77 BCMA-transfected target cells (10B5) in the presence of various concentrations of antibody at an E:T ratio of 5:1 for 2 hours. Europium release from the target cells was measured and specific lysis was calculated.

[0290] Results: All four antibodies tested showed ADCC activity against ARH77 10B5 cells. See Figure 10.

[0291] 5.4 Antibody-Drug Conjugate (ADC) Activity of Chimeric CA8 ADCs The ADCC activity of the chimeric CA8 antibody, the chimeric CA8-mcMMAF antibody-drug conjugate, and the chimeric CA8-vcMMAE antibody-drug conjugate against human multiple myeloma cell lines was measured. The multiple myeloma cell lines were treated with the chimeric CA8 antibody-drug conjugates to determine the ADC concentration required for growth inhibition and death.

[0292] The antibody-drug conjugates tested were added to wells containing multiple myeloma cells at concentrations ranging from 1 μg / ml to 5 ng / ml. Plates were incubated at 37°C for 96 hours, at which point viable cells were quantified using Cell titre Glo. Unconjugated chimeric CA8 antibody did not exhibit significant growth inhibitory activity at the antibody concentrations tested. The chimeric CA8-mcMMAF antibody-drug conjugate exhibited greater growth inhibitory activity than the chimeric CA8-vcMMAE antibody-drug conjugate in all four of the multiple myeloma cell lines tested. See Figure 11 and Table 13. [Table 13]

[0293] 5.5 Measurement of cell cycle arrest activity of chimeric CA8 antibody, chimeric CA8-mcMMAF antibody-drug conjugate, and chimeric CA8-vcMMAE antibody-drug conjugate against human multiple myeloma cell line H929.

[0294] To determine the mechanism by which chimeric CA8 antibody-drug conjugates (ADCs) cause growth inhibition in multiple myeloma cells, NCI-H929 cells were monitored by measuring cellular DNA content by fixed-cell propidium iodide staining at multiple time points after chimeric CA8 antibody and chimeric CA8 ADC treatment.

[0295] At the chimeric CA8 ADC concentration tested (50 ng / mL), the chimeric CA8-mcMMAF ADC induced significant G2 / M cell cycle arrest (4N DNA content), which was maximal at 48 hours. Subsequently, at 48, 72, and 96 hours, treatment with the chimeric CA8-mcMMAF ADC resulted in the accumulation of a cell population containing sub-2N DNA content, indicative of cell death. At the 50 ng / mL concentration tested, the chimeric CA8-vcMMAE ADC had no significant effect on G2 / M cell cycle arrest or sub-G1 accumulation. See Figure 12.

[0296] 5.6 Phospho-Histone-H3 (Thr11) Staining as a Marker for Mitotic Arrest Induced by Chimeric CA8-mcMMAF and Chimeric CA8-vcMMAE Antibody Drug Conjugates To determine whether the accumulation of cells with 4N DNA content was a specific consequence of mitotic arrest induced by chimeric CA8, ADC NCI-H929 cells were stained with an anti-phospho-histone H3 antibody after 48 hours of treatment with increasing concentrations of unconjugated chimeric CA8, chimeric CA8-vcMMAE, or chimeric CA8-mcMMAF.

[0297] Treatment with the chimeric CA8 ADC resulted in a dose-dependent accumulation of NCI-H929 cells that stained positive for 65eroxidi-Histone H3 (Thr11), a specific marker of mitotic cells. The chimeric CA8-mcMMAF ADC resulted in the accumulation of 65eroxidi-Histone H3-positive cells at lower concentrations than the chimeric CA8-vcMMAE ADC. See Figure 13.

[0298] 5.7 Measuring apoptosis in NCI-H929 cells in response to chimeric CA8 ADC by staining for Annexin V To determine whether the accumulation of cells with sub-2N DNA content was a specific consequence of apoptosis induced by the chimeric CA8 ADC, NCI-H929 cells were stained with anti-annexin-V antibody after 48 hours of treatment with increasing concentrations of unconjugated chimeric CA8, chimeric CA8-vcMMAE, or chimeric CA8-mcMMAF. Treatment with the chimeric CA8 ADC resulted in a dose-dependent accumulation of NCI-H929 cells that stained positive for annexin-V, a specific marker of apoptosis. The chimeric CA8-mcMMAF ADC caused accumulation of annexin-V-positive cells at lower concentrations than the chimeric CA8-vcMMAE ADC. See Figure 14.

[0299] 5.8 Antibody-Drug Conjugate (ADC) Activity of Humanized Variants of CA8 Anti-BCMA Antibody-Drug Conjugates Cells were seeded in 96-well plates (4,000 cells per well in 100 μL of RPMI + 10% FBS). Naked antibody or ADC was added 6 hours after cell seeding, and plates were incubated for 144 hours. Growth inhibition in the presence of antibody or ADCC was measured at 144 hours using CellTiterGlo. Data points represent the mean of triplicate CellTiterGlo measurements. Error bars represent standard error.

[0300] The multiple myeloma cell lines NCI-H929 and OPM2 were treated with humanized CA8 anti-BCMA antibody-drug conjugates to determine the ADC concentrations required for growth inhibition and death. The mcMMAF and vcMMAE antibody-drug conjugate forms of these antibodies demonstrated significant growth inhibitory activity compared to that found with the CA8 chimera. Variant J6M0 demonstrated greater potency than the chimera, and data are shown for H929 and OPM2 cells in Figure 15. The mcMMAF antibody-drug conjugates demonstrated greater growth inhibitory activity than the vcMMAE antibody-drug conjugates for all antibodies in both cell lines tested. Results for all humanized variants are shown in Table 14. [Table 14]

[0301] 5.9 Antibody-Drug Conjugate (ADC) Activity of Other Murine Antibody-Drug Conjugates Cells were seeded in 96-well plates (4,000 cells per well in 100 μL RPMI + 10% FBS). Antibodies or ADCs were added 6 hours after cell seeding and plates were incubated for 144 hours. Growth inhibition in the presence of ADC was measured at 144 hours using CellTiterGlo. The mean of triplicate CellTiterGlo measurements is represented. Tables 15a and 15b are from experiments performed at different times with different series of antibodies. The multiple myeloma cell lines NCI-H929 and U266-B1 were used for the antibodies in Table 15a.

[0302] The mcMMAF and vcMMAE antibody-drug conjugate forms of murine antibodies S322110D07, S332121F02, and S332136E04 demonstrated significant growth inhibitory activity. The mcMMAF antibody-drug conjugate demonstrated greater growth inhibitory activity than the vcMMAE antibody-drug conjugate in all of the murine anti-BCMA antibodies tested for which activity was observed. IC50 plots are shown in Table 15a. See Figure 16 for dose-response curves for these three antibodies and also S107118G03. Error bars represent standard error. NCI-H929, U266-B1, JJN3, and OPM2 cells for the antibodies in Table 15b were treated with a different series of murine anti-BCMA antibody-drug conjugates to determine the ADC concentrations required for growth inhibition and death. IC50 plots are shown in Table 15b. All five antibodies shown in that table had significant ADC activity. [Table 15]

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[0304] 5.10 ADCC Potency of Conjugated, Afucosylated J6M0 (Potelligent) Afucosylated J6M0 conjugated to MMAE or MMAF was tested in an ADCC assay using BCMA transfectants to ensure that its ADCC activity was not compromised by conjugation. Europium-labeled ARH77-10B5 cells were incubated with various J6M0 WT and Potelligent BCMA antibodies at concentrations up to 10,000 ng / ml for 30 minutes before adding PBMCs (PBMC:target cell ratio 50:1). After 2 hours, an aliquot of cell culture medium was sampled and mixed with enrichment solution. After 30 minutes on a plate shaker, europium release was monitored using a Victor 2 1420 multilabel reader. Data points represent the average of triplicate values. Data are representative of two experiments.

[0305] There was no significant difference in ADCC potency between the unconjugated and ADC forms of J6M0 Potelligent. In the same experiment, wild-type J6M0 was included to demonstrate how potency compared to the afucosylated form. As expected, defucosylation resulted in a lower EC50 and higher maximal lysis. No lysis was observed with the Fc-disabled form of J6M0 (Figure 17).

[0306] 5.11 ADCC Potency of Afucosylated J6M0 against MM Cell Lines Human PBMCs were incubated with multiple myeloma target cells at an E:T ratio of 50:1 in the presence of various concentrations of afucosylated (Potelligent) J6M0. The percentage of target cells remaining in the effector + target cell mixture after 18 hours was determined by FACS using a fluorescently labeled anti-CD138 antibody to detect target cells, and the percentage of lysis was calculated. This is representative of several experiments.

[0307] The J6M0 Potelligent antibody demonstrated ADCC activity against all five multiple myeloma cell lines tested. This was important to test since initial studies were performed using transfected cells. The results are shown in Figure 18. The complete data set, including multiple donors, is shown in Table 16. Potencies were all in the same range as those found with transfectants. ADCC activity was not directly related to BCMA surface expression on these cell lines. [Table 16]

[0308] Example 6: Xenograft data 6.1 Mouse xenografts of human MM cell lines were tested to ensure that antibody efficacy detected in vitro could also be demonstrated in vivo. The cell line selected for xenograft studies was NCI-H929, which is susceptible to in vitro killing ADC and ADCC. Studies were performed in immunodeficient CB.17 SCID mice, which lack T and B cells but maintain NK cells that enable ADCC activity. However, it should be noted that while human IgG1 can engage mouse Fc receptors, Potelligent enhancement does not improve affinity when performed with human Fc receptors.

[0309] 6.2 Effect of unconjugated and MMAE- or MMAF-conjugated J6M0 on NCI-H929 tumor growth To independently analyze both the ADCC and ADC activities of J6M0, we tested the J6M0 antibody in the presence and absence of MMAF or MMAE conjugation. By testing unconjugated J6M0, some antitumor effects appeared to be due to some combination of ADCC and functional inhibitory activity.

[0310] 200mm on average 3Mice bearing NCI-H929 tumors reaching a volume of 100 μg were tested with either a human IgG1 control or the J6M0 antibody (unconjugated MMAE or MMAF) at doses of 50 μg or 100 μg twice weekly for two weeks. Results from this study show that a 100 μg dose of the J6M0-MMAF conjugate resulted in tumor elimination in these mice that completed the dosing. After the final dose, J6M0-MMAF mice were maintained for 40 days with no recurrence of tumor development. These results from this experiment demonstrate that MMAF conjugation increased antitumor activity over both the unconjugated J6M0 antibody and the J6M0-MMAE conjugate. See Figure 19.

[0311] [Example 7] Evaluation of soluble BCMA levels from MM patient serum 7.1 It is currently unknown whether BCMA exists extracellularly and can be detected in the blood. In this study, we determined human BCMA serum levels from MM patients. Serum samples from 54 MM and plasma cell dyscrasia patients and 20 normal control samples were analyzed by ELISA. Approval for human subjects was obtained from the Western Institutional Review Board.

[0312] 7.2 Assessment of serum human BCMA levels Blood from patients and normal controls was collected in the clinic into serum collection tubes. MM patient samples were from various stages (progressive, remission, relapse, newly diagnosed, and others). Blood samples were spun at 10,000 rpm for 10 minutes, and serum was transferred into sterile microcentrifuge tubes.

[0313] BCMA was detected using a human BCMA / TNFRSF17 ELISA kit from R&D Systems (catalog no. DY193E), which measures soluble human BCMA levels, following the standard protocol supplied with the kit.

[0314] Briefly, a 96-well microplate was coated with 100 μl of capture antibody per well and incubated overnight at 4°C. The plate was washed three times with wash buffer (0.05% Tween® 20 in PBS, pH 7.2) and blocked with 300 μl of 1% BSA in PBS for 2 hours at room temperature. The plate was washed three times with wash buffer. 100 μl of serum sample or standard was added to each well and incubated for 2 hours at room temperature. The plate was washed three times with wash buffer, and then 100 μl of detection antibody was added to each well and incubated for 2 hours at room temperature. After washing the plate three times, 100 μl of streptavidin-HRP was added to each well and incubated for 20 minutes in the dark. The plate was washed three times, 50 μl of stop solution was added, and then the plate was measured using a microplate reader at a wavelength of 570 nM.

[0315] A series of assays were performed to determine an appropriate serum dilution factor for the level of BCMA that was present. A dilution factor of 1:500 was found to be appropriate for most samples and is the dilution factor used for the data shown in Figure 20. The complete data set is shown in Table 17.

[0316] Patient and normal control serum samples were diluted and run in triplicate and had BCMA levels measured. BCMA serum levels were significantly increased in serum from MM patients compared to normal controls in this study. When disease subsets were further separated, there was a trend for increased serum BCMA levels in serum from progressive MM patients compared to those in remission. This is the first report identifying serum BCMA in any human disease, suggesting that these levels may be a novel biomarker for monitoring disease status and treatment response in MM patients and other patients suffering from plasma cell-mediated diseases. [Table 17]

[0317] P-value (one-tailed T-test, 95% significance) ~1-500 single Normal vs. progressive: p=.0010 * Progressive vs. remission: p=.0146 * ~1-500 Triple Normal vs. progressive: p=.0004 * Progressive vs. remission: p=.0091 * ~1-50 attempts 1 Normal vs. progressive: p=.0171 * Progressive vs. remission: p=.0777 ~1-50 attempts 2 Normal vs. progressive: p=.0184 * Progressive vs. remission: p=.0876 * indicates significance.

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Claims

1. 1. An antigen binding protein that specifically binds to BCMA and inhibits binding of BAFF and / or APRIL to BCMA, wherein the antigen binding protein is capable of binding to FcγRIIIA or FcγRIIIA-mediated effector function and is capable of internalization.

2. 2. The antigen-binding protein of claim 1, which has enhanced binding to FcγRIIIA or has enhanced FcγRIIIA-mediated effector function.

3. The antigen-binding protein of claim 2 , wherein the antigen-binding fragment has an enhanced ADCC effector function.

4. 4. The antigen-binding protein of any one of claims 1 to 3, which is defucosylated.

5. 5. The antigen-binding protein of any one of claims 1 to 4, wherein the antigen-binding fragment does not bind to Taci.

6. 6. The antigen-binding protein of any one of claims 1 to 5, comprising a CDRH3 of SEQ ID NO: 3 or a variant of SEQ ID NO:

3.

7. 7. The antigen binding protein of claim 6, further comprising one or more of CDRH1 of SEQ ID NO: 1, CDRH2: SEQ ID NO: 2, CDRL1: SEQ ID NO: 4, CDRL2: SEQ ID NO: 5 and / or CDRL3: SEQ ID NO:

6.

8. i) CDRH3 as set forth in SEQ ID NO: 3; ii) CDRH1 as set forth in SEQ ID NO: 1, and iii) CDRH2 as set forth in SEQ ID NO: 2; 8. The antigen-binding protein of claim 7, comprising:

9. i) CDRH3 as set forth in SEQ ID NO: 3; ii) CDRH1 as set forth in SEQ ID NO: 1; iii) CDRH2 as set forth in SEQ ID NO: 2; iv) CDRL1 as set forth in SEQ ID NO: 4; v) CDRL2 as set forth in SEQ ID NO:5, and vi) CDRL3 set forth in SEQ ID NO: 6; 9. The antigen-binding protein of claim 8, comprising:

10. 10. The antigen-binding protein of any one of claims 1 to 9, comprising a heavy chain variable region encoded by any one of SEQ ID NO:23 or SEQ ID NO:27 or SEQ ID NO:

29.

11. 11. The antigen-binding protein of any one of claims 1 to 10, comprising a light chain variable region encoded by any one of SEQ ID NO: 31 or SEQ ID NO:

33.

12. 12. The antigen-binding protein of any one of claims 1 to 11, comprising a heavy chain variable region encoded by SEQ ID NO:23 and a light chain variable region encoded by SEQ ID NO:

31.

13. 12. The antigen-binding protein of any one of claims 1 to 11, comprising a heavy chain encoded by SEQ ID NO:27 and a light chain encoded by SEQ ID NO:

31.

14. 14. The antigen-binding protein of any one of claims 1 to 13, which is a humanized monoclonal antibody.

15. 15. The antigen-binding protein of claim 14, wherein the antibody is an IgG1 isotype.

16. Fab, Fab', F(ab') comprising the CDR of any one of claims 6 to 9 2 , an Fv, a diabody, a triabody, a tetrabody, a miniantibody, a minibody, an isolated VH, or an isolated VL fragment.

17. 17. The antigen-binding protein of any one of claims 1 to 16, which further binds to non-human primate BCMA.

18. 18. The antigen-binding protein of any one of claims 1 to 17, which binds to BCMA with an affinity of greater than 150 pM.

19. An immunoconjugate comprising the antigen-binding protein of any one of claims 1 to 18 and a cytotoxic agent.

20. 20. The immunoconjugate of claim 19, wherein the antigen-binding protein is conjugated to the cytotoxic agent via a linker.

21. 21. The immunoconjugate of claim 19 or 20, wherein the cytotoxic agent is auristatin or dolostatin.

22. The immunoconjugate of any one of claims 19 to 21, wherein the cytotoxic agent is selected from MMAE and MMAF.

23. The immunoconjugate of any one of claims 19 to 22, wherein the cytotoxic agent is covalently attached to the antigen-binding protein.

24. The immunoconjugate of any one of claims 20 to 23, wherein the linker is a cleavable linker.

25. The immunoconjugate of any one of claims 20 to 23, wherein the linker is a non-cleavable linker.

26. 26. The immunoconjugate of any one of claims 20 to 25, wherein the linker is selected from 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), N-succinimidyl 4-(2-pyridylthio)pentanoate (SPP), N-succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), and N-succinimidyl(4-iodo-acetyl)aminobenzoate (SIAB).

27. The immunoconjugate of any one of claims 19 to 26, wherein the immunoconjugate is phagocytosed by tumor cells when contacted with the tumor cells.

28. 28. A pharmaceutical composition comprising the antigen-binding protein or immunoconjugate of any one of claims 1 to 27 and a pharmaceutically acceptable carrier.

29. 30. A method of treating a human patient suffering from an inflammatory disorder or disease, comprising administering the composition of claim 28.

30. 28. Use of the composition of claim 27 in the treatment of a human patient suffering from a B-cell lymphoma, such as multiple myeloma (MM) or chronic lymphocytic leukemia (CLL).

31. 28. An antigen-binding protein or immunoconjugate according to any one of claims 1 to 27 for use in treating a human patient suffering from a B-cell lymphoma, such as multiple myeloma (MM) or chronic lymphocytic leukemia (CLL).

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