BCMA(CD269 / TNFRSF17) binding protein

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 antigens (BCMAs), particularly human BCMAs (hBCMAs).

[0002] The present invention also relates to a method for treating a disease or disorder using the antigen-binding fragment, a pharmaceutical composition comprising the antigen-binding fragment, and a method for producing such a composition. Other embodiments of the present invention will become apparent from the following details. [Background technology]

[0003] BCMA (CD269 or TNFRSF17) is a member of the TNF receptor superfamily. It is a nonglycosylated intrinsic membrane receptor for the ligands BAFF and APRIL. BCMA's ligands can also bind to APRIL and BAFF, as well as to BAFF-R (BAFF receptor or BR3), which exhibits a limited but high affinity for BAFF, and to further receptors: TACI (transmembrane activators, calcium regulators, and cyclophylline ligand interacting substances). In summary, these receptors and their corresponding ligands regulate various aspects of humoral immunity, B cell development, and homeostasis.

[0004] BCMA expression is typically restricted to B cell lineages and has been reported to increase in response to terminal B cell differentiation. BCMA is expressed by human plasma blasts, tonsils, spleens, and bone marrow-derived plasma cells, but also by tonsil memory B cells and germinal center B cells with TACI-BAFFR low phenotype (Darce et al, 2007). BCMA is barely present on unsensitized cells and memory B cells (Novak et al, 2004a and b). BCMA antigens are expressed on the cell surface and can be accessed by antibodies, but they are 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 is also important 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, the BCMA-APRIL signaling axis is dominant in the later stages of B cell differentiation and is suggested to be perhaps the most physiologically important interaction.

[0005] Multiple myeloma (MM) is a clonal B-cell malignancy that arises in multiple locations within the bone marrow, either de novo or as an exacerbation from monoclonal immunoglobulinemia (MGUS) of unknown significance, before spreading into the bloodstream. It is generally characterized by increased paraprotein and osteoclast activity, as well as hypercalcemia, cytopenia, renal dysfunction, hyperviscosity, and peripheral neuropathy. Decreased levels of both normal antibodies and neutrophil counts are also common, leading to life-threatening susceptibility to infection. BCMA has been implicated 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, the TACI and BCMA genes were demonstrated to be overexpressed in multiple myeloma cells (MMCs) compared to 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 normal bone marrow, as well as primary tumor cells from B-cell leukemia (Bellucci et al, 2005). The BCMA gene was highly expressed in all myeloma samples. Purified plasma cells from patients with MGUS expressed BCMA less, but there was no significant difference compared to 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 that overexpress BAFF or APRIL via gene transfer significantly increased the incidence of B-cell lymphomas (Batten et al, 2004-BAFF; Planelles et al, 2004-APRIL). In humans, excessive BAFF and APRIL have been detected in the serum and microenvironment of patients with multiple B-cell malignancies and other B-cell injuries.

[0007] All patents and references disclosed herein are incorporated herein expressly and entirely by reference. [Overview of the project] [Problems that the invention aims to solve]

[0008] The present invention provides an antigen-binding protein that binds to a membrane-bound target and can be internalized. In a further embodiment, the present invention provides an immunoconjugate comprising the antigen-binding protein 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 an antigen-binding protein that specifically binds to BCMA, for example, an antibody that specifically binds to BCMA and inhibits the binding of BAFF and / or APRIL to the BCMA receptor. The present invention also provides an antigen-binding protein that specifically binds to BCMA and inhibits the binding of BAFF and / or APRIL to BCMA, and which can bind to FcγRIIIA or have an FcγRIIIA-mediated effector function.

[0010] The antigen-binding protein of the present invention specifically binds to BCMA, inhibits the binding of BAFF and / or APRIL to BCMA, and has enhanced binding to FcγRIIIA or enhanced FcγRIIIA-mediated effector function. In one embodiment, the antigen-binding protein can be internalized.

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

[0012] In one embodiment of the present invention, an immune conjugate comprising the antigen-binding protein and the cytotoxic agent of the present invention is provided.

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

[0014] In further embodiments, the drug conjugate is vcMMAE or mcMMAF. In one embodiment, the immune conjugate is also enhanced ADCC.

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

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

[0017] Other mouse monoclonal antibodies (from which the antigen-binding protein 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 include the following CDRs or variants of those CDRs (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 include the following CDRs or variants of those CDRs (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 a polynucleotide sequence encoding the heavy chain variable region of any of the antigen-binding proteins described herein, and a polynucleotide encoding the light chain variable region of any of the antigen-binding proteins described herein.

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

[0022] Such polynucleotides represent the coding sequences corresponding to the equivalent polypeptide sequences, and it will be understood that such polynucleotide sequences can be cloned within an expression vector together with a start codon, an appropriate signal sequence and a stop codon.

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

[0024] The present invention provides a method for producing any of the antigen-binding proteins described herein, the method comprising culturing a host cell comprising a first and a second vector in a suitable medium (e.g., a serum-free medium), wherein the first vector comprises a polynucleotide encoding the heavy chain of any of the antigen-binding proteins described herein, and the 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 embodiment, the present invention provides a method for treating or preventing a disease or disorder that responds to inhibition or blockade of BCMA, such as by modulating the interaction between BCMA and its ligand, BAFF, or APRIL, the method comprising the step of administering a therapeutically effective amount of the antigen-binding protein described herein to the patient.

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

[0028] In another embodiment of the present invention, a method is provided for 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), the method comprising the step of administering to the patient a therapeutically effective amount of an antigen-binding protein described herein.

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

[0030] [Figure 1] This figure shows the results of an FMAT assay for CA8 antibody binding to human and cyno BCMA-expressing HEK293 cells. Human chimeric CA8 binds well to human and cyno BCMA-expressing cells. [Figure 2]This figure shows the ELISA binding assay results for CA8 antibodies that bind to human and cyno-BCMA recombinant proteins. This clearly demonstrates that human chimeric CA8 antibodies bind equally to human and cyno-BCMA proteins. [Figure 3] This figure shows the binding of CA8 to BCMA-Fc, TACI-Fc, and BAFF-R-Fc proteins in the BiaCore binding assay-BiaCore experiment. CA8 chimeric antibodies do not bind to TACI or BAFF-R proteins. [Figure 4] This figure shows the binding of mouse S307118G03, S3222110D07, S332121F02, and S332126E04 to H929 multiple myeloma cells as determined by cell binding assay-FACS, and the binding of S3322110D07, S332121F02, and S332126E04 to BCMA-transfected ARH77 cells. Transformed cells expressing the multiple myeloma cell line H929 or ARH77-hBCMA 10B5 BCMA were stained with either mouse anti-BCMA antibody (solid histogram) or mouse IgG2a isotype control (open histogram). Cells were analyzed by FACS to detect antibodies bound to the cells. [Figure 5] This figure shows the binding of chimeric CA8 to a panel of multiple myeloma cell lines as determined by cell binding assay-FACS. Binding to H929, OPM-2, JJN-3, and U266 was tested by flow cytometry, and binding was determined by measuring the mean fluorescence intensity (MFI) value. Synagis was used as an unrelated isotype control. [Figure 6] This figure shows the binding curves of humanized CA8 variants to BCMA-transfected ARH77 cells (A) and multiple myeloma H929 cells (B), as determined by cell binding assay-FACS. Humanized variants J6M0, J6M1, J6M2, J9M0, J9M1, and J9M2 were tested by flow cytometry, and their binding was determined by measuring the mean fluorescence intensity (MFI) value compared to the CA8 chimera. [Figure 7-1]Ligand Neutralization Assays - (A and B) Figures showing the ability of CA8 and J6M0 to neutralize the binding of recombinant BAFF or APRIL to recombinant BCMA coated on ELISA plates. The antibody-mediated inhibition of the maximum signal achieved by the binding of the relevant ligand alone to recombinant BCMA was calculated using OD values. Data are reported as a percentage of inhibition of the maximum signal. The antibodies tested were chimeric CA8 and humanized CA8-type J6M0 in both wild-type and afucosylated (potelligent) forms. (A) Neutralization of BAFF ligand binding, (B) Neutralization of APRIL ligand binding. (C) Figure showing the ability of the J6M0 BCMA antibody in inhibiting BAFF or APRIL-induced phosphorylation of NF kappa B in H929 cells. H929 cells were washed three times to remove some sBCMA and resuspended in serum-free medium. J6M0 Potelligent antibody was added to a 96-well plate and combined with BAFF or APRIL ligand to obtain final well concentrations up to 100 ug / ml, resulting in final well concentrations of 0.6 ug / ml or 0.2 ug / ml, respectively. H929 cells were then seeded in serum-free medium at 7.5 × 10⁴ cells / well. After 30 minutes, the cells were lysed and phosphorylated NF kappa B levels were measured using the MSD pNF kappa B assay (MSD Reader 502819). This data is from a single 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 - This figure shows the ADCC activity of chimeric CA8 and defucosylated (Fc-enhanced) CA8 together with target cells expressing BCMA. Human NK cells were incubated with target cells transfected with europium-labeled ARH77 10B5 BCMA in the presence of various antibody concentrations. Europium release from target cells was measured and specific lysis was calculated. (A) ADCC dose-response curve of chimeric CA8 compared to isotype control. (B) ADCC dose-response curve of chimeric CA8 and defucosylated chimeric CA8 (Fc-enhanced) against the BCMA-expressing cell line ARH77 10B5. [Figure 9]This figure shows an ADCC assay using CA8 humanized antibodies with target cells expressing ARH77 BCMA. 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 ug / ml. [Figure 10] This figure shows the ADCC activity of chimeric cells S332121F02(A), S3322110D07(B), S307118G03(C), and humanized S307118G03 H3L0(D) against ARH7710B5 target cells together with purified NK cells as effector cells in an ADCC assay. Human NK target cells were incubated with target cells transfected with europium-labeled ARH77 10B5 BCMA in the presence of various antibody concentrations. Europium release from target cells was measured and specific lysis was calculated. [Figure 11] This figure shows dose-response curves in a cell survival assay for chimeric CA8 antibodies, 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 measured using CellTiterGlo. Data points represent the mean of three consecutive CellTiterGlo measurements. Error bars represent the standard error. [Figure 12]This figure shows the effect of CA8 chimeric antibodies on the cell cycle. (A) Histograms of the cell cycle of NCI-H929 cells treated with unconjugated chimeric CA8, chimeric CA8-vcMMAE ADC, or chimeric CA8-mcMMAF ADC at 50 ng / mL at 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 shown in the graph. (B) Quantification of 4N DNA cell populations showing G2 / M arrest and (C) Sub-2N DNA cell populations showing cell death for each of the indicated treatments. Cells were seeded in 12-well plates (2 × 10⁵ cells / well in 1 mL of RPMI + 10% FBS). Antibody or ADC was added 6 hours after cell seeding. [Figure 13] This figure shows the effect of chimeric CA8 on phosphohistone H3. Chimeric CA8 ADC treatment results in increased phosphohistone 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-phosphohistone H3 (Thr11) antibody (FL1-H) on the y axis after treatment with either control IgG (A) or chimeric CA8-mcMMAF (B). (C) Quantification of phosphohistone H3-positive NCI-H929 cells after 48 hours of treatment with chimeric CA8 ADC at the indicated concentrations. 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 × 10⁵ cells / well in 1 mL of RPMI + 10% FBS). Antibody or ADC was added 6 hours after cell seeding. [Figure 14]This figure 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 high 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 × 10⁵ cells / well in 1 mL of RPMI + 10% FBS). Antibody or ADC was added 6 hours after cell seeding. [Figure 15] This figure shows the dose-response curves for survival assays—both unconjugated (naked) and vcMMAE and mcMMAF antibody-drug conjugates. Antibody-drug conjugates were tested against human multiple myeloma cell lines NCI-H929 and OPM2. [Figure 16] This figure shows the dose-response curves of mouse anti-BCMA antibodies S332121F02, S322110D07, S332126E04, and S307118G03 in human multiple myeloma cell lines NCI-H929 and U266-B1, for unconjugated antibodies, vcMMAE, and mcMMAF antibody-drug conjugates. [Figure 17] This figure shows the ADCC assay using the ADC J6M0 molecule and target cells expressing ARH77 BCMA. Human PBMCs were incubated with target cells transfected with europium-labeled ARH77 BCMA 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 with a Victor 2 1420 multi-label reader. [Figure 18]This figure shows the ADCC dose-response curves of CA8 J6M0 Potelligent for a panel of five multiple myeloma cell lines. Human PBMCs were incubated with multiple myeloma target cells for 18 hours at an E:T ratio of 50:1 in the presence of various concentrations of CA8 J6M0 Potelligent antibody. 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 for the five multiple myeloma cell lines tested. Each data point is from the singlicate value. [Figure 19] This figure shows the effect of dose-increasing of J6M0 and drug-conjugated J6M0 on the proliferation and establishment of NCI-H929 cells in CB.17 SCID mice. The calculated tumor volume of NCI-H929 tumors in CB17 SCID mice after two weekly intraperitoneal doses of either unconjugated or MMAE- or MMAF-conjugated 50 or 100 ug of J6M0 anti-BCMA or IgG1 isotype control over a two-week period. Data points represent the mean tumor volume per group (n=5). [Figure 20] This figure shows the determination of soluble BCMA levels in serum from healthy volunteers and myeloma patients. Serum samples were collected from MM patients, representing various stages (progressive, sedated, relapsed, newly diagnosed, and others). The samples shown in the figure are serum dilutions of 1 / 500 before assay. BCMA was detected using the R&D Systems Human BCMA / TNFRSF17 sandwich ELISA kit to measure soluble human BCMA levels, following the standard protocol provided with the kit. [Modes for carrying out the invention]

[0031] The present invention provides an antigen-binding protein that binds to a membrane-bound target and is internalizable. In a further embodiment, an immunoconjugate comprising the antigen-binding protein of the present invention and a cytotoxic agent is provided. 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, an antigen-binding protein or fragment thereof is provided that specifically binds to BCMA, for example, human BCMA (hBCMA), and inhibits the 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, inhibiting the binding of BAFF and / or APRIL to BCMA, and the antigen-binding protein or fragment has the ability to bind to FcγRIIIA and mediate FcγRIIIA-mediated effector function, or has enhanced FcγRIIIA-mediated effector function. In one embodiment provided in the present invention, the antigen-binding protein can be internalized.

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

[0035] In one embodiment of the present invention, an antigen-binding protein described herein is provided, comprising CDRH3 of SEQ ID NO: 3 or a variant of SEQ ID NO: 3.

[0036] In a further embodiment of the present invention, an antigen-binding protein described herein is provided, further comprising one or more of the following: 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 embodiment of the present invention, an antigen-binding protein described herein is provided, comprising CDRH3 of SEQ ID NO: 184 or a variant of SEQ ID NO: 184.

[0038] In a further embodiment of the present invention, an antigen-binding protein described herein is provided, further comprising one or more of the following: 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 further embodiment, the antigen-binding protein includes CDRH3:CDRH2:CDRH2:CDRH1:CDRL1:CDRH4:CDRL2:CDRL3:CDRL3:CDRL6 (as of SEQ ID NO: 3).

[0040] In a further embodiment, the antigen-binding protein includes CDRH3 (SEQ ID NO: 184), CDRH2 (SEQ ID NO: 183), CDRH1 (SEQ ID NO: 182), CDRL1 (SEQ ID NO: 185), CDRL2 (SEQ ID NO: 186), and CDRL3 (SEQ ID NO: 187).

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

[0042] The antigen-binding protein of the present invention may include heavy-chain and light-chain variable regions of the present invention that can be formatted to the structure of a natural antibody or a functional fragment or equivalent thereof. Accordingly, the antigen-binding protein of the present invention may include the VH region of the present invention, which, when paired with a suitable light chain, can be formatted to a full-length antibody, a (Fab')2 fragment, a Fab fragment, or equivalent thereof (e.g., scFV, bi-body, tri-body, or tetra-body, Tandab, etc.). 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, the antigen-binding protein may include all classes, e.g., IgG dimers that no longer bind to the Fc receptor or no longer mediate C1q binding, modified Fc variants, etc. The antigen-binding protein may also be a chimeric antibody of the type described in WO86 / 01533, which includes an antigen-binding region and a non-immunoglobulin region.

[0043] The constant region is selected according to the desired functionality. For example, IgG1 can demonstrate lysis ability by binding to complement and / or mediate ADCC (antibody-dependent cell-mediated cytotoxicity).

[0044] The antigen-binding proteins of the present invention are derived from mouse antibodies having the variable regions described in SEQ ID NOs: 7 and SEQ ID NOs: 9, or their non-mouse equivalents, such as their rat, human, chimeric, or humanized variants. For example, they are derived from antibodies having the variable heavy chain sequences described in SEQ ID NOs: 11, 13, 15, 17, 19, 21, 23, 25, 27, and 29, and / or the variable light chain sequences described in SEQ ID NOs: 31, 33, and / or 35.

[0045] In another embodiment, the antigen-binding protein of the present invention is derived from an antibody having a variable heavy chain sequence described in SEQ ID NO: 116 or SEQ ID NO: 118 and / or a variable light chain sequence described 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 a variable heavy chain sequence described in SEQ ID NO: 140 and / or a variable light chain sequence described in SEQ ID NO: 144.

[0047] In one embodiment of the present invention, an antigen-binding protein is provided that includes 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 embodiment of the present invention, an antigen-binding protein is provided comprising an isolated light chain variable domain selected from any one of 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 embodiment of the present invention, an antigen-binding protein is provided 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 embodiment, the antigen-binding protein of the present invention includes 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 embodiment, the antigen-binding protein of the present invention includes 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 embodiment, the antigen-binding protein of the present invention includes 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 embodiment, the antigen-binding protein of the present invention includes 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 embodiment, the antigen-binding protein of the present invention includes 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 embodiment, a polynucleotide encoding an isolated variable heavy chain is provided, comprising SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 117, SEQ ID NO: 119, or SEQ ID NO: 141.

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

[0055] In a further embodiment, polynucleotides encoding an isolated variable heavy chain, comprising SEQ ID NO: 24, SEQ ID NO: 28, or SEQ ID NO: 30, and polynucleotides encoding an isolated variable light chain, comprising SEQ ID NO: 32, or SEQ ID NO: 34, are provided.

[0056] In a further embodiment, a polynucleotide encoding an isolated variable heavy chain, comprising SEQ ID NO: 24, and a polynucleotide encoding an isolated variable light chain, comprising SEQ ID NO: 32 are provided.

[0057] In a further embodiment, a polynucleotide encoding an isolated variable heavy chain, comprising SEQ ID NO: 117, and a polynucleotide encoding an isolated variable light chain, comprising SEQ ID NO: 121 are provided.

[0058] In a further embodiment, a polynucleotide encoding an isolated variable heavy chain, comprising SEQ ID NO: 119, and a polynucleotide encoding an isolated variable light chain, comprising SEQ ID NO: 123, are provided.

[0059] In a further embodiment, a polynucleotide encoding an isolated variable heavy chain, comprising SEQ ID NO: 141, and a polynucleotide encoding an isolated variable light chain, comprising SEQ ID NO: 145 are provided.

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

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

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

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

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

[0065] In one embodiment of the present invention, an antibody having the heavy chain sequence described in SEQ ID NO: 55, SEQ ID NO: 59, or SEQ ID NO: 61 is provided.

[0066] In one embodiment of the present invention, an antibody having the light chain sequence described in SEQ ID NO: 63 or SEQ ID NO: 65 is provided.

[0067] In a further embodiment of the present invention, an antibody having the heavy chain sequence of SEQ ID NO: 55 and the light chain sequence described in SEQ ID NO: 63 is provided.

[0068] In one embodiment, an antigen-binding protein is provided that competes with the antigen-binding protein of the present invention as described herein. In one such embodiment, therefore, an antigen-binding protein is provided that competes with the 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, an antigen-binding protein is provided that competes with an antigen-binding protein comprising a heavy chain variable sequence selected from one of SEQ ID NOs: 27, 29, 116, 118, and 140, and a light chain variable region selected from one of SEQ ID NOs: 31, 120, 122, and 144.

[0070] In another embodiment, the antigen-binding protein binds to human BCMA with high affinity, and when measured by Biacore, for example, the antigen-binding protein binds to human BCMA with affinity of 20 nM or less, or 15 nM or less, or 5 nM or less, or 1000 pM or less, or 500 pM or less, or 400 pM or less, or 300 pM or less, or for example, about 120 pM. In a further embodiment, the antigen-binding protein binds to human BCMA 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, when measured by Biacore. In one embodiment of the present invention, the antigen-binding protein binds to BCMA with 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 embodiment, the antigen-binding protein binds to human BCMA and neutralizes the binding of ligand BAFF and / or APRIL to the BCMA receptor in a cell neutralization assay, wherein the antigen-binding protein has an IC50 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 present invention, the antigen-binding protein binds to BCMA and neutralizes BCMA in a cell neutralization 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, as described, for example, in Example 4.6.

[0073] Antigen-binding proteins, such as the antibodies of the present invention, can be produced by transfection of host cells with an expression vector containing the coding sequence for the antigen-binding protein of the present invention. The expression vector or recombinant plasmid is produced by incorporating these coding sequences for the antigen-binding protein operably bound to conventional regulatory sequences that can control replication and expression in and / or secretions derived from host cells. The regulatory sequences include promoter sequences, such as the CMV promoter and signal sequences that may be derived from other known antibodies. Similarly, a second expression vector having DNA sequences encoding the complementary antigen-binding protein light or heavy chain can be produced. In certain embodiments, this second expression vector is identical to the first, except that the coding sequences and selectable markers are relevant to ensure that each polypeptide chain is functionally expressed as much as possible. Alternatively, the heavy and light chain coding sequences for the antigen-binding protein may be present in a single vector.

[0074] Selected host cells are co-transfected (or simply transfected with a single vector) using both the first and second vectors by conventional techniques to generate transfected host cells of the present 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 present invention. Antigen-binding proteins containing binding of both recombinant heavy and / or light chains are screened from the culture by appropriate assays such as ELISA or RIA. Similar conventional techniques can be used to construct other antigen-binding proteins.

[0075] Those skilled in the art can select a suitable vector for the cloning and subcloning steps used in the method 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 is easily replicated, has numerous 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 can also be characterized by heterologous DNA sequences, such as genes suitable for amplifying the expression of the mammalian dihydrofolate reductase gene (DHFR). Other vector sequences include poly(A) signaling sequences derived from bovine growth hormone (BGH) and the beta-taglobin promoter sequence (betaglopro). 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, select genes, enhancers, promoters, and signal sequences, may be obtained from commercial or natural sources, or they may be synthesized by known procedures for use in driving the expression and / or secretion of recombinant DNA products within a selected host. Many other suitable expression vectors 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 sequence of the antigen-binding protein of the present invention. There are conventional host cells useful for cloning and other operations of these cloning vectors. However, cells derived from various strains of Escherichia coli (E. coli) may also be used for replication of the cloning vectors and other steps in the construction of the antigen-binding protein of the present invention.

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

[0080] Alternatively, other eukaryotic cell lines may be used. Methods for selecting appropriate mammalian host cells and for transforming, culturing, amplifying, screening, producing, and purifying the products are known in the art. See, for example, Sambrook et al. cited above.

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

[0082] If desired, yeast cell strains known to those skilled in the art can also be used as host cells and insect cells, such as Drosophila and Lepidoptera, and as virus expression systems. See, for example, Miller et al, Genetic Engineering, 8:277-298, Plenum Press (1986) and the references cited therein.

[0083] The general methods by which vectors can 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 are all prior art. Typically, the culture method of the present invention is a serum-free culture method, usually by culturing cells in a 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, and gel electrophoresis. Such techniques are within the scope of the art and do not limit the present invention. For example, the preparation of the modified antibodies is described in WO99 / 58679 and WO96 / 16990.

[0084] Another method for expressing antigen-binding proteins may utilize expression in transgenic animals, as described in U.S. Patent No. 4,873,316. ​​This relates to an expression system using an animal casein promoter that, when incorporated into a mammal by gene transfer, allows the female to produce the desired recombinant protein in her milk.

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

[0086] According to the present invention, a method for producing an anti-BCMA antibody that binds to human BCMA and neutralizes the activity of human BCMA, The steps include providing a first vector encoding the heavy chain of an antibody, The steps include providing a second vector encoding the light chain of the antibody, The steps include transforming mammalian host cells (e.g., CHO) with the first and second vectors, Step (c) is to culture the host cells of step (c) under conditions that induce the secretion of antibodies from the host cells into the culture medium, Step (d) is the step of collecting the secreted antibodies and A method is provided that includes this.

[0087] Once expressed in the desired manner, the antibody is then tested for in vitro activity using an appropriate assay. Currently, conventional ELISA assay formats are used to evaluate the qualitative and quantitative binding of antibodies to BCMA. Furthermore, other in vitro activities can be used to verify neutralizing effects before subsequent human clinical studies to assess the persistence of antibodies in the body despite normal clearance mechanisms.

[0088] The dosage and duration of treatment may be adjusted by those skilled in the art, depending on the condition being treated and the patient's overall health, with respect to the duration of involvement of the molecule of the present invention in the human circulation. It is also intended that repeated administration over a long period (e.g., 4 to 6 months) (e.g., once a week, once every two weeks, or once every three weeks) may be necessary to achieve the maximum therapeutic effect.

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

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

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

[0092] In another embodiment of the present invention, a method is provided for producing an antibody comprising a constant region or an antigen-binding fragment thereof ligated to a constant region according to the present invention as described herein, the method comprising the step of culturing host cells in a culture medium, for example, a serum-free medium.

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

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

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

[0096] The mode of administration of the therapeutic agent of the present invention may be any suitable route for delivering the drug to the host. The antigen-binding protein and pharmaceutical composition of the present invention are particularly useful for parenteral administration, i.e., subcutaneous (sc), intrathecal, intraperitoneal, intramuscular (im), or intravenous (iv). In one such embodiment, the antigen-binding protein of the present invention is administered intravenously or subcutaneously.

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

[0098] Thus, the pharmaceutical composition of the present invention for intravenous injection can be configured to contain about 250 ml of sterile Ringer's solution and about 1 to about 30 or 5 mg to about 25 mg of the antigen-binding protein of the present invention per 1 ml of Ringer's solution. Practical methods for preparing parenterally administered compositions are well known or evident to those skilled in the art, for example, Remington's Pharmaceutical Science, 15 th The preparation of the antigen-binding protein formulation of the present invention, which can be administered intravenously, 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) See 1033-1039, Johnson, R, "Mannitol-sucrose mixtures-versatile formulations for protein peroxidise 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 these are incorporated herein by reference and are explicitly provided to the reader).

[0099] In one embodiment, the therapeutic agent of the present invention, when present in a pharmaceutical formulation, exists in the form of a single dose. A suitable therapeutic effective dose can be easily determined by those skilled in the art. The appropriate dose can be calculated for the patient according to the patient's weight. For example, the appropriate dose may be in the range of about 0.1 to about 20 mg / kg, for example about 1 to about 20 mg / kg, for example about 10 to about 20 mg / kg, or for example about 1 to about 15 mg / kg, for example about 10 to about 15 mg / kg, or for example 1 to 5 mg / kg. In one embodiment, the antibody is administered at a dose of 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 of the antigen-binding protein of the present invention may be within the range of approximately 0.1 to approximately 1000 mg, for example, approximately 0.1 to approximately 500 mg, for example, approximately 500 mg, for example, approximately 0.1 to approximately 100 mg, or approximately 0.1 to approximately 80 mg, or approximately 0.1 to approximately 60 mg, or approximately 0.1 to approximately 40 mg, or for example, approximately 1 to approximately 100 mg, or approximately 1 to approximately 50 mg, and the antigen-binding protein can be administered parenterally, for example subcutaneously, intravenously, or intramuscularly. Such doses can be repeated at appropriate time intervals as needed by a physician.

[0100] The antigen-binding proteins described herein may be freeze-dried for storage and reconstituted in a suitable carrier before use. This technique has been shown to be more effective than conventional immunoglobulins and peroxides known in the art, and the reconstitution technique can be utilized.

[0101] In another embodiment of the present invention, antigen-binding proteins described herein for pharmaceutical use are provided.

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

[0103] One embodiment of the present invention provides a method for treating cancer in a human, comprising the step of administering to a human a antigen-binding protein that specifically binds to BCMA. In some examples, the antigen-binding protein is part of an immune conjugate.

[0104] In another embodiment of the present invention, a patient develops neutralizing antibodies against recombinant protein replacement therapy in multiple myeloma (MM), chronic lymphocytic leukemia (CLL), nonsecretory multiple myeloma, and smoldering multiple myeloma. The antigen-binding proteins described herein are provided for use in the treatment of B-cell-mediated or plasma cell-mediated diseases or antibody-mediated diseases or disorders selected from any non-Hodgkin lymphoma B-cell leukemia or Hodgkin lymphoma (HL) or any disease having BCMA expression, and the method comprises the step of administering a therapeutically effective amount of the antigen-binding protein described herein to the patient.

[0105] B-cell disorders can be divided into defects in B-cell development / immunoglobulin production (immune deficiency) and excessive / uncontrolled proliferation (lymphoma, leukemia). As used herein, B-cell disorder refers to both types of diseases, and methods for treating B-cell disorders using antigen-binding proteins are provided.

[0106] In a particular embodiment, the disease or disorder is selected from the group consisting of multiple myeloma (MM), chronic lymphocytic leukemia (CLL), solitary plasmacytoma (bone, extramedullary), and Waldenström macroglobulinemia.

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

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

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

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

[0111] The use of antigen-binding proteins described herein in the manufacture of pharmaceuticals for the treatment of diseases and disorders described herein is also provided.

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

[0113] In one aspect of the present invention, the use of an antigen-binding protein described herein for 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 is provided.

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

[0115] In one embodiment, the present invention relates to the development of neutralizing antibodies against recombinant protein replacement therapy in patients with rheumatoid arthritis, type 1 diabetes, multiple sclerosis or psoriasis, or multiple myeloma (MM), chronic lymphocytic leukemia (CLL), monoclonal immunoglobulinemia of unknown significance (MGUS), smoldering multiple myeloma (SMM), solitary plasmacytoma (bone, extramedullary), Waldenström macroglobulinemia, primary amyloidosis (AL), heavy chain disease, systemic lupus erythematosus (SLE), POEMS syndrome / osteosclerosing myeloma, type I and type II cryoglobulins. The present invention provides a pharmaceutical composition comprising an antigen-binding protein or a functional fragment thereof and a pharmaceutically acceptable carrier for treating or preventing antibody-mediated or plasma cell-mediated diseases or disorders selected from hememia, light chain deposition disease, Goodpasture syndrome, idiopathic thrombocytopenic purpura (ITP), acute glomerulonephritis, pemphigus and bullous pemphigoid disorders, and acquired epidermolysis bullosa, any non-Hodgkin lymphoma and leukemia having BCMA expression, or any other disease, the method comprising the step of administering a therapeutically effective amount of the antigen-binding protein described herein to the patient.

[0116] Another embodiment of the present invention provides a method for 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 neutralizing antibodies to recombinant protein replacement therapy, the method comprising the step of administering a therapeutically effective amount of the antigen-binding protein according to the present invention as described herein, for example, a method for 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 against recombinant protein replacement therapy in multiple myeloma (MM), chronic lymphocytic leukemia (CLL), monoclonal immunoglobulinemia of unknown significance (MGUS), smoldering multiple myeloma (SMM), solitary plasmacytoma (bone, extramedullary), Waldenström macroglobulinemia, primary amyloidosis (AL), heavy chain disease, systemic lupus erythematosus (SLE), POEMS syndrome / osteosclerosing myeloma, type I and type II cryoglobulinemia, light chain deposition disease, and other conditions. The antigen-binding proteins according to the present invention are provided herein for use in the treatment of antibody-mediated or plasma cell-mediated diseases or disorders selected from dopasture syndrome, idiopathic thrombocytopenic purpura (ITP), acute glomerulonephritis, pemphigus and bullous pemphigoid disorders, and acquired epidermolysis bullosa, any non-Hodgkin lymphoma and leukemia having BCMA expression, or any other disease, the method comprising the step of administering a pharmaceutical composition comprising the antigen-binding proteins according to the present invention in conjunction with a pharmaceutically acceptable carrier.

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

[0118] definition As used herein, the terms “cancer,” “neoplasm,” and “tumor” are interchangeable and refer to cells that have undergone malignant transformation, either singular or plural, that have become pathological to a host organism. Primary cancer cells can be readily distinguished from non-cancerous 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 cancer cell ancestors. 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 due to 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) scans, magnetic resonance imaging (MRI), X-rays, ultrasound, or palpation during a physical examination. Tumors may also be hematopoietic (or hematologic, hematological, or blood-related) cancers, which may be referred to as “humoral tumors,” and may originate from blood cells or immune cells. Specific examples of clinical symptoms based on hematological malignancies include leukemias such as chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, and acute lymphoblastic leukemia; plasma cell malignancies such as multiple myeloma, MGUS, and Waldenström macroglobulin; and lymphomas such as non-Hodgkin lymphoma and Hodgkin lymphoma.

[0119] Cancer may be any cancer diagnosed as a hematological malignancy, including both lymphoid and myeloid malignancies, characterized by the presence of an abnormal number of blast cells or undesirable cell proliferation. Myeloid malignancies include, but are not limited to, acute myeloid (or myeloid, myeloid, or myeloblastic) leukemia (undifferentiated or differentiated), acute promyelolytic (or promyelocytic, promyelogenous, or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia, and megakaryoblastic (or megakaryoblastic) leukemia. These leukemias are sometimes collectively referred to as acute myeloid (or myeloid, or myeloid) leukemia (AML). Myeloid malignancies also include, but are not limited to, chronic myeloid (or myeloid) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and myeloproliferative disorders (MPD), including polycythemia vera (PCV). Myeloid malignancies also include myelodysplasia (or myelodysplastic syndromes or MDS), which may also be called refractory anemia (RA), refractory anemia with superblasts (RAEB), and refractory anemia with superblasts in transformation (RAEBT), as well as myelofibrosis (MFS), with or without primary myelofibrosis.

[0120] Hematopoietic carcinomas also include lymphoid malignancies that may affect lymph nodes, spleen, bone marrow, peripheral blood, and / or extranodal sites. Lymphoid carcinomas include, but are not limited to, B-cell malignancies, including B-cell non-Hodgkin lymphoma (B-NHL). B-NHL may be painless (or low-grade), intermediate-grade (or aggressive), or high-grade (very aggressive). Painless 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 hairy lymphocytes, lymphoplasmacytic lymphoma (LPL), and mucosa-associated lymphoid tissue (MALT or extranodal marginal zone) lymphoma. Intermediate-grade B-NHL includes leukemia-associated or unassociated mantle cell lymphoma (MCL), diffuse large cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade 3B) lymphoma, and primary mediastinal lymphoma (PML). Grade B-NHL includes Burkitt lymphoma (BL), Burkitt-like lymphoma, small non-incisional nuclear cell lymphoma (SNCCL), and lymphoblastic lymphoma. Other B-NHL include immunoblastic lymphoma (or immunocytoma), primary exudate, HIV-associated (or AIDS-associated) lymphoma, and post-transplant lymphoproliferative disorder (PTLD) or lymphoma. Other examples of B-cell malignancies include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenström macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocyte (LGL) leukemia, acute lymphoblastic (or lymphocytic or lymphoblastic) leukemia, and Castlman's disease. Other examples of NHLs include, but are not limited to, T-cell non-Hodgkin lymphoma, unless 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 T-cell non-Hodgkin lymphoma (T-NHL), including Sézary syndrome.

[0121] Hematopoietic carcinomas also include Hodgkin lymphomas (or diseases), including classical Hodgkin lymphoma, nodular sclerotic Hodgkin lymphoma, mixed cell Hodgkin lymphoma, lymphocyte-dominant (LP) Hodgkin lymphoma, nodular LP Hodgkin lymphoma, and lymphopenic Hodgkin lymphoma. Hematopoietic carcinomas also include plasma cell diseases or cancers such as smoldering MM, monoclonal immunoglobulinemia of unknown significance (MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenström macroglobulinemia, plasma cell leukemia, and multiple myeloma (MM), including primary amyloidosis (AL). Hematopoietic carcinomas may also include other cancers of further 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 tissue," include bone marrow, peripheral blood, thymus and spleen, lymph nodes, mucosal lymphoid tissues (such as intestinal lymphoid tissue), tonsils, Peyer's patches and appendages, and other mucosal lymphoid tissues, such as peripheral lymphoid tissues like the bronchial lining.

[0122] As used herein, the term “antigen-binding protein” refers to antibodies, antibody fragments, and other protein constructs that can bind to and neutralize human BCMA.

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

[0124] The term "antibody" is used herein in a broad sense and includes, in particular, monoclonal antibodies (full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies).

[0125] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of individual antibodies that are substantially homogeneous, i.e., identical except for possible naturally occurring mutants that may exist in small amounts. Monoclonal antibodies are highly specific, directed to a single antigen-binding site. Furthermore, in contrast to polyclonal antibody preparations, which contain different antibodies directed to typically different determinants (epitopes), each monoclonal antibody is directed to a single determinant on an antigen.

[0126] A "chimeric antibody" refers to a type of manipulated antibody in which a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular donor antibody class or subclass, but which originates from a different species or belongs to a different antibody class or subclass, and insofar as they exhibit the desired biological activity, the remaining chain(s) in such an antibody fragment are identical or homologous to the corresponding sequence (U.S. Patent No. 4,816,567 and Morrison et al. Proc. Natl. Acad. Sci. USA 81:6851-6855) (1984).

[0127] A “humanized antibody” refers to a type of modified antibody that has its CDR derived from a non-human donor immunoglobulin, with the remaining immunoglobulin-derived portion of the molecule derived from one (or more) human immunoglobulins. In addition, framework-supporting 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)). A suitable human acceptor antibody may be selected from conventional databases, such as the KABAT® database, the Los Alamos database, and the Swiss Protein Database, based on homology with the nucleotide and amino acid sequences of the donor antibody. Human antibodies characterized by homology with the framework region of the donor antibody (based on amino acids) may be suitable for providing a heavy chain constant region and / or heavy chain variable framework region for donor CDR insertion. A suitable acceptor antibody capable of donating a constant or variable framework region of the light chain can also be selected. It should be noted that the heavy and light chains of the acceptor antibody do not need 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 specified sequences thereof, that, when optimally aligned and compared, are identical in at least approximately 80% of nucleotides, approximately 90% to approximately 95% of nucleotides, or at least approximately 98% to approximately 99.5% of suitable nucleotide insertions or deletions. Alternatively, substantially identical segments exist if they hybridize with the chain complement under selective hybridization conditions. “Identical” means, in some cases, a comparison calculated using the algorithms given in (1) and (2) below for polynucleotides and polypeptides: (1) Identity of a polynucleotide is determined by multiplying the total number of nucleotides in a given sequence by an integer (divided by 100) that defines the identity percentage, and then subtracting the product from the total number of nucleotides in the sequence, or: nn ≤ xn - (xn·y) [In the formula, 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%, and · is the symbol for the multiplication operator, and any non-integer product of xn and y is rounded down to the nearest integer before being subtracted from xn.] This is calculated by [the following method]. Modifications to the polynucleotide sequence encoding a polypeptide may result in nonsense, missense, or frameshift mutations in this encoding sequence, thereby altering the polypeptide encoded by the polynucleotide after such modification.

[0129] (2) Identity with respect to a polypeptide is determined by multiplying the total number of amino acids by an integer (divided by 100) that defines the identity percentage, and then subtracting the product from the aforementioned total number of amino acids, or: na ≤ xa - (xa·y) [In the formula, na is the number of amino acid modifications, 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 symbol for the multiplication operator. Any non-integer product of xa and y is rounded down to the nearest integer before being subtracted from xa.] It is calculated by [this method].

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

[0131] "Isolated" means that it has been modified "by human hands" from its natural state, and has been altered 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 that has been separated from its naturally occurring coexisting material is "isolated," including, but not limited to, cases where such polynucleotide or polypeptide has been reintroduced into a cell, even if the cell is of the same species or type (from which the polynucleotide or polypeptide was isolated).

[0132] Throughout this specification and the accompanying claims, the terms “contains” and “include” are confined to “consist of” and “consist of.” That is, these terms are intended to convey the possible inclusion of other elements or integers not specifically enumerated, where the context allows.

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

[0134] When used throughout this specification with respect to the antigen-binding proteins of the present invention, the term “inhibits” means that the biological activity of BCMA is reduced in the presence of the antigen-binding protein of the present invention compared to the activity of BCMA in the absence of such antigen-binding protein. Inhibition may result from, but not limited to, blocking ligand binding of one or more ligands, preventing ligands from activating receptors, and / or downregulation of BCMA. Inhibition may also refer to the binding of antigen-binding proteins to BCMA and the resulting cell apoptosis or ADCC. The antibodies of the present invention can neutralize the activity of BCMA ligand BAFF and / or APRIL binding to BCMA. The level of neutralization can be measured by several methods, for example, by using the assay described in 4.4 in the following examples, for example, in the H929 cell NFκB signaling assay. BCMA ligands BAFF and APRIL can induce NFκB signaling and downstream events after binding to BCMA. Neutralization of BCMA in this assay is measured by evaluating the ability of an anti-BCMA monoclonal antibody to inhibit NFκB induction driven by BAFF or APRIL.

[0135] If an antibody or its antigen-binding fragment can neutralize the target, 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, less than 20 micrograms / ml, less than 10 micrograms / ml, less than 5 micrograms / ml, 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 sequence of an antibody, which is the hypervariable domain of the immunoglobulin heavy chain and light chain. Three heavy chain and three light chain CDRs (or CDR regions) exist in the variable region of the immunoglobulin. Thus, as used herein, "CDR" may refer to all three heavy chain CDRs, or all three light chain CDRs (or, where appropriate, both all heavy chains and all light chain CDRs).

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

[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 by the Chothia numbering system (Al-Lazikani et al., (1997) JMB 273, 927-948), the method for defining contact points (MacCallum RM and Martin ACR and Thornton JM, (1996), Journal of Molecular Biology, 262(5), 732-745), or by any other established method for numbering residues in antibodies and determining CDRs that is 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 smallest overlapping region using at least two of the Kabat, Chothia, AbM, and Contact methods can be determined to provide a "minimal binding unit." The minimal binding unit may be a subpart of the CDR.

[0140] Table A below shows one definition using each numbering rule 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 CDR definitions may differ depending on the individual publication used.

[0141] Throughout 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 described 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 substitutions are preferred. In one such embodiment, the substitution is a conservative substitution.

[0143] In an alternative embodiment, the variant sequence contains at least one substitution while maintaining the canonicality of the antigen-binding protein.

[0144] Complementarity-determining regions (CDRs) L1, L2, L3, H1, and H2 tend to structurally represent one of a finite number of main chain conformations. A particular canonical structural class of CDRs is defined by both the length of the CDR and the loop packing (which structurally determines the residues or SDRs) determined by residues in critical positions in both the CDR and framework regions. Martin and Thornton (1996, J Mol Biol 263:800-815) developed an automated method to define canonical templates for “critical residues.” Cluster analysis is used to define canonical classes for sets of CDRs, and then canonical templates are identified by analyzing embedded hydrophobic hydrogen-bonding residues and, for example, conserved glycine. CDRs of antibody sequences can be assigned to canonical classes by comparing their sequences to 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 chain variable domain and light chain variable domain of the antibody, respectively.

[0146] As used herein, the term “domain” refers to a folded protein structure having a tertiary structure independent of the rest of the protein. Generally, domains are responsible for distinct functional properties of a protein and can often be added to, removed from, or transferred to other proteins without losing the function of the rest of the protein and / or the domain. A “single antibody-variable domain” is a folded polypeptide domain containing sequences characteristic of an antibody-variable domain. Thus, it includes complete antibody-variable domains and modified variable domains (e.g., one or more loops replaced with sequences not characteristic of the antibody-variable domain), or antibody-variable domains in which the N- or C-terminal extension is cleaved or contains such sequences, 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 monovariate domain” refers to an antibody monovariate domain (VH, VHH, VL) that specifically binds to an antigen or epitope independently of a different V region or domain. An immunoglobulin monovariate domain can exist in a format (e.g., homo or heteromultimer) with other different variable regions or domains, in which case the other regions or domains are not required for antigen binding by the monoimmunoglobulin monovariate domain (i.e., the immunoglobulin monovariate domain binds to the antigen independently of any new variable domain). “Domain antibody” or “dAb” is the same as “immunoglobulin monovariate domain” capable of binding to an antigen, as used herein. An immunoglobulin monovariate domain may be a human antibody monovariate domain, but may also include monoantibody monovariate domains from other species, such as rodent (e.g., disclosed in WO00 / 29004), nurse shark, and camel VHH dAbs. Camel VHH is an immunoglobulin monovariate domain polypeptide derived from species including camels, llamas, alpacas, dromedaries, and guanacos, which naturally produce heavy-chain antibodies lacking light chains. Such VHH domains can be humanized by 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 the camel VHH domain. NARV is another type of immunoglobulin monovariate domain identified in cartilaginous fish, including nurse sharks. These domains are also known as novel antigen receptor variable regions (usually 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 of a different V region or domain. This may be a domain antibody (dAb), such as a single variable domain of human, camel, or shark immunoglobulin, 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 GroEl and GroES, 29eroxidise29g (trans-body), ankyrin repeat protein (DARPin), peptide aptamers, C-type lectin domains (tetranectin), human γ-crystallin and human ubiquitin (affilin), PDZ domains, or the human protease inhibitor scorpion toxinknitz. The domains may be derivatives of scaffolds selected from the group consisting of toxinkunitz type domains and fibronectin (adnectin), and these are subjected to protein manipulation to obtain binding to ligands other than innate ligands.

[0149] CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is a CD28- family receptor primarily expressed on CD4+ T cells. Its extracellular domain has a variable domain-like Ig fold. The loop corresponding to the antibody's CDR can be substituted with heterologous sequences to confer different binding properties. CTLA-4 molecules engineered to have different binding specificities are also known as organisms. For further details, see Journal of Immunological Methods 248(1-2), 31-45(2001).

[0150] Lipocalin is a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bilines, retinoids, and lipids. They possess a robust β-sheet secondary structure with many loops at the open end of a conical structure, which can be manipulated to bind to various target antigens. Antikarin is an amino acid between 160 and 180 in size, derived from lipocalin. For further details, see Biochim Biophys Acta 1482:337-350 (2000), U.S. Patent No. 7,250,297B1, and U.S. Patent Application Publication No. 20070224633.

[0151] The affibody is a scaffold derived from Staphylococcus aureus protein A that can be manipulated to bind to antigens. This domain consists of three helical bundles of approximately 58 amino acids. The library is generated by randomization of surface residues. For further details, see Protein Eng. Des. Sel. 17, 455-462 (2004) and European Patent No. 1641818A1.

[0152] Abimer is a multidomain protein derived from the A-domain scaffold family. Its undenatured domain, approximately 35 amino acids long, adopts a specific disulfide bond structure. Diversity is generated by the mixture of spontaneous 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 manipulated to bind to various target antigens by inserting peptide sequences into its permissible surface loop. Examples of manipulated transferrin scaffolds include transbodies. For further details, see J. Biol. Chem 274, 24066-24073 (1999).

[0154] Designer ankyrin repeat proteins (DARPin) are derived from ankyrin, a family of proteins that mediate the linkage of membrane-bound proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif consisting of two α-helices and β-turns. These can be manipulated to bind to various target antigens by randomizing the residues in the first α-helix and β-turn of each repeat. These binding surfaces can be increased by increasing the number of molecules (affinity maturation method). 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 manipulated to bind to antigens. Adnectin consists of a native amino acid sequence backbone of the 10th domain of 15 repeat units in human type III fibronectin (FN3). Three loops at one end of the β-sandwich can be manipulated to allow adnectin to specifically recognize the therapeutic target 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. 6,818,418B1.

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

[0157] Microbodies are derived from naturally occurring microproteins with a length of 25–50 amino acids, containing 3–4 cysteine ​​bridges. Examples of microproteins include Kalata B1, and conotoxins and nottin. Microproteins have loops that can be manipulated to contain up to 25 amino acids without affecting the overall folding of the microprotein. For further details on the manipulated nottin domain, see WO2008098796.

[0158] Other epitope-binding domains include proteins that have been used as scaffolds to manipulate various target antigen-binding properties, such as human γ-crystallin and human ubiquitin (affilin), the Kunitz-type domain of human protease inhibitors, the PDZ-domain of the Ras-binding protein AF-6, scorpion venom (caribudotoxin), and the C-type lectin domain (tetranectin), which have been 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 domain 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 that can specifically bind to an antigen, which may be a single domain, such as an epitope-binding domain, or a paired VH / VL domain, as can be found in standard antibodies. In some embodiments of the present invention, a single-stranded Fv(ScFv) domain can provide an antigen-binding site.

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

[0161] As used herein, the term “antigen-binding protein” refers to antibodies, antibody fragments, e.g., domain antibodies (dAb), ScFv, Fab, Fab2, and other protein constructs. An antigen-binding molecule may include at least one Ig variable domain, e.g., 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 monovariate domain, e.g., VH, VHH, or VL, which specifically binds an antigen or epitope independently of different V regions or domains. An antigen-binding molecule can bind to two targets; i.e., they may be bitargeted proteins. An antigen-binding molecule may be a combination of an antibody and an antigen-binding fragment, e.g., one or more domain antibodies and / or one or more ScFv linked to a monoclonal antibody. The antigen-binding molecule may also include domains that are derivatives of scaffolds selected from the group consisting of non-Ig domains, e.g., CTLA-4 (epibody), lipocalin, protein A-derived molecules such as the Z-domain (afibody, SpA) and A-domain (avimer / maxibody) of protein A, heat shock proteins such as GroEl and GroES, 31eroxidize 31g (transbody), ankyrin repeat protein (DARPin), peptide aptamers, C-type lectin domains (tetranectin), human γ-crystallin and human ubiquitin (afirin), PDZ domains, scorpion toxin Knitz-type domains of human protease inhibitors, and fibronectin (adnectin), which are subjected to protein manipulation to obtain binding to OSM. As used herein, the "antigen-binding protein" can antagonize and / or neutralize human OSM. In addition, antigen-binding proteins can inhibit and block OSM activity by binding to OSM and preventing native ligands from binding to and / or activating the gp130 receptor.

[0162] As used herein, the term “effector function” means one or more of the following: antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC)-mediated reactions, Fc-mediated phagocytosis, and antibody reuse via FcRn receptors. For IgG antibodies, effector functions, including ADCC and ADCP, are mediated by the interaction between a family of Fcγ receptors present on the surface of immune cells and their heavy chain constant regions. 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 antigen-binding proteins and various Fc receptors (FcRs) is thought to mediate the effector function of antigen-binding proteins. Significant biological effects may result from effector function, particularly antibody-dependent cell-mediated cytotoxicity (ADCC), complement fixation (complement-dependent cell-mediated cytotoxicity or CDC), and the half-life / clearance of antigen-binding proteins. Typically, the ability to mediate effector function requires the binding of the antigen-binding protein to the antigen, and not all antigen-binding proteins mediate all effector functions.

[0164] Effector function can be measured in numerous ways, including, for example, by measuring ADCC effector function via the binding of FcγRIII to natural killer cells or via the binding of FcγRI to monocytes / macrophages. For example, the antigen-binding protein of the present invention can be evaluated 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] An example of an assay for measuring CDC function is described in 1995 J Imm Meth 184:29-38.

[0166] Several isotypes of the human constant region, particularly the IgG4 and IgG2 isotypes, essentially lack the functions of a) classical pathway complement activation and b) antibody-dependent cell-mediated cytotoxicity. Depending on the desired effector properties, various modifications may be made to the heavy chain constant region of antigen-binding proteins. The constant region of IgG1 containing specific mutations has been separately reported to reduce binding to the Fc receptor and thus decrease 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] One embodiment of the present invention provides an antigen-binding protein comprising a constant region such that the antigen-binding protein reduces ADCC and / or complement activity or effector function. In one such embodiment, the heavy chain constant region may include an innately non-existent constant region of an IgG2 or IgG4 isotype or a mutant IgG1 constant region. An example of a suitable modification is described in European Patent No. 0307434. One example involves the substitution of an alanine residue at positions 235 and 237 (EU index numbering).

[0168] Human IgG1 constant regions containing specific mutations or modified glycosylations at residue Asn297 have also been reported to enhance binding to the Fc receptor. In several 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 present invention, such mutations are located at one or more positions selected from 239, 332, and 330 (IgG1), or at equivalent positions in other IgG isotypes. Examples of suitable mutations are S239D, I332E, and A330L. In one embodiment, the antigen-binding protein of the present invention as described herein is mutated at positions 239 and 332, for example, S239D and I332E, or in a further embodiment, it is mutated at three or more positions selected from 239, 332, and 330, for example, S239D, I332E, and A330L (EU index numbering).

[0170] In alternative embodiments of the present invention, an antigen-binding protein is provided comprising a heavy chain constant region having a modified glycosylation profile to have 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 generating antigen-binding proteins having modified glycosylation profiles are described in WO2003011878, WO2006014679 and European Patent No. 1229125, all of which are applicable to the antigen-binding proteins of the present invention.

[0171] The present invention also relates to a method for producing an antigen-binding protein according to the present invention, a) A step of culturing recombinant host cells comprising an expression vector containing isolated nucleic acids as described herein, wherein the FUT8 gene encoding alpha-1,6-fucosyltransferase is inactivated in the recombinant host cells, b) A step of recovering antigen-binding protein and This provides a method that includes [something].

[0172] Such a method for producing antigen-binding proteins can be carried out, for example, using the Potelligent® technical system available from BioWa, Inc. (Princeton, NJ), in which CHOK1SV cells lacking a functional copy of the FUT8 gene produce a monoclonal antibody with enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) activity that is increased against the same monoclonal antibody produced in cells possessing the functional FUT8 gene. Embodiments of the Potelligent® technical system are described in U.S. Patents 7,214,775, 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, an antigen-binding protein is provided that includes a chimeric heavy chain constant region, for example, an antigen-binding protein that includes a chimeric heavy chain constant region having at least one CH2 domain derived from IgG3 so that the antigen-binding protein has enhanced effector function, for example, enhanced ADCC or enhanced CDC, or enhanced ADCC and CDC function. In one such embodiment, the antigen-binding protein may include one CH2 domain derived from IgG3, or both CH2 domains may be derived from IgG3.

[0174] A method for producing an antigen-binding protein according to the present invention, a) A step of culturing recombinant host cells comprising an expression vector comprising isolated nucleic acids as described herein, wherein the expression vector comprises a nucleic acid sequence encoding an Fc domain having IgG1 and IgG3Fc domain amino acid residues, b) A step of recovering antigen-binding protein and Methods including the above are also provided.

[0175] Such a method for producing antigen-binding proteins can be carried out, for example, using the Compligent® technical system available from BioWa, Inc. (Princeton, NJ) and Kyowa Hakko Kogyo Co., Ltd. (now Kyowa Hakko Kirin Co., Ltd.). In this system, recombinant host cells containing an expression vector encoding a chimeric Fc domain having both IgG1 and IgG3 Fc domain amino acid residues in the nucleic acid sequence are expressed to produce an antigen-binding protein having enhanced complement-dependent cytotoxicity (CDC) activity, which is increased compared to other identical antigen-binding proteins lacking such a chimeric Fc domain. Embodiments of the Compligent® technical system are described in WO2007011041 and U.S. Patent Application Publication No. 20070148165 (each of which is incorporated herein by reference). In alternative embodiments, 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 understand that such modifications can be used individually, as well as in combination with each other to further enhance the effects function.

[0177] In one such embodiment of the present invention, an antigen-binding protein is provided comprising a heavy chain constant region including a mutant and chimeric heavy chain constant region, wherein the antigen-binding protein comprises at least one CH2 domain derived from IgG3 and one CH2 domain derived from IgG1, and the antigen-binding protein has an 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, wherein the IgG1 CH2 domain has one or more mutations (for example, the mutations can be selected from S239D, I332E, and A330L) at positions selected from 239, 332, and 330. In one embodiment, the IgG1 CH2 domain has mutations S239D and I332E.

[0178] In an alternative embodiment of the present invention, an antigen-binding protein is provided that comprises a chimeric heavy chain constant region and has a modified glycosylation profile. In one such embodiment, the heavy chain constant region comprises at least one CH2 domain derived from IgG3 and one CH2 domain derived from IgG1, and the fucose-to-mannose ratio is 0.8:3 or less, for example, the antigen-binding protein is defucosylated so that the antigen-binding protein has enhanced effector function compared to an equivalent antigen-binding protein having an immunoglobulin heavy chain constant region lacking the mutation and modified glycosylation profile, for example, it has one or more of the following functions: enhanced ADCC or enhanced CDC, for example, it has an enhanced ADCC and an enhanced CDC, thus having a modified glycosylation profile.

[0179] In alternative embodiments, 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 limitations described herein.

[0180] In one aspect of the present invention, a method for producing an antigen-binding protein according to the present invention as described herein, a) A step of culturing recombinant host cells 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 the FUT8 gene encoding alpha-1,6-fucosyltransferase is inactivated in the recombinant host cells, b) A step of recovering antigen-binding protein and A method is provided that includes this.

[0181] Such a method for producing antigen-binding proteins can be used, for example, with the acritamab™ technology system available from BioWa, Inc. (Princeton, NJ), which combines the Potelligent™ and Compligent™ technology systems, to produce antigen-binding proteins with enhanced activity for both ADCC and CDC, which are increased compared to other identical monoclonal antibodies that lack a chimeric Fc domain and have fucose on an oligosaccharide.

[0182] In yet another embodiment of the present invention, an antigen-binding protein is provided comprising a mutant and a chimeric heavy chain constant region, wherein the antigen-binding protein has a modified glycosylation profile such that the antigen-binding protein has enhanced effector function, for example, one or more of the following functions: enhanced ADCC function or enhanced CDC function. In one embodiment, the mutation is selected from positions 239, 332, and 330, for example, the mutation is selected from S239D, I332E, and A330L. In a further embodiment, the heavy chain constant region comprises at least one CH2 domain derived from IgG3 and one Ch2 domain derived from IgG1. In one embodiment, the fucose-to-mannose ratio is 0.8:3 or less, for example, the antigen-binding protein is defucosylated, so that the heavy chain constant region has a modified glycosylation profile such that the antigen-binding protein has enhanced effector function compared to an equivalent non-chimeric antigen-binding protein or an immunoglobulin heavy chain constant region lacking the mutant and modified glycosylation profile.

[0183] Immunoconjugate Also provided are immunoconjugates (also interchangeably referred to as “antibody-drug conjugates” or “ADCs”) comprising antigen-binding proteins according to the present invention as described herein, including, but not limited to, antibodies conjugated to one or more cytotoxic agents such as chemotherapeutic agents, drugs, growth inhibitors, toxins (e.g., protein toxins, enzymatically active toxins or fragments thereof of bacterial, fungal, plant, or animal origin), or radioisotopes (i.e., radioconjugates).

[0184] Immunoconjugates are 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. Patent No. 4,975,278). Immunoconjugates enable targeted delivery of drug components to tumors and intracellular accumulation there when systemic administration of non-conjugated drugs may result in unacceptable toxicity to normal cells and tumor cells to be 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 vindecine (Rowland et al., (1986) above).Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins such as lysine, 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), meitansinoids (European Patent No. 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. Examples of small molecule toxins include those listed in 53:3336-3342.

[0185] In one embodiment, the present invention has the following general structure: ABP-((linker) n -Ctx) m (In the formula, ABP is an antigen-binding protein, The linker is either absent or is one of the severable or non-severable linkers 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) Includes an immunoconjugate having [specific properties].

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

[0187] It is shown here.

[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 in the production of immunoconjugates are described herein. Enzymatically active toxins and fragments thereof that can be used include the A chain of diphtheria, nonbinding active fragments of diphtheria toxin, the A chain of exotoxin A (from Pseudomonas aeruginosa), the A chain of ricin, the A chain of abrin, the A chain of modeccin, alpha-sarcin, Aleurites fordii protein, dianthin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricothecene. See, for example, WO93 / 21232, published Oct. 28, 1993. A variety of radionuclides are available for the production of radiolabeled antibodies. Examples include 211 At, 212 Bi, 131 I, 131 In, 90 Y, and 186 Re.

[0189] The antigen-binding proteins of the present invention can also be conjugated to one or more toxins, including, but not limited to, calicheamycin, meitansinoids, drastatin, aurostatin, trichothecene, and CC1065, and derivatives of these toxins having toxic activity. Suitable cytotoxic agents include, but not limited to, dovaline-valine-dolaisoleunine-dolaproin-phenylalanine (MMAF) and monomethyl auristatin E (MMAE), auristatin including ester forms of MMAE, DNA minor groove binders, DNA minor groove alkylating agents, engine, lexitropsin, duocalmycin, taxanes including paclitaxel and docetaxel, puromycin, drastatin, meitansinoids, and vinca alkaloids. Specific cytotoxic agents include topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino-doxorubicin, drastatin-10, ethinomycin, combretatostatin, chalicheamicin, meitansin, DM-1, DM-4, ​​and netropsin. Other suitable cytotoxic agents include antitubulin agents such as auristatin, vinca alkaloids, podophyllotoxin, taxanes, baccatin derivatives, cryptophysins, meitansinoids, combretastatin, or drastatin. Examples of antitubulin agents include dimethylvaline-valine-drysoloiin-dlaproin-phenylalanine-p-phenylenediamine, MMAF, MMAE, auristatin E, vincristatin, vinblastine, vindesine, vinorelbine, VP-16, camptothecin, paclitaxel, docetaxel, epotilon A, epotilon B, nocodazole, cohirtine, colcimid, estramustine, semadolin, discodermolide, meitansine, DM-1, DM-4, ​​or eryuterobin.

[0190] Antibody-drug conjugates were produced by conjugating antibodies with the small molecule antitubulin agents monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF). In the case of MMAE, the linker consists of a thiol-reactive maleimide, a caproyl spacer, a dipeptide valine-citrulline, and p-aminobenzyloxycarbonyl, a 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, altering both the number of drugs bound to each antibody molecule (molar ratio [MR]) and the binding sites. The most common species has an MR of 4, while less common are substances with MRs of 0, 2, 6, and 8. The overall average drug-antibody MR is approximately 4.

[0191] Production of immune conjugates The binding site is cysteine ​​produced by a mild reduction of the interchain disulfide of the antibody that occurs while the antibody is immobilized on the protein G affinity resin (this allows for the use of a large excess of reagent without purifying the intermediate). During immobilization, the large excess of TCEP completely reduces the interchain disulfide but does not affect the binding of the antibody to the resin.

[0192] The number of thiols per antibody produced by this procedure depends on the antibody source and isotype. For example, human (and mouse-human chimeric) IgG1 has 4 reducing disulfides and produces 8 thiols upon complete reduction, while mouse IgG1 has 5 reducing disulfides and produces 10 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 an excess sufficient to ensure complete conjugation. However, ADCs with a small amount of drug per antibody can also be prepared from a fully reduced antibody by including a biologically inactive capping agent such as N-ethylmaleimide (NEM), which accounts for some of the thiols available on the antibody. When the maleimide-drug-linker and capping agent are added simultaneously to a fully reduced antibody in a large excess (at least 3 times), the two maleimide electrophiles compete for a limited number of available thiols. In this configuration, drug loading can be considered rate-controlled, as it is determined by the relative thiol reaction rates of the drug-linker and capping agent. Since the relative reaction rates of maleimide-drug-linker change significantly, the molar ratio of drug-linker to NEM present in the reaction mixture must be experimentally determined to reach a panel of ADCs with the desired level of drug loading. Table 2 summarizes the molar fractions of drug linkers SGD-1006 (vcMMAE) and SGD-1269 (mcMMAF) in NEM mixtures that produce ADCs with approximately 4 drugs per antibody for common human and mouse IgG isotypes.

[0193] Auristatin and Dorastatin In some embodiments, the immunoconjugate includes an antigen-binding protein or antibody conjugated to auristatin (U.S. Patent No. 5,635,483, U.S. Patent No. 5,780,588), which is a peptide analog or derivative of drastatin. Drastatin and auristatin 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 possess anticancer activity (U.S. Patent No. 5,663,149) and antifungal activity (Pettit et al. (1998) Antimicrob. Agents Chemother. 42:2961-2965). The drug components of drastatin or auristatin (which is a pentaptide derivative of drastatin) can be attached to antibodies via the N (amino) or C (carboxyl) terminus of the peptide drug component (WO02 / 088172).

[0194] Exemplary embodiments of auristatin include the N-terminally conjugated monomethyl auristatin drug components DE and DF disclosed in "Monomethylvaline Compounds Capable of Conjugation to Ligands," U.S. Patent No. 7,498,298 (this disclosure 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 dovalin-valine-dry soloin-dlaproin-phenylalanine.

[0195] Typically, peptide-based drug components can be prepared by forming peptide bonds between two or more amino acids and / or peptide fragments. Such peptide bonds can be prepared, for example, by liquid-phase synthesis methods well known in the field of peptide chemistry (see E. Schroder and K. Lubke, "The Peptides," volume 1, pp 76-136, 1965, Academic Press). The auristatin / drastatin drug component can be prepared according to the methods described in U.S. Patent No. 5,635,483, U.S. Patent 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 Linkers,” U.S. Patent No. 7,498,298, filed November 5, 2004 (which is 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). Bioactive organic compounds acting as cytotoxic agents, particularly as pentapeptides, are disclosed in U.S. Patents No. 6,884,869, No. 7,498,298, No. 7,098,308, No. 7,256,257, and No. 7,423,116. Monoclonal antibodies conjugated to MMAE, MMAF, and various derivatives of auristatin, as well as methods for producing them, are described in U.S. Patent No. 7,964,566.

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

[0197] Maytansine and Maytansinoids Maytansinoids are mitototic inhibitors that act by inhibiting tubulin polymerization. Maytansin was first isolated from the East African shrub Maytenus serrata (U.S. Patent No. 3,896,111). Subsequently, it was discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol ester (U.S. Patent No. 4,151,042). Highly cytotoxic maytansinoid drugs can be prepared from anthamitosin precursors produced by fermentation of microorganisms such as Actinosynnema. A method for isolating anthamitosin is described in U.S. Patent No. 6,573,074. Synthetic meitansinol and its derivatives and analogs are, for example, 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, 4,309, This information is disclosed in Nos. 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 conjugating an antibody to a maytansinoid molecule without significantly reducing the biological activity of either the antibody or the maytansinoid molecule. See, for example, U.S. Patent No. 5,208,020. An average of 3-4 maytansinoid molecules conjugated per antibody molecule has been shown to be effective in enhancing cytotoxicity to target cells without adversely affecting antibody function or solubility, although even a single molecule of toxin / antibody is expected to enhance cytotoxicity compared to the use of a naked antibody.

[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 in other literature and non-patent literature referenced herein above. Maytansinoids are maytansinol and maytansinol analogs modified within or at other positions of the aromatic ring of maytansinol molecules, such as various maytansinol esters. Methods for preparing maytansinoids for binding to antibodies are disclosed in U.S. Patents No. 6,570,024 and No. 6,884,874.

[0200] Kalicheamycin The calicheamicin family of antibiotics can cause double-stranded DNA disruption at sub-picomole concentrations. For preparations of calicheamicin family conjugates, see U.S. Patents 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 from the American Cyanamid Company). Structural analogues of calicheamicin that may be used include, but are not limited to, gamma-1I, alpha-2I, alpha-3I, N-acetyl-gamma-1I, PSAG, and theta-I1 (Hinman et al., Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research 58:2925-2928 (1998), and the aforementioned US patent by American Cyanamid). Another antitumor agent that can be conjugated with antibodies is the folate antagonist QFA. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, the uptake of these drugs into cells by antibody-mediated internalization enhances their cytotoxic effects.

[0201] Other cytotoxic agents Other antitumor agents that can be conjugated to antibodies include BCNU, streptoycin, vincristine, and 5-fluorouracil, a family of drugs known collectively as the LL-E33288 conjugate, as described in U.S. Patents 5,053,394 and 5,770,710, respectively, as well as esperamicine (U.S. Patent 5,877,296).

[0202] Enzymatically active toxins and their fragments that may be used include diphtheria A chain, unbound active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), lysine A chain, abrin A chain, modexin A chain, alpha-sarcin, aleurites fordi protein, dianthin protein, phytraca americana protein (PAPI, PAPII, and PAP-S), Momordica charantia inhibitors, curcin, crotin, Sapaonaria officinalis inhibitors, geronin, mitogenin, restrictosin, phenomycin, enomycin, and trichothecenes. See, for example, WO93 / 21232, published October 28, 1993.

[0203] The present invention further intends to provide an immunoconjugate formed between an antibody and a compound having nucleic acid degradation activity (for example, a ribonuclease such as deoxyribonuclease or DNase, or a DNA endonuclease).

[0204] To selectively destroy tumors, antibodies may contain atoms with high radioactivity. Various radioisotopes are available to produce radioconjugated antibodies. Examples include the radioisotopes 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 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 suitable amino acid precursors containing, for example, fluorine-19 instead of hydrogen. Labels such as tc99m or I123, Re186, Re188, and In111 can be conjugated via cysteine ​​residues in the peptide. Yttrium-90 can be conjugated via lysine residues. The IODOGEN method (Fraker et al. (1978) Biochem. Biophys. Res. Commun. 80: 49-57) may be used to introduce iodine-123. "Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press 1989) describes other methods in detail.

[0206] ADC preparation In antibody-drug conjugates, antibodies can be conjugated directly to cytotoxic agents or via linkers. 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 that can be cleaved by intracellular proteases such as lysosomal proteases 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 that can be hydrolyzed at pH less than 5.5, such as hydrazone linkers. Further suitable cleavable linkers include disulfide linkers.

[0207] Bristol-Myers Squibb describes specific lysosomal enzyme-cleavable antitumor 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 those applications are limited to aminobenzyl ether compositions.

[0208] Conjugates of antigen-binding proteins and cytotoxic agents can be produced using various bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), difunctional derivatives of imide esters (e.g., dimethylHCl adipimidoate), active esters (disaxinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azide compounds (bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (1,5-difluoro-2,4-dinitrobenzene).

[0209] Furthermore, the linker may consist of one or more linker components. Examples of linker components 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-iodoacetyl)aminobenzoate ("SIAB"). Further linker components are known in the art and some are described herein. See also U.S. Patent No. 7,498,298, filed November 5, 2004, “Monomethylvaline Compounds Capable of Conjugation to Ligands” (the contents of which are incorporated herein by reference in their entirety).

[0210] The linker may also contain amino acids and / or amino acid analogs. Examples of 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 containing amino acid linker components include naturally occurring amino acids, as well as a small number of amino acids and unnaturally occurring amino acid analogs, such as citrulline. The amino acid linker components can be designed and optimized in terms of 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 may react to conjugate with linker reagents. Nucleophiles in antibodies include, but are not limited to, (i) N-terminal amine groups, (ii) side-chain amine groups, e.g., lysine, (iii) side-chain thiol groups, e.g., cysteine, and (iv) sugar hydroxyl or amino groups to which the antibody is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and can react to form covalent bonds with electrophiles in the linker moiety and linker reagents, including (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides, (ii) alkyl halides and benzyl halides such as haloacetamides, and (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducing interchain disulfides, i.e., cysteine ​​crosslinks. Antibodies may react to conjugate with linker reagents by treatment with reducing agents such as DTT (dithiothreitol). Therefore, each cysteine ​​crosslink theoretically forms two reactive thiol nucleophiles. Reaction of lysine with 2-iminothiolane (Traut's reagent) allows for the introduction of further nucleophiles into the antibody, resulting in the conversion of amines to thiols. Reactive thiol groups can be introduced into an antibody (or fragment thereof) by introducing one, two, three, four, or more cysteine ​​residues (for example, by preparing a mutant antibody containing one or more non-natural 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 sugar in a glycosylated antibody can be oxidized, for example, with a periodic acid oxidizing agent, to form an aldehyde or ketone group that can react with an amine group on the linker reagent or drug moiety. The resulting imine Schiff base can form a stable bond, or can be reduced, for example, by hydrogen boride to form a stable amine bond. In one embodiment, the reaction of the carbohydrate moiety of a glycosylated antibody with either galactose oxidase or sodium metaperiodate can produce a carbonyl group (aldehyde and ketone) in a protein that can react with a suitable group on the drug (Hermanson, Bioconjugate techniques). In another embodiment, a protein containing an N-terminal serine or threonine residue can react with sodium metaperiodate to produce an aldehyde instead of the initial amino acid (Geoghegan & Stroh, (1992) Bioconjugate Chem. 3:138-146, U.S. Patent No. 5,362,852). Such an aldehyde can react with a drug moiety or a linker nucleophile.

[0213] Examples of nucleophiles on the drug moiety include, but are not limited to, (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl halides and benzyl halides such as haloacetamides; and (iii) amines, thiols, hydroxyls, hydrazides, oximes, hydrazines, thiosemicarbazones, hydrazine carboxylates, and arylhydrazide groups that can react to form covalent bonds with the linker moiety containing aldehydes, ketones, carboxyls, and maleimide groups and with electrophiles on the linker reagent.

[0214] In some embodiments, the linker can be cleaved by cleavage agents present in the intracellular environment (e.g., within lysosomes, endosomes, or caveolae). The linker may be a peptidyl linker cleaved by intracellular peptidase or protease enzymes, including, but not limited to, lysosomal or endosomal proteases. Typically, the peptidyl linker is at least two amino acid long or at least three amino acid long. Examples of cleavage agents include cathepsins B and D and plasmin, all of which are known to hydrolyze dipeptide drug derivatives to release active drugs into target cells (see, e.g., Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). The peptidyl linker may be cleaved by enzymes 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., Phe-Leu or Gly-Phe-Leu-Gly (SEQ ID NO: 50) linker). Other such linkers are described, for example, in U.S. Patent No. 6,214,345. In certain embodiments, the intracellular protease-cleavable peptidyl linker is the Val-Cit linker or the Phe-Lys linker (see, for example, U.S. Patent No. 6,214,345, which describes the synthesis of doxorubicin by the val-cit linker). One advantage of using intracellular proteolytic release of a therapeutic agent is that when conjugated, the agent is typically attenuated and the serum stability of the conjugate is typically high.

[0215] In other embodiments, the cleavable linker is pH-sensitive, i.e., susceptible to hydrolysis at a specific pH value. Typically, pH-sensitive linkers hydrolyze under acidic conditions. For example, acid-unstable linkers that are hydrolyzable in lysosomes (e.g., hydrazone, semicarbazone, thiosemicarbazone, cis-aconitamide, orthoester, acetal, ketal, etc.) may be used. (See, for example, U.S. Patents 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 in blood, but unstable at pH 5.5 or below, which is the approximate pH of lysosomes. In certain embodiments, the hydrolyzable linker is a thioether linker (for example, a thioether bonded to the therapeutic agent via an acylhydrazone linkage (see, for example, U.S. Patent No. 5,622,929)).

[0216] In yet another embodiment, the linker is cleavable under reducing conditions (e.g., a disulfide linker). For example, various disulfide linkers are known in the art, including 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-pyridyldithio)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 (CW Vogel ed., Oxford U. Press, 1987; see also U.S. Patent No. 4,880,935)).

[0217] In further, other specific embodiments, the linker is a malonic acid linker (Johnson et al., 1995, Anticancer Res. 15:1387-93), a maleimide benzoyl 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 the linker means that when the ADC or ADC derivative is present in an extracellular environment (e.g., plasma), about 20% or less, typically about 15% or less, more typically about 10% or less, even more typically about 5% or less, about 3% or less, or about 1% or less of the linker is cleaved in a sample of the ADC or ADC derivative. Whether the linker is substantially insensitive to the extracellular environment can be determined, for example, by incubating both (a) an ADC or ADC derivative ("ADC sample") and (b) an equimolar amount of an unconjugated antibody or therapeutic agent ("control sample") independently with plasma for a predetermined period (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 intracellular integration. In certain embodiments, when conjugated to a therapeutic agent (i.e., in the environment of the linker-therapeutic agent portion of the ADC or ADC derivative described herein), the linker promotes intracellular integration. In yet another embodiment, when conjugated to both a therapeutic agent and an antigen-binding protein or its antibody or derivative (i.e., in the environment of the ADC or ADC derivative described herein), the linker promotes intracellular integration.

[0220] The compositions and methods and various linkers that can be used 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 in 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" (their disclosures are incorporated herein by reference).

[0221] Alternatively, a fusion protein containing an antigen-binding protein and a cytotoxic agent can be prepared, for example, by recombinant technology or peptide synthesis. The length of the DNA may include each region encoding two parts of the conjugate, either adjacent to each other or separated by a region encoding a linker peptide that does not disrupt 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 pre-targeting, and the antibody-receptor conjugate is administered to the patient, after which the unbound conjugate is removed from circulation using a washing agent, and then a "ligand" (e.g., avidin) that is conjugated to a cytotoxic agent (e.g., radioactive nucleotide) is administered.

[0223] As used herein, the term "non-human antibody or antibody fragment" means an antibody or fragment of which 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 provides a modified immunoglobulin coding region to a first immunoglobulin partner, thereby contributing to the amino acid sequence of its variable domain, CDR, or other functional fragment or analogue, so as to express a modified antibody having the antigen specificity and neutralizing activity properties of the donor antibody.

[0225] The term "acceptor antibody" refers to an antibody (monoclonal and / or recombinant) that is non-homologous to a donor antibody and contributes to all (or any, preferably all) of the amino acid sequence encoding the heavy chain and / or light chain framework region and / or the heavy chain and / or light chain constant region of a first immunoglobulin partner. Human antibodies are acceptor antibodies.

[0226] As used herein, the term "human acceptor sequence" means a framework of an antibody or antibody fragment that includes an amino acid sequence of a human consensus sequence framework, which may incorporate a VH or VL framework derived from a human antibody or antibody fragment, or a CDR derived from a non-human species.

[0227] As used herein, the terms "incorporation" of a CDR or hypervariable region encompass any means by which a non-human CDR is arranged with a human acceptor framework. It will be understood that this can be achieved in various ways. For example, a nucleic acid encoding a desired amino acid sequence may be generated by mutating a nucleic acid encoding a non-human variable domain sequence, thereby changing its framework residues to human acceptor framework residues; or a nucleic acid encoding a human variable domain sequence may be generated by mutating a nucleic acid, thereby changing the CDR to non-human residues; or a nucleic acid encoding a desired sequence may be synthesized. In one embodiment, the final sequence is generated in a computer.

[0228] The present invention is described herein by illustrative purposes only. The appended claims may include generalizations of one or more of the following embodiments. [Examples]

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

[0230] The anti-human BCMA mAb mouse parent S307118G03 was identified from hybridomas derived from SJL mice immunized with recombinant human BCMA / TNFRSF17-Fc chimera (R&D 193-Fc) using the RIMMS (Rapid Multi-site Immunization) method. On day 0, 5 ug of protein per mouse was emulsified in AS02a adjuvant at the dorsal (upper buttocks and shoulders) and at two sites below the major lymph nodes in four anterior sites. On days 6 and 11, 2.5 ug of protein in RIBI adjuvant per mouse was injected below the major lymph nodes in four anterior sites. The animals were sacrificed on day 14. Lymph nodes and spleens were excised, lysed, and PEG1500-inducible somatic cell fusion was performed using a 3:1 ratio containing mouse myeloma cells X63 AG8 653.GFP.Bcl-2.11 (BioCat 112754;R17209 / 58). The fusions were deposited in 10 × 96 well plates and screened directly from them.

[0231] The anti-human BCMA mAb mouse parent S336105A07 was identified from a hybridoma derived from the same immunization. Lymph nodes and spleens were resected and lysed on day 14, and cytopulse electrofusion was performed using a 1:1 ratio of mouse myeloma cells X63 AG8 653.GFP.Bcl-2.11 (BioCat 112754;R17209 / 58). The fusions were deposited in omnitrays containing semi-solid medium before being collected in 10 × 96 well plates, and these were screened directly 5 days later.

[0232] The anti-human BCMA mouse parental mAbs S332121F02 and S332126E04 were identified from hybridomas derived from SJL mice immunized with recombinant Fc fusions of the extracellular domain of human BCMA(4-53)BCMA using the RIMMS (Rapid Immunization Mass Refining) method. On day 0, 5 ug of protein per mouse was emulsified in AS02a adjuvant at the dorsal (upper buttocks and shoulders) and at two sites below the major lymph nodes in four anterior sites. On day 6, 5 ug of recombinant cyno BCMA-Fc protein in RIBI adjuvant per mouse was injected below the major lymph nodes in four anterior sites. On day 11, 2.5 ug of recombinant human BCMA-Fc and 2.5 ug of recombinant cyno BCMA-Fc in RIBI adjuvant per mouse were injected below the major lymph nodes in four anterior sites. On the 14th, the animals were slaughtered and the cells were processed in the same way as for S307118G03.

[0233] Anti-human BCMA mouse parental mAb S322110D07 was identified from hybridomas derived from SJL mice immunized with a recombinant FC fusion of the extracellular domain of human BCMA (4-53) conjugated with recombinant human April (R&D 5860-AP / CF) premixed in a 1:1 molar ratio. Mice were immunized via ip with 5 ug of April / Cyno BCMA-Fc complex in PBS suspended in RIBI adjuvant (100 ul dose) per mouse, and three additional immunizations were administered at 3-4 week intervals using 2.5 ug of April / Cyno BCMA-Fc complex in PBS suspended in RIBI adjuvant (100 ul dose) per mouse injected via the intraperitoneal route. A pre-fusion immunization with the same immunogen was administered one day before fusion, and the mice were treated in the same manner as for S307118G03.

[0234] Anti-human BCMA mAb mouse parents 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 Multi-site Immunization) method. On day 0, 2.5 ug of each protein per mouse was emulsified in AS02a adjuvant and injected at four sites on the dorsal side (above the rump and shoulder) and two sites on the anterior side below the major lymph nodes. On days 6 and 11, 2.5 ug of each protein in RIBI adjuvant per mouse was injected at four sites on the anterior side below the major lymph nodes. Animals were sacrificed on day 14. Lymph nodes and spleens were excised, lysed, and site-pulsed electrofusion was performed using a 1:1 ratio of mouse myeloma cells X63 AG8 653.GFP.Bcl-2.11 (BioCat 112754;R17209 / 58). The fusions were deposited in omni trays containing semi-solid medium before being collected in 32×96 well plates, and these were screened directly from 5 days later.

[0235] [Example 2] Humanization 2.1 Cloning of the CA8 hybridoma variable region 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). For RT-PCR, the forward primers were a mixture of degenerate primers specific to the mouse immunoglobulin gene reader sequence, while the reverse primers were specific to the antibody constant region. Since the isotypes were unknown, reverse primers for IgG1, IgG2a, and IgG2b were used in this case. To design the primers, mouse V H and V k DNA multi-sequence alignments of the gene leader sequences were generated.

[0236] 2.2 Cloning of Chimera CA8 A DNA expression construct encoding a chimeric antibody was newly prepared by constructing duplicate oligonucleotides containing restriction enzyme recognition sites for cloning within a mammalian expression vector and a human signal sequence. By introducing HindIII and SpeI restriction enzyme recognition sites, a VH domain containing a signal sequence for cloning within a mammalian expression vector containing the human γ1 constant region was constructed. By introducing HindIII and BsiWI restriction enzyme recognition sites, a VL domain containing a signal sequence for cloning within a mammalian expression vector containing the human kappa constant region was constructed.

[0237] 2.3 Cloning of humanized CA8 variants A DNA expression construct encoding a humanized antibody variant was newly prepared by constructing duplicate oligonucleotides containing restriction enzyme recognition sites and human signal sequences for cloning within a mammalian expression vector. By introducing HindIII and SpeI restriction enzyme recognition sites, a VH domain containing a signal sequence for cloning within a mammalian expression vector containing the human γ1 constant region was constructed. By introducing HindIII and BsiWI restriction enzyme recognition sites, a VL domain containing a signal sequence for cloning within a mammalian expression vector containing the human kappa constant region was constructed.

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

[0239] 2.5 Production of defucosylated antibodies To generate defucosylated antibodies, the heavy and light chains were co-transfected into CHO DG44 MS705 BioWa cells and expressed at a level sufficient to produce antibodies. Briefly, 30 μg of DNA was linearized overnight using Not1, the DNA was precipitated with ethanol, and redissolved in TE buffer. From the culture, 2.4 × 10⁷ BioWa DG44 cells were obtained and washed in 14 ml of warm PBS-sucrose. The cells were rotated, and the pellet was resuspended in 1.6 ml of PBS-sucrose. Half of the cells suspended in PBS-sucrose (0.8 ml) were added to a BioRad cuvette along with 30 μg of linearized DNA (in 50 μl of TE buffer). The BioRad GenePulser was programmed to 380 V using a 25 μF capacitance, and the cuvette was placed in for electroporation. The resulting 850 μl of electroporated cells and DNA were added to (80 ml) warm SFM512 medium (containing phenol red, 2XHT (nucleoside), glutamax, and Gibco Supplement 4). Finally, the resulting 80 ml cell suspension was transferred to each well of one 4 × 96-well plate (150 μl / well). After 48 hours, the medium was changed to be nucleoside-free by removing approximately 130 μl of acclimatization medium and replaced with 150 μl of fresh selective medium, SFM512 medium (containing phenol red and glutamax). Every 3-4 days, 130-150 μl of acclimatization medium was removed and replaced with fresh selective medium. Wells were monitored for color changes and assays were performed for IgG concentration as previously described.

[0240] 2.6 Further cloning of antibody-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). For RT-PCR, the forward primers were a mixture of degenerate primers specific to the mouse immunoglobulin gene reader sequence, while the reverse primers were specific to the antibody constant region, in this case, isotype IgG2a. The 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 for subsequent validation of the accurate V-region sequences. DNA sequence data were obtained for the V-region products generated by RT-PCR.

[0241] 2.7 Further cloning of antibody-chimeric models A novel DNA expression construct encoding a chimeric antibody was prepared by infusion-advantage PCR cloning (Clonetech) of the V gene PCR product within a mammalian expression vector. This cloning method enabled the fusion of the mouse variable region with the constant regions of the human IgG1 H chain and kappa L chain.

[0242] 2.8 S307118G03 - Cloning of humanized variants Cloning was performed in the same manner as described in paragraph 2.3.

[0243] 2.9 Expression of recombinant antibody S307118G03 Expression plasmids encoding the associated heavy and light chains (listed in Table 8 below) were transiently co-transfected into HEK293 6E cells and expressed at a small level to produce antibodies. The antibodies were purified protein A from the supernatant and quantified using a nanodrop spectrophotometer.

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

[0245] [Example 3] Conjugation of antibodies against vcMMAE and mcMMAF for generating antibody-drug conjugates (ADCs) Table B: Chemical Structure of Drug-Linker [ka]

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

[0247] The conjugated antibody was then reduced by adding 10 mM TCEP, 150 mM NaCl, pH 7, and 500 μL of 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 washing solution was removed by centrifugation, and the process was repeated three times for a total of four washes. The conjugated and reduced antibody was then conjugated using a mixture of NEM and drug linker prepared according to the mole 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 to be added to each well. For example, for mouse IgG1 with 5 reducing disulfides (10 available thiols when reduced), 0.5 mg of antibody at 150 kD is 3.33 nmol, corresponding to 33.3 nmol of thiols. Therefore, a 3-fold excess is 100 nmol of total maleimide or 10 μl of a 10 mM drug linker / NEM mixture. For the SGD-1269 conjugate, this mixture was then prepared with 5.86 μL of SGD-1269 and 4.14 μL of NEM. Next, the maleimide mixture was diluted in 500 μL of PBS before being added to the immobilized reductive antibody. In practice, since multiple antibodies for each isotype were conjugated simultaneously with a single SGD-1269 / NEM, a mixture for each isotype was prepared by multiplying the number of wells containing that isotype by 10 μL / well, and then diluted in a volume of PBS equal to 500 μL of 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 of the mixtures were then added to the reductive antibody (500 μL / well), and the plate was shaken at 22°C for 30 minutes. The plate was then centrifuged as described above to remove excess reaction solution, and then washed four times with PBS as before. The conjugated ADCs were 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 μL of neutralizing buffer (1 M potassium phosphate, pH 7.4, 500 mM NaCl, 0.2% Tween®-20) was added to each well of a 1 mL collection plate. The solution was then eluted in the collection plate by rotating the ADC at 1500 × g for 6 minutes. The collection plate was then briefly shaken to ensure complete mixing of the neutralizing buffer.

[0249] Next, the solution was transferred into a UV assay plate (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-1 cm-1 was used to give an appropriate estimate of the ADC concentration across the panel. To confirm successful conjugation, the drug loading of the isotype controls was estimated using reverse-phase protein HPLC (described below). For plates containing the humanized variant of CA8, this method was used to directly estimate the loading of all ADCs.

[0250] The reverse-phase protein chromatography method for determining drug loading utilizes a PLRP-S polymer stationary phase (Agilent Technologies). Since the antibody was fully reduced during the conjugation process, all antibody subunits eluted from the column as single polypeptide chains, allowing for the separate evaluation of subpopulations of light and heavy chain species with various levels of drug loading. Therefore, the analysis of these data enables the calculation of the average light chain drug loading and the average heavy chain drug loading as independent factors, which can then be combined, based on the fundamental knowledge that each antibody consists of two light chains and two heavy chains, to determine the average antibody drug loading. The chromatographic conditions were as follows: a PLRP-S column, 1000 Å, 50 × 2.1 mm, 8 um particle size (Agilent Technologies), with mobile phase A being water + 0.05% TFA and mobile phase B being acetonitrile + 0.01% TFA, and elution with a linear gradient of 27% B to 42% B at 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 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 loads of each isotype control, determined again by PLRP chromatography and data, are summarized in Table 4. [Table 4]

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

[0254] The mean drug load and %CV for each isotype are shown at the bottom. Large, uncharacteristic variability in drug load was observed for SGD-1269 ADC prepared with mIgG2b antibody, the reason for which is unknown. Furthermore, Fc-enhanced CA8 antibodies produced somewhat lower drug load levels than other CA8 human variants. To address this, further Fc-enhanced CA8 was conjugated in a solution-layer reaction to better match the drug load achieved with other antibodies.

[0255] [Example 4] Combined data 4.1 FMAT binding assay demonstrating the binding of chimeric CA8 to human or cyno-BCMA expressing cells Cryopreserved transfected human, cyno-BCMA, and sham-transfected HEK293 cells were collected from LN2 storage. Assay wells were prepared with human chimeric CA8 antibody at a wide range of concentrations and mixed with human BCMA HEK293, cyno-BCMA HEK293, and sham-transfected cells, respectively. Anti-human IgG FMAT blue secondary conjugate was added to detect human chimeric CA8. Assay plates were left standing for at least 90 minutes before reading the results with an ABI8200 (FMAT) plate reader.

[0256] This demonstrated that the chimeric CA8 antibody successfully bound to both human and cyno BCMA proteins expressed in HEK293 cells.

[0257] The results are shown in Figure 1.

[0258] 4.2 ELISA experiment demonstrating the binding of chimeric CA8 to recombinant BCMA protein Chimeric CA8 antibodies were 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 using BSA to reduce nonspecific binding. CA8 chimeric antibodies were added to ELISA plates coated with human and cyno BCMA at concentrations ranging from 5 ug / ml to 0.1 ug / ml. If necessary, some conjugated human chimeric CA8 antibodies were detected using a secondary antibody conjugated with anti-human IgG HRP. The ELISA was further developed by adding HRP substrate (TMB). This demonstrated that the CA8 antibodies bound to recombinant human and cyno BCMA in the ELISA assay.

[0259] The results are shown in Figure 2.

[0260] 4.3 Biacore experiments demonstrating CA8 antibody binding to BCMA and TACI proteins to determine cross-reactivity with TACI proteins The CA8 chimeric antibody was injected and captured on Protein A. (A Protein A-derivatized sensor chip was used). The remaining Protein A binding was blocked by injection of a high concentration of human IgG solution. Then, BCMA-Fc, TACI-Fc or BAFF-R-Fc solution was tested for binding to the antibody. Three proteins were injected in sequence and the binding events were measured. The surface was regenerated between injections of each protein.

[0261] The sensorgram was analyzed in the Biaevaluation program. Double reference subtraction was performed to remove instrument noise and any non-specific binding from the sensorgram curve.

[0262] This showed that CA8 was specific for binding to BCMA and not specific for TACI and BAFFR.

[0263] The binding of the 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 mouse anti-BCMA antibodies to multiple myeloma cells and BCMA-expressing cells Multiple myeloma cell lines H929 and transfected cells expressing ARH77-hBCMA 10B5 BCMA were stained with mouse S332211D07, S3332121F02 or S332126E04 at 5 μg / mL or a mouse isotype control. Multiple myeloma cell line H929 was stained with mouse S307118G03. The cells were incubated at room temperature (RT) for 20 minutes and then washed with FACS buffer (PBS + 0.5% BSA + 0.1% sodium azide) to remove unbound antibody. The cells were incubated at RT for 15 minutes with a secondary PE-labeled anti-mouse IgG antibody and then washed with FACS buffer to remove unbound antibody. The cells were analyzed by FACS to detect the antibody bound to the cells.

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

[0266] 4.4.2 Binding curves of chimeric CA8 to multiple myeloma cells as determined by FACS The binding of chimeric CA8 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 unrelated antibody (Synagis) at various concentrations for 20 minutes in RT. The cells were then washed with FACS buffer (PBS + 0.5% BSA + 0.1% sodium azide) to remove unbound antibodies. The cells were incubated with secondary PE-labeled anti-human IgG antibody for 15 minutes in RT, and then washed with FACS buffer to remove unbound antibodies. The cells were analyzed by FACS, and binding was determined by measuring the mean fluorescence intensity (MFI) value.

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

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

[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 the binding of BAFF or APRIL to recombinant BCMA. The objective of this assay was to evaluate the ability of antibody CA8 and its humanized form J6M0, in both wild-type and afcosylated (potelligent) forms, at various concentrations to neutralize the binding ability of either BCMA ligand, BAFF, or APRIL.

[0271] A 96-well flat-bottom plate was coated overnight with a 1 μg / mL solution of recombinant human BCMA Fc4-53 in PBS. After washing with 0.05% TWEEN® 20, the plate was blocked at room temperature for 1 hour with a 2% bovine serum albumin solution in PBS. The plate was washed as described above, and 40 μL of each antibody (mouse IgG, mouse CA8, and chimeric CA8), titrated in a 1 / 2 ratio starting at 10 μg / mL in a double well, was added to the relevant wells and incubated at room temperature for 1 hour. 40 μl of 2% BSA was added to the relevant control well. 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 obtain final concentrations of 6 ng / mL and 150 ng / mL in each well. An equivalent volume of 2% BSA was added to the corresponding control wells. The plates were incubated at room temperature for 2 hours, and then washed as described above. Biotinylated anti-human ligand (BAFF BAF124 or APRIL BAF884, R&D Systems) was added to the corresponding wells at 50 ng / mL and incubated for 1 hour. After the washing step, a 1:4000 dilution of 50 μL of streptavidin-HRP (Amersham RPN4401) was added to each well and incubated at room temperature for 30 minutes. The washing process was repeated, and then 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. Optical density was determined at 450 nm using a Spectromax reader. See Figures 7A and B.

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

[0273] 4.6 Effects of chimeric CA8 and humanized J6M0 BCMA antibodies on BAFF or APRIL-induced phosphorylation of NFκB in H929 cells In one set of experiments, H-929 cells were seeded at 75,000 cells / well in serum-free medium in a 96-well plate. Chimeric CA8 antibody was added after 24 hours to obtain a final well concentration up to 200 ug / ml. After 10 minutes, BAFF or APRIL ligand was added to the cells to obtain a final well concentration of 0.6 or 0.3 ug / ml, respectively. After 30 minutes, the cells were lysed and phosphorylated Nf-kappa B levels were measured using the MSD pNF-kappa B assay.

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

[0275] Averaged data from two experiments. The IC50 was 10 nM for BAFF-induced Nf-kappa B neutralization and 257 nM for APRIL-induced Nf-kappa B neutralization (average of two independent experiments), as shown in Table 7. [Table 7]

[0276] Further sets of experiments were conducted to understand the reasons for such differences in the potency of APRIL and BAFF neutralization in cell-based systems. After discovering the soluble form of BCMA, the experimental design was modified to include a step of washing H929 cells before 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 a 96-well plate and obtained final concentrations of ≤100 ug / ml with BAFF or APRIL ligand, resulting in final concentrations of 0.6 or 0.2 ug / ml, respectively. H-929 cells were then seeded in serum-free medium at 7.5 × 10⁴ cells / well. After 30 minutes, the 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 IC50s required to inhibit BAFF and APRIL signaling were 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 using ProteON XPR36 (Biorad). The method was as follows: Protein A was immobilized on a GLC tip (Biorad, catalog number: 176-5011) by primary amine coupling, and then CA8 variants were captured on this surface. Recombinant human BCMA (in-house or commercially available US Biological, B0410) (performed only twice) was passed through at 256, 64, 16, 4, and 1 nM using 0 nM injection (i.e., buffer only) to double-reference the binding curve. The buffer used was HBS-EP buffer. The capture surface was regenerated using 50 mM NaOH. The data were fitted to a 1:1 model using the analytical software built into the ProteOn XPR36. Practice 1 corresponds to the first screening of humanized CA8 variants (J0-J5 series), and Practice 2 corresponds to the second screening of humanized CA8 variants (J5-J9 series). Both tests were conducted at 25°C.

[0278] Data from Experiment 1 are shown in Table 8, and data from Experiment 2 are shown in Table 9. In Experiment 2 (Table 9), some molecules did not yield measurable affinity values ​​by ProteOn, which was due to an off-rate exceeding the instrument's sensitivity in this assay. However, this indicates that all of these molecules bind strongly to recombinant human BCMA. From Experiment 1, the data shows that some constructs did not bind to recombinant cyno BCMA at all. [Table 8] [Table 9]

[0279] 4.8 BIAcore analysis of anti-BCMA CA8 chimeras and humanized constructs (J7-J9 series) Protein A was immobilized on a CM5 chip (GE Healthcare, catalog number: BR-1005-30) by primary amine coupling, and antibody molecules were then captured using this surface. Recombinant human BCMA (US Biological, B0410) was used as the sample at concentrations of 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 by buffer injection (i.e., 0 nM), and the data were fitted using the 1:1 model inherent in the T100 evaluation software. The procedure was performed at 37°C using HBS-EP as the running buffer.

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

[0281] 4.9 BIAcore analysis of anti-BCMA CA8 chimeras and humanized constructs J6M0 and J9M0 Protein A was immobilized on a CM5 chip (GE Healthcare, catalog number: BR-1005-30) by primary amine coupling, and antibody molecules were then captured using this surface. Recombinant human BCMA (US Biological, B0410) was used as the sample at concentrations of 256 nM, 64 nM, 16 nM, 4 nM, and 1 nM. The capture surface was regenerated using 50 mM NaOH. All binding curves were doubly referenced by buffer injection (i.e., 0 nM), and the data were fitted using the 1:1 model inherent in the T100 evaluation software. HBS-EP was used as the running buffer, and Experiment 1 was performed at 25°C and 37°C, while Experiment 2 was performed at 37°C only.

[0282] Both experiments identified J9M0 as the optimal molecule in terms of overall affinity to human BCMA. The data generated from this experiment are 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 using ProteOn XPR36 (Biorad). The method was as follows: Protein A was immobilized on a GLM tip (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 used for dual reference of the binding curve, and the mixture was passed through at 256, 64, 16, 4, and 1 nM using 0 nM injection (i.e., buffer only). The buffer used was HBS-EP buffer. The capture surface was regenerated using 50 mM NaOH. The data were fitted to a 1:1 model using the analytical software built into ProteOn XPR36. The procedure was performed at 25°C.

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

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

[0286] Results: Chimeric CA8 and defucosylated chimeric CA8 killed BCMA-expressing target cells via ADCC. Defucosylated chimeric antibodies showed sufficiently effective ADCC activity when measured by a higher lysis rate achieved in all tested target cells, and exhibited a 10-fold lower EC50 in the high-BCMA-expressing target cell line 10B5 compared to the parental chimeric antibody. See Figures 8A and 8B.

[0287] 5.2 ADCC activity of CA8 humanized antibodies using ARH77 BCMA-expressing target cells and PBMCs as effectors Human PBMCs were incubated for 2 hours with europium-labeled ARH77 BCMA-transfected target cells (10B5) in the presence of various concentrations of humanized CA8 antibody (5 ug / ml to 0.005 ug / ml) at a 5:1 E:T ratio. Europium release from the target cells was measured, and specific lysis was calculated.

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

[0289] 5.3 ADCC efficacy 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 for 2 hours with europium-labeled ARH77 BCMA-transfected target cells (10B5) in the presence of various antibody concentrations at a 5:1 E:T ratio. 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 chimeric CA8 antibodies, chimeric CA8-mcMMAF antibody drug conjugates, and chimeric CA8-vcMMAE antibody drug conjugates against human multiple myeloma cell lines was measured. Multiple myeloma cell lines were treated with chimeric CA8 antibody-drug conjugates to determine the ADC concentrations required for growth inhibition and cell death.

[0292] The tested antibody-drug conjugates were added to wells containing multiple myeloma cells at concentrations ranging from 1 ug / ml to 5 ng / ml. The plates were incubated at 37°C for 96 hours, at which point viable cells were quantified using Cell Titre Glo. The unconjugated chimeric CA8 antibody did not show significant growth inhibitory activity at the tested antibody concentrations. The chimeric CA8-mcMMAF antibody-drug conjugate showed higher growth inhibitory activity than the chimeric CA8-vcMMAE antibody-drug conjugate in all four tested multiple myeloma cell lines. 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 (of ADCs) induce growth inhibition in multiple myeloma cells, NCI-H929 cells were monitored by measuring cellular DNA content by propidium iodide staining of fixed cells at multiple time points after treatment with chimeric CA8 antibody and chimeric CA8 ADC.

[0295] At the tested chimeric CA8 ADC concentration (50 ng / mL), chimeric CA8-mcMMAF ADC induced significant G2 / M cell cycle arrest (4N DNA content), which peaked at 48 hours. Subsequently, at 48, 72, and 96 hours, treatment with chimeric CA8-mcMMAF ADC resulted in the accumulation of cell populations containing sub-2N DNA content, indicating cell death. At the tested concentration of 50 ng / mL, chimeric CA8-vcMMAE ADC did not have a significant effect on G2 / M cell cycle arrest or sub-G1 accumulation. See Figure 12.

[0296] 5.6 Phosphohistone H3 (Thr11) staining as a marker for mitotic arrest induced by chimeric CA8-mcMMAF antibody-drug conjugate and chimeric CA8-vcMMAE antibody-drug conjugate To determine whether the accumulation of cells with 4N DNA content is a specific consequence of chimeric CA8-induced mitotic arrest, ADC NCI-H929 cells were stained with an anti-phosphohistone H3 antibody after 48 hours of treatment with increased concentrations of unconjugated chimeric CA8, chimeric CA8-vcMMAE, or chimeric CA8-mcMMAF.

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

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

[0299] 5.8 Antibody-drug conjugate (ADC) activity of humanized variant of CA8 anti-BCMA antibody-drug conjugate 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 the plates were incubated for 144 hours. Growth inhibition in the presence of antibody or ADC was measured at 144 hours using CellTiterGlo. Data points represent the mean of three CellTiterGlo measurements. Error bars represent the standard error.

[0300] 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 cell death. The mcMMAF and vcMMAE antibody-drug conjugate forms of these antibodies showed significantly greater growth inhibitory activity compared to those found with CA8 chimeras. Variant J6M0 showed higher potency than the chimera, with data for H929 and OPM2 cells shown in Figure 15. The mcMMAF antibody-drug conjugate showed higher growth inhibitory activity than the vcMMAE antibody-drug conjugate 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 mouse anti-BCMA antibody-drug conjugates Cells were seeded in 96-well plates (4,000 cells per well in 100 μL of RPMI + 10% FBS). Antibodies or ADCs were added 6 hours after cell seeding, and the plates were incubated for 144 hours. Growth inhibition in the presence of ADCs was measured at 144 hours using CellTiterGlo. The mean of three CellTiterGlo measurements is shown. Tables 15a and 15b are from experiments performed at different times with different series of antibodies. 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 the mouse antibodies S322110D07, S332121F02, and S332136E04 showed significant growth inhibitory activity. The mcMMAF antibody-drug conjugate showed higher growth inhibitory activity than the vcMMAE antibody-drug conjugate for all mouse anti-BCMA antibodies tested in which activity was observed. The IC50 figures are shown in Table 15a. See Figure 16 for dose-response curves for these three antibodies and also for S107118G03. Error bars represent standard error. NCI-H929, U266-B1, JJN3, and OPM2 cells were treated with different series of mouse anti-BCMA antibody-drug conjugates for the antibodies in Table 15b to determine the ADC concentrations required for growth inhibition and death. The IC50 figures are shown in Table 15b. All five antibodies listed in that table had significant ADC activity. [Table 15]

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[0304] 5.10 ADCC efficacy of conjugated afcosylated J6M0 (Potelligent) Afcosylated J6M0 conjugated to MMAE or MMAF was tested in an ADCC assay using BCMA transformants to ensure that its ADCC activity was not impaired by conjugation. Europium-labeled ARH77-10B5 cells were incubated with various J6M0 WT and Potelligent BCMA antibodies at concentrations of ≤10,000 ng / ml for 30 minutes before adding PBMCs (PBMC:target cell ratio 50:1). After 2 hours, aliquots of cell medium were sampled and mixed with fortification solution. After 30 minutes in a plate shaker, europium release was monitored with a Victor 2 1420 multi-label reader. The data point represents the average of three consecutive values. This data represents 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 show how comparable its potency was to that of the afucosylated form. As expected, defucosylation resulted in a lower EC50 and higher maximal solubility. No solubility was observed in the Fc-inactive form of J6M0 (Figure 17).

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

[0307] The J6M0 Potelligent antibody exhibited ADCC activity against all five multiple myeloma cell lines tested. This was important to test, as earlier studies had been conducted using transfected cells. The results are shown in Figure 18. The complete dataset, including multiple donors, is shown in Table 16. The potency was all within the same range as that observed in transformants. ADCC activity was not directly related to BCMA surface expression in these cell lines. [Table 16]

[0308] [Example 6] Xenograft data 6.1 To ensure that antibody efficacy detected in vitro could also be demonstrated in vivo, mouse xenografts of human MM cell lines were tested. The cell line selected for the xenograft study was NCI-H929, which is sensitive to ADCs and ADCCs that die in vitro. The study was conducted in immunodeficient CB.17 SCID mice, which lack T and B cells but retain NK cells that enable ADCC activity. However, it should be noted that while human IgG1 can associate with the mouse Fc receptor, potelligent enhancement does not improve affinity when performed using the human Fc receptor.

[0309] 6.2 Effects of unconjugated and MMAE or MMAF-conjugated J6M0 on NCI-H929 tumor growth To independently analyze the ADCC and ADC activity 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 a combination of ADCC and functional inhibitory activity.

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

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

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

[0313] BCMA was detected using the R&D Systems Human BCMA / TNFRSF17 ELISA kit (catalog number DY193E), which measures soluble human BCMA levels, following the standard protocol for which the kit is supplied.

[0314] In short, a 96-well microplate was coated with 100 μl / well of capture antibody and incubated overnight at 4°C. The plate was washed three times with washing 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 washing buffer. 100 μl of serum sample or standard was added to each well and incubated at room temperature for 2 hours. The plate was washed three times with washing buffer, then 100 μl of detection antibody was added to each well and incubated at room temperature for 2 hours. After washing the plate three times, 100 μl of streptavidin-HRP was added to each well and incubated in a dark room for 20 minutes. The plate was washed three times, 50 μl of stop solution was added, and then measured using a microplate reader at a wavelength of 570 nM.

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

[0316] Triple dilution was performed, and patient and normal control serum samples had the measured BCMA levels. Serum levels of BCMA were significantly increased in serum from MM patients compared to normal controls in this study. When the disease subset was further divided, there was a tendency for serum levels of BCMA to be increased in serum from progressive MM patients compared to those in remission. This is the first report to identify serum BCMA in some human diseases, suggesting that these levels may be novel biomarkers for monitoring disease status and treatment response in MM patients and other patients with plasma cell-mediated diseases. [Table 17]

[0317] P-value (one-sided t-test, 95% significance) ~1-500 Single Normal vs. progressive: p = .0010 * Progressive vs. Remission: p=.0146 * ~1-500 Triple Normal vs. progressive: p = 0.0004 * 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 * This indicates significance. This application provides the following: 1. 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 can bind to FcγRIIIA, or can have an FcγRIIIA-mediated effector function, and can be internalized. 2. The antigen-binding protein described in 1 above, having enhanced binding to FcγRIIIA or enhanced FcγRIIIA-mediated effector function. 3. The antigen-binding protein described in 2 above, wherein the antigen-binding fragment has enhanced ADCC effector function. 4. An antigen-binding protein described in any of items 1-3 above, which is defucosylated. 5. An antigen-binding protein as described in any of items 1-4 above, wherein the antigen-binding fragment does not bind to Taci. 6. An antigen-binding protein according to any one of items 1 to 5 above, comprising CDRH3 of SEQ ID NO: 3 or a variant of SEQ ID NO: 3. 7. The antigen-binding protein described in 6 above, further comprising one or more of the following: 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 described in Sequence ID No. 3, ii) CDRH1 as described in Sequence ID No. 1, and iii) CDRH2 as described in Sequence ID No. 2, The antigen-binding protein described in item 7 above, including the antigen-binding protein described in item 7 above. 9. i) CDRH3 as described in Sequence ID No. 3, ii) CDRH1 as described in Sequence ID No. 1, iii) CDRH2 as described in Sequence ID No. 2, iv) CDRL1 as described in Sequence ID No. 4, v) CDRL2 as described in Sequence ID 5, and vi) CDRL3 described in sequence number 6, The antigen-binding protein described in item 8 above, including the antigen-binding protein described in item 8 above. 10. An antigen-binding protein according to any one of 1 to 9 above, comprising a heavy chain variable region encoded by any one of SEQ ID NO: 23, SEQ ID NO: 27, or SEQ ID NO: 29. 11. An antigen-binding protein according to any one of items 1 to 10 above, comprising a light chain variable region encoded by either SEQ ID NO: 31 or SEQ ID NO: 33. 12. An antigen-binding protein according to any one of 1 to 11 above, 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. An antigen-binding protein according to any one of 1 to 11 above, comprising a heavy chain encoded by SEQ ID NO: 27 and a light chain encoded by SEQ ID NO: 31. 14. An antigen-binding protein described in any of items 1 to 13 above, which is a humanized monoclonal antibody. 15. The antigen-binding protein described in 14 above, wherein the antibody is of the IgG1 isotype. 16. An antigen-binding protein comprising a CDR as described in any of 6-9 above, and which is a fragment that is Fab, Fab', F(ab')2, Fv, diabody, triabody, tetrabody, miniantibody, minibody, isolated VH, or isolated VL. 17. An antigen-binding protein described in any of items 1-16 above, which further binds to non-human primate BCMA. 18. An antigen-binding protein described in any of items 1-17 above, which binds to BCMA with an affinity stronger than 150 pM. 19. An immune conjugate comprising an antigen-binding protein and a cytotoxic agent as described in any of items 1 to 18 above. 20. The immunoconjugate described in 19 above, wherein an antigen-binding protein is bound to a cytotoxic agent via a linker. 21. The immunoconjugate according to 19 or 20 above, wherein the cytotoxic agent is auristatin or drostatin. 22. An immunoconjugate according to any one of items 19 to 21 above, wherein the cytotoxic agent is selected from MMAE and MMAF. 23. An immunoconjugate according to any one of items 19 to 22 above, wherein a cytotoxic agent is covalently bound to an antigen-binding protein. 24. An immunoconjugate according to any of items 20-23 above, wherein the linker is a cleavable linker. 25. An immunoconjugate as described in any of items 20-23 above, wherein the linker is not cleavable. 26. An immunoconjugate according to any one of 20-25 above, 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-iodoacetyl)aminobenzoate (SIAB). 27. An immune conjugate according to any of items 19 to 26 above, wherein the immune conjugate is phagocytosed by tumor cells upon contact with them. 28. A pharmaceutical composition comprising an antigen-binding protein or immunoconjugate described in any of items 1 to 27 above and a pharmaceutically acceptable carrier. 29. A method for treating a human patient suffering from an inflammatory disorder or disease, comprising the step of administering the composition described in 28 above. 30. Use of the composition described in item 27 above in the treatment of human patients suffering from B-cell lymphoma such as multiple myeloma (MM) or chronic lymphocytic leukemia (CLL). 31. An antigen-binding protein or immune conjugate described in any of items 1 to 27 above, for use in treating human patients with B-cell lymphoma, such as multiple myeloma (MM) or chronic lymphocytic leukemia (CLL).

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Claims

1. A method for producing an anti-B cell maturation antigen (BCMA) antibody, comprising the step of culturing recombinant host cells containing a first vector and a second vector, (a) The first vector is CDRH1 of sequence number 1, CDRH2 of sequence number 2, and The 5th position N is replaced with D in sequence number 3, CDRH3. It contains a polynucleotide that codes for, (b) The second vector is CDRL1 of sequence number 4, CDRL2 of sequence number 5, and CDRL3 of sequence number 6 Contains polynucleotides that code for The above method.

2. (a) The first vector contains a polynucleotide encoding the heavy chain variable region shown in SEQ ID NO: 23, and (b) The second vector contains a polynucleotide encoding the light chain variable region shown in SEQ ID NO: 31, The method according to claim 1.

3. (a) The first vector contains a polynucleotide encoding the heavy chain amino acid sequence shown in SEQ ID NO: 55, and (b) The second vector contains a polynucleotide encoding the light chain amino acid sequence shown in SEQ ID NO: 63, The method according to claim 1.

4. A method for producing an immunoconjugate, comprising attaching one or more cysteine ​​residues of an anti-BCMA antibody according to any one of claims 1 to 3 to a linker bound to a cytotoxic agent.

5. The method according to claim 4, wherein the cytotoxic agent is selected from calicheamycin, meitansinoids, drastatin, auristatin, trichoceten, DNA minor groove binders, DNA minor groove alkylating agents, enediyne, lexitropsin, duocalmycin, taxane, puromycin, vinca alkaloids, antitubulin agents, podophyllotoxin, baccatin derivatives, cryptophysin, and combretastatin.

6. The method according to claim 4, wherein the cytotoxic agent is selected from auristatin and drastatin.

7. The method according to claim 4, wherein the cytotoxic agent is monomethyl auristatin F (MMAF).

8. The method according to claim 4, wherein the cytotoxic agent is monomethyl auristatin E (MMAE).

9. The method according to any one of claims 4 to 8, wherein the linker is a severable linker.

10. The method according to any one of claims 4 to 8, wherein the linker is a linker that cannot be cut.

11. The method according to any one of claims 4 to 8, 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-iodoacetyl)aminobenzoate (SIAB).

12. The method according to any one of claims 4 to 8, wherein the linker is 6-maleimidocaproyl (MC).

13. The method according to claim 12, wherein the antibody is defucosylated.

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